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2009 Annual Report I. Table of Contents

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Cover 1

Table of Contents 2

Overview of the Activities of the Taube-Koret Center for HD Research 3–5

Oversight of the Taube-Koret Center for HD Research Biographies of our Advisors 6 Report from Dr. Pagno Paganetti 7–10 Report from Dr. Norbert Bischofberger 11–13

Publications and Presentations of the Taube-Koret Center for HD Research Bibliography of Publications 14 HD-related Academic Seminars 15 HD-related Industry Consultations and Seminars 16

Taube-Koret Center for HD Research and the Community Press releases 17–22 News stories 23–33 The Taube-Koret Center for HD Research and 34 HD Families

Appendix of Publications 35–113

2 II. Overview of the Activities of the Taube-Koret Center for Huntington’s Disease Research for 2009

We are pleased to provide this annual report describing the activities of the Taube-Koret Center for Huntington’s Disease Research during 2009. The Center was established in 2009 with a joint gift from Taube Philanthropies and the Koret Foundation. It has been a very exciting year. I am delighted to say that we exceeded all five of the scientific and financial goals we set for the first year of operation. Our progress in each area is described in detail below.

Goal 1. Establish the Taube-Koret Center for Huntington’s Disease Our initial goal was to establish a Center focused on developing therapeutics for Huntington’s disease (HD). We proposed to develop an infrastructure that would be capable of identifying and validating drug targets for HD and of discovering compounds that modify HD and have the potential to become drugs. The new Center is housed in rented space within the Gladstone Center for Translational Research at 1700 Owens Street and in existing space within the main research building of the J. David Gladstone Institutes at 1650 Owens Street. The new laboratories have been outfitted with equipment to evaluate potential HD drug targets and to synthesize potential new therapeutics. Substantial capabilities, including special robotics, have been added to our existing laboratories to carry out high-throughput screens to find new therapeutics. One silver lining of the global financial crisis last year was that it enabled us to purchase equipment and set up these laboratories for less than it would otherwise have cost.

Goal 2. Integrate industrial experience and capability into the academic framework In addition to the physical resources necessary to find HD therapeutics, we added critical human resources. Dr. Stephen Freedman provides assistance in prioritizing drug targets, designing screens, developing hits into lead programs, and negotiating relationships with potential industry partners. His decades of drug experience with Merck and Elan have proven to be extremely helpful. In addition, we recruited experts in medicinal chemistry to help us develop leads into potential drugs and established relationships with an array of contract research organizations that can perform critical steps in drug development that are not cost- effective to establish in house. We also recruited two external advisors of international reputation and drug discovery experience to provide a detailed scientific review of our program. Throughout the year, they have provided advice and oversight. In December, at our request, they made a site visit to review the program. The review meeting with Dr. Paolo Paganetti (Novartis) and Dr. Norbert Bischofberger (Gilead) was highly successful and added considerable input to our future direction. The detailed reports are provided below.

Goal 3. Implement a critical review process and focus on programs most likely to succeed Recognizing that our resources are limited, we implemented a hard-nosed strategy to periodically re-prioritize our programs as results from our experiments become available. Programs that fail to meet performance criteria are dropped, and resources are redeployed to more promising leads. Programs that meet performance criteria and progress to the point that they interest industry are favored. They lead to partnerships that bring in additional resources from our industry partners, which also allow us to redeploy resources of the Center to other

3 promising leads. Industry partners will eventually be needed to carry leads forward into clinical trials, which require resources that are currently beyond those of the Center. We began the year with 11 programs, spanning target identification, validation, and lead development. By year’s end, one program was dropped because it failed to meet performance milestones. Another program had progressed to the point that it garnered interest by two competing biotechnology companies, who delivered term sheets to form a partnership. Three new lead programs have been added.

Goal 4. Use a publication strategy to validate the scientific excellence of the Center, stimulate scientific discussion and promote scientific awareness in the Huntington’s disease field The scientific productivity of the Center during its first year has been exceptional. The Muchowski and Finkbeiner laboratories published 10 peer-reviewed papers describing results from their HD research programs. These studies revealed a range of pathogenic mechanisms in HD and therapeutic strategies. These include ground-breaking work on misfolding and abnormal clearance of huntingtin, critical neurobiology of cellular mechanisms to rid cells of protein aggregates, excessive neuroinflammation, new potential drugs to protect neurons against neurodegeneration induced by polyglutamine stretches, and new methods to use neurons to find therapeutics. A bibliography and copies of all the original publications from the Center in 2009 are enclosed.

Publication is the major mechanism for achieving international renown for our HD research program. Other mechanism are to accept invitations to speak about the work from the Center all over the world and to participate in service to the National Institutes of Health (NIH) and on scientific advisory boards (SABs) of drug companies working on HD. Drs. Muchowski and Finkbeiner both helped to guide NIH HD programs in 2009 and provided SAB service and consultation to 11 biotechnology and pharmaceutical companies. As a result of these and other activities, the Center has been featured in the popular press. Some of these news stories can be found in this annual report.

Goal 5. Leverage additional external funding to support the overall mission of the center Another important strategic feature of Center is our commitment to attract additional resources to leverage the investment by our donors. We were unusually successful in 2009, raising an additional $7.85M to support our HD therapeutics programs. A $1.7M grant from the prestigious Keck Foundation will enable us to establish a facility to study electrical activity in the region of the brain affected by HD in mice while they are awake and behaving. A $3.7M Grand Opportunity grant from the NIH will enable us to generate inducible pluripotent stem cells from skin tissue of adults with HD and use them to create human neurons we will use to search for new therapeutics. Further, the award itself provides additional recognition for the Center as one of the world’s leading sites for HD research. The remaining $2.45M came from the NIH in a series of smaller grants. We might never duplicate the fund raising success we experienced in 2009, but it was an encouraging start for the new Center nonetheless.

The Taube-Koret Center for Huntington’s Disease Research was established to facilitate the development of therapeutics for HD. We proposed a novel strategy to bridge the gap between academia and industry and to create a pipeline for therapeutics. This year, we expected to be heavily focused on building the infrastructure to develop therapeutics. However, we are pleased to report the unexpected news that two of our lead programs have already attracted industry interest. The fact that these programs have warranted industry interest is an important validation for the overall strategy of the Taube-Koret Center for Huntington’s Disease Research.

The need for HD therapeutics is clear. Overall, we are very pleased with the success of the Taube-Koret Center for HD Research during its first year of operation. We remain as committed as ever to the primary goal of the Center—to develop therapies that prevent, treat, and eventually cure HD.

Steven Finkbeiner, M.D., Ph.D. Paul Muchowski, Ph.D. Director, Taube-Koret Center Co-Director, Taube-Koret Center for Huntington’s Disease Research for Huntington’s Disease Research Associate Director, Senior Investigator Associate Investigator Gladstone Institute of Neurological Gladstone Institute of Neurological Disease Disease Professor, Departments of Neurology Associate Professor, Department and Physiology of Biochemistry and Biophysics UCSF UCSF

5 III. Reports of the External Advisors to the Taube-Koret Center for Huntington’s Disease Research

We seek to be transparent and accountable in our management of the gifts entrusted to us by the donors, which enabled us to establish the Taube-Koret Center for Huntington’s Disease Research. As part of this effort, we recruited expert external advisors to the Center to provide an outside perspective on our performance. Short biographies of our advisors can be found below. The advisors provided advice and oversight throughout 2009. On December 15, 2009, we organized a day-long meeting on-site with our external advisors, who reviewed the structure of the Center and our major lead programs. Their reports are reproduced verbatim below.

Biographies of the External Advisors to the Taube-Koret Center for Huntington’s Disease Research

Paolo Paganetti, PhD Head of Huntington’s Disease Research Novartis

Dr. Paganetti received his PhD from the University of Zurich, Switzerland in the lab of Prof. M.E. Schwab, in the Brain Research Institute. His postdoctoral research was done with Prof. Schwab and Prof. R.H. Scheller, HHMI and Stanford University. He joined Novartis in 1992 as a lab head and has occupied positions with increasing responsibilities. Within the neuroscience disease area, Dr. Paganetti was part of the Alzheimer’s disease team responsible for drug discovery programs for compounds reducing Ab-peptide secretion and inhibiting the aspartic protease BACE. Currently, he leads the Huntington’s disease team and is involved in several external research collaborations. He was mentor for six postdoctoral fellows, four PhD students and seven research assistants and leads a lab with five associates. Dr. Paganetti received the Novartis Leading Scientist award in 2003 and was appointed senior research investigator II in 2006. He is author of over 60 scientific publications.

Norbert W. Bischofberger, PhD Executive Vice President, Research and Development and Chief Scientific Officer Gilead Sciences

Dr. Bischofberger joined Gilead Sciences in 1990 and has served as executive vice president for research and development since 2000 and chief scientific officer since 2007. He oversees all of Gilead’s research discovery, preclinical & clinical development, pharmaceutical development and API manufacturing. Before joining Gilead, Dr. Bischofberger was a senior scientist in Genentech’s DNA Synthesis Group from 1986 until 1990. He received a PhD in organic chemistry from Zurich's Eidgenossische Technische Hochschule and performed postdoctoral research in steroid chemistry at Syntex. He also performed additional research in organic chemistry and applied enzymology in Professor George Whiteside’s lab at Harvard University.

6 Paolo Paganetti, PhD Senior Research Investigator II Novartis Institutes for BioMedical Research Novartis Pharma AG Basel, Switzerland

External Evaluation Taube-Koret Center for Huntington’s Disease Advisory Meeting of December 15, 2009

I had the great pleasure to actively participate at the advisory board meeting of the Taube-Koret Center for Huntington’s Disease as an external advisor. I was astonished by the clear and concise presentations of top scientific quality made by Dr. Steve Finkbeiner and Dr. Paul Muchowski and the other presenters, as well as by the focused drug discovery activities and the quality of the translational research advancing rapidly at the Center. The objective of the Taube-Koret Center is to find a cure for Huntington’s disease (HD) by 2020. HD is a progressive neurodegenerative genetic disorder that affects muscle coordination and some cognitive functions. Caused by a dominant mutation in a gene located on chromosome 4 encoding for the huntingtin protein, HD is inherited with a 50% risk by any child of an affected parent. Mutated huntingtin with a CAG repeat expansion (for polyglutamine) provokes a gradual damage to the brain by mechanisms not fully understood. Clinical symptoms usually begin with subtle changes in physical skills, personality, or cognition in middle age. Lethal complications such as pneumonia or heart disease result in a life expectancy of ~20 years after onset of clinical symptoms. HD is an orphan disease with no cure available, but with treatments improving some symptoms. Approved in 2008, Tetrabenazine has specific use for reducing the severity of chorea in HD. There is a lot of confidence that a pharmacological intervention reducing the amount of mutant huntingtin in the brain would lead to an effective cure for HD. On the other hand, the length of the CAG repeat accounts for only 50% of the variation in age of onset and rate of disease progression, implying that other “modifying” genes or to environmental factors influence the disease mechanism and explain the remaining variation. The drug discovery activities progressing at the Taube-Koret Center are targeting both intervention nodes making the aim to find a cure for HD within the proposed timeline an achievable mission. Fulfilling this goal requires a deep understanding of the pathogenic mechanisms of HD and the application of this knowledge to develop more effective methods of early detection and treatment. This is crucially dependent on advances in genomics, cell biology, chemistry and computational science. The most modern tools and techniques in these areas have been developed by the scientists of the Taube-Koret Center or are accessible through affiliated Institutes (Gladstone and UCSF to only mention the two most important) or well established scientific and technical collaborations. This is an excellent basis for propelling basic science and drug discovery, in particular because

7 the Taube-Koret Center will bridge these two disciplines and fill an historic gap in the discovery of new therapies. The Taube-Koret Center has been created this year and is directed by is by Dr. Steven Finkbeiner and Dr. Paul Muchowski, two world-wide recognized scientists who have made critical contributions to advancing basic knowledge by dissecting pathomechanisms underlying the development and progression of Huntington’s disease. This is not only evident by an impressive number of recent peered reviewed publications in top-ranked scientific journals, but also by a well running network of collaborations that is among the most impressive existing in the field. Clear recognition for this achievement is demonstrated by the fact that their work has attracted financial support through a handful of grants for a yearly funding that surpasses by more than fivefold the initial investment made by the donors who made the creation of the Taube- Koret Center possible. In this report, I would like to give a feedback on different projects that attracted my attention during the meeting and include some recommendations. Drug Target Identification Identification of new drug targets for a cure of HD at the Taube-Koret Center is based on well-established unbiased screening capabilities in cultured cells. Dr. Muchowski has long-standing expertise in successfully applying yeast to identify genetic modifiers of the toxic properties of mutant huntingtin. Dr. Finkbeiner has developed over the last 10 years a powerful automated microscopy screening model with mammalian neurons in cultures that not only has proven its use as a screening assay but represent a worldwide unique test paradigm for drug target validation in vitro. In addition to other target screening and validation techniques, already these two models (yeast and primary neurons) led the researchers at the Center promising starting point for drug discovery. Such candidate drug target genes are currently validated not only with the mentioned in vitro test assays but through a battery of in vivo mouse lines. These models are recognized by the scientific community as golden standard for HD-relevant pathological and clinical measures and thus of robust translational medicine potential. In this contest, at The Gladstone Institute there are excellent facilities for neurobehavioral and neuropathological studies to which as good access. Medicinal Chemistry Medicinal chemistry with best pharmaceutical practice and decades of know-how is present at the Center including computational chemistry and other modern techniques. Although small, these capacities have already delivered series of proprietary small molecular weight compounds with proven in vitro and in vivo activities. It is suggested to make appropriate use of these assets in the different programs and seek external partners with the adequate resources to accelerate the most advanced programs. Partnering will also allow access capabilities not yet available at the Center and leverage the investments made to date by the donors as pointedly recognized by the presenters.

8 Animal Models Use of animal experiments needs careful evaluation. Their importance as a powerful translational medicine tool is obvious. On the other hand, in the field of neurodegenerative disorders efficacy studies in mouse models often acquire proportion similar to those of clinical trials with long study length, substantial costs and often requiring a large number of animals because only few measurable endpoints are available. The scientists at the Center are well aware of these issues and beside pharmacokinetic studies of compound distribution, gave high priority to demonstrate target engagement, as well as adequate safety margins by the experimental drugs. For programs directly aiming at reducing the load of toxic huntingtin in the brain, the link with mechanism of action and efficacy is well accepted. In contrast, for putative toxicity modifiers the link between brain pathology, animal behavioral endpoints and clinical efficacy is weaker and may require significant tailoring for each program. The search for powerful biomarkers of disease onset and progression is one of the priority activities in the HD field and the Center has established privileged relationship with the most important HD center in the US and Europe. KMO Program Dr. Muchowski has demonstrated a relation between this target and HD in several cellular and animal models by tenaciously championing this program to steady progress. This year, the Center has unequivocally validated the target in vivo making KMO world-wide one of maybe two-three preclinically validated targets. This contribution is outstanding and of excellent quality. The animal data indicate that KMO inhibition will affect disease progression, prolonging survival and rescuing some of the pathological measurements. Further morphological analysis of brain atrophy and striatal markers, such as DARP32, may represent an additional asset of the program, as well as attempts to better understand the mechanism of action possibly also in peripheral tissues. Dose chronically one or more of the KMO metabolites may also contribute in elucidating the mechanism. Overall, there was good agreement on the path forward, such as integrating the key enzymatic tests within the Center, convincing enzymatic studies, the need for an efficient measure for short-term mouse compound screen and a mechanistic readout in corticospinal fluid. In the near future, the established IP position needs an aggressive protection strategy as the design of adequate partnering plans. The preliminary positive outcome in animal models of other neurodegenerative disorders, such as Alzheimer’s disease, is remarkable and of wonderful potential. Autophagy Program Macroautophagy is a cellular defense mechanism for degradation of defective organelles and toxic protein aggregates that has attracted recently a lot of attention by the scientific community and drug discovery researchers. Also neurons make us of autophagy but the regulatory mechanisms in these cells are poorly understood as the classical inducing treatment paradigms are ineffective. Dr. Finkbeiner has made perfect use of his automated microscopy technique by screening a large number of marketed drugs and identifying a small molecular weight drug which efficiently induce mutant huntingtin degradation by autophagy in neurons. This discovery is of upmost importance and combined with the identification of a marketed drug with proven safe clinical use,

9 this program pushes the Center in unique competitive advantage. The path forward was endorsed by all participants: a concise medicinal chemistry program with the aim of obtaining a small increase in potency to allow validation of the hypothesis in vivo, proof of concept could also be envisaged in peripheral tissues and thus limit the program should not be limited to CNS active compounds, demonstration of a specific mechanism and not related to the known biology of the current leads. The well progressed partnering negotiation for licensing biology and chemistry to one of the two companies Proteostatis or LINK is fully supported. Additional Programs IDO/TDO represents a very attractive back-up program to KMO. It is expected that the identified modulatory compounds, as well as use of the knock-out mice, are adequate to rapidly validate this program in vivo. Additional exploratory activities to assess the possible role of inflammatory cytokines in the brain with the potential to produce a biomarker strategy as well as therapeutic approach are well founded. Mgmt, a DNA repair enzyme identified in the yeast screen, if validated, has a lot of potential since compounds in advance clinical trials exist for oncology indications. Also here, compound treatment and knock-out mice are adequate to rapidly validate this program in vivo. CB2 and Nrf2 are in an early exploratory phase, and their potential as drug targets difficult to assess at the present date. Huntingtin modifying strategies have an excellent rationale, and the programs on polyglutamine conformation and phosphorylation have great potential. It is unfortunate that the compound leads identified in the screen can not be pursued with the necessary determination for lack of resources. If a reprioritization would endanger more advanced program, then partnering seems the best solution. General comment When testing strategies reducing toxic huntingtin, it is advised to analyze additional neurodegeneration-linked proteins, such as alpha synuclein or tau, in the HD models. Integration of human models in the current screen would further increase the value of the screening models developed at the Center.

MEMORANDUM

TO: Steven Finkbeiner, M.D., Ph.D. Professor, Departments of Neurology and Physiology University of California, San Francisco Senior Investigator and Associate Director, Gladstone Institute of Neurological Disease Director, Taube-Koret Center for Huntington’s Disease Research 1650 Owens St., Office 308 San Francisco, CA 94158

CC: Paul Muchowski, Ph.D. Stephen Freedman, Ph.D.

FROM: Norbert Bischofberger, Ph.D. Executive Vice President, R&D and Chief Scientific Officer Gilead Sciences, Inc. 333 Lakeside Drive Foster City, CA 94404

DATE: January 29, 2010

Re: Report of the December 15, 2009 External Advisory Meeting of the Taube-Koret Center for HD Research

The following constitutes my report following The External Advisory meeting of the Taube-Koret Center for HD Research which took place December 15th 2009 at the Gladstone Institute in San Francisco. I was one of two external advisors attending the meeting. My expertise is mainly in drug discovery and drug development including regulatory issues and translational medicine.

Overall, I was very impressed with the progress that is being made with the work by Paul Muchowski and Steve Finkbeiner. I sensed a high awareness and desire to advance basic scientific findings into therapeutics which in my experiences is not at all common in academic settings. Both Paul and Stephen are very much aware of the issues that have to be addressed and the hurdles that have to be overcome in early lead optimization, preclinical development and in human clinical studies. The progress made so far is impressive particularly

more extensive and expensive phase III studies. Also, the design and nature of the POC studies can shape discovery and preclinical development strategies.

In summary, I was impressed by the efforts of the group. With Paul and Stephen, The Taube-Koret Center has two world-class biologists and experts in CNS biology. The choice of targets is judicious and there is a goal oriented approach to research. Near term, some of the advanced projects have to be pushed further to answer basic questions, earlier projects need to be focused and the promise has to be further defined.

I am looking forward to reviewing the progress at our next meeting.

Sincerely,

Norbert Bischofberger, Ph.D. Executive Vice President, Research & Development Chief Scientific Officer Gilead Sciences, Inc.

3 IV. Publications and Presentations of the Taube-Koret Center for Huntington’s Disease Research

A. Bibliography of Publications

Daub A, Sharma P, Finkbeiner S. High content screening in primary neurons, Curr. Opin. Neurobiol. 2009, 19, 1–7. (Advanced online publication doi:10.1016).

Gu X, Greiner ER, Mishra R, Kodali R, Osmand A, Finkbeiner S, Steffan JS, Thompson LM, Wetzel R, and Yang XW. Ser13 and Ser16 are critical determinants of full-length human mutant huntingtin induced disease pathogenesis in HD mice. Neuron. 2009, 64:828–840.

Legleither J, Lotz GP, Miller J, Ko J, Ng C, Williams GL, Finkbeiner S, Patterson PH, Muchowski PJ. Monoclonal antibodies recognize distinct conformational epitopes formed by polyglutamine in a mutant huntingtin fragment. J. Biol. Chem. 2009, 284: 21647–21658.

Miller J, Rutenber E, Muchowski P. Polyglutamine dances the conformational cha-cha-cha. Structure. 2009, 17: 1151–1153.

Mitra S, Tsvetkov A, Finkbeiner S. Single-neuron ubiquitin-proteasome dynamics accompanying inclusion body formation in Huntington’s disease. J. Biol. Chem. 2009, 284: 4398–4403.

Mitra S, Tsvetkov, AS, Finkbeiner S. Protein turnover and inclusion body formation. Autophagy 2009, 5: 1037–1038.

Montie HL, Cho MS, Holder L, Liu Y, Tsvetkov AS, Finkbeiner S, Merry DE. Cytoplasmic retention of polyglutamine-expanded androgen receptor ameliorates disease via autophagy in a mouse model of spinal and bulbar muscular atrophy. Hum. Mol. Gen. 2009, 18: 1937–1950.

Thompson LM, Aiken CT, Agrawal N, Kaltenbach LS, Illes K, Khoshnan A, Martinez-Vincente M, Arrasate M, O’Rourke JG, Lukacsovich T, Zhu Y-Z, Lau AL, Massey A, Hayden MR, Zeitlin SO, Finkbeiner S, Huang L, Lo DC, Patterson PH, Cuervo AM, Marsh JL, and Steffan JS. The IKK complex phosphorylates huntingtin and targets it for degradation by the proteasome and lysosome, J. Cell Bio. 2009, 187:1083–1099.

Tsvetkov A, Wong J, Rao V, Finkbeiner S. Differential regulation of autophagy in neuronal and non-neuronal cells. Autophagy 2009, PMID: 19411824.

Wacker JL, Huang SY, Steele AD, Aron R, Lotz GP, Nguyen Q, Giorgini F, Roberson ED, Lindquist S, Masliah E, Muchowski PJ.Loss of Hsp70 exacerbates pathogenesis but not levels of fibrillar aggregates in a mouse model of Huntington's disease.J Neurosci. 2009 Jul 15;29(28):9104-14.

14 B. Huntington’s Disease-Related Academic Seminars

Discussion leader, Gordon Research Conference on CAG Triplet Repeat Disorders, Science Session: Inflammation in CAG Triplet Repeat Disorders, Waterville Valley, Vermont (Muchowski).

Keynote speaker, "The pathomechanisms of brain diseases: new technologies and approaches" (sponsored by RIKEN), Sapporo, Japan (Muchowski).

Moderator, World Congress of Huntington’s Disease (HD), Science Session: Inflammatory and Metabolic Changes in HD, Vancouver, Canada (Muchowski).

Invited speaker, The Fourth International Congress on Stress Responses in Biology and Medicine, Sapporo, Japan (Muchowski).

Keynote speaker, Protein Misfolding and Neurological Disorders Conference, Port Douglas, Australia (Muchowski).

Invited speaker, Adler Symposium on Proteotoxicity in Neurodegeneration; Salk Institute, Torrey Pines, California 2009 (Muchowski).

Invited speaker, Huntington’s Disease Society of America, Coalition for the Cure; Vancouver, Canada (Finkbeiner).

Invited speaker, Towards Treatment of Spinocerebellar Ataxia (EuroSCA) Conference; Tübingen, Germany (Finkbeiner).

Symposium chair, Society for Neuroscience; Nanomedicine Symposium, Chicago (Finkbeiner).

Invited speaker, Washington University School of Medicine, Department of Neurobiology; St. Louis (Finkbeiner).

Invited speaker, Cornell Medical Center, New York Presbyterian Hospital, Department of Neurology and Neuroscience; New York (Finkbeiner).

Invited speaker, High Impact Science Seminar, Burnham Institute; La Jolla (Finkbeiner).

Invited speaker, Institute for Systems Biology; Seattle (Finkbeiner).

Invited talk, Cytometry Development Workshop; Asilomar (Finkbeiner).

Invited talk, University of California Irvine, Departments of Neurobiology and Behavior, Irvine (Finkbeiner).

C. Huntington’s Disease-related Industry Consultations and Seminars

FivePrime Therapeutics; San Francisco (Finkbeiner).

Vertex Pharmaceuticals, Inc.; San Diego (Finkbeiner).

LINK Medicine Corporation; Cambridge (Finkbeiner). iPierian; San Francisco (Finkbeiner).

Pescadero Technologies; San Francisco (Finkbeiner).

Valla Technologies; San Diego (Finkbeiner).

Amgen; Thousand Oaks (Muchowski).

Genentech; South San Francisco (Muchowski).

Lundbeck; San Francisco (Muchowski).

Merck; San Francisco (Muchowski).

Proteostasis; Cambridge (Muchowski and Finkbeiner).

16 V. Taube-Koret Center for Huntington’s Disease Research and the Community

A. Press releases in 2009

Contact: For Immediate Release Valerie Tucker 415-734-2019 [email protected]

GLADSTONE INSTITUTES ESTABLISHES TAUBE-KORET CENTER FOR HUNTINGTON’S DISEASE RESEARCH Targeted program to cure Huntington’s by 2020

SAN FRANCISCO, CA – March 25, 2009 – The J. David Gladstone Institutes has joined forces with Taube Philanthropies and the Koret Foundation to initiate a groundbreaking research program aimed at preventing, treating, or curing Huntington’s disease (HD) by the year 2020. The new Taube-Koret Center for Huntington’s Disease Research has been established at the Gladstone Center for Translational Research at Mission Bay, with $3.6 million in funding from the two organizations.

HD, also called ‘Huntington’s chorea’ and ‘Woody Guthrie’s disease,’ is a devastating inherited, degenerative brain disorder. More than 100,000 Americans and more than 10 times that number worldwide have HD or are at risk of inheriting the disease from a parent.

Investigators Steven Finkbeiner, MD, PhD, and Paul Muchowski, PhD, of the Gladstone Institute of Neurological Disease (GIND) will build on their leading-edge research, which has led to the development of powerful assays for the identification of potential drug targets and a pipeline of several molecular targets that may modulate HD progression. Taube Philanthropies has supported the work of Drs. Finkbeiner and Muchowski, as well as other researchers for several years. This new research program is called “HD Cure 2020.”

“We believe that the focus and evolving new technologies of the HD Cure 2020 program provide a real chance to close in on a cure,” said Tad Taube, chairman of Taube Philanthropies and president of the Koret Foundation. “It is our hope that Gladstone’s depth of understanding about how Huntington’s progresses, combined with a well-defined and integrated therapeutic screening strategy, will enable real progress to be made toward treating or curing this devastating disease.” -more-

Taube-Koret Center Page 2

“While so much is known about Huntington’s disease, it remains an unsolved mystery,” said GIND Associate Director Steven Finkbeiner. “Over the last few years, we have been able to find new points of entry into how the disease progresses and where we might possibly intervene.”

Dr. Finkbeiner has pioneered new technologies that have added important new understanding to HD etiology and pathology. Dr. Muchowski has focused his work on identifying key intracellular pathways that modify progression of the disease. Both investigators have developed innovative technological and biological approaches for finding and screening small molecules that may work to modulate the disease.

“While Gladstone brings a unique and impressive foundation of Huntington’s research to this program, we are extremely grateful for the visionary leadership of the Koret Foundation and the Taube Philanthropies for their creation of this center and their support of our approach,” said Andrew S. Garb, Trustee of The J. David Gladstone Institutes.

The Taube-Koret Center is located in Gladstone’s Center for Translational Research where Gladstone is collaborating with several pharmaceutical companies on potential therapies for Alzheimer’s disease (Merck), HIV (Gilead Sciences and JT Pharma), and for applying induced pluripotent stem (iPs) cell technology to cardiovascular disease (iZumi Bio).

About Taube Philanthropies Guided by a long-term commitment to both secular and Jewish life, Taube Philanthropies provide direct and indirect support to projects and institutions that advance the philosophies and vision of founder Tad Taube. Central to these are the concepts and principles of a free, democratic society, including open economic enterprise, self-reliance, academic freedom of inquiry and limited government, and programs that support Jewish heritage, survival and cultural celebration.

About the Koret Foundation An entrepreneurial spirit guides Koret in addressing societal challenges and strengthening Bay Area life. In the San Francisco Bay Area, Koret adds to the region’s vitality by promoting educational opportunity, contributing to a diverse cultural landscape, and bolstering organizations that are innovative in their approaches to meeting community needs. With roots in the Jewish community, Koret embraces the community of Israel, especially through Koret Israel Economic Development Funds, believing that economic stability and free market expansion offer the best hope for a prosperous future -more-

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About the Gladstone Institutes Established in 1979, The J. David Gladstone Institutes is an independent, nonprofit biomedical research organization that operates in close affiliation with the University of California, San Francisco (UCSF). Gladstone is dedicated to the health and welfare of humankind through research into the causes and prevention of some of the world’s most devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology, the Gladstone Institute of Neurological Disease, and the Gladstone Center for Translational Research. More information can be found at: www.gladstone.ucsf.edu

About Huntington’s disease Huntington’s disease (HD), also called Woody Guthrie’s disease, is a devastating degenerative brain disorder that is inherited from a parent with the disease. Over a period of 10 to 25 years, HD slowly but steadily reduces a person’s ability to walk, think, talk, and reason. Ultimately, HD renders its victims totally dependent upon others for their care. Patients with HD ultimately die from complications, such as choking, infection, or heart failure. Men and women of all racial and ethnic groups are equally susceptible to contracting HD. A child of a parent with HD is 50% likely to inherit the fatal “huntingtin” gene. Tragically, every person who carries the HD gene ultimately develops the disease.

The typical patient with HD is aged 30 to 50, although manifestations of the disease may arise in children as young as 2 years of age. Young people who are afflicted with the juvenile form of HD rarely live to adulthood. Today, more than 250,000 Americans—and more than 10 times that number worldwide—have HD or are at risk of inheriting the disease from a parent with HD. The disease affects as many people as hemophilia, cystic fibrosis, and muscular dystrophy.

The HD gene was successfully isolated in 1993. Subsequently, a genetic blood test was developed to determine precisely whether a person has inherited the HD gene. However, no test can predict when HD symptoms will begin. As with other diseases that are inherited, many of those who have a parent with HD elect not to take the HD gene test.

Over the years, biomedical research involving HD has yielded a wealth of knowledge about the disease and its basic mechanisms. However, no effective method exists for preventing, treating, or curing HD. In fact, no validated drug targets for HD, besides the huntingtin gene itself, have been discovered. Although HD is one of the most cruel and devastating diseases, those afflicted are too few in number to interest most major pharmaceutical companies in developing relevant HD-targeted drug discovery programs.

# # #

Valerie Tucker (415) 734-2019 FOR IMMEDIATE RELEASE E-mail: [email protected] Web:www.gladstone.ucsf.edu

GLADSTONE AND PARTNERS RECEIVE $3.7 MILLION TO USE STEM CELL TECHNOLOGY FOR HUNTINGTON’S DISEASE RESEARCH

NIH Funds Effort to Develop Disease Models for Pathogenesis and Drug Discovery

SAN FRANCISCO, CA – October 13, 2009 – The National Institutes of Health (NIH) has awarded a “Grand Opportunity” grant of $3.7 million to a consortium formed with the Gladstone Institute of Neurological Disease (GIND) and the Taube-Koret Center for Huntington’s Disease Research to use stem cell technology to better understand Huntington’s disease (HD) and to develop potential therapies. The consortium comprises a partnership of five leading Huntington’s research laboratories at the University of Wisconsin, Massachusetts General Hospital, the University of California at Irvine, Johns Hopkins and the Gladstone Institutes. The consortium will use induced pluripotent stem (iPS) cell technology pioneered by Gladstone and Kyoto University’s Shinya Yamanaka, MD, PhD, to develop human neurons with Huntington’s disease characteristics. iPS technology enables stem cells to be generated from skin samples from adults and avoids the ethical issues surrounding the use of fetal stem cells.

“One of the challenges of Huntington’s (and many other neurological diseases) is that many of the potential therapies that show promise in animal models are ineffective in people. We think that molecular differences between mice and humans may be an important cause for this failure,” said Steven Finkbeiner MD, PhD, consortium co-leader and Director of the Taube-Koret Center for Huntington’s Disease Research and Associate Director of GIND. -more-

Huntington’s Consortium 2-2-2

“One of the promises of iPS technology is to be able to develop models from Huntington’s disease patients that can give us more detailed information about the disease and better predict how therapies could work in humans,” he said.

HD, which is also called “Huntington’s chorea” and “Woody Guthrie’s disease,” is a devastating inherited, degenerative brain disorder. More than 100,000 Americans and more than 10 times that number worldwide have HD or are at risk of inheriting the disease from a parent. iPS cells are generated by reprogramming adult cells from skin or other tissues. They are almost identical to human embryonic stem cells with the ability to self-renew for long periods and to differentiate into all cell lineages. More importantly, iPS cells can be generated from adult patients with genetically inherited and sporadic diseases making it possible to study some diseases, such as Alzheimer’s and Parkinson’s disease, for which the causes remain largely unknown.

“HD is caused by a single mutation, which provides an ideal paradigm to generate a panel of patient-specific lines,” Finkbeiner explained. “This offers hope that these models can teach us why some patients experience certain symptoms and why some family members develop symptoms later rather than sooner, which then can potentially be used to develop treatments that can act before symptoms appear.”

Finkbeiner added, “the convergence of a dedicated, collaborative group of committed investigators targeting HD, the need for new treatments based on the root causes of the disease, and the emergence of powerful new technologies herald a truly grand opportunity to make a real difference for those afflicted with Huntington’s.”

Dr. Finkbeiner’s primary affiliation is with the Gladstone Institute of Neurological Disease where his laboratory is located and all of his research is conducted. He is also associate professor of neurology and physiology at the University of California, San Francisco.

-more-

21 Huntington’s Disease Consortium 3-3-3

About the Taube-Koret Center for Huntington’s Disease Research. The Center was established in 2009 with gifts from Taube Philanthropies and the Koret Foundation for the sole purpose of identifying strategies and developing therapeutics to treat people with Huntington’s disease and related neurodegenerative diseases. # # #

22 B. The Taube-Koret Center for Huntington’s Disease Research in the News

23 New HD Research Center Tasked with Preventing, Treating, or Curing Disease by 2020 10/17/09 11:58 PM

Mar 26 2009, 11:30 AM EST

New HD Research Center Tasked with Preventing, Treating, or Curing Disease by 2020

GEN News Highlights

The J. David Gladstone Institutes, Taube Philanthropies, and the Koret Foundation joined forces to initiate a research program aimed at preventing, treating, or curing Huntington's disease (HD) by 2020.

The new Taube-Koret Center for Huntington's Disease Research has been established at the Gladstone Center for Translational Research at Mission Bay, CA, with $3.6 million in funding from the two organizations. The program is called HD Cure 2020.

The center will build on research from investigators Steven Finkbeiner, M.D., Ph.D., and Paul Muchowski, Ph.D., of the Gladstone Institute of Neurological Disease (GIND) related to assay development and molecular targets that may modulate HD progression.

Dr. Finkbeiner’s technologies reportedly aid in the understanding of HD etiology and pathology. Dr. Muchowski’s studies have identified intracellular pathways that modify progression of the disease. Together they have also developed methods to find and screen small molecules that may work to modulate the disease.

“While so much is known about Huntington's disease, it remains an unsolved mystery,” notes Dr. Finkbeiner. “Over the last few years, we have been able to find new points of entry into how the disease progresses and where we might possibly intervene.”

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Gladstone Institutes Establishes Taube-Koret Center For Huntington's Disease Research, Aims To Cure Huntington's By 2020

30 Mar 2009 Click to Print

The J. David Gladstone Institutes has joined forces with Taube Philanthropies and the Koret Foundation to initiate a groundbreaking research program aimed at preventing, treating, or curing Huntington's disease (HD) by the year 2020. The new Taube-Koret Center for Huntington's Disease Research has been established at the Gladstone Center for Translational Research at Mission Bay, with $3.6 million in funding from the two organizations.

HD, also called 'Huntington's chorea' and 'Woody Guthrie's disease,' is a devastating inherited, degenerative brain disorder. More than 100,000 Americans and more than 10 times that number worldwide have HD or are at risk of inheriting the disease from a parent.

"We believe that the focus and evolving new technologies of the HD Cure 2020 program provide a real chance to close in on a cure," said Tad Taube, chairman of Taube Philanthropies and president of the Koret Foundation. "It is our hope that Gladstone's depth of understanding about how Huntington's progresses, combined with a well-defined and integrated therapeutic screening strategy, will enable real progress to be made toward treating or curing this devastating disease."

"While so much is known about Huntington's disease, it remains an unsolved mystery," said GIND Associate Director Steven Finkbeiner. "Over the last few years, we have been able to find new points of entry into how the disease progresses and where we might possibly intervene."

"While Gladstone brings a unique and impressive foundation of Huntington's research to this program, we are extremely grateful for the visionary leadership of the Koret Foundation and the Taube Philanthropies for their creation of this center and their support of our approach," said Andrew S. Garb, Trustee of The J. David Gladstone Institutes.

The Taube-Koret Center is located in Gladstone's Center for Translational Research where Gladstone is collaborating with several pharmaceutical companies on potential therapies for Alzheimer's disease (Merck), HIV (Gilead Sciences and JT Pharma), and for applying induced pluripotent stem (iPs) cell technology to cardiovascular disease (iZumi Bio).

About Taube Philanthropies

Guided by a long-term commitment to both secular and Jewish life, Taube Philanthropies provide http://www.medicalnewstoday.com/printerfriendlynews.php?newsid=144184 Page 1 of 3 Medical News Today News Article - Printer Friendly 10/30/09 8:47 PM

direct and indirect support to projects and institutions that advance the philosophies and vision of founder Tad Taube. Central to these are the concepts and principles of a free, democratic society, including open economic enterprise, self-reliance, academic freedom of inquiry and limited government, and programs that support Jewish heritage, survival and cultural celebration.

About the Koret Foundation

An entrepreneurial spirit guides Koret in addressing societal challenges and strengthening Bay Area life. In the San Francisco Bay Area, Koret adds to the region's vitality by promoting educational opportunity, contributing to a diverse cultural landscape, and bolstering organizations that are innovative in their approaches to meeting community needs. With roots in the Jewish community, Koret embraces the community of Israel, especially through Koret Israel Economic Development Funds, believing that economic stability and free market expansion offer the best hope for a prosperous future

About the Gladstone Institutes

Established in 1979, The J. David Gladstone Institutes is an independent, nonprofit biomedical research organization that operates in close affiliation with the University of California, San Francisco (UCSF). Gladstone is dedicated to the health and welfare of humankind through research into the causes and prevention of some of the world's most devastating diseases. Gladstone is comprised of the Gladstone Institute of Cardiovascular Disease, the Gladstone Institute of Virology and Immunology, the Gladstone Institute of Neurological Disease, and the Gladstone Center for Translational Research.

About Huntington's disease

Huntington's disease (HD), also called Woody Guthrie's disease, is a devastating degenerative brain disorder that is inherited from a parent with the disease. Over a period of 10 to 25 years, HD slowly but steadily reduces a person's ability to walk, think, talk, and reason. Ultimately, HD renders its victims totally dependent upon others for their care. Patients with HD ultimately die from complications, such as choking, infection, or heart failure. Men and women of all racial and ethnic groups are equally susceptible to contracting HD. A child of a parent with HD is 50% likely to inherit the fatal "huntingtin" gene. Tragically, every person who carries the HD gene ultimately develops the disease.

The typical patient with HD is aged 30 to 50, although manifestations of the disease may arise in children as young as 2 years of age. Young people who are afflicted with the juvenile form of HD rarely live to adulthood. Today, more than 250,000 Americans-and more than 10 times that number worldwide-have HD or are at risk of inheriting the disease from a parent with HD. The disease affects as many people as hemophilia, cystic fibrosis, and muscular dystrophy.

Source Gladstone Institutes

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Main News Category: Huntingtons Disease

Also Appears In: Neurology / Neuroscience,

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Posted on April 4, 2009

Koret Foundation, Taube Philanthropies Award $3.6 Million for Huntington's Disease Research

The Koret Foundation has announced a joint $3.6 million grant with Taube Philanthropies to establish a center for Huntington's disease (HD) research at the Gladstone Center for Translational Research in Mission Bay, California.

The new Taube-Koret Center for Huntington's Disease Research will house a program designed to help prevent, treat, and cure HD by 2020. The program will build on previous research by the center's investigators that has led to the development of powerful assays for the identification of potential drug targets and a pipeline of molecular targets that could modulate HD progression.

Also called Huntington's chorea and Woody Guthrie's disease, HD is an inherited, degenerative brain disorder. More than 100,000 Americans — and more than one million worldwide — have HD or are at risk of inheriting the disease from a parent.

“Gladstone Institutes Establishes Taube-Koret Center for Huntington's Disease Research.” Koret Foundation Press Release 4/25/09.

Primary Subject: Health Secondary Subject(s): Medical Research Location(s): California

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1 of 1 4/6/09 9:59 AM Gladstone, Stanford share $3.9M to study Huntington’s - San Francisco Business Times: 10/30/09 8:47 PM

Members: Log in | Not Registered? Register for free extra services. San Francisco Business Times - March 30, 2009 /sanfrancisco/stories/2009/03/30/story18.html

Friday, March 27, 2009 Gladstone, Stanford share $3.9M to study Huntington’s San Francisco Business Times - by Ron Leuty

Taube Philanthropies and the Koret Foundation have donated a total $3.9 million to the J. David Gladstone Institutes and Stanford University to find a treatment or cure for Huntington’s disease.

The bulk of that money — $3.6 million from both Taube and Koret — is earmarked over three years to the Gladstone Institutes in San Francisco.

The money will create the Taube-Koret Center for Huntington’s Disease Research at the Gladstone Center for Translational Research at Mission Bay.

Dr. Steven Finkbeiner and Paul Muchowski will hire at least five new staffers to help translate their basic research into promising drug candidates and — perhaps as soon as the next 12 months — ink a partnership with a biopharmaceutical company like Merck & Co., Novartis or Elan.

That makes the gifts critical for crossing the so-called “valley of death” between basic research funded largely by the National Institutes of Health and the point where a biotech or pharmaceutical company would be interested in pursuing a drug.

“There’s a critical gap,” Finkbeiner said.

Huntington’s, a genetic disorder that strikes seven in every 100,000 people globally, is marked by progressively uncoordinated, jerky body movements of the hands, feet, face and trunk and the loss of some mental abilities. There is no cure.

At least one Bay Area company, Medivation Inc., has undertaken a Phase II trial of its drug, Dimebon, as a potential Huntington’s treatment.

The other $300,000 — from Taube Philanthropies alone — will be used over two years by Dr. Frank Longo, who leads Stanford’s department of neurology and neurological sciences. He is undertaking a massive trial-and-error process testing thousands of potential drugs on mice.

The Taube-Koret Center is looking at small molecules that Finkbeiner and Muchowski hope will stop or even roll back Huntington’s damage, Finkbeiner said.

Hladstone and Stanford are working toward a Huntington’s cure by 2020.

“That’s our collective light,” said Tad Taube, chairman of Taube Philanthropies in Belmont and president of the Koret Foundation in San Francisco. “They hope and are optimistic that by 2020 there should be some results that lead to a positive drug therapy or a cure.” [email protected] / (415) 288-4939

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Find more articles on taube-koret-center Gladstone and partners receive $3.7 million for Huntington's disease research

October 13th, 2009

The National Institutes of Health (NIH) has awarded a "Grand Opportunity" grant of $3.7 million to a consortium formed with the Gladstone Institute of Neurological Disease (GIND) and the Taube-Koret Center for Huntington's Disease Research to use stem cell technology to better understand Huntington's disease (HD) and to develop potential therapies. The consortium comprises a partnership of five leading Huntington's research laboratories at the University of Wisconsin, Massachusetts General Hospital, the University of California at Irvine, Johns Hopkins and the Gladstone Institutes. The consortium will use induced pluripotent stem (iPS) cell technology pioneered by Gladstone and Kyoto University's Shinya Yamanaka, MD, PhD, to develop human neurons with Huntington's disease characteristics. iPS technology enables stem cells to be generated from skin samples from adults and avoids the ethical issues surrounding the use of fetal stem cells.

"One of the challenges of Huntington's (and many other neurological diseases) is that many of the potential therapies that show promise in animal models are ineffective in people. We think that molecular differences between mice and humans may be an important cause for this failure," said Steven Finkbeiner MD, PhD, consortium co-leader and Director of the Taube-Koret Center for Huntington's Disease Research and

http://www.physorg.com/wire-news/16901103/gladstone-and-partners-receive-37-million-for-huntingtons-diseas.html Page 1 of 8 Gladstone and partners receive $3.7 million for Huntington's disease research 10/30/09 8:52 PM

Associate Director of GIND.

"One of the promises of iPS technology is to be able to develop models from Huntington's disease patients that can give us more detailed information about the disease and better predict how therapies could work in humans," he said.

HD, which is also called "Huntington's chorea" and "Woody Guthrie's disease," is a devastating inherited, degenerative brain disorder. More than 100,000 Americans and more than 10 times that number worldwide have HD or are at risk of inheriting the disease from a parent.

iPS cells are generated by reprogramming adult cells from skin or other tissues. They are almost identical to human embryonic stem cells with the ability to self-renew for long periods and to differentiate into all cell lineages. More importantly, iPS cells can be generated from adult patients with genetically inherited and sporadic diseases making it possible to study some diseases, such as Alzheimer's and Parkinson's disease, for which the causes remain largely unknown.

"HD is caused by a single mutation, which provides an ideal paradigm to generate a panel of patient- specific lines," Finkbeiner explained. "This offers hope that these models can teach us why some patients experience certain symptoms and why some family members develop symptoms later rather than sooner, which then can potentially be used to develop treatments that can act before symptoms appear."

Finkbeiner added, "the convergence of a dedicated, collaborative group of committed investigators targeting HD, the need for new treatments based on the root causes of the disease, and the emergence of powerful new technologies herald a truly grand opportunity to make a real difference for those afflicted with Huntington's."

Source: Gladstone Institutes

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Public release date: 13-Oct-2009 [ Print | E-mail | Share ] [ Close Window ]

Contact: Valerie Tucker [email protected] 415-734-2019 Gladstone Institutes Gladstone and partners receive $3.7 million for Huntington's disease research

NIH funds effort to develop disease models for pathogenesis and drug discovery

###

http://www.eurekalert.org/pub_releases/2009-10/gi-gap100909.php Page 1 of 2 Gladstone and partners receive $3.7 million for Huntington's disease research 10/30/09 8:45 PM

Dr. Finkbeiner's primary affiliation is with the Gladstone Institute of Neurological Disease where his laboratory is located and all of his research is conducted. He is also associate professor of neurology and physiology at the University of California, San Francisco.

About the Gladstone Institutes

About the Taube-Koret Center for Huntington's Disease Research.

The Center was established in 2009 with gifts from Taube Philanthropies and the Koret Foundation for the sole purpose of identifying strategies and developing therapeutics to treat people with Huntington's disease and related neurodegenerative diseases.

[ Print | E-mail | Share ] [ Close Window ]

http://www.eurekalert.org/pub_releases/2009-10/gi-gap100909.php Page 2 of 2 C. The Taube-Koret Center for Huntington’s Disease Research and HD Families

1. Huntington’s Disease Education. People who are newly diagnosed with HD often have many questions. Nowadays, the internet is a common place to look for information, and the first result from a Google search for Huntington’s disease is an entry from Wikipedia.

To help improve the quality and access to information about HD, we collaborated with Lee van- Jackson, an author at Wikipedia, to develop and improve their entry. The result was an article that got promoted to featured article. Less than 0.1% of articles in Wikipedia receive that distinction, which is given by their editors based on the quality and accuracy of the article. Wikipedia gets 65 million visitors a month, so we think this is a worthwhile investment of our effort. The Taube-Koret Center is acknowledged as the source of the image that first appears as the Wikipedia web page on HD opens.

2. Supporting Families with Huntington’s Disease. This year, members of the Center participated in the annual “Walk for Hope” sponsored by the Huntington’s Disease Society of America. The event brings HD families from all over Northern California to San Francisco, and it gave us an opportunity to answer questions about the Center. The members of the Taube-Koret Center are committed to showing our support for HD families and we raised funds from our friends and family. Overall we raised nearly $6,000.

One of the most moving experiences of establishing the Taube-Koret Center has been the outpouring of gratitude from the HD community for the hope that it offers patients and their families. We have included an example of the sort of encouragement we receive from HD families.

Michelle from Denver wrote:

Hello, I just found out about the grant establishing the Taube-Koret Center for Huntington's Disease Research and your involvement in this project. Your research into the cause, treatment and dare I say, cure of this disease is the most fabulous kernel of hope that I have come across on this subject. My family has been affected for generations by HD and I just want to thank you for your efforts. I am in no way able to put into words how much this means to me. Thank you.

Shellie

34 VI. Appendix of Publications

35 This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy

Available online at www.sciencedirect.com

High-content screening of primary neurons: ready for prime time Aaron Daub1,2,3,*, Punita Sharma1,3,* and Steven Finkbeiner1,3,4,5

High-content screening (HCS), historically limited to drug- We are only beginning to understand the benefits — in development companies, is now a powerful and affordable fact, the necessity — of studying biological systems with technology for academic researchers. Through automated large-scale unbiased screens [1]. Here we focus on high- routines, this technology acquires large datasets of content screening (HCS) and considerations needed to fluorescence images depicting the functional states of use this method effectively to study normal and disease thousands to millions of cells. Information on shapes, textures, physiology in primary cells, currently the most biologi- intensities, and localizations is then used to create unique cally relevant models. representations, or ‘phenotypic signatures,’ of each cell. These signatures quantify physiologic or diseased states, for Why high-content screening? example, dendritic arborization, drug response, or cell coping HCS is a multiplexed, functional screening method based strategies. Live-cell imaging in HCS adds the ability to on extracting multiparametric fluorescence data from correlate cellular events at different points in time, thereby multiple targets in intact cells [2,3]. By temporally and allowing sensitivities and observations not possible with fixed spatially resolving fluorescent readouts within individual endpoint analysis. HCS with live-cell imaging therefore cells, HCS yields an almost unlimited number of kinetic provides an unprecedented capability to detect and morphometric outputs. HCS was developed to facili- spatiotemporal changes in cells and is particularly suited for tate drug-target validation and lead optimization before time-dependent, stochastic processes such as costly animal testing [4]. Today it is broadly used to neurodegenerative disorders. catalog cellular, subcellular, and intercellular responses Addresses to multiple systematic perturbations and is applicable to 1 Gladstone Institute of Neurological Disease, San Francisco, CA 94158, basic science, translational research, and drug develop- United States ment [5–8]. We distinguish HCS from high-content 2 Medical Scientist Training Program and Program in Bioengineering, analysis (HCA). HCA refers to extracting information University of California, San Francisco, 94143, United States 3 Taube-Koret Center for Huntington’s Disease Research and the from image data. HCS is the automated, high-throughput Consortium for Frontotemporal Dementia Research, San Francisco, CA application of HCA. 94158, United States 4 Program in Biomedical Sciences, Neuroscience Graduate Program, HCS can fill a gap in academic research. Our growing Biomedical Sciences Program, University of California, San Francisco, awareness of biological complexity underscores the need 94143, United States 5 Departments of Neurology and Physiology, San Francisco, CA 94143, to examine more than one variable at a fixed point in time. United States Traditional low-throughput methods have severe limitations. In complex systems with many interacting genes, * These authors contributed equally to this work. measuring any single perturbation is not very informative. Corresponding author: Finkbeiner, Steven In gain-of-function diseases, especially those with late (sfi[email protected]) onset, a toxic protein effect may not be related to the protein’s normal function. Unbiased screens therefore identify potential pathogenic mechanisms faster and Current Opinion in Neurobiology 2009, 19:537–543 more comprehensively, and the large datasets are less This review comes from a themed issue on prone to sampling error when analyzing stochastic events. New technologies Edited by Ehud Isacoff and Stephen Smith HCS assays capture cell-system dynamics and exploit typically confounding cell-to-cell variability. For Available online 4th November 2009 example, a recent study used simultaneous tracking of 0959-4388/$ – see front matter 1000 proteins in lung carcinoma cells after drug treat- # 2009 Elsevier Ltd. All rights reserved. ment to detect time-dependent proteomic changes that DOI 10.1016/j.conb.2009.10.002 predicted individual cell fate [9 ]. Hypotheses in HCS are used to design tracked variables and outputs that maximize the likelihood of meaningful results. We labeled mutant huntingtin and measured cell survival Introduction to determine the role of inclusion bodies in Huntington’s Biological research is entering a new era. Molecular disease (HD) [10], a question unanswered by 10 years of biology will be combined with novel engineering tech- time-invariant, low-throughput approaches. HCS pro- nologies and increased computational power to examine vides large datasets that unveil multiple, often nonintui- living systems in exciting new ways. tive, correlations that seed subsequent lines of thought. www.sciencedirect.com Current Opinion in Neurobiology 2009, 19:537–543 Author's personal copy

538 New technologies

Table 1

Neuronal cell models for HCS

Property Immortalized cells Primary neurons Embryonic stem cells Induced pluripotent stem cells Current use in HCS Ubiquitous Limited Differentiation screens Differentiation screens Ready for HCS Yes Yes No No Source Specific to cell line Animal tissue Established or new cell line Established or new cell line Specific brain From human or animal From human or animal regions embryos fibroblasts (most common) Freeze/Thaw Yes Once Yes Yes Proliferative capacity Very High Post-mitotic High High Murine better than human Murine better than human Differentiation required In some cases No Yes Yes Population type Clonal or Heterogeneous Clonal ! Heterogeneous Clonal ! Heterogeneous Heterogeneous Handling Durable Sensitive Sensitive Sensitive Ability to be engineered High Limited Medium to high Medium to high Cost Low High Medium Medium Physiologic relevance Low High Medium to high Medium to high Major challenge for HCS Physiologic Limited human Limited human source Dedifferentiation relevance source Labor intensive Differentiation Differentiation Quality control Quality control Major benefits for HCS Quantity Physiologic Quantity Quantity relevance Engineering Diversity of cell types Diversity of cell types Patient-specific screening

The advantages and disadvantages of different cell types are summarized for their use in HCS. Adapted from Eglen et al. [10].

Thus, HCS accelerates the iterative process of classical For example, iPS cells from patients with spinal mus- hypothesis-driven research [11]. cular atrophy differentiated into motor neurons retained pathological deficits and drug responses consistent with Primary cells or cell lines? the disease. More work is needed to characterize iPS cell Choosing the best cell type for a particular HCS assay is lines, and better dedifferentiation protocols will avoid challenging. Each option comes with inherent benefits viral vectors and oncogenes [21–24]. Ultimately, HCS and drawbacks (Table 1). Primary cells provide high- will place additional demands on dedifferentiation and quality models for several reasons. They are more physiologically relevant than immortalized cell lines [12]. They form synapses, thus incorporating significant neuro- Figure 1 modulatory and trophic inputs. Neuronal physiology and disease are also notoriously cell-type specific, and neurons differentiated in vivo best recapitulate actual neuronal subpopulations. One study found that hepa- toma cell lines differ profoundly from primary hepatocytes, consistent with a shift from oxidative to anaerobic metabolism, upregulation of mitotic proteins, and downregulation of typical hepatocyte functions [13]. High attrition rates for candidate neuropharmacologics (Figure 1) suggest even more striking differences in neurons.

Most screenings have involved cell lines, but future screenings will use primary and stem cells [14,15]. Embryonic stem (ES) cells can be differentiated into Success rates and millions of dollars spent from first-in-man clinical motor neurons in large numbers [16]. Mouse and trials to registration by therapeutic area. The overall clinical success rate is 11% with 900 million dollars spent. However, when the analysis is human induced pluripotent stem (iPS) cells [17,18] carried out by therapeutic area, big differences emerge, with central may better predict in vivo drug side effects and are nervous system (CNS) and oncology trailing far behind cardiovascular particularly attractive for disease-focused HCS [15–21]. diseases in the % success rate versus the dollars spent [54,55].

Current Opinion in Neurobiology 2009, 19:537–543 www.sciencedirect.com Author's personal copy

Screening of primary neurons Daub, Sharma and Finkbeiner 539

Table 2

Recommended fluorescent proteins

Fluorescent Spectral Excitation Emission Brightnessb Photostabilityc pKac Association Filter setd proteina class peak (nm) peak (nm) statec EBFP2 Blue 383 448 18 55 5.3 Weak dimer DAPI/BFP mCerulean Cyan 433/445 475/503 27/24 36 4.7 Monomer CFP mEGFP Green 488 507 34 174 6.0 Monomer FITC/GFP mEmerald Green 487 509 39 101e 6.0 Monomer FITC/GFP EYFP Yellow 514 527 51 60 6.9 Weak dimer FITC/YFP mCitrine Yellow 516 529 59 49 5.7 Monomer FITC/YFP mOrange2 Orange 549 565 35 228 6.5 Monomer TRITC/DsRed TagRFP-T Orange 555 584 33 337 4.6 Monomer TRITC/DsRed mCherry Red 587 610 17f 96 <4.5 Monomer TxRed mKate2 Far-red 588 633 25 118 5.4 Monomer TxRed

Physical properties for fluorescent proteins (FPs) in each spectral class. a Common abbreviation. b Product of the molar extinction coefficient and the quantum yield (mM cm)1. c Literature values except as noted. d Specialized applications may require choosing filter combinations that closely match the spectral profiles [56]. e Measured in live cells with mEGFP (t1/2 = 150 s) as a control. f Averages of literature values. Adapted from Shaner et al.[27,30]. redifferentiation, including high efficiency and reprodu- fection all have benefits and drawbacks [33]. Primary cibility. High throughput screens are already helping to neurons pose additional challenges: they are susceptible address these needs [25,26]. to transfection toxicities and plagued by low transfection efficiency [34]. We found Lipofectamine 2000 (Invitro- Despite technical challenges in isolating, culturing, and gen) best for efficiency, cell viability, and automation in transfecting primary neurons, their use decreases false assays that require transfection after cell plating. With this negatives and saves time and money wasted on pursuing reagent, most transfection variability results from cell- false positives. Until protocols are improved for differ- plating density, total mass of DNA, and ratio of transfec- entiating ES and iPS cells into many neuronal cell types, tion reagent to DNA. These factors must be optimized for primary cells will remain the most physiologically specific cells and DNAs. Reverse transfection with this relevant model for large-scale screens. reagent now makes arrayed libraries of transfection-ready DNA and siRNA a reality for HCS [35,36]. Although HCS planning for live-cell imaging biochemical assays utilizing large numbers of pooled cells Assay development encompasses selecting fluorophores rely on high transfection efficiencies, this actually com- and proteins to label, choosing a transfection method, plicates microscopy-based screening of individual cells. migrating to 96-well or 384-well formats, upgrading auto- Identifying the same cell over time can be confounded by mation, and completing preliminary experiments to cell movement. The researcher must strike a balance determine the robustness of readouts. None of these between maximizing transfected cell number per field steps are trivial. Migrating to a new format alone requires and verifying the ability of image-analysis algorithms to re-optimizing labware, intra-well and inter-well cell accurately track the cells. distributions, and transfection and image-acquisition protocols. During this time, a lab data management system Automation. Automation can be applied to each step of must also be integrated. HCS, including sample preparation, image acquisition and analysis, quality-control measures, and data reporting. Fluorophores. Excellent reviews describe fluorophores for Highly capable liquid-handling robots are increasingly HCA [27,28]. Notably, mKate [29] (now mKate2), mOr- affordable for individual labs. They provide scalable ange2 and TagRFP-T [30], and EBFP2 [31] provide options for liquid aspiration and dispensing of large improved brightness and photostability. After balancing and small volumes. Multiple high-content microscopy these features, the best options for live-cell imaging are systems are now available [37]. The most popular use listed in Table 2. HCS allows up to four fluorophores with confocal or wide-field microscopes, and all offer hardware sufficient spectral separation to avoid crosstalk. In the autofocus, options for environmental control, and data future, more channels will be simultaneously acquired management and image-analysis software. They provide with spectral imaging [32]. out-of-the-box access to HCS for many scientific applications. Downsides to these solutions include expense, Transfection. Lipid-based methods, Ca2+-phosphate co- proprietary image formats and algorithms, and the inability precipitation, viral infection, electroporation, and nucleo- to write ground-level scripts for true user customization. www.sciencedirect.com Current Opinion in Neurobiology 2009, 19:537–543 Author's personal copy

540 New technologies

Lab automation upgrades should be integrated early into used 300 unbiased parameters and a multivariate clus- low-throughput assay development so quality measures are tering algorithm to determine separation between drug- determined from datasets reflecting the automation. treated HeLa cells and controls [40]. The redundancy of this parameter set was reduced, resulting in a minimal Robustness. Minimizing assay variability is essential for phenotypic signature of the treated cells at various drug HCS. The Z0-factor is a useful way to estimate assay dosages. With the signatures, a drug class could be pre- quality and is calculated as a signal detection window dicted, and therapeutic windows could also be deduced. between positive and negative controls scaled by the The close relationship of neuronal morphology and func- dynamic range [38]. It is an excellent measure of tional state [48] holds promise for similar phenotypic single-output assays. Since HCS allows powerful multi- signatures to emerge from HCS focused on neuronal parametric analyses with potentially hundreds of quanti- development, physiology, and disease. For instance, an fied parameters, a Z0-factor can be calculated individually HCS study of cultured rat primary cortical neurons ident- for each parameter [39]. Alternatively, multivariate ified Ab1–42 induced reduction in neurite outgrowth with criteria without informational losses due to averaging no apparent effect on neuron number, pointing to more can be instituted from the beginning [40]. In either subtle morphological changes that can precede cell death. case, large datasets from positive and negative controls These studies used fixed-cell imaging; however, the full should be used to determine assay quality before screen- potential of HCS will be realized by imaging live cells ing is initiated. over time [49,50].

Data Management. HCS datasets are large. Live-cell ima- HCS and live-cell imaging of primary neurons: ging of a single 96-well plate with three channels and nine putting it all together images per well yields 30 GB of raw image data. A HCS with live-cell imaging in relevant neuronal models reliable informatics infrastructure is needed. Data should promises to elucidate physiologic and pathophysiologic flow seamlessly from acquisition to storage on a server processes with unprecedented sensitivity and correlative where it can be accessed for offline image analysis. power. Live-cell imaging captures changes in cellular Initially, hierarchical file structures can be used, but phenotypes. Thus, previously static features are trans- optimal management should include a central database formed into dynamic features where timed occurrences for storing images and metadata that can be accessed by and rates of change generate more informative phenoty- both acquisition and image-analysis software [41]. pic signatures. Imaging in live cells also permits cause- and-effect relationships to be determined. We use this Image analysis: the new bottleneck novel approach to investigate pathogenic mechanisms of Automation advancements have been valuable for HCS, neurodegenerative disorders, including HD, Parkinson’s but extracting meaningful data from complex image sets disease, amyotrophic lateral sclerosis, and frontotemporal poses major challenges. These challenges arise from a dementia. Our system (Figure 2) allows us to correlate combination of microscopy and image-processing limita- events in thousands of neurons to individual cell fates — tions and the need for new statistical tools. Neuroscience enabling us to determine if the events are adaptive, poses particular difficulties due to complexities in pathogenic, or incidental to disease progression [51]. neuronal morphology and subcellular trafficking. Most For instance, we used live-cell imaging in a primary laboratories use image-analysis algorithms and manual neuron model of HD to establish a mitigating role for labor to analyze images, but the throughput is too low inclusion bodies [6] and reveal the interplay between for HCS. More robust and accurate image-analysis algor- ubiquitin-proteasome system function and inclusion body ithms that can be applied to entire datasets with minimal formation [52]. Such studies necessitate large sample user intervention are necessary [42]. Zhang et al. pub- sizes and the ability to follow individual neurons over lished a neurite extraction algorithm [43] for HCS, and time. They highlight the power of HCS, when coupled multiple commercial packages quantify neuronal bodies with live-cell imaging, to reveal causal relationships in and neurites. To understand HCS informatics problems biological processes. more fully, we refer you to excellent reviews [44–46]. Repeated measures of individual cells by automated HCA uniquely provides multiplexed quantification of microscopy allow use of powerful statistical techniques, individual cell features with temporal and spatial resol- such as Cox proportional hazards (CPH) analysis [53]. ution. Image analysis comprises image segmentation and CPH integrates a user-defined number of parameters to cell tracking, extraction of individual cell features, and determine whether they explain time-to-event outcomes, data modeling and classification [46]. Image-analysis pro- for instance cell survival. Much as in a prospective cohort grams routinely measure size, shape, intensity, texture, study, we allow cells, through stochastic diversification, to moments, and subcellular localization that, when com- ‘take on’ certain traits and then retrospectively determine bined, yield hundreds of parameters that characterize a how significant these traits are in predicting outcomes. specific cellular phenotype [47]. For example, Loo et al. Our goal is to find robust, disease-specific phenotypic

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Screening of primary neurons Daub, Sharma and Finkbeiner 541

Figure 2

Workflow of our second-generation high-content screening system for live-cell imaging. Our system uses primary neurons from embryonic mice. A Microlab STARlet (Hamilton, Reno, CA) automated pipetting workstation prepares and transfects cells in 96-well plates, which are then transferred to the plate stacker of a KiNEDx 4-axis robot (Peak Robotics, Colorado Springs, CO). The plates are loaded onto an MS-2000 stage (Applied Scientific Instruments, Eugene, OR) fixed to a Nikon TE-2000 (Nikon, Melville, NY) microscope. The robot and microscope are enclosed in an environmental chamber (InVivo Scientific, St Louis, MO) to enable around-the-clock imaging for six to seven days. Wide-field images are acquired according to in-house scripts. At each time point, montage images are generated for each well and fluorophore channel. Image analysis algorithms then extract cell-based information. Metadata generated from image acquisition and analysis flows into a central database for data modeling, mining and classification. signatures for screening small-molecule pharmacological HCS can be applied to diverse assay types, depending agents and genome-wide siRNA libraries. CPH takes on the experimental conditions and labeled proteins. advantage of inherent cell-to-cell heterogeneity, and Challenges still remain in image analysis and data the increased sensitivity resulting from temporal analysis interpretation, and new statistical tools will be necessary permits fewer cells to be analyzed. We therefore avoid to analyze time-dependent processes of millions of cells two main drawbacks of screening in primary cells — across thousands of conditions. Advances in HCS will decreased transfection efﬁciency and lack of cell hom- result from new microscopy techniques, such as spectral ogeneity. imaging, better ﬂuorescence proteins, and the maturation of stem cell technology. Greater knowledge of which Conclusion proteins to probe for particular physiologic and disease HCS is a technology with vast potential for academic processes will increase HCS sensitivity. HCS with live- researchers and particularly neuroscientists. Large-scale cell imaging in primary neurons is practical and will help screens are strategically essential in understanding com- answer some of the most elusive questions in neurobiol- plex biological systems and gain-of-function diseases. ogy and related disease. www.sciencedirect.com Current Opinion in Neurobiology 2009, 19:537–543 Author's personal copy

542 New technologies

Acknowledgements The authors used stable isotope labeling and mass spectrometry to compare the proteomes of cell lines to primary cells. The Hep1–6 liver We thank the members of the Finkbeiner Lab for their generous support cell line showed downregulation of proteins involved in complement and and advice. We thank G. Howard and S. Ordway for editorial assistance and coagulation factor production along with the important P450 family of K. Nelson for administrative assistance. This work was supported by the enzymes. There was also a drastic shift from oxidative to anaerobic Consortium for Frontotemporal Dementia Research, the Taube-Koret metabolism. Center for Huntington’s Disease Research, National Institutes of Health (NIH) grants 2R01 NS039074 and 2R01045491 from the National Institutes 14. Eglen RM, Gilchrist A, Reisine T: An overview of drug screening of Neurological Disorders and Stroke and 2P01 AG022074 from the using primary and embryonic stem cells. Comb Chem High National Institutes of Aging and by the J. David Gladstone Institutes (to Throughput Screen 2008, 11:566-572. S.F.). Support was also provided by the NIH-NIGMS UCSF Medical Scientist Training Program (to A.C.D.) and the California Institute of 15. Rubin LL: Stem cells and drug discovery: the beginning of a new era? Cell 2008, 132:549-552. Regenerative Medicine (P.S.). 16. Di Giorgio FP, Carrasco MA, Siao MC, Maniatis T, Eggan K: Non- References and recommended reading cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci 2007, Papers of particular interest, published within the period of review, 10:608-614. have been highlighted as: An in vitro model based on ES cells is presented for studying amyotrophic lateral sclerosis (ALS). 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30. Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, 42. Jones TR, Carpenter AE, Lamprecht MR, Moffat J, Silver SJ, Davidson MW, Tsien RY: Improving the photostability of bright Grenier JK, Castoreno AB, Eggert US, Root DE, Golland P et al.: monomeric orange and red fluorescent proteins. Nat Meth Scoring diverse cellular morphologies in image-based 2008, 5:545-551. screens with iterative feedback and machine learning. Proc A novel screening method is presented that increased the photostability Natl Acad Sci U S A 2009, 106:1826-1831. of bright red and orange fluorescent proteins TagRFP and mOrange to create TagRFP-T and mOrange2. Through a combination of random and 43. Zhang Y, Zhou X, Degterev A, Lipinski M, Adjeroh D, Yuan J, site-directed mutagenesis, the new proteins became 9 and 25 times more Wong ST: Automated neurite extraction using dynamic photostable, respectively. 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Monoclonal Antibodies Recognize Distinct Conformational Epitopes Formed by Polyglutamine in a Mutant Huntingtin Fragment*□S Received for publication, March 24, 2009, and in revised form, May 4, 2009 Published, JBC Papers in Press, June 2, 2009, DOI 10.1074/jbc.M109.016923 Justin Legleiter‡§1,2, Gregor P. Lotz‡§, Jason Miller‡¶ʈ3, Jan Ko**, Cheping Ng‡, Geneva L. Williams‡, Steve Finkbeiner‡§ʈ‡‡¶¶, Paul H. Patterson**, and Paul J. Muchowski‡§ §§¶¶4 From the ‡Gladstone Institute of Neurological Disease, Departments of §Neurology and ¶Chemistry and the Chemical Biology Graduate Program, ʈMedical Scientist Training Program, and Departments of ‡‡Physiology and §§Biochemistry and Biophysics, University of California, San Francisco, California 94158 and the ¶¶Taube-Koret Center for Huntington’s Disease Research and **Biology Division, California Institute of Technology, Pasadena, California 91125

Huntington disease (HD) is a neurodegenerative disorder that are classified as “conformational diseases,” which include caused by an expansion of a polyglutamine (polyQ) domain in Alzheimer disease (AD), Parkinson disease (PD), the prion the N-terminal region of huntingtin (htt). PolyQ expansion encephalopathies, and many more (2–4). The length of polyQ Downloaded from above 35–40 results in disease associated with htt aggregation expansion in HD is tightly correlated with disease onset, and a into inclusion bodies. It has been hypothesized that expanded critical threshold of 35–40 glutamine residues is required for polyQ domains adopt multiple potentially toxic conformations disease manifestation (5). Biochemical and electron micro- that belong to different aggregation pathways. Here, we used scopic studies with htt fragments demonstrated that expanded atomic force microscopy to analyze the effect of a panel of anti- polyQ repeats (Ͼ39) form detergent-insoluble aggregates that www.jbc.org htt antibodies (MW1–MW5, MW7, MW8, and 3B5H10) on share characteristics with amyloid fibrils (6–8), and the forma- aggregate formation and the stability of a mutant htt-exon1 tion of amyloid-like fibrils by polyQ was confirmed by studies fragment. Two antibodies, MW7 (polyproline-specific) and with synthetic polyQ peptides (9). Collectively, these studies 3B5H10 (polyQ-specific), completely inhibited fibril formation demonstrated a correlation between polyQ length and the at UCSF Library & CKM, on April 8, 2010 and disaggregated preformed fibrils, whereas other polyQ-spe- kinetics of aggregation. This phenomenon has been recapitu- cific antibodies had widely varying effects on aggregation. These lated in cell-culture models that express htt fragments (10–12). results suggest that expanded polyQ domains adopt multiple Although it is clear that proteins with expanded polyQ repeats conformations in solution that can be readily distinguished by assemble into fibrils in vitro, recent studies have reported that monoclonal antibodies, which has important implications for htt fragments can also assemble into spherical and annular oli- understanding the structural basis for polyQ toxicity and the gomeric structures (13–16) similar to those formed by A␤ and development of intrabody-based therapeutics for HD. ␣-synuclein, which are implicated in AD and PD, respectively. While the major hallmark of HD is the formation of intranuclear and cytoplasmic inclusion bodies of aggregated htt (17), 5 Huntington disease (HD) is a fatal neurodegenerative disor- the role of these structures in the etiology of HD remains con- der that is caused by an expansion of a polyglutamine (polyQ) troversial. For instance, the onset of symptoms in a transgenic domain in the protein huntingtin (htt), which leads to its aggre- mouse model of HD follows the appearance of inclusion bodies gation into fibrils (1). HD is part of a growing group of diseases (18), while other studies indicate that inclusion body formation may protect against toxicity by sequestering diffuse, soluble * This work was supported, in whole or in part, by National Institutes of Health forms of htt (10, 19, 20). Based on the direct correlation Grants R01NS047237 and R01NS054753 (to P. J. M.), P01AG022074 (to between polyQ length, htt aggregation propensity, and toxicity S. F.), R01NS039074 (to S. F.), and R01NS045091 and R01NS055298 (to P. H. P.). This work was also supported by the Hereditary Disease Founda- (6), it has been hypothesized that the aggregation of htt may tion and the Cure Huntington’s Disease Initiative. mediate neurodegeneration in HD. However, there is no clear □S The on-line version of this article (available at http://www.jbc.org) contains consensus on the aggregate form(s) that underlie toxicity, and supplemental Fig. 1 and Movies S1 and S2. 1 Supported by a postdoctoral fellowship from the Hereditary Disease there likely exist bioactive, oligomeric aggregates undetectable Foundation. by traditional biochemical and electron microscopic ap- 2 Current address: The C. Eugene Bennett Dept. of Chemistry, Wes Virginia proaches whose formation precedes disease symptoms. University, Morgantown, WV 26505. 3 Supported by the National Institutes of Health-NIGMS UCSF Medical Scien- Although identification of the one or more toxic species of htt tist Training Program and a fellowship from the University of California at that trigger neurodegeneration in HD remains elusive, such San Francisco Hillblom Center for the Biology of Aging. species might exist in a diffuse, mobile fraction rather than in 4 To whom correspondence should be addressed: Gladstone Institute of Neu- rological Disease,1650 Owens St., San Francisco, CA 94158. Tel.: 415-734- inclusion bodies (19). A thioredoxin-polyQ fusion protein was 2515; Fax: 415-355-0824; E-mail: [email protected]. recently reported to exhibit toxicity in a meta-stable, ␤-sheet-rich, 5 The abbreviations used are: HD, Huntington disease; polyQ, polyglutamine; monomeric conformation (21), suggesting that polyQ can adopt htt, huntington; PD, Parkinson disease; polyP, polyproline; AFM, atomic force microscopy; GST, glutathione S-transferase; GFP, green fluorescent multiple monomeric conformations, only some of which may be protein. toxic. Consistent with such a scenario, molecular dynamic simu-

AUGUST 7, 2009•VOLUME 284•NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 21647 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin lations and fluorescence correlation spectroscopy experiments while another (mEM48) ameliorates neurological symptoms in with synthetic polyQ peptides indicate that polyQ domains can a mouse model of HD (48). adopt a heterogeneous collection of collapsed conformations that Three of the antibodies examined in this study (MW1, MW2, are in equilibrium before aggregation (22–25). and MW7) modulate htt-induced cell death when co-trans- Although biochemical, biophysical, and computational fected as single-chain variable region fragment antibodies approaches have yielded insight into the structures formed by (scFvs) in 293 cells with htt exon 1 containing an expanded polyQ in vitro, whether such structures form in vivo remains polyQ domain (46). In these studies MW1 and MW2, which largely unknown. Indeed, determining the conformational state bind to the polyQ repeat in htt, increased htt-induced toxicity of any misfolded/aggregated protein in situ and/or in vivo and aggregation (46). Conversely, MW7, which binds to the remains a major technical challenge. polyproline (polyP) regions adjacent to the polyQ repeat in htt, Toward this goal, antibodies have been explored as a poten- decreased its aggregation and toxicity (46). Interestingly, MW7 tially powerful tool for detecting specific conformations or mul- has also been shown to increase the turnover of mutant htt in timeric states of aggregated proteins in situ. Antibodies specific cultured cells and reduce its toxicity in corticostriatal brain for amyloid fibrils often do not react with natively folded glob- slice explants (49). ular proteins from which they are derived, suggesting that such Given the difficulty in understanding which specie(s) of htt antibodies recognize a conformational epitope (26, 27). Several exist and mediate pathogenesis in the putative toxic diffuse antibodies display conformation-dependent interactions with fraction of neurons, we sought to rigorously characterize the amyloids, aggregation intermediates, or natively folded precur- conformational specificity of a panel of anti-htt antibodies, the Downloaded from sor proteins. For example, there are antibodies specific for best in situ probes currently available for distinguishing spe- paired helical filaments of Tau (28–31), of aggregated forms of cie(s) of htt. We reasoned that if htt can adopt multiple confor- A␤ ranging from dimers to fibrils (32–34), and of native (35) or mations that mediate different aggregation pathways, then disease-related (36) forms of the prion protein. Antibodies have anti-htt antibodies should differentially alter htt aggregation also been developed that are specific for common structural pathways by stabilizing or sequestering the specific conformers motifs associated with amyloid diseases, such as oligomers (37) or aggregates they recognize. We therefore examined the www.jbc.org and fibrils (38), independent of the peptide sequence of the effects of various antibodies on mutant htt fragment fibril for- amyloid forming protein from which they are derived, suggest- mation and stability by atomic force microscopy (AFM). Our

ing the potential for a common mechanism of aggregation and results are consistent with the hypothesis that monoclonal anti- at UCSF Library & CKM, on April 8, 2010 toxicity for these diseases. bodies recognize distinct conformational epitopes formed by With regard to htt, several antibodies (MW1, MW2, MW3, polyQ in a mutant htt fragment. MW4, MW5, IC2, and IF8), which are specific for polyQ repeats, stain Western blots of htt with expanded polyQ repeats EXPERIMENTAL PROCEDURES much more strongly than htt with normal polyQ length (39, 40), Protein Purification—GST-HD53Q fusion proteins were suggesting that these antibodies may recognize abnormal purified as described (52). Cleavage of the GST moiety by Pre- polyQ conformations. Furthermore, these polyQ-specific anti- Scission Protease (Amersham Biosciences) initiates aggrega- bodies have distinct staining patterns in immunohistochemical tion. Fresh, unfrozen GST-HD53Q was used for each experi- studies of brain tissue sections (39). In one study, the affinity ment. GST-HD53Q was centrifuged at 20,000 ϫ g for 30 min at and stoichiometry of MW1 binding to htt increased with polyQ 4 °C to remove any preexisting aggregates before the addition of length, suggesting a “linear lattice” model for polyQ (41). This the PreScission protease. MW series of antibodies were model is supported by a crystal structure of polyQ bound to obtained as described previously (39). 3B5H10 was purified as MW1, which showed that polyQ can adopt an extended, coil- described before (53). like structure (42). However, an independent structural study Western Blot Analysis—For Western blotting analysis, puri- showed that the anti-polyQ antibody 3B5H10 binds to a com- fied GST-HD53Q proteins were incubated at 37 °C with shak- pact ␤-sheet-like structure of polyQ in a monomeric htt frag- ing at 1400 rpm. Solutions were sampled at 0, 5, and 20 h after ment.6 These results clearly indicate that polyQ domains can the addition of PreScission Protease. Proteins and aggregates fold into at least two unique, stable, monomeric conformations were separated by SDS-PAGE and then transferred onto Prot- and suggest that the “linear lattice” model is not generally appli- ran BA85 nitrocellulose membranes (pore-size ϭ 0.45 ␮m, cable to all polyQ structures. Whatman) by standard Western transfer techniques. The Not only are antibodies useful for understanding what polyQ membranes were incubated for 1 h at 37 °C with MW1, MW2, structures exist in situ, especially in the diffuse htt fraction of MW3, MW4, MW5, MW7, MW8, or 3B5H10 at a dilution of neurons, but antibodies and/or intrabodies may also have 1:1000. The membranes were then incubated with horseradish potential as therapeutic agents. For example, several studies peroxidase-conjugated rabbit anti-mouse IgG or IgM (Jackson showed that intrabodies reduce htt toxicity in cellular models ImmunoResearch) at a 1:5000 dilution for1hatroom temper- (44–49). Moreover, one intrabody (C4) slows htt aggregation ature. The horseradish peroxidase was detected using an ECL and prolongs lifespan in a Drosophila model of HD (50, 51), Advance Western blotting Detection System (Amersham Bio- sciences), and the membranes were exposed to x-ray films. Neuronal Culture, Transfection, and Immunocytochemistry— 6 C. Peters-Libeu, E. Rutenber, J. Miller, Y. Newhouse, P. Krishnan, K. Cheung, E. Brooks, K. Widjaja, T. Tran, D. Hatters, S. Mitra, M. Arrasate, L. Mosquera, D. Primary cultures of rat striatal neurons were prepared from Taylor, K. Weisgraber, and S. Finkbeiner, submitted for publication. embryos (embryonic days 16–18) and transfected with plas-

21648 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 32•AUGUST 7, 2009 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin mids (6–7 days in vitro) as described (10). Neurons were co- fibrils and were compared using a t test. Aggregate populations ex1 transfected with pGW1-Htt -Q46 or 97-GFP in a 1:1 molar based on height were compared using a Spearman’s rank cor- ratio, using a total of 1–4 ␮g of DNA in each well of a 24-well relation performed with GraphPad Prism. plate. After transfection, neurons were maintained in serum- free medium. All immunocytochemistry was performed as RESULTS described (54). Cy3-conjugated secondary antibodies targeted Anti-htt Antibodies Recognize a Variety of SDS-stable Oligo- to the appropriate primary antibody were acquired from Jack- meric Species of HD53Q—All experiments in this study, with son Immunolabs. the exception of the immunocytochemistry studies described Atomic Force Microscopy—For experiments on monomeric later, were performed with a mutant htt fragment that preparations, GST-HD53Q was incubated at 20 ␮M alone or expresses exon 1 with 53Q (HD53Q). HD53Q was purified with anti-htt antibodies (MW1, MW2, MW3, MW4, MW5, from Escherichia coli as a soluble fusion with glutathione MW7, MW8, or 3B5H10) in a 1:1 ratio of protein to antigen S-transferase (GST) (Fig. 1) (52). After purification, GST- binding sites in buffer A (50 mM Tris-HCl, pH 7, 150 mM NaCl, HD53Q appeared non-aggregated as determined by AFM anal- 1mM dithiothreitol). PreScission protease (4 units/100 ␮gof ysis and size-exclusion chromatography (data not shown). The fusion protein) was added at time zero to initiate GST cleavage HD53Q fragment contains epitopes specifically recognized by and aggregation. Samples were incubated at 37 °C with shaking the panel of eight independent monoclonal anti-htt antibodies at 1400 rpm for the duration of the experiment. At time 1, 5, 8, (Fig. 1A) used in this study. MW1, MW2, MW3, MW4, MW5, and 24 h after cleavage of the GST, a sample (5 ␮l) of each and 3B5H10 are specific for the polyQ domain. MW7 is specific Downloaded from incubation solution was deposited onto freshly cleaved mica for the polyP domains. MW8 is specific for the last seven resi- (Ted Pella Inc., Redding, CA) and allowed to sit for 1 min. The dues of the C terminus of htt exon 1. substrate was washed with 200 ␮l of ultrapure water and dried Cleavage of a unique peptide sequence between the GST under a gentle steam of air. For experiments on preformed moiety and HD53Q with a site-specific protease (PreScission fibrils, 40 ␮M solutions of HD53Q were incubated alone for 5–6 protease) released the HD53Q fragment, initiating aggregation h after the removal of the GST tag to allow the formation of in a time-dependent manner as reported (7, 15). Western blots www.jbc.org fibrils. Buffer or anti-htt antibodies (MW1, MW2, MW3, of HD53Q were used to monitor cleavage 0, 5, and 20 h after the MW4, MW5, MW7, MW8, or 3B5H10) were added so that the addition of the protease (Fig. 1B). Before proteolytic cleavage ␮ ϭ final concentration of HD53Q was 20 M, and the ratio of (t 0 h), most antibodies specific for the polyQ domain at UCSF Library & CKM, on April 8, 2010 HD53Q to anti-htt antigen binding sites was 1:1. These solu- detected a prominent band of intact htt-GST fusion protein tions were sampled immediately and 3 h after the addition of that migrated at an apparent molecular mass of ϳ53 kDa, and a the buffer or anti-htt antibody. Dose dependence studies of less intense band that migrated at an apparent molecular mass fibril disaggregation by MW7 and 3B5H10 were performed of a dimer of the fusion protein (ϳ106 kDa). At later time similarly, except that the ratio of HD53Q to antibody binding points, MW1 and MW3 recognized the intact fusion protein site varied (10:1, 5:1, and 1:1) and solutions were sampled at 0, 1, and a band that migrated at a lower apparent molecular mass and 3 h after the addition of the antibodies. that may represent monomeric HD53Q (ϳ40 kDa). MW2 did Each sample was imaged ex situ using an MFP3D scanning not recognize this ϳ40-kDa species after proteolytic cleavage probe microscope (Asylum Research, Santa Barbara, CA). but did react with a larger, potentially dimeric species (ϳ80 Images were taken with silicon cantilevers with nominal spring kDa) at later time points. MW4, MW5, and 3B5H10 recognized constants of 40 newtons (N)/m and resonance frequency of a ϳ40-kDa species and a variety of SDS-stable bands of HD53Q, ϳ300 kHz. Typical imaging parameters were: drive amplitude some of which may be fragments of HD53Q. Only antibodies 150–500 kHz with set points of 0.7–0.8 V, scan frequencies of that were not specific for the polyQ domain (MW7 and MW8) 2–4 Hz, image resolution 512 by 512 points, and scan size of 5 recognized large aggregated forms of HD53Q that remained in ␮m. All experiments were performed in triplicate. the wells of the gel, indicating that the polyQ epitopes recog- For in situ AFM experiments tracking individual fibrils, solu- nized by these anti-polyQ antibodies are not accessible or tions containing preformed fibrils of HD53Q were allowed to absent in large aggregates. Of the two antibodies that bound the rest on mica until several fibrils were present on the surface. large aggregated form, only MW7 stained the ϳ40-kDa species Then, the substrate was washed with buffer A to remove proof HD53Q. These results indicate quite remarkably that six teins remaining in solution. The deposited fibrils were either independent anti-polyQ antibodies (MW1–5 and 3B5H10) imaged in clean buffer as a control or in the presence of anti-htt detect a variety of stable polyQ epitopes formed by HD53Q, antibodies (2.5 ␮M final concentration). Images were taken with even after apparent htt denaturation in SDS. Two antibodies V-shaped oxide-sharpened silicon nitride cantilevers with a against regions outside the polyQ stretch of htt exon1 (MW7 nominal spring constants of 0.5 N/m. Scan rates were set at 1–2 and -8) appear to expand the repertoire of recognizable htt Hz with cantilever drive frequencies ranging from ϳ8–12 kHz. species further. Statistics—All error bars in quantification of ex situ AFM Anti-htt Antibodies Recognize a Variety of htt Species in Neu- experiments (number of fibrils or oligomers per ␮m2) represent rons in Situ—To determine if these anti-htt antibodies could the standard error of at least three independent experiments distinguish different htt epitopes in neurons, we applied immu- and were compared using a t test. All error bars in quantifica- nocytochemistry to an established neuronal model (19) in tion of in situ AFM experiments (change in fibril length) repre- which primary striatal neurons are transiently transfected with sent the standard error measured from at least eight individual a mutant htt exon1 fragment fused to enhanced green fluores-

AUGUST 7, 2009•VOLUME 284•NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 21649 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

fibril formation, AFM images from all incubations were analyzed by counting the number of fibrils per ␮m2 (Fig. 3A). For this analysis, the number of fibrils in the AFM images for a given sample was divided by the total area covered by the AFM images. Fibrils were defined as objects with a height larger than 5 nm and a length-to-width (aspect) ratio Ͼ3. The AFM images of HD53Q incubated alone displayed fibril growth and an increase in fibril abundance per unit area over the 24-h time course of the experiment (Figs. 2 and 3A). At 1 h after removal

of GST, only a small number of Downloaded from fibrils were present, and these increased in number and grew from several hundred nanometers to ϳ1 ␮m in length at later time points. The fibrils were ϳ6–8 nm tall and 12 nm wide (measured at half www.jbc.org height). Fibril formation in solutions of HD53Q co-incubated with

FIGURE 1. Anti-htt antibodies recognize a variety species of HD53Q in vitro and in situ. A, a schematic MW1, MW2, or MW4 altered at UCSF Library & CKM, on April 8, 2010 representation of the GST-htt exon 1 fusion protein with 53Q (HD53Q) shows a PreScission protease site aggregation similarly (Figs. 2 and located between GST and the htt fragment (not drawn to scale) and the locations of epitopes for the antibodies that were used in this study. B, Western blots of HD53Q after incubation with protease for varying times, 3A). After 1 h of incubation, the probed with antibodies as labeled. The location of bands representing intact GST-HD53Q fusion protein at ϳ53 number of fibrils/␮m2 significantly kDa is indicated by a green arrow. A band that migrated at an apparent molecular mass of a dimer of the fusion increased in the presence of these protein (ϳ106 kDa) is indicated by a red arrow.Ablue arrow indicated the location of the wells of the gel where larger HD53Q aggregates are observed. C, primary cultures of rat striatal neurons expressing a GFP-labeled antibodies. Despite this early mutant htt-exon1 fragment with 97Q were analyzed by immunocytochemistry with antibodies as labeled. increase in the number of fibrils, MW1, MW2, and MW4 all had sig- cent protein (GFP) (Fig. 1C). We compared the GFP signal, nificantly fewer fibrils than the controls at later time points. which exhibited fluorescence in a diffuse cytoplasmic localiza- Co-incubation of HD53Q with MW8 also resulted in an initial tion and in inclusion bodies, to that detected by specific anti- increase in the number of fibrils formed, with a significant bodies. Consistent with the results with Western blots, only reduction compared with controls at later time points. How- MW7 and MW8 labeled large htt inclusion bodies based on ever, MW8 appeared to be the least effective antibody in reduc- co-localization with the GFP signal from htt. MW7 also stained ing fibril formation after 24 h. At early time points, the number diffuse htt. PolyQ-specific antibodies did not stain inclusion of fibrils formed in the presence of MW3 and MW5 did not bodies; rather, they recognized a diffuse population of htt pro- significantly differ from controls (Figs. 2 and 3A). By 24 h of teins. All of these results were consistent with Western blots co-incubation, however, both MW3 and MW5 had signifi- from Fig. 1B. This diffuse population might contain a heteroge- cantly inhibited HD53Q fibril formation. These results suggest neous mix of monomeric conformers and soluble, oligomeric that MW1–5 may recognize one or more conformers of mutant aggregates. The Western blot and immunocytochemistry stud- htt that are required for efficient fibril formation. ies suggest that these antibodies recognized different conform- Unlike all other antibodies tested, MW7 and 3B5H10 com- ers or oligomeric forms of HD53Q. pletely prevented fibril formation of HD53Q over the entire Anti-htt Antibodies Modulate htt Aggregation Differentially— time course of the experiment (Figs. 2 and 3A). Instead of fibrils, We next used AFM to analyze the effects of anti-htt antibodies compact globular structures were observed in co-incubations on HD53Q aggregation. Co-incubation experiments were per- of HD53Q with MW7 or 3B5H10. The height of individual formed with monomeric preparations of HD53Q and each anti- globular structures was analyzed at all time points for HD53Q body. Representative AFM images of aliquots removed from with or without MW7 or 3B5H10 (Fig. 3, B–D). Height was solutions of HD53Q in the presence and absence of anti-htt chosen for analysis because it is the most accurately measured antibodies after 1, 5, 8, and 24 h of incubation are shown in Fig. dimension in AFM, and it does not contain artifacts due to the 2. The concentration of HD53Q in all solutions was 20 ␮M, and finite shape and size of the probe tip. Fibrillar structures were the ratio of antigen binding sites on the antibody to HD53Q was not included in the analysis with HD53Q alone. In incubations 1:1. In an effort to quantify the effect the anti-htt antibodies on of HD53Q alone, globular oligomers gradually increased in

21650 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 32•AUGUST 7, 2009 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

distributions under each condition did not change over time based on Spearman’s rank correlation coefficient (p Ͻ 0.001). That is, the size of globular species formed upon co-incubation of HD53Q with MW7 was the same at all time points, as was true for co-incubations of HD53Q with 3B5H10. This indicated that globular species observed in these co-incubations were different from those formed in incubations of HD53Q alone. Overall, the quantitative AFM analyses demonstrate that antibodies specific for the polyQ domain modulate HD53Q aggregation differentially and that antibodies

with specificity for other domains of Downloaded from htt can also alter this process. We next performed biochemical experiments to confirm the AFM results, in which antibodies were added to monomeric preparations of GST-HD53Q before ini- www.jbc.org tiating aggregation with protease. 20 ␮M HD53Q solutions were

sampled after 8 h for Western blot at UCSF Library & CKM, on April 8, 2010 analysis of aggregate formation by staining with MW8 (supplemental Fig. S1). Before addition of protease (t ϭ 0 h), no aggregated HD53Q was detected. Aggregated HD53Q was detected in the wells for HD53Q alone after8hofincuba- tion; however, there appeared to be fewer aggregates detected for HD53Q incubated with MW1– FIGURE 2. Anti-htt antibodies modulate htt aggregation differentially. Representative 2 ␮m ϫ 2 ␮m AFM images of 20 ␮M HD53Q incubated in the absence or presence of antibodies as labeled for 1, 5, 8, and 24 h after MW5 and MW8. For co-incuba- cleavage of the GST moiety. The ratio of antigen binding sites to HD53Q was 1:1. For HD53Q alone and with tions of HD53Q with MW7 and MW1-MW5 or MW8, fibrillar structures (black arrows) appeared after 1–5 h of incubation. The number of fibrils 3B5H10, no aggregates were de- increased at 8 and 24 h. However, it appeared that there were more fibrils for HD53Q alone. For incubations of HD53Q with MW7 or 3B5H10, no fibrillar structures appeared throughout the 24-h experiment. In incubations tected in the well, confirming the with MW7, globular aggregates (blue arrows) around ϳ3.5 nm tall were the dominant species observed at all complete inhibition of aggregate time points. For incubation with 3B5H10, smaller globular species (green arrows) ϳ2.5 nm tall were present at all time points. Shown are representative AFM images. Quantification of the number of fibrils per ␮m2 in these formation by these antibodies. experiments is shown in Fig. 3. Anti-htt Antibodies MW7 and 3B5H10 Disassemble htt Aggregates— height as a function of time (Figs. 2 and 3B). The oligomers To test the effects of different antibodies on pre-aggregated observed at 1 and 24 h represented distinct populations of HD53Q, GST was first removed from HD53Q by proteolytic HD53Q aggregates, because the height distributions were no cleavage, and then HD53Q was incubated for 6–8 h prior to longer similar based on a Spearman’s rank correlation coeffi- addition of anti-htt antibodies. The preincubation resulted in a cient (p ϭ 0.37). MW7 and 3B5H10 appeared to stabilize dis- large population of HD53Q fibrils (time point0hinFig. 4). tinct globular structures, which likely are complexes of anti- After the initial incubation time, aliquots were deposited on body and HD53Q, with globular structures observed for mica, dried, and imaged. Approximately 10–20 fibrils were co-incubations of HD53Q with MW7 being slightly larger than observed per 5 ␮m2 by ex situ AFM. These pre-aggregated those observed with 3B5H10 (compare Fig. 3C with 3D). HD53Q solutions were divided into several aliquots to which Whereas the mean height of HD53Q oligomers observed in buffer (for control) or antibodies were added to a final antigen controls at 24 h was 5.3 Ϯ 1.65 nm, globular species observed binding site to HD53Q ratio 1:1, with a final HD53Q concen- from co-incubations of HD53Q with MW7 and 3B5H10 were tration of 20 ␮M. Immediately after buffer or antibody were 4.4 Ϯ 1.76 nm and 2.6 Ϯ 0.74 nm tall, respectively. The height added, the HD53Q solutions were re-sampled and imaged to

AUGUST 7, 2009•VOLUME 284•NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 21651 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

verify that fibrils were still present to obtain a time point 0-h measurement (Fig. 4). The solutions were then incubated for an additional 3 h, sampled, and imaged (Fig. 4). Pre- formed fibrils that were treated with buffer, MW1, MW2, MW3, MW4, MW5, or MW8 appeared to be stable, as the number of fibrils per ␮m2 was unchanged between 0 and 3 h (Figs. 4 and 5A). Importantly, the two anti-htt antibodies that prevented fibril formation (MW7 and 3B5H10) also significantly reduced the number of preformed fibrils. At the 1:1 ratio of antigen binding sites to HD53Q, MW7 and 3B5H10 com-

pletely disaggregated preformed Downloaded from FIGURE 3. Quantification over time of HD53Q aggregates in the absence and presence of anti-htt fibrils. antibodies. A, the number of fibrils/␮m2 was calculated from AFM images of HD53Q incubated in the We next evaluated the dose absence and presence of anti-htt antibodies analyzed at 1, 5, 8, and 24 h of incubation. Compared with control experiments of HD53Q alone, all of the antibodies significantly reduced the number of fibrils dependence of HD53Q fibril disag- formed at later time points. However, there was a significant increase in the number of fibrils formed after gregation by MW7 and 3B5H10 1 h for incubations with MW1, MW2, MW4, and MW8. MW7 and 3B5H10 completely inhibited the forma- # (Fig. 5, B and C). Preformed fibrils of tion of fibrils over the time course of the experiments. , a significant increase (p Ͻ 0.05) in the number of www.jbc.org fibrils/␮m2 in comparison to HD53Q alone at the same time point (Student’s t test). * and denote HD53Q were treated with MW7 or significant decreases (* ϭ p Ͻ 0.01, ϭ p Ͻ 0.05) in the number of fibrils/␮m2 in comparison to HD53Q 3B5H10 at an antigen binding site to ᭜ alone at the same time point (Student’s t test). indicates that no fibrils were observed. The experiment HD53Q ratio of 1:10, 1:5, and 1:1. was replicated six times, and the error bars represent standard error. B–D, height histograms for globular structures observed in HD53Q alone (B) and with MW7 (C) or 3B5H10 (D) as a function of time. Whereas the Controls consisting of preformed at UCSF Library & CKM, on April 8, 2010 height of HD53Q oligomers gradually increased over time, both MW7 and 3B5H10 stabilized distinct HD53Q fibrils treated with buffer globular structures that likely represent complexes of HD53Q and antibody. The legend applies to all panels in the figures. were also prepared. The final concentration of HD53Q was 20 ␮M in all experiments. These solutions were sampled at 0, 1, and 3 h after the addition of buffer, MW7, or 3B5H10 and imaged with AFM. Preformed fibrils present on mica were significantly reduced at all ratios of antibody:htt, with a clear antibody dose dependence for the disaggregation. Tracking the Fates of Individual HD53Q Fibrils Exposed to Anti-htt Antibodies in Situ—To further explore the stability of preformed fibrils of HD53Q, we took advantage of the ability of AFM in solution to track morphological changes of individual fibrils as a function of time (Fig. 6 and supplemental movies S1 and S2). Preformed HD53Q fibrils were deposited on mica and imaged continuously. Buffer (control) or anti-htt antibodies were injected directly into the fluid cell of the AFM. This allowed for the tracking of the fate of individual fibrils exposed to different anti-htt antibodies. Fibrils that were treated with buffer remained stable with no apparent change in length for FIGURE 4. Ex situ AFM analysis indicates that the anti-htt antibodies MW7 and 3B5H10 disassemble htt aggregates. Samples of HD53Q were over 300 min, verifying that the continual scanning of the AFM incubated for 6–8 h after removal of the GST moiety to form a large pop- probe tip was not sufficient to invoke mechanical disruption of ulation of fibrils. Then, buffer (as control), MW1-MW5, MW7, MW8, or 3B5H10 was added. The ratio of antigen binding sites to HD53Q was 1:1. fibril integrity (supplemental movie S1). Similarly, the majority The solutions were sampled directly after the addition of buffer/antibod- of fibrils treated with MW1, MW2, MW3, MW4, MW5, or ies (t ϭ 0 h) and deposited on mica for AFM imaging. Fibrils (black arrows) MW8 did not exhibit large morphological changes for up to 300 were present in all samples at this time. The solutions were incubated for 3 h after the addition of buffer or antibodies and re-sampled. Fibrils (black min during continuous imaging (data not shown). Consistent arrows) were still present in samples that had been treated with buffer, with the co-incubation experiments described above, fibrils MW1-MW5 or MW8. However, fibrils were no longer detected in samples treated with MW7 or 3B5H10. Treatment with MW7 resulted in a large exposed to MW7 and 3B5H10 gradually shortened in length population of globular species (blue arrows) that varied greatly in size with (supplemental movie S2). In the case of MW7, some fibrils the majority of species ranging in height from 4 to 8 nm. Treatment with completely disappeared from the surface. We then quantified 3B5H10 resulted in globular species (green arrows) that were only ϳ2.5 nm tall. Shown are representative 2 ␮m ϫ 2 ␮m AFM images. Quantification of the change in length of individual fibrils as a function of time the number of fibrils per ␮m2 in these experiments is shown in Fig. 5. (Fig. 7, A–I) by subtracting the length at time 0 from the length

21652 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 32•AUGUST 7, 2009 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin Downloaded from www.jbc.org

FIGURE 6. Monitoring disassembly of single htt aggregates incubated at UCSF Library & CKM, on April 8, 2010 with MW7 or 3B5H10 by in situ AFM. Samples of HD53Q were incubated for 6–8 h after removal of the GST moiety to form a large population of fibrils. These fibrils were deposited on mica and imaged using in situ AFM, which allows for the tracking of the fate of individual fibrils as a function of time. These fibrils were imaged in the absence or presence of anti-htt antibodies. Fibrils appeared to be stable after treatment with buffer, MW1-MW5, or MW8 (location of stable fibrils indicated by black arrows). However, treatment with MW7 or 3B5H10 caused fibrils to disaggregate and/or shorten in length (location of disaggregating fibrils indicated by green arrows). Scale bar represents 500 nm and is applicable to all images. See also supplemental movies S1 and S2.

of the fibril at any given time. While the length of fibrils did not vary as a function of time for HD53Q treated with buffer or MW1-MW5 or MW8 (Fig. 7, A–F and H), all fibrils treated with MW7 or 3B5H10 displayed a negative change in length. The average rate of change in fibril length was calculated based on measurements on individual fibrils under all conditions (Fig. 7J). Fibrils exposed to MW7 or 3B5H10 exhibited significant FIGURE 5. Quantification of the number of fibrils/␮m2 for pre-aggre- rates of decreasing contour length compared with control gated HD53Q treated with buffer or anti-htt antibodies. A, the number fibrils, with MW7 disaggregating fibrils at a faster rate than of fibrils/␮m2 was calculated from AFM images of incubations of fibrillar 3B5H10. The other antibodies did not differ significantly from preparations of HD53Q taken immediately after (t ϭ 0 h) and 3 h after the addition of buffer, MW1-MW5, MW7, MW8, or 3B5H10. The ratio of antigen the buffer control. These results indicate that some, but not all, binding sites to HD53Q was 1:1. For comparison, all bars are normalized to anti-htt antibodies can disassemble fibrils in solution. the number of fibrils/␮m2 at t ϭ 0 h for that sample. With the addition of buffer (control), MW1-MW5, or MW8, there was no change in the number MW7 and 3B5H10 Disassemble Fibrils by Forming Different of fibrils present after 3 h. With MW7 and 3B5H10, the number of fibrils was Complexes with htt—Because MW7 and 3B5H10 both pre- significantly reduced, indicating that these antibodies were able to disas- vented fibril formation and destabilized preformed fibrils, we semble preformed fibrils. *, p Ͻ 0.001 (Student’s t test). Error bars represent standard error. B and C, the dose dependence of fibril disaggregation next compared the height of the globular complexes formed by was studied by quantitative analysis of AFM images of fibrillar preps of htt with the antibodies when the antibodies were added to HD53Q taken immediately after (t ϭ 0 h), 1 h, and 3 h after the addition of monomeric or fibrillar HD53Q (Fig. 8). Globular species B, MW7 or C, 3B5H10. The ratio of antigen binding sites to HD53Q was 10:1, 5:1, and 1:1. For comparison, all bars are normalized to the number of formed after incubation of HD53Q in the absence of antibodies fibrils/␮m2 at t ϭ 0 h for that sample. The disaggregation of fibrils by MW7 were predominately 4–5 nm tall with a large number of oli- (B) and 3B5H10 (C) appeared to be dose-dependent. *, p Ͻ 0.01; **, p Ͻ 0.001 (Student’s t test). gomers taller than 6 nm (Fig. 8A). In contrast, globular species observed from co-incubations of MW7 or 3B5H10 with mono-

AUGUST 7, 2009•VOLUME 284•NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 21653 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

meric HD53Q were only 3–4 and 2–3 nm tall, respectively (Fig. 8, B and C). Interestingly, when MW7 was added to preformed fibrillar HD53Q and allowed to completely disaggregate the fibrils (3 h after addition MW7), the resulting oligomeric species were much larger than those observed following incubation of this antibody with monomeric HD53Q (Fig. 8B). These globular structures were predominately 5–6 nm tall with a large number of globular structures taller than 6 nm. Based on a Spearman’s rank correlation coefficient, this difference in size was statistically significant, demonstrating that the final size of the complex formed between MW7 and HD53Q can vary, depending upon the initial aggregation state of HD53Q. This result may indicate that MW7 can recognize both monomeric and aggregated forms of htt, consistent with the immunocytochemical experiments and Western blot analysis (Fig. 1). Sur- prisingly, the globular structures observed from the complete disaggregation (3 h after the addition of antibody) of preformed

HD53Q fibrils by 3B5H10 were precisely the same size as those Downloaded from formed when 3B5H10 was added to monomeric HD53Q, based on Spearman’s rank correlation coefficient. This indicates that, in contrast to MW7, 3B5H10, which has been previously shown to bind a monomer of htt,6 forms the same complex with HD53Q regardless of its initial aggregation state (Fig. 8C). This suggests that 3B5H10 is incapable of recognizing oligomeric www.jbc.org species of htt. Because MW7 apparently recognizes both aggregated and diffuse forms of htt, MW7 may be physically disrupt-

ing fibril structure by stabilizing a population of oligomeric at UCSF Library & CKM, on April 8, 2010 structures. However, as 3B5H10 only recognizes soluble, non- aggregated forms of htt, it may be tightly binding and sequestering a monomeric conformation of htt that is in direct equilibrium with fibril ends. DISCUSSION Expanded polyQ repeats in htt have been postulated to adopt multiple conformations, but it is unclear which conformations may exist in neurons and are pathogenic. To study the existence and effects of different htt conformations in neurons, appropriate conformational probes must be first be established and characterized. The ability of antibodies to be used in situ makes them attractive tools to measure htt conformations in neurons and to ultimately determine their functional significance in HD pathogenesis. We therefore set out to characterize the range of htt conformations that can be detected by a panel of anti-htt antibodies, including many that are specific for expanded polyQ repeats. Because various htt conformations have been linked to different aggregation pathways in vitro (15), we reasoned that different anti-htt antibodies may have disparate effects on aggregation if the antibodies are recognizing different htt conformational epitopes. In this study we showed that a panel of antibodies (MW1– MW5 and 3B5H10) that are all specific for polyQ sequences detected different aggregated species of HD53Q in Western blots and in cultured neurons. These antibodies also had widely

FIGURE 7. Quantification of change in length and rate of change of fibrils treated with anti-htt antibodies. A–I, the change in length (⌬length) of individual fibrils imaged in the absence and presence of anti-htt antibodies was (G) or 3B5H10 (I). J, the average rate of change of fibril length for fibrils treated tracked as a function of time as measured by in situ AFM. Fibril length with buffer (as control), MW1-MW5, MW7, MW8, or 3B5H10 was calculated, appeared stable with the addition of buffer (A), MW1-MW5 (B–F), or MW8 (H). showing that only MW7 and 3B5H10 caused a significant change in fibril The length of individual fibrils steadily decreased after treatment with MW7 length (*, p Ͻ 0.01 with a Student’s t test).

21654 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 32•AUGUST 7, 2009 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

varying effects on HD53Q aggregation, and some even disas- sembled preformed htt fibrils. MW1, MW2, and MW4 initially increased fibril formation before suppressing it at later time points. MW3, MW5, and MW8 slowed fibril formation. MW7 (polyP-specific) and 3B5H10 (polyQ-specific) completely prevented the formation of fibrillar structures. These two antibodies also destabilized preformed fibrils despite being specific for different regions of htt. These results are consistent with the hypothesis that expanded polyQ repeats can adopt multiple conformation-specific epitopes that can be easily discriminated by the immune system. While compared with controls at later time points, all of the polyQ-specific antibodies at least partially inhibited the formation of fibrils. MW1, MW2, and MW4 appeared to initially boost fibril formation. This initial increase in aggregation is consistent with previous reports that MW1 and MW2 enhanced aggregation, which was associated with increased

htt-induced toxicity, when they were expressed as scFvs in a Downloaded from cellular model of HD (46). Among the polyQ-specific antibodies we tested, 3B5H10 appears to recognize a unique polyQ conformation, because it was the only polyQ-specific antibody to completely prevent fibril formation and destabilize preformed fibrils. Recent structural studies lend further support to the notion that polyQ repeats can exist in stable conformations www.jbc.org of different structure. For example, a crystal structure of a polyQ peptide bound to MW1 showed that polyQ can adopt an

extended, coil-like structure (42). However, an independent at UCSF Library & CKM, on April 8, 2010 structural study showed that 3B5H10 binds to a compact ␤-sheet-like structure of polyQ.6 We speculate that MW1 binding to a range of conformations on single-stranded polyQ may initially catalyze the collapse of polyQ into aggregation-prone structures, accounting for the early increase in fibril formation for HD53Q incubated with MW1 compared with HD53Q incubated in buffer. However, as aggregation starts, the accumulation of MW1 antibody on each HD53Q molecule may eventually sterically hinder further aggregation, accounting for the late attenuation in fibril formation for HD53Q incubated with MW1 compared with HD53Q incubated in buffer. In contrast, 3B5H10’s binding to a compact, double-stranded structure of polyQ may fully bury the edges of the polyQ conformation that seeds aggregation, accounting for 3B5H10’s ability to completely block aggregation. Therefore, our results indicate unequivocally that polyQ domains can sample at least two FIGURE 8. Size analysis of aggregate observed with MW7 or 3B5H10 unique monomeric conformations, but the polyQ domains are added to monomeric or fibrillar HD53Q. A, HD53Q oligomers (HD53Q likely to adopt other stable or meta-stable structures as well. For incubated alone) after 5 h of incubation were predominantly 4–5 nm in height with several as tall as 6–8 nm. B, when MW7 was incubated (added example, fluorescence correlation spectroscopy experiments at t ϭ 0 h) with monomeric HD53Q (black diamonds), the height of glob- and molecular dynamics simulations (23) indicate that polyQ ular aggregates formed after5hofco-incubation were predominantly peptides can form a heterogeneous population of collapsed 3–4 nm tall, although there was a large portion of taller globular aggregates (shoulder on the right of the histogram). In contrast, when MW7 was structures in aqueous solution. In the absence of antibodies, htt incubated with pre-aggregated fibrillar HD53Q (gray circles), globular appears to be able to sample multiple conformations; however, aggregates (conditions where fibrils disaggregated) observed when a collapsed conformation appears to be the dominant species as imaged 3 h after addition of MW7 were much taller (4–5 nm) in compari- 6 son to those formed by adding MW7 to monomeric HD53Q, with a larger detected by small-angle x-ray scattering. portion of aggregates being 5–10 nm tall. C, when 3B5H10 was incubated The antibodies MW7 (anti-polyP) and 3B5H10 (anti-polyQ) (added at t ϭ 0 h) with monomeric HD53Q (black diamonds), the majority of globular aggregates observed after 5 h co-incubation were 2–3 nm in both destabilized polyQ fibrils. However, the mechanisms height. Similarly, when 3B5H10 was incubated with pre-aggregated fibril- appear to be different, based on size analysis of the aggregate/ lar HD53Q (gray circles), globular species (conditions where fibrils disag- complex after disaggregation. Although MW7 and 3B5H10 are gregated) observed 3 h after the addition of 3B5H10 again were predominantly 2–3 nm tall. specific for different regions of htt, there are other notable differences between the two antibodies. MW7 is an IgM while

AUGUST 7, 2009•VOLUME 284•NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 21655 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

3B5H10 is an IgG. MW7 recognizes aggregated and diffused sequence to the C terminus of a polyQ peptide altered both forms of htt by Western blot and immunocytochemistry, aggregation kinetics and conformational properties of the whereas 3B5H10 does not recognize aggregates of htt. MW7 polyQ tract (56). Flanking polyP sequences can also inhibit the can block fibril formation from monomeric HD53Q by binding formation of ␤-sheet structure in polyQ peptides by inducing a to a specific conformer, resulting in a stable complex with a PPII-like helix structure, extending the length of the polyQ narrow size distribution. However, MW7 can also bind aggre- domain necessary to induce fibril formation (57). Flanking gates and may physically bind to fibrils, disrupting their stabil- sequences in htt exon1 of various polyQ domain lengths mod- ity, and resulting in a different population of oligomeric com- ulate toxicity in yeast models, not only in cis, but also in trans plexes with a broader size distribution. Although we did not during aggregation (58, 59). Interestingly, the proline-rich observe any direct binding of MW7 to htt fibrils by in situ AFM, regions of htt exon1 reduced polyQ-related toxicity in these this possibility cannot be ruled out because the ϳ8-min interval studies (58, 59). between images may not be fast enough to capture such an Protein interactions with the polyP sequence in htt may have event. Although 3B5H10 also formed a stable complex with a major influence on the conformation of the adjacent polyQ monomeric HD53Q, it did not appear to bind to large htt aggre- domain. Other studies have demonstrated that the polyP gates observed by Western blot, biochemical, and immunocy- domain of htt interacts with vesicle trafficking proteins (i.e. tochemical methods, suggesting that 3B5H10 disaggregates HIP1, SH3GL3, and dynamin), which may lead to sequestration fibrils by sequestering monomeric HD53Q and shifting the of these proteins in inclusion bodies (61). By analogy, MW7 7 equilibrium toward soluble forms of HD53Q. This notion is binding to the polyP domains of HD53Q may stabilize a con- Downloaded from supported by the finding that 3B5H10 forms stable complexes formation of the polyP domains that can, in turn, prevent the of the same size regardless of whether it was added to mono- necessary conformational changes in the polyQ domain that meric or fibrillar HD53Q. Our AFM data also suggest that lead to fibril formation. Such findings underscore the critical 3B5H10 is unable to bind oligomeric species of htt, consistent importance of protein context in polyQ aggregation and aggre- with 3B5H10’s demonstrated conformational specificity for a gate stability. There are currently nine diseases related to polyQ compact, double-stranded conformation of monomeric htt.6 expansions in proteins that are broadly expressed, and the www.jbc.org Of the polyQ-specific antibodies used in this study, only nature of the proteins that contain the polyQ domain and their MW1 and 3B5H10 are IgG-type antibodies; the rest are IgM. associated pathologies differ substantially. That is, each mutant

We attempted to control for this difference by calculating the polyQ protein causes a distinct neurodegenerative disease that at UCSF Library & CKM, on April 8, 2010 ratio of HD53Q to antibody in all experiments based on antigen is associated with a different population of affected neurons. It binding sites on the respective antibody. Antibody type did not is likely that the protein context of the expanded polyQ appear to have a clear impact on htt aggregation. For instance, domains associated with each disease, and concomitant protein co-incubation of MW1 (IgG) or MW2 (IgM) with monomeric interactions that vary due to protein context, could help HD53Q resulted in very similar aggregation profiles; whereas, explain, at least in part, the striking cell specificity that is 3B5H10 (IgG) prevented fibril formation. In regards to fibril observed in each disease. destabilization, antibody type did not appear to play a role, Because MW7 and MW8 displayed similar behavior in rec- because 3B5H10 reduced the number of fibrils even at a ratio of ognizing aggregated forms of htt by Western blot analysis and five peptides per antigen binding site, which is analogous to the immunocytochemical studies of a HD neuronal model, it is dilution factor used to control for IgM type antibodies. 3B5H10 interesting that MW7 was much more effective in preventing was still effective in destabilizing fibrils even at a dilution of htt aggregation from monomers. This provides further evi- 10:1, yet none of the polyQ-specific IgM type antibodies desta- dence that the polyP region plays an important role in htt aggre- bilized fibrils. The other IgG-type polyQ-specific antibody, gation compared with the specific motif recognized my MW8. MW1, did not disaggregate fibrils even at a ratio of 1:1. There- Further, it appears that binding of an antibody to aggregated fore, the effects of these antibodies on htt aggregation and forms of htt is not sufficient to disrupt aggregate stability as aggregate stability cannot be simply correlated to their anti- MW8, which recognized aggregated forms of htt, was not able body type. This notion is further supported by the observations to disaggregate preformed fibrils. that MW7 (polyP-specific), which is an IgM type antibody, was The AFM studies presented here are consistent with previ- able to completely prevent fibril formation and destabilize pre- ous reports that MW7 suppresses aggregation and toxicity formed fibrils. when it is expressed as a scFv in cellular (46, 49) and Drosophila The ability of MW7 to prevent fibril formation and destabi- (60) models of HD. Co-transfection of MW7 with mutant htt lize preformed HD53Q fibrils provides additional support for exon 1 in corticostriatal rat brain slices increased the number of the importance of the polyP domains in htt aggregation. More healthy medium spinal neurons (49). Interestingly, expression broadly, it also indicates the critical importance of flanking of the MW7 scFV promotes turnover of htt in cellular models of sequences on polyQ structure and aggregation. Studies on syn- HD (49). These results indicate that the ability of MW7 to pre- thetic peptides revealed that the addition of a 10-residue polyP vent htt aggregation and destabilize htt fibrils, observed in this study, may play a pivotal role in the ability of MW7 to reduce cellular toxicity in a variety of HD models. 7 M. Arrasate, J. Miller, E. Brooks, C. Peters-Libeu, J. Legleiter, D. Hatters, J. An important finding in the present study is that htt aggre- Curtis, K. Cheung, P. Krishnana, S. Mitra, K. Widjaja, B. Shaby, Y. Newhouse, G. Lotz, V. Thulasiramin, F. Sandou, P. J. Muchowski, M. Segal, K. Weisgraber, gation can be reversed by antibodies. There is a great deal of and S. Finkbeiner, submitted for publication. interest in the use of antibodies and intrabodies as potential

21656 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 32•AUGUST 7, 2009 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin therapeutic agents to treat HD and other polyQ disorders (44– 9. Chen, S., Berthelier, V., Hamilton, J. B., O’Nuallain, B., and Wetzel, R. 47, 50). Our observations point to the potential for preventing (2002) Biochemistry 41, 7391–7399 aggregation and also destabilizing pre-existing aggregated 10. Saudou, F., Finkbeiner, S., Devys, D., and Greenberg, M. E. (1998) Cell 95, 55–66 forms of htt. By promoting formation of soluble forms of htt, 11. Lunkes, A., and Mandel, J. L. (1998) Hum. Mol. Genet. 7, 1355–1361 antibodies and intrabodies may increase htt turnover, as was 12. Hackam, A. S., Singaraja, R., Wellington, C. L., Metzler, M., McCutcheon, shown with a htt fusion protein system in HEK293 cells K., Zhang, T., Kalchman, M., and Hayden, M. R. (1998) J. Cell Biol. 141, cotransfected with a scFv of the antibody MW7 (49). This ob- 1097–1105 servation is consistent with MW7-promoting soluble forms of 13. Tanaka, M., Morishima, I., Akagi, T., Hashikawa, T., and Nukina, N. HD53Q when added to monomeric or fibrillar forms of the (2001) J. Biol. Chem. 276, 45470–45475 14. Poirier, M. A., Li, H., Macosko, J., Cai, S., Amzel, M., and Ross, C. A. (2002) protein, as demonstrated here. Such a notion is supported by J. Biol. Chem. 277, 41032–41037 mouse models, which demonstrate that continuous expression 15. Wacker, J. L., Zareie, M. H., Fong, H., Sarikaya, M., and Muchowski, P. J. of mutant htt is required to maintain inclusion integrity and (2004) Nat. Struct. Mol. Biol. 11, 1215–1222 disease symptoms (62). However, without clear knowledge of 16. Dahlgren, P. R., Karymov, M. A., Bankston, J., Holden, T., Thumfort, P., what constitutes a toxic species or conformation, altering the Ingram, V. M., and Lyubchenko, Y. L. (2005) Nanomedicine 1, 52–57 Annu. Rev. Neurosci. 23, aggregation process could also conceivably lead to detrimental 17. Zoghbi, H. Y., and Orr, H. T. (2000) 217–247 18. Davies, S. W., Turmaine, M., Cozens, B. A., DiFiglia, M., Sharp, A. H., Ross, effects. For example, if a particular antibody recognizes a non- C. A., Scherzinger, E., Wanker, E. E., Mangiarini, L., and Bates, G. P. (1997) toxic htt conformer, in principle it might actually shift the equi- Cell 90, 537–548 librium of aggregate species in a manner that would increase 19. Arrasate, M., Mitra, S., Schweitzer, E. S., Segal, M. R., and Finkbeiner, S. Downloaded from the concentration of a toxic conformer(s). Although we show (2004) Nature 431, 805–810 here that the equilibrium of htt aggregation can be altered in 20. Muchowski, P. J., Ning, K., D’Souza-Schorey, C., and Fields, S. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 727–732 vitro by antibodies, other exogenous factors, including molec- 21. Nagai, Y., Inui, T., Popiel, H. A., Fujikake, N., Hasegawa, K., Urade, Y., ular chaperones, may possess similar activities (15, 43, 55). Goto, Y., Naiki, H., and Toda, T. (2007) Nat. Struct. Mol. Biol. 14, 332–340 Because our panel of anti-polyQ antibodies displayed dra- 22. Wang, X., Vitalis, A., Wyczalkowski, M. A., and Pappu, R. V. (2006) Pro- www.jbc.org matically different properties, we speculate that polyQ teins 63, 297–311 repeats can display a wide variety of conformation-specific 23. Crick, S. L., Jayaraman, M., Frieden, C., Wetzel, R., and Pappu, R. V. (2006) epitopes in vivo and that polyQ misfolding and aggregation Proc. Natl. Acad. Sci. U.S.A. 103, 16764–16769 24. Vitalis, A., Wang, X., and Pappu, R. V. (2007) Biophys. J. 93, 1923–1937 within the context of the htt protein may be a far more com- 25. Vitalis, A., Wang, X., and Pappu, R. V. (2008) J. Mol. Biol. 384, 279–297 at UCSF Library & CKM, on April 8, 2010 plex process than previously imagined. Thus, additional 26. 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AUGUST 7, 2009•VOLUME 284•NUMBER 32 JOURNAL OF BIOLOGICAL CHEMISTRY 21657 Supplemental Material can be found at: http://www.jbc.org/content/suppl/2009/06/02/M109.016923.DC1.html Antibodies Recognize Distinct Conformers of Huntingtin

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50. Wolfgang, W. J., Miller, T. W., Webster, J. M., Huston, J. S., Thompson, Soc. Neurosce. Abstr. 30:938.5 Downloaded from L. M., Marsh, J. L., and Messer, A. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 61. Qin, Z. H., Wang, Y., Sapp, E., Cuiffo, B., Wanker, E., Hayden, M. R., Kegel, 11563–11568 K. B., Aronin, N., and DiFiglia, M. (2004) J. Neurosci. 24, 269–281 51. McLear, J. A., Lebrecht, D., Messer, A., and Wolfgang, W. J. (2008) FASEB 62. Yamamoto, A., Lucas, J. J., and Hen, R. (2000) Cell 101, 57–66 J. 22, 2003–2011 63. Nekooki-Machida, Y., Kurosawa, M., Nukina, N., Ito, K., Oda, T., and 52. Muchowski, P. J., Schaffar, G., Sittler, A., Wanker, E. E., Hayer-Hartl, Tanaka, M. (2009) Proc. Nat. Acad. Sci. U.S.A. 106, 9679–9684 www.jbc.org at UCSF Library & CKM, on April 8, 2010

21658 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 32•AUGUST 7, 2009 Structure Previews

required by the fact they are regulated by the on-off switching of a kinase active Falke, J.J., Bass, R.B., Butler, S.L., Chervitz, S.A., small molecule binding. The somewhat site, or trigger a larger structural rearrange- and Danielson, M.A. (1997). Annu. Rev. Cell Dev. Biol. 13, 457–512. larger displacement proposed for TorS ment in a signal conversion module such as (Moore and Hendrickson, 2009)isnot the HAMP domain. Thus, it appears likely Falke, J.J., and Hazelbauer, G.L. (2001). Trends Biochem. Sci. 26, 257–265. unreasonable since it binds a regulatory that chemoreceptors and His kinase protein, TorT, and the resulting protein- receptors have retained the same piston Hazelbauer, G.L., Falke, J.J., and Parkinson, J.S. protein interaction could perhaps generate mechanism of transmembrane signaling (2008). Trends Biochem. Sci. 33, 9–19. enough binding free energy to drive larger for good biophysical reasons. Hughson, A.G., and Hazelbauer, G.L. (1996). Proc. changes in side chain and ridges-grooves Natl. Acad. Sci. USA 93, 11546–11551. interactions. Second, transmembrane ACKNOWLEDGMENTS Marina, A., Waldburger, C.D., and Hendrickson, signals in bacterial receptors must span W.A. (2005). EMBO J. 24, 4247–4259. distances of 150 A˚ or more from the peri- Support provided by NIH R01 GM-040731. Milburn, M.V., Prive, G.G., Milligan, D.L., Scott, plasmic ligand binding site to the cyto- W.G., Yeh, J., Jancarik, J., Koshland, D.E., Jr., plasmic domain, and thus must be trans- REFERENCES and Kim, S.H. (1991). Science 254, 1342–1347. mitted over a remarkably long distance. Miller, A.S., and Falke, J.J. (2004). Biochemistry To a ﬁrst approximation, the H-bonding Chervitz, S.A., and Falke, J.J. (1996). Proc. Natl. Acad. Sci. USA 93, 2545–2550. 43, 1763–1770. framework of an a helix is incompressible along the helix axis, ensuring that a piston Chervitz, S.A., Lin, C.M., and Falke, J.J. (1995). Moore, J.O., and Hendrickson, W.A. (2009). Struc- ture 17, this issue, 1195–1204. force pushing on one end of a helix will be Biochemistry 34, 9722–9733. faithfully transmitted throughout the entire Cheung, J., and Hendrickson, W.A. (2009). Struc- Ottemann, K.M., Xiao, W., Shin, Y.K., and Kosh- helix length. By contrast, helix bends, rota- ture 17, 190–201. land, D.E., Jr. (1999). Science 285, 1751–1754. tions, or tilts can be more easily damped by Draheim, R.R., Bormans, A.F., Lai, R.Z., and Man- Szurmant, H., White, R.A., and Hoch, J.A. (2007). long-range helix ﬂexibility over these son, M.D. (2005). Biochemistry 44, 1268–1277. Curr. Opin. Struct. Biol. 17, 706–715. ˚ distances. Third, a small 1-2 A displace- Erbse, A.H., and Falke, J.J. (2009). Biochemistry Ward, S.M., Delgado, A., Gunsalus, R.P., and ment is large enough to directly regulate 48, 6975–6987. Manson, M.D. (2002). Mol. Microbiol. 44, 709–719.

Polyglutamine Dances the Conformational Cha-Cha-Cha

Jason Miller,1,2,3 Earl Rutenber,1 and Paul J. Muchowski1,4,* 1Gladstone Institute of Neurological Disease 2Chemistry and Chemical Biology Graduate Program 3Medical Scientist Training Program 4Departments of Biochemistry and Biophysics, and Neurology University of California, San Francisco, CA 94158, USA *Correspondence: [email protected] DOI 10.1016/j.str.2009.08.004

While polyglutamine repeats appear in dozens of human proteins, high-resolution structural analysis of these repeats in their native context has eluded researchers. Kim et al. now describe multiple crystal structures and demonstrate that polyglutamine in huntingtin dances through multiple conformations.

There are 66 human proteins with a homo- generative diseases. Although the struc- structures of a Q17-containing exon1 frag- polymeric stretch of ﬁve glutamines or tural basis that underlies the toxicity of ment of wild-type huntingtin (Httex1), a more. The overrepresentation of polyglut- proteins with expanded polyQ repeats is multifunctional protein that, when mutated amine (polyQ)-containing proteins in tran- not clear, numerous laboratories have in the polyQ stretch (>Q36), causes scription-related processes suggests hypothesized that a variety of misfolded a devastating neurodegenerative disorder a critical function for these repeats (But- conformers, including monomers, oligo- called Huntington’s chorea (chorea, land et al., 2007). At least 9 of these 66 mers, and ﬁbrils, are the toxic culprits. derived from Greek, describes the invol- proteins have a polyQ stretch that, when Into this debate enters the heroic crys- untary dance-like movements of Hunting- expanded beyond a critical threshold, tallography feat of Kim et al. (2009). The ton’s patients). Reminiscent of the dance- misfold, aggregate, and cause neurode- authors solved seven independent crystal like contortions of affected patients, the

wild-type polyQ stretch in proteins in transcription- Httex1 was surprisingly crys- related processes suggest tallized in multiple confor- conformational flexibility is mational contortions, most especially important for these convincingly forming a helices processes? Another interesting that varied from 1–15 polyQ question raised by this study is residues in length (Figure 1A). whether the polyQ stretch Although the structure of the jumps between defined confor- polyQ sequences C-terminal mations (Nagai et al., 2007; to these helices was not Tuinstra et al., 2008)orfluidly always well resolved in the flows through conformational crystal structures, the authors space. Because Kim et al. suggest that these sequences (2009) observed a wide range likely adopted a random coil or of conformations for the polyQ extended-loop conformation. stretch, one may assume that The sequences surrounding fluid conformational sampling the polyQ stretch, the struc- may predominate. On the other tures of which have also been hand, it is hard to imagine how contested, generally demon- Httex1 crystallized if there was strated less conformational not at least a limited set of flexibility. The 17 amino acids conformations that the polyQ N-terminal to the polyQ stretch samples. sequence in Httex1 (N17)were From the perspective of invariably a-helical in every neurodegenerative diseases, structure that was solved, it is interesting to speculate consistent with structure pre- Figure 1. Conformational Cha-cha-cha: X-Ray Crystallography whether the conformational diction programs and circular Reveals That PolyQ and Polyproline Adopt Multiple Conformations sampling of space by the polyQ dichroism (CD) spectroscopy in Htt Exon1 region increases, decreases, (A) Four a helices are shown. Each extends from the N-terminal residue of the N17 (Atwal et al., 2007). C-terminal region(Met 371-Phe387) ofHttExon1 (blue)andcontinues as a helix for a varying or stays the same when the to the polyQ region is a poly- number of glutamine residues (cyan = 5, yellow = 9, magenta = 12, and salmon = polyQ stretch expands into a proline stretch, which formed 15). Glutamines C-terminal to the -helical structured residues may adopt other the mutant (>Q36) range. For a classic proline helix, also as conformations, including random coil, extended loop, or b strand. example, while the structure (B) Five of the seven observed polyproline regions of Htt Exon1 are shown suggested by CD experiments superimposed on their five C-terminal residues. Note that all demonstrate of fully aggregated fibrillar (Darnell et al., 2007). Interest- a proline-helix conformation, but some are kinked while others are extended. polyQ in many proteins is ingly, the polyproline stretch This figure was generated using PyMol (www.pymol.org). composed predominantly of was either straight or kinked b sheet, Kim et al. (2009) did (Figure 1B), suggesting that not observe this conformation this sequence in huntingtin may itself by recognizing that the structures of the in the crystal structures of wild-type Httex1. exhibit some conformational flexibility. N17 and polyproline regions are relatively Does this conformation exist among the Before interpreting and digesting this constant, while the polyQ region varied. portions of polyQ in Httex1, whose electron wealth of structural information, it is worth The conformational flexibility of the density was unresolved by Kim et al. reflecting upon this astounding technical polyQ region in Httex1 raises several inter- (2009)? Alternatively, does this b strand/ feat. Since the huntingtin gene was esting questions about the functional role sheet conformation emerge only in mono- ex1 cloned more than sixteen years ago, of these stretches. For example, of the 66 mers of mutant Htt (>Q36) or only upon numerous laboratories have attempted human proteins with R Q5 stretch, aggregation? Notably, there is evidence and failed to determine the structure of approximately half (including all proteins that polyQ in monomeric mutant Httex1 various huntingtin fragments. Indeed, associated with polyQ-expansion disease) can adopt a collapsed b sheet conforma- this is the first crystal structure of any demonstrate significant length polymor- tion (Nagai et al., 2007). Further, while polyQ-containing (>Q10) protein in its phisms in the polyQ stretch in the normal a wide range of aggregate morphologies native protein context. The fact that the human population. Are polyQ stretches for mutant Httex1 species exists (Wacker polyQ stretch in the Httex1 fragment only conformationally flexible in the et al., 2004), it is unknown whether a single adopts different conformations within the proteins with length polymorphism? A conformation of polyQ in monomeric asymmetric unit of each crystal that the protein that must be functional within mutant Httex1 leads to a single type of authors solved, combined with the fact a wide range of polyQ lengths may have aggregated species or, alternatively, that Kim et al. (2009) analyzed diffraction to consequently demonstrate significant whether a single monomeric conformation from 30 crystals and obtained structures conformational flexibility in this region. can produce all observed aggregate for seven crystal forms, speaks to the How does this conformational flexibility species. While a recent study with mono- daunting nature of the entire effort. The assist in cellular functions? For example, clonal antibodies strongly implicated the authors demonstrated significant insight does the overrepresentation of polyQ existence of multiple monomeric polyQ

conformations in mutant Httex1 (Legleiter ingly, the N17 a-helix appears to ‘‘bleed’’ Bhattacharyya, A., Thakur, A.K., Chellgren, V.M., et al., 2009), Kim et al. (2009) provide direct into the C-terminal adjacent polyQ region, Thiagarajan, G., Williams, A.D., Chellgren, B.W., Creamer, T.P., and Wetzel, R. (2006). J. Mol. Biol. structural evidence of this, suggesting that, causing 1–15 glutamines to participate in 355, 524–535. at least in principle, each conformation the extended a helix (Figure 1A). The struc- may seed a unique type of aggregate. tural data from Kim et al. (2009) also hint Butland, S.L., Devon, R.S., Huang, Y., Mead, C.L., Even if we fully understood how different that the polyQ repeat in Httex1 may be inﬂu- Meynert, A.M., Neal, S.J., Lee, S.S., Wilkinson, A., Yang, G.S., Yuen, M.M., et al. (2007). BMC Geno- monomeric conformations of polyQ in enced by the C-terminal polyproline region. mics 8, 126. Httex1 lead to various aggregated species, Because Httex1 may be more aggregation the questions of which species contribute prone (and possibly more toxic) when the Darnell, G., Orgel, J.P., Pahl, R., and Meredith, S.C. to neurotoxicity and how they do it are still polyQ region is more compact, it is inter- (2007). J. Mol. Biol. 374, 688–704. open questions. Kim et al. (2009) propose esting to speculate whether the polyproline two general mechanisms for polyQ-medi- region may serve both its known function as Duennwald, M.L., Jagadish, S., Muchowski, P.J., and Lindquist, S. (2006). Proc. Natl. Acad. Sci. ated toxicity. By one mechanism, the a protein-interaction domain and a less- USA 103, 11045–11050. expanded polyQ stretch adopts a de novo appreciated function as a protector against conformation that mediates toxicity or is polyQ conformational collapse. Indeed, this Kim, M.W., Chelliah, Y., Kim, S.W., Otwinowski, Z., the precursor to a toxic species. By the structural explanation may account for why and Bezprozvanny, I. (2009). Structure 17, this second mechanism, the expanded polyQ Httex1 with the polyproline stretch is less issue, 1205–1212. stretch is largely unstructured but presents toxic and aggregation prone than Httex1 Legleiter, J., Lotz, G.P., Miller, J., Ko, J., Ng, C., a very large linear binding surface for without this sequence (Bhattacharyya Williams, G.L., Finkbeiner, S., Patterson, P.H., proteins witha polyQ afﬁnity. The structures et al., 2006; Darnell et al., 2007; Duennwald and Muchowski, P.J. (2009). J. Biol. Chem. 284, from Kim et al. (2009) leave open the possi- et al., 2006). Thus, N17 and polyproline 21647–21658. bility that either mechanism may be correct. dance partners may keep the Cha-cha- The study by Kim et al. (2009) also cha-prone polyQ stretch of hunting- Nagai, Y., Inui, T., Popiel, H.A., Fujikake, N., Hase- gawa, K., Urade, Y., Goto, Y., Naiki, H., and Toda, provides interesting insight into the relation- tin in step, and thereby prevent a toxic T. (2007). Nat. Struct. Mol. Biol. 14, 332–340. ship between the polyQ stretch and the conformational stumble. ex1 17 surrounding sequences in Htt .TheN Tuinstra, R.L., Peterson, F.C., Kutlesa, S., Elgin, sequence, which is important for the E.S., Kron, M.A., and Volkman, B.F. (2008). Proc. subcellular localization of Httex1 and is highly REFERENCES Natl. Acad. Sci. USA 105, 5057–5062. conserved (100% similarity) in all vertebrate Atwal, R.S., Xia, J., Pinchev, D., Taylor, J., Epand, Wacker, J.L., Zareie, M.H., Fong, H., Sarikaya, M., species (Atwal et al., 2007), was invariably R.M., and Truant, R. (2007). Hum. Mol. Genet. 16, and Muchowski, P.J. (2004). Nat. Struct. Mol. Biol. a-helical in all solved structures. Interest- 2600–2615. 11, 1215–1222.

Keeping an Eye on Membrane Transport by TR-WAXS

Jeff Abramson1,* and Vincent Chaptal1 1Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles 90095, CA *Correspondence: [email protected] DOI 10.1016/j.str.2009.08.003

In this issue of Structure, Andersson et al. apply time-resolved wide angle X-ray scattering (TR-WAXS) to follow light-induced conformational changes for both bacteriorhodopsin and proteorhodopsin and probe real-time dynamics at atomic resolution.

Membrane transport proteins perform remains a critical objective for basic and conformations. What is lacking is the abil- a multitude of cellular reactions, including medical research. It is well established ity to capture the transition between these energy and signal transduction, regulation that membrane transport proteins require conformations and to probe the role of of ion concentrations, and transport of distinct temporally regulated structural speciﬁc domains and ligands in the pro- metabolites into the cell and noxious sub- rearrangements to carry out their biolog- cess as they proceed through the mem- stances out. Altered membrane protein ical functions. However, structural details brane. function underlies many human diseases, of these dynamic macromolecules have In recent years, our knowledge of and thus, a deeper understanding of mem- only been studied as snapshots of indi- membrane protein structure has dramati- brane protein structure and dynamics vidual static (and, in most cases, stable) cally increased, providing unforeseen

Structure 17, September 9, 2009 ª2009 Elsevier Ltd All rights reserved 1153 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 7, pp. 4398–4403, February 13, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Single Neuron Ubiquitin-Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Disease*□S Received for publication, August 14, 2008, and in revised form, December 9, 2008 Published, JBC Papers in Press, December 10, 2008, DOI 10.1074/jbc.M806269200 Siddhartha Mitra‡§1, Andrey S. Tsvetkov‡¶2, and Steven Finkbeiner‡§¶3 From the ‡Gladstone Institute of Neurological Disease, San Francisco, California 94158 and the §Biomedical Sciences Program, Medical Scientist Training Program, and ¶Neuroscience Program, Departments of Neurology and Physiology, University of California, San Francisco, California 94143

The accumulation of mutant protein in intracellular aggre- has implicated the ubiquitin-proteasome system (UPS) in the gates is a common feature of neurodegenerative disease. In pathogenesis of HD, amyotrophic lateral sclerosis, Parkinson Huntington disease, mutant huntingtin leads to inclusion body disease, and polyQ-mediated disorders (3). (IB) formation and neuronal toxicity. Impairment of the ubiq- The UPS is a major pathway of intracellular protein degrada- uitin-proteasome system (UPS) has been implicated in IB for- tion. After a series of three reactions, each catalyzed by a differ- mation and Huntington disease pathogenesis. However, IBs ent set of enzymes, ubiquitin, a 76-amino acid polypeptide, form asynchronously in only a subset of cells with mutant hun- forms an isopeptide bond with the amino group of lysine resi- tingtin, and the relationship between IB formation and UPS dues on substrate proteins. Several lysine residues within ubiq- function has been difficult to elucidate. Here, we applied single- uitin are sites for more ubiquitin additions. Once a protein cell longitudinal acquisition and analysis to monitor mutant accumulates four or more ubiquitins, it is efficiently targeted to huntingtin IB formation, UPS function, and neuronal toxicity. the proteasome for degradation. The proteasome binds poly- We found that proteasome inhibition is toxic to striatal neurons ubiquitinated substrates and hydrolyzes ubiquitin isopeptide in a dose-dependent fashion. Before IB formation, the UPS is bonds, releasing ubiquitin moieties before degrading substrate more impaired in neurons that go on to form IBs than in those proteins through chymotrypsin-like, trypsin-like, and post-glu- that do not. After forming IBs, impairment is lower in neu- tamyl peptidase activities (3). rons with IBs than in those without. These findings suggest Increased polyubiquitin levels and changes in ubiquitin link- IBs are a protective cellular response to mutant protein medi- ages accompany the accumulation of UPS substrates in the ated in part by improving intracellular protein degradation. brains of HD patients and transgenic mice and in cellular HD models (4). UPS substrates accumulate throughout the cell in polyQ models, even before IB formation (5, 6). This has added Huntington disease (HD)4 is a progressive incurable neuro- to the confusion over whether polyQ expansion leads to toxicity degenerative disorder caused by the expansion of a polyglu- through direct impairment of proteasomal degradation. Pro- tamine (polyQ) stretch in the N-terminal end of the huntingtin teasomes have been reported to cleave polyQ stretches effi- (htt) protein above a threshold length of ϳ36 (1). The deposi- ciently (7), inefficiently (8), or essentially not at all (9). In vivo, tion of polyQ-expanded aggregated mutant htt in inclusion polyQ-dependent degeneration occurs with no detectable pro- bodies (IBs) is a hallmark of HD, and IBs are found in human teasome inhibition (10, 11) or is tightly linked to it (12, 13). The post-mortem samples, transgenic mouse brain, and cell-culture inability of some studies to detect UPS impairment in HD mod- models (2). The accumulation of ubiquitinated proteins in IBs els may be due to the limited sensitivity of conventional approaches to identify cell-to-cell variations in UPS function. The relationship between IB formation and UPS function has * This work was supported, in whole or in part, by National Institutes of Health Grants R01 2NS039074 and R01 NS045191 from the NINDS (to S. F.) and been difficult to determine. Protein turnover in cells with IBs is Grant P01 AG022074 from the NIA. This work was also supported by the evidently reduced and accompanied by the accumulation of Taube Family Foundation Program in Huntington Disease, and the Glad- cellular proteins (14–16); HEK293 cells containing mutant htt stone Institutes (to S. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must there- IBs have a greater degree of UPS impairment than those with- fore be hereby marked “advertisement” in accordance with 18 U.S.C. Sec- out IBs (5). Proteasome subunits and heat shock proteins colo- tion 1734 solely to indicate this fact. □ calize with IBs, but it is unclear if this colocalization facilitates S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. protein delivery or unfolding at the mouth of active protea- 1 Supported by NIH-NIGHMS UCSF Medical Scientist Training Program and a somes, or if it harms proteasome function by sequestering fellowship from the UC-wide adaptive biotechnology (GREAT) program. essential cellular machinery (18). Some IBs are relatively static 2 Supported by the Milton Wexler fellowship from the Hereditary Disease Foundation. (8, 25), but the proteins in others are dynamically exchanged 3 To whom correspondence should be addressed: Gladstone Institute of Neu- with cytoplasmic and nuclear pools (19, 20). rological Disease, 1650 Owens St., San Francisco, CA 94158. Tel.: 415-734- UPS function is critical to cellular homeostasis. Deletion of 2508; Fax: 415-355-0824; E-mail: [email protected]. 4 The abbreviations used are: HD, Huntington Disease; UPS, ubiquitin-protea- one of the two inducible polyubiquitin genes in mice leads to some system; IB, inclusion body. lower intracellular ubiquitin levels in germ cells and hypotha-

4398 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 7•FEBRUARY 13, 2009 Ubiquitin-Proteasome Dynamics in Huntington Disease lamic neurons. These same populations undergo cell-cycle (Sigma-Aldrich) were added in 1 ml of conditioned NCM per arrest and hypothalamic neurodegeneration, respectively (22, well 12–60 h after transfection. 23). Cell lines expressing mutant huntingtin accumulate ubiq- Colocalization of fluorescence was calculated using Meta- uitinated proteins and undergo cell-cycle arrest in G2/M (5). In morph. Briefly, images of fluorescence from CFP-htt, ubiquitin neurons, UPS impairment may lead to cell death through an staining, or LMP-YFP were analyzed, and pixels were classified accumulation of signals for apoptosis, a decrease in NF-␬B sig- as “positive” if their intensity was 3ϫ greater than background naling, sensitization to other toxic stimuli, remodeling of syn- pixels. The fraction of positive pixels for CFP-htt IBs that over- apses, retraction of neurites, or other unidentified mechanisms lapped positive pixels of ubiquitin staining or LMP-YFP fluo- (24). The effect of UPS impairment depends on cell type and rescence was calculated with Metamorph for (n ϭ 20 neurons). cell cycle, and the relationship between UPS impairment and Live-Cell Imaging and Analysis—Images of cells were striatal neuronal survival is largely unknown. obtained with a robotic microscope system as described (2, 32). Diffuse species of mutant htt induce IB formation and neu- Briefly, the imaging was performed with a Nikon TE300 ronal death in a protein concentration-dependent manner (2). inverted microscope with a long working-distance Nikon 20ϫ IB formation delays neuronal death, suggesting that IB forma- (NA 0.45) objective. Stage movements and focusing were per- tion helps neurons cope with toxic diffuse mutant htt. Whether formed using a Proscan II stage controller (Prior Scientific, the effect of IB formation on survival is mediated through UPS Rockland, MA). Samples were illuminated with a 175 watt function has been difficult to determine. IB formation and neu- Xenon Lambda LS illuminator (Sutter Instruments, Novato, ronal death occur asynchronously in overlapping but distinct CA). Blue, green, and red fluorescent protein (BFP, GFP, and subsets of neurons that express mutant htt. The observation RFP, respectively) images were captured using an 86014 beam- ϫ ϫ that IB formation is not required for UPS impairment also com- splitter and 350/50 ; 465/30m, 480/40 ; 517/30m and 580/ ϫ plicates population analysis (6, 26). 20 ; 630/60m fluorescence filters respectively. CFP, Venus, To explore this problem, we applied single-cell analysis. We and RFP images were captured using a 86006 beamsplitter and ϫ ϫ ϫ tracked single neurons over their entire lifetimes, gaining spa- 420/35 ; 470/30m, 500/20 ; 535/30m, and 580/20 ; tial and temporal resolution while simultaneously monitoring 630/60m fluorescence filters (Chroma Corp, Rockingham, VT). IB formation, UPS inhibition, and neuronal toxicity. Algorithms for plate registration, stage movements, filter movements, focusing, and acquisition were generated with EXPERIMENTAL PROCEDURES Metamorph imaging software (Molecular Devices, Sunnyvale, CA). Images were analyzed manually using Metamorph soft- Plasmids—mRFP (27), pCS2-Venus (28), and pEGFP- ware. Fully automated acquisition and analysis algorithms have CL1(5), pGW1-GFP, pGW1-httQ72-eGFP, pGW1-mRFP (2) been created (Media Cybernetics, Bethesda, MD). Survival have been described. pGW1-httQ72-CFP was generated from analysis was performed with the Statview software package u u pGW1-httQ72-eGFP. pGW1-mRFP (mRFP ) was generated (SAS Institute, Cary, NC); t tests for comparisons of means and ϩ by subcloning mRFP1 from pcDNA3.1( ) into pEGFP-CL; two-sample Kolmogorov-Smirnov tests for comparisons of dis- u mRFP1-CL1 was then subcloned into pGW1. pGW1-GFP was tributions were performed with Prism (Graphpad Software, constructed by excising EGFP-CL1 from pEGFP-CL1 and San Diego, CA). inserting it into pGW1. pGW1-Venus-CL1 (Venusu) was generated by subcloning Venus from pCS2-Venus into RESULTS ϩ pcDNA3.1( ). The stop codon from Venus was removed and Longitudinal Live-Cell Monitoring of UPS Function in Pri- replaced by the sequence AGATCTCG. The CL1 sequence (5) mary Neurons—To monitor dynamic changes in protein deg- was introduced at the 3Ј-end of Venus. Venus-CL1 was then radation in live cells, we used a unique high-throughput image G76V G76V subcloned into pGW1. pCS2-Ub -Venus (Ub -Venus) acquisition platform (2, 32) and fluorescent protein substrates G76V G76V was generated by PCR of Ub from Ub -GFP (29). of UPS degradation. We used fluorescent proteins with the CL1 Cell Culture—Striata from rat embryos (E17–18) were dis- peptide fused to the C terminus (34) or a non-hydrolyzable sected, dissociated, and plated on 24-well tissue-culture plates ubiquitin moiety (UbG76V) fused to the N terminus (35) to tar- 5 (5.8 ϫ 10 /well) coated with poly-D-lysine and laminin (BD Bio- get them to the UPS for degradation. These destabilized fluo- sciences, San Jose, CA) as described (2, 30). The cells were rescent proteins were transfected into primary neurons and grown in 1 ml of modified neuronal culture medium (NCM). fluorescence in individual cells was monitored for hours or days Cells were fed every 5–7 days by replacement with equal meas- to detect changes in the degradation of UPS substrates. To con- ures of conditioned and fresh neuronal culture medium. trol for nonspecific changes in transcription and protein han- Transfection, Pharmacology, and Colocalization—Primary cul- dling while monitoring cell survival (2), we co-transfected and tures were transfected 5–7 days in vitro with combinations of tracked the fluorescence of unmodified fluorescent proteins in pGW1-GFPu and pGW1-mRFP, pGW1-mRFPu, and pGW1- the same cells. GFP, pGW1-Venusu, or pCS2-UbG76V-Venus, and pGW1-CFP, Destabilized Fluorescent Proteins Accumulate after Protea- and pGW1-httQ72-eGFP, pGW1-mRFPu, and pGW1-BFP, or some Inhibition in Primary Neurons—Fluorescence intensity in pGW1-httQ72-CFP and pYFP-LMP2 in a 1:1 or 1:1:1 molar ratio live cells is an accurate indicator of the amount of fluorescent with 2–4 ␮g of total plasmid DNA per well. Our transfection protein within the cell (2). Fluorescence levels in primary stri- protocol was described (2). MG132 (Sigma-Aldrich), epoxomi- atal neurons of a destabilized form of enhanced GFPu (5) (Fig. cin (Boston Biochem, Cambridge, MA), and Bafilomycin A1 1A), monomeric mRFPu (27) (Fig. 1C), or two forms of the

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FIGURE 2. Limited interaction between the UPS and autophagic pathways in neurons. A, 24 h after cotransfection with UbG76V-Venus and CFP, striatal neurons were treated with vehicle or 50 nM BafA1. BafA1 treatment caused a significant amount of toxicity above control (p Ͻ 0.03, top line). Mean UbG76V-Venus/CFP ratio (B) and the distribution of the single-cell changes in UbG76V-Venus/CFP (C) in these cells did not increase above control in 20 h after BafA1 addition. D, neurons or HEK293 cells (E) were treated with BafA1 or epoxomycin, followed by Western blotting with an LC3 antibody. FIGURE 1. Levels of proteasome reporters increase after inhibition of pro- u While BafA1 caused accumulation of LC3-II in both neurons and HEK293 cells, teasome. A, after transfection with GFP and mRFP, striatal neurons were epoxomycin increased LC3-II levels only in HEK293 cells. Unlabeled lanes in E ␮ treated with 50 M MG132 for 12 h. GFP fluorescence (A) and the ratio of are lysates from cells transfected with LC3. GFPu/mRFP fluorescence (B) both increase after treatment relative to control. C, after transfection with mRFPu and GFP, striatal neurons were treated with 50 ␮M MG132 for 12 h. The mRFPu/GFP ratio is significantly greater than the Proteasome Inhibition Does Not Change LC3-II Levels in Pri- control (p Ͻ 0.02). D, after transfection with Venusu and CFP, striatal neurons mary Neurons—Proteasome inhibition can increase flux were treated with 2 ␮M epoxomycin (solid lines) or vehicle (broken lines) for 10 h. Both mean change in Venusu/CFP fluorescence (D) and single-cell dis- through the autophagic pathway in some cells (13). To deter- tributions of Venusu/CFP fluorescence (E) are increased relative to control mine if autophagic activity could be confounding fluorescent (p Ͻ 0.05, p Ͻ 0.05). F, after transfection with UbG76V-Venus and CFP, striatal reporter measurements of UPS function, we examined the neurons were treated with 2 ␮M epoxomycin for 10 h. Both mean change in UbG76V-Venus/CFP fluorescence (F) and single-cell distributions of UbG76V- activity of the autophagic pathway after proteasome inhibition. Venus/CFP fluorescence (G) are increased (p Ͻ 0.05, p Ͻ 0.01). Experiments The level of LC3-II is commonly used as a surrogate for the were repeated twice with over 50 cells analyzed in each condition. number of autophagosomes and flux through the macroautophagic pathway. After treatment with epoxomicin, primary enhanced yellow fluorescent protein variant Venus (UbG76V- neurons showed no change in LC3-II levels (Fig. 2D), though as Venus and Venusu) (28) (Fig. 1, D–G) increased after treatment seen in previous reports, LC3-II accumulated in HEK293 cells with proteasome inhibitor, even when changes in fluorescence (Fig. 2E). UPS Reporter Fluorescence Demonstrates a Graded Response of unmodified spectrally distinct fluorescent proteins in the to Proteasome Inhibition—Having validated the use of destabi- same cells was controlled for (Fig. 1, B, C, E, G). The significant lized fluorescent proteins as reporters of UPS function in pri- and rapid increase in fluorescence of these reporters from low mary neurons, we then examined the nature of their response to baseline levels after proteasome inhibition in primary neurons varying levels of proteasome impairment. We co-transfected is in agreement with previous work in cell lines (5, 6, 26). Addi- mRFPu and GFP into primary striatal neurons and treated the G76V degron to fluorescent pro- tion of the CL1 peptide or Ub cells with increasing doses of the proteasome inhibitor MG132. teins did not cause the proteins to aggregate when they were Though fluorescent UPS reporters have been reported to relo- expressed in neurons, unlike observations from cell lines (36). calize to IBs, we found that mRFPu fluorescence remained dif- Inhibiting Autophagy Does Not Result in Accumulation of fuse in striatal neurons after proteasome inhibitor treatment UPS Reporters in Primary Neurons—To ensure that these (6). As early as 2.5 h after addition of MG132, reporter fluores- destabilized proteins were targeted primarily to the UPS for cence increased in proportion to the amount of MG132 added degradation, we used Bafilomycin A1 (BafA1) to inhibit auto- (Fig. 3A), and reporter fluorescence continued to increase over phagy. BafA1, a vacuolar ATPase inhibitor, prevents autopha- time (Fig. 3B). Thus, in primary neurons, the increase in fluo- gosome-lysosome fusion and causes the accumulation of sub- rescence of these proteins faithfully reports the extent of pro- strates targeted for macroautophagy (37). BafA1 caused a rapid teasome impairment (5, 6). accumulation of the membrane-bound form of microtubule- By monitoring individual cells treated with MG132 over associated protein 1 light chain 3 (LC3-II) and was toxic to days, we determined the effect of increasing proteasome inhi- primary neurons (Fig. 2, A and D), but BafA1 did not increase bition on the survival of primary striatal neurons. When the levels of UPS reporters (Fig. 2, B and C). dose of MG132 increased, neurons died faster (Fig. 3C). These

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UPS Impairment Decreases after IB Formation—To determine if IB formation improves or worsens UPS function, we examined UPS reporter fluorescence in neurons during and after IB formation. We compared these measurements to UPS reporter fluorescence in an otherwise matched cohort of neurons that did not form IBs over the same interval. To again reduce FIGURE 3. Inhibition of proteasome activity is toxic in a dose-dependent fashion. A, UPS reporter fluores- potential biases introduced by using cence shows a dose-dependent response to MG132 treatment. MG132 at the indicated doses was added to striatal neurons 24 h after transfection with mRFPu and GFP. The change in mRFPu/GFP ratio over the first 2.5 h IB formation as a selection criterion, after MG132 administration is shown. B, UPS reporter fluorescence continues to increase up to 12 h after the neurons from each cohort were addition of MG132. Note the difference in scale with A. Measurements from 80 ␮M were excluded due to noticeable toxicity. C, MG132 is toxic to neurons in a dose-dependent fashion. The same neurons shown in A matched for the length of time they and B were observed with the risk of death as shown. Longitudinal analysis was repeated twice on different lived in vitro. We found that neu- transfections, with n Ͼ 50 for each treatment in each experiment. rons that formed IBs had significantly smaller increases in UPS reporter fluorescence (Fig. 4, E and neurons demonstrated a proportional relationship between F), indicating that less UPS impairment occurs in cells after IB proteasome impairment and the accumulation of UPS sub- formation than in cells that did not form IBs. strates; similarly, there was a proportional relationship between IB Formation Improves Neuronal Survival—To determine if proteasome impairment and neuronal toxicity. this reduced UPS impairment changes neuronal survival, we Longitudinal Live Cell Detection of UPS Function in a Pri- compared the survival of neurons that we analyzed for UPS mary Neuronal Model of HD—We then examined a primary function. When we examined matched cohorts of neurons ex1 u striatal model of HD (2, 30) and prospectively followed visual transfected with htt -Q72-GFP, mRFP , and BFP that formed markers of UPS function, IB formation, and neuronal viability or did not form IBs, those cells that formed IBs survived longer in single cells. This model reproduces key features of HD, (Fig. 4, G and H). This finding agrees with previous results including neuronal subtype specificity (30) and polyQ length- showing that neurons survive longer if they form IBs (2). dependent toxicity (2, 30). To induce the HD disease phenotype in this model, we transiently transfected an N-terminal htt frag- DISCUSSION ex1 ment with 72 glutamines fused to GFP (htt -Q72-GFP). We By applying a high-throughput single-cell longitudinal imag- simultaneously introduced mRFPu and BFP into the same neu- ing platform to a neuronal model, we were able to examine the rons to monitor UPS impairment and cell viability, respectively. events in the cellular pathogenesis of HD with improved sensi- Virtually all IBs in this model stain with ubiquitin and colocalize tivity and temporal resolution. Through the use of spectrally with proteasome subunits (supplemental Fig. S1). From series distinct fluorescent species, we simultaneously monitored neu- of images of individual neurons, we quantified single-cell ronal viability, htt IB formation, and intracellular protein deg- changes in UPS reporter fluorescence over the lifetimes of cells radation. We found that neurons that form IBs have increased ex1 expressing the htt -Q72-GFP protein (Fig. 4A). UPS impairment preceding IB formation and less UPS impair- Would UPS function differ in neurons that do and do not ment after IB formation than cells that do not form IBs. Though form IBs? By reviewing images from our longitudinal analy- tonic UPS inhibition is toxic to primary striatal neurons, neu- sis experiments, we identified neurons that had or had not rons that formed IBs survived better than those that did not. formed an IB at some point over the course of the experi- These results support a model in which IB formation reflects a ment. From these two groups, we then chose neurons that beneficial cellular response to mutant protein, mediated in part were from the same well of the culture dish to form two by restoring UPS function. cohorts based on IB formation. To reduce potential biases Though multiple pathways of intracellular protein degrada- introduced by using IB formation as a selection criterion, we tion may handle aggregation-prone protein, we found that included only neurons that had already lived the same length some proteins are likely targeted primarily to the UPS for deg- of time in vitro. We then monitored UPS reporter fluores- radation. In our experience with fluorescent UPS reporters, we cence in neurons before, during, and after IB formation and found little evidence that they are routinely degraded by auto- compared it to that in the cohort of age-matched neurons phagy. Though it is clear that autophagy modulates the turn- that did not form IBs. over and toxicity of aggregation prone-proteins, the addition of UPS Impairment Precedes IB Formation—Those cells that the CL1 or UbG76V degron does not cause fluorescent proteins would go on to form IBs had significantly larger increases in to aggregate in neurons. This discrepancy with other reports in UPS reporter fluorescence before IB formation, both in the sin- cell lines may be due to lower expression levels in neurons after gle-cell distribution of reporter fluorescence (Fig. 4B) and in transient transfection. mean reporter fluorescence (Fig. 4C). This relationship was The finding that proteasome inhibition is not sufficient to independent of the time at which IBs formed (Fig. 4D). change the flux through the autophagic pathway in primary

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proteins normally targeted to the UPS to other pathways of intracellular protein degradation. In both yeast and mammalian cells, misfolded and aggregation-prone proteins may be targeted to different intracellular compartments depending on the availability of ubiquitin (31). Differential localization may be one component of targeting proteins to the autophagic pathway of protein degradation, which has been implicated in the clearance of aggregation-prone protein, including mutant htt (17, 21). If expanded polyQ tracts impair the ability of the proteasome to degrade other cellular proteins (9) or if ubiquitination is inadequate due to ubiquitin sequestration by IBs, shifting polyQ degradation from the UPS to the autophagic pathway could effectively increase the flux of other proteins through the UPS. A second possibility that is not mutually exclusive is that IB formation is part of a cellular program to more efficiently degrade protein through the UPS. The recruitment of chaperones and proteasomal machinery to intracellular inclusions varies based on protein and cell type (19, 25). Though the IBs in our primary FIGURE 4. IB formation and UPS function in primary neurons. A, GFP-htt, BFP, and mRFPu were imaged over neuronal model are long-lived, the course of days to follow htt IB formation, UPS impairment, and neuronal survival. Single-cell distributions with fewer than 2% disappearing (B) or population means (C) of the change in mRFPu/GFP fluorescence in the interval preceding IB formation at 54 h. The increase in mRFPu/GFP ratio was higher in neurons that went on to form IBs (p Ͻ 0.05, p Ͻ 0.05). D,in before the neuron that contains a parallel experiment, single-cell distributions of the change in mRFPu/GFP fluorescence in the interval preced- them dies (2), a small proportion Ͻ ing IB formation at 76 h also show higher UPS impairment in those neurons that will go on to form IBs (p 0.05). of cells can clear IBs, and a detailed After 54 h, single-cell distributions (E) or population means (F) show a greater increase in mRFPu/GFP fluorescence in those cells that did not form IBs (p Ͻ 0.05, p Ͻ 0.05). The survival of those neurons that formed htt IBs longitudinal analysis of these cells at 18 h (G)or27h(H) was better than the survival of neurons that survived at least that long but never formed will likely be informative. IBs (p Ͻ 0.01, p Ͻ 0.03). Longitudinal analysis was repeated twice in different experiments with over 300 cells analyzed in each experiment, with n Ͼ 30 for each cohort. Previous work suggested that IB formation safely sequesters more neurons also highlights possible differences between mamma- toxic forms of mutant htt to improve neuronal survival. This lian neurons and other model systems. The difference in behav- study suggests two additional mechanisms by which IB forma- ior of the autophagic pathway in mammalian neurons may be tion might contribute to improved cell survival after IB forma- due to a difference in constitutive activity (39). While most tion. First, we found that tonic UPS inhibition is toxic and that non-neuronal cells upregulate autophagy after 24 h of starva- IB formation is associated with a relative improvement in UPS tion, neurons do not in vivo (40) or in vitro5 even after longer function. Thus, IB formation may partially restore longevity by starvation periods. The finding that the deletion of essential improving UPS throughput and consequently lowering the autophagic machinery results in a neurodegenerative pheno- overall cellular burden of misfolded proteins. A second but type points to a critical role in neuronal function and survival related possibility is suggested by reports that transient suble- (38, 41). thal proteasome inhibition can induce cells to adapt in ways Though it remains unclear how IB formation is functionally that protect them against further insults (33). Transient protea- linked to an improvement in UPS function, one possibility is some inhibition might trigger a cell-wide adaptive response in that IB formation is a step toward shunting aggregation-prone neurons that may involve coordinated changes in molecular chaperones and protein turnover pathways. If so, such an 5 A. Tsvetkov and S. Finkbeiner, unpublished observations. adaptive response may be important in a variety of neurodegen-

4402 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 7•FEBRUARY 13, 2009 Ubiquitin-Proteasome Dynamics in Huntington Disease erative diseases that result from misfolded intracellular 18. Waelter, S., Boeddrich, A., Lurz, R., Scherzinger, E., Lueder, G., Lehrach, proteins. H., and Wanker, E. E. (2001) Mol. Biol. Cell 12, 1393–1407 19. Stenoien, D. L., Mielke, M., and Mancini, M. A. (2002) Nat Cell Biol. 4, Acknowledgments—We thank R. Kopito for pEGFP-CL1, V. Rao for 806–810 20. Taylor, J. P., Tanaka, F., Robitschek, J., Sandoval, C. M., Taye, A., Mark- pGW1-GFPu, A. Miyawaki for pCS2-Venus, M. Mancini for pYFP- ovic-Plese, S., and Fischbeck, K. H. (2003) Hum. Mol. Genet. 12, 749–757 LMP2, and N. Dantuma for UbG76V-GFP; members of the Finkbeiner 21. Rubinsztein, D. C. (2006) Nature 443, 780–786 laboratory for insightful discussions; S. Ordway and G. Howard for 22. Ryu, K. Y., Sinnar, S. A., Reinholdt, L. G., Vaccari, S., Hall, S., Garcia, M. A., editorial assistance; and K. 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FEBRUARY 13, 2009•VOLUME 284•NUMBER 7 JOURNAL OF BIOLOGICAL CHEMISTRY 4403 FIGURE S1. Inclusion bodies are ubiquitinated and co-localized with proteasomes. (A) Striatal neurons were transfected with CFP-htt (middle panel), LMP2-YFP (proteasome subunit) (left panel), fixed after 48 h, and stained with an antibody against ubiquitin (right panel). (B) Colocalization of CFPhtt fluorescence with ubiquitin staining and with proteasomes indicated by LMP2-YFP fluorescence. Colocalization for IBs/ubiquitin: 92.7%+/-7.6; for IBs/proteasomes 73.8%+/-15.8. The bar is 50 ⎧m.

Autophagic Punctum Protein turnover and inclusion body formation

Siddhartha Mitra,1,4 Andrey S. Tsvetkov1-3 and Steven Finkbeiner1-4,*

1Gladstone Institute of Neurological Disease; San Francisco, CA USA; 2Taube-Koret Center for Huntington’s Disease Research; San Francisco, CA USA; 3Neuroscience Program; Departments of Neurology and Physiology; 4Biomedical Sciences Program and Medical Scientist Training Program; University of California; San Francisco, CA USA Key words: huntington disease, huntingtin, polyglutamine, autophagy, neurodegeneration, ubiquitin, proteasome

In a recent study, we investigated the relationship between UPS impairment is toxic to many cell types, including neurons. inclusion body (IB) formation and the activity of the ubiq- In our study, neuronal toxicity increased with increasing levels of uitin-proteasome system (UPS) in a primary neuron model of pharmacological UPS inhibition. Yet cells expressing mutant htt Huntington disease. We followed individual neurons over the that formed IBs—those with higher levels of UPS inhibition— course of days and monitored the level of mutant huntingtin survived longer than cells that did not form IBs and had lower levels (which causes Huntington disease), IB formation, UPS function, of UPS impairment after IB formation. One explanation is that a and neuronal toxicity. The accumulation of UPS substrates and compensatory process accompanies IB formation. Alternatively, neuronal toxicity increased with increasing levels of proteasome the IB itself may afford protection, perhaps by sequestering toxic inhibition. The UPS was more impaired in neurons that subse- hard-to-degrade intracellular proteins. The improvement in UPS quently formed IBs than in those that did not; however, after IBs function after IB formation is consistent with both hypotheses formed, UPS function improved. These findings suggest that IB (Fig. 1). formation is a protective cellular response mediated in part by Increasing evidence has implicated the autophagic pathway increased degradation of intracellular protein. in Huntington disease and other neurodegenerative disorders. To determine whether concurrent changes in autophagy affected Many aggregation-prone proteins responsible for neurodegen- our measurement of UPS activity, we examined the activity of eration inhibit the UPS, but the effect of IB formation on UPS the autophagic pathway after treatment with the UPS inhibitor function has been difficult to study. IBs form asynchronously epoxomicin. LC3-II levels, a surrogate marker of macroautophagic in only a subset of cells that express aggregation-prone proteins. flux, are unchanged in primary striatal neurons. In HEK293 cells, Some of this variation likely arises from cell-to-cell differences in however, proteasome inhibition leads to LC3-II accumulation, the balance between protein production and protein degradation. consistent with previous reports. Unfortunately, traditional biochemical and imaging approaches What might account for this surprising difference between give a static picture of different populations of cells and combine neuronal and non-neuronal cells? One possibility is the death measurements from cells with and without IBs. A single-cell longi- of neurons that upregulate autophagy; however, the inhibitor tudinal approach has been invaluable in elucidating the physiology treatment did not cause significant toxicity, a finding supported of stochastic cellular events. Using this approach previously, we by the similar levels of LC3-I in the two cell types. The absence showed that the amount of intracellular mutant protein predicts of increased flux through the autophagic pathway may reflect IB formation. In this study, we found that cells that eventually the inability of neurons to upregulate autophagy. Alternatively, formed IBs had higher levels of UPS impairment than cells that autophagosome-lysosome fusion may not be rate-limiting in some did not. After IBs formed, UPS impairment improved relative to cell types and, as a result, LC3-II levels may be an insensitive that in cells without IBs. marker of autophagic flux in neurons. Autophagy has been characterized mostly in yeast and mammalian non-neuronal cells, and the few studies in neurons reached different conclusions. Further This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. the issue is complete and page numbers have Once to printing. has been published online, prior This manuscript characterization of neuronal responses to autophagy-inducing *Correspondence to: Steven Finkbeiner; Gladstone Institute of Neurological Disease; stimuli will be helpful. 1650 Owens Street; San Francisco, CA 945158 USA; Tel.: 415.734.2000; Fax: 415.355.0824; Email: [email protected] Why do some cells form IBs and survive longer? Although intracellular mediators of IB formation have been identified, answering Submitted: 06/11/09; Revised: 06/15/09; Accepted: 06/16/09 this question will require knowledge about how the UPS and the Previously published online as an Autophagy E-publication: autophagic pathway interact in handling toxic aggregation-prone http://www.landesbioscience.com/journals/autophagy/article/9291 proteins. Particular substrates are often preferentially targeted to Punctum to: AUTHOR: please provide the citation information for the one of the two pathways. After cell stress, the concerted action of paper to which this paper is commenting both pathways is clearly important for cellular homeostasis. Since

1 Autophagy 2009; Vol. 5 Issue 7 Protein turnover and inclusion body formation

Figure 1. The effect of IB formation on UPS function and neurodegeneration. Mutant aggregation-prone protein leads to toxic UPS impairment. A subset of neurons with higher levels of UPS impairment form IBs. UPS function subsequently improves, and these cells survive longer than cells that do not form IBs.

UPS inhibition alone does not increase autophagy in neurons, IB formation may be necessary to induce autophagy in certain cell types. Further investigation of both the molecular mediators of autophagy and the dynamic changes in autophagic activity during IB formation will help to reveal the roles of the UPS and the autophagic pathway in preventing cell death. Much of the machinery and physiology may vary with the cell type and, in the case of neurodegenerative disease, the neuronal subtype. Without a better understanding of cell-type-specifc variations in the UPS and autophagic activity, it will be difficult to determine the role of protein degradation in the pathogenesis of neurodegenerative disease. Acknowledgements This work was supported by R01 2NS039746 and 2R01 NS045191 from the National Institute of Neurological Disease and Stroke, P01 2AG022074 from the National Institute on Aging, the Taube-Koret Center for Huntington’s Disease Research, and the J. David Gladstone Institutes (S.F.); a Milton Wexler Award and a fellowship from the Hereditary Disease Foundation (A.T.); NIH-NIGHMS UCSF Medical Scientist Training Program and a fellowship from the UC-wide adaptive biotechnology (GREAT) program (S.M.); and RR018928 from the National Center for Research Resources. Kelley Nelson provided administrative assistance, and Gary C. Howard edited the manuscript.

www.landesbioscience.com Autophagy 2 Human Molecular Genetics, 2009, Vol. 18, No. 11 1937–1950 doi:10.1093/hmg/ddp115 Advance Access published on March 11, 2009 Cytoplasmic retention of polyglutamine-expanded androgen receptor ameliorates disease via autophagy in a mouse model of spinal and bulbar muscular atrophy

Heather L. Montie1, Maria S. Cho1, Latia Holder1, Yuhong Liu1, Andrey S. Tsvetkov2, Steven Finkbeiner2,3,4,5 and Diane E. Merry1,

1Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA, 2Gladstone Institute of Neurological Disease, San Francisco, CA, USA, 3Taube-Koret Center for Huntington’s Disease Research, San Francisco, CA, USA, 4Department of Neurology and 5Department of Physiology, University of California, San Francisco, CA, USA

Received January 13, 2009; Revised February 19, 2009; Accepted March 9, 2009

The nucleus is the primary site of protein aggregation in many polyglutamine diseases, suggesting a central role in pathogenesis. In SBMA, the nucleus is further implicated by the critical role for disease of androgens, which promote the nuclear translocation of the mutant androgen receptor (AR). To clarify the importance of the nucleus in SBMA, we genetically manipulated the nuclear localization signal of the polyglutamine- expanded AR. Transgenic mice expressing this mutant AR displayed inefficient nuclear translocation and substantially improved motor function compared with SBMA mice. While we found that nuclear localization of polyglutamine-expanded AR is required for SBMA, we also discovered, using cell models of SBMA, that it is insufficient for both aggregation and toxicity and requires androgens for these disease features. Through our studies of cultured motor neurons, we further found that the autophagic pathway was able to degrade cytoplasmically retained expanded AR and represents an endogenous neuroprotective mechanism. Moreover, pharmacologic induction of autophagy rescued motor neurons from the toxic effects of even nuclear-residing mutant AR, suggesting a therapeutic role for autophagy in this nucleus-centric disease. Thus, our studies firmly establish that polyglutamine-expanded AR must reside within nuclei in the presence of its ligand to cause SBMA. They also highlight a mechanistic basis for the requirement for nuclear localization in SBMA neurotoxicity, namely the lack of mutant AR removal by the autophagic protein degradation pathway.

INTRODUCTION accumulation of misfolded proteins is most likely due to the lack of a secondary degradation mechanism within nuclei Nuclear residing proteins are normally directed to the nucleus and this accumulation of mutant protein is toxic to neurons. by a signaling sequence, a particular folding pattern and/or a Spinal and bulbar muscular atrophy (SBMA, Kennedy’s post-translational modiﬁcation. After they have served their disease) is an X-linked neurodegenerative disease resulting function, nuclear proteins are either degraded by nuclear pro- from the expansion of a polyglutamine (polyQ)-encoding teasomes or exported to the cytoplasm for degradation. A CAG tract in the 50 end of the androgen receptor (AR) gene mutation within a protein, such as the expansion of a polyglu- (1). When containing more than 40 CAG repeats, the AR tamine tract, causes it to accumulate within particular cellular causes slowly progressive proximal limb and bulbar compartments, as it is refractory to degradation. Nuclear muscle weakness, fasciculations and atrophy in men (2,3).

To whom correspondence should be addressed at: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 228 Bluemle Life Sciences Building, 223 S. 10th Street, Philadelphia, PA 19107, USA. Tel: þ1 2155034907; Fax: þ1 2159239162; Email: [email protected]

Patients may also suffer some sensory loss (4,5) and display slight androgen insensitivity (2). While partial loss of AR function exists in SBMA, this does not represent the primary disease etiology (6,7); rather accumulation of toxic AR protein species leads to motor neuron dysfunction and death (8–10). SBMA is one of a family of nine polyQ-expansion diseases (reviewed by 11), with a common pathological hallmark; the accumulation of misfolded and aggregated species of mutant protein in the cytoplasm or nuclei of vulnerable neurons. Figure 1. Protein expression of ARdNLS mice. Five-week-old male mice Although there are conflicting views in the field concerning were sacrificed and whole brain and spinal cord lysates evaluated for AR by the correlation of aggregates with disease, considerable data western blot. AR protein was detected with antibody AR(N-20). GAPDH indicate that inclusions themselves are not toxic (12,13). was used as a loading control. nTG, non-transgenic. Instead, species that are produced in early stages of the aggregation cascade (likely proteolyzed AR monomer and oligo- aggregation and toxicity. Furthermore, we present evidence mer) induce toxicity. Nonetheless, the presence of inclusions that the lack of access to the autophagic degradation pathway in a population of neurons reveals the late stage of a patho- represents one explanation for the enhanced toxicity of nuclear- genic process. confined mutant AR. The common finding of nuclear inclusions in polyQ diseases suggests a central role for the nucleus in pathogenesis. While inclusions of polyQ-expanded huntingtin are found in RESULTS both the cytoplasm and nucleus, the accumulation of nuclear ARdNLS112Q transgenic mice express greater levels mutant huntingtin causes the greatest neuronal toxicity of AR than AR112Q mice (13,14). In SCA-1 and SCA-3, inclusions of the mutant protein are found only within nuclei (15,16) and mutation of In order to understand the role of the nucleus in disease, we the endogenous nuclear localization signal (NLS) within created transgenic mice bearing an AR with a mutation in the each of these respective proteins, to sequester them within NLS. We previously created a transgenic mouse model of the cytoplasm, has proved to be neuroprotective (17,18). SBMA expressing full-length (human) polyQ-expanded AR These findings highlight an important role for the nucleus in (112Q) driven by the prion protein promoter (PrP) (22). The the toxicity induced by polyQ-expanded proteins, although new transgenic mice (ARdNLS112Q and ARdNLS24Q) were the mechanistic basis for this role has remained elusive. created to express transgenic AR with a deletion of amino In SBMA, inclusions of aberrantly cleaved polyQ-expanded acids 628–640 within the bipartite NLS of the AR. A line of AR are also present primarily in nuclei (19), although neuropil ARdNLS112Q was established which expresses over 2-fold accumulation of 1C2-positive material has been observed (20). more AR protein, in brain and spinal cord, than AR112Q In cell and rodent models of SBMA, nuclear aggregation and mice (Fig. 1). In addition, ARdNLS24Q mice expressed equiv- disease are dependent on the presence of AR ligands [testoster- alent levels of AR as ARdNLS112Q mice (Fig. 1), and both one or dihydrotestosterone (DHT)] (10,21–27), which drive lines were used for behavioral analysis. nuclear translocation of the AR. As a type I nuclear hormone receptor transcription factor, the unliganded AR resides primarily within the cytosol, where it is associated with heat shock and Motor deficits associated with SBMA are ameliorated accessory proteins (28,29). Upon hormone binding, the AR in ARdNLS112Q male mice undergoes a conformational change that exposes its bipartite We previously determined that the rotarod assay is a sensitive NLS, directing it to the nucleus, where it regulates transcription measure of motor function in SBMA mice (22). Therefore, we of its target genes. measured latency to fall from an accelerating rotarod, as well The localization of inclusions within nuclei and the depen- as grip strength, every 4 weeks beginning at 8 weeks of age. dence of disease on androgens suggest a central role for the This behavioral analysis of a large age-matched cohort of nucleus in SBMA. Moreover, the finding that some alternative ARdNLS112Q transgenic males (n ¼ 18) and AR112Q males ligands that direct AR to the nucleus also cause disease sup- (n ¼ 10) revealed delayed onset and reduction of motor deficits ports this idea (24,30). In a Drosophila model of SBMA, associated with SBMA, when compared with non-transgenic lit- retention of a polyQ-expanded AR fragment in the cytoplasm termates (n ¼ 18). While AR112Q males showed significant ameliorated disease (26). However, in contrast to these results, and progressive deficits in maintaining themselves on an accel- in a cell model of SBMA, fast axonal transport was reduced erating rotarod at 8, 12 and 16 weeks of age, ARdNLS112Q by expanded AR in a hormone-independent manner (31), males performed as well as non-transgenics (Fig. 2A). At 20, suggesting a cytoplasmic site of pathology, and making uncer- 24 and 28 weeks of age ARdNLS112Q males had significantly tain the role of the nucleus in disease. In this study, we sought reduced rotarod function compared with non-transgenics, but to determine, using transgenic mouse and cell models, whether still performed substantially better than AR112Q males mammalian systems reveal a necessity for nuclear localization (Fig. 2A). Female ARdNLS112Q (n ¼ 10) performed as well and whether nuclear localization is sufficient for disease. as non-transgenic littermates (n ¼ 15) until 24 weeks of age, Our results firmly establish that nuclear localization of polyQ- when they had a slight reduction in rotarod performance expanded AR is necessary, but not sufficient for nuclear (Fig. 2B). ARdNLS24Q (n ¼ 15) males did not develop any Human Molecular Genetics, 2009, Vol. 18, No. 11 1939

Figure 2. Motor deficits associated with SBMA are ameliorated in ARdNLS112Q mice. Latency to fall from an accelerating rotarod was measured every 4 weeks from 8 to 28 weeks of age in male (A)[#¼ P , 0.05 between AR112Q males and both non-transgenic (nTG) and ARdNLS112 males only; ¼ P , 0.05 between all groups] and female (B) transgenic mice. 8 to 28 ¼ age in weeks; Tick numbers on x-axis represent trials 1–4 for each age. Error bars represent standard deviation for each trial for each group. (C) Forepaw and all paw grip strength were measured every 4 weeks from 8 to 28 weeks of age. Error bars represent standard deviation. ¼ P 0.05. rotarod deficits up to 28 weeks of age (Supplementary Material, age, while both measures of grip strength of ARdNLS112Q Fig. S1). mice were similar to non-transgenic males (Fig. 2C). At 16, Both forepaw and all paw grip strength was significantly 20 and 24 weeks of age (data not shown), grip strength reduced in AR112Q male mice beginning at 12 weeks of results resembled those shown at 12 weeks of age. At 28 1940 Human Molecular Genetics, 2009, Vol. 18, No. 11 weeks of age, results of grip strength reflected those of rotarod analysis in that AR112Q males had significantly reduced grip strength compared with both ARdNLS112Q and non- transgenic males and ARdNLS112Q males were somewhat weaker than non-transgenic males. Female ARdNLS112Q mice showed grip strength similar to non-transgenic females up to 28 weeks of age (Fig. 2C). AR112Q male mice fail to gain weight after 6 months of age (22). Analysis of the present cohort showed a failure of AR112Q male mice to gain weight after 28 weeks of age, while ARdNLS112Q males continued to gain weight in the same manner as non-transgenic male littermates (Supplemen- tary Material, Fig. S2A). Female ARdNLS112Q mice had slightly greater weight gain over time compared with non- transgenic littermates (Supplementary Material, Fig. S2B). ARdNLS24Q male mice also showed no decrease in weight gain (Supplementary Material, Fig. S2C). Additional tests of motor function, including footprint and rearing analysis, revealed similar results as rotarod and grip strength analysis (data not shown). From 8 to 16 weeks of age, ARdNLS112Q male mice demonstrated a normal gait, while AR112Q males exhibited a wider and shorter gait. From 20 to 28 weeks of age, ARdNLS112Q males had a slightly lower and wider stance compared with nTG males, but their gait was substantially better than that of AR112Q males. Analysis of vertical activity (during a 5-min period) was performed using a Versamax activity monitor (AccuScan Instruments, Columbus, OH). At ages when ARdNLS112Q males performed as well as nTG males on the rotarod, their vertical activity was also normal, while AR112Q males showed significant deficits. After 20 weeks of age, ARdNLS112Q males had decreased vertical activity compared with nTG males but increased vertical activity compared with AR112Q males. Female AR112Q mice had normal gait and vertical activity at all ages evaluated. As previously described, survival of the AR112Q mice was not substantially compromised; survival of ARdNLS112Q mice was also unchanged (data not shown).

ARdNLS112Q has delayed nuclear accumulation and forms oligomers later than AR112Q Neuropathological analysis of AR112Q, ARdNLS112Q and non-transgenic male mice was performed at 8, 16 and 24 weeks of age. In AR112Q males at 8 weeks of age, AR112Q protein was localized primarily within nuclei of spinal motor neurons (Fig. 3A and Supplementary Material, Fig. S4) and these males had significant deficits in motor function (Fig. 2A). In contrast, at this age in ARdNLS112Q mice, transgenic AR protein was observed primarily within the cytoplasmic compartment, as observed both by immuofluores-

Figure 3. Analysis of AR aggregation in spinal cord. (A) Anterior horn from lumbar spinal cord of 8, 16 and 24 week old mice immunostained for AR (ARH280) and stained with Hoechst to reveal nuclei. Left panel, immunostain- ing of AR; middle panel, Hoechst staining; right panel, merged image. Arrow in image of 16 week ARdNLS112Q male indicates a small intranuclear inclusion. Arrow in image of 37 week ARdNLS112Q female indicates cytoplasmic AR. (B) Protein lysates from the same spinal cords as in (A) were prepared to evaluate oligomeric species of AR (ARH280) by western blot. Human Molecular Genetics, 2009, Vol. 18, No. 11 1941 cence and fractionation (Fig. 3A and Supplementary Material, is capable of forming nuclear inclusions, we targeted ARdNLS Fig. S4) and males had normal motor function (Fig. 2A). In to the nucleus with an exogenous NLS (NLSX3- addition, western blot analysis of brain and spinal cord revealed ARdNLS63Q). This resulted in the hormone-dependent for- substantially more SDS-insoluble, high-molecular weight oli- mation of nuclear inclusions of full-length AR (Fig. 4B and gomeric species of AR112Q than ARdNLS112Q at this age data not shown). In these cell lines, AR was expressed at com- (Fig. 3B). At 16 weeks of age oligomeric species of AR112Q parable levels and was stabilized by DHT (Fig. 4C). were increased (Fig. 3B), although inclusions were not detected Hormone treatment of AR112Q-expressing cells resulted (Fig. 3A). Sixteen week-old male ARdNLS112Q revealed in toxicity (Fig. 4D). However, polyQ-expanded ARdNLS accumulated mutant AR within nuclei (Fig. 3A) and a small (ARdNLS78Q)-expressing cells (Fig. 4D) failed to die in proportion of neurons contained small punctate intranuclear response to hormone, indicating that nuclear localization is inclusions (Fig. 3A). Western analysis revealed oligomeric necessary, not only for AR aggregation, but for cell death as forms of ARdNLS112Q, although these were substantially well. Targeting polyQ-expanded ARdNLS to the nucleus less abundant than those from AR112Q mice (Fig. 3B), (NLSX3-ARdNLS63Q) resulted in DHT-dependent death despite the abundance of AR protein. By 24 weeks of age, (Fig. 4D), confirming that the deletion in the NLS does not AR112Q male mice had a considerable proportion of neurons affect the capacity of the polyQ-expanded AR to confer toxicity in the anterior horn of the spinal cord with large intranuclear when localized to the nucleus. Thus, the possibility that deletion inclusions (Fig. 3A); as previously shown, inclusions consisted of the NLS alters AR conformation and prevents toxicity for of proteolyzed AR (data not shown) (22). At this age, reasons unrelated to its localization is unlikely, due to our ARdNLS112Q males also had a significant number of finding that nuclear targeting confers on the mutant AR neurons with large intranuclear inclusions of mutant AR protein the capability of forming inclusions and causing toxicity. (Fig. 3A); intranuclear inclusions of ARdNLS112Q were also composed of fragmented AR, as they lacked the epitopes for antibodies AR441 and ARC-19 (data not shown). Similar Nuclear localization of polyglutamine-expanded AR results were observed in cortical neurons from these animals is insufficient for aggregation and toxicity (Supplementary Material, Fig. S3A and B). In females, Our results indicate a requirement for nuclear localization in ARdNLS112Q was found largely within the cytoplasm at all both the nuclear aggregation and toxicity of polyQ-expanded ages, although by 37 weeks, a small number of neurons with AR. We next sought to determine whether nuclear localization large nuclear inclusions were observed (Fig. 3A). In males, is sufficient for these events. To accomplish this, we created ARdNLS24Q had also accumulated within neuronal nuclei by PC12 inducible cell lines that express an AR targeted to the 37 weeks of age, but did not form intranuclear inclusions (Sup- nucleus in the absence of hormone (NLSX3-AR). In the plementary Material, Fig. S5). Inclusions of ARdNLS112Q absence of DHT, NLSX3-AR76Q was localized within were confirmed by confocal microscopy to be contained nuclei, while AR112Q was diffusely distributed within cyto- within nuclei (data not shown). In addition, our previous plasm and nuclei (Fig. 5). No intranuclear inclusions of studies revealed decreased immunoreactivity of unphosphory- NLSX3-AR76Q were observed in the absence of DHT; lated neurofilament heavy chain (NF-H) in soma of both inclusions consisting of N-terminal AR fragments were spinal motor neurons and neurons of the cerebral cortex (22). formed exclusively in response to DHT (Fig. 5). These data This alteration was absent from neurons of ARdNLS112Q indicate that nuclear localization of the polyQ-expanded AR mice (data not shown). is insufficient for nuclear aggregation. Moreover, nuclear localization is insufficient for toxicity, as NLSX3-AR76Q PC12 cells failed to die in the absence of DHT, and only Polyglutamine-expanded ARdNLS fails to cause nuclear did so in the presence of DHT (Fig. 4C). It was also noted aggregation or toxicity in a cell model of SBMA that NLSX3AR, containing an even shorter polyQ-tract We previously created an inducible cell model of SBMA that (76Q) than non-NLS-tagged AR (112Q), induced a greater expresses full-length human AR with 112 glutamines and level of toxicity, despite equivalent protein levels (Fig. 4C), forms intranuclear inclusions in response to DHT (27). consistent with a role for the nucleus in mediating toxicity. Notably, as in patients’ tissue, nuclear inclusions in this NLSX3-AR35Q cells showed no aggregation or toxicity in model consist of proteolyzed N-terminal fragments of AR. response to DHT treatment (data not shown). In the present studies, we established a cell line expressing ARdNLS78Q to evaluate the metabolism of cytoplasmically retained polyQ-expanded AR in culture. In contrast to Primary motor neurons from ARdNLS112Q mice are AR112Q-expressing cells, ARdNLS78Q-expressing cells protected from DHT-dependent toxicity by autophagy showed a diffuse cytoplasmic distribution of AR in the pre- In order to evaluate SBMA motor neuron toxicity in response to sence of hormone (DHT), and failed to form intranuclear DHT, we initiated spinal cord cultures from non-transgenic, inclusions (Fig. 4A). Over time, these cells formed large cyto- AR112Q and ARdNLS112Q transgenic mice. Monomeric plasmic aggregates of full-length AR [detected with antibodies levels of both AR112Q and ARdNLS112Q were increased to the N-terminus (AR(N-20))], an internal epitope (AR441) (stabilized) in the presence of DHT; in addition, ARdNLS112Q (Fig. 4A) and the C-terminus [AR(C-19)] (data not shown). was expressed at significantly higher levels than AR112Q ARdNLS10Q cells also contained cytoplasmic AR in the pre- (Fig. 6A). While DHT caused the loss of 40% of sence of DHT and never formed nuclear or cytoplasmic aggre- AR112Q-expressing motor neurons, it failed to cause the death gates (data not shown). To confirm that mutant ARdNLS78Q of ARdNLS112Q-expressing motor neurons (Fig. 6B). 1942 Human Molecular Genetics, 2009, Vol. 18, No. 11

Figure 4. Polyglutamine-expanded ARdNLS fails to produce intranuclear inclusions or toxicity in a cell model of SBMA. (A) Immunoﬂuorescence of stably transfected tet-inducible PC12 cells treated with doxycycline to express either AR112Q or ARdNLS78Q and DHT for 48 h. Cells were immunostained with antibodies to the N-terminus (AR(N-20)), and an internal epitope (AR441) of the AR and stained with Hoechst to reveal nuclei. Arrow in AR112Q panel indicates intranuclear inclusions that lack the epitope for AR441. Arrow in ARdNLS78Q panel indicates cytoplasmic inclusion that contains the epitope for AR441. (B) NLSX3-AR76Q and NLSX3-ARdNLS63Q PC12 cells were treated with doxycycline to express AR in the presence of DHT and immunostained as in (A). Arrow in NLSX3-AR76Q panel indicates intranuclear inclusions that lack the epitope for AR441. Arrow in NLSX3-ARdNLS63Q panel indicates intranuclear inclusions that also contain the epitope for AR441. (C) AR112Q, ARdNLS78Q, NLSX3-AR76Q and NLS-ARdNLS63Q PC12 cells were treated with doxycycline (DOX) to induce AR in the absence or presence of DHT. Cells were harvested after 48 h and evaluated for AR protein levels via western blot analysis. (D) Analysis of PC12 cell toxicity. Cells expressing AR10Q, AR112Q and ARdNLS78Q were treated with doxycycline to express AR in the presence or absence of DHT for 12 days, and cell death determined by trypan blue uptake. Two-hundred cells were counted and the percentage of trypan blue-positive cells determined. Student’s t-tests were performed. ¼ P 0.05.

Figure 5. Nuclear localization of polyglutamine-expanded AR is insufﬁcient for the formation of nuclear inclusions in a cell model of SBMA. Stably transfected tet-inducible PC12 cells were treated with doxycycline to express either AR112Q or NLSX3-AR76Q in the presence or absence of DHT for 48 h. Cells were immunostained with antibodies to the N-terminus (AR(N-20)), and an internal epitope (AR441) of AR and stained with Hoechst to reveal nuclei. White arrow in AR112Q panel indicates diffuse cytoplasmic AR in the absence of DHT. White arrow in NLSX3AR76Q panel indicates diffuse nuclear AR in the absence of DHT. Student’s t-tests were performed. ¼ P 0.05.

We next sought to determine the mechanism by which protein diseases (reviewed in 32). Therefore, we evaluated ARdNLS112Q motor neurons resist DHT-dependent death. the essential autophagy marker LC3B (33) in primary motor Immunofluorescence staining revealed the presence of cyto- neurons. Immunofluorescence analysis of LC3B in plasmic puncta consisting of mutant AR in ARdNLS112Q ARdNLS112Q motor neurons revealed punctate cytoplasmic motor neurons (Fig. 6C). With the knowledge that staining of LC3B following treatment with DHT (Fig. 7A), ARdNLS112Q enters the nucleus with reduced efficiency in indicating the activation of autophagy in these neurons. In the presence of hormone (Figs 3A and 4A), and that it forms addition, LC3B puncta were found to co-localize with AR cytoplasmic inclusions (Figs 3A and 6C), we considered (Fig. 7A). Punctate staining of LC3B was not detected in autophagy to be a likely candidate. It is well established that nTG or AR112Q motor neurons following DHT treatment activation of autophagy is neuroprotective in misfolded (data not shown). Human Molecular Genetics, 2009, Vol. 18, No. 11 1943

Figure 7. Autophagy protects ARdNLS112Q motor neurons from DHT-dependent death. (A) Primary motor neuron cultures were initiated from ARdNLS112Q transgenic mouse embryo spinal cords. Cells were treated with or without DHT for 7 days, and immunostained for neurofilament heavy chain (SMI32) to reveal motor neurons, and LC3B (LC3B) to detect autophagosomes. ARdNLS112Q motor neuron shown was double- Figure 6. Primary motor neurons from SBMA mice die in response to immunostained for AR (AR-318) and LC3B, then immunostained using hormone treatment while motor neurons from ARdNLS112Q mice survive. SMI32. (B) Additional ARdNLS112Q motor neurons were treated with (A) Primary motor neuron cultures were initiated from AR112Q and or without 3-methyladenine (3-MA), to inhibit autophagy, for the last 3 ARdNLS112Q transgenic mouse embryo spinal cords. Cultures were treated days of the 7-day treatment period with DHT. Protein lysates were ana- with or without DHT for 7 days and protein lysates evaluated by western lyzed by western blot with antibodies to AR (AR(N-20)), LC3B and blot for AR and GAPDH. (B) Cultures were treated as in (A) and additional GAPDH. ARdNLS112Q and AR112Q motor neuron cultures were treated with 3- methyladenine to inhibit autophagy. Ten random fields of immunostained (SMI32) motor neuron cultures were counted under a fluorescent Leica microscope. Counts from three separate wells for each cell line and treatment group form of ARdNLS112Q following treatment with DHT and were graphed. Student’s t-tests were performed. ¼P 0.05. (C) Cultures 3-MA (Fig. 7B), well above the stabilization of AR seen were treated as in (A) and immunostained for AR (AR(N-20)) and neurofila- with DHT alone. In addition, the active form of LC3B ment heavy chain (SMI32) to reveal motor neurons. Note the presence of cytoplasmic inclusions of ARdNLS112Q. Nuclear ARdNLS112Q protein is also (LC3B II) was decreased in the presence of 3-MA (Fig. 7B), observed. validating the inhibitory effects of 3-MA on autophagy.

Given the suggestion that autophagy was activated in DHT-dependent death of motor neurons from AR112Q ARdNLS112Q motor neurons, we determined the role of mice is prevented by activation of autophagy autophagy in the resistance of ARdNLS112Q motor neurons The observation that endogenous autophagy can protect motor to DHT-dependent death. To this end, we treated spinal cord neurons from DHT-dependent death when polyQ-expanded cultures with 3-methyladenine (3-MA), a well-known inhibitor AR is retained within the cytoplasm (ARdNLS112Q) conﬁrms of autophagy (34). 3-MA failed to cause toxicity of non- the importance of this degradation pathway in clearing mis- transgenic motor neurons (data not shown); moreover, it did folded cytoplasmic proteins. We next sought to determine not enhance DHT-dependent toxicity of AR112Q-expressing whether pharmacologic activation of autophagy could rescue motor neurons (Fig. 6B). In contrast, 3-MA induced nuclear polyQ-expanded AR (AR112Q)-expressing motor DHT-dependent death of ARdNLS112Q motor neurons neurons from DHT-dependent death. We used an AKT inhibi- (Fig. 6B). Biochemical analysis of protein extracts from tor (AKTi) to activate autophagy in spinal cord cultures from these cultures showed a large increase in the monomeric our SBMA (AR112Q) mice. Previous studies demonstrated 1944 Human Molecular Genetics, 2009, Vol. 18, No. 11

Figure 8. DHT-dependent death of motor neurons from SBMA mice is prevented by activation of autophagy. (A) Primary motor neuron cultures were initiated from AR112Q transgenic mouse embryo spinal cords. Cells were treated with or without DHT for 7 days, in the presence or absence of an AKT inhibitor (AKTi) for the last 3 days. Counts from three separate wells for each cell line and treatment group were graphed. AKTi treatment rescued AR112Q motor neurons from DHT-dependent death. (B) Cultures were immunostained for neurofilament-heavy chain (SMI32) to reveal motor neurons and immunostained for LC3B to reveal autophagosomes. (C) Cells treated in parallel to those described in (A) were harvested and protein lysates analyzed by western blot with antibodies against AR (AR(N-20)), LC3B and GAPDH. (D) AR112Q motor neurons were treated with trehalose for the last 3 days of a 7-day treatment period with or without DHT. Motor neurons were immunostained for neurofilament-heavy chain (SMI32) and LC3B. (E) Cells treated as in (D) were harvested and protein lysates analyzed by western blot for AR (AR(N-20)), LC3B and GAPDH. (F) Motor neurons were counted as in (A) following trehalose treatment, and trehalose was found to protect AR112Q motor neurons from DHT-dependent death. Student’s t-tests were performed. ¼ P 0.05. the ability of the AKT inhibitor, phenoxazine, to activate cultures also showed an increase in LC3B II following autophagy in primary neurons expressing mutant huntingtin treatment with AKTi (data not shown). To confirm these (Tsvetkov and Finkbeiner, unpublished results). Treatment of findings, we evaluated another activator of autophagy, treha- AR112Q motor neurons with AKTi for the last 3 days of a lose, which was previously shown to activate mTOR- 7-day DHT treatment resulted in a substantial rescue from independent autophagy (35) and relieve the neurotoxicity of DHT-dependent death (Fig. 8A). As expected, AKTi-treated polyQ-expanded huntingtin (36,37). Treatment of AR112Q motor neurons contained cytoplasmic puncta of LC3B spinal cord cultures with trehalose resulted in the formation (Fig. 8B). Moreover, western analysis of AKTi-treated of LC3B-positive cytoplasmic puncta (Fig. 8D), an increase cultures revealed a significant increase in the active form of in LC3B II (Fig. 8E) and rescue from DHT-dependent death LC3B (LC3B II) (Fig. 8C). Non-transgenic motor neuron (Fig. 8F). Non-transgenic cultures also showed increased Human Molecular Genetics, 2009, Vol. 18, No. 11 1945

LC3B II levels following trehalose treatment (data not shown). accumulation ARdNLS112Q in male mice was somewhat sur- No effect on monomeric levels of AR112Q by either prising, based on our data in PC12 cells, but it was also not com- autophagy-inducing regimen was observed (Fig. 8C and E). pletely unexpected. A similar, but more substantial, deletion of the AR NLS (D628–657) allowed partial nuclear entry upon androgen binding (39). These results suggest that an alternative hormone-dependent signal may be utilized in the absence of a DISCUSSION functional bipartite NLS. It is also important to note that the A critical role for the nucleus in polyglutamine disease has ARdNLS112Q likely translocated to the nucleus as full-length emerged in recent years. In SBMA, this is evidenced by the pre- monomer rather than as a proteolyzed fragment. In support of sence of inclusions of polyQ-expanded AR within nuclei and this, we observed substantial levels of full-length the dependence of disease upon androgens, which enable the ARdNLS112Q by western analysis at ages when this protein AR to translocate into the nucleus following binding. Previous was visualized within nuclei by immunofluorescence. In studies of Drosophila and mammalian cell culture models to addition, we observed the localization of normal delineate the role of nuclear versus cytoplasmic AR in SBMA ARdNLS24Q within nuclei of male mice in the absence of have raised questions due to conflicting results (26,31). To pathologic inclusions, confirming that full-length ARdNLS is clarify the importance of the nucleus in SBMA using mamma- capable of eventual nuclear translocation. It is curious that, lian systems, we created transgenic mouse and cell models that despite higher levels of ARdNLS112Q protein and its eventual express polyQ-expanded AR with a deletion in a portion of its accumulation within nuclei, ARdNLS112Q mice developed bipartite NLS (amino acids D628–640; ARdNLS112Q), to only modest motor impairments with age. Data from PC12 reduce its androgen-dependent nuclear transit. We hypoth- cells expressing expanded ARdNLS demonstrate that this esized that nuclear localization of the mutant AR is essential mutant AR is benign when retained within the cytoplasm, but for disease and that cytoplasmic retention of mutant AR causes substantial toxicity when directed to the nucleus with would be neuroprotective in these models. an exogenous NLS. Thus, in the case of the mice, it may be We observed that DHT-dependent polyQ-induced toxicity that simply delaying onset of disease by reducing nuclear was ameliorated in three mammalian models of SBMA. transit of mutant AR minimizes its overall impact on neuronal First, even temporary retention of polyQ-expanded AR function. within the cytoplasm ameliorated motor deficits in male trans- Our finding that ARdNLS112Q formed inclusions earlier than genic mice. At 8 weeks of age, when ARdNLS112Q was loca- AR112Q was somewhat surprising. The formation of lized within the cytoplasm, male mice were completely ARdNLS112Q inclusions may be due to the higher levels of the normal, while AR112Q male mice, with exclusively nuclear protein, once it has accumulated within nuclei, compared with AR, displayed substantial motor deficits. With age, older AR112Q. We also observed that, although ARdNLS112Q male mice accumulated nuclear ARdNLS112Q, despite formed inclusions earlier than AR112Q (at 16 weeks, with oligo- mutation of the NLS. This nuclear localization was also mers also present at this time), AR112Q formed oligomers much observed in male ARdNLS24Q mice, but not in female trans- earlier (8 weeks of age) than ARdNLS112Q. The efficient nuclear genic mice, demonstrating that ARdNLS is capable of localization of AR112Q likely resulted in the earlier formation hormone-dependent nuclear translocation, albeit with substan- and sustained presence of oligomers and thus earlier and more tially reduced efficiency. Only when ARdNLS112Q had accu- substantive disease. mulated within nuclei and formed both oligomeric and The requirement in SBMA for nuclear mutant AR localiz- aggregated species did male mice begin to display signs of ation defined by our transgenic mouse and cell culture motor deficits, consistent with the previous demonstration studies led us to evaluate if nuclear localization is sufficient that oligomeric AR species precede disease symptoms (38). for disease. Our cell studies revealed that nuclear localization However, despite the eventual nuclear localization and aggre- alone is not sufficient for disease, and that androgen binding by gation of mutant ARdNLS112Q protein, male ARdNLS112Q the AR is essential for its aberrant metabolism and ability to mice exhibited substantially improved motor function. These induce toxicity. Targeting of a polyQ-expanded AR with a results indicate that (i) retention of a significant portion of shorter polyglutamine tract (76Q) to the nucleus led to toxicity polyQ-expanded AR within the cytoplasm is sufficient to in a hormone-dependent manner. Moreover, we observed both delay and ameliorate disease and (ii) nuclear localization enhanced toxicity of this protein over normally trafficked enhances the formation of oligomeric AR species that precede AR112Q, despite the shorter polyglutamine length, confirming motor deficits. In addition to the amelioration of motor deficits the importance of nuclear localization in toxicity. in mice by cytoplasmic AR retention, motor neurons from In SBMA, nuclear inclusions consist of an N-terminal ARdNLS112Q mice were resistant to DHT-dependent death. fragment(s) of AR (19,22,27). Fragmented polyQ-expanded Finally, our studies in PC12 cells indicate that the mutant proteins have been documented by numerous groups, and AR must enter the nucleus both for nuclear aggregation and may be a result of normal or aberrant protease cleavage, or toxicity. Therefore, nuclear localization is essential for inefficient processing by the proteasome. These fragments polyQ-expanded AR to elicit its primary toxic effects. have been shown to be refractory to degradation (40) and Complete and efficient nuclear localization of polyQ- are more toxic than intact, full-length, polyglutamine- expanded AR (AR112Q) caused early, severe and progressive expanded proteins (22,41–45). In our present studies, the motor deficits in male mice; these deficits were significantly cytoplasmic retention of polyQ-expanded AR led to the for- worse than those eventually observed in older ARdNLS112Q mation of large cytoplasmic inclusions that contained full- mice, which exhibited aggregated nuclear AR. Nuclear length AR, unlike the nuclear inclusions of patients’ tissue, 1946 Human Molecular Genetics, 2009, Vol. 18, No. 11 which contain only N-terminal AR species (19). When the more, the inhibition of autophagy led to DHT-dependent tox- mutant expanded ARdNLS was directed to the nucleus with icity, indicating that the cytoplasmic mutant AR is capable of an exogenous NLS, intranuclear inclusions were detected causing toxicity when autophagy is inhibited. Finally, the that contained the epitope for antibody AR441. It is unclear increase in mutant AR protein upon autophagy inhibition sup- whether there is any fragmented AR within these aggregates ports our conclusion that the mutant AR is, at least in part, or whether complete loss of the AR441 epitope would occur degraded by this pathway. While inhibition of autophagy with more time. Our aggregation studies were carried out caused a substantial increase in DHT-dependent toxicity in after 2 days of hormone treatment, while toxicity was evalu- ARdNLS112Q motor neurons, it had no effect on the toxicity ated after 12 days of hormone treatment. In mice, nuclear of AR112Q motor neurons (Fig. 7B) or of non-transgenic accumulated ARdNLS112Q was found to form intranuclear motor neurons (data not shown), indicating that endogenous inclusions of fragmented AR, and thus we hypothesize that autophagy does not substantially modulate toxicity in fragmented AR represents the most toxic species. In all, AR112Q motor neurons, in which AR112Q is confined to these data suggest that nuclear localization of polyQ-expanded the nucleus. Thus, the results of our studies shown here estab- AR is a prerequisite for its proteolysis and nuclear accumu- lish that the differential toxicity of nuclear versus cytoplasmic lation. This feature places the nucleus at a central point of mutant AR can be explained, in part, by the differential acti- pathology, the aberrant cleavage of mutant AR to a form vation of, and AR degradation by, autophagy. that is both toxic and aggregation-prone. The data presented here reveal that cytoplasmically retained While our studies place the location of mutant AR toxicity polyQ-expanded AR (ARdNLS112Q) can be degraded by in the nucleus, the mechanism by which the polyglutamine- autophagy, protecting motor neurons from DHT-dependent expanded AR confers toxicity within the nucleus is unclear. death. The high levels of ARdNLS112Q protein, even in the While AR transcriptional activity is not required for toxicity face of robust and efficient autophagic degradation, are consist- (24), transcriptional dysregulation occurs in the presence of ent with the increased transgene copy number in ARdNLS112Q the mutant AR (46,47). In addition, proteasome function is mice. Despite this increased protein, however, ARdNLS112Q impaired in mutant AR-expressing cells (our unpublished mice showed reduced motor symptoms. Thus, the increased results) and flies (48). Mitochondrial dysfunction has also ARdNLS112Q protein in the cytoplasm represents a form that been described in the face of nuclear mutant AR (49), conco- is less toxic than nuclear-confined AR. Whether this form is mitant with the altered transcription of genes involved in mito- non-toxic due to its lack of amino-terminal fragment-producing chondrial function. In addition to representing a major site for proteolysis or to other aspects of AR metabolism that occur the toxic cellular sequelae of expanded-polyglutamine AR, the within the nucleus is an active area of investigation. In all, nucleus also represents a major site of altered metabolism of our observations indicate one mechanism by which cytoplasmic the mutant AR. One of the distinctive features of nuclear retention of polyQ-expanded AR is neuroprotective; the mutant mutant AR when compared with cytoplasmic AR is the host protein is available to be degraded by autophagy. In accordance, of AR post-translational modifications, protein–protein inter- nuclear localization of polyQ-expanded AR likely limits its actions and structural AR changes that occur in response to access to the autophagic pathway and thus is one mechanism hormone binding (50,51). Our current and future studies will by which this localization contributes to its toxic effects address alterations in these pathways and their role in within motor neurons. nuclear polyQ-expanded AR toxicity. The potent neuroprotective effects of autophagy in Upon determining that a critical role for the nucleus exists ARdNLS112Q motor neurons led us to evaluate whether in SBMA pathogenesis, we investigated the mechanistic enhanced activation of the autophagic pathway would basis for the neuroprotective role of cytoplasmic retention of protect neurons from a nuclear localized polyQ-expanded polyQ-expanded AR. Previous studies have revealed failure protein, AR112Q. Pharmacologic induction of both mTOR- of the proteasome to efficiently and appropriately degrade mis- dependent and -independent pathways of autophagy rescued folded proteins, specifically those containing polyQ expan- AR112Q motor neurons from DHT-dependent death. This sions (40), unless chaperone-mediated therapies are initiated intervention, however, had no effect on monomeric levels of (52–59). In addition, it has become increasingly clear that AR112Q. This lack of an effect on AR112Q levels is similar the ubiquitin proteasome system is reduced in neuronal to findings of a previous study in which autophagy was inef- nuclei compared with the cytoplasmic compartment (60,61), fective at eliminating nuclear inclusions of mutant protein suggesting one explanation for the differential toxicities con- (63), but is contrary to results in a fly model of SBMA, in ferred by misfolded proteins in these two locations. which HDAC6 over-expression (which enhances autophagy) However, another important and emerging feature of cyto- led to lower steady-state levels of monomeric and aggregated plasmic localization of misfolded proteins is their availability polyQ-expanded AR (48). It may be that, while monomeric to activate a second method of degradation, the autophagic/ AR was unchanged in our study, oligomeric and nuclear lysosomal pathway, which has been shown to degrade aggregated forms of AR112Q were altered; these species polyQ-expanded proteins (62). When pharmacologically acti- were not evaluated in our spinal cord culture model due to dif- vated, autophagy can effectively degrade misfolded proteins ficulties with their detection. This would be in keeping with and is neuroprotective (reviewed by 32,35). earlier studies showing that nuclear aggregates may be Our studies of cultured, transgenic motor neurons revealed dynamic in nature (64–66). Alternatively, the effects of autop- that ARdNLS112Q motor neurons failed to die in response hagy on motor neuron viability may be independent of direct to DHT (Fig. 6). The observation of LC3 puncta indicates effects on mutant AR. It may be that activation of autophagy that autophagy was activated in these motor neurons. Further- alleviates proteasomal inhibition induced by mutant AR, in Human Molecular Genetics, 2009, Vol. 18, No. 11 1947 turn enhancing cell viability, as described by Pandey et al. length via sequence analysis. Cells were maintained in (48). It may also be that autophagy plays a more general normal growth media [Dulbecco’s modified Eagle’s medium role, relieving proteotoxic stress induced by polyQ-expanded with 10% heat-inactivated horse serum, 5% fetal bovine nuclear AR, perhaps by promoting the autophagic degradation serum, 2 mM L-glutamine, 100 units/ml penicillin/streptomy- of misfolded metastable proteins (67). cin, 200 mg/ml hygromycin (Invitrogen) and 100 mg/ml In all, these findings indicate that hormone binding and G418 (Mediatech, Manassas, VA)] at 378C, 10% CO2. nuclear localization are essential for the polyQ-expanded AR to aggregate and induce toxicity within motor neurons. There- fore, nuclear hormone-dependent AR events will be key in understanding the specific modifications, interactions and Treatment of inducible PC12 cell lines metabolic products responsible for causing disease. Although Stable Tet-On PC12 cell lines were treated with doxycycline hormone withdrawal has proved neuroprotective in mouse to express AR for various times and with various concen- models of SBMA, its effects in SBMA patients have yet to trations of DHT in charcoal-stripped serum-containing cell- be firmly established. Moreover, it is expected that therapies culture media. directed at the specific events that lead to the formation of a toxic AR species within motor neurons will prove to be more beneficial to patients and cause less side effects, as they will allow for normal AR function that is otherwise inter- PC12 cell toxicity assay rupted by hormone withdrawal. The studies herein highlight a Stable Tet-On PC12 cell lines (AR10Q, AR112Q, need to focus on the nucleus in SBMA, as well as on the ARdNLS78Q, NLSX3-AR76Q and NLSX3-ARdNLS63Q) autophagic pathway when developing these therapies. were treated with doxycycline to express equivalent levels of AR, in the absence and presence of 10 nM DHT for 12 days. At the end of treatment, cells were harvested and MATERIALS AND METHODS stained with trypan blue. Two hundred cells were counted and the percentage of trypan blue-positive cells determined. ARdNLS and NLSX3-AR inducible PC12 cell lines Significance was determined with Student’s t-test. Site-directed mutagenesis (Quick Change II XL, Stratagene) was performed on a pTRE plasmid (Clontech, Mountain View, CA), previously engineered to contain full-length ARdNLS PC12 cell and transgenic mouse constructs human AR cDNA, bearing 10 or 112 CAGs, to delete the nucleic acids encoding amino acids 628–640 of the AR The human AR gene, bearing either 24 CAGs (normal) or 112 (within the NLS) (ARdNLS). Mutation and CAG repeat CAGs (expanded), was previously cloned into the prion length were confirmed by sequence analysis. protein promoter (PrP) construct deleted of coding sequences NLSX3-AR was created as follows: The SV40 NLS in tri- (22). The deleted portion of the nuclear localization sequence plicate (NLSX3) was PCR-amplified from pShooterTM pEF/ of the AR (deleted of nucleic acids encoding amino acids myc/nuc (Invitrogen, Carlsbad, CA) vector, and an EcoRI 628–640) was cloned into the NruI and BstBI sites of both restriction digest site engineered on both the 50 and 30 ends. the 24 CAG and 112 CAG containing PrP-AR constructs. An NheI restriction was also engineered just upstream of the DNAs were linearized and the plasmid backbone (pBS) EcoRI site at the 50 end. The PCR product was cloned into removed by digestion with NotI, gel-purified and injected plasmid pCMVAR (16-CAG)DHA (9) (EcoRI site is just 50 into fertilized oocytes (C57Bl/6), by the Kimmel Cancer of the CTG start of the AR cDNA). The pCMV-NLSX3-AR Center Transgenic Facility at Thomas Jefferson University. (16-CAG)DHA was then digested with NheI and NarI, and Both ARdNLS and AR112Q mice were maintained on a pTRE-AR (112-CAG) was linearized with NheI and partially C57Bl/6 background (Charles River, Wilmington, MA). Foun- digested with NarI. The NLSX3-AR fragment from ders were screened by genotyping tail clips and brain and pCMVAR(16-CAG)DHA was ligated to pTRE-AR(112-CAG) spinal cord lysates from 5-week-old male mice were analyzed (containing full length AR), resulting in pTRE-NLSX3- for ARdNLS protein expression compared with those from AR(112-CAG). ARdNLS was then cloned into this construct age-matched AR112Q SBMA male mice. Additionally, CAG using NruI and BstBI. All constructs were sequenced to repeat length was determined by sequence analysis of PCR verify mutation and CAG length. products (Laragene, Inc., Los Angeles, CA). Stable transfections of Tet-On PC12 cells (Clontech) were performed using LipofectAMINE Plus (Invitrogen) with a plasmid conferring hygromycin resistance (pTK-hygromycin). Stable transformants were selected with 200 mg/ml hygromy- Genotyping mice cin. Single colonies were isolated and expanded and screened DNA from mice was prepared from tail or ear biopsies using for doxycycline-inducible AR protein expression by slot blot Red Extract-N-Amp Kit (Sigma). Transgenic animals were and western blot analysis using AR(N-20) antibody (Santa identified by PCR of the human AR: forward primer from Cruz, Santa Cruz, CA). AR expression levels were adjusted the PrP promoter region (50-ACTGAACCATTTCAACC with various doxycycline concentrations to achieve protein GAGC-30) coupled with a reverse primer from the AR levels equivalent to AR112Q PC12 cells. Genomic DNA sequence 50 to the CAG repeat (50AGGTGCTGCGCTCGC was extracted from each clone to verify mutation and CAG GGCCTCT-30). 1948 Human Molecular Genetics, 2009, Vol. 18, No. 11

Western blot analysis ARdNLS112Q or non-transgenic spinal cords were pooled separately, plated for culture and incubated for 3 weeks in Freshly dissected tissue was flash-frozen in liquid nitrogen. media conditioned by glial culture from 13.5-day-old non- Frozen sections were pulverized in a mortar and pestle on dry ice and homogenized in either 10 volumes of Triton-DOC transgenic brain [MEM, 35 mM NaHCO3, 0.5% dextrose, 1% buffer (1% sodium deoxycholate and 0.5% Triton X-100 in N3, 10 nM 2.5 S NGF (added after conditioning)]. During PBS with protease inhibitors) or RIPA buffer (50 mM Tris– the development of this culture system, motor neurons were HCl, pH 8.0, 0.15 M NaCl, 0.1% Nonidet P-40, 0.5% identified using antibodies to choline acetyltransferase, sodium deoxycholate, 0.1% SDS and protease inhibitors). neuron-specific enolase and neurofilament heavy chain PC12 cells and cells from primary spinal cord cultures were (SMI32). Motor neurons were identified to have much larger lysed with Triton-DOC buffer. All lysates were sonicated cell bodies relative to other spinal neurons and large, tapering, three times for 10 s using a Branson cup sonifier. A portion highly branched dendrites with a fibrillar appearance. In our of tissue lysates in RIPA was centrifuged at 15 000g for experiments presented here, SMI32 immunoreactivity and 5 min at 48C for detection of oligomeric species of AR (38). morphology were used to identify motor neurons. Three A DC protein assay (BioRad, Hercules, CA) was performed weeks after initiation, cultures were treated with or without m to determine protein concentration and lysates were electro- 10 M DHT for 7 days. Additional reagents/drugs were admi- phoresed by SDS–PAGE and transferred to 0.45 mm PVDF nistered for the last 3 days of the 7 day treatment period [5 mM 3-Methyladenine (3-MA), 100 mM Trehalose (Sigma), 2.5 mM (Immobilon-P). Western hybridization was performed using AKT inhibitor X [10-(40-N-diethylamino)butyl)-2-chlorophe- the following antibodies: AR(N-20), GAPDH (1:1000) noxazine] (AKTi) (Calbiochem, San Diego, CA)]. Three (Santa Cruz Biotechnology) and LC3B (1:500) (NB600- wells of each motor neuron culture line and treatment group 1384) (Novus Biologicals, Littleton, CO). Detection was were immunostained as described in what follows. Motor performed with ECL (Amersham Biosciences, Arlington neurons were determined by SMI32 stain and morphology Heights, IL). and counted. Significance was determined by a Student’s t-test. Behavioral analysis Every 4 weeks, beginning at 8 weeks of age, an age-matched Immunofluorescence cohort of ARdNLS24Q males, AR112Q males, ARdNLS112Q males, ARdNLS112Q females, non-transgenic males and non- Dissected fresh whole brain and spinal cord were frozen in transgenic females was subject to various measures of motor OCT, then sectioned with a cryostat (7 mm). Tissue sections, function. Mice were tested during the light phase of a 12 h motor neuron cultures and PC12 cells were fixed with 4% par- light/dark cycle for their latency to fall off a steadily acceler- aformaldehyde for 10 min, washed in PBS, permeabilized with ating rotarod (4–40 rpm over 10 min) (Ugo Basile, Comerio, 0.3% Triton X-100 for 15 min (cells only), blocked in 1.5% VA-Italy). During the first week of testing, mice were tested goat serum (Jackson ImmunoResearch, West Grove, PA) for four times per day for 3 consecutive days. The first 2 days con- 20 min and incubated for 60 min in primary antibody diluted stituted a learning period and data collected on the third day in 1.5% goat serum. Tissue sections or cells were washed in were used for analysis. For subsequent testing sessions, mice PBS and incubated for 30 min with secondary antibodies were only subjected to rotarod for 1 day, as statistical analysis (FITC- or Texas Red-conjugated) (Jackson ImmunoResearch, revealed no loss of statistical power under this regimen West Grove, PA), washed in PBS, incubated for 10 min with (unpublished data). Mice were allowed a rest period of at Hoechst (2 mg/ml), washed in PBS and mounted in Vecta- least 15 min between testing sessions. Scores were analyzed shield (Vector Laboratories, Burlingame, CA). Fluorescence by two-way repeated measures ANOVA with a Tukey post was visualized with a Leica (Leica Microsystems GmbH, hoc analysis using SigmaStat 3.0 software (SPSS Inc., Wetzlar, Germany) microscope; images were captured with Chicago, IL). A grip strength meter (Columbus Instruments, a Leica camera and compiled with IP Lab software (BD Bio- Columbus, OH) was used to measure the force exerted by a sciences, Rockville, MD). Antibodies used include AR(N-20), mouse as it was pulled across a grid by its tail. Grip strength ARH280, AR441 (1:100) (Santa Cruz), AR-318 (Vector Lab- was measured for forepaws only or hindpaws and forepaws oratories Burlingame, CA), SMI32 (1:1,000) (Sternberger together. Six measures were taken for both measures of grip Monoclonal, Baltimore, MD) and LC3B (NB600-1384) strength and the lowest and highest scores for each animal (1:200) (Novus Biologicals). dropped. An average for each animal was used for statistical analysis. Significance was determined with two-way repeated measures ANOVA and a Tukey post hoc analysis (SigmaStat). SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online. Primary motor neuron cultures Dissociated spinal cord cultures were established according to ACKNOWLEDGEMENTS Roy et al. (68). In brief, spinal cords were dissected on ice from 13.5-day-old embryos in dissection media (0.1% dex- We are grateful to Carlisle Landel, Ph.D., Director, Transgenic trose, 2% sucrose, 1.4 mM NaCl, 5.4 mM KCl, 0.17 mM and Gene Targeting Facility at Thomas Jefferson University Na2HPO4,22mMKH2PO4, 9.9 mM HEPES). Genotyping for creation of transgenic mice and for thoughtful discussions. was performed from a tail biopsy. Transgenic AR112Q, We also thank Heather Durham, Ph.D., Montreal Neurological Human Molecular Genetics, 2009, Vol. 18, No. 11 1949

Institute, McGill University, Montreal, for helpful advice on 15. Schmidt, T., Landwehrmeyer, G.B., Schmitt, I., Trottier, Y., Auburger, G., the initiation of spinal cord cultures. Laccone, F., Klockgether, T., Volper, M., Epplen, J.T., Schols, L. et al. (1998) An isoform of ataxin-3 accumulates in the nucleus of neuronal cells in affected brain regions of SCA3 patients. Brain Pathol., 8, 669– Conflict of Interest statement. None declared. 679. 16. Skinner, P.J., Koshy, B.T., Cummings, C.J., Klement, I.A., Helin, K., Servadio, A., Zoghbi, H.Y. and Orr, H.T. (1997) Ataxin-1 with an FUNDING expanded glutamine tract alters nuclear matrix-associated structures. Nature, 389, 971–974. This work was supported by the National Institutes of Health 17. Bichelmeier, U., Schmidt, T., Hubener, J., Boy, J., Ruttiger, L., Habig, K., (NS047381 and NS32214 to D.E.M.); (2NS045191 and Poths, S., Bonin, M., Knipper, M., Schmidt, W.J. et al. 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IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome

Leslie Michels Thompson,1,2,3 Charity T. Aiken,4 Linda S. Kaltenbach,7 Namita Agrawal,4 Katalin Illes,1 Ali Khoshnan,8 Marta Martinez-Vincente,9,10 Montserrat Arrasate,11 Jacqueline Gire O’Rourke,3 Hasan Khashwji,2 Tamas Lukacsovich,4 Ya-Zhen Zhu,1 Alice L. Lau,1 Ashish Massey,9 Michael R. Hayden,12 Scott O. Zeitlin,13 Steven Finkbeiner,14 Kim N. Green,2 Frank M. LaFerla,2 Gillian Bates,15 Lan Huang,4,5 Paul H. Patterson,8 Donald C. Lo,7 Ana Maria Cuervo,9 J. Lawrence Marsh,4,6 and Joan S. Steffan1

1Department of Psychiatry and Human Behavior, 2Department of Neurobiology and Behavior, 3Department of Biological Chemistry, 4Department of Developmental and Cell Biology, 5Department of Physiology and Biophysics, and 6Department of Pathology and Developmental Biology Center, University of California, Irvine, Irvine, CA 92697 7Center for Drug Discovery and Department of Neurobiology, Duke University, Durham, NC 27704 8California Institute of Technology, Pasadena, CA 91125 9Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, NY 10461 10Institute of Neuropathology, IDIBELL-Hospital Universitari de Bellvitge, L’Hospitalet de Llobregat, 08907 Barcelona, Spain 11Division of Neuroscience, Center for Applied Medical Research, University of Navarra, E-31008 Pamplona, Spain 12University of British Columbia, Vancouver, BC, Canada V6T 1Z4 Downloaded from 13Department of Neuroscience, University of Virginia, Charlottesville, VA 22908 14Departments of Neurology and Physiology, Gladstone Institute of Neurological Disease, University of California, San Francisco, San Francisco, CA 94158 15Department of Medical and Molecular Genetics, King’s College London School of Medicine, King’s College London, London SE1 9RT, England, UK

xpansion of the polyglutamine repeat within the post-translational modifications, including Htt ubiquitina- protein Huntingtin (Htt) causes Huntington’s dis- tion, SUMOylation, and acetylation, and increases Htt nu- jcb.rupress.org E ease, a neurodegenerative disease associated with clear localization, cleavage, and clearance mediated by aging and the accumulation of mutant Htt in diseased lysosomal-associated membrane protein 2A and Hsc70. neurons. Understanding the mechanisms that influence We propose that IKK activates mutant Htt clearance until Htt cellular degradation may target treatments designed an age-related loss of proteasome/lysosome function pro- on April 8, 2010 to activate mutant Htt clearance pathways. We find motes accumulation of toxic post-translationally modified that Htt is phosphorylated by the inflammatory kinase IKK, mutant Htt. Thus, IKK activation may modulate mutant Htt enhancing its normal clearance by the proteasome and neurotoxicity depending on the cell’s ability to degrade lysosome. Phosphorylation of Htt regulates additional the modified species. THE JOURNAL OF CELL BIOLOGY Introduction Abnormal accumulation of misfolded and aggregated protein (polyQ) disease protein Huntingtin (Htt) in Huntington’s in affected neurons is a hallmark of many neurodegenerative disease (HD), tau in frontotemporal dementias (FTD), -synuclein diseases associated with aging. The major pathways of protein in Parkinson’s disease (PD), ataxin-1 in spinocerebellar ataxia clearance in the cell are performed by the proteasome and the 1 (SCA1), and SOD1 in amyotrophic lateral sclerosis (ALS). lysosome, which both become compromised with age (Cuervo Post-translational modification of target proteins can et al., 2005; Martinez-Vicente and Cuervo, 2007; Chondrogianni regulate their clearance from cells. Phosphorylation regulates and Gonos, 2008; Tonoki et al., 2009). Parallel with reduced protein degradation, alters subcellular localization, and/or creates turnover, proteins mutated in familial neurodegenerative phosphodegrons/binding motifs for interactors that regulate diseases accumulate and cause dysfunction and death, and secondary modifications such as ubiquitination, SUMOylation, accompanying symptoms. Examples include the polyglutamine and acetylation. For instance, phosphorylation of HSF1, MEF2,

Correspondence to Joan S. Steffan: [email protected] © 2009 Thompson et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication Abbreviations used in this paper: CMA, chaperone-mediated autophagy; HD, date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Huntington’s disease; Htt, Huntingtin; LAMP-2A, lysosomal-associated membrane Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, protein 2A; polyQ, polyglutamine; wt, wild type. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Supplemental Material can be found at: http://jcb.rupress.org/content/suppl/2009/12/21/jcb.200909067.DC1.html The Rockefeller University Press $30.00 J. Cell Biol. Vol. 187 No. 7 1083–1099 www.jcb.org/cgi/doi/10.1083/jcb.200909067 1083 Published December 28, 2009

and GATA-1 activates their SUMOylation (Hietakangas et al., Results 2006), phosphorylation of p53 and RelA activates their acetyl ation (D’Orazi et al., 2002; Hofmann et al., 2002; Chen et al., The IKK complex directly 2005), and phosphorylation of IB and FOXO3a activates phosphorylates Htt their ubiquitination (Karin and Ben-Neriah, 2000; Karin et al., The N-terminal 17 amino acids of Htt contain a number of 2002; Hu et al., 2004). In turn, these modifications may ulti- potentially modifiable residues (Fig. 1 A). In addition to the mately target the protein for degradation (Hernandez-Hernandez lysines at 6, 9, and 15, which can be SUMO modified and ubiq- et al., 2006; Hietakangas et al., 2006; Hunter, 2007; Wu uitinated (Steffan et al., 2004), three residues are possible phos- et al., 2007; Zuccato et al., 2007; Jeong et al., 2009). As protein phorylation targets (T3, S13, and S16). clearance mechanisms become impaired upon aging, modi- Sequence evaluation for conserved motifs revealed that fied proteins normally targeted for degradation by post- Htt residues 11–18 share sequence similarity with residues translational modification may accumulate and disease-causing 642–649 of FOXO3a (Fig. 1 B), a substrate of IKK at S644 proteins take on toxic functions (Orr and Zoghbi, 2007; Shao analgous to Htt S13 (Hu et al., 2004). Because expanded polyQ and Diamond, 2007). Httex1p activates the IKK complex in cell culture and transgenic HD is a member of a family of polyQ repeat expansion mice, and interacts with IKK- in vitro (Khoshnan et al., 2004), diseases characterized by the accumulation and aggregation of IKK emerged as a candidate kinase that might target Htt S13. mutant Htt protein in diseased neurons (Orr and Zoghbi, 2007). IB kinase (IKK) is composed of three subunits: IKK- and In HD, when the repeat expands above 40, disease will mani- IKK- are homologous catalytic subunits, and IKK- is a regu- fest, typically striking in mid-life (Walker, 2007). Above 65 latory subunit. As a first step to evaluate phosphorylation of this Downloaded from repeats, a juvenile form of the disease occurs. The polyQ domain and a potential role for IKK, mass spectrometry was expansion exists within the context of a large 350-kD protein; used. 25QP-HBH, a His-tagged unexpanded form of Httex1p however, expressing just the N-terminal fragment of Htt en- was transiently cotransfected with IKK- into ST14A cells, coded by exon 1 (Httex1p), which contains a highly expanded purified by nickel enrichment under denaturing conditions, di- polyQ repeat, can precipitate an aggressive HD-like disease in gested with chymotrypsin, and analyzed by reverse-phase liquid transgenic mice and flies (Mangiarini et al., 1996; Steffan chromatography coupled to tandem mass spectrometry. Phos- phorylation of both S13 and S16 was observed in the presence et al., 2001). The first 17 amino acids of Htt can mediate aggre- jcb.rupress.org gation, subcellular localization and membrane association, sta- of IKK (Fig. 1 C). bility, and cellular toxicity, each of which are implicated in HD Using Htt peptides phosphorylated at either S13 or S16 as pathogenesis (Steffan et al., 2004; Luo et al., 2005; Warby antigens, affinity-purified rabbit polyclonal antisera, designated et al., 2005, 2009; Anne et al., 2007; Rockabrand et al., 2007; anti–S13-P and anti–S16-P, were generated (Fig. S1). Over- Atwal and Truant, 2008). The potential for Htt post-translational expression of IKK- or IKK- but not IKK- increased phos- modification to have a disease-modifying role has recently phorylation of both unexpanded (25QP) and expanded (46QP) on April 8, 2010 emerged as a consistent theme, with regulatory functions im- forms of Httex1p with a C-terminal His-HA-HA-His (H4) plicated for other sites within the full-length protein as well, tag in ST14A cells cotransfected with Httex1p-H4 and IKK including phosphorylation at S421 by Akt and S434, S1181, subunits (Fig. 1 D). Phosphorylation of 46QP-H4 appears less and S1201 by Cdk5 (Humbert et al., 2002; Luo et al., 2005; efficient than 25QP-H4 and phosphorylation of 25QP-H4 is Warby et al., 2005; Anne et al., 2007), SUMOylation and ubiq- associated with its reduced abundance. To first determine uitination at K6, K9, and K15 (Steffan et al., 2004), palmitoylation whether IKK can directly phosphorylate Htt and determine the at C214 (Yanai et al., 2006), and acetylation at K444 (Jeong specific residue involved, recombinant IKK phosphorylation of et al., 2009). The regulatory properties of post-translational purified Htt was tested in vitro. S13 of both purified unexpanded modifications extend to other polyQ repeat diseases, most (25QP) and expanded (46QP) Httex1p was phosphorylated by notably phosphorylation of S776 in expanded ataxin-1, the both IKK- and IKK- (Fig. 1 E), whereas phosphorylation of mutant protein in SCA1 (Orr and Zoghbi, 2007). S16 was not observed (not depicted). These results suggest that We evaluated the effect of phosphorylation within the S13 is a direct target of IKK and that the phosphorylation of first 17 amino acids of Htt on its subcellular localization, down- S16 observed with IKK overexpression in cell culture may be stream post-translational modifications, and protein clearance. primed by phosphorylation of S13 by IKK. Alternatively, the This domain contains two serines at positions 13 and 16, which sensitivity of anti–S16-P antisera may be inadequate to detect are adjacent to the lysines found to be modified by SUMO and the in vitro modification. ubiquitin (Steffan et al., 2004). We demonstrate that the IKK complex, previously shown to directly interact with Htt (Khoshnan IKK-activated phosphorylation of et al., 2004), phosphorylates Htt S13 and may activate phos- Httex1p regulates its post-translational phorylation of S16. Phosphorylation of these residues promotes modification and subcellular localization modification of the adjacent lysine residues and activates Htt As described above, phosphorylation can regulate post- clearance in a manner requiring both the proteasome and translational modification of adjacent lysine residues (Hunter, lysosome. We find that expansion of the Htt polyQ repeat 2007). The role of Htt S13 and S16 phosphorylation on Htt ubiq- may reduce the efficiency of this phosphorylation, potentially uitination, SUMOylation, and acetylation of Httex1p was tested. contributing to the accumulation of mutant Htt. We previously found that 97QP Httex1p is polyubiquitinated

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Figure 1. IKK directly phosphorylates Htt. (A) The first 17 amino acids of the Htt protein contain three residues that may be phosphorylated (red) and three modifiable lysine residues (blue). (B) Htt S13 is within a domain similar to FOXO3a S644. (C) Mass spectrometry analysis shows that Htt serines 13 and 16 can be phosphorylated. 25QP-HBH was purified under denaturing conditions from St14A cells cotransfected with IKK-. ESI-MS/MS spectra were obtained after chymotryptic digestion and collision-induced dissociation (CID) for N-terminally acetylated and diphosphorylated peptide on S13 and +2 +2 S16 Ac-ATLEKLMKAFEpSLKpSF, with the parent ion, [MH2] , at m/z 1023.03 (M = 2044.06 D). (D) Htt phosphorylation of S13 and S16, detected with phospho-specific antibodies, is activated with coexpression of IKK- or IKK-. Httex1p was purified from St14A cells transiently transfected with 25QP-H4 or 46QP-H4 with vector control or plasmids encoding subunits of IKK. Total Htt was detected with CAG53b antibody, and myc-actin transfection control detected with anti-myc antibody. (E) IKK- and IKK- directly phosphorylate Htt S13 in vitro. An in vitro kinase assay was performed with 75 ng recombinant IKK- or IKK- protein, and wt (SS) or S13,16A (AA) mutant 25Q or 46Q purified Httex1p-H4. Htt was detected with CAG53b or anti-S13-P.

(Steffan et al., 2004). Making a mutant that cannot be phos- In addition to the detection of phosphorylation at both phorylated on S13 (S13A) reduces this polyubiquitination, S13 and 16, acetylation of lysine K9 was detected together whereas mimicking phosphorylation of S13 (S13D) retains its with modification of S13 by mass spectrometry upon exoge- polyubiquitination (Fig. 2 A). If the dual phosphorylation we nous IKK overexpression (Fig. 2 C). To evaluate this acetyl see activated by IKK is mimicked (S13,16D), a reduction in ation further and the possible influence of phosphorylation, Htt polyubiquitination is again observed (Fig. 2 A). Consistent affinity-purified polyclonal antiserum was generated against a with this, overexpression of IKK reduces polyubiquitination of K9-acetyl, S13-phospho, S16-phospho Htt peptide (anti–K9-Ac; 97QP Httex1p (Fig. S2 A). The S13A and S13,16D mutants Fig. S1). The antibody recognizes Htt 25 or 46QP in the also demonstrate reduced mono-SUMOylation of 97QP presence of exogenous IKK-; however, it shows little to no Httex1p, whereas S13D retains its SUMOylation (Fig. 2 B). immunoreactivity without IKK coexpression (Fig. 2 D). Acetyl Overexpression of IKK leads to a reduction in 97QP mono- ation can be mimicked by a lysine (K) to glutamine (Q) substi- SUMOylation and an increase in its poly-SUMOylation tution and phosphorylation mimicked by either a serine (S) to (Fig. S2 B). Therefore, IKK may modulate ubiquitin and aspartic acid (D) or to glutamic acid (E) substitution. Using a SUMO addition to Httex1p, two modifications globally tied to K9Q, S13,16E (QEE) mimic, immunoreactivity was observed protein clearance mechanisms. in the absence of IKK, confirming the specificity of the antibody

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Figure 2. Phosphorylation of Httex1p regulates its ubiquitination, SUMOylation, acetylation, and nuclear localization. (A and B) Phosphorylation of serines 13 and 16 regulates mutant Httex1p ubiquitination and SUMOylation. St14A (A) or HeLa (B) cells were transiently transfected with vector or HIS-ubiquitin (A) or HIS-SUMO-1 (B) and control and mutant 97QP VL* Httex1p. Conjugated proteins were purified under denaturing conditions by Ni-NTA magnetic nickel beads and Htt detected with anti-Htt CAG53b by Western analysis. (C) Mass spectrometry analysis shows that Htt S13 phosphorylation can occur with K9 acetylation. 25QP-HBH was purified from St14A cells cotransfected with IKK- and CBP and treated for 2 h with histone deacetylase inhibitors 200 mM Trichostatin A/5 mM Nicotinamide. ESI-MS/MS spectra were obtained after chymotryptic digestion and collision-induced dissociation +2 +2 (CID) for a peptide acetylated at K9 and phosphorylated at S13 MAcKAFEpSLKSF, [MH2] at m/z 655.30 (M = 1308.60 D). (D) IKK- overexpression increases phosphorylation of Htt S13 and acetylation of K9. St14A cells were transiently transfected with Httex1p-H4 with 25 or 46Qs, +/ IKK- or with 46QP QEE-H4. Htt was purified and subjected to Western analysis with anti-K9-Ac, anti-S13-P, and CAG53b. (E) Mimicking phosphorylation significantly increases nuclear localization in primary neurons. Primary cortical neurons were cotransfected with pcDNA3.1-mRFP and 97QP-GFP or 97QP-DD-GFP plasmids. The subcellular distribution of these polypeptides was examined by measuring the fluorescence intensity of GFP, to which they are fused, in regions of the nucleus and cytoplasm for each cell. The extent to which these polypeptides localized preferentially to the nucleus or the cytoplasm was determined by calculating the ratio of nuclear/cytoplasmic GFP fluorescence intensity and comparing the distribution of the two polypeptides byt test. Error bars indicate SEM in arbitrary units.

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for this Htt species (Fig. 2 D). Collectively, these data suggest reduction in Httex1p abundance (Fig. 3 A), which may at least that IKK-mediated phosphorylation may regulate ubiquitina- partially be the result of its less efficient phosphorylation by tion, SUMOylation, and acetylation within the first 17 amino IKK- (Fig. 1 D). acids of Htt. The abundance of Httex1p AA, EE, and QEE mutants Because the first 17 amino acids of Htt can function as were next compared with control Httex1p or to Httex1p in a cytoplasmic retention signal and regulate the association which all the lysines were mutated to arginine (K6,9,15R or 3R), of Htt with mitochondria, Golgi, endoplasmic reticulum, late also in the presence of epoxomicin or ammonium chloride/ endosomes, and autophagic vesicles (Steffan et al., 2004; leupeptin. Consistent with the potential destabilization of un- Atwal et al., 2007; Rockabrand et al., 2007), and because expanded polyQ by phosphorylation, unexpanded (25QP) overexpression of IKK- was previously demonstrated to pro- EE and QEE Httex1p levels were lower than control and AA mote expanded polyQ Httex1p nuclear localization (Khoshnan (Fig. 3 C). In contrast, elimination of modifiable lysines (3R et al., 2004), we tested whether phosphorylation of this 25QP) had no effect on soluble Htt levels, but did increase lev- domain could influence its cellular localization. Either phospho- els of insoluble Htt. This result is in contrast to what we previ- mimetic (S13,16D/S13,16E or DD/EE) or phosphoresistant ously reported for a highly expanded 3R mutant (3R97QP; (S13,16A, or AA) forms of Httex1p were used to assess the Steffan et al., 2004); however, we did not use filter retardation consequence of phosphorylation on soluble cellular localiza- assays together with Western analysis to examine levels of in- tion. Fluorescence of mutant Httex1p with 97Qs fused to soluble Htt in those studies, and now understand that this 97QP GFP was first assessed in NIH-3T3 cells (Fig. S3 A), demon- 3R mutant is detectable, however mostly in the insoluble frac- strating largely cytoplasmic localization for both 97QP-GFP tion. Inhibition of the proteasome or the lysosome increased Downloaded from and 97QP-AA-GFP. In contrast, phosphomimetic 97QP- levels of the unexpanded phosphomimetics (25QP EE and DD-GFP and 97QP-EE-GFP displayed increased nuclear QEE), suggesting that the proteasome and lysosome may both localization. Similarly, a statistically significant increase in be involved in clearance of phosphorylated and acetylated nuclear localization of expanded 97QP-DD-GFP over control forms of unexpanded Htt fragments. 97QP-GFP was observed upon transfection into primary In contrast to results with unexpanded Htt, expanded con- cortical neurons (Fig. 2 E). Nuclear localization was also trol and mutant 46QP proteins accumulated in both soluble and

enhanced using phophomimetics of unexpanded Httex1p insoluble fractions (Fig. 3 C, bottom); this accumulation was jcb.rupress.org (25QP-EE-GFP) compared with wild type (wt) or AA in 3T3 also influenced by proteasomal and lysosomal inhibition. The cells, suggesting this process may extend to normal Htt or Htt 46QP EE phosphomimetic consistently ran on SDS-PAGE as a fragments (Fig. S3 B). doublet, suggesting the presence of a phosphorylated intermediate that is not well cleared in the presence of the expanded re- Phosphorylation of Htt by peat. The doublet was not observed with the acetylation mimetic IKK activates Httex1p and 586aa Htt QEE, implicating lysine 9 as a critical residue in this clearance on April 8, 2010 fragment clearance mechanism and possibly suggesting an alternative lysine 9 post- We then asked whether phosphorylation by IKK might also reg- translational modification other than acetylation in doublet for- ulate Htt stability. Unexpanded (25QP) and expanded (46QP) mation. We conclude that expansion of the polyQ repeat in polyQ Httex1p were cotransfected into ST14A cells with IKK-, Httex1p reduces the efficiency of phosphorylation-activated as this subunit of IKK had the greatest Htt phosphorylation IKK-mediated clearance. activity in cell culture (Fig. 1 D), and levels of Htt evaluated. To further characterize IKK-mediated Htt phosphory Unexpanded Htt showed a dependence on IKK- for enhanced lation and clearance, a larger Htt fragment comprised of 586 clearance relative to a myc-actin transfection control, and this amino acids (aa) with and without IKK- (Fig. 3 D) was ex- effect persisted in the presence of either the specific proteasome pressed in ST14A cells. Co-expression of IKK- significantly inhibitor epoxomicin or the lysosome inhibitors ammonium reduced levels of unexpanded Htt (586 aas with 15Qs). Al- chloride/leupeptin (Fig. 3, A and B). Proteasome inhibitors though there was a reduction in levels of total unexpanded 586 were able to block basal and IKK-–induced degradation of (15Q) with IKK- overexpression, as determined using anti-Htt 25QP; however, they were not able to abolish the differences 3B5H10 (Fig. 3 D) or anti-Htt EM48 (Fig. S4 A), a significant between both types of degradation, suggesting that the reduc- increase in an immunoreactive species was observed when the tion in cellular levels of unexpanded 25QP Httex1p mediated anti–S13-P antibody was used for detection, particularly upon by IKK involves proteasome-dependent and -independent longer-term lysosomal inhibition. Expanded repeat 128Q 586aa degradation of the protein. Inhibition of the lysosome reduced fragment levels were not reduced with overexpression of IKK-, the effect of IKK- on 25QP clearance, implicating lysosomal and S13-phosphorylated, expanded 128Q fragment was not involvement. Interestingly, the IKK-–induced degradation detectible above background levels, again suggesting a reduced of Httex1p was largely reduced for the expanded form of the ability of the mutant Htt protein to be phosphorylated and protein, although the basal degradation of the protein was uncleared. Collectively, these data show that IKK- can increase perturbed and still dependent on the proteasome system. Phos- phosphorylation and reduce levels of unexpanded polyQ phorylation of unexpanded polyQ Httex1p may therefore target Htt fragments in a manner dependent on both the proteasome it for degradation by both the proteasome and lysosome. Expan- and the lysosome, and that expansion of the polyQ repeat sion of the polyQ repeat to 46Qs inhibited this IKK-–mediated inhibits this effect.

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Figure 3. Phosphorylation of unexpanded polyQ Httex1p and 586aa Htt is associated with its reduced abundance in cell culture. (A) Levels of un expanded polyQ Httex1p are reduced with overexpression of IKK-; this effect is inhibited with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4 was cotransfected with myc-actin and with vector or IKK- into St14A cells. Cells were treated for 16 h with DMSO, 100 nM epoxomicin in DMSO, or 20 mM ammonium chloride/100 µM leupeptin in DMSO. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc to detect myc-actin, and anti-HA to detect Httex1p. (B) IKK- overexpression reduces levels of unexpanded polyQ Httex1p in the presence of proteasome or lysosome inhibition. Scion software was used to quantitate triplicate levels of 25QP-H4 from the experiment represented in A, normalized to levels of myc-actin transfection control, within each treatment group: control, epoxomicin, or ammonium chloride/leupeptin. (C) Mimicking phosphorylation of unexpanded polyQ Httex1p reduces its abundance in cell culture; this effect is reduced with expansion of the polyQ repeat. 25QP-H4 or 46QP-H4, wt control or QEE, EE, AA, or 3R were cotransfected with myc-actin into St14A cells. Cells and lysates were treated as in A. (D) Levels of phosphorylated unexpanded polyQ 586aa Htt accumulate with inhibition of the proteasome or the lysosome; phosphorylation is reduced with expansion of the polyQ repeat. 15Q or 128Q 586aa Htt constructs were cotransfected into St14A cells with myc-actin and with vector or IKK-. Cells were treated for 4 h with DMSO or 100 nM epoxomicin in DMSO (to eliminate any possible effect on the lysosome by epoxomicin), or for 16 h with water or 20 mM ammonium chloride/100 µM leupeptin in water. Lysates were subjected to filter-retardation assay and Western analysis using anti-myc, anti–S13-P, and anti-Htt 3B5H10.

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Endogenous wt full-length Htt is expression of IKK-, whereas MAB2166 does not, supporting phosphorylated by IKK- the idea that the cleared Htt species may not be not recognized The above results show that Htt fragments can be phosphory- well by this antibody (Fig. S4 A). lated by IKK in cells and in vitro. We next examined whether In addition to genetic modulation of IKK, pharmacologi- exogenous expression of IKK subunits could phosphorylate cal activation of IKK with IL-1 or TNF- was tested for acti- full-length, endogenous wt Htt in ST14A cells to determine vation of Htt S13 phosphorylation. ST14A cells were treated whether this type of regulation may be involved in an endoge- with these standard IKK-activating cytokines for 15, 30, 60, and nous clearance mechanism. IKK- overexpression increased the 120 min (Fig. 4 C). Both Il-1 and TNF- increased levels levels of acetylated and S13-phosphorylated full-length Htt of phosphorylated full-length and fragmented Htt at 60 and and Htt fragments in ST14A cells (Fig. 4 A). To ensure that this 120 min, similar to phosphorylated IB. S13-phosphorylated effect is specific to IKK, the ability of IKK- to enhance immuno Htt and phosphorylated IB were both reduced at 15 and 30 min, reactivity to both antibodies was eliminated in the presence paralleling the increased clearance of total IB at these time of an shRNA pool against IKK- (Fig. 4 A). As a control, levels points. Total full-length Htt levels were not reduced as assessed of phosphorylated IB, a defined IKK substrate targeted for by MAB2166 on Western analysis, but an 180-kD-sized set degradation by its phosphorylation (Karin and Ben-Neriah, of N-terminal fragments recognized by anti-Htt Ab1 showed a 2000), were also evaluated in this experiment, and levels (Fig. 4 A, possible decrease in abundance at 15 and 30 min and increased boxed) paralleled that of phosphorylated and acetylated Htt. abundance at 60 and 120 min, similar in trend to robust effects Full-length acetylated and phosphorylated Htt, as well as on total IB. S13-phosphorylated Htt and Htt fragments accu- phosphorylated Htt fragments, were again increased by inhibi- mulated in cells treated for 60 and 120 min with Il-1. These Downloaded from tion of either the proteasome or the lysosome (Fig. 4 B), sug- phosphorylated Htt species accumulated even further at 120 min gesting that they may serve as intermediates in the wt Htt of IL-1 treatment with either epoxomicin or ammonium degradation process. High molecular weight full-length S13- chloride/leupeptin, suggesting both proteasomal and lysosomal phosphorylated Htt species were observed upon IKK- involvement (Fig. 4 D). overexpression (Fig. 4 B), which may reflect full-length Htt Endogenous S13-phosphorylated Htt was next analyzed post-translational modification by SUMO or ubiquitin as we by immunofluorescence. Without exogenous IKK-, only mi-

observed for Httex1p (Fig. 2, A and B). Using a well-described totic cells stained with anti–S13-P (Fig. 5 A), representing jcb.rupress.org anti-Htt antibody, MAB2166 (Millipore), which is uniquely a small fraction of the cell population. In cells that were tran- sensitive enough to detect endogenous rat Htt in ST14A siently transfected with FLAG–IKK-, the phosphorylated whole-cell lysate, we did not observe either a loss of endoge- species of Htt was detected in cells with exogenous IKK- nous Htt in cells overexpressing IKK- or an accumulation expression in a variety of localization patterns (Fig. 5 B and with inhibition of the proteasome or lysosome, possibly sug- Fig. S3 C). This staining was specific for phosphorylated S13, as gesting that the levels of modified Htt that are modulated it could only be competed away with a 1–17aa peptide phosphory on April 8, 2010 represent a small percentage of the total. However, MAB2166 lated on serine 13, but not with the corresponding unmodified may not interact well with the phosphorylated Htt species. peptide (not depicted). S13-phosphorylated Htt and K9-acetyl Within the epitope recognized by MAB2166, Htt residues Htt immunoreactivity was also detected in FLAG–IKK- 414–503, S421 (Humbert et al., 2002), and S434 (Luo et al., nucleofected mouse striatal progenitor cells, Hdh7/7 (Fig. 5 B; 2005) are phosphorylated by Akt and Cdk5, respectively, and unpublished data). K444 is acetylated (Jeong et al., 2009). This acetylation at K444, involved in Htt lysosomal clearance, destroys the Proteins involved in lysosomal and MAB2166 epitope (Krainc, D., personal communication), proteasomal clearance mechanisms modify supporting the possibility that modifications eliminate reactiv- levels of phosphorylated Htt ity with MAB2166. Although we have not determined whether The pronounced effect of the lysosomal inhibitors on the intra- these modifications occur at the same time as phosphorylation cellular levels of phosphorylated S13 Htt compared with the of S13 by IKK, Akt acts upstream of IKK activation (Dan unmodified protein (Fig. 3 D and Fig. 4, B and D), and the et al., 2008) and it is possible that modification of this epitope distinctive punctuate pattern observed in the immunofluores- concurrent with S13 phosphorylation may reduce the ability cence studies with the anti–S13-P antibody compatible with of MAB2166 to recognize the Htt species being cleared. lysosomal association of the modified protein (Fig. 5 and Fig. S3 C), We find that MAB2166 does not strongly recognize immuno- led us to further characterize the mechanism mediating the precipitated S13/S16-phosphorylated or K9-acetylated Htt lysosomal degradation of S13-phophorylated Htt. Because species in cells overexpressing IKK-, whereas Ab1 and lysosomal inhibition increases levels of the S13 phospho- MAB5490/1H6, antibodies that recognize the N-terminal species, the expectation is that proteins involved in regulating domain of Htt, do recognize these modified forms (Fig. S4 B). lysosomal activity could also influence levels of phosphorylated In addition, Htt antibodies EM48 (recognizing the first 256 aa Htt. The lysosomal-associated membrane protein 2A (LAMP-2A) of human Htt with a deletion of the polyQ stretch [Gutekunst mediates selective autophagy of proteins that contain KFERQ- et al., 1999]) and 3B5H10 (raised against GST-human Htt like Hsc70 binding sequences in mammalian cells through 171aa-66Q [Peters-Libeu et al., 2005]) detect reduced abundance a process known as chaperone-mediated autophagy (CMA; of unexpanded human 15Q 586aa fragment with exogenous Massey et al., 2006b). Similarly, Atg7 is essential for autophagy

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Figure 4. Phosphorylated and acetylated endogenous wt Htt accumulates with inhibition of the proteasome and lysosome. (A) Overexpression of IKK- increases phosphorylation of endogenous Htt and IB in cell culture. St14A cells were transiently transfected with vector, IKK-, or IKK-, together with vector or two different pools of anti–IKK- shRNAs; a control pool that did not silence IKK well (pool 1) and one pool that was effective in silencing IKK (pool 2), as assessed by levels of FLAG-IKK- and - by Western. Lysates were subjected to Western analysis with anti-Htt MAB2166, anti–S13-P, anti–K9-Ac, anti-phosphoserine 32 IB (IB-P), anti–IB, anti–-tubulin, and anti-FLAG to detect FLAG-tagged IKK- and IKK-. Bands the size of full-length endogenous Htt (350 kD) are shown by the arrow on the left. Boxed bands show the reduction in phosphorylated IB with IKK shRNA. (B) Phosphory- lated and acetylated Htt accumulate with inhibition of the proteasome or the lysosome. St14A cells were transiently transfected with IKK- or vector, and were treated as in Fig. 3 D. Lysates were subjected to Western analysis with anti–-tubulin, and anti-Htt antibodies MAB2166, anti–S13-P or anti–K9-Ac. (C) Pharmacological activation of the IKK complex modulates levels of phosphorylated Htt and IB. St14A cells were incubated with 20 ng/ml TNF- or IL-1 over a time course. Lysates were subjected to Western analysis as in A with additional detection by anti-Htt Ab1. (D) IL-1–induced Htt S13-phosphorylated species accumulate with inhibition of the proteasome or lysosome. St14A cells were treated for 2 h with 20 ng/ml Il-1 before lysis. The proteasome and lysosome were inhibited, and Western analysis performed as in B.

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Figure 5. Phosphorylated Htt can be detected by immunofluorescence in cell culture. (A) S13-phosphorylated Htt is present in untransfected mitotic rat jcb.rupress.org S14A cells. (B) S13-phosphorylated Htt colocalizes with FLAG immunoreactivity in rat St14A cells lipofected with FLAG–IKK-, and S13-phosphorylated Htt colocalizes with FLAG immunoreactivity in mouse Hdh7/7 cells nucleofected with FLAG–IKK-.

and a loss of Atg7 function in mouse brain causes neuro Htt-mediated toxicity (Fig. 6 D) was tested. Overexpression of degeneration (Mizushima et al., 1998; Komatsu et al., 2006). Hsc70 increased the survival of HdhQ111/Q111-expressing cells We therefore examined whether LAMP-2A or Atg7 might be in- more than overexpression of Hsp70, the latter being 85% on April 8, 2010 volved in the clearance of phosphorylated Htt by the lysosome. identical to Hsc70. Exogenous expression of Hsp70 or Hsc70 When either endogenous LAMP-2A or Atg7 is knocked down showed that Hsc70, but not Hsp70, increased levels of Htt by shRNA, accumulation of endogenous S13-phosphorylated acetylation and S13 phosphorylation, suggesting that Hsc70 Htt and Htt fragments is observed (Fig. 6 A), suggesting that may specifically activate an IKK-regulated Htt degradation both proteins may be involved in lysosomal clearance of phos- process (Fig. S5). Minor differences in heat-shock proteins phorylated Htt. Likewise, shRNA against rat LAMP-2A increased have previously been demonstrated to define their function. Httex1p levels and aggregation, whereas overexpression of For instance, in yeast, the homologues of Hsp70/Hsc70, Ssa1p, human LAMP-2A had the opposite effect (Fig. 6 B). These and Ssa2p are 97% identical and yet only Ssa2p is required to combined data are consistent with a role for LAMP-2A in the target protein substrates to the yeast vacuole, the functional lysosomal clearance of Htt. equivalent of the mammalian lysosome (Brown et al., 2000). Although Httex1p does not contain a bonafide “KFERQ”- We propose that Hsc70 may increase Htt clearance by the pro- like Hsc70-binding motif in its sequence, phosphorylation of teasome and the lysosome by activating S13 phosphorylation Htt serine 16 (14-LKpSFQ-18) could provide the negative and K9 acetylation. charge required to convert this sequence to an Hsc70-binding motif (mimic 14-LKEFQ-18, where the phosphorylated serine Mimicking phosphorylation of mutant Htt in at residue 16 resembles a glutamic acid [E]). We therefore tested rat brain slice cultures reduces its toxicity the ability of phosphomimetic Httex1p to interact with GST- The data presented show that IKK- can enhance the level of Hsc70 in vitro and found that mimicking phosphorylation of a phosphorylated form of Htt that appears to be more readily Htt serines 13 and 16 on unexpanded polyQ Httex1p (25QP-EE) cleared. To test whether this phosphorylation is functionally increased the in vitro binding of Httex1p to Gst-Hsc70 by a significant, toxicity of Htt phosphomimetics was compared specific ADP-dependent mechanism (Fig. 6 C, left), whereas with control expanded repeat Htt in an acutely transfected rat expansion of the polyQ repeat to 46Qs reduced this interaction cortico-striatal slice culture model where toxicity is dependent (Fig. 6 C, right). on expansion of the polyQ repeat (Khoshnan et al., 2004). Because an interaction with Hsc70 could regulate clearance Phosphomimetic (DD) or phosphoresistant (AA) forms of of phosphorylated mutant Htt, the ability of Hsc70 to reduce expanded polyQ Httex1p (97QP) were tested for their effects

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Figure 6. LAMP-2A, Atg7, and Hsc70 may modulate Htt clearance and toxicity. (A) Reducing levels of LAMP-2A or Atg7 in cell culture increases abundance of S13-phosphorylated Htt. St14A cells were transiently transfected with shRNA for rat LAMP-2A or Atg7, or pSUPER vector control; antibodies used for Western analysis are shown to the left of the Western panels. (B) LAMP-2A levels modulate Httex1p abundance. St14A cells were transiently cotransfected with LAMP-2A shRNA, human HA-tagged LAMP-2A or vector control, and myc-actin transfection control. Lysates were subjected to Western analysis and filter-retardation assay, detected with anti-Htt CAG53b, anti–rat/mouse LAMP-2A, anti-HA to detect HA-hLAMP-2A, anti-myc, and MemCode protein stain. (C) Hsc70 interacts with phosphomimetic unexpanded polyQ Httex1p in vitro. Purified 25QP-H4 or 46QP-H4 wt and mutant proteins radiolabeled with 35S were incubated with isolated GST-Hsc70 or GST protein bound to glutathione-agarose beads. Where indicated, 5 mM ATP or ADP was added to the reaction. Bound proteins were washed, subjected to SDS-PAGE, and detected by phosphoimager autoradiography. (D) Hsc70 reduces Htt-mediated toxicity in Hdh111/111 cells. Hdh7/7 or Hdh111/111 cells were nucleofected with vector, Hsp70, or Hsc70 together with GFP. 47 h later, XTT cell viability assays were performed, and the percentage of relative survival was calculated correcting for transfection efficiency of the GFP control ± SEM from triplicates.

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Figure 7. Phosphorylation may reduce toxicity but increase insolubility of Htt. (A) Mimicking 97QP Httex1p phosphorylation reduces its toxicity in acutely transfected rat cortico-striatal slice explants. Plasmids encoding YFP and 97QP-CFP, 97QP DD-CFP, 97QP AA-CFP, or CFP control were biolistically cotransfected into rat cortico-striatal brain slices. The number of healthy medium spiny neurons in the striatal region of each slice was scored 5 d after transfection. n = 9 for each condition. Error bars represent SEM. Asterisk = difference from 97QP at P < 0.0001. (B) Immuno precipitated phosphorylated/acetylated wt Htt purified from mouse brain runs in an insoluble fraction in the SDS-PAGE stacking gel. 12 independent wt control (W) or R6/2 (R) mouse brains were collected at 4, 8, and 12 wk of age, snap frozen, and lysed. 500 µg of lysate was subjected to immunoprecipitation with PW0595 anti-Htt antibody or zero antibody control (lysate from lanes 12 and 13 are identical). Western analysis was performed with a series of antibodies listed to the right of the panel. Immunoprecipitated Htt is present as an insoluble species in the stacking gel and at the top of the separating gel, and as a soluble form at the standard 350-kD size, marked with an arrow. MAB2166 does not detect the insoluble spe- Downloaded from cies well. jcb.rupress.org on April 8, 2010

on Htt-mediated toxicity in this model. The phosphomimetic is toxic, nuclear localization facilitated by phosphorylation 97QP-DD displayed significantly reduced toxicity compared could be part of a normal process of protein degradation that with either control or 97QP-AA (Fig. 7 A), suggesting that phos becomes impaired upon expansion of the polyQ repeat, thus phorylation of Httex1p may reduce neurotoxicity. Because promoting the accumulation of nuclear, toxic Htt that is impli- we showed that the phosphomimetic 97QP-DD Httex1p is cated in HD. Confirming a potential in vivo role for this modi- more nuclear localized (Fig. 2 E), but is less toxic than 97QP fication, mutant Htt-mediated neurotoxicity was significantly in the slice cultures, the data present a potential contradiction reduced when the phosphomimetic was expressed compared to the extensive studies showing that nuclear accumulation of with control or phosphoresistant mutant Htt in BACHD mice, mutant Htt significantly enhances neurodegeneration (Saudou suggesting that Htt phosphorylation may slow the progres- et al., 1998; Schilling et al., 1999; Cornett et al., 2005). These sion of HD in vivo and represents a valid therapeutic target results suggest that while nuclear accumulation of mutant Htt (Gu et al., 2009).

Phosphorylation activates Htt degradation • Thompson et al. 1093 Published December 28, 2009

Phosphorylated and acetylated Htt can be and Fig. 3 C), which may result in reduced clearance of mutant detected in mouse brain Htt by inhibiting this phosphorylation-driven mechanism, and We next examined whether modified Htt species could be de- ultimately contribute to disease. Finally, modified species rec- tected in an extensively studied HD mouse model, R6/2, which ognized by phospho- and acetyl-specific antibodies are present contains two wt copies of the mouse HD gene and is transgenic in mouse brain. for exon 1 of the human huntingtin gene originally carrying Mimicking phosphorylation of Htt serines 13 and 16 150 CAG repeats (Mangiarini et al., 1996). R6/2 mice have a increases soluble Httex1p-GFP nuclear localization (Fig. 2 E). rapid phenotypic progression (are severely impaired by 14 wk) In previous studies, we showed that Htt interacts with the acetyl and intranuclear inclusions throughout the brain. Htt was transferase CBP (Steffan et al., 2000, 2001); therefore CBP, a immunoprecipitated from whole brain tissue from 4-, 8-, and nuclear protein, is a candidate acetyltransferase for Htt lysine 12-wk R6/2 versus wt control mice (Fig. 7 B; antibody PW0595, 9, as was demonstrated for lysine 444 (Jeong et al., 2009). Enzo Life Sciences, Inc.). S13- and S16-phosphorylated and CBP/p300 contains ubiquitin ligase activity which regulates K9-acetylated endogenous mouse Htt showed reactivity to rela- protein degradation (Grossman et al., 2003); therefore, this E3 tively insoluble Htt species in both R6/2 and wt control brain. ligase activity of CBP could also be involved in regulating Htt A major percentage of modified Htt remained in the stacking ubiquitination. A futher connection between the IKK-mediated gel and at the top of the separating gel with less migrating to the phosphorylation of Htt and CBP activites may exist, as CBP size of the standard 350-kD full-length Htt band. No fragments interacts directly with IKK- in the nucleus (Verma et al., originating from the transgene were visualized using this 2004) and is a substrate for IKK- (Huang et al., 2007), sug- method with the modification-specific antibodies, suggesting gesting that CBP and the IKK complex could function together Downloaded from that the transgene was not efficiently phosphorylated and acetyl to modulate Htt stability. ated, although it could be detected after immunoprecipititation Relevant to the potential role for S13 and S16 phosphory- with the Enzo Life Sciences, Inc. antibody (unpublished data). lation in nuclear localization, nuclear caspase-6 cleavage of Wt Htt antibodies Ab1, MAB5490/1H6, and the Enzo Life Sci- mutant Htt has been implicated in its pathogenic potential ences, Inc. antibody, each raised against unique Htt species, rec- (Graham et al., 2006; Warby et al., 2008). We find that IKK ognized wt-soluble and the insoluble Htt species in the stacking activates phosphorylation of Htt fragments, one of which is

gel, whereas MAB2166 did not recognize the insoluble Htt, consistent with the predicted size of a wt Htt caspase-6 cleavage jcb.rupress.org consistent with our data above suggesting that MAB2166 does product (Fig. 4 B) and the fragment recently shown to be gener- not substantially detect the phosphorylated/acetylated Htt spe- ated in neurons with activation of IKK- (Khoshnan et al., cies (Fig. S4) or that the insoluble fraction consists primarily of 2009). It is therefore an intriguing possibility that phosphory truncated N-terminal Htt species, which do not contain the lation by IKK ultimately promotes Htt nuclear localization, poly- MAB2166 epitope. We also find that Htt phosphorylated on SUMOylation, acetylation by CBP, and subsequent caspase-6 threonine 3 (T3) is present in the insoluble fraction, consistent cleavage, which all facilitate a regulated form of clearance. on April 8, 2010 with our recent observation that mimicking phosphorylation of Phosphorylation of Htt by IKK appears to activate its deg- T3 increases Htt aggregation (Aiken et al., 2009). Overall, our radation at least in part by the lysosome, dependent on LAMP-2A data suggest that the S13/S16-phosphorylated and K9-acetylated levels (Fig. 6), the integral membrane receptor protein that forms of wt endogenous Htt are detectable in both wt and R6/2 can directly import proteins across the lysosomal membrane mice, and that these modified forms represent relatively insolu- for CMA (Massey et al., 2006b). The CMA chaperone Hsc70 ble species. preferentially interacts with phosphomimetic wt Httex1p and reduces Htt-mediated toxicity (Fig. 6, C and D). Combined, this Discussion data may suggest that phosphorylated Htt is degraded in a LAMP-2A–dependent mechanism through CMA. Because Delineation of Htt clearance mechanisms is of great signifi- phosphorylation can trigger nuclear localization and acetylation cance because an accumulation of mutant Htt is implicated in of specific Htt species (Fig. 2, C–E) and overexpression of HD pathogenesis. We demonstrate that the IKK complex phos- Hsc70 increases levels of acetylated and phosphorylated en- phorylates Htt at S13 and may activate its degradation, similar dogenous full-length and fragmented Htt (Fig. S5), the findings to IKK-mediated degradation of IB and FOXO3a (Karin and implicate Hsc70 in either mediating an interaction of Htt with Ben-Neriah, 2000; Karin et al., 2002; Hu et al., 2004). The pro- the IKK complex, or alternatively activating the IKK complex, posed selective degradation of phosphorylated wt Htt, which as has been demonstrated for the ubiquitin ligase parkin, which involves both the proteasome and lysosome, may include a is mutated in Parkinson’s disease (Henn et al., 2007). transient nuclear localization mediated by phosphorylation of CMA activity declines with age due to a gradual decrease Htt, where it is subsequently acetylated, ubiquitinated, and of LAMP-2A levels in lysosomes (Cuervo and Dice, 2000), SUMOylated in an order that remains to be established. Proteins whereas artificially maintaining LAMP-2A levels in aging rat involved in lysosomal degradation pathways, Hsc70, LAMP-2A, liver similar to those in young animals can restore CMA activity and Atg7, appear to modulate the levels of these modified to youthful levels and improve organ function (Zhang and forms of Htt in mammalian cells. Our data also suggest that Cuervo, 2008). Because HD is a neurodegenerative disease asso- IKK-–mediated Htt S13 phosphorylation is more efficient for ciated with aging and we have found clearance of phosphorylated wt than for expanded polyQ truncated Htt polypeptides (Fig. 1 D Htt dependent on LAMP-2A, a reduction in LAMP-2A levels

1094 JCB • VOLUME 187 • NUMBER 7 • 2009 Published December 28, 2009

lysosome might actually increase HD pathogenesis through increased nuclear accumulation and aggregation of the mutant protein. At this stage of disease, serine-modified Htt would accumulate, leading to increased pathology as a result of the intrinisic toxicity of the modified Htt. Consistent with this, we find that mimicking Httex1p phosphorylation increases its toxicity and aggregation in Drosophila photoreceptor neurons where components of the mammalian machinery to degrade phosphorylated Htt, specifically LAMP-2A, are not present (unpublished data). Thus, during late-stage HD, it may be harmful to increase pathways involved in IKK activation, SUMOylation, and acetylation, whereas in presymptomatic stages, these pathways may be protective. The Htt protein itself may play an integral role in autopha- Figure 8. Proposed molecular mechanism for the development of HD. gic clearance of proteins. Conditional knockout of Htt in the Normal neuronal function: IKK phosphorylates wt Htt activating its post- translational modification, caspase cleavage, and clearance by the protea- mouse central nervous system results in an accumulation of some and lysosome. Presymptomatic HD neuronal function: With chronic neuropil protein aggregates containing ubiquitin and p62/ expression of mutant Htt, proteasome activity is reduced, and lysosomal SQSTM1 (unpublished data). Htt has been shown to associate degradation of mutant Htt becomes essential. Mutant Htt triggers activa- with autophagosomes (Atwal and Truant, 2008) and lysosomes tion of the IKK complex; however, it is less efficiently phosphorylated than Downloaded from wt Htt. With the clearance mechanism activated, mutant and wt Htt in the (unpublished data) and may therefore play a functional and reg- presymptomatic cell are degraded by the lysosome. Symptomatic HD neu- ulatory role in a selective protein clearance mechanism ulti- ronal function: Lysosomal degradation of Htt is impaired through reduction of LAMP-2A levels or other loss of lysosomal function caused by aging and mately involved in its own processing. Extensive investigation by mutant Htt expression. Uncleared mutant Htt and Htt fragments accumu- will be necessary to test this possibility and elucidate the pre- late and take on toxic functions, enhancing HD pathogenesis. cise mechanisms involved.

over time may be tied to HD pathogenesis. We propose a hypo- Materials and methods jcb.rupress.org thetical model for the progression of HD at the molecular level (Fig. 8), where IKK phosphorylates Htt and activates a cascade Plasmid constructs of Htt post-translational modifications and caspase cleavage pcDNA3.1-based plasmids (Invitrogen) containing the Htt exon 1 DNA between the HindIII and BamHI sites were used as described previously (Khoshnan et al., 2009) associated with rapid Htt degradation (Steffan et al., 2004). These plasmids contained alternating CAG/CAA by the proteasome and the lysosome in unaffected neurons. In a repeats, coding for either a normal range (25) or expanded (46 or 97)

presymptomatic HD neuron, IKK could be induced by the pres- polyglutamine tracts followed by the proline-rich region of Htt ending with on April 8, 2010 the amino acids HRP to create Httex1p. The plasmids were opened at ence of the mutant protein (Khoshnan et al., 2004), thus stimu- BamHI and XbaI and DNA encoding various tags was inserted in frame lating an IKK-mediated mechanism of Htt clearance, consistent with Httex1p to create C-terminal tagged Httex1p. The following tags were with the innate immune activation that occurs in premanifest used: GS*, VL*, HBH (a gift from Peter Kaiser and Christian Tagwerker, University of California, Irvine, Irvine, CA [Tagwerker et al., 2006]), EGFP, patients well before symptom onset (Björkqvist et al., 2008). and H4 (HIS-HA-HA-HIS: GSHHHHHHMGYPYDVPDYAEFYPYDVPDYAVH- As long as LAMP-2A levels remain high, patients can degrade HHHHH*) where a stop codon is denoted by the asterisk. The H4 tag was mutant Htt before it can cause toxicity, despite progressively created by a two-step double-stranded oligonucleotide ligation. Mutations in Httex1p were created using double-stranded oligo inhibited proteasome activity and reduced efficiency of mutant nucleotides containing HindIII-compatible ends encoding the first 17 amino Htt phosphorylation. As aging and mutant Htt together progres- acids of Huntingtin, which were ligated between the HindIII site of  sively impair proteasome and overall lysosomal activity, and as pcDNA3.1 in the polylinker and the HindIII site in exon I, immediately 5 to the CAG repeat. K6R K9R K15R (3R) was used as described previously LAMP-2A levels decline with age, modified Htt may accumu- (Steffan et al., 2004). To create S16 mutations, the HindIII site was shifted late, enhancing HD pathogenesis. using oligonucleotides containing BspMI (BfuAI) sites, then double-stranded oligonucleotides were again used to create S13 together with S16 mu- From this model, it follows that increasing the efficiency tants. pHsc70 was constructed by ligating the BamHI fragment from pGST- of Htt clearance by the lysosome, or increasing levels or mobil- Hsc70 encoding Hsc70 into the BamHI site of pcDNA3.1. For plasmids ity (Kaushik et al., 2006) of functional LAMP-2A in the lyso- used in the acutely transfected striatal slice culture assay, 97QP Httex1p wt and mutants were cloned into pGWIZ (Gene Therapy Systems) in frame somal membrane early in the disease process, could delay HD with CFP using the PstI site. onset and serve as a therapeutic strategy. Treatment choice may The following plasmids were obtained collaboratively or as gifts: vary depending on the stage of HD. In mammals, early treatment pHis-SUMO-1 (A. Dejean, Institut Pasteur, Paris, France); pHis-Ubiquitin (D. Bohmann, University of Rochester, Rochester, NY); p-human-HA-LAMP-2A to increase Htt phosphorylation and acetylation may be useful (pAMC1) and pGST-Hsc70 (A.M. Cuervo, Albert Einstein College of Medi- when levels of LAMP-2A are adequate, as suggested by the cine, Bronx, NY; Cuervo and Dice, 1996); pCMV-Hsp70 (P. Muchowski reduced toxicity in acutely transfected rat slice cultures (Fig. 7 A) [University of California, San Francisco, San Francisco, CA] and H. Kamp- inga [University of Groningen, Groningen, Netherlands; Michels et al., and complete lack of neurotoxicity in BACHD mice when Htt is 1997); pMyc-Actin (H. Rommelaere, Ghent University, Ghent, Belgium); mutated to mimic the phosphorylated form (Gu et al., 2009). pFLAG-IKK, pFLAG-IKK, and pHA-IKK (A. Khoshnan and P. Patterson, However, when LAMP-2A levels are low or its function is im- California Institute of Technology, Pasadena, CA); pYFP (L. Kaltenbach and D. Lo; Duke University, Durham, NC); 586aa Htt constructs 15Q (pCI- paired, drugs that activate the formation of the post-translationally NeoHtttt586-15) and 128Q (pCINeoHtt 586–128; M. Hayden, University modified Htt species normally targeted for degradation by the of British Columbia, Vancouver, Canada); pcDNA3.1-CBP (A. Kazantsev

Phosphorylation activates Htt degradation • Thompson et al. 1095 Published December 28, 2009

and D. Housman, Massachusetts Institute of Technology, Cambridge, MA; Research Laboratories, Inc.) and anti–mouse conjugated with FITC (Jackson Kazantsev et al., 1999); pcDNA3.1-mRFP (S. Finkbeiner, University of ImmunoResearch Laboratories, Inc.), and they were used at 1:1,000. California, San Francisco, San Francisco, CA); and pcDNA3-CHIP and Slides were stained with DAPI to detect nuclei. ProLong Gold Antifade pcDNA-CHIPUbox (C. Patterson, University of North Carolina at Chapel (Invitrogen) imaging medium was used. Images were collected at room Hill, Chapel Hill, NC; Jiang et al., 2001). temperature with an inverted microscope (Observer.Z1; Carl Zeiss, Inc.) shRNA for rat Atg7 5-GAAGTACCACTTCTACTAC-3was cloned with a Plan-Apochromat 63X objective, NA 1.40. AxioVision AxioVs40 into pSUPER vector (Appllied Biosystems). shRNA for rat LAMP-2A, v 4.7.1.0 software (Carl Zeiss, Inc.) was used to generate 3D deconvoluted 5-GACTGCAGTGCAGATGAAG-3 in pSUPER was previously constructed images. The camera used was an AxioCam MRm (Carl Zeiss, Inc.). (Massey et al., 2006a). IKK pool 1 contained the following shRNAs in pLKO.1 (Addgene): 5-CCGGGCACTGGGAAAGTATCTGAAACTCGAGT Protein purification and Western analysis TTCAGATACTTTCCCAGTGCTTTTT-3, 5-CCGGCCAGCCAAGAAGAGTG His-tagged proteins were purified under denaturing conditions using AAGAACTCGAGTTCTTCACTCTTCTTGGCTGGTTTTT-3, 5-CCGGCTTAC magnetic Ni-NTA nickel beads (QIAGEN) as described previously (Steffan CTGAATCAGACAAGAACTCGAGTTCTTGTCTGATTCAGGTAAGTTTTT-3, et al., 2004) for Fig. 1 D, Fig. 2, A and B, and Fig. S2, A and B. Western 5-CCGGGCATCTAGTAGAGCGGATGATCTCGAGATCATCCGCTCTACT blots were processed with SuperSignal West Pico and Dura reagents AGATGCTTTTT-3, 5-CCGGCGTTGTTAGTGAAGACTTGAACTCGAGTTC (Thermo Fisher Scientific). Quantitative densiometric analyses (Fig. 6A AAGTCTTCACTAACAACGTTTTT-3. IKK pool 2 shRNAs in pLKO.1 were: and Fig. S4) were performed on digitalized images of immunoblots using 5-CCGGGCATCATAAGGAGTTGGTGTACTCGAGTACACCAACTCCT Scion Image 4.0 software (Scion Corporation) and SEM calculated from TATGATGCTTTTT-3, 5-CCGGCCAGATTATGAAGAAGTTGAACTCGAGT densiometric levels of Western signal from triplicate preparations of protein TCAACTTCTTCATAATCTGGTTTTT-3, 5-CCGGCCAGCCTCTCAATGT extracts. Densitometric levels of phosphorylated or acetylated Htt protein GTTCTACTCGAGTAGAACACATTGAGAGGCTGGTTTTT-3, 5-CCGGGC were normalized to levels of -tubulin loading control. Filter retardation as- AAATGAGGAACAGGGCAATCTCGAGATTGCCCTGTTCCTCATTTGC says were performed as follows: Cell debris pellets were taken after centrif- TTTTT-3, 5-CCGGGCGTGCCATTGATCTATATAACTCGAGTTATATAGATC ugation for 10 min at 16,000 g. Pellets were resuspended in 100 µl of Tris AATGGCACGCTTTTT-3. buffer (20 mM Tris and 15 mM MgCl2 at pH 8.0) and 100 µl of 4% SDS- 100 mM DTT in PBS was added. These samples were boiled for 5 min and Cell culture and transfections then filtered through nitrocellulose membrane via a dot-blot apparatus. The The Hela, St12.7, ST14A, and N548mu and the wild-type STHdhQ7/HdhQ7 membranes were then dried at room temperature for 30 min, stained with Downloaded from and homozygous mutant STHdhQ111/HdhQ111 cell lines were propagated MemCode reversible protein stain (Thermo Fisher Scientific), blocked, and as described previously (Steffan et al., 2004; Apostol et al., 2008). NIH- primary antibodies were added for Western analysis. Native buffer A, 3T3 cells were grown in DMEM and 10% newborn calf serum (Hyclone). used for lysis of cells in Figs. 3, 4, 6, S3, S4, and S5: 10 mM Tris-HCl, All cells were transfected with Lipofectamine 2000 according to the manu- pH 7.5, 10% glycerol, 400 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.5% facturer’s instructions (Invitrogen) except STHdhQ7/HdhQ7, which were nucleo- NP-40, 20 mM N-ethylmaleimide, 1 mM PMSF, phosphatase inhibitors 1 and 2 fected. rhTNF- and rmIl-1 (R&D Systems) were used to pharmacologically (Sigma-Aldrich), complete mini protease inhibitor pellet (Roche), 10 ng/ml activate IKK. aprotenin, 10 ng/ml leupeptin, 5 mM nicotinamide and 5 mM butyrate, pH 7.5. Native buffer B, used for purification in Fig. 2 D: 50 mM NaH2PO4, Primary antibodies pH 8.0, 150 mM NaCl, 0.1% Tween 20, 1 mM DTT, 5 mM ADP, 10 mM jcb.rupress.org Three affinity-purified rabbit polyclonal antibodies were generated Imidazole, 1 mM PMSF, 10 ng/ml aprotenin, 10 ng/ml leupeptin, com- (New England Peptide) against post-translationally modified 1–17aa plete mini protease inhibitor pellet (Roche), and phosphatase inhibitors Htt peptides. Antibodies were generated against the following peptides: 1 and 2 (Sigma-Aldrich). All St14A cells used in Westerns showing phos- anti-S16-P: H2N-CMATLEKLMKAFESLK(pS)F-amide; anti-S13-P: H2N- phorylated Htt were treated with phosphatase inhibitor Calyculin A (Enzo CMATLEKLMKAFE(pS)LKSF-amide; anti-K9-Ac: Ac-CLEKLM(Ac-K)AFE(pS)LK Life Sciences, Inc.) 10–30 min (20 nM) before lysis. (pS)F-amide. Two rabbits were immunized with each peptide; antisera were

pooled and run over an unmodified Htt 1–17aa peptide column (affinity In vitro kinase assay on April 8, 2010 matrix 20401; Thermo Fisher Scientific) to remove antibodies recognizing The in vitro kinase assay was performed as described previously (Liu et al., unmodified Htt species. The flow-through from each was then run over the 2007) using 75 ng recombinant IKK- or IKK- protein (Millipore) and modified peptide column for each respective project. The elutions from 25QP-H4 or 46QP-H4 purified from St14A cells using magnetic Ni-NTA these columns were used as the modification-specific antibodies for this nickel beads (QIAGEN) under native conditions with the following buffers. study, and tested for specificity using a peptide dot blot (Fig. S1). These Lysis buffer: 50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10 mM imidazole, three antibodies were also tested on Westerns of lysates from Hdh7/7 and 0.05% Tween 20, 1 mM PMSF, 10 ng/ml aprotenin, 10 ng/ml leupeptin, Hdh111/111 cells nucleofected with IKK or vector +/ siRNA for Htt (a gift and phosphatase inhibitors 1 and 2 (Sigma-Aldrich). Wash buffer: 50 mM from R. Friedlander, Brigham and Women’s Hospital, Boston, MA); levels NaH2PO4, pH 8.0, 300 mM NaCl, 20 mM imidazole, 0.05% Tween 20, of antigenic species were reduced with Htt siRNA in both cell lines, dem- and phosphatase inhibitors 1 and 2 (Sigma-Aldrich). Recombinant IKK was onstrating modified Htt specificity. JG1 is another rabbit polyclonal anti-Htt diluted in enzyme dilution buffer: 20 mM MOPS/NaOH, pH 7.0, 1 mM 1–17aa antibody generated by our laboratory. EDTA, 5% glycerol, 0.01% Brij35, 0.1% -mercaptoethanol, and 1 mg/ml We also used the following antibodies for this work: CAG53b (a gift BSA. The kinase assay was performed in 5X kinase buffer: 40 mM MOPS/ from E. Wanker, Max Delbrueck Center for Molecular Medicine, Berlin, NaOH, pH 7.0, and 1 mM EDTA. Before the assay, purified Htt bound to Germany); anti-Htt PW0595 (Enzo Life Sciences, Inc.); anti-Myc 9E10 Ni-NTA beads was incubated at 95°C for 3 min, and then placed on ice (Millipore); anti-FLAG (Sigma-Aldrich), anti-HA 16B12 (Covance); anti– for 5 min. The assay was performed at 30°C with light agitation for 10 min -tubulin clone B-5-1-2 (Sigma-Aldrich); anti-Htt Ab1 (M. DiFiglia, Harvard under the following conditions: 2.5 µl recombinant IKK subunit, 5 µl 5X University, Cambridge, MA); anti-Htt EM48 MAB5374 (Millipore); anti-Htt kinase buffer, 2.5 µl 1 mM ATP, 2.5 µl 0.1 M MgAc, and 12.5 µl purified 1H6 (Abnova; Fig. S4); anti-Htt MAB2166 (Millipore); anti-Htt MAB5490 Htt in water. The assay was stopped with addition of Western sample load- (Millipore; Fig. 7); anti-Htt 3B5H10 (S. Finkbeiner, Univeristy of California, ing buffer, boiled 10 min, run on 12% SDS-PAGE, and Western analysis San Francisco); anti–rat LAMP-2A Igp96 (Invitrogen); anti-nestin (Millipore); was performed. anti-Atg7 (Abcam); anti–SUMO-1 (PW9460; Enzo Life Sciences, Inc.), anti-ubiquitin (13–1600, Invitrogen; and sc8017, Santa Cruz Biotechnol- Mass spectrometry analysis ogy, Inc.); anti-IB clone IB-245 (Invitrogen); anti–phospho-IB Ser 32 ST14A cells were transiently transfected with Htt25QP-HBH + IKK- 14D4 (Cell Signaling Technology); and anti-phosphorylated Htt threonine 3 (Fig. 1 C) or Htt25QP-HBH + CBP + IKK- (Fig. 2 C); 48 h after transfection, [anti-T3-P; Aiken et al. [2009]). Secondary antibodies used for Western the cells reached confluency. Cells were then treated with fresh media contain- analysis were goat anti–mouse HRP (The Jackson Laboratory) and goat ing 10 nM Calyculin A (EMD) for 30 min, or 20 nM Calyculin A (Enzo Life anti–rabbit HRP (Thermo Fisher Scientific). Sciences, Inc.) for 10 min. Cells were washed with cold 1x PBS, then harvested and lysed in 1 ml lysis buffer each (50 mM Tris-HCl, pH 8.0, 8 M Immunofluorescence analysis urea, 500 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, 0.5% Triton Cells were transfected (Lipofectamine 2000) or nucleofected (Lonza) with X-100, and complete mini protease inhibitor [Roche]). The DNA was sheared IKK- (1/2 IKK- and 1/2 pcDNA) and 24 h later fixed at room tempera- and the cells further lysed by passing through a 20-guage needle 20 times, ture with 4% PFA, permeabilized, and blocked with 5% BSA and 4% don- and cellular debris was removed by centrifugation. Clarified lysates were key serum. Primary antibodies were diluted at 1:1,000. Secondary then incubated with 25 µl Ni-Sepharose 6 Fast Flow (GE Healthcare) or antibodies were anti–rabbit conjugated with Cy3 (Jackson Immuno Ni-NTA magnetic nickel (QIAGEN) bead slurry for 3 h or overnight at

1096 JCB • VOLUME 187 • NUMBER 7 • 2009 Published December 28, 2009

room temperature. The beads were then washed twice in the lysis buffer, phosphate with plasmids at 5 d in vitro, as described previously (Xia et al., and four times with wash buffer (50 mM Tris-HCl, pH 6.3, 8 M urea, 500 mM 1996; Finkbeiner et al., 1997). Specifically, neurons were cotransfected NaCl, 50 mM NaH2PO4, 20 mM imidazole, and 0.5% Triton X-100). The with pcDNA3.1-mRFP (Arrasate et al., 2004) and 97QP-GFP or 97QP-PP- beads of the same condition were pooled and the urea buffer was GFP in a 1:1 molar ratio using a total of 3 µg of DNA in each well of a replaced with 50 mM NH4CO3 before digestion. 24-well plate. After transfection, neurons were maintained in serum-free Chymotryptic digestion (2% by weight) of 25QP-HBH was per- medium with Forskolin (10 µM; Sigma-Aldrich) and IBMX (100 µM; Sigma- formed on the Ni beads used to purify the protein to maximize peptide re- Aldrich). Neurons were fixed with 4% paraformaldehyde in PBS (15 min) covery. The digestion occurred overnight at 37°C. Resulting peptides were 20 h after transfection, permeabilized with 0.1% Triton X-100 in PBS extracted from the beads with 25% acetonitrile, 0.1% formic acid three (30 min), and incubated with 1 M glycine in PBS (20 min). Finally, neurons times. The extracts were pooled, concentrated using a SpeedVac, and were washed twice for 5 min each with 2.5 µg/ml of Hoechst 33258 in acidified by 0.1% formic acid before mass spectrometric analysis. Resul- PBS dye to stain the nuclei. tant peptides were then separated and analyzed by reverse-phase liquid Robotic microscope imaging system. The microscope imaging system chromatography coupled to tandem mass spectrometry (LC MS/MS) on a quadropole-orthogonoal-time-of-flight tandem (Guerrero et al., 2008) has been described previously (Arrasate et al., 2004; Arrasate and (QSTAR XL; Applied Biosystems/PE Sciex) or an ultra-high performance Finkbeiner, 2005). Basically, the system is based on an inverted microscope Thermo Electron linear trap quadropole (LTQ)-Orbitrap hybrid (Fang et al., (TE300 Quantum; Nikon). Xenon lamp (175 W) illumination was supplied 2008) (Thermo Fisher Scientific) mass spectrometer. by a liquid light guide. The Nikon working distance objective 20X (NA Extraction of the monoisotopc masses (m/z) of parent ions, their 0.45) was used. Fluorescence excitation and emission filters were moved charge states, and their corresponding fragment ions was performed auto- into or out of the optical path by two ten position filter wheels (Sutter Instru- matically using Analyst software (Applied Biosystems) for QSTAR data or ment Co.) under computer control. Images were collected with a 12/14 bit using extract MSn (Matrix Science) for LTQ-Orbitrap data. These data were digital cooled CCD camera (Orca II; Hamamatsu Photonics) and digitized then submitted for automated database searching for protein identification with MetaMorph software (Universal Imaging). Before image acquisition, using the Protein Prospector (University of California, San Francisco) search care was taken to adjust the gain and offset of the camera and the analogue- engine. Post-translational modifications were confirmed by manual inspec- to-digital converter to ensure that the intensities of all the pixels of each im- tion of the MS/MS spectra. age were within the detection range of the instrument, and that the settings Downloaded from were the same across the samples that were examined. The whole system GST pull-down assay is mounted on a vibration isolation table. For GST pull-down assays, 35S-labeled His-tagged Httex1p-H4 proteins Image analysis. Measurements of htt expression were extracted from were synthesized in a TNT coupled reticulocyte lysate system (Promega), images generated with the microscope imaging system described above. purified, and eluted under native conditions using magnetic Ni-NTA nickel The validity of estimating htt expression levels in live cells from images of beads (QIAGEN) in the buffers suggested by the manufacturer. The eluted the fluorescence of the GFP fusion tag to which it is fused has been demon- proteins were dialyzed using SlideALyzer 3.5K Dialysis Casettes (Thermo strated previously by directly comparing this approach to other methods Fisher Scientific) against 20 mM MOPS, pH 7.3/0.25 M sucrose buffer. that are highly quantitative but unsuitable for live-cell imaging (Arrasate

GST-Hsc70 was purified from Escherichia coli using glutathione-agarose et al., 2004). The expression of 97QP-GFP or 97QP-DD-GFP was estimated jcb.rupress.org beads, and incubated with purified radioactive Httex1p-H4 proteins in by measuring GFP fluorescence intensity within a region of interest from the 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.1% Tween 20, and 1 mM DTT image that corresponded to a portion of the cytoplasm or the nucleus in supplemented with ATP or ADP corresponding to the incubation conditions, neurons that had not developed inclusion bodies. Hoechst staining was washed five times, and subjected to SDS-PAGE and autoradiography. used to localize the nucleus. Pixel intensities within a similar sized region from an adjacent acellular portion of the image were collected as a mea- Toxicity assays surement of background signal. Pixel values from these background For XTT cell viability assays, STHdhQ7 and STHdhQ111 and cell lines were measurements were subtracted from the corresponding pixel intensity on April 8, 2010 plated in 24-well plates (0.75 × 105 cells per well) in complete media as measurements made from the nucleus and the cytoplasm. These calcula- described previously (Apostol et al., 2008). The next day, cells were shifted tions produced background-corrected measurements of htt 97QP-GFP or to nonpermissive conditions (i.e., 39°C and low serum media) for 48 h 97P-DD-GFP from the cytoplasm and nucleus. for STHdhQ111 and STHdhQ7 lines followed by incubation for 4 h with XTT and phenazine methosulfate (0.2 mg/ml and 0.2 µg/ml, respectively; Statistical analysis. The ratio of GFP intensity in the nucleus over Sigma-Aldrich) for 4 h, and plates were read at 450 nM. the cytoplasm was calculated for individual neurons by dividing the background-corrected pixel intensities from the region of interest within the Rat cortico-striatal brain slice neurodegeneration assay nucleus by the background-corrected pixel intensities from the region of in- All animal experiments were performed in accordance with the Institu- terest within the cytoplasm of the same neuron. Differences in the mean of tional Animal Care and Use Committee and Duke University Medical Cen- these ratio measurements were compared by t test with commercially avail- ter Animal Guidelines. Brain slice preparation and biolistic transfection able software (Prism 3.0; GraphPad Software, Inc.). were performed as described previously (Lo et al., 1994; Southwell et al., 2008) with some modifications. In brief, brain tissue was dissected from Htt immunoprecipitation from mouse brain postnatal day 10 CD Sprague Dawley rats (Charles River Laboratory) and Age-matched R6/2 and wt control brains were collected and snap frozen. placed in ice-cold Neurobasal A culture medium containing 10% heat- Whole brain tissue was dounced 20 times on ice in T-Per lysis buffer inactivated pig serum, 5% heat-inactivated rat serum (Lampire), 10 mM KCl, (Thermo Fisher Scientific) containing an EDTA-free mini protease inhibitor 10 mM Hepes (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), pellet (Roche), a Phos-Stop pellet (Roche), 5 mM sodium fluoride, and 100 U/ml penicillin/streptomycin, and 1 mM l-glutamine. All media 1 mM sodium orthovanadate. Lysates were microfuged at 16,000 g at 4°C reagents were obtained from Invitrogen unless otherwise noted. Brain for 15 min, and the supernatant saved. Htt was immunoprecipitated from tissue was cut into 250-µm-thick coronal slices using a Vibratome and in- the 500 µg of supernatant using Protein G-Plus Agarose (Santa Cruz cubated for 30 min at 32°C/5% CO before biolistic transfection. Gold 2 Biotechnology, Inc.) with 1 l PW0595 antibody (Enzo Life Sciences, Inc.) particles (1.6 µm; Bio-Rad Laboratories) were coated with the indicated or zero antibody control, and run on 8% SDS-PAGE and blotted to nitro plasmids, loaded into Tefzel tubing (McMaster-Carr), and transfected with cellulose for standard Western analysis. the Helios Gene Gun (Bio-Rad Laboratories) at 95 psi. Brain slices were incubated at 32°C/5%CO2 until analysis at 5 d post-transfection. Medium spiny neurons were visualized by fluorescence microscopy of YFP and Online supplemental material scored by neuron morphology. Medium spiny neurons were considered Fig. S1 shows the specificity of the modification-specific antibodies. Fig. S2 healthy if they were of uniform size and shape and contained visible den- shows the role of IKK in the regulation of mutant Httex1p ubiquitination and drites. Data were analyzed with Prism software (GraphPad Software, Inc.) SUMOylation. Fig. S3 shows cellular localization of phosphorylated Htt. and significance was determined by unpaired Student’s t test. Fig. S4 shows loss of an epitope for popular anti-Htt antibody MAB2166 with post-translational modification of Htt. Fig. S5 shows the role of Hsc70 Calculation of the nucleus/cytoplasm ratio for 97QP-GFP versus 97QP-DD-GFP and the ubiquitin ligase CHIP in the regulation of levels of phosphorylated Cell culture and transfection. Primary cultures of rat cortical neurons were and acetylated Htt. Online supplemental material is available at http:// prepared from embryonic rats (E 19–20) and transfected using calcium www.jcb.org/cgi/content/full/jcb.200909067/DC1.

Phosphorylation activates Htt degradation • Thompson et al. 1097 Published December 28, 2009

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Autophagic Punctum Protein turnover differences between neurons and other cells

Andrey S. Tsvetkov,1-3 Siddhartha Mitra1,4 and Steven Finkbeiner1-4,*

1Gladstone Institute of Neurological Disease; San Francisco, CA USA; 2Taube-Koret Center for Huntington’s Disease Research; San Francisco, CA USA; 3Neuroscience Program; Departments of Neurology and Physiology; 4Biomedical Sciences Program and Medical Scientist Training Program; University of California; San Francisco, CA USA Key words: huntington disease, autophagy, neurodegeneration, rapamycin, everolimus, LC3

In a recent study, we investigated the relationship between fusion of autophagosomes with lysosomes. LC3-II levels increased formation of an inclusion body (IB) and activity of the ubiq- in primary rat neurons and HeLa cells, indicating autophagosome uitin-proteasome system (UPS) in a primary neuron model of accumulation (Fig. 1A). These results suggest that autophagy is Huntington disease. We applied single-cell longitudinal acquisi- constitutively active in neurons and that fusion of autophagosomes tion and analysis to simultaneously monitor mutant huntingtin, to lysosomes is similar in all cells. which causes Huntington disease, IB formation, UPS function We then determined if autophagy is induced similarly in and neuronal toxicity. We found that proteasome inhibition is neurons and non-neuronal cells. Others showed that GFP-LC3 toxic to striatal neurons in a dose-dependent fashion. The UPS transgenic mice exhibited no autophagy in the brain after starva- is more impaired in neurons that go on to form IBs than in those tion. But was this because the brain is protected from starvation? that do not; however, after IBs form, UPS function improves. To ensure that neurons were deprived of nutrients, we eliminated Our findings suggest that IBs are a protective cellular response to contributions from glial cells (our cultures are 95% neurons) and mutant protein that also improves intracellular protein degrada- homeostatic mechanisms outside the central nervous system. The tion The study also revealed some surprising differences in the pathways that mediate starvation-induced autophagy in neurons ways that neurons regulate protein turnover compared with non- evidently differ from those in non-neuronal cells; starvation in neuronal cells, which we discuss further in this article. Hank’s balanced salt solution increased LC3-II levels in HeLa cells, but not in neurons (Fig. 1B). To determine if concurrent changes in autophagy affected our We also assessed the effects of rapamycin. In neurons, rapamycin measurement of UPS activity, we examined the activity of the pretreatment blocked BDNF-induced phosphorylation of p70S6K autophagic pathway after treatment with the UPS inhibitor epoxo- (Fig. 1C), indicating mTOR inhibition. Next we determined micin. We found that levels of LC3-II, which indicate the extent of if rapamycin or everolimus induced autophagy. As expected, autophagy, were unchanged in primary striatal neurons, suggesting rapamycin potently increased LC3-II levels in HeLa cells. However, no upregulation of autophagy. However, consistent with previous neither chemical increased LC3-II levels in primary neurons (Fig. reports, proteasome inhibition in HEK293 cells led to LC3-II 1D). While surprising, this result is consistent with our obser- accumulation. We concluded that autophagy regulation in neurons vations in nutrient-deprived neurons. Even in non-neuronal might be different than in other cell types. cells, rapamycin effects are complex. Nanomolar concentrations Although implicated in neurodegeneration, autophagy has been completely inhibit mTOR activity, but only vast excesses induce characterized mostly in yeast and mammalian non-neuronal cells, autophagy in non-neuronal cells, suggesting rapamycin may act and the few studies in neurons reach different conclusions. We on additional cellular targets. In fly and mouse HD models, sought to determine if common autophagy enhancers would stim- it attenuates mutant htt toxicity and promotes htt clearance. ulate autophagy in cultured primary striatal and cortical neurons. However, more experiments are needed to show that the benefits First, we treated primary neurons with bafilomycin A1 to block the of rapamycin are due only to autophagy. In autophagy-deficient fibroblasts, for example, rapamycin apparently inhibits huntingtin aggregation by reducing protein synthesis. This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly. the issue is complete and page numbers have Once to printing. has been published online, prior This manuscript *Correspondence to: Steven Finkbeiner; Gladstone Institute of Neurological Lithium chloride induces autophagy in non-neuronal cells by Disease; 1650 Owens Street; San Francisco, California 945158 USA; Tel.: inhibiting inositol monophosphatase—a mechanism that is inde- 415.734.2000; Fax: 415.355.0824; Email: [email protected] pendent of mTOR. We found that lithium chloride effectively Submitted: 03/18/09; Revised: 04/03/09; Accepted: 04/09/09 induced autophagy in control cells, but LC3-II did not accumulate Previously published online as an Autophagy E-publication: in primary neurons (Fig. 1E), suggesting that neurons may differ http://www.landesbioscience.com/journals/autophagy/article/8705 somewhat from other cells in both mTOR-dependent and -independent mechanisms of autophagy. Punctum to: Mitra S, Tsvetkov A S, Finkbeiner S. Single Neuron Ubiquitin- Proteasome Dynamics Accompanying Inclusion Body Formation in Huntington Western blots show steady-state LC3-II levels but not the flux Disease. J Biol Chem 2009; 284:4398-403. Epub 2008 Dec 10. through the autophagic pathway. If autophagy basal rates are high

1 Autophagy 2009; Vol. 5 Issue 5 Protein turnover differences between neurons and other cells

Figure 1. Common inducers of autophagy in non-neuronal cells fail to stimulate autophagy in primary neurons. Relative intensities of LC3-I and LC3-II

bands reflect levels of autophagy. (A) Bafilomycin A1 (bafA; 4 h, 1 nM) induced LC3-II accumulation in striatal and cortical neurons and in HeLa cells. (cont), control untreated cells. (B) Striatal and cortical neurons were incubated in Hanks’ solution. Starvation (2 h in Hanks’ solution) induced autophagy in HeLa cells but not neurons. Longer incubations gave similar results. (C) Pretreatment with rapamycin (2 µM) blocked BDNF-induced phosphorylation of p70S6K in striatal neurons. (D) Striatal and cortical neurons incubated in medium with the mTOR inhibitors 2 µM rapamycin (rap) or everolimus (everol) for 48 h. Shorter or longer incubations and higher and lower concentrations (2 nM to 20 µM) gave similar results (not shown). Rapamycin (2 µM, 24 h) induced autophagy in HeLa cells. (E) Lithium chloride (LiCl, 10 mM) induced autophagy in COS-7 cells but not neurons. (F) Autophagy induc-

ers did not increase the flux through the autophagic pathway. In striatal neurons, inhibition of lysosomal degradation with bafilomycin A1 (overnight) led to LC3-II accumulation (compare lanes 1 and 2). Starvation of these treated cells did not increase LC3-II levels (compare lanes 2–4). Incubation of these treated cells with 2 µM rapamycin (compare 2, 5, 6), 2 µM everolimus (compare 2, 7, 8), or 10 mM LiCl (compare 2, 9, 10) did not increase LC3-II levels. (G) Striatal and cortical neurons were incubated in medium with niguldipine (nig, 4 µM), trifluoperazine (3F, 8 µM), or loperamide (lop, 5 µM) (overnight). Note LC3-II accumulation. (H) Starvation (starv, Hanks’ solution, 8 h), rapamycin (rap, 2 µM, 24 h), and lithium chloride (LiCl, 10 mM) induced autophagy in astrocytes.

in neurons, steady-state levels of autophagosomes and LC3-II We tested other small molecules that induce autophagy in non- might not change in neurons under autophagy-inducing condi- neuronal cells. Niguldipine, trifluoroperazine and loperamide tions. To determine if flux through autophagy changes with drugs, robustly induced autophagy in primary neurons (Fig. 1G). we examined LC3-II levels with these drugs and bafilomycin Therefore, we conclude that the upregulation of autophagy, in A1, which blocks fusion of autophagosomes and lysosomes. We principle, can be induced and detected in our system. incubated primary neurons with bafilomycin A1, under starvation Is there another way to reconcile the different results? They conditions, or with rapamycin, everolimus, or lithium chloride might be affected by contaminating non-neuronal cells in the (Fig. 1F). Simultaneous application of bafilomycin A1 and drugs mixed primary cultures. To test this, we prepared pure primary that induce autophagy in non-neuronal cells did not increase cultures of astrocytes and starved them or treated them with LC3-II accumulation in neurons over conditions in which only rapamycin or lithium chloride. Like the cell lines, astrocytes fusion is inhibited. Thus, starvation, rapamycin, everolimus and responded by inducing autophagy (Fig. 1H). lithium chloride do not significantly increase autophagic flux in These results raise the intriguing possibility that autophagy in primary neurons. neurons is regulated by mechanisms that differ, at least in part, Since at least one study reports LC3-II accumulation in from those in non-neuronal cells. Our results underscore the rapamycin-treated neurons, we thought perhaps our western potential importance of using primary neurons to study the role blots might not detect LC3-II in neurons undergoing autophagy. of autophagy in neurodegeneration and the consideration of these

www.landesbioscience.com Autophagy 2 Protein turnover differences between neurons and other cells potential differences in any efforts to target this pathway thera- peutically. Acknowledgements This work was supported by R01 2NS039746 and 2R01 NS045191 from the National Institute of Neurological Disease and Stroke, P01 2AG022074 from the National Institute on Aging, the Taube-Koret Center for Huntington’s Disease Research, and the J. David Gladstone Institutes (S.F.); a Milton Wexler Award and a fellowship from the Hereditary Disease Foundation (A.T.); NIH-NIGHMS UCSF Medical Scientist Training Program and a fellowship from the UC-wide adaptive biotechnology (GREAT) program (S.M.); and RR018928 from the National Center for Research Resources. We thank Dr. Walter Schuler (Novartis) for everolimus, helpful discussions, and bringing to our attention the difference in the doses of rapamycin that inhibit mTOR and those that are typically used to induce autophagy. We also thank Jayanta Debnath for LC3 antibodies and helpful discussions, Dr. Ana Maria Cuervo for helpful advice, and members of the Finkbeiner laboratory for helpful discussions. Kelley Nelson provided administrative assistance, and Gary C. Howard edited the manuscript.

3 Autophagy 2009; Vol. 5 Issue 5 9104 • The Journal of Neuroscience, July 15, 2009 • 29(28):9104–9114

Neurobiology of Disease Loss of Hsp70 Exacerbates Pathogenesis But Not Levels of Fibrillar Aggregates in a Mouse Model of Huntington’s Disease

Jennifer L. Wacker,1 Shao-Yi Huang,2 Andrew D. Steele,6 Rebecca Aron,2 Gregor P. Lotz,2 QuangVu Nguyen,1 Flaviano Giorgini,1 Erik D. Roberson,2 Susan Lindquist,6 Eliezer Masliah,7 and Paul J. Muchowski1,2,3,4,5 1Department of Pharmacology, University of Washington, Seattle, Washington 98195, 2Gladstone Institute of Neurological Disease, 3The Taube-Koret Center for Huntington’s Disease Research, and Departments of 4Biochemistry and Biophysics and 5Neurology, University of California, San Francisco, San Francisco, California 94158, 6Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, and 7Department of Neurosciences, University of California, San Diego, La Jolla, California 92093

Endogenous protein quality control machinery has long been suspected of influencing the onset and progression of neurodegenerative diseases characterized by accumulation of misfolded proteins. Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin (htt), which leads to its aggregation and accumulation in inclusion bodies. Here, we demonstrate in a mouse model of HD that deletion of the molecular chaperones Hsp70.1 and Hsp70.3 significantly exacerbated numerous physical, behavioral and neuropathological outcome measures, including survival, body weight, tremor, limb clasping and open field activities. Deletion of Hsp70.1 and Hsp70.3 significantly increased the size of inclusion bodies formed by mutant htt exon 1, but surprisingly did not affect the levels of fibrillar aggregates. Moreover, the lack of Hsp70s significantly decreased levels of the calcium regulated protein c-Fos, a marker for neuronal activity. In contrast, deletion of Hsp70s did not accelerate disease in a mouse model of infectious prion-mediated neurodegeneration, ruling out the possibility that the Hsp70.1/70.3 mice are nonspecifically sensitized to all protein misfolding disorders. Thus, endogenous Hsp70s are a critical component of the cellular defense against the toxic effects of misfolded htt protein in neurons, but buffer toxicity by mechanisms independent of the deposition of fibrillar aggregates.

Introduction shock proteins (Hsps) that function as molecular chaperones to Many neurodegenerative diseases, including Alzheimer’s disease help to restore cellular homeostasis (Lindquist, 1986). Postmi- (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis totic neurons, unable to dilute misfolded and/or aggregated (ALS), prion disease and HD, are characterized by conformational proteins through cell division, are particularly vulnerable to the changes in disease-causing proteins that result in misfolding and aggre- deleterious effects of misfolded proteins (Muchowski and gation and have collectively been termed protein-conformational Wacker, 2005). Accordingly, the endogenous protein quality control disorders. In contrast to AD, PD, and ALS, in which the vast system is speculated to be critical in controlling the onset and severity majority of cases are idiopathic, HD is one of a number of inher- of protein-conformational diseases that affect the brain. ited neurodegenerative disorders, collectively termed polyQ The 70 kDa Hsps (Hsp70s) are abundantly expressed molec- diseases, which are caused by an expansion of CAG repeats, cod- ular chaperones that participate in a variety of fundamental cel- ing for glutamine, in their respective disease proteins. The depo- lular processes. Hsp70s promote the renaturation of misfolded sition of aggregation-prone proteins that contain expanded and/or aggregated proteins through ATP-dependent cycles of polyQ repeats in inclusion bodies is a neuropathological hall- binding and release and are likely to provide a first line of defense mark of the majority of these disorders. against aggregation-prone disease proteins in vivo (Hartl and The accumulation of misfolded proteins in cells triggers a Hayer-Hartl, 2002). Indeed, genetic screens and directed studies protective stress response that includes the upregulation of heat have shown that Hsp70 and its partners potently modulate the aggregation and/or suppresses the toxicity of mutant polyQ pro- Received May 13, 2009; accepted June 11, 2009. teins in cell-, yeast-, worm- and fly-based models of polyQ aggre- Support for this study was provided by National Institute of Neurological Disease Grants NS47237 and NS054753 (P.J.M.) and National Institute of Aging Grant AG022074 (P.J.M., E.M.). We thank S. Ordway and G. Howard for gation and disease (Warrick et al., 1998; Chai et al., 1999; Warrick editorial assistance and Artur Topolszki for assistance with mouse colony management for PrP studies. S.L. is an et al., 1999; Jana et al., 2000; Krobitsch and Lindquist, 2000; Mu- investigator of the Howard Hughes Medical Institute. chowski et al., 2000; Kobayashi and Sobue, 2001; Zhou et al., 2001; Correspondence should be addressed to Paul J. Muchowski, Gladstone Institute of Neurological Disease, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158. E-mail: Gunawardena et al., 2003; Nollen et al., 2004). Hsp70 overexpression [email protected]. also conferred a dose-dependent improvement in behavioral pheno- F. Giorgini’s current address: Department of Genetics, University of Leicester, Leicester LE1 7RH, UK. types of transgenic mouse models of Spinocerebellar ataxia-1 E. D. Roberson’s current address: Department of Neurology, University of Alabama at Birmingham, Birmingham, (SCA1) and Spinal and bulbar muscular atrophy (SBMA) (Cum- AL 35294. DOI:10.1523/JNEUROSCI.2250-09.2009 mings et al., 2001; Adachi et al., 2003). Conversely, overexpression of Copyright © 2009 Society for Neuroscience 0270-6474/09/299104-11$15.00/0 Hsp70 in the R6/2 mouse model of HD had only a marginal effect on Wacker et al. • Hsp70 and Huntington’s Disease J. Neurosci., July 15, 2009 • 29(28):9104–9114 • 9105

weight loss and no effect on other behavioral and neuropathological assays of this study had a CAG repeat length of ϳ185. Genetic deletion of features (Hansson et al., 2003; Hay et al., 2004). Hsp70.1/3 did not have a dramatic effect on CAG repeat length, which tg/Ϫ Ϫ/Ϫ The goal of this study was to determine whether endogenous was ϳ181 in the R6/2 ;Hsp70 mice used in the behavioral assays. tg/Ϫ ϩ/ϩ Hsp70s can modulate the onset, progression and/or severity of R6/2 ;Hsp70 mice used in the neuropathological assays of this study had a CAG repeat length of 115. pathogenesis in a mouse model of HD. We used the well charac- Ϫ Ϫ Prion studies. Hsp70.1/3 knock-out mice (Hsp70 / ) used for the terized R6/2 transgenic model of HD, in which expression of htt ϳ prion studies were the same mice used for the R6/2 study, other than exon 1 with 150 CAG repeats causes a progressive HD-like being maintained on a 129Sv/Ev pure background (Hampton et al., behavioral phenotype, including a robust decline in motor per- 2003). The Hsp70 overexpressing transgenic mouse was maintained on a formance, alterations in activity level, weight loss and premature hybrid C57BL/6-SJL background and expresses the rat inducible Hsp70 death (Mangiarini et al., 1996). R6/2 mice also accumulate mu- gene of a rat under a ␤-actin promoter (Marber et al., 1995). Hsp70 Ϫ/Ϫ tant htt exon 1 in intranuclear and cytoplasmic inclusion bodies (n ϭ 19) and Hsp70 ϩ/ϩ (n ϭ 12) mice were injected intracranially with (Davies et al., 1997), a feature of HD brains (DiFiglia et al., 1997). 30 ␮l of the Rocky Mountain Laboratory (“RML”) strain of murine ϳ ␮ ϩ/Ϫ To determine whether inducible Hsp70s play a protective role in prions, corresponding to a dose of 3.5 log LD50/30 l. Hsp70 Tg Ϫ/Ϫ the R6/2 model of HD, we crossed transgenic R6/2 mice with (n ϭ 14) and Hsp70 Tg (n ϭ 10) mice were injected intracranially ␮ Ϫ/Ϫ ϭ ϩ/ϩ ϭ knock-out mice that lack both Hsp70.1 and Hsp70.3. with 5.5 log LD50/30 l. Hsp70 (n 19) and Hsp70 (n 11) mice were also injected with “22L” strain of murine prions at a dose of ϳ ␮ ϩ/Ϫ ϭ Ϫ/Ϫ ϭ 3.5 log LD50/30 l. Hsp70 Tg (n 12) and Hsp70 Tg (n 11) ␮ Materials and Methods mice were injected intraperitoneally with 100 l of 4.5 log LC50 RML. Animals and breeding strategy. The University of Washington Animal Mice were monitored daily for typical prion symptoms, such as imbal- Care and Use Committee, the University of California San Francisco ance, priapism (males), and weight loss. IACUC Committee, or the Massachusetts Institute of Technology (MIT) Survival. For the R6/2 study (performed at the University of Washing- Committee on Animal Care approved all experiments and procedures ton) mice were observed twice daily, in the early morning and late after- involving mice. Mice were maintained and bred in accordance with Na- noon. Survival was evaluated as the time to which the mice either died tional Institutes of Health guidelines. Hemizygous transgenic R6/2 tg/Ϫ spontaneously, or exceeded a defined endpoint criterion. Motor perfor- male founder mice were kindly provided by Dr. James Olson (Fred mance, neurobehavioral and physical symptoms, weight, and ability to Hutchinson Cancer Research Center, Seattle, WA). The R6/2 tg/Ϫ male feed were closely monitored. Mice were killed when they had lost Ͼ20% mice were backcrossed five times to C57BL/6 female mice to generate a of their maximal weight, and were no longer actively eating or drinking. colony of R6/2 tg/Ϫ mice. The Hsp70.1/3 knock-out mice were originally For the prion studies (performed at MIT), mice were closely monitored generated by simultaneously targeting the Hsp70.1 and Hsp70.3 genes so and killed when they were unable to reach the food bin or water spout or that homologous recombination with the targeting construct resulted in regain posture after being placed on their side. a 12 kb deletion of both Hsp70.1 and Hsp70.3 coding regions as well as Rotarod experiments. A Rotamex rotarod (Rotamex 4/8, Columbus insertion of a neomycin-resistance gene (Hampton et al., 2003). A breed- Instruments International) was programmed to accelerate from 4 to 40 ing pair of double knock-out Hsp70.1/3 Ϫ/Ϫ mice (herein referred to as rpm over a period of 10 min and measure the latency to fall. Testing was Hsp70 Ϫ/Ϫ mice) were obtained with the permission of Dr. David Dix performed every 2 weeks, starting at week 4. During the first week of from Dr. Philip Mirke (University of Washington, Seattle, WA) and used testing (week 4) the mice performed three trials per day on four consec- to establish a colony of Hsp70 Ϫ/Ϫ mice that was maintained on a utive days. Data from day 1 of week 4 was excluded from the analysis as C57BL/6 background for R6/2 studies and on 129Sv/Ev for prion studies. the mice were learning the task. During the subsequent weeks of testing Hsp70 Ϫ/Ϫ females were mated with R6/2 tg/Ϫ males. Resulting R6/2 tg/Ϫ; (6, 8, 10, 12, and 14 weeks), the mice were tested on three consecutive Hsp70 Ϫ/ϩ males were mated with Hsp70 Ϫ/Ϫ females to yield four geno- days for three trials per day and all of the trial data were included in the types: R6/2 Ϫ/Ϫ;Hsp70 Ϫ/ϩ, R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ, R6/2 Ϫ/Ϫ;Hsp70 Ϫ/Ϫ, analysis. For each week, the trials were pooled and used to calculate the and R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ. Female mice of these four genotypes were an- average latency to fall for each mouse. alyzed alongside female R6/2 tg/Ϫ;Hsp70 ϩ/ϩand R6/2 Ϫ/Ϫ;Hsp70 ϩ/ϩ Weight loss. Starting at 28 d, the mice were weighed twice weekly, at the mice for a total of six genotypes. The number of mice in each cohort that same time of day, to the nearest 0.1 g. was analyzed in the behavioral paradigms was as follows: R6/2 tg/Ϫ; Neurobehavioral and physical phenotype assessment. Beginning week 6, Hsp70 ϩ/ϩ (n ϭ 21), R6/2 tg/Ϫ;Hsp70 ϩ/ϩ (n ϭ 18), R6/2 Ϫ/Ϫ;Hsp70 Ϫ/ϩ mice were evaluated once a week, as described (Ditzler et al., 2003) to (n ϭ 27), R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ (n ϭ 22), R6/2 Ϫ/Ϫ;Hsp70 Ϫ/Ϫ(n ϭ 18), extensively characterize their neurobehavioral and physical phenotype. and R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ (n ϭ 18). The experimenter was blind to the Each mouse was removed from its home cage and placed into a new, genotype during all testing paradigms. At 4 weeks of age the mice were sterile cage where it was observed for 2 min. Briefly, to assess the neu- weaned and housed randomly in groups of five. Mice were allowed access robehavioral phenotype mice were scored for grooming, spontaneous to water and food ad libitum and maintained on a 12 h light-dark cycle. At activity, and locomotor activity. During the same 2 min period the phys- 10 weeks of age, mice were given powdered chow mixed with water ical phenotype of each mouse was scored for palpebral closure, coat (mash) to provide adequate nutrition and hydration. appearance, body position and tail position. The scoring protocol for the Genotyping. Mouse tail DNA was analyzed by PCR to determine the neurobehavioral and physical assessment is detailed in supplemental Ta- genotype. The R6/2 transgene was identified as described using the ble 1, available at www.jneurosci.org as supplemental material. At the following primer sequences to identify the R6/2 transgene: forward- end of the 2 min period the mouse was removed and suspended by the tail CGCAGGCTAGGGCTGTCAATCATGCT and reverse-TCATCAG- ϳ10 cm above the cage for 30 s to analyze pathogenic clasping behavior. CTTTTCCAGGGTCGCCAT (Hockly et al., 2003). Hsp70 Ϫ/Ϫ and Paw clasping behavior was scored from 0 to 2 points as described in Hsp70 Ϫ/ϩ mice were genotyped using a protocol established by the mu- supplemental Table 1, available at www.jneurosci.org as supplemental tant mouse regional resource center at UC Davis (http://www. material. mmrrc.org/strains/372/0372.html). The primer sequences used to iden- Statistics. All data are expressed at the mean Ϯ SEM. For each outcome tify the targeted knock-out Hsp70.1/3-neo were: forward-GAACGGAG- measure a two-way ANOVA was performed to determine whether there GATAAAGTTAGG and reverse-AGTACACAGTGCCAAGACG. The primer was a significant interaction between the R6/2 transgene and the sequences used to identify the wild-type (WT) Hsp70.3 allele were: forward- Hsp70.1/3 genes. Specifically, the Mixed Models ANOVA in SPSS 13 was GTACACTTTAAACTCCCTCC and reverse-CTGCTTCTCTTGTCTTCG. used with week as a repeated variable, mouse as a subject variable and the We used GeneMapper techniques to determine the CAG repeat num- R6/2 transgene or Hsp70 deletion as factors. An unstructured repeated ber by measuring the size of fluorescently labeled PCR products that covariance was used to analyze weight, rotarod, clasping, tremor, body cover the CAG repeat region in the exon 1 of HD gene. GeneMapper postion, tail position, grooming, locomotor activity, and spontaneous results showed that the R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice used in the behavioral activity outcome measures. A compound asymmetry repeated covari- 9106 • J. Neurosci., July 15, 2009 • 29(28):9104–9114 Wacker et al. • Hsp70 and Huntington’s Disease

ance was used to evaluate eye closure and fur phenotypes. In cases in which differences between the various genotypes were examined at a single time-point, a one-way ANOVA in con- junction with the Bonferroni post hoc test was performed in GraphPad Prism. The Kaplan– Meier method was used to evaluate survival, followed by the log rank test to identify significant changes in GraphPad Prism. Biochemical experiments. At 14 weeks of age

mice were killed with CO2. The brains were removed and homogenized with 5 ␮l/mg tissue RIPA buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM ␤-mercaptoethanol, 1 mM Figure 1. Deletion of Hsp70.1 and Hsp70.3 decreases survival in the R6/2 mouse, but not in mice infected with prions. A, PMSF, and a protease inhibitor cocktail (Roche Kaplan–Meier survival curve for the indicated genotypes [R6/2 Ϫ/Ϫ;Hsp70 ϩ/ϩ (n ϭ 21), R6/2 tg/Ϫ;Hsp70 ϩ/ϩ (n ϭ 18), Diagnostics) and centrifuged at 10,000 ϫ g for R6/2 Ϫ/Ϫ;Hsp70 Ϫ/ϩ (n ϭ 27), R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ (n ϭ 22), R6/2 Ϫ/Ϫ;Hsp70 Ϫ/Ϫ(n ϭ 18), and R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ 90 min at 4°C. A Bradford assay was used to (n ϭ 18)] demonstrates that the absence of Hsp70.1/3 significantly decreased survival of R6/2 mice (log rank: p ϭ 0.033). No determine protein concentration of the super- nontransgenic, Hsp70 heterozygous knock-out, or Hsp70 homozygous knock-out mice died during the 14 week time course. B, C, natant fraction. For Western blots, 3ϫ SDS Kaplan–Meier survival curves for Hsp70 ϩ/ϩ (n ϭ 11, and 12, respectively) and Hsp70 Ϫ/Ϫ (n ϭ 19) mice inoculated intracra- sample buffer was added, and the samples were ϭ nially with 3.5 LC50 22L prion or 3.5 LC50 RML prion indicate that deletion of Hsp70.1/3 did not affect survival (log rank: p 0.207 heated at 95°C for 5 min. Equal amounts of and 0.495, respectively). protein (25 ␮g) were loaded in each well, separated by 4–20% SDS/PAGE, transferred to ni- ated secondary antibody, avidin coupled to horseradish peroxidase and trocellulose membranes, blocked for 30 min at room temperature in 5% reacted with DAB. Sections were analyzed and the numbers of Iba-1- milk/TBST. After overnight incubation with primary antibody (made in positive microglia were averaged and expressed as total number per 0.1 5% milk/TBST, blots were rinsed three times in TBST, incubated with mm 3. Ten digital images per field were obtained and analyzed with secondary antibodies for2hatroom temperature, rinsed three times in Image-Pro Plus (MediaCybernetics) to determine the number of micro- TBST and detected with enhanced chemiluminescence (GE Healthcare). glia per unit area. Similar immunohistochemical methods were per- Antibodies and concentrations were as follows: EM48 (1:500, a kind gift formed to quantify astrocyte activation with a mouse monoclonal from Dr. Xiao-Jiang Li, Emory University), GAPDH (1:200, Millipore antibody against GFAP (1:1000, Abcam), c-Fos with a rabbit poly- Bioscience Research Reagents), secondary antibodies (1:5000, Jackson clonal antibody (1:500, Abcam) and synaptophysin with a mouse ImmunoResearch). All chaperone antibodies were from Stressgen Bio- monoclonal antibody (1:200, Sigma). From each animal at least three technologies, and dilutions were as follows: Hsp70 (1:1000), Hsp40 (1: blind-coded random sections were analyzed, and the results were 10,000), Hsp27 (1:1000), Hsp25 (1:5000), Hsc70 (1:1000), Hsp90 (1: averaged and expressed as mean value. Two sets of mice were used for ␮ 5000). To detect formic acid-sensitive monomer/oligomers, 100 gof the pathology experiments. For the first analysis, the mice that remained ␮ total protein lysate was incubated with 100 l of formic acid at room alive after the 14 week behavioral study were perfused and the brains were temperature for 1 h. Treating the lysate with formic acid releases mutant harvested. For the second analysis, mice were bred specifically for the htt species that migrate at the approximated molecular weight of a tri- biochemical and pathology experiments so that a more comprehensive mer/tetramer, although it is possible that this species is an aberrantly analysis could be performed with a larger number of mice/genotype. migrating monomer. Formic acid was removed in a speed vacuum and 30 Shown here are the results of the second analysis, but results were similar ␮ l of SDS loading buffer was added. The samples were neutralized with 2 in both groups of animals. ␮lof5M NaOH and heated at 95°C for 5 min. For the filter assay, 30 ␮lof 1ϫ SDS sample buffer (4% SDS) were incubated with 100 ␮g of total protein lysate at 95°C for 5 min and then filtered onto a cellulose acetate Results membrane with a slot blot manifold. For densitometry films were Deletion of Hsp70.1 and Hsp70.3 decreases survival in the scanned using ArcSoft PhotoStudio 5.5, and analyzed with ImageQuant R6/2 mouse, but not in prion-infected mice V2005 (GE Healthcare). To determine whether endogenous Hsp70s play an important Neuropathology. At 14 weeks of age, mice were deeply anesthetized role in combating the toxic effects induced by a mutant htt with halothane and perfused with 100 ml of phosphate buffer, followed fragment, we crossed the R6/2 mouse model of HD to knock- by 100 ml of 4% paraformaldehyde in phosphate buffer, pH 7.4. Brains out mice lacking the inducible Hsp70.1 and Hsp70.3 genes were removed, cryoprotected overnight in 30% sucrose and frozen in cooled isopentane. To investigate the effects of Hsp70.1/Hsp70.3 deletion (herein referred to as Hsp70.1/3). We subsequently analyzed a on levels of mutant htt immunoreactivity in R6/2 mutant mice, the sec- number of physical and behavioral outcome measures in six Ϫ/Ϫ ϩ/ϩ Ϫ/Ϫ Ϫ/ϩ tions were immunolabeled overnight with a rabbit polyclonal antibody genotypes of mice: R6/2 ;Hsp70 , R6/2 ;Hsp70 , Ϫ/Ϫ Ϫ/Ϫ tg/Ϫ ϩ/ϩ tg/Ϫ Ϫ/ϩ (EM48, Millipore Bioscience Research Reagents) against a glutathione R6/2 ;Hsp70 , R6/2 ;Hsp70 , R6/2 ;Hsp70 , Ϫ Ϫ Ϫ S-transferase fusion protein containing the first 256 aa of htt lacking the and R6/2 tg/ ;Hsp70 / . polyQ and polyproline stretches. Sections were washed in PBS and then Kaplan–Meier survival analysis demonstrated that deletion of placed in biotinylated secondary antibody (1:100) (Vector Laboratories) one copy of Hsp70.1/3 did not alter the lifespan of the R6/2 mouse for 2 h. Sections were placed in 20% diaminobenzidine (DAB) (Vector (details of all statistical analyses used in this study can be found in Laboratories), mounted, dried, and coverslipped with Entillin (Fisher). Materials and Methods). The survival curves of the R6/2 tg/Ϫ; Three immunostained sections per mouse were imaged with an Olympus Hsp70 ϩ/ϩ and the R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ mice were indistinguish- digital microscope. A total of 10 digital images per section and region of able, as were the endpoint survival rates of 83% and 82%, respec- interest were analyzed with Image-Pro Plus (MediaCybernetics) to de- tively (Fig. 1A). Strikingly, deletion of both copies of Hsp70.1/3 termine the optical density per field and the mean diameter and number tg/Ϫ of intranuclear inclusions. Individual values were averaged and ex- profoundly affected R6/2 survival: only 50% of the R6/2 ; Ϫ/Ϫ pressed as mean value. To quantify microglial activation, microtome Hsp70 mice were alive at the study endpoint. Survival analy- tg/Ϫ sections from R6/2 mice were immunostained with a mouse monoclonal sis demonstrated a significant decrease in the lifespan of the R6/2 ; Ϫ Ϫ Ϫ ϩ ϩ antibody against Iba-1 (1:1000, DakoCytomation) followed by biotinyl- Hsp70 / mice relative to the R6/2 tg/ ;Hsp70 / ( p ϭ 0.033, Wacker et al. • Hsp70 and Huntington’s Disease J. Neurosci., July 15, 2009 • 29(28):9104–9114 • 9107

performances of wild-type and R6/2 mice were well matched. In contrast, the R6/ 2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice were already significantly impaired at 4 weeks ( p Ͻ 0.001), demonstrating that the absence of inducible Hsp70s decreases the age of onset of the R6/2 motor phenotype. The intermediate motor phenotype of the R6/2 tg/Ϫ; Hsp70 Ϫ/ϩ mice, when compared with the R6/2 tg/Ϫ;Hsp70 ϩ/ϩ and the R6/2 tg/Ϫ; Hsp70 Ϫ/Ϫ mice, suggests that the relative expression levels of inducible Hsp70s modulate both the progression and severity of motor abnormalities in R6/2 mice.

Deletion of Hsp70.1 and Hsp70.3 exacerbates neurobehavioral phenotypes in R6/2 mice To characterize the neurobehavioral and physical decline of our mice we used a modified SHIRPA assessment (Rogers et al., 1997). This behavioral protocol was recently refined to provide a rapid, reproducible and quantitative means of examining numerous outcome mea- Figure 2. Deletion of Hsp70.1/3 worsens motor deficits in R6/2 mice. A–D, Deletion of Hsp70.1/3 decreases the latency to fall sures that clearly distinguish R6/2 trans- of R6/2 mice (two-way ANOVA: p Ͻ 0.05) (A), and increases severity of clasping (two-way ANOVA: p Ͻ 0.001) (B), tremor genic mice from their wild-type litter- (two-way ANOVA: p Ͻ 0.001) (C), and grooming (two-way ANOVA: p Ͻ 0.03) (D). E, F, Deletion of Hsp70.1/3 decreases R6/2 mates (Ditzler et al., 2003). The spontaneous activity (two-way ANOVA: p Ͻ 0.001) but has only a moderate effect on locomotor activity. Error bars indicate SEM. protocol includes a number of neurobe- NotethatintheabsenceoftheR6/2transgene,thelossofoneorbothcopiesof Hsp70.1/3doesnotinfluenceanyofthepresented havioral (clasping, tremor, grooming, outcome measures. spontaneous and locomotor activities) and physical (weight, palpebral closure, log rank test) and the R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ mice ( p ϭ 0.026). An coat appearance, body and tail position) outcome measures intact endogenous Hsp70 response, thus, appears to be critical for (supplemental Table 1, available at www.jneurosci.org as sup- survival in the R6/2 mice. plemental material). Hsp70s are presumed to be broadly protective against the Progressive clasping of the front and hind limbs that is trig- toxic effects of misfolded protein in the CNS. Therefore, we ex- gered by a tail suspension test is a conserved motor abnormality amined the effect of deleting endogenous Hsp70s on survival in observed in numerous mouse models of neurological disease and two mouse models of prion disease. A dose corresponding to is widely used as a marker of neuronal dysfunction (Mangiarini et ϳ 3.5 log LD50 of the 22L strain of murine prions was injected al., 1996; Carter et al., 1999; Stack et al., 2005). We analyzed intracranially into Hsp70 ϩ/ϩ and Hsp70 Ϫ/Ϫ mice. Surpris- clasping behavior once a week by suspending each mouse above ingly, the absence of endogenous Hsp70s had no effect on lifes- its cage for 30 s and scoring 0 for no clasp, 1 for a mild clasp in pan (Fig. 1B). The survival curves of the Hsp70 ϩ/ϩ and which only the fore or hind-limbs press into the stomach, and 2 Hsp70 Ϫ/Ϫ mice were indistinguishable, and the median survival for a severe clasp in which both fore and hind-limbs touch and times of the Hsp70 ϩ/ϩ and Hsp70 Ϫ/Ϫ mice injected with the 22L press into the stomach. Deletion of Hsp70.1/3 significantly wors- prion strain were 25.0 and 24.1 weeks, respectively. Similarly, the ened ( p Ͻ 0.001) the average clasping score of the R6/2 mice (Fig. median survival times of the Hsp70 ϩ/ϩ and Hsp70 Ϫ/Ϫ mice in- 2B). In contrast to the R6/2 tg/Ϫ;Hsp70 ϩ/ϩmice, the R6/2 tg/Ϫ; jected with the RML prion strain were identical at 26.4 weeks Hsp70 Ϫ/ϩ and the R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice already displayed post-prion inoculation, and the two survival curves were indis- significant clasping by 6 weeks ( p Ͻ 0.01). Moreover, the R6/2 tg/Ϫ; tinguishable (Fig. 1C). Moreover, transgenic overexpression of Hsp70 Ϫ/Ϫ mice consistently exhibited the most severe clasping Hsp70 did not prolong survival of prion-infected mice (data not score, followed by the R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ mice and finally the shown). R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice, suggesting a gene dose-dependent effect on the onset, progression and severity of this R6/2 phenotype Deletion of Hsp70.1/3 worsens motor deficits in R6/2 mice (Fig. 2B). We used a panel of diverse outcome measures to systematically R6/2 mice develop a progressive, resting tremor in the limbs, characterize the effect of deleting endogenous Hsp70s on the phe- trunk and head, which was scored as 0 (no tremor,) 1 (mild notypes of R6/2 mice. We first evaluated the effects of Hsp70 tremor), or 2 (severe tremor) (Mangiarini et al., 1996; Ditzler et deletion on motor performance, as measured by rotarod analysis, al., 2003). Tremor analysis showed that deletion of Hsp70.1/3 which is widely used to characterize the progressive decline in significantly increased ( p Ͻ 0.001) the score of the R6/2 mice motor performance of R6/2 mice (Carter et al., 1999; Hockly et (Fig. 2C). At 6 weeks, the R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice had a negligi- al., 2002). We found that deletion of Hsp70.1/3 significantly en- ble tremor score, whereas the R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ and the R6/2 tg/Ϫ; hanced ( p Ͻ 0.05) the severity of rotarod deficits in R6/2 mice Hsp70 Ϫ/Ϫ mice exhibited a significantly higher average score of (Fig. 2A). As expected, at the early time point of 4 weeks the 0.5 ( p Ͻ 0.001), demonstrating that deletion of one or both 9108 • J. Neurosci., July 15, 2009 • 29(28):9104–9114 Wacker et al. • Hsp70 and Huntington’s Disease alleles of Hsp70.1/3 decreased the age of tremor onset. The consistently intermediate score of the R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ mice relative to the R6/2 tg/Ϫ;Hsp70 ϩ/ϩ and R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice suggests a gene dose-dependent effect of Hsp70.1/3 on the R6/2 tremor phenotype. As R6/2 mice become symptomatic, either a complete lack of grooming or a stereotypic, repetitive grooming behavior is often observed (Mangiarini et al., 1996; Carter et al., 1999). Repetitive hindlimb grooming is thought to mimic the choreiform movements displayed by HD patients (Mangiarini et al., 1996). Mice received a score of 1 for normal grooming and a score of 2 for abnormal grooming. Analysis of cumulative grooming scores revealed that the deletion of Hsp70.1/3 genes significantly worsened ( p Ͻ 0.03) the abnormal grooming behavior of the R6/2 at later time points (Fig. 2D). In this case, however, the loss of both alleles of Hsp70.1/3 was required to enhance the progression and endpoint severity of the R6/2 grooming phenotype. The progressive development of abnormalities in the activity level of R6/2 HD mice has been well characterized (Dunnett et al., 1998; Bolivar et al., 2003; Stack et al., 2005), and our modified SHIRPA protocol included two measures of activity. We first measured spontaneous activity by scoring the coverage of four delineated cage quadrants by each mouse during a 2 min testing period. A score of 1 denoted movement into all four quadrants, 2 denoted slow movement in three or less quadrants, 3 denoted no movement or stereotypic darting/circling movements. We found that deletion of Hsp70.1/3 significantly exacerbated ( p Ͻ 0.001) Figure 3. Deletion of Hsp70.1/3 exacerbates the physical phenotypes of R6/2 mice. The the spontaneous activity phenotype of the R6/2 mouse, most no- absence of Hsp70.1/3 significantly exacerbates the weight loss phenotype (two-way ANOVA: p Ͻ 0.05) (A), and worsens the coat appearance (two-way ANOVA: p Ͻ 0.001) (B), body ticeably after 8 weeks of age (Fig. 2E). The absence of both alleles position (two-way ANOVA: p Ͻ 0.02) (C), and tail position (two-way ANOVA p Ͻ 0.001) (D)of of Hsp70.1/3 had a marked effect on the onset, progression and R6/2 mice. Error bars indicate SEM. Note that in the absence of the R6/2 transgene, the loss of endpoint severity. Despite the fact that deletion of one allele of one or both copies of Hsp70.1/3 does not influence any of the presented outcome measures. Hsp70.1/3 also had a more moderate effect, there was still a trend toward gene dose dependence for this outcome measure. We also performed the locomotor test as a second measure of activity by coat. Deletion of Hsp70.1/3 significantly worsened ( p Ͻ 0.001) scoring the number of times that each mouse touched the side of the R6/2 coat appearance phenotype (Fig. 3B). A decrease in the the cage during a 2 min observation period. The locomotor ac- age of onset, enhanced progression and increase in endpoint se- tivity test did not reveal a significant effect of the inducible verity were observed with a trend toward Hsp70.1/3 gene dose Hsp70s on the R6/2 phenotype, although there was a trend to- dependence. We also scored body position and tail position to ward a gene dose-dependent effect of Hsp70.1/3 deletion to en- further evaluate the effect of Hsp70.1/3 on the decline of the R6/2 hance motor abnormalities (Fig. 2F). Thus, two distinct outcome physical phenotype. The R6/2 body position phenotype was measures showed that Hsp70.1/3 affects the development, pro- scored as 1 for normal, and 2 for a hunched and rounded stature. gression and severity of activity deficits in the R6/2 mouse. Tail position was scored as 1 for normal or horizontally extended, and 2 for dragging/straub. Deletion of Hsp70.1/3 significantly Deletion of Hsp70.1/3 exacerbates the physical phenotypes of enhanced the severity of the body position outcome measure R6/2 mice ( p Ͻ 0.02, Fig. 3C) and the tail position outcome measure ( p Ͻ To characterize decline in the physical phenotypes of the R6/2 0.001, Fig. 3D). In both cases a trend toward an Hsp70.1/3 gene- mice, we measured weight and scored for coat appearance, body dose dependent enhancement of phenotypic severity was ob- position, tail position and palpebral closure. Female R6/2 mice served. The only component of the physical phenotype test that show a characteristic weight loss pattern: weight plateaus around was unaffected by the deletion of Hsp70.1/3 was palpebral closure week 8 and declines significantly at 12 weeks (Hockly et al., 2003). (data not shown). Importantly, all outcome measures included in The weights of the R6/2 Ϫ/Ϫ;Hsp70 ϩ/ϩ, R6/2 Ϫ/Ϫ;Hsp70 Ϫ/ϩ, and the neurobehavioral and physical phenotype assessment showed the R6/2 Ϫ/Ϫ;Hsp70 Ϫ/Ϫ mice were indistinguishable, demon- that there were no significant differences between the wild-type strating that the absence of inducible Hsp70s alone does not in- nontransgenic mice and the Hsp70.1/3 heterozygous or homozy- fluence body weight (Fig. 3A). Analysis of the weight of the gous knock-out mice. R6/2 tg/Ϫ;Hsp70 ϩ/ϩ and R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice showed a significant interaction ( p Ͻ 0.05) on this phenotype and a Deletion of Hsp70.1/3 increases the size of inclusion bodies in Hsp70.1/3 gene dose-dependent trend on weight loss was ob- R6/2 mice served, suggesting that the inducible Hsp70s may modulate the To determine whether the exacerbated behavioral and physical onset, progression and endpoint severity of the R6/2 weight loss phenotypes observed in R6/2 mice that lacked inducible Hsp70s phenotype. correlated with changes in the density or size of inclusion bodies The coat appearance of the R6/2 mice declines as the disease formed by mutant htt exon 1, we examined serial sections from state progresses and is characterized by a score of 1 for a shiny, the neocortex of 14-week-old mice with immunohistochemistry well groomed coat and a score of 2 for a scruffy and/or piloerected and the EM48 anti-htt antibody. As expected, the R6/2 Ϫ/Ϫ; Wacker et al. • Hsp70 and Huntington’s Disease J. Neurosci., July 15, 2009 • 29(28):9104–9114 • 9109

Western blots probed with the EM48 antibody on total brain homogenates from R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice showed that all of the reactivity corresponded to large SDS-insoluble aggregates that were retained in the stacking portion of the gel (Fig. 5A) as described (Davies et al., 1997). An identical pattern of reactivity is observed on Western blots that contain purified mutant htt exon 1 with 53Q that has been aggregated into fibrillar protein assemblies (Wacker et al., 2004). Surpris- ingly, analysis of the pixel intensities relative to the GAPDH loading controls showed that the average EM48 reactivities for the R6/2 tg/Ϫ;Hsp70 ϩ/ϩ and R6/2 tg/Ϫ; Hsp70 Ϫ/Ϫ mice were indistinguishable (Fig. 5A, B). Previous studies showed that HD brain homogenates treated with formic acid liberate a SDS-resistant oligomer Figure 4. Deletion of Hsp70.1/3 increases the size of inclusion bodies in R6/2 mice. A, Representative images (600ϫ)of as analyzed by Western immunoblots (Iu- inclusion bodies in the neocortex of R6/2 mice as detected with the anti-htt antibody EM48. B, Quantification of inclusion body chi et al., 2003; Hoffner et al., 2005). Sim- tg/Ϫ Ϫ/Ϫ numberintheneocortexshowsthatR6/2 ;Hsp70 micehaveanincreaseinthedensityofinclusionbodiescomparedwith ilar to the results observed in HD brain R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice, although this difference only showed a trend toward statistical significance ( p ϭ 0.086). C, Repre- ϫ homogenates, we found that treatment of sentative images (1000 ) illustrating the size of inclusion bodies in the neocortex of R6/2 mice as detected with the anti-htt total brain homogenates from R6/2 tg/Ϫ; antibody EM48.D, Quantification of inclusion body size shows that the average size of the inclusion bodies was significantly larger ϩ/ϩ ( pϽ0.001)inR6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ micecomparedwithR6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice.Statisticalcomparisonswereperformedby Hsp70 mice with formic acid released one-way ANOVA (n ϭ 6–11 mice per group). two bands that reacted with EM48 which migrated at an apparent molecular mass of 70–85 kDa, while concomitantly leading ϩ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩ Hsp70 / , R6/2 / ;Hsp70 / and R6/2 / ;Hsp70 / mice to nearly a complete loss of reactivity in the stacking gel (Fig. 5C). did not display EM48-positive inclusion bodies (data not Based on their apparent molecular mass, these bands may reflect shown). Immunohistochemical analyses on cortical brain sec- a low molecular mass SDS-resistant oligomer or aberrantly mi- tg/Ϫ Ϫ/Ϫ tions with EM48 suggested R6/2 ;Hsp70 had an increased grating monomers of mutant htt exon 1. The levels of formic tg/Ϫ ϩ/ϩ density of inclusion bodies compared with R6/2 ;Hsp70 acid-sensitive monomers/oligomers (normalized to GAPDH re- mice (Fig. 4A). However, quantification of the number of inclu- activity) appeared to increase in the absence of Hsp70.1/3, but did sion bodies in a defined brain volume indicated this difference not reach statistical significance (Student’s t test p ϭ 0.15). (Fig. ϭ only showed a trend toward statistical significance ( p 0.086) 5D). Identical findings were observed using the 3B5H10 antibody (Fig. 4B). generated by the Finkbeiner laboratory (data not shown). Analysis of average inclusion body diameter demonstrated that the We next used filter-trap assays as an independent method to R6/2 tg/Ϫ;Hsp70 ϩ/ϩ (4.22 Ϯ0.55 ␮m) and R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ (3.81 Ϯ ␮ Ͼ evaluate total SDS-insoluble material formed by mutant htt exon 0.34 m) mice were indistinguishable ( p 0.05, Fig. 4D). In 1 in R6/2 brains in the presence and absence of inducible Hsp70s. tg/Ϫ Ϫ/Ϫ Ϯ comparison, the R6/2 ;Hsp70 inclusion bodies (7.68 In this assay, total brain homogenates were boiled in SDS and 0.44 ␮m) stained with EM48 were dramatically and significantly Ϫ ϩ ϩ filtered through a cellulose acetate membrane that contains 0.2 larger ( p Ͻ 0.001) than in R6/2 tg/ ;Hsp70 / mice (Fig. 4C, D). ␮m pores. Previous studies with brains from R6/2 mice showed The pixel intensity of EM48 staining in R6/2 mice lacking both that this method traps large (Ͼ0.2 ␮m) SDS-insoluble aggregates alleles of Hsp70.1/3 also appeared greater than in R6/2 mice alone. of fibrillar material (Scherzinger et al., 1997). Consistent with the In summary, these results indicate that the complete absence of Western immunoblots, we found that the levels of SDS-insoluble inducible Hsp70s increased the size of inclusion bodies formed by mutant htt exon 1 in R6/2 mice, consistent with in vitro data material detected by EM48 in filter-trap assays were not significantly different between R6/2 tg/Ϫ;Hsp70 ϩ/ϩ and R6/2 tg/Ϫ; indicating Hsp70 can directly modulate the misfolding and ag- Ϫ/Ϫ gregation of mutant htt (Muchowski et al., 2000; Wacker et al., Hsp70 mice (Fig. 5E, F). Interestingly, total brain homoge- 2004). nates treated with formic acid and SDS were still detected by EM48 in filter assays (Fig. 5G), suggesting that this treatment ␮ Deletion of Hsp70.1/3 does not modulate levels of releases oligomeric species larger than 0.2 m in size. However, as SDS-insoluble fibrillar protein aggregates formed by mutant with the other assays, no significant differences were observed in tg/Ϫ Htt exon 1 in R6/2 mice brain homogenates analyzed in this manner between R6/2 ; ϩ/ϩ tg/Ϫ Ϫ/Ϫ We next sought to determine whether the increased size of inclu- Hsp70 and R6/2 ;Hsp70 mice (Fig. 5G, H). Similar sion bodies in the absence of inducible Hsp70s in R6/2 mice was results were also obtained when agarose native gels were used to Ϫ attributed to increased levels of aggregates formed by mutant htt detect oligomeric species in total brain homogenates from R6/2 tg/ ; ϩ ϩ Ϫ Ϫ Ϫ exon 1. We used Western immunoblots, filter-trap assays and Hsp70 / and R6/2 tg/ ;Hsp70 / mice (Fig. 5I). Thus, three agarose gels to measure the relative levels of SDS-insoluble aggre- independent approaches used to evaluate mutant htt exon 1 ag- gates and formic acid–sensitive htt species in the brains of 14- gregates in 14-week-old R6/2 brain homogenates showed no sig- week-old R6/2 tg/Ϫ;Hsp70 ϩ/ϩ and R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice. nificant differences in aggregate levels in the presence or absence 9110 • J. Neurosci., July 15, 2009 • 29(28):9104–9114 Wacker et al. • Hsp70 and Huntington’s Disease of inducible Hsp70s. Similar results were obtained in 7-week-old R6/2 brain homogenates (data not shown). Previous studies demonstrated changes in the relative levels of chaperone proteins in mouse models over the course of polyQ disease (Cummings et al., 2001; Hay et al., 2004). For example, expression of mutant ataxin-1 in a mouse model of SCA1 elicits an increase in Hsp70 expression (Cummings et al., 2001), whereas levels of Hsp70 and other chaperones decreased progressively in R6/2 mice (Hay et al., 2004). To test whether deletion of Hsp70.1/3 caused compensatory changes in the relative levels of other heat shock proteins, we performed Western immunoblots on brain homogenates from R6/ 2 tg/Ϫ;Hsp70 ϩ/ϩ, R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ, R6/ 2 Ϫ/Ϫ;Hsp70 ϩ/ϩ and R6/2 Ϫ/Ϫ;Hsp70 Ϫ/Ϫ mice. At 14 weeks of age, no significant changes were detected in the levels of Hsp27 and Hsp90 (supplemental Fig. 1, available at www.jneurosci.org as supplemental material), or Hsp25, Hsp40 and Hsc70 (data not shown), relative to a GAPHD loading control. Thus, the lack of both Hsp70 alleles on a wild-type or R6/2 strain background did not appear to confer compensatory changes in levels of other major heat shock proteins.

Deletion of Hsp70.1/3 exacerbates the loss of c-Fos immunoreactivity and other neuropathological deficits in R6/2 mice To determine the effect of Hsp70 deletion on neuronal loss in R6/2 mice we used immunohistochemistry and unbiased ste- reology with an antibody against the Figure 5. Deletion of Hsp70.1/3 does not modulate levels of SDS-insoluble fibrillar protein aggregates formed by mutant htt neuronal-specific protein NeuN in brain exon1inR6/2mice.A,B,DeletionofHsp70.1/3doesnotalterthelevelsofEM48reactiveSDS-insolubleaggregates(normalizedto sections from 14-week-old mice. These GAPDHreactivity)measuredwithWesternimmunoblotsin14-week-oldR6/2brainhomogenates(Student’sttestpϭ0.96).C,D, analyses showed no significant change in Treatment of brain homogenates with formic acid liberates an SDS-resistant monomeric/oligomeric mutant huntingtin exon 1 species. The levels of formic acid-sensitive monomers/oligomers (normalized to GAPDH reactivity) appeared to increase in the NeuN immunoreactivity in the cortex or ϭ striatum of R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice absence of Hsp70.1/3, but did not reach statistical significance (Student’s t test p 0.15). E, F, The levels of SDS-insoluble EM48-positive fibrillar aggregates in brain measured by a filter-trap assay do not change in the absence of Hsp70.1/3 (Student’s t compared with WT animals, and no test p ϭ 0.89). G, H, Formic acid-treated brain homogenates were subjected to the filter-trap assay, which showed no change in significant difference in NeuN levels be- EM48 immunoreactivity in the absence of Hsp70.1/3 (Student’s t test p ϭ 0.90). I, A native agarose gel used to examine EM48 tg/Ϫ ϩ/ϩ tg/Ϫ tween R6/2 ;Hsp70 and R6/2 ; immunoreactive oligomeric species in R6/2 brain homogenates shows no apparent change in the absence of Hsp70.1/3. Hsp70 Ϫ/Ϫ mice (data not shown). In an independent study, we recently found that and this loss was further and significantly exacerbated ( p Ͻ 0.05) immunoreactivity for the presynaptic protein synaptophysin and in R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice (Fig. 6A, B). Importantly, levels of the calcium regulated immediate early gene product c-Fos, a sur- c-Fos were not significantly different between R6/2 Ϫ/Ϫ;Hsp70 ϩ/ϩ rogate marker for neuronal activity, were decreased in R6/2 mice Ϫ Ϫ Ϫ Ϫ and R6/2 / ;Hsp70 / mice. Levels of synaptophysin immuno- relative to nontransgenic littermate controls, and that these reactivity also appeared to be decreased in the cortex and striatum changes were attenuated in R6/2 mice treated with a small- Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ of R6/2 tg/ ;Hsp70 / mice compared with R6/2 / ;Hsp70 / molecule inhibitor of kynurenine 3-monooxygenase in a manner that correlated with survival (P. Guidetti, W. Kwan, S.-Y. Huang, mice, although this decrease did not reach statistical significance J. Lee, C. Patrick, F. Giorgini, T. Mo¨ller, C. S. Cheah, T. Wu, K. possibly explained by the lack of statistical power due to the small ϭ Scearce-Levie, J. M. Muchowski, E. Masliah, R. Schwarcz, and P. numbers of mice analyzed (n 4–6 per group) (Fig. 6C, D). A J. Muchowski, unpublished observations). In the current study recent study showed increased levels of immunoreactivity for the immunohistochemical analysis of brain sections from R6/2 tg/Ϫ; microglia-specific protein Iba1 in R6/2 mice (Simmons et al., Hsp70 ϩ/ϩ mice showed a significant ( p Ͻ 0.05) decrease in c-Fos 2007). We observed a significant ( p ϭ 0.0297) increase in immu- immunoreactivity relative to WT mice in the cortex and striatum, noreactivity for Iba1, and a trend toward an increase in the Wacker et al. • Hsp70 and Huntington’s Disease J. Neurosci., July 15, 2009 • 29(28):9104–9114 • 9111

ing, activity, weight, coat appearance, and body position). The absence of both alleles of Hsp70.1/3 profoundly enhanced the onset, severity and progression of behavioral phenotypes in R6/2 mice, including a significant decrease in median lifespan. R6/2 mice completely lacking inducible Hsp70s showed an increase in the number and size of inclusion bodies, although these findings did not correlate with a biochemical changes in the relative levels of SDS-insoluble fibrillar aggregates as measured by multiple independent approaches. Finally, we found that deletion of Hsp70.1/3 exacerbated the loss of c-Fos, a surrogate marker for neuronal activity, in a highly significant manner. These findings indicate that the absence of inducible Hsp70s increased neuronal sensitivity to mutant htt exon 1 in the R6/2 mouse model of HD, without affecting htt expression or its accumulation into SDS- insoluble aggregates. Deletion of Hsp70.1/3 had no significant effect on lifespan in two mouse models of transmissible prion disease. This is not simply because prion diseases are so extreme that they can not be modified. In- deed, deletion of HSF1, a master regulator of homeostatic stress responses, has a pronounced effect on the course of these same prion models (Steele et al., 2008). Thus, the striking effect of the absence of inducible Hsp70s on R6/2 mice indicates that a specific genetic interaction occurs between the inducible Hsp70s and the mutant htt fragment in vivo. Although the inducible Hsp70s may play a pivotal role in prion propagation in yeast (Tutar et al., Figure 6. Deletion of Hsp70.1/3 exacerbates the loss of c-Fos immunoreactivity and other neuropathological deficits in R6/2 2006; Loovers et al., 2007), our results mice. A, B, Quantification of c-Fos immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2 tg/Ϫ; suggest that the inducible Hsp70s do not Hsp70 Ϫ/Ϫ mice have a significant decrease ( p Ͻ 0.05) in c-Fos levels compared with R6/2 tg/Ϫ;Hsp70 ϩ/ϩ mice. C, D, Quanti- influence toxicity in mice infected with es- Ϫ ϩ Ϫ Ϫ Ϫ fication of synaptophysin immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2 tg/ ;Hsp70 / and / tablished strains of prions. Importantly, mice have a significant decrease ( p Ͻ 0.05) in synaptophysin levels compared with R6/2 Ϫ/Ϫ;Hsp70 Ϫ/Ϫ mice. E, F, Quantification of tg/Ϫ ϩ/Ϫ and Ϫ/Ϫ unlike HD, which is an autosomal domi- Iba1 and GFAP immunohistochemistry in the neocortex from 14-week-old mice shows that R6/2 ;Hsp70 mice have a nant inherited neurodegenerative disor- significant increase ( p ϭ 0.0297) in Iba1 levels, and a trend toward increased GFAP levels ( p ϭ 0.1048), respectively, compared with Ϫ Ϫ Ϫ Ϫ der, prion disease encompasses diverse R6/2 / ;Hsp70 / mice. etiologies in addition to acute infection (Kingsbury et al., 1983). The inducible astrocyte-specific marker GFAP in the cortex of R6/2 tg/Ϫ; Hsp70s may possibly play a role in sup- Hsp70 Ϫ/Ϫ mice compared with R6/2 Ϫ/Ϫ;Hsp70 ϩ/Ϫ and Ϫ/Ϫ mice pressing toxicity in other mouse models of spontaneous and/or (Fig. 6E, F). Insufficient brain material unfortunately precluded genetically derived prion disease. the analysis of other genotypes in these studies. Our results dem- The loss of one copy of Hsp70.1/3 did not decrease the lifespan onstrate that endogenous Hsp70s protect against the loss of c-Fos of R6/2 mice, suggesting a potent activity of endogenous Hsp70 in a highly significant manner, and suggest these chaperones are chaperones, even when present at half of their normal concentra- critical regulators of neuronal activity and inflammatory re- tion, to mitigate pathogenic cascades and modulate disease onset, sponses in R6/2 mice. progression and severity in vivo. However, the decrease in the age of onset observed for the majority of behavioral and physical testing Discussion parameters in both the R6/2 tg/Ϫ;Hsp70 Ϫ/ϩ and the R6/2 tg/Ϫ; Here we showed that endogenous Hsp70s critically regulate the Hsp70 Ϫ/Ϫ mice demonstrates that an intact inducible Hsp70s re- toxicity of a disease-causing misfolded protein in a mouse model sponse is required to limit mutant htt toxicity at the earliest stages of of HD. The absence of even one allele of the Hsp70.1/Hsp70.3 pathogenesis in R6/2 mice. genes significantly exacerbated the severity of a number of out- The absence of both alleles of Hsp70.1/3 significantly in- come measures for the R6/2 mouse model of HD (rotarod, clasp- creased the average size and appeared to increase the number of 9112 • J. Neurosci., July 15, 2009 • 29(28):9104–9114 Wacker et al. • Hsp70 and Huntington’s Disease inclusion bodies in R6/2 brains, yet paradoxically did not alter the leads to the accumulation of toxic monomers/oligomers, the in- total load of fibrillar aggregates detected biochemically. What ducible Hsp70s may also buffer the toxicity of mutant htt mono- mutant htt species detected by EM48 in brain sections can ac- mers/oligomers by masking surfaces that promote pathogenic count for the increased size and abundance of inclusion bodies? interactions with essential cellular proteins. For example, in one Our biochemical studies excluded the possibility that the in- study, a mutant htt monomer underwent an intramolecular tran- creased size and abundance of inclusion bodies were due to any sition that facilitated an interaction with the Tata binding protein significant changes in fibrillar and/or large oligomeric species (TBP) and ultimately resulted in the functional inactivation of that are insoluble in SDS. In addition, formic acid-treated R6/2 this important transcription factor (Schaffar et al., 2004). Addi- brain lysates had similar levels of mutant htt monomers and oli- tion of Hsp70 to the in vitro system prevented the conformational gomers in the presence or absence of Hsp70s. Our previous in rearrangement of mutant htt and thus inhibited the pathogenic vitro studies used atomic force microscopy and biochemical ap- interaction with TBP, suggesting that the activity of Hsp70 to proaches to demonstrate that the cooperative activity of Hsp70 bind and hold mutant htt monomers can prevent aberrant and Hsp40 stabilized a monomeric conformation of a mutant htt protein-protein interactions that lead to neuronal dysfunction. fragment (HD53Q), while concomitantly suppressing the accu- Mutant htt, in addition to causing transcriptional repression, has mulation of annular and spherical oligomeric assemblies also been shown to upregulate p53 associated transcriptional (Wacker et al., 2004). However, a recent study indicated Hsp70 events in neuronal cultures (Bae et al., 2005). p53 is a strong and Hsp40 can also partition onto SDS-soluble mutant htt oli- suppressor of Hsp70 expression in specific neuronal subtypes gomers in an ATP-dependent manner (G. P. Lotz, J. Legleiter, E. that are affected in HD (Tagawa et al., 2007), and, moreover, Mitchell, S.-Y. Huang, C.-P. Ng, C. Glabe, L. M. Thompson, and genetic deletion of p53 ameliorates behavioral abnormalities in P. J. Muchowski, unpublished observations). Therefore we spec- the N171-82Q mouse model of HD (Schilling et al., 1999; Bae et ulate that, in the absence of inducible Hsp70s in R6/2 mice, small, al., 2005). Thus, it is tempting to speculate that the effect of p53 SDS-soluble mutant htt exon 1 assemblies that accumulate may on HD pathogenesis may be at least partially mediated by changes account for the increase in inclusion body density and size in the in the expression of inducible Hsp70s. R6/2 tg/Ϫ;Hsp70 Ϫ/Ϫ mice. Consistent with this interpretation, The exacerbation of R6/2 phenotypes in mice lacking Hsp70s deletion of C terminus of Hsp70 interacting protein (CHIP) in a may also be due to an overall disruption in the protein homeosta- mouse model of Spinocerebellar Ataxia Type 3 (SCA3) markedly sis network, as suggested from studies in Caenorhabditis elegans increased levels of ataxin-3 microaggregates in a manner that by Morimoto and colleagues (Gidalevitz et al., 2006). Consistent correlated with exacerbated behavioral phenotypes in these mice with this scenario, we observed that levels of the calcium regu- (Williams et al., 2009). We hypothesize that inducible Hsp70s lated immediate early gene c-Fos and the presynaptic protein buffer toxicity by binding monomeric and/or low molecular mass synaptophysin were decreased in R6/2 mice lacking Hsp70s rela- SDS-soluble oligomers that are likely off-pathway to fibril forma- tive to controls, whereas levels of protein markers for inflammation, but may be potentially pathogenic. However, based on the tory responses (Iba1 and GFAP) were increased. Although the multifunctional nature of Hsp70 it is very likely that this chaper- functional significance of these changes in R6/2 mice has not yet one can also suppress protein misfolding toxicity by multiple been investigated, the levels of c-Fos, which is used a surrogate mechanisms independent of its direct effects on misfolded pro- marker for neuronal activity, are tightly linked to cognitive defi- tein (see below). cits in mouse models of AD (Palop et al., 2003). The apparent loss Although larger inclusion bodies were observed in R6/2 mice of synaptophysin in R6/2 mice lacking Hsp70 is consistent with in the absence of Hsp70s, this does not necessarily suggest that previous studies in R6/2 mice (Cepeda et al., 2003) and, more inclusion bodies are toxic entities. Indeed, in direct contrast to broadly, with studies that suggest synaptic loss may be important the current study, we recently observed a strong positive correla- for pathogenesis in HD (Li et al., 2003). Interestingly, as mutant tion between survival and inclusion body size in mice treated htt inhibits the acetyltransferase activity of CREB-binding pro- with an inhibitor of the mitochondrial enzyme kynurenine tein (CBP) (Steffan et al., 2000; Steffan et al., 2001), which itself 3-monooxygenase (P. Guidetti, W. Kwan, S.-Y. Huang, J. Lee, C. controls c-Fos expression (Yuan et al., 2009), it is possible that Patrick, F. Giorgini, T. Mo¨ller, C. S. Cheah, T. Wu, K. Scearce- aberrant protein interactions between mutant htt and CBP, and Levie, J. M. Muchowski, E. Masliah, R. Schwarcz, and P. J. Mu- suppression of these interactions by Hsp70 (Schaffar et al., 2004), chowski, unpublished observations). Furthermore, systematic mediate in part the effects we observed on c-Fos expression in analysis of the effects of genetic enhancers (Willingham et al., R6/2 mice. Our results also indicate endogenous Hsp70s may 2003) or suppressors (Giorgini et al., 2005) of mutant htt exon 1 influence inflammatory responses, consistent with previous re- toxicity in yeast showed no correlations between toxicity and ports (Van Molle et al., 2002; Hampton et al., 2003; Singleton and inclusion bodies. These experiments underscore the inherent Wischmeyer, 2006; Mycko et al., 2008). Collectively these studies limitations of quantifying inclusion body size and abundance in strongly suggest that Hsp70s may modulate pathogenesis of pro- mouse brain sections using immunohistochemistry to draw tein misfolding diseases in vivo by direct and indirect effects in meaningful deductions of the role of these abnormal brain de- multiple cell types that may only be dissected by modulating posits on in vivo pathogenesis. We propose that the molecular levels of these proteins and their interacting proteins in specific composition of SDS-soluble conformers that may exist in a dif- cell types in vivo. fuse fraction or in inclusion bodies, be they monomers or small The majority of the behavioral outcome measures that we oligomers, will be the key to understanding which structures me- examined in R6/2 mice showed a trend toward Hsp70.1/3 gene diate pathogenesis. Thus, tools to identify and track such struc- dose dependence, demonstrating that the relative levels of induc- tures in situ, such as antibodies, will be required before unequiv- ible Hsp70s can dramatically alter pathogenesis in vivo. A recent ocal experiments can determine which are the toxic species of study used RNA interference (RNAi) to show that the expression mutant htt in mouse models of HD. levels of Hsp70 dictate the susceptibility of primary neurons to Although a primary function of Hsp70s in animal models of mutant htt toxicity (Tagawa et al., 2007). Thus, even a modest polyQ disease may be to counteract the assembly process that increase in the levels of molecular chaperones may suffice to de- Wacker et al. • Hsp70 and Huntington’s Disease J. Neurosci., July 15, 2009 • 29(28):9104–9114 • 9113 crease the severity of protein-conformational disorders. 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