Characterization of Survival Motor Neuron Protein Degradation
Deborah Y. Kwon B.S. Virginia Polytechnic Institute and State University, 2005
A dissertation submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Neuroscience at Brown University
Providence, Rhode Island May 2013
© 2013 By Deborah Y. Kwon
This dissertation by Deborah Y. Kwon is accepted in its present form by the Department of Neuroscience as satisfying the dissertation requirements for the degree of Doctor of Philosophy.
Date ______Kenneth H. Fischbeck, M.D., Advisor National Institute of Neurological Disorders and Stroke
Recommended to the Graduate Council
Date ______Ajay Chitnis, M.B.B.S., Ph.D., Reader National Institute of Child Health and Human Development
Date ______Justin Fallon, Ph.D., Reader Brown University
Date ______Anne Hart, Ph.D., Reader Brown University
Date ______Richard Youle, Ph.D., Reader National Institute of Neurological Disorders and Stroke
Date ______Judith Steen, Ph.D., Reader Children’s Hospital Boston
Approved by the Graduate Council
Date ______Peter Weber, Ph.D. Dean of the Graduate School
iii
Deborah Y. Kwon
35 Convent Drive, Building 35, Room 2A1008, Bethesda, MD 20892 (301) 435-9288 • [email protected]
Education
Virginia Polytechnic Institute and State University, Blacksburg, VA B.S. Biological Sciences, Biotechnology concentration, 2005
Brown University, Providence, RI Ph.D. candidate, Neuroscience Dissertation: “Characterization of survival motor neuron (SMN) protein degradation” Advisor: Kenneth H. Fischbeck, M.D.
Research Experience
Undergraduate Research Assistant, May 2003-May 2005 Fralin Biotechnology Center, Virginia Tech Advisor: Brenda S.J. Winkel, Ph.D. • Collaborated with the Virginia Bioinformatics Institute in studying metabolic function in transgenic Arabidopsis thaliana. Project involved isolating T-DNA mutants that are genetically altered at genes known to code flavanoid biosynthetic enzymes. Analyzed resultant lines by liquid chromatography linked to mass spectrophotometry (LC-MS) to study the effect of the mutations on the production of flavanoids in Arabidopsis. • Studied the protein-protein interaction between the chalcone synthase and chalcone isomerase enzymes in the flavanoid pathway.
Intern Analyst, June 2004-August 2004 Drug Control Centre, King’s College London Advisor: David Cowan, Ph.D. • Certified to perform the assays testing for the presence of the controlled substance, human chorionic gonadotrophin, in athletes competing in the Summer 2004 Olympics. • Trained in the assay for detection of diuretics.
Postbaccalaurate Fellow, August 2005-May 2007 National Institute of Child Health and Human Development, National Institutes of Health Advisor: Judith A. Kassis, Ph.D. • Utilized genetic and molecular techniques to determine which sequences of a previously identified Polycomb response element are necessary for transposon homing in Drosophila melanogaster. • Studied enhancer-promoter interactions at the Drosophila engrailed locus.
Graduate Student, July 2007 - Present National Institute of Neurological Disorders and Stroke, National Institutes of Health Advisor: Kenneth H. Fischbeck, M.D. • Investigating the molecular mechanisms underlying spinal muscular atrophy (SMA), a genetically inherited motor neuron disorder, using cell culture and animal model systems.
iv
• Characterized the degradation of the survival of motor neuron (SMN) protein by the ubiquitin proteasome pathway.
Honors and Awards
2003-2005 Phi Sigma Biological Sciences Honor Society, Virginia Tech 2007 NIH Post-Baccalaureate Research Festival Award for Best Poster Presentation 2008 NIH Graduate Student Research Award recipient (travel award) 2011 NIH Graduate Student Research Award recipient (travel award) 2011 The Fellows Award for Research Excellence recipient (travel award) 2012 NIH Graduate Student Research Award recipient (travel award)
Teaching and Mentoring Activities
Student mentor 2009 College undergraduate- Brown University 2010-Present College undergraduate- Eastern Mennonite University* 2012 College undergraduate- Arizona State University* 2012 College undergraduate- University of Michigan* * NINDS Exceptional Summer Student Award recipients
Lecturer, National Institutes of Health, Fall 2010 “Protein Biology: Protein Basics” Designed and gave a lecture in the graduate course, “Molecular Approaches to Studying Diseases” at the Foundation for Advanced Education in the Sciences Graduate School.
Leadership/Professional Service
Virginia Tech, Blacksburg, Va., 2002-2004 Dean’s Student Advisory Committee Member • Worked with the dean of the College of Sciences to address student issues.
National Institutes of Health, Bethesda, MD, 2009-Present • Mentored and trained summer interns and a postbaccalaureate fellow on skills in molecular biology, biochemistry and preclinical studies.
National Institutes of Health, 2010-Present NIH Graduate Student Research Symposium Committee Member • Organized the 2011 and 2012 Graduate Student Research Symposium, the largest graduate student event of the year, to highlight the research of the 500+ graduate students performing their Ph.D. dissertation research at the NIH.
Publications
1. DeVido SK, Kwon D, Brown JL, Kassis JA. The role of polycomb-group response elements in regulation of engrailed transcription in Drosophila. Development. 2008 Feb;135(4):669-76.
v
2. Burnett BG, Muñoz E, Tandon A, Kwon DY, Sumner CJ, Fischbeck KH. Regulation of SMN protein stability. Mol Cell Biol. 2009 Mar;29(5):1107-15.
3. Kwon D, Mucci D, Langlais KK, Americo JL, DeVido SK, Cheng Y, Kassis JA. Enhancer- promoter communication at the Drosophila engrailed locus. Development. 2009 Sep;136(18):3067- 75.
4. Kwon DY, Motley WW, Fischbeck KH, Burnett BG. Increasing the expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy disease phenotype in mice. Hum Mol Genet. 2011 Sep 15;20(18):3667-77. Selected for cover (image)
5. Cheng Y, Kwon DY, Arai AL, Mucci D, Kassis JA. P-Element homing is facilitated by engrailed polycomb-group response elements in Drosophila melanogaster. PLoS ONE 2012 7(1): e30437
6. Kwon DY, Dimitriadi M, Cable C, Hart A, Chitnis A, Fischbeck KH, Burnett BG. The E3 ubiquitin ligase mind bomb 1 ubiquitinates and promotes the degradation of survival of motor neuron protein. Under review
Oral Presentations
1. Kwon DY, Burnett BG, Fischbeck KH. Survival of Motor Neuron protein degradation and its effect on spinal muscular atrophy. NIH Graduate Student Seminar Series, September 2009.
2. Kwon, DY, Chitnis, A., Fischbeck KH and Burnett, BG. Mind Bomb 1 is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of survival of motor neuron protein. Families of SMA International Research Group Meeting, June 2011. Selected for platform presentation
Selected Abstracts
1. Kwon DY, Burnett BG, Fischbeck KH. The effect of proteasome inhibitors on spinal muscular atrophy. 5th Annual NIH Graduate Student Research Symposium. November 2008.
2. Kwon DY, Burnett BG, Fischbeck KH. The effect of TSA and bortezomib on spinal muscular atrophy. 6th Annual NIH Graduate Student Research Symposium. January 2009.
3. Kwon DY, Motley WW, Burnett BG, Fischbeck KH. The effect of TSA and bortezomib on spinal muscular atrophy. International Spinal Muscular Atrophy Research Group Meeting, Cincinnati, OH. June 2009.
4. Kwon DY, Motley WW, Burnett BG, Fischbeck KH. The effect of TSA and bortezomib on spinal muscular atrophy. Society for Neuroscience Annual Meeting, Chicago, Il. October 2009.
5. Kwon DY, Motley WW, Fischbeck KH, Burnett BG. Upregulation of the SMN2 gene with stabilization of the SMN protein ameliorates the SMA phenotype. International Spinal Muscular Atrophy Research Group Meeting, Santa Clara, CA. June 2010.
vi
6. Kwon DY, Chitnis A, Burnett BG, Fischbeck KH. Mind bomb 1 is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of the survival of motor neuron protein. 7th Annual NIH Graduate Student Research Symposium. January 2011.
7. Kwon DY, Dimitriadi M, Hart A, Chitnis A, Fischbeck KH, Burnett BG. Mind bomb 1 is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of the survival of motor neuron protein. International Spinal Muscular Atrophy Research Group Meeting, Orlando, FL. June 2011.
8. Kwon DY, Dimitriadi M, Hart A, Chitnis A, Fischbeck KH, Burnett BG. Mind bomb 1 is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of the survival of motor neuron protein. 8th Annual NIH Graduate Student Research Symposium. January 2012.
9. Kwon DY, Nofziger JH, Hall M, Fischbeck KH, Burnett BG. Stabilization of the SMN protein by modulating the blood-brain barrier. International Spinal Muscular Atrophy Research Group Meeting, Minneapolis, MN. June 2012.
Membership in Professional Societies
2003-2005 American Institute of Biological Sciences 2003-2005 Phi Sigma Biological Sciences Honor Society, Virginia Tech 2007-Present Society for Neuroscience 2011-Present Sigma Xi
vii
Preface
Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder and a leading inherited cause of infant mortality. SMA is a consequence of a deletion or mutation of the survival of motor neuron-1 (SMN1) gene with preservation of the nearly identical SMN2 gene. The over- arching aim of this dissertation is to characterize the degradation of the SMN protein. A detailed understanding of this mechanism could provide insight into methods of increasing levels of functional SMN protein as an approach to treatment of SMA. Based on a previously published report which showed that the ubiquitin proteasome system (UPS) is primarily responsible for degrading
SMN in cell culture, we hypothesized that the UPS could be targeted to increase SMN protein levels.
Chapter 1 of this thesis provides a brief history of the characterization of SMA and an overview of the genetic and molecular etiology underlying the disease. Chapter 2 examines the effect of proteasome inhibition on SMN protein levels and the SMA disease phenotype in vivo. In chapter 3, we discuss the characterization of a novel E3 ubiquitin ligase for SMN and investigate whether its targeted knockdown can improve the SMA phenotype in an animal model. Chapter 4 summarizes these findings, presents a mechanism by which SMN is degraded, and discusses future directions.
Delineating the mechanism underlying SMN protein stability may indicate how deficiencies in SMN cause disease and may demonstrate how this system may be controlled for therapeutic use.
viii
Acknowledgements
The work presented in this dissertation was supported by Brown University’s Neuroscience
Graduate Program, the Graduate Partnerships Program at the National Institutes of Health, and the
National Institute of Neurological Disorders and Stroke. First and foremost, I thank my graduate advisor, Dr. Kurt Fischbeck, for the opportunity to perform my thesis research under his guidance and support. His experienced knowledge of neurology and the inner workings of the spinal cord have made him an invaluable resource during my graduate career. I would also like to thank the members of the Fischbeck lab, both past and present. I owe a debt of gratitude to Dr. Barrington Burnett, whose wisdom, mentorship, and patience made it possible for me to develop this thesis, and I thank fellow graduate students, Katherine Bricceno and William Motley, both of whom always willingly dispensed scientific advice and celebrated and commiserated with me during the highs and lows of my graduate career. I also would like to thank the students who gave me the opportunity to grow my own mentorship and leadership skills. In particular, Jonathan Nofziger’s efforts were indispensable in expanding part of this thesis and saved me from exacerbating a developing allergy to rodent dander.
Vania Cao made the whole graduate school experience more enjoyable and helped me perfuse many mice, even creating a perfusion rig small enough to handle tiny SMA-affected pups. I also thank my thesis committee for guiding me through my thesis research—their advice and discussions were essential to the evolution of this dissertation.
Finally, I would like to dedicate this thesis to my parents, 권순엽 and 서승진, and my sister,
Sarah, for their love and support.
ix
Table of Contents
Curriculum vitae...... iv
Preface ...... viii
Acknowledgements...... ix
List of Figures...... xii
Chapter 1: Overview of spinal muscular atrophy ...... 1
1.1 Clinical History ...... 2
1.2 Genetics of SMA ...... 3
1.2.1 SMN gene structure ...... 4
1.3 SMN Protein Function ...... 5
1.3.1 SMN function in snRNP biogenesis ...... 5
1.3.2 Another role for SMN in neurons ...... 6
1.3.3 SMN function in the neuromuscular junction...... 7
1.4 Modulating SMN expression and levels...... 8
1.4.1 SMN degradation by the ubiquitin proteasome system...... 8
1.4.2 SMN upregulation by HDAC inhibitors...... 9
1.5 Overview of Thesis ...... 10
Chapter 2: Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice ...... 12
2.1 Introduction...... 13
2.2 Methods ...... 14
2.3 Results ...... 18
2.4 Discussion...... 22
x
Chapter 3: The E3 ubiquitin ligase Mind bomb 1 ubiquitinates and promotes the degradation of
SMN ...... 42
3.1 Introduction...... 43
2.2 Methods ...... 44
3.3 Results ...... 48
3.4 Discussion...... 53
Chapter 4: Summary, discussion and conclusions ...... 65
4.1 SMN in peripheral tissues ...... 66
4.2 SMN independent mechanisms of disease phenotype improvement ...... 66
4.3 Characterizing Mind bomb 1 as an E3 ligase for SMN...... 67
4.4 Current and future studies...... 69
4.5 Implications ...... 71
Bibliography ...... 76
xi
List of Figures
Figure 1.1 SMA is caused by mutations in the SMN1 gene ...... 4
Figure 1.2 The ubiquitin proteasome system...... 9
Figure 2.1 SMN is degraded by the UPS...... 28
Figure 2.2 SMN is increased in bortezomib treated cells ...... 29
Figure 2.3 Bortezomib increases SMN protein levels in vivo ...... 30
Figure 2.4 Bortezomib reduces proteasome activity in peripheral tissues of SMA mice...... 31
Figure 2.5 Bortezomib treatment does not increase SMN staining in anterior horn cells in lumbar spinal cord sections...... 32
Figure 2.6 Bortezomib improves motor function; does not affect survival ...... 33
Figure 2.7 Synergistic increase in SMN protein levels following TSA and bortezomib treatment...... 34
Figure 2.8 TSA increases SMN protein levels in vivo ...... 35
Figure 2.9 Combining TSA and bortezomib increases SMN in vivo ...... 36
Figure 2.10 TSA with bortezomib extends survival and improves the SMA phenotype in transgenic mice ...... 37
Figure 2.11 TSA with bortezomib ameliorates muscle pathology in SMA model mice...... 38
Figure 2.12 TSA with bortezomib delays motor neuron death in lumbar spinal cord tissues of SMA model mice...... 39
Figure 2.13 TSA with bortezomib improves the NMJ ...... 40
Figure 2.14 Co-administration of TSA and bortezomib continues to improve the NMJ ...... 41
Figure 3.1 Overexpression of Mib1 increases SMN ubiquitination and turnover...... 57
Figure 3.2 Effects of Mib1 overexpression on SMN protein levels and gem number ...... 58
xii
Figure 3.3 Decreasing Mib1 expression increases SMN protein levels ...... 59
Figure 3.4 Mib1 and SMN associate ...... 60-61
Figure 3.5 shRNA knockdown of Mib1 decreases Mib1 expression in pulse-chase experiments...... 62
Figure 3.6 Mib1 binds but does not ubiquitinate SMNΔ7 with mutant degron (S270A)...... 63
Figure 3.7 Knockdown of the C. elegans ortholog of Mib1 ameliorates the pharyngeal pumping neuromuscular defects of smn-1 loss of function animals with increased RNAi neuronal sensitivity ..64
Figure 4.1 Mib1 is increased in spinal cords of SMA mice ...... 72
Figure 4.2 P-glycoprotein structure and function ...... 73
Figure 4.3 Tariquidar and bortezomib increase SMN and decrease proteasome activity in both CNS and peripheral tissues and improve body weight and motor function in SMA mice...... 74
Figure 4.4 Schematic of SMN protein degradation ...... 75
xiii
Chapter 1
Overview of Spinal Muscular Atrophy
1
SMA is an autosomal recessive neurological disorder characterized by loss of lower alpha- motor neurons and skeletal muscle atrophy and it is one of the leading genetic causes of infant death.
Approximately 1 in 40 people in the United States and Europe are carriers for SMA, and an estimated 1 in 8,000 to 10,000 live births result in disease. SMA is associated with reduced levels of the SMN protein, which has the fundamental role of assembling small nuclear ribonucleoproteins
(snRNPs)-- molecules involved in pre-mRNA splicing (Battle et al, 2006; Lefebvre et al, 1995). In accordance with this function, loss of SMN generates defects in splicing and affects the range of snRNAs and mRNAs produced in tissues (Gabanella et al, 2007; Zhang et al, 2008). While SMN’s role in snRNP biogenesis has been well characterized, it remains uncertain why deletion of this ubiquitously expressed gene causes a progressive loss of alpha-motor neurons and a specific neuromuscular phenotype. Insight into SMN degradation in affected tissues may help to explain motor neuron susceptibility to SMN loss and provide a target for treatment of this debilitating disease.
Clinical History
The first published case of SMA was reported in 1891 by the Austrian neurologist, Guido
Werdnig. Werdnig described two brothers, both of whom had an onset of weakness at around 10 months of age and had died at the ages of 3 and 6. At autopsy, Werdnig discovered degeneration of ventral horn cells in the spinal cords of both children (Dubowitz, 2009). Much of the early literature describing the disorder was separately published by Johann Hoffmann from Heidelberg University, a neurologist who reviewed Werdnig’s two cases and added seven of his own. In a series of 3 papers,
Hoffmann included illustrations of muscle and spinal cord histology from SMA patients, which depicted the atrophy of spinal cord ventral cells Werdnig had first reported in his seminal paper
(Dubowitz, 2009). Detailed descriptions of the findings of SMA followed in 1903, when Charles E.
Beevor first documented the intercostal weakness and sparing of the diaphragm that are now known to be characteristic of the disease. Neurologists Kugelberg and Welander later described the
2
hallmarks of the mild, ambulatory form of SMA, which they observed in 12 cases from several families, and they were first to suggest that their disease might be a mild variant of Werdnig-
Hoffmann disease (Kugelberg & Welander, 1956).
The location of the gene defective in SMA (5q13) was first discovered in 1990 by a group in
New York studying a group of large Amish families with the mild Kugelberg-Welander form of SMA
(Brzustowicz et al, 1990)and subsequently by Judith Melki’s group in Paris (Melki et al, 1990). Both additionally confirmed the same gene locus for Werdnig-Hoffmann’s disease, thereby linking both disorders into one, and Melki’s group went on to isolate and characterize the gene itself, which they named survival motor neuron (SMN) (Lefebvre et al, 1995).
Because of the wide range of severity that is observed in patients, SMA has been categorized into 3 types based on age of onset and disease manifestations:
Type I: Severe SMA (Werdnig-Hoffmann disease); characterized by early onset (< 6
months), severe muscular weakness, inability to sit or stand, and early death (~2 years)
Type II: Intermediate form; characterized by an ability to sit but not stand.
Type III: Juvenile SMA (Kugelberg-Welander); patients generally survive into adulthood
and experience muscular weakness yet develop the capability to stand without assistance.
Genetics of SMA
Two types of SMN are normally present at chromosome 5q. SMA is caused by deletion and other mutations of the telomeric copy of SMN1, with all patients retaining at least one copy of the centromeric homologue, SMN2 to produce a truncated protein, termed SMNΔ7, that is unstable and rapidly degraded (Coovert et al, 1997; Lorson & Androphy, 2000; Lorson et al, 1999; Monani, 2005;
Monani et al, 1999). The nucleotide change disrupts an SF2/ASF splicing factor binding site in
SMN2, converting an exon splice enhancer in exon 7 into a silencer (Cartegni & Krainer, 2002).
3
Figure 1.1 SMA is caused by mutations in the SMN1 gene. Patients with SMA have complete loss of SMN1 but at least one copy of SMN2, which produces insufficient levels of the full- length SMN protein.
The SMN2 gene is located in a highly unstable region of chromosome 5q13 and is often present in multiple copies. Multiple copies of SMN2 in transgenic mice can alleviate severity of the
SMA disease phenotype on a SMN1 null background, consistent with a gene dosage effect (Hsieh-Li et al, 2000). Indeed, patients with mild SMA phenotypes (SMA type III) have often been found to have multiple SMN2 copies and individuals with as many as 4 or 5 SMN2 genes have been found to be phenotypically normal (Lefebvre et al, 1995; McAndrew et al, 1997; Prior et al, 2004). These findings suggest a possibility for treatment of SMA in increasing levels of existing functional SMN protein.
SMN gene structure
The SMN gene consists of nine exons (exons 1 through 8, with two exons, 2a and 2b, composing exon 2) and eight introns, totaling approximately 20 kb in length (Burglen et al, 1996). A
4
stop codon at the end of exon 7 precludes translation of exon 8; however, alternative splicing of
SMN2 results in the loss of the normal 16 amino acids of exon 7 and gains instead four amino acids,
EMLA, encoded by exon 8 (Cho & Dreyfuss, 2010). The acquisition of these four amino acids to the
C-terminal of exon 6 has been proposed to create a degron, a protein destabilizing sequence that results in the rapid degradation of SMNΔ7 (Cho & Dreyfuss, 2010).
Regions of the SMN protein encoded by various exons in the transcript have been found to play distinct roles in SMN protein function. Exon 2b is known to encode a domain responsible for
Gemin 2 binding (Young et al, 2000), and the Tudor domain encoded by exon 3 interacts with Sm proteins, both of which are critical for SMN’s role in snRNP biogenesis (Buhler et al, 1999). Exon 5 encodes a proline-rich region and has been found to be important for the interaction with the actin- binding protein, profilin (Giesemann et al, 1999). Exon 6 encodes the self-oligomerization domain of
SMN; mutations or deletions in this region prevents SMN self-association and increases SMN protein turnover (Burnett et al, 2009b; Lorson et al, 1998a).
SMN Protein Function
SMN function in snRNP biogenesis
Full-length SMN is a 38 kDa protein that is concentrated in puncta-like structures in the nucleus and diffusely throughout the cytoplasm of all cells. As previously mentioned, SMN is best characterized as a critical mediator of snRNP biogenesis. snRNPs are essential for splicing out introns from pre-mRNA, and are assembled by the SMN complex, which consists of the SMN, gemins 2-8 and UNR-interacting proteins. SMN’s ability to self-associate and form multimeric structures stabilizes the protein and prevents its degradation via the ubiquitin proteasome system
(UPS) (Burnett et al, 2009b; Lorson et al, 1998a; Paushkin et al, 2002). Mutations in SMN that affect its ability to self-associate and incorporate into the gemin complex, such as the loss of exon 7, reduce
SMN half-life (Burnett et al., 2009). Missense mutations have been found in SMA patients that
5
impair SMN complex formation may, therefore, cause a rapid degradation of unassociated SMN in cells and result in low SMN levels, a disruption of snRNP biogenesis, and an alteration in splicing.
Motor neurons, as large cells with high-energy demand, may be particularly sensitive to loss of SMN and consequently altered snRNP biogenesis and defective pre-mRNA splicing of genes that are critical for proper motor neuron circuitry may also underlie the degeneration of these cells.
Another novel role for SMN in neurons
Immunocytochemistry experiments have also revealed the presence of SMN in granules along the axons and growth cones of chick cortical neurons and rat spinal motor neurons that move bidirectionally and localize with microtubules (Zhang et al, 2003). These studies point to a neuron- specific function for the SMN protein and may provide an alternate explanation for the progressive motor neuron degeneration seen in SMA. It is thought that SMN in these granular structures helps to traffic specific RNAs to the axon tip of developing motor neurons in response to local cues during development. Support for this hypothesis comes from data which show that SMN reduction leads to decreased growth cone size, shorter neurites in primary motor neurons from SMA mice (Rossoll et al, 2003), and aberrantly truncated motor neurons in SMA zebrafish (McWhorter et al, 2003a). Loss of SMN may decrease the transport of mRNAs to the synapse, where their translation may be required for the formation and maintenance of the synapse or for proper axonal outgrowth and pathfinding. It is known that SMN interacts with β-actin RNA-binding protein, heterogeneous nuclear ribonucleoprotein R, and co-localization of SMN-Gemin2-Gemin3 positive granules with β- actin mRNA has been detected in the axons of differentiated human-derived neuroblastoma cells
(Rossoll et al, 2003; Todd et al, 2010). SMN has also been shown to associate with the neuron- specific RNA-binding protein, HuD, and its known mRNA interactor, candidate plasticity-related gene 15 (cpg15), in the axons and growth cones of cortical neurons. Over-expression of cpg15 mRNA partially rescues motor neuron axon growth defects in an SMA zebrafish model, lending support to a role for SMN in mRNA transport and axonal growth (Akten et al, 2011; Fallini et al,
6
2011). Other target transcripts have yet to be identified, and it is still unclear whether loss of an SMN function mediating translation in nerve terminals or pre-mRNA splicing is the primary cause for the motor neuron death.
SMN function in the neuromuscular junction
In support of a neuron-specific role for SMN, a recent study reported a list of defects observed in the neuromuscular junctions (NMJs) of SMA mice which included their reduced arborization, abnormal synaptic transmission, and delayed maturity (Kong et al, 2009). While the authors noted that the NMJs of affected mice remained well-connected even late in the disease course, electrophysiological recordings indicated a reduction in quantal content. Corresponding with this result, electron microscopy studies showed a decrease in synaptic vesicles that most likely contributed to the observed reduced release probability. Furthermore, NMJs of SMA mice showed a delayed switch in the subunit composition of acetylcholine receptors (AChRs) from the embryonic subunit to the adult subunit and SMA NMJ postsynaptic terminals remained small and simplified compared to those in control littermates. Similarly, results from another group demonstrated that while muscle fibers from SMA mutant mice fired normal action potentials, there were severe defects in evoked neurotransmission in the presynaptic terminals, resulting in a slowed, asynchronous release of neurotransmitters (Ruiz et al, 2010). The kinetics of the postsynaptic responses, which normally increase during postnatal development of the NMJ (due to the switch from the fetal subunit to the adult unit of the nicotinic receptor), remained slow and did not mature. The same group also performed immunostaining of the NMJs from which they recorded and reported plaque-like structures that did not progress to the perforated, pretzel-like formation typical of a mature NMJ
(Ruiz et al., 2010). Furthermore, depletion of SMN in motor neuron progenitor cells alone can cause neuromuscular weakness in mice, specifically resulting in NMJ defects and muscle atrophy (Park et al, 2010).
7
In addition, analysis of conditional SMA mice with tissue-specific correction of SMN deficiency showed that increasing SMN in motor neurons alone improved NMJ synaptic function but that SMN expression exclusively in muscle failed to affect either NMJ physiology or structure
(Martinez et al, 2012). The impaired neurotransmission at NMJs and delayed maturation of motor units in SMA mice indicate how reduced levels of SMN produce a neuromuscular disease phenotype and underscore a critical role for SMN in nerve terminals. However, SMN deficiency has also been reported to intrinsically affect muscle development and defects in the muscle may also compromise
NMJ integrity (Braun et al, 1995; Rajendra et al, 2007). Nerve-muscle co-culture studies in which motor neurons were normal and skeletal muscle cells were acquired from SMA patients resulted in degeneration of myotubes, suggesting an inherent abnormality in SMA-affected muscle tissue (Braun et al., 1995). Muscle-specific SMN expression in SMA conditional mice was also found to improve myofiber size, body weight, motor function and survival (Martinez et al., 2012). These findings highlight the role of the postsynaptic components of the NMJ, indicating that SMN may have important functions in both muscle and motor neurons. Restoration of SMN in both tissues is likely necessary for full recovery of the motor unit.
Modulating SMN gene expression and protein levels
SMN degradation by the ubiquitin proteasome system
Mammalian cells have a system dedicated to proteolysis called the ubiquitin-proteasome system.
In this system, proteins destined for degradation are targeted by linkage to the cofactor ubiquitin through the action of three enzymes termed E1 (ubiquitin-activating), E2 (ubiquitin-conjugating), and E3 (ubiquitin ligase) (Petroski, 2008). A ubiquitin molecule is first activated in an ATP- dependent manner by a single E1 enzyme before its transfer onto E2 conjugating enzymes. It is then further transferred and linked to the substrate protein by the E3 ligase, which binds and recruits the protein to be degraded (Figure 1.2; Petroski 2008). E3 ligases are thought to confer the substrate- specificity in this process and thus have been considered potential therapeutic targets. One such E3
8
ligase, called murine double minute 2 (MDM2), regulates the tumor suppressor protein p53, and represses p53’s role in cell cycle arrest and apoptosis (Fakharzadeh et al, 1993; Haupt et al, 1997).
Inhibitors of the human homologue of MDM2 have been shown to inhibit tumor growth and induce tumor cell apoptosis in vitro, providing an incentive for targeting MDM2 function to treat cancer in vivo (Issaeva et al., 2004; Koblish et al., 2006).
Figure 1.2 The UPS. As a major degradation pathway in the cell, the UPS functions to destroy proteins that are marked with a chain of ubiquitin molecules. The enzymes that aid in this process are termed E1, E2 (which activate and conjugate the ubiquitin molecule respectively) and E3, which is responsible for binding the substrate protein and catalyzing its ubiquitination. The ubiquitinated protein is then shuttled to the 26S proteasome where it is degraded into oligopeptides (Meusser et al, 2005).
Several findings indicate that SMN is ubiquitinated and degraded by the ubiquitin proteasome system (Chang et al., 2004; Burnett et al., 2009). Treatment of SMA patient fibroblasts with the proteasome inhibitor, MG132, produced a >2-fold increase in SMN protein levels while inhibition of other degradatory pathways had no effect. Moreover, incubation of cells with proteasomal inhibitors increased ubiquitinated SMN residues and in-vitro ubiquitination assays further confirmed SMN as a substrate of the UPS (Burnett et al. 2009). These results point to the
UPS as a potential target for increasing SMN levels in cells.
9
SMN upregulation by HDAC inhibition
Another well-studied approach to increasing SMN is to augment SMN gene expression, specifically in increasing SMN mRNA levels with histone deacetylase (HDAC) inhibitors. These drugs have been proposed as a treatment for SMA and are a point of discussion in this thesis.
DNA in cells is densely packaged around histones, which form spool-like structures in the nucleus (Richmond & Davey, 2003). Post-translational modification of these histones, the essential components of chromatin, forms the basis of the ‘histone code’ and histone modifications known to affect transcriptional activity of the genome include acetylation and deacetylation. The former is associated with euchromatin and promotes DNA transcription while the latter, mediated by HDACs, is generally associated with heterochromatin and gene repression (Minucci & Pelicci, 2006). Studies have shown that the SMN gene shows a developmental pattern of histone acetylation and associates with HDACs 1 and 2 (Kernochan et al., 2005). Moreover, several HDAC inhibitors upregulate the
SMN2 transcript and increase SMN protein levels, including valproic acid, sodium butyrate, trichostatin A (TSA), phenylbutyrate, and vorinostat (Andreassi et al, 2004; Brichta et al, 2003;
Brichta et al, 2006; Chang et al, 2001; Hahnen et al, 2006; Riessland et al, 2006; Sumner et al, 2003).
As described in further detail in chapter 2, TSA increases both SMN transcript and protein levels in
SMA mice, enhancing motor function and extending lifespan (Avila et al., 2007).
Overview of Thesis
The work described in this thesis explores the regulation of SMN protein degradation by the ubiquitin proteasome system. The second chapter analyzes the effect of the proteasome inhibiting drug, bortezomib, which increases SMN protein levels in cell culture but is unable to cross the blood brain barrier. While the genetic cause of SMA has been well characterized, it remains to be determined how much the loss of SMN has a pathogenic effect in the CNS versus in peripheral tissues. Resolving this question would help focus future therapies for SMA and their targets. Using bortezomib in SMA model mice, we addressed whether increased SMN in peripheral tissues
10
ameliorates the SMA disease phenotype. In the same chapter, we also describe experiments in which we attempted to upregulate the SMN gene expression while inhibiting degradation of the SMN protein, with the hypothesis that this combined approach would have a synergistic effect on SMN levels.
The third chapter of this thesis looks upstream of the proteasome to modulate SMN protein turnover. We identified and characterized the role of Mind bomb 1, an E3 ubiquitin ligase, in the ubiquitination and degradation of SMN. Several drugs that target E3 ligases involved in cell cycle regulation and cell proliferation are under development as potential treatment for various types of cancers, demonstrating this class of enzymes may be pharmacologically tractable.
The final discussion chapter summarizes these results and presents a model for the mechanism by which SMN is degraded.
11
Chapter 2
Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice
Sections of this chapter have been published:
Burnett BG, Muñoz E, Tandon A, Kwon DY, Sumner CJ, Fischbeck KH. Regulation of SMN protein stability. Mol Cell Biol. 2009 Mar;29(5):1107-15.
Kwon DY, Motley WW, Fischbeck KH, Burnett BG. 2011. Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice. Hum Mol Genet. 2011 Sep 15;20(18):3667-77.
Contributions:
In this chapter, I replicated the data in Figure 2.1, performed all of the experiments presented in Figures 2.2 to 2.12 and prepared animals for NMJ analysis in Figures 2.13 and 2.14.
12
Introduction
Spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disorder, is one of the leading genetic causes of infant death. SMA results from deletion of the survival of motor neuron-1 (SMN1) gene and a consequent deficiency of the SMN protein. Although a second nearly identical gene, SMN2, is retained in SMA patients, the SMN2 gene primarily produces an alternatively spliced isoform lacking exon 7, which encodes a protein that is largely unstable and rapidly degraded. As discussed in chapter 1, multiple copies of SMN2 in transgenic mice and SMA patients can alleviate severity of the SMA disease phenotype. Thus, a promising approach to treating
SMA could include increasing levels of functional SMN protein by increasing SMN2 transcript levels and blocking the degradation of its gene product.
SMN is degraded through the ubiquitin proteasome system (UPS) (Burnett et al, 2009b;
Chang et al, 2004). In this system, proteins destined for degradation are targeted by linkage to ubiquitin through the action of various enzymes. Once a chain of four or more ubiquitin moieties is assembled on a protein, it is delivered to the 26S proteasome for proteolysis. Contained within the central core of each 26S proteasome are six active proteolytic sites; two cleave after hydrophobic residues (chymotrypsin-like), two after basic residues (trypsin-like), and two others after acidic residues (caspase-like) (Kisselev et al, 2006). Together, these sites catalyze the breakdown of proteins into short oligopeptides. Because the proteolytic sites in the 26S proteasome function by different mechanisms, it has been possible to develop drugs that inhibit one or two active sites in the 20S core without rendering the entire proteasome nonfunctional. The clinically used dipeptide boronic acid, bortezomib, is one such drug, which reversibly inhibits chymotrypsin cleavage in the proteasome without affecting the other active sites. Bortezomib was selected from an in vitro screen for its pro- apoptotic and anti-tumor profile and is currently approved for treatment of multiple myeloma.
(Adams et al, 1999)
13
We confirmed the SMN protein as a substrate of the UPS by targeting this pathway to block the degradation of SMN protein. Treatment of cultured cells with proteasome inhibitors MG132 and lactacystin increased SMN protein levels; however these drugs are not amenable for therapeutic use due to their off-target effects and toxicity (Burnett et al, 2009b) In this chapter, we characterized bortezomib’s effect on SMN protein levels in SMA patient-derived cell lines and on the SMA phenotype in SMA model mice. We asked whether we could synergistically increase SMN protein levels by combining bortezomib with trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor previously shown to upregulate SMN gene transcription (Avila et al, 2007; Narver et al, 2008). Our study demonstrates that a two-drug regimen that increases gene transcription and stabilizes protein levels may be a promising therapy for SMA, and that peripheral tissues, such as the muscle, may be targeted to improve the SMA phenotype.
Methods
Cell culture and drug treatment
Human fibroblast cell lines from a 3-year old type I SMA patient (GM03813) and a 2-year old type I SMA patient (GM09677) were obtained from Coriell Cell Repository. For proteasome inhibitor studies, cells were either plated in growth media alone or with MG132 (Biomol
International), lactacystin (Sigma-Aldrich), or bortezomib (Millenium), which was dissolved in
DMSO or water before use. TSA was dissolved in DMSO to a concentration of 50 nM and used to treat cells alone or in combination with bortezomib. The cells were incubated with either bortezomib alone (16 hrs) or with TSA for 8 hrs twice, or a combination of TSA and bortezomib overnight. The cells were then harvested for protein isolation and quantification as previously described (Burnett et al, 2009b).
14
Mice and drug treatment
Studies were approved by the National Institute of Neurological Diseases and Stroke Animal
Care Committee and performed in accordance with the National Institutes of Health Guide for the
Care and Use of Laboratory Animals. Transgenic SMA mice on the FVB background
(hSMN2/delta7SMN/mSmn-/-) were purchased from Jackson Laboratories. Animals were genotyped using polymerase chain reaction (PCR) on tail DNA as previously reported (Kernochan et al, 2005).
Bortezomib was dissolved in sterile water to a concentration of 0.15 µg/µl. Pups at postnatal day 5 (P5) were administered 1 µl of bortezomib per gram (for a concentration of 0.15 mg/kg) intraperitoneally using a 33-gauge needle and treated every other day until P13. Control animals received equal volumes of water.
TSA was dissolved in DMSO to a concentration of 4 µg/µl. Pups at P5 were administered 1
µl of TSA per gram daily for a total concentration of 4 mg/kg in a manner similar to bortezomib administration. In order to reduce toxicity with the drug combination, mice were given TSA in the mornings and bortezomib in the evenings.
For one-time measures, pups were weighed and tested for their ability to right themselves.
Righting time was defined as the average of two trials of the time required for a pup to turn over after being placed on its back (maximum 30 seconds). Mice that lost 30% of their body weight and were unable to right themselves were euthanized.
For biochemical studies, the mice were anesthetized with isofluorane and sacrificed by cervical dislocation. Fifty-100 mg of various tissues (brains, livers, spinal cords, kidneys, limb skeletal muscle) were dissected, flash-frozen in liquid nitrogen, and stored at -80°C.
For survival studies, litters were maintained and kept with the mother until P21, after which they were weaned and tested.
15
Protein extraction and quantification
Tissues were homogenized and incubated in 500 ul of lysis buffer (0.1% NP-40 and 0.5% sodium deoxycholate) for 15 minutes on ice, and the collected supernatant was sonicated for 10 seconds before another 15 minute incubation. Supernatant was collected after a 15 min centrifuge step at 4°C and stored at -80°C. Protein concentrations were determined by the BCA Protein Assay kit (Pierce) according to the manufacturer’s protocol.
Western blotting
Protein lysates (25 ug for wild type and heterozygous mice, 50-100 ug for SMA mice) were run and separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. These were then probed with a mouse anti-SMN antibody (BD Transduction Laboratories, diluted 1:1000), anti-ChAT antibody (Millipore, dilution 1:1000) and a mouse anti-B-actin antibody (Sigma-Aldrich, diluted
1:10,000).
Pathological analysis
The mice were transcardially perfused with 4% paraformaldehyde. Lumbar spinal cords and distal hind limbs were dissected and postfixed in the same fixative overnight. Hind limb tissues were decalcified, embedded in paraffin, and cross-sectioned at the midpoint of the tibilias anterior muscle.
Sections (10 μm) were mounted on slides and stained with hematoxylin and eosin. Digital images were captured using a Zeiss Axiovert 100M microscope and analyzed with NIH ImageJ software for total TA cross sectional area (original magnification, ×5), total tibilias anterior myofiber number
(original magnification, ×10), and myofiber diameter (original magnification, ×40). Myofiber diameter was determined by measuring the largest diameter of at least 300 neighboring myofibers per animal. Paraffin-embedded lumbar spinal cord was serially sectioned at 5-μm steps, mounted on slides, and stained with Nissl. Images of contiguous sections, 170 μm apart (original magnification,
16
×10) were analyzed with NIH ImageJ software. The diameter and number of all neurons greater than
25 μm in the region below a line drawn horizontally at the level of the spinal canal were determined.
Neuromuscular junction staining
Tibialis anterior muscles were isolated from P13 or P19 mice and fixed in freshly prepared
2% paraformaldehyde in phosphate buffered saline (PBS) for four hours on ice. Samples were then transferred to a blocking and permeabilizing solution of 5% normal goat serum and 0.5% Triton-X
100 in PBS for one hour before they were pressed between two glass slides using a binder clip for 15 minutes, after which the samples were returned to the blocking and permeabilizing solution. The samples were then incubated overnight at 4°C with 1:1000 dilutions of SV2 and 2H3 primary antibodies (Developmental Studies Hybridoma Bank, Iowa City, IA), which target synaptic vesicle protein 2 and neurofilament, respectively. After at least three one-hour washes in PBS with 0.5%
Triton-X 100, the samples were transferred to blocking and permeabilizing solution with Alexa Fluor
488 goat anti-mouse IgG1 (y1) (Invitrogen, Carlsbad, CA) and α-bungarotoxin conjugated with Alexa
Fluor 594. After incubation overnight at 4°C samples were washed three times for one hour and mounted with Vectashield mounting media (Vector Labs, Burlingame, CA) and imaged using an
LSM 710 laser scanning microscope (Carl Zeiss, Oberkochen, Germany). Neuromuscular junction size measurements were determined using the Volocity software (Improvision, Perkin Elmer,
Waltham, MA). The objects were found using standard deviation of intensity with a lower limit of two. The objects were then sorted to exclude those with a volume below 300 µm3 and those touching the edge of the image before they were measured. This staining protocol was adapted from (Le et al,
2005).
Statistics
Survival and biochemical data were analyzed using the GraphPad Prism software package
(version 3; GraphPad Software) and compared statistically with the log-rank test (Kaplan-Meier
17
survival curves), the 2-tailed Student’s t test or 1-way analysis of variance followed by the Newman-
Keuls multiple comparison post-hoc test. Pathological data were analyzed using STATA version 9 software. To compare differences among the three groups, a nonparametric equality of medians test was performed, because the data were not normally distributed. If this was statistically significant, then a pairwise comparison between the two treatment groups was performed using a Mann-Whitney
U test. P ≤ 0.05 was considered significant.
Results
SMA patient derived fibroblasts were treated with the commercially available proteasome inhibitor, bortezomib (Velcade®, previously PS-341), to determine whether it would increase SMN protein levels. Similar to previously published results using other proteasome inhibitors, SMN levels increased in a dose-dependent manner in the presence of bortezomib (Figure 2.2A). This correlated with an accumulation of ubiquitinated SMN, indicating that the increase in SMN protein was due to proteasome inhibition rather than to off-target effects of the drug (Figure 2.2B). Given bortezomib’s ability to increase SMN and its low toxicity profile compared to that of other proteasome inhibitors, we then proceeded to test the drug in SMA model mice.
To study bortezomib’s efficacy in vivo, we treated SMA pups (SMN2 +/+, SMNΔ7, Smn-
/-) by intraperitoneal (I.P.) injections (0.15 mg/kg) starting at P5 and continuing every other day. We found this was the maximum tolerated dose for mice at this age, as the doses we tested exceeding
0.15 mg/kg resulted in gastrointestinal toxicity (diarrhea) and premature death. SMA pups were treated until P13 and then sacrificed to examine SMN levels in various tissues by western blot. We observed an approximately two-fold SMN increase in the liver, muscle, and kidney tissues of bortezomib-treated mice, while levels of SMN in the spinal cord and brain remained unchanged
(Figure 2.3). Bortezomib has low central nervous system (CNS) penetrance, and brain and spinal cord tissues from bortezomib-treated animals showed no change in proteasome activity (Figure 2.4).
Since SMN levels in the peripheral nervous system could be increased by bortezomib treatment and
18
affect SMN levels in the anterior horn cells (AHC) of the spinal cord, we stained lumbar spinal cord sections in vehicle and bortezomib treated mice. However, we did not observe a statistically significant change in SMN with bortezomib treatment in these cells (Figure 2.5).
Bortezomib improves the motor function of SMA model mice but does not extend lifespan.
We then asked whether the increase in SMN protein levels we observed in peripheral tissues is sufficient to ameliorate the SMA phenotype. SMA mice were given bortezomib (0.15 mg/kg) as described above, but with treatment continuing until weaning at P21. Motor function was assessed daily as the average time it took for the mice to right themselves once placed on their backs. The righting times of bortezomib-treated animals were improved starting at P8 (Figure 2.6B; P < 0.05).
However, bortezomib had no overall effect on the survival of these mice, although some decrease in early deaths was observed (Figure 2.6A). These results suggest that an increase in SMN protein levels in peripheral tissues may improve motor behavior, but that increased SMN in the central nervous system may be required for an extension of lifespan.
Bortezomib and TSA synergistically increase SMN protein levels and improve survival and motor function in SMA model mice.
Since our data showed that bortezomib treatment blocks SMN degradation in the peripheral tissues of SMA mice, we next asked whether upregulating the SMN2 gene throughout these animals might synergize with bortezomib and confer a greater therapeutic effect on the SMA phenotype in vivo. To test this hypothesis, we used a combined regimen of bortezomib and TSA, a CNS-penetrant
HDAC inhibitor that has been previously shown to increase SMN transcript levels in vitro and in vivo
(Avila et al, 2007). Treatment of SMA patient-derived fibroblasts with either drug alone increased
SMN protein levels but giving both drugs together was additive, consistent with the actions of the drugs on distinct pathways (Figure 2.7).
19
We next sought to determine whether delivery of both bortezomib and TSA affects the
SMA phenotype in the mice. Since inhibition of the proteasome can last for up to 48 hours (data not shown), mice were treated every other day with bortezomib and daily with TSA, starting at P5. In order to mitigate the gastrointestinal toxicity we initially observed administering both drugs together, we reduced the dose of TSA to 4 mg/kg and bortezomib to 0.075 mg/kg. We found that even at this lower dose SMN protein levels were increased in tissues from TSA treated mice compared to those treated with vehicle (Figure 2.8). In all tissues isolated, SMN protein levels were significantly increased in TSA and bortezomib treated animals at P13 compared to those treated with vehicle alone (Figure 2.9). SMN was increased in peripheral tissues above levels observed with either bortezomib or TSA alone when both drugs were co-administered.
Combining TSA and bortezomib treatment also extended the survival of SMA mice more than TSA alone. Mice treated with 4 mg/kg of TSA lived approximately 3 days longer than vehicle
(DMSO)-treated mice similar to effects reported with 10 mg/kg of TSA (Avila et al, 2007), however average lifespan was increased to 6 days when both TSA and bortezomib were given in combination
(Figure 2.10). While we observed a similar improvement in the righting times of TSA only and TSA and bortezomib treated mice, we found that animals treated with both drugs gained more weight over the course of treatment on average than those treated with either vehicle or TSA alone (Figure
2.10B).
Bortezomib and TSA improve the motor unit pathology in SMA mice
Based on the motor improvement we observed with TSA and bortezomib treatment we next examined whether there was structural improvement of the motor unit. Cohorts of vehicle-, bortezomib-, TSA-, and TSA plus bortezomib-treated mice were sacrificed at P13, and muscle and spinal cord tissues were isolated for histological examination (n=3, each group). Hematoxylin- and eosin-stained cross sections of tibialis anterior (TA) muscles from P13 SMA mice showed a
20
significant increase in myofiber size with either bortezomib (P<0.05) or TSA treatments alone
(P<0.01) compared to vehicle-treated mice (Figure 2.11). We did not observe a statistically significant increase in myofiber size with the combination of TSA and bortezomib, however, treatment with both drugs increased myofiber number over both bortezomib (P<0.05) and TSA alone (P<0.01) treatments.
We next analyzed the number of neurons greater than 25 µm in diameter in the anterior horn of the lumbar spinal cord. We found a nearly two-fold increase in the number of anterior horn cells (AHCs) per section in mice treated with bortezomib alone or bortezomib in combination with
TSA (Figure 2.12). TSA treatment alone did not increase AHC numbers, consistent with previously published data (Avila et al, 2007). Furthermore, there was no significant difference in AHC numbers between mice treated with bortezomib alone and those treated with TSA with bortezomib, indicating that the observed increase was likely an effect of bortezomib alone. We have previously reported that expression levels of choline acetyltransferase (ChAT), a motor neuron marker, are reduced in SMA mice compared to heterozygous littermates (Avila et al, 2007). To further validate the increase in
AHCs observed in drug treated mice, we also analyzed ChAT levels in these animals by western blot.
ChAT levels were only slightly increased in TSA-treated mice (Figure 2.8; P=0.066). However, we observed an approximate four-fold increase in ChAT protein levels in animals treated with bortezomib alone (Figure 2.12B; P=0.055) or with the combination of bortezomib and TSA
(Figure 2.12B; P=0.029), suggesting that these drugs reduce motor neuron loss in SMA mice.
Given the low availability of bortezomib in the CNS, the increase in AHC number in mice treated with the drug was surprising. Recent studies have identified a role for SMN in the maturation of NMJs, with poor terminal arborization, denervation, impaired synaptic vesicle release, neuronal antigen accumulation, and decreased NMJ size in SMA mice. We therefore examined the effect of bortezomib treatment on the NMJ, comparing NMJs in the TA muscles of affected mice at P13.
Alpha-bungarotoxin was used to identify the postsynaptic side of the NMJ, and antibodies to
21
synaptic vesicle protein 2 and neurofilament were used to visualize presynaptic morphology. As previously reported, we found that SMA NMJ postsynaptic terminals were small and simplified compared with those in control littermates (Figure 2.13). Quantification of the size indicated that the morphology of SMA NMJs was significantly increased with bortezomib treatment alone. TSA alone also improved the mean surface area of SMA NMJs, and an additive effect was observed when both drugs were delivered together. Qualitative comparison also showed that there were incremental improvements in NMJ maturity as indicated by an increase in invaginations in the post-synaptic staining (Figure 2.13, white arrows). We then compared the NMJ morphology from SMA mice treated with TSA and bortezomib and unaffected littermates at P19. The NMJs in TSA and bortezomib treated mice continued to develop and mature between P13 and P19, similar to control littermates, even as the condition of these animals began to decline (Figure 2.14). Our results suggest that the motor improvements we observed in SMA mice treated with bortezomib alone could be due to improvement at the NMJ and that extended survival in TSA and bortezomib treated mice may be due to amelioration of motor unit pathology. We note that TSA also increases NMJ size but does not increase AHC number, suggesting that TSA and bortezomib affect the CNS by different mechanisms.
Discussion
Our results validate the UPS as a major degradative pathway for SMN. Steady-state SMN protein levels were increased in cells treated with proteasome inhibitors, but were unchanged when other degradative pathways were inhibited (Burnett et al., 2009). While it has been reported that the
SMN protein is degraded by calpains in muscle cells (Fuentes et al, 2010; Walker et al, 2008) we did not observe any change in SMN protein levels when SMA patient fibroblasts were treated with the calpain inhibitor, calpeptin. Calpains are proteases activated by calcium and are known to be involved in many cellular processes. Unlike the proteasome, which destroys proteins, calpains cleave their substrate proteins and may regulate their activity rather than degrade them (Goll et al, 2003). There
22
are several possible reasons for the difference in our results. It may be due to indirect or off-target effects of calpeptin, a nonspecific calpain inhibitor, or it may be due to a cell-specific interaction between calpains and SMN in muscle cells, which were not tested in our study. More importantly, the same study also showed that only a small population of SMN protein interacts with endogenous calpains—the SMN C-terminal cleavage product only accounted for a small percentage of total SMN in cells after the addition of calcium—and is nearly exclusively cleaved by cytoplasmic calpain alone
(Fuentes et al., 2010). Nuclear SMN was found to be resistant to the addition of either calcium or exogenous calpain (Fuentes et al., 2010). It is likely that these reasons accounted for the inability of calpeptin to modulate SMN in our experiments.
Intraperitoneal administration of the proteasome inhibitor, bortezomib, resulted in a two- fold increase in SMN protein levels in peripheral tissues of SMA model mice. Affected mice treated with bortezomib alone showed improved righting times but no significant change in lifespan. The lack of a survival benefit in the absence of CNS availability of bortezomib highlights the critical role of SMN in the CNS. Nevertheless, we observed increased myofiber size and number in bortezomib- treated mice and, surprisingly, an increased number of AHCs in the spinal cord.
Immunohistochemical examination of the NMJ of bortezomib-treated mice showed increased NMJ size compared to vehicle-treated mice. This improvement in NMJ size could explain the increase in
AHC number and the improvement in motor function we observed in treated animals despite the lack of increased SMN levels in the CNS with bortezomib treatment and suggests that treatments that target peripheral tissues may contribute to improving the disease phenotype.
It remains unclear whether SMA is cell autonomous, i.e. caused by the effects of reduced
SMN in other multiple tissues or solely in motor neurons. In Drosophila, a null mutation in Smn is partially rescued by maternal SMN expression, which allows development to the larval stage (Chan et al, 2003). The eventual depletion of SMN in all tissues results in death and can only be rescued by providing SMN to both muscles and neurons. Depletion of SMN in muscle in flies and mice results in muscle degeneration, indicating that SMN is an essential protein in muscle, as in other tissues
23
(Cifuentes-Diaz et al, 2001). Recent work with transgenic mice expressing SMN in muscle under the control of the human skeletal muscle actin (HSA) promoter showed that SMN restoration in skeletal muscle alone has no appreciable impact on the SMA phenotype (Gavrilina et al, 2008). However, this does not rule out the possibility that increasing SMN levels in muscle may enhance whatever beneficial effect repletion of motor neuron SMN may have. Two studies examined inhibitors of the myostatin pathway in SMA mice with contrasting results, one showing a modest extension in lifespan and gross motor function, with delivery of recombinant follistatin, the other detecting no phenotypic improvement in the SMA mice with ActRIIB-Fc treatment or transgenic overexpression of follistatin
(Rose et al, 2009; Sumner et al, 2009). The basis for this discrepancy is unclear; however, the possibility remains that motor neurons may require additional support in peripheral tissues to respond optimally to SMN-based therapeutics. In this study we found that a 2-fold increase in SMN in peripheral tissues had no effect on the survival of SMA mice, but a similar increase in SMN in animals treated with the CNS penetrant drug, TSA, was sufficient to significantly improve survival.
Taken together, these studies would indicate the need for adequate levels of SMN in the CNS for survival.
While this study was ongoing, it was reported that the hydroxamic acid-derived HDAC inhibitor LBH589 markedly increases SMN levels in SMA patient-derived fibroblasts by both increasing SMN2 gene expression and blocking SMN protein degradation (Garbes et al, 2009).
Similarly, we show here that TSA and bortezomib delivered together synergistically increased SMN protein levels in cultured cells and tissues of SMA mice. Mice treated with both drugs lived longer and showed increased body weight. Immunohistochemical analysis of NMJs in mice treated with both drugs showed a cumulative effect on their maturation. Although TSA alone increases SMN protein levels, mitigates muscle and nerve pathophysiology and extends the lifespan of SMA model mice (Avila et al, 2007), co-administering TSA with bortezomib approximately doubled SMN levels in affected mice compared to TSA alone and further extended survival from 3 to 6 days.
24
Of particular interest in this study was the finding that bortezomib alone significantly improved the motor function of SMA mice. We observed an increase in SMN protein levels in muscle tissue of treated mice and increases in the number of myofibers and myofiber diameter size.
AHC loss was also delayed in bortezomib-treated animals, indicating a central effect of this peripherally acting drug. These SMA mice show motor deficits as early as postnatal day 2, although they do not have significant spinal motor neuron loss at this stage and there is evidence to suggest that defects at the NMJ precede motor neuron loss (Le et al, 2005). We found that the NMJs of bortezomib-treated mice were larger than those of vehicle-treated animals. We presume that increased SMN in muscle positively influences the maturation of the NMJ and survival of motor neurons. It is also possible that inhibition of the proteasome contributes to muscle improvement by another mechanism independent of SMN. Muscle is a source of neurotrophic factors that are protective of motor neurons (Griesbeck et al, 1995). Muscle-derived NT-4 is an activity-dependent neurotrophic signal for growth and remodeling of adult motor nerve terminals, and muscle-derived neurotrophic factors are retrogradely transported by motor neurons with effects at the level of the cell body (DiStefano et al, 1992). Recently, muscle-specific IGF-1 expression was shown to reduce spinal cord and muscle pathology in spinal and bulbar muscle atrophy demonstrating that muscle cell signaling can have an effect on motor neuron survival. These data indicate that drugs acting peripherally may ameliorate SMA.
The UPS is involved in key events in neuronal development such as neuronal migration and synaptogenesis (Hegde & Upadhya, 2007; Muralidhar & Thomas, 1993; Yi & Ehlers, 2007), operating at both the presynaptic and at the postsynaptic level. Alteration of the ubiquitination of specific substrates due to mutations of ubiquitin conjugating enzymes and ligases may be associated with neurological disease (Liu et al, 2002; Nawaz et al, 1999; Trockenbacher et al, 2001). Recently, X- linked infantile spinal muscular atrophy (XL-SMA) has been linked to changes in the E1 ubiquitin- activating enzyme (Ramser et al, 2008), suggesting that altered ubiquitination in motor neurons could cause defective development of the motor unit. Maturation of the NMJ involves the pruning of
25
axons from muscle fibers that are innervated by multiple motor neurons, the formation of a highly- branched motor axon terminal and the coordinated expansion of the nerve terminal and muscle fiber
(Sanes & Lichtman, 1999). These events occur during the first two weeks of postnatal development in mice with effects on both neuronal development and the growth and maturation of muscle fibers.
Interestingly, the proteasome-associated deubiquitinating enzyme Usp14 has been reported to be crucial for the postnatal maturation of the peripheral nervous system by regulating ubiquitin pools at the nerve terminal (Chen et al, 2009). These and other findings are consistent with a critical role for ubiquitin homeostasis and proteasome activity at the NMJ and could, along with increased SMN levels in peripheral tissues, explain the improvement we observed in the motor unit of SMA mice treated with bortezomib.
Proteasome inhibitors have potential as treatment for a variety of diseases, including immunologic, inflammatory, metabolic, and neurological disorders, viral diseases, muscular dystrophies, and tuberculosis (Jung et al, 2009; Meiners et al, 2008). Dystrophin and other proteins of the dystrophin glycoprotein complex are degraded by the UPS in the muscle of Duchenne muscular dystrophy patients. Treatment with the proteasome inhibitor MG‑132 restores the presence and cellular localization of dystrophin and associated proteins in mdx mice and in skeletal muscle explants from DMD patients (Assereto et al, 2006; Bonuccelli et al, 2003). Similar results were obtained with bortezomib, a more specific proteasome inhibitor (Bonuccelli et al, 2007). Bortezomib has been approved for clinical use in multiple myeloma and is currently being tested in phase II clinical trials as a possible therapeutic agent for other malignancies (Armand et al, 2007; Davis et al,
2004; Faderl et al, 2006; Nencioni et al, 2007). Two phase I studies involving bortezomib were conducted in children affected by refractory leukemia and solid tumors (Blaney et al, 2004; Horton et al, 2007). Considerable efforts have been made over the past few years to identify and optimize structurally different proteasome inhibitors using medicinal chemistry or isolating natural compounds with the long-term goal of increasing the beneficial effects and reducing side-effects and toxicity.
26
In summary, our results provide preclinical support for the UPS as a potential therapeutic target for SMA therapy. Strategies to inhibit SMN degradation can be used in combination with stimulation of SMN gene expression and may enable the use of lower doses of the latter, possibly increasing efficacy and reducing toxicity. The next chapter provides further validation of the role of the UPS in SMN degradation and discusses investigations into the regulatory factors that target SMN upstream of the proteasome.
27
Figure 2.1 SMN is degraded by the UPS. (A) Treatment of 3813 SMA patient-derived fibroblast with the proteasome inhibitor, MG132 (10 µM); the cell-permeable calpain inhibitor, Calpeptin; the lysosome inhibitor, ammonium chloride (NH4Cl); and an autophagy inhibitor, 3-MA. Quantification is shown in lower panel. (B) 3813 cells treated with 1, 5, or 10 µM concentrations of the proteasome inhibitor lactacystin. The data represent mean the standard error of the mean (SEM) of five independent experiments. *, P <0.05.
28
Figure 2.2 SMN is increased in bortezomib treated cells. A) SMA patient fibroblasts treated for 16 hours with increasing doses (1, 10, 50 and 100 µM respectively) of the proteasome inhibitor bortezomib showed dose-dependent increased SMN protein levels. B) HEK 293T cells treated with bortezomib have increased SMN ubiquitination in a dose-dependent manner.
29
Figure 2.3 Bortezomib increases SMN protein levels in vivo. Mice at P5 were treated with either bortezomib (0.15mg/kg) or vehicle (water) every other day and sacrificed at P13. Brain, spinal cord, liver, kidney, and muscle tissues were removed, and protein lysates were isolated for biochemical analysis. A densitometry analysis was performed on the resulting western blots to ascertain relative SMN protein levels in each tissue. Values represent mean ± SEM. **, P < 0.01.
30
Figure. 2.4 Bortezomib reduces proteasome activity in peripheral tissues of SMA mice. Aliquots of tissue homogenates were incubated with 50 μM Suc-LLVY-AMC and assay buffer (20 mM Tris–HCl, pH 8.0, 1 mM ATP and 2 mM MgCl2) for 20 min at 37 °C. The reaction was stopped by the addition of cold ethanol and fluorescence was measured in a fluorometer (380 nm excitation and 440 nm emission). Proteasome activity was measured as the units of fluorescence released per µg of protein. Graph represents relative proteasome activity in tissues of three bortezomib-treated animals with three vehicle-treated animals as controls. *, P < 0.05
31
Figure. 2.5 Bortezomib treatment does not increase SMN staining in anterior horn cells in lumbar spinal cord sections. Paraffin-embedded lumbar spinal cords of vehicle (n=3) and bortezomib-treated mice (n=3) were serially sectioned at 5 μm steps, mounted on slides, and processed for Nissl staining. Images of 6 contiguous sections, 150 μm apart (original magnification, ×10) were analyzed with NIH ImageJ software. The tissue sections were stained with a mouse anti-SMN antibody (BD Transduction Laboratories, diluted 1:500) using Envision-plus kit (Dako K2006) according to the manufacturer's instructions. Sections were counterstained with Mayer's hematoxylin. SMN staining intensity in anterior horn cells was quantitated using Image J software. We observed no significant difference in SMN staining between bortezomib-treated and vehicle-treated mice. *, P < 0.05
*, P < 0.05
32
Figure 2.6 Bortezomib improves motor function; does not affect survival. SMA mice were treated with intraperitoneal injections of bortezomib (0.15 mg/kg) or vehicle (water) every other day starting on P5. (A) Survival curves of SMA mice treated with bortezomib (n=12) or vehicle (n=10) (B) Righting time in SMA mice treated with bortezomib (n=12) or vehicle (n=10).
33
Figure. 2.7 Synergistic increase in SMN protein levels following TSA and bortezomib treatment. Western blot showing SMA patient fibroblasts (GM09677; Coriell Cell Repository) treated with TSA (50 nm), bortezomib (1 um), and a combination of TSA and bortezomib for 16 hours. Quantitation of western blots on right
*, P < 0.05; **, P<0.01
34
Figure 2.8 TSA increases SMN protein levels in vivo. Mice at P5 were treated with either TSA (4 mg/kg; n=4) or vehicle (DMSO; n=4) every day and sacrificed at P13. A) Brain, spinal cord, liver, kidney, and muscle tissues were removed, and protein lysates were isolated for biochemical analysis. A densitometry analysis was performed on the resulting western blots to ascertain relative SMN protein levels in each tissue. Values represent mean ± SEM. *, P < 0.05. B) Choline acetyltransferase (ChAT) protein levels in spinal cord lysates from SMA mice treated with either vehicle or TSA were measured by western blot and densitometry analysis (p= 0.066). *, P < 0.05
35
Figure 2.9 Combining TSA and bortezomib increases SMN in vivo. SMA mice aged P5 were treated daily with TSA (4 mg/kg) and bortezomib (0.075 mg/kg) or with vehicle (DMSO and water). Mice were sacrificed for biochemical analysis at P13. Brain, spinal cord, liver, kidney and muscle tissues were removed and protein lysates from these tissues were isolated to examine SMN protein levels. The ratio of SMN to actin protein levels was determined by a densitometry analysis. Values represent mean ± SEM. *, P < 0.05; **, P<0.01; ***, P,0.001.
36
Figure 2.10 TSA with bortezomib extends survival in SMA mice and improves the SMA phenotype in transgenic mice. SMA mice were treated with daily I.P. injections of TSA (4 mg/kg) and bortezomib (0.075 mg/kg) or vehicle (equal amounts of DMSO and water). A control group was treated with vehicle (DMSO) and TSA alone (4 mg/kg) in the same manner. (A) Kaplan- Meier survival curves of SMA mice treated with TSA alone (n=12; median survival=16 days) or vehicle (n=10; median survival=18.5 days) (B) Survival curves of SMA mice treated with TSA with bortezomib (n=21; median survival=20 days) or vehicle (DMSO and water; n=21; median survival=14 days). (C) Righting times of SMA mice treated with TSA and bortezomib, TSA alone, and vehicle. (D) Body weights of SMA mice treated with TSA with bortezomib, TSA alone, and vehicle.
37
Figure 2.11 TSA with bortezomib ameliorates muscle pathology in SMA model mice. SMA mice were treated with vehicle (n = 3), bortezomib (n=3), TSA (n = 3), TSA + bortezomib (n = 3) from P5 to P13. (A) Hematoxylin and eosin staining of quadriceps muscles from vehicle and drug-treated mice. Scale bars: 50 μm. (B) Average myofiber diameter was increased with bortezomib treatment (P < 0.05), TSA treatment (P < 0.01) and TSA + bortezomib (P < 0.01). The total TA myofiber number increased with individual bortezomib (P < 0.01) and TSA treatments (P < 0.05) and increased further with TSA + bortezomib (P < 0.001). Values represent mean ± SEM . *, P < 0.05; **, P<0.01; ***; P<0.001.
38
Figure 2.12 TSA with bortezomib delays motor neuron death in lumbar spinal cord tissues of SMA model mice. SMA mice treated with vehicle (n=3), bortezomib alone (0.15 mg/kg; n = 3), TSA alone (4mg/kg) or bortezomib (0.075 mg/kg) plus TSA (4 mg/kg; n = 3) were sacrificed at P13. (A) Nissl-stained sections of ventral spinal cords. Quantitative analysis showed an increase in motor neurons/section in mice treated with bortezomib alone (P<0.01) and a combination of TSA and bortezomib (P<0.01) but not with TSA alone. (B) Western blot analysis showed an increase in the choline acetyltransferase protein levels in spinal cord of bortezomib (p= 0.056) and TSA+bortezomib- treated mice compared to vehicle-treated mice (P < 0.05). Values represent mean ± SEM. *, P < 0.05; **, P<0.01; ***; P<0.001.
39
Figure 2.13 TSA with bortezomib improves the NMJ. Neuromuscular junctions were isolated from the TA muscles of vehicle and drug treated animals at P13. While TSA and bortezomib (n = 3) both independently increased NMJ surface area, combining both TSA and bortezomib further improved NMJ size in SMA mice (P<0.01). Values represent mean ± SEM. **, P<0.01.
40
Figure 2.14 Co-administration of TSA and bortezomib continues to improve the NMJ. Neuromuscular junctions were isolated from the TA muscles of drug treated animals at P19. TSA plus bortezomib treatment (n=2) increased NMJ size to approximately the same as control littermates.
41
Chapter 3
The E3 ubiquitin ligase Mind bomb 1 ubiquitinates and promotes the degradation of SMN
Sections of this chapter have been submitted for publication:
Kwon DY, Dimitriadi M, Cable C, Hart A, Chitnis A, Fiscbeck KH, Burnett BG. The E3 ubiquitin ligase Mind bomb 1 ubiquitinates and promotes the degradation of Survival of Motor Neuron protein.
Contributions:
In this chapter, I performed the ubiquitination assay in Figure 3.1A, mentored a student who ran the ubiquitination assay in 3.1B, prepared cells for pulse-chase analysis in 3.1C, and performed all of the experiments presented in Figures 3.2 to 3.6.
42
Introduction
We have shown in Chapter 2 that SMN protein levels increase in cell culture and animal models with proteasome inhibition (Burnett et al., 2009; Kwon et al., 2011). However, because protein degradation by the UPS is an essential process in cellular homeostasis and general inhibition may have undesired effects, we looked at regulatory events upstream of the proteasome for a more specific strategy in inhibiting SMN degradation.
E3 ubiquitin ligases are known to confer the substrate-specificity in the UPS and thus have been considered candidates for targeted inhibition of protein degradation. In this chapter, we sought to identify and characterize the E3 ligase(s) responsible for targeting SMN for degradation. We chose to evaluate mind bomb 1 (Mib1) as a candidate ligase for SMN based on their opposing roles in neuronal outgrowth and NMJ development. Overexpression of Mib1 inhibits the outgrowth of neurites in primary cultured neurons while SMN reduction decreases neurite and axonal length in both cultured cells and zebrafish embryos (Choe et al, 2007). In Drosophila melanogaster, loss of Mib1 promotes synaptic overgrowth by increasing the number of synaptic boutons at NMJs (Choe et al.,
2007). This is in contrast to SMN loss of function in the same animal model, which has been found to diminish NMJ bouton numbers (Chang et al, 2008; Choe et al, 2007; McWhorter et al, 2003b).
Mib1 is a multidomain enzyme with three carboxyl-terminal RING finger domains (Itoh et al, 2003; Jin et al, 2002b; Yoo et al, 2006; Zhang et al, 2007). Its best-characterized role is in activating the Delta-Notch signaling pathway, which controls differentiation by regulating cell-cell communication (Barsi et al, 2005; Chen & Casey Corliss, 2004; Itoh et al, 2003; Koo et al, 2005a; Lai et al, 2005). Recently, other functions for Mib1 have also emerged. Mib1 has been identified in postsynaptic densities, where it has been shown to act as a binding partner of the cyclin dependent kinase 5 (CDK5) complex in regulating morphogenesis in post-mitotic neurons (Choe et al, 2007) and in regulating apoptosis by promoting the proteasomal degradation of the anti-apoptotic factor death associated protein kinase (Jin et al, 2002a). Overexpression of Mib1 was also shown to
43
promote the ubiquitination of receptor-like tyrosine kinase (RYK), reducing its steady-state levels at the plasma membrane (Berndt et al, 2011).
In this chapter, we present a novel function for Mib1. We show that SMN interacts with
Mib1 and that overexpression of Mib1 in cultured cells increases SMN ubiquitination and degradation. Importantly, Mib1 knockdown results in increased SMN levels in cultured cells and ameliorates the neuromuscular pharyngeal pumping defect in Caenorhabditis elegans deficient in SMN, indicating a physiological role for Mib1 in modulating SMN.
Materials and Methods
Plasmids and miRNA
Plasmids encoding human full-length and truncated Mib1 were constructed as previously described (Itoh et al, 2003). Mib1 shRNA and non-silencing control were purchased from
SABiosciences (KM26177G) and pre-miR miRNA precursors (control and miR-137) were purchased from Ambion (PM10513).
Cell Culture, Transfection, and Western Blotting
Human embryonic kidney (HEK) 293T and mouse motor neuron-like (NSC-34) cells were maintained at 37°C with 5% CO2 in DMEM (Invitrogen) supplemented with 10% FBS and penicillin/streptomycin/glutamine (Invitrogen). Cells were plated into a 6-well culture plate and transiently transfected with 0.5-4 μg of plasmid DNA per well using FuGENE HD transfection reagent (Roche Applied Science) according to the manufacturer’s protocol. The total amount of
DNA used for transfection was kept constant in each experiment by adding an appropriate amount of empty vector plasmid. Cells were harvested 24-48 hrs post-transfection.
For siRNA, shRNA and miRNA transfections, HEK 293T cells were plated sub-confluently in a 6-well culture dish and transfected with 100 pmol of siRNA/miRNA and 10 μl of Lipofectamine
44
2000 (Invitrogen) or 2-3 µg of shRNA. After 48 h of transfection, cells were again transfected with siRNA/miRNA/shRNA for 24 h and were harvested after a total 72 h of transfection.
Western Blotting, Immunoprecipitation and Pull-Down Assays
Transfected cells were lysed in either NP-40 lysis buffer (1% NP-40, 50 mM Tris-HCl, 150 mM NaCl, PH 8.0), or RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl,
0.1% SDS, PH 8.0) and quantified using the BCA Protein Assay Kit (Pierce) for Western blotting.
Equal amounts of protein lysate (15 μg) were run and separated on a 10% SDS-polyacrylamide gel electrophoresis (PAGE, Invitrogen) and transferred to a polyvinylidene fluoride membrane
(Invitrogen). These were then probed with the appropriate antibodies in 5% milk and washed with
Tris-buffered saline and Tween 20. Antibodies: anti-SMN (BD Transduction), anti-Mib1 (Epitomics) and anti-Mib2 (Sigma) were used at a 1:1000 dilution; anti-FLAG M2 (Sigma), anti-Myc 9E10 (Santa
Cruz), anti-HA (Sigma), and anti-actin (Sigma) were used at a 1:5000 dilution. Anti-Mib1 serum was a gift from Dr. Junmin Peng. The signal was visualized with a secondary antibody (anti-mouse or - rabbit immunoglobulin-horseradish peroxidase, both at 1:3,000-10,000 dilution) using a chemiluminescence detection system (Perkin-Elmer).
For immunoprecipitation experiments, cell lysates were clarified by centrifugation and pre- cleared with pan-mouse IgG Dynabeads (Invitrogen) for 1 h at 4°C. The lysates were then incubated with 1ug of antibody for 2 h to overnight then incubated with pan-mouse IgG Dynabeads for 1 h on a rotator at 4°C. The beads were washed with NP-40 buffer 4-5 times and boiled in SDS gel loading buffer. Eluted proteins were run on an SDS-polyacrylamide gel for Western blotting.
For the pull-down assay, 5 µg of purified recombinant Mib1 protein purchased from
Origene (TP321377) was incubated with GST or SMN-GST beads at 4°C on a rotator overnight.
Samples were spun down and the beads were washed 5 times with NP-40 buffer. Following the last wash, the beads were boiled in SDS gel loading buffer for 5 minutes and eluted proteins were processed for western blotting.
45
In vitro ubiquitination
Recombinant SMN protein (Enzo) was incubated with ubiquitin, an E1 ubiquitin-activating enzyme (Boston Biochem, Cambridge, MA), E2 ubiquitin-conjugating enzyme, UBCH5B, and recombinant Mib1 (OriGene Technologies Inc, Rockville, MD) in reaction buffer (50 mM Tris-HCl, pH 7.5, 2 mM ATP, 4 mM MgCl2, 2 mM dithiothreitol) for 1 hour at 37°C. Reactions were quenched with 30 µl of SDS gel loading buffer, boiled for 5 min, and separated via SDS-PAGE.
SMN ubiquitination was analyzed by western blotting, using an antibody against polyubiquitin (FK1;
Enzo Life Sciences, Farmingdale, NY).
RNA Extraction and Quantification
Cells were homogenized in 500 μl TRIzol reagent (Invitrogen) and spun for 10 min at 4°C.
One hundred μl of chloroform was added to the supernatant, and the mixture was vortexed and spun down again. After the aqueous upper phase was transferred to another tube, 250 μl of isopropanol and 50 μl 3M sodium acetate was added. The RNA was then pelleted, washed with 70% ethanol, and resuspended in 100 μl DNAse and RNAse free water. Total RNA was quantified by absorption at 260 nm, and 450 ng of each RNA sample was converted to cDNA using the High
Capacity cDNA Reverse Transcription kit (Applied Biosystems). Aliquots of 20 μl quantitative PCR reactions were run in triplicate using the ABI Prism 7900 sequence detection system (Applied
Biosystems). The level of HPRT (control), SMN and Mib1 transcripts were quantified by the threshold cycle (CT) using primers purchased from Applied Biosystems.
Immunohistochemical Analysis of Gem Numbers
The gem number was assayed 24 h after transfection with myc-Mib1 or myc-Mib1-C1009S.
The cells were fixed, permeabilized, and immunostained with anti-SMN and anti-myc antibodies.
Blinded counts of gems were performed in more than 300 nuclei for each condition.
46
Pulse-chase Protein Labeling
Pulse-chase analysis was performed as previously described (34). In brief, HEK 293T cells were incubated in cysteine-methionine-free medium (Sigma-Aldrich) for 2 h or 12 h, followed by incubation in cysteine-methionine-free medium containing 100 µCi of 35S-labeled cysteine- methionine (GE Healthcare, Piscataway, NJ) for 1 h. After labeling, the cells were washed once with culture medium containing a 10-fold excess of unlabeled methionine and cysteine (5 mM each) and then incubated further in the same medium. The cells were collected at the indicated time points and processed for immunoprecipitation with anti-SMN antibody (BD Transduction Laboratories).
Immunoprecipitations were carried out for 6 h at 4°C with antibodies bound to protein G-Sepharose
(Sigma-Aldrich). After three washes with lysis buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.4], 2.5 mM MgCl2, 0.1% NP-40, and protease (Roche)) and phosphatase inhibitor cocktails (Sigma-Aldrich), bound proteins were eluted by boiling in SDS-PAGE sample buffer. Immunocomplexes were separated on 10% SDS-PAGE. The gels were dried and exposed to a phosphorimager screen, and the signal visualized with a Storm phosphorimager system (Molecular Dynamics, Piscataway, NJ).
Densitometric analysis of the protein bands was carried out using ImageQuant PhosphorImager software (Molecular Dynamics).
C. elegans Strains
TU3311 uIs60 [unc-119p::yfp, unc-119p::sid-1] (Calixto et al, 2010), HA2258 smn-
1(ok355)/hT2[bli-4(e937) let-?(q782) qIs48] (I;III); uIs60 (Briese et al, 2009) and HA2201 smn-
1(ok355)/hT2[bli-4(e937) let-?(q782) qIs48] (I;III); osm-11(rt142)X were maintained at 20°C according to standard protocols (Brenner, 1974).
Construction of RNAi Feeding Clone for Y47D3A.22/mib-1
A 796 bp PCR product corresponding to the C. elegans mib-1 cDNA (Berndt et al, 2011) was cloned into the L4440 RNAi feeding vector, followed by transformation into the HT115(DE3)
47
bacterial strain (Timmons et al, 2001; Timmons & Fire, 1998). Primers for amplification were:
ACH72 5’- tgatgctagcgcagcttctcctgttgcgtg-3’ and ACH73 5’- tgatctcgaggcgggagccgatttgaactg-3’.
C. elegans Pharyngeal Pumping Assay
The pharyngeal pumping assay was performed as previously described (Dimitriadi et al,
2010). Briefly, eggs hatched on L4440 control vector [L4440 (RNAi)] or [Y47D3A.22/mib-1(RNAi)] bacterial feeding strains (Kamath & Ahringer, 2003) were reared after two days at 25°C and one day at 20°C. Pumping rates were determined using an AxioCam ICc1 camera on a Zeiss Stemi SV11 microscope. A pumping event was scored as a pharyngeal grinder movement in any axis and average pumping rates (± SEM) were derived in a blinded fashion from at least three independent trials (n ≥
30 animals in total). Heterozygous and homozygous smn-1 loss of function animals were distinguished by GFP fluorescence. Both wild type (N2) and smn-1 loss of function heterozygous animals showed similar pumping rates that were not significantly different. Hence, heterozygous smn-1 loss of function animals were used as controls in this study.
Statistics
The biochemical data were analyzed with the GraphPad Prism software package (GraphPad
Software, Inc., San Diego, CA) and compared statistically by either t test or one-way analysis of variance, with the Newman-Keuls multiple comparison post hoc correction. To compare differences among the three groups, a nonparametric equality of medians test was performed, since the data were not normally distributed. If this was statistically significant, then a pair-wise comparison between the two treatment groups was done using a Mann-Whitney test.
48
Results
Mib1 Increases SMN Ubiquitination and Protein Turnover
E3 ligases promote protein degradation by catalyzing the transfer of ubiquitin molecules from the E2 enzyme onto substrate proteins. To determine whether Mib1 ubiquitinates SMN we co- transfected HEK-293T cells with HA-tagged ubiquitin and full-length or selected domains of myc- tagged Mib1. The ubiquitination of SMN, as indicated by a high molecular weight ubiquitin-positive smear on Western blotting, was increased in cells expressing full-length, but not truncated or active site mutant forms of Mib1 (Figure 3.1A and B). To ascertain whether Mib1 directly ubiquitinates
SMN we performed an in vitro ubiquitination assay using recombinant Mib1 and SMN. In this cell- free assay we found Mib1 ubiquitinated SMN, consistent with the results we obtained in cultured cells (Figure 3.1C) Given that Mib1 ubiquitinates SMN, we next sought to quantitate the effect of
Mib1 on SMN protein turnover. We performed pulse-chase analysis using HEK 293T cells transfected with wild-type Mib1-myc or an active site mutant, Mib1-C1009S-myc, to determine whether Mib1 expression alters SMN protein half-life. We found that overexpressing wild type Mib1 decreased the half-life of SMN by half, from about 4 h to 2 h (Figure 3.1D). In addition, overexpressing Mib1 reduced SMN protein levels, an effect that was blocked by the proteasome inhibitor bortezomib (Figure 3.2A), indicating that Mib1 targets SMN for proteasomal degradation.
SMN is normally localized to nuclear Cajal bodies or gems, and the number of SMN- positive gems has been used as a measure for evaluating changes in functional SMN protein levels
(Coovert et al, 1997; Jarecki et al, 2005; Wolstencroft et al, 2005). We counted the number of gems per nucleus and assessed their fluorescent intensity in cells overexpressing either wild type Mib1 or mutant Mib1-C1009S. We found that the number and intensity of SMN-positive gems were significantly reduced in cells overexpressing wild-type Mib1 compared to untransfected cells (GFP negative) and to those expressing Mib1-C1009S (P < 0.001, Figure 3.2B). Together these data demonstrate that Mib1 determines SMN protein levels by promoting its ubiquitination and subsequent degradation.
49
Reducing Mib1 Expression Increases SMN Protein Levels
Since Mib1 overexpression decreased SMN levels we next investigated whether decreasing
Mib1 increases SMN levels. We transfected HEK 293T cells with Mib1 siRNA or a scrambled control, and confirmed by RT-PCR that Mib1 levels were reduced by more than half 72 h after transfection with Mib1 siRNA (Figure 3.3A). Importantly, we found that SMN protein levels were significantly increased when Mib1 was knocked down (P < 0.001, Figure 3.3A). It has previously been reported that the microRNA miR-137 targets Mib1 mRNA and regulates its protein expression
(Smrt et al, 2010). We next sought to determine whether overexpressing miR-137 affects SMN protein levels. We confirmed that transfection of cells with miR-137 decreased Mib1 mRNA and protein levels (Figure 3.3B), and found that SMN levels were increased in these cells compared to those overexpressing a scrambled microRNA control (Figure 3.3B). Decreasing Mib1 in SMA patient-derived fibroblasts by shRNA knockdown also increased SMN in these cells (Figure 3.3C).
RT-PCR analysis showed that SMN transcript levels were unchanged by expression of either Mib1 siRNA or shRNA or by expression of miR-137 (Figure 3.3D), indicating that Mib1’s effect on SMN is limited to enhancing the protein’s degradation. Hence, using three independent techniques we have shown that reducing Mib1 expression results in increased SMN protein levels.
Interaction of Mib1 and SMN is Mediated by the N-terminal Domain of Mib1 and the Segment of SMN Encoded by Exon 6
We next investigated whether Mib1 associates with SMN. Using a monoclonal antibody to
SMN, we found that endogenous Mib1 co-immunoprecipitated with endogenous SMN, indicating that these proteins interact (Figure 3.4A). We also tested for an interaction between SMN and mind bomb 2 (Mib2), a homolog of Mib1 possessing similar structural domain organization and redundant function in activating the Delta-Notch signaling pathway (Koo et al, 2005b). Endogenous Mib2 failed to immunoprecipitate with endogenous SMN, indicative of Mib1’s specificity in binding SMN
50
(Figure 3.4B). To further characterize this interaction, we used purified recombinant Mib1 in pulldown assays with purified GST-SMN or GST alone. We found that GST-SMN but not GST alone pulled down Mib1 (Figure 3.4C), confirming a direct association in which other factors, including members of the SMN complex, are not required.
We then sought to identify the requirements for interactions between Mib1 and SMN. To map the domains of Mib1 that mediate SMN binding, we performed co-immunoprecipitation assays with SMN and truncated or mutant forms of Mib1. While all forms of Mib1 containing the N- terminal region of the protein (full-length, C1009S, m132-myc) co-immunoprecipitated with SMN, constructs containing the middle ankyrin repeat and C-terminal RING domain showed weak or no interaction (Figure 3.4D). To determine the regions of SMN required for binding Mib1 we co- transfected a FLAG-tagged Mib1 construct with plasmids expressing either full length SMN or SMN lacking each of its seven translated exons. Full-length SMN and each deletion mutant, with the exception of SMNΔ6, co-immunoprecipitated with Mib1, suggesting that the region encoded by
SMN exon 6 is essential for interaction with Mib1 (Figure 3.4E). Consistent with this observation, we found that Mib1 was also unable to promote the ubiquitination of SMNΔ6 (Figure 3.4F).
Because exon 6 is known to mediate SMN oligomerization, we examined if SMN oligomerization was required for Mib1 to interact with SMN. We found that Mib1 co-immunoprecipitated with wild- type SMN as well as the patient-derived SMN mutants, SMN-G279V and SMN-Y272C, which do not efficiently self-associate or oligomerize (Figure 3.4G). Thus, SMN oligomerization is not required for Mib1 association.
Recently, a putative degradation signal (degron) was identified in the SMNΔ7 protein; this putative degron spans a portion of the protein encoded by exon 6 and may account for the instability of the truncated SMN protein encoded by SMN2 (Cho & Dreyfuss, 2010). Mutating a conserved serine residue to alanine (S270A) in this region slows turnover of SMNΔ7, suggesting that this residue is important in targeting SMNΔ7 for degradation. We sought to determine whether Mib1 contributes to the instability of SMNΔ7 and whether mutating the conserved serine residue affects
51
binding or the rate of degradation of SMNΔ7. We found that overexpressing Mib1 resulted in greater ubiquitination of SMNΔ7 than full-length SMN, and that knocking down Mib1 increased SMNΔ7 stability (Figure 3.4H and I; Figure 3.5). However, the interaction between Mib1 and SMNΔ7, as detected by co-immunoprecipitation analysis, was not disrupted by mutating serine 270 to alanine
(Figure 3.6A). Nevertheless, the rate of degradation of SMNΔ7-S270A, as measured by pulse-chase analysis, was not affected by Mib1 overexpression (Figure 3.6B). We then examined whether the
S270A mutation hinders the ability of Mib1 to ubiquitinate SMNΔ7. While we found increased ubiquitination of SMNΔ7 when Mib1 and SMNΔ7 were co-expressed, there was no increase in ubiquitination of SMNΔ7-S270A in the presence of Mib1 (Figure 3.6C). These data suggest that the instability of the SMNΔ7 is due in part to ubiquitination by Mib1 and that while the conserved residue S270 within exon 6 may promote the ubiquitination of SMNΔ7 by Mib1, it does not mediate binding to this E3 ligase.
Loss of the C. elegans Ortholog mib-1 Ameliorates the Neuromuscular Defects Caused by smn-1 Loss of Function.
Potential cross-species conservation of the SMN-Mib1 interaction was examined in C. elegans. The C. elegans genome has a single ortholog of SMN, smn-1. Homozygous smn-1 loss of function causes developmental delay, impaired locomotion, reduced pharyngeal pumping, and larval lethality (Briese et al, 2009; Dimitriadi et al, 2010). Although complete loss of SMN function causes lethality, smn-1 animals can survive for several days due to partial maternal rescue. The C. elegans ortholog of human MIB1 is the gene mib-1 (Berndt et al, 2011). To assess genetic interactions, we decreased Mib-1 levels in smn-1 deficient animals by RNA interference (RNAi) through feeding
(Timmons et al, 2001; Timmons & Fire, 1998). Knocking down the Mib-1 C. elegans ortholog increased the pharyngeal pumping rates of smn-1 deficient animals, but this did not reach statistical significance (P = 0.06, Figure 3.7A). RNAi by feeding is generally less effective in neurons than in other tissues (Kamath et al, 2001; Rual et al, 2004; Timmons et al, 2001). To circumvent this
52
difficulty, we increased neuronal sensitivity to RNAi feeding by ectopic neuronal expression of the
SID-1 double stranded RNA channel in smn-1 animals (Calixto et al, 2010). In this sensitized background, mib-1 RNAi significantly ameliorated the smn-1 pharyngeal pumping defects (Figure
3.7B; P = 0.04), suggesting that Mib1 is a conserved cross-species modifier of SMN loss of function defects. Since Mib1 plays an important role in Notch function (Itoh et al, 2003; Lai et al, 2005; Le
Borgne et al, 2005), we examined whether the Mib-1 impact was due to perturbations in Notch signaling. Loss of the Notch co-ligand function (Komatsu et al, 2008; Singh et al, 2011) in smn-1 mutants did not ameliorate the pharyngeal pumping defect of smn-1(ok355);osm-11(rt142) animals
(Figure 3.7C), suggesting that Mib-1 directly impacts SMN-1 function independent of Notch signaling.
Discussion
In this study, we provide evidence that the E3 ligase Mib1 ubiquitinates and catalyzes SMN protein degradation. These effects are not associated with changes in SMN mRNA expression and are not observed when a ligase-defective point mutant of Mib1 is expressed. Knocking down Mib1 increases SMN protein levels in cultured cells and partially rescues a neuromuscular defect due to
SMN deficiency in C. elegans. These data indicate that Mib1 modulates SMN protein stability by targeting it for degradation by the proteasome and represents a new modifier of the SMA phenotype.
The high correlation between SMN2 copy number and SMA severity indicates that increasing SMN protein levels may ameliorate the disease phenotype. Mechanisms to increase SMN protein levels have been actively pursued as therapeutic options for SMA. Inhibiting the degradation of SMN is one way to increase its steady-state level. Inhibiting the proteasome increases SMN levels in cultured cells and SMA model mice (Kwon et al, 2011), and it ameliorates the disease phenotype.
Identifying enzymes that specifically target SMN for degradation could thus provide new therapeutic targets. We show here that overexpression of Mib1 increases ubiquitination of SMN, as indicated by increased ubiquitin-SMN conjugates. We found that overexpressed Mib1 accelerates the rate of SMN
53
degradation, whereas knockdown of Mib1 by siRNAs, shRNAs or the microRNA mir-137 stabilizes
SMN. Our data demonstrates that Mib1-mediated ubiquitination of SMN enhances its degradation and decreases steady-state SMN protein levels.
While most SMA patients have homozygous deletion of the SMN1 gene, ll retain at least one copy of SMN2. The major SMN2 gene product is the truncated SMNΔ7 protein which, based on its ability to extend the survival of SMA mice, likely retains some function but is unstable and rapidly degraded. The instability of SMNΔ7 is thought to be due to a number of factors, including its inability to self-associate (Burnett et al, 2009a; Lorson et al, 1998b) and the presence of a degradation signal that promotes its rapid turnover (Cho & Dreyfuss, 2010). Here, we show that Mib1 ubiquitinates SMNΔ7 to a greater extent than full-length SMN. Mutating a critical serine residue within the putative degradation signal does not affect binding but reduces ubiquitination of SMNΔ7 by Mib1. Given that such serine residues are potential sites for post-translational modification, these findings suggest that SMN ubiquitination and degradation may be inhibited by targeting the kinase or phosphatase that modifies this residue. We propose that the increased Mib1-mediated ubiquitination of SMNΔ7 could at least in part account for the relative instability of the SMNΔ7 protein. Consistent with this, reducing Mib1 levels results in increased stability of SMNΔ7 protein.
We found that the interaction of SMN with Mib1 is likely mediated by exon 6. Missense mutations in exon 6 and deletion of exon 7 disrupt SMN’s ability to oligomerize (Le et al, 2005;
Lorson et al, 1998b; Pellizzoni et al, 1999; Zhang et al, 2003), resulting in severe SMA; this may be because SMN that fails to oligomerize is rapidly degraded (Burnett et al, 2009a). A mutation in SMN exon 6 that disrupts oligomerization (Y272C) also destabilizes the SMN protein, and cells from a patient with this mutation have similar SMN levels to those with homozygous deletion of SMN1
(Lefebvre et al, 1997). One possibility is that Mib1 only interacts with SMN that is oligomerized, since exon 6 mediates SMN oligomerization. However, this is not likely because Mib1 interacts with
SMN that has the patient-derived mutations G279V and Y272C, which do not efficiently oligomerize
(Figure 3.4G). Thus, it is more likely that Mib1 binds the region of the SMN protein encoded by
54
exon 6 and that the inability of SMN to oligomerize increases its likelihood of binding Mib1. Since the exon 6 domain mediates SMN self-association (Lorson et al, 1998b), this domain may not be available for interaction with Mib1 when SMN oligomerizes or is in a complex. This could explain the increased stability of complexed SMN, which is inaccessible to Mib1 and thus unlikely to be ubiquitinated and degraded.
Two genetic modifiers of SMA have been reported in humans: SMN2 and plastin3. A genome-wide RNAi screen in C. elegans and Drosophila deficient in SMN identified other genes that modify the disease phenotype in these model systems (Dimitriadi et al, 2010). SMN deficiency in C. elegans is characterized by marked reduction in the rate of pharyngeal pumping. We show here that
Mib1 knock-down by RNAi partially rescues the SMN deficient phenotype in C. elegans, indicating that Mib1 may be a modifier of SMA. Our biochemical data suggests that Mib1 promotes SMN degradation by the proteasome. However, we cannot rule out the possibility that SMN-independent pathways involving other functions of Mib1 are responsible for the partial rescue of the neuromuscular phenotype. Nonetheless, the identification of a new modifier of the SMA phenotype is of particular importance, given the currently limited number of targets with a favorable therapeutic index.
The steady-state level of a protein is governed by the balance between protein synthesis and degradation. Strategies to increase SMN protein synthesis by upregulating the gene, stabilizing the mRNA, and altering splicing to promote inclusion of exon 7 have been pursued in SMA therapeutic development. That the SMN2 gene produces some full-length SMN protein suggests that strategies to slow its degradation may also be therapeutic options. Since SMN is ubiquitinated and degraded by the proteasome, retarding its turnover either through blocking the proteasome or inhibiting SMN ubiquitination may be useful in treating SMA. We have shown that SMA mice treated with the proteasome inhibitor bortezomib show reduced pathology and improved motor function (Kwon et al, 2011). However, the toxicity of available proteasome inhibitors may preclude chronic use in SMA patients. While small molecule inhibitors of the proteasome have been developed for research and
55
clinical use, E3 ubiquitin ligases are emerging as targets for disease treatment, because they confer the substrate-specificity of the UPS. Drugs that inhibit E3 ligases involved in cell cycle regulation and cell proliferation are currently under study to treat various types of cancer. One such E3 ligase, murine double minute 2 (MDM2), regulates the tumor suppressor p53, and represses its role in cell cycle arrest and apoptosis (Fakharzadeh et al, 1993; Haupt et al, 1997). Inhibitors of the human homologue of MDM2 have been shown to reduce tumor growth and induce tumor cell apoptosis in vitro, providing an incentive for targeting MDM2 function to treat cancer in vivo (Issaeva et al, 2004;
Koblish et al, 2006). High throughput small molecule compound screens have been used to identify inhibitors of E3 ligases (Davydov et al, 2004; Huang et al, 2005). These screens may be adapted to identify molecules that disrupt the interaction between a ligase and its protein substrate, which would confer greater selectivity and reduce off-target effects. Nutlin, which inhibits the interaction between
MDM2 and p53, was identified in a small molecule screen (Vassilev et al, 2004) and is currently being evaluated in early phase clinical trials. While new, less toxic, general proteasome inhibitors are being developed, targeting an E3 ligase that selectively tags SMN for degradation may be safer and more effective. Small molecule screens to identify inhibitors of Mib1 may thus lead to novel therapeutics for SMA.
56
Figure 3.1 Overexpression of Mib1 increases SMN ubiquitination and turnover. (A) HEK 293T cells were transfected with 2 µg of Mib1 and 1 µg HA-Ub cDNAs. The cells were harvested 48 h later and endogenous SMN was immunoprecipitated. Immunoprecipitated proteins were resolved by SDS PAGE and the proteins analyzed by Western blotting. The blots were probed with an HA antibody to detect ubiquitinated SMN. (B) Schematic representation of Mib1 protein domains. (C) Cell-free SMN ubiquitination assay. Recombinant SMN was incubated with equal amounts of E1 and E2 enzymes with or without Mib1 and ubiquitin for 1 hour at 37°C. (D) Pulse-chase analysis of endogenous SMN, in the presence of 2 µg of Mib1-myc or Mib1-C1009S-myc. The data represent mean ± the SEM of three independent experiments.
57
Figure 3.2 Effects of Mib1 overexpression on SMN protein levels and gem number. (A) HEK 293T cells were transfected with either 1 or 2 µg of Mib1-myc cDNA. 24 hours after transfection cells were treated with either vehicle (water) or 1 µM bortezomib and harvested 6 hours later. Cell lysates were resolved by SDS-PAGE, and densitometry of the resulting bands was performed using NIH Image software. SMN protein levels were corrected based on actin values. The data represent mean ± the SEM of three independent experiments. * P < 0.05. (B) Overexpression of Mib1 decreases gem number and intensity in cultured cells. Hela cells were transfected with Mib1-myc or Mib-C1009S-myc for 24 h. The cells were fixed and stained with antibodies to SMN and myc and the appropriate Alexa secondary antibodies for visualization by microscopy. The number and intensity of nuclear gems were quantitated using Nikon NIS Elements software. At least 300 cells were counted for each transfection condition. That data represent mean ± the SEM of three independent experiments. *** P < 0.001.
58
Figure 3.3 Decreasing Mib1 expression increases SMN protein levels. (A) HEK 293T cells were transfected with either 100 pmol of scrambled siRNA or Mib1 siRNA for 72 hours. Mib1 and SMN protein levels were determined after 72 h by Western blotting. (B) HEK 293T cells were transfected with either 100 µM negative control miRNA or miR-137 for 72 h. SMN protein levels were determined by Western blotting. The data represent the mean ± SEM of three independent experiments. (C) SMA patient derived fibroblasts (3813) were transfected with Mib1 siRNA or scrambled control. Cells were harvested 72 hours post-transfection and Mib-1 mRNA and SMN protein levels assessed by RT-PCR and Western blot analysis, respectively. (D) HEK 293T cells were transfected with Mib1 siRNA, miR-137, or scrambled control. Cells were harvested 72 hours post- transfection, and SMN mRNA level was analyzed by RT-PCR. * P < 0.05; ** P < 0.01; *** P < 0.001
59
60
Figure 3.4 Mib1 and SMN associate. (A) Endogenous SMN was immunoprecipitated with an antibody to SMN and immunoprecipitates resolved by SDS-PAGE. Association with Mib1 was determined by Western blot analysis using an antibody to Mib1. (B) Protein lysates from HEK 293T cells were immunoprecipitated with an SMN antibody. While Mib1 co-immunoprecipitated with SMN, Mib2 was not detected by western blot analysis. Membranes were probed with anti- SMN, anti-Mib1 and anti-Mib2 antibodies. (C) GST or GST-SMN was used to pull down purified recombinant Mib1 protein. (D) Mib1 constructs containing either full-length or truncated transcripts of the Mib1 gene were transiently transfected into HEK 293T cells. Immunoprecipitation of endogenous SMN in these cells showed binding to Mib1 proteins containing its N-terminal region. (E) HEK 293T cells were transfected with 0.5 µg of full- length SMN cDNA or various SMN constructs with a single exon deletion and 2 µg of Mib1- FLAG. Mib1-FLAG was immunoprecipitated and resolved on a SDS-PAGE gel. Western blots were probed with antibodies to myc and HA to determine binding of Mib1 to SMN. (F) HEK 293T cells were transfected with 2 µg of Mib1-FLAG, 1 µg HA-Ub and 1 µg of either full length SMN-myc or SMNΔ6-myc. The cells were harvested 48 h later, and endogenous SMN was immunoprecipitated and the proteins separated on a Western blot. The blots were probed with an HA antibody to detect ubiquitinated SMN. (G) HEK 293T cells were transfected with 2 µg of Mib1-FLAG and either 0.5 µg of GFP-tagged wild-type SMN, SMN G279V or SMN Y272C. The cells were harvested 48 h later and SMN was immunoprecipitated with an anti-myc antibody and resolved on a SDS-PAGE gel. Western blots were probed with antibodies to myc and GFP to determine interaction of Mib1 and wild-type SMN or the disease-associated mutants. (H) HEK 293T cells were transfected with 2 µg of full-length SMN or SMNΔ7 and 1 µg Mib1-FLAG and HA-Ub cDNAs. The cells were harvested 48 h later and SMN was immunoprecipitated and resolved by SDS PAGE and the proteins analyzed by Western blotting. The blots were probed with an HA antibody to detect ubiquitinated SMN. (I) Pulse-chase analysis of full-length SMN and SMNΔ7 in the
absence or presence of 2 µg of Mib1-myc. The data represent the mean ± SEM of three
experiments.
61
Figure 3.5 shRNA-knockdown of Mib1 decreases Mib1 expression in pulse-chase experiments. HEK 293T cells were transfected with 3 mg of either scrambled control or Mib1 shRNA (Applied Biosystems) and 0.5 mg of SMN∆7-myc for 48 hours. Cells were harvested for RT- PCR and Western blot analyses. (A) Mib1 shRNA transfection resulted in a 50% decrease in Mib1 mRNA. (B) Western blotting showed a decrease in Mib1 protein by approximately 40%. **P<0.01
62
Figure 3.6 Mib1 binds but does not ubiquitinate SMNΔ7 with mutant degron (S270A). (A) HEK 293T cells were transfected with 2 µg Mib1-FLAG, and 0.5 µg SMN-myc, SMNΔ6, SMNΔ7, SMN- S270A-myc, or SMNΔ7-S270A-myc. The cells were harvested 24 h post- transfection and immunoprecipitated with anti-FLAG antibody; the samples were run on SDS-PAGE, and Western blots probed with an anti-myc antibody. (B) Pulse-chase analysis of SMNΔ7-S270A, in the presence of 2 µg of Mib1-myc or Mib1-C1009S- myc. The data represent the mean ± SEM of three experiments. (C) HEK 293T cells were transfected with 1 µg HA-Ub, SMNΔ7 or SMNΔ7-S270A and either Mib1 cDNA or an empty vector. The cells were harvested 48 h later, SMN was immunoprecipitated using an anti-myc antibody (Sigma), and the proteins separated on a Western blot. The blots were probed with an HA antibody to detect ubiquitinated SMNΔ7 or SMNΔ7- S270A.
63
Figure 3.7 Knockdown of the C. elegans ortholog of Mib1 ameliorates the pharyngeal pumping neuromuscular defects of smn-1 loss of function animals with increased RNAi neuronal sensitivity. (A) mib-1 knockdown does not significantly increase pharyngeal pumping in smn-deficient animals without SID-1 sensitization. Decreasing expression of the C. elegans ortholog of Mib1 by RNAi increased pharyngeal pumping, but this did not reach statistical significance. (B) mib- 1 was decreased in smn-1 deficient animals overexpressing neuronal SID-1 by RNAi feeding. (C) Loss of the Notch co-ligand function in smn-1 mutants did not ameliorate the pharyngeal pumping defect of smn-1(ok355);osm-11(rt142) animals. Pharyngeal pumping events were measured in both control and mib-1 RNAi fed animals. All error bars are standard error of the mean (SEM); *P< 0.05
64
Chapter 4
Summary, Discussion and Conclusions
65
SMN deficiency causes a debilitating neuromuscular disease; and a promising avenue for treatment involves increasing levels of the SMN protein. The goals of this thesis were to characterize the molecular mechanisms underlying SMN protein degradation and to determine whether targeting the ubiquitin proteasome pathway ameliorates the SMA disease phenotype in SMA model mice.
SMN in peripheral tissues
The proteasome inhibitor bortezomib is non-CNS penetrant and was unable to increase
SMN protein levels in the brain and spinal cord tissues of SMA mice. Bortezomib improved motor function, muscle, spinal cord and NMJ pathology in the SMA mice; however, it was unable to extend lifespan, underscoring the importance of SMN in the CNS. While these experiments suggest that future therapies for SMA may need to effectively target the CNS, they also highlight the beneficial effects of a peripherally acting drug on the SMA phenotype.
Chapter 1 discussed evidence highlighting SMN’s role in muscle, and there is a growing body of evidence to support a critical role for SMN in other peripheral tissues as well. Recently, systemic delivery of an antisense oligonucleotide (ASO) correcting SMN2 splicing was found to improve the lifespan of the SMA mice more than intracerebroventricular (ICV) administration (Hua et al, 2011).
The same study observed decreased levels of circulating insulin-like growth factor (IGF), a neurotrophic factor, in SMA mice, and the levels were restored with the ASO treatment (Hua et al,
2011). Although a direct role for SMN in IGF production has yet to be determined, these results indicate a function for SMN in the liver and other peripheral tissues, suggesting that ideal treatments for correction of SMA target all tissues.
SMN independent mechanisms of disease phenotype improvement
One of the hallmarks of muscle atrophy is an increase in protein breakdown by the proteasome (Hasselgren, 2002). Several E3 ligase atrogenes involved in this process were found to be upregulated in the muscle tissues of SMA mice and patients (Bricceno et al, 2012). Proteasome
66
inhibition by bortezomib and other agents has also been shown to reduce skeletal muscle atrophy caused by denervation and muscle disuse (Beehler et al, 2006; Hasselgren et al, 2002; Jamart et al,
2011). The improvement in muscle morphology we observed with bortezomib treatment may therefore have been due a combination of effects that include increased SMN protein stability and reduction in the degradation of other muscle proteins with atrophy.
Another SMN-independent consequence of proteasome inhibition may have led to the improvement in NMJ structure in bortezomib treated SMA mice. The UPS is a key regulator of synapse development and controls local protein abundance in pre- and postsynaptic densities to remodel the synapse and modulate neurotransmission. Inhibiting proteasomal degradation has been found to increase synaptic growth and transmission by increasing the concentration of various postsynaptic factors, including glutamate receptors, in the neuromuscular synapses of Drosophila
(Haas et al, 2007; van Roessel et al, 2004). It would be interesting to determine whether systemic administration of bortezomib can directly target peripheral nerves extending from the spinal cord and act to decrease protein degradation at the NMJ and increase its growth. Whatever the mechanism of bortezomib action in increasing NMJ size, it is unlikely to be entirely due to SMN in spinal motor neurons, as SMN levels were unchanged in the spinal cords of bortezomib treated mice compared to vehicle treated littermates. A more detailed study of proteasome function at the NMJ, during and after synapse formation, could further elucidate the role of protein degradation in regulating NMJ size and transmission.
Characterizing Mind bomb 1 as an E3 ligase for SMN
There are only a few identified modifiers of the SMA disease phenotype in humans, underscoring the importance of discovering others. Mib1 associates with SMN and mediates its degradation by the proteasome. Our study showed that a neuromuscular defect characteristic of
SMN loss in worms could only be significantly rescued when Mib1 RNAi uptake was facilitated in neurons, suggestive of an interaction between Mib1 and SMN in the CNS. This is supported by
67
evidence of a role for Mib1 and SMN in neurite and axonal outgrowth (Choe et al, 2007; Rossoll et al, 2003). One hypothesis is that Mib1 and SMN must be properly regulated to ensure appropriate growth of motor neurons. Upregulation of Mib1 could lead to decreased levels of SMN, resulting in the truncated axons that have been observed in motor neurons cultured from SMA mice. This is supported by our finding of increased Mib1 protein levels in the spinal cord tissues of SMA mice
(Figure 4.1). However, Notch signaling is also known to affect axon growth and it is still unclear whether the function of Mib1 in this process is related to its canonical role in Delta-Notch signaling or to its direct effects on SMN turnover (Sestan et al., 1999). Decreasing key factors in the Delta-
Notch pathway did not improve the C. elegans SMA phenotype, suggesting that the partial rescue of the neuromuscular defect observed by decreasing Mib1 in C. elegans was probably independent of
Delta-Notch. Although we did not test whether Mib1 knockdown in neurons alone is sufficient for rescue, our findings in C. elegans and the inability of bortezomib to extend the lifespan of SMA mice may indicate a critical role for SMN in the CNS.
Although Mib1 is ubiquitously expressed during embryonic development, it is most highly concentrated in the brain and the spinal cord, underscoring its essential function in CNS development. Knock out of Mib1 causes embryonic lethality and gross abnormalities of the CNS, indicative of its effect on activating the Delta-Notch signaling pathway and regulating neuronal differentiation (Itoh et al., 2005). The function of Mib1 in the mature nervous system has yet to be ascertained, however. While decreasing Mib1 function in embryos would certainly cause CNS defects, inhibiting it in a child, adolescent, or adult with a developed nervous system, may not be detrimental. Moreover, a homologue of Mib1, Mib2, possesses redundant ability to ubiquitinate Delta and rescue the neuronal and vascular defects observed in Mib1 mutant zebrafish embryos (Koo et al.,
2005). While Mib1 is highly expressed in both embryonic and adult tissues, Mib2 is nearly exclusively expressed in adult tissues (Koo et al., 2005). This further validates Mib1 as an attractive target for
SMA therapy, as Mib2’s presence in mature tissues suggests that it could help compensate to regulate
Delta-Notch signaling and reduce side effects if Mib1 function is inhibited in patients.
68
Another interesting finding was the discovery of increased ubiquitination of SMNΔ7 by
Mib1, promoting its degradation by the proteasome. Not only does it offer a potential mechanism behind SMNΔ7 ’s marked instability, it also underscores Mib1’s ability to degrade both full-length and SMNΔ7 proteins. Future SMA therapies would ideally target both SMN protein isoforms, as
SMNΔ7 is thought to be at least partially functional. The SMNΔ7 protein retains the ability to complex with full-length SMN, thereby partially compensating for SMN deficiency. In SMA transgenic mice, overexpression of SMNΔ7 can extend the lifespan of SMA mice only expressing human SMN2. Thus, decreasing the degradation of both full-length SMN and SMNΔ7 may further improve the SMA disease phenotype, and this is achievable by decreasing ubiquitination of SMN by
Mib1 or by directly inhibiting the proteasome.
While we identified one E3 ubiquitin ligase for SMN, we cannot exclude the possibility of other E3 ubiquitin ligases for SMN. Recently, a study identified ubiquitin carboxyl-terminal hydrolase
L1 (UCHL1) as a potential regulator of SMN protein turnover (Hsu et al., 2010). UCHL1 has two opposing functions acting as both a deubiquitinating enzyme and an E3 ligase (Hurst-Kennedy et al,
2012; Liu et al, 2002). Overexpression of UCHL1 was found to decrease steady-state levels of SMN and increase its ubiquitination, suggesting it acts as an E3 ligase for SMN. UCHL1, Mib1 and other yet-to-be identified E3 ligases for SMN may work in concert to mediate SMN protein turnover, or they may interact individually with SMN in specific cell-types and at specific points in development.
Future work identifying these other E3 ligases, which may have structural similarity to Mib1, could provide additional potential targets for increasing SMN protein levels.
Current and future studies
Pharmacotherapy for neurodegenerative disorders is constrained by the inability of many small molecules to cross the blood-brain barrier (BBB). We are currently addressing this problem with a strategy to test the effect of proteasome inhibition in the CNS on the SMA phenotype. To overcome bortezomib’s low CNS penetrance we co-administered bortezomib with the P-
69
glycoprotein (P-gp) inhibitor, tariquidar. P-gp is a multidrug efflux transporter that is present at the
BBB and implicated in central nervous system resistance to drugs (Figure 4.2).
We treated a small cohort of SMA model mice with tariquidar in combination with bortezomib, and using a fluorescent reporter assay we found evidence of proteasome inhibition in the brain and spinal cord 24 hours after treatment (Figure 4.3B). SMN levels were also increased in the CNS tissues of the treated mice (Figure 4.3A). These data suggest that tariquidar increases the
CNS penetrance of bortezomib by blocking the active efflux of the drug. We also observed improved righting times and increased body weight (Figure 4.3C and Figure 4.3D). Histological, biochemical and physiological parameters will be examined. These experiments will test the hypothesis that inhibiting the UPS in the CNS as well as the periphery will increase SMN levels and correct the phenotype in SMA mice. It would also be interesting to compare the effects of tariquidar and bortezomib to animals given ICV injections of bortezomib. The results could further elucidate the contribution of increasing SMN in peripheral tissues versus the CNS.
We also plan to validate the role of Mib1 in SMN degradation in vivo in SMA mice. Early administration of a single injection of self-complementary adeno-associated virus 9 (scAAV9) carrying SMN has been found to rescue the SMA phenotype in mice (Bevan et al, 2010; Dominguez et al, 2011; Foust et al, 2010; Valori et al, 2010). Using the same viral vector, we have made constructs with either a non-specific scrambled shRNA sequence or an shRNA to Mib1. These constructs will be packaged into scAAV9 viruses and injected ICV into neonatal mice at P2, when the skulls of the animals are still thin enough to puncture. Knockdown of Mib1 in Mib1 shRNA- scAAV9 animals will be assessed for rescue of the disease phenotype. We have chosen to use ICV injection as a route to deliver our Mib1 shRNA as results described earlier in this thesis have shown that Mib1 knockdown is essential in neurons to improve the SMA phenotype in C. elegans.
Another on-going project includes developing a high throughput small molecule screen for inhibitors of Mib1. Such drugs currently do not exist and are needed to show that Mib1 can be targeted pharmacologically to increase SMN. We plan to screen for compounds that decrease the
70
ability of Mib1 to ubiquitinate SMN and will optimize and validate these compounds in cell culture and animal models.
Implications
There is currently no treatment for SMA. Increasing SMN protein availability as a strategy for SMA therapy requires an understanding of SMN protein degradation. Work in this thesis identified the ubiquitin proteasome pathway as the cellular system by which SMN is degraded and discovered a novel modifier of SMN, the E3 ligase Mib1 (Figure 4.4). Targeting either the proteasome or Mib1 increased SMN and improved the SMA phenotype in animal models. Our work also indicates that increasing SMN availability in both CNS and peripheral tissues leads to maximal correction of SMA, pointing to a role for SMN in all tissues. In summary, we have validated stabilizing the SMN protein as a therapeutic option for SMA, providing incentive for future work identifying other factors involved in its degradation.
71
Figure 4.1 Mib1 increased in spinal cords of SMA mice. Protein lysates from the spinal cords of P13 heterozygous (Het) and SMA mice were analyzed for Mib1 protein and mRNA levels by western blotting and RT-PCR, respectively. ** P <0.01
72
Figure 4.2. P-glycoprotein structure and function. (A) P-gp is a transmembrane protein localized on the apical side of cells that prevents the ingress of molecules. These polarized cells are connected by tight junctions that prevent
paracellular diffiusion and ensure that the passage of small molecules is transporter-regulated.
(B) A model of P-gp in the lipid bilayer extruding a chemical. P-gp binds and hydrolyzes ATP which initiates substrate extrusion. Substrates can be intercepted and extruded directly from the lipid bilayer or be drawn from the intracellular pool. (C) P-gp is expressed in various organs in the human body. Direction of substrate transport by P-gp is indicated by arrows. (D) Representative positron emission tomograph and magnetic resonance imaging images of brains after injection of radiolabeled loperamide, a P-gp inhibitor, under baseline and blocked conditions in three species (Kannan et al, 2009).
73
Figure 4.3. Tariquidar and bortezomib increase SMN and decrease proteasome activity in both CNS and peripheral tissues and improve body weight and motor function in SMA mice. SMA mice were treated with intraperitoneal injections of tariquidar (15 mg/kg) bortezomib
(0.15 mg/kg) or vehicle every other day starting on P5. (A) Western blot of tissues from mice
treated with either tariquidar alone or a combination of tariquidar and bortezomib (B) Proteasome activity assay in the same tissues. (C) Body weights of mice treated with either tariquidar alone, bortezomib alone or a combination of tariquidar and bortezomib. (D) Percent of mice from each treatment group able to right themselves within 10 seconds.
74
Figure 4.4. Schematic of SMN protein degradation. After a ubiquitin molecule is activated and conjugated by E1 and E2 enzymes, respectively, SMN is ubiquitinated by Mib1, an E3 ubiquitin ligase that binds both SMN and the E2 enzyme. The process is repeated until a chain of ubiquitin molecules is formed and SMN is shuttled into the proteasome and digested into short oligopeptides.
75
Bibliography
Adams J, Palombella VJ, Sausville EA, Johnson J, Destree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott PJ (1999) Proteasome inhibitors: a novel class of potent and effective antitumor agents. Cancer Res 59: 2615-2622
Akten B, Kye MJ, Hao le T, Wertz MH, Singh S, Nie D, Huang J, Merianda TT, Twiss JL, Beattie CE, Steen JA, Sahin M (2011) Interaction of survival of motor neuron (SMN) and HuD proteins with mRNA cpg15 rescues motor neuron axonal deficits. Proc Natl Acad Sci U S A 108: 10337-10342
Andreassi C, Angelozzi C, Tiziano FD, Vitali T, De Vincenzi E, Boninsegna A, Villanova M, Bertini E, Pini A, Neri G, Brahe C (2004) Phenylbutyrate increases SMN expression in vitro: relevance for treatment of spinal muscular atrophy. Eur J Hum Genet 12: 59-65
Armand JP, Burnett AK, Drach J, Harousseau JL, Lowenberg B, San Miguel J (2007) The emerging role of targeted therapy for hematologic malignancies: update on bortezomib and tipifarnib. Oncologist 12: 281-290
Assereto S, Stringara S, Sotgia F, Bonuccelli G, Broccolini A, Pedemonte M, Traverso M, Biancheri R, Zara F, Bruno C, Lisanti MP, Minetti C (2006) Pharmacological rescue of the dystrophin- glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment. Am J Physiol Cell Physiol 290: C577-582
Avila AM, Burnett BG, Taye AA, Gabanella F, Knight MA, Hartenstein P, Cizman Z, Di Prospero NA, Pellizzoni L, Fischbeck KH, Sumner CJ (2007) Trichostatin A increases SMN expression and survival in a mouse model of spinal muscular atrophy. J Clin Invest 117: 659-671
Barsi JC, Rajendra R, Wu JI, Artzt K (2005) Mind bomb1 is a ubiquitin ligase essential for mouse embryonic development and Notch signaling. Mech Dev 122: 1106-1117
Battle DJ, Kasim M, Yong J, Lotti F, Lau CK, Mouaikel J, Zhang Z, Han K, Wan L, Dreyfuss G (2006) The SMN complex: an assembly machine for RNPs. Cold Spring Harb Symp Quant Biol 71: 313- 320
Beehler BC, Sleph PG, Benmassaoud L, Grover GJ (2006) Reduction of skeletal muscle atrophy by a proteasome inhibitor in a rat model of denervation. Exp Biol Med (Maywood) 231: 335-341
Berndt JD, Aoyagi A, Yang P, Anastas JN, Tang L, Moon RT (2011) Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/{beta}-catenin signaling. J Cell Biol 194: 737-750
Bevan AK, Hutchinson KR, Foust KD, Braun L, McGovern VL, Schmelzer L, Ward JG, Petruska JC, Lucchesi PA, Burghes AH, Kaspar BK (2010) Early heart failure in the SMNDelta7 model of spinal muscular atrophy and correction by postnatal scAAV9-SMN delivery. Hum Mol Genet 19: 3895- 3905
Blaney SM, Bernstein M, Neville K, Ginsberg J, Kitchen B, Horton T, Berg SL, Krailo M, Adamson PC (2004) Phase I study of the proteasome inhibitor bortezomib in pediatric patients with refractory solid tumors: a Children's Oncology Group study (ADVL0015). J Clin Oncol 22: 4804-4809
76
Bonuccelli G, Sotgia F, Capozza F, Gazzerro E, Minetti C, Lisanti MP (2007) Localized treatment with a novel FDA-approved proteasome inhibitor blocks the degradation of dystrophin and dystrophin-associated proteins in mdx mice. Cell Cycle 6: 1242-1248
Bonuccelli G, Sotgia F, Schubert W, Park DS, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, Lisanti MP (2003) Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins. Am J Pathol 163: 1663-1675
Braun S, Croizat B, Lagrange MC, Warter JM, Poindron P (1995) Constitutive muscular abnormalities in culture in spinal muscular atrophy. Lancet 345: 694-695
Brenner S (1974) The genetics of Caenorhabditis elegans. Genetics 77: 71-94
Bricceno KV, Sampognaro PJ, Van Meerbeke JP, Sumner CJ, Fischbeck KH, Burnett BG (2012) Histone deacetylase inhibition suppresses myogenin-dependent atrogene activation in spinal muscular atrophy mice. Hum Mol Genet
Brichta L, Hofmann Y, Hahnen E, Siebzehnrubl FA, Raschke H, Blumcke I, Eyupoglu IY, Wirth B (2003) Valproic acid increases the SMN2 protein level: a well-known drug as a potential therapy for spinal muscular atrophy. Hum Mol Genet 12: 2481-2489
Brichta L, Holker I, Haug K, Klockgether T, Wirth B (2006) In vivo activation of SMN in spinal muscular atrophy carriers and patients treated with valproate. Ann Neurol 59: 970-975
Briese M, Esmaeili B, Fraboulet S, Burt EC, Christodoulou S, Towers PR, Davies KE, Sattelle DB (2009) Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan. Hum Mol Genet 18: 97-104
Brzustowicz LM, Lehner T, Castilla LH, Penchaszadeh GK, Wilhelmsen KC, Daniels R, Davies KE, Leppert M, Ziter F, Wood D, et al. (1990) Genetic mapping of chronic childhood-onset spinal muscular atrophy to chromosome 5q11.2-13.3. Nature 344: 540-541
Buhler D, Raker V, Luhrmann R, Fischer U (1999) Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy. Hum Mol Genet 8: 2351- 2357
Burglen L, Lefebvre S, Clermont O, Burlet P, Viollet L, Cruaud C, Munnich A, Melki J (1996) Structure and organization of the human survival motor neurone (SMN) gene. Genomics 32: 479-482
Burnett BG, Munoz E, Tandon A, Kwon DY, Sumner CJ, Fischbeck KH (2009a) Regulation of SMN Protein Stability. Mol Cell Biol 29: 1107-1115
Burnett BG, Munoz E, Tandon A, Kwon DY, Sumner CJ, Fischbeck KH (2009b) Regulation of SMN Protein Stability. Molecular and Cellular Biology 29: 1107-1115
Calixto A, Chelur D, Topalidou I, Chen X, Chalfie M (2010) Enhanced neuronal RNAi in C. elegans using SID-1. Nat Methods 7: 554-559
Cartegni L, Krainer AR (2002) Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet 30: 377-384
77
Chan YB, Miguel-Aliaga I, Franks C, Thomas N, Trulzsch B, Sattelle DB, Davies KE, van den Heuvel M (2003) Neuromuscular defects in a Drosophila survival motor neuron gene mutant. Hum Mol Genet 12: 1367-1376
Chang HC, Dimlich DN, Yokokura T, Mukherjee A, Kankel MW, Sen A, Sridhar V, Fulga TA, Hart AC, Van Vactor D, Artavanis-Tsakonas S (2008) Modeling spinal muscular atrophy in Drosophila. PLoS One 3: e3209
Chang HC, Hung WC, Chuang YJ, Jong YJ (2004) Degradation of survival motor neuron (SMN) protein is mediated via the ubiquitin/proteasome pathway. Neurochem Int 45: 1107-1112
Chang JG, Hsieh-Li HM, Jong YJ, Wang NM, Tsai CH, Li H (2001) Treatment of spinal muscular atrophy by sodium butyrate. Proc Natl Acad Sci U S A 98: 9808-9813
Chen PC, Qin LN, Li XM, Walters BJ, Wilson JA, Mei L, Wilson SM (2009) The proteasome- associated deubiquitinating enzyme Usp14 is essential for the maintenance of synaptic ubiquitin levels and the development of neuromuscular junctions. J Neurosci 29: 10909-10919
Chen W, Casey Corliss D (2004) Three modules of zebrafish Mind bomb work cooperatively to promote Delta ubiquitination and endocytosis. Dev Biol 267: 361-373
Cho S, Dreyfuss G (2010) A degron created by SMN2 exon 7 skipping is a principal contributor to spinal muscular atrophy severity. Genes Dev 24: 438-442
Choe EA, Liao L, Zhou JY, Cheng D, Duong DM, Jin P, Tsai LH, Peng J (2007) Neuronal morphogenesis is regulated by the interplay between cyclin-dependent kinase 5 and the ubiquitin ligase mind bomb 1. J Neurosci 27: 9503-9512
Cifuentes-Diaz C, Frugier T, Tiziano FD, Lacene E, Roblot N, Joshi V, Moreau MH, Melki J (2001) Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy. J Cell Biol 152: 1107-1114
Coovert DD, Le TT, McAndrew PE, Strasswimmer J, Crawford TO, Mendell JR, Coulson SE, Androphy EJ, Prior TW, Burghes AH (1997) The survival motor neuron protein in spinal muscular atrophy. Hum Mol Genet 6: 1205-1214
Davis NB, Taber DA, Ansari RH, Ryan CW, George C, Vokes EE, Vogelzang NJ, Stadler WM (2004) Phase II trial of PS-341 in patients with renal cell cancer: a University of Chicago phase II consortium study. J Clin Oncol 22: 115-119
Davydov IV, Woods D, Safiran YJ, Oberoi P, Fearnhead HO, Fang S, Jensen JP, Weissman AM, Kenten JH, Vousden KH (2004) Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2. J Biomol Screen 9: 695-703
Dimitriadi M, Sleigh JN, Walker A, Chang HC, Sen A, Kalloo G, Harris J, Barsby T, Walsh MB, Satterlee JS, Li C, Van Vactor D, Artavanis-Tsakonas S, Hart AC (2010) Conserved genes act as modifiers of invertebrate SMN loss of function defects. PLoS Genet 6: e1001172
DiStefano PS, Friedman B, Radziejewski C, Alexander C, Boland P, Schick CM, Lindsay RM, Wiegand SJ (1992) The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron 8: 983-993
78
Dominguez E, Marais T, Chatauret N, Benkhelifa-Ziyyat S, Duque S, Ravassard P, Carcenac R, Astord S, Pereira de Moura A, Voit T, Barkats M (2011) Intravenous scAAV9 delivery of a codon- optimized SMN1 sequence rescues SMA mice. Hum Mol Genet 20: 681-693
Dubowitz V (2009) Ramblings in the history of spinal muscular atrophy. Neuromuscul Disord 19: 69-73
Faderl S, Rai K, Gribben J, Byrd JC, Flinn IW, O'Brien S, Sheng S, Esseltine DL, Keating MJ (2006) Phase II study of single-agent bortezomib for the treatment of patients with fludarabine-refractory B- cell chronic lymphocytic leukemia. Cancer 107: 916-924
Fakharzadeh SS, Rosenblum-Vos L, Murphy M, Hoffman EK, George DL (1993) Structure and organization of amplified DNA on double minutes containing the mdm2 oncogene. Genomics 15: 283- 290
Fallini C, Zhang H, Su Y, Silani V, Singer RH, Rossoll W, Bassell GJ (2011) The survival of motor neuron (SMN) protein interacts with the mRNA-binding protein HuD and regulates localization of poly(A) mRNA in primary motor neuron axons. J Neurosci 31: 3914-3925
Foust KD, Wang X, McGovern VL, Braun L, Bevan AK, Haidet AM, Le TT, Morales PR, Rich MM, Burghes AH, Kaspar BK (2010) Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol 28: 271-274
Fuentes JL, Strayer MS, Matera AG (2010) Molecular determinants of survival motor neuron (SMN) protein cleavage by the calcium-activated protease, calpain. PLoS One 5: e15769
Gabanella F, Butchbach ME, Saieva L, Carissimi C, Burghes AH, Pellizzoni L (2007) Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs. PLoS One 2: e921
Garbes L, Riessland M, Holker I, Heller R, Hauke J, Trankle C, Coras R, Blumcke I, Hahnen E, Wirth B (2009) LBH589 induces up to 10-fold SMN protein levels by several independent mechanisms and is effective even in cells from SMA patients non-responsive to valproate. Hum Mol Genet 18: 3645-3658
Gavrilina TO, McGovern VL, Workman E, Crawford TO, Gogliotti RG, DiDonato CJ, Monani UR, Morris GE, Burghes AH (2008) Neuronal SMN expression corrects spinal muscular atrophy in severe SMA mice while muscle-specific SMN expression has no phenotypic effect. Hum Mol Genet 17: 1063-1075
Giesemann T, Rathke-Hartlieb S, Rothkegel M, Bartsch JW, Buchmeier S, Jockusch BM, Jockusch H (1999) A role for polyproline motifs in the spinal muscular atrophy protein SMN. Profilins bind to and colocalize with smn in nuclear gems. J Biol Chem 274: 37908-37914
Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system. Physiol Rev 83: 731-801
Griesbeck O, Parsadanian AS, Sendtner M, Thoenen H (1995) Expression of neurotrophins in skeletal muscle: quantitative comparison and significance for motoneuron survival and maintenance of function. J Neurosci Res 42: 21-33
Haas KF, Miller SL, Friedman DB, Broadie K (2007) The ubiquitin-proteasome system postsynaptically regulates glutamatergic synaptic function. Mol Cell Neurosci 35: 64-75
79
Hahnen E, Eyupoglu IY, Brichta L, Haastert K, Trankle C, Siebzehnrubl FA, Riessland M, Holker I, Claus P, Romstock J, Buslei R, Wirth B, Blumcke I (2006) In vitro and ex vivo evaluation of second- generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy. J Neurochem 98: 193-202
Hasselgren PO, Wray C, Mammen J (2002) Molecular regulation of muscle cachexia: it may be more than the proteasome. Biochem Biophys Res Commun 290: 1-10
Haupt Y, Maya R, Kazaz A, Oren M (1997) Mdm2 promotes the rapid degradation of p53. Nature 387: 296-299
Hegde AN, Upadhya SC (2007) The ubiquitin-proteasome pathway in health and disease of the nervous system. Trends Neurosci 30: 587-595
Horton TM, Pati D, Plon SE, Thompson PA, Bomgaars LR, Adamson PC, Ingle AM, Wright J, Brockman AH, Paton M, Blaney SM (2007) A phase 1 study of the proteasome inhibitor bortezomib in pediatric patients with refractory leukemia: a Children's Oncology Group study. Clin Cancer Res 13: 1516-1522
Hsieh-Li HM, Chang JG, Jong YJ, Wu MH, Wang NM, Tsai CH, Li H (2000) A mouse model for spinal muscular atrophy. Nat Genet 24: 66-70
Hua Y, Sahashi K, Rigo F, Hung G, Horev G, Bennett CF, Krainer AR (2011) Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model. Nature 478: 123-126
Huang J, Sheung J, Dong G, Coquilla C, Daniel-Issakani S, Payan DG (2005) High-throughput screening for inhibitors of the e3 ubiquitin ligase APC. Methods Enzymol 399: 740-754
Hurst-Kennedy J, Chin LS, Li L (2012) Ubiquitin C-Terminal Hydrolase L1 in Tumorigenesis. Biochem Res Int 2012: 123706
Issaeva N, Bozko P, Enge M, Protopopova M, Verhoef LG, Masucci M, Pramanik A, Selivanova G (2004) Small molecule RITA binds to p53, blocks p53-HDM-2 interaction and activates p53 function in tumors. Nat Med 10: 1321-1328
Itoh M, Kim CH, Palardy G, Oda T, Jiang YJ, Maust D, Yeo SY, Lorick K, Wright GJ, Ariza- McNaughton L, Weissman AM, Lewis J, Chandrasekharappa SC, Chitnis AB (2003) Mind bomb is a ubiquitin ligase that is essential for efficient activation of Notch signaling by Delta. Dev Cell 4: 67-82
Jamart C, Raymackers JM, Li An G, Deldicque L, Francaux M (2011) Prevention of muscle disuse atrophy by MG132 proteasome inhibitor. Muscle Nerve 43: 708-716
Jarecki J, Chen X, Bernardino A, Coovert DD, Whitney M, Burghes A, Stack J, Pollok BA (2005) Diverse small-molecule modulators of SMN expression found by high-throughput compound screening: early leads towards a therapeutic for spinal muscular atrophy. Hum Mol Genet 14: 2003- 2018
Jin Y, Blue EK, Dixon S, Shao Z, Gallagher PJ (2002a) A death-associated protein kinase (DAPK)- interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factor-induced apoptosis and regulates the cellular levels of DAPK. J Biol Chem 277: 46980-46986
80
Jin Y, Blue EK, Dixon S, Shao Z, Gallagher PJ (2002b) A death-associated protein kinase (DAPK)- interacting protein, DIP-1, is an E3 ubiquitin ligase that promotes tumor necrosis factor-induced apoptosis and regulates the cellular levels of DAPK. J Biol Chem 277: 46980-46986
Jung T, Catalgol B, Grune T (2009) The proteasomal system. Mol Aspects Med 30: 191-296
Kamath RS, Ahringer J (2003) Genome-wide RNAi screening in Caenorhabditis elegans. Methods 30: 313-321
Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J (2001) Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol 2: RESEARCH0002
Kannan P, John C, Zoghbi SS, Halldin C, Gottesman MM, Innis RB, Hall MD (2009) Imaging the function of P-glycoprotein with radiotracers: pharmacokinetics and in vivo applications. Clin Pharmacol Ther 86: 368-377
Kernochan LE, Russo ML, Woodling NS, Huynh TN, Avila AM, Fischbeck KH, Sumner CJ (2005) The role of histone acetylation in SMN gene expression. Hum Mol Genet 14: 1171-1182
Kisselev AF, Callard A, Goldberg AL (2006) Importance of the different proteolytic sites of the proteasome and the efficacy of inhibitors varies with the protein substrate. J Biol Chem 281: 8582- 8590
Koblish HK, Zhao S, Franks CF, Donatelli RR, Tominovich RM, LaFrance LV, Leonard KA, Gushue JM, Parks DJ, Calvo RR, Milkiewicz KL, Marugan JJ, Raboisson P, Cummings MD, Grasberger BL, Johnson DL, Lu T, Molloy CJ, Maroney AC (2006) Benzodiazepinedione inhibitors of the Hdm2:p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo. Mol Cancer Ther 5: 160-169
Komatsu H, Chao MY, Larkins-Ford J, Corkins ME, Somers GA, Tucey T, Dionne HM, White JQ, Wani K, Boxem M, Hart AC (2008) OSM-11 facilitates LIN-12 Notch signaling during Caenorhabditis elegans vulval development. PLoS Biol 6: e196
Kong L, Wang X, Choe DW, Polley M, Burnett BG, Bosch-Marce M, Griffin JW, Rich MM, Sumner CJ (2009) Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice. J Neurosci 29: 842-851
Koo BK, Lim HS, Song R, Yoon MJ, Yoon KJ, Moon JS, Kim YW, Kwon MC, Yoo KW, Kong MP, Lee J, Chitnis AB, Kim CH, Kong YY (2005a) Mind bomb 1 is essential for generating functional Notch ligands to activate Notch. Development 132: 3459-3470
Koo BK, Yoon KJ, Yoo KW, Lim HS, Song R, So JH, Kim CH, Kong YY (2005b) Mind bomb-2 is an E3 ligase for Notch ligand. J Biol Chem 280: 22335-22342
Kugelberg E, Welander L (1956) Heredofamilial juvenile muscular atrophy simulating muscular dystrophy. AMA Arch Neurol Psychiatry 75: 500-509
Kwon DY, Motley WW, Fischbeck KH, Burnett BG (2011) Increasing expression and decreasing degradation of SMN ameliorate the spinal muscular atrophy phenotype in mice. Hum Mol Genet 20: 3667-3677
81
Lai EC, Roegiers F, Qin X, Jan YN, Rubin GM (2005) The ubiquitin ligase Drosophila Mind bomb promotes Notch signaling by regulating the localization and activity of Serrate and Delta. Development 132: 2319-2332
Le Borgne R, Remaud S, Hamel S, Schweisguth F (2005) Two distinct E3 ubiquitin ligases have complementary functions in the regulation of delta and serrate signaling in Drosophila. PLoS Biol 3: e96
Le TT, Pham LT, Butchbach ME, Zhang HL, Monani UR, Coovert DD, Gavrilina TO, Xing L, Bassell GJ, Burghes AH (2005) SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal muscular atrophy and associates with full- length SMN. Hum Mol Genet 14: 845-857
Lefebvre S, Burglen L, Reboullet S, Clermont O, Burlet P, Viollet L, Benichou B, Cruaud C, Millasseau P, Zeviani M, et al. (1995) Identification and characterization of a spinal muscular atrophy-determining gene. Cell 80: 155-165
Lefebvre S, Burlet P, Liu Q, Bertrandy S, Clermont O, Munnich A, Dreyfuss G, Melki J (1997) Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 16: 265- 269
Liu Y, Fallon L, Lashuel HA, Liu Z, Lansbury PT, Jr. (2002) The UCH-L1 gene encodes two opposing enzymatic activities that affect alpha-synuclein degradation and Parkinson's disease susceptibility. Cell 111: 209-218
Lorson CL, Androphy EJ (2000) An exonic enhancer is required for inclusion of an essential exon in the SMA-determining gene SMN. Hum Mol Genet 9: 259-265
Lorson CL, Hahnen E, Androphy EJ, Wirth B (1999) A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci U S A 96: 6307-6311
Lorson CL, Strasswimmer J, Yao JM, Baleja JD, Hahnen E, Wirth B, Le T, Burghes AH, Androphy EJ (1998a) SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat Genet 19: 63-66
Lorson CL, Strasswimmer J, Yao JM, Baleja JD, Hahnen E, Wirth B, Le T, Burghes AH, Androphy EJ (1998b) SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat Genet 19: 63-66
Martinez TL, Kong L, Wang X, Osborne MA, Crowder ME, Van Meerbeke JP, Xu X, Davis C, Wooley J, Goldhamer DJ, Lutz CM, Rich MM, Sumner CJ (2012) Survival Motor Neuron Protein in Motor Neurons Determines Synaptic Integrity in Spinal Muscular Atrophy. J Neurosci 32: 8703-8715
McAndrew PE, Parsons DW, Simard LR, Rochette C, Ray PN, Mendell JR, Prior TW, Burghes AH (1997) Identification of proximal spinal muscular atrophy carriers and patients by analysis of SMNT and SMNC gene copy number. Am J Hum Genet 60: 1411-1422
McWhorter ML, Monani UR, Burghes AH, Beattie CE (2003a) Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J Cell Biol 162: 919-931
82
McWhorter ML, Monani UR, Burghes AH, Beattie CE (2003b) Knockdown of the survival motor neuron (Smn) protein in zebrafish causes defects in motor axon outgrowth and pathfinding. J Cell Biol 162: 919-931
Meiners S, Ludwig A, Stangl V, Stangl K (2008) Proteasome inhibitors: poisons and remedies. Med Res Rev 28: 309-327
Melki J, Sheth P, Abdelhak S, Burlet P, Bachelot MF, Lathrop MG, Frezal J, Munnich A (1990) Mapping of acute (type I) spinal muscular atrophy to chromosome 5q12-q14. The French Spinal Muscular Atrophy Investigators. Lancet 336: 271-273
Meusser B, Hirsch C, Jarosch E, Sommer T (2005) ERAD: the long road to destruction. Nat Cell Biol 7: 766-772
Minucci S, Pelicci PG (2006) Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat Rev Cancer 6: 38-51
Monani UR (2005) Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor neuron- specific disease. Neuron 48: 885-896
Monani UR, Lorson CL, Parsons DW, Prior TW, Androphy EJ, Burghes AH, McPherson JD (1999) A single nucleotide difference that alters splicing patterns distinguishes the SMA gene SMN1 from the copy gene SMN2. Hum Mol Genet 8: 1177-1183
Muralidhar MG, Thomas JB (1993) The Drosophila bendless gene encodes a neural protein related to ubiquitin-conjugating enzymes. Neuron 11: 253-266
Narver HL, Kong L, Burnett BG, Choe DW, Bosch-Marce M, Taye AA, Eckhaus MA, Sumner CJ (2008) Sustained improvement of spinal muscular atrophy mice treated with trichostatin A plus nutrition. Ann Neurol 64: 465-470
Nawaz Z, Lonard DM, Smith CL, Lev-Lehman E, Tsai SY, Tsai MJ, O'Malley BW (1999) The Angelman syndrome-associated protein, E6-AP, is a coactivator for the nuclear hormone receptor superfamily. Mol Cell Biol 19: 1182-1189
Nencioni A, Grunebach F, Patrone F, Ballestrero A, Brossart P (2007) Proteasome inhibitors: antitumor effects and beyond. Leukemia 21: 30-36
Park GH, Maeno-Hikichi Y, Awano T, Landmesser LT, Monani UR (2010) Reduced survival of motor neuron (SMN) protein in motor neuronal progenitors functions cell autonomously to cause spinal muscular atrophy in model mice expressing the human centromeric (SMN2) gene. J Neurosci 30: 12005-12019
Paushkin S, Gubitz AK, Massenet S, Dreyfuss G (2002) The SMN complex, an assemblyosome of ribonucleoproteins. Curr Opin Cell Biol 14: 305-312
Pellizzoni L, Charroux B, Dreyfuss G (1999) SMN mutants of spinal muscular atrophy patients are defective in binding to snRNP proteins. Proc Natl Acad Sci U S A 96: 11167-11172
Petroski MD (2008) The ubiquitin system, disease, and drug discovery. BMC Biochem 9 Suppl 1: S7
83
Prior TW, Swoboda KJ, Scott HD, Hejmanowski AQ (2004) Homozygous SMN1 deletions in unaffected family members and modification of the phenotype by SMN2. Am J Med Genet A 130: 307-310
Rajendra TK, Gonsalvez GB, Walker MP, Shpargel KB, Salz HK, Matera AG (2007) A Drosophila melanogaster model of spinal muscular atrophy reveals a function for SMN in striated muscle. J Cell Biol 176: 831-841
Ramser J, Ahearn ME, Lenski C, Yariz KO, Hellebrand H, von Rhein M, Clark RD, Schmutzler RK, Lichtner P, Hoffman EP, Meindl A, Baumbach-Reardon L (2008) Rare missense and synonymous variants in UBE1 are associated with X-linked infantile spinal muscular atrophy. Am J Hum Genet 82: 188-193
Richmond TJ, Davey CA (2003) The structure of DNA in the nucleosome core. Nature 423: 145-150
Riessland M, Brichta L, Hahnen E, Wirth B (2006) The benzamide M344, a novel histone deacetylase inhibitor, significantly increases SMN2 RNA/protein levels in spinal muscular atrophy cells. Hum Genet 120: 101-110
Rose FF, Jr., Mattis VB, Rindt H, Lorson CL (2009) Delivery of recombinant follistatin lessens disease severity in a mouse model of spinal muscular atrophy. Hum Mol Genet 18: 997-1005
Rossoll W, Jablonka S, Andreassi C, Kroning AK, Karle K, Monani UR, Sendtner M (2003) Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta- actin mRNA in growth cones of motoneurons. J Cell Biol 163: 801-812
Rual JF, Ceron J, Koreth J, Hao T, Nicot AS, Hirozane-Kishikawa T, Vandenhaute J, Orkin SH, Hill DE, van den Heuvel S, Vidal M (2004) Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14: 2162-2168
Ruiz R, Casanas JJ, Torres-Benito L, Cano R, Tabares L (2010) Altered intracellular Ca2+ homeostasis in nerve terminals of severe spinal muscular atrophy mice. J Neurosci 30: 849-857
Sanes JR, Lichtman JW (1999) Development of the vertebrate neuromuscular junction. Annu Rev Neurosci 22: 389-442
Singh K, Chao MY, Somers GA, Komatsu H, Corkins ME, Larkins-Ford J, Tucey T, Dionne HM, Walsh MB, Beaumont EK, Hart DP, Lockery SR, Hart AC (2011) C. elegans Notch signaling regulates adult chemosensory response and larval molting quiescence. Curr Biol 21: 825-834
Smrt RD, Szulwach KE, Pfeiffer RL, Li X, Guo W, Pathania M, Teng ZQ, Luo Y, Peng J, Bordey A, Jin P, Zhao X (2010) MicroRNA miR-137 regulates neuronal maturation by targeting ubiquitin ligase mind bomb-1. Stem Cells 28: 1060-1070
Sumner CJ, Huynh TN, Markowitz JA, Perhac JS, Hill B, Coovert DD, Schussler K, Chen X, Jarecki J, Burghes AH, Taylor JP, Fischbeck KH (2003) Valproic acid increases SMN levels in spinal muscular atrophy patient cells. Ann Neurol 54: 647-654
Sumner CJ, Wee CD, Warsing LC, Choe DW, Ng AS, Lutz C, Wagner KR (2009) Inhibition of myostatin does not ameliorate disease features of severe spinal muscular atrophy mice. Hum Mol Genet 18: 3145-3152
84
Timmons L, Court DL, Fire A (2001) Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263: 103-112
Timmons L, Fire A (1998) Specific interference by ingested dsRNA. Nature 395: 854
Todd AG, Shaw DJ, Morse R, Stebbings H, Young PJ (2010) SMN and the Gemin proteins form sub-complexes that localise to both stationary and dynamic neurite granules. Biochem Biophys Res Commun 394: 211-216
Trockenbacher A, Suckow V, Foerster J, Winter J, Krauss S, Ropers HH, Schneider R, Schweiger S (2001) MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat Genet 29: 287-294
Valori CF, Ning K, Wyles M, Mead RJ, Grierson AJ, Shaw PJ, Azzouz M (2010) Systemic delivery of scAAV9 expressing SMN prolongs survival in a model of spinal muscular atrophy. Sci Transl Med 2: 35ra42 van Roessel P, Elliott DA, Robinson IM, Prokop A, Brand AH (2004) Independent regulation of synaptic size and activity by the anaphase-promoting complex. Cell 119: 707-718
Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303: 844-848
Walker MP, Rajendra TK, Saieva L, Fuentes JL, Pellizzoni L, Matera AG (2008) SMN complex localizes to the sarcomeric Z-disc and is a proteolytic target of calpain. Hum Mol Genet 17: 3399-3410
Wolstencroft EC, Mattis V, Bajer AA, Young PJ, Lorson CL (2005) A non-sequence-specific requirement for SMN protein activity: the role of aminoglycosides in inducing elevated SMN protein levels. Hum Mol Genet 14: 1199-1210
Yi JJ, Ehlers MD (2007) Emerging roles for ubiquitin and protein degradation in neuronal function. Pharmacol Rev 59: 14-39
Yoo KW, Kim EH, Jung SH, Rhee M, Koo BK, Yoon KJ, Kong YY, Kim CH (2006) Snx5, as a Mind bomb-binding protein, is expressed in hematopoietic and endothelial precursor cells in zebrafish. FEBS Lett 580: 4409-4416
Young PJ, Man NT, Lorson CL, Le TT, Androphy EJ, Burghes AH, Morris GE (2000) The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding. Hum Mol Genet 9: 2869-2877
Zhang C, Li Q, Jiang YJ (2007) Zebrafish Mib and Mib2 are mutual E3 ubiquitin ligases with common and specific delta substrates. J Mol Biol 366: 1115-1128
Zhang HL, Pan F, Hong D, Shenoy SM, Singer RH, Bassell GJ (2003) Active transport of the survival motor neuron protein and the role of exon-7 in cytoplasmic localization. J Neurosci 23: 6627- 6637
Zhang Z, Lotti F, Dittmar K, Younis I, Wan L, Kasim M, Dreyfuss G (2008) SMN deficiency causes tissue-specific perturbations in the repertoire of snRNAs and widespread defects in splicing. Cell 133: 585-600
85
86