NUCLEAR RECEPTORS AS THERAPEUTIC TARGETS FOR ALZHEIMER’S DISEASE
by
REBECCA COURTNEY
Submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
Department of Neurosciences
CASE WESTERN RESERVE UNIVERSITY
January, 2017
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
Rebecca Courtney
Candidate for the degree of Doctor of Philosophy*.
Committee Chair
Evan Deneris
Committee Member
Gary Landreth
Committee Member
Bruce Lamb
Committee Member
Heather Broiher
Date of Defense
November 22, 2016
*We also certify that written approval has been obtained for any proprietary material
contained therein.
2 Contents List of Tables ...... 5
List of Figures ...... 6
Acknowledgements...... 8
Abstract ...... 10
Chapter 1: Introduction ...... 12
Alzheimer’s disease ...... 13
Pathogenesis of Alzheimer’s disease ...... 15
APP processing ...... 17
Amyloid hypothesis ...... 20
Preclinical models of AD ...... 22
Late-onset Alzheimer’s ...... 25
Aβ clearance mechanisms ...... 25
ApoE and LOAD ...... 29
Nuclear receptor structure and function ...... 32
RXRs ...... 33
LXRs ...... 34
PPARs ...... 36
NR4As ...... 37
Nuclear receptors in inflammation ...... 38
Nuclear receptors in Alzheimer’s disease ...... 40
Research Goals ...... 46
3 Chapter 2: Combined Liver X Receptor/Peroxisome Proliferator-Activated Receptor γ Agonist
Treatment Reduces Amyloid-β Levels and Improves Behavior in Amyloid Precursor
Protein/Presenilin 1 Mice ...... 57
Abstract ...... 58
Introduction ...... 59
Experimental Procedures ...... 62
Results ...... 68
Discussion ...... 74
Chapter 3: Nurr1 is neuroprotective in the 5XFAD subiculum...... 91
Abstract ...... 92
Introduction ...... 92
Experimental Procedures ...... 95
Results ...... 98
Discussion ...... 101
Chapter 4: Discussion ...... 111
Nuclear receptor agonists act in combination on apoE and microglia ...... 114
The amelioration of hypertriglyceridemia by combining pioglitazone with GW3965 introduces
new therapeutic options for AD ...... 116
Nurr1 represents a novel target for neuroprotection in AD ...... 117
4 List of Tables
Chapter 1:
Table 1: Nuclear receptor agonist treatments of neurodegenerative diseases…49
5 List of Figures
Chapter 1:
Figure 1.1: Nuclear receptors are ligand-activated transcription factors…………..48
Figure 1.2: Cell-type specific actions of nuclear receptors in the brain……………..56
Chapter 2:
Figure 2.1: Combination therapy increases target gene expression and Aβ
degradation and decreases inflammatory markers in vitro……………………………….81
Figure 2.2: Nuclear receptor agonists stimulate transcription of LXR target genes
in AD mice……………………..…………..…………..…………..…………..…………..………………….83
Figure 2.3: Nuclear receptor agonists reduce the pro-inflammatory environment
in APP/PS1 mice……………………………..…………..…………..…………..…………………..………85
Figure 2.4: Combination therapy significantly reduces amyloid burden in AD
mice…………………………..…………..…………..…………..…………..…………..……………….……..87
Figure 2.5: Nuclear receptor agonists promote microglial colocalization with
plaques………………………..…………..…………..…………..…………..…………..………….………..89
Figure 2.6: Nuclear receptor agonists ameliorates cognitive deficits in AD mice.90
6 Chapter 3:
Figure 3.1: shRNA specific to Nurr1 decreases neuron survival in the 5XFAD
mouse…………………..…………..…………..…………..…………..…………..…………..…………….105
Figure 3.2: Nurr1 overexpression in the 5XFAD model preserves subicular
neurons and decreases dystrophic neurites…………………..…………..…………..…..….107
Figure 3.3: Nurr1 overexpression decreases intraneuronal and deposited forms
of amyloid…………………..…………..…………..…………..…………..…………..…………..……...108
Figure 3.4: Microglial activation is unaffected by Nurr1 modulation in
neurons……………..…………..…………..…………..…………..…………..…………..…………....…110
Chapter 4:
Figure 4.1. Cultured neurons from 5XFAD animals exhibit glutamate
excitotoxicity in vitro, and this is ameliorated with bexarotene treatment…..…120
7 Acknowledgements
There are many people I would like to thank for what has been an amazing journey. First, I’d like to thank my advisor, Dr. Gary Landreth, for sharing his truly inspiring excitement about science especially at the times when my own was lagging. He always managed to make sense out of everything, and encouraged me both professionally and personally with his boundless enthusiasm. I would like to thank my committee members, Drs. Evan Deneris, Bruce Lamb, and Heather Brioher, for their insightful comments and advice over the years both as my committee and as members of a wonderful and collaborative Neurosciences department. I would especially like to thank Dr. Noa Noy in memoriam; as a teacher and as a committee member she was a guiding force and an inspiration.
I would also like to thank all of the Landreth lab members that have touched my life, past and present. It truly takes a village. Special thanks goes to Dr. Julie Savage,
Brad Casali, Drs. Monica Mariani and Angela Corona, and Taylor Jay for being my technical and emotional support both in and out of the lab, and to Drs. Joanna
Pucilowska and Erin Reed-Geaghan for excellent discussions, advice and assistance. I’d also like to thank Drs. Shweta Mandrekar-Collucci, Daniel Lee, Paige Cramer, and Brent
Cameron for their patience in showing me the ropes, and Dr. Joe Vithayathil for literally always being around when I had a question or needed to chat. Thanks as well to Donna
Kirsch for always keeping us all on track, and Colleen Karlo for solving every lab-related problem any of us ever had, and thousands more we never even knew existed. You have
8 all been such an incredibly wonderful group of people to travel this road with and learn from.
Finally, I’d like to thank my friends and family who have made this possible.
Thank you to my parents and sister for their endless support and constant encouragement, and for always putting things in perspective when I can’t. And a very special thank you to my husband Nick, who has been on this journey with me the entire way. You have been the most patient, kind and supportive human being I have ever met. I look forward to always taking the next step with you.
9
NUCLEAR RECEPTORS AS THERAPEUTIC TARGETS FOR ALZHEIMER’S DISEASE
Abstract
by
REBECCA COURTNEY
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (Aβ) species in the brain. This is followed by robust neuroinflammation, the development of neurofibrillary tangles composed of tau, synaptic dysfunction, neuronal death, and the progressive development of cognitive and behavioral impairments. We demonstrate that nuclear receptors make useful therapeutic targets to address several aspects of this pathology.
Using agonists for PPARγ and LXR, we show that a short-term treatment of 9 days is sufficient to ameliorate behavioral impairments, attenuate neuroinflammation, and induce partial clearance of Aβ from the brain in an AD mouse model. This combination therapy acted on astrocytes to increase the expression and lipidation of apoE, and on microglia to increase their rate of Aβ degradation and ability to associate with Aβ plaques. We also demonstrate that some nuclear receptors, such as Nurr1, are valuable targets for addressing the neuronal pathology associated with AD. Knocking down Nurr1
10 decreased the survival of vulnerable neurons in an AD mouse model. Conversely, increasing Nurr1 expression increased neuronal survival, decreased dystrophic neurites, and ameliorated local Aβ pathology including decreasing intraneuronal Aβ by about
30%. Together, these data demonstrate that drugs targeting multiple nuclear receptors can be valuable to treat various aspects of AD pathology. Interestingly, LXR, PPARγ, and
Nurr1 can all be activated by the RXR agonist bexarotene. These data provide insight into the mechanisms by which bexarotene might act, and suggest several novel therapeutic approaches to the treatment of AD.
11
Chapter 1: Introduction
12 Alzheimer’s disease
Alzheimer’s disease was first identified as a distinct form of dementia in a 1906 lecture by Dr. Alois Alzheimer, who described the case of a woman in her 50’s presenting with a “peculiar severe disease process of the cerebral cortex” (Hippius &
Neundörfer, 2003). The patient in question, Auguste D., had been admitted to the psychiatric hospital at Frankfurt am Main in 1901 at the age of 51, after exhibiting an unusual jealousy of her husband (paranoia) followed by a rapid loss of memory, disorientation, and delusions (Stelzmann, Norman Schnitzlein, & Reed Murtagh, 1995).
Her mental condition progressively deteriorated during her time in the facility, and after four and a half years, Auguste D. died. Upon post-mortem examination of the brain, Dr.
Alzheimer noted general brain atrophy, along with an unusual accumulation of intracellular neurofibrils, “miliary foci” deposited throughout the cortex, and changes in glial cells (Stelzmann et al., 1995). Although Alzheimer was one of the first to credit that these pathological changes had also been observed in various other forms of dementia, the early onset and severity of clinical symptoms and the aggressive number of histological changes described by Alzheimer indicated a unique disease. In 1910 Emil
Kraepelin was the first to report on this disease using the name that would become a household phrase in the current day, and “Alzheimer’s Disease” was born (Hippius &
Neundörfer, 2003).
Today, Alzheimer’s disease affects an estimated 5.4 million Americans, and the aging population could cause that number to increase to 13.8 million by 2050
(Alzheimer’s Association, 2016). Alzheimer’s is the most common cause of dementia,
13 accounting for 60-80% of all cases. In 2016, it is estimated that 700,000 Americans older than 65 will die with Alzheimer’s disease, making it the sixth leading cause of death overall in the US. Alzheimer’s is also a significant budgetary concern for the US government, with Medicare and Medicaid expected to cover 68% of the $236 billion total cost of caring for Alzheimer’s patients in 2016. Additionally, Alzheimer’s patients generate a huge caregiver burden emotionally and financially, with unpaid caregivers providing an estimated $221 billion worth of care over 18.1 billion hours in 2015. By
2050, the yearly cost of care for Alzheimer’s patients is expected to break $1 trillion dollars, including a 5-fold increase in both government spending and out-of-pocket expenses for patients and family members (Alzheimer’s Association, 2016).
Current FDA-approved treatments for AD include cholinesterase inhibitors
(donepezil, galantamine, rivastigmine) and the NMDAR antagonist memantine, however, these treatments are only effective in a subset of the population, and the beneficial effects can be temporary (“About Alzheimer’s Disease: Treatment,” n.d.).
None of the currently approved drugs address the neuronal dysfunction and death that underlies cognitive impairment in AD patients. Currently 67 therapeutics are undergoing phase 2-3 clinical trials for AD or mild cognitive impairment (“Therapeutics Search |
ALZFORUM,” n.d.), but since 2003 only one new AD medication has been FDA approved
(Namzaric, a combination of donepezil and memantine, in 2014). There are several reasons the success rate for new drug approval is so low, including a lack of accurate preclinical animal models for this complex disease, late-stage diagnoses that make preventative or early-stage treatments difficult to test, a large amount of heterogeneity
14 in AD pathology and disease progression between patients, and the possibility of misdiagnosis, with current clinical diagnostic criteria for a probable AD diagnosis exhibiting a sensitivity and specificity of about 71% when compared to postmortem pathological diagnosis (Beach, Monsell, Phillips, & Kukull, 2010).
Pathogenesis of Alzheimer’s disease
Today, we know that the “intracellular neurofibrils” Alois Alzheimer described are largely composed of the microtubule-binding protein tau (Delacourte & Defossez,
1986; Grundke-Iqbal, Iqbal, Quinlan, et al., 1986; Kosik, Joachim, & Selkoe, 1986; Wood,
Mirra, Pollock, & Binder, 1986), especially hyperphosphorylated forms (p-tau) (Grundke-
Iqbal, Iqbal, Tung, et al., 1986; Ihara, Nukina, Miura, & Ogawara, 1986), and the “miliary foci” deposited throughout the cortex are mainly deposited amyloid beta (Aβ) protein
(Glenner & Wong, 1984a, 1984b; Masters et al., 1985). Glenner and Wong capitalized on observations that Down’s Syndrome patients obligatorily developed Alzheimer’s disease in the course of aging (Olson & Shaw, 1969) and isolated the amyloid β protein from the amyloid-laden microvasculature in both Down Syndrome brains (Glenner & Wong,
1984a) and the brains of AD patients (Glenner & Wong, 1984b). This observation was corroborated by Masters et al. (Masters et al., 1985) using Aβ isolated directly from plaques in Down’s and AD patients rather than the microvasculature. Shortly after, the gene encoding Aβ was cloned and mapped to chromosome 21(Goldgaber, Lerman,
McBride, Saffiotti, & Gajdusek, 1987; Kang et al., 1987; Robakis et al., 1987) and
15 identified as the amyloid precursor protein (Goldgaber et al., 1987; R. E. Tanzi et al.,
1987), validating the linkage between Down’s Syndrome, with its characteristic triplication of the 21st chromosome, and AD.
Less than 5% of AD patients have a dominantly inherited form of AD, which is known as Familial Alzheimer’s Disease (FAD) and generally presents prior to 60 years of age. The late-onset (>60 years of age), or sporadic, form of AD (LOAD) is much more common and is not generally linked to a specific genetic disruption, although genetic risk factors for LOAD will be discussed later. Because the disease progression, apart from age of onset, is extremely similar in both LOAD and FAD, the mutations that lead to
Familial Alzheimer’s Disease (FAD) have been instrumental in our understanding of AD pathogenesis. The first FAD mutation, termed the London mutation, was identified in
1991 (Goate et al., 1991) as a point mutation resulting in a V717I substitution in the APP gene. APP codes for the amyloid precursor protein (APP), which is proteolytically processed by α-, β-, and γ-secretases to generate a variety of cleavage products including Aβ. Fifty one mutations linked to FAD have since been discovered in the APP gene, along with 219 mutations in PSEN1 and 16 mutations in PSEN2 (“AD&FTD
Mutation Database,” n.d.), which code for subunits of the γ-secretase complex.
Together, these mutations account for the majority of FAD cases, and they are functionally linked by altering aggregation of Aβ or its production from APP.
16 APP processing
APP is a member of the APP family of proteins, a group of evolutionarily conserved single-pass transmembrane proteins which, in mammals, also includes APP- like proteins 1 and 2 (APLP1 and APLP2) (van der Kant & Goldstein, 2015). The intracellular C-terminus is highly conserved among these proteins, but the N-terminal extracellular region varies widely in structure, with APP the only family member to include the Aβ sequence in its extracellular domain. APP is an 18-exon gene with several splice variants that are generated in a tissue- and cell-type specific manner. Among APP family members, APLP1 and the 695 amino acid-long splice variant of APP (APP695) are specifically expressed in the human brain, while APLP2 and other APP isoforms are variably expressed in other tissues. The endogenous function of APP in the adult brain is not well understood, but knockout of APP in mice results in both motor and cognitive neurological impairments as well as axonal defects (G. R. Dawson et al., 1999; Kamal,
Almenar-Queralt, LeBlanc, Roberts, & Goldstein, 2001; Magara et al., 1999; Seabrook et al., 1999; Smith, Kallhoff, Zheng, & Pautler, 2007; Zheng et al., 1995), and knockdown of
APP results in impairments in cortical neuron migration during development (Dawkins &
Small, 2014; Young-Pearse et al., 2007). Interestingly, overexpression of human APP695 in mice increases synaptogenesis without leading to Aβ plaques or other AD-related pathology (L. Mucke et al., 1994), although Herzig et al. (Herzig et al., 2004) observed mild parenchymal and vascular Aβ deposition in a mouse overexpressing the human
APP751 splice variant. It remains difficult to define the role of APP protein in the normal
CNS due to the potential for compensation by other APP family members when APP
17 levels are decreased, as well as potentially different roles for splice variants or processing products of APP.
APP can normally enter one of two proteolytic processing pathways, which here will be termed non-amyloidogenic or amyloidogenic. Which pathway predominates is an incredibly complex issue that depends not only on relative protein concentrations but possibly on subcellular localization of APP, its association with lipid rafts, and of course the presence or absence of amyloidogenic mutations in either APP or its secretases
(Haass, Kaether, Thinakaran, & Sisodia, 2012). Full-length APP can undergo non- amyloidogenic cleavage by α-secretases, which are members of the A disintegrin and metalloproteinase family (notably ADAM9, ADAM10, and ADAM17) that cleave APP within the Aβ region between residues 687 and 688, releasing a secreted ectodomain
(sAPPα) while the C-terminal fragment (αCTF or C83) remains membrane-associated
(Nhan, Chiang, & Koo, 2015; van der Kant & Goldstein, 2015). Alternatively, APP can undergo amyloidogenic cleavage by the β-secretase (β-site APP-cleaving enzyme 1 or
BACE1) between residues 671 and 672, releasing sAPPβ from the membrane and leaving
β-CTF or C99 associated with the membrane. Following this initial cleavage step by α- or
β-secretase, the remaining C83 or C99 fragment can be cleaved within the transmembrane domain by a γ-secretase to generate either P3 (non-amyloidogenic) or
Aβ (amyloidogenic) and an APP intracellular domain (AICD).
γ-Secretase consists of a complex of four proteins, including presenilin, nicastrin, anterior pharynx defective (APH1) and presenilin enhancer (PEN2) (Nhan 2015). A
18 complex can contain either presenilin 1 (PS1) or 2 (PS2), which contain the aspartate residues that form the catalytic site of γ-secretase transmembrane cleavage activity (De
Strooper et al., 1998; Wolfe et al., 1999). This cleavage activity is not specific to one site within the APP transmembrane domain, but rather can take place at varying locations between Aβ residues 36-43, generating a pool of various Aβ species (Kang et al., 1987;
Qi-Takahara et al., 2005; Takami et al., 2009). The most common Aβ species are Aβ1-40
(Aβ40), which accounts for the majority of Aβ, and Aβ1-42(43) (Aβ42), which normally accounts for less than 10% of Aβ species in human CSF (Bibl et al., 2012; Scheuner et al.,
1996) and in cell-based assays (Sinha et al., 1999; Suzuki et al., 1994). Aβ42 more readily forms oligomers and fibrils (Burdick et al., 1992; Jarrett, Berger, & Lansbury, 1993) than
Aβ40 and is the major Aβ species that is deposited in plaques in the AD brain (Gravina et al., 1995), leading to the hypothesis that Aβ42 is the more pathogenic species.
Additionally, a large number of FAD mutations in APP and the presenilins have been observed to alter the production of Aβ species to favor Aβ42 (Bentahir et al., 2006;
Borchelt et al., 1996; Kumar-Singh et al., 2006; Scheuner et al., 1996; Rudolph E. Tanzi,
2012), although some APP mutations increase overall production of Aβ, such as the
Swedish mutation (Citron et al., 1992; Mullan et al., 1992) and the K16N mutation
(Kaden et al., 2012) or change Aβ structure to increase Aβ aggregation, such as the
Arctic (Nilsberth et al., 2001) and Dutch (Levy et al., 1990) mutations (reviewed by
(Haass et al., 2012)). Interestingly, one mutation adjacent to the site of β-secretase cleavage in APP was recently identified (A673T) that results in a 40% reduction of amyloidogenic β-cleavage in vitro and confers a decreased risk of AD in elderly
19 populations (Jonsson et al., 2012). Recently it has become understood that soluble or oligomeric species of Aβ are neurotoxic (Lambert et al., 1998; L. Mucke et al., 2000;
Lennart Mucke & Selkoe, 2012; Shankar et al., 2007, 2008), but the exact roles of these species, or their post-translationally modified derivatives, and their relative contributions to pathology remain difficult to determine.
Amyloid hypothesis
Interestingly, although there is a strong body of genetic evidence linking production of Aβ species to FAD, amyloid plaque formation correlates poorly with cognitive impairment. Aβ plaques can appear 15-20 years before the onset of AD symptoms in human patients (Bateman et al., 2012), and appear in subjects that never develop AD symptoms (Perez-Nievas et al., 2013). Instead, the development of neurofibrillary tangles has been found to correlate more closely with neuronal dysfunction and cognitive decline than plaque development both temporally (Bennett,
Schneider, Wilson, Bienias, & Arnold, 2004; Duyckaerts et al., 1998; Giannakopoulos et al., 2003; Gómez-Isla et al., 1997; Ingelsson et al., 2004; Jack et al., 2011) and spatially
(Brettschneider, Del Tredici, Lee, & Trojanowski, 2015; Guillozet, Weintraub, Mash, &
Mesulam, 2003). However, mutations in MAPT, the gene encoding microtubule- associated protein tau, do not lead to AD but rather frontotemporal dementia (Goedert
& Jakes, 2005) and no FAD mutations have been discovered in MAPT. As is the case for
Aβ plaques, neurofibrillary tangles can be observed in cognitively normal individuals, but
20 in these individuals tangles are generally sparser than in those diagnosed with MCI or
AD, and remain confined to the limbic system. Tau pathology in individuals with AD is comparatively prolific throughout the limbic system and spreads to the neocortex
(Braak & Braak, 1991), and this aggressive pathology appears to be dependent on the co-occurrence of Aβ aggregation (Knopman et al., 2003; Petersen et al., 2006; Price &
Morris, 1999). These findings support the “amyloid hypothesis,” which proposes that increased Aβ42 accumulation and deposition is the initiating event in a pathogenic cascade that includes tau hyperphosphorylation and aggregation into tangles, activation of astrocytes and microglia, and synaptic dysfunction and neuronal loss, resulting in symptomatic AD (Selkoe & Hardy, 2016). This hypothesis fits well with the genetic mutations involved in FAD, which center around the Aβ production pathway, as well as the previously discussed predisposition of Down’s syndrome patients to develop AD.
However, the amyloid hypothesis leaves several important questions unanswered. The relationship between Aβ and AD cognitive symptoms is unclear, with
25-30% of individuals positive for amyloid plaques retaining normal cognitive function
(Perez-Nievas et al., 2013; Villemagne et al., 2013). Similarly, the relationship between
Aβ and tau pathology remains poorly defined. Although it has been suggested that Aβ can exert a prion-like effect on tau to cause misfolding (“cross seeding”), Aβ can disrupt neuronal signaling and lead to aberrant kinase activity and cause p-tau downstream, and neuroinflammation caused by Aβ deposits can create an environment that favors neurofibrillary tangle formation (Stancu, Vasconcelos, Terwel, & Dewachter, 2014), the extent to which any or all of these mechanisms contribute to AD pathology remains
21 unknown. The amyloid hypothesis has also received mixed support from recent clinical trials for AD therapeutics targeting Aβ, prominently immunotherapies and β- and γ- secretase inhibitors. All of these therapies appear to be extremely effective in preclinical models (Eketjall et al., 2013; Mulder et al., 2016; Schenk, Basi, & Pangalos, 2012), yet the translation of these therapies to human patients has so far met with limited success
(Godyń, Jończyk, Panek, & Malawska, 2016). Reasons for the failure of so many trials include the difficulty of identifying appropriate study populations for Aβ-directed treatment, which several trials have indicated are probably limited to very early AD or pre-AD subjects, and the limitations of preclinical models currently available for AD research.
Preclinical models of AD
Mammals that spontaneously develop AD pathology in the course of aging include, of course, humans, but also primates, dolphins, dogs, and an unusual rodent called the degu (Braidy et al., 2012). Mice possess an APP gene that is 96.8% homologous with human APP, with only 22 amino acid substitutions in the brain isoform
APP695 and only three of those found in the Aβ region (Yamada et al., 1987), yet do not naturally develop Aβ plaques or tau tangles. Due to the availability of inbred strains, relative ease and speed of breeding, and receptiveness to genetic manipulation, mice are highly desirable mammals for use in laboratory studies, and a large amount of resources have been directed toward developing a suitable mouse model of AD. The
22 first genetic mouse models of AD overexpressed human APP and had only limited success in recapitulating the amyloid pathology of human AD (Lamb et al., 1993; L.
Mucke et al., 1994; Pearson & Choi, 1993; Quon et al., 1991; Sandhu, Salim, & Zain,
1991). In 1995 (Games et al., 1995) published the first mouse model utilizing the brain- expressed platelet-derived growth factor (PDGF)-β promoter to drive high expression of
APP with a human FAD mutation (V717F). This mouse, termed the PD-APP mouse, developed more aggressive Aβ pathology, including deposition of dense-core plaques, synaptic loss, and astrocytosis and microgliosis (Games et al., 1995). Since then, a large number of AD mouse models have been developed by overexpressing human APP,
PSEN1, or both, with FAD mutations. These occur on several different background strains and use a variety of promoters, which results in a wide disparity in onset and speed of pathological progression, differing levels and localizations of plaque deposition, and a range of behavioral phenotypes (Webster, Bachstetter, Nelson, Schmitt, & Van
Eldik, 2014).
Notably, although these models commonly exhibit hyperphosphorylaytion of tau, frequently observed in dystrophic neurites surrounding plaques, none of them recapitulates the NFT pathology observed in human AD (Spires-Jones & Hyman, 2014).
Mouse models that utilize a frontotemporal dementia-linked mutation in MAPT (P301L or P301S) in combination with FAD mutations (Bolmont et al., 2007; Grueninger et al.,
2010; Hurtado et al., 2010; Lewis et al., 2001; Oddo et al., 2003; Saul, Sprenger, Bayer, &
Wirths, 2013; Seino et al., 2010; Stancu, Ris, et al., 2014) have been developed to study neurofibrillary tangle pathology in an AD context, but the artificiality of such a system is
23 a large caveat to these studies. The neuronal loss that is characteristic of human AD is also typically absent or mild in AD mouse models, with the exception of the 5XFAD model (Oakley et al., 2006). This model overexpresses human APP with the Swedish
(K670N/M671L), Florida (I716V), and London (V717I) mutations and PSEN1 with M146L and L286V mutations, for a total of five FAD mutations. The extremely aggressive amyloid pathology in these mice can be observed as early as 1.5 months and widespread neuronal death has been observed by 4 months of age in the subiculum
(Moon et al., 2012) and by 10 months of age in layer 5/6 of the cortex (Oakley et al.,
2006). While the presence of neurodegeneration in this model makes it a unique and valuable resource for the study of neuropathology in an AD context, the artificiality of overexpressing five FAD mutations and the lack of significant NFT pathology cannot be ignored. The shortcomings of current AD models have likely contributed to the high failure rate of AD drugs in clinical trials. The generation of more biologically relevant
FAD models, generated by using a knock-in approach to humanize mouse APP and insert
FAD mutations (Saito et al., 2014), and models that can better recapitulate the human disease is a major goal of the AD field. At best, current models only reflect pathology in the extremely small population of patients with FAD, and there are currently no models that represent the much more common late-onset form of AD.
24 Late-onset Alzheimer’s
In FAD, the case for overproduction of Aβ and its subsequent accumulation in the brain is relatively clear-cut. In late-onset AD (LOAD), which arises sporadically in patients >65 and accounts for 95% of all AD cases (Alzheimer’s Association, 2016), the initiating factor for Aβ accumulation is postulated to be impairments in the endogenous
Aβ clearance mechanisms in the brain, resulting in a loss of homeostasis and deposition of Aβ into plaques (Wildsmith, Holley, & Savage, 2013). The exact mechanism behind this dysfunction in Aβ clearance is unknown, and indeed, a variety of genetic and environmental factors are likely to influence Aβ homeostasis during aging. In this section we will address mechanisms of Aβ clearance and genes that have been associated with increased risk of LOAD, most notably APOE4.
Aβ clearance mechanisms
Aβ is secreted by neurons as part of their normal physiological activity (Haass et al., 1993), especially during synaptic transmission (Bero et al., 2011; Cirrito et al., 2005,
2008; Kamenetz et al., 2003; Tampellini et al., 2010), resulting in the constant presence of various Aβ species in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) of the
CNS. An elegant study by Bateman et al. (Bateman et al., 2006) demonstrated that in normal individuals, newly generated Aβ species are added to this pool at a rate of about
7.6% per hour while endogenous clearance mechanisms remove about 8.3% of Aβ. A large number of Aβ-degrading proteases are involved in endogenous Aβ clearance
25 (Saido & Leissring, 2012), including: zinc metalloproteinases neprilysin (NEP), insulin- degrading enzyme (IDE), endothelin-converting enzymes 1 and 2 (ECE-1, ECE-2), angiotensin –converting enzyme (ACE), and matrix metalloproteinases 2 and 9 (MMP-2,
MMP-9); the serine proteases plasmin, acylpeptide hydrolase (APH), and myelin basic protein (MBP); the cysteine protease cathepsin B (CatB); and the aspartyl protease cathepsin D (CatD).
Studies focusing on NEP have established the importance of proteolytic clearance pathways in Aβ homeostasis. Iwata et al. (Nobuhisa Iwata et al., 2000) first identified NEP as a major Aβ-degrading enzyme, and reported that treatment of normal rats with a NEP inhibitor for just 30 days was sufficient to result in parenchymal Aβ deposition in the form of diffuse plaques. The same group also used NEP-/- and NEP -/+ mice to establish that deficiency of NEP increases brain Aβ levels in a dose-dependent manner and decreases Aβ clearance efficiency (N. Iwata et al., 2001), and similar results were obtained in AD-transgenic mice that were NEP-/- or NEP -/+ (Farris et al., 2007).
Leissring et al. (Leissring et al., 2003) and Meilandt et al. (Meilandt et al., 2009) later demonstrated that, conversely, overexpression of NEP in AD-transgenic mice decreased
Aβ deposition. Other studies have used viral vectors to overexpress NEP in AD- transgenic mice with similar results (Nobuhisa Iwata et al., 2004; Marr et al., 2003).
However, evidence indicates that NEP may not be an effective degrader of Aβ oligomers
(Farris et al., 2007; Huang et al., 2006; Meilandt et al., 2009), so further investigation into both the relative toxicity of Aβ oligomeric species and the contribution of NEP to
AD-related outcomes not directly linked to Aβ, such as neuronal dysfunction and
26 cognitive impairment, is warranted. No clinical trials specifically targeting Aβ-degrading proteases are currently underway for AD, but suggested therapies to increase Aβ degradation have included inhibitors of plasminogen activator inhibitor-1 (PAI-1), which would lead to downstream activity increases in the Aβ-degrading protease plasmin
(Jacobsen et al., 2008), and increasing somatostatin levels, which has been observed to increase brain NEP activity (Saito et al., 2005).
Aβ can also be internalized and degraded by both microglia (Aguzzi, Barres, &
Bennett, 2013; Prinz, Priller, Sisodia, & Ransohoff, 2011) and by astrocytes (Basak,
Verghese, Yoon, Kim, & Holtzman, 2012; Cramer et al., 2012; Koistinaho et al., 2004;
Nicoll & Weller, 2003/7; Wyss-Coray et al., 2003). Microglia, the brain’s resident phagocytes (Perry & Gordon, 1988), are observed in close apposition to plaques in human AD (Perlmutter, Barron, & Chui, 1990) and in many mouse models. Microglia can internalize soluble Aβ species via fluid phase macropinocytosis (Mandrekar et al., 2009), and also express several cell-surface receptors that can interact with fibrillar Aβ, including class A scavenger receptor (J. El Khoury et al., 1996; Paresce, Ghosh, &
Maxfield, 1996), RAGE (Du Yan et al., 1996), and a large receptor complex consisting of
α6β1 integrin, CD36, CD47, CD14, and toll-like receptors (TLRs) 6, 4 and 2 (Bamberger,
Harris, McDonald, Husemann, & Landreth, 2003; Chen et al., 2006; Fassbender et al.,
2004; Hickman, Allison, & El Khoury, 2008; Koenigsknecht & Landreth, 2004; Kopec &
Carroll, 1998; Liu et al., 2005; Reed-Geaghan, Savage, Hise, & Landreth, 2009; Stewart et al., 2010) that mediates microglial phagocytosis of Aβ. After Aβ is internalized by microglia, it is trafficked to lysosomes for degradation (C. Y. D. Lee, Tse, Smith, &
27 Landreth, 2012). Proteases that function intracellularly, such as NEP, facilitate this process (Jiang et al., 2008). The exact role of microglia in AD pathogenesis and Aβ clearance is hotly debated, as are the relative contributions and respective roles of endogenous brain microglia and infiltrating peripheral macrophages (Heneka et al.,
2015; Malm, Jay, & Landreth, 2015). Additionally, neurons are able to internalize Aβ via low-density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1) (Kanekiyo et al., 2011, 2013), although further study is required to determine the impact of this mechanism on overall Aβ clearance. These receptors are also expressed on microglia and astrocytes, and at the blood-brain barrier (BBB), where they have an established role in mediating transport of Aβ from the brain parenchyma to the periphery (Sagare,
Bell, & Zlokovic, 2013).
In addition to proteolytic degradation and cellular internalization, Aβ can also be cleared from the brain by active or passive transport across the BBB. LRP1 is a cell- signaling receptor and transporter expressed by neurons, astrocytes, endothelial cells of the BBB and epithelial cells of the choroid plexus (Sagare et al., 2013). Several studies have shown that LRP1 is capable of interacting directly with Aβ, or complexes formed by
Aβ and other proteins such as apoE, and mediates Aβ clearance from the parenchyma into the blood where it can be degraded in the periphery (Zlokovic, Deane, Sagare, Bell,
& Winkler, 2010). Conversely, Aβ can be transported from the periphery into the brain by receptor for advanced glycation end products (RAGE). The idea that plasma Aβ levels and Aβ in the CSF/ISF are maintained in homeostasis by these transporters and that decreasing Aβ levels in the plasma will allow greater efflux of Aβ from the brain is
28 known as the “peripheral sink” mechanism. Aβ can also reach the peripheral sink through interstitial fluid bulk-flow clearance methods, such as perivascular drainage and glymphatic clearance that are mediated by vessel pulsation. Decreased BBB integrity and vessel stiffening are observed during aging and associated with late-onset AD, and
AD patients have decreased LRP1 (Zlokovic et al., 2010), but the relative importance of these changes in the pathogenesis of AD has not been well-defined (Tarasoff-Conway et al., 2015).
ApoE and LOAD
Population studies have demonstrated that APOE genotype is the strongest risk factor for late-onset Alzheimer’s disease. Three common isoforms of apoE occur in humans, differing from each other at two amino acids: apoE2 (cys112, cys158), apoE3
(cys112, arg158), and apoE4 (arg112, arg158). Possession of one ε4 allele imparts a 3- fold increase in risk for LOAD, and two alleles impart a 12-fold increased risk (Farrer et al., 1997), while the ε2 allele decreases the likelihood of developing LOAD (Corder et al.,
1993). With a prevalence of about 15 percent in the population, the ε4 allele has been estimated to account for 50 percent of all AD cases (Ashford, 2004). The ε4 allele is also associated with an earlier age of onset (Corder et al., 1993; Khachaturian, Corcoran,
Mayer, Zandi, & Breitner, 2004) and increased Aβ deposition both in animal models of
AD (Bales et al., 2009; Castellano et al., 2011; D. M. Holtzman et al., 2000) and in human
AD (Mann et al., 1997).
29 ApoE is the predominant apoprotein in the brain, where it is secreted primarily by astrocytes, but also by microglia, in high-density lipoprotein (HDL)-like particles (Bu,
2009). Lipidation of ApoE is mediated primarily by ATP-binding cassette A1 (ABCA1) and secondarily by ABCG1 (Hirsch-Reinshagen et al., 2004; Zelcer et al., 2007) and the lipidation status of ApoE has been shown to regulate its Aβ-binding properties (Tokuda et al., 2000). Direct evidence that ABCA1-mediated lipidation influences amyloid degradation has been demonstrated in multiple transgenic models of AD. Deletion or overexpression of ABCA1 results in increased or decreased Aβ deposition, respectively
(Hirsch-Reinshagen et al., 2005; R. Koldamova, Staufenbiel, & Lefterov, 2005; Wahrle et al., 2005). Both intracellular and extracellular degradation of Aβ is also dramatically enhanced by lipidated ApoE (Jiang et al., 2008). ApoE4 is less stable (Bales et al., 2009;
D. M. Holtzman et al., 2000) and a less effective lipid carrier under physiological conditions than ApoE3 or ApoE2 (Hara et al., 2003; Michikawa, Fan, Isobe, &
Yanagisawa, 2000), and this probably contributes to its influence in AD pathogenesis.
The effects of the various ApoE isoforms on Aβ clearance were further investigated in targeted-replacement mice expressing human ApoE isoforms at the murine locus. Aβ deposition and cognitive deficits are exacerbated in APP/ABCA+/− targeted-replacement mice expressing ApoE4 but not ApoE3 (Fitz et al., 2012).
It has been proposed that ApoE4 modulates amyloid pathology by enhancing Aβ deposition into plaques and reducing clearance of Aβ from the brain (Bales et al., 1999;
D. M. Holtzman, 2001; D. M. Holtzman et al., 2000; Shibata et al., 2000; Strittmatter et al., 1993). One of the first pieces of evidence linking ApoE to AD pathology was ApoE
30 immunoreactivity in amyloid deposits and neurofibrillary tangles (Namba, Tomonaga,
Kawasaki, Otomo, & Ikeda, 1991). It has since been shown that ApoE forms complexes with Aβ, with ApoE2 and E3 binding Aβ more efficiently than E4 (Aleshkov, Abraham, &
Zannis, 1997; LaDu et al., 1994; D.-S. Yang, Smith, Zhou, Gandy, & Martins, 1997), and these complexes are thought to influence both seeding of fibrillar Aβ and transport of soluble Aβ. It has been shown that AD transgenic mice lacking APOE have decreased plaque deposition and increased levels of soluble Aβ in the cerebrospinal fluid and ISF
(Bales et al., 1999; DeMattos, 2004). Crosses between AD transgenic mice and human
ApoE targeted-replacement mice exhibit Aβ accumulation in an isoform-dependent manner, with greater Aβ deposition observed in ApoE4-expressing mice than those expressing E2 and E3 (Bales et al., 2009; Castellano et al., 2011). The cause of the accumulation is most likely due to the degree to which the isoforms impact Aβ clearance and deposition (Castellano et al., 2011; Deane et al., 2008). However, a recent study by
Holtzman and colleagues (Verghese et al., 2013) has provided conflicting evidence that
Aβ does not directly interact with ApoE under physiological conditions to any significant extent. Instead, ApoE competes with Aβ in an isoform- and concentration-dependent manner for binding to lipoprotein LRP1, and this could impact Aβ clearance by glia and across the BBB (Verghese et al., 2013).
31 Nuclear receptor structure and function
The expression of ApoE and its lipidating transporters is transcriptionally regulated by nuclear receptors. Nuclear receptors are a large superfamily of transcription factors that mediate biological responses to steroid hormones and other lipophilic molecules, and they are broadly grouped into four categories based on their activation and DNA-binding properties (Mangelsdorf et al., 1995). Class I nuclear receptors, such as estrogen receptor (ER), progesterone receptor, and glucocorticoid receptor (GR), are ligand-activated in the cytoplasm and translocate to the nucleus, where they interact with inverted repeat DNA response sites as homodimers. Class II nuclear receptors are generally activated in the nucleus, and form obligate heterodimers with the retinoid x receptor (RXR) at direct repeat DNA response sites
(DRs). These receptors are characterized as permissive or non-permissive depending on whether RXR targeted ligands will activate a heterodimer complex with that nuclear receptor (M. I. Dawson & Xia, 2012). Depending on the heterodimer partner, RXRs interact with DRs spaced one to five nucleotides apart (DR1-5) (Evans & Mangelsdorf,
2014). Class III receptors are similar to class I receptors in that they form homodimers, but they interact with direct repeat rather than inverted repeat DNA sequences. Class IV receptors bind response sites as monomers, and are generally orphan receptors, meaning that their endogenous ligands are unknown (Mangelsdorf et al., 1995). Nuclear receptors all share a similar structure, consisting of a variable N-terminal region, a zinc finger-containing DNA-binding domain, a variable hinge region, a C-terminal ligand-
32 binding domain that allows receptor activation in response to a stimulus, and a variable
C-terminal region (Mangelsdorf et al., 1995).
This thesis will focus on type II nuclear receptors, which act broadly in the brain to regulate lipid metabolism, inflammation, and neuroprotection. The predominant type
II nuclear receptors in the brain are the peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs). PPARs and LXRs form obligate heterodimers with RXRs α, β and γ to create a functional transcription factor. These heterodimers associate with DNA in the nucleus regardless of their ligand binding status. When unbound to ligand, PPAR:RXR or LXR:RXR complexes mediate transcriptional repression by interacting with corepressors such as nuclear receptor corepressor (NCoR) or silencing mediator for retinoid and thyroid receptors (SMRT) and histone deacetylase 3 (HDAC3) (Saijo, Crotti, & Glass, 2013), functionally silencing the expression of target genes. In the presence of ligand, the corepressor complex is exchanged for a coactivator complex, thereby promoting transcription (Figure 1.1).
RXRs
In addition to acting as heterodimerization partners for type II nuclear receptors,
RXRs can act as homotetramers to modulate DNA 3D architecture or as homodimers that associate with their target genes in the presence or absence of ligand (Dawson and
Xia, 2012). Only a few genes have been shown to be regulated by the RXR homodimer complex (IJpenberg et al., 2004), most prominently a subset of chemokines (Nunez et al., 2010). RXR heterodimers are characterized as ‘permissive’ or ‘non-permissive’ based
33 on whether ligation of either member of the heterodimer can elicit transcription. In the brain, permissive receptors include the PPARs, LXRs and the NR4A receptors (Rőszer et al., 2013) while non-permissive complexes are formed with RARs, thyroid and vitamin D receptors. Thus, RXR agonists can elicit pleiotropic actions through their actions on RXR homodimers as well as heterodimers containing permissive receptors, and this complexity is poorly understood. Recent work has shown that RXR agonists only regulate a subset of genes controlled by permissive receptors and that this subset is cell type-specific (Széles et al., 2010). The basis for this restriction is unclear but explains why a broader range of effects of RXR agonists in the brain is not observed. Additionally,
RXRs act in microglia to facilitate phagocytosis of fAβ, and this effect is reliant upon
RXRα (Yamanaka et al., 2012).
LXRs
LXRs are cholesterol sensors that play an essential modulatory role in cholesterol metabolism, lipogenesis, and the regulation of inflammation. Two isoforms of LXR exist,
LXRα, which is prominently expressed in liver, adipose tissue, adrenal glands, intestine, kidney, and macrophages, and LXRβ, which is expressed 2–5 times higher than LXRα in the brain (Whitney et al., 2001) and ubiquitously expressed at low levels throughout the body. The two isoforms are activated by the same endogenous ligands, namely 24(S)- hydroxycholesterol, 22(R)-hydroxycholesterol 24(S), 25-epoxycholesterol, and 27- hydroxycholesterol, and transcriptionally regulate genes involved in reverse cholesterol
34 transport (RCT). LXRs directly regulate the transcription of apoE and its lipidating transporters ABCA1 and ABCG1 (Courtney & Landreth, 2016). In addition to regulating cholesterol efflux via RCT, LXRs also regulate the uptake of cholesterol by members of the low-density lipoprotein receptor (LDLR) family of cell surface receptors (Zhang,
Reue, Fong, Young, & Tontonoz, 2012). Activated LXRs also exhibit anti-inflammatory action, as they are sumoylated and repress transcription at NFκB target genes (J. H. Lee et al., 2009) and can also transactivate genes in microglia which are important for the resolution of inflammation, similarly to PPARγ. The role of nuclear receptors in regulating microglial inflammation will be discussed in further detail below.
LXRs have been shown to play an essential role in the normal CNS. Neuronal development, synaptogenesis, and learning and memory are dependent on cholesterol, and dysregulation of cholesterol metabolism has been implicated in several neurodegenerative disorders (Brown, 2004). LXR double knockout mice have CNS abnormalities, including increased lipid deposition and neurodegeneration (Wang et al.,
2002), and LXRβ knockout mice exhibit adult-onset motor neuron degeneration
(Andersson, Gustafsson, Warner, & Gustafsson, 2005). Two widely used synthetic ligands, T0901317 and GW3965, have been developed as tools for the study of LXRs.
LXRs have been studied in a number of neurodegenerative diseases and CNS injury models and the development of new LXR agonists with acceptable side effect profiles remains an active area of interest.
35 PPARs
PPARs function as lipid sensors which bind dietary lipids or their metabolites, most prominently fatty acids and eicosanoids. There are three PPAR isoforms in mammals, each of which has a distinct expression pattern, unique preferential ligands, and a subset of metabolic functions to regulate. PPARα is mainly expressed in the liver, heart, and brown adipose tissue, and activates a gene program resulting in increased catabolism of fatty acids. PPARβ, also known as PPARδ, is the most ubiquitously expressed PPAR but prominently occurs in skeletal muscle, liver, and heart, where it participates in glucose homeostasis and also regulates the expression of proteins involved in fatty acid oxidation. PPARγ is highly expressed in white and brown adipose tissue, and is a master regulator of adipogenesis and a critical player in glucose homeostasis and lipid storage (Grygiel-Górniak, 2014). In addition to their roles in the periphery all three PPAR isoforms are expressed in the brain, where immunostaining for
PPARα colocalizes with neurons, astrocytes, and microglia, and PPARβ/δ and γ are mainly expressed by neurons and astrocytes in both mouse and human brain (Warden et al., 2016). PPARγ becomes upregulated by microglia upon induction of inflammation
(Warden et al., 2016) and has a well-established role in the regulation of inflammatory responses in peripheral macrophages (Hong & Tontonoz, 2008) and microglia (Bernardo
& Minghetti, 2006). Interestingly, ligands that target PPARγ also produce increases in
LXR target genes, including apoE and ABCA1, likely by inducing the expression of LXRα as well as interaction with LXR enhancer elements (Daniel et al., 2014; Mandrekar-Colucci,
Karlo, & Landreth, 2012; Yue & Mazzone, 2009).
36
NR4As
The NR4A family of nuclear receptors are also broadly expressed throughout the nervous system and they have been recently reported to play major roles in learning and memory (Josh D. Hawk & Abel, 2011). The NR4A receptors were first identified as immediate-early genes induced in the nervous system in response to a wide variety of extracellular stimuli such as seizures (de Ortiz & Jamieson, 1996), stress (García-Yagüe,
Rada, Rojo, Lastres-Becker, & Cuadrado, 2013) and neurotransmitters (Barneda-
Zahonero et al., 2012; Debernard, Mathisen, & Paulsen, 2012). The NR4A family, including Nur77 (NR4A1; NGF-IA), Nurr1 (NR4A2; NGF-IB) and Nor-1 (NR4A3), is unique among type II nuclear receptors because the steric hindrance in their nominal ligand binding domains prevents them from accepting ligands. They were long thought to be constitutively active, but it has been recently shown that they can also play a role in transcriptional repression in a context dependent manner. NR4A transcriptional activity depends mainly on gene expression, miRNA targeting, alternative splicing, posttranslational modification, subcellular localization, and interactions with other nuclear receptors (Maxwell & Muscat, 2006; Michelhaugh et al., 2005; Mohan et al.,
2012; Sacchetti, Carpentier, Ségard, Olivé-Cren, & Lefebvre, 2006; D. Yang et al., 2012).
All three family members can signal at NBREs (or NurREs) as monomers or homo- or heterodimers with other NR4As. Importantly, Nurr1 and Nur77 can also signal in complex with RXRs at DR5 repeats, and NR4A/RXR heterodimers can be activated by
37 RXR ligands. Synthetic ligands that bind directly to NR4A:RXR heterodimers and drive transcriptional activity have also been described (Ishizawa, Kagechika, & Makishima,
2012; Morita et al., 2005). Development of specific ligands for NR4As could be therapeutically relevant for a variety of diseases. Aside from their critical role in nervous system function, NR4As are implicated as regulators of glucose homeostasis (Close,
Rouillard, & Buteau, 2013), fatty acid metabolism (Holla, Wu, Shi, Menter, & DuBois,
2011; Volakakis, Joodmardi, & Perlmann, 2009), cellular proliferation (Sirin, Lukov, Mao,
Conneely, & Goodell, 2010), cancer (Mohan et al., 2012), and immune regulation both in the periphery and in the brain.
Nuclear receptors in inflammation
The brain is densely and uniformly populated by resident immune cells, the microglia (Nayak, Roth, & McGavern, 2014). In neurodegenerative diseases, the microglia increase in number and transform from a surveillant, tissue maintenance mode to a protective host-defense mode while upregulating proinflammatory genes
(Heneka et al., 2015). In AD, these ‘activated’ microglia are found associated with fibrillar Aβ plaques (Serrano-Pozo et al., 2013), and have increased cytokine production
(J. B. El Khoury et al., 2003; Stewart et al., 2010) while their phagocytic capabilities are impaired (Hickman et al., 2008). Compromised microglial function in AD is likely a significant contributor to pathology, as their reduced ability to phagocytose and clear Aβ contributes to its further accumulation and aggregation, while their production of
38 proinflammatory molecules such as IL1-β, TNF and iNOS can have neurotoxic effects
(Barger & Harmon, 1997; Meda et al., 1995).
In the periphery, macrophages resolve this response to chronic proinflammatory stimuli (e.g. parasitic infections) with the inhibition of inflammatory gene expression as well as the induction of a genetic program associated with tissue repair and enhanced phagocytosis (Gordon & Martinez, 2010). It has only recently been appreciated that nuclear receptors act as master regulators of this resolution of inflammation (Odegaard & Chawla, 2011). Macrophages in which PPARγ (Odegaard et al., 2007), PPARδ (Mukundan et al., 2009), LXRs (A-Gonzalez et al., 2009) and RXRα
(Núñez et al., 2010) have been genetically inactivated exhibit reduced phagocytosis and are unable to attenuate their immune response (Saijo et al., 2013).
Nuclear receptor activation can act to suppress proinflammatory gene expression. Activated PPAR:RXR and LXR:RXR heterodimers transrepress inflammatory gene expression by recruiting nuclear receptor corepressors NCoR/SMRT to inflammatory NFκB target genes (Glass & Saijo, 2010). Nurr1 is known to act via a similar mechanism where it recruits the corepressor COREST to NFκB promoters (Saijo et al.,
2009). PPARs, LXRs, and Nurr1 also regulate microglial activation state by activating transcription of genes associated with tissue repair and enhanced phagocytosis
(Mandrekar-Colucci & Landreth, 2010; McMorrow & Murphy, 2011). These genes help prime microglia for phagocytosis of amyloid plaques and other debris (Colton, 2009).
39 Nuclear receptors in Alzheimer’s disease
RXR
LXR agonists and PPARγ agonists are valuable tools for helping elucidate the role of apoE and mechanism of Aβ clearance in AD. RXR agonists, which stimulate both LXR and PPARγ pathways, also are able increase Aβ clearance in an apoE-dependent manner. RXR agonist bexarotene reduced the half-life of Aβ in APP/PS1 and C57Bl/6 mice, but had no effect on Aβ clearance in apoE-null mice (Cramer et al., 2012). The reduction in soluble Aβ species was associated with improved neural network function and reversal of behavioral deficits. This effect on soluble Aβ levels was also reported by Fitz et al. (2013), Veeraraghavalu et al. (2013) and Ulrich et al. (2013), but not others
(Table 1). Importantly, bexarotene-mediated behavioral improvement was observed by Fitz et al. (2013), Boehm-Cagan and Michaelson (2014) and Tesseur et al. (2013).
Cramer et al. reported that bexarotene treatment also resulted in the rapid reduction in amyloid plaque burden (Cramer et al., 2012) and this finding has been controversial
(Landreth et al., 2013). The literature on RXR activation is confused by a number of studies which investigate the actions of docasohexanoic acid (DHA), an omega 3- polyunsaturated fatty acid, and attribute its actions to its binding to RXRs (de Urquiza et al., 2000). However, DHA also binds to PPARs (Kliewer et al., 1997) and two GPCRs (Im,
2012), thus it is not possible to conclude that these are RXR-specific actions.
LXR
40 LXR, due to its activity as a direct transcriptional regulator of apoE, ABCA1, and
ABCG1 and a regulator of microglial inflammation, is an attractive therapeutic target for
AD treatment. In the past 10 years, LXR agonists have been investigated in twelve separate studies (Table 1) which demonstrate their efficacy in improving behavioral impairments and amyloid clearance in AD models, with some mixed results in the stimulation of plaque clearance (Cui et al., 2012; Donkin et al., 2010; Fitz et al., 2010,
2014; Y. Hu et al., 2013; Jiang et al., 2008; R. P. Koldamova et al., 2005; Lefterov et al.,
2007; Riddell et al., 2007; Terwel et al., 2011; Vanmierlo et al., 2011; Wesson et al.,
2011). Vanmierlo et al. observed improvements in hippocampal dependent memory but no change in plaque load with T0901317 treatment in APPSLxPS1mut mice (Vanmierlo et al., 2011). However, Riddell et al. only observed decreases in hippocampal Aβ42 with no change in cortical Aβ levels, although T0901317 treatment of Tg2576 mice mediated behavioral improvement (Riddell et al., 2007). The ability of LXR agonists to stimulate Aβ clearance and behavioral improvements is dependent on ABCA1 (Donkin et al., 2010;
Fitz et al., 2010), indicating that LXR-mediated regulation of apoE lipidation state plays an important role in amyloid pathology. Additionally, apoE secreted from primary astrocytes treated with LXR agonists was able to stimulate phagocytosis of Aβ in primary microglia. If LXR or apoE were deleted from the primary astrocytes, the microglia lost their ability to remove fibrillar Aβ in response to LXR activation (Terwel et al., 2011).
Their anti-inflammatory properties and stimulation of reverse cholesterol transport make LXRs an attractive therapeutic candidate for AD treatment, and one that is supported by an extensive literature in AD models. However, the poor side effect
41 profile of LXR ligands precludes their clinical use. Better targeted or tissue-specific agonists of LXRs are currently in development (Y. Hu et al., 2013) as potential AD therapeutics.
PPARγ
Similar to LXR, peroxisome proliferator-activated receptor γ (PPARγ) activation can mediate degradation of Aβ (Chawla et al., 2001; Heneka & Landreth, 2007). In addition to its ability to increase apoE and ABCA1 levels, PPARγ activation also has been shown to induce the expression of the scavenger receptor CD36 on microglia, which increased the uptake of Aβ (Yamanaka et al., 2012). PPARγ agonist treatment of murine models of AD has been associated with the reversal of transgene-induced behavioral impairments, as evaluated in a number of different assays of cognition, memory and neural network function. It remains enigmatic exactly how the PPARγ-mediated improvement of behavior is effected. One proposed mechanism is that the robust anti- inflammatory effects of PPARγ suppress the levels of proinflammatory cytokines that have been linked to cognitive impairment. Whether anti-inflammatory effects are entirely responsible for reversal of the behavioral deficits remains to be convincingly demonstrated. Recent studies of PPARγ signaling in neurons argue that other mechanisms likely participate in the PPARγ-mediated enhancement of cognition and memory. The actions of PPARγ agonists on neurons have received comparatively little attention. PPARγ agonists are reported to stimulate Wnt signaling (Toledo & Inestrosa,
2010). Recent work by Dinely and colleagues has dissected the neuronal effects of
42 PPARγ agonists and their underlying mechanisms (Denner et al., 2012; Nenov et al.,
2014; Rodriguez-Rivera, Denner, & Dineley, 2011). It has also been reported that PPAR agonists act to normalize synaptic function in AD mouse models (Searcy et al., 2012).
The salutary effects of PPARγ agonists in AD mice have been postulated to arise from their ability to improve peripheral insulin sensitivity in type II diabetes and by extension work in analogous ways in the brain (Craft, Cholerton, & Baker, 2013). There is no direct evidence to support the view that neurons are insensitive to insulin action, but they have been reported to exhibit changes in signal transduction pathways reflective of impaired insulin receptor signaling in AD models of rodents, monkeys and in humans
(Ferreira, Clarke, Bomfim, & De Felice, 2014).
One of the most compelling effects of chronic PPARγ agonist treatment documented in earlier work is the reduction of amyloid plaque burden owing to induction of microglial phagocytosis of Aβ deposits (Denner et al., 2012; Escribano et al.,
2009, 2010; Masciopinto et al., 2012; O’Reilly & Lynch, 2012; Pedersen et al., 2006;
Rodriguez-Rivera et al., 2011; Searcy et al., 2012; Toledo & Inestrosa, 2010; Yamanaka et al., 2012). Recently, Mandrekar-Collucci reported that pioglitazone treatment as brief as
9 days was sufficient to clear up to 50% of plaques in 6 or 12 month old APP/PS1 mice and was associated with the appearance of amyloid-laden microglia in the cortex and hippocampus of the drug-treated mice (Mandrekar-Colucci et al., 2012). Similarly,
Yamanaka reported that pioglitazone and a new PPARγ agonist, DSP-8658, stimulated the recruitment of microglia to plaques and promoted their clearance (Yamanaka et al.,
2012). In vitro studies demonstrated that PPARγ agonists stimulated Aβ phagocytosis
43 through induction of CD36 expression. The effect of the PPARγ agonists was dependent upon RXRα expression, and was additively enhanced by simultaneous treatment with an
RXR agonist. The same study observed that DSP-8658 was also able to stimulate microglial recruitment to plaques and increase their phagocytosis of Aβ in an AD mouse model. In each of these latter studies behavioral improvement was observed.
There have been several phase I/II trials of pioglitazone and rosiglitazone in AD patients. A large phase III trial of rosiglitazone in mild/moderate AD failed to show clinical benefit (Gold et al., 2010). Currently, a phase III trial of pioglitazone is underway.
Nurr1
While the activation of NR4A receptors has been recently implicated in a wide variety of processes including fatty acid metabolism and inflammation, they are best known for a broadly neuroprotective spectrum of actions. Nurr1 is essential during development for the survival of dopaminergic neurons, and Nurr1 dysfunction is implicated in decreased survival of dopaminergic neurons in Parkinson’s disease (W. Le et al., 2008; W.-D. Le et al., 2003; Thomas Perlmann & Wallén-Mackenzie, 2004; Saijo et al., 2013). The role of Nurr1 in general neuronal survival is probably due to a combination of actions, including induction of BDNF expression (Volakakis et al., 2010), inhibition of proapoptotic gene induction via several signaling pathways (Mohan et al.,
2012), and suppression of inflammatory NFκB target genes in microglia and astrocytes
(Saijo et al., 2009).
44 Nurr1 mutations are associated with rare genetic forms of PD, but are not a major genetic risk factor for PD (Decressac, Volakakis, Björklund, & Perlmann, 2013). A substantial body of evidence indicates that Nurr1 is downregulated in sporadic PD patients. The role of Nurr1 in mdDA neuronal survival is probably due to a combination of factors, including maintenance of dopaminergic neurotransmission machinery expression and facilitating the response of mdDAs to GDNF (Decressac et al., 2012).
Nurr1 has also been shown to mediate cAMP-response element binding protein (CREB)- induced neuroprotection in response to stress, by upregulating an anti-apoptotic gene program in hippocampal neurons (Volakakis et al., 2010) and increasing BDNF expression in cerebellar granule cells (CGCs) (Barneda-Zahonero et al., 2012). In fact,
Nurr1 is transcriptionally regulated in a CREB-dependent manner (Altarejos &
Montminy, 2011). Additionally, Nurr1 plays a role in DNA repair of double strand breaks in neurons (Malewicz et al., 2011) and nucleotide excision repair in melanoma cells
(Jagirdar et al., 2013). In both cell types Nurr1 is recruited to nuclear foci containing
DNA repair proteins via a mechanism involving poly(ADP-ribose) polymerase-1 (PARP-1) and has a non-transcriptional critical role in DNA repair. Nurr1 is also induced by inflammatory stimuli in microglia and astrocytes and downregulates inflammatory gene transcription by CoREST-dependent transrepression at NFκB target gene promoters
(Saijo et al., 2009). Knockdown of Nurr1 in the SN resulted in an enhanced glial inflammatory response to LPS or α-synuclein and increased death of dopaminergic neurons, suggesting that anti-inflammatory activities of Nurr1 may also be important for
PD pathogenesis.
45 Recent evidence from the Saura group indicates that Nurr1 levels are decreased in an AD mouse model as well as in late-stage AD patients (Espana et al., 2010; Parra-
Damas et al., 2014), and it has been reported that Nur77 levels decrease with age in an
APP/PS1 mouse model of AD (Dickey et al., 2003). Moon et al. also recently published that Nurr1 is normally expressed in subiculum neurons, and it is decreased over time selectively in subicular neurons in 5XFAD mice that accumulate intraneuronal Aβ (Moon et al. 2015)
The involvement of Nurr1 in regulating neuronal survival, neuroinflammation, and hippocampal function and plasticity (Bridi & Abel, 2013; Joshua D. Hawk et al., 2012;
Volakakis et al., 2010), however, makes it an attractive target for further study in AD and other neurodegenerative diseases.
Research Goals
In this thesis we examine novel therapeutic approaches to targeting nuclear receptors in an AD context. A large variety of studies have previously used nuclear receptor agonists in AD models with mixed success. Of recent interest was a study by
Cramer et al. (Cramer et al., 2012) that reported that an extremely short treatment of
AD mouse models with an RXR agonist was able to dramatically reduce soluble and insoluble Aβ and reverse behavioral deficits. This effect was apoE-dependent, and postulated to occur through permissive activation of PPAR:RXR and LXR:RXR complexes.
Our goal was to investigate the heterodimer partners of RXR, and specifically to
46 determine if combinatorial activation of PPARγ and LXR could be of therapeutic benefit, and if other RXR partners, such as Nurr1, represented additional therapeutic targets.
In chapter two, we propose a novel method of therapeutic treatment for AD using two nuclear receptor agonists in combination. LXR and PPARγ function in a linked metabolic pathway that ameliorates amyloid pathology by stimulating apoE production and lipidation, and modulating the brain’s inflammatory state. We demonstrate that combining agonists for LXR and PPARγ in the same treatment paradigm enhances the efficacy of this metabolic pathway, increasing apoE lipidation and Aβ clearance, decreasing inflammation while enhancing the localization of microglia to Aβ deposits, and improving behavioral performance while decreasing harmful off-target drug effects.
In Chapter 3, we propose a novel target for AD therapeutics, the orphan nuclear receptor Nurr1. We demonstrate that Nurr1 acts in a neuron-intrinsic manner to promote survival of a vulnerable neuron population in our AD mouse model, and present evidence that this receptor can also influence intracellular and extracellular Aβ deposition. Recently developed therapeutics have been shown to increase Nurr1 transcriptional activation, as have RXR-directed therapies. We propose that Nurr1 is a potential novel therapeutic target in AD treatment.
47 Figure 1: Nuclear receptors are ligand-activated transcription factors.
48 Table 1: Nuclear receptor agonist treatments of neurodegenerative diseases
Key:
IP: intraperitoneal; i.c.v.: intracerebroventricular; s.c.: subcutaneous; sol Aβ: soluble amyloid β; insol Aβ: insoluble amyloid β; n.c.: no change; ISF: interstitial fluid; TH: tyrosine hydroxylase; DA: dopamine; MWM: Morris water maze; CFC: contextual fear conditioning; NOR: novel object recognition; RWM: radial arm water maze.
49 Table 1. Effects of nuclear receptor agonists in neurodegenerative disease models.
Nuclear Inflam Neurodegenerat Length of Pathology Behavioral Receptor Ligand Dosage Animal model matory ive disease treatment effects Outcomes target Effects Alzheimer's disease
20 mg/kg/ Yan et al., 2003 PPARγ Pioglitazone day 16 wks Tg2576 ↓ sol Aβ 18 Lacombe et al. mg/kg/ 2004 Pioglitazone day 2 months TGFbeta OE ↓ sol Aβ42 ↓ 40 Heneka et al., mg/kg/ 2005 Pioglitazone day 7 days APPV717I ↓ plaques, sol Aβ ↓ 40 Sastre et al., mg/kg/ ↓ intracellular 2006 Pioglitazone day 7 days APPV717I Aβ
20 Nicolakakis et mg/kg/ al., 2008 Pioglitazone day 6-8 wks hAPP swe-ind no effect ↓ n.c. MWM
Mandrekar- 80 Collucci et al., mg/kg/ APPswe/PSEN1 2012 Pioglitazone day 9 days dE9 ↓ plaques, sol Aβ ↓ ↑ CFC
18 Searcy et al. mg/kg/ ↓ intracellular ↑ active avoidance 2012 Pioglitazone day 14 wks 3xTg-AD Aβ, ↓ p-tau learning
20 Masciopinto et mg/kg/ ↑ MWM, NOR in al., 2012 Pioglitazone day 9 months PS1-KIM146V females
20 mg/kg/ Pioglitazone day 9 months 3xTg-AD n.c.
20 Papadopoulos et mg/kg/ hAPP swe- al., 2013 Pioglitazone day 6 months ind/TGF-b1 n.c. ↓ n.c.
20 mg/kg/ hAPP swe- Pioglitazone day 3 months ind/TGF-b1 n.c. ↓ n.c.
4 Pedersen et al., mg/kg/ 2006 Rosiglitazone day 15 wks Tg2576 ↓ sol Aβ42 ↑ radial arm maze
5 Escribano et al. mg/kg/ 2009 Rosiglitazone day 10 wks hAPP swe-ind ↑ NOR
5 mg/kg/ Rosiglitazone day 4 wks hAPP swe-ind ↑ NOR
50 3 Toledo and mg/kg/ APPswe/PSEN1 Inestrosa, 2010 Rosiglitazone day 12 wks dE9 ↓ plaques ↓ ↑ MWM
5 Escribano et al., mg/kg/ ↓ plaques, sol 2010 Rosiglitazone day 4-16 wks hAPP swe-ind Aβ, p-tau ↓ ↑ NOR, MWM
Rodriguez- ↑ CFC, age Rivera et al., 0.18 dependent (9m 2011 Rosiglitazone mg/day 1 month Tg2576 only)
6 O'Reilly and mg/kg/ APPswe/PSEN1 ↓ plaques, insol Lynch, 2012 Rosiglitazone day 4 wks dE9 Aβ42 ↓ ↑ MWM
Denner et al., 0.18 2012 Rosiglitazone mg/day 1 month Tg2576 n.c. ↑ CFC
150 ↑ Yamanaka et al., mg/kg/ APPswe/PSEN1 phagocy 2012 DSP-8658 day 3 months dE9 ↓ plaques, sol Aβ tosis ↑ MWM
Inestrosa et al., APPswe/PSEN1 2013 PPARα WY-14643 0.2 g/l 60 days dE9 ↓ plaques, p-tau ↓ ↑ MWM
APPswe/PSEN1 4-PB 10 mg/l 60 days dE9 ↓ plaques, p-tau ↓ ↑ MWM Kalinin et al., 2009 PPARδ GW742 5XFAD ↓ plaques ↓
↓ locomotor Dumont et al. pan- 0.5% ↓ tau pathology, deficits and 2012 PPAR bezafibrate chow 9 months P301S p-tau ↓ anxiety
33 mg/kg/ Jiang et al., 2008 LXR GW3965 day 4 months Tg2576 ↓ plaques, sol Aβ ↑ CFC
2.5 Donkin et al., mg/kg/ 8 or 24 APPswe/PSEN1 2010 GW3965 day wks dE9 ↑ sol Aβ ↑ NOR/MWM
33 mg/kg/ APPswe/PSEN1 ↓ plaques, ↑ sol GW3965 day 8 wks dE9 Aβ ↑ NOR/MWM
33 Wesson et al., mg/kg/ ↑ olfactory 2011 GW3965 day 2 wks Tg2576 ↓ plaques, sol Aβ behavior 50 Koldamova et mg/kg/ al., 2005 TO901317 day 6 days APP23 ↓ sol Aβ 10 Riddell et al., mg/kg/ 2007 TO901317 day 7 days Tg2576 n.c. 30 mg/kg/ TO901317 day 7 days Tg2576 ↓ sol Aβ42
51 50 mg/kg/ TO901317 day 7 days Tg2576 ↓ sol Aβ42 ↑ CFC 50 Lefterov et al., mg/kg/ 2007 TO901317 day 1 day APP23 n.c. 20 mg/kg/ TO901317 day 4 wks APP23 ↓ insol Aβ ↓
25 mg/kg/ Fitz et al., 2010 TO901317 day 4 months APP23 ↓ plaques, sol Aβ ↑ MWM
30 Vanmierlo et al., mg/kg/ ↑ NOR and object 2011 TO901317 day 6-9 wks APPSLxPS1mut n.c. in plaques location
50 Terwel et al., mg/kg/ 2011 TO901317 day 7 wks APP23 ↓ plaques, sol Aβ (↑) MWM ↑ glia/pla 50 que mg/kg/ associat TO901317 day 6 days APP23 ion
30 mg/kg/ APPswe/PSEN1 Cui et al., 2012 TO901317 day 30 days dE9 ↓ plaques ↓ ↑ MWM 25 mg/kg/ Fitz et al., 2014 TO901317 day 15 days APP23 ↓ ISF Aβ42
25 mg/kg/ n.c. in plaques, TO901317 day 50 days APP23 sol Aβ ↑ CFC and RWM 10 mg/kg; APPswe/PSEN1 Hu et al., 2013 19 3x/wk 6 wks dE9 ↓ plaques, sol Aβ
100 Cramer et al., mg/kg/ 3, 7 or 14 APPswe/PSEN1 ↓ plaques, ↓sol 2012 RXR bexarotene day days dE9 Aβ ↑ CFC/MWM
100 mg/kg/ APPswe/PSEN1 ↓ sol Aβ, n.c. bexarotene day 90 days dE9 plaques ↑ CFC/MWM
100 mg/kg/ bexarotene day 20 days APPPS1-21 ↓ plaques, sol Aβ ↑ CFC/MWM
100 mg/kg/ ↑ olfactory bexarotene day 3 or 9 days Tg2576 behavior/nesting 100 mg/kg/ APPswe/PSEN1 n.c. in plaques Price et al., 2013 bexarotene day 3 or 7 days dE9 or sol Aβ
52 100 mg/kg/ APPswe/PSEN1 ↓ ISF Aβ, n.c. in Fitz et al., 2013 bexarotene day 15 days dE9 plaques ↑ RWM 100 Veeraraghavalu mg/kg/ APPswe/PSEN1 (↓) sol Aβ, n.c. in et al., 2013 bexarotene day 7 days dE9 plaques 100 ↓ sol Aβ40, (↓) mg/kg/ sol Ab4β, n.c. in bexarotene day 7 days 5XFAD plaques 100 mg/kg/ (↓) sol Aβ, n.c. in bexarotene day 7 days APPPS1-21 plaques
100 unclear, possibly Tesseur et al., mg/kg/ n.c. plaques/sol due to drug 2013 bexarotene day 19 days APPPS1-21 Aβ40 toxicity 100 Ulrich et al., mg/kg/ APPswe/PSEN1 2013 bexarotene day 1 day dE9 ↓ ISF Aβ40
100 LaClair et al., mg/kg/ 3, 7 or 14 APPswe/PSEN1 2013 bexarotene day days dE9 n.c. plaques n.c. n.c. in CFC
Boehm-Cagan and Michaelson, 2.5mg/d ↓ neuronal 2014 bexarotene ay 10 days ApoE4-TR Aβ42, tau ↑ MWM, NOR
Parkinson's disease
20 Breidert et al., mg/kg/ ↓ TH+ neuron 2002 PPARγ Pioglitazone day 6-14 days MPTP loss ↓ 20 Dehmer et al., mg/kg/ ↓ TH+ neuron 2004 Pioglitazone day 6-16 days MPTP loss ↓
20 Quinn et al., mg/kg; ↓ TH+ neuron ↑ motor 2008 Pioglitazone 2x/day 7 days MPTP loss performance
10 or 30 Kumar et al., mg/kg/ ↓ oxidative ↑ MWM/passive 2009 Pioglitazone day 35 days MPTP (rat) stress avoidance
2.5 Swanson et al., mg/kg/ 2011 Pioglitazone day 3 months MPTP (monkey) n.c. n.c. n.c.
5 mg/kg/ ↓ TH+ neuron ↑ motor Pioglitazone day 3 months MPTP (monkey) loss ↓ performance
53 10 Ulusoy et al., mg/kg/ ↑ motor 2011 Pioglitazone day 15 days rotenone ↑ striatal DA performance
50 Laloux et al, mg/kg/ ↓ TH+ neuron ↑ motor 2012 Pioglitazone day 14 days MPTP loss performance
50 mg/kg/ Pioglitazone day 14 days 6-OHDA (rat) n.c. n.c. 20 Sadeghian et al., mg/kg; ↓ TH+ neuron 2012 Pioglitazone 2x/day 7 days 6-OHDA (rat) loss ↓ 15 mg/kg; ↓ TH+ neuron GW855266X 2x/day 7 days 6-OHDA (rat) loss ↓
10 Schintu et al., mg/kg/ ↓ TH+ neuron ↑ motor/olfactory 2009 Rosiglitazone day 5 wks MPTPp loss ↓ performance 10 mg/kg/ ↓ TH+ neuron Carta et al., 2011 Rosiglitazone day 1.5 wk MPTPp loss ↓ 10 Martin et al., mg/kg/ 2012 Rosiglitazone day 29 days MPTP 3 mg/kg; ↓ TH+ neuron Lee et al., 2012 Rosiglitazone 2x/day 1 day 6-OHDA (rat) loss ↓/↑ 30 Swanson et al., mg/kg/ ↓ TH+ neuron 2013 LSN862 day 29 days MPTP loss ↓
100 Barbiero et al., mg/kg/ ↓ TH+ neuron ↑ motor 2014 PPARα fenofibrate day 1 day MPTP (rat) loss performance 10 Uppalapati et al., mg/kg/ 2014 fenofibrate day 30 days MPTP (rat) ↓ TH+ cell loss ↓
30 mg/kg/ fenofibrate day 30 days MPTP (rat) ↓ TH+ cell loss ↓ ↑ MWM
100 mg/kg/ ↑ passive fenofibrate day 30 days MPTP (rat) ↓ TH+ cell loss ↓ avoidance, MWM 10 Sadeghian et al., mg/kg/ 2012 PPARδ GW610742X day 7 days 6-OHDA (rat) n.c. ↓ Martin et al., 84 ↓ TH+ neuron 2013 GW0742 ug/day 14 days MPTP loss 24 or Iwashita et al., 240 2007 L-165041 ug/day 2 days MPTP ↑ striatal DA
54 24 or 240 GW501516 ug/day 2 days MPTP ↑ striatal DA 20 mg/kg/ ↓ TH+ neuron Dai et al., 2012 LXR GW3965 day 7 days MPTP loss ↓
6 McFarland et al., RXR/Nur ug/kg/d ↓ TH+ neuron ↑ motor 2013 r1 bexarotene ay 28 days 6-OHDA (rat) loss performance
0.3 mg/kg/ bexarotene day 28 days 6-OHDA (rat) n.c.
1 or 3 mg/kg/ ↓ TH+ neuron ↑ motor bexarotene day 28 days 6-OHDA (rat) loss performance
ALS
1200 6 wks - ↓ motor neuron ↑ motor Kiaei et al., 2005 PPARγ Pioglitazone ppm death G93A SOD1 loss ↓ performance
40 Schutz et al., mg/kg/ day 57 - ↓ motor neuron ↑ motor 2005 Pioglitazone day death G93A SOD1 loss ↓ performance Shibata et al., 1200 6 wks - ↓ motor neuron 2008 Pioglitazone ppm death G93A SOD1 loss ↓
Huntington's disease
↑ motor Chiang et al., 0.01% 4 wks - n.c. in neuronal performance/survi 2010 PPARγ Rosiglitazone in chow death R6/2 death val
10 ↓ mg/kg/ neurodegenerati ↑ motor Jin et al., 2013 Rosiglitazone day 24 wks N171-82Q on performance
↓ ↑ motor pan- 0.5% in neurodegenerati performance/survi Johri et al., 2012 PPAR bezafibrate chow 9 wks R6/2 on val
55 Figure 2: Cell-type specific actions of nuclear receptors in the brain
56
Chapter 2: Combined Liver X Receptor/Peroxisome Proliferator-Activated Receptor γ Agonist Treatment Reduces Amyloid-β Levels and Improves Behavior in Amyloid Precursor Protein/Presenilin 1 Mice
Skerrett, R., Pellegrino, M. P., Casali, B. T., Taraboanta, L., & Landreth, G. E. (2015).
Combined Liver X Receptor/Peroxisome Proliferator-activated Receptor γ Agonist
Treatment Reduces Amyloid β Levels and Improves Behavior in Amyloid Precursor
Protein/Presenilin 1 Mice. The Journal of Biological Chemistry, 290(35), 21591–21602
57 Abstract
Alzheimer’s disease (AD) is characterized by the extracellular accumulation of amyloid β
(Aβ) that is accompanied by a robust inflammatory response in the brain. Both of these pathogenic processes are regulated by nuclear receptors, including the liver X receptors
(LXRs) and peroxisome-proliferator receptor γ (PPARγ). Agonists of LXRs have been previously demonstrated to reduce Aβ levels and improve cognitive deficits in AD mouse models by inducing the transcription and lipidation of apolipoprotein E (apoE). Agonists targeting PPARγ reduce microglial expression of proinflammatory genes, and have also been shown to modulate apoE expression. Here we investigate whether a combination therapy with both LXR and PPARγ agonists results in increased benefits in an AD mouse model. We found that the LXR agonist GW3965 and the PPARγ agonist pioglitazone were individually able to increase levels of apoE and related genes, decrease expression of proinflammatory genes, and facilitate Aβ decreases in the hippocampus. Combined treatment with both agonists provoked a further increase in expression of apoE and decrease of soluble and deposited forms of Aβ. The decrease in plaques was associated with increased co-localization between microglia and plaques. In addition, the PPARγ agonist in the combined treatment paradigm was able to counteract the elevation in plasma triglycerides that is a side effect of LXR agonist treatment. These results suggest that a combined LXR/PPARγ agonist treatment merits further investigation for the treatment of AD.
58 Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid beta (Aβ) peptides in the brain. In cases of late-onset AD, which is the predominant form of the disease, the elevation of soluble forms of Aβ and their subsequent deposition into plaques arises principally from a deficiency in Aβ clearance (Mawuenyega et al., 2010; Wildsmith et al., 2013). Apolipoprotein E (apoE) modulates Aβ deposition and clearance in an isoform-dependent manner (Bales et al.,
2009; Castellano et al., 2011; Deane et al., 2008). Possession of an apoE4 allele, the strongest genetic risk factor for late-onset AD (Corder et al., 1993; Schmechel et al.,
1993), has been linked to decreased efficiency of Aβ clearance (Bales et al., 2009;
Castellano et al., 2011; Deane et al., 2008). ApoE is the principal apolipoprotein in the brain, where it acts as a scaffold for HDL-like particles and facilitates the trafficking of lipids throughout the CNS. Cholesterol and phospholipids are loaded onto nascent apoE primarily by the lipid transporter ATP-binding cassette A1 (ABCA1) and additionally by
ATP-binding cassette G1 (ABCG1) (David M. Holtzman, Herz, & Bu, 2012). Lipidated apoE is produced in the brain by astrocytes, and to a lesser extent, microglia. We have previously shown that lipidated apoE facilitates the proteolytic clearance of soluble forms of Aβ (Jiang et al., 2008), which in microglia is effected by increasing the rate of
Aβ trafficking to lysosomes for degradation (C. Y. D. Lee et al., 2012). The ability of apoE to facilitate soluble Aβ clearance is strongly dependent on the apoE isoform (Bales et al.,
2009; Castellano et al., 2011; Deane et al., 2008) and its lipidation state, with ABCA1- dependent lipidation playing an important role (Donkin et al., 2010; R. P. Koldamova et
59 al., 2005; Wahrle et al., 2008). The production of apoE is transcriptionally regulated by liver X receptors (LXRs) α and β (Lehmann et al., 1997; Tall, 2008). LXRs are type II nuclear receptors that are activated by oxysterol ligands and function as cholesterol sensors. Upon ligand binding, LXRs form obligate heterodimers with retinoid X receptors
(RXRs) and promote transcription of apoE, ABCA1, and ABCG1, among other genes involved in cholesterol metabolism (Hong & Tontonoz, 2014). Treatment of murine models of AD with synthetic LXR ligands such as GW3965 or TO901317 has been previously shown to result in increased apoE expression and lipidation in the brain, memory improvements, and reduced levels of Aβ (Burns et al., 2006; Cui et al., 2012;
Donkin et al., 2010; Fitz et al., 2010, 2014; Jiang et al., 2008; R. P. Koldamova et al.,
2005; Lefterov et al., 2007; Riddell et al., 2007; Terwel et al., 2011; Vanmierlo et al.,
2011; Wesson et al., 2011). Ligands that target peroxisome proliferator-activated receptor γ (PPARγ) also produce increases in LXR target genes and the associated benefits, likely by inducing the expression of LXRα as well as interaction with enhancer elements (Daniel et al., 2014; Mandrekar-Colucci et al., 2012; Yue & Mazzone, 2009). A large body of literature indicates that activation of LXR, PPARγ, or their heterodimeric partner RXR is able to ameliorate Aβ pathology and mediate behavioral improvements in mouse models of AD (Cramer et al., 2012; Skerrett, Malm, & Landreth, 2014).
Deposition of Aβ induces a robust inflammatory response in microglia, resulting in their migration and subsequent association with the Aβ deposits and increased cytokine and chemokine production (Heneka, O’Banion, Terwel, & Kummer, 2010; Hickman et al.,
2008; Mandrekar-Colucci & Landreth, 2010). PPARγ activates a program of gene
60 expression that promotes phagocytosis and tissue repair in macrophages (Chawla, 2010;
Chinetti-Gbaguidi & Staels, 2011), and has been shown to mediate a similar phenotypic conversion in microglia (Mandrekar-Colucci et al., 2012; Saijo et al., 2013; Yamanaka et al., 2012). LXR agonists also modulate microglial function and act as regulators of phagocytosis in AD models (Terwel et al., 2011; Zelcer et al., 2007), partially through transcriptional control of phagocytic proteins such as MerTK and Axl (A-Gonzalez et al.,
2009). PPARγ and LXR agonists have been reported to mitigate neuroinflammation in AD models by exerting robust anti-inflammatory activity (Ghisletti et al., 2007; Saijo et al.,
2013). Additionally, ligand activation of PPARγ or LXR results in their sumoylation and sumoylated PPARγ or LXR interacts with corepressor complexes at NFκB and AP1 promoters to prevent their clearance (Ghisletti et al., 2007; Saijo et al., 2013), suppressing proinflammatory gene expression. LXR and PPARγ each act independently to transrepress an overlapping but distinct subset of proinflammatory genes (Bensinger
& Tontonoz, 2008; Ghisletti et al., 2007; Saijo et al., 2013). In this study, we evaluated the therapeutic potential of combining LXR and PPAR agonists in treating
APPswe/PSEN1dE9 (APP/PS1) mice, which express familial human mutations in both
APP and presenilin. We reproduce previous findings that LXR and PPAR agonists individually are able to decrease Aβ plaques in the hippocampi of AD mice, produce anti-inflammatory effects, and mediate behavioral improvements. Additionally, we demonstrate that a combination of LXR and PPARγ agonists elicits biochemical and behavioral improvements while ameliorating LXR-mediated plasma hypertriglyceridemia. Combination treatment was able to effect a further increase in
61 LXR target gene production and effectively reduced inflammatory markers. Importantly, combination treatment was more effective than individual agonist treatments at stimulating the colocalization of microglia and plaques in APP/PS1 mice and promoting
Aβ intracellular degradation. These data provide a rationale for further investigation of combination therapies utilizing LXR and PPARγ agonists in AD.
Experimental Procedures
Animals and treatment
APPswe/PS1Δe9 (APP/PS1) mice (Jackson Laboratories) co-express a chimeric human- mouse amyloid precursor protein containing the APPswe mutations (K595N/M596L) and human presenilin 1 with an exon 9 deletion (deltaE9) from the mouse prion promoter.
Male 6-month-old APP/PS1 mice or non-transgenic (NonTg) littermates were orally gavaged daily for 9 days with 50 mg/kg/day GW3965, 80 mg/kg/day pioglitazone, both
50mg/kg/day GW3965 and 80mg/kg/day pioglitazone, or vehicle control (0.5% DMSO in sesame oil). Behavioral analysis was performed during the last two days of treatment.
The animals were then sacrificed, and one hemisphere was fixed in 4% PFA and processed for immunohistochemistry. The hippocampus and cortex were removed from the other hemisphere and snap-frozen until subject to RNA and protein extraction. For analysis of plasma triglycerides, male 2-month-old C57Bl/6 mice were treated with
GW3965, pioglitazone, both, or vehicle as above, with plasma collected on day 9 of treatment within 3 hours of the last dose. All experiments involving animals followed
62 protocols approved by the Case Western Reserve University School of Medicine’s
Institutional Animal Care and Use Committee.
Behavioral testing
Contextual Fear Conditioning - Freezing behavior was monitored by automated tracking system (Coulbourn Instruments, USA). On day 8 of drug treatment mice underwent training, which consisted of 2 minutes of free exploration in the shock chamber followed by 30 sec of the conditioned stimulus (CS: an 85dB sound at 2800Hz). After a 2 second delay, the unconditioned stimulus (US: 0.56mA) was delivered and the freezing response was measured for 30 sec. The training paradigm was repeated 4 times. Twenty four hours later the retention test was performed, during which mice were returned to the same shock chamber for 5 min for contextual freezing measurement in the absence of
CS and US. The percent of time frozen and number of freezes was recorded.
Protein extraction
Cortex and hippocampus dissected from hemibrains were homogenized in 800μL of tissue homogenization buffer (250mM sucrose, 20mM Tris pH7.4, 1mM EDTA, 1mM
EGTA in diethylpyrocarbonate-treated water). Homogenates were centrifuged at 5000xg for 10 minutes at 4°C, and supernatants were stored at - 80°C for Western analysis. For extraction of soluble Aβ species, 250μL of homogenate was added to an equal volume
0.4% diethylamine in 100mM NaCl, and the samples were mechanically homogenized again. Samples were then centrifuged at 135,000xg for 1 hour at 4°C. 0.5M Tris-HCl
63 pH6.8 was added to the supernatant, which was stored at -80°C for analysis of soluble
Aβ species by ELISA. The remaining pellet was sonicated in cold 70% formic acid and centrifuged at 109,000xg for 1 hour at 4°C. The supernatant was neutralized and the samples stored at -80°C for analysis of insoluble Aβ species by ELISA.
Cell culture
Primary microglia and astrocytes were prepared from P0-P3 mice as previously described (9). Purified microglia and astrocytes were maintained in DMEM/F12
(Invitrogen) containing 5% heat inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin for 3 days. Twenty four hours before treatment, media was changed to serum free DMEM/F12 containing 1% penicillin/streptomycin. For Western blot and qPCR analysis, cells were plated in 6 well plates at 1x106 cells/well and treated for 24 hours with GW3965, pioglitazone, or GW3965 and pioglitazone or vehicle (DMSO) at the indicated concentrations.
Intracellular Aβ Degradation Assay – Soluble Aβ was prepared by dissolving lyophilized
Aβ1-42 in DMSO to a final concentration of 1mg/ml to create a solution of mostly monomeric Aβ species with very few oligomers (Shen & Murphy, 1995). Primary microglia were plated in 12-well plates at a density of 4 × 105 cells/well. Microglia were then pre-treated for 24 hours with GW3965, pioglitazone, or GW3965 and pioglitazone or vehicle (DMSO) at the indicated concentrations, and then incubated with 2 μg/ml
Aβ1–42 (American Peptide Company) for 18 h. Plates were washed with PBS and cells
64 were lysed in 1% SDS with protease inhibitor cocktail (PIC) (Roche). Remaining intracellular Aβ was measured by ELISA.
Aβ ELISA
For the intracellular Aβ degradation assay, ELISAs were performed using 6E10 as the capture antibody and 4G8-HRP as the detection antibody (Covance). To analyze levels of soluble and insoluble Aβ in brain homogenates, ELISAs were performed using 6E10 as the capture antibody and Aβ1-40-HRP or Aβ1-42-HRP (Covance) for detection. The results were read using a Spectramax colorimetric plate reader (Molecular Devices) and normalized to the total protein.
Western Blot Analysis
Cell lysates or brain homogenates were resolved on Bis-Tris 4-12% gels (Invitrogen), transferred to PVDF membranes, and immunodetected using anti-ABCA1 (Novus
Biologicals), anti-ABCG1 (Novus Biologicals), anti-apoE (Santa Cruz Biotechnology), and anti-β-actin (Santa Cruz Biotechnology). Band intensities were quantified using NIH
Image J software.
Native PAGE
Cell lysates or brain homogenates were resolved on Tris-Glycine 4-12% gels (Invitrogen), transferred to PVDF membranes, and immunodetected using an anti-ApoE antibody
(Santa Cruz Biotechnology). Native high molecular weight standards (GE High Molecular
65 Weight Native Marker Kit 17044501) were run on each gel and used to determine the
Stokes diameter of samples. Intensity of the bands above 8nm in size was quantified using NIH Image J software to determine the apoE lipidation index.
RNA extraction, reverse transcription, and quantitative PCR
Quantification of pro- or anti-inflammatory gene expression was performed as previously described (Mandrekar-Colucci et al., 2012). For qPCR analysis of cells, RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. For qPCR analysis of brain homogenate, 200μL homogenate was combined with an equal volume of RNA-Bee (Tel-Test), chloroform was added, and samples were shaken vigorously. Samples were incubated on ice for 15 minutes followed by centrifugation at 13,000xg for 15 minutes at 4°C. The aqueous phase was collected, and
RNA extraction was performed as above. RNA samples (0.5μg) were reverse transcribed using QuantiTect Reverse Transcription kit (Quiagen). The Taqman PreAmp Master Mix
(Life Technologies) was then used to preamplify cDNA for 14 cycles according to the manufacturer’s instructions. Pre-amplified cDNA was run in a 10μL reaction for 40 cycles on the StepOne Plus Real Time PCR system (Applied Biosystems) using the TaqMan
Gene Expression Master Mix (Life Technologies). Primers labeled with FAM probes were from Life Technologies and included Iba1 (Mm00479862_g1), CD45 (Mm01293575_m1),
Tnfα (Mm99999068_m1), Il-1β (Mm01336189_m1), Nos2 (Mm01309902_m1), IL6
(Mm00446190_m1), and GAPDH (4352339E-0904021). The comparative CT method
(ΔΔCT) was used to analyze gene expression.
66 Immunohistochemistry and image analysis
Coronal sections (30μm) were taken from postfixed hemispheres using a cryostat.
Alternate sections were immunostained for analysis of plaque area, microglial area, and microglia/plaque colocalization. Antigen retrieval was performed with 20μL/mL
Proteinase K in TE buffer (50mM Tris,1mM EDTA, 0.5% Triton X-100 pH 8) and slides were blocked in 5% normal goat serum in PBS 0.1% Triton X-100. Primary antibodies
(6E10 1:1000, Covance; Iba1 1:500, Wako) were incubated overnight at 4°C. Analysis was performed on 2 sections per slide on 3 slides spaced evenly throughout the hippocampus. Images were analyzed by a blinded observer using Image Pro-Plus software (Media Cybernetics) for percentage area of 6E10-positive plaques in the cortex or hippocampus and percentage area occupied by Iba1-positive microglia. The percentage of 6E10 positive area also positive for Iba1 immunostaining was determined for every individual plaque in each section, and the results normalized to Iba1 intensity in a non-plaque area in each image to determine Iba1 enrichment at plaques.
Triglyceride Assay
Plasma was collected using 3.8% sodium citrate in water as an anticoagulant. Blood was centrifuged at 1,000 x g for 10 minutes at 4°C. The plasma was removed and stored at -
80°C for further analysis. Levels of plasma triglycerides were determined using the
Triglyceride Colorimetric Assay Kit (Cayman Chemical Company) according to the manufacturer’s instructions. The assays were read using a Spectramax colorimetric plate reader (Molecular Devices).
67 Statistics
All statistical analyses were performed using Prism software (GraphPad, San Diego, CA).
Twotailed Student’s t test, one-way analysis of variance (ANOVA) with a Tukey post-test, or two-way ANOVA was used to determine p values.
Results
Enhanced effects of combined activation of PPARγ and LXR in vitro
Because PPARγ and LXR agonists have been independently reported to increase expression of LXR target genes (Hong & Tontonoz, 2014; Mandrekar-Colucci et al.,
2012), we analyzed whether a combined activation of PPARγ and LXR would lead to a greater increase in LXR target gene expression. We cultured primary astrocytes and quantified target gene expression after a 24-hour treatment with an LXR agonist
(GW3965), a PPARγ agonist (pioglitazone) or a combined treatment with both.
Consistent with our previous findings, we observed that LXR and PPARγ agonists were able to increase protein expression of apoE and its lipidating proteins ABCA1 and ABCG1 in cultured astrocytes (Fig 2.1A) (Jiang et al., 2008; Mandrekar-Colucci et al., 2012).
Combined treatment with GW3965 and pioglitazone did not produce an additive effect in the expression of apoE protein, but significantly increased the expression of its lipid transporter ABCA1 over pioglitazone alone (Fig 2.1A). To determine how this upregulation in ABCA1 affected apoE, we assessed the quantity of large diameter apoE
68 particles secreted by astrocytes into the extracellular media by native-PAGE (Fig 2.1B).
Detection of large-diameter apoE particles by native-PAGE has been shown to correlate with increases in apoE lipidation (J. Hu et al., 2015), but we are unable to exclude the possibility that native-PAGE detects some amount of apoE aggregation. As expected,
GW3965 and pioglitazone independently were able to increase the amount of large- diameter apoE secreted, and a combination treatment induced a further increase in large-diameter apoE particles (Fig 2.1B). Microglia internalize soluble Aβ from the media and degrade it intracellularly, and this process is facilitated by agonists of LXR, PPARγ and RXR. (Cramer et al., 2012; Jiang et al., 2008; C. Y. D. Lee et al., 2012; Mandrekar-
Colucci et al., 2012). Microglia were incubated with Aβ for 18 hours resulting in the uptake and degradation of Aβ. The amount of intracellular Aβ remaining after that time was measured as an indicator of Aβ degradation efficiency. Pretreatment with GW3965 or pioglitazone for 24 hours enhanced the degradation efficiency in microglia, and pretreatment with a combination of both drugs further enhanced this effect (Fig 2.1C).
LXR and PPARγ are also known to have anti-inflammatory effects in microglia due to transrepression of NFκB at the promoters of inflammatory genes (Saijo et al., 2013). To determine whether combination therapy targeting both receptors could enhance these anti-inflammatory effects, we pretreated primary cultured microglia for 24 hours with
DMSO, GW3965, pioglitazone, or both nuclear receptor agonists, then induced an inflammatory response with LPS. Induction of transcription for pro-inflammatory cytokines TNFα and IL1β was less in microglia pretreated with either GW3965 or pioglitazone (Fig 2.1D). Microglia pretreated with a combination therapy exhibited
69 enhanced suppression of these proinflammatory genes (Fig 2.1D). Pretreatment with
GW3965 or pioglitazone alone did not decrease transcript levels of iNOS, but the combination pretreatment produced a significant decrease in iNOS transcript (Fig 2.1D).
Combination therapy increases apoE particle size in APP/PS1 mice
Previously published data indicates that increasing apoE protein levels and lipidation was associated with reversal of the behavioral deficits and some aspects of pathology in
AD mouse models (Skerrett et al., 2014). Based on our in vitro data indicating that combination therapy enhances the transcriptional effects of LXR and PPARγ agonists and their facilitation of Aβ degradation (Fig 2.1), we chose to treat APP/PS1 mice with the combination therapy. Four treatment groups of age-matched transgenic male six month old animals were generated and treated for 9 days by oral gavage. The first group was treated with 50mg/kg/day GW3965, the second with 80mg/kg/day pioglitazone, the third with 50mg/kg/day GW3965 and 80mg/kg/day pioglitazone in the same volume, and the fourth with a vehicle of DMSO in sesame oil. Western blot analysis on hemibrain cortical/hippocampal homogenate indicated that GW3965 and pioglitazone increased transcription of LXR target genes ABCA1, ABCG1, and apoE, with combination therapy providing a significant increase in apoE over GW3965 alone (Fig
2.2, A and B). Non-denaturing gel electrophoresis was performed on cortical/hippocampal homogenates and detected with apoE antibody to determine the size distribution of apoE. GW3965 and pioglitazone were both able to increase the
70 proportion of large-diameter apoE particles, and combination therapy further increased this effect (Fig 2.2C). Pioglitazone treatment reduces GW3965-mediated elevation of plasma triglycerides LXRs agonists have been reported to upregulate the synthesis of fatty acids in the liver due to LXR-mediated stimulation of a gene expression program which includes SREBP-1c and fatty acid synthase, among other lipogenic enzymes
(Bensinger & Tontonoz, 2008). In a preliminary study in a cohort of WT mice, we observed a significant increase in plasma triglycerides after 9 days of treatment with
GW3965 compared to vehicle, which was not observable after 9 days of treatment with pioglitazone or with both GW3965 and pioglitazone (data not shown). In 6 month old
APP/PS1 mice, we observed a strong trend for GW3965 to increase plasma triglycerides, which was significantly reduced by the addition of pioglitazone in our combination treatment (Fig 2D). Treatment with either GW3965, pioglitazone, or both agonists did not correspond to an increased liver weight (Fig 2E).
Combination therapy reduces inflammatory markers in APP/PS1 mice
APP/PS1 animals have increased expression of Iba1 and CD45, microglial markers which are reflective of a proinflammatory phenotype (Fig 2.3, A-D). GW3965 and pioglitazone were able to reduce expression of Iba1, as assessed by qPCR on cortical/hippocampal homogenate (Fig 2.3A) and immunostaining on hemicortical sections, significantly reducing Iba1 immunostaining compared to vehicle or GW3965 alone (Fig 2.3, B and C).
Interestingly, combination therapy effected the largest decrease in Iba1, reducing Iba1
71 levels to a point below baseline wild-type levels (Fig 2.3A). The levels of CD45 were also decreased by combination therapy (Fig 2.3D). Our in vitro data indicate that treating microglia with GW3965 and pioglitazone together decreases the inflammatory response they exhibit upon challenge with LPS (Fig 2.1D). In the APP/PS1 mouse model, mice treated with GW3965, pioglitazone, or both, all exhibited different levels of pro- inflammatory gene expression as measured by qPCR (Fig 2.3, E-G). IL6 was the only cytokine significantly increased in APP/PS1 animals at 6 months of age, and pioglitazone and GW+pio were able to significantly decrease IL6 gene expression (Fig 2.3E). IL1β was significantly decreased by GW3965 and GW+pio (Fig 2.3F), and TNFα was decreased by pioglitazone but increased by GW+pio treatment (Fig 2.3G), suggesting that GW3965, pioglitazone, and GW+pio act on microglia to differently regulate their inflammatory state.
Combination therapy reduces Aβ deposition by increasing co-localization between microglia and plaques
To evaluate the ability of nuclear receptor agonists to reduce Aβ species in 6 month old
APP/PS1 mice, we sequentially extracted soluble and insoluble Aβ species from cortical and hippocampal homogenates. Treatment with GW3965 alone did not significantly reduce soluble or insoluble Aβ levels, while pioglitazone treatment significantly decreased only levels of soluble Aβ40 by about 25% (Fig 2.4, A and B). However, when we evaluated Aβ levels in treated mice by IHC (Fig 2.4, C-E) we were able to observe
72 significant decreases in the amount of hippocampal (Fig 2.4E) but not cortical (Fig 2.4D)
6E10 staining, indicating that GW3965 and pioglitazone were able to facilitate the clearance of deposited Aβ in the hippocampus. In contrast, mice treated with combination therapy exhibited decreased levels of both soluble Aβ40 and 42 as measured by ELISA (Fig 2.4A). Insoluble Aβ40 levels were also decreased by about 50%, but the decreases in insoluble Aβ42 were not significant (Fig 2.4B). These findings correlated with an approximate 60% decrease in cortical plaques and a 50% decrease in hippocampal plaques as quantified by IHC (Fig 2.4, C-E). Decreases in insoluble species of Aβ and plaque are likely mediated principally by microglia, which are capable of taking up and degrading fibrillar forms of Aβ. We found that the overall Iba1+ area in the brain is decreased following nuclear receptor agonist treatments (Fig 2.3C), and thus follows overall plaque burden. However, we observed there are more microglia at sites of plaque deposition (Fig 2.5). To determine the amount of colocalization between microglia and plaques, we quantified the amount of 6E10/Iba1 double positive area in the cortex (Fig 2.5B) and hippocampus (Fig 2.5C) of treated and control mice by IHC.
GW3965, pio, and GW+pio treatments significantly increased the amount of microglia/plaque co-localization in the cortex, and GW3965 and GW+pio were able to significantly increase co-localization in the hippocampus. These observations suggest that nuclear receptor agonists mediate reductions in insoluble Aβ by altering the interaction between microglia and plaques.
73 Treatment with nuclear receptor agonists reverses the cognitive deficits in APP/PS1 mice
To determine whether treatment with nuclear receptor agonists is able to improve behavioral impairments in APP/PS1 animals, we evaluated hippocampal-dependent memory in the contextual fear conditioning test. APP/PS1 animals exhibited a decreased freezing response as compared to WT animals during the training period of the task, but drug-treated animals did not exhibit this impairment in learning during the training period (Fig 2.6A). As expected, WT age-matched littermate controls froze for about 25% of the testing period, while at 6 months of age APP/PS1 mice exhibited deficits in freezing behavior, freezing about 15% less than WT littermate animals (Fig 2.6B).
Consistent with previously published studies, 9 day treatments with GW3965 and pioglitazone were able to restore freezing behavior in APP/PS1 animals to WT levels. A 9 day treatment with GW+pio was able to achieve an equivalent cognitive improvement
(Fig 2.6B).
Discussion
LXR and PPAR agonists have previously been demonstrated to have beneficial effects in AD mouse models. The first study utilizing nuclear receptor agonists in AD was published in 2003 by Yan et al. (Yan et al., 2003), and reported that long-term treatment of Tg2576 mice with pioglitazone was able to decrease levels of soluble Aβ. However, subsequent studies using PPAR agonists in several AD mouse models had variable success in observing changes in Aβ levels. Several studies using various treatment
74 paradigms with PPARγ agonists observed decreases in soluble Aβ species (Lacombe et al., 2004; Pedersen et al., 2006), plaques (O’Reilly & Lynch, 2012; Toledo & Inestrosa,
2010) or both (Escribano et al., 2010; Heneka et al., 2005; Mandrekar-Colucci et al.,
2012; Yamanaka et al., 2012), and others reported decreased intracellular Aβ (Sastre et al., 2006; Searcy et al., 2012). However, there were also cases where PPARγ agonists exhibited no measurable effect on Aβ pathology (Denner et al., 2012; Nicolakakis et al.,
2008; Papadopoulos, Rosa-Neto, Rochford, & Hamel, 2013). LXR agonists have had a similarly mixed success since 2005, when Koldamova et al. reported that TO901317 was able to reduce soluble Aβ levels in APP23 mice (R. P. Koldamova et al., 2005). Since then,
LXR agonists have been reported to decrease soluble Aβ (Fitz et al., 2010, 2014; Y. Hu et al., 2013; Jiang et al., 2008; Riddell et al., 2007; Terwel et al., 2011; Wesson et al., 2011), decrease insoluble Aβ or plaques (Cui et al., 2012; Fitz et al., 2010; Y. Hu et al., 2013;
Jiang et al., 2008; Lefterov et al., 2007; Terwel et al., 2011; Wesson et al., 2011), or in one case decrease plaques while increasing soluble Aβ species (Donkin et al., 2010). One study observed no biochemical changes in Aβ pathology in aged animals (Vanmierlo et al., 2011), but reported behavioral improvements. While the majority of studies report that PPARγ and LXR agonists are able to reduce Aβ pathology, the variability in the clearance of soluble Aβ species and plaques remains unexplained. The genotype, age of the mice, differences in diet, and drug formulation are all likely to influence drug efficacy, but these factors have not been systematically explored.
We found that treatment with GW3965 did not significantly stimulate clearance of soluble and insoluble species of Aβ, while pioglitazone was only able to stimulate
75 clearance of soluble Aβ40 as measured in whole brain homogenates. We also visualized plaques using IHC to evaluate regional effects on Aβ deposition. Interestingly, although plaques did not significantly decrease in the cortices of pioglitazone or GW3965 treated animals, both drugs were able to induce significant decreases in hippocampal plaque load in the same animals. Our results are similar to those of Riddel et al. (Riddell et al.,
2007), who reported that an LXR agonist selectively induced apoE and ABCA1 and reduced Aβ42 in the hippocampus of Tg2576 mice. Donkin et al. (Donkin et al., 2010) also reported that GW3965 mediated a hippocampal-specific reduction in dense-core plaques in APP/PS1 mice, although when Aβ deposition across the whole brain was quantified no significant reduction was observed.
Combination therapy with GW+pio significantly reduced overall levels of soluble
Aβ40 and Aβ42. It is likely that the reduction in soluble Aβ we observe in GW+pio treatment is due in part to the enhanced transcription of apoE and its lipid transporters, as levels of lipidated apoE have been previously shown to increase the rate of degradation of Aβ by microglia (Jiang et al., 2008; C. Y. D. Lee et al., 2012). However, we also observe an acute reduction of insoluble Aβ40 and deposited Aβ plaques in both the cortex and hippocampus over the course of our 9 day treatment with GW+pio, which is more likely due to active phagocytic clearance by microglia (Savage et al., 2015). We postulate that the reductions in Aβ we observe are due mainly to modulation of Aβ clearance rather than Aβ production because previous studies using GW3965 (Jiang et al., 2008) and pioglitazone (Mandrekar-Colucci et al., 2012) have established that these drugs do not affect the production or processing of APP in transgenic mouse models of
76 AD. This agrees with our in vitro observations that GW+pio treatment is able to enhance microglial degradation of Aβ to a greater degree than either agonist alone. The mechanistic basis of the enhanced effectiveness of the combined drug treatment may be the ability of the drugs to target independent enhancer sequences to which the nuclear receptors bind and act combinatorially to promote gene expression (Schmechel et al., 1993). Future studies will be able to determine whether the Aβ clearance resulting from engaging PPARγ and LXR together via a combination therapy will be more reproducible than the effects of individual PPARγ and LXR agonists.
It is possible that the facilitation of microglial phenotypic conversion to an anti- inflammatory, phagocytic phenotype contributes to the increased benefits of GW+pio treatment. APP/PS1 mice at 6 months of age already exhibit increased microgliosis and astrogliosis as well as proinflammatory alterations in their brain cytokine profile
(Mandrekar-Colucci et al., 2012). Importantly, PPARγ agonists have been shown not only to reduce inflammation, but also to increase phagocytosis of Aβ in vitro and in vivo
(Mandrekar-Colucci et al., 2012; Yamanaka et al., 2012). The effects of LXR agonists on inflammation in AD models have not been studied as thoroughly, but treatment with
LXR agonists has been reported to be anti-inflammatory (Cui et al., 2012; Lefterov et al.,
2007), increase co-localization between glia and plaques (Terwel et al., 2011), and regulate transcription of phagocytic genes (A-Gonzalez et al., 2009). Together with evidence that, in macrophages, PPARγ and LXR are able to transrepress overlapping but distinct sets of proinflammatory genes (Bensinger & Tontonoz, 2008; Saijo et al., 2013;
Zelcer et al., 2007), this body of evidence led us to hypothesize that co-stimulation with
77 LXR and PPARγ agonists would have a greater anti-inflammatory effect than either agonist alone. We found that GW+pio treatment effectively decreased pro- inflammatory markers in vitro and in vivo with an interesting exception – GW+pio reduced TNFα transcript levels in cultured primary microglia but not in whole brains in
APP/PS1 mice. This could indicate that GW+pio treatment acts differently on non- microglial brain cells that produce TNFα, such as astrocytes, or imply that environment is an important factor for determining the effects of these drugs on microglia.
Importantly, GW+pio also increased the co-localization between plaques and microglia.
We propose that this enhanced co-localization facilitates improved phagocytosis and together with the increased degradation of Aβ by microglia accounts for the reduction of Aβ species we observe with GW+pio treatment.
Our investigation also reports behavioral improvements in APP/PS1 mice after
LXR or PPARγ agonist treatment, which is consistent with the vast majority of previous studies. LXR (Cui et al., 2012; Donkin et al., 2010; Fitz et al., 2010; Jiang et al., 2008;
Riddell et al., 2007; Terwel et al., 2011; Vanmierlo et al., 2011; Wesson et al., 2011) and
PPARγ (Denner et al., 2012; Escribano et al., 2010; Mandrekar-Colucci et al., 2012;
Masciopinto et al., 2012; O’Reilly & Lynch, 2012; Pedersen et al., 2006; Rodriguez-Rivera et al., 2011; Searcy et al., 2012; Yamanaka et al., 2012) agonists have been reported to ameliorate memory deficits using a variety of tests in a range of mouse models. It should be noted, however, that Nicolakakis et al. (Nicolakakis et al., 2008), Masciopinto et al. (Masciopinto et al., 2012), and Papadopoulos et al. (Papadopoulos et al., 2013) reported no behavioral improvements after treating mice with pioglitazone. We find
78 that APP/PS1 mice at 6 months of age are impaired in performance of the contextual fear conditioning task, and that this impairment is rectified by treatment with GW, pio, or both agonists. Our dosage paradigm did not allow for closer examination of this effect, since we utilized high doses of GW+pio that have been previously shown to restore freezing behavior to wild-type levels. It will be important in future studies to treat at lower doses of GW+ pio that are less individually effective to determine if combination treatment is able to further enhance memory improvements.
Importantly, although pioglitazone has entered phase III clinical trials for the treatment of AD, no LXR agonists have reached clinical trials due to an unfavorable side- effect profile that includes hypertriglyceridemia (Baranowski, 2008; Li & Glass, 2004).
This is likely due to LXR-dependent upregulation of the sterol response element binding protein-1c (SREBP1C) pathway in the liver. PPARα agonists have been shown to decrease hypertriglyceridemia, possibly through stimulating β-oxidation of fatty acids
(Shen & Murphy, 1995), and a PPARα agonist was shown to counteract LXR agonist- induced hypertriglyceridemia in mice (Beyer et al., 2004). There is evidence that PPARγ activation could also normalize LXR-mediated hypertriglyceridemia, possibly by mediating the redistribution of triglycerides from plasma to storage in fatty tissue by inducing, genes involved in lipoprotein uptake and hydrolysis (Evans, Barish, & Wang,
2004; Schoonjans et al., 1996). Our finding that the addition of a PPARγ agonist is able to normalize the hypertriglyceridemia observed in LXR agonist treated mice renews the potential for LXR agonists to enter clinical trials for AD. Additionally, we did not observe changes in liver weight in mice treated with GW3965, pioglitazone, or GW+pio, which is
79 consistent with previously reported work (Kruit et al., 2005; Miao et al., 2004; Quinet et al., 2006; van der Hoorn et al., 2011; Ye et al., 2001).
In this study we provide support for the beneficial actions of LXR and PPARγ agonists in the treatment of amyloid pathology, inflammation, and behavioral impairments in AD mouse models. LXR and PPARγ activation in combination more effectively improves several biochemical markers and alleviates cognitive impairments in AD mice, with an additional reduction in side effects. Our results demonstrate potential for therapies targeting multiple heterodimer partners of RXR for the treatment of AD.
FOOTNOTES *This work was supported by NIH grant R01 AG030482 (to G.E.L.), NIH
Grant 5T32NS067431-13 (to R.R.S.), the Gail & Elliott Schlang Philanthropic Fund, and
CAPES Foundation Grant 5758/12-2 (to M.P.P.).
80 Figure 2.1. Combination therapy increases target gene expression and Aβ degradation and decreases inflammatory markers in vitro. (A) Cultured primary astrocytes were incubated for 24 hours with DMSO (uM), 500nM GW3965, 100nM pioglitazone, or both doses combined, then LXR target genes were quantified by immunoblotting and normalized to actin. Representative blots are shown on right. (B) Conditioned medium from the same treated primary astrocytes was collected and separated by native PAGE, then immunoblotted for apoE. (C) Primary microglia were treated for 24 hours with GW, pio, or GW+pio, followed by the addition of 2μg/mL Aβ1-42 for 18 hours. Remaining intracellular Aβ was quantified using ELISA and normalized to total protein. (D) Cultured primary microglia were incubated for 24 hours with the indicated concentrations of drug or DMSO for controls, then 100ng/mL LPS was introduced to the media for 12 hours.
RNA was extracted and expression of pro-inflammatory genes examined by qPCR analysis. The dotted line indicates baseline transcript levels in DMSO only control. The
LPS-treated control was preincubated with DMSO for 24 hours then 100ng/mL LPS for
12 hours. n=4. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle-treated control; ##p < 0.01, ###p < 0.001 compared with the LPStreated control; student’s t test.
Abbreviations: nm, nanometers.
81
82 Figure 2.2. Nuclear receptor agonists stimulate transcription of LXR target genes in AD mice
(A) 6 month old transgenic APPswe/PSENΔE9 mice were orally gavaged with 50 mg/kg/day GW3965, 80 mg/kg/day pioglitazone, or both for 9 days.
Cortical/hippocampal homogenate was immunoblotted for ABCA1, ABCG1, and apoE.
Representative blots are shown. (B) Each sample was normalized to actin, and results are expressed as fold difference compared to vehicle controls. (C) Equal volumes whole brain homogenate from each treatment group were analyzed by native PAGE and immunoblotted for apoE. n=7-11 animals/group. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the vehicle-treated control. (D and E) 6 month old transgenic
APPswe/PSENΔE9 or non-Tg littermate control animals were orally gavaged with doses described above for 9 days. (D) Plasma was collected and analyzed for triglyceride content by colorimetric assay. n=6 animals/group. (E) Complete livers were removed from each animal and weighed, and liver weight is normalized to total body weight for each animal. *p < 0.05 compared with vehicle-treated control; #p < 0.05 compared with
GW-treated group; student’s t test. Abbreviations: nm, nanometers.
83
84 Figure 2.3. Nuclear receptor agonists reduce the pro-inflammatory environment in
APP/PS1 mice.
Nuclear receptor combination therapy benefits AD mice 15 Transcript levels from RNA isolated from whole-brain homogenate (A) and quantification of immunofluorescence in cortex and hippocampus (B) of the microglia marker Iba1 in 6 month old
APPswe/PSENΔE9 orally gavaged with the indicated drugs for 9 days. 9 hemicoronal sections per animal were immunostained for Iba1 and Iba1 area analysis was performed as described in “Experimental Procedures.” (C) Representative images of Iba1 immunostaining in the cortex are shown. (D-G) Transcript levels of pro-inflammatory markers CD45 (D) IL6 (E) IL1β (F) and TNFα (G) were analyzed by qPCR on RNA isolated from whole-brain homogenate. n=7-11 animals/group. *p < 0.05, **p < 0.01 compared with vehicle-treated APP/PS1 mice; #p < 0.05, ##p < 0.01, ###p < 0.001 compared with nonTg littermate control mice. A, D-G: One way ANOVA with Tukey post-test, B: student’s t test.
85
86 Figure 2.4. Combination therapy significantly reduces amyloid burden in AD mice.
Aβ was sequentially extracted using DEA for soluble Aβ (A) and formic acid for insoluble
Aβ (B) from half-brain cortex/hippocampal homogenates. Samples were analyzed by
ELISA and Aβ values were normalized for total protein loaded and to vehicle treated animals. To quantify plaque load by immunohistochemistry, 9 hemicoronal sections per animal were immunostained for 6E10 and plaque area analysis was performed as described in “Experimental Procedures.” (C) Representative images are shown from the cortex. Aβ plaque area was quantitated in the cortex (D) and hippocampus (E). n=7-11 animals/group. *p < 0.05, **p < 0.01 compared with vehicle-treated APP/PS1 mice; student’s t test.
87
88 Figure 2.5. Nuclear receptor agonists promote microglial colocalization with plaques.
(A) Hemicoronal sections from each treatment group were co-immunostained with Iba1
(green) for microglia and 6E10 (red) for Aβ plaques. Representative images from the cortex are shown. Colocalization between 6E10 and Iba1 was analyzed according to
“Experimental Procedures” in the cortex (B) and hippocampus (C). n=7-11 animals/group. *p < 0.05, **p < 0.01 compared with vehicle treated APP/PS1 mice; student’s t test.
89 Figure 2.6. Nuclear receptor agonists ameliorates cognitive deficits in AD mice.
Training for the fear conditioning assay was performed on day 8 of drug treatment and mice were tested on day 9. (A) The number of freezes by each treatment group during training is shown as a function of periods. #p < 0.05, ###p < 0.001 between non-TG control mice and TG Veh mice by two-way ANOVA. (B) The percentage of time spent freezing by each treatment group during the contextual fear conditioning test period. *p
< 0.05 compared with vehicle-treated APP/PS1 mice; #p < 0.05 compared with non-Tg littermate control mice by one way ANOVA with Tukey post-test. n=7-11 animals/group.
90
Chapter 3: Nurr1 is neuroprotective in the 5XFAD subiculum.
91 Abstract
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly and affects millions of people worldwide. AD pathology includes characteristic amyloid plaques and neurofibrillary tangles composed of tau and at later stages synaptic loss and significant neurodegeneration. Major symptoms of AD, including memory loss and cognitive decline, correlate closely in their severity to the degree of neuronal loss an individual experiences. Previous work from our lab and others has proposed that nuclear receptors are important players in the clearance of amyloid pathology and inflammation in AD mouse models, but the role of NRs in neurodegeneration has not yet been addressed. The NR4A subfamily of NRs are known to have neuroprotective effects, and dysregulation of Nurr1 (NR4A2) has been implicated in Parkinson’s disease and more recently in AD. We observe that decreased expression of neuronal Nurr1 correlates with increased neuronal loss in the 5XFAD mouse model of AD, while increased Nurr1 expression is neuroprotective, and decreases neuritic dystrophy and amyloid deposition. These results indicate that Nurr1 has a neuroprotective role in AD, and suggest that drugs targeting Nurr1 activation could be of therapeutic value.
Introduction
Alzheimer’s disease (AD) is the most common cause of dementia in the elderly, accounting for 60-80% of cases (Alzheimer’s Association, 2016). AD is characterized by a progressive loss of neuron function and integrity, which eventually results in memory
92 loss, cognitive impairment, and other behavioral changes. Pathologically, the hallmarks of AD include extracellular amyloid plaques and intracellular neurofibrillary tangles, which are accompanied by the activation of microglia and astrocytes into a proinflammatory state. Nuclear receptors, especially RXR, LXR, and PPARγ, have previously been shown to play important roles in the pathogenesis of Alzheimer’s disease by regulating the clearance of Aβ and the inflammatory state of the brain, and it has been suggested that some neuroprotective functions of these receptors may also be beneficial in AD. Recently it has been found that members of a related family of nuclear receptors, the NR4A receptors, are dysregulated in AD mouse models and in human patients (Josh D. Hawk & Abel, 2011; Montarolo et al., 2016; Moon et al., 2015).
The NR4A family of nuclear receptors are highly expressed in neurons, where they were first identified as immediate-early genes that respond to a wide variety of stimuli including stress, seizures, and neurotransmitter signaling (Skerrett et al., 2014).
The NR4A family includes Nur77 (NR4A1, NGFI-B, TR3), Nur-related factor 1 (NR4A2,
Nurr1) and Neuron-Derived Orphan Receptor 1 (NR4A3, Nor-1, MINOR), which can signal as monomers, homodimers, or heterodimers at NGFIB response elements (NBREs) or Nur response elements (NurREs) (Close et al., 2013). Additionally, Nurr1 and Nur77 are known to heterodimerize with the retinoid x receptor (RXR) and bind to DR5 repeats, and these heterodimer complexes are responsive to RXR ligands (de Urquiza et al., 2000; Ishizawa et al., 2012; Morita et al., 2005; T. Perlmann & Jansson, 1995;
Sacchetti, Dwornik, Formstecher, Rachez, & Lefebvre, 2002; Volakakis et al., 2015, 2009;
Wallen-Mackenzie, 2003). The regulation of NR4A receptor signaling is complex and
93 largely depends on gene expression levels, posttranslational modifications, and interactions with other nuclear receptor partners such as RXR (Johnson, Michelhaugh,
Bouhamdan, Schmidt, & Bannon, 2011; Sacchetti et al., 2002). This indicates that NR4A activity could be exquisitely sensitive to perturbations in receptor expression, such as those recently observed in the AD context.
NR4A receptors have been shown to play important roles in learning and memory (Joshua D. Hawk et al., 2012; McNulty et al., 2012) as well as neuroprotective roles in response to stress or disease pathology (Decressac et al., 2012; Volakakis et al.,
2010). NR4A receptors are also expressed in microglia and astrocytes in response to inflammatory stimuli (Lallier, Graf, Waidyarante, & Rogers, 2016; Pei, Castrillo, Chen,
Hoffmann, & Tontonoz, 2005; Saijo et al., 2009) and act as repressors of pro- inflammatory signaling (Saijo et al., 2009). Nurr1 is of particular interest for neuroprotection, as it has been shown to participate in cAMP-response element binding protein (CREB)-mediated neuroprotection (Volakakis et al., 2010) and is a regulator of brain derived neurotrophic factor (BDNF) (Barneda-Zahonero et al., 2012; Koo et al.,
2015; Volpicelli et al., 2007). It has been established that Nurr1 mediates neuroprotection in a variety of cell types, including dopaminergic cells (Decressac et al.,
2012), cerebellar granule cells (Barneda-Zahonero et al., 2012), and hippocampal neurons (Volakakis et al., 2010). Recently it has been found that Nurr1 expression is decreased in in late stage AD patients and in mouse models of AD (Espana et al., 2010;
Moon et al., 2015; Parra-Damas et al., 2014).
94 In this study, we evaluated the neuroprotective function of Nurr1 in the 5XFAD model of Alzheimer’s disease, an aggressive model of amyloidosis expressing three familial AD human mutations in amyloid precursor protein (APP) and two in presenilin.
These mice accumulate intraneuronal Aβ by 1 month of age, especially in the subicular region of the hippocampus, and by four months of age about 50% of subicular neurons degenerate (Eimer & Vassar, 2013; Moon et al., 2012; Oakley et al., 2006); our unpublished observations). We found that knockdown of Nurr1 in the subiculum decreased neuron survival during this period, while overexpression of Nurr1 increased neuron survival. Interestingly, we also demonstrate that Nurr1 overexpression is able to modulate subicular Aβ levels without affecting overall microglial activation, implying a relationship between Nurr1-mediated neuroprotection and modulation of the larger AD pathological state.
Experimental Procedures
Viral constructs
An AAV expressing Nurr1 under the control of the CBA promoter was generated by inserting 6-His tagged full-length mouse Nurr1 into the multi-cloning site of a pAAV vector kindly provided by Todd Golde (University of Florida, Gainesville, FL). Packaging, production and titering of AAV (serotype 1) was performed by the Golde lab. An AAV1-
GFP construct was generously provided by the Deneris lab (Case Western Reserve
University, Cleveland, OH). pGIPZ lentivirus containing shRNA targeted against Nurr1
95 (Gene target sequence: CTGTCACTCTTCTCCTTTA, source clone ID V2LMM_44007) or scrambled shRNA was purchased from Dharmacon.
Animals and Treatment
5XFAD mice (The Jackson Laboratory) co-express a human amyloid precursor protein containing the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations and a human presenilin 1 containing the M146L and L286V mutations from the mouse
Thy1 promoter. Male and female 5XFAD mice were treated at 2 months of age with either shNurr1 lentivirus and scramble lentivirus (females n=4, males n=5), or AAV-
Nurr1 and AAV-GFP (females n=3, males n=3). A total volume of 1uL shNurr1 (108 particles/μL) or AAV-Nurr1 (109 particles/μL) virus was injected unilaterally into the subiculum (AP -3.5 from Bregma, ML 2.5 from midline, DV 1.35 from dura) at a rate of
1μL/10 minutes, and the respective control was injected into the contralateral subiculum in the same manner. All experiments involving animals followed protocols approved by the Case Western Reserve University School of Medicine’s Institutional
Animal Care and Use Committee.
Cell culture
Neuro2A mouse neuroblastoma cells were obtained from ATCC (#CCL-131) and maintained in DMEM (Gibco #11965092) with 50% OptiMEM (Gibco #31985088), 10%
FBS and 1% penicillin/streptomycin (N2a growth medium). Five days before treatment, differentiation was initiated by switching medium to N2a medium containing 2% FBS
96 (N2a differentiation medium). Differentiation was monitored by evaluating morphological changes, including development of neuronal-like processes. Cells were transfected using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol.
RNA extraction, reverse transcription, and quantitative PCR
Cells were lysed and RNA was isolated using the RNeasy Mini Kit (Qiagen). RNA samples
(0.5μg) were reverse transcribed using the QuantiTect Reverse Transcription kit
(Quiagen). cDNA was run for 40 cycles on the StepOne Plus Real Time PCR system
(Applied Biosystems) using the TaqMan Gene Expression Master Mix (Thermo Fisher) and TaqMan Gene Expression Assays (Thermo Fisher) for Nurr1 (Mm00443060_m1) and
Nur77 (Mm01300401_m1).
Immunohistochemistry and Image Analysis
Mice were perfused with 4% PFA, and free-floating saggital brain sections (30um) were prepared using a cryostat. Sections were collected in PBS and stored at 4C. Antigen retrieval was performed with sodium citrate buffer (10mM sodium citrate, 0.5% Tween
20) at pH 6, and sections were blocked in 5% normal goat serum in PBS (pH 7.4) with
0.3% Triton X-100 for 1 hour. For each set of staining, 10-12 sections were stained and analyzed, 330um apart, evenly spaced through the brain hemisphere. The following primary antibodies were added overnight at 4C: GFP, 1:1000 (Abcam); Ubiqutin, 1:1000
(Cell Signaling); NeuN, 1:1000 (Millipore); 6e10, 1:1000 (BioLegend); Iba1, 1:1000
97 (Wako). Image J was used for all image analysis. For neuronal counts in the subiculum, each subiculum was outlined, the total subiculum area in that image recorded, and
NeuN positive neurons were manually counted within the selected area using the Cell
Counter plugin and normalized to the subicular area. For analysis of ubiquitin positive area and Iba positive area, staining was only measured within the subiculum and normalized to the subicular area. For analysis of 6e10 positive area, total 6e10 area was first measured in the subiculum, then plaques were isolated as a region of interest and measured separately. Remaining non-plaque positive 6e10 staining was determined to be intracellular Aβ.
Results
Nurr1 is neuroprotective in subiculum neurons in the 5XFAD model
It has been established that Nurr1 plays an important role in the differentiation and maintenance of dopaminergic neurons, and studies have demonstrated that Nurr1 plays a neuroprotective role in PD models by acting as a transcription factor in either neurons
(Decressac 2012) or glia (Saijo 2009). The discovery that Nurr1 is also expressed in subiculum neurons, and that Nurr1-expressing neurons decrease over time in the 5XFAD mouse model (Moon et al. 2014) raised the question of whether Nurr1 is neuroprotective in the context of Alzheimer’s disease, and specifically if its loss contributes to the cell death readily observable in the subiculum of 5XFAD animals.
Since Nurr1-deficient mice exhibit perinatal lethality, we performed stereotaxic
98 injections of a lentivirus encoding shRNA directed against Nurr1 or a control lentivirus containing scrambled shRNA into the dorsal subiculum of 2-month-old 5XFAD mice. In
N2a cells, the shNurr1 efficiently reduced Nurr1 mRNA levels selectively (Fig 3.1A), without affecting mRNA levels for the closely related receptor Nur77 (Fig 3.1B).
Loss of subicular neurons in the 5XFAD mouse occurs between 2 and 4 months of age
(Moon et al., 2012). Stereological analysis revealed that knockdown of Nurr1 induced about a 10% increase in the loss of NeuN+ neurons in 5XFAD animals by 4 months of age
(Fig 3.1C, E+G). Additionally, neurons surrounding plaques in animal models of AD have been observed to develop dystrophic neurites which contain conglomerated ubiquitin.
By 4 months of age, 5XFAD animals exhibit ubiquitin expression concentrated around plaques. When shNurr1 was injected into the subiculum, no significant increase in ubiquitin-positive dystrophic neurites was detected (Fig 3.1D, F+H).
To determine if increasing Nurr1 expression in the subiculum is able to rescue the neurodegenerative phenotype of the 5XFAD mouse, we performed similar stereotaxic injections of an AAV1 virus encoding a genetic copy of Nurr1, or a control AAV-GFP, into the dorsal subiculum of 5XFAD mice. Overexpression of Nurr1 in subiculum neurons increased the number of NeuN+ neurons surviving at 4 months (Fig 3.2 A, C+E), and decreased dystrophic neurites by about 50% as evaluated by ubiquitin staining (Fig 3.2
B, D+F).
99 Overexpression of Nurr1 in the subiculum decreases Aβ pathology
Neurons are the main source of Aβ in the brain, which in the 5XFAD model can be observed frequently both as extracellular plaques and as intracellular accumulations in neurons. It has been proposed that accumulation of intracellular Aβ is one of the main contributing factors to the neuronal death observed in the 5XFAD model (Moon et al.,
2012, 2015). To determine the relationship between Nurr1 expression levels in the subiculum and the Aβ burden, we analyzed the total area of 6e10+ Aβ staining in the subiculum, as well as the area occupied by plaques or intraneuronal Aβ. We found no overall effect on Aβ levels in subicula injected with the shNurr1 expressing construct (Fig
3.3 A-D). However, when mice were injected with the Nurr1-OE virus, significant decreases were observed in overall 6e10+ area, as well as Aβ plaques and intracellular
Aβ (Fig 3.3 E-H).
Nurr1 overexpression does not modify microglial activity in the subiculum
Nurr1 has been observed to promote an anti-inflammatory phenotype in microglia and astrocytes (C.-H. Kim et al., 2015; Saijo et al., 2009), and in peripheral inflammatory cells as well (Glass & Saijo, 2010; Montarolo, Perga, Martire, & Bertolotto, 2015). Neurons have also been observed to play an important role in regulating their surrounding inflammatory environment (Eyo & Wu, 2013). Because the progression of amyloid pathology in the 5XFAD is associated with rampant inflammation, we examined the effect of shNurr1 or Nurr1-OE on Iba1 expression by microglia, which is upregulated during inflammation. We observed that neither knockdown nor overexpression of Nurr1
100 induced a change in Iba1+ area in the subiculum or overt microglial morphology (Fig
3.4).
Discussion
Nurr1 has previously been demonstrated to play a neuroprotective role in the context of Parkinson’s disease, where it is a critical differentiation and survival factor for the vulnerable population of dopaminergic neurons in the substantia nigra. It has recently been suggested by Moon et al. (Moon et al., 2015) that Nurr1 is linked to the survival of subiculum neurons, a vulnerable population that undergoes significant death early in the pathological development of the 5XFAD model of Alzheimer’s. Here we demonstrate that Nurr1 plays a neuron-intrinsic role in cell survival and neurite integrity in the 5XFAD mouse, although the role of Nurr1 in modulating amyloid pathology and inflammation in this model is unclear
To investigate the role of Nurr1 in neuron survival, we employed viral methods that would primarily target neurons to overexpress or knock down Nurr1 transcript. We found that knocking down Nurr1 decreased subiculum neuron survival over the period between 2-4 months, while overexpressing Nurr1 increased the number of surviving neurons. We hypothesize that Nurr1 acts in a neuroprotective manner in the 5XFAD subiculum, as has been shown in a variety of other contexts. Although it has been recently suggested that activation of Nurr1 enhances adult hippocampal neurogenesis
(J.-I. Kim et al., 2016), adult neurons are born in the dentate gyrus (DG) and not known to migrate to the subiculum, making neuroprotection a more likely mechanism in this
101 case than neurogenesis. Additionally, our observation that dystrophic neurites, which become loaded with ubiquitin and dysfunctional, are decreased after Nurr1-OE argues that already established neurons benefit from Nurr1 expression.
Interestingly, Nurr1-OE produced a reduction in Aβ deposition in the subiculum, while the converse increase in Aβ was not observed with expression of shNurr1. This could reflect that since Nurr1 in 4 month old 5XFAD animals is already decreased compared to controls (Moon et al., 2015), further decreasing Nurr1 has no effect on Aβ while restoring Nurr1 expression is able to alter Aβ deposition. The potential mechanism behind this effect is intriguing, and several distinct possibilities arise. First, Nurr1 could influence APP processing directly in neurons, causing them to produce less Aβ, although there is currently no evidence that such an interaction exists. Second, Nurr1 could facilitate a neuron-intrinsic mechanism for the uptake and degradation of Aβ, allowing neurons to participate in Aβ clearance and decreasing the presence of intracellular and extracellular amyloid deposits. Previous work has suggested that the low-density lipoprotein receptor (LDLR) and its relative LDLR-related protein 1 (LRP1) are expressed on neurons and mediate Aβ uptake and its subsequent degradation (Kanekiyo et al.,
2011, 2013). A third potential mechanism involves Nurr1-mediated regulation of inflammation, either by regulating neuron-microglia communication or through off- target effects of our viral constructs directly in microglia or astrocytes. While intriguing because of Nurr1’s established anti-inflammatory activity, this last possibility seems unlikely, since our viral constructs showed minimal non-neuronal transfection and
102 neither shNurr1 nor Nurr1-OE produced overt effects on microglial morphology in the subiculum.
For a more thorough analysis of the neuron-intrinsic effects of Nurr1 expression in this model, it will be essential to evaluate Nurr1 protein levels in the subiculum after viral manipulation. The lack of a readily available specific and robust Nurr1-detecting antibody is unfortunate, as it precludes examination of Nurr1 expression at the cellular level by IHC. The development of such an antibody has been recently reported (Moon et al., 2015), and will be of utmost value for future studies if it proves reliable. Alternative approaches that could aid in our understanding of Nurr1 function in subicular neurons include in-situ hybridization, which has previously been used to examine Nurr1 expression with success (Watakabe et al., 2007), and potentially Western blotting or qPCR, although the small size of the subiculum and difficulty of its dissection precluded such analysis from this study.
In concert, these experiments indicate that Nurr1 exhibits neuroprotective activity in subiculum neurons in the context of the 5XFAD mouse model of AD, and can also modify Aβ pathology in this model through an unknown mechanism. These actions establish the therapeutic potential for Nurr1, which is of special interest since it can be activated by a known set of drugs that include RXR agonists as well as Nurr1-targeted synthetic ligands. Future studies examining the cellular expression of Nurr1 and modulating its expression in glial cells will advance our understanding of its activity in
103 microglia and astrocytes in AD models, and further our understanding of the relationship between its neuron-intrinsic and anti-inflammatory activities.
104 Figure 3.1: shRNA specific to Nurr1 decreases neuron survival in the 5XFAD mouse.
Lentivirus containing an shRNA directed against Nurr1 was used to transfect N2a mouse neuroblastoma cells. qPCR analysis confirmed that the shNurr1 knocked down (A) Nurr1 transcript levels ~70%, but the closely related family member (B) Nur77 was not affected. 2 month old 5XFAD mice were injected with shNurr1-expressing lentivirus in one dorsal subiculum, and scramble-expressing lentivirus in the contralateral subiculum.
At 4 months of age, both hemispheres were stained with (E,G) NeuN and the surviving neurons counted using unbiased stereology (C), or slices were stained with (F,H)
Ubiquitin and the total area of ubiquitin+ dystrophic neurites was determined (D). *p <
0.05, student’s t test.
105
106 Figure 3.2: Nurr1 overexpression in the 5XFAD model preserves subicular neurons and decreases dystrophic neurites.
AAV containing a full-length Nurr1 construct was injected into 2 month old 5XFAD mice in one dorsal subiculum, and AAV-GFP in the contralateral subiculum. At 4 months of age, both hemispheres were stained with (C,E) NeuN and the surviving neurons counted using unbiased stereology (A), or slices were stained with (D,F) Ubiquitin and the total area of ubiquitin+ dystrophic neurites was determined (B). *p < 0.05, student’s t test.
107 Figure 3.3: Nurr1 overexpression decreases intraneuronal and deposited forms of amyloid.
2 month old 5XFAD mice were injected with shNurr1-expressing lentivirus and respective control (A-D) or AAV1-Nurr1 and its respective control (E-H). At 4 months of age, both hemispheres were stained with (D, H) 6e10 and the area of 6e10+ plaques determined (A,E), area of intracellular Aβ determined (B, F) or the total 6e10+ area containing plaques and intracellular Aβ quantified (C,G). All of these measurements were normalized to the area of the subiculum in each image. *p < 0.05, student’s t test.
108
109 Figure 3.4: Microglial activation is unaffected by Nurr1 modulation in neurons.
2 month old 5XFAD mice were injected with shNurr1-expressing lentivirus and respective control (A,B) or AAV1-Nurr1 and its respective control (C,D). At 4 months of age, both hemispheres were stained with (B,D) Iba1 and the total Iba1+ area measured and normalized to the area of the subiculum in each image.
110
Chapter 4: Discussion
111 This thesis draws on previous work establishing that treatment of AD mouse models with the RXR agonist bexarotene induces a robust amelioration of pathology that not only includes apoE-mediated clearance of Aβ but also the rapid correction of aberrant network activity and behavioral deficits (Cramer et al., 2012). Agonists of LXR and PPARγ have been broadly used in AD models to increase the expression and lipidation of apoE, and these studies have resulted in a wide variety of outcomes regarding the ability of nuclear receptor agonists to stimulate Aβ clearance. A clear example of the variability of plaque reduction is provided by the 12 studies examining the effect of LXR agonists as described in the introduction and Table 1. Plaque loss varied from 0–65% in these studies. These studies differed in the animal models used, the LXR agonist, and the formulation used to treat the mice, as well as the length of the treatment period. The most reproducible effect was improvement in performance on behavioral tasks, which was observed in all but one study (Skerrett et al., 2014). It has been postulated that bexarotene acts by stimulating both LXR:RXR and PPARγ:RXR transcriptional activity, and that the activation of both these pathways could act synergistically via a linked metabolic loop to result in more efficient apoE production and lipidation than activation of either pathway alone. However, it was not determined if bexarotene preferentially activated one or both of these pathways in AD models. We hypothesized that a combination therapy with LXR and PPARγ agonists would reproduce or exceed the dramatic effects of bexarotene on plaque clearance, and possibly reduce some of the harmful triglyceride effects that have kept LXR agonists from becoming a viable option for clinical trials.
112 Additionally, it has been unclear if the behavioral improvements mediated by nuclear receptor agonists in AD models are mediated through an apoE-dependent pathway or an unrelated neuronal effect. LXR, PPARs and RXR have established roles in neuronal function (Courtney & Landreth, 2016; Skerrett et al., 2014), although it is entirely possible that increasing trafficking of cholesterol to neuronal membranes via lipid-rich apoE could also allow improved neuronal plasticity and ameliorate behavioral impairments (Mauch et al., 2001). In investigating the effect of bexarotene on cultured neurons, we found that neurons cultured from mice with the 5XFAD transgenes represent a population especially susceptible to excitotoxic challenge. Treatment of these neurons with 10nm bexarotene for 24 hours prior to the introduction of 250um glutamate significantly improved neuronal survival (Fig 4.1). This effect of bexarotene is unlikely to be due to an RXR-mediated induction of apoE, since neurons do not express apoE under normal conditions (Xu et al., 2006), although such a possibility cannot be ruled out in the artificiality of an in vitro preparation. We hypothesized that in neurons, bexarotene could have a non-apoE related neuroprotective effect, which may be mediated by the RXR-heterodimerization capable Nurr1 nuclear receptor.
The primary goal of this thesis was to determine if drugs targeting multiple nuclear receptors can be valuable to treat various aspects of AD pathology.
Interestingly, LXR, PPARγ, and Nurr1 can all be activated by the RXR agonist bexarotene.
We determined that a combination therapy targeting LXR and PPARγ is able to increase apoE lipidation, decrease inflammation, and improve behavioral deficits. We also observed, similar to previous studies, an inconsistent tendency for these treatments to
113 facilitate clearance of soluble and fibrillar Aβ. Interestingly, combining the LXR agonist with the PPARγ agonist had the beneficial effect of ameliorating the hypertriglyceridemia associated with LXR agonist treatment. We also investigated the role of RXR-heterodimer partner Nurr1 in facilitating neuronal survival in a mouse model of AD that exhibits cell death. We observed that stimulation of Nurr1 activity might be a valuable option for neuron-directed therapy. These data provide insight into the mechanisms by which bexarotene might act, and suggest several novel therapeutic approaches to the treatment of AD.
Nuclear receptor agonists act in combination on apoE and microglia
Although the clearance of soluble and insoluble Aβ we observed in mice treated with combination therapy was not as dramatic as that observed with bexarotene
(Cramer et al., 2012), and, in fact, in most cases not different from the clearance induced by either LXR (GW3965 or GW) or PPARγ (Pioglitazone or PIO) agonists alone, we did observe several notable effects of combination therapy. First, GWPIO treatment resulted in the greatest amount of apoE lipidation, and this was true both in cultures of primary astrocytes and in vivo. It is possible that activation of PPARγ by PIO stimulated increased LXR transcription (Chawla et al., 2001), allowing a greater activation of the LXR transcriptional response by GW. It would be informative to examine levels of LXR by western blot, and also the levels of some non-apoE related LXR (Mertk) and PPARγ
(Cd36) target genes. Second, GWPIO treatment provided the largest enhancement of
114 microglial Aβ assay in vitro, and was the most effective in decreasing microgliosis in vivo as measured by immunostaining for total area covered by Iba1 in the cortex, and qPCR on cortical homogenate for genes upregulated in proinflammatory microglia, such as
Iba1, CD45, and IL6.
The next step should be to examine the cellular mechanisms by which these nuclear receptor agonists act on microglia. Because we also observed that nuclear receptor agonist treatment increases association of microglia with Aβ plaques, it would be informative to assay microglia treated with GW, PIO, or both, for their chemotactic abilities. Interestingly, such an assay using a microfluidic chamber with an Aβ gradient to record the chemotaxis of microglia in response to amyloid species has recently been developed (Cho et al., 2013). This assay could be combined with the treatment of microglia directly with nuclear receptor agonists, and indirectly with the conditioned media of astrocytes that have been drug-treated. Such a study would inform our understanding of the relative impact of microglia-intrinsic actions of nuclear receptors, such as transrepression of genes at NFκB promoters, versus astrocyte-mediated actions like the release of lipidated apoE, which can then be sensed by LDLR-family receptors on microglia. An alternative approach would be to use an ex vivo slice preparation from an
AD mouse model brain to track the movements of specific microglia and their relationship with amyloid plaques, a system which has the advantage of being more biologically relevant but is not as easily manipulated.
115 The amelioration of hypertriglyceridemia by combining pioglitazone with GW3965 introduces new therapeutic options for AD
LXRs are expressed not only in the brain, but regulate cholesterol metabolism in a host of peripheral tissues, particularly in the liver. The unfavorable side effect profile of LXR agonists in the periphery, which includes liver steatosis and hypertriglyceridemia induced by upregulation of the LXR target gene SREBP-1c in the liver (Baranowski, 2008), has been prohibitive to the use of LXR agonists in clinical trials for AD. PPARs have been previously observed to inhibit LXR agonist-induced upregulation of SREBP-1c in the liver.
Although there is only a very small amount of literature indicating that PPARγ would participate in such a mechanism, our results in this study are promising. The effects of
GWPIO treatment should be carefully evaluated in the periphery, with special attention paid to the fat content of the liver to track possible development of hepatic steatosis.
Based on the results we obtained, additional studies that could be of benefit to inform future clinical trial paradigms include the use of an LXR agonist in combination with poly-unsaturated fatty acids (Pawar, Botolin, Mangelsdorf, & Jump, 2003) such as docosahexaenoic acid (DHA), which is found in fish oil. Additionally, it might be beneficial to adjust the dosage of GW and PIO in relation to one another. We used a high dose of each of these drugs in relation to other LXR and PPAR agonist studies that have been performed in AD mouse models (Table 1). The benefits of combination therapy might be achieved with lower doses of one or both drugs, which would help reduce off target effects, although the number of mice needed for such an experiment is fairly cost prohibitive.
116
Nurr1 represents a novel target for neuroprotection in AD
Nurr1 has mostly been studied for its role in the differentiation and maintenance of dopaminergic cells, especially in the substantia nigra, but it has become apparent that this nuclear receptor plays a critical part in the function and survival, especially in response to stress, of other types of neurons as well (Josh D. Hawk & Abel, 2011;
Skerrett et al., 2014). Our data from in vitro studies (Fig 4) indicate that the RXR agonist bexarotene is able to prevent excitotoxicity in cultured neurons, and this could occur through bexarotene’s ability to stimulate Nurr1:RXR heterodimer activity (McFarland et al., 2013; Volakakis et al., 2015) to drive transcription of neuroprotective genes such as
BDNF (Barneda-Zahonero et al., 2012). Additionally, unpublished work from our lab by
Dr. Monica Mariani indicates that bexarotene treatment of 5XFAD mice from 2.5 to 3.5 months of age is able to prevent the loss of about 15% of subicular neurons. This effect is equal to or greater than the loss (~10%) or preservation (also ~10%) we observe by respectively knocking down or increasing Nurr1 levels in subicular neurons during this same time period.
Studies combining viral knockdown and overexpression of Nurr1 in the subiculum combined with bexarotene treatment will be essential to examine our hypothesis. Additionally, many other questions have been raised about the function of
Nurr1 in AD that remain unanswered. Because Nurr1 has an established role in mediating anti-inflammatory responses in microglia and astrocytes, it will be important
117 to determine the contribution of these cell types to the role of Nurr1 in AD. Visualizing cell-specific Nurr1 expression in AD mouse models during pathogenesis is an excellent first step, which Moon et al. (2014) have already engaged. Ideally, mouse models will be generated which can target Nurr1 function in astrocytes, microglia, or neurons, but this approach meets with some difficulty because no NR4A2-floxed mice are commercially available. Ted Abel’s laboratory has developed an elegant solution to this problem that utilizes a well-characterized NR4A-dominant negative (NR4Adn) protein with conditional expression to selectively impair NR4A signaling in specific cell types. A truncated version of Nur77 with DNA binding and dimerization domains, but no transactivation domain, has been previously found to bind to all three NR4A family members to create nonfunctional dimers [51, 52]. This conditional inhibition of NR4A function has an advantage, since NR4A receptors have been shown to have redundant functions and multiple NR4As can be activated by the same stimulus [45].
Of course, the use of the NR4Adn model brings up another caveat – Nur77 and
Nor-1 are also expressed in the CNS and have been ascribed similar roles to Nurr1. Nor-
1 is less studied, and does not heterodimerize with RXR, but there is a wide body of literature, largely from the cancer field, ascribing important roles in inflammatory regulation to Nur77 (Pei, Castrillo, & Tontonoz, 2006).
Nur77 is also necessary for proper function of dopaminergic neurons (Thomas
Perlmann & Wallén-Mackenzie, 2004), and is induced in the hippocampi of mice during memory tasks (Malkani & Rosen, 2000; von Hertzen & Giese, 2005). Deletion of all
118 NR4A receptors significantly increases the death of hippocampal neurons in response to kainic acid (Volakakis et al., 2010), and a pan-NR4A dominant negative receptor decreases the ability of mice to consolidate hippocampal-dependent memories (Joshua
D. Hawk et al., 2012). This indicates that NR4A receptors have broadly neuroprotective roles not restricted to dopaminergic neurons, and play a prominent role in hippocampal function as well. It should be noted, additionally, that a role for Nur77 unrelated to its transcriptional activity has been defined where, on translocation to the cytoplasm, it interacts with mitochondria to promote apoptosis (Boldingh Debernard, Mathisen, &
Paulsen, 2012; Cao et al., 2004; Han et al., 2006; Mohan et al., 2012).
In summary, we have described a novel way of treating AD pathology using a combination of nuclear receptor agonists, and how further exploration of this study can contribute to our scientific understanding of nuclear receptor function and role in AD.
By describing Nurr1 receptor activity in a novel disease setting, we have additionally contributed to the scientific understanding of AD pathogenesis and NR4A receptor function in the brain. This thesis demonstrates that drugs targeting multiple nuclear receptors can be valuable to treat various aspects of AD pathology. Interestingly, LXR,
PPARγ, and Nurr1 can all be activated by the RXR agonist bexarotene. These data provide insight into the mechanisms by which bexarotene might act, and suggest several novel therapeutic approaches to the treatment of AD.
119 Figure 4.1. Cultured neurons from 5XFAD animals exhibit glutamate excitotoxicity in vitro, and this is ameliorated with bexarotene treatment.
120 References
About Alzheimer’s Disease: Treatment. (n.d.). Retrieved November 7, 2016, from
https://www.nia.nih.gov/alzheimers/topics/treatment
AD&FTD Mutation Database. (n.d.). Retrieved October 24, 2016, from
http://www.molgen.ua.ac.be/ADMutations/default.cfm?MT=1&ML=6&Page=StatP
erGene
A-Gonzalez, N., Bensinger, S. J., Hong, C., Beceiro, S., Bradley, M. N., Zelcer, N., …
Castrillo, A. (2009). Apoptotic cells promote their own clearance and immune
tolerance through activation of the nuclear receptor LXR. Immunity, 31(2), 245–
258.
Aguzzi, A., Barres, B. A., & Bennett, M. L. (2013). Microglia: scapegoat, saboteur, or
something else? Science, 339(6116), 156–161.
Aleshkov, S., Abraham, C. R., & Zannis, V. I. (1997). Interaction of nascent ApoE2, ApoE3,
and ApoE4 isoforms expressed in mammalian cells with amyloid peptide beta (1-
40). Relevance to Alzheimer’s disease. Biochemistry, 36(34), 10571–10580.
Altarejos, J. Y., & Montminy, M. (2011). CREB and the CRTC co-activators: sensors for
hormonal and metabolic signals. Nature Reviews. Molecular Cell Biology, 12(3),
141–151.
Alzheimer’s Association. (2016). 2016 Alzheimer’s disease facts and figures. Alzheimer’s
& Dementia: The Journal of the Alzheimer's Association, 12(4), 459–509.
Andersson, S., Gustafsson, N., Warner, M., & Gustafsson, J.-A. (2005). Inactivation of
liver X receptor beta leads to adult-onset motor neuron degeneration in male mice.
121 Proceedings of the National Academy of Sciences of the United States of America,
102(10), 3857–3862.
Ashford, J. W. (2004). APOE genotype effects on Alzheimer’s disease onset and
epidemiology. Journal of Molecular Neuroscience: MN, 23(3), 157–165.
Bales, K. R., Liu, F., Wu, S., Lin, S., Koger, D., DeLong, C., … Paul, S. M. (2009). Human
APOE Isoform-Dependent Effects on Brain β-Amyloid Levels in PDAPP Transgenic
Mice. The Journal of Neuroscience: The Official Journal of the Society for
Neuroscience, 29(21), 6771–6779.
Bales, K. R., Verina, T., Cummins, D. J., Du, Y., Dodel, R. C., Saura, J., … Paul, S. M. (1999).
Apolipoprotein E is essential for amyloid deposition in the APPV717F transgenic
mouse model of Alzheimer’s disease. Proceedings of the National Academy of
Sciences, 96(26), 15233–15238.
Bamberger, M. E., Harris, M. E., McDonald, D. R., Husemann, J., & Landreth, G. E. (2003).
A cell surface receptor complex for fibrillar beta-amyloid mediates microglial
activation. The Journal of Neuroscience: The Official Journal of the Society for
Neuroscience, 23(7), 2665–2674.
Baranowski, M. (2008). Biological role of liver X receptors. Journal of Physiology and
Pharmacology: An Official Journal of the Polish Physiological Society, 59 Suppl 7,
31–55.
Barger, S. W., & Harmon, A. D. (1997). Microglial activation by Alzheimer amyloid
precursor protein and modulation by apolipoprotein E. Nature, 388(6645), 878–
881.
122 Barneda-Zahonero, B., Servitja, J.-M., Badiola, N., Miñano-Molina, A. J., Fadó, R., Saura,
C. A., & Rodríguez-Alvarez, J. (2012). Nurr1 protein is required for N-methyl-D-
aspartic acid (NMDA) receptor-mediated neuronal survival. The Journal of
Biological Chemistry, 287(14), 11351–11362.
Basak, J. M., Verghese, P. B., Yoon, H., Kim, J., & Holtzman, D. M. (2012). Low-density
lipoprotein receptor represents an apolipoprotein E-independent pathway of Aβ
uptake and degradation by astrocytes. The Journal of Biological Chemistry, 287(17),
13959–13971.
Bateman, R. J., Munsell, L. Y., Morris, J. C., Swarm, R., Yarasheski, K. E., & Holtzman, D.
M. (2006). Human amyloid-β synthesis and clearance rates as measured in
cerebrospinal fluid in vivo. Nature Medicine, 12(7), 856–861.
Bateman, R. J., Xiong, C., Benzinger, T. L. S., Fagan, A. M., Goate, A., Fox, N. C., …
Dominantly Inherited Alzheimer Network. (2012). Clinical and biomarker changes in
dominantly inherited Alzheimer’s disease. The New England Journal of Medicine,
367(9), 795–804.
Beach, T. G., Monsell, S. E., Phillips, L. E., & Kukull, W. (2010). Accuracy of the Clinical
Diagnosis of Alzheimer Disease. Centers, 2, 005.
Bennett, D. A., Schneider, J. A., Wilson, R. S., Bienias, J. L., & Arnold, S. E. (2004).
Neurofibrillary tangles mediate the association of amyloid load with clinical
Alzheimer disease and level of cognitive function. Archives of Neurology, 61(3),
378–384.
Bensinger, S. J., & Tontonoz, P. (2008). Integration of metabolism and inflammation by
123 lipid-activated nuclear receptors. Nature, 454(7203), 470–477.
Bentahir, M., Nyabi, O., Verhamme, J., Tolia, A., Horré, K., Wiltfang, J., … De Strooper, B.
(2006). Presenilin clinical mutations can affect gamma-secretase activity by
different mechanisms. Journal of Neurochemistry, 96(3), 732–742.
Bernardo, A., & Minghetti, L. (2006). PPAR-gamma agonists as regulators of microglial
activation and brain inflammation. Current Pharmaceutical Design, 12(1), 93–109.
Bero, A. W., Yan, P., Roh, J. H., Cirrito, J. R., Stewart, F. R., Raichle, M. E., … Holtzman, D.
M. (2011). Neuronal activity regulates the regional vulnerability to amyloid-[beta]
deposition. Nature Neuroscience, 14(6), 750–756.
Beyer, T. P., Schmidt, R. J., Foxworthy, P., Zhang, Y., Dai, J., Bensch, W. R., … Cao, G.
(2004). Coadministration of a liver X receptor agonist and a peroxisome proliferator
activator receptor-alpha agonist in Mice: effects of nuclear receptor interplay on
high-density lipoprotein and triglyceride metabolism in vivo. The Journal of
Pharmacology and Experimental Therapeutics, 309(3), 861–868.
Bibl, M., Gallus, M., Welge, V., Lehmann, S., Sparbier, K., Esselmann, H., & Wiltfang, J.
(2012). Characterization of cerebrospinal fluid aminoterminally truncated and
oxidized amyloid-β peptides. Proteomics. Clinical Applications, 6(3-4), 163–169.
Boldingh Debernard, K. A., Mathisen, G. H., & Paulsen, R. E. (2012). Differences in NGFI-
B, Nurr1, and NOR-1 expression and nucleocytoplasmic translocation in glutamate-
treated neurons. Neurochemistry International, 61(1), 79–88.
Bolmont, T., Clavaguera, F., Meyer-Luehmann, M., Herzig, M. C., Radde, R., Staufenbiel,
M., … Jucker, M. (2007). Induction of tau pathology by intracerebral infusion of
124 amyloid-beta -containing brain extract and by amyloid-beta deposition in APP x Tau
transgenic mice. The American Journal of Pathology, 171(6), 2012–2020.
Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., Ratovitsky, T., …
Sisodia, S. S. (1996). Familial Alzheimer’s Disease–Linked Presenilin 1 Variants
Elevate Aβ1–42/1–40 Ratio In Vitro and In Vivo. Neuron, 17(5), 1005–1013.
Braak, H., & Braak, E. (1991). Neuropathological stageing of Alzheimer-related changes.
Acta Neuropathologica, 82(4), 239–259.
Braidy, N., Muñoz, P., Palacios, A. G., Castellano-Gonzalez, G., Inestrosa, N. C., Chung, R.
S., … Guillemin, G. J. (2012). Recent rodent models for Alzheimer’s disease: clinical
implications and basic research. Journal of Neural Transmission , 119(2), 173–195.
Brettschneider, J., Del Tredici, K., Lee, V. M.-Y., & Trojanowski, J. Q. (2015). Spreading of
pathology in neurodegenerative diseases: a focus on human studies. Nature
Reviews. Neuroscience, 16(2), 109–120.
Bridi, M. S., & Abel, T. (2013). The NR4A orphan nuclear receptors mediate
transcription-dependent hippocampal synaptic plasticity. Neurobiology of Learning
and Memory, 105, 151–158.
Brown, J. (2004). Differential Expression of Cholesterol Hydroxylases in Alzheimer’s
Disease. The Journal of Biological Chemistry, 279(33), 34674–34681.
Bu, G. (2009). Apolipoprotein E and its receptors in Alzheimer’s disease: pathways,
pathogenesis and therapy. Nature Reviews. Neuroscience, 10(5), 333–344.
Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., … Glabe, C.
(1992). Assembly and aggregation properties of synthetic Alzheimer’s A4/beta
125 amyloid peptide analogs. The Journal of Biological Chemistry, 267(1), 546–554.
Burns, M. P., Vardanian, L., Pajoohesh-Ganji, A., Wang, L., Cooper, M., Harris, D. C., …
Rebeck, G. W. (2006). The effects of ABCA1 on cholesterol efflux and A$\beta$
levels in vitro and in vivo. Journal of Neurochemistry, 98(3), 792–800.
Cao, X., Liu, W., Lin, F., Li, H., Kolluri, S. K., Lin, B., … Zhang, X.-K. (2004). Retinoid X
Receptor Regulates Nur77/Thyroid Hormone Receptor 3-Dependent Apoptosis by
Modulating Its Nuclear Export and Mitochondrial Targeting. Molecular and Cellular
Biology, 24(22), 9705–9725.
Castellano, J. M., Kim, J., Stewart, F. R., Jiang, H., DeMattos, R. B., Patterson, B. W., …
Holtzman, D. M. (2011). Human apoE isoforms differentially regulate brain
amyloid-β peptide clearance. Science Translational Medicine, 3(89), 89ra57.
Chawla, A. (2010). Control of macrophage activation and function by PPARs. Circulation
Research, 106(10), 1559–1569.
Chawla, A., Boisvert, W. A., Lee, C.-H., Laffitte, B. A., Barak, Y., Joseph, S. B., … Tontonoz,
P. (2001). A PPARγ-LXR-ABCA1 Pathway in Macrophages Is Involved in Cholesterol
Efflux and Atherogenesis. Molecular Cell, 7(1), 161–171.
Chen, K., Iribarren, P., Hu, J., Chen, J., Gong, W., Cho, E. H., … Wang, J. M. (2006).
Activation of Toll-like receptor 2 on microglia promotes cell uptake of Alzheimer
disease-associated amyloid beta peptide. The Journal of Biological Chemistry,
281(6), 3651–3659.
Chinetti-Gbaguidi, G., & Staels, B. (2011). Macrophage polarization in metabolic
disorders: functions and regulation. Current Opinion in Lipidology, 22(5), 365–372.
126 Cho, H., Hashimoto, T., Wong, E., Hori, Y., Wood, L. B., Zhao, L., … Irimia, D. (2013).
Microfluidic chemotaxis platform for differentiating the roles of soluble and bound
amyloid-β on microglial accumulation. Scientific Reports, 3, 1823.
Cirrito, J. R., Kang, J.-E., Lee, J., Stewart, F. R., Verges, D. K., Silverio, L. M., … Holtzman,
D. M. (2008). Endocytosis is required for synaptic activity-dependent release of
amyloid-beta in vivo. Neuron, 58(1), 42–51.
Cirrito, J. R., Yamada, K. A., Finn, M. B., Sloviter, R. S., Bales, K. R., May, P. C., …
Holtzman, D. M. (2005). Synaptic activity regulates interstitial fluid amyloid-beta
levels in vivo. Neuron, 48(6), 913–922.
Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., … Selkoe, D.
J. (1992). Mutation of the beta-amyloid precursor protein in familial Alzheimer’s
disease increases beta-protein production. Nature, 360(6405), 672–674.
Close, A. F., Rouillard, C., & Buteau, J. (2013). NR4A orphan nuclear receptors in glucose
homeostasis: a minireview. Diabetes & Metabolism, 39(6), 478–484.
Colton, C. A. (2009). Heterogeneity of microglial activation in the innate immune
response in the brain. Journal of Neuroimmune Pharmacology: The Official Journal
of the Society on NeuroImmune Pharmacology, 4(4), 399–418.
Corder, E. H., Saunders, A. M., Strittmatter, W. J., Schmechel, D. E., Gaskell, P. C., Small,
G. W., … Pericak-Vance, M. A. (1993). Gene dose of apolipoprotein E type 4 allele
and the risk of Alzheimer’s disease in late onset families. Science, 261(5123), 921–
923.
Courtney, R., & Landreth, G. E. (2016). LXR Regulation of Brain Cholesterol: From
127 Development to Disease. Trends in Endocrinology and Metabolism: TEM.
https://doi.org/10.1016/j.tem.2016.03.018
Craft, S., Cholerton, B., & Baker, L. D. (2013). Insulin and Alzheimer’s disease: untangling
the web. Journal of Alzheimer’s Disease: JAD, 33 Suppl 1, S263–75.
Cramer, P. E., Cirrito, J. R., Wesson, D. W., Lee, C. Y. D., Karlo, J. C., Zinn, A. E., …
Landreth, G. E. (2012). ApoE-directed therapeutics rapidly clear β-amyloid and
reverse deficits in AD mouse models. Science, 335(6075), 1503–1506.
Cui, W., Sun, Y., Wang, Z., Xu, C., Peng, Y., & Li, R. (2012). Liver X receptor activation
attenuates inflammatory response and protects cholinergic neurons in APP/PS1
transgenic mice. Neuroscience, 210, 200–210.
Daniel, B., Nagy, G., Hah, N., Horvath, A., Czimmerer, Z., Poliska, S., … Nagy, L. (2014).
The active enhancer network operated by liganded RXR supports angiogenic
activity in macrophages. Genes & Development, 28(14), 1562–1577.
Dawkins, E., & Small, D. H. (2014). Insights into the physiological function of the β-
amyloid precursor protein: beyond Alzheimer’s disease. Journal of Neurochemistry,
129(5), 756–769.
Dawson, G. R., Seabrook, G. R., Zheng, H., Smith, D. W., Graham, S., O’Dowd, G., …
Sirinathsinghji, D. J. (1999). Age-related cognitive deficits, impaired long-term
potentiation and reduction in synaptic marker density in mice lacking the beta-
amyloid precursor protein. Neuroscience, 90(1), 1–13.
Dawson, M. I., & Xia, Z. (2012). The retinoid X receptors and their ligands. Biochimica et
Biophysica Acta, 1821(1), 21–56.
128 Deane, R., Sagare, A., Hamm, K., Parisi, M., Lane, S., Finn, M. B., … Zlokovic, B. V. (2008).
apoE isoform-specific disruption of amyloid beta peptide clearance from mouse
brain. The Journal of Clinical Investigation, 118(12), 4002–4013.
Debernard, K. A. B., Mathisen, G. H., & Paulsen, R. E. (2012). Differences in NGFI-B,
Nurr1, and NOR-1 expression and nucleocytoplasmic translocation in glutamate-
treated neurons. Neurochemistry International, 61(1), 79–88.
Decressac, M., Kadkhodaei, B., Mattsson, B., Laguna, A., Perlmann, T., & Björklund, A.
(2012). α-Synuclein-induced down-regulation of Nurr1 disrupts GDNF signaling in
nigral dopamine neurons. Science Translational Medicine, 4(163), 163ra156.
Decressac, M., Volakakis, N., Björklund, A., & Perlmann, T. (2013). NURR1 in Parkinson
disease—from pathogenesis to therapeutic potential. Nature Reviews. Neurology,
9(11), 629–636.
Delacourte, A., & Defossez, A. (1986). Alzheimer’s disease: Tau proteins, the promoting
factors of microtubule assembly, are major components of paired helical filaments.
Journal of the Neurological Sciences, 76(2-3), 173–186.
DeMattos, R. B. (2004). Apolipoprotein E dose-dependent modulation of β-amyloid
deposition in a transgenic mouse model of Alzheimer’s disease. Journal of
Molecular Neuroscience: MN. Retrieved from
http://link.springer.com/article/10.1385/JMN:23:3:255
Denner, L. A., Rodriguez-Rivera, J., Haidacher, S. J., Jahrling, J. B., Carmical, J. R.,
Hernandez, C. M., … Dineley, K. T. (2012). Cognitive enhancement with
rosiglitazone links the hippocampal PPARγ and ERK MAPK signaling pathways. The
129 Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 32(47),
16725–35a. de Ortiz, S. P., & Jamieson, G. A., Jr. (1996). HZF-3, an immediate-early orphan receptor
homologous to NURR1/NOT: induction upon membrane depolarization and
seizures. Brain Research. Molecular Brain Research, 38(1), 1–13.
De Strooper, B., Saftig, P., Craessaerts, K., Vanderstichele, H., Guhde, G., Annaert, W., …
Van Leuven, F. (1998). Deficiency of presenilin-1 inhibits the normal cleavage of
amyloid precursor protein. Nature, 391(6665), 387–390. de Urquiza, A. M., Liu, S., Sjöberg, M., Zetterström, R. H., Griffiths, W., Sjövall, J., &
Perlmann, T. (2000). Docosahexaenoic acid, a ligand for the retinoid X receptor in
mouse brain. Science, 290(5499), 2140–2144.
Dickey, C. A., Loring, J. F., Montgomery, J., Gordon, M. N., Eastman, P. S., & Morgan, D.
(2003). Selectively reduced expression of synaptic plasticity-related genes in
amyloid precursor protein + presenilin-1 transgenic mice. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 23(12), 5219–
5226.
Donkin, J. J., Stukas, S., Hirsch-Reinshagen, V., Namjoshi, D., Wilkinson, A., May, S., …
Wellington, C. L. (2010). ATP-binding cassette transporter A1 mediates the
beneficial effects of the liver X receptor agonist GW3965 on object recognition
memory and amyloid burden in amyloid precursor protein/presenilin 1 mice. The
Journal of Biological Chemistry, 285(44), 34144–34154.
Du Yan, S., Chen, X., Fu, J., Chen, M., Zhu, H., Roher, A., … Schmidt, A. M. (1996). RAGE
130 and amyloid-β peptide neurotoxicity in Alzheimer’s disease. Nature, 382(6593),
685–691.
Duyckaerts, C., Colle, M. A., Dessi, F., Grignon, Y., Piette, F., & Hauw, J. J. (1998). The
progression of the lesions in Alzheimer disease: insights from a prospective
clinicopathological study. Journal of Neural Transmission. Supplementum, 53, 119–
126.
Eimer, W. A., & Vassar, R. (2013). Neuron loss in the 5XFAD mouse model of Alzheimer’s
disease correlates with intraneuronal Aβ42 accumulation and Caspase-3 activation.
Molecular Neurodegeneration, 8, 2.
Eketjall, S., Janson, J., Jeppsson, F., Svanhagen, A., Kolmodin, K., Gustavsson, S., …
Falting, J. (2013). AZ-4217: A High Potency BACE Inhibitor Displaying Acute Central
Efficacy in Different In Vivo Models and Reduced Amyloid Deposition in Tg2576
Mice. Journal of Neuroscience, 33(24), 10075–10084.
El Khoury, J. B., Moore, K. J., Means, T. K., Leung, J., Terada, K., Toft, M., … Luster, A. D.
(2003). CD36 mediates the innate host response to beta-amyloid. The Journal of
Experimental Medicine, 197(12), 1657–1666.
El Khoury, J., Hickman, S. E., Thomas, C. A., Cao, L., Silverstein, S. C., & Loike, J. D. (1996).
Scavenger receptor-mediated adhesion of microglia to beta-amyloid fibrils. Nature,
382(6593), 716–719.
Escribano, L., Simón, A.-M., Gimeno, E., Cuadrado-Tejedor, M., López de Maturana, R.,
García-Osta, A., … Frechilla, D. (2010). Rosiglitazone rescues memory impairment in
Alzheimer’s transgenic mice: mechanisms involving a reduced amyloid and tau
131 pathology. Neuropsychopharmacology: Official Publication of the American College
of Neuropsychopharmacology, 35(7), 1593–1604.
Escribano, L., Simón, A.-M., Pérez-Mediavilla, A., Salazar-Colocho, P., Del Río, J., &
Frechilla, D. (2009). Rosiglitazone reverses memory decline and hippocampal
glucocorticoid receptor down-regulation in an Alzheimer’s disease mouse model.
Biochemical and Biophysical Research Communications, 379(2), 406–410.
Espana, J., Valero, J., Minano-Molina, A. J., Masgrau, R., Martin, E., Guardia-Laguarta, C.,
… Saura, C. A. (2010). -Amyloid Disrupts Activity-Dependent Gene Transcription
Required for Memory through the CREB Coactivator CRTC1. Journal of
Neuroscience, 30(28), 9402–9410.
Evans, R. M., Barish, G. D., & Wang, Y.-X. (2004). PPARs and the complex journey to
obesity. Nature Medicine, 10(4), 355–361.
Evans, R. M., & Mangelsdorf, D. J. (2014). Nuclear Receptors, RXR, and the Big Bang. Cell,
157(1), 255–266.
Eyo, U. B., & Wu, L.-J. (2013). Bidirectional microglia-neuron communication in the
healthy brain. Neural Plasticity, 2013, 456857.
Farrer, L. A., Cupples, L. A., Haines, J. L., Hyman, B., Kukull, W. A., Mayeux, R., … van
Duijn, C. M. (1997). Effects of age, sex, and ethnicity on the association between
apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and
Alzheimer Disease Meta Analysis Consortium. JAMA: The Journal of the American
Medical Association, 278(16), 1349–1356.
Farris, W., Schütz, S. G., Cirrito, J. R., Shankar, G. M., Sun, X., George, A., … Selkoe, D. J.
132 (2007). Loss of neprilysin function promotes amyloid plaque formation and causes
cerebral amyloid angiopathy. The American Journal of Pathology, 171(1), 241–251.
Fassbender, K., Walter, S., Kühl, S., Landmann, R., Ishii, K., Bertsch, T., … Beyreuther, K.
(2004). The LPS receptor (CD14) links innate immunity with Alzheimer’s disease.
FASEB Journal: Official Publication of the Federation of American Societies for
Experimental Biology, 18(1), 203–205.
Ferreira, S. T., Clarke, J. R., Bomfim, T. R., & De Felice, F. G. (2014). Inflammation,
defective insulin signaling, and neuronal dysfunction in Alzheimer’s disease.
Alzheimer’s & Dementia: The Journal of the Alzheimer's Association, 10(1 Suppl),
S76–83.
Fitz, N. F., Castranio, E. L., Carter, A. Y., Kodali, R., Lefterov, I., & Koldamova, R. (2014).
Improvement of memory deficits and amyloid-β clearance in aged APP23 mice
treated with a combination of anti-amyloid-β antibody and LXR agonist. Journal of
Alzheimer’s Disease: JAD, 41(2), 535–549.
Fitz, N. F., Cronican, A. A., Saleem, M., Fauq, A. H., Chapman, R., Lefterov, I., &
Koldamova, R. (2012). Abca1 deficiency affects Alzheimer’s disease-like phenotype
in human ApoE4 but not in ApoE3-targeted replacement mice. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 32(38), 13125–
13136.
Fitz, N. F., Cronican, A., Pham, T., Fogg, A., Fauq, A. H., Chapman, R., … Koldamova, R.
(2010). Liver X receptor agonist treatment ameliorates amyloid pathology and
memory deficits caused by high-fat diet in APP23 mice. The Journal of
133 Neuroscience: The Official Journal of the Society for Neuroscience, 30(20), 6862–
6872.
Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., …
Gillespie, F. (1995). Alzheimer-type neuropathology in transgenic mice
overexpressing V717F beta-amyloid precursor protein. Nature, 373(6514), 523–
527.
García-Yagüe, Á. J., Rada, P., Rojo, A. I., Lastres-Becker, I., & Cuadrado, A. (2013).
Nuclear import and export signals control the subcellular localization of Nurr1
protein in response to oxidative stress. The Journal of Biological Chemistry, 288(8),
5506–5517.
Ghisletti, S., Huang, W., Ogawa, S., Pascual, G., Lin, M.-E., Willson, T. M., … Glass, C. K.
(2007). Parallel SUMOylation-Dependent Pathways Mediate Gene- and Signal-
Specific Transrepression by LXRs and PPARγ. Molecular Cell, 25(1), 57–70.
Giannakopoulos, P., Herrmann, F. R., Bussière, T., Bouras, C., Kövari, E., Perl, D. P., …
Hof, P. R. (2003). Tangle and neuron numbers, but not amyloid load, predict
cognitive status in Alzheimer’s disease. Neurology, 60(9), 1495–1500.
Glass, C. K., & Saijo, K. (2010). Nuclear receptor transrepression pathways that regulate
inflammation in macrophages and T cells. Nature Reviews. Immunology, 10(5),
365–376.
Glenner, G. G., & Wong, C. W. (1984a). Alzheimer’s disease and Down's syndrome:
sharing of a unique cerebrovascular amyloid fibril protein. Biochemical and
Biophysical Research Communications, 122(3), 1131–1135.
134 Glenner, G. G., & Wong, C. W. (1984b). Alzheimer’s disease: initial report of the
purification and characterization of a novel cerebrovascular amyloid protein.
Biochemical and Biophysical Research Communications, 120(3), 885–890.
Goate, A., Chartier-Harlin, M. C., Mullan, M., Brown, J., Crawford, F., Fidani, L., … James,
L. (1991). Segregation of a missense mutation in the amyloid precursor protein
gene with familial Alzheimer’s disease. Nature, 349(6311), 704–706.
Godyń, J., Jończyk, J., Panek, D., & Malawska, B. (2016). Therapeutic strategies for
Alzheimer’s disease in clinical trials. Pharmacological Reports: PR, 68(1), 127–138.
Goedert, M., & Jakes, R. (2005). Mutations causing neurodegenerative tauopathies.
Biochimica et Biophysica Acta, 1739(2-3), 240–250.
Goldgaber, D., Lerman, M. I., McBride, O. W., Saffiotti, U., & Gajdusek, D. C. (1987).
Characterization and chromosomal localization of a cDNA encoding brain amyloid
of Alzheimer’s disease. Science, 235(4791), 877–880.
Gold, M., Alderton, C., Zvartau-Hind, M., Egginton, S., Saunders, A. M., Irizarry, M., …
Sawchak, S. (2010). Rosiglitazone monotherapy in mild-to-moderate Alzheimer’s
disease: results from a randomized, double-blind, placebo-controlled phase III
study. Dementia and Geriatric Cognitive Disorders, 30(2), 131–146.
Gómez-Isla, T., Hollister, R., West, H., Mui, S., Growdon, J. H., Petersen, R. C., … Hyman,
B. T. (1997). Neuronal loss correlates with but exceeds neurofibrillary tangles in
Alzheimer’s disease. Annals of Neurology, 41(1), 17–24.
Gordon, S., & Martinez, F. O. (2010). Alternative activation of macrophages: mechanism
and functions. Immunity, 32(5), 593–604.
135 Gravina, S. A., Ho, L., Eckman, C. B., Long, K. E., Otvos, L., Jr, Younkin, L. H., … Younkin, S.
G. (1995). Amyloid beta protein (A beta) in Alzheimer’s disease brain. Biochemical
and immunocytochemical analysis with antibodies specific for forms ending at A
beta 40 or A beta 42(43). The Journal of Biological Chemistry, 270(13), 7013–7016.
Grueninger, F., Bohrmann, B., Czech, C., Ballard, T. M., Frey, J. R., Weidensteiner, C., …
Ozmen, L. (2010). Phosphorylation of Tau at S422 is enhanced by Abeta in
TauPS2APP triple transgenic mice. Neurobiology of Disease, 37(2), 294–306.
Grundke-Iqbal, I., Iqbal, K., Quinlan, M., Tung, Y. C., Zaidi, M. S., & Wisniewski, H. M.
(1986). Microtubule-associated protein tau. A component of Alzheimer paired
helical filaments. The Journal of Biological Chemistry, 261(13), 6084–6089.
Grundke-Iqbal, I., Iqbal, K., Tung, Y. C., Quinlan, M., Wisniewski, H. M., & Binder, L. I.
(1986). Abnormal phosphorylation of the microtubule-associated protein tau (tau)
in Alzheimer cytoskeletal pathology. Proceedings of the National Academy of
Sciences of the United States of America, 83(13), 4913–4917.
Grygiel-Górniak, B. (2014). Peroxisome proliferator-activated receptors and their
ligands: nutritional and clinical implications - a review. Nutrition Journal, 13(1), 17.
Guillozet, A. L., Weintraub, S., Mash, D. C., & Mesulam, M. M. (2003). Neurofibrillary
tangles, amyloid, and memory in aging and mild cognitive impairment. Archives of
Neurology, 60(5), 729–736.
Haass, C., Hung, A. Y., Schlossmacher, M. G., Oltersdorf, T., Teplow, D. B., & Selkoe, D. J.
(1993). Normal Cellular Processing of the β-Amyloid Precursor Protein Results in
the Secretion of the Amyloid β Peptide and Related Moleculesa. Annals of the New
136 York Academy of Sciences, 695(1), 109–116.
Haass, C., Kaether, C., Thinakaran, G., & Sisodia, S. (2012). Trafficking and proteolytic
processing of APP. Cold Spring Harbor Perspectives in Medicine, 2(5), a006270.
Han, Y.-H., Cao, X., Lin, B., Lin, F., Kolluri, S. K., Stebbins, J., … Zhang, X.-K. (2006).
Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt. Oncogene,
25(21), 2974–2986.
Hara, M., Matsushima, T., Satoh, H., Iso-o, N., Noto, H., Togo, M., … Tsukamoto, K.
(2003). Isoform-dependent cholesterol efflux from macrophages by apolipoprotein
E is modulated by cell surface proteoglycans. Arteriosclerosis, Thrombosis, and
Vascular Biology, 23(2), 269–274.
Hawk, J. D., & Abel, T. (2011). The role of NR4A transcription factors in memory
formation. Brain Research Bulletin, 85(1-2), 21–29.
Hawk, J. D., Bookout, A. L., Poplawski, S. G., Bridi, M., Rao, A. J., Sulewski, M. E., … Abel,
T. (2012). NR4A nuclear receptors support memory enhancement by histone
deacetylase inhibitors. The Journal of Clinical Investigation, 122(10), 3593–3602.
Heneka, M. T., Carson, M. J., El Khoury, J., Landreth, G. E., Brosseron, F., Feinstein, D. L.,
… Kummer, M. P. (2015). Neuroinflammation in Alzheimer’s disease. Lancet
Neurology, 14(4), 388–405.
Heneka, M. T., & Landreth, G. E. (2007). PPARs in the brain. Biochimica et Biophysica
Acta, 1771(8), 1031–1045.
Heneka, M. T., O’Banion, M. K., Terwel, D., & Kummer, M. P. (2010). Neuroinflammatory
processes in Alzheimer’s disease. Journal of Neural Transmission , 117(8), 919–947.
137 Heneka, M. T., Sastre, M., Dumitrescu-Ozimek, L., Hanke, A., Dewachter, I., Kuiperi, C., …
Landreth, G. E. (2005). Acute treatment with the PPARγ agonist pioglitazone and
ibuprofen reduces glial inflammation and Aβ1–42 levels in APPV717I transgenic
mice. Brain: A Journal of Neurology, 128(6), 1442–1453.
Herzig, M. C., Winkler, D. T., Burgermeister, P., Pfeifer, M., Kohler, E., Schmidt, S. D., …
Jucker, M. (2004). Aβ is targeted to the vasculature in a mouse model of hereditary
cerebral hemorrhage with amyloidosis. Nature Neuroscience, 7(9), 954–960.
Hickman, S. E., Allison, E. K., & El Khoury, J. (2008). Microglial Dysfunction and Defective
β-Amyloid Clearance Pathways in Aging Alzheimer’s Disease Mice. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 28(33), 8354–
8360.
Hippius, H., & Neundörfer, G. (2003). The discovery of Alzheimer’s disease. Dialogues in
Clinical Neuroscience, 5(1), 101–108.
Hirsch-Reinshagen, V., Maia, L. F., Burgess, B. L., Blain, J.-F., Naus, K. E., McIsaac, S. A., …
Wellington, C. L. (2005). The absence of ABCA1 decreases soluble ApoE levels but
does not diminish amyloid deposition in two murine models of Alzheimer disease.
The Journal of Biological Chemistry, 280(52), 43243–43256.
Hirsch-Reinshagen, V., Zhou, S., Burgess, B. L., Bernier, L., McIsaac, S. A., Chan, J. Y., …
Wellington, C. L. (2004). Deficiency of ABCA1 impairs apolipoprotein E metabolism
in brain. The Journal of Biological Chemistry, 279(39), 41197–41207.
Holla, V. R., Wu, H., Shi, Q., Menter, D. G., & DuBois, R. N. (2011). Nuclear orphan
receptor NR4A2 modulates fatty acid oxidation pathways in colorectal cancer. The
138 Journal of Biological Chemistry, 286(34), 30003–30009.
Holtzman, D. M. (2001). Role of apoe/Abeta interactions in the pathogenesis of
Alzheimer’s disease and cerebral amyloid angiopathy. Journal of Molecular
Neuroscience: MN, 17(2), 147–155.
Holtzman, D. M., Bales, K. R., Tenkova, T., Fagan, A. M., Parsadanian, M., Sartorius, L. J.,
… Paul, S. M. (2000). Apolipoprotein E isoform-dependent amyloid deposition and
neuritic degeneration in a mouse model of Alzheimer’s disease. Proceedings of the
National Academy of Sciences of the United States of America, 97(6), 2892–2897.
Holtzman, D. M., Herz, J., & Bu, G. (2012). Apolipoprotein E and apolipoprotein E
receptors: normal biology and roles in Alzheimer disease. Cold Spring Harbor
Perspectives in Medicine, 2(3), a006312.
Hong, C., & Tontonoz, P. (2008). Coordination of inflammation and metabolism by PPAR
and LXR nuclear receptors. Current Opinion in Genetics & Development, 18(5), 461–
467.
Hong, C., & Tontonoz, P. (2014). Liver X receptors in lipid metabolism: opportunities for
drug discovery. Nature Reviews. Drug Discovery, 13(6), 433–444.
Huang, S.-M., Mouri, A., Kokubo, H., Nakajima, R., Suemoto, T., Higuchi, M., … Iwata, N.
(2006). Neprilysin-sensitive Synapse-associated Amyloid-β Peptide Oligomers
Impair Neuronal Plasticity and Cognitive Function. The Journal of Biological
Chemistry, 281(26), 17941–17951.
Hu, J., Liu, C.-C., Chen, X.-F., Zhang, Y.-W., Xu, H., & Bu, G. (2015). Opposing effects of
viral mediated brain expression of apolipoprotein E2 (apoE2) and apoE4 on apoE
139 lipidation and Abeta metabolism in apoE4-targeted replacement mice. Molecular
Neurodegeneration, 10(1), 6.
Hurtado, D. E., Molina-Porcel, L., Iba, M., Aboagye, A. K., Paul, S. M., Trojanowski, J. Q.,
& Lee, V. M.-Y. (2010). A{beta} accelerates the spatiotemporal progression of tau
pathology and augments tau amyloidosis in an Alzheimer mouse model. The
American Journal of Pathology, 177(4), 1977–1988.
Hu, Y., Yang, Y., Yu, Y., Wen, G., Shang, N., Zhuang, W., … Bu, X. (2013). Synthesis and
identification of new flavonoids targeting liver X receptor β involved pathway as
potential facilitators of Aβ clearance with reduced lipid accumulation. Journal of
Medicinal Chemistry, 56(15), 6033–6053.
Ihara, Y., Nukina, N., Miura, R., & Ogawara, M. (1986). Phosphorylated tau protein is
integrated into paired helical filaments in Alzheimer’s disease. Journal of
Biochemistry, 99(6), 1807–1810.
Ingelsson, M., Fukumoto, H., Newell, K. L., Growdon, J. H., Hedley-Whyte, E. T., Frosch,
M. P., … Irizarry, M. C. (2004). Early Abeta accumulation and progressive synaptic
loss, gliosis, and tangle formation in AD brain. Neurology, 62(6), 925–931.
Ishizawa, M., Kagechika, H., & Makishima, M. (2012). NR4A nuclear receptors mediate
carnitine palmitoyltransferase 1A gene expression by the rexinoid HX600.
Biochemical and Biophysical Research Communications, 418(4), 780–785.
Iwata, N., Mizukami, H., Shirotani, K., Takaki, Y., Muramatsu, S.-I., Lu, B., … Saido, T. C.
(2004). Presynaptic localization of neprilysin contributes to efficient clearance of
amyloid-beta peptide in mouse brain. The Journal of Neuroscience: The Official
140 Journal of the Society for Neuroscience, 24(4), 991–998.
Iwata, N., Tsubuki, S., Takaki, Y., Shirotani, K., Lu, B., Gerard, N. P., … Saido, T. C. (2001).
Metabolic regulation of brain Abeta by neprilysin. Science, 292(5521), 1550–1552.
Iwata, N., Tsubuki, S., Takaki, Y., Watanabe, K., Sekiguchi, M., Hosoki, E., … Saido, T. C.
(2000). Identification of the major Aβ1–42-degrading catabolic pathway in brain
parenchyma: Suppression leads to biochemical and pathological deposition. Nature
Medicine, 6(2), 143–150.
Jack, C. R., Jr, Vemuri, P., Wiste, H. J., Weigand, S. D., Aisen, P. S., Trojanowski, J. Q., …
Alzheimer’s Disease Neuroimaging Initiative. (2011). Evidence for ordering of
Alzheimer disease biomarkers. Archives of Neurology, 68(12), 1526–1535.
Jacobsen, J. S., Comery, T. A., Martone, R. L., Elokdah, H., Crandall, D. L., Oganesian, A.,
… Pangalos, M. N. (2008). Enhanced clearance of Abeta in brain by sustaining the
plasmin proteolysis cascade. Proceedings of the National Academy of Sciences of
the United States of America, 105(25), 8754–8759.
Jagirdar, K., Yin, K., Harrison, M., Lim, W., Muscat, G. E. O., Sturm, R. A., & Smith, A. G.
(2013). The NR4A2 Nuclear Receptor Is Recruited to Novel Nuclear Foci in Response
to UV Irradiation and Participates in Nucleotide Excision Repair. PloS One, 8(11),
e78075.
Jarrett, J. T., Berger, E. P., & Lansbury, P. T., Jr. (1993). The carboxy terminus of the beta
amyloid protein is critical for the seeding of amyloid formation: implications for the
pathogenesis of Alzheimer’s disease. Biochemistry, 32(18), 4693–4697.
Jiang, Q., Lee, C. Y. D., Mandrekar, S., Wilkinson, B., Cramer, P., Zelcer, N., … Landreth,
141 G. E. (2008). ApoE promotes the proteolytic degradation of Abeta. Neuron, 58(5),
681–693.
Johnson, M. M., Michelhaugh, S. K., Bouhamdan, M., Schmidt, C. J., & Bannon, M. J.
(2011). The Transcription Factor NURR1 Exerts Concentration-Dependent Effects on
Target Genes Mediating Distinct Biological Processes. Frontiers in Neuroscience, 5,
135.
Jonsson, T., Atwal, J. K., Steinberg, S., Snaedal, J., Jonsson, P. V., Bjornsson, S., …
Stefansson, K. (2012). A mutation in APP protects against Alzheimer’s disease and
age-related cognitive decline. Nature, 488(7409), 96–99.
Kaden, D., Harmeier, A., Weise, C., Munter, L. M., Althoff, V., Rost, B. R., … Multhaup, G.
(2012). Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ
oligomers. EMBO Molecular Medicine, 4(7), 647–659.
Kamal, A., Almenar-Queralt, A., LeBlanc, J. F., Roberts, E. A., & Goldstein, L. S. (2001).
Kinesin-mediated axonal transport of a membrane compartment containing beta-
secretase and presenilin-1 requires APP. Nature, 414(6864), 643–648.
Kamenetz, F., Tomita, T., Hsieh, H., Seabrook, G., Borchelt, D., Iwatsubo, T., … Malinow,
R. (2003). APP processing and synaptic function. Neuron, 37(6), 925–937.
Kanekiyo, T., Cirrito, J. R., Liu, C.-C., Shinohara, M., Li, J., Schuler, D. R., … Bu, G. (2013).
Neuronal clearance of amyloid-β by endocytic receptor LRP1. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 33(49), 19276–
19283.
Kanekiyo, T., Zhang, J., Liu, Q., Liu, C.-C., Zhang, L., & Bu, G. (2011). Heparan sulphate
142 proteoglycan and the low-density lipoprotein receptor-related protein 1 constitute
major pathways for neuronal amyloid-beta uptake. The Journal of Neuroscience:
The Official Journal of the Society for Neuroscience, 31(5), 1644–1651.
Kang, J., Lemaire, H. G., Unterbeck, A., Salbaum, J. M., Masters, C. L., Grzeschik, K. H., …
Müller-Hill, B. (1987). The precursor of Alzheimer’s disease amyloid A4 protein
resembles a cell-surface receptor. Nature, 325(6106), 733–736.
Khachaturian, A. S., Corcoran, C. D., Mayer, L. S., Zandi, P. P., & Breitner, J. C. S. (2004).
Apolipoprotein E ϵ4 Count Affects Age at Onset of Alzheimer Disease, but Not
Lifetime Susceptibility: The Cache County Study. Archives of General Psychiatry,
61(5), 518–524.
Kim, C.-H., Han, B.-S., Moon, J., Kim, D.-J., Shin, J., Rajan, S., … Kim, K.-S. (2015). Nuclear
receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits
in an animal model of Parkinson’s disease. Proceedings of the National Academy of
Sciences of the United States of America, 112(28), 8756–8761.
Kim, J.-I., Jeon, S. G., Kim, K. A., Kim, Y. J., Song, E. J., Choi, J., … Chung, H. (2016). The
pharmacological stimulation of Nurr1 improves cognitive functions via
enhancement of adult hippocampal neurogenesis. Stem Cell Research.
https://doi.org/10.1016/j.scr.2016.09.027
Knopman, D. S., Parisi, J. E., Salviati, A., Floriach-Robert, M., Boeve, B. F., Ivnik, R. J., …
Petersen, R. C. (2003). Neuropathology of cognitively normal elderly. Journal of
Neuropathology and Experimental Neurology, 62(11), 1087–1095.
Koenigsknecht, J., & Landreth, G. (2004). Microglial phagocytosis of fibrillar beta-
143 amyloid through a beta1 integrin-dependent mechanism. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 24(44), 9838–
9846.
Koistinaho, M., Lin, S., Wu, X., Esterman, M., Koger, D., Hanson, J., … Paul, S. M. (2004).
Apolipoprotein E promotes astrocyte colocalization and degradation of deposited
amyloid-beta peptides. Nature Medicine, 10(7), 719–726.
Koldamova, R. P., Lefterov, I. M., Staufenbiel, M., Wolfe, D., Huang, S., Glorioso, J. C., …
Lazo, J. S. (2005). The Liver X Receptor Ligand T0901317 Decreases Amyloid β
Production in Vitro and in a Mouse Model of Alzheimer’s Disease. The Journal of
Biological Chemistry, 280(6), 4079–4088.
Koldamova, R., Staufenbiel, M., & Lefterov, I. (2005). Lack of ABCA1 considerably
decreases brain ApoE level and increases amyloid deposition in APP23 mice. The
Journal of Biological Chemistry, 280(52), 43224–43235.
Koo, J. W., Mazei-Robison, M. S., LaPlant, Q., Egervari, G., Braunscheidel, K. M., Adank,
D. N., … Nestler, E. J. (2015). Epigenetic basis of opiate suppression of Bdnf gene
expression in the ventral tegmental area. Nature Neuroscience, 18(3), 415–422.
Kopec, K. K., & Carroll, R. T. (1998). Alzheimer’s $\beta$-Amyloid Peptide 1--42 Induces
a Phagocytic Response in Murine Microglia. Journal of Neurochemistry, 71(5),
2123–2131.
Kosik, K. S., Joachim, C. L., & Selkoe, D. J. (1986). Microtubule-associated protein tau
(tau) is a major antigenic component of paired helical filaments in Alzheimer
disease. Proceedings of the National Academy of Sciences of the United States of
144 America, 83(11), 4044–4048.
Kruit, J. K., Plösch, T., Havinga, R., Boverhof, R., Groot, P. H. E., Groen, A. K., & Kuipers, F.
(2005). Increased fecal neutral sterol loss upon liver X receptor activation is
independent of biliary sterol secretion in mice. Gastroenterology, 128(1), 147–156.
Kumar-Singh, S., Theuns, J., Van Broeck, B., Pirici, D., Vennekens, K. ’l, Corsmit, E., … Van
Broeckhoven, C. (2006). Mean age-of-onset of familial alzheimer disease caused by
presenilin mutations correlates with both increased Abeta42 and decreased
Abeta40. Human Mutation, 27(7), 686–695.
Lacombe, P., Mathews, P. M., Schmidt, S. D., Breidert, T., Heneka, M. T., Landreth, G. E.,
… Galea, E. (2004). Effect of anti-inflammatory agents on transforming growth
factor beta over-expressing mouse brains: a model revised. Journal of
Neuroinflammation, 1(1), 11.
LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., & Frail, D. E.
(1994). Isoform-specific binding of apolipoprotein E to beta-amyloid. The Journal of
Biological Chemistry, 269(38), 23403–23406.
Lallier, S. W., Graf, A. E., Waidyarante, G. R., & Rogers, L. K. (2016). Nurr1 expression is
modified by inflammation in microglia. Neuroreport, 27(15), 1120–1127.
Lamb, B. T., Sisodia, S. S., Lawler, A. M., Slunt, H. H., Kitt, C. A., Kearns, W. G., …
Gearhart, J. D. (1993). Introduction and expression of the 400 kilobase precursor
amyloid protein gene in transgenic mice. Nature Genetics, 5(1), 22–30.
Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed, R., Liosatos, M., … Klein,
W. L. (1998). Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent
145 central nervous system neurotoxins. Proceedings of the National Academy of
Sciences of the United States of America, 95(11), 6448–6453.
Lee, C. Y. D., Tse, W., Smith, J. D., & Landreth, G. E. (2012). Apolipoprotein E promotes β-
amyloid trafficking and degradation by modulating microglial cholesterol levels.
The Journal of Biological Chemistry, 287(3), 2032–2044.
Lee, J. H., Park, S. M., Kim, O. S., Lee, C. S., Woo, J. H., Park, S. J., … Jou, I. (2009).
Differential SUMOylation of LXRalpha and LXRbeta mediates transrepression of
STAT1 inflammatory signaling in IFN-gamma-stimulated brain astrocytes. Molecular
Cell, 35(6), 806–817.
Lefterov, I., Bookout, A., Wang, Z., Staufenbiel, M., Mangelsdorf, D., & Koldamova, R.
(2007). Expression profiling in APP23 mouse brain: inhibition of Abeta amyloidosis
and inflammation in response to LXR agonist treatment. Molecular
Neurodegeneration, 2, 20.
Lehmann, J. M., Kliewer, S. A., Moore, L. B., Smith-Oliver, T. A., Oliver, B. B., Su, J. L., …
Willson, T. M. (1997). Activation of the nuclear receptor LXR by oxysterols defines a
new hormone response pathway. The Journal of Biological Chemistry, 272(6),
3137–3140.
Leissring, M. A., Farris, W., Chang, A. Y., Walsh, D. M., Wu, X., Sun, X., … Selkoe, D. J.
(2003). Enhanced proteolysis of beta-amyloid in APP transgenic mice prevents
plaque formation, secondary pathology, and premature death. Neuron, 40(6),
1087–1093.
Levy, E., Carman, M. D., Fernandez-Madrid, I. J., Power, M. D., Lieberburg, I., van
146 Duinen, S. G., … Frangione, B. (1990). Mutation of the Alzheimer’s disease amyloid
gene in hereditary cerebral hemorrhage, Dutch type. Science, 248(4959), 1124–
1126.
Le, W.-D., Xu, P., Jankovic, J., Jiang, H., Appel, S. H., Smith, R. G., & Vassilatis, D. K.
(2003). Mutations in NR4A2 associated with familial Parkinson disease. Nature
Genetics, 33(1), 85–89.
Lewis, J., Dickson, D. W., Lin, W. L., Chisholm, L., Corral, A., Jones, G., … McGowan, E.
(2001). Enhanced neurofibrillary degeneration in transgenic mice expressing
mutant tau and APP. Science, 293(5534), 1487–1491.
Le, W., Pan, T., Huang, M., Xu, P., Xie, W., Zhu, W., … Jankovic, J. (2008). Decreased
NURR1 gene expression in patients with Parkinson’s disease. Journal of the
Neurological Sciences, 273(1-2), 29–33.
Li, A. C., & Glass, C. K. (2004). PPAR- and LXR-dependent pathways controlling lipid
metabolism and the development of atherosclerosis. Journal of Lipid Research,
45(12), 2161–2173.
Liu, Y., Walter, S., Stagi, M., Cherny, D., Letiembre, M., Schulz-Schaeffer, W., …
Fassbender, K. (2005). LPS receptor (CD14): a receptor for phagocytosis of
Alzheimer’s amyloid peptide. Brain: A Journal of Neurology, 128(Pt 8), 1778–1789.
Magara, F., Müller, U., Li, Z.-W., Lipp, H.-P., Weissmann, C., Stagljar, M., & Wolfer, D. P.
(1999). Genetic background changes the pattern of forebrain commissure defects
in transgenic mice underexpressing the β-amyloid-precursor protein. Proceedings
of the National Academy of Sciences, 96(8), 4656–4661.
147 Malewicz, M., Kadkhodaei, B., Kee, N., Volakakis, N., Hellman, U., Viktorsson, K., …
Perlmann, T. (2011). Essential role for DNA-PK-mediated phosphorylation of NR4A
nuclear orphan receptors in DNA double-strand break repair. Genes &
Development, 25(19), 2031–2040.
Malkani, S., & Rosen, J. B. (2000). Induction of NGFI-B mRNA following contextual fear
conditioning and its blockade by diazepam. Brain Research. Molecular Brain
Research, 80(2), 153–165.
Malm, T. M., Jay, T. R., & Landreth, G. E. (2015). The evolving biology of microglia in
Alzheimer’s disease. Neurotherapeutics: The Journal of the American Society for
Experimental NeuroTherapeutics, 12(1), 81–93.
Mandrekar-Colucci, S., Karlo, J. C., & Landreth, G. E. (2012). Mechanisms underlying the
rapid peroxisome proliferator-activated receptor-γ-mediated amyloid clearance
and reversal of cognitive deficits in a murine model of Alzheimer’s disease. The
Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 32(30),
10117–10128.
Mandrekar-Colucci, S., & Landreth, G. E. (2010). Microglia and inflammation in
Alzheimer’s disease. CNS & Neurological Disorders Drug Targets, 9(2), 156–167.
Mandrekar, S., Jiang, Q., Daniel Lee, C. Y., Koenigsknecht-Talboo, J., Holtzman, D. M., &
Landreth, G. E. (2009). Microglia Mediate the Clearance of Soluble Aβ through Fluid
Phase Macropinocytosis. The Journal of Neuroscience: The Official Journal of the
Society for Neuroscience, 29(13), 4252–4262.
Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schütz, G., Umesono, K., …
148 Evans, R. M. (1995). The nuclear receptor superfamily: the second decade. Cell,
83(6), 835–839.
Mann, D. M., Iwatsubo, T., Pickering-Brown, S. M., Owen, F., Saido, T. C., & Perry, R. H.
(1997). Preferential deposition of amyloid beta protein (Abeta) in the form Abeta40
in Alzheimer’s disease is associated with a gene dosage effect of the apolipoprotein
E E4 allele. Neuroscience Letters, 221(2-3), 81–84.
Marr, R. A., Rockenstein, E., Mukherjee, A., Kindy, M. S., Hersh, L. B., Gage, F. H., …
Masliah, E. (2003). Neprilysin gene transfer reduces human amyloid pathology in
transgenic mice. The Journal of Neuroscience: The Official Journal of the Society for
Neuroscience, 23(6), 1992–1996.
Masciopinto, F., Di Pietro, N., Corona, C., Bomba, M., Pipino, C., Curcio, M., … Sensi, S. L.
(2012). Effects of long-term treatment with pioglitazone on cognition and glucose
metabolism of PS1-KI, 3xTg-AD, and wild-type mice. Cell Death & Disease, 3, e448.
Masters, C. L., Simms, G., Weinman, N. A., Multhaup, G., McDonald, B. L., & Beyreuther,
K. (1985). Amyloid plaque core protein in Alzheimer disease and Down syndrome.
Proceedings of the National Academy of Sciences of the United States of America,
82(12), 4245–4249.
Mauch, D. H., Nägler, K., Schumacher, S., Göritz, C., Müller, E. C., Otto, A., & Pfrieger, F.
W. (2001). CNS synaptogenesis promoted by glia-derived cholesterol. Science,
294(5545), 1354–1357.
Mawuenyega, K. G., Sigurdson, W., Ovod, V., Munsell, L., Kasten, T., Morris, J. C., …
Bateman, R. J. (2010). Decreased clearance of CNS beta-amyloid in Alzheimer’s
149 disease. Science, 330(6012), 1774.
Maxwell, M. A., & Muscat, G. E. O. (2006). The NR4A subgroup: immediate early
response genes with pleiotropic physiological roles. Nuclear Receptor Signaling, 4,
e002.
McFarland, K., Spalding, T. A., Hubbard, D., Ma, J.-N., Olsson, R., & Burstein, E. S. (2013).
Low dose bexarotene treatment rescues dopamine neurons and restores
behavioral function in models of Parkinson’s disease. ACS Chemical Neuroscience,
4(11), 1430–1438.
McMorrow, J. P., & Murphy, E. P. (2011). Inflammation: a role for NR4A orphan nuclear
receptors? Biochemical Society Transactions, 39(2), 688–693.
McNulty, S. E., Barrett, R. M., Vogel-Ciernia, A., Malvaez, M., Hernandez, N.,
Davatolhagh, M. F., … Wood, M. A. (2012). Differential roles for Nr4a1 and Nr4a2 in
object location vs. object recognition long-term memory. Learning & Memory ,
19(12), 588–592.
Meda, L., Cassatella, M. A., Szendrei, G. I., Otvos, L., Baron, P., Villalba, M., … Rossi, F.
(1995). Activation of microglial cells by β-amyloid protein and interferon-γ. Nature,
374(6523), 647–650.
Meilandt, W. J., Cisse, M., Ho, K., Wu, T., Esposito, L. A., Scearce-Levie, K., … Mucke, L.
(2009). Neprilysin overexpression inhibits plaque formation but fails to reduce
pathogenic Abeta oligomers and associated cognitive deficits in human amyloid
precursor protein transgenic mice. The Journal of Neuroscience: The Official Journal
of the Society for Neuroscience, 29(7), 1977–1986.
150 Miao, B., Zondlo, S., Gibbs, S., Cromley, D., Hosagrahara, V. P., Kirchgessner, T. G., …
Mukherjee, R. (2004). Raising HDL cholesterol without inducing hepatic steatosis
and hypertriglyceridemia by a selective LXR modulator. Journal of Lipid Research,
45(8), 1410–1417.
Michelhaugh, S. K., Vaitkevicius, H., Wang, J., Bouhamdan, M., Krieg, A. R., Walker, J. L.,
… Bannon, M. J. (2005). Dopamine neurons express multiple isoforms of the
nuclear receptor nurr1 with diminished transcriptional activity. Journal of
Neurochemistry, 95(5), 1342–1350.
Michikawa, M., Fan, Q. W., Isobe, I., & Yanagisawa, K. (2000). Apolipoprotein E exhibits
isoform-specific promotion of lipid efflux from astrocytes and neurons in culture.
Journal of Neurochemistry, 74(3), 1008–1016.
Mohan, H. M., Aherne, C. M., Rogers, A. C., Baird, A. W., des Winter, C., & Murphy, E. P.
(2012). Molecular pathways: the role of NR4A orphan nuclear receptors in cancer.
Clinical Cancer Research: An Official Journal of the American Association for Cancer
Research, 18(12), 3223–3228.
Montarolo, F., Perga, S., Martire, S., & Bertolotto, A. (2015). Nurr1 reduction influences
the onset of chronic EAE in mice. Inflammation Research: Official Journal of the
European Histamine Research Society ... [et Al.], 64(11), 841–844.
Montarolo, F., Perga, S., Martire, S., Navone, D. N., Marchet, A., Leotta, D., & Bertolotto,
A. (2016). Altered NR4A Subfamily Gene Expression Level in Peripheral Blood of
Parkinson’s and Alzheimer's Disease Patients. Neurotoxicity Research.
https://doi.org/10.1007/s12640-016-9626-4
151 Moon, M., Hong, H.-S., Nam, D. W., Baik, S. H., Song, H., Kook, S.-Y., … Mook-Jung, I.
(2012). Intracellular amyloid-β accumulation in calcium-binding protein-deficient
neurons leads to amyloid-β plaque formation in animal model of Alzheimer’s
disease. Journal of Alzheimer’s Disease: JAD, 29(3), 615–628.
Moon, M., Jeong, I., Kim, C.-H., Kim, J., Lee, P. K. J., Mook-Jung, I., … Kim, K.-S. (2015).
Correlation between orphan nuclear receptor Nurr1 expression and amyloid
deposition in 5XFAD mice, an animal model of Alzheimer’s disease. Journal of
Neurochemistry, 132(2), 254–262.
Morita, K., Kawana, K., Sodeyama, M., Shimomura, I., Kagechika, H., & Makishima, M.
(2005). Selective allosteric ligand activation of the retinoid X receptor heterodimers
of NGFI-B and Nurr1. Biochemical Pharmacology, 71(1-2), 98–107.
Mucke, L., Masliah, E., Johnson, W. B., Ruppe, M. D., Alford, M., Rockenstein, E. M., …
Abraham, C. R. (1994). Synaptotrophic effects of human amyloid β protein
precursors in the cortex of transgenic mice. Brain Research, 666(2), 151–167.
Mucke, L., Masliah, E., Yu, G. Q., Mallory, M., Rockenstein, E. M., Tatsuno, G., …
McConlogue, L. (2000). High-level neuronal expression of abeta 1-42 in wild-type
human amyloid protein precursor transgenic mice: synaptotoxicity without plaque
formation. The Journal of Neuroscience: The Official Journal of the Society for
Neuroscience, 20(11), 4050–4058.
Mucke, L., & Selkoe, D. J. (2012). Neurotoxicity of amyloid β-protein: synaptic and
network dysfunction. Cold Spring Harbor Perspectives in Medicine, 2(7), a006338.
Mukundan, L., Odegaard, J. I., Morel, C. R., Heredia, J. E., Mwangi, J. W., Ricardo-
152 Gonzalez, R. R., … Chawla, A. (2009). PPAR-delta senses and orchestrates clearance
of apoptotic cells to promote tolerance. Nature Medicine, 15(11), 1266–1272.
Mulder, C. K., Dong, Y., Brugghe, H. F., Timmermans, H. A. M., Tilstra, W., Westdijk, J., …
Eisel, U. L. M. (2016). Immunization with Small Amyloid-β-derived Cyclopeptide
Conjugates Diminishes Amyloid-β-Induced Neurodegeneration in Mice. Journal of
Alzheimer’s Disease: JAD, 52(3), 1111–1123.
Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L., Winblad, B., & Lannfelt, L.
(1992). A pathogenic mutation for probable Alzheimer’s disease in the APP gene at
the N-terminus of beta-amyloid. Nature Genetics, 1(5), 345–347.
Namba, Y., Tomonaga, M., Kawasaki, H., Otomo, E., & Ikeda, K. (1991). Apolipoprotein E
immunoreactivity in cerebral amyloid deposits and neurofibrillary tangles in
Alzheimer’s disease and kuru plaque amyloid in Creutzfeldt-Jakob disease. Brain
Research, 541(1), 163–166.
Nayak, D., Roth, T. L., & McGavern, D. B. (2014). Microglia development and function.
Annual Review of Immunology, 32, 367–402.
Nenov, M. N., Laezza, F., Haidacher, S. J., Zhao, Y., Sadygov, R. G., Starkey, J. M., …
Denner, L. (2014). Cognitive enhancing treatment with a PPARγ agonist normalizes
dentate granule cell presynaptic function in Tg2576 APP mice. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 34(3), 1028–
1036.
Nhan, H. S., Chiang, K., & Koo, E. H. (2015). The multifaceted nature of amyloid
precursor protein and its proteolytic fragments: friends and foes. Acta
153 Neuropathologica, 129(1), 1–19.
Nicolakakis, N., Aboulkassim, T., Ongali, B., Lecrux, C., Fernandes, P., Rosa-Neto, P., …
Hamel, E. (2008). Complete rescue of cerebrovascular function in aged Alzheimer’s
disease transgenic mice by antioxidants and pioglitazone, a peroxisome
proliferator-activated receptor $\gamma$ agonist. The Journal of Neuroscience:
The Official Journal of the Society for Neuroscience, 28(37), 9287–9296.
Nicoll, J. A. R., & Weller, R. O. (2003/7). A new role for astrocytes: β-amyloid
homeostasis and degradation. Trends in Molecular Medicine, 9(7), 281–282.
Nilsberth, C., Westlind-Danielsson, A., Eckman, C. B., Condron, M. M., Axelman, K.,
Forsell, C., … Lannfelt, L. (2001). The “Arctic” APP mutation (E693G) causes
Alzheimer’s disease by enhanced Abeta protofibril formation. Nature Neuroscience,
4(9), 887–893.
Núñez, V., Alameda, D., Rico, D., Mota, R., Gonzalo, P., Cedenilla, M., … Ricote, M.
(2010). Retinoid X receptor alpha controls innate inflammatory responses through
the up-regulation of chemokine expression. Proceedings of the National Academy
of Sciences of the United States of America, 107(23), 10626–10631.
Oakley, H., Cole, S. L., Logan, S., Maus, E., Shao, P., Craft, J., … Vassar, R. (2006).
Intraneuronal β-Amyloid Aggregates, Neurodegeneration, and Neuron Loss in
Transgenic Mice with Five Familial Alzheimer’s Disease Mutations: Potential Factors
in Amyloid Plaque Formation. The Journal of Neuroscience: The Official Journal of
the Society for Neuroscience, 26(40), 10129–10140.
Oddo, S., Caccamo, A., Shepherd, J. D., Murphy, M. P., Golde, T. E., Kayed, R., … LaFerla,
154 F. M. (2003). Triple-transgenic model of Alzheimer’s disease with plaques and
tangles: intracellular Abeta and synaptic dysfunction. Neuron, 39(3), 409–421.
Odegaard, J. I., & Chawla, A. (2011). Alternative macrophage activation and metabolism.
Annual Review of Pathology, 6, 275–297.
Odegaard, J. I., Ricardo-Gonzalez, R. R., Goforth, M. H., Morel, C. R., Subramanian, V.,
Mukundan, L., … Chawla, A. (2007). Macrophage-specific PPARgamma controls
alternative activation and improves insulin resistance. Nature, 447(7148), 1116–
1120.
Olson, M. I., & Shaw, C. M. (1969). Presenile dementia and Alzheimer’s disease in
mongolism. Brain: A Journal of Neurology, 92(1), 147–156.
O’Reilly, J.-A., & Lynch, M. (2012). Rosiglitazone Improves Spatial Memory and
Decreases Insoluble A$\beta$ 1--42 in APP/PS1 Mice. Journal of Neuroimmune
Pharmacology: The Official Journal of the Society on NeuroImmune Pharmacology,
7(1), 140–144.
Papadopoulos, P., Rosa-Neto, P., Rochford, J., & Hamel, E. (2013). Pioglitazone improves
reversal learning and exerts mixed cerebrovascular effects in a mouse model of
Alzheimer’s disease with combined amyloid-β and cerebrovascular pathology. PloS
One, 8(7), e68612.
Paresce, D. M., Ghosh, R. N., & Maxfield, F. R. (1996). Microglial cells internalize
aggregates of the Alzheimer’s disease amyloid beta-protein via a scavenger
receptor. Neuron, 17(3), 553–565.
Parra-Damas, A., Valero, J., Chen, M., España, J., Martín, E., Ferrer, I., … Saura, C. A.
155 (2014). Crtc1 activates a transcriptional program deregulated at early Alzheimer’s
disease-related stages. The Journal of Neuroscience: The Official Journal of the
Society for Neuroscience, 34(17), 5776–5787.
Pawar, A., Botolin, D., Mangelsdorf, D. J., & Jump, D. B. (2003). The role of liver X
receptor-alpha in the fatty acid regulation of hepatic gene expression. The Journal
of Biological Chemistry, 278(42), 40736–40743.
Pearson, B. E., & Choi, T. K. (1993). Expression of the human beta-amyloid precursor
protein gene from a yeast artificial chromosome in transgenic mice. Proceedings of
the National Academy of Sciences of the United States of America, 90(22), 10578–
10582.
Pedersen, W. A., McMillan, P. J., Kulstad, J. J., Leverenz, J. B., Craft, S., & Haynatzki, G. R.
(2006). Rosiglitazone attenuates learning and memory deficits in Tg2576 Alzheimer
mice. Experimental Neurology, 199(2), 265–273.
Pei, L., Castrillo, A., Chen, M., Hoffmann, A., & Tontonoz, P. (2005). Induction of NR4A
orphan nuclear receptor expression in macrophages in response to inflammatory
stimuli. The Journal of Biological Chemistry, 280(32), 29256–29262.
Pei, L., Castrillo, A., & Tontonoz, P. (2006). Regulation of macrophage inflammatory gene
expression by the orphan nuclear receptor Nur77. Molecular Endocrinology , 20(4),
786–794.
Perez-Nievas, B. G., Stein, T. D., Tai, H.-C., Dols-Icardo, O., Scotton, T. C., Barroeta-Espar,
I., … Gómez-Isla, T. (2013). Dissecting phenotypic traits linked to human resilience
to Alzheimer’s pathology. Brain: A Journal of Neurology, 136(Pt 8), 2510–2526.
156 Perlmann, T., & Jansson, L. (1995). A novel pathway for vitamin A signaling mediated by
RXR heterodimerization with NGFI-B and NURR1. Genes & Development, 9(7), 769–
782.
Perlmann, T., & Wallén-Mackenzie, A. (2004). Nurr1, an orphan nuclear receptor with
essential functions in developing dopamine cells. Cell and Tissue Research, 318(1),
45–52.
Perlmutter, L. S., Barron, E., & Chui, H. C. (1990). Morphologic association between
microglia and senile plaque amyloid in Alzheimer’s disease. Neuroscience Letters,
119(1), 32–36.
Perry, V. H., & Gordon, S. (1988). Macrophages and microglia in the nervous system.
Trends in Neurosciences, 11(6), 273–277.
Petersen, R. C., Parisi, J. E., Dickson, D. W., Johnson, K. A., Knopman, D. S., Boeve, B. F.,
… Kokmen, E. (2006). Neuropathologic features of amnestic mild cognitive
impairment. Archives of Neurology, 63(5), 665–672.
Price, J. L., & Morris, J. C. (1999). Tangles and plaques in nondemented aging and
“preclinical” Alzheimer’s disease. Annals of Neurology, 45(3), 358–368.
Prinz, M., Priller, J., Sisodia, S. S., & Ransohoff, R. M. (2011). Heterogeneity of CNS
myeloid cells and their roles in neurodegeneration. Nature Neuroscience, 14(10),
1227–1235.
Qi-Takahara, Y., Morishima-Kawashima, M., Tanimura, Y., Dolios, G., Hirotani, N.,
Horikoshi, Y., … Ihara, Y. (2005). Longer forms of amyloid beta protein: implications
for the mechanism of intramembrane cleavage by gamma-secretase. The Journal of
157 Neuroscience: The Official Journal of the Society for Neuroscience, 25(2), 436–445.
Quinet, E. M., Savio, D. A., Halpern, A. R., Chen, L., Schuster, G. U., Gustafsson, J.-Å., …
Nambi, P. (2006). Liver X Receptor (LXR)-β Regulation in LXRα-Deficient Mice:
Implications for Therapeutic Targeting. Molecular Pharmacology, 70(4), 1340–
1349.
Quon, D., Wang, Y., Catalano, R., Scardina, J. M., Murakami, K., & Cordell, B. (1991).
Formation of beta-amyloid protein deposits in brains of transgenic mice. Nature,
352(6332), 239–241.
Reed-Geaghan, E. G., Savage, J. C., Hise, A. G., & Landreth, G. E. (2009). CD14 and toll-
like receptors 2 and 4 are required for fibrillar A{beta}-stimulated microglial
activation. The Journal of Neuroscience: The Official Journal of the Society for
Neuroscience, 29(38), 11982–11992.
Riddell, D. R., Zhou, H., Comery, T. A., Kouranova, E., Lo, C. F., Warwick, H. K., …
Jacobsen, J. S. (2007). The LXR agonist TO901317 selectively lowers hippocampal
Aβ42 and improves memory in the Tg2576 mouse model of Alzheimer’s disease.
Molecular and Cellular Neurosciences, 34(4), 621–628.
Robakis, N., Wisniewski, H., Jenkins, E., Devine-Gage, E., Houck, G., Yao, X.-L., … Brown,
W. T. (1987). Chromosome 21q21 sublocalisation of gene encoding beta-amyloid
peptide in cerebral vessels and neuritic (senile) plaques of people with Alzheimer
disease and Down syndrome. The Lancet, 329(8529), 384–385.
Rodriguez-Rivera, J., Denner, L., & Dineley, K. T. (2011). Rosiglitazone reversal of Tg2576
cognitive deficits is independent of peripheral gluco-regulatory status. Behavioural
158 Brain Research, 216(1), 255–261.
Sacchetti, P., Carpentier, R., Ségard, P., Olivé-Cren, C., & Lefebvre, P. (2006). Multiple
signaling pathways regulate the transcriptional activity of the orphan nuclear
receptor NURR1. Nucleic Acids Research, 34(19), 5515–5527.
Sacchetti, P., Dwornik, H., Formstecher, P., Rachez, C., & Lefebvre, P. (2002).
Requirements for heterodimerization between the orphan nuclear receptor Nurr1
and retinoid X receptors. The Journal of Biological Chemistry, 277(38), 35088–
35096.
Sagare, A. P., Bell, R. D., & Zlokovic, B. V. (2013). Neurovascular defects and faulty
amyloid-β vascular clearance in Alzheimer’s disease. Journal of Alzheimer’s Disease:
JAD, 33 Suppl 1, S87–100.
Saido, T., & Leissring, M. A. (2012). Proteolytic degradation of amyloid β-protein. Cold
Spring Harbor Perspectives in Medicine, 2(6), a006379.
Saijo, K., Crotti, A., & Glass, C. K. (2013). Regulation of microglia activation and
deactivation by nuclear receptors. Glia, 61(1), 104–111.
Saijo, K., Winner, B., Carson, C. T., Collier, J. G., Boyer, L., Rosenfeld, M. G., … Glass, C. K.
(2009). A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic
neurons from inflammation-induced death. Cell, 137(1), 47–59.
Saito, T., Iwata, N., Tsubuki, S., Takaki, Y., Takano, J., Huang, S.-M., … Saido, T. C. (2005).
Somatostatin regulates brain amyloid beta peptide Abeta42 through modulation of
proteolytic degradation. Nature Medicine, 11(4), 434–439.
Saito, T., Matsuba, Y., Mihira, N., Takano, J., Nilsson, P., Itohara, S., … Saido, T. C. (2014).
159 Single App knock-in mouse models of Alzheimer’s disease. Nature Neuroscience,
17(5), 661–663.
Sandhu, F. A., Salim, M., & Zain, S. B. (1991). Expression of the human beta-amyloid
protein of Alzheimer’s disease specifically in the brains of transgenic mice. The
Journal of Biological Chemistry, 266(32), 21331–21334.
Sastre, M., Dewachter, I., Rossner, S., Bogdanovic, N., Rosen, E., Borghgraef, P., …
Heneka, M. T. (2006). Nonsteroidal anti-inflammatory drugs repress β-secretase
gene promoter activity by the activation of PPARγ. Proceedings of the National
Academy of Sciences of the United States of America, 103(2), 443–448.
Saul, A., Sprenger, F., Bayer, T. A., & Wirths, O. (2013). Accelerated tau pathology with
synaptic and neuronal loss in a novel triple transgenic mouse model of Alzheimer’s
disease. Neurobiology of Aging, 34(11), 2564–2573.
Savage, J. C., Jay, T., Goduni, E., Quigley, C., Mariani, M. M., Malm, T., … Landreth, G. E.
(2015). Nuclear receptors license phagocytosis by trem2+ myeloid cells in mouse
models of Alzheimer’s disease. The Journal of Neuroscience: The Official Journal of
the Society for Neuroscience, 35(16), 6532–6543.
Schenk, D., Basi, G. S., & Pangalos, M. N. (2012). Treatment strategies targeting amyloid
β-protein. Cold Spring Harbor Perspectives in Medicine, 2(9), a006387.
Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., … Younkin, S.
(1996). Secreted amyloid beta-protein similar to that in the senile plaques of
Alzheimer’s disease is increased in vivo by the presenilin 1 and 2 and APP
mutations linked to familial Alzheimer's disease. Nature Medicine, 2(8), 864–870.
160 Schmechel, D. E., Saunders, A. M., Strittmatter, W. J., Crain, B. J., Hulette, C. M., Joo, S.
H., … Roses, A. D. (1993). Increased amyloid beta-peptide deposition in cerebral
cortex as a consequence of apolipoprotein E genotype in late-onset Alzheimer
disease. Proceedings of the National Academy of Sciences of the United States of
America, 90(20), 9649–9653.
Schoonjans, K., Peinado-Onsurbe, J., Lefebvre, A. M., Heyman, R. A., Briggs, M., Deeb, S.,
… Auwerx, J. (1996). PPARalpha and PPARgamma activators direct a distinct tissue-
specific transcriptional response via a PPRE in the lipoprotein lipase gene. The
EMBO Journal, 15(19), 5336–5348.
Seabrook, G. R., Smith, D. W., Bowery, B. J., Easter, A., Reynolds, T., Fitzjohn, S. M., …
Hill, R. G. (1999). Mechanisms contributing to the deficits in hippocampal synaptic
plasticity in mice lacking amyloid precursor protein. Neuropharmacology, 38(3),
349–359.
Searcy, J. L., Phelps, J. T., Pancani, T., Kadish, I., Popovic, J., Anderson, K. L., … Thibault,
O. (2012). Long-term pioglitazone treatment improves learning and attenuates
pathological markers in a mouse model of Alzheimer’s disease. Journal of
Alzheimer’s Disease: JAD, 30(4), 943–961.
Seino, Y., Kawarabayashi, T., Wakasaya, Y., Watanabe, M., Takamura, A., Yamamoto-
Watanabe, Y., … Shoji, M. (2010). Amyloid β accelerates phosphorylation of tau and
neurofibrillary tangle formation in an amyloid precursor protein and tau double-
transgenic mouse model. Journal of Neuroscience Research, 88(16), 3547–3554.
Selkoe, D. J., & Hardy, J. (2016). The amyloid hypothesis of Alzheimer’s disease at 25
161 years. EMBO Molecular Medicine, 8(6), 595–608.
Serrano-Pozo, A., Muzikansky, A., Gómez-Isla, T., Growdon, J. H., Betensky, R. A., Frosch,
M. P., & Hyman, B. T. (2013). Differential relationships of reactive astrocytes and
microglia to fibrillar amyloid deposits in Alzheimer disease. Journal of
Neuropathology and Experimental Neurology, 72(6), 462–471.
Shankar, G. M., Bloodgood, B. L., Townsend, M., Walsh, D. M., Selkoe, D. J., & Sabatini,
B. L. (2007). Natural oligomers of the Alzheimer amyloid-beta protein induce
reversible synapse loss by modulating an NMDA-type glutamate receptor-
dependent signaling pathway. The Journal of Neuroscience: The Official Journal of
the Society for Neuroscience, 27(11), 2866–2875.
Shankar, G. M., Li, S., Mehta, T. H., Garcia-Munoz, A., Shepardson, N. E., Smith, I., …
Selkoe, D. J. (2008). Amyloid-beta protein dimers isolated directly from Alzheimer’s
brains impair synaptic plasticity and memory. Nature Medicine, 14(8), 837–842.
Shen, C. L., & Murphy, R. M. (1995). Solvent effects on self-assembly of beta-amyloid
peptide. Biophysical Journal, 69(2), 640–651.
Shibata, M., Yamada, S., Kumar, S. R., Calero, M., Bading, J., Frangione, B., … Zlokovic, B.
V. (2000). Clearance of Alzheimer’s amyloid-β1-40 peptide from brain by LDL
receptor–related protein-1 at the blood-brain barrier. The Journal of Clinical
Investigation, 106(12), 1489–1499.
Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R., Davis, D., … John, V.
(1999). Purification and cloning of amyloid precursor protein beta-secretase from
human brain. Nature, 402(6761), 537–540.
162 Sirin, O., Lukov, G. L., Mao, R., Conneely, O. M., & Goodell, M. A. (2010). The orphan
nuclear receptor Nurr1 restricts the proliferation of haematopoietic stem cells.
Nature Cell Biology, 12(12), 1213–1219.
Skerrett, R., Malm, T., & Landreth, G. (2014). Nuclear receptors in neurodegenerative
diseases. Neurobiology of Disease, 72 Pt A, 104–116.
Smith, K. D. B., Kallhoff, V., Zheng, H., & Pautler, R. G. (2007). In vivo axonal transport
rates decrease in a mouse model of Alzheimer’s disease. NeuroImage, 35(4), 1401–
1408.
Spires-Jones, T. L., & Hyman, B. T. (2014). The intersection of amyloid beta and tau at
synapses in Alzheimer’s disease. Neuron, 82(4), 756–771.
Stancu, I.-C., Ris, L., Vasconcelos, B., Marinangeli, C., Goeminne, L., Laporte, V., …
Dewachter, I. (2014). Tauopathy contributes to synaptic and cognitive deficits in a
murine model for Alzheimer’s disease. FASEB Journal: Official Publication of the
Federation of American Societies for Experimental Biology, 28(6), 2620–2631.
Stancu, I.-C., Vasconcelos, B., Terwel, D., & Dewachter, I. (2014). Models of $\beta$-
amyloid induced Tau-pathology: the long and ``folded’' road to understand the
mechanism. Molecular Neurodegeneration, 9(1), 51.
Stelzmann, R. A., Norman Schnitzlein, H., & Reed Murtagh, F. (1995). An english
translation of alzheimer’s 1907 paper, “über eine eigenartige erkankung der
hirnrinde.” Clinical Anatomy , 8(6), 429–431.
Stewart, C. R., Stuart, L. M., Wilkinson, K., van Gils, J. M., Deng, J., Halle, A., … Moore, K.
J. (2010). CD36 ligands promote sterile inflammation through assembly of a Toll-
163 like receptor 4 and 6 heterodimer. Nature Immunology, 11(2), 155–161.
Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L. M., Salvesen, G. S., Pericak-
Vance, M., … Roses, A. D. (1993). Binding of human apolipoprotein E to synthetic
amyloid beta peptide: isoform-specific effects and implications for late-onset
Alzheimer disease. Proceedings of the National Academy of Sciences of the United
States of America, 90(17), 8098–8102.
Suzuki, N., Cheung, T. T., Cai, X. D., Odaka, A., Otvos, L., Jr, Eckman, C., … Younkin, S. G.
(1994). An increased percentage of long amyloid beta protein secreted by familial
amyloid beta protein precursor (beta APP717) mutants. Science, 264(5163), 1336–
1340.
Takami, M., Nagashima, Y., Sano, Y., Ishihara, S., Morishima-Kawashima, M., Funamoto,
S., & Ihara, Y. (2009). gamma-Secretase: successive tripeptide and tetrapeptide
release from the transmembrane domain of beta-carboxyl terminal fragment. The
Journal of Neuroscience: The Official Journal of the Society for Neuroscience, 29(41),
13042–13052.
Tall, A. R. (2008). Cholesterol efflux pathways and other potential mechanisms involved
in the athero-protective effect of high density lipoproteins. Journal of Internal
Medicine, 263(3), 256–273.
Tampellini, D., Capetillo-Zarate, E., Dumont, M., Huang, Z., Yu, F., Lin, M. T., & Gouras, G.
K. (2010). Effects of synaptic modulation on beta-amyloid, synaptophysin, and
memory performance in Alzheimer’s disease transgenic mice. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 30(43), 14299–
164 14304.
Tanzi, R. E. (2012). The genetics of Alzheimer disease. Cold Spring Harbor Perspectives in
Medicine, 2(10). https://doi.org/10.1101/cshperspect.a006296
Tanzi, R. E., Gusella, J. F., Watkins, P. C., Bruns, G. A., St George-Hyslop, P., Van Keuren,
M. L., … Neve, R. L. (1987). Amyloid beta protein gene: cDNA, mRNA distribution,
and genetic linkage near the Alzheimer locus. Science, 235(4791), 880–884.
Tarasoff-Conway, J. M., Carare, R. O., Osorio, R. S., Glodzik, L., Butler, T., Fieremans, E.,
… de Leon, M. J. (2015). Clearance systems in the brain[mdash]implications for
Alzheimer disease. Nature Reviews. Neurology, 11(8), 457–470.
Terwel, D., Steffensen, K. R., Verghese, P. B., Kummer, M. P., Gustafsson, J.-Å.,
Holtzman, D. M., & Heneka, M. T. (2011). Critical role of astroglial apolipoprotein E
and liver X receptor-α expression for microglial Aβ phagocytosis. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 31(19), 7049–
7059.
Therapeutics Search | ALZFORUM. (n.d.). Retrieved November 7, 2016, from
http://www.alzforum.org/therapeutics/search?fda_statuses=&target_types%5B%5
D=170&therapy_types%5B%5D=166&conditions=&keywords-entry=&keywords=
Tokuda, T., Calero, M., Matsubara, E., Vidal, R., Kumar, A., Permanne, B., … Ghiso, J.
(2000). Lipidation of apolipoprotein E influences its isoform-specific interaction
with Alzheimer’s amyloid beta peptides. Biochemical Journal, 348 Pt 2, 359–365.
Toledo, E. M., & Inestrosa, N. C. (2010). Activation of Wnt signaling by lithium and
rosiglitazone reduced spatial memory impairment and neurodegeneration in brains
165 of an APPswe/PSEN1$\Delta$E9 mouse model of Alzheimer’s disease. Molecular
Psychiatry, 15(3), 272–285. van der Hoorn, J., Lindén, D., Lindahl, U., Bekkers, M., Voskuilen, M., Nilsson, R., …
Princen, H. (2011). Low dose of the liver X receptor agonist, AZ876, reduces
atherosclerosis in APOE*3Leiden mice without affecting liver or plasma triglyceride
levels. British Journal of Pharmacology, 162(7), 1553–1563. van der Kant, R., & Goldstein, L. S. B. (2015). Cellular functions of the amyloid precursor
protein from development to dementia. Developmental Cell, 32(4), 502–515.
Vanmierlo, T., Rutten, K., Dederen, J., Bloks, V. W., van Vark-van der Zee, L. C., Kuipers,
F., … Mulder, M. (2011). Liver X receptor activation restores memory in aged AD
mice without reducing amyloid. Neurobiology of Aging, 32(7), 1262–1272.
Verghese, P. B., Castellano, J. M., Garai, K., Wang, Y., Jiang, H., Shah, A., … Holtzman, D.
M. (2013). ApoE influences amyloid-β (Aβ) clearance despite minimal apoE/Aβ
association in physiological conditions. Proceedings of the National Academy of
Sciences of the United States of America, 110(19), E1807–16.
Villemagne, V. L., Burnham, S., Bourgeat, P., Brown, B., Ellis, K. A., Salvado, O., …
Australian Imaging Biomarkers and Lifestyle (AIBL) Research Group. (2013).
Amyloid β deposition, neurodegeneration, and cognitive decline in sporadic
Alzheimer’s disease: a prospective cohort study. Lancet Neurology, 12(4), 357–367.
Volakakis, N., Joodmardi, E., & Perlmann, T. (2009). NR4A orphan nuclear receptors
influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty
acid binding protein 5. Biochemical and Biophysical Research Communications,
166 390(4), 1186–1191.
Volakakis, N., Kadkhodaei, B., Joodmardi, E., Wallis, K., Panman, L., Silvaggi, J., …
Perlmann, T. (2010). NR4A orphan nuclear receptors as mediators of CREB-
dependent neuroprotection. Proceedings of the National Academy of Sciences of
the United States of America, 107(27), 12317–12322.
Volakakis, N., Tiklova, K., Decressac, M., Papathanou, M., Mattsson, B., Gillberg, L., …
Perlmann, T. (2015). Nurr1 and Retinoid X Receptor Ligands Stimulate Ret Signaling
in Dopamine Neurons and Can Alleviate α-Synuclein Disrupted Gene Expression.
The Journal of Neuroscience: The Official Journal of the Society for Neuroscience,
35(42), 14370–14385.
Volpicelli, F., Caiazzo, M., Greco, D., Consales, C., Leone, L., Perrone-Capano, C., … di
Porzio, U. (2007). Bdnf gene is a downstream target of Nurr1 transcription factor in
rat midbrain neurons in vitro. Journal of Neurochemistry, 102(2), 441–453. von Hertzen, L. S. J., & Giese, K. P. (2005). Memory reconsolidation engages only a
subset of immediate-early genes induced during consolidation. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 25(8), 1935–
1942.
Wahrle, S. E., Jiang, H., Parsadanian, M., Hartman, R. E., Bales, K. R., Paul, S. M., &
Holtzman, D. M. (2005). Deletion of Abca1 Increases Aβ Deposition in the PDAPP
Transgenic Mouse Model of Alzheimer Disease. The Journal of Biological Chemistry,
280(52), 43236–43242.
Wahrle, S. E., Jiang, H., Parsadanian, M., Kim, J., Li, A., Knoten, A., … Holtzman, D. M.
167 (2008). Overexpression of ABCA1 reduces amyloid deposition in the PDAPP mouse
model of Alzheimer disease. The Journal of Clinical Investigation, 118(2), 671–682.
Wallen-Mackenzie, A. (2003). Nurr1-RXR heterodimers mediate RXR ligand-induced
signaling in neuronal cells. Genes & Development, 17(24), 3036–3047.
Wang, L., Schuster, G. U., Hultenby, K., Zhang, Q., Andersson, S., & Gustafsson, J.-A.
(2002). Liver X receptors in the central nervous system: from lipid homeostasis to
neuronal degeneration. Proceedings of the National Academy of Sciences of the
United States of America, 99(21), 13878–13883.
Warden, A., Truitt, J., Merriman, M., Ponomareva, O., Jameson, K., Ferguson, L. B., …
Harris, R. A. (2016). Localization of PPAR isotypes in the adult mouse and human
brain. Scientific Reports, 6, 27618.
Watakabe, A., Ichinohe, N., Ohsawa, S., Hashikawa, T., Komatsu, Y., Rockland, K. S., &
Yamamori, T. (2007). Comparative analysis of layer-specific genes in Mammalian
neocortex. Cerebral Cortex , 17(8), 1918–1933.
Webster, S. J., Bachstetter, A. D., Nelson, P. T., Schmitt, F. A., & Van Eldik, L. J. (2014).
Using mice to model Alzheimer’s dementia: an overview of the clinical disease and
the preclinical behavioral changes in 10 mouse models. Frontiers in Genetics, 5, 88.
Wesson, D. W., Borkowski, A. H., Landreth, G. E., Nixon, R. A., Levy, E., & Wilson, D. A.
(2011). Sensory network dysfunction, behavioral impairments, and their
reversibility in an Alzheimer’s β-amyloidosis mouse model. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 31(44), 15962–
15971.
168 Whitney, K. D., Watson, M. A., Goodwin, B., Galardi, C. M., Maglich, J. M., Wilson, J. G.,
… Kliewer, S. A. (2001). Liver X Receptor (LXR) Regulation of the LXR Gene in Human
Macrophages. The Journal of Biological Chemistry, 276(47), 43509–43515.
Wildsmith, K. R., Holley, M., & Savage, J. C. (2013). Evidence for impaired amyloid beta
clearance in Alzheimer’s disease. Alzheimer’s Research & Therapy, 5(33), 1–6.
Wolfe, M. S., Xia, W., Ostaszewski, B. L., Diehl, T. S., Kimberly, W. T., & Selkoe, D. J.
(1999). Two transmembrane aspartates in presenilin-1 required for presenilin
endoproteolysis and gamma-secretase activity. Nature, 398(6727), 513–517.
Wood, J. G., Mirra, S. S., Pollock, N. J., & Binder, L. I. (1986). Neurofibrillary tangles of
Alzheimer disease share antigenic determinants with the axonal microtubule-
associated protein tau (tau). Proceedings of the National Academy of Sciences of
the United States of America, 83(11), 4040–4043.
Wyss-Coray, T., Loike, J. D., Brionne, T. C., Lu, E., Anankov, R., Yan, F., … Husemann, J.
(2003). Adult mouse astrocytes degrade amyloid-beta in vitro and in situ. Nature
Medicine, 9(4), 453–457.
Xu, Q., Bernardo, A., Walker, D., Kanegawa, T., Mahley, R. W., & Huang, Y. (2006). Profile
and regulation of apolipoprotein E (ApoE) expression in the CNS in mice with
targeting of green fluorescent protein gene to the ApoE locus. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience, 26(19), 4985–
4994.
Yamada, T., Sasaki, H., Furuya, H., Miyata, T., Goto, I., & Sakaki, Y. (1987).
Complementary DNA for the mouse homolog of the human amyloid beta protein
169 precursor. Biochemical and Biophysical Research Communications, 149(2), 665–
671.
Yamanaka, M., Ishikawa, T., Griep, A., Axt, D., Kummer, M. P., & Heneka, M. T. (2012).
PPARγ/RXRα-induced and CD36-mediated microglial amyloid-β phagocytosis
results in cognitive improvement in amyloid precursor protein/presenilin 1 mice.
The Journal of Neuroscience: The Official Journal of the Society for Neuroscience,
32(48), 17321–17331.
Yang, D., Li, T., Wang, Y., Tang, Y., Cui, H., Tang, Y., … Le Weidong. (2012). miR-132
regulates the differentiation of dopamine neurons by directly targeting Nurr1
expression. Journal of Cell Science, 125(Pt 7), 1673–1682.
Yang, D.-S., Smith, J. D., Zhou, Z., Gandy, S. E., & Martins, R. N. (1997). Characterization
of the Binding of Amyloid-β Peptide to Cell Culture-Derived Native Apolipoprotein
E2, E3, and E4 Isoforms and to Isoforms from Human Plasma. Journal of
Neurochemistry, 68(2), 721–725.
Yan, Q., Zhang, J., Liu, H., Babu-Khan, S., Vassar, R., Biere, A. L., … Landreth, G. (2003).
Anti-inflammatory drug therapy alters beta-amyloid processing and deposition in
an animal model of Alzheimer’s disease. The Journal of Neuroscience: The Official
Journal of the Society for Neuroscience, 23(20), 7504–7509.
Ye, J. M., Doyle, P. J., Iglesias, M. A., Watson, D. G., Cooney, G. J., & Kraegen, E. W.
(2001). Peroxisome proliferator-activated receptor (PPAR)-alpha activation lowers
muscle lipids and improves insulin sensitivity in high fat-fed rats: comparison with
PPAR-gamma activation. Diabetes, 50(2), 411–417.
170 Young-Pearse, T. L., Bai, J., Chang, R., Zheng, J. B., LoTurco, J. J., & Selkoe, D. J. (2007). A
critical function for beta-amyloid precursor protein in neuronal migration revealed
by in utero RNA interference. The Journal of Neuroscience: The Official Journal of
the Society for Neuroscience, 27(52), 14459–14469.
Yue, L., & Mazzone, T. (2009). Peroxisome Proliferator-activated Receptor γ Stimulation
of Adipocyte ApoE Gene Transcription Mediated by the Liver Receptor X Pathway.
The Journal of Biological Chemistry, 284(16), 10453–10461.
Zelcer, N., Khanlou, N., Clare, R., Jiang, Q., Reed-Geaghan, E. G., Landreth, G. E., …
Tontonoz, P. (2007). Attenuation of neuroinflammation and Alzheimer’s disease
pathology by liver x receptors. Proceedings of the National Academy of Sciences of
the United States of America, 104(25), 10601–10606.
Zhang, L., Reue, K., Fong, L. G., Young, S. G., & Tontonoz, P. (2012). Feedback Regulation
of Cholesterol Uptake by the LXR–IDOL–LDLR Axis. Arteriosclerosis, Thrombosis,
and Vascular Biology, 32(11), 2541–2546.
Zheng, H., Jiang, M., Trumbauer, M. E., Sirinathsinghji, D. J., Hopkins, R., Smith, D. W., …
Van der Ploeg, L. H. (1995). beta-Amyloid precursor protein-deficient mice show
reactive gliosis and decreased locomotor activity. Cell, 81(4), 525–531.
Zlokovic, B. V., Deane, R., Sagare, A. P., Bell, R. D., & Winkler, E. A. (2010). Low-density
lipoprotein receptor-related protein-1: a serial clearance homeostatic mechanism
controlling Alzheimer’s amyloid $\beta$-peptide elimination from the brain.
Journal of Neurochemistry, 115(5), 1077–1089.
171