Degradation of the Oncoprotein Mdmx in Neurodegenerative States: Evidence for a Pro-Survival Role of Mdmx in Neurons

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Publicly Accessible Penn Dissertations

2014

Daniel James Colacurcio University of Pennsylvania, [email protected]

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Recommended Citation Colacurcio, Daniel James, "Degradation of the Oncoprotein Mdmx in Neurodegenerative States: Evidence for a Pro-Survival Role of Mdmx in Neurons" (2014). Publicly Accessible Penn Dissertations. 1241. https://repository.upenn.edu/edissertations/1241

This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1241 For more information, please contact [email protected]. Degradation of the Oncoprotein Mdmx in Neurodegenerative States: Evidence for a Pro-Survival Role of Mdmx in Neurons

Abstract Neurodegenerative diseases, such as Alzheimer Disease (AD) and HIV–associated neurocognitive disorder (HAND) represent a tremendous burden to healthcare and a devastating impact on society. A common feature of neurodegenerative diseases is progressive dysfunction and death of neurons in key regions of the brain. Observations of dysregulated cell cycle proteins in the brains of patients, along with research in animal models and cultured cells, suggest that aberrant functions of cell cycle proteins contribute to neuronal death and progression of neurodegenerative diseases.

The p53 tumor suppressor is a key component in cell cycle signaling and cell death. p53 maintains multiple functions related to cellular homeostasis and cell fate determination, and is negatively regulated by two E3 ubiquitin ligases, MDM2 and MDMX (murine double minute X). MDM2 and MDMX promote cell survival and are implicated in several types of cancer, but the functions of MDMX in neurons are largely unknown. MDMX promotes survival in cultured neurons, but becomes degraded in response to neurotoxic stress, leading to a conclusion that MDMX acts as a “node” for stress factors in neuronal death.

This thesis examines mechanisms by which MDMX promotes neuronal survival, and how MDMX functions become disrupted during neurodegenerative disease. Utilizing human tissue samples, rodent models, and pharmacological experiments in cultured neurons, we determined how MDMX is affected in AD and HAND, and how these changes relate to neuronal damage. First, we demonstrate that calpain–mediated loss of MDMX contributes to neuronal damage in a model of HAND, and that MDMX overexpression protects neurons in this model. In our second body of work, we demonstrate that neuronal MDMX is reduced in AD and in AD models due to amyloid–induced caspase activation, and that loss of MDMX function contributes to pathological processes observed in AD, including mitochondrial damage, calpain activation, and Cdk5 hyperactivity. Finally, we examine p53 as a downstream effector of MDMX in neurons.

We conclude that the oncoprotein MDMX promotes neuronal survival through regulation of mitochondria, calpains, and Cdk5. Our work has identified mechanisms of MDMX degradation in two neurodegenerative diseases, whereby MDMX activity is reduced, causing dysregulation of downstream factors and neuronal damage.

Degree Type Dissertation

Degree Name Doctor of Philosophy (PhD)

Graduate Group Neuroscience

First Advisor Kelly L. Jordan-Sciutto

Keywords Alzheimer Disease, calpain, caspase, HIV-Associated Neurocognitive Disorder, MDMX, Mitochondria Subject Categories Cell Biology | Molecular Biology | Neuroscience and Neurobiology

This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1241

DEGRADATION OF THE ONCOPROTEIN MDMX IN NEURODEGENERATIVE

STATES: EVIDENCE FOR A PRO-SURVIVAL ROLE OF MDMX IN NEURONS

Daniel James Colacurcio

A DISSERTATION

Neuroscience

Presented to the Faculties of the University of Pennsylvania

Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy

2014

Supervisor of Dissertation

______

Kelly L. Jordan-Sciutto, Ph.D., Chair and Professor of Pathology, School of Dental Medicine

Graduate Group Chairperson

______

Joshua I. Gold, Ph.D., Professor of Neuroscience, Perelman School of Medicine

Dissertation Committee

David R. Lynch, M.D., Ph.D. Professor of Neurology, Perelman School of Medicine

(Committee Chair)

Dennis L. Kolson, M.D., Ph.D. Professor of Neurology, Perelman School of Medicine

Eric J. Brown, Ph.D. Associate Professor of Cancer Biology, Perelman School of Medicine

Steven E. Arnold, M.D. Professor of Psychiatry, Penn Memory Center

DEGRADATION OF THE ONCOPROTEIN MDMX IN NEURODEGENERATIVE STATES:

EVIDENCE FOR A PRO-SURVIVAL ROLE OF MDMX IN NEURONS

2014

Daniel James Colacurcio

ACKNOWLEDGMENTS

First, I would like to express my deepest gratitude to my thesis advisor, Dr. Kelly L.

Jordan-Sciutto, for her exemplary mentorship and guidance. Dr. Jordan-Sciutto has been incredibly generous with her time, and her advice, and she has helped me immensely in my development as a scientist. I am also indebted to the outstanding members of the Jordan-Sciutto laboratory who have been my role models, co-workers, and close friends, and who have helped me in countless ways. I also thank Drs. Virginia M.-Y. Lee, John Q. Trojanowski, and Kurt R.

Brunden at the Center for Neurodegenerative Disease Research, for including me in their journal club, for their assistance, and for productive scientific discussions. I also thank Drs. Dennis L.

Kolson, Samantha S. Soldan, and the members of their laboratories for their critical discussions of research during Neurovirology Journal Club meetings. Additionally, I am grateful to my thesis committee; Drs. David R. Lynch, Dennis L. Kolson, Eric J. Brown, and Steven E. Arnold, for their exceptional commitment and guidance. I am also grateful to the members of the Neuroscience

Graduate Group, particularly Drs. Michael P. Nusbaum, Rita J. Balice-Gordon, Joshua I. Gold, as well as Jane Hoshi, and my rotation supervisors: Drs. Harry Ischiropoulos and Michael Granato.

Also, I am especially thankful to Dr. Ronald L. Schnaar for giving me my first opportunity to work in a laboratory, and inspiring me to pursue neuroscience research.

Lastly, I would like to give special thanks to my family; my mother, my father, my step parents, and my little brother; for a lifetime of constant love, encouragement, and support. Finally,

I give special thanks to my life partner, Denise, for her support, her humor, her positivity, and for her music playlists, which were greatly appreciated during the writing of this dissertation.

iii

ABSTRACT

DEGRADATION OF THE ONCOPROTEIN MDMX IN NEURODEGENERATIVE STATES:

EVIDENCE FOR A PRO-SURVIVAL ROLE OF MDMX IN NEURONS

Daniel James Colacurcio

Kelly L. Jordan-Sciutto

Neurodegenerative diseases, such as Alzheimer Disease (AD) and HIV-associated neurocognitive disorder (HAND) represent a tremendous burden to healthcare and a devastating impact on society. A common feature of neurodegenerative diseases is progressive dysfunction and death of neurons in key regions of the brain. Observations of dysregulated cell cycle proteins in the brains of patients, along with research in animal models and cultured cells, suggest that aberrant functions of cell cycle proteins contribute to neuronal death and progression of neurodegenerative diseases.

MDM2 and MDMX promote cell survival and are implicated in several types of cancer, but the functions of MDMX in neurons are largely unknown. MDMX promotes survival in cultured neurons, but becomes degraded in response to neurotoxic stress, leading to a conclusion that

MDMX acts as a "node" for stress factors in neuronal death.

This thesis examines mechanisms by which MDMX promotes neuronal survival, and how

MDMX functions become disrupted during neurodegenerative disease. Utilizing human tissue samples, rodent models, and pharmacological experiments in cultured neurons, we determined how MDMX is affected in AD and HAND, and how these changes relate to neuronal damage.

First, we demonstrate that calpain-mediated loss of MDMX contributes to neuronal damage in a

model of HAND, and that MDMX overexpression protects neurons in this model. In our second body of work, we demonstrate that neuronal MDMX is reduced in AD and in AD models due to amyloid-induced caspase activation, and that loss of MDMX function contributes to pathological processes observed in AD, including mitochondrial damage, calpain activation, and Cdk5 hyperactivity. Finally, we examine p53 as a downstream effector of MDMX in neurons.

TABLE OF CONTENTS

ACKNOWLEDGEMENTS...... iii

ABSTRACT...... iv

LIST OF TABLES...... ix

LIST OF FIGURES...... x

CHAPTER 1: INTRODUCTION...... 1

1.1: INTRODUCTION...... 1

1.2: CELL CYCLE PROTEINS IN ALZHEIMER DISEASE...... 1

1.3: CELL CYCLE PROTEINS IN HIV-ASSOCIATED NEUROCOGNITIVE DISORDER

(HAND)...... 7

1.4: p53 AND THE p53/MDM2/MDMX NETWORK...... 10

1.5: THE MDMX ONCOPROTEIN...... 15

1.6: FUNCTIONS OF p53 IN NEURONS...... 19

1.7: FUNCTIONS OF MDMX IN NEURONS...... 22

1.8: RATIONALE, HYPOTHESIS, AND OBJECTIVES...... 25

1.9: BIBLIOGRAPHY...... 27

CHAPTER 2: Calpain-mediated degradation of MDMX/MDM4 contributes to HIV-induced neuronal damage...... 43

2.1: ABSTRACT...... 44

2.2: INTRODUCTION...... 45

2.3: EXPERIMENTAL METHODS...... 47

2.4: RESULTS...... 52

MDMX levels are decreased in the mid-frontal cortices of

neurocognitively impaired HIV (+) patients...... 52

MDMX is decreased in a time- and dose-dependent manner

in an in vitro model of HIV-associated neurodegeneration...... 53

Degradation of MDMX is calpain-dependent...... 54

Overexpression of MDMX attenuates HIV M/M-induced

neurotoxicity...... 54

Inhibition of MDMX activity leads to calpain-dependent

neuronal death...... 55

2.5: DISCUSSION...... 55

2.6: ACKNOWLEDGEMENTS...... 60

2.7: REFERENCES...... 69

CHAPTER 3: Amyloid-β-induced degradation of the MDMX oncoprotein triggers mitochondrial damage and calpain activation in neurons...... 78

3.1: ABSTRACT...... 79

3.2: INTRODUCTION...... 80

3.3: MATERIALS & METHODS...... 82

3.4: RESULTS...... 87

MDMX protein is reduced in frontal cortices of aged Tg2576 mice...... 87

Loss of MDMX-expressing cholinergic neurons in aged Tg2576 mice...... 87

Aβ induces caspase-mediated degradation of MDMX in primary cortical

neurons...... 88

Inhibition of MDMX causes mitochondrial depolarization, increased calpain

activation and p35 cleavage in primary cortical neurons...... 89

MDMX protein is reduced in brain tissue from AD patients...... 90

3.5: DISCUSSION...... 91

3.6: ACKNOWLEDGEMENTS...... 96

vii

3.7:REFERENCES...... 104

CHAPTER 4: MDMX Promotes Neuronal Survival in a p53-Independent

Manner...... 111

4.1: ABSTRACT...... 112

4.2: INTRODUCTION...... 113

4.3: MATERIALS & METHODS...... 114

4.4: RESULTS...... 115

Neuronal damage induced by MDMX inhibition is p53-independent...... 115

4.5: DISCUSSION...... 116

4.6: REFERENCES...... 120

CHAPTER 5: Conclusions & Future Directions...... 123

5.1: Overview...... 123

5.2: MDMX Degradation Mechanisms in Neurons...... 124

5.3: MDMX and p53 Functions in Neurons...... 126

5.4: MDMX and Mitochondrial Functions in Neurons...... 130

5.5: MDMX and Calpain/Cdk5 Activation...... 132

5.6: E2F1 and Alternative MDMX Effectors in Neurons...... 136

5.7: Functions of MDM2 in Neurons...... 138

5.8: Clinical Perspectives on Neuronal MDMX...... 139

5.9: Summary & Closing Remarks...... 142

5.10: References...... 143

viii

LIST OF TABLES

Chapter 2

Table 1: The cases used for western immunoblotting in Fig. 1A...... 61

Chapter 3

Table 1: Patient Data for Samples used...... 97

LIST OF FIGURES

Chapter 2

Figure 1: Decreased MDMx expression in the HAND cortex...... 62

Figure 2: HIV M/M leads to decreases in MDMx in a time- and dose-dependent manner.....63

Figure 3: HIV M/M-induced decrease in MDMx is dependent on NMDA receptor and calpain activation...... 64

Figure 4: Overexpression of MDMx in neurons provides partial protection from HIV M/M- induced neuronal damage...... 66

Figure 5: Inhibition of MDMx activity leads to calpain-dependent neuronal death...... 68

Chapter 3

Figure 1: MDMX protein is reduced in frontal cortices of aged Tg2576 mice...... 98

Figure 2: Loss of MDMX-expressing cholinergic neurons in aged Tg2576 mice...... 99

Figure 3: Aβ induces caspase-mediated degradation of MDMX in primary cortical neurons...... 100

Figure 4: Inhibition of MDMX causes mitochondrial depolarization, increased calpain activation and p35 cleavage in primary cortical neurons...... 101

Figure 5: MDMX protein is reduced in brain tissue from AD patients...... 103

Chapter 4

Figure 4.1:Neuronal damage induced by MDMX inhibition is p53-dependent...... 119

APPENDIX I

ILLUSTRATION 1:Graphical model of MDMX functions in dividing cells and in neurons..150

APPENDIX II

FIGURE 6.1: Neuronal damage curve of MDM2 inhibitor Nutlin-3a...... 151

CHAPTER 1

INTRODUCTION

1.1: INTRODUCTION

Neurodegenerative diseases encompass a variety of debilitating neurologic conditions, each with specific causes, symptoms, and pathologies. Despite these differences, many neurodegenerative diseases share common features, such as progressive onset, inflammation, oxidative damage, mitochondrial dysfunction, accumulation of pathological protein aggregates, synaptic damage, and ultimately, loss of neurons. Among these diseases, Alzheimer Disease

(AD) and HIV-associated neurocognitive disorder (HAND) are two neurodegenerative diseases with notably different etiologies that exhibit a subset of common pathological features, particularly inflammation, oxidative damage, mitochondrial dysfunction, excitotoxic damage, synaptodendritic damage, and neuronal death. Of interest to our group, the dysregulation of cell cycle proteins in post-mitotic neurons is an additional common feature of both AD and HAND. This body of work investigates the contributions of dysregulated cell cycle proteins to neuronal damage during both

AD and HAND, as an introduction to our research on murine double minute X (MDMX) in neurodegenerative diseases.

1.2: CELL CYCLE PROTEINS IN ALZHEIMER DISEASE

Alzheimer Disease (AD) was originally described in 1907 by Alois Alzheimer following clinical and neuropathological examination of a 51-year old patient with dementia [1]. Once considered to be a rare disorder, AD is now understood to be the leading cause of dementia in the elderly, affecting approximately 5.5 million individuals in the Unites States, and one in every nine Americans over the age of 65 [2, 3]. AD is also the 6th leading cause of death in the US, with an average time of 3.8 years between diagnosis and death [4]. The majority of AD cases (82%) occur in those age 75 or older, with prevalence reaching 32% by age 85 [3, 5]. In the context of increasing life spans, AD represents a growing problem for healthcare systems. AD exerts a massive personal and financial burden on hospitals, caretakers, and families, and this crisis is

expected to worsen, based on the prediction that the number of AD patients will triple by 2050 [5,

6]. Current therapeutic options for AD are highly limited; pharmacological treatments can alleviate symptoms but are unable to prevent the progression of the disease [7]. Thus, a more complete understanding of the origins, risk factors, and pathogenesis of AD is imperative to developing more effective diagnostic tools and therapeutic interventions.

AD is characterized by memory loss, and a gradual, but progressive onset [8, 9].

Diagnosis of "probable AD" can be made based on clinical examination, but AD can only be confirmed based on post-mortem analysis of neuropathology [10-12]. Pathologically, the key markers of AD observed in the brain include: 1) Senile plaques [13-15], which are extracellular inclusions formed primarily of amyloid-β (Aβ); 2) intracellular neurofibrillary tangles (NFTs) formed from hyperphosphorylated tau protein (p-tau) [16-19]; and 3) progressive neuronal death beginning in the hippocampus, and progressing throughout the cortex and other regions of the brain [20, 21]. Biomarker studies indicate that pathological markers typically appear in a sequential manner over time; beginning with amyloid, which is followed by changes in p-tau, and then followed by synaptic loss and ultimately neuronal death [22, 23]. The long (~20 year) period between the initial changes in amyloid biomarkers and the observation of behavioral/memory impairments [24] illustrates that AD pathology develops for many years, reaching advanced stages before symptoms are noticed, representing a fundamental obstacle for therapeutic interventions.

AD can be caused by genetic and non-genetic factors [9]. Early-onset dominantly inherited (EODI) AD and familial AD (FAD) are caused by mutations in the genes encoding amyloid precursor protein (APP) [25], presenilin-1 (PS1) [26], and presenilin-2 (PS2) [27-29]. The discovery of these genetic causes, and further study in both in vitro and in vivo models, provided evidence for the "amyloid hypothesis" of AD, which contends that defects in the production of

APP lead to the accumulation of Aβ, causing pathological changes in tau phosphorylation and neuronal death; thus driving the progression of AD [30-32]. In addition to genetic studies

implicating APP processing in the pathogenesis of AD [33], the amyloid hypothesis has also been supported by multiple lines of evidence that Aβ drives tau pathology during AD [34-39], as well as the discovery of synaptic damage being caused by Aβ [40-43]. Further research has clarified the amyloid hypothesis, demonstrating that the soluble, oligomeric forms of Aβ40 and Aβ42 are the primary toxic species [43-48], while insoluble amyloid plaques are considered to be less toxic

[49]. Additionally, recent patient data places changes in cerebral and CSF amyloid as the earliest biomarkers in the development of AD [23].

However, the amyloid hypothesis has not been proven as a complete explanation for the neuropathology of AD, particularly with consideration for late-onset sporadic AD (LOSAD), the predominant form of the disease [50]. Unlike EODI and FAD, LOSAD is currently thought to be caused by a combination of environmental [51, 52], genetic [53, 54], and epigenetic factors [2, 55,

56]. Principally, animal models of AD, which are largely based on mutations in APP, PS1, and

PS2, do not completely recapitulate the other pathological hallmarks and behavioral symptoms of

AD observed in human patients [57, 58]. Alternatively to amyloid, the development of neurofibrillary pathology has been found to associate more closely with the cognitive defects observed in AD than amyloid plaque [59]. Targeting amyloid in the clinic has also been ineffective thus far in combating AD, as the vast majority of drugs and treatments tested in clinical trials based on reducing amyloid (beta-secretase inhibitors, anti-amyloid immunotherapy) have not been successful [60]. It is worthy of note that many of these trials were conducted in patients with later stages of AD, and the current approach is focused on earlier intervention in hope of improved outcomes [9, 60]. While amyloid is currently the earliest detectable AD biomarker [23], and a well-recognized part of AD pathology [15], the aforementioned results cast doubt on the amyloid hypothesis, as changes in amyloid metabolism may not be the initiating factor which fully explains LOSAD [50, 61, 62]. Thus, multiple alternative or complementary hypotheses have been put forth to explain the cause and progression of AD.

Multiple initiating factors independent of amyloid have been proposed to be the fundamental cause of LOSAD. Defects of neuronal autophagy [63], mitochondrial failure [64-66], oxidative damage [67, 68], inflammation [69-71], and insulin signaling [72] have all been proposed as primary causes of LOSAD. Another potential mechanism behind AD pathogenesis is the "two-hit" hypothesis; that multiple types of stress accumulate to stimulate neurons into entering a pathological state [73, 74]. It is conceivable that AD is a more heterogeneous disease than originally considered, and that the pathological markers observed are the endpoints of multiple stressors that affect neuronal health throughout aging, and are particularly exacerbated in AD. This view of AD progression also implies that multiple types of pathology occur over time, stemming from the initiating dysfunction as the disease worsens.

One explanation of neuronal dysfunction during AD is that dysregulated functions of cell cycle proteins in neurons contribute to long-term neuronal damage and ultimately cell death [75-

80]. Cell cycle proteins are primarily known for their roles in mediating replication of DNA, cytoskeletal changes during cell division, and passage of mitotic cell types through the cell cycle to produce two daughter cells. Cell cycle proteins are a diverse group of proteins including cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, the E2F family of transcription factors, retinoblastoma protein, and the p53 tumor suppressor family, among others [81, 82]. Although mature neurons have exited the cell cycle, the actions of cell cycle proteins are not entirely repressed in neurons, and many cell cycle proteins have been demonstrated to perform neuron- specific functions [83]. Cell cycle proteins maintain a variety of non-mitotic functions in mature neurons [84], including roles in synaptic plasticity [85, 86], growth of axons and dendrites [87], and neuronal death [88, 89]. Initial evidence for a role for cell cycle proteins in AD was first shown in studies on tissue from AD patients, demonstrating ectopic expression of several cell cycle proteins in neurons, including p53, E2F1, Cdk1, Cdk4, Cyclin D, Cyclin E, p21, PCNA

(proliferating cell nuclear antigen), p16, and Ki-67, suggesting that the dysregulation of neuronal cell cycle proteins is associated with the pathogenesis of AD [76, 90-97]. Studies on mild

cognitive impairment (MCI), often considered a developmental stage of AD, have shown that these alterations to neuronal cell cycle proteins occur early in the disease process, and promote neuronal death [80, 98, 99]. Several cell cycle proteins have been shown to be increased in patients with MCI, including p53, Cdk2, Cdk5, and cyclin G1, lending further evidence to cell cycle dysregulation as an early event in the development of AD [100-102]. It is thought that a significant percentage of the neuronal loss in AD is caused by dysregulated cell cycle events [103]. In addition to changes in the expression of cell cycle proteins, there is evidence that these changes lead to cell cycle re-entry in post-mitotic neurons; the increased number of aneuploid neurons in the AD brain suggests that post-mitotic neurons re-activate DNA replication machinery [104, 105], which renders neurons increasingly vulnerable to death over time [100, 106, 107]. As observed with neuronal cell cycle proteins, DNA replication in neurons is also observed in MCI [100].

Since the initial observations in patients, evidence from animal models and in vitro systems has given support for a "cell cycle/mitotic stress hypothesis" of AD [108]. Changes in neuronal cell cycle proteins have been connected with the major pathological hallmarks of AD:

Aβ-mediated neuronal damage, hyperphosphorylation of tau, and neuronal death [103]. First, four different transgenic mouse models of AD display similar changes in the activity of neuronal cell cycle proteins as those observed in human AD, including changes in cyclin A and aneuploid neurons, which preceded the appearance of amyloid plaques [109]. A subsequent study identified patterns of spatial and temporal changes in neuronal cell cycle proteins in the brains of five different mouse models of AD [110]. Aβ oligomers have been shown to drive neuronal cell cycle events, including DNA replication, in a murine AD model and in cultured neurons [111]. In vitro studies show that these inappropriate neuronal cell cycle events can be induced by amyloid peptide and mediated by Cdk5 [112, 113], GSK3-β [114], as well as the PI3K-Akt-mTOR pathway

[115]; all of which have been implicated in AD [116-119]. Together, these results strongly support the involvement of neuronal cell cycle proteins in amyloid-induced neuronal dysfunction in the early stages of AD.

In addition to amyloid, changes in neuronal cell cycle proteins are also related to aberrant phosphorylation of tau [120], potentially positioning cell cycle proteins as mediators of amyloid- driven tau phosphorylation which occurs throughout the progression of AD [39, 121]. Neuronal cell cycle events were observed in htau mice, which overexpress wild-type human tau and display neurofibrillary pathology [122]. AAV-mediated introduction of tau into the hippocampus of wild- type mice was also found to promote neurodegeneration in conjunction with dysregulation of cell cycle markers, without the formation of tangles [123]. In vitro studies have also connected the activity of cell cycle proteins to tau phosphorylation [124]. A central culprit in tau hyperphosphorylation is Cdk5, itself a neuron-specific member of the cyclin-dependent kinase family and suppressor of the neuronal cell cycle [125-127], which maintains a variety of functions in neurons unrelated to cell cycle regulation, including regulation of neuronal development, neurite outgrowth, and synaptic functions [118, 128, 129]. Cdk5 hyperactivity has been implicated in tau hyperphosphorylation as well as neuronal death in AD [118, 130, 131], and Cdk5 acts as a linking factor between amyloid and subsequent neuronal cell cycle events [112] and tau hyperphosphorylation [132-135]. The role of Cdk5 in AD is strongly supported by studies demonstrating amyloid-induced Cdk5 hyperactivity through calpain-mediated generation of its p25 subunit in cultured neurons [136] and in transgenic animals [137, 138]. Pathological functions of Cdk5 are also supported by evidence from inducible p25 mice, in which the p25 subunit of

Cdk5 is selectively expressed in the brain, leading to development of neurofibrillary pathology and a neurodegenerative phenotype [139]. Interestingly, one study determined that tau is a necessary component in driving amyloid-induced cell cycle events in cultured neurons [140].

While there is abundant evidence for amyloid as the initiating stimulus which drives neuronal cell cycle dysregulation, tau and Cdk5 appear to play essential roles in the process as well. But further studies are necessary to fully understand the interplay between amyloid, tau, and cell cycle re-activation in the context of neurodegeneration in AD.

As key mediators of cell fate across different cell types, it is not surprising that cell cycle proteins also mediate neuronal cell fate decisions. Neuronal cell cycle events render neurons particularly vulnerable to cell death [89, 106], and this process is potentially mediated by cell cycle-related proteins such as p53 and E2F1, which promote a variety of pro-apoptotic functions through pro-apoptotic gene transcription as well as mitochondria-dependent mechanisms in multiple models [141-143]. It is hypothesized that DNA replication in post-mitotic neurons could lead to p53 activation, as p53 protects the genome from errors by activating programmed cell death [144, 145]. Studies in animal models also suggest that inflammation, which is implicated in

AD [69-71], stimulates dysfunction of neuronal cell cycle proteins in AD [146, 147]. These observations correlate with studies indicating reduced risk for AD in patients given high-dose

NSAIDs [148]. Unfortunately, the results in animal models suggest that that NSAIDs can prevent pathological cell cycle reentry, but cannot reverse the process once it has begun [146], which may explain the lack of improvement in clinical tests of NSAIDs [149].

While dysregulated functions of neuronal cell cycle proteins are not the current primary focus of drug development in AD, the evidence supporting the cell cycle hypothesis of AD is compelling, particularly the observation that ectopic expression of cell cycle proteins and neuronal

DNA replication is an early event in AD pathogenesis [100-103]. Further understanding of cell cycle proteins and their functions in post-mitotic neurons could uncover new therapeutic angles by which we may be able to prevent or slow the progression of AD.

1.3: CELL CYCLE PROTEINS IN HIV-ASSOCIATED NEUROCOGNITIVE DISORDER (HAND)

According to the World Health Organization (WHO) the Human Immunodeficiency Virus

(HIV) affects 35 million people worldwide (as of 2012) [150], and is the cause of Acquired

Immunodeficiency Syndrome (AIDS). The progressive infection of immune cells by HIV leads to destruction of host cells and damage to the immune system over time. Because of this, patients with AIDS become vulnerable to "opportunistic" infections which are often fatal. Prior to the

development of highly-active antiretroviral therapy (HAART) [151], The life expectancy of an untreated AIDS patient was as short as 20 months [152]. Today, because of HAART, the lifespan of the average HIV patient has significantly increased, largely due to suppression of HIV replication in the periphery, but lack of treatment remains a major concern; as HIV/AIDS was still responsible for 1.6 million deaths worldwide in 2012 [150, 152, 153]. HIV is a retrovirus which infects T lymphocytes cells via CD4 receptors, and utilizes the transcriptional machinery of the host cell to reproduce [154]. These activities lead to the destruction of host cells, and reduce the

T-cell count, thus, damaging the immune system [155]. In addition to its harmful effects on the immune system, HIV has also been shown to cause damage to the CNS [156, 157]. HIV- associated dementia (HAD) was observed in up to 30% of HIV-infected patients before the advent of HAART, but in the current post-HAART era, the primary neurological condition associated with HIV infection is a collection of impairments collectively termed HIV-associated neurocognitive disorder (HAND) [158]. HAND is characterized clinically by cognitive, behavioral/emotional, and motor dysfunctions, and manifests as a spectrum of deficits that may involve impairment of one or more of these domains [159]. Despite the success of HAART in suppressing HIV, HAND remains a serious and growing problem for HIV-infected individuals, considering that HAND affects 30-50% of patients in the post-HAART era [160]. Although HAART reduces plasma viral load [161], it has been shown that HIV is still able to maintain viral reservoirs in the CNS. In the post-HAART era, the incidence of HAND has decreased, but the prevalence has increased, partially because of the longer average lifespan of HIV-infected individuals [162].

Despite the vast benefits of HAART, HAND remains a serious problem moving forward for HIV- infected individuals, and there is a need for CNS-targeted therapeutic interventions complementary to HAART [163].

HAND is characterized pathologically by inflammatory markers, astrogliosis, myelin pallor, and the presence of multinucleated giants cells (derived from macrophages) [164].

Neuronal loss is observed in HAND, but neuronal death is not considered the major contributor to

HAND; rather, damage to synapses is implicated as the key factor underlying cognitive deficits

[165, 166]. Additionally, some studies have noted pathological markers similar to those observed in AD [167, 168]. Peripheral monocytes, which can cross the blood-brain-barrier (BBB), are the main carrier of HIV into the brain, initiating the neurotoxic effects of HIV. As a "Trojan Horse" mechanism, these monocytes can differentiate into macrophages after entering the brain parenchyma, and subsequently infect resident cell types of the brain [159, 169]. However, neurons are not infected by HIV due to the lack of the necessary HIV co-receptors (CD4).

Instead, HIV (secreted from infected monocytes) is able to productively infect a subset of glial cells in the brain, including microglia and astrocytes, although astrocytes are not productively infected, integrating the HIV genome into chromosomal DNA and silencing expression of the viral transcripts [170, 171]. HIV-infected monocytes/macrophages, as well as HIV-infected microglia, can release a variety of harmful factors, including: reactive oxygen species, glutamate, inflammatory cytokines, quinolinic acid, and viral proteins such as gp120 and tat [156, 172].

These factors are released into the extracellular space, leading to a toxic extracellular environment which causes synaptic damage and neurotoxicity via NMDA-receptor-mediated excitotoxic damage [173].

Post-mitotic neurons utilize cell cycle proteins for a variety of functions not related to mitosis (see section 1.2). A pertinent role of cell cycle proteins in post-mitotic neurons is their regulation of neuronal death pathways during neurodegenerative diseases. Altered expression, localization, and function of cell cycle proteins have all been observed in brain tissue in neurodegenerative diseases such as AD [89, 103, 174]. In HAND, there is also evidence for inappropriate activation of cell cycle proteins in neurons [175-180]. These lines of evidence indicate that dysfunctional cell cycle proteins could mediate the neuronal damage observed in

HAND. Observations of neurofibrillary [167] and amyloid pathology [168] in HAND also suggests dysregulation of common factors between AD and HAND. Intriguingly, many of the same cell cycle proteins (Cdk5, E2F1, p53) which are dysregulated in AD and also aberrantly expressed in

HAND, suggesting that neuronal cell cycle events are not a disease-specific phenomenon, but rather, a more general response to toxic stimuli. Evidence for dysregulation of Cdk5 via increased

Cdk5:p25 complex formation, which contributes to tau hyperphosphorylation and neurodegeneration in AD [181], is also observed in brain tissue of HAND patients [182], while neurotoxic effects of calpain-mediated p35 cleavage to p25 are observed in in vitro models of

HAND-induced neurotoxicity [182]. The cell cycle regulator and transcription factor, E2F1 [183], is ectopically expressed in AD tissue [97, 184], and neuronal E2F1 expression is increased across multiple regions of the brain during HAND [177]. Increased neuronal E2F1 is also observed in simian models of HAND [176, 177], and is accompanied by increased expression of the retinoblastoma susceptibility gene product (pRb), another key cell cycle protein which becomes dysregulated (in its hyperphosphorylated, inactive state; ppRb) in neurons in both HAND [176-

178] and AD [97, 185]. Finally, another key cell cycle regulator, the p53 tumor suppressor (see section 1.4) [186], is a transcription factor known for pro-apoptotic functions in neurons [145], which is also increased in brain tissue during HAND [176, 179, 187] and AD [94, 96, 188, 189]. It is thought that these changes in cell cycle proteins represent a fundamental dysregulation of cell cycle machinery in post-mitotic neurons during neurodegenerative diseases, such as AD and

HAND. Many cell cycle proteins, such as p53 and E2F1 [142], are known to regulate broad cellular functions, interacting extensively with intricate regulatory networks of proteins, many of which are primarily studied in dividing cell types. Through further research on the functions of cell cycle-related proteins in mature neurons, novel pathways of neuronal survival may be identified.

Further understanding of how dysregulated cell cycle proteins contribute to long-term neuronal dysfunction and death is a compelling area of research for the development of therapeutic interventions against neurodegenerative diseases.

1.4: p53 AND THE p53/MDM2/MDMX NETWORK

Each cell must be able to maintain its homeostasis while reacting to its extracellular environment. Paramount to homeostasis is the protection of the genome, as the stability and propagation of genetic information is crucial for life. Thus, eukaryotic cells have developed multiple ways to maintain genomic integrity while living in environments with ever-present harmful stimuli. In multicellular organisms, the response to DNA damage is typically cell cycle arrest, which halts the progression of the cell cycle, and allows DNA repair machinery to correct genomic errors before corrupted DNA is replicated. If cell cycle arrest and DNA repair are not sufficient to prevent DNA damage, an apoptotic cell death program is triggered. Together, these mechanisms prevent cells with excessive DNA mutations from dividing and propagating defective genes, thereby protecting the integrity of the genome. Impairments in the intracellular signaling pathways which underlie cell cycle arrest and apoptosis can cause a cell to carry on successive divisions with genomic errors, leading to the development of tumors and potentially cancer.

A critical protein in this process is the tumor suppressor p53 [186, 190]. p53 was initially discovered in 1979 as a binding partner to the SV40 large T-antigen; and was initially touted as the first example of a cellular oncogene interacting with a known viral oncoprotein [191, 192]. This assumption was further supported by observations that p53 was found in multiple types of tumors and tumor-derived cell lines at super-physiologic levels, consistent with the concept of oncogene amplification as part of the carcinogenesis pathway [193]. Additionally, DNA-damaging factors, such as UV radiation, were shown to induce rapid increases in p53. Soon after, genetic studies in p53-/- mice shed light on the functions of p53 in vivo. It was revealed that p53-/- mice are highly prone to developing tumors with age, leading to the conclusion that p53 functions as a tumor suppressor [194]. Since these pioneering discoveries, p53 has been at the focus of the cancer biology field, often referred to as "The Guardian of the Genome" [186]. Roughly 50% of all human cancers harbor mutations in p53 [190, 195], and 70% of human cancer mutations are estimated to affect p53 signaling, making inactivation of the p53 pathway the most common defect in cancer

[196]. Loss-of-function mutations in p53 underlie a large number of cancers [193, 197], and p53

reactivation has become a major target in cancer therapy [198, 199]. However, p53 is now known to have numerous physiologic roles in a diverse range of tissues, and has been implicated in processes from development [200, 201] to aging and neurodegenerative disease [145, 202].

Our best understanding of p53 indicates that it acts as an arbiter of cell fate, which executes its duties by receiving a large amount of signaling inputs [203], and responding through a wide variety of transcriptional and non-transcriptional outputs [204]. p53 regulates multiple functions related to cell cycle progression, cellular proliferation, differentiation, metabolism [205], mitochondrial function [206, 207], DNA repair, senescence, and cell fate determination [208]. p53 functions as a transcription factor, allowing it to regulate the gene expression of multiple genes, including apoptotic regulators, PUMA, BAX, and the cyclin-dependent kinase inhibitor, p21 [209].

Many of the p53-dependent mechanisms of cell death also involve destabilization of the mitochondria [207]. p53 can activate the transcription of pro-apoptotic genes, such as BAX, which lead to mitochondrial damage and cell death [210], and p53 can interact with the mitochondria directly and trigger apoptosis in a transcription-independent manner [211]. p53 is typically localized to the nucleus, but can be shuttled into the cytoplasm and is also able to interact with mitochondrial proteins [207]. p53 has an N-terminal transactivation domain which mediates its interactions with DNA [204], and is typically found in tetrameric conformations when interacting with DNA and transcriptional machinery. The tetrameric organization of p53 appears to be integral to its transcriptional functions, and may explain why p53 mutants have such a potent impact on p53 function in cancer [212]. p53 is the most well-known member of the "p53 family" which includes homologues p63 and p73, both of which are structurally similar to p53, but maintain functions which are more closely related to development and cell differentiation [213-

215]. p53 is also subject to alternative forms of transcription, leading to the expression of four p53 isoforms, all of which are truncated versions of the full-length p53 protein [216, 217].

Because of the widespread impact of p53 on a variety of cellular functions, and its potency towards determining cell fate, p53 must be responsive to changes in cellular

homeostasis which occur throughout the lifetime of a cell, allowing it to react appropriately to stress. The sensitivity of p53 is maintained by an intricate network of p53-regulating proteins

[203]. The primary negative regulator of p53 is the E3 ubiquitin ligase murine double minute 2

(MDM2) [218, 219]. MDM2 was originally discovered as a gene which was amplified 50-fold in a spontaneously transformed 3T3DM murine cell line, and it was subsequently shown that overexpression of MDM2 in NIH3T3 and Rat2 cell lines induced tumorigenicity, demonstrating

MDM2 as an oncogene [218]. The gene was named due to the location of the MDM2 locus on extrachromosomal "minutes". The mechanism by which MDM2 promotes tumorigenesis was later found to occur through negative regulation of p53 [220, 221]. This p53-regulating function of

MDM2 has since been rigorously studied in vitro and in vivo, and MDM2 has also been found to be amplified in a variety of human cancers which exhibit wild-type p53 [222]. MDM2 has also been shown to be essential for development, as mdm2-/- mice display embryonic lethality, but this lethality is prevented when mdm2-/- mice are crossed with p53-/- mice [223, 224], further implicating MDM2 as a critical p53 regulator. MDM2 can inhibit p53 through several mechanisms

[222, 225]. MDM2 can directly bind p53 via its N-terminal p53-binding domain, disrupting p53-

DNA interactions and preventing p53 transactivation [220]. MDM2 also has NLS/NES sequences, and is able to shuttle p53 out of the nucleus, preventing p53-mediated transcriptional functions.

Additionally, as an E3 ligase, MDM2 is able to ubiquitinate p53 via its RING domain [226], which promotes the degradation of p53 by the proteasome [227]. The ubiquitination capability of MDM2 is enhanced by the ability of MDM2 to facilitate the interaction between ubiquitinated p53 and the proteasome [228], further expediting the proteasomal degradation of p53.

MDM2 was also shown to be one of the transcriptional targets promoted by p53, allowing for a negative regulatory feedback loop whereby excessive p53 activity can induce increased expression of MDM2 via interaction with the mdm2 promoter [229], balancing the levels of both proteins to prevent tumorigenesis (caused by hyperactive MDM2) or premature apoptosis

(caused by hyperactive p53). This feedback mechanism underlies an oscillation between MDM2

and p53 which occurs in dividing cells [230]. This relationship with p53 makes MDM2 an avenue by which various cellular factors can exert changes in p53 function, thus endowing p53 with sensitivity to a wide variety of cellular events. Under stress conditions, MDM2 becomes proteolytically degraded [231, 232], leading to increased p53 activity. The degradation of MDM2 can be mediated by caspase-2 cleavage of MDM2 at its 367DVPD370 caspase-recognition sequence [233]. Anti-oncoproteins, such as p19/ARF and ATM, can inhibit MDM2 as a mechanism of indirectly activating p53 [234].

MDMX (murine double minute X; also known as MDM4 or HDMX/HDM4 in humans) was originally discovered as a homologue of MDM2 (see section 1.5 for further discussion of MDMX)

[235, 236]. As an MDM2 homologue, MDMX possesses similar p53-regulating functions as

MDM2, but unlike MDM2, MDMX is not able to ubiquitinate p53 in cells [237]. MDM2 and MDMX have overlapping, but distinctly different functions in the regulation of p53 [39]. Broadly, MDM2 is involved in regulating the stability of p53, while MDMX regulates p53 activity [214]. Both act as p53 inhibitors, but MDMX regulates p53 primarily through direct binding, which prevents p53 transactivation. MDMX is also able to inhibit p53 function by increasing the ubiquitin ligase activity of MDM2. MDM2 and MDMX form heterodimers through interactions of their RING domains [238,

239], and these heterodimers are able to ubiquitinate p53 more effectively than MDM2 homodimers. This interaction between MDM2 and MDMX is critical for effective p53 regulation during development [240].

In clinical research, MDM2 and MDMX are implicated as pro-oncogenes, which promote cancer through inhibition of p53 [222, 225]. The role of MDM2 and MDMX in cancer is supported by the observation that the frequency of MDM2/MDMX protein deregulation is greater in tumors with wild-type p53 than tumors with mutant forms of p53 [222]. Deregulation of MDM2 and

MDMX, primarily in the form of amplifications, is found in a variety of different human cancers

[222, 241]. These observations have led to the undertaking of drug discovery programs to interfere with p53-MDM interactions [242]. Out of the several pharmacological compounds found

to disrupt p53-MDM2 interactions, Nutlin-3 was found to promote apoptosis in cancer cell lines

[243, 244] and mouse models, and has been tested in clinical trials for the treatment of retinoblastoma [245]. Thus, further understanding of the p53/MDM2/MDMX network is a major prospect for drug development in the cancer field.

The p53-MDM2-MDMX network has evolutionary roots reaching back roughly 780 million years [246, 247]. MDM2 and MDMX are both present in the majority of vertebrate species, while seven invertebrate species only possess the one MDM gene [246]. Comparative genetic studies have shown that the first appearance of a "p53 family" gene was in unicellular flagellates over one billion years ago, and the function of this primitive p53 in unicellular flagellates is protection of the genome, a function which p53 maintains all the way through modern vertebrates [248].

Selection pressure caused a differentiation of p53 forms (into p53, p63, and p73) over time around 440 million years ago, evidenced by the fact that almost all jawed vertebrates possess the three p53 forms [249]. While some invertebrates possess p53 family members without an MDM homolog, these genes were later accompanied by a p53-regulating "proto-MDM" in placazoans

(sharing a common ancestor with humans 780 million years ago) which eventually became

MDM2 and MDMX due to a duplication event, prior to the appearance of bony vertebrates roughly

440 million years ago [246]. It is thought that this "proto-MDM" evolved to prevent inappropriate cell death mediated by p53. Interestingly, there is evidence that co-adapting point mutations in p53 correspond with changes in MDM proteins over time, suggesting a co-evolution of the genes underlying the p53-MDM2-MDMX network [247].

1.5: THE MDMX ONCOPROTEIN

Originally discovered as a homologue of MDM2, MDMX was found to be a p53-regulating protein with a high degree of structural/sequence similarity to MDM2, but with notably different functions [225, 235, 236, 250-252]. MDMX has been a protein of interest in cancer biology because it is frequently found to be amplified in various types of tumors [253]. Since the initial

detection of MDMX amplification and overexpression in malignant gliomas in the absence of p53/MDM2 mutations [254, 255], overexpression of MDMX has been found in cancers of the colon, lung, stomach, larynx, testicle, breast, and uterus; as well as melanoma [256]. MDMX gene amplification is also observed in soft-tissue sarcomas [257], astrocytic tumors [258], retinoblastoma [259], head and neck squamous carcinoma [260], hepatocellular carcinomas

[261], and urothelial cell carcinoma [262]. These changes are typically found in tumors without mutations in MDM2 or p53, strongly suggesting that gain-of-function amplifications of MDMX are sufficient to induce cancer in the absence of p53 mutations [254, 256, 263], earning MDMX the title of a pro-oncoprotein.

MDMX was identified as a 490 amino acid protein with a high degree of homology with

MDM2 [235], which is ubiquitously expressed across different cell types [236, 264]. Like MDM2,

MDMX is a RING finger protein and a structural E3 ubiquitin ligase. But unlike MDM2, MDMX lacks intrinsic E3 ligase activity, and is not able to ubiquitinate targets in cells [237, 265]. MDMX migrates at 80 kDa in most cell types, which is higher than its predicted molecular weight. This discrepancy is largely due to post-translational modifications of MDMX, which include glycosylation, SUMOlaytion, phosphorylation, acetylation, and ubiquitination [253]. MDMX is also subject to differential splicing, and truncated MDMX splice variants are detected in cells [264,

266, 267]. In mitotic cells, MDMX is typically localized in the cytoplasm, but is able to shuttle between the nucleus and cytoplasm, in a process largely dependent upon interaction with MDM2 or other shuttling proteins, as MDMX lacks a NLS sequence [268]. A significant portion of cytoplasmic MDMX is localized in the mitochondria (10% in A2780 cells; 29% in RKO cells), where it is found within the outer membrane, and mediates a pro-apoptotic function dependent on the regulation of mitochondrial p53 and its interaction with Bcl-2 [269, 270]. The regulatory mechanisms of mitochondrial MDMX are not know, but it is thought that mitochondrial MDMX exists in balance with cytoplasmic MDMX, as siRNA knockdown of MDMX reduces MDMX in the mitochondrial fraction [269, 270].

The most well-known functions of MDMX relate to the regulation of p53. MDMX functions in a cooperative manner with MDM2 to negatively regulate p53 [271], but MDMX-mediated regulation of p53 can also occur in an MDM2-independent manner [250]. In the absence of

MDM2, MDMX is able to bind p53 and prevent p53 transactivation via an N-terminal p53-binding domain, leading to reduced expression of p53 target genes [235]. Additionally, MDMX is able to shuttle p53 out of the nucleus, further preventing p53 transactivation. Despite some controversy on the ability of p53 to regulate transcription of MDMX mRNA, recent studies have indicated that p53 does indeed mediate MDMX expression [272] (similar to the MDM2-p53 feedback loop

[273]). Beyond its typical pro-survival functions, MDMX has been shown to regulate the activity of mitochondrial p53, but in a pro-apoptotic manner [269, 270]. Genetic studies have shown that

MDMX is necessary for proper development, evidenced by the embryonic lethality of mdmx-/- mice[274]. However, crossing mdmx-/- mice with p53-/- mice rescues this phenotype, and these double knockout animals have none of the deficits observed in mdmx-/- mice [274, 275]. This discovery strongly supports the argument that the functions of MDMX are p53-dependent, and also that MDMX promotes specific functions, which cannot be fully compensated by MDM2, indicating non-overlapping mechanisms of p53 regulation by MDM2 and MDMX.

MDMX is targeted by multiple enzymes which modify its function, often as a mechanism of regulating p53; including c-Abl [276], Cdk2 [277], and Akt [278], which reduce the p53-binding ability of MDMX. Conversely, CK1α-mediated phosphorylation of MDMX promotes MDMX-p53 interaction [279]. Additionally, MDMX is regulated by ubiquitination by MDM2 and PIRH2 [280,

281]; and ubiquitination of MDMX can be mediated by the ARF tumor suppressor [282]. The activities of these ubiquitin ligases are opposed by HAUSP and USP2a, which de-ubiquitinate

MDMX [283, 284]. Like MDM2, MDMX can also be cleaved by caspases at a DVPD recognition site, and both full-length MDMX and cleavage products are both subject to proteasomal degradation [285]. MDMX degradation is particularly relevant to p53 activation by stress factors; under conditions of genotoxic stress, MDMX is ubiquitinated by MDM2, and is then degraded by

the proteasome, thereby reducing MDMX activity and simultaneously activating p53 and reducing the activity of MDM2 [250, 286]. Phosphorylation of MDMX also contributes to its stability, as phosphorylation of MDMX at S367 promotes p53 activation by targeting MDMX for ubiquitination by MDM2 and subsequent degradation [287]. 14-3-3 has also been shown to bind MDMX when

MDMX is phosphorylated under DNA damage conditions, triggering p53-dependent apoptosis

[278, 288-290]. MDMX can be phosphorylated at multiple sites by ATM and Chk2 kinases following DNA damage [291]. Altogether, post-translational modifications of MDMX, particularly ubiquitination and phosphorylation, appear to be closely linked with MDMX degradation. Because

MDMX negatively regulates p53, stress-induced MDMX degradation is a mechanism of p53 activation and apoptosis.

In addition to negative regulation of p53, MDMX also mediates p53-independent functions, interacting with a variety of other proteins [292]. MDMX forms heterodimers with

MDM2, increasing the ubiquitin ligase ability of MDM2 and facilitating enhanced p53 regulation

[238] (described in section 1.4). MDMX negatively regulates the transcription factor, E2F1 [293-

295]. E2F1 is a cell cycle protein which, like p53, can act as a transcription factor and promote apoptosis [142, 296]. MDMX binds E2F1, thereby reducing E2F1-DNA interaction [293], and

MDMX is also shown to reduce E2F1 transactivation [294] (the MDMX-E2F1 relationship is discussed further in section 1.7). MDMX regulates the cyclin-dependent kinase inhibitor, p21, via promotion of proteasomal degradation during the progression of the cell cycle [289], and MDMX also negatively regulates SMAD transactivation, potentially connecting MDMX to the TGF-β signaling pathway [297, 298]. Further demonstrating p53-indepdentent functions of MDMX,

MDMX was shown to promote multiple functions related to DNA damage and chromosomal stability (e.g. centrosome clustering and bipolar mitosis) in p53-null cells [299-301]. While the majority of studies on MDMX address its p53-regulating capabilities, MDMX maintains a wider repertoire of functions related to cell cycle regulation and DNA damage.

1.6: FUNCTIONS OF p53 IN NEURONS

Neurons, like all eukaryotic cells, still possess the necessary proteins needed to activate

DNA repair mechanisms [302], as well as programmed cell death (apoptosis). Because mature neurons do not divide, one might ask why neurons require DNA repair mechanisms or apoptotic mechanisms? Whether neurons have these signaling pathways as artifacts left over from earlier stages in development, or if neuronal apoptotic programs exist as a favorable alternative to nonapoptotic cell death is not known. One hypothesis is that damaged or defective neurons are best removed through apoptosis and subsequent digestion by microglia, rather than facing the problem of a defective neuron interfering with synaptic signaling of the entire network of connected neighboring neurons or causing "bystander" cell death of neighboring neurons.

Regardless of origin, p53 plays significant roles in neuronal function and neuronal cell fate determination in addition to its well-established roles in cancer [303].

p53 has been demonstrated to mediate neuronal death in a variety of models [143, 304].

Predominantly studied for its pro-apoptotic effects in dividing cells, it was later shown the p53 can promote apoptosis in post-mitotic neurons as well [305-307]. p53 is implicated in pro-apoptotic pathways which are activated during a variety of neurological injuries (stroke [308, 309], ischemia

[310, 311], traumatic brain injury [312], spinal cord injury [313, 314]), and in neurodegenerative diseases (Alzheimer Disease [145, 315], Parkinson Disease [316-318], and amyotrophic lateral sclerosis [319]). Middle cerebral artery occlusion and kianic acid-induced neurotoxicity both cause p53 induction in damaged brain areas [320, 321]. Additionally, overexpression of p53 in cultured hippocampal neurons induced neuronal death and the appearance of apoptotic markers [305].

The ability of p53 to mediate neuronal cell death brought p53 into the crosshairs of drug development research [322]. A small-molecule inhibitor of p53, pifithrin-α (PFT-α) was synthesized to test the neuroprotective potential of p53 inhibition. PFT-α was able to protect cultured neurons from a variety of toxic insults, including DNA damaging agents, glutamate, and

Aβ [323-325]. Remarkably, mice given PFT-α exhibited increased neuronal survival in models of focal ischemic injury and excitotoxic injury [323].

Considering its role in neuronal death, it is not unexpected that excessive neuronal p53 is observed in neurodegenerative disease. p53 is increased in the brain tissue from AD patients [94,

96, 101, 188, 189, 326], notably in the superior temporal gyrus [189] in the inferior parietal lobe

[101], a region known to display oxidative, as well as neurodegenerative pathology during AD

[327]. p53 is also increased in the brains of patients with MCI [101, 326], suggesting that changes in p53 activity occur early in the progression of AD. The increases in p53 were associated with markers of oxidative stress in both MCI and AD [101, 326], suggesting that p53 activation is caused by, or contributes to, oxidative damage during the early stages of AD. Post-translational modifications to p53, rather than direct change in p53 level, may be a contributor to pathological p53 activity, suggesting that mechanisms of p53 regulation in neurons is affected during AD.

Abnormal glutathionylation of p53 has been observed in tissue from AD patients [328]. This study also noted that p53 from AD brain tissue is significantly more monomeric/dimeric than p53 from control patients; this could indicate a potential mechanism of how normal tetrameric formation of p53 is impaired in AD, preventing the normal transcriptional functions of p53 and contributing to downstream neuronal dysfunction [328]. Other conformational alterations and modifications of p53, such as oxidative modification, have been associated with the progression of MCI towards

AD [101, 326], suggesting that p53 contributes to early changes underlying cognitive decline. p53 is also increased in brain tissue in murine AD models [188, 329]. Studies on peripheral changes in p53 folding observed in MCI and AD patients has led to studies addressing the potential of unfolded p53 as a predictive blood-based biomarker for MCI conversion to AD [330, 331].

Similarly, another group identified increased amounts of unfolded p53 in fibroblasts from patients with sporadic AD [332]. Altogether, there is abundant evidence that p53 is increased and altered in the brain during AD, which could have implications for AD pathogenesis, and may even allow for the development of p53-based biomarkers.

Mechanistic studies have implicated p53 in Aβ-associated neuronal death, as well as amyloid processing in animal and cell culture models of AD [145]. p53 can mediate Aβ-induced neuronal death through transcription-dependent and transcription-independent mechanisms

[143]. Cultured neurons lacking p53 are resistant to excitotoxicity and DNA damage [333].

Additionally, inhibition of p53 with PFT-α prevented Aβ-induced mitochondrial damage and caspase activation, and neuronal death in cultured hippocampal neurons [323, 334]. Intriguingly, p53 has been shown to play a role in Aβ-induced disruption of lysosomal processing in neurons, based on Aβ-induced lysosomal destabilization, cathepsin-L activation, and subsequent cell death all being prevented by pifithrin-α or knockdown of p53 [335]. One study determined that intracellular Aβ42 and Aβ40 can activate the p53 promoter and increase p53 expression as an apoptotic mechanism in cultured neurons [188]. p53 can mediate neuronal death downstream of

Aβ42 toxicity through transcriptional upregulation of PUMA [336]. Experiments performed in cultured human neurons demonstrated that Aβ42 (but not Aβ40) peptides activate a p53-dependent programmed death pathway mediated by BAX and caspase activation [337]. Independently of direct cell death-related functions, p53 binds the amyloid precursor protein (APP) promoter and regulates APP expression [338]. p53 also regulates (and is itself regulated by) multiple members of the gamma-secretase complex [339-341]. These observations indicate that p53 can contribute to the progression of AD pathology without necessarily mediating neuronal death. The involvement of p53 in both the processing of amyloid, and amyloid-induced neuronal death, makes p53 an intriguing target for therapy in AD.

p53 has also been implicated as a mediator of neuronal damage HIV-associated neurocognitive disorder (HAND) [307]. One study determined that p53 was upregulated in brain tissue from HAND patients, and that wild-type p53 is necessary for HIV-induced neuronal death, and for the production of soluble neurotoxic factors by HIV-infected microglia [179]. p53 is also increased in the basal ganglia in a simian model of HAND [176]. Silencing of p53 via RNAi was shown to prevent HIV-1 replication [342], which could be mediated by interactions between p53

and HIV-1 viral proteins [343], suggesting a role for p53 in the progression of HIV infection and

HAND. There is also evidence for p53 mediating neuronal death in PD [318]. Neuronal p53 was found to be stabilized in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a model of inducible PD [344]. and the effects of MPTP are prevented by PFT-α [316] or genetic deletion of p53 [345] in vivo. p53 also maintains a functional relationship with Parkin, whereby the two proteins regulate each other [346]. Additionally, increased p53 is detected in brain tissue from

PD patients [347]. Altogether, these studies represent significant evidence that p53 acts as a fundamental regulator of neuronal death in multiple neurodegenerative diseases.

p53 also maintains functions in neurons which are not directly related to neurodegenerative disease. As a broad regulator of cell fate, it comes as little surprise that p53 regulates the proliferation and differentiation of neural progenitor cells in the developing CNS

[348]. p53 signaling is shown to mediate neurite outgrowth and growth cone remodeling by promoting the transcription of several cytoskeleton-remodeling genes [349]. The role of p53 in axonal regeneration is supported by studies which show impaired axonal regeneration in p53-null mice [348]. Despite its original discovery in the context of cancer biology, p53 performs a variety of functions in post-mitotic neurons, particularly in regard to neuronal death in AD. As p53 becomes increased in AD, it is possible that defects of p53 regulation cause the changes observed in tissue samples, and understanding the networks which regulate p53 in post-mitotic neurons could allow for elucidation of pro-survival signaling pathways in neurons.

1.7: FUNCTIONS OF MDMX IN NEURONS

MDMX's p53-inhibiting functions position MDMX as a protein of interest in promoting neuronal survival. While p53 has been implicated a variety of neuronal processes [350], and

MDMX has been studied extensively as a p53-regulating protein in dividing cells [250, 252], there are notably few studies on how MDMX mediates these activities in neurons. However, the previous work MDMX in neurons has yielded several interesting discoveries. The initial studies

addressing the functions of MDMX in the CNS examined the effects of genetic deletion of MDMX in mice. Two separate studies have shown that mdmx-null mice display a p53-depednent embryonic lethal phenotype [274, 275]. One key study published in 2002 by Migliorini and colleagues determined that in addition to embryonic lethality, deletion of MDMX (via "gene trap") in mice leads to excessive apoptosis in the developing CNS (measured by caspase-3 activation)

[351]. These changes were accompanied by improper neural tube closure, dilated lateral ventricles, and altered expression of cell cycle proteins (p21 and PCNA) in the developing CNS

[351]. Interestingly, this phenotype was rescued when the mdm4-/- mice were crossed with p53-/- mice, similar to previous results in non-neuronal cell types [274, 275]. These observations led to the conclusions that MDMX is a critical regulator of p53 in neurons during development, and that neuronal survival decisions mediated by MDMX are completely p53-dependent in the developing

CNS. The changes in PCNA induced by deletion of MDMX also suggest a role in the regulation of cell cycle machinery in neurons. This study was the first to demonstrate in vivo evidence that

MDMX is critical for neuronal survival during development. The implications of this study were that the functions of MDMX in neurons were fundamentally dependent on the negative regulation of p53.

In addition to its relationship with p53, MDMX interacts with E2F1, another transcription factor known to play multiple roles in neuronal function [293, 352]. Following observations by

Strachan and colleagues of MDMX negatively regulating the DNA-binding ability of E2F1 in dividing cells [293], the interactions between MDMX and E2F1 were explored in brain tissue of

SIV-infected macaques [295]. This study determined that increased cytoplasmic E2F1 in neurons was inversely related to neuronal MDMX in this model of HIV-associated neurotoxicity.

Underlying this relationship, the investigators examined how E2F1 induces the degradation of

MDMX protein in vitro, demonstrating E2F1-induced degradation of MDMX in 293 cells. This study also showed that E2F1 can induce degradation of MDMX in a calpain-dependent manner in

293 cells, suggesting calpain activation as a potential mechanism of MDMX regulation in the

brain during neurodegenerative disease. The interactions between MDMX and E2F1 are of particular interest in neurons because E2F1 has been shown to promote neuronal death [353-

355], and is ectopically expressed in neurons in AD brain tissue [97]. Deletion of E2F1 was shown to prevent Aβ-induced neuronal death [141, 356]. Multiple studies have also demonstrated a role for E2F1 in adult neurogenesis [357], synaptic plasticity and dendritic arborization [358].

A key study addressing the functions of MDMX in cultured neurons was published by

Benosman and colleagues in 2007. This study yielded several novel observations regarding the role of MDMX in neuronal survival. First, it was shown that knockdown of MDMX expression using antisense oligonucleotides induced apoptosis in cultured neurons. Additionally, it was shown that various types of toxic insults (including DNA-damaging agents, excitotoxic stress, and potassium deprivation) induced proteolytic degradation of the MDMX protein in cultured neurons.

In addition to implicating MDMX in regulating of p53- and E2F1-mediated transcriptional activities in neurons, this study also demonstrated that overexpression of MDMX was neuroprotective in cultured neurons. The authors concluded that MDMX acts as a "node" for neurotoxic stress; whereby multiple toxic factors induce the degradation of MDMX protein, which leads to neuronal cell death [359]. It was speculated that degradation of MDMX could underlie neuronal cell death in neurodegenerative diseases, such as AD, considering that p53 and E2F1 are both dysregulated in neurons in AD [97, 145], and both p53 and E2F1 can be negatively regulated by

MDMX [235, 293].

Interestingly, one recent study examined changes in gene expression of p53 regulators and found a small but significant increase in MDMX mRNA in the superior temporal gyrus of patients with dementia compared to asymptomatic control patients [360]. However, changes in

MDMX mRNA did not significantly associate with neuritic plaque density or Braak stage.

Additionally, no changes in MDMX mRNA were detected in patients with schizophrenia. These results provide some basis to suggest that during dementia, neurons activate MDMX expression, potentially as a protective mechanism. However, the lack of association between MDMX mRNA

and pathological markers suggests that these changes are not directly altered due to neurodegenerative pathology. Previous results demonstrating stress-induced MDMX degradation in neurons [359] did not observe changes in MDMX mRNA in vitro, but transcriptional upregulation of MDMX may occur in the neurons of dementia patients as a mechanism of replacing MDMX protein lost to proteolytic degradation.

1.8: RATIONALE, HYPOTHESIS, AND OBJECTIVES

The initial studies of MDMX as a neuroprotective protein suggest that multiple types of neurotoxic stress induce degradation of MDMX, and that MDMX promotes neuronal survival by opposing the pro-death functions of p53 and/or E2F1. We expanded upon this previous work by examining the changes in MDMX in two disease models, and also employed a pharmacological approach to determine downstream effectors of MDMX in neurons, based on the small-molecule inhibitor of MDMX, SJ-172550, a compound designed to prevent MDMX functions as a potential chemotherapeutic agent [361]. We hypothesized that MDMX maintains essential pro-survival functions in neurons, which are disrupted due to proteolytic degradation of MDMX during neurodegenerative diseases. We examined: a) How extracellular stress factors lead to the degradation of neuronal MDMX, and b) How loss of MDMX function contributes to intracellular signaling pathways related to neuronal death. These objectives were addressed in two separate neurodegenerative disease models: HAND (Chapter 2) and in AD (Chapter 3).

First, we examined the role of MDMX in HAND; both in patient tissue samples and in an in vitro model of HAND-induced neurotoxicity (Chapter 2). These studies not only implicated

MDMX in the neuronal death process in a model of HAND, but also identified MDMX as a novel calpain substrate in neurons. We also demonstrate calpain-dependent neuronal death induced by pharmacological inhibition of MDMX.

Next, we examined how MDMX is affected in AD using patient tissue samples, a transgenic mouse model of AD, and an in vitro model of Aβ toxicity (Chapter 3). We showed that

neuronal MDMX is reduced in neurons of the frontal cortex in AD and in AD models, and that the degradation of MDMX is induced by Aβ peptide in a caspase-dependent manner. Notably, we also observed MDMX loss in cholinergic neurons in a murine AD model. Additionally, we determined novel roles for neuronal MDMX in the regulation of mitochondrial stability, calpain activity, and Cdk5 kinase activity. Together our results from this study suggest that amyloid- induced loss of MDMX contributes to mitochondrial damage, calpain activation, and Cdk5 hyperactivity during the pathogenesis of AD.

We also studied p53 as a downstream effector of MDMX in cultured neurons (Chapter 4), and found that while previous results indicate that MDMX regulates p53 in cultured neurons, the pro-survival effects of MDMX are not directly mediated by p53, representing a significant departure from previously observed functions of MDMX and p53 in dividing cells.

Finally, in Chapter 5 we discuss the conclusions and implications of our data. We summarize findings from previous research and our collective body of work, describing our current conceptual model of how MDMX promotes neuronal survival, and how the functions of

MDMX become altered during neurodegenerative diseases. Lastly, we also discuss potential directions for future studies towards further understanding the role of MDMX in neuronal function.

Altogether, our work has identified the degradation of MDMX oncoprotein in neurons as a contributing factor in neuronal damage in two neurodegenerative diseases (AD and HAND). We have identified mechanisms of disease-specific proteolytic MDMX degradation in neurons which underlie MDMX loss in AD and HAND. We have also characterized novel functions and downstream effectors of MDMX, which contribute to its neuroprotective abilities. It is hoped that our finding will provide a groundwork for further investigation of MDMX as a survival node in neurons that is disrupted during neurodegenerative disease.

1.9: BIBLIOGRAPHY

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CHAPTER 2

"Calpain-mediated degradation of MDMx/MDM4 contributes to HIV-induced neuronal

damage"

Daniel J. Colacurcioa, Alyssa Yeagerb, Dennis L. Kolsonb, Kelly L. Jordan-Sciuttoa, Cagla Akaya

a Department of Pathology, School of Dental Medicine, University of Pennsylvania, 312 Levy

Building, 240 South 40th Street, Philadelphia, PA 19104, United States

b Department of Neurology, The Perelman School of Medicine, University of Pennsylvania, 280C

Clinical Research Building, 415 Curie Boulevard, Philadelphia, PA 19104, United States

Originally Published in:

Molecular and Cellular Neuroscience: Volume 57, November 2013, Pages 54–62

Received 29 April 2013; Revised 10 September 2013; Accepted 5 October 2013

This work was supported by the following grants: NS41202 (KJS) and MH095671 (DLK).

2.1: ABSTRACT

Neuronal damage in HIV-associated Neurocognitive Disorders (HAND) has been linked to inflammation induced by soluble factors released by HIV-infected, and non-infected, activated macrophages/microglia (HIV M/M) in the brain. It has been suggested that aberrant neuronal cell cycle activation determines cell fate in response to these toxic factors. We have previously shown increased expression of cell cycle proteins such as E2F1 and phosphorylated pRb in HAND midfrontal cortex in vivo and in primary neurons exposed to HIV M/M supernatants in vitro. In addition, we have previously shown that MDMx (also referred to as MDM4), a negative regulator of E2F1, was decreased in the brain in a primate model of HIV-induced CNS neurodegeneration.

Thus, we hypothesized that MDMx provides indirect neuroprotection from HIV-induced neurodegeneration in our in vitro model. In this report, we found significant reductions in MDMx protein levels in the mid-frontal cortex of patients with HAND. In addition, treatment of primary rat neuroglial cultures with HIV M/M led to NMDA receptor- and calpain-dependent degradation of

MDMx and decreased neuronal survival, while overexpression of MDMx conferred partial protection from HIV M/M toxicity in vitro. Further, our results demonstrate that MDMx is a novel and direct calpain substrate. Finally, blocking MDMx activity led to neuronal death in vitro in the absence of toxic stimulus, which was reversed by calpain inhibition. Overall, our results indicate that MDMx plays a pro-survival role in neurons, and that strategies to stabilize and/or induce

MDMx can provide neuroprotection in HAND and in other neurodegenerative diseases where calpain activation contributes to neuropathogenesis.

2.2: INTRODUCTION

Approximately 50% of HIV (+) patients suffer from a spectrum of deficits in cognition, motor function, behavior and emotion, collectively termed HIV-associated Neurocognitive Disorders

(HAND) (Masliah et al., 2000 and McArthur et al., 2004). The areas of cognition affected in HAND patients include concentration, executive functioning, memory, and attention; and usually are accompanied by depression, psychosis, and anxiety of varying severity (McArthur, 2004).

Extensive in vitro and in vivo studies indicate that HIV-infected monocytes and macrophages

(M/Ms) serve as viral reservoirs during the chronic course of disease. These cells act as a source for viral proteins and other secreted mediators of neuronal damage and toxicity, as supported by the presence of infected macrophages and microglia along with pathological indicators of neuronal damage and death, dendritic simplification, axonal damage, and synaptic loss observed in the CNS of HAND patients ( Ellis, 2010, Gonzalez-Scarano and Martin-Garcia, 2005, Kaul et al., 2005 and Masliah et al., 1997). Additionally, the levels of macrophage activation markers, neopterin and β2-microglobulin, are elevated in the cerebrospinal fluid (CSF) of HIV- infected patients ( Edén et al., 2010, Hagberg et al., 2010 and Yilmaz et al., 2006).

CNS viral reservoirs, established primarily in HIV-infected macrophages, are an important source of a wide variety of toxic factors which ultimately contribute to the neuropathological changes in the HAND brain (Gonzalez-Scarano and Martin-Garcia, 2005). Extensive studies have shown that infected and/or activated macrophages can release viral proteins such as gp120 and Tat, as well as soluble factors includingglutamate, TNF-α, quinolinic acid, reactive oxygen species (ROS), and cytokines such as CCL2 (monocyte chemotactic protein-1, MCP-1) and interleukin-6 (IL-6), all of which have been shown to adversely affect neurons. Additionally, these viral and soluble factors also induce secretion of more of these and other neurotoxic factors, such as excitatory amino acids, from macrophages/microglia as well as neighboringastrocytes (Giulian et al.,

1996, Gonzalez-Scarano and Martin-Garcia, 2005, Gorry et al., 2003, Lindl et al., 2010, Price et al., 1988 and Soontornniyomkij et al., 1998). Consistent with the role of macrophages in HAND

neuropathogenesis, levels of neuroinflammatory factors (e.g. TNF-α, CCL2, and IL-6) are increased in the cerebrospinal fluid (CSF) of HIV-infected patients ( Achim et al., 1996, Achim and Wiley, 1996, Conant et al., 1998, Gisolf et al., 2000 and Sippy et al., 1995). These findings underscore the central role macrophages play in HIV-associated neuropathogenesis.

There are several non-mutually exclusive mechanisms by which neuroinflammation can lead to synaptic damage and neuronal death in HAND, including oxidative stress (Hu et al.,

2009 and Reynolds et al., 2007),N-methyl-D-aspartate (NMDA) receptor (NMDAR) activation

( Giulian et al., 1996 and O'Donnell et al., 2006), caspase and/or calpain activation ( O'Donnell et al., 2006 and Wang et al., 2007), and aberrant cell cycle regulation ( Akay et al., 2011b, Jordan-

Sciutto et al., 2002b, Jordan-Sciutto et al., 2001, Wang et al., 2010 and Wang et al., 2007).

Interestingly, many reports have suggested altered expression of several cell cycle proteins, including E2F1, pRb, p53, and cyclin-dependent kinase 5 (cdk5), in several neurodegenerative diseases including Alzheimer Disease, Parkinson Disease, amyotrophic lateral sclerosis and

HAND ( Jordan-Sciutto et al., 2001, Jordan-Sciutto et al., 2003, Jordan-Sciutto et al.,

2002a, Jordan-Sciutto et al., 1999, Jordan-Sciutto et al., 2002b, Jordan-Sciutto et al.,

2000, Ranganathan and Bowser, 2003 and Wang et al., 2010). We have previously shown increased expression of E2F1 and phosphorylated pRb (ppRb) in several brain regions in macaques with simian immunodeficiency virus encephalitis (SIVE) in an in vivo model of HIV- associated CNS disease ( Jordan-Sciutto et al., 2000 and Jordan-Sciutto et al., 2002b). We have also observed decreased expression of a negative regulator of E2F1, murine double minute x/4

(MDMx), in the SIVE brain ( Strachan et al., 2005).

MDMx is a homologue of the E3 ligase murine double minute 2 (MDM2), which has been originally identified and studied as a p53-regulating protein (Shvarts et al., 1996). In dividing cells,

MDMx acts with MDM2 to inhibit the pro-apoptotic functions of p53 by directly binding p53, and by enhancing the E3 ligase activity of MDM2, while lacking intrinsic ligase activity itself (Sharp et al.,

1999 and Wang et al., 2011). Additionally, MDM2 and MDMx inhibit the pro-apoptotic activity of

E2F1 in mitotic cells (Loughran and La Thangue, 2000 and Strachan et al., 2003). Within the mouse CNS, MDMx is necessary for normal development, as mdmx−/− mice display significant neuronal apoptosis and are embryonically lethal ( Migliorini et al., 2002). Additionally, MDMx knockdown in vitro damages neurons in the absence of toxic stress ( Benosman et al., 2007).

Finally, DNA-damaging agents, glutamate and extracellular potassium depletion lead to decreased levels of MDMx protein in cultured neurons ( Benosman et al., 2007).

We previously demonstrated decreased MDMx expression and increased cytoplasmic E2F1 expression in the frontal cortex, basal ganglia and hippocampus of SIV-infected macaques with encephalitis (SIVE), and increased cytoplasmic E2F1 expression in HIVE (Strachan et al., 2005).

We further showed that cytoplasmic E2F1 in HEK293 cells is associated with calpain activation and transcription-independent degradation of MDMx (Strachan et al., 2005). Using an in vitro model of HIV-associated neurodegeneration in which soluble factors released by HIV- infected monocyte-derived macrophages induce neuronal damage, we investigated the role of

MDMx as a determinant of neuronal survival in HAND (Y. Wang et al., 2007). Our results show that MDMx expression is decreased in brain tissue of HAND patients. Further, we show, for the first time, that MDMx is a direct calpain substrate, and that calpain-mediated MDMx degradation following NMDA receptor activation partially contributes to neuronal death in vitro. Lastly, we demonstrate that overexpression of MDMx partially protects neurons in an in vitro model of

HAND, and that pharmacological inhibition of MDMx induces calpain-mediated neuronal damage.

2.3: EXPERIMENTAL METHODS

Chemicals and reagents

N-Methyl-D-aspartic acid (NMDA), (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine maleate (MK801) and SJ-172550 were purchased from Tocris Bioscience (Ellisville, MO).

MDL-28170 and protease inhibitor cocktail were from Sigma Aldrich (St. Louis, MO) and native calpain-1 from porcine erythrocytes was from EMD Chemicals (Gibbstown, NJ). Q-VD-OPh

(quinolyl-valyl-O-methylaspartyl-[-2, 6-difluorophenoxy]-methyl ketone) was purchased from R&D

Systems, Inc. (Minneapolis, MN). Rabbit polyclonal antibody to MDMx, donkey anti-goat HRP, and propidium iodide were from Santa Cruz Biotechnology (Santa Cruz, CA), and goat anti-rabbit

HRP antibody was purchased from Thermo Scientific (Waltham, MA). Poly-L-lysine was from

Peninsula Laboratories (San Carlos, CA). DAPI was from Molecular Probes (Carlsbad, CA).

Neurobasal media, B27 supplement, goat anti-mouse β-lactamase and fluorocillin green substrate were from Invitrogen (Carlsbad, CA). The antibody that recognizes calpain-cleaved spectrin was a generous gift from Dr. Robert Siman at the University of Pennsylvania.

HIV-1 infection of macrophages and collection of supernatants

The supernatants from primary human monocyte-derived macrophages were prepared as described previously (O'Donnell et al., 2006). For the generation of supernatants from HIV- infected monocyte-derived macrophages (MDM), primary monocytes were isolated from the peripheral blood of HIV (−) healthy volunteers in accordance with protocols approved by the

University of Pennsylvania Committee on Studies Involving Human Beings. Monocytes were differentiated into macrophages for 7 days at a density of 1 × 106per well in 6-well CellBind plates

(Corning, Lowell, MA), as described previously (Akay et al., 2011b). On 7 days in vitro (DIV), the monocyte-derived macrophages were incubated with a specific primary HIV isolate, Jago (100 ng of p24 per well), for 24 h. This primary isolate was acquired from cell-free cerebrospinal fluid

(CSF) harvested by lumbar puncture from a patient undergoing diagnostic testing at the

University of Pennsylvania, who was confirmed to have HIV-associated dementia (HAD) by established clinical and neuropsychological criteria at the time of virus isolation, as defined by the

Dana Consortium ( Chen et al., 2002; “ Clinical confirmation of the American Academy of

Neurology algorithm for HIV-1-associated cognitive/motor disorder. The Dana Consortium on

Therapy for HIV Dementia and Related Cognitive Disorders,” 1996). At the end of incubation, the virus was removed from the media by two washes with Dulbecco's modified eagle medium

(DMEM). The cultures were incubated in fresh macrophage media for up to 15–17 days post-

infection. HIV infection was monitored by reverse transcriptase activity in the supernatants (HIV

M/M) and the supernatants with peak reverse transcriptase activity that consistently induced ~

40–50% neuronal damage at a dilution of 1:20 were used for all experiments. The supernatants from monocyte-derived macrophages of the same donors that were cultured in the absence of

HIV-1 infection served as mock controls (Mock).

Primary neuroglial cultures

Rat pups were harvested at embryonic day 17 from timed-pregnant Sprague–Dawley adults euthanized by CO2 inhalation and cervical dislocation, in accordance with the Institutional Animal

Care and Use Committee (IACUC) protocols. Primary cortical neuroglial cultures were prepared, as described previously (Akay et al., 2011b). The cells were plated at a density of 2 × 106 cells in

60 mm Petri dishes or at a density of 1 × 105cells per well in 96-well tissue culture plates coated with poly-L-lysine. The cultures were maintained in neurobasal media with B27 supplement at 37

°C with 5% CO2. Half of the media were replaced with fresh media every 7 days and the experiments were performed at 21 days in vitro (DIV). HIV-1-infected (HIV M/M) or mock-infected

(Mock) monocyte-derived macrophage supernatants were added to cultures at the indicated dilutions.

Assessment of neuronal damage

Primary rat neuroglial cultures in 96-well plates were assessed for MAP2 fluorescence using a

MAP2 cell-based ELISA, as described previously (Akay et al., 2011b, Wang et al., 2007 and

White et al., 2011). In parallel, cultures grown on coverslips were confirmed for neuronal viability under the same experimental conditions, by hand counting using a propidium iodide (PI) exclusion assay. Briefly, PI is a red fluorescent stain that binds nucleic acids by intercalating between bases for use as a label for nucleic acids. PI is also membrane impermeable, and is excluded from viable cells while it accumulates in the nuclei of cells with damaged plasma and nuclear membranes, indicative of irreversible cell damage. In our experiments, 15 μM PI was

added to tissue culture media 15 min before the fixation step followed by subsequent staining for

MAP2 and DAPI (nuclear stain) using standard immunofluorescence techniques for the identification of dead cells in culture via fluorescent microscopy ( White et al., 2011). Dead cells are identified as those which are negative for MAP2, positive for PI, and showing a fragmented and/or dense nuclear morphology as detected by DAPI, whereas live cells were identified as those which are negative for MAP2, positive for PI, and showing intact nuclei by DAPI. 1000 cells on average were counted for each condition.

Immunoblotting for primary neuroglial cultures

Whole cell protein extracts were prepared by scraping cells in lysis buffer (50 mM Tris pH 7.5,

120 mM NaCl, 0.5% NP-40, 5 mM NaF and 0.5 mM Na Orthovanadate supplemented with a protease inhibitor cocktail). The lysates were incubated on ice for 10 min and cleared by centrifugation at 14,000 g for 10 min. The protein concentrations of supernatants were determined by the Bradford method. 4–12% SDS-polyacrylamide gradient gels (Invitrogen) and

Immun-Blot polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA) were used for electrophoresis and transfer, respectively. Following standard protocols for immunoblotting ( Akay et al., 2011b), the membranes were visualized using enhanced chemiluminescence (ECL) reagent (Thermo Scientific, Rockford, IL) and autoradiography.

Subcellular fractionation

Cytoplasmic protein extractions were prepared by scraping the cultures in ice-cold cytoplasmic lysis buffer (10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), 10 mM KCl,

10 mM Ethylenediaminetetraacetic acid (EDTA), 4% Nonidet P-40 and 10 mM dithiothreitol

(DTT), supplemented with a protease inhibitor cocktail). The cellular debris was cleared by centrifugation at 14,000 g for 3 min. The supernatants were saved as cytoplasmic fractions. The remaining pellets were resuspended in the nuclear lysis buffer (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 10% glycerol and 10 mM DTT, supplemented with a protease inhibitor cocktail) and

were incubated on a rocking platform at 4 °C for 2 h, followed by centrifugation at 14,000 g for 5 min. The collected supernatants were the nuclear extracts. These fractions were analyzed by routine immunoblotting techniques.

Human brain tissue

Tissue samples from the mid-frontal cortices of HIV (−) control and HIV (+) human autopsy cases used for this study were obtained from the tissue banks of the National NeuroAIDS Tissue

Consortium (NNTC) (Table 1). For western immunoblotting, whole cell lysates were prepared as described previously, and 50 μg protein was loaded to each lane of 10% Bis–Tris gels and processed further (Akay et al., 2011a and Lindl et al., 2007).

In vitro calpain cleavage assay pcDNA3.1-MDMx (human) was transcribed and translated using TNT quick coupled transcription kit (Promega, Madison, WI). 1 μg of MDMx was incubated in the assay buffer which contained 50 mM Tris–HCl pH 7.4, 100 mM KCl, 2 mM DTT with 2.5 U/ml of recombinant calpain I in the presence or absence of the indicated amounts of CaCl2 for 90 min at 37 °C. Incubation was stopped by the addition of the SDS loading buffer to the reaction tubes. The samples were then boiled for 10 min at 95 °C, separated on 4–12% SDS-polyacrylamide gradient gels and transferred to PVDF membranes. The membranes were immunoblotted and visualized, as described above.

Construction of viral vectors pcDNA3.1-MDMx (human) was cloned into the recombinant adeno-associated virus (AAV) cloning plasmid, pZac2.1 (University of Pennsylvania Vector Core), using BamHI–BamHI restriction enzyme sites. The AAV was packaged into recombinant AAV particles using pPACK-H plasmid that encodes adenovirusglycoprotein and helper plasmid (University of Pennsylvania

Vector Core).

Infection of primary neuroglial cultures with viral vectors

Fifty percent of culture media of 21 DIV primary rat neuroglial cultures in 60-mm dishes or 96-well plates were changed one day prior to infection. The infections were carried out by addition of thawed virus directly into the culture media. The amount of AAV particles was empirically titrated in 21 DIV primary neuronal cultures at dilutions not exceeding 1:40. Experiments were carried out three days post-infection.

Statistical analysis

One-way ANOVA with post-hoc Newman–Keuls multiple comparison test was used for statistical analysis of in vitro immunoblot quantifications, MAP2 ELISA and neuronal viability assays. For immunoblot studies of human tissue, Student's t-test was performed. In experiments using human tissue samples, additional statistical analyses were performed between the post-mortem interval

(PMI) and age, and the relative MDMx protein levels detected by immunoblotting, to investigate possible correlations between these parameters. For all statistical analyses performed, p values of < 0.05 were considered significant.

2.4: RESULTS

MDMx levels are decreased in the mid-frontal cortices of neurocognitively impaired HIV (+) patients

In this study, we examined MDMx protein expression in brain tissue from HIV-infected patients by immunoblotting. MDMx migrated as a doublet in immunoblots of whole cell lysates from human mid-frontal cortical samples, as shown in Fig. 1A (see Table 1 for a summary of cases). The quantification of the MDMx immunoblotting data, as shown in Fig. 1B, revealed that total MDMx protein levels were significantly decreased in the mid-frontal cortices of HIV-infected patients, as compared with those in uninfected controls (*p < 0.05). Additionally, when we compared MDMx levels according to the neurocognitive status of the cases, we observed statistically significant

decreases in MDMx levels in HAND patients, as compared with those in HIV-infected, neurocognitively normal (NcN) patients ( Fig. 1C, *p < 0.05). These findings suggest that MDMx protein is decreased in the HAND cortex, in agreement with our previous findings in the macaque model of HIV-induced neurodegeneration ( Strachan et al., 2005). Of note, analysis of immunoblots for correlations between MDMx protein levels and age ( Fig. 1D) or post-mortem interval (PMI, Fig. 1E) showed no significance.

MDMx is decreased in a time- and dose-dependent manner in an in vitro model of HIV- associated neurodegeneration

We have previously utilized supernatants secreted from primary human monocyte-derived macrophages infected with a primary HIV isolate (Jago) to treat primary rat neuroglial cultures, and have shown that these supernatants (HIV M/M) consistently lead to dose- and time- dependent neuronal damage in this in vitro model of HIV-induced neuronal damage ( Akay et al.,

2011b, O'Donnell et al., 2006, Wang et al., 2007 and White et al., 2011). To begin to investigate the mechanism(s) underlying reduced MDMx levels detected in HAND brain tissue, we assessed

MDMx protein levels in HIV M/M-treated primary neuroglial cultures. As seen in Fig. 2A, we observed multiple MDMx isoforms in these cultures. Importantly, HIV M/M that was diluted 1:40 or

1:20 in culture media led to significant decreases in all MDMx isoforms after 20 h, whereas the treatment of cultures with supernatants from mock-treated monocyte-derived macrophages

(Mock) at similar dilutions did not cause significant loss of MDMx relative to untreated cultures

( Fig. 2B, *p < 0.05). Additionally, significant decreases in MDMx protein levels were detectable as early as 4 h after exposure to HIV M/M at a dilution of 1:20 ( Fig. 2C and D, *p < 0.05).

Further, the analysis of the cytoplasmic and nuclear extracts of cultures exposed to Mock or HIV

M/M revealed that HIV M/M-induced, significant losses of MDMx isoforms were induced by HIV

M/M in both compartments ( Fig. 2E and F, *p < 0.05). NMDA was used as a positive control for neurotoxicity. These results suggest that MDMx is decreased in a dose- and time-dependent manner in our in vitro model of HIV-associated neurotoxicity.

Degradation of MDMx is calpain-dependent

We have previously shown that HIV M/M-induced neurotoxicity occurs through the activation of

N-methyl-D-aspartic acid (NMDA) receptor (NMDAR) and subsequent calpain activation in our in vitro model ( O'Donnell et al., 2006 and Wang et al., 2007). In this study, treatment of cultures with NMDA decreased MDMx protein, at levels comparable to those observed in HIV M/M-treated cultures ( Fig. 2E and F). Additionally, pre-incubation of neuroglial cultures with the NMDAR inhibitor, MK801, prevented HIV M/M-induced MDMx degradation ( Fig. 3A and B, *p < 0.05).

Further, pre-treatment of cultures with a calpain inhibitor, MDL28170, prevented MDMx loss, suggesting a role for calpains in MDMx loss ( Fig. 3A and B, *p < 0.05). Next, we performed an in vitro calpain cleavage assay to determine if MDMx is a direct calpain substrate. As seen in

Fig. 3C, in vitro transcribed and translated full-length MDMx (1–491) was a substrate for two types of calpain: μ-calpain, which is activated by nanomolar concentrations of CaCl2, and m- calpain, which is activated by micromolar concentrations of CaCl2. However, a truncated form of

MDMx, MDMx (1–90) is not degraded by m-calpain under the same conditions ( Fig. 3D). These data collectively suggest that activated calpains induced by HIV M/M directly degrade MDMx at a site (or sites) located in the internal region and/or carboxyl terminus.

Overexpression of MDMx attenuates HIV M/M-induced neurotoxicity

Based on our findings which suggest that excitotoxic injury led to calpain-mediated MDMx degradation, we determined whether overexpression of MDMx would provide neuroprotection from HIV M/M in vitro. We overexpressed full-length MDMx in neuroglial cultures via an adeno- associated virus (AAV) expression system. Using this system, we achieved a viral titer-dependent overexpression of MDMx in neuroglial cultures ( Fig. 4A). Fig. 4B shows representative immunofluorescence images of cultures overexpressing MDMx. Neuronal viability as measured by cell-based MAP2 ELISA and PI exclusion assays showed that HIV M/M-induced neurotoxicity in our in vitro model was attenuated in cultures that overexpressed MDMx (Fig. 4C,

*p < 0.05 vs. HIV M/M 1:40, #p < 0.05 vs. HIV M/M 1:20). Importantly, overexpressed MDMx protein was still degraded in cultures exposed to NMDA ( Fig. 4D). These results suggest a neuroprotective function for MDMx in our in vitro HIV-associated neurotoxicity model, and suggest that additional strategies to stabilize MDMx protein or block MDMx degradation may promote neuroprotection.

Inhibition of MDMx activity leads to calpain-dependent neuronal death

Finally, we questioned whether pharmacological inhibition of MDMx activity would impact neuronal viability in vitro. We used a cell-permeable and MDMx-specific inhibitor, SJ-172550, which binds to the p53-binding pocket at the N terminus of MDMx and blocks p53 docking ( Reed et al., 2010). We first determined that the exposure of primary neuroglial cultures to SJ-172550 at

20 μM for 20 h caused 50% neuronal death in the absence of external stimuli (data not shown).

Next, we determined whether blocking caspases or calpains would prevent neuronal death induced by MDMx inhibition. As seen in Fig. 5, the pre-treatment of cultures with a pan-caspase inhibitor, Q-VD-OPh before the addition of SJ-172550 did not prevent neuronal loss. However, the pre-treatment of cultures with MDL28170 prevented neuronal loss induced by SJ-172550

(n = 3, *p < 0.05). Taken together, our findings suggest that blocking MDMx activity causes caspase-independent, calpain-mediated death in primary neurons in vitro, and further, suggest an important pro-survival role for MDMx in post-mitotic neurons.

2.5: DISCUSSION

Multiple studies have demonstrated that altered/aberrant expression or modification of cell cycle proteins and cell cycle-related transcription and regulatory factors play important roles in neuronal viability in various neurodegenerative diseases, including HIV-associated Neurocognitive

Disorders (HAND) (Jordan-Sciutto et al., 1999, Jordan-Sciutto et al., 2000, Jordan-Sciutto et al.,

2001, Jordan-Sciutto et al., 2002a, Jordan-Sciutto et al., 2002b, Jordan-Sciutto et al.,

2003, Ranganathan and Bowser, 2003 and Wang et al., 2010). In the current study, we demonstrate that MDMx, an important regulator of a major cell cycle-related transcription factor,

E2F1, is decreased significantly in the mid-frontal cortex of HAND patients compared to that in neurocognitively normal, HIV-infected patients. Further, we show that these changes in MDMx protein are not correlated with either age or post-mortem interval. We have previously shown that

E2F1 levels were increased in the neuronal cytoplasm of the HAND brain as well as in the basal ganglia and the cortical neurons of SIV encephalitic macaques, whereas we have detected reciprocal decreases in MDMx levels in the neurons of SIVE brain (Jordan-Sciutto et al., 2002b).

While E2F1 overexpression has been shown to be pro-apoptotic in post-mitotic neurons (O'Hare et al., 2000), E2F1 does not induce the expression of classical E2F1 transcriptional targets in the

HAND cortex, as we have recently reported (Wang et al., 2010). These differences in subcellular localization of E2F1, as well as the non-classical roles of E2F1 in post-mitotic neurons, suggest that the regulatory mechanisms that govern E2F1:MDMx interactions described in dividing cells might not apply to post-mitotic neurons.

In the current study, we demonstrate that all MDMx isoforms were decreased in our in vitro model of HIV-associated neurodegeneration. Several mechanisms have been shown to contribute to the presence of multiple MDMx isoforms, including pre-mRNA alternative splicing, phosphorylation and ubiquitination, as determined in human fibroblasts and cancer cells ( de Graaf et al.,

2003, Jeyaraj et al., 2009, Markey, 2011,Pereg et al., 2005, Rallapalli et al., 1999 and Rallapalli et al., 2003). However, the mechanism(s) underlying the presence of MDMx isoforms in primary neurons are not known. Future studies investigating these pathways will be instrumental towards delineating the function of specific MDMx isoforms in post-mitotic neurons.

In our in vitro model of disease, MDMx protein levels are decreased in response to calpain activation in neurons ( O'Donnell et al., 2006, Wang et al., 2007 and White et al., 2011). Our findings regarding calpain activation are in agreement with our previous findings, in which we have shown that calpains, but not caspases, are the major death proteases activated in neurons

exposed to HIV M/M or NMDA ( O'Donnell et al., 2006, Wang et al., 2007 and White et al., 2011).

Additionally, we have recently reported that calpain levels in untreated neuroglial cultures increased with age while caspase-3 levels decreased ( Wang et al., 2012). Calpains are intracellular Ca2 +-dependent cysteine proteases that degrade many proteins ( Sorimachi et al.,

2011). While we had previously shown that overexpression of an endogenous calpain inhibitor, calpastatin, stabilized MDMx in HEK293 cells, to date there is no report suggesting MDMx as a direct calpain substrate in dividing cell types, or in neurons ( Strachan et al., 2005). In this report, we demonstrate that MDMx is a direct calpain substrate. Although previous studies in dividing cell types demonstrated a caspase cleavage site within the internal region of MDMx by which it can be degraded and subsequently be targeted for proteasomal degradation ( Gentiletti et al., 2002); to date, a calpain cleavage site within MDMx has not been identified. The examination of calpain cleavage sites of a number of known calpain target proteins reveals certain characteristics for these preferred cleavage sites, such as a proline-rich region (Tompa et al.,

2004). Based on these shared characteristics, several algorithms are used to more accurately predict such sites ( duVerle et al., 2010). The analysis of the primary amino acid sequence of

MDMx using “Calpain for Modulatory Proteolysis Database” (calpain.org) predicts the region surrounding threonine at position 347 as a calpain cleavage site. Although a substantial undertaking, it will be important to identify the calpain cleavage site(s) on MDMx in future investigations for several reasons. First, the overexpression of MDMx provided partial protection from neuronal death in our in vitro model, as well as in other in vitro models of neurotoxicity

( Benosman et al., 2007). The full-length MDMx used in our overexpression studies is still a calpain substrate; and as degradation is likely to be more efficient than protein synthesis, MDMx overexpression in our studies attenuates damage and death, but does not completely prevent it, as demonstrated by our results showing NMDA-mediated degradation in MDMx still occurring in cultures overexpressing MDMx. Future studies will be instrumental towards determining whether overexpression of a calpain-resistant MDMx mutant will provide further neuroprotection in our in

vitro model. Secondly, the majority of MDMx's functions are determined by protein:protein interactions ( Lenos and Jochemsen, 2011). While we did not detect any accumulation of distinct

MDMx cleavage products in vitro or in vivo, transient MDMx cleavage products may still form, altering downstream pathways by disrupting specific protein:protein interactions or potentially acting as a dominant-negative MDMx. Thus, by identifying the domain(s) on MDMx that are targeted by calpain for cleavage, we can begin to understand the specific pathways downstream from MDMx that may be disrupted in neurons in response to neurotoxic stimuli. Finally, strategies that can stabilize MDMx and promote its accumulation within neurons will be useful in treatment approaches for HAND, during which decreased MDMx and increased calpain activation contribute to neuronal damage.

The in vitro calpain cleavage assay demonstrated that MDMx is a direct target of calpain in the absence of additional cellular factors. In addition to calpain, there is evidence that other factors may still be involved in MDMx degradation, in parallel with, or subsequent to, calpain activation.

For example, cathepsins, downstream effectors of calpains, have been demonstrated to cause

MDMx degradation in a previous study ( Strachan et al., 2001). Another possible mechanism of

MDMx loss is dysregulation at the mRNA level. Previous studies have shown that MDMx regulation occurs primarily at the protein level, suggesting that changes at the transcriptional level are not as likely ( Benosman et al., 2007). However, future studies using human macrophages and HIV M/M-exposed neurons in our in vitro model will be contributory to address both of these possibilities.

Studies exploring the specific roles of MDMx in post-mitotic neurons are limited. The phenotype of mdmx knockout mice reveals that MDMx is necessary for CNS development ( Xiong et al.,

2006). Interestingly, the severe cell death observed in the brains of these embryos is rescued by concurrent silencing of p53. Multiple studies demonstrate that MDMx:MDM2 heterodimers bind and ubiquitinate p53, targeting p53 for degradation by the proteasome, and thus, allowing cells to maintain a low basal level of this pro-apoptotic protein under normal conditions ( Wade et al.,

2010). Additionally, MDMx silencing in cerebellar granule neurons caused caspase-3 activation and cell death in the absence of a toxic insult ( Benosman et al., 2007). Thus, it is highly likely that the massive cell loss observed in mdmx−/− mice during development is due to p53-driven and caspase-mediated apoptosis. In our studies, we demonstrate that treatment of neurons with SJ-

172550, which has been shown to inhibit binding of p53 to MDMx in other cell types, leads to death in post-mitotic neurons in the absence of toxic stimuli. However, our findings differ from the abovementioned studies in that neither the inhibition of p53 transcriptional activity by pre- treatment of neurons with Pifithrin-α (data not shown), nor the inhibition of caspase activation via a pan-caspase inhibitor, Q-VD-OPh, conferred protection from SJ-172550-mediated neurotoxicity, while SJ-172550-induced neuronal death was reversed by calpain inhibition. These results suggest that neuronal death observed in our cultures involves a p53- and caspase- independent mechanism, contrary to findings from previous studies. There are several factors that need to be considered for these differences. First, we utilized SJ-172550 to determine the implications of the disruption of MDMx:p53 binding on neuronal viability, while previous studies have examined the effect of silencing of MDMx ( Benosman et al., 2007). Secondly, SJ-172550 binding to MDMx at the amino terminus may disrupt its binding to a protein other than/in addition to p53, triggering the activation of a downstream pro-death pathway involving calpains. The effect of SJ-172550 on MDMx association with other proteins in post-mitotic differentiating neurons using co-immunoprecipitation approaches will begin to address this possibility, and will provide additional information on pathways downstream from MDMx in neurons. Finally, while SJ-172550 has been shown to specifically block MDMx:p53 interaction upon binding to MDMx without affecting its stability, those findings were not verified in cells. In vitro, p53 binding may be masking a calpain cleavage site on MDMx, which may become accessible by calpains in SJ-172550- treated neurons, leading to increased calpain-mediated degradation. The identification of calpain cleavage site(s) on MDMx, as mentioned above, will be instrumental to assess this possibility.

In summary, our findings show that MDMx stability and activity are crucial for the survival of post- mitotic neurons, and support current literature involving other tissue and cell types which suggests that MDMx is regulated mainly at the protein level by post-translational modifications that impact its stability and protein:protein interactions. Further, MDMx overexpression provided partial protection from HIV-associated neurodegeneration in our in vitro model. Finally, we identify

MDMx as a novel calpain substrate. These findings suggest that strategies to stabilize MDMx will have therapeutic implications which are applicable, not only in HAND, but also in other neurodegenerative diseases in which calpain activation is an underlying mechanism of neuronal death and dysfunction.

2.6: ACKNOWLEDGEMENTS

We would like to thank Dr. Kathryn A. Lindl and Jacob Zyskind for their contributions to the discussions during the preparation of this manuscript.

Fig. 1

Decreased MDMx expression in the HAND cortex. A, B and C. Whole cell tissue lysates from the midfrontal cortex of a cohort of NcN (n = 13) and HAND (n = 12) cases (Table 1) were used for immunoblotting. The membranes were blotted for MDMx, which were later stained with

Coomassie to control for loading (A). Statistically significant decreases in MDMx expression are observed in HIV (+) cases compared with those observed in the HIV (−) group (B, Student's t- test, Mann–Whitney U post-hoc, *p < 0.05); as well as in HAND cases compared with those observed in the NcN group (C, Student's t-test, Mann–Whitney U post-hoc, *p < 0.05).

Fig. 2

HIV M/M leads to decreases in MDMx in a time- and dose-dependent manner. A–D. Primary neuroglial cultures that were 21 days in vitro (DIV) were treated with mock-infected macrophage supernatants (Mock) or HIV-infected macrophage supernatants (HIV M/M) at the indicated doses for 20 h. Western immunoblotting of whole cell protein lysates shows a dose-dependent (A, B) and a time-dependent (C, D) decrease in MDMx levels (n = 4, one-way ANOVA, post-hoc

Newman–Keuls, *p < 0.05 vs. untreated). E–F. Cytoplasmic and nuclear extracts from primary neuroglial cultures treated with either Mock (1:20 dilution), HIV M/M (1:20 dilution) or NMDA (10

μM) for 1, 4, or 8 h were immunoblotted for MDMx. HIV M/M treatment led to statistically significant decreases in MDMx protein levels in both cytoplasmic and nuclear fractions. Likewise,

NMDA treatments led to significant decreases in nuclear MDMx levels starting at 4 h following treatment (n = 3, one-way ANOVA, post-hoc Newman–Keuls, *p < 0.05 vs. untreated). Mock did not cause a change in MDMx levels in either cytoplasmic or nuclear fractions. Representative

immunoblots of at least three biological replicates are shown. Note that the antibody used against

MDMx detects at least three isoforms in protein lysates.

Fig. 3.

HIV M/M-induced decrease in MDMx is dependent on NMDA receptor and calpain activation. A and B. Primary neuroglial cultures were treated with either Mock (1:20 dilution) or

HIV M/M (1:20 dilution) for 20 h in the presence or absence of the calpain inhibitor, MDL28170

(20 μM) or an NMDA receptor antagonist, MK801 (10 μM). The inhibitors were added to the culture media 30 min prior to Mock or HIV M/M treatments. The nuclear lysates were immunoblotted for MDMx. HIV M/M exposure lead to a decrease in MDMx, which was blocked by

pre-treatment with MDL28170 or MK801 (n = 5, one-way ANOVA, post-hoc Newman–Keuls, *p <

0.05 vs. untreated, vs. Mock, vs. HIV M/M + MDL, and vs. HIV M/M + MK801). Lamin A/C was used to show successful nuclear protein extraction. C. In vitro transcribed and translated human

MDMx was incubated with native calpain-1 in the presence of different concentrations of CaCl2 and the lysates were immunoblotted for MDMx. μ-Calpain activation by lower mM doses of

CaCl2, or m-calpain activation by higher mM concentrations of CaCl2 led to degradation of

MDMx. D. In vitro transcribed and translated human full-length MDMx (1–491) (lanes 1–3) or truncated MDMx (1–90) (lanes 4–6) were incubated with native calpain-1 in the presence of increasing concentrations of CaCl2 and the lysates were immunoblotted for MDMx. Calpain activation led to degradation of MDMx (1–491), while MDMx (1–90) was not degraded by calpain under the same conditions.

Fig. 4.

Overexpression of MDMx in neurons provides partial protection from HIV M/M-induced neuronal damage. An adeno-associated virus expression system was utilized for overexpression of human MDMx in primary neurons in vitro. A. Infection with AAV-MDMx led to increased MDMx expression in cultures in a dose-dependent manner, as assessed by immunoblotting for MDMx.

B. Following three days after infection with AAV-eGFP-MDMx or AAV-eGFP, primary rat cortical cultures on coverslips were immunostained for MAP2 and DAPI. Images were captured by epifluorescent microscopy. Representative images are shown. Magnification 40 ×. C. Attenuation of HIV M/M-induced MAP2 loss in neurons overexpressing MDMx, as compared with those observed in HIV M/M-treated cultures, as assessed by cell-based MAP2 ELISA (black bars), and

PI exclusion (gray bars) (*p < 0.05 vs. HIV M/M 1:40, #p < 0.05 vs. HIV M/M 1:20, one way-

ANOVA, Newman–Keuls post-hoc). D. Three days after infection with AAV-eGFP-MDMx or AAV- eGFP, neuroglial cultures were treated with either 20 μM of NMDA, or were left untreated. The lysates were immunoblotted for MDMx, and calpain activation was assessed by immunoblotting for the calpain-cleaved spectrin. Pound sign denotes the bands for overexpressed MDMx, arrowheads mark the bands for endogenous MDMx, and asterisk denotes the band for GFP.

Fig. 5.

Inhibition of MDMx activity leads to calpain-dependent neuronal death. 21 DIV primary neuroglial cultures were treated with SJ-172550 (20 μM) for 20 h in the presence or absence of either the calpain inhibitor MDL28170 (20 μM) or the pan-caspase inhibitor Q-VD-OPh (50 μM).

The inhibitors were added to the culture media 30 min prior to SJ-172550 treatments. Neuronal viability was assessed by cell-based MAP2 ELISA (vehicle: 0.04% DMSO, n = 3, *p < 0.05, ns = not significant, Student's t-test).

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CHAPTER 3

“Amyloid-β-induced degradation of the MDMX oncoprotein triggers mitochondrial damage

and calpain activation in neurons”

Daniel J. Colacurcio, Jacob W. Zyskind, and Kelly L. Jordan-Sciutto

Department of Pathology, School of Dental Medicine, University of Pennsylvania, 312 Levy

Building, 240 South 40th Street, Philadelphia, PA 19104, United States

This work was supported by the following grants: NIH grant # NS41202

3.1: ABSTRACT

MDMX is a p53-regulating protein associated with cancer due to its anti-apoptotic functions in dividing cells. MDMX also promotes survival in mature neurons. It has been shown that MDMX is necessary for neuronal survival, and that proteolytic degradation of MDMX occurs in response to neuronal stress. Thus, we hypothesized that amyloid-β induces degradation of

MDMX as a potential mechanism of neuronal damage during the progression of Alzheimer

Disease (AD).

Utilizing a murine model of AD and pharmacological treatments of primary neurons, we investigated pathways that mediate Aβ-induced degradation of MDMX, as well as intracellular factors downstream of MDMX in neurons. We observed reduction of cortical MDMX in aged

APPswe mice. Our results demonstrate that amyloid peptides trigger caspase-mediated degradation of MDMX in cultured neurons. We also show that MDMX function is necessary for mitochondrial stability in neurons. Additionally, our data suggest that MDMX loss contributes to calpain-dependent Cdk5 activation in neurons. Lastly, we determine that MDMX protein is reduced in brain tissue from AD patients. Altogether, our findings suggest that amyloid-induced degradation of the MDMX oncoprotein causes mitochondrial depolarization and excessive activity of calpain and Cdk5, contributing to neuronal damage during the progression of AD.

3.2: INTRODUCTION

Alzheimer Disease (AD) is an aging-associated neurodegenerative disease and the leading cause of dementia, affecting 5.2 million Americans(1-3). AD is characterized by the presence of extracellular senile plaques composed predominantly of amyloid-β (Aβ), intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein, and progressive synaptic loss and neuronal death throughout the cortex and hippocampus(4, 5). The pathogenesis of AD underlies progressive memory loss and dementia. Biomarker studies suggest that these pathological markers develop in a sequential manner; amyloid pathology appears earliest, and is followed by accumulation of NFTs, synaptic damage, and neuronal death(6).

Previous evidence from patient studies and transgenic animals has led to the "amyloid hypothesis" of AD which states that aberrant metabolism of amyloid precursor protein (APP) leads to accumulation of extracellular Aβ, which causes subsequent pathological changes (tau hyperphosphorylation, synaptic damage, neuronal death) observed in AD(7-9).

While the role of amyloid in the pathogenesis of AD is not fully understood, multiple studies suggest that Aβ contributes to the neuronal damage in various ways. Aβ can disrupt the stability of neuronal membranes(10, 11), and also trigger activation of intraneuronal caspases, leading to neuronal death(12, 13). Oligomeric forms of Aβ can interact with neuronal cell surface receptors, causing intracellular changes leading to synaptic damage and neuronal death(9, 14,

15). Aβ drives the development of tau pathology by activating intracellular signaling pathways in neurons which convert tau from a normal state to a pathogenic one(16-18). Aβ-induced dysregulation of tau occurs via multiple mechanisms, many of which involve activation of kinases(19), particularly Cdk5(20, 21) and GSK3β(22, 23), leading to hyperphosphorylation of tau. Calpain activity is also implicated in the progression of AD(24, 25). Aβ can induce calpain- mediated cleavage of the p35 subunit of Cdk5 to its more active p25 form(26, 27), leading to increased Cdk5 activity and hyperphosphorylation of tau. Various apoptotic factors have been shown to mediate Aβ-induced neuronal damage, including the p53 tumor suppressor protein(28).

p53 regulates a variety of functions related to cell cycle progression(29), metabolism(30), cell fate determination(31), DNA repair(32), and mitochondrial functions(33, 34). The multiple pro- apoptotic roles of p53 underlie the observation that a significant portion (~50%) of human cancers harbor p53 mutations(29, 35). While p53 is often studied in the context of cancer biology, there is evidence for increased p53 activity contributing to neuronal death in neurodegenerative disease(36-38), particularly AD(39). Increased p53 protein has been observed in brain tissue from AD patients(40-44), and changes in p53 could precede the progression of mild cognitive impairment towards AD(44). p53 has also been implicated in multiple models of Aβ-induced neuronal death (39, 45).

p53 operates downstream of multiple regulatory proteins, allowing it to react to various changes in cellular homeostasis. One p53 regulator is MDMX (or MDM4), an E3 ubiquitin ligase and homologue of the p53-regulating protein MDM2 (murine double minute 2) (46-48). In dividing cells, MDMX promotes cell survival and proliferation primarily via inhibition of p53 (46). MDMX functions in a cooperative manner with MDM2 to promote inactivation, nuclear export, ubiquitination, and proteasomal degradation of p53, although MDMX itself does not ubiquitinate p53 in cells (49, 50). MDMX has been shown to regulate both transcriptional and mitochondria- related functions of p53(46, 51). MDMX has also been shown to negatively regulate the transcription factor E2F1 (52), another cancer-related pro-apoptotic protein(53), in a p53- independent manner (54). Several human cancers are linked to MDMX gain-of-function; multiple types of tumors have been shown to carry MDMX amplifications and/or increased amounts of

MDMX protein (55). Thus, MDMX has become a target for drug development in cancer research

(56).

p53 and E2F1 both promote neuronal death in AD models, and are increased in tissue samples from AD patients (39, 43, 57-59), but functions of neuronal MDMX in AD are not well- known. Several studies have shown that MDMX acts as a pro-survival protein in neurons.

Experiments in mdmx-/- mice show that MDMX is necessary for neuronal survival during CNS

development, and that functions of MDMX in the developing CNS are p53-dependent (60-63).

One study demonstrated that various toxic stimuli (including potassium deprivation or DNA- damaging agents) induce proteolytic degradation of MDMX in cultured neurons, and that overexpression of MDMX protects neurons from aforementioned stimuli(64). This study also determined that reducing MDMX expression causes neuronal death, suggesting that MDMX is necessary for, and supportive of, neuronal survival; and that stress-induced MDMX degradation contributes to activation of downstream cell death factors(64). Previous results from our group also demonstrated toxicity-induced degradation of MDMX in cultured primary neurons, in an in vitro model of HIV-associated neurocognitive disorder (HAND)(65). Overexpression of MDMX was shown to partially protect neurons in an in vitro model of HAND toxicity. We also determined that a small-molecule inhibitor of MDMX induces neuronal damage in vitro(65).

We sought to determine the effects of amyloid on neuronal MDMX, and determine mechanism(s) by which MDMX promotes neuronal survival. We report that extracellular Aβ induces caspase-mediated degradation of MDMX in neurons. Our results also suggest novel roles of MDMX in the stabilization of mitochondria and calpain/Cdk5 activity in neurons. Aβ- induced loss of neuronal MDMX, which we observe in cultured neurons, transgenic animal models, and human tissue, contributes to mitochondrial dysfunction, calpain activation, and Cdk5 hyperactivity, which contribute to the progression of AD.

3.3: MATERIALS AND METHODS

Animals: Hemizygous Tg2576 mice harboring the amyloid precursor protein Swedish (APPswe) mutation (K670N/M671L) and wild-type (WT) mice of the same genetic background (BL6SJL/J) were used. Only female mice were used. Protein samples from the frontal cortices were collected in RIPA buffer with PMSF and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) added, briefly sonicated, centrifuged at 40,000 x g for 30 min at 4ºC, and stored at -80° C prior to use. All

animals were housed at University of Pennsylvania School of Medicine. All animal protocols of the University of Pennsylvania were followed.

Primary culture of cortical neurons: Primary cortical neurons were isolated from brains of embryonic day 16 Sprague Dawley rats, using previously described protocols(65). Briefly, neuroglial cultures were isolated and plated at a density of 750k cells/ml on tissue culture dishes coated with poly-L-lysine (Peptides International, Louisville, KY). Cultured neurons were maintained in neurobasal medium with B27 supplement (Invitrogen, Carlsbad, CA) at 37ºC and

5% CO2. 10 µM Cytosine β-D-arabinofuranoside (AraC; Sigma-Aldrich) was added to cultured cells 72 hours after plating to remove non-neuronal cells. Half of the media volume was replaced with fresh neurobasal/B27 at 7DIV. All neuronal cultures were treated at 14DIV and examined via light microscopy to ensure health prior to treatments.

Human tissue: Tissue samples from patients with AD and age-matched controls were obtained from the Alzheimer Disease Core Center tissue bank at the University of Pennsylvania. Midfrontal cortical tissue was dissected from fresh-frozen tissue from 7 AD cases and 7 control cases.

Diagnosis was defined by clinical history, neurological examination, and neuropathological assessment of Braak staging (AD:Braak stage >4; control: Braak stage = 1) Average ages between AD and control cases were not significantly different (79.1 +/- 3.8 and 81.3 +/- 4.3, respectively). Postmortem interval (PMI) was also not significantly different between AD and control cases (8.0 +/- 0.9 and 14.2 +/- 3.4, respectively). Patient data is shown in Table 1. Protein extracts were prepared for immunoblotting from tissue via manual homogenization on ice in two tissue volumes of a buffer containing: 10 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM

DTT, and protease inhibitor cocktail (Sigma-Aldrich). Homogenates were spun at 3000 rpm for 5 minutes at 4°C. The supernatant was removed and the pellet was resuspended in 4 pellet

volumes of lysis buffer containing 0.1% NP-40, 10 mM Tris (pH 8.0), 10 mM MgCl2, 15 mM NaCl) with protease inhibitor cocktail (Sigma-Aldrich).

In vitro amyloid-β toxicity model: Aβ treatments were performed based on techniques described previously(66). Aβ 1-40 peptide (Bachem, Torrence, CA) was reconstituted in 1% NH4OH and immediately diluted to 1 mg/ml peptide in sterile PBS. This solution was aliquoted and stored at -

20 C prior to use. To induce aggregation and formation of higher-molecular weight oligomers, the peptide solution was diluted to 0.2 ml/ml in neurobasal media, vortexed, and sonicated for 1 min.

This solution was then incubated under gentle agitation for 48 hours at 37°C prior to storage at -

80°C and application to cultured neurons at a final concentration of 10 µM.

Immunohistochemistry: Mouse brains were collected and paraffin embedded, and 6 µm sections were then cut, and mounted on slides. Immunohistochemistry techniques were performed as described previously(67). Prior to immunohistochemistry, tissue samples were heated at 50ºC overnight, and deparaffinized in histoclear (3 times, 15 minutes each). Following rehydration and incubation in 3% hydrogen peroxide, tissue slides were placed in antigen retrieval solution at

95ºC for one hour. After cooling to room temperature, 10% normal goat serum in PBS was used to block the slides. Slides were then incubated overnight at 4ºC in primary antibody, diluted in normal antibody diluent (Scytek, Logan, UT). Antibodies used for IFA included MDMX (H-130 antibody, 1:50; Santa Cruz, Dallas, TX), MAP2 (1:20,000; Covance, Princeton, NJ), and choline acetyltransferase (ChAT, 1:50; Abcam, Cambridge, MA). Primary antibodies were titered to confirm effects prior to use in experiments. FITC- or Cy3- conjugated goat secondary antibodies

(Jackson Immunoresearch, West Grove, PA) were added to slides, with DAPI, and incubated for

30 min at room temperature. Tyramide amplification, described previously(68), was used for IFA of ChAT. Tissue samples were then visualized via confocal microscopy.

Western Blotting: Western blots were performed in the same manner as shown previously(65).

Briefly, neuronal cell lysates were collect by scraping cells in lysis buffer containing 50 mM tris-

HCl, 200 mM NaCl, 0.5% NP-40, 10% glycerol, 1 mM DTT, and 80 µM KCl. The lysates were centrifuged at 20,000 x g for 20 min at 4ºC, and protein concentrations were determined using the

Bradford method (Bio-Rad, Hercules, CA). Proteins were then denatured in LDS and Reducing

Agent (Invitrogen) for 10 min at 70ºC, separated via SDS-PAGE in 4-12% Bis-Tris gels

(Invitrogen), transferred to PVDF membranes (Bio-Rad), and blocked in 5% bovine serum albumin (BSA) in TBS-T. Membranes were incubated with primary antibodies diluted in 5% BSA in TBS-T overnight at 4ºC, and incubated with horseradish peroxidase-conjugated secondary antibodies prior to development using enhanced chemiluminescence. Densitometry analysis was performed using NIH ImageJ software.

TMRM Assay: Cortical neurons were cultured for 14DIV in 96-well poly-L-lysine-coated plates.

Following treatment, cultures were treated with 1 µM tetramethylrhodamine methyl ester (TMRM).

A mixture of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (2 µM) and oligomycin (12 µM) in PBS was used as positive control for mitochondrial damage. After addition of CCCP/oligomycin and TMRM, culture plates were incubated for 30 minutes at 37°C before being washed three times with PBS. A solution of 50% DMSO in dH20 was then added to the wells, and TMRM fluorescence was measured using a Fluoroskan Ascent microplate fluorometer (ThermoFisher

Scientific, Waltham, MA) (Excitation wavelength:544 nm, emission wavelength: 590 nm).

Mitochondrial-Cytoplasmic Fractionation: Primary rat cortical neurons (DIV 21) were harvested from 60mm plates using chilled sucrose extraction buffer (250 mM sucrose, 10 mM HEPES, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 1:20 Protease Inhibitor cocktail (Sigma-Aldrich) and pelleted by centrifugation at 1200 RPM, 4°C. After washing the pellet with ice-cold PBS, the supernatant was discarded and the pellet was resuspended in 150 μL of chilled sucrose extraction buffer. The

resuspended pellet was then homogenized by passing the solution through a 20 gauge needle.

Following low-speed centrifugation at 1000 RPM, the supernatant was collected in an microcentrifuge tube and then spun at high speed (9000 RPM). The resulting supernatant was then transferred to a fresh tube mixed with 5 μL of 10% SDS, and vortexed to produce the cytosolic fraction. The remaining pellet from the high speed centrifugation step was resuspended in 50 μL of ice-cold extraction buffer lacking sucrose (0.5% NP-40, 0.1% SDS, 1 mM DTT, 1 mM

PMSF, 2X PBS, 1:20 protease inhibitor cocktail [Sigma-Aldrich]) to produce the mitochondrial fraction.

Peptides, antibodies, and reagents: All reagents were dissolved in dimethyl sulfoxide

(DMSO)(Sigma-Aldrich), aliquoted, and stored at -20º C prior to use. All treatments were performed by diluting the agent(s) in conditioned media from cultured neurons, and re-applying the diluted agent to the cultured neurons. SJ-172550 (Tocris, Bristol, UK) was used at 20 µM. Q-

VD-OPh (Tocris) was used at 50 µM. Roscovitine (Sigma-Aldrich) was used at 1 µM. MDL-28170

(MDL; Tocris) was used at 20 µM. 1 µM staurosporine (Sigma-Aldrich) was used to induce caspase activation. Pifithrin-α and pifithrin-µ (Tocris) were both used at 10 µM. Antibodies used for immunoblotting include MDMX (C-19 and H-130; Santa Cruz), amyloid precursor protein

(mab314; Millipore, Temecula, CA), cleaved caspase-3 (Cell Signaling, Danvers, MA), COX4 (BD biosciences, Franklin Lakes, NJ), p35/25 (Cell Signaling), and A38 antibody (a generous gift from

Dr. Robert Siman at the University of Pennsylvania) was used to detect calpain-cleaved spectrin.

Data analysis: An unpaired Student’s t-test was used for comparisons between two groups.

ANOVA with Tukey's post-hoc analysis was used for comparisons across multiple groups/conditions. All experiments shown were replicated in at least 3 independent experiments. p values lower than 0.05 were interpreted as statistically significant. Averages are expressed as mean +/- SEM. Prism 5.0 (Graphpad; La Jolla, CA) was used for all data analysis.

3.4: RESULTS

MDMX protein is reduced in frontal cortices of aged Tg2576 mice

To study the effects of amyloid toxicity on neuronal MDMX, we utilized a murine model of

AD amyloid pathology. Tg2576 (APPswe) mice display age-dependent deficits in learning and memory, increased cortical Aβ1-40 and Aβ1-42, and amyloid plaque formation in the cortex and limbic system (69-71). We used immunofluorescence labeling of MDMX to examine changes in

MDMX associated with amyloid pathology in Tg2576 mice. It was noted that MDMX was primarily localized to soma and nuclei in the neurons of the frontal cortex. There was a significant loss of

MDMX-expressing neurons in the cortices of aged Tg2576 mice compared to those of wild-type animals (Fig. 1A-B). We observed no significant difference in the number of cortical MDMX- positive neurons between 4 month-old Tg2576 mice and aged wild-type mice, demonstrating that the loss of MDMX-positive neurons coincides with age-dependent pathology and is not directly caused by the genetic background of Tg2576 mouse. We also determined, via immunoblot of tissue lysates, that full-length MDMX protein is significantly reduced in the frontal cortices of aged

(14 month-old) Tg2576 mice relative to wild-type animals of the same age (Fig. 1C-D). These results demonstrate that age-dependent amyloid pathology phenotype of Tg2576 mice induces a) loss of MDMX-expressing neurons in the frontal cortex, and b) loss of total cortical MDMX protein.

Loss of MDMX-expressing cholinergic neurons in aged Tg2576 mice

Considering the reduced number of MDMX-positive neurons induced by amyloid pathology, we sought to determine the identity of these neuronal populations. One pathological feature of AD is the progressive loss of cholinergic neuronal populations in the cortex and hippocampus(72), and defects in cholinergic signaling have been demonstrated in human AD cases as well as in Tg2576 mice(73). Using immunofluorescence labeling of MDMX and choline acetyltransferase (ChAT), we determined that the majority (~80%) of cholinergic neurons in the frontal cortices of wild type mice express MDMX (Fig. 2D).

Next, we examined the distribution of these MDMX-expressing cholinergic neurons in the frontal cortex of Tg2576 mice to determine the effects of amyloid accumulation on these neuronal populations. Our goal was to determine whether cholinergic neurons expressing MDMX die due to amyloid toxicity, or if they remain intact while losing MDMX immunoreactivity, presumably due to degradation. Using immunofluorescence labeling of MDMX and ChAT, we were able to determine that cholinergic neuronal populations are significantly reduced in aged Tg2576 mice relative to aged wild-type and 4 month-old Tg2576 animals (Fig. 2B). We then determined that the number of MDMX-expressing cholinergic neurons are reduced in aged Tg2576 mice in a similar fashion (Fig. 2C), suggesting that progressive death of MDMX-expressing cholinergic neurons occurs during aging as amyloid pathology develops. Our results suggest that amyloid toxicity contributes to the death of MDMX-expressing cholinergic neurons in the frontal cortex.

Aβ induces caspase-mediated degradation of MDMX in primary cortical neurons

After examining loss of neuronal MDMX protein in a murine model of amyloid pathology, we examined the effects of Aβ on MDMX in cultured neurons. Prior studies (64) have shown that multiple types of toxic insult contribute to caspase-mediated and proteasomal degradation of

MDMX. There is also evidence for degradation of neuronal MDMX by calpain proteases in excitotoxic conditions(65). We thus utilized an in vitro model of amyloid toxicity (66) to test the effects of Aβ1-40 oligomers on MDMX in primary neurons. To test the role of caspases in the degradation of MDMX, we pre-treated cultured neurons with Q-VD-OPh (OPH), a pan-caspase inhibitor, prior to addition of Aβ. The appearance of cleaved caspase-3 via immunoblot was used as a marker for caspase activation. Hydrogen peroxide was used as a positive control for caspase activation. We observed that Aβ causes loss of MDMX protein in neurons in conjunction with increased caspase-3 cleavage. We also determined that Aβ-induced MDMX loss is reversed by inhibition of caspases (Fig. 3A). Our results suggest that Aβ activates caspases, causing

degradation of MDMX in neurons. Similarly, the caspase-inducing agent staurosporine was also shown to induce degradation of MDMX in neurons (Fig. 3B).

Inhibition of MDMX causes mitochondrial depolarization, increased calpain activation and p35 cleavage in primary cortical neurons

Evidence from diving cells indicates that MDMX localizes to the mitochondria, where it interacts with Bcl-2 and mitochondrial p53, facilitating pro-apoptotic functions of p53(51, 74). It is unknown if/how MDMX interacts with mitochondria in neurons, or if these functions are related to neuronal p53. Therefore, we analyzed mitochondrial fractions from cultured cortical neurons for

MDMX and p53. Our results indicate that MDMX is present in mitochondrial fractions of neurons

(Fig.4A). The presence of higher- and lower-molecular weight forms of MDMX in the mitochondria suggests post-translational modification of mitochondrial MDMX, or possibly that specific isoforms of MDMX are selectively enriched in mitochondria. The presence of MDMX in the mitochondria could provide basis for neuronal death-related functions of MDMX dependent on mitochondrial function.

Based on previous studies demonstrating a role of MDMX in apoptotic signaling via interaction with mitochondrial proteins and p53(51), we sought to determine if MDMX functions relate to mitochondrial stability in neurons. We utilized the MDMX inhibitor, SJ-172550(75), to determine the effects of MDMX inhibition on mitochondrial membrane potential, which was measured via TMRM assay of cultured neurons. Our results indicate that inhibition of MDMX causes significant mitochondrial depolarization as early as 2 hours after treatment (Fig. 4B). After

16 hours of treatment with the MDMX inhibitor, the loss of TMRM signal was similar to that induced by direct mitochondrial-damaging agents (carbonyl cyanide m-chlorophenyl hydrazone and oligomycin).

To determine which downstream effectors of MDMX might mediate SJ-172550-induced mitochondrial damage, we pre-treated cultured neurons with a) a calpain inhibitor (MDL), b) a

Cdk5 inhibitor (roscovitine), and c) p53 inhibitors (PFT-µ and PFT-α). Our previous results demonstrated that inhibition of calpains prevents SJ-172550-induced neuronal damage(65).

Surprisingly, MDL had no significant effect on mitochondrial damage induced by SJ-172550, nor did roscovitine, or either of the p53 inhibitors used (Fig. 4C). This data demonstrates that MDMX function is necessary for mitochondrial stability in neurons, and that this function occurs independently of p53, calpains, and Cdk5.

Previous work indicates that calpain-mediated cleavage of the p35 subunit of Cdk5 to a p25 form causes hyperactivity of Cdk5(26, 76). In neurons, Cdk5 hyperactivity has been implicated in neuronal death in multiple disease models, including AD(21) and HAND(77, 78).

Based on our observations of Aβ-induced degradation of MDMX in transgenic mice (Figs. 1-2) and in cultured neurons (Fig. 3A), and that pharmacological block of MDMX induces calpain- dependent neuronal damage(65), we hypothesized that loss of MDMX could contribute to changes in Cdk5 activity via activation of calpains. To assess changes in Cdk5 and calpain downstream of MDMX, we treated cultured cortical neurons with SJ-172550(75), a small- molecule inhibitor of MDMX. Treatment of cultured cortical neurons with the MDMX inhibitor SJ-

172550 induced accumulation of calpain-cleaved spectrin, a product of calpain activation (Fig.

4D). We also observed evidence of Cdk5 hyperactivation in response to MDMX inhibition, based on cleavage of p35 to p25.

To test if inhibition of calpains could reverse p25 generation caused by pharmacological block of MDMX, we pre-treated neurons with the calpain inhibitor MDL prior to addition of SJ-

172550. Inhibition of calpains lead to partial reversal of p35 cleavage in SJ-treated neurons (Fig.

4E), suggesting that p35 cleavage downstream of MDMX loss is calpain-mediated.

MDMX protein is reduced in brain tissue from AD patients

Based on our previous experiments demonstrating that MDMX protein is degraded in transgenic animals, as well as in vitro models of Aβ toxicity, we examined MDMX in brain tissue

from AD patients and age-matched controls to test how our experimental models resemble effects observed in human patients. We collected whole-cell lysates from human brain tissue

(Table 1) using an antibody targeted to the C-terminus (H-130; amino acids 361-490) of MDMX.

Immunoblotting for MDMX revealed a 55 kDa band consistent with previous observations of

MDMX in human brain tissue(65). Analysis of tissue samples from AD and non-AD patients showed a significant loss of MDMX protein in AD brain tissue (Fig. 5). We observed no significant independent effects from postmortem interval or age on MDMX protein level in human tissue

(Table 1). These findings demonstrate that MDMX is reduced in the brain during AD, and corroborate our observations of amyloid-induced degradation of MDMX in aged APPswe mice and in cultured cortical neurons.

3.5: DISCUSSION

MDMX as a Neuroprotective Protein & Node for Neurotoxic Stress

Our results support the hypothesis that MDMX promotes, and is necessary for, cell survival in neurons as well as dividing cell types. We show, in agreement with previous studies, that 1) toxic factors induce the proteolytic degradation of MDMX, and 2) inhibition of MDMX induces cell death in cultured neurons (64, 65). Collectively, these studies suggest that MDMX is subject to multiple types of degradation in neurons, acting as a "node" with sensitivity to a variety of cellular stress signals. In dividing cells, this “node” function of MDMX allows cells to initiate apoptosis in response to DNA damage; but in neurons, this function contributes to neuronal death following exposure to extracellular stressors. The type of MDMX degradation which occurs is likely dependent on the type of toxic factor present, and the type(s) of proteases which subsequently become activated. While caspase- and proteasome-mediated MDMX degradation occur in neurons and in dividing cells (64, 79), calpain-mediated MDMX degradation could be a neuron-specific event which occurs only due to excitotoxic stimuli (i.e. NMDA, Glutamate, or HIV-

M/M supernatants (65)) as no studies in dividing cells have observed degradation of MDMX by

calpains. It is also noteworthy that caspase and calpain activation are not mutually exclusive mechanisms in neuronal death processes, and cross-talk between caspases and calpains can occur through a variety of mechanisms (80).

When MDMX is degraded, or inactivated pharmacologically, downstream pro-death factors are activated, and neuronal death follows. Knockdown of MDMX using antisense oligonucleotides was shown to induce neuronal apoptosis(64), genetic deletion of MDMX in the

CNS causes an apoptotic phenotype and embryonic lethality(62), and the MDMX inhibitor SJ-

172550 induces neuronal damage in a calpain-dependent, caspase-independent manner(65).

Our findings implicate mitochondrial depolarization and calpain-mediated Cdk5 hyperactivation in neuronal damage downstream of MDMX inactivation. Mitochondrial damage could mediate the effect of MDMX loss on calpain activation due to released calcium leaked form damaged mitochondria, which has been observed previously(81, 82). Research in dividing cells determined that mitochondrial MDMX promotes p53-based apoptotic functions by mediating interactions between p53 and Bcl-2(51). It is not yet known precisely how MDMX promotes mitochondrial stability in neurons, but our results suggest that mitochondrial MDMX is vital to mitochondrial function, and functions in a p53-independent manner.

MDMX in AD

We observe loss of MDMX-expressing neurons and total cortical MDMX protein in a transgenic murine model of age-dependent amyloid pathology. We also observe Aβ-induced

MDMX degradation in cultured neurons, and reduced MDMX in human AD brain tissue. A previous study observed a small, but significant increase in MDMX mRNA in patients with dementia, but this change did not associate with changes in plaque density or Braak staging(83).

Our observations suggest that the regulation of MDMX occurs primarily at the protein level in neurons, as observed previously(64). Nonetheless, changes in MDMX expression in AD could

indicate MDMX upregulation as a protective mechanism, and more study is needed to determine how neurons regulate MDMX expression.

We determined that the majority of cholinergic neurons in the frontal cortex express

MDMX, and that this neuronal population, which is particularly vulnerable to damage in AD(72), displays a significant loss of MDMX-expressing neurons. Damage to cholinergic populations in

AD mouse models is consistent with previous observations(84). Our assessment of MDMX in brain tissue of transgenic mice is not without limitations; analysis of monthly changes in cortical

MDMX over the murine lifespan would allow more precise tracking of changes. Further studies of this nature could help determine the changes in neuronal MDMX in AD models and normal aging.

Nonetheless, our study identifies MDMX loss in neurons in an age-dependent amyloid toxicity model, suggesting that Aβ induces degradation of MDMX in neurons of the frontal cortex, including cholinergic neuronal populations(72). Our experiments in cultured neurons suggest that the loss of MDMX protein in aged APPswe mice is caused by amyloid-induced caspase activation.

Considering that mitochondrial defects are observed of AD(85-88), and also implicated in aging and aging-related neurodegenerative diseases(89-91), the necessity of MDMX in regulating mitochondrial function provides a novel mechanism by which Aβ-induced MDMX degradation could contribute to neuronal dysfunction or death. We determined that pharmacological inhibition of MDMX caused mitochondrial depolarization, and that MDMX localizes to mitochondria in neurons. This effect was not altered by pharmacological block of calpains, Cdk5, nor p53, suggesting that mitochondrial dysfunction is one of the earliest pathological events which occurs following MDMX loss. While our previous data show that neuronal damage induced by MDMX inhibition is calpain-dependent(65), mitochondrial damage caused by MDMX inhibition occurs independently of calpain activity. MDMX degradation could cause destabilization of mitochondria, leakage of mitochondrial calcium(92, 93), and subsequent calpain/Cdk5 activation during Aβ- induced neuronal stress. In addition to direct neurotoxicity(94), and further dysregulation of intracellular calcium(95), increased activity of calpain and Cdk5 can also lead to tau

hyperphosphorylation, favoring the formation of neurofibrillary tangles(96, 97). Because mitochondrial damage, increased calpain activity, and hyperactivity of Cdk5 are all implicated in

AD(21, 24, 25, 88, 98-100), our results suggest that degradation of MDMX is a mechanism by which Aβ stimulates these pathogenic intracellular events. Further understanding of the mechanisms underlying the degradation of MDMX in neurons, and subsequent changes in downstream signaling pathways could yield potential targets for neuroprotective interventions.

Our studies have implicated loss of MDMX protein as part of the neurodegenerative process in both AD and HAND(65). In our in vitro model of HAND neurotoxicity, calpain-induced p25 generation, MDMX degradation, and neuronal death were also observed (65, 77).

Intriguingly, there is evidence for AD-like pathology in HAND patients, including amyloid(101) and neurofibrillary(102) pathology, suggesting that dysregulation of MDMX and its downstream targets is a common factor underlying neurodegeneration in AD, HAND, and potentially other neurodegenerative diseases.

MDMX negatively regulates p53 in neurons(64), which is implicated in neuronal death in

AD(39), the regulation of the gamma-secretase complex(103), and expression of APP(104).

Thus, it is possible that Aβ-induced MDMX loss could cause excessive p53 activity, leading to dysregulated APP expression and/or processing. Aβ-induced MDMX degradation could create a positive feedback loop in which Aβ is progressively generated in a p53-dependent fashion, triggering additional MDMX degradation in neighboring neurons, and further propagation of pathology. Interestingly, an isoform of p53, p44(105), was found to promote accelerated aging, neurodegeneration, and memory defects in mice(106, 107). p44 can also cause tau hyperphosphorylation via transcriptional upregulation of Cdk5 and related tau kinases(108).

MDMX-mediated regulation of p53, particularly the p44 isoform, represents an intriguing target for future investigation.

Cancer and Neurodegenerative Disease

In contrast to cancer, which can be caused by defects in apoptotic signaling pathways, neuronal death in AD has been connected with activation of pro-apoptotic proteins. It is notable that

MDMX, a protein which prevents apoptosis in cancer models via p53 regulation, is reduced in neurons in AD and AD models; while tumor suppressors (p53 and E2F1) are increased in neurons during AD(40, 43, 44, 58, 109). Considering that MDMX can inhibit both p53 and

E2F1(46, 52), loss of MDMX could be a mechanism by which p53 and/or E2F1 become increased in AD. MDMX amplifications are associated with several types of cancer(55); and it is possible that such mutations could also contribute to a neuroprotective phenotype. Similarly, a mutation that reduces the stability or activity of MDMX could render a phenotype resistant to some forms of cancer, but with increased vulnerability for neurodegenerative disease. Clinical studies have shown that an inverse relationship exists between the occurrence of cancer and neurodegenerative disease(110, 111). Our work suggests that MDMX is part of a group of proteins implicated in both cancer and neurodegenerative disease(112). The interactions between MDMX and other proteins in this group, particularly p53, E2F1, and Cdk5, provide potential avenues for future study.

Summary

We observe loss of MDMX in the frontal cortex, and cholinergic populations, of aged APPswe mice. Similar loss of MDMX protein is also observed in tissue from AD patients. We demonstrate that MDMX degradation is mediated by caspases in an in vitro model of amyloid toxicity. We also show that inhibition of MDMX function in neurons leads to mitochondrial depolarization, calpain activation, and Cdk5 hyperactivity. Aβ-induced degradation of neuronal MDMX is a mechanism which could connect extracellular amyloid to dysregulation of critical intracellular processes in neurons during the progression of AD.

3.6: ACKNOWLEDGEMENTS

The authors thank Margaret Manowski for preparation of primary neuronal cultures. We also thank Chenere Ramsey for aid in processing tissue samples. We thank Dr. Cagla Akay, Dr.

Michelle Erickson, and Patrick Gannon for critical discussions. We are also grateful to Dr. Virginia

M.-Y. Lee, Dr. Jenna Carroll, and the Center for Neurodegenerative Disease Research (CNDR) for tissue samples, as well as productive discussions.

Figure 1: MDMX protein is reduced in frontal cortices of aged Tg2576 mice.

(A) Representative gray matter images from frontal cortical sections of mice labeled for MDMX

(green), MAP2 (red), and DAPI (blue). (B) Quantification of MDMX-positive neuron counts.

Values are expressed as mean +/- S.E.M. ANOVA with Tukey’s post-hoc analysis; ***p<0.001, n=5. (C) Frontal cortices from 14-month old wild-type and Tg2576 mice were homogenized, proteins were separated via SDS-PAGE, and proteins were analyzed via immunoblot for MDMX

(H-130 antibody), APP (mab314 antibody), and actin (loading control). (D) Quantification of

MDMX protein level in wild-type and Tg2576 mice. Values are expressed as mean +/- S.E.M.

Student's T-test; **p<0.01, n=5.

Figure 2: Loss of MDMX-expressing cholinergic neurons in aged Tg2576 mice.

(A) Representative gray matter images from frontal cortical sections of mice labeled for MDMX

(green), ChAT (red), and DAPI (blue). (B) Quantification of ChAT-positive cells. (C) Total number of cells positive for both MDMX and ChAT. (D) Percent of ChAT-positive cells expressing MDMX.

All values are expressed as mean +/- S.E.M. ANOVA with Tukey’s post-hoc analysis; **p<0.01,

***p<0.001, n=5.

Figure 3: Aβ induces caspase-mediated degradation of MDMX in primary cortical neurons.

(A) Cultured cortical neurons were treated with OPH (50 µM) for 2 hours prior to addition of aggregated Aβ1-40 peptide (10 µM) for 24 hours. Neuronal lysates were collected and separated via SDS-PAGE, and proteins were analyzed via immunoblot for MDMX, cleaved caspase-3, and actin (loading control). (B) Staurosporine (1 µM) was added to cultured neurons and proteins were analyzed via immunoblot for MDMX, cleaved caspase-3, and actin (loading control).

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Figure 4: Inhibition of MDMX causes mitochondrial depolarization, increased calpain activation and p35 cleavage in primary cortical neurons.

(A) Cytoplasmic (cyto) and mitochondrial (mito) fractions collected from cultured neurons were immunoblotted for MDMX. COX4 and GAPDH were used as mitochondrial and cytoplasmic markers. Coomassie gel staining was used to control for loading.

(B) Cultured neurons were treated with SJ-172550 (20 µM) or DMSO (veh). Following treatment, mitochondrial membrane potential was measured via TMRM assay. A solution of carbonyl cyanide m-chlorophenyl hydrazone (2 µM) and oligomycin (12 µM) (cccp/oligomycin) was used as a positive control for mitochondrial damage. All values are expressed as mean +/- S.E.M.;

ANOVA with Tukey’s post-hoc analysis; ***p<0.001, ns=no significant change, n=6.

(C) After a 1 hour pre-treatment with MDL (20 µM), roscovitine (1 µM), pifithrin-µ (10 µM), or pifithrin-α (10 µM), cultured neurons were treated with SJ-172550 (20 µM) or DMSO (veh) for 24

101

hours. Following treatment, mitochondrial membrane potential was measured via TMRM assay.

All values are expressed as mean +/- S.E.M.; ANOVA with Tukey’s post-hoc analysis;

***p<0.001, ns=no significant change, n=6.

(D) Effects of SJ-172550 (20 µM) on calpain-cleaved spectrin and p35/25 were assessed in a time-dependent manner via immunoblot. Actin was used as a loading control. Representative blot shown.

(E) MDL (20 µM) or DMSO (veh) was added to cultured neurons 2 hrs prior to SJ-172550 treatment (20 µM) or treatment with DMSO (veh). Neuronal lysates were collected 8 hrs later.

Effects on calpain-cleaved spectrin and p35/p25 were measured via immunoblot. Actin was used as a loading control. Representative blot shown.

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Figure 5: MDMX protein is reduced in brain tissue from AD patients.

(A) Protein samples from normal (N) and Alzheimer Disease-diagnosed (AD) patients were separated via SDS-PAGE and immunoblotted for MDMX. Coomassie gel staining was used as a loading control. (B) MDMX immunoreactivity was measured using NIH ImageJ and normalized to loading. Values are expressed as mean +/- S.E.M. and normalized as a % of normal MDMX level.

Significance determined via Student's T-test. *p<0.05, n=7 per group.

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CHAPTER 4

"MDMX Promotes Neuronal Survival in a p53-Independent Manner"

Daniel J. Colacurcio and Kelly L. Jordan-Sciutto

Department of Pathology, School of Dental Medicine, University of Pennsylvania, 312 Levy

Building, 240 South 40th Street, Philadelphia, PA 19104, United States

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4.1: ABSTRACT

The p53 tumor suppressor has been implicated in neuronal death during neurodegenerative diseases, including AD and HAND. In dividing cells, MDMX acts as a pro- oncogene by negatively regulating p53. Recent studies have shown that MDMX promotes neuroprotection in post-mitotic neurons. While previous studies have demonstrated that MDMX regulates transcriptional functions of p53 in cultured neurons, it remains unclear if MDMX promotes neuronal survival via p53 regulation. Because p53 is implicated in neuronal death in multiple neurodegenerative diseases, we sought to determine if MDMX promotes neuronal survival via inhibition of p53. Using pharmacological techniques, we demonstrate that MDMX-p53 interactions do not directly relate to neuronal viability in our model, and that the pro-survival functions of MDMX occur in a p53-independent manner in mature neurons.

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4.2: INTRODUCTION

The p53 tumor suppressor has been implicated in neuronal death in multiple neurodegenerative diseases, including Alzheimer Disease (AD) and also HIV-associated neurocognitive disorder (HAND) [1-6]. (See Chapter 1.5 for further discussion of p53 in neurodegenerative disease). Increased p53 is observed in brain tissue from AD patients [7-10], and mechanistic studies demonstrate interactions between p53 and amyloid processing [11-13]. p53 can also promote neuronal death [14], both as a transcription factor, and as a regulator of mitochondrial function [1, 15-18]. Due to the multiple roles of p53 in neurodegeneration and AD pathogenesis, an understanding of p53-regulating proteins in neurons could be beneficial for the potential development of effective neuroprotective therapies. p53 is a fundamental regulator of a diverse variety of processes in all cell types [19, 20], and thus, is subject to regulation by a large number of proteins [21]. The primary p53 regulators in dividing cells are murine double minute 2

(MDM2), and murine double minute X (MDMX) [22, 23]. (See Chapter 1.3 for further discussion of the MDM2-MDMX-p53 network).

The primary functions of MDMX in dividing cells are related to the regulation of p53 [24].

MDMX was originally discovered as a homologue to the p53-binding protein [25], MDM2, and since, the majority of the studies on MDMX have addressed its ability to negatively regulate p53 activity in the context of tumor formation [23, 25-29]. Unlike MDM2, MDMX is not able to directly ubiquitinate p53 (except in a highly specialized cell-free system [30]), but MDMX is still able to regulate p53 through a variety of mechanisms. These include direct binding, which prevents p53 transactivation in the nucleus, shuttling of p53 from the nucleus, and allosteric activation of MDM2

[24, 31, 32]. These mechanisms allow MDMX to prevent p53-mediated apoptosis in dividing cells, and endow the cell with sensitivity to stress factors, such as DNA damage, which disinhibits p53 via MDMX degradation, leading to apoptosis [33].

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But while MDMX has been shown to promote neuronal survival during development [34-

36] and in cultured neurons [37], it is not known if these functions occur through the regulation of p53 in mature neurons. Examination of the developing CNS of mdm4-/- mice demonstrated improper neural tube closure, dilated lateral ventricles, and caspase-3 activation, in addition to premature embryonic lethality [35]. These effects were not observed when the mdm4-/- mice were crossed with p53-/- mice, leading to the conclusion that MDMX is a critical regulator of p53 during development, and that neuronal cell fate decisions mediated by MDMX are p53-dependent in the developing CNS [35]. In cultured cerebellar granule neurons, it was shown that the same stress factors which induce neuronal MDMX degradation (glutamate, cisplatin, and neocarzinostatin) also induce increased p53 protein [37]. It was not determined if alterations in MDMX are the cause of the observed increases in p53 level, but in this same neuronal culture model, overexpression of MDMX was shown to reduce the transcriptional activity of p53 [37]. Taken together, these results suggest that MDMX promotes neuronal survival in a p53-dependent manner during CNS development, and that MDMX also regulates p53 transcriptional activity in cerebellar granule neurons, providing evidence that MDMX and p53 maintain a functional relationship in neurons. However, it remains unknown if the pro-survival functions of MDMX in mature neurons occur through inhibition of p53 as they do in dividing cells. We utilized a small- molecule inhibitor of MDMX-p53 interactions, SJ-172550 [38], in conjunction with a neuronal damage assay in cultured neurons to test the potential of MDMX to regulate p53 in neurons.

4.3: MATERIALS & METHODS

Primary Neuronal Cultures: All neuronal cultures were obtained via dissection from frontal cortices of Sprague–Dawley rat pups from embryonic day 17 from timed-pregnant adult rats, as described previously [39]. Rat cortical neurons (with glia present) were cultured in a 37°C

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incubator with 5% CO2 for 21 DIV in 96-well cell culture plates at an initial plating density of

50,000 cells per well. 50% of media volume was replaced with fresh media every 7 days.

Treatments, Antibodies, and Reagents: For MAP2 ELISA, cultured neurons were pre-treated with 10 µM Pifithrin-α (Tocris, Bristol, UK), 10 µM Pifithrin-µ (Tocris, Bristol, UK), or dimethyl sulphoxide (DMSO; ThermoFischer, MA) vehicle for 2 hours prior to treatment with 30 µM SJ-

172550 (Tocris, Bristol, UK) or DMSO for 24 hours. For all treatments, stock solutions of all small molecules were made up in DMSO, and added to cultures by dilution to final concentration in conditioned culture media. Treatments were replicated in two independent experiments.

MAP2 ELISA: MAP2 ELISA for neuronal viability was performed as demonstrated previously

[40]. Briefly, Following treatments, media was removed and cells were fixed in 4% paraformaldehyde for 30 minutes, blocked using 5% normal goat serum in PBS, and incubated in

(1:1000) MAP2 antibody (Covance, Princeton, NJ) at 4°C overnight. The cells were then incubated for 30 min with a β-lactamase TEM-1-conjugated goat anti-mouse secondary antibody

(Molecular Probes, Eugene, OR), and washed with PBS-T three times, and then once with PBS.

Cells were then incubated (in the dark) with 0.5μg/well Fluorocillin Green substrate (Molecular

Probes), while gently rocking at room temperature for 1 hr. Fluorescence intensity was measured using a Fluoroskan Ascent plate reader (Thermo Electron, Waltham, MA) with excitation at 485 nm and emission at 527 nm.

4.4: RESULTS

Neuronal damage induced by MDMX inhibition is p53-independent

To address the question of whether MDMX promotes neuronal survival through negative regulation of p53, as observed previously in dividing cells [24], we employed a pharmacological approach in cultured cortical neurons. We utilized the MDMX inhibitor, SJ-172550, which was

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previously shown to induce p53-dependent apoptosis in cell lines via interacting with the p53- binding site of MDMX [38]. SJ-172550 was applied to cultured neurons following pre-treatment with p53 inhibitors. The first was pifithrin-α, which prevents p53-mediated transcriptional functions

[41], and has been shown to promote neuroprotection [42-44]. We also pre-treated our cultures with pifithrin-µ, which prevents the apoptotic effects of p53 by reducing p53 binding affinity with mitochondrial proteins [45]. We tested the effects of these inhibitors in cultured cortical neurons using a MAP2 ELISA as an assay of neuronal viability [40]. Our results showed that SJ-172550- induced significant neuronal damage, which was previously observed [39], but we also demonstrate that SJ-172550-induced damage was not significantly altered by pre-treatment with either of two p53 inhibitors (Fig. 4.1). Thus, our data suggest that the pro-survival effects of

MDMX occur independently of p53, with regard to both transcriptional and mitochondrial functions of p53.

4.5: DISCUSSION

Our findings suggest that the neuronal cell death activated following pharmacological block of MDMX is not affected by the inhibition of p53, suggesting that the pro-survival functions of MDMX in neurons are not mediated by p53. These results demonstrate a key difference in the functions of MDMX in neurons as opposed to dividing cells. These findings also raise questions regarding the p53-independent mechanism(s) by which MDMX promotes neuroprotection, which is observed in multiple models [37, 39]. Previous work has demonstrated that activation of the calpain-Cdk5 pathway is sufficient to cause death in the absence of p53 and "classical" apoptotic factors [46]. Our previous results indicate that inhibition of MDMX leads to rapid mitochondrial depolarization, calpain activation, and Cdk5 activation (Chapter 3), suggesting that MDMX mediates critical functions in the maintenance of mitochondrial stability and calpain activity. We also observed that neuronal damage induced by SJ-172550 is reversible by inhibition of calpains,

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but not caspases [39]. Calpain activation following MDMX inhibition leads to hyperactive Cdk5

(based on cleavage of the p35 subunit) (Chapter 3). Cdk5 hyperactivation [47], in conjunction with calpain-mediated MDMX degradation [39] was also observed in our model of HIV-associated neurotoxicity.

While our results suggest that MDMX does not promote neuronal survival via negative regulation of p53, MDMX still maintains a functional relationship with p53 in neurons, based on previous observations [37]. This relationship could have significant implications for long-term neuronal homeostasis and progression of amyloid pathology in the context of AD. The interactions between p53, APP expression, and members of the APP processing network [11, 12] provide a potential mechanism by which MDMX loss, which is observed in AD and AD models,

(Chapter 3) could influence further p53-mediated amyloidogenic APP processing in the development of AD. Degradation of MDMX in AD could be the cause of neuronal p53 accumulation observed in MCI (mild cognitive impairment) and AD tissue [7-10, 16]. The MDMX- p53 relationship in mature neurons could have implications to neurodegenerative conditions other than AD [1, 4, 5]. p53 is implicated in neuronal death in Parkinson Disease [48], as well as HIV- associated Neurocognitive Disorder (HAND) [6]. Loss of MDMX function could affect p53- mediated APP processing in HAND, during which amyloid pathology has also been observed

[49]. (Future directions of research regarding the MDMX-p53 relationship in neurons, as well as downstream effectors of MDMX-mediated neuroprotection, are discussed further in Chapter 5.)

In summary, our results indicate that neuronal damage induced by MDMX loss-of- function is not prevented by inhibition of p53. Our results suggest that the MDMX-p53 relationship, predominantly studied for its role in tumorigenesis, is fundamentally different in post- mitotic mature neurons than in dividing cell types, in which MDMX promotes cell survival via negative regulation of p53. Further understanding of the mechanisms of MDMX-mediated neuroprotection, as well as the cell death-independent functions of MDMX-p53 interactions in

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neurons, could potentially identify novel therapeutic targets for promoting neuroprotection in the context of neurodegenerative disease.

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Figure 4.1: Neuronal damage induced by MDMX inhibition is p53-independent. Prior to 24 hour treatment with 30 µM SJ-172550 (SJ), 21DIV rat cortical neurons were pre-treated for 2 hours with the p53 inhibitor, pifithrin-α (PFT-α, 10 µM), pifithrin-µ (PFT-µ), or DMSO (Veh).

Following treatment, neuronal damage was measured via MAP2-based ELISA. Values shown represent mean +/- S.E.M.. Significance was determined via ANOVA and Tukey's post-hoc analysis. ***p<0.001; NS=not significant.

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4.6: BIBLIOGRAPHY

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CHAPTER 5

CONCLUSIONS & FUTURE DIRECTIONS

5.1: OVERVIEW

Our studies have added to the understanding of the functions of the oncoprotein MDMX in neurons. While previous work demonstrated a neuroprotective function of MDMX in cultured neurons [1], and the necessity of MDMX in the developing CNS [2], our work has built upon these initial discoveries; identifying mechanisms of proteolytic MDMX degradation which occur in two neurodegenerative diseases (AD and HAND), and elucidating downstream mechanisms by which

MDMX promotes neuronal survival. We determine that degradation of neuronal MDMX, which can be mediated by calpains (during HAND) and by caspases (during AD), is a permissive step in the neuronal death process, which is likely to contribute to neuronal death observed in both

HAND and AD. Additionally, we demonstrate novel mechanisms by which loss of MDMX function contributes to neuronal damage via mitochondrial dysfunction, as well as calpain activation and pathological Cdk5 activity, suggesting that MDMX degradation is a trigger for multiple harmful intracellular processes which occur during the pathogenesis of AD and HAND. Finally, we have shown the neuroprotective functions of MDMX are p53-independent in cultured neurons, representing a significant departure from the p53-dependent functions of MDMX observed in mitotic cells.

However, multiple questions remain regarding the functions of MDMX in neurons. The loss of MDMX protein during neurodegenerative diseases and the involvement of MDMX in neuronal survival raise new questions regarding the specific functions of MDMX in neurons and potential pro-survival functions which remain unknown. More broadly, our research on MDMX suggests further research is necessary to address the functions of similar cell cycle- and cancer- related proteins in post-mitotic neurons, which may represent a valuable route for neuroprotective therapeutic interventions in neurodegenerative diseases such as AD and HAND.

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5.2: MDMX DEGRADATION MECHANISMS IN NEURONS

Degradation of neuronal MDMX has been demonstrated to occur as a reaction to cellular stress factors in cultured neurons (Chapter 3)[1, 3]. Our data suggest that neuronal MDMX is subject to proteolytic degradation by both caspases and calpain proteases[3]. While caspase- mediated MDMX degradation has been observed in dividing cells [4], it is possible that calpain- mediated MDMX degradation is a neuron-specific event. Previous work determined that MDMX is degraded by the proteasome in cultured neurons [1]. It is possible that proteasomal degradation of MDMX could be the "default" processing pathway (as is common for many proteins), but during neurotoxic conditions, MDMX becomes degraded by calpains or caspases, depending on the type of toxic insult. In our studies, calpains are activated in our HIV-M/M model [3], and caspases are activated in a model of Aβ toxicity (Chapter 3). While both proteases are able to degrade

MDMX in neurons, neuronal damage downstream of MDMX loss-of-function is mediated by calpains and is caspase-independent [3]. Thus, we conclude that the neuroprotective functions of

MDMX occur upstream of fatal calpain activation. In our model, HIV-M/Ms initiate neuronal death in a calpain-dependent manner, consistent with the predominant toxic components in HIV-M/M supernatants being excitotoxic factors, such as glutamate [5].

It is noteworthy, considering that neuronal death does not conform strictly to classical definitions of apoptosis [6, 7], calpain- and caspase-dependent MDMX degradation and subsequent neuronal death may not be mutually exclusive events. As the progression of neuronal death occurs, one family of proteases is able to activate the other [8]. For instance, calpistatin is an endogenous calpain inhibitor, which is also a caspase substrate. Calpistatin is but one of many proteins which interacts with both caspases and calpains[9]. A variety of signaling-related and cytoskeletal proteins are directly cleaved by both caspases and calpains (including amyloid precursor protein and tau) [6]. Intriguingly, MDMX may be another protein which connects caspase and calpain activation in neurons, as MDMX is degraded by both calpains [3] and caspases (Chapter 3), yet mediates neuronal survival by negatively regulating calpain activity [3].

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Future studies regarding MDMX stability in neurons are informed by previous work performed in cell lines. In dividing cells, it was determined that cellular stress factors (such as

DNA-damaging agents) induce caspase-mediated cleavage of MDMX [4]. The targeting of MDMX by caspases was found to be dependent on a caspase-recognition sequence on MDMX

(361DVPD364). Caspase cleavage of MDMX was shown to occur at this site, leading to a 361- amino acid N-terminal cleavage product (~60 kDa), which is then processed by the proteasome.

Mutation of this caspase-recognition site (to D361A) conferred increased intracellular stability of

MDMX due to its resistance to caspase cleavage [4]. Considering the neuroprotective functions of

MDMX, and our evidence that MDMX is degraded by caspases in models of AD (Chapter 3), it is possible that expression of a caspase-resistant MDMX mutant in neurons would confer increased neuronal protection from Aβ, compared to expression of wild-type MDMX. In experiments where overexpression of MDMX is shown to protect cultured neurons from toxic stress, the protection observed is not a complete reversal of toxic effects [1, 3]. The reason for incomplete rescue is likely that stressed cells have already begun to activate caspases and/or calpains, and that the rate of MDMX translation, even when overexpressed, cannot outpace the rate of MDMX degradation by proteases in the environment of a dying neuron. Previous studies on Aβ-induced neuronal death have shown that both caspases and calpains are activated in Aβ-treated cultured neurons [10-12]. Aβ can activate caspase-2 to promote neuronal death [13], which is particularly relevant to MDMX degradation because caspase-2 is also able to target MDM2 for cleavage [14].

Overexpression of MDMX in cultured neurons was partially protective from HIV-M/M toxicity [3], but overexpression of MDMXD361A would likely have negligible effects on neuronal survival in this model because the caspase-resistant MDMX mutant would presumably still be vulnerable to degradation by calpains. Identification of the amino acid sites on MDMX which are targeted by calpains would shed light on the similarities and differences between caspase- and calpain-mediated degradation of MDMX in neurons. If feasible, a site-directed mutagenesis approach to (as used with the MDMX caspase recognition site [4]) could potentially be employed

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to stabilize MDMX and prevent or retard its cleavage by calpains. This approach may be limited by the difficulty in identifying calpain-cleavage sites, which are generally not as well-defined as caspase-targeted sites with regards to amino acid sequence specificity.

In addition to increased neuronal survival, it is expected that enhanced MDMX stability would reduce calpain activation downstream of MDMX, and reduce conversion of the Cdk5 subunit p35 to p25, based on our results (Chapter 3). Increased expression and/or stability of

MDMX may also have positive effects on mitochondrial stability during toxic conditions, based on our observation that MDMX function is necessary for mitochondrial stability in neurons (Chapter

3). Parallel to the effects of a degradation-resistant MDMX mutant on neuronal survival and mitochondrial stability, intracellular stability of neuronal MDMX could be studied using green fluorescent protein (GFP)-fusion constructs. Expression of GFP-MDMXwt and GFP-MDMXD361A in neurons, followed by treatment with oligomeric Aβ peptide, could be visualized via live imaging of

GFP. The results of such an experiment would allow a precise measurement of the intracellular half-life of MDMX in neurons in normal conditions, and when exposed to amyloid toxicity.

Additionally, the use of the GFP-MDMXD361A would determine if the effects of Aβ on neuronal

MDMX are completely caspase-dependent.

5.3: MDMX AND p53 FUNCTIONS IN NEURONS

While the canonical functions of MDMX in dividing cells are primarily related to its ability to prevent p53 activity [15], the MDMX-p53 relationship is less well-defined in neurons. Our evidence, which suggests that MDMX does not prevent neuronal death through regulation of p53

(Chapter 4) is partially conflicting with previous observations of the MDMX-p53 relationship in neuronal development and in cell death. First, studies in the developing CNS of mdmx-/- mice determined that MDMX maintains critical p53-dependent functions determining cell fate of developing neurons [2]. However, it is likely that the apoptotic pathways activated in mature neurons are highly divergent from those active during development [6, 16, 17]. The other piece of

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evidence suggesting that MDMX prevents neuronal cell death via negative regulation of p53 is the observation that MDMX overexpression in cerebellar granule neurons caused reduced p53 transcriptional activity [1]. While this observation supports MDMX-mediated regulation of p53 activity in neurons, it is well-established that p53 is a multifunctional protein in neurons, and not all p53 transcriptional functions necessarily relate to apoptosis. Indeed, two of the p53 transcriptional targets found to be reduced by MDMX overexpression were MDM2 and p21, neither of which has been shown to have a direct pro-apoptotic role in neurons[18]. Based on previous work, MDMX is able to regulate p53 transcriptional activity in neurons [1]; however; our observations (Chapter 4), suggest that MDMX-mediated neuroprotective functions occur independently of p53. Together, these results suggest that the MDMX-p53 relationship in neurons is limited to the regulation of transcriptional functions of p53. These findings represent a major difference between the functions of MDMX in dividing cells, which are highly p53-dependent and survival-related, and the functions of MDMX in post-mitotic neurons.

The MDMX-p53 relationship in neurons, while not directly implicated in neuronal cell death in mature neurons, still implies several relevant mechanisms by which the loss of MDMX observed in AD and AD models (Chapter 3) may contribute to the progression of AD. Loss of neuronal MDMX in AD could lead to increased p53 transcriptional activity. p53 has been shown to regulate APP secretion machinery in multiple ways: 1) p53 binds the promoter for APP [19], 2) p53 regulates gamma secretase [20, 21], and 3) p53 is increased in AD brain tissue [22-26] (see section 1.6). Thus, loss of MDMX could allow for p53 hyperactivity in neurons, contributing to dysregulation of APP processing over time. This potential mechanism could potentially underlie progression of amyloid pathology in AD, creating a positive feedback loop in which reduced

MDMX leads to increased Aβ secretion, which induces degradation of MDMX in neighboring neurons, thereby propagating amyloid pathology. p53 has also been shown to interact with Cdk5 in neurons; our results demonstrating Cdk5 activation following inhibition of MDMX suggest that increased Cdk5 expression by p53 and increased p35 conversion to p25 following MDMX loss

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could be cumulative in nature, causing rapid upregulation of Cdk5 activity in neurons, which has been associated with tau hyperphosphorylation and neurodegeneration in AD [27], as well as in

HAND [28].

Further studies are necessary to determine the downstream effects of the MDMX-p53 relationship in mature neurons. The role of p53 in amyloid processing[21] provides several interesting prospects to assess how MDMX loss could relate to amyloid processing in AD.

Cultured neurons could be used as a model, in conjunction with qPCR- and ELISA-based assays to determine the effects of MDMX inhibition (or MDMX overexpression) on the expression and enzymatic activity of components of the gamma-secretase complex. As an additional measurable outcome, ELISAs for Aβ40 and Aβ42 could be used in conjunction with pharmacological inhibitors of MDMX and p53 to assess potential changes in amyloid secretion by neurons. If performed in cultured neurons, such assays could be complemented by performed parallel studies in neurons from p53-/- mice. Such studies could determine if the interaction between MDMX and p53 in neurons could have implications towards amyloid processing in the context of AD. In vivo experiments in animal models of AD (such as the Tg2576 or 3xTgAD mice) could be used in conjunction with an inducible MDMX neuron-specific knockout or AAV-mediated overexpression.

Such experiments could determine if introduction of MDMX counteracts or slows the development of pathological markers and behavioral outcomes in murine AD models.

Pharmacological p53 inhibitors could be added to this experimental design to elucidate the role of p53 in MDMX-mediated neuroprotection in vivo.

The potential implications of the MDMX-p53 relationship in neurons are not limited to full- length p53. A transcriptional variant of p53, p44, is expressed as a truncated form of p53 lacking one N-terminal transactivation domain [29-31]. Interestingly, p44 has been found to play significant roles in murine aging and CNS function, largely demonstrated in animal models. p44 was shown to regulate lifespan, evidenced by accelerated aging in p44-overexpressing mice [30,

32-34]. It was shown that this progeroid phenotype was dependent on the presence of wild-type

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p53, suggesting that p44 functions in part by modulating the activity of wild-type p53. Later studies demonstrated an AD-like phenotype of p44 mice, characterized by synaptic and memory defects, and altered tau metabolism [35]. This phenotype was obviated by Igf1r or Mapt haploinsufficiency, but it was also exacerbated by crossing the mice with a human APP mutant mouse line. This cross resulted in marked neurodegeneration in the hippocampus and corpus callosum, along with increased oligomeric Aβ, tau hyperphosphorylation, astrogliosis, markers of paraptosis- and autophagy-mediated neuronal death, as well as significantly reduced lifespan. p44 was also found to activate transcription of multiple tau kinases, including Dyrk1A, GSK3β,

Cdk5, p35, and p39, by binding to their respective promoters [36]. This study also demonstrated that nuclear p44 levels increase in the brain throughout the lifespan of wild-type mice, which may be caused by loss-of-function changes in p53 regulators such as MDMX. The AD-like phenotype of p44-overexpressing mice suggests a potential pathway by which MDMX may promote neuroprotection and oppose the progression of AD.

The potential of MDMX to regulate p44 has not been studied in dividing cells or in neurons, and the aging-related functions of p44 make it an intriguing target. Future directions determining pro-survival functions of MDMX could address p44 as a downstream target of

MDMX. Neurodegenerative/progeroid phenotypes in p44+ mice could potentially be rescued by introduction of MDMX (via AAV), and neuropathological/behavioral outcomes could subsequently be quantified. p44 expression could be modified in cultured neurons using siRNA. qPCR, in conjunction with genetic or pharmacological modulators of MDMX (such as SJ-172550) could be utilized to determine the effects of MDMX on p44-mediated transcription of tau kinases, which may underlie the effects of p44 overexpression on aging and AD-like pathology in mice. The protein-protein interactions between MDMX and p44 could also be investigated in cultured neurons using co-immunoprecipitation. Considering that MDMX interacts with the N-terminal transactivation domains of p53, one of which is absent in the p44 isoform, differences between

MDMX-p53 and MDMX-p44 interactions may be apparent, and MDMX may not interact with p44.

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However, MDMX has been shown to interact with p53 homologues p63 [37] and p73 [38], which have similar transactivation domains as p53, so there is precedent for MDMX regulating alternative forms of p53 via binding of the transactivation domain [39]. Pertinent to this concept, one study demonstrated that MDM2 cannot directly bind, ubiquitinate, or shuttle p44 from the nucleus in the absence of wild-type p53 [40]. However, when wild-type p53 is expressed, p44 and p53 form oligomers, permitting MDM2-mediated ubiquitination, shuttling, and proteasomal processing of p44 [40]. These results demonstrate that MDM2 can interact with p44 and reduce p44 activity and stability, but only in an indirect manner which is dependent on wild type p53. The high degree of homology between MDM2 and MDMX raises the possibility that MDMX regulates p44 in a similar manner.

5.4: MDMX AND MITOCHONDRIAL FUNCTIONS IN NEURONS

The relationship between MDMX and mitochondrial function, which we observed in cultured neurons, could have significant implications to aging and neurodegenerative disease, considering the widely studied importance of mitochondrial function [41-43]. Evidence from dividing cells indicates that MDMX regulates mitochondrial p53 [44], which plays multiple roles related to mitochondrial function, both in dividing cells and neurons [45-49]. MDMX has been shown to interact with Bcl-2, anchoring p53 to the mitochondria as part of the p53-mediated mitochondrial death pathway [44]. However, the MDMX-mitochondria relationship is shown to be pro-apoptotic in dividing cells, while our evidence suggests that mitochondrial functions of MDMX are beneficial for neuronal survival. While the regulation of mitochondrial p53 by MDMX is a potential angle for future study, our pharmacological data suggests that the mitochondrial functions of MDMX are p53-independent (Chapter 3). Pifithrin-µ, which reduces the binding affinity of p53 for mitochondrial proteins [50], and prevents neuronal apoptosis [51, 52], did not prevent neuronal damage or mitochondrial depolarization induced by MDMX inhibition (Chapters

3 & 4). Our data suggest that while MDMX function is necessary for baseline mitochondrial

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stability in neurons, this function is not p53-mediated, representing a departure from the observations of mitochondrial MDMX function in dividing cells, and leaving open possibilities for studies regarding the mechanism(s) by which MDMX promotes mitochondrial stability and how degradation of MDMX triggers mitochondrial damage. It would be relevant for future studies to determine which protein(s) interact with MDMX to mediate its mitochondrial functions.

Future studies could utilize genetic and pharmacological techniques to further determine the role(s) of MDMX in mitochondrial function. It is not known how inhibition of MDMX causes mitochondrial depolarization. MDMX has been shown to interact with Bcl-2 [44], a mitochondrial protein implicated in mitochondrial membrane permeability and apoptotic signaling [53, 54].

Examining the effects of MDMX overexpression or MDMX inhibition on protein levels of Bcl-2 family members and their activities could determine if MDMX promotes mitochondrial permeability through regulation of Bcl-2-related proteins. Because long-term mitochondrial dysfunction is a feature common to aging-related neurodegenerative disease, markers of mitochondrial damage could be used as endpoints to assess the effects of AAV-mediated overexpression of MDMX in

AD mouse models. Live imaging techniques to detect intraneuronal calcium released from mitochondria following inhibition or knockdown of MDMX could be a valuable tool in determining how MDMX regulates mitochondrial permeability, and how mitochondrial calcium could stimulate calcium-activated proteases such as calpain, which are implicated in neuronal damage in our results [3] and in the pathogenesis of AD [55]. Beyond mitochondrial permeability, other aspects of mitochondrial regulation by MDMX could be alternatively be investigated in vivo. Defects in mitogenesis or mitophagy, which is implicated in mitochondrial dysfunction in AD [41, 56-59], could be examined as an experimental endpoint in aforementioned AD animal models, in conjunction with neuronal MDMX overexpression. Long-term changes in mtDNA damage could also be examined in AD animal models, as mtDNA damage is already observed in murine models of neurodegeneration and aging [60, 61]. The ability of MDMX to a) interact with mitochondria[44], and b) regulate p53, which is involved in maintenance of mtDNA [49], suggests a potential

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relationship between long-term MDMX loss (observed in AD models; Chapter 3) and mtDNA damage in aging. Genetic experiments overexpressing MDMX in neurons of AD mouse models could examine the potential of MDMX in the p53-mediated prevention of long-term mtDNA damage in vivo. Thus, the potential of MDMX to promote stable mitochondrial function represents an intriguing area for future study, particularly in light of the paucity of studies on mitochondrial

MDMX.

5.5: MDMX AND CALPAIN/CDK5 ACTIVATION

Our data indicate that Cdk5 is activated following pharmacological block of MDMX in cultured neurons. Cdk5 becomes hyperactive when its p35 subunit is cleaved to the more stable p25 subunit by calpain [62]. This conversion precedes multiple pathological functions in neurons, including cytoskeletal damage, hyperphosphorylation of tau, and neuronal death [63]. Our results suggest that MDMX maintains a baseline function in neurons related to the negative regulation of calpain activity, due to the observations that neuronal damage and p35 cleavage induced by

MDMX inhibition is prevented by calpain inhibition (Chapter 3) [3]. The role of MDMX in the regulation of calpain and Cdk5, suggests that the loss of MDMX in AD could be a contributor to calpain activation and hyperactivity of Cdk5; both of which are implicated in AD and other neurodegenerative conditions, including HAND.

Calpains have been shown to mediate a host of intracellular processes in the progression of AD [11, 55, 64]. Observations from AD tissue [65], as well as studies in animal models [66], have implicated calpains in synaptic damage, neurofibrillary pathology [67], and neuronal death in

AD. Calpastatin, the endogenous calpain inhibitor, is reduced in AD tissue, and induction of calpastatin was shown to reverse pathogenic APP processing in multiple AD mouse models [68,

69], further demonstrating the importance of calpains in AD pathogenesis. Specific inhibition of calpain activity can prevent neurofibrillary pathology and neurodegeneration, and also improve life spans in transgenic mouse models of tauopathy [70]. These lines of evidence have led to the

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consideration of pharmacological calpain inhibition of as a candidate for therapeutic use in AD

[71, 72]. Calpains target and cleave a variety of proteins in neurons, including cytoskeletal proteins, various receptors, synaptic proteins [73], and intracellular signaling proteins [74].

Calpains exacerbate neuronal damage in AD following disruptions in calcium homeostasis caused by excitotoxic stress, leakage of calcium from the ER and lysosomes [75]. Following their activation, calpains can cleave IP3 receptors on the ER, leading to increased disruption of calcium homeostasis [76]. Tau itself is a target of calpain cleavage, which can cause tau to take on a more aggregate-prone conformation following cleavage [77]. Additionally, calpains can activate a variety of kinases which can mediate further pathological functions, including ERK1/2,

GSK3β, and Cdk5, which can contribute to tau phosphorylation and neurodegeneration [78].

Cdk5, a cyclin-dependent kinase, is implicated in a variety of neuronal functions, including neuronal development and synaptic plasticity, but Cdk5 is also known for its role in AD, where Cdk5 dysregulation is implicated in neuronal damage [79]. Cdk5 functions based on its interaction with its co-factor, the p35 subunit (or p39 in neurons) [80]. As described previously, calpain can activate Cdk5 by cleaving p35 to its p25 form [62, 81]. Because the p25 form is more stable than the full-length form, p25-bound Cdk5 is hyperactive, which causes multiple harmful effects in neurons [62]. Cdk5 hyperactivity is implicated in neurodegeneration in multiple models

[63]. Mice overexpressing the p25 subunit have constitutive Cdk5 hyperactivity, causing a neurodegenerative phenotype [82], and a similar study determined that Cdk5 activity led to increased cell cycle markers in the brain [83]. Cdk5 has also been implicated in amyloid- associated cell cycle re-entry in cultured neurons [84], and is known to interact with p53 [85, 86].

A major area of interest in AD research is the role of Cdk5 as a tau kinase [27, 79, 80, 87, 88].

Cdk5 activity becomes altered early in AD development [89]. Hyperphosphorylation of tau is a noted factor in AD (phospho-tau is 3- 4-fold increased in AD brain tissue compared to controls

[90]), and is known to contribute to the formation of neurofibrillary tangles [91]. Cdk5-mediated tau phosphorylation has been observed in patient tissue [92], animal models [88, 93], and in

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cultured neurons [94, 95]. Cdk5 is also implicated in HAND neurotoxicity based on HAND toxicity models; both in gp120 transgenic mice [96], and in cultured neurons [28]. Intriguingly, there is evidence for increased phospho-tau and neurofibrillary pathology in HAND brain tissue [97], indicating common mechanisms of neurodegeneration, possibly related to calpain/Cdk5 activation, which could underlie the cognitive deficits in HAND as well as AD.

Our results indicate that MDMX negatively regulates calpain activity, and by extension,

Cdk5 (Chapter 3). These observations imply that MDMX degradation, observed in AD (Chapter 3) and in HAND [3] (Chapter 2) allows for pathological calpain activation and subsequent Cdk5 hyperactivity, both of which are observed in previous studies of AD [27, 84] and HAND [28]. Our results also suggest that MDMX is necessary for mitochondrial stability; as inhibition of MDMX led to mitochondrial depolarization in neurons (Chapter 3). It is possible that increased mitochondrial permeability following MDMX loss leads to increased calpain activation. This outcome is partially supported by our evidence that calpain inhibitors prevent p35 cleavage and neuronal damage induced by an MDMX inhibitor, but calpain inhibitors do not prevent MDMX inhibitor-induced mitochondrial damage (Chapter 3). The timing of mitochondrial depolarization compared to changes in calpain-cleaved spectrin and p35 cleavage also suggest that mitochondrial damage precedes changes in calpain activity and Cdk5 (Chapter 3). Together, these observations suggest that MDMX loss causes mitochondrial damage prior to calpain activation and p35 cleavage. This sequence allows the possibility that MDMX loss causes mitochondrial permeability to increase, and leaked calcium from mitochondria could activate neuronal calpains, and subsequently, Cdk5

[98]. Changes in both intracellular calcium and calpain activity can initiate a deleterious feed- forward mechanism; calpain-mediated cleavage of IP3 receptors on the endoplasmic reticulum causes further dysregulation of intraneuronal calcium [76], and amplifies the adverse functions of both calpains and caspases en route to neuronal death.

Both calpain and Cdk5 are implicated in tau phosphorylation and NFT formation in AD models, and Aβ-induced degradation of MDMX protein could be a triggering mechanism which

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leads to increased phosphorylation of tau. MDMX could be one of a growing number of proteins involved in amyloid-induced tau phosphorylation, thought to be a critical process in the progression of AD [99, 100]. Tau processing in neurons is highly dependent on its interaction with the Hsp90 chaperone [101]. Hsp90 interacts with a large percentage of intracellular enzymes

(~50% of all kinases, ~30% of all ubiquitin ligases [102]). Both MDMX and tau are known to interact with Hsp90, which could provide scaffolding for enzymatic interactions between tau,

Cdk5, and MDMX.

Future experiments to determine the relationship between MDMX function and tau phosphorylation would address multiple downstream effectors of MDMX; mitochondrial damage, the calpain/Cdk5 pathway, and p44 as well (see section 5.3). MDMX's potential role in tau phosphorylation is an experimental angle we initially investigated as part of our larger study on the role of MDMX in AD, but these experiments were inconclusive. A more comprehensive approach would examine if MDMX mediates amyloid-induced tau phosphorylation. Because pharmacological inhibition of MDMX causes neuronal damage [3], it is likely that toxicity occurred too quickly to observe significant changes in phospho-tau in our neuronal culture model. Amyloid- driven changes in tau phosphorylation are thought to drive tau pathology in AD [99]. Because we demonstrate that amyloid induces the degradation of neuronal MDMX (Chapter 3), genetic approaches could be employed to compare the effects of amyloid peptides on tau phosphorylation in neurons overexpressing MDMX. Such an experiment could determine if

MDMX degradation represents a mechanistic link between amyloid toxicity and downstream tau hyperphosphorylation. Alternatively, by knocking down MDMX in cultured neurons, changes in

Cdk5 activity and GSK3β could be quantified via immunofluorescence and immunoblotting in conjunction with changes in site-specific tau phosphorylation (using site-specific phospho-tau antibodies). Potential changes in the "pattern" of tau phosphorylation caused by MDMX loss of function could give insight to the possibility that MDMX regulates tau kinases (in addition to

Cdk5). Other tau kinases, such as CamKII and fyn could also be studied as potential downstream

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effectors of MDMX [78]. Individual kinases could be targeted with RNAi or small-molecular inhibitors to differentiate their respective roles in tau phosphorylation, as previous studies have shown [103].

Because neurofibrillary pathology is a long-term age-related process with many contributing factors, the most worthwhile experiments might ideally be performed in animal models. Experimental methods to induce MDMX loss-of-function, whether by toxic factors, genetic techniques, or small-molecule inhibitors, are all shown to cause neuronal death in cultured neurons [1, 3]. Therefore, long-term studies in animal models would allow more relevant assessment of changes in tau phosphorylation and the development of neurofibrillary pathology.

Genetic models of AD which include the neurofibrillary component of the disease, such as the

3xtgAD mouse or the P301S tau mouse [104], could be used as a pathological model in comparison to wild-type animals. AAV-mediated overexpression of MDMX (under the control of a neuron-specific promoter) could be used to examine the effects of increased MDMX protein on tau phosphorylation and NFT formation during aging in the transgenic and wild-type murine brains respectively. Further, the effects of neuronal MDMX overexpression on behavioral/learning/memory outcomes could determine if the neuroprotective effects of MDMX promote measurable outcomes related to cognitive function. Alternatively, MDMX loss-of- functions could be investigated in various aforementioned murine AD models. While MDMX-null animals are not viable, an inducible MDMX knockout mouse, again under a neuron-specific promoter, could potentially allow us to reduce MDMX expression and measure subsequent changes in pathological markers, which would potentially include: neuronal cell death, mitochondrial instability, calpain activation markers, p35 cleavage, amyloid pathology, tau phosphorylation and NFT formation.

5.6: E2F1 AND ALTERNATIVE MDMX EFFECTORS IN NEURONS

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p53 and E2F1 are both implicated in neuronal death in a variety of cell culture and animal models. Increased p53 and ectopic E2F1 expression are also observed in neurons in AD tissue.

Research in cell lines has demonstrated that MDMX maintains E2F1-regulating functions [105,

106], complimenting the often-studied roles of MDMX in p53 regulation. Previous results in cultured neurons indicate that MDMX overexpression reduces the transcriptional activity of E2F1

[1]. Because the MDMX-p53 relationship in neurons, while potentially relevant to long-term p53 functions, is not directly involved in neuronal death (see section 5.3), it is possible that negative regulation of E2F1 is a mechanism by which MDMX promotes neuroprotection. A previous experiment was attempted measuring the effects of the MDMX inhibitor (SJ-172550 [107]) on neuronal viability in cultured neurons from wild-type and e2f1-/- mice, but was confounded by technical difficulties and incomplete deletion of e2f1 in the mutant mouse line. Future experiments could benefit from improved genetic techniques to fully elucidate the role of E2F1 in MDMX- mediated neuroprotection. Neurons could be cultured from mice with conditional E2F1knockouts

(using a "floxed" E2F1), and the expression of E2F1 could be modulated in cultured neurons.

Alternatively, novel gene-editing approaches such as CRISPR/Cas9 [108] could be used to delete

E2F1 and prevent its expression. This technique, in conjunction with pharmacological and genetic modifications of MDMX function, would determine if the pro-survival functions of MDMX in neurons are mediated by negative regulation of E2F1. It is also relevant to determine if the pro- apoptotic functions of E2F1 in neurons are transcription-dependent. While MDMX was shown to reduce the transcriptional functions of E2F1 in neurons [1], these functions may not directly relate to neuronal death.

E2F1 has been found to play neuron-specific roles in neuroplasticity [109], and the potential of MDMX to regulate these recently-discovered E2F1 functions is a potential angle for future research, particularly with regard to synaptic stability in murine models of neurodegenerative disease. Additionally, our observation of mitochondrial MDMX in neurons

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suggests that E2F1-dependent effects on mitochondrial function and expression of mitochondrial proteins[110] may also be regulated by MDMX [111].

Further studies are necessary to determine potential novel downstream effectors of

MDMX functions in neurons. Our research has already implicated mitochondrial depolarization and calpain-mediated cleavage of p35 in neuronal death induced by MDMX inhibition (Chapter 3), and the potential protein-protein interactions which allow MDMX to mediate neuronal calpains would be a subject of interest. The relationships of neuronal MDMX with mitochondrial proteins with known roles in apoptosis would be another intriguing potential angle for experiments, based on previous observations of MDMX binding of mitochondrial Bcl-2 [44]. A screen of protein- protein interactions would allow the assembly of an "interactome" of MDMX-interacting proteins in mature neurons. A comparison of MDMX-interacting proteins in neurons compared to dividing cells would 1) highlight the interactions underlying the divergent functions of MDMX in different cell types, and 2) potentially determine novel neuron-specific MDMX binding partners which could mediate the neuroprotective functions of MDMX. A yeast two-hybrid screen of MDMX could be performed using a panel of human brain proteins, and GST-MDMX fusion proteins could be used in pulldowns to verify individual protein-protein interactions. This approach may be a valuable tool in determining novel interactions of neuronal MDMX, and potentially identifying targets for intervention.

5.7: FUNCTIONS OF MDM2 IN NEURONS

Our work has identified novel functions of MDMX in neurons relating to neuronal death in the context of neurodegenerative diseases. However, many of the functions of MDMX observed in dividing cells are related to the functions of its homologue, MDM2. Similarly to MDMX, few studies have addressed the functions of MDM2 in neurons, but there is evidence that MDM2 maintains neuron-specific functions. A recent study also demonstrated that MDM2 is localized to the axon initial segment in rodent neurons [112]. A study in a neuron-derived cell line also

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determined a relationship between GSK3β and the p53-MDM2 pathway in neuronal response to radiation. Dysregulation of MDM2-p53 signaling has been observed in animal models of epilepsy

[113], while decreased MDM2 and increased p53 proteins are observed in hippocampal tissue from patients with temporal lobe epilepsy [114]. One study demonstrated that MDM2 interacts with the synaptic scaffolding protein PSD-95, and mediates the ubiquitination and proteasomal turnover of PSD-95 at synapses [115]. It was later shown that this process is regulated by Cdk5 in cultured neurons and in mdm2/p53-null mice [116]. This research suggests a p53-independent role for MDM2 in the regulation of synaptic plasticity. Considering our observations of MDMX degradation in AD, and the ability of MDMX to increase the ubiquitin ligase activity of MDM2, long-term loss of neuronal MDMX could reduce MDM2 activity at the synapse and potentially cause impaired turnover of synaptic proteins. We observed that Nutlin-3a, a small-molecule inhibitor of p53-MDM2 interactions [117], induced damage in cultured cortical neurons in a similar fashion as the MDMX inhibitor, SJ-172550 (unpublished data). Experiments in neuron-derived cell lines showed a similar effect of Nutlin-3a [118]. Future studies utilizing RNA interference in conjunction with small molecule inhibitors of MDM2 and/or MDMX could help determine if loss of

MDMX and/or MDM2 contributes to synaptic loss observed in AD, and also determine if MDM2 and MDMX maintain similar relationships as they do in dividing cells [119].

5.8: CLINICAL PERSPECTIVES ON NEURONAL MDMX

Epidemiological studies have shown that an inverse relationship exists between cancer and AD [120-124]. The risk for dementia is reduced in patients who suffer from cancer. More recent studies have implicated certain types of cancer and their respective effects on AD risk. It has been proposed that this observation is caused by individual variations in the functions of various genes which determine cell fate [125], such as p53 [126].

The fundamental cellular mechanisms which mediate autophagy [127], metabolism, oxidative stress, mitochondrial function [128], epigenetic modification [129], and inflammation all

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have relevance to both cancer biology and neurodegenerative disease, especially considering that broadly speaking, (most types of) cancer and neurodegenerative diseases (AD, PD) are both aging-related diseases which develop based on complex intersections between genetic and environmental risk factors. Many of these genetic factors with dual roles in neurodegeneration and cancer are cell cycle proteins, such as p53 [126, 130]. For instance, an individual who carries a loss-of-function p53 mutation would likely be at a higher risk for cancer (50% of human cancers have p53 mutations[131]), but this individual's neurons would be less inclined to initiate p53- dependent cell death programs in the context of a long-term neurodegenerative disease. p53 is one of a group of cell cycle proteins which are implicated in both cancer and neurodegenerative disease, including E2F1, Cdk5, ATM, mTOR, Cdk5 [132, 133]; and even proteins strongly linked to neurodegenerative diseases such as APP, PS-1, PS-2, Huntingtin, Parkin, and α-synuclein are implicated in various types of cancer [125]. Tau has been implicated in the cell cycle [134], playing a role in mitotic spindle formation in a phosphorylation-dependent manner [135], and tau expression is also connected with certain types of cancer [136, 137]. We propose that MDMX is a member of this group, considering its demonstrated roles promoting cancer [138], and our findings implicating MDMX loss in neurodegeneration in AD (Chapter 3) and HAND [3]. An intriguing prospect would be to examine the prevalence of dementia in patients with tumors caused by MDMX amplifications, in the same style as previous epidemiological studies on the cancer-AD relationship [139]. We predict that patients with increased MDMX protein in CNS neurons would be more resistant to neuronal death in neurodegenerative diseases such as AD.

Populations of patients who develop the pathological markers of AD without notable cognitive impairments (referred to as AD-resilient [140-142]) may be such individuals with mutations related to the functions of neuronal cell cycle proteins. If the rates of various types of cancer were calculated in AD-resilient individuals, it is possible that an overall higher rate of cancer may be caused by the same group of genes which allow for AD resilience. One study investigated contributors to AD resilience and screened for protein abnormalities [143].

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Interestingly, a number of the proteins significantly altered between normal AD and resilient AD groups were cell cycle-related proteins, including E2F1. Further understanding of the genetic variants which allow for "natural" resilience to cognitive decline could provide the basis for developing effective therapeutics.

The use of MDMX inhibitors in the treatment of cancer is a therapeutic angle which is currently moving from drug development towards clinical trials [144]. The function of MDMX as a neuroprotective protein suggests the need for extra consideration towards potential side effects of

MDMX inhibitors. Several DNA-damage-related proteins, which are targets in cancer treatment are currently being shown to play important roles in the CNS and PNS [145, 146]. As cancer treatments continue to improve, and patient life expectancies continue increasing, the long-term effects of these treatments on the brain could become a more relevant concern. Physicians have noted cases of memory loss and behavioral impairments associated with cancer treatments, often referred to as "chemo brain" [147]. Initial observations have been corroborated by neuroimaging studies [148, 149]. Complicating the therapeutic outcome, these impairments are often compounded by co-morbidities associated with aging. Chemotherapy-induced neuropathy is also observed clinically [150]. It has been proposed that the effects of chemotherapy drugs on memory and cognition are caused by inhibition of DNA repair proteins in neurons and oxidative DNA damage has been linked to cognitive impairment in breast cancer survivors who underwent chemotherapy [151].

As MDMX-targeted small molecules continue to be pursued in cancer drug development, as the MDM2 inhibitor Nutlin-3 already has [152], it will be a matter of importance to consider side effects on the CNS potentially caused by systemic inhibition of MDMX. Long-term studies on the effects of MDM2/MDMX inhibitors in rodents (utilizing both wild-type and models of cancer development) with emphasis on cognitive function and neuropathological markers will be necessary to determine the long-term effects of MDM2/MDMX-targeted treatments on the CNS.

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Studies on adjunctive therapies, with the goal of managing potential side effects from such treatments, could be beneficial towards promoting the best possible quality of life post-treatment.

5.9: SUMMARY & CLOSING REMARKS

In summary, the work discussed in this thesis has advanced our understanding of the neuronal functions of MDMX, a protein previously studied primarily for its role in cancer. We describe novel functions of MDMX in neurons, and address how MDMX is affected during neurodegenerative diseases. Fundamentally, the functions of MDMX are related to the promotion of cell survival. These functions, which have implicated MDMX in the development of multiple types of cancers, also allow MDMX to promote neuronal survival; and become disrupted during neurodegenerative diseases such as AD and HAND. Our data demonstrate degradation of

MDMX in HAND and HAND models, and we also identify MDMX as a novel substrate and regulator of calpains. We observe loss of neuronal MDMX in aged APPswe mice and in human AD tissue, and demonstrate amyloid-induced MDMX degradation via caspase cleavage. Loss of

MDMX function is linked to downstream intracellular events in the neuronal death process, including mitochondrial depolarization and calpain-mediated Cdk5 activation. Further, we determine that MDMX-mediated pro-survival functions in neurons occur in a p53-independent manner.

In closing, our results represent a significant progression from previous studies addressing the functions of the MDMX oncoprotein in neurons. Application of our findings towards future research can improve the understanding of neuroprotective functions of MDMX, as well as other cancer-related proteins, during both health and disease.

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APPENDIX I

ILLUSTRATION 1:

Graphical model of MDMX functions in dividing cells and in neurons.

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APPENDIX II

FIGURE 6.1: Neuronal damage curve of MDM2 inhibitor Nutlin-3a.

FIGURE 6.1: Neuronal damage curve of MDM2 inhibitor Nutlin-3a. 21 DIV cultured rat cortical neurons were treated with escalating doses of the MDM2 inhibitor Nutlin-3a (Selleck Chemical,

Boston, MA). DMSO was used as a vehicle control. After 24 hr treatment, neuronal viability was assessed using MAP2 viability assay (See Chapter 4).

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