INDUCED-PLURIPOTENT STEM-DERIVED NEURONAL PROGENITOR CELLS AS A NOVEL TREATMENT FOR NEURODEGENERATIVE DISEASES

Cellular therapies, as neuronal progenitor (NP) cells grafting, are promising therapies for patients affected with neurodegenerative diseases like Creutzfeldt-Jakob Disease (CJD). At this time there is no effective treatment or cure for CJD. The disease is inevitably fatal and affected people usually die within months of the appearance of the first clinical symptoms. Compelling evidence indicate that the hallmark event in the disease is the conversion of the normal prion protein (termed PrPC) into the disease-associated, misfolded form (called PrPSc). Thus, a reasonable therapeutic target would be to prevent PrP misfolding and prion replication. This strategy has been applied with poor results since at the time of clinical intervention substantial brain damage has been done. It seems that a more effective treatment aimed at patients with established symptoms of CJD would need to stop further brain degeneration or even recover some of the previously lost brain tissue. The most promising possibility to recover brain tissue is the use of NPs that have the potential to replenish the nerve cells lost during the early stages of the disease. Advanced cellular therapies, beside their potential for cell replacement, might be used as biomaterials for drug delivery in order to stimulate cell survival or the resolution the disease. Also, implanted cells can be genetically manipulated to correct abnormalities causing disease or to make them more resistant to the toxic microenvironments present in damaged tissue. In recent years cell engineering has been within the scope of the scientific and general community after the development of technologies able to “de-differentiate” somatic cells into induced-pluripotent stem (IPS) cells. This new tool permits the use of easy-to-reach cells like skin or blood cells as a primary material to obtain embryonic stem-like cells for cellular therapies, evading all ethical issues regarding the use of human embryos as a source of embryonic stem cells. The complete work proposes to implant IPS-derived NP cells into the brain of prion-infected animals to evaluate their therapeutic potential. Since it is well known that the expression of prion protein in the cell membrane is necessary for PrPSc mediated toxicity, we also want to determine if NPs lacking the prion protein have better survival rates once implanted into sick animals. The main objective of this work is to develop implantable neural precursor from IPS coming from animals lacking the prion protein. Specific aim 1: To develop and characterize cellular cultures of IPS cells from prp-/- mice. Fibroblasts from prp-/- animals will be reprogrammed using the four Yamanaka factors. IPS colonies will be selected and characterized by immunohistochemistry for markers of pluripotency. Their developmental capabilities will be evaluated by teratoma and embryoid body formation assays. Specific aim 2: To differentiate IPS cells to a neuronal lineage. IPS cells will be differentiated to a NP stage by the use of defined media culture conditions. NP cells will be characterized by their immunohistochemical profile as well as by their ability to differentiate into neuronal cells. Specific aim 3: Cellular labeling of neuronal progenitors cells for in vitro traceability. In order to track the cells once implanted in the host brain, they will be tagged with different methods such as lipophilic fluorescent tracers and transduction with GFP protein expression.

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Activation of heat shock and antioxidant responses by the natural product celastrol: transcriptional signatures of a thiol-targeted molecule.

Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.

The Role of the Androgen Receptor Cofactor p44/WDR77 in Astrocyte Activation

Astrogliosis is induced by neuronal damage and is also a pathological feature of the major aging-related neurodegenerative disorders. The mechanisms that control the cascade of astrogliosis have not been well established. In a previous study, we identified a novel androgen receptor (AR)-interacting protein (p44/WDR77) and found that it plays a critical role in the control of proliferation and differentiation of prostate epithelial cells. In the present study, we found that deletion of the p44 gene in the mouse brain caused accelerated aging with dramatic astrogliosis. The p44/WDR77 is expressed in astrocytes and loss of p44/WDR77 expression in astrocytes leads to astrogliosis. Our results reveal a novel role of p44/WDR77 in astrocytes, which may explain the well-documented role of androgens in suppression of astrogliosis. While many of detailed mechanisms of astrocyte activation remain to be elucidated, a number pathways have been implicated in astrocyte activation including p21Cip1 and the NF-kB pathway. Astrocytic activation induced by p44/WDR77 gene deletion was associated with a significant increase of p21Cip1 expression and NF-kB activation characterized by p65 nuclear localization. We found that down-regulation of p21Cip1 expression inhibited astrocyte activation induced by the p44/WDR77 deletion and was accompanied by a decreased p65 nuclear localization. While p21Cip1 role in astrocyte activation and NF-kB activation is not well understood, studies of other cell cycle regulators have implicated cell cycle control systems as modulators of astrocyte activation, thus p21Cip1 could induce secondary effect to induce p65 nuclear localization. However, p65 knockdown completely relieved the inhibition of astrocyte growth induced by the p44/WDR77 deletion, while p21Cip1 knockdown only partially recovered this inhibition. Thus, NF-kB activity performs additional regulatory actions not mediated by p21Cip1. These analyses imply that p4/WDR77 suppresses astrocyte activation through modulating p21Cip1 expression and NF-kB activation.

Annexin A5-Conjugated Polymeric Micelles For Dual SPECT and Optical Detection of Apoptosis

Apoptosis, a form of programmed cell death, is critical to homoeostasis, normal development, and physiology. Dysregulation of apoptosis can lead to the accumulation of unwanted cells, such as occurs in cancer, and the removal of needed cells or disorders of normal tissues, such as heart, neurodegenerative, and autoimmune diseases. Noninvasive detection of apoptosis may play an important role in the evaluation of disease states and response to therapeutic intervention for a variety of diseases. It is desirable to have an imaging method to accurately detect and monitor this process in patients. In this study, we developed annexin A5-conjugated polymeric micellar nanoparticles dual-labeled with a near-infrared fluorescence fluorophores (Cy7) and a radioisotope (111In), named as 111In-labeled annexin A5-CCPM. In vitro studies demonstrated that annexin A5-CCPM could strongly and specifically bind to apoptotic cells. In vivo studies showed that apoptotic tissues could be clearly visualized by both single photon emission computed tomography (SPECT) and fluorescence molecular tomography (FMT) after intravenous injection of 111In-labeled Annexin A5-CCPM in 6 different apoptosis models. In contrast, there was little signal in respective healthy tissues. All the biodistribution data confirmed imaging results. Moreover, histological analysis revealed that radioactivity count correlated with fluorescence signal from the nanoparticles, and both signals co-localized with the region of apoptosis. In sum, 111In-labeled annexin A5-CCPM allowed visualization of apoptosis by both nuclear and optical imaging techniques. The complementary information acquired with multiple imaging techniques should be advantageous in improving diagnostics and management of patients.

DEFINITION OF THE LANDSCAPE OF CHROMATIN STRUCTURE AT THE FRATAXIN GENE IN FRIEDREICH’S ATAXIA

Friedreich’s ataxia (FRDA) is caused by the transcriptional silencing of the frataxin (FXN) gene. FRDA patients have expansion of GAA repeats in intron 1 of the FXN gene in both alleles. A number of studies demonstrated that specific histone deacetylase inhibitors (HDACi) affect either histone modifications at the FXN gene or FXN expression in FRDA cells, indicating that the hyperexpanded GAA repeat may facilitate heterochromatin formation. However, the correlation between chromatin structure and transcription at the FXN gene is currently limited due to a lack of more detailed analysis. Therefore, I analyzed the effects of the hyperexpanded GAA repeats on transcription status and chromatin structure using lymphoid cell lines derived from FRDA patients. Using chromatin immunoprecipitation and quantitative PCR, I observed significant changes in the landscape of histone modifications in the vicinity of the GAA tract in FRDA cells relative to control cells. Similar epigenetic changes were observed in GFP reporter construct containing 560 GAA repeats. Further, I detected similar levels of FXN pre-mRNA at a region upstream of hyperexpanded GAA repeats in FRDA and control cells, indicating similar efficiency of transcription initiation in FRDA cells. I also showed that histone modifications associated with hyperexpanded GAA repeats are independent of transcription progression using the GFP reporter system. My data strongly support evidence that FXN deficiency in FRDA patients is consequence of defective transition from initiation to elongation of FXN transcription due to heterochromatin-like structures formed in the proximity of the hyperexpanded GAAs.

ROLE OF THE GCN5 HISTONE ACETYLTRANSFERASE IN SPINOCEREBELLAR ATAXIA TYPE 7 AND IN IMMATURE NEURONS

Spinocerebellar Ataxia type 7 (SCA7) is a neurodegenerative disease caused by expansion of a CAG repeat encoding a polyglutamine tract in ATXN7, a component of the SAGA histone acetyltransferase (HAT) complex. Previous studies provided conflicting evidence regarding the effects of polyQ-ATXN7 on the activity of Gcn5, the HAT catalytic subunit of SAGA. Here I showed that reducing Gcn5 expression accelerates both cerebellar and retinal degeneration in a mouse model of SCA7. Deletion of Gcn5 in Purkinje cells in mice expressing wild type Atxn7, however, causes only mild ataxia and does not lead to the early lethality observed in SCA7 mice. Reduced Gcn5 expression strongly enhances retinopathy in SCA7 mice, but does not affect the transcriptional targets of Atxn7, as expression of these genes is not further altered by Gcn5 depletion. These findings demonstrate that loss of Gcn5 functions can contribute to the time of onset and severity of SCA7 phenotypes, but suggest that non-transcriptional functions of SAGA may play a role in neurodegeneration in this disease.

Amyotrophic lateral sclerosis and Parkinsonism-dementia complex of Guam: Descriptive epidemiology, secular trends, and birth cohort effects on incidence, 1950--1989

This study describes the patterns of occurrence of amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC) of Guam during 1950-1989. Both ALS and PDC occur with high frequency among the indigenous Chamorro population, first recognized in the early 1950's. Reports in the early 1980's indicated that both ALS and PDC were disappearing, due to a purported reduction in exposure to harmful environmental factors as a result of the dramatic changes in lifestyle that took place after World War II. However, this study provides compelling evidence that ALS and PDC have not disappeared on Guam and that rates for both are higher during 1980-1989 than previously reported.^ The patterns of occurrence for both ALS and PDC overlap in most respects: (1) incidence and mortality are decreasing; (2) median age at onset is increasing; (3) males are at increased risk for developing disease; (4) risk is higher for those residing in the south compared to the non-south; and (5) age-specific incidence is decreasing over time except in the oldest age groups.^ Age-specific incidence of ALS and PDC, separately and together, is generally higher for cohorts born before 1920 than for those born after 1920. A significant birth cohort effect on the incidence of PDC for the 1906-1915 birth cohort was found, but not for ALS and for ALS and PDC together. Whether or not a cohort effect, period effect, or both are associated with incidence of ALS and PDC cannot be determined from the data currently available and will require additional follow-up of individuals born after 1920.^ The epidemiological data amassed over this 40-year period provide evidence that supports an environmental exposure model for disease occurrence as opposed to a simple genetic or infectious disease model. Whether neurodegenerative disease in this population occurs as a consequence of a single exposure or is explained by a multifactorial model such as a genetic predisposition with some environmental interaction is yet to be determined. However, descriptive studies such as this can provide clues concerning timing and location of potential adverse exposures but cannot determine etiology, underscoring the urgent need for analytic studies of ALS and PDC to further investigate existing etiologic hypotheses and to test new hypotheses. ^

The illness experience of persons with Parkinson's Disease

Background. Parkinson's disease is a chronic, progressive, age-related, neurodegenerative disorder with no known cause or promising cure. While substantial information is known about the pathophysiology of Parkinson's disease, little is known about the illness experience of persons living with the disease. The purpose of this study was to understand how persons with Parkinson's disease construct their illness experience and manage living with their illness on a daily basis. ^ Method. A qualitative study with an ethnographic approach employed the strategies of participant observations and fieldwork. Field data were generated from a two year exposure to two Parkinson's disease support groups in east Texas. Open-ended semi-structured interviews with seven men and seven women with Parkinson's disease were also conducted. These data were combined and analyzed using thematic analysis. ^ Findings. The illness experience is described through the metaphor "Sailing the Sea in The Eye of the Storm." This metaphor served as the overarching theme that covered the two interacting content themes of the voyage of Daily Negotiations in the Midst of Uncertainty and Reconstruction of the Self with Parkinson's Disease. Daily negotiations incorporated navigating daily activities with the uncertainty of both the progression and daily vicissitudes of the disease. Participants described their symptoms as progressive imprisonment that interfered with daily activities. The progressive nature of the disease required the participants to reconstruct their perceptions of themselves. Reconstructing the self involved the paradoxical balancing of preserving the self while simultaneously releasing aspects of the former self to reconstruct the self with Parkinson's disease. This process was reflected in four exemplars: I Know Me." "It's Still Me," "See Me." and "Remember Me." ^ Conclusions. This qualitative study illuminated the struggle of persons in dealing with the uncertainties and fluctuations of Parkinson's disease and the process of reconstructing their perceptions of themselves. The meaning and reconstruction of the illness experience expressed by participants will inform understanding beyond the disease itself to the illness experience that these participants must deal with on a daily basis. ^

The eukaryotic cellular stress response: Biochemical and genetic analyses in Saccharomyces cerevisiae

All cells must have the ability to deal with a variety of environmental stresses. Failure to correctly adapt to and/or protect against adverse stress conditions can lead to cell death. In humans, stress response defects have been linked to a number of neurodegenerative diseases and cancer, underscoring the importance of developing a fundamental understanding of the eukaryotic stress response.^ In an effort to characterize cellular response to high temperature stress, I identified and described one member of a novel gene family— RTR1. I show that the RTR1 gene and its protein product genetically and biochemically interact with core subunits of the RNA polymerase II enzyme. Appropriately, loss of RTR1 results in defective transcription from multiple promoters. These data provide evidence that Rtr1, which is essential under stress conditions, acts as a key regulator of transcription.^ In addition to transcriptional regulation, cells deal with many stressors by inducing molecular chaperones. Molecular chaperones are ubiquitous in all living cells and bind unfolded or damaged proteins and catalyze refolding or degradation. Hsp90 is a unique chaperone because it targets specific clients—typically signaling proteins—for maturation. While it has been shown that Sse1, the yeast Hsp110, is a critical regulator of the Hsp90 chaperone cycle, this work describes the molecular basis for that regulation. I show that Sse1 modulates Hsp90 function through regulation of Hsp70 nucleotide exchange. Further, Hsp110-type nucleotide exchange factors (NEFs) appear to have a specific role in modulating Hsp90 function in this manner. Finally, in addition to Hsp110, the eukaryotic cytosol contains two other types of Hsp70 NEF: Snl1 (BAG-domain protein) and Fes1 (HspBP1-like protein). I investigated the cellular roles of these NEFs to better understand the reason that eukaryotic cells contain three distinct protein families that perform the same biochemical function. I show that while cytsolic Hsp70 NEFs have some degree of functional overlap, they also exhibit striking divergence. Taken together, the work presented in this dissertation provides a more detailed understanding of the eukaryotic stress response. ^

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

Regulation of the heat shock response by thiol-reactive compounds in the yeast Saccharomyces cerevisiae

Cells govern their activities and modulate their interactions with the environment to achieve homeostasis. The heat shock response (HSR) is one of the most well studied fundamental cellular responses to environmental and physiological challenges, resulting in rapid synthesis of heat shock proteins (HSPs), which serve to protect cellular constituents from the deleterious effects of stress. In addition to its role in cytoprotection, the HSR also influences lifespan and is associated with a variety of human diseases including cancer, aging and neurodegenerative disorders. In most eukaryotes, the HSR is primarily mediated by the highly conserved transcription factor HSF1, which recognizes target hsp genes by binding to heat shock elements (HSEs) in their promoters. In recent years, significant efforts have been made to identify small molecules as potential pharmacological activators of HSF1 that could be used for therapeutic benefit in the treatment of human diseases relevant to protein conformation. However, the detailed mechanisms through which these molecules drive HSR activation remain unclear. In this work, I utilized the baker's yeast Saccharomyces cerevisiae as a model system to identify a group of thiol-reactive molecules including oxidants, transition metals and metalloids, and electrophiles, as potent activators of yeast Hsf1. Using an artificial HSE-lacZ reporter and the glucocorticoid receptor system (GR), these diverse thiol-reactive compounds are shown to activate Hsf1 and inhibit Hsp90 chaperone complex activity in a reciprocal, dose-dependent manner. To further understand whether cells sense these reactive compounds through accumulation of unfolded proteins, the proline analog azetidine-2-carboxylic acid (AZC) and protein cross-linker dithiobis(succinimidyl propionate) (DSP) were used to force misfolding of nascent polypeptides and existing cytosolic proteins, respectively. Both unfolding reagents display kinetic HSP induction profiles dissimilar to those generated by thiol-reactive compounds. Moreover, AZC treatment leads to significant cytotoxicity, which is not observed in the presence of the thiol-reactive compounds at the concentrations sufficient to induce Hsf1. Additionally, DSP treatment has little to no effect on Hsp90 functions. Together with the ultracentrifugation analysis of cell lysates that detected no insoluble protein aggregates, my data suggest that at concentrations sufficient to induce Hsf1, thiol-reactive compounds do not induce the HSR via a mechanism based on accumulation of unfolded cytosolic proteins. Another possibility is that thiol-reactive compounds may influence aspects of the protein quality control system such as the ubiquitin-proteasome system (UPS). To address this hypothesis, β-galactosidase reporter fusions were used as model substrates to demonstrate that thiol-reactive compounds do not inhibit ubiquitin activating enzymes (E1) or proteasome activity. Therefore, thiol-reactive compounds do not activate the HSR by inhibiting UPS-dependent protein degradation. I therefore hypothesized that these molecules may directly inactivate protein chaperones, known as repressors of Hsf1. To address this possibility, a thiol-reactive biotin probe was used to demonstrate in vitro that the yeast cytosolic Hsp70 Ssa1, which partners with Hsp90 to repress Hsf1, is specifically modified. Strikingly, mutation of conserved cysteine residues in Ssa1 renders cells insensitive to Hsf1 activation by cadmium and celastrol but not by heat shock. Conversely, substitution with the sulfinic acid and steric bulk mimic aspartic acid led to constitutive activation of Hsf1. Cysteine 303, located in the nucleotide-binding/ATPase domain of Ssa1, was shown to be modified in vivo by a model organic electrophile using Click chemistry technology, verifying that Ssa1 is a direct target for thiol-reactive compounds through adduct formation. Consistently, cadmium pretreatment promoted cells thermotolerance, which is abolished in cells carrying SSA1 cysteine mutant alleles. Taken together, these findings demonstrate that Hsp70 acts as a sensor to induce the cytoprotective heat shock response in response to environmental or endogenously produced thiol-reactive molecules and can discriminate between two distinct environmental stressors.

STUDYING AGGREGATE FORMATION BY AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTANT SOD1 PROTEIN IN DROSOPHILA MODEL

A common pathological hallmark of most neurodegenerative disorders is the presence of protein aggregates in the brain. Understanding the regulation of aggregate formation is thus important for elucidating disease pathogenic mechanisms and finding effective preventive avenues and cures. Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a selective neurodegenerative disorder predominantly affecting motor neurons. The majority of ALS cases are sporadic, however, mutations in superoxide dismutase 1 (SOD1) are responsible for about 20% of familial ALS (fALS). Mutated SOD1 proteins are prone to misfold and form protein aggregates, thus representing a good candidate for studying aggregate formation. The long-term goal of this project is to identify regulators of aggregate formation by mutant SOD1 and other ALS-associated disease proteins. The specific aim of this thesis project is to assess the possibility of using the well-established Drosophila model system to study aggregation by human SOD1 (hSOD1) mutants. To this end, using wild type and the three mutant hSOD1 (A4V, G85R and G93A) most commonly found among fALS, I have generated 16 different SOD1 constructs containing either eGFP or mCherry in-frame fluorescent reporters, established and tested both cell- and animal-based Drosophila hSOD1 models. The experimental strategy allows for clear visualization of ectopic hSOD1 expression as well as versatile co-expression schemes to fully investigate protein aggregation specifically by mutant hSOD1. I have performed pilot cell-transfection experiments and verified induced expression of hSOD1 proteins. Using several tissue- or cell type-specific Gal4 lines, I have confirmed the proper expression of hSOD1 from established transgenic fly lines. Interestingly, in both Drosophila S2 cells and different fly tissues including the eye and motor neurons, robust aggregate formation by either wild type or mutant hSOD1 proteins was not observed. These preliminary observations suggest that Drosophila might not be a good experimental organism to study aggregation and toxicity of mutant hSOD1 protein. Nevertheless this preliminary conclusion implies the potential existence of a potent protective mechanism against mutant hSOD1 aggregation and toxicity in Drosophila. Thus, results from my SOD1-ALS project in Drosophila will help future studies on how to best employ this classic model organism to study ALS and other human brain degenerative diseases.

PHENOTHIAZINES INDUCE ABORTIVE AUTOPHAGY LEADING TO CANCER CELL DEATH

Autophagy is an evolutionarily conserved process that functions to maintain homeostasis and provides energy during nutrient deprivation and environmental stresses for the survival of cells by delivering cytoplasmic contents to the lysosomes for recycling and energy generation. Dysregulation of this process has been linked to human diseases including immune disorders, neurodegenerative muscular diseases and cancer. Autophagy is a double edged sword in that it has both pro-survival and pro-death roles in cancer cells. Its cancer suppressive roles include the clearance of damaged organelles, which could otherwise lead to inflammation and therefore promote tumorigenesis. In its pro-survival role, autophagy allows cancer cells to overcome cytotoxic stresses generated the cancer environment or cancer treatments such as chemotherapy and evade cell death. A better understanding of how drugs that perturb autophagy affect cancer cell signaling is of critical importance toimprove the cancer treatment arsenal. In order to gain insights in the relationship between autophagy and drug treatments, we conducted a high-throughput drug screen to identify autophagy modulators. Our high-throughput screen utilized image based fluorescent microscopy for single cell analysis to identify chemical perturbants of the autophagic process. Phenothiazines emerged as the largest family of drugs that alter the autophagic process by increasing LC3-II punctae levels in different cancer cell lines. In addition, we observed multiple biological effects in cancer cells treated with phenothiazines. Those antitumorigenic effects include decreased cell migration, cell viability, and ATP production along with abortive autophagy. Our studies highlight the potential role of phenothiazines as agents for combinational therapy with other chemotherapeutic agents in the treatment of different cancers.