Nancy Mara Bonini - US grants

Inappropriate cell death is associated with many human eye diseases, including retinitis pigmentosa and aniridia; abnormal cell survival contributes to oncogenic diseases, like retinoblastoma. The long term goal of this research is to understand the molecular mechanisms by which cell survival is controlled during eye development, using combined genetic and molecular methods. The Drosophila eye provides an ideal system in which to approach the molecular control of survival pathways, due to its accessibility to genetic and molecular techniques, and the striking conservation of many developmental genes with human genes. A molecular and genetic analysis of the novel Drosophila gene eyes absent (eya) is proposed. Preliminary studies indicate a gene with striking homology to eya is expressed in human retina. The Drosophila eya gene is required early in eye development for the differentiation of eye progenitor cells. Upon loss of function of the gene, eye progenitor cells die by programmed cell death. These data have led to the hypothesis that the eya gene is either a developmentally-regulated survival factor that functions early in eye progenitor cells, or that the gene is involved in a step of differentiation that is coincidentally or subsequently linked to cell survival control. We will pursue two lines of investigation that elucidate the mechanisms of eya action to link eye differentiation with cell death. First, we will distinguish between the models of eya function by (1a) defining the relationship of eya gene function and that of cell death genes, (1b) defining the relationship of eya function to known genes of eye differentiation conserved to humans, including eyeless the homolog of the human Aniridia gene, and (1c) defining new genes that link cell survival control with eye differentiation, by analysis of six newly-identified suppressors of the eya reduced eye phenotype. Second, we will define the molecular mechanisms of eya action to link cell death and differentiation by (2a) defining in detail the novel nuclear scaffold localization of the eya protein, (2b) defining functional domains of the gene by molecular analysis of eya mutations that affect expression and activity in eye progenitor cells, and (2c) defining domains of eya conserved in human retinal homologs. It is anticipated that the eya gene represents a gene of conserved function from invertebrates to humans. The results gained from this research will contribute to the molecular understanding and design of therapeutic strategies for treating diseases of the human eye that involve aberrant cell death.

DESCRIPTION (From the applicant's abstract): Research proposed in this application seeks to continue the applicant's genetic and molecular studies on Presenilin function and its role in the intracellular trafficking and proteolytic processing of its substrate proteins. Mutant human Presenilins influence the proteolysis of amyloid precursor protein (APP), resulting in an accelerated accumulation of the neurotoxic amyloid peptides during Alzheimer's disease. In the model organisms Caenorhabditis and Drosophila, Presenilins are required for Notch/Lin-12 developmental signaling. Presenilins have recently been shown to regulate proteolytic processing events during Notch receptor maturation and signaling that may be analogous to the Presenilin-dependent cleavages of APP in Alzheimer's disease. Finally, Presenilins have also been implicated in the cellular response to apoptotic stimuli in both mammalian cells and Drosophila. Mosaic tissue studies will be performed in vivo using newly isolated Presenilin gene mutants. Preliminary experiments have revealed integrin-like phenotypes in the mutant tissue clones, prompting the applicants to analyze the role of Presenilin in integrin cleavage using the genetic and biochemical approaches that have been used previously to demonstrate the effects of Presenilin on Notch processing. These studies may reveal shared feature of Presenilin substrates and lead to a better understanding of the specific pathway of protein processing controlled by Presenilin. A central goal of this proposal is to develop an extensive collection of new molecular probes to dissect Notch processing at much higher resolution than is currently possible. These reagents, including new antibodies and epitope-tagged constructs that can discriminate among Notch cleavage products, will be combined with mutational and proteolysis inhibition studies to identify the biochemical steps of Notch processing that involve Presenilin. Genetic and molecular screens for Presenilin-interacting factors will also be performed, taking advantage of the applicant's recent finding that the conserved C-terminus of Presenilin is a crucial functional domain. Finally, detailed parallel studies on the trafficking of Notch and other proteins will be undertaken in tissues lacking either Presenilin or another protein with known effect of subcellular trafficking, the SERCA-type Calcium-ATPase. These experiments are made possible by the applicant's recent isolation of Calcium-ATPase mutants, and they will address the unresolved issue of whether Presenilin is required for protein trafficking or only proteolysis. The studies proposed here will clarify the biochemical activity of Presenilin in the processing of Notch, APP and other proteins, and may ultimately increase our understanding of the molecular causes of Alzheimer's disease.

DESCRIPTION (provided by applicant): Many human neurodegenerative diseases are poorly understood as well as untreatable, including Parkinson's, Alzheimer's and Huntington's diseases. For some familial forms of these diseases, mutations in specific genes products associated with disease are known, allowing the possibility to model the disease in simple systems in order to address mechanisms of degeneration and to pioneer novel treatments. Toward this end, we applied a new approach to the problem of polyglutamine-induced neurodegeneration by developing a model for this class of human disease in the fruit fly Drosophila melanogaster. These experiments demonstrated that fundamental molecular mechanisms of polyglutamine-induced neurodegeneration are conserved in Drosophila, such that Drosophila genetics can be applied to investigate these human diseases in order to address mechanisms of degeneration and define new means of treatment. Using this model, we have shown that the molecular chaperones, which are highly conserved proteins, are potent modulators of neurodegeneration in vivo. We now propose to apply the powerful molecular genetics of Drosophila in genetic screens to uncover additional modulators of neurodegeneration. The advantage of genetic screens is that they provide the ability to define genes that can influence and modulate pathogenesis without requiring previous knowledge of the mechanisms involved. The specific aims are to define novel modulators of neurodegeneration in mis-expression and loss-of-function genetic screens, and to molecularly define and biologically characterize these modifiers in order to address their molecular and biological modes of action. By applying the power of Drosophila molecular genetics to address conserved features of polyglutamine-induced degeneration, these studies provide the foundation for new approaches to cures and treatments for human neurodegenerative disease.

Many human neurodegenerative diseases are poorly understood and untreatable, including Parkinson's (PD), Alzheimer's and Huntington's diseases. For some familial forms, mutations in specific genes are known, allowing pathology to be modeled in simpler systems in order to define mechanisms and pioneer novel treatments. Toward this end, we applied the power of fly genetics to the problem of neurodegeneration by developing models for human disease in Drosophila. Here, we propose to further characterize mechanisms of alpha-synuclein (alpha-syn) associated loss of dopaminergic (DA) neuron integrity, and to study a newly-identified PD-like disorder gene, DJ-1, and its role in protection from oxidative stress. Mutations in alpha-syn are pathogenic for hereditary PD, and wild-type alpha-syn is the major protein component of the pathological aggregates called Lewy bodies that characterize sporadic PD and other synucleinopathies. In Drosophila, expression of alpha-syn compromises the integrity of DA neurons. To further investigate this, in Aim 1 we propose to generate modified forms of alpha-syn and then characterize in detail their toxicity in vivo. Modifications of interest include new hereditary mutations, truncation, and phosphorylation, which are associated with PD or have striking effects on alpha-syn fibrillization in vitro. In Aim 2, given strong links between environmental toxins and development of PD, we will expand our studies of Drosophila models to the detailed characterization of a new PD-like disorder gene, DJ-1. Preliminary studies reveal that null mutation of fly DJ-1 homologues strikingly enhances sensitivity to select oxidative toxins associated with PD, including paraquat and rotenone. Since oxidative damage is thought to be critically involved in both genetic and sporadic forms of PD, study of DJ-1 homologues will reveal new insight into these links. In Aim 3, we will apply the power of Drosophila genetics to address conserved features of alpha-syn and DJ-1-associated pathology, in order to pioneer new approaches to understand and treat human neurodegeneration.

Project Summary - Neuronal injury, such as spinal cord damage, is devastating and typically leads to extreme reduction in life quality. Spinal cord injuries alone affect ~15,000 people/year in the US;about 10,000 of these patients will be permanently paralyzed and many others die due to their injuries. Few approaches are available to treat such devastating injuries, in large part because little is clear about biological pathways &genes that influence regeneration, due to the complexity of the process and the limited systems available for attacking the problem. New insight may be applicable not only to spinal cord injury, but also to brain trauma, stroke and neurodegenerative disease. We propose to develop one of the premier model organism systems-Drosophila-as a new experimental paradigm for adult-stage neural regeneration. In pilot studies in living adult animals, we have severed the wing nerve bundle with a laser and observed degeneration of the nerve tract. Striking preliminary observations show that after a 3-week quiescent period, the nerve bundle regenerates in about 40% of the animals. We will perform detailed characterization of this nerve injury experimental paradigm, including extent and speed of degeneration, as well as extent, speed and accuracy of regeneration. Then, using this system, we will exploit the available sets of molecularly-defined gene mutants in the fly to identify new genes &pathways. Because the baseline regeneration appears partial, this experimental paradigm will reveal both pathways that influence degeneration, as well as those that modulate regeneration. These studies will establish a new experimental paradigm for regeneration studies, and reveal new molecules &pathways, providing basic biological insight and the foundation for discovery of novel therapeutics for nerve injury, damage and degenerative disease.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that wreaks havoc on motor neurons. Recently, a central role for RNA binding proteins and RNA metabolism pathways has emerged. TDP-43 is an RNA binding protein that has been identified as a component of pathological inclusions in neurons of most ALS patients. Further, pathogenic mutations in the gene encoding TDP-43 (TARDBP) are associated with familial and sporadic ALS cases. These data argue that pathological activities of TDP-43 are critical to ALS. Despite the central importance of TDP-43, however, little is understood about how it causes disease and protein interaction partners that lead to ALS. This Co-PI research proposal brings together two experts in yeast and fly genetics who have made fundamental discoveries into mechanisms of devastating human age-related diseases such as Parkinson's disease and ALS using these simple but powerful systems. Importantly, discoveries made in yeast are translatable to Drosophila disease models, and from yeast and flies to human patients. Using these systems, we have found that ataxin-2, a polyglutamine (polyQ) protein, whose polyQ repeat expansions cause spinocerebellar ataxia type 2 (SCA2), is a potent modifier of TDP-43 toxicity in yeast and fly. Launching from this finding, we tested and found that polyQ expansions of 27-33Qs in the human ataxin-2 gene, ATXN2, are significantly associated with ALS. These data argue that ATXN2 is a new and relatively common ALS susceptibility disease gene, and that the TDP-43/Atx2 interaction is a critical target in ALS and perhaps other diseases associated with TDP-43 pathology. With the goal to reveal mechanistic insight into this interaction, we propose three Specific Aims. In Aim 1, we will use mammalian cell culture and Drosophila to define the domains of the TDP-43 and ataxin-2 proteins that are critical for the synergistic interaction in degeneration and function. These studies will provide insight into the activity of the proteins and processes affected in the pathological situation. In Aim 2, we will use the power of fly genetics to define additional genes critical for the synergy between TDP-43 and ataxin-2. This will reveal understanding of the processes perturbed and new pathways for therapeutic targeting. Finally, in Aim 3, we will address the greater role of ataxin-2 in neurodegeneration. ALS shares a disease spectrum with frontotemporal lobar degeneration (FTLD) and TDP- 43 pathology characterizes other neurodegenerative diseases. We will assess polyQ repeat lengths of ataxin-2 in other diseases to assess whether expansions occur in related disorders. Taken together, these findings will reveal key aspects of the TDP-43/ataxin-2 interaction central to ALS, the broader role of ataxin-2 in neurodegenerative disease, and the foundation for novel therapeutic insights.

DESCRIPTION (provided by applicant): Many fundamental cellular processes are affected by epigenetic modulation, and in recent years it has become evident that chromatin-based epigenetic mechanisms underlie important aspects of the aging process. However, despite the fact that age is a prominent risk factor in neurodegenerative disease (ND), there is remarkably little information on the role of epigenetic alterations in mechanisms of ND such as Alzheimer's disease (AD), Parkinson's dementia (PD), frontotemporal degeneration (FTLD) or amyotrophic lateral sclerosis (ALS). We believe that a detailed biological, mechanistic and molecular understanding of the epigenetic factors that are altered in human ND holds promise for an improved understanding of disease pathogenesis and for the development of novel therapeutic interventions. The goals of this Project are to: (1) investigate whether major epigenetic modifications (histone post-translational modifications) change in the context of different NDs using an extensive bank of human samples available from the Penn Center for Neurodegenerative Disease Research (CNDR), (2) use our model systems to discover new epigenetic modifications that underlie ND disease, and (3) test the relevance of novel changes seen in human ND using models of ND. The proposed studies of this multiple-PI and co-Investigator effort will leverage complementary and intersecting interests from our laboratories concerning epigenetics and aging (Berger, Bonini, Johnson), ND (Bonini, Torres and Trojanowski), and the generation and bioinformatic analysis of genome-wide data obtained by chromatin immunoprecipitation followed by second generation sequencing (Gregory, Wang, Berger). The application of our combined expertise to the analysis of the rich collection of CNDR ND brain samples will launch a major new effort in the field of epigenetics in ND. In the broader scientific and medical communities, this effort will promote discoveries of epigenetic mechanisms of ND to provide the foundation for new insights and novel clinical approaches to treat ND. )

DESCRIPTION (provided by applicant): Project Summary - Neuronal injury, such as spinal cord damage, is devastating and typically leads to extreme reduction in life quality. Spinal cord injuries alone affect ~15,000 people/year in the US; about 10,000 of these patients will be permanently paralyzed and many others die due to their injuries. Few approaches are available to treat such devastating injuries, in large part because little is clear about biological pathways & genes that influence regeneration, due to the complexity of the process and the limited systems available for attacking the problem. New insight may be applicable not only to spinal cord injury, but also to brain trauma, stroke and neurodegenerative disease. We propose to develop one of the premier model organism systems-Drosophila-as a new experimental paradigm for adult-stage neural regeneration. In pilot studies in living adult animals, we have severed the wing nerve bundle with a laser and observed degeneration of the nerve tract. Striking preliminary observations show that after a 3-week quiescent period, the nerve bundle regenerates in about 40% of the animals. We will perform detailed characterization of this nerve injury experimental paradigm, including extent and speed of degeneration, as well as extent, speed and accuracy of regeneration. Then, using this system, we will exploit the available sets of molecularly-defined gene mutants in the fly to identify new genes & pathways. Because the baseline regeneration appears partial, this experimental paradigm will reveal both pathways that influence degeneration, as well as those that modulate regeneration. These studies will establish a new experimental paradigm for regeneration studies, and reveal new molecules & pathways, providing basic biological insight and the foundation for discovery of novel therapeutics for nerve injury, damage and degenerative disease.

DESCRIPTION (provided by applicant): One of most unexpected and surprising recent areas of scientific investigation are interactions of the intestinal microbiome on health and disease. Interesting findings include the impact of the microbiome on heart disease due to metabolites generated by bacterial flora promoted by meat consumption, and the impact of the microbiome on obesity. The interactions between the microbiome and the host are likely bidirectional, with the host state impacting the diversity and range of the intestinal microbiome, as well as the intestinal microbiome impacting host health and disease state. Understanding these interactions and how they influence and are influenced by disease is an exploding area of biology with profound implications for health. However, a huge challenge in this work is the difficulty and expense in performing the experiments, in either humans or mammalian models like mice. Diseases, especially chronic neurodegenerative diseases, are especially difficult to investigate for these interactions because of the advanced age of the individuals and their physical condition. We propose to use Drosophila for pioneering studies to assess if the microbiome is impacted by degenerative disease in the animal, and if the microbiome impacts the presentation of disease. Our laboratory has a special focus on amyotrophic lateral sclerosis/frontotemporal dementia and Parkinson's disease, and we propose to initiate studies with robust models for these disorders. In Aim 1, we will assess the impact of neurodegenerative disease on the microbiota of the animal. We will define the gut microbiome of animals over progression of disease longitudinally in the adult, using a range of critical models for these diseases. These studies will define if the microbiome is altered upon disease onset or progression, as well as provide a comparison of this impact between different models of disease. In Aim 2, we will investigate the reciprocal interaction, to determine if the gut microbiome impacts progression and severity of neurodegenerative disease. We will grow disease models germ free on axenic medium and assess key benchmarks and features of the disease phenotype. These experiments will define whether the gut microbiota of the animal itself has an impact on the onset, progression or severity of disease. Here we take advantage of the fact that experiments with gnotobiotic flies are straightforward, while experiments with gnotobiotic vertebrates are quite difficult and expensive. The findings of these Aims will define the reciprocal impact between the microbiome and the neurodegenerative disease state of the animal, to assess these inter-related connections. These studies will establish a simple model foundation for microbiota-animal interactions in neurodegenerative disease, providing predictions for more complex-but slower and far more expensive-animals.

Neurodegenerative disease is among the greatest unmet challenges being faced in healthcare today. Such disease is devastating to families and an enormous economic burden. These disorders include Alzheimer's disease, Parkinson's disease, and the clinically- and pathologically-related disorders frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Despite tremendous medical advances that have extended lifespan, degenerative disease remains formidable with a huge impact on healthspan. Novel approaches and innovative applications are needed to provide biological insight into these diseases and the foundation for therapeutic advances. Drosophila melanogaster is a remarkable model organism with biological pathways highly conserved to humans, a complex brain and nervous system, and a staggering array of genetic and molecular biological approaches for gene and pathway discovery and manipulation. We propose to apply the power of Drosophila to understand genes and mechanisms that underlie neurodegenerative disease. Our special current focus is on the genetic and biological underpinnings of ALS/FTD. ALS is a devastating motor neuron disease that leads to rapid paralysis and death. FTD is the second most common form of dementia. Drosophila research has already provided many striking insights into the biological mechanisms of these diseases, while more basic insights are still needed. We have developed, and will continue to develop, models for familial disease. Our unique, interdisciplinary approach launches from a fly model, which we use to identify pathways of interest by performing genetics screens for modifiers of the disease toxicity. We then extend the findings from Drosophila into human patient tissue, mammalian cells, and primary neurons in culture, ultimately returning our in vivo fly model for detailed mechanistic insight. In addition to genetic studies, we currently plan on using the fly to assess the impact of critical risk factors, such as traumatic brain damage and the gut microbiota, the impacts of which can be difficult, or impossible, to interrogate in mammalian models or cells. Thus, launching from Drosophila, our research program strives to provide novel avenues for the understanding of disease and the foundation for therapeutic insight toward the enormous burden of neurodegenerative disease facing society today.

PROJECT SUMMARY Accumulating evidence strongly supports the idea that sleep is a crucial variable in neurodegenerative disease: disease progression disrupts sleep, and disrupted sleep worsens brain degeneration. Sleep is thought to represent a powerful untapped therapeutic modality through which neurodegeneration can be modified. Yet how sleep and neurodegeneration are coupled at a mechanistic level is poorly understood. Defining cellular and molecular links between sleep and neurodegeneration has been difficult, limiting the ability to pursue sleep modification as a therapeutic avenue. We propose leveraging a neurodegeneration model in Drosophila to dissect mechanisms of disrupted sleep in detail, including with high throughput genetic screens available in simple systems, with the goal of defining molecular pathways linking sleep and brain integrity. We have found that expression of the human neurodegenerative disease protein TDP43 (linked to frontotemporal dementia, Alzheimer?s and motor neuron disease) causes a robust sleep impairment. Our initial data suggest that the Drosophila sleep phenotype results from dysfunction in glia, which are known to be critically involved in sleep regulation. Moreover, TDP43-dependent sleep disturbances can be mitigated by specific modifier genes. Here we will investigate the molecular mechanisms linking TDP43-associated brain toxicity and sleep. In Aim 1, we propose to define the glial subtype critical for the sleep effect and examine how sleep loss affects the subcellular localization and accumulation of TDP43. A genetic screen for modifiers of TDP43-induced sleep dysfunction has already defined several suppressors, including Ataxin-2, a known human disease gene that interacts with TDP43 in neurons. In Aim 2, we will examine this suppressor and others in detail to define molecular and cellular mechanisms of the interaction. Finally, our preliminary data indicate that restriction of sleep opportunity (Sleep Restriction Therapy, SRT) can reverse sleep defects in TDP43 flies. In Aim 3 we will examine SRT in TDP43 flies, and conduct a genetic screen to define the molecular pathways through which SRT improves sleep in this brain degeneration model. Taken together these aims will shed new light on the molecular and genetic links between sleep dysfunction and brain degeneration, and provide the foundation for novel therapeutic targets that leverage sleep to promote brain integrity.