Ai Yamamoto - US grants

[unreadable] DESCRIPTION (provided by applicant): Project Summary Over the last several years, macroautophagy has been implicated in a wide array of neurodegenerative disorders from the aggregation prone disorder, Huntington's disease to the lysosomal storage disorders, Neiman-Pick Type C. Despite its prevalence however, macroautophagy is still poorly understood, making it difficult to define how it contributes towards pathogenesis. Perhaps unsurprisingly, in different disorders, macroautophagy has been considered both as a potentially causative and potentially ameliorative element in disease progression. If we are to target this complex degradative pathway for therapeutics, we need to better define the autophagic process in a means we can apply it towards the brain. In this grant submission, we propose to gain new insights into macroautophagy by focusing on the key organelle involved: the autophagic vacuole (AV). Defined as an onion-like multilamellar vesicle that is positive for the marker MAP1LC3 (a mammalian homologue of ATG8), the formation and maturation of this structure is at the heart of the autophagic process and is by far the least understood. Using a novel approach which we have developed that can isolate specific populations of AV for proteomic and lipid-based analyses, we will: 1) characterize AVs from neuronal cells and brain; 2) compare and contrast MAP1LC3- labeled AVs from vesicles labeled with the other four ATG8 mammalian homologues; and 3) use functional cell based assays to further define how the various ATG8- proteomes impact macroautophagy. PUBLIC HEALTH RELEVANCE: Macroautophagy is a poorly understood process that is important for allowing cells, such as neurons to get rid of proteins that no longer function. Interestingly, this process has been implicated to be at the heart of many neurodegenerative diseases such as Huntington's disease, Parkinson's disease, Alzheimer's disease, many lysosomal storage diseases and others. Here we propose to study macroautophagy as it pertains to the brain so that we can use this information to design effective treatment for these many diseases. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Accumulation and aggregation of mutant proteins are common traits across different neurodegenerative disorders, such as the polyglutamine expansion disorder Huntington's disease (HD). A recently emerging theme is that if mutant protein accumulation is eliminated, symptomatic progression not only halts but also recovers. For example, in an inducible model of Huntington's disease, loss of mutant protein accumulation in symptomatic animals led to complete reversion of the disease-like symptoms. Therefore, if we can accelerate the clearance of a disease-causing mutant protein, there exists the tantalizing possibility of recovery from disease. But how do cells clear these mutant proteins? And how does clearance of the mutant proteins lead to recovery from symptoms? To gain insight into these questions we have run Affymetrix gene arrays on stably transfected cell lines that carry mutant huntingtin protein. The comparison of the different genetic profiles revealed surprisingly robust changes in pathways indicating lysosome-mediated degradation and vesicular trafficking. These two areas are little explored in Huntington's disease and polyglutamine diseases in general, and is thus a rich source of questions. In this proposal we will therefore systematically test the following hypotheses: 1) Lysosome-mediated degradation has a significant impact on the degradation of mutant huntingtin proteins; and 2) Aggregation leads to reversible deficits in vesicular trafficking. Using a combination of biochemical and genetic techniques we also propose to identify regulators of protein aggregation and clearance using a functional cell-based assay: a stable cell line that conditionally expresses mutant proteins fused to variants ol GFP. In sum, during this grant period we will reveal targets that directly alter the level of mutant proteins in a cell, elucidate the basic degradation pathways crucial for handling these difficult proteins, and examine how deficits in protein degradation alters vesicular trafficking in the cell. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Over the last decade, studies have revealed that diminishing mutant huntingtin levels in mouse models of Huntington's disease (HD) leads to the amelioration of pre-existing symptoms, raising the tantalizing possibility for a cure. One of the primary events that accompany symptomatic reversal is the concomitant clearance of the aggregated mutant protein. Despite the intense debate that surrounds the role of protein aggregation in the pathogenesis of HD, a great amount of effort has been put forward to inhibit or disaggregate these proteinaceous intracellular deposits, with limited success. More recently, efforts to drive the turnover of these structures have been proposed, with some promise. One difficulty with these studies has been the inability to target protein degradation pathways in such a way to either enhance or impede the selective elimination of the aggregates, often leading to unwanted, nonspecific consequences that obscure the interpretation of the studies outcome. Recent studies have emerged demonstrating that the protein degradation pathway macroautophagy is capable of the selective degradation of various cargo including ubiquitinated protein aggregates. We have identified the Autophagy linked FYVE domain protein (Alfy) as essential to this process: Importantly not only does depletion of Alfy inhibit the macroautophagic clearance of aggregated mutant htt, but it does so without inhibiting basal and starvation-mediated macroautophagy. Moreover, over-expression of Alfy in neurons led to fewer mutant huntingtin inclusions. In response to the PAS-10-183 Validation of Novel Therapeutic Targets for Huntington's disease, we propose to use Alfy to genetically determine whether the clearance of aggregated mutant huntingtin represents a valid therapeutic approach in HD.

Project Summary Parkinson's disease (PD) is a chronic progressive neurologic disease that is the most common degenerative cause of impaired movement. It is estimated to affect 1 million Americans and its prevalence is predicted to double in developed nations by 2030 as the average age of these populations increases. PD has been the prototypic neurodegenerative disorder for which effective medical and surgical therapies exist to alleviate symptoms. However these treatments neither protect nor restore neural systems and there is a progressive deterioration in the quality of life. There is therefore a compelling need to develop therapies for this disease that prevent ongoing degeneration. The development of such therapies depends on a better understanding of the molecular mechanisms underlying the disease process such that critical mediators can be targeted. Since the discovery that mutations in the gene for ?-synuclein can cause PD, it has become a principal molecule of interest in the pathogenesis. There is now a growing consensus that, at a cellular level, ?-synuclein pathology first appears in axons. Furthermore, in PD it is clear that substantial damage has occurred to the axon projections by the time of diagnosis. The fact that ?-synuclein pathology first appears in axons does not necessarily mean that that they are the site of the earliest disease-related events at the molecular level. There is a substantial body of evidence that the earliest molecular events may occur within the nucleus. Where ?- synuclein first acts at a molecular level to initiate the pathological events underlying PD has not previously been addressed in a living mammalian system. The purpose of this proposal therefore is to address this fundamentally important question in vivo in mice using an AAV 2/7 h-?-synuclein(WT) model to deliver differentially targeted forms of ?-synuclein. We hypothesize that ?-synuclein acts first within the nucleus to initiate the pathologic events that underlie the onset of PD. We will test this hypothesis in two Specific Aims in which we will examine the differential effects of targeted forms of synuclein on the number and morphology of axons in the medial forebrain bundle. Our proposal is unique not only for being the first to examine the initial site of action of ?-synuclein in vivo, but also for using novel methodologies, developed in our lab, for monitoring axonal populations as the most sensitive indicators of early disease-related events. Our results will provide a much-needed fundamental advance in our understanding of how synuclein causes this disease.

In Parkinson's disease (PD), not only has macroautophagy been proposed to be a potential therapeutic target, but its dysfunction has also been implicated in disease pathogenesis both indirectly and directly. Macroautophagy (MA) is a lysosome-mediated degradation pathway that first sequesters cytosolic constituents into a transient, multimembranous vesicle known as an autophagosome (AP), and then fuses into the endolysosomal system for degradation. Although classically known to promote bulk degradation in response to starvation, MA also promotes the selective turnover of defined substrates in response to different stressors, such as protein misfolding and mitochondrial damage. These selective MA pathways achieve selectivity using adaptor proteins which scaffold cargo to the core autophagic machinery and the nascent AP membrane. Two selective autophagy pathways have been particularly relevant in PD; aggrephagy, the selective degradation of protein aggregates, and mitophagy, the selective degradation of mitochondria. If selective MA pathways are to be considered in global therapeutic strategies for the treatment of PD, it is essential that we apply these questions to the mammalian brain and models of PD. With this in mind, we will use newly created mouse models and methods to examine and validate mechanistically the relevance of these two pathways on pathogenesis in mouse models of genetics PD. First, building upon our identification of the selectivity adaptor for aggrephagy, we will establish the mechanism by which ?-synuclein oligomers enter aggrephagy and whether affecting its degradation might influence the phenotype, with a special emphasis on axonal pathology, in a series of transgenic models of ?-synuclein. Next, building upon our findings that mitochondrial are by far the most prevalent autophagic cargo in the brain, we will examine the mechanism by which PINK1 and Parkin might exert their function in the brain. First, we will establish if compensatory changes in macroautophagic pathways might be responsible for masking the constitutive loss of PINK1 or Parkin in vivo, then use mouse genetics to establish better the relationship between these two PD genes and selective macroautophagy.

PROJECT SUMMARY Although a common theme across adult onset neurodegenerative diseases, the pathogenic role of aggregated proteins is a continuous topic of debate. For the incurable familial neurodegenerative disorder Huntington's disease (HD), resolving the accumulation of mutant huntingtin (Htt) (neuronal or cytoplasmic) is highly correlated with favorable therapeutic outcomes. Whether targeting aggregate clearance per se is beneficial, however, has remained unclear. We have previously identified a pathway by which aggregated proteins are selectively eliminated by the lysosome-mediated pathway macroautophagy. We found that the protein Alfy is central for the selective turnover of aggregates in cell based systems. During the previous funding period, we used a mouse genetics and cell biology to determine that Alfy is indeed essential turnover of aggregated proteins in adult brain, and diminishing Alfy levels in vivo modifies disease onset. In this renewal application, we will use genetic and molecular based approaches to determine if augmenting Alfy levels promotes the elimination of aggregated nuclear and cytoplasmic proteins, the mechanism by which a genetic variant of Alfy might delay the age of onset of MD, and the molecular mechanism by which Alfy permits aggregate clearance. .

PROJECT SUMMARY Macroautophagy (MA) is a cellular response to stress whose dysfunction is implicated in several neurodegenerative diseases. Our limited understanding about how MA is used by distinct CNS cell types has hindered our ability to fully understand the implications of its dysfunction for CNS diseases. A fundamental and well-defined physiologic stress linked to MA is acute nutrient starvation. In vertebrates, starvation-induced activation of MA in peripheral organs such as liver, heart and muscle is essential for maintaining amino acid and glucose blood levels. However, prolonged MA results in loss of organ mass, ultimately leading to breakdown and irreparable damage of these vital organs. It has long been appreciated that under conditions of prolonged starvation, peripheral organs are sacrificed to maintain brain function; however, the mechanisms remain poorly understood. In contrast to most cells in the body, neurons receive nutrients indirectly from the blood through a tightly coordinated signalling with endothelial cells (ECs), pericytes and astrocytes that collective form the neurovascular unit (NVU). Given the critical role that these cells play in forming the blood- brain barrier (BBB), that tightly regulates the influx of nutrients into the brain, they are likely to be highly sensitive to the nutrient status of the periphery, and play a key role in prioritizing the brain during starvation. In this proposal, we will test the hypothesis that MA is upregulated in astrocytes upon starvation and plays a central role in triggering an essential, positive-feedback loop with brain ECs to maintain neuronal function during physiologic starvation. In Aim 1, we will characterize the metabolic function of mice lacking MA in discrete CNS cell types. In Aim 2 we will establish the autophagic response to starvation in astrocytes and how this influences ECs. Upon successful completion of the proposed studies, we will gain new understanding into the molecular and cell-specific response of MA in NVU cells of the CNS before and during starvation, and how they coordinate their efforts to ensure that neuronal function is maintained. In addition to gaining insight into this important and critical physiologic response, these studies will provide conceptual and methodological advances regarding how discrete cell types of the CNS use MA during conditions of stress and disease.

On the sporadic Amyotrophic Lateral Sclerosis-Frontotemporal Dementia (sALS/FTD) clinical spectrum, the aggregation and accumulation of disease-associated proteins such as TDP-43 is a notable neuropathological hallmark, yet we know little about why this highly abnormal event might occur. Although disruptions in multiple cellular processes have been implicated in ALS, 3 critical gaps in knowledge remain: 1) What triggers the aggregation of wildtype proteins in sporadic disease? Is protein aggregation sufficient to drive pathology? 2) What drives the cell-specific vulnerabilities and variable clinical manifestation from ALS to FTD? 3) How do disease-associated alterations in protein homeostasis perturb communication in the tissue microenvironment? Given that more than 95% of ALS arises sporadically, and that the mechanisms of sporadic disease remain unknown, we will look beyond individual mutations, and establish a novel conceptual framework that examines the cellular changes that occur during disease states. We posit that by focusing on why TDP-43 aggregation occurs, especially in sporadic ALS, we will gain insights into pathogenic mechanisms underlying this spectrum of disorders. Our central hypothesis is that there are physical changes at the cell and tissue scale that initiate ALS/FTD. We propose that altered biophysical properties within cells (predominantly altered molecular crowding), which are linked to mechanical perturbations to the tissue microenvironment (stiffening, inflammation, edema causing osmotic stress), lead to age-dependent cellular dysfunction by altering the dynamics of assembly, disassembly and transport of macromolecular protein machines. We will test this hypothesis in cellular models, animal models, and patient tissue by (1) using novel tools to probe the intracellular biophysical environment of cells; (2) integrating these findings using novel genomics technologies applied to mouse models to study (i) how intracellular changes in crowding and extracellular changes in the tissue microenvironment may drive pathogenesis in vivo, and (ii) how such perturbations disrupt cell-cell communication in vulnerable regions of tissue; and (3) relating our findings to human disease by re-examining these findings in the context of a clinically and neuropathologically deeply curated cohort of ALS/FTD patients. These studies will allow us to address the following questions: 1) Why does abnormal protein aggregation and accumulation occur in sporadic disease, and how might this contribute to disease pathogenesis; 2) Do these alterations in protein homeostasis perturb intercellular communication in the tissue microenvironment; and 3) What drives the cell type vulnerability that makes ALS/FTD unique? The proposed work will accomplish the following: A) represent the first detailed survey of molecular crowding in neural cells; B) uncover whether a causal link between intracellular crowding, protein aggregation, and neurodegeneration exists, C) establish whether the impacts of intracellular crowding show cell type specific signatures including changes in protein- protein interactions, and D) provide a new framework to explore therapeutic strategies for treating ALS/FTD.