Richard I. Morimoto, Ph.D. - US grants

The goal of our research is to understand the mechanism of translational control of protein synthesis in eukaryotic cells. We have developed a system that combines the advantages of reticulocytes to study and dissect protein synthesis and heat shock to rapidly alter translational specificity. We have found that chicken reticulocytes respond to heat shock by the increased synthesis of only one heat shock protein (HSP70) and the rapid repression of globin synthesis. The globin mRNA is neither degraded nor structurally modified in heat shocked cells and can be isolated and in vitro translated. HSP70 mRNA is transcribed in normal reticulocytes and apparently maintained in a translationally repressed state in the cytoplasm. Heat shock induces the selective synthesis of HSP70 even though there is no significant increase in the amount of HSP70 mRNA in the cytoplasm. We suggest that the increased synthesis of HSP70 is due to selective utilization of HSP70 mRNA in heat shocked cells. The selectivity may be due to heat shock induced modifications of the translational apparatus or other factors that discriminate between HSP70 mRNA and other cellular mRNAs. We expect that HSP70 mRNA contains features that are recognized by these discriminatory factors. Our system is ideally suited for studying translational control because the parameters of protein synthesis are well characterized in reticulocytes and these cells from chickens respond to heat shock by the increased synthesis of only HSP70. This provides the opportunity to focus on the expression of a single species of mRNA, that for HSP70, and the regulation of its interaction with the translational apparatus. The experiments proposed in this grant will elucidate the mechanism by which translational control operates, preferentially utilizes HSP70 mRNA and results in the selective translation of HSP70 mRNA in heat shocked cells. These goals will be accomplished by identifying the changes in the protein synthetic apparatus that affect translational specificity by using a chicken reticulocyte in vitro translation system. We will also determine whether HSP70 mRNA contains information necessary for its preferential translation by mutational analysis of the cloned chicken HSP70 gene and introduction of the modified genes into chicken lymphoid and erythroid cells. The studies will increase our knowledge about basic cellular control mechanisms, in particular how vertebrate cells respond to alterations in their environment.

nucleic acid sequence; DNA replication; genetic promoter element; gene expression; genetic transcription; cell growth regulation; Adenoviridae; heat; virus genetics; environmental stressor; heavy metals; evolution; serum; chromatin; oncogenes; tissue /cell culture; mutant; laboratory rabbit; radiotracer; synchronous cell division; HeLa cells; human tissue; immunofluorescence technique; gel electrophoresis;

biomedical equipment purchase;

Common among all organisms is an essential, highly conserved and exquisitely regulated cellular response to stressful environments. The heat shock response represents an adaptative mechanism that involves the elevated synthesis of a family of proteins commonly referred to as heat shock or stress-induced proteins. The HSP70 family participates in numerous protein biosynthetic reactions including the synthesis, translocation and folding of many cytoplasmic, organellar, membrane associated and secreted proteins. In this proposal we will investigate the properties and structure of the human HSP70 proteins. To accomplish this goal we will identify and characterize the peptide-binding domain of HSP70 by the construction of deletion and point mutants. In vitro studies will include peptide binding assays to examine and compare substrate specificities. Chimeric fusions between dnaK and human HSP70 and between the various members of the HSP70 family (p72/HSC70, GRP78/BiP and mitochondrial p75) will also be assayed in mammalian cells and in E. coli. The complementation assays in E. coli include lambda replication, growth of E. coli at elevated temperatures and autoregulation of the heat shock response. The function of the mutant HSP70 proteins, chimeric 7OkD stress proteins, HSP70-related proteins and bacterial dnaK will be examined in mammalian cells by redirecting their subcellular locale of these stress proteins and the analysis of subsequent in vivo interactions by immunofluorescence and immunoprecipitation assays. The collection of mutant heat shock proteins will be used to identify the amino acid sequence requirements for the cell cycle and stress-dependent reversible translocation of HSP70 between the cytosolic, nuclear and nucleolar compartments of the mammalian cell. Finally, we will clone the remaining (uncloned) HSP70-related genes in the human genome using sequence homology and monoclonal antibody reagents. This will allow us to establish whether distinct members of the HSP70 family differ in their biological and biochemical function.

Common among all organisms is an essential, highly conserved and exquisitely regulated cellular response to stressful environments. The heat shock response represents an adaptative mechanism that involves the elevated synthesis of a family of proteins commonly referred to as heat shock or stress-induced proteins. The HSP70 family participates in numerous protein biosynthetic reactions including the synthesis, translocation and folding of many cytoplasmic, organellar, membrane associated and secreted proteins. In this proposal we will investigate the properties and structure of the human HSP70 proteins. To accomplish this goal we will identify and characterize the peptide-binding domain of HSP70 by the construction of deletion and point mutants. In vitro studies will include peptide binding assays to examine and compare substrate specificities. Chimeric fusions between dnaK and human HSP70 and between the various members of the HSP70 family (p72/HSC70, GRP78/BiP and mitochondrial p75) will also be assayed in mammalian cells and in E. coli. The complementation assays in E. coli include lambda replication, growth of E. coli at elevated temperatures and autoregulation of the heat shock response. The function of the mutant HSP70 proteins, chimeric 7OkD stress proteins, HSP70-related proteins and bacterial dnaK will be examined in mammalian cells by redirecting their subcellular locale of these stress proteins and the analysis of subsequent in vivo interactions by immunofluorescence and immunoprecipitation assays. The collection of mutant heat shock proteins will be used to identify the amino acid sequence requirements for the cell cycle and stress-dependent reversible translocation of HSP70 between the cytosolic, nuclear and nucleolar compartments of the mammalian cell. Finally, we will clone the remaining (uncloned) HSP70-related genes in the human genome using sequence homology and monoclonal antibody reagents. This will allow us to establish whether distinct members of the HSP70 family differ in their biological and biochemical function.

DESCRIPTION (adapted from the applicant's abstract): The molecular response to environmental and physiological stress alerts cells to imminent danger and serves to protect the genetic and biosynthetic apparatus from sustaining potentially lethal proteotoxic damage. The heat shock response adjusts the levels of molecular chaperones to prevent the appearance and accumulation of aggregation-prone polypeptides during cell stress. At the center of this regulatory network, in larger eukaryotes, is a family of heat shock transcription factors (HSFs). Under conditions of normal cell growth, HSFs are repressed. In response to stress, HSFs are rapidly activated to induce the transcription of heat shock genes. The levels of chaperones are carefully balanced to reflect the levels of misfolded proteins and heat shock factor activity, such that the heat shock response is autoregulated and attenuates during recovery. This is a proposal for continued funding to: (1) Order the events associated with activation and attenuation of the heat shock response. The experiments would elucidate the process of intramolecular negative regulation of both the DNA binding and transactivation domains, the role of the molecular chaperone Hsp90 in maintenance of the inert monomer, the role of Hsp70/Hdj1 in transcriptional repression, the function of HSBP1 in the negative regulation of the heat shock response, and the characterization of other trans-regulators of HSF1. (2) Characterize the function of HSF1 stress granules in the cell biology of the heat shock response. Experiments are proposed to identify the targeting and retention signals involved in localization of HSF1 to stress granules, to identify the stress-regulated nuclear transport mechanism, to identify the chromosomal location of HSF1 granules, and to establish the role of HSF1 stress granules in the heat shock response. (3) Examine how different members of the family of heat shock factors coordinate their activities. Experiments are proposed to understand how the activities of HSF1 and HSF2 are coordinated under normal conditions of cell growth and stress and to establish the regulatory link between HSF2 lability and control by general protein degradation and the ubiquitin-dependent proteasome. (4) Characterize the C. elegans heat shock response to understand how stress responses are regulated in a multicellular differentiating organism. Dr. Morimoto will focus on tissue specific, developmental, and aging signals which modulate the heat shock response in C. elegans; the function of C. elegans HSF and trans-regulators, including heat shock proteins and HSB-1; and the relationship of the heat shock response to diseases of protein misfolding and protein aggregation, including Huntington's Disease and ALS. Proteins containing beta-hairpin stacked sheets will be expressed in C. elegans and human tissue culture cells (neuronal and non-neuronal), to understand the molecular basis of the "stress signaling".

oxidative stress; chemoprevention; arachidonate; salicylate; indomethacin; enzyme activity; virus; phosphoproteins; hydrogen peroxide; transcription factor; tumor necrosis factor alpha; phosphorylation; protein kinase; stress proteins; virus infection mechanism; tissue /cell culture;

A. Summary Aging is a major risk factor for degenerative diseases, and associated with cumulative protein damage and the decline of cellular function. Our previous work demonstrated that the expression of aggregation-prone proteins in C. elegans models of neurodegenerative disease initiates a cascade of protein damage that results in misfolding of other metastable proteins. Using a computation approach, we showed that proteins at-risk for aggregation are not randonri, but rather have in common sequence elements that predict their intrinsic metastability and tendency to aggregate. Moreover, misfolding and aggregation in various neuronal and nonneuronal tissues is not sporadic throughout lifespan, but rather occurs at a much earlier point in C. elegans adulthood coincident with a dramatic decline in the robustness of inducible cell stress responses. In this MERIT extension application, we propose to test the hypothesis that the composition of the proteome. and alterations in the balance of soluble and insoluble species during aging are accelerated by acute and chronic proteotoxic stress and protected by selective induction of proteostasis network and lifespan pathways. This will be demonstrated using two complementary model systems: an organismal approach using C. elegans that affords a precise model for aging and the ability to test genetic pathways that control proteostasis and lifespan, .and adult human primary cells and inducible pluripotent cells to assess whether changes in the aging proteome and mechanisms that control these processes are conserved. The Aims are: (1) Assessing the specificity and selectivity of proteome aggregation in acute and chronic proteotoxic stress and aging. We will perform a proteomic analysis at multiple points of C. elegans adulthood and aging, and quantify the soluble and aggregated fractions in animals challenged by acute (heat shock) and chronic (expression of polyglutamine. Aß, and tau) proteotoxic stress. Proteins that shift to the aggregated state will be validated by generating a corresponding series of transgenic protein-GFP reporter lines and used to assess folding transitions in different compartments and tissues in living animals. Comparative proteomic analysis of human primary cells from donors over a wide range of chronological age will reveal whether the same proteins or pathways undergo similar changes in solubility and aggregation. The proteomic data will be integrated with a corresponding set of RNA-seq data and used to develop models to establish whether proteomic risk, failure, or protection can be assessed at the tissue-level to establish an organismal understanding of proteome-wide networks. (2) Establishing whether proteome stability can be selectively altered by regulation of the proteostasis networif and lifespan pathways. We will determine the consequences to the soluble and aggregated proteome of enhancing or inhibiting different arms of the PN. for example by constitutive activation or inhibition of the heat shock response and the organellar unfolded protein responses. Likewise, are the consequences to the proteome the same or distinct, by activating or inhibiting the lifespan pathways regulated by caloric restriction, germline stem cell signaling, insulin-signaling, and the heat shock response, and '(3) Developing a multi-dimensional systems network map of the stressed and aging proteome. To develop a visual image of proteome dynamics over chronological age using data from Aims 1 and 2. together with archival data on expression of the proteome and the PN. This map will be used to predict the proteins, compartments, or tissues that are affected by aging and stress, and protected by chaperone networks and the PN.

[unreadable] DESCRIPTION (provided by applicant): Elevated expression of molecular chaperones has been shown to suppress protein misfolding/aggregation and toxicity in various model systems of Huntington's disease, Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis (ALS). Mutations in the respective proteins associated with these diseases results in the appearance of misfolded species that adopt alternate conformations. These observations have led to the proposal that a common feature of mutant huntingtin, tau, alpha-synuclein, and superoxide dismutase (SOD1) is the appearance of alternate folded states that self-associate and form toxic species and protein aggregates. Molecular chaperones offer intriguing targets for therapeutics because of their unique characteristic to recognize and sequester damaged and misfolded species. Consequently, chaperones may have a central role in protein homeostasis to prevent the deleterious consequences of chronic misfolded species, that, over time results in cell dysgenesis and pathologies as occurs in neurodegenerative diseases and other diseases associated with protein misfolding. However, because chaperones function in vivo as networks, it has also become increasingly evident that the expression of individual chaperones alone is either ineffective or much less effective than the coordinated expression of multiple chaperones to achieve maximal network functionality. We propose three Specific Aims: Aim 1. To establish a robust primary screen for small molecule regulators of the heat shock response; Aim 2. To characterize the candidates for cytotoxicity and kinetics of induction of HS gene expression and chaperone levels; and Aim 3. To test small molecule regulators of the heat shock response in secondary assays of cells expressing mutant huntingtin and SOD1. Although our experiments will only examine the consequences of novel small molecule regulators of chaperone expression on model systems of neurodegenerative disease, we anticipate that our results may well extend to other pathologies associated with the flux of misfolded proteins in the cytoplasm. [unreadable] [unreadable] [unreadable]

We propose to develop a Center for Systems Biology to promote interdisciplinary scientific investigation and education in Chicago. Faculty in the Institute for Genomics and Systems Biology at the University of Chicago will take a leadership role and together with collaborators at other Chicago institutions will create a broad outreach to the community. The Centers scientific program will focus on the robustness of transcriptional networks in physiological, developmental and evolutionary time scales. We propose to go beyond mapping Network topologies to develop dynamical models of the behavior of transcriptional regulatory networks during physiological stress, during cellular and organismal development, and during the evolution of species. These goals will be achieved by bringing together experts in genomics, developmental biology, evolutionary biology, stress and physiology, network modeling, high performance and grid computing, chemistry, and physics. The overarching aim of the Centers research is to uncover the organizational principles that transcriptional regulatory networks share as they respond to physiological, development and evolutionary inputs and pressures. These principles are expected to reveal structure-function relationships in networks that lead to physiological and evolutionary robustness or, its complement, flexibility.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topics, 06-AG-108 Technologies for obtaining genomic, proteomic, and metabolomic data from individual viable cells in complex tissues, and 06-DK-105 Enabling technologies for cell biology and macromolecular analyses. The long-term health of all metazoan cells is inextricably linked to protein quality control. This is achieved by proteostasis, a complex network of molecular interactions that determines the health of the proteome. Proteostasis balances protein biosynthesis, folding, translocation, assembly/disassembly, and clearance with the challenges imposed by environmental or physiological stress that results in a flux of misfolded and damaged proteins. An imbalance in homeostasis, if left unattended can result in severe molecular damage to the cell, dysregulation of key tissues leading to pathology, and susceptibility to diseases of aging. Adaptation and survival requires an ability to sense damaged proteins and to coordinate induction of protective stress response pathways, chaperone, and clearance networks. Despite the abundance and apparent capacity of chaperones and other components of the proteostasis network to restore folding equilibrium, the cell is poorly adapted for chronic proteotoxic stress as occurs when certain aggregation-prone proteins are expressed in metabolic disease, cancer, and neurodegenerative disease. This decline in repair activities that challenges the integrity of the proteome is influenced strongly by genes that control aging thus linking stress biology, metabolism, and protein homeostasis with the health and lifespan of the organism. This proposal brings together the complementary strengths of two groups, the Dillin laboratory at the Salk Institute and the Morimoto laboratory at Northwestern University to develop and test a new set of molecular tools that will report on the health of the proteome. These "proteostasis sensors" are designed to provide real-time assessment of the capabilities of protein folding quality control in each compartment of the cell and to assess the consequences of protein damage, cell stress, aging, and diseases of protein conformation. These tools will be initially developed for use in C. elegans to obtain a rapid test of hypothesis and the ability to assess functional capacities using genetic approaches and then extended to mammalian tissue culture cells and eventually in transgenic mice. The impact of these studies is very broad and extends across all areas of biology and medicine, for protein quality control is fundamental to the health and protection of the proteome in all cells and tissues of eukaryotes. The tools that we develop will be made available to all other researchers upon request upon publication. PUBLIC HEALTH RELEVANCE: The expression of damaged proteins is associated with hundreds of human diseases associated with aging. How protein misfolding in one compartment of the cell affects another and variability among tissues represents key questions that have not been resolved. This proposal is to develop a molecular toolbox of folding sensors that provide real-time living cell imaging to quantify the health of the proteome in the face of acute stress, chronic expression of damaged proteins, and aging.

DESCRIPTION (provided by applicant): This GO grant addresses the critical need for development of biosensors that detect protein dysfunction during aging. The long-term health of all cells is inextricably linked to protein folding and sustainability of function. This is achieved by protein homeostatasis or 'proteostasis'(Balch et al. (2008) Science 319: 916), a complex network of molecular interactions that determines the health of the proteome. Proteostasis balances protein biosynthesis, folding, translocation, assembly/disassembly and clearance with the challenges imposed by environmental or physiological stress that results in a continual flux of misfolded and damaged proteins that the cell must manage. An imbalance, if left unattended can result in severe molecular damage to the cell, dysregulation of key tissues leading to pathology, and susceptibility to nearly all of diseases of aging. Adaptation and survival requires an ability to sense these damaged proteins and to coordinate induction of protective stress response pathways, chaperone and clearance networks. Despite the abundance and apparent capacity of the proteostasis network to restore the folding equilibrium, the cell appears to be poorly adapted for chronic proteotoxic stress as occurs when certain aggregation-prone proteins are expressed, for instance, in neurodegenerative aging diseases. We have hypothesized that this decline in repair activities, that challenges the integrity of the proteome, is influenced strongly by genes that control aging- thus linking stress biology, metabolism (diet), and protein homeostasis with health and human lifespan. The proposal brings together the complementary strengths of the Balch, Kelly and Wiseman laboratories at The Scripps Research Institute, the Dillin laboratory at the Salk Institute and the Morimoto laboratory at Northwestern University, to develop and test a new set of molecular tools that will globally report on the health of the proteome during aging. These groups form the Proteostasis Aging Sensor Consortium (PASC) to develop 'proteostasis sensors", innovative molecular reporters that will provide real- time assessment of the capabilities of protein folding quality control in each compartment of the cell, and in tissue and organismal models. These innovative probes will assess the consequences of protein damage, cell stress, aging and diseases of protein conformation that influence human longevity. The impact of these studies on the aging field is very broad and extends across all areas of biology and medicine. The combined collaborative efforts from the members of the PASC will leverage the tools, techniques and knowledge of protein homeostasis and aging to gauge the folding environment within cells and animals, and provide the next generation tools that will considerably accelerate efforts in the aging sciences. PUBLIC HEALTH RELEVANCE: The long-term health of mankind during aging is inextricably linked to protein folding and sustainability of protein function in spite of the many challenges imposed by environmental and/or physiological stress. Longevity requires an ability to sense damaged proteins and to coordinate induction of protective pathways and clearance networks responsive to genes that control aging, stress biology and metabolism (diet). This proposal by the Proteostasis Aging Sensor Consortium (PASC) consisting of the Balch-Dillin-Kelly-Morimoto- Wiseman groups will develop and test a new set of innovative molecular tools, referred to as proteostasis sensors that will globally report in real-time on the health of the human proteome during aging. The impact of these studies on the aging field is necessarily very broad and extends across all areas of biology and medicine related to human health.

DESCRIPTION (provided by applicant): The health of the proteome is of central importance to the cell and contributes significantly to the health and lifespan of the organism. The proteome is constantly challenged by external physiological and environmental stress, and places demands upon the protein quality control machinery and the proteostasis network, to sense and respond to the expression of misfolded and damaged proteins. It is increasingly clear that acute proteotoxicity, associated with the chronic expression of disease-associated aggregation-prone proteins, is daunting to the cell. When proteostatic capacity is exceeded, the consequence can be neurodegeneration, cancer, immunological disease, or metabolic diseases. Our studies have shown that expression of an aggregation-prone protein imbalances proteostasis and destabilize other conformationally challenged, metastable proteins. During ageing, the collapse of proteostasis leads to the disruption of multiple cellular activities leading to cell dysfunction and organismal failure. The studies proposed here are to understand how diverse stress signals are sensed by individual cells and tissues in the intact metazoan animal and the roles of stress-inducible transcription factors, HSF1 and Daf-16, to integrate stress biology to enhance cytoprotective networks that suppress the deleterious consequences of ageing and disease. We propose three integrated aims: (1) At the level of the organism, to understand how stress response's in the intact metazoan are regulated by specific neurons that sense and transmit environmental and physiological stress signals to control expression of chaperones in somatic cells. We will identify the signaling pathways that transmit the thermosensory signal from the AFD neurons to regulate the cell non-autonomous control of the heat shock response and HSF1 activity in somatic cells, (2) At the cellular level, to characterize the tissue-specific expression of the family of genes encoding molecular chaperones to elucidate the underlying strategy for chaperone networks in response to stress, during development, and ageing. These studies will provide a network-level understanding of the organizational properties of the eukaryotic chaperome, and (3) At the molecular level, to characterize the regulation of HSF1 by stress-inducible acetylation and deacetylation by the NAD-dependent sirtuin, SIRT1, and the role(s) of this post-translational regulatory pathway in metabolic control of cell stress and lifespan. PUBLIC HEALTH RELEVANCE: Chaperone networks and cytoprotective stress responses regulate the stability of the proteome, and consequently the health of the cell, and lifespan of the organism. An understanding of how these networks are organized and regulated has relevance to a multitude of diseases associated with protein misfolding and aggregation. The studies proposed here are to understand how diverse stress signals are sensed by individual cells and tissues in the intact animal and the roles of stress-inducible transcription factors to integrate stress biology to enhance cytoprotective networks that suppress the deleterious consequences of ageing and disease.

PROJECT SUMMARY This proposal is to examine the basis of intertissue signaling in the regulation of organismal proteostasis. We posit that tissue health is influenced by signals between peripheral tissues and neurons to establish an integrated proteostasis network (PN) in metazoans. This ensures that the proteome expressed by each tissue is engaged with its corresponding tissue PN for balanced synthesis, folding and function, and degradation. In addition to coordinating proteome health within each tissue, intertissue signaling affects the organismal response to proteotoxicity during aging. Our previous work provided some of the key observations to suggest a role for peripheral proteostasis in C. elegans. We showed that the organismal heat shock response (HSR) is regulated by a specific sensory neuron that communicates the cell stress signal via serotonin to the somatic tissues, that an imbalance in proteostasis within any single tissue sends a stress response signal to peripheral receiving tissues resulting in a compensatory transcellular chaperone response, and that germ line stem cells regulate the HSR in peripheral somatic tissues at reproductive maturity using a global chromatin repression signal to initiate PN failure in aging. These studies provide the basis for this proposal to understand the regulation and properties of peripheral proteostasis. We propose the following Aims: (1) To establish the functional properties of the tissue PN in neurons, body wall muscle cells, and intestine of C. elegans, (2) To identify the signaling pathways for communication between neurons, body wall muscle cells, and intestine for peripheral proteostasis, and (3) To examine how proteotoxic proteins Abeta, tau or polyQ affects peripheral proteostasis, and the effects of aging on aggregation and toxicity.

Project Summary One of the most challenging problems facing society is the mixed benefit of longer lifespan that is also accompanied by the increased risk for neurodegenerative diseases. A central theme of this proposal is that aging is associated with the declining capability of the protein quality control machinery, leading to protein misfolding and aggregation, and resulting in cell and tissue failure and neurodegenerative disease. In this PPG, we have assembled the team of S. Finkbeiner (UCSF), D. Finley (Harvard), J. Frydman (Stanford), J. Kelly (Scripps) and R. Morimoto (Northwestern) to examine proteostasis failure in aging as the basis for misfolding and aggregation of Tau, SOD1 and expanded polyQ in Alzheimer's disease (AD), ALS, Huntington's disease and Ataxias, respectively. A distinctive strength of this PPG team is our expertise with multiple biological systems including S. cerevisiae, C. elegans, mice, patient derived cells and differentiated neurons, and multiple experimental approaches from biochemical and biophysical, live cellular imaging of aggregation phenotypes, and small molecule high-throughput screens. We posit that the unique richness of approaches afforded by this team will provide novel insights that will uncover how aging affects the proteostasis network (PN) of protein synthesis and molecular chaperones, transport machineries, and the degradative arms of the PN comprised of the ubiquitin-proteasome and autophagy lysosomal pathways. An understanding of how aging affects the PN at the cellular, tissue, and organismal level will provide a mechanistic understanding on the events during proteostasis failure that contributes to and accelerates aggregation of Tau, SOD1 and expanded polyQ proteins leading to AD and other neurodegenerative diseases. Through four Projects and four Cores, our team will explore how aging affects the function of molecular chaperones to influence nascent- chain synthesis and the off-pathway aggregation properties of Tau and polyglutamine in yeast (Proj. 1) and in different tissues of short-and-long-lived C. elegans (Proj. 4). We will examine the degradative arms of the PN in transgenic mice with altered levels of proteasome activity and the effects on proteotoxicity of Tau and mutant SOD1 (Proj. 2), and in human iPSCs derived patient neurons by monitoring the autophagic lysosomal pathways (Proj. 3) challenged by aging and expression of Tau or TDP43. These Projects will be supported by: the coordinating Administrative Core A, Proteostasis Sensors Core B that develops PN reporters to quantify different PN activities, Proteostasis Proteomics Core C, and the Proteostasis Regulator Pharmacology Core D to develop a small molecule strategy to restore PN functionality in aging and neurodegenerative disease. Working together, the Cores and Projects will generate PN reagents and tools, datasets and small molecules to quantify and perturb different components of the PN, to generate a comparative analysis of aging and neurodegeneration across all biological systems, and to develop a small molecule strategy to prevent or reverse the age-and-disease dependent failures in the PN leading to neurodegenerative disease.

Project 4 Summary- Morimoto Aging leads to proteostasis failure at the organismal level and is associated with the increased risk for misfolding, aggregation, and neurodegenerative disease. The emphasis of this Project is on organismal proteostasis, to understand how intertissue regulation of the proteostasis network (PN) between neurons and non-neuronal tissues of C. elegans. Our goals are to establish how communication between tissues ensures healthy proteostasis, to identify the basis for failure in quality control in aging, and how expression of Tau, SOD1, and polyglutamine causes amplification of proteotoxicity directly relevant to neurodegenerative disease. C. elegans has the advantages of transparency, detailed lineage relationships, and powerful genetic and molecular tools that are ideal to assess synthesis, folding, the ubiquitin-proteasome and autophagy lysosome pathway in tissues of wild type animals during normal aging. These results will be compared to short-and-long lived animals, and exposure to acute (heat shock) and chronic (Tau, SOD1, and polyglutamine) proteotoxic stress to identify the critical components of the PN that are essential for rapid response and protection. In C. elegans and other metazoans, the PN is regulated by cell non-autonomous control; for example, the organismal heat shock response (HSR) is regulated by sensory neurons that control induction of the HSR and properties of the PN in distal somatic tissues, moreover inducibility of the HSR declines in early adulthood by signal(s) from germ line stem cells that results in reduced tissue resilience post fecundity. The Aims are to: (1): Examine the effects of aging and proteotoxic stress on PN composition and properties. We will use proteostasis reporters (Core B) to assess and quantify folding, transport, and degradation in different tissues during development and aging, and upon exposure to physiological and proteotoxic stress, relate PN functionality to PN composition in tissues using cell type-specific transcriptomic profiling and proteomics (Cores B, C), and establish how the PN adjusts in short-and long-lived animals, (2): Examine how intertissue stress signaling in aging is affected by Tau, SOD1, and polyglutamine proteins. We will use genetic approaches to perturb the PN in neurons, muscle, intestine to identify tissue circuits and directionality of stress signaling across tissues, determine the effects of aging and expression of Tau, SOD1, and polyglutamine proteins on intertissue communication, and whether PN modulation in sending or receiving tissues can restore proteostasis against proteotoxicity, and (3): Deploy proteostasis regulators to restore organismal proteostasis in aging and neurodegeneration. With Core D, we will develop strategies for PN modulation by small molecule Proteostasis Regulators to prevent proteostasis failure during aging and proteotoxicity of Tau, SOD1, and polyglutamine. The targets will be validated using genetic approaches (RNAi and CRISPR) and proteostasis sensors (Core B). We will establish chemical strategies to reset the PN, restore stress resilience, counteract the age-dependent decline in proteostasis, and prevent proteotoxicity.

Project Summary/Abstract for Administrative Core The objective of the Administrative Core (Core A-Morimoto) is to manage the interactions and progress of the Projects and Cores of this proposal; to ensure they are in constant and convenient communication, and to maximize the benefits of Project and Core collaborations to enhance the scientific output and impact. Due to the highly interdisciplinary nature of our approach and the distance separating the teams, we have placed a priority on communication (virtual and in person exchanges) to ensure that there are productive interactions among all team members. To help achieve these goals, we propose three Specific Aims for the Administrative Core: 1) To maximize communications among the Project and Core Leaders; 2) To organize the annual external review; 3) To strengthen collaborations and maximize interactions among Projects and Cores.

Project Summary / Abstract (Project 2) A marked decline in proteasome activity is observed as humans and other mammals age. This has been observed in many tissues, including the brain and motor neurons. Aging is characterized by compromised proteostasis, and declining proteasome activity may play a significant role in aging-associated deficiencies of proteostasis, given the pivotal role of the proteasome in protein dynamics. By degrading ubiquitin conjugates, the proteasome controls protein stability on a global level. There has historically been little interest in the potential role of the proteasome in determining the overall output of the ubiquitin-proteasome pathway (UPS). Through recent work, however, it is now recognized that the proteasome is on the contrary a focal point of regulation of the UPS. Indeed, the level of proteasome activity controls protein breakdown rates and stress resistance. Remarkably, the proteasome can in general be up-regulated without toxicity. Extensive work has also shown that the proteasome is compromised in many disease states, particularly in aging-associated and neurodegenerative diseases. However, a deeper and more reliable understanding of the relevance of the proteasome to aging and neurodegeneration in humans clearly requires the use of in vivo mammalian model systems. To directly test whether the proteasome becomes limiting in aged animals, three transgenic mouse lines have been designed to allow conditional elevation of proteasome levels; two of these lines have already been generated. Each mouse line will conditionally express the wild-type murine form of a different proteasome subunit?either Rpn6, Rpn11, or ?5. These subunits were chosen because they are expected from existing data to be rate-limiting for proteasome assembly. The multiplicity of strategies to elevate proteasome levels increases the likelihood of success, and if more than one method succeeds it will enhance confidence in subsequent results. For each transgene, a floxed and dox-suppressible construct is integrated into the Rosa26 locus via CRISPR/Cas9. The transgenes should be expressed in a tissue-specific and temporally-controlled manner. Our first objective will be to validate the strategy for elevating proteasome levels in the brain, spinal cord, and in Flp-In?-3T3 cells with transgenes similarly targeted to Rosa26. Excellent biochemical and proteomic methods exist for quantifying alterations in proteasome levels. Remarkably, global proteomics can now quantify the impact of elevated proteasome levels on the control of hundreds of substrate proteins in these settings. We will proceed to assess the effects of elevated proteasome expression on the health and aging of these animals. We will test whether elevated proteasome levels influence autophagy and the proteostasis network (PN) as a whole, and seek to identify age-dependent vulnerabilities in the PN by applying specific stresses to the system. A central focus of the work on transgenic mice will be to employ disease models to assess the effect of elevated proteasome levels on the progression of neurodegenerative diseases, particularly tauopathies, Huntington?s disease, and ALS.