Rockland Luke Wiseman, Ph.D. - US grants

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): Extracellular protein aggregation is inextricably linked to human neurodegenerative diseases such as Alzheimer's disease, Creutzfeldt-Jakob disease and the transthyretin amyloidoses. The importance of protein aggregation in these disorders has led to significant experimental effort focused on characterizing the cellular pathways that regulate extracellular protein homeostasis (or proteostasis). Two primary determinants in defining extracellular proteostasis identified through these efforts are the efficiency of endoplasmic reticulum (ER) quality control pathways and the spectrum and activity of extracellular chaperones. A primary function of the ER is to facilitate the proper folding of proteins for trafficking to downstream environments of the secretory pathway such as the extracellular environment, while preventing the secretion of destabilized, misfolding-prone proteins. Through this so-called quality control mechanism, the ER indirectly influences extracellular proteostasis by reducing the extracellular population of misfolding prone proteins available for concentration- dependent aggregation. Alternatively, extracellular chaperones such as clusterin directly influence extracellular proteostasis by binding misfolding prone proteins, preventing their aggregation. While the combined activity of ER quality control pathways and extracellular chaperones efficiently regulate extracellular proteostasis under normal conditions, imbalances in ER quality control efficiency induced by environmental, genetic or aging- related insults can lead to increased secretion of aggregation-prone proteins that challenge extracellular chaperoning capacity. To confront this challenge, cells activate the unfolded protein response (UPR) - a stress-responsive signaling pathway that translationally and transcriptionally remodels ER quality control pathways. Thus, UPR activation restores ER function and attenuates the secretion of aggregation-prone proteins. Despite the importance of the UPR in regulating proteostasis in the ER and downstream environments of the secretory pathway, no link between UPR activation and extracellular chaperoning capacity has been established. We hypothesize that UPR activation, in response to stress, increases secretion of extracellular chaperones to prevent the aberrant, extracellular aggregation of misfolding prone proteins. Herein, we identify ERdj3 (DNAJB11) as a previously uncharacterized, UPR-regulated extracellular chaperone. We propose to evaluate the capacity for ERdj3 to attenuate pathologic protein aggregation in the extracellular environment, characterize the biological pathways responsible for stress-induced ERdj3 secretion, and identify the subset of secreted proteins that interacts with ERdj3 during normal physiology and in response to stress. Experimental success herein will provide proof-of-principle of a direct, functional role for the UPR in regulating extracellular proteostasis capacit, catalyzing research to both identify other UPR-regulated extracellular proteostasis factors and explore the potential for inducing the UPR-dependent increase in extracellular chaperone capacity to treat aggregation-associated degenerative disorders.

DESCRIPTION (provided by applicant): Systemic amyloid diseases such as the transthyretin (TTR) amyloidoses are a class of devastating disorders caused by the pathologic aggregation and deposition of specific destabilized proteins as amyloid fibrils on tissues distal from the site of protein synthesis. Currently, no non-invasive therapies exist to treat the majority of these diseases, making systemic amyloidoses a large unmet medical need. A primary factor defining the pathologic extracellular protein aggregation central to these disorders is the secretion of destabilized, amyloidogenic proteins from effector tissues such as the liver. The efficient secretion of these proteins increases serum concentrations of amyloidogenic protein available for pathologic, concentration-dependent aggregation, directly impacting disease pathogenesis in patients. Clinical results from liver transplant recipients show that reducing serum concentrations of amyloidogenic proteins can decrease pathologic protein aggregation, attenuate peripheral proteotoxicity and improve prognosis for patients presenting with a variety of distinct systemic amyloidoses. We hypothesize that activating the endogenous Unfolded Protein Response (UPR) signaling pathways that regulate protein secretion from effector tissues is a non-invasive strategy to similarly decrease secretion and reduce extracellular concentrations of amyloidogenic proteins available for pathologic extracellular aggregation. Consistent with this prediction, we show that activating the UPR- associated transcription factor ATF6 reduces secretion of destabilized, amyloidogenic TTR mutants, but does not affect the secretion of wild-type TTR or the endogenous secreted proteome. Here, we employ TTR as a model amyloidogenic protein to show that ATF6 activation has therapeutic potential to reduce pathologic extracellular aggregation and proteotoxicity of amyloidogenic TTR mutants using a novel patient-derived, multi- system induced pluripotent stem cell model of TTR amyloid disease that recapitulates nearly all aspects of TTR amyloid disease pathology observed in patients. Furthermore, we are extending this analysis to show that ATF6 activation similarly reduces the secretion and proteotoxicity of amyloidogenic proteins involved in other systemic amyloid diseases including Light Chain Amyloidosis - an acquired systemic amyloid disease that affects >1 million individuals worldwide. Through these efforts, we will show that the stress-independent activation of UPR-associated signaling pathways such as that regulated by ATF6 is a broadly-applicable therapeutic strategy to reduce the secretion and pathologic extracellular aggregation of amyloidogenic proteins associated with multiple systemic amyloid diseases. These results will further motivate our ongoing high- throughput screening efforts to identify ATF6 activators, as a single small molecule ATF6 activator has the potential to treat multiple systemic amyloidoses (i.e. a one-drug:multiple-disease therapeutic paradigm) dramatically improving the economics of translating selective ATF6 activators into the clinic to ameliorate pathologic extracellular aggregation associated with these diseases.

? DESCRIPTION (provided by applicant): The misfolding and extracellular aggregation of destabilized, amyloidogenic proteins is inextricably linked to degenerative phenotypes in over 30 protein aggregation (i.e., amyloid) diseases including Alzheimer's disease, Creutzfeldt-Jakob disease and the systemic amyloidoses. Significant pharmacologic and genetic evidence confirms a causal relationship between protein aggregation and degeneration of post-mitotic tissues in these diseases. The importance of extracellular protein aggregation in amyloid disease pathology has stimulated significant experimental effort focused on defining the organismal and cellular pathways that regulate protein homeostasis (or proteostasis) in the extracellular environment. One such pathway is the Unfolded Protein Response (UPR) - the stress-responsive signaling pathways responsible for regulating proteostasis within the secretory pathway in response to endoplasmic reticulum (ER) stress. Previous results from our lab and others have shown that the UPR indirectly influences extracellular aggregation of destabilized, amyloidogenic proteins by reducing their secretion from mammalian cells, thus decreasing extracellular protein levels available for concentration-dependent aggregation. Here, we hypothesize that UPR activation also directly regulates extracellular proteostasis through the increased expression and secretion of extracellular chaperones that prevent the proteotoxic aggregation of destabilized, aggregation-prone proteins. We have identified the ER-targeted HSP40 co-chaperone ERdj3 as a UPR regulated, secreted chaperone that promotes extracellular proteostasis in response to ER stress. We show that ERdj3 attenuates the aggregation and proteotoxicity of disease-associated, aggregation-prone secreted proteins including Aß40 and toxic prion protein (TPrP). Additionally, we show that ERdj3 is co-secreted in a complex with destabilized, aggregation-prone proteins under conditions where ER proteostasis pathways are overwhelmed, providing a mechanism to preemptively protect the extracellular environment from proteotoxic extracellular protein aggregation. In this application, we expand on these findings using biophysical, biochemical and cell biological approaches to define the molecular mechanisms by which UPR-dependent ERdj3 secretion protects the extracellular environment against proteotoxic protein conformations. Through these efforts, we will identify specific aspects of UPR-regulated ERdj3 secretion directly involved in preventing the proteotoxic aggregation of destabilized secreted proteins associated with amyloid disease pathology. These results will demonstrate that altered UPR signaling, such as those that occur during normal aging or in response to amyloid-disease associated genetic mutations, can facilitate aging-dependent extracellular protein aggregation involved in amyloid disease pathogenesis. Furthermore, we will identify components of UPR signaling pathways that can be therapeutically targeted to promote extracellular proteostasis and prevent extracellular protein aggregation, revealing a new strategy to attenuate proteotoxicity of secreted proteins involved in the pathology of amyloid diseases.

? DESCRIPTION (provided by applicant): Light chain amyloidosis (AL) is a devastating disease caused by the clonal expansion of a malignant plasma cell that secretes a destabilized, amyloidogenic immunoglobulin light chain (LC). Amyloidogenic LCs undergo misfolding and concentration-dependent aggregation into toxic oligomers and amyloid fibrils that deposit on distal tissues such as the heart. Thus, AL patients suffer from both a plasma cell malignancy and a systemic amyloid disease. Current AL treatments use chemotherapy and autologous stem cell replacement to decrease the clonal plasma cell population, only indirectly affecting AL amyloid pathology. While this approach is efficient for 70% of patients, the remaining 30% of patients are too sick from LC proteotoxicity to tolerate this treatment. In order to treat these patients, new therapies must be developed to treat the LC proteotoxicity in AL pathology. A challenge in developing such strategies is the heterogeneity of AL-associated, amyloidogenic LC sequences. Thus, any strategy to ameliorate AL amyloid pathology must target a fundamental biologic mechanism that mediates toxicity of heterogeneous amyloidogenic LCs, but does not globally compromise organismal immunity or secretion of the endogenous secreted proteome. We hypothesize that the activity of endoplasmic reticulum (ER) proteostasis pathways is a critical determinant in toxic LC aggregation that can be targeted to ameliorate AL amyloid pathology. ER proteostasis pathways can facilitate secretion of destabilized, amyloidogenic proteins, increasing their serum concentrations available for toxic misfolding and aggregation. Thus, modulating the activity of ER proteostasis pathways offers a unique opportunity to reduce serum concentrations of destabilized, amyloidogenic LCs and thus decrease proteotoxic LC aggregation. We show that adapting ER proteostasis pathways through stress-independent activation of select Unfolded Protein Response (UPR)-associated transcription factors reduces the secretion and extracellular aggregation of a destabilized, amyloidogenic LC, without affecting secretion of a non-amyloidogenic LC, IgGs or the global endogenous secreted proteome. Here, we define the contribution of ER proteostasis pathways in toxic LC aggregation by identifying pathways preferentially involved in the secretion, extracellular aggregation and subsequent toxicity of amyloidogenic LCs. Furthermore, we will demonstrate that small molecule ER proteostasis regulators that alter the activity of these ER proteostasis pathways reduce secretion and toxic aggregation of destabilized, amyloidogenic LCs in AL patient-derived plasma cells. Through these efforts, we will show that the activity of ER proteostasis pathways is a fundamental determinant in dictating AL amyloid pathology. Furthermore, we will identify first-in-class small molecule ER proteostasis regulators that target these pathways to attenuate secretion and toxic aggregation of amyloidogenic LCs. Our results will establish ER proteostasis regulation as the first strategy to ameliorate AL amyloid pathology that can then be used in combination with chemotherapeutics to treat the AL patient cohort suffering from severe LC proteotoxicity.

? DESCRIPTION (provided by applicant): Oxidative stress and mitochondria dysfunction are inextricably linked in the onset and pathology of human diseases including neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and Huntington's disease. Currently, the underlying molecular mechanisms that define the relationship between oxidative stress and mitochondria dysfunction in these diseases remain poorly defined. Mitochondria inner membrane (IM) proteases such as YME1L and OMA1 coordinate to regulate many aspects of mitochondrial function including energy metabolism, organellar morphology and apoptotic signaling. Imbalances in the activity of these proteases induced by genetic or environmental factors disrupt mitochondria function and predispose individuals to etiologically diverse human diseases including many neurodegenerative disorders. Despite the importance of these proteases for mitochondria function, how the activity of IM proteases is impacted by pathologic insults are poorly understood. We hypothesize that stress-induced alterations in mitochondria IM proteases directly influence mitochondrial function and dictate cell survival in response to pathologic insults. Consistent with this prediction, we have identified YME1L and OMA1 as stress-sensitive mitochondrial proteases that undergo reciprocal regulation in response to oxidative and pathologic insults. OMA1, but not YME1L, is degraded in response to cellular insults that depolarize the mitochondria membrane through a mechanism involving YME1L. In contrast, YME1L, but not OMA1, is degraded in response to cellular insults that depolarize the mitochondria membrane and induce metabolic crisis by reducing cellular ATP through a mechanism involving activated OMA1. In this proposal, we will define the impact of YME1L or OMA1 degradation on mitochondria functions including regulation of mitochondrial morphology, inner membrane proteostasis maintenance, electron transport chain activity and neuronal sensitivity to oxidative and proteotoxic insults associated with neurodegenerative disease pathology. Through these efforts, we will demonstrate that the differential stress-sensitivity of YME1L and OMA1 distinctly impacts IM proteolytic capacity and alters mitochondria function in response to oxidative insults. Thus, our work will reveal YME1L or OMA1 degradation as a new molecular mechanism involved in defining the relationship between oxidative stress, mitochondria dysfunction and cell death associated with diseases such as the neurodegenerative disorders. Additionally, our work will identify YME1L and OMA1 activity as new therapeutic targets that can be modulated to attenuate pathologic mitochondria dysfunction associated with human disease.

The maintenance of secreted protein homeostasis, or proteostasis, involves balancing protein biosynthesis, translocation across membranes, folding, degradation, etc., which we hypothesize is critical for healthy aging. Since the demands on secretory compartments to maintain proteostasis change with development, aging, and environmental stresses, mammals evolved the Unfolded Protein Response (UPR) stress-responsive signaling pathway, which transcriptionally adjusts secretory proteostasis network capacity to meet demand. Recent human genetic, chemical biologic, and in vivo evidence shows that activating the protective IRE1/XBP1s or ATF6 arms of the UPR has significant promise to ameliorate age-related declines in secretory proteostasis and correct imbalances associated with etiologically-diverse diseases, including systemic amyloid diseases, cardiovascular disorders, diabetes, and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Few compounds exist to achieve arm-selective UPR activation, and those that do suffer from limitations that prevent their translational development. We have leveraged cell-based transcriptional reporter assays miniaturized for high-throughput screening (HTS), along with whole cell transcriptional and proteomic profiling to understand the selectivity of the transcriptional and translational response generated by our screening hits. We have elaborated promising compounds using medicinal chemistry to establish first-in- class small molecule `proteostasis regulators' that selectively activate the protective IRE1/XBP1s or ATF6 signaling arms of the UPR with improved potency and selectivity, and we seek their mechanism of action through multiple approaches. We will assess whether our proteostasis regulators can induce protective, arm- selective UPR activation in young and old animals. We have established collaborations to test the hypothesis that our IRE1/XBP1s and ATF6 activators will be useful for ameliorating pathologic imbalances in secretory proteostasis associated with multiple diseases, including the systemic amyloidoses, degenerative eye diseases, cardiovascular disease, and neurodegenerative disorders. Furthermore, we will show that these compounds pharmacologically ameliorate two pathologic phenotypes associated with Alzheimer's disease in cell culture models: i.e., the pathologic production of A? and A? oligomer-associated neuronal cytotoxicity. We will deliver to the scientific community the first well-characterized small molecules that preferentially activate the IRE1/XBP1s or the ATF6 UPR transcriptional programs with a defined potency and selectivity. These compounds have the potential to be widely employed as therapeutics for a spectrum of age-associated diseases. Importantly, these compounds will be made available to all scientists with disease models wherein pharmacologic IRE1/XBP1s or ATF6 activation has the potential to influence pathogenesis. The availability of these compounds offers the promise to broadly influence multiple aspects of scientific endeavor funded by the NIH, including basic science such as stem cell biology.

PROJECT SUMMARY Systemic amyloid diseases are a class of disorders pathologically associated with the aggregation and deposition of destabilized, amyloidogenic proteins on peripheral target tissues distal from the site of protein synthesis such as the kidney, gut, heart, and peripheral nerves. Over 1 million individuals have been diagnosed with these deadly diseases, although this is likely a significant underestimate of disease prevalence as many undiagnosed renal and cardiac disorders are now being shown to involve systemic amyloid disease pathology. Destabilizing mutations in over 15 different proteins, the majority of which are synthesized in the liver, predispose individuals to systemic amyloid diseases. Since the pathology of these diseases is tightly linked to the site of deposition, systemic amyloid diseases have traditionally been viewed as disorders of the affected peripheral target tissues (e.g. renal disease, cardiomyopathy, and neuropathy). However, over the past 10 years, significant clinical and biological evidence has revealed a critical role for the liver in dictating the extracellular aggregation and deposition of amyloidogenic proteins. These results have challenged the traditional view of systemic amyloid diseases and indicate that these disorders may be best viewed as diseases of the liver. However, the mechanisms by which the liver contributes to systemic amyloid disease pathogenesis remain poorly defined. Here, we hypothesize that imbalances in liver endoplasmic reticulum (ER) protein homeostasis (or proteostasis) promotes the toxic extracellular aggregation of amyloidogenic proteins implicated in systemic amyloid disease pathogenesis. Significant published results from our groups strongly support a critical role for liver ER proteostasis in the toxic aggregation of liver-derived amyloidogenic proteins such as transthyretin (TTR). We showed that disrupting or enhancing ER proteostasis in cells secreting destabilized, amyloidogenic proteins such as TTR directly impacts their extracellular aggregation into toxic oligomers and amyloid fibrils implicated in systemic amyloid disease pathogenesis. Here, we will show that imbalances in liver ER proteostasis promotes the extracellular aggregation and peripheral toxicity of multiple amyloidogenic proteins. These results will demonstrate that diverse genetic, environmental, or aging-related factors that impact ER proteostasis in the liver can promote peripheral toxicity of multiple liver-synthesized amyloidogenic proteins, establishing a molecular framework to explain the clinical importance of the liver in the pathogenesis of these diseases. Furthermore, we will show that enhancing ER proteostasis in the liver through pharmacologic activation of endogenous unfolded protein response (UPR)-associated signaling pathways that regulate ER proteostasis offers a broadly-applicable strategy to ameliorate the secretion, extracellular aggregation, and peripheral toxicity of multiple amyloidogenic proteins. These results will establish pharmacologic UPR activation as a new therapeutic opportunity to treat this largely untreatable class of disease through a one drug:multiple disease therapeutic paradigm.

SUMMARY Mitochondrial dysfunction is a pathologic hallmark in the onset and pathogenesis of nearly all neurodegenerative diseases. One of the primary determinants in dictating mitochondrial function is the activity of inner membrane (IM) proteases including the ATP-dependent AAA+ zinc metalloproteases YME1L and AFG3L2 and the ATP-independent zinc metalloprotease OMA1. These proteases regulate many different aspects of mitochondrial biology and function to protect mitochondria from pathologic insults. However, imbalances in the activity of IM proteases induced by genetic or environmental factors are implicated in the pathogenesis of etiologically-diverse diseases including many neurodegenerative disorders. Despite this, the molecular mechanisms by which IM proteases regulate mitochondrial biology remain poorly understood. Here, we are applying a structure-driven approach to determine the molecular mechanisms by which IM proteases regulate mitochondria in the context of health and disease. We previously solved the first high-resolution structures of the IM AAA+ proteases YME1 and AFG3L2. Our structures showed that these two proteases employ a conserved nucleotide-driven, hand-over-hand mechanism to translocate substrates into a privileged proteolytic chamber for proteolysis. Surprisingly, we also identified unique structural features of YME1 and AFG3L2 that integrate into this conserved translocation mechanism to distinctly influence protease activity and stability. Here, we hypothesize that these unique structural differences endow IM proteases with different mechanistic and biologic functions important for their regulation of mitochondria. To address this, we are using a combination of cryo-electron microscopy and cell biology to determine how structural differences in IM AAA+ proteases influence their mechanochemical cycle and enable proteases to perform distinct biological functions. This will reveal new insights into the molecular mechanisms by which IM AAA+ proteases regulate mitochondria in health and disease. Furthermore, we are extending this study utilizing both functional genomic and structural approaches to establish a structure-function relationship that explains the activation and proteolytic activity of the ATP-independent, stress-activated IM protease OMA1 ? a protease whose dysregulation is implicated in the pathologic mitochondrial dysfunction associated with many human diseases. Through these efforts, we will define how IM proteases utilize distinct structural features to perform the myriad of biological functions required for the proper regulation of mitochondrial proteostasis and function. Furthermore, we will reveal new insights into the pathologic and potentially therapeutic implications of altered mitochondrial IM protease activity in human disease and identify new opportunities to pharmacologically target IM proteases to mitigate mitochondrial dysfunction associated with many neurodegenerative disorders.