Showing papers on "Proteotoxicity published in 2020"

β-amyloid redirects norepinephrine signaling to activate the pathogenic GSK3β/tau cascade

Abstract: The brain noradrenergic system is critical for normal cognition and is affected at early stages in Alzheimer's disease (AD). Here, we reveal a previously unappreciated direct role of norepinephrine signaling in connecting β-amyloid (Aβ) and tau, two key pathological components of AD pathogenesis. Our results show that Aβ oligomers bind to an allosteric site on α2A adrenergic receptor (α2AAR) to redirect norepinephrine-elicited signaling to glycogen synthase kinase 3β (GSK3β) activation and tau hyperphosphorylation. This norepinephrine-dependent mechanism sensitizes pathological GSK3β/tau activation in response to nanomolar accumulations of extracellular Aβ, which is 50- to 100-fold lower than the amount required to activate GSK3β by Aβ alone. The significance of our findings is supported by in vivo evidence in two mouse models, human tissue sample analysis, and longitudinal clinical data. Our study provides translational insights into mechanisms underlying Aβ proteotoxicity, which might have strong implications for the interpretation of Aβ clearance trial results and future drug design and for understanding the selective vulnerability of noradrenergic neurons in AD.

Islet Amyloid Polypeptide: A Partner in Crime With Aβ in the Pathology of Alzheimer's Disease.

Abstract: Diabetes affects hundreds of millions of patients worldwide. Despite the advances in understanding the disease and therapeutic options, it remains a leading cause of death and of comorbidities globally. Islet amyloid polypeptide (IAPP), or amylin, is a hormone produced by pancreatic β-cells. It contributes to the maintenance of glucose physiological levels namely by inhibiting insulin and glucagon secretion as well as controlling adiposity and satiation. IAPP is a highly amyloidogenic polypeptide forming intracellular aggregates and amyloid structures that are associated with β-cell death. Data also suggest the relevance of unprocessed IAPP forms as seeding for amyloid buildup. Besides the known consequences of hyperamylinemia in the pancreas, evidence has also pointed out that IAPP has a pathological role in cognitive function. More specifically, IAPP was shown to impair the blood-brain barrier; it was also seen to interact and co-deposit with amyloid beta peptide (As), and possibly with Tau, within the brain of Alzheimer's disease (AD) patients, thereby contributing to diabetes-associated dementia. In fact, it has been suggested that AD results from a metabolic dysfunction in the brain, leading to its proposed designation as type 3 diabetes. Here, we have first provided a brief perspective on the IAPP amyloidogenic process and its role in diabetes and AD. We have then discussed the potential interventions for modulating IAPP proteotoxicity that can be explored for therapeutics. Finally, we have proposed the concept of a "diabetes brain phenotype" hypothesis in AD, which may help design future IAPP-centered drug developmentstrategies against AD.

Mitochondrial Quality Control and Cellular Proteostasis: Two Sides of the Same Coin.

Abstract: Mitochondrial dysfunction is a hallmark of cardiac pathophysiology. Defects in mitochondrial performance disrupt contractile function, overwhelm myocytes with reactive oxygen species (ROS), and transform these cellular powerhouses into pro-death organelles. Thus, quality control (QC) pathways aimed at identifying and removing damaged mitochondrial proteins, components, or entire mitochondria are crucial processes in post-mitotic cells such as cardiac myocytes. Almost all of the mitochondrial proteins are encoded by the nuclear genome and the trafficking of these nuclear-encoded proteins necessitates significant cross-talk with the cytosolic protein QC machinery to ensure that only functional proteins are delivered to the mitochondria. Within the organelle, mitochondria contain their own protein QC system consisting of chaperones and proteases. This system represents another level of QC to promote mitochondrial protein folding and prevent aggregation. If this system is overwhelmed, a conserved transcriptional response known as the mitochondrial unfolded protein response is activated to increase the expression of proteins involved in restoring mitochondrial proteostasis. If the mitochondrion is beyond repair, the entire organelle must be removed before it becomes cytotoxic and causes cellular damage. Recent evidence has also uncovered mitochondria as participants in cytosolic protein QC where misfolded cytosolic proteins can be imported and degraded inside mitochondria. However, this process also places increased pressure on mitochondrial QC pathways to ensure that the imported proteins do not cause mitochondrial dysfunction. This review is focused on discussing the pathways involved in regulating mitochondrial QC and their relationship to cellular proteostasis and mitochondrial health in the heart.

Intracellular amyloid toxicity induces oxytosis/ferroptosis regulated cell death.

Abstract: Amyloid beta (Aβ) accumulates within neurons in the brains of early stage Alzheimer’s disease (AD) patients. However, the mechanism underlying its toxicity remains unclear. Here, a triple omics approach was used to integrate transcriptomic, proteomic, and metabolomic data collected from a nerve cell model of the toxic intracellular aggregation of Aβ. It was found that intracellular Aβ induces profound changes in the omics landscape of nerve cells that are associated with a pro-inflammatory, metabolic reprogramming that predisposes cells to die via the oxytosis/ferroptosis regulated cell death pathway. Notably, the degenerative process included substantial alterations in glucose metabolism and mitochondrial bioenergetics. Our findings have implications for the understanding of the basic biology of proteotoxicity, aging, and AD as well as for the development of future therapeutic interventions designed to target the oxytosis/ferroptosis regulated cell death pathway in the AD brain.

Widespread remodeling of proteome solubility in response to different protein homeostasis stresses.

Abstract: The accumulation of protein deposits in neurodegenerative diseases has been hypothesized to depend on a metastable subproteome vulnerable to aggregation. To investigate this phenomenon and the mechanisms that regulate it, we measured the solubility of the proteome in the mouse Neuro2a cell line under six different protein homeostasis stresses: 1) Huntington’s disease proteotoxicity, 2) Hsp70, 3) Hsp90, 4) proteasome, 5) endoplasmic reticulum (ER)-mediated folding inhibition, and 6) oxidative stress. Overall, we found that about one-fifth of the proteome changed solubility with almost all of the increases in insolubility were counteracted by increases in solubility of other proteins. Each stress directed a highly specific pattern of change, which reflected the remodeling of protein complexes involved in adaptation to perturbation, most notably, stress granule (SG) proteins, which responded differently to different stresses. These results indicate that the protein homeostasis system is organized in a modular manner and aggregation patterns were not correlated with protein folding stability (ΔG). Instead, distinct cellular mechanisms regulate assembly patterns of multiple classes of protein complexes under different stress conditions.

Extracellular proteostasis prevents aggregation during pathogenic attack

Abstract: In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1–5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6–9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity. A systematic analysis of the proteostasis network of secreted proteins in Caenorhabditis elegans identifies numerous regulators of protein homeostasis outside the cell, and highlights the contribution of extracellular proteostasis to host defence.

Neuronal specific and non-specific responses to cadmium possibly involved in neurodegeneration: A toxicogenomics study in a human neuronal cell model

Abstract: Epidemiological data have linked cadmium exposure to neurotoxicity and to neurodegenerative diseases (e.g., Alzheimer's and Parkinson's disease), and to increased risk of developing ALS. Even though the brain is not a primary target organ, this metal can bypass the blood brain barrier, thus exerting its toxic effects. The coordination chemistry of cadmium is of strong biological relevance, as it resembles to zinc(II) and calcium(II), two ions crucial for neuronal signaling. A toxicogenomics approach applied to a neuronal human model (SH-SY5Y cells) exposed to cadmium (10 and 20 μM) allowed the identification of early deregulated genes and altered processes, and the discrimination between neuronal-specific and unspecific responses as possible triggers of neurodegeneration. Cadmium confirmed its recognized carcinogenicity even on neuronal cells by activating the p53 signaling pathway and genes involved in tumor initiation and cancer cell proliferation, and by down-regulating genes coding for tumor suppressors and for DNA repair enzymes. Two cadmium-induced stress responses were observed: the activation of different members of the heat shock family, as a mechanism to restore protein folding in response to proteotoxicity, and the activation of metallothioneins (MTs), involved in zinc and copper homeostasis, protection against metal toxicity and oxidative damage. Perturbed function of essential metals is suggested by the mineral absorption pathway, with MTs, HMOX1, ZnT-1, and Ferritin genes highly up-regulated. Cadmium interferes also with Ca2+ regulation as S100A2 is one of the top up-regulated genes, coding for a highly specialized family of regulatory Ca2+-binding proteins. Other neuronal-related functions altered in SH-SY5Y cells by cadmium are microtubules dynamics, microtubules motor-based proteins and neuroprotection by down-regulation of NEK3, KIF15, and GREM2 genes, respectively.

Proteotoxicity and Neurodegenerative Diseases.

Abstract: Neurodegenerative diseases are a major burden for our society, affecting millions of people worldwide. A main goal of past and current research is to enhance our understanding of the mechanisms underlying proteotoxicity, a common theme among these incurable and debilitating conditions. Cell proteome alteration is considered to be one of the main driving forces that triggers neurodegeneration, and unraveling the biological complexity behind the affected molecular pathways constitutes a daunting challenge. This review summarizes the current state on key processes that lead to cellular proteotoxicity in Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis, providing a comprehensive landscape of recent literature. A foundational understanding of how proteotoxicity affects disease etiology and progression may provide essential insight towards potential targets amenable of therapeutic intervention.

MEF2 impairment underlies skeletal muscle atrophy in polyglutamine disease

Abstract: Polyglutamine (polyQ) tract expansion leads to proteotoxic misfolding and drives a family of nine diseases. We study spinal and bulbar muscular atrophy (SBMA), a progressive degenerative disorder of the neuromuscular system caused by the polyQ androgen receptor (AR). Using a knock-in mouse model of SBMA, AR113Q mice, we show that E3 ubiquitin ligases which are a hallmark of the canonical muscle atrophy machinery are not induced in AR113Q muscle. Similarly, we find no evidence to suggest dysfunction of signaling pathways that trigger muscle hypertrophy or impairment of the muscle stem cell niche. Instead, we find that skeletal muscle atrophy is characterized by diminished function of the transcriptional regulator Myocyte Enhancer Factor 2 (MEF2), a regulator of myofiber homeostasis. Decreased expression of MEF2 target genes is age- and glutamine tract length-dependent, occurs due to polyQ AR proteotoxicity, and is associated with sequestration of MEF2 into intranuclear inclusions in muscle. Skeletal muscle from R6/2 mice, a model of Huntington disease which develops progressive atrophy, also sequesters MEF2 into inclusions and displays age-dependent loss of MEF2 target genes. Similarly, SBMA patient muscle shows loss of MEF2 target gene expression, and restoring MEF2 activity in AR113Q muscle rescues fiber size and MEF2-regulated gene expression. This work establishes MEF2 impairment as a novel mechanism of skeletal muscle atrophy downstream of toxic polyglutamine proteins and as a therapeutic target for muscle atrophy in these disorders.

Challenging Proteostasis: Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease.

Abstract: Protein homeostasis (Proteostasis) is essential for correct and efficient protein function within the living cell. Among the critical components of the Proteostasis Network (PN) are molecular chaperones that serve widely in protein biogenesis under physiological conditions, and prevent protein misfolding and aggregation enhanced by conditions of cellular stress. For Alzheimer's, Parkinson's, Huntington's diseases and ALS, multiple classes of molecular chaperones interact with the highly aggregation-prone proteins amyloid-β, tau, α-synuclein, huntingtin and SOD1 to influence the course of proteotoxicity associated with these neurodegenerative diseases. Accordingly, overexpression of molecular chaperones and induction of the heat shock response have been shown to be protective in a wide range of animal models of these diseases. In contrast, for cancer cells the upregulation of chaperones has the undesirable effect of promoting cellular survival and tumor growth by stabilizing mutant oncoproteins. In both situations, physiological levels of molecular chaperones eventually become functionally compromised by the persistence of misfolded substrates, leading to a decline in global protein homeostasis and the dysregulation of diverse cellular pathways. The phenomenon of chaperone competition may underlie the broad pathology observed in aging and neurodegenerative diseases, and restoration of physiological protein homeostasis may be a suitable therapeutic avenue for neurodegeneration as well as for cancer.

CHIP phosphorylation by protein kinase G enhances protein quality control and attenuates cardiac ischemic injury.

Abstract: Proteotoxicity from insufficient clearance of misfolded/damaged proteins underlies many diseases. Carboxyl terminus of Hsc70-interacting protein (CHIP) is an important regulator of proteostasis in many cells, having E3-ligase and chaperone functions and often directing damaged proteins towards proteasome recycling. While enhancing CHIP functionality has broad therapeutic potential, prior efforts have all relied on genetic upregulation. Here we report that CHIP-mediated protein turnover is markedly post-translationally enhanced by direct protein kinase G (PKG) phosphorylation at S20 (mouse, S19 human). This increases CHIP binding affinity to Hsc70, CHIP protein half-life, and consequent clearance of stress-induced ubiquitinated-insoluble proteins. PKG-mediated CHIP-pS20 or expressing CHIP-S20E (phosphomimetic) reduces ischemic proteo- and cytotoxicity, whereas a phospho-silenced CHIP-S20A amplifies both. In vivo, depressing PKG activity lowers CHIP-S20 phosphorylation and protein, exacerbating proteotoxicity and heart dysfunction after ischemic injury. CHIP-S20E knock-in mice better clear ubiquitinated proteins and are cardio-protected. PKG activation provides post-translational enhancement of protein quality control via CHIP.

Organismal Protein Homeostasis Mechanisms.

Abstract: Sustaining a healthy proteome is a lifelong challenge for each individual cell of an organism. However, protein homeostasis or proteostasis is constantly jeopardized since damaged proteins accumulate under proteotoxic stress that originates from ever-changing metabolic, environmental, and pathological conditions. Proteostasis is achieved via a conserved network of quality control pathways that orchestrate the biogenesis of correctly folded proteins, prevent proteins from misfolding, and remove potentially harmful proteins by selective degradation. Nevertheless, the proteostasis network has a limited capacity and its collapse deteriorates cellular functionality and organismal viability, causing metabolic, oncological, or neurodegenerative disorders. While cell-autonomous quality control mechanisms have been described intensely, recent work on Caenorhabditis elegans has demonstrated the systemic coordination of proteostasis between distinct tissues of an organism. These findings indicate the existence of intricately balanced proteostasis networks important for integration and maintenance of the organismal proteome, opening a new door to define novel therapeutic targets for protein aggregation diseases. Here, we provide an overview of individual protein quality control pathways and the systemic coordination between central proteostatic nodes. We further provide insights into the dynamic regulation of cellular and organismal proteostasis mechanisms that integrate environmental and metabolic changes. The use of C. elegans as a model has pioneered our understanding of conserved quality control mechanisms important to safeguard the organismal proteome in health and disease.

The Multifaceted Role of HSF1 in Tumorigenesis.

Abstract: Heat Shock Factor 1 (HSF1), the master transcriptional regulator of the heat shock response (HSR), was first cloned more than 30 years ago. Most early research interrogating the role that HSF1 plays in biology focused on its cytoprotective functions, as a factor that promotes the survival of organisms by protecting against the proteotoxicity associated with neurodegeneration and other pathological conditions. However, recent studies have revealed a deleterious role of HSF1, as a factor that is co-opted by cancer cells to promote their own survival to the detriment of the organism. In cancer, HSF1 operates in a multifaceted manner to promote oncogenic transformation, proliferation, metastatic dissemination, and anti-cancer drug resistance. Here we review our current understanding of HSF1 activation and function in malignant progression and discuss the potential for HSF1 inhibition as a novel anticancer strategy. Collectively, this ever-growing body of work points to a prominent role of HSF1 in nearly every aspect of carcinogenesis.

Frondoside A Attenuates Amyloid-β Proteotoxicity in Transgenic Caenorhabditis elegans by Suppressing Its Formation.

Abstract: Oligomeric assembly of Amyloid-β (Aβ) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of Aβ oligomers could halt the disease progression. In this study, by using transgenic Caenorhabditis elegans model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber Cucumaria frondosa saponin with anti-cancer activity, on Aβ aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 µM significantly delayed the worm paralysis caused by Aβ aggregation as compared with control group. In addition, the number of Aβ plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of Aβ species in the transgenic C. elegans were significantly reduced upon treatment with frondoside A, whereas the level of Aβ monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of Aβ. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express Aβ. Taken together, these data suggested that low dose of frondoside A could protect against Aβ-induced toxicity by primarily suppressing the formation of Aβ oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-Aβ aggregation should be studied and elucidated in the future.

Cross organelle stress response disruption promotes gentamicin-induced proteotoxicity

Abstract: Gentamicin is a nephrotoxic antibiotic that causes acute kidney injury (AKI) primarily by targeting the proximal tubule epithelial cell. The development of an effective therapy for gentamicin-induced renal cell injury is limited by incomplete mechanistic insight. To address this challenge, we propose that RNAi signal pathway screening could identify a unifying mechanism of gentamicin-induced cell injury and suggest a therapeutic strategy to ameliorate it. Computational analysis of RNAi signal screens in gentamicin-exposed human proximal tubule cells suggested the cross-organelle stress response (CORE), the unfolded protein response (UPR), and cell chaperones as key targets of gentamicin-induced injury. To test this hypothesis, we assessed the effect of gentamicin on the CORE, UPR, and cell chaperone function, and tested the therapeutic efficacy of enhancing cell chaperone content. Early gentamicin exposure disrupted the CORE, evidenced by a rise in the ATP:ADP ratio, mitochondrial-specific H2O2 accumulation, Drp-1-mediated mitochondrial fragmentation, and endoplasmic reticulum–mitochondrial dissociation. CORE disruption preceded measurable increases in whole-cell oxidative stress, misfolded protein content, transcriptional UPR activation, and its untoward downstream effects: CHOP expression, PARP cleavage, and cell death. Geranylgeranylacetone, a therapeutic that increases cell chaperone content, prevented mitochondrial H2O2 accumulation, preserved the CORE, reduced the burden of misfolded proteins and CHOP expression, and significantly improved survival in gentamicin-exposed cells. We identify CORE disruption as an early and remediable cause of gentamicin proteotoxicity that precedes downstream UPR activation and cell death. Preserving the CORE significantly improves renal cell survival likely by reducing organelle-specific proteotoxicity during gentamicin exposure.

Reductive stress promotes protein aggregation and impairs neurogenesis.

Abstract: Redox homeostasis regulates key cellular signaling in both physiology and pathology. While perturbations result in shifting the redox homeostasis towards oxidative stress are well documented, the influence of reductive stress (RS) in neurodegenerative diseases and its mechanisms are unknown. Here, we postulate that a redox shift towards the reductive arm (through the activation of Nrf2 signaling) will damage neurons and impair neurogenesis. In proliferating and differentiating neuroblastoma (Neuro 2a/N2a) cells, sulforaphane-mediated Nrf2 activation resulted in increased transcription/translation of antioxidants and glutathione (GSH) production along with significantly declined ROS in a dose-dependent manner leading to a reductive-redox state (i.e. RS). Interestingly, this resulted in endoplasmic reticulum (ER) stress leading to subsequent protein aggregation/proteotoxicity in neuroblastoma cells. Under RS, we also observed elevated Tau/α-synuclein and their co-localization with other protein aggregates in these cells. Surprisingly, we noticed that acute RS impaired neurogenesis as evidenced from reduced neurite outgrowth/length. Furthermore, maintaining the cells in a sustained RS condition (for five consecutive generations) dramatically reduced their differentiation and prevented the formation of axons (p < 0.05). This impairment in RS mediated neurogenesis occurs through the alteration of Tau dynamics i.e. RS activates the pathogenic GSK3β/Tau cascade thereby promoting the phosphorylation of Tau leading to proteotoxicity. Of note, intermittent withdrawal of sulforaphane from these cells suppressed the proteotoxic insult and re-activated the differentiation process. Overall, this results suggest that either acute or chronic RS could hamper neurogenesis through GSK3β/TAU signaling and proteotoxicity. Therefore, investigations identifying novel redox mechanisms impacting proteostasis are crucial to preserve neuronal health.

Cellular Protein Quality Control in Diabetic Cardiomyopathy: From Bench to Bedside.

Abstract: Heart failure is a serious comorbidity and the most common cause of mortality in diabetes patients. Diabetic cardiomyopathy (DCM) features impaired cellular structure and function, culminating in heart failure; however, there is a dearth of specific clinical therapy for treating DCM. Protein homeostasis is pivotal for the maintenance of cellular viability under physiological and pathological conditions, particularly in the irreplaceable cardiomyocytes; therefore, it is tightly regulated by a protein quality control (PQC) system. Three evolutionarily conserved molecular processes, the unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and autophagy, enhance protein turnover and preserve protein homeostasis by suppressing protein translation, degrading misfolded or unfolded proteins in cytosol or organelles, disposing of damaged and toxic proteins, recycling essential amino acids, and eliminating insoluble protein aggregates. In response to increased cellular protein demand under pathological insults, including the diabetic condition, a coordinated PQC system retains cardiac protein homeostasis and heart performance, on the contrary, inappropriate PQC function exaggerates cardiac proteotoxicity with subsequent heart dysfunction. Further investigation of the PQC mechanisms in diabetes propels a more comprehensive understanding of the molecular pathogenesis of DCM and opens new prospective treatment strategies for heart disease and heart failure in diabetes patients. In this review, the function and regulation of cardiac PQC machinery in diabetes mellitus, and the therapeutic potential for the diabetic heart are discussed.

USP7 regulates ALS-associated proteotoxicity and quality control through the NEDD4L-SMAD pathway.

Abstract: An imbalance in cellular homeostasis occurring as a result of protein misfolding and aggregation contributes to the pathogeneses of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here, we report the identification of a ubiquitin-specific protease, USP7, as a regulatory switch in a protein quality-control system that defends against proteotoxicity. A genome-wide screen in a Caenorhabditis elegans model of SOD1-linked ALS identified the USP7 ortholog as a suppressor of proteotoxicity in the nervous system. The actions of USP7 orthologs on misfolded proteins were found to be conserved in Drosophila and mammalian cells. USP7 acts on protein quality control through the SMAD2 transcription modulator of the transforming growth factor β pathway, which activates autophagy and enhances the clearance of misfolded proteins. USP7 deubiquitinates the E3 ubiquitin ligase NEDD4L, which mediates the degradation of SMAD2. Inhibition of USP7 protected against proteotoxicity in mammalian neurons, and SMAD2 was found to be dysregulated in the nervous systems of ALS patients. These findings reveal a regulatory pathway of protein quality control that is implicated in the proteotoxicity-associated neurodegenerative diseases.

Rapid Alpha-Synuclein Toxicity in a Neural Cell Model and Its Rescue by a Stearoyl-CoA Desaturase Inhibitor.

Abstract: Genetic and biochemical evidence attributes neuronal loss in Parkinson's disease (PD) and related brain diseases to dyshomeostasis of the 14 kDa protein α-synuclein (αS). There is no consensus on how αS exerts toxicity. Explanations range from disturbed vesicle biology to proteotoxicity caused by fibrillar aggregates. To probe these mechanisms further, robust cellular toxicity models are needed, but their availability is limited. We previously reported that a shift from dynamic multimers to monomers is an early event in αS dyshomeostasis, as caused by familial PD (fPD)-linked mutants such as E46K. Excess monomers accumulate in round, lipid-rich inclusions. Engineered αS '3K' (E35K+E46K+E61K) amplifies E46K, causing a PD-like, L-DOPA-responsive motor phenotype in transgenic mice. Here, we present a cellular model of αS neurotoxicity after transducing human neuroblastoma cells to express yellow fluorescent protein (YFP)-tagged αS 3K in a doxycycline-dependent manner. αS-3K::YFP induction causes pronounced growth defects that accord with cell death. We tested candidate compounds for their ability to restore growth, and stearoyl-CoA desaturase (SCD) inhibitors emerged as a molecule class with growth-restoring capacity, but the therapeutic window varied among compounds. The SCD inhibitor MF-438 fully restored growth while exerting no apparent cytotoxicity. Our αS bioassay will be useful for elucidating compound mechanisms, for pharmacokinetic studies, and for compound/genetic screens.

Therapeutic genetic variation revealed in diverse Hsp104 homologs.

Abstract: The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.