Showing papers on "Proteotoxicity published in 2015"

Targeting protein aggregation for the treatment of degenerative diseases.

Abstract: The aggregation of specific proteins is hypothesized to underlie several degenerative diseases, which are collectively known as amyloid disorders. However, the mechanistic connection between the process of protein aggregation and tissue degeneration is not yet fully understood. Here, we review current and emerging strategies to ameliorate aggregation-associated degenerative disorders, with a focus on disease-modifying strategies that prevent the formation of and/or eliminate protein aggregates. Persuasive pharmacological and genetic evidence now supports protein aggregation as the cause of postmitotic tissue dysfunction or loss. However, a more detailed understanding of the factors that trigger and sustain aggregate formation and of the structure-activity relationships underlying proteotoxicity is needed to develop future disease-modifying therapies.

The unfolded protein response and cellular senescence. A review in the theme: cellular mechanisms of endoplasmic reticulum stress signaling in health and disease.

Abstract: The endoplasmic reticulum (ER) is a multifunctional organelle critical for the proper folding and assembly of secreted and transmembrane proteins. Perturbations of ER functions cause ER stress, which activates a coordinated system of transcriptional and translational controls called the unfolded protein response (UPR), to cope with accumulation of misfolded proteins and proteotoxicity. It results in ER homeostasis restoration or in cell death. Senescence is a complex cell phenotype induced by several stresses such as telomere attrition, DNA damage, oxidative stress, and activation of some oncogenes. It is mainly characterized by a cell enlargement, a permanent cell-cycle arrest, and the production of a secretome enriched in proinflammatory cytokines and components of the extracellular matrix. Senescent cells accumulate with age in tissues and are suspected to play a role in age-associated diseases. Since senescence is a stress response, the question arises of whether an ER stress could occur concomitantly with senescence and participate in the onset or maintenance of the senescent features. Here, we described the interconnections between the UPR signaling and the different aspects of the cellular senescence programs and discuss the implication of UPR modulations in this context.

Regulated degradation of Chk1 by chaperone-mediated autophagy in response to DNA damage

Abstract: Chaperone-mediated autophagy (CMA) is activated in response to cellular stressors to prevent cellular proteotoxicity through selective degradation of altered proteins in lysosomes. Reduced CMA activity contributes to the decrease in proteome quality in disease and ageing. Here, we report that CMA is also upregulated in response to genotoxic insults and that declined CMA functionality leads to reduced cell survival and genomic instability. This role of CMA in genome quality control is exerted through regulated degradation of activated checkpoint kinase 1 (Chk1) by this pathway after the genotoxic insult. Nuclear accumulation of Chk1 in CMA-deficient cells compromises cell cycle progression and prolongs the time that DNA damage persists in these cells. Furthermore, blockage of CMA leads to hyperphosphorylation and destabilization of the MRN (Mre11-Rad50-Nbs1) complex, which participates in early steps of particular DNA repair pathways. We propose that CMA contributes to maintain genome stability by assuring nuclear proteostasis.

KEAP1-NRF2 signalling and autophagy in protection against oxidative and reductive proteotoxicity.

Abstract: Maintaining cellular redox status to allow cell signalling to occur requires modulation of both the controlled production of oxidants and the thiol-reducing networks to allow specific regulatory post-translational modification of protein thiols. The oxidative stress hypothesis captured the concept that overproduction of oxidants can be proteotoxic, but failed to predict the recent finding that hyperactivation of the KEAP1–NRF2 system also leads to proteotoxicity. Furthermore, sustained activation of thiol redox networks by KEAP1–NRF2 induces a reductive stress, by decreasing the lifetime of necessary oxidative post-translational modifications required for normal metabolism or cell signalling. In this context, it is now becoming clear why antioxidants or hyperactivation of antioxidant pathways with electrophilic therapeutics can be deleterious. Furthermore, it suggests that the autophagy–lysosomal pathway is particularly important in protecting the cell against redox-stress-induced proteotoxicity, since it can degrade redox-damaged proteins without causing aberrant changes to the redox network needed for metabolism or signalling. In this context, it is important to understand: (i) how NRF2-mediated redox signalling, or (ii) the autophagy-mediated antioxidant/reductant pathways sense cellular damage in the context of cellular pathogenesis. Recent studies indicate that the modification of protein thiols plays an important role in the regulation of both the KEAP1–NRF2 and autophagy pathways. In the present review, we discuss evidence demonstrating that the KEAP1–NRF2 pathway and autophagy act in concert to combat the deleterious effects of proteotoxicity. These findings are discussed with a special emphasis on their impact on cardiovascular disease and neurodegeneration.

20S proteasome activation promotes life span extension and resistance to proteotoxicity in Caenorhabditis elegans

Abstract: Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to str...

Loss of hepatic chaperone-mediated autophagy accelerates proteostasis failure in aging

Abstract: Chaperone-mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver-specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.

Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

Abstract: The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis.

Proteotoxicity and Cardiac Dysfunction

Abstract: Baseline physiological function of the mammalian heart is under the constant threat of environmental or intrinsic pathological insults. Cardiomyocyte proteins are thus subject to unremitting pressure to function optimally, and this depends on them assuming and maintaining proper conformation. This review explores the multiple defenses a cell may use for its proteins to assume and maintain correct protein folding and conformation. There are multiple quality control mechanisms to ensure that nascent polypeptides are properly folded and mature proteins maintain their functional conformation. When proteins do misfold, either in the face of normal or pathological stimuli or because of intrinsic mutations or post-translational modifications, they must either be refolded correctly or recycled. In the absence of these corrective processes, they may become toxic to the cell. Herein, we explore some of the underlying mechanisms that lead to proteotoxicity. The continued presence and chronic accumulation of misfolded or unfolded proteins can be disastrous in cardiomyocytes because these misfolded proteins can lead to aggregation or the formation of soluble peptides that are proteotoxic. This in turn leads to compromised protein quality control and precipitating a downward spiral of the cell’s ability to maintain protein homeostasis. Some underlying mechanisms are discussed and the therapeutic potential of interfering with proteotoxicity in the heart is explored.

Specification of Hsp70 Function by Type I and Type II Hsp40

Abstract: Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing nonnative proteins to facilitate protein folding, refolding and degradation. Hsp40 family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. Type I and Type II Hsp40s direct Hsp70 to preform multiple functions in protein homeostasis. This review describes the mechanisms by which Type I and Type II sub-types of Hsp40 bind and deliver substrates to Hsp70.

Proteasome Dysfunction Associated to Oxidative Stress and Proteotoxicity in Adipocytes Compromises Insulin Sensitivity in Human Obesity

Abstract: Aims: Obesity is characterized by a low-grade systemic inflammatory state and adipose tissue (AT) dysfunction, which predispose individuals to the development of insulin resistance (IR) and metabolic disease. However, a subset of obese individuals, referred to as metabolically healthy obese (MHO) individuals, are protected from obesity-associated metabolic abnormalities. Here, we aim at identifying molecular factors and pathways in adipocytes that are responsible for the progression from the insulin-sensitive to the insulin-resistant, metabolically unhealthy obese (MUHO) phenotype. Results: Proteomic analysis of paired samples of adipocytes from subcutaneous (SC) and omental (OM) human AT revealed that both types of cells are altered in the MUHO state. Specifically, the glutathione redox cycle and other antioxidant defense systems as well as the protein-folding machinery were dysregulated and endoplasmic reticulum stress was increased in adipocytes from IR subjects. Moreover, proteasome activity ...

Disrupting SUMOylation enhances transcriptional function and ameliorates polyglutamine androgen receptor–mediated disease

Abstract: Expansion of the polyglutamine (polyQ) tract within the androgen receptor (AR) causes neuromuscular degeneration in individuals with spinobulbar muscular atrophy (SBMA). PolyQ AR has diminished transcriptional function and exhibits ligand-dependent proteotoxicity, features that have both been implicated in SBMA; however, the extent to which altered AR transcriptional function contributes to pathogenesis remains controversial. Here, we sought to dissociate effects of diminished AR function from polyQ-mediated proteotoxicity by enhancing the transcriptional activity of polyQ AR. To accomplish this, we bypassed the inhibitory effect of AR SUMOylation (where SUMO indicates small ubiquitin-like modifier) by mutating conserved lysines in the polyQ AR that are sites of SUMOylation. We determined that replacement of these residues by arginine enhances polyQ AR activity as a hormone-dependent transcriptional regulator. In a murine model, disruption of polyQ AR SUMOylation rescued exercise endurance and type I muscle fiber atrophy; it also prolonged survival. These changes occurred without overt alterations in polyQ AR expression or aggregation, revealing the favorable trophic support exerted by the ligand-activated receptor. Our findings demonstrate beneficial effects of enhancing the transcriptional function of the ligand-activated polyQ AR and indicate that the SUMOylation pathway may be a potential target for therapeutic intervention in SBMA.

Silymarin Extends Lifespan and Reduces Proteotoxicity in C. elegans Alzheimer’s Model

Abstract: Aging is a process of progressive decline in physiological functions resulting in increased vulnerability to diseases and death. Aging results in increased rates of age related disorders like neurodegenerative diseases, cardiovascular diseases, diabetes, cancer, arthritis etc. Modulation of insulin signaling, protein aggregation, stress, free radical damage and inflammation are the major causes for deleterious changes resulting in aging. Many studies are being undertaken to find novel compounds which can improve a typical human life span and aid in healthy aging. We investigated the potential of one such compound silymarin for its anti-aging effect. Silymarin is a flavanone derivative extracted from the seeds of the milk thistle Silybum marianum. It is widely used for the treatment of liver diseases in clinical practice. We tested the anti-aging efficacy of silymarin using the Caenorhabditis elegans model system. Our results demonstrate that C. elegans treated with 25μM and 50μM silymarin concentration resulted in an increase in mean lifespan by 10.1% and 24.8% respectively compared to untreated control. Besides increased lifespan, silymarin treated aged animals showed better locomotion rate, higher response to stimuli and improved tolerance to stress compared to untreated control. We also checked the potential of silymarin to slow the progression of neurodegenerative disorder like Alzheimer's disease (AD) by using CL4176 C. elegans model for AD. C. elegans CL4176 transgenic animal induces expression of amyloid beta-protein (Aβ1-42) in muscle tissues when subjected to temperature of 23°C and above resulting in worm paralysis. CL4176 animals treated with silymarin showed delayed paralysis via enhancing resistance to oxidative stress. These results suggested that silymarin is a potential hormetin for preventing aging and age-related diseases.

HTT/Huntingtin in selective autophagy and Huntington disease: A foe or a friend within?

Abstract: Huntington disease (HD) is caused by a unique mutation, an abnormal expansion of a polyglutamine (polyQ) tract in the HTT (huntingtin) protein. It has long been hypothesized that in addition to gained toxicity from the expanded polyQ, loss of HTT's normal cellular functions also contributes to HD pathogenesis. Consistently, wild-type HTT has a well-documented neuronal protective activity and is essential for postnatal neuronal survival. However, despite its relatively simple genetic cause, the etiology of HD remains unclear, due in part to the poor understanding of the physiological cellular functions of HTT. The perplexing HTT is a large ∼3,144 amino acid-long protein with ubiquitous localization, but it lacks any known functional domain that could provide clues about its cellular activities. The phenotypes of HTT knockout (KO) mice are equally puzzling: homozygote HTT KO mice die by day 7.5, but this early embryonic lethality is due to a critical role of HTT in extraembryonic membranes, not in the embryo per se, as it can be rescued if wild-type HTT is provided in extraembryonic tissues. HTT has a vast number of reported interacting partners that have been used to infer some of the growing list of cellular pathways that HTT could participate in. However, it is still uncertain how HTT achieves its well-documented neuroprotective role. Using both Drosophila and mammalian experimental models, we recently demonstrated that HTT has an essential function in selective autophagy where it serves as a scaffold by modulating the activities of the cargo receptor SQSTM1/p62 and the autophagy initiation kinase ULK1. We previously created a null deletion mutant (htt-ko) of the single HTT homolog (htt) in Drosophila and showed that in contrast to mouse, htt-ko flies, which develop ex utero, are fully viable with only mild aging-related defects. Unexpectedly, ectopic expression of a truncated form of the mammalian MAPT induced severe defects in htt-ko flies, suggesting that htt protects against pathogenic MAPT toxicity. Further, in genetic screens using this MAPT-induced phenotype as a functional readout, we detected dosage-sensitive genetic interaction between htt-ko and several components of the autophagy pathway, including atg8a (MAP1LC3A homolog), atg1 (ULK1 homolog) and ref(2)P (SQSTM1/p62 homolog), thus unveiling a functional link between HTT and autophagy. HTT is not a foreigner to the autophagy field, although the main emphasis has been on the toxic effect of mutant HTT on this cellular clearance mechanism. In fact, our earlier work in HD demonstrated that reduced autophagy in the disease context, also reported by many other groups, originated from diminished ability to “trap” cytosolic cargo. We attributed the “empty autophagosomes” phenotype in HD cells to defective cargo recognition due to the presence of mutant polyQ-HTT in the inner part of the closing autophagosomes. However, our genetic studies ablating the wild-type HTT made us reconsider this proposed gain-of-toxicity function. In this recent study, we have found that in both htt-ko flies and HTT-depleted mammalian cells, autophagic abnormalities are present but surprisingly, starvation-induced autophagy is largely normal. However, loss of HTT compromised autophagic induction when challenged by several other stresses, including proteotoxicity, lipotoxicity, and mitochondria damage. In all these instances, mammalian cells depleted of HTT also showed an “empty autophagosomes” defect, suggesting that wild-type HTT is required for efficient cargo recognition by the autophagosome. We found that HTT and SQSTM1/p62, as well as their Drosophila counterparts Htt and Ref(2)P, physically interact and that proteotoxic stress promotes this interaction. Depletion of HTT reduces the association of SQSTM1 with MAP1LC3A and also compromises the binding of SQSTM1 with proteins with lysine-63-linked ubiquitin (K63-Ub) chains, which are preferential substrates of autophagy. However, loss of HTT does not affect self-polymerization of SQSTM1 or its association with K48-Ub-modified proteins mainly cleared by the proteasome. Collectively, these findings support the idea that HTT facilitates cargo recognition by modulating the assembly of the cargo receptors and autophagy proteins. We also found that the dependence on HTT for the initiation of stress-induced selective autophagy is related to its physical interaction with ULK1, whose kinase activity is essential for autophagy initiation. HTT depletion does not affect starvation-induced activation of ULK1 kinase, but compromises ULK1 activation upon proteotoxicity challenge, revealing a contribution of HTT to differentially regulating autophagosome biogenesis under basal or stress conditions. We found that the regulation of HTT on ULK1 is at the level of its interaction with the MTORC1 complex, another main regulator of ULK1 that binds to and inactivates ULK1 under nutrient-rich conditions. We showed that (1) ULK1 exists in 2 mutually exclusive complexes, ULK1-HTT and ULK1-MTORC1; (2) HTT does not affect the kinase activity of MTORC1; (3) HTT competes with MTORC1 for binding with ULK1; (4) stresses that induce selective autophagy (e.g., proteotoxicity, lipotoxicity, or mitophagy), but not starvation, result in an increased association of ULK1 with HTT at the expense of MTOR, thus freeing ULK1 from its inhibition by MTORC1. Collectively, HTT promotes selective autophagy by activating and bringing together SQSTM1/p62 and ULK1 to assure spatial proximity between the cargo and autophagy initiation components, thereby orchestrating 2 major autophagy steps: cargo recognition and autophagy induction (Fig. 1). Figure 1. Model of HTT in promoting selective autophagy. HTT serves as a scaffolding for selective autophagy by bringing together cargo bound through SQSTM1 and an initiator of autophagy, ULK1 kinase. Basal autophagy: under basal conditions, binding of HTT to the ... Increasing evidence supports a tangled relationship between HTT, HD, and autophagy. Thus, after the first report of altered autophagy in HD by DiFiglia, the Holzbaur lab showed that HTT facilitates axonal trafficking of autophagosomes, a function that is compromised by polyQ-HTT; Hayden's group reported an autophagy-inducing domain within HTT; Steffan with Thompson and colleagues also recently reported physical interactions between HTT and several autophagy proteins including SQSTM1 and ULK1. Our finding places HTT in the core of the autophagic process, bringing together components of the cargo recognition and autophagy initiation complexes, and raising many new interesting questions. How is HTT differentially activated upon different stress challenges? Does polyQ expansion directly compromise the endogenous activities of HTT, as implicated by the very similar “empty autophagosomes” phenotypes in both HD and HTT-depleted cells, and if so, will this effect be partially responsible for the toxicity of mutant polyQ-HTT in long-lived neurons? Last, how can we exploit HTT's role in selective autophagy to modulate this process in the fight against this devastating brain disease called HD?

Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

Abstract: Protein quality control is essential for clearing misfolded and aggregated proteins from the cell, and its failure is associated with many neurodegenerative disorders. Here, we identify two genes, ufd-2 and spr-5, that when inactivated, synergistically and robustly suppress neurotoxicity associated with misfolded proteins in Caenorhabditis elegans. Loss of human orthologs ubiquitination factor E4 B (UBE4B) and lysine-specific demethylase 1 (LSD1), respectively encoding a ubiquitin ligase and a lysine-specific demethylase, promotes the clearance of misfolded proteins in mammalian cells by activating both proteasomal and autophagic degradation machineries. An unbiased search in this pathway reveals a downstream effector as the transcription factor p53, a shared substrate of UBE4B and LSD1 that functions as a key regulator of protein quality control to protect against proteotoxicity. These studies identify a new protein quality control pathway via regulation of transcription factors and point to the augmentation of protein quality control as a wide-spectrum antiproteotoxicity strategy.

COP9 signalosome controls the degradation of cytosolic misfolded proteins and protects against cardiac proteotoxicity.

Abstract: Rationale:Impaired degradation of misfolded proteins is associated with a large subset of heart diseases. Misfolded proteins are degraded primarily by the ubiquitin-proteasome system, but the ubiquitin ligases responsible for the degradation remain largely unidentified. The cullin deneddylation activity of the COP9 signalosome (CSN) requires all 8 CSN subunits (CSN1 through CSN8) and regulates cullin-RING ligases, thereby controlling ubiquitination of a large number of proteins; however, neither CSN nor cullin-RING ligases is known to regulate the degradation of cytosolic misfolded proteins. Objective:We sought to investigate the role of CSN8/CSN in misfolded protein degradation and cardiac proteinopathy. Methods and Results:Cardiac CSN8 knockout causes mouse premature death; hence, CSN8 hypomorphism (CSN8hypo) mice were used. Myocardial neddylated forms of cullins were markedly increased, and myocardial capacity of degrading a surrogate misfolded protein was significantly reduced by CSN8 hypomorphism. Wh...

Functional Amyloid Signaling via the Inflammasome, Necrosome, and Signalosome: New Therapeutic Targets in Heart Failure.

Abstract: As the most common cause of death and disability, globally, heart disease remains an incompletely understood enigma. A growing number of cardiac diseases are being characterized by the presence of misfolded proteins underlying their pathophysiology, including cardiac amyloidosis and dilated cardiomyopathy (DCM). At least nine precursor proteins have been implicated in the development of cardiac amyloidosis, most commonly caused by multiple myeloma light chain disease and disease-causing mutant or wildtype transthyretin (TTR). Similarly, aggregates with PSEN1 and COFILIN-2 have been identified in up to one-third of idiopathic DCM cases studied, indicating the potential predominance of misfolded proteins in heart failure. In this review, we present recent evidence linking misfolded proteins mechanistically with heart failure and present multiple lines of new therapeutic approaches that target the prevention of misfolded proteins in cardiac TTR amyloid disease. These include multiple small molecule pharmacological chaperones now in clinical trials designed specifically to support TTR folding by rational design, such as tafamidis, and chaperones previously developed for other purposes, such as doxycycline and tauroursodeoxycholic acid. Last, we present newly discovered non-pathological "functional" amyloid structures, such as the inflammasome and necrosome signaling complexes, which can be activated directly by amyloid. These may represent future targets to successfully attenuate amyloid-induced proteotoxicity in heart failure, as the inflammasome, for example, is being therapeutically inhibited experimentally in autoimmune disease. Together, these studies demonstrate multiple novel points in which new therapies may be used to primarily prevent misfolded proteins or to inhibit their downstream amyloid-mediated effectors, such as the inflammasome, to prevent proteotoxicity in heart failure.

Regulation of protein homeostasis in neurodegenerative diseases: the role of coding and non-coding genes

Abstract: Protein homeostasis is fundamental for cell function and survival, because proteins are involved in all aspects of cellular function, ranging from cell metabolism and cell division to the cell’s response to environmental challenges. Protein homeostasis is tightly regulated by the synthesis, folding, trafficking and clearance of proteins, all of which act in an orchestrated manner to ensure proteome stability. The protein quality control system is enhanced by stress response pathways, which take action whenever the proteome is challenged by environmental or physiological stress. Aging, however, damages the proteome, and such proteome damage is thought to be associated with aging-related diseases. In this review, we discuss the different cellular processes that define the protein quality control system and focus on their role in protein conformational diseases. We highlight the power of using small organisms to model neurodegenerative diseases and how these models can be exploited to discover genetic modulators of protein aggregation and toxicity. We also link findings from small model organisms to the situation in higher organisms and describe how some of the genetic modifiers discovered in organisms such as worms are functionally conserved throughout evolution. Finally, we demonstrate that the non-coding genome also plays a role in maintaining protein homeostasis. In all, this review highlights the importance of protein and RNA homeostasis in neurodegenerative diseases.

Understanding Caffeine's Role in Attenuating the Toxicity of α-Synuclein Aggregates: Implications for Risk of Parkinson's Disease.

Abstract: Epidemiological studies report a beneficial relationship between drinking coffee and the risk of developing Parkinson's disease (PD). This is likely due to caffeine, a constituent of coffee, acting as an adenosine A2A receptor antagonist. This study was planned to investigate whether caffeine has any effect on the aggregation of α-synuclein, present in Lewy bodies, the pathological hallmark of PD, which may account for this positive association. Aggregation of recombinant α-synuclein was followed in vitro and in a well-validated yeast proteotoxicity model of PD. Caffeine was found to have twin effects: it accelerated the process of aggregation and also altered the nature of mature aggregates. Aggregates formed in the presence of caffeine displayed amorphous as well as fibrillar morphology. In the presence of caffeine, the toxicity of oligomers and aggregates was diminished, with concomitant reduction in intracellular oxidative stress, decreased oxidative proteome damage, and increased cell survival. Caffeine-treated samples showed improved binding to phospholipids, a property likely to be important in cellular functioning of α-synuclein. Far-UV CD spectroscopy and fluorescence quenching analysis revealed that caffeine induced transient changes in this intrinsically disordered protein, forming a non-native species that enhanced the rate of aggregation of α-synuclein and modified the population of mature aggregates, introducing a higher fraction of amorphous, less toxic species. Increasingly, it is felt that the process of fibrillation itself, along with the nature of mature aggregates, dictates the cytotoxicity of the process. Our results provide a rationale for the observed epidemiological link between drinking coffee and developing PD.

Conformational switch of polyglutamine-expanded huntingtin into benign aggregates leads to neuroprotective effect.

Abstract: The abundant accumulation of inclusion bodies containing polyglutamine-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington’s disease (HD). Here, we demonstrate that FKBP12, an isomerase that exhibits reduced expression in HD, decreases the amyloidogenicity of mHTT, interrupts its oligomerization process, and structurally promotes the formation of amorphous deposits. By combining fluorescence-activated cell sorting with multiple biophysical techniques, we confirm that FKBP12 reduces the amyloid property of these ultrastructural-distinct mHTT aggregates within cells. Moreover, the neuroprotective effect of FKBP12 is demonstrated in both cellular and nematode models. Finally, we show that FKBP12 also inhibit the fibrillization process of other disease-related and aggregation-prone peptides. Our results suggest a novel function of FKBP12 in ameliorating the proteotoxicity in mHTT, which may shed light on unraveling the roles of FKBP12 in different neurodegenerative diseases and developing possible therapeutic strategies.

Inducible HSP70 Is Critical in Preventing the Aggregation and Enhancing the Processing of PMP22

Abstract: Chaperones, also called heat shock proteins (HSPs), transiently interact with proteins to aid their folding, trafficking, and degradation, thereby directly influencing the transport of newly synthesized molecules. Induction of chaperones provides a potential therapeutic approach for protein misfolding disorders, such as peripheral myelin protein 22 (PMP22)-associated peripheral neuropathies. Cytosolic aggregates of PMP22, linked with a demyelinating Schwann cell phenotype, result in suppression of proteasome activity and activation of proteostatic mechanisms, including the heat shock pathway. Although the beneficial effects of chaperones in preventing the aggregation and improving the trafficking of PMP22 have been repeatedly observed, the requirement for HSP70 in events remains elusive. In this study, we show that activation of the chaperone pathway in fibroblasts from PMP22 duplication-associated Charcot-Marie-Tooth disease type 1A patient with an FDA-approved small molecule increases HSP70 expression and attenuates proteasome dysfunction. Using cells from an HSP70.1/3(-/-) (inducible HSP70) mouse model, we demonstrate that under proteotoxic stress, this chaperone is critical in preventing the aggregation of PMP22, and this effect is aided by macroautophagy. When examined at steady-state, HSP70 appears to play a minor role in the trafficking of wild-type-PMP22, while it is crucial for preventing the buildup of the aggregation-prone Trembler-J-PMP22. HSP70 aids the processing of Trembler-J-PMP22 through the Golgi and its delivery to lysosomes via Rab7-positive vesicles. Together, these results demonstrate a key role for inducible HSP70 in aiding the processing and hindering the accumulation of misfolded PMP22, which in turn alleviates proteotoxicity within the cells.

Inhibition of Cytohesins Protects against Genetic Models of Motor Neuron Disease

Abstract: Mutant genes that underlie Mendelian forms of amyotrophic lateral sclerosis (ALS) and biochemical investigations of genetic disease models point to potential driver pathophysiological events involving endoplasmic reticulum (ER) stress and autophagy. Several steps in these cell biological processes are known to be controlled physiologically by small ADP-ribosylation factor (ARF) signaling. Here, we investigated the role of ARF guanine nucleotide exchange factors (GEFs), cytohesins, in models of ALS. Genetic or pharmacological inhibition of cytohesins protects motor neurons in vitro from proteotoxic insults and rescues locomotor defects in a Caenorhabditis elegans model of disease. Cytohesins form a complex with mutant superoxide dismutase 1 (SOD1), a known cause of familial ALS, but this is not associated with a change in GEF activity or ARF activation. ER stress evoked by mutant SOD1 expression is alleviated by antagonism of cytohesin activity. In the setting of mutant SOD1 toxicity, inhibition of cytohesin activity enhances autophagic flux and reduces the burden of misfolded SOD1. These observations suggest that targeting cytohesins may have potential benefits for the treatment of ALS.

Loss of rad-23 protects against models of motor neuron disease by enhancing mutant protein clearance

Abstract: Misfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate in amyotrophic lateral sclerosis. Loss of RAD-23 suppresses the locomotor deficit of Caenorhabditis elegans engineered to express mutTDP-43 or mutSOD1 and also protects against aging and proteotoxic insults. Knockdown of RAD-23 is further neuroprotective against the toxicity of SOD1 and TDP-43 expression in mammalian neurons. Biochemical investigation indicates that RAD-23 modifies mutTDP-43 and mutSOD1 abundance, solubility, and turnover in association with altering the ubiquitination status of these substrates. In human amyotrophic lateral sclerosis spinal cord, we find that RAD-23 abundance is increased and RAD-23 is mislocalized within motor neurons. We propose a novel pathophysiological function for RAD-23 in the stabilization of mutated proteins that cause neurodegeneration. SIGNIFICANCE STATEMENT In this work, we identify RAD-23, a component of the protein homeostasis network and nucleotide excision repair pathway, as a modifier of the toxicity of two disease-causing, misfolding-prone proteins, SOD1 and TDP-43. Reducing the abundance of RAD-23 accelerates the degradation of mutant SOD1 and TDP-43 and reduces the cellular content of the toxic species. The existence of endogenous proteins that act as “anti-chaperones” uncovers new and general targets for therapeutic intervention.

Heat shock protein responses to aging and proteotoxicity in the olfactory bulb

Abstract: The olfactory bulb is one of the most vulnerable brain regions in age-related proteinopathies. Proteinopathic stress is mitigated by the heat shock protein (Hsp) family of chaperones. Here, we describe age-related decreases in Hsc70 in the olfactory bulb of the female rat and higher levels of Hsp70 and Hsp25 in middle and old age than at 2-4 months. To model proteotoxic and oxidative stress in the olfactory bulb, primary olfactory bulb cultures were treated with the proteasome inhibitors lactacystin and MG132 or the pro-oxidant paraquat. Toxin-induced increases were observed in Hsp70, Hsp25, and Hsp32. To determine the functional consequences of the increase in Hsp70, we attenuated Hsp70 activity with two mechanistically distinct inhibitors. The Hsp70 inhibitors greatly potentiated the toxicity of sublethal lactacystin or MG132 but not of paraquat. Although ubiquitinated protein levels were unchanged with aging in vivo or with sublethal MG132 in vitro, there was a large, synergistic increase in ubiquitinated proteins when proteasome and Hsp70 functions were simultaneously inhibited. Our study suggests that olfactory bulb cells rely heavily on Hsp70 chaperones to maintain homeostasis during mild proteotoxic, but not oxidative insults, and that Hsp70 prevents the accrual of ubiquitinated proteins in these cells. The olfactory bulb is affected in the early phases of many age-related neurodegenerative disorders. Here, we described the impact of aging on multiple heat shock proteins (Hsps), such as Hsp70, in the female rat olfactory bulb in vivo. Using multiple proteasome and Hsp70 inhibitors (see schematic), we found that proteotoxicity elicited a compensatory increase in Hsp70 in primary olfactory bulb cells in vitro. Hsp70 then reduced the proteotoxic buildup of ubiquitinated proteins and robustly protected against cell death according to three independent viability assays. Thus, olfactory bulb neurons can mount impressive natural adaptations to proteotoxic injury, perhaps explaining why neurodegenerative disorders are so delayed in onset and so slow to progress.

In Vivo Profiling Reveals a Competent Heat Shock Response in Adult Neurons: Implications for Neurodegenerative Disorders.

Abstract: The heat shock response (HSR) is the main pathway used by cells to counteract proteotoxicity. The inability of differentiated neurons to induce an HSR has been documented in primary neuronal cultures and has been proposed to play a critical role in ageing and neurodegeneration. However, this accepted dogma has not been demonstrated in vivo. We used BAC transgenic mice generated by the Gene Expression Nervous System Atlas project to investigate the capacity of striatal medium sized spiny neurons to induce an HSR as compared to that of astrocytes and oligodendrocytes. We found that all cell populations were competent to induce an HSR upon HSP90 inhibition. We also show the presence and relative abundance of heat shock-related genes and proteins in these striatal cell populations. The identification of a competent HSR in adult neurons supports the development of therapeutics that target the HSR pathway as treatments for neurodegenerative disorders.

Selective autophagy and Huntingtin: learning from disease

Abstract: Autophagy, the process responsible for degradation and recycling of intracellular components in lysosomes, is essential for both the maintenance of cellular energetics and in quality control of proteins and organelles.1 Failure of such functions is detrimental in non-dividing differentiated cells such as neurons, explaining why autophagy malfunctioning has been associated with severe neurodegenerative diseases such as Alzheimer, Parkinson and Huntington's disease. The challenge is now to discriminate between pathological conditions in which the defect in autophagy is primary and those in which failure originates from the toxic effect of pathogenic proteins over the autophagy machinery. Surprisingly, we have recently found that in the case of Huntington's disease (HD), autophagy malfunctioning occurs through a combination of these 2 possibilities, as we have identified a key role in autophagy of huntingtin (HTT), the protein mutated in this devastating disorder.2 This study represents an interesting example of how, by learning about disease conditions, we can gain a better understanding of fundamental cellular processes such as, in this case, selective autophagy. HD is a genetic neurodegenerative disorder linked to a single mutation in the HTT gene that generates a prone-to-aggregate HTT protein with an abnormally large number of glutamine repeats (polyQ) in its N-terminus.3 Although neurological symptoms, due to neuronal loss, are the most noticeable ones, patients also develop systemic deficiencies, as HTT is ubiquitously expressed. Early studies demonstrating changes in intracellular protein degradation and alterations in the endolysosomal system4 pointed toward a possible malfunctioning of autophagy in HD. Our early work demonstrated that cells from HD animal models and from patients contain abnormally looking autophagosomes that seem to lack content inside.5 Proteomic analysis revealed that these apparently “empty autophagosomes” contain cytosolic proteins but fail to sequester organelles in a specific manner. Because we found mutant HTT decorating the inner part of autophagosomes, we hypothesized that the extended polyQ track was somehow compromising recognition of cargo by the forming autophagosome. Consequently, we proposed that the autophagic failure in HD was secondary to mutant HTT toxicity on this system.5 However, our recent work has made us realize that, while mutant HTT is still behind the autophagic defect in HD, the reason for autophagy malfunctioning is not just a mere non-specific toxic effect of the polyQ on the cargo recognition machinery. On the contrary, it raises a provocative possibility that the extended polyQ interferes with a previously unknown physiological function of HTT in selective autophagy.2 The key finding in support of this conceptual change was the fact that depletion of wild-type HTT in flies and mammalian cells was sufficient to reproduce a very similar autophagic phenotype to the one observed in HD but now in the absence of the pathogenic protein. Complementation studies, also in the fly, helped us to identify functional interactions between HTT and 2 important components of the autophagic machinery, SQSTM1/p626 and ULK1, involved in cargo recognition and autophagy initiation, respectively. We found that the appearance of “empty” autophagosomes in HTT-defective cells relates to the function of HTT as a scaffold protein in the interaction of SQSTM1/p62 with lysine 63 (K63)-ubiquitin chains in the cargo and with LC3, the major constituent of forming autophagosomes.2 In the absence of HTT, this binding is compromised preventing recruitment of LC3 to selective cargo. However, the role of HTT extends beyond this stabilizing function, as HTT also contributes to bring to the cargo ULK1, a kinase required for initiation of the autophagic process. Interestingly, this HTT-mediated recruitment of ULK1 to the site of autophagosome formation answers a long standing question in the autophagy field, that was, how is it possible to activate autophagy in response to non-nutrient related stressors? ULK1 is normally maintained inactive through its phosphorylation and direct sequestration by mTORC1.7 During starvation, the lack of nutrients renders mTORC1 inactive with the subsequent release of ULK1 to contribute to autophagosome formation. However, activation of autophagy by stressors, such as proteotoxicity or organelle damage, was puzzling, as those conditions do not affect mTORC1 activity and ULK1 should remain sequestered by this kinase complex. We have identified that ULK1 interacts with both mTOR and HTT but in exclusive complexes and that HTT and mTOR compete each other out for binding with ULK1. Thus, upon stress conditions that require activation of selective autophagy, HTT will compete ULK1 out of the inhibitory effect of mTORC1 and bring it to the specific cargo, thanks to its dual interaction with cargo receptors (Fig.1). Figure 1. Novel function of HTT in selective autophagy. Left: In response to starvation “in bulk” autophagy is initiated through inactivation of mTORC1 and subsequent release of the ULK1 kinase complex toward sites of autophagosome biogenesis. ... This novel function of HTT in selective autophagy identifies HTT itself as an interesting therapeutic target for the modulation of autophagy in the fight against neurodegeneration.