Characterization of a brain-enriched chaperone, MRJ, that inhibits Huntingtin aggregation and toxicity independently

Molecular chaperones are involved in a wide range of cellular events, such as protein folding and oligomeric protein complex assembly. DnaK- and DnaJ-like proteins are the two major classes of molecular chaperones in mammals. Recent studies have shown that DnaJ-like family proteins can inhibit polyglutamine aggregation, a hallmark of many neurodegenerative diseases, including Huntington's disease (HD). Although most DnaJ-like proteins studied are ubiquitously expressed, some have restricted expression, so it is possible that some specific chaperones may affect polyglutamine aggregation in specific neurons. In this report, we describe the isolation of a DnaJ-like protein MRJ and the characterization of its chaperone activity. Tissue distribution studies showed that MRJ is highly enriched in the central nervous system. In an in vitro cell model of HD, overexpressed MRJ effectively suppressed polyglutamine-dependent protein aggregation, caspase activity, and cellular toxicity. Collectively, these results suggest that MRJ has a relevant functional role in neurons.     

Chaperone suppression of cellular toxicity of huntingtin is independent of polyglutamine aggregation

Polyglutamine protein aggregation is associated with eight inherited neurodegenerative disorders. In Huntington's disease, N-terminal fragments of mutant huntingtin form intracellular aggregates and mediate cellular toxicity. Recent studies have shown that chaperones inhibit polyglutamine-mediated aggregation and cellular toxicity. Because chaperones also inhibit caspase activation to protect cells from death, it remains unclear whether the protective effect of chaperones on polyglutamine-mediated cellular toxicity is dependent on their inhibition of protein aggregation. In this study, we show that several chaperones including HSP 40, HSP 70, and N-ethylmaleimide-sensitive factor can inhibit cellular toxicity caused by N-terminal mutant huntingtin fragments. However, only HSP 40 is able to inhibit huntingtin aggregation. Furthermore, time-course study suggests that the protection of chaperones against huntingtin toxicity is not the result of their suppression of huntingtin aggregation. Chaperones inhibit caspase-3 and caspase-9 activation mediated by mutant huntingtin, and this inhibition is independent of huntingtin aggregation. We propose that the inhibition of caspase activity by chaperones is involved in their suppression of polyglutamine toxicity.     

Polyglutamine-mediated neurodegeneration: Use of chaperones as prevention strategy

Polyglutamine diseases are a class of inherited neurodegenerative disorders caused by the expansion of a polyglutamine tract within the respective proteins. Clinical studies have revealed that the forming of neuronal intranuclear inclusions by the disease protein is a common pathological feature of polyglutamine diseases. Although there has been considerable progress in understanding polyglutamine diseases, many questions regarding their mechanism are still unanswered. The finding that molecular chaperones are associated with ubiquitinated intranuclear inclusions clearly indicates a crucial role of molecular chaperones in the generation of these fatal diseases. Molecular and chemical chaperones have been found to be a good agent for suppressing many polyglutamine diseases in several animal models. In this review, I discuss the roles of the ubiquitin-proteasome pathway and molecular chaperones in the development of polyglutamine diseases and probable approach for the prevention of many of these fatal disorders using molecular chaperones as a therapeutic agent. Newly found chemical chaperones have been demonstrated to be potentially useful and could be used as a therapeutic strategy in preventing many versions of polyglutamine diseases.     

Polyglutamine diseases and molecular chaperones

The polyglutamine diseases, a group of diseases currently thought to consist of nine inherited neurodegenerative diseases, are caused by the expansion of unstable CAG trinucleotide repeats that code for polyglutamine tracts in the responsible genes. These diseases are now recognized as being of a type with conformationally abnormal or amyloid-related proteins, and thus are called 'conformational diseases'. Recently, many studies using cell cultures and model organisms have suggested that the two major machineries for protein quality control (the molecular chaperone and the protein degradation machineries) play important roles in the pathogenesis of the polyglutamine diseases. Interestingly, molecular chaperones have been shown to behave in totally different ways in these studies, namely in suppressing as well as enhancing neurodegeneration or cell death. These apparently opposite actions of molecular chaperones suggest that a certain balance between the activities of molecular chaperones and the expression level of polyglutamine is an important determinant of the pathogenesis. In this review, we summarize recent findings on such ambiguous effects of molecular chaperones on polyglutamine diseases, and discuss possible mechanisms by which molecular chaperones, especially VCP, are involved in the pathogenesis.     

Modulation of prion-dependent polyglutamine aggregation and toxicity by chaperone proteins in the yeast model

In yeast, aggregation and toxicity of the expanded polyglutamine fragment of human huntingtin strictly depend on the presence of the endogenous self-perpetuating aggregated proteins (prions), which contain glutamine/asparagine-rich domains. Some chaperones of the Hsp100/70/40 complex, modulating propagation of yeast prions, were also reported to influence polyglutamine aggregation in yeast, but it was not clear whether they do it directly or via affecting prions. Our data show that although some chaperone alterations indeed act on polyglutamines via curing endogenous prions, other alterations decrease size and ameliorate toxicity of polyglutamine aggregates without affecting prion propagation. Therefore, the role of yeast chaperones in polyglutamine aggregation and toxicity is not restricted only to their effects on the endogenous prions. Moreover, chaperone interactions with prion and polyglutamine aggregates appear to be of a highly specific nature. One and the same chaperone alteration, substitution A503V in the middle region of the chaperone Hsp104, exhibited opposite effects on one of the endogenous prions ([PSI(+)], the prion form of Sup35) and on polyglutamines, increasing aggregate size and toxicity in the former case and decreasing them in the latter case. On the other hand, different members of a single chaperone family exhibited opposite effects on one and the same type of aggregates: excess of the Hsp40 chaperone Ydj1 increased polyglutamine aggregate size and toxicity, whereas excess of the other Hsp40 chaperone, Sis1, decreased them. As many stress-defense proteins are conserved between yeast and mammals, these data shed light on possible mechanisms modulating polyglutamine aggregation and toxicity in mammalian cells.     

Critical role of the proline-rich region in Huntingtin for aggregation and cytotoxicity in yeast

Nine neurodegenerative diseases, such as Huntington, are caused by a polyglutamine (poly(Q)) expansion in otherwise unrelated proteins. Although poly(Q) expansion causes aggregation of the affected proteins, the protein context might determine the selective neuronal vulnerability found in each disease. Here we have report that, although expression of Huntingtin derivatives with a pathological poly(Q) expansion are innocuous in yeast, deletion of the flanking proline-rich region alters the shape and number of poly(Q) inclusions and unmasks toxic properties. Strikingly, deletion of Hsp104 increases the size of inclusions formed by expanded poly(Q) lacking the proline-rich region and abolishes toxicity. Overexpression of the chaperones Hsp104 or Hsp70 rescues growth defects in affected cells without resolving inclusions. However, aggregates formed by nontoxic Huntingtin derivatives or by toxic derivatives cured by chaperones are physically distinct from aggregates formed by toxic proteins. This study identifies the proline-rich region in Huntingtin as a profound cis-acting modulator of expanded poly(Q) toxicity and distinguishes between aggregates of toxic or non-toxic proteins.     

Association of Bcl-2 with misfolded prion protein is linked to the toxic potential of cytosolic PrP

Protein misfolding is linked to different neurodegenerative disorders like Alzheimer's disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K-resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115-156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.     

Hsp105alpha suppresses the aggregation of truncated androgen receptor with expanded CAG repeats and cell toxicity

Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The N-terminal fragment of AR containing the expanded polyglutamine tract aggregates in cytoplasm and/or in nucleus and induces cell death. Some chaperones such as Hsp40 and Hsp70 have been identified as important regulators of polyglutamine aggregation and/or cell death in neuronal cells. Recently, Hsp105alpha, expressed at especially high levels in mammalian brain, has been shown to suppress apoptosis in neuronal cells and prevent the aggregation of protein caused by heat shock in vitro. However, its role in polyglutamine-mediated cell death and toxicity has not been studied. In the present study, we examined the effects of Hsp105alpha on the aggregation and cell toxicity caused by expansion of the polyglutamine tract using a cellular model of SBMA. The transient expression of truncated ARs (tARs) containing an expanded polyglutamine tract caused aggregates to form in COS-7 and SK-N-SH cells and concomitantly apoptosis in the cells with the nuclear aggregates. When Hsp105alpha was overexpressed with tAR97 in the cells, Hsp105alpha was colocalized to aggregates of tAR97, and the aggregation and cell toxicity caused by expansion of the polyglutamine tract were markedly reduced. Both beta-sheet and alpha-helix domains, but not the ATPase domain, of Hsp105alpha were necessary to suppress the formation of aggregates in vivo and in vitro. Furthermore, Hsp105alpha was found to localize in nuclear inclusions formed by ARs containing an expanded polyglutamine tract in tissues of patients and transgenic mice with SBMA. These findings suggest that overexpression of Hsp105alpha suppresses cell death caused by expansion of the polyglutamine tract without chaperone activity, and the enhanced expression of the essential domains of Hsp105alpha in brain may provide an effective therapeutic approach for CAG repeat diseases.     

Nuclear aggregation of huntingtin is not prevented by deletion of chaperone Hsp104

Polyglutamine expansion causes the disease proteins to aggregate, resulting in stable insoluble aggregates in the nucleus. The in vitro aggregation and cellular toxicity of polyglutamine proteins are reduced by chaperone heat shock proteins (Hsp). In polyglutamine disease animal models, however, polyglutamine inclusions remain in the nucleus despite the suppression of neurodegeneration by Hsp. Studies using yeast genetic approach revealed that the balance of Hsp is important for regulating protein aggregation in the cytoplasm of yeast cells. Here we report that N-terminal fragments of huntingtin with an expanded polyglutamine tract form aggregates only in the cytoplasm of yeast cells and, when tagged with nuclear localization sequences (NLS), are able to aggregate in the nucleus. Deletion of the Hsp104 gene prevents the aggregation of huntingtin in the cytoplasm but is unable to eliminate the aggregation of NLS-tagged huntingtin in the nucleus. The inhibitory effect of Hsp104 deletion on the cytoplasmic aggregation of huntingtin only occurs in viable yeast cells, as aggregates can be formed in Hsp104 deletion cells that have been frozen for 72 h. Fresh cytosolic extracts of the Hsp104 deletion strain inhibit the aggregation of huntingtin in vitro, suggesting that the deletion of Hsp104 may alter the activities of other cytoplasmic factors to inhibit polyglutamine aggregation in the cytoplasm. We propose that the regulatory effects of chaperones may mainly be restricted to the cytoplasm and have much less influence on polyglutamine-containing aggregates in the nucleus.     

Chaperones Hsp70 and Hsp40 suppress aggregate formation and apoptosis in cultured neuronal cells expressing truncated androgen receptor protein with expanded polyglutamine tract

Spinal and bulbar muscular atrophy (SBMA) is one of a group of human inherited neurodegenerative diseases caused by polyglutamine expansion. We have previously demonstrated that the SBMA gene product, the androgen receptor protein, is toxic and aggregates when truncated. Heat shock proteins function as molecular chaperones, which recognize and renaturate misfolded protein (aggregate). We thus assessed the effect of a variety of chaperones in a cultured neuronal cell model of SBMA. Overexpression of chaperones reduces aggregate formation and suppresses apoptosis in a cultured neuronal cell model of SBMA to differing degrees depending on the chaperones and their combinations. Combination of Hsp70 and Hsp40 was the most effective among the chaperones in reducing aggregate formation and providing cellular protection, reflecting that Hsp70 and Hsp40 act together in chaperoning mutant and disabled proteins. Although Hdj2/Hsdj chaperone has been previously reported to suppress expanded polyglutamine tract-formed aggregate, Hsdj/Hdj2 showed little effect in our system. These findings indicate that chaperones may be one of the key factors in the developing of CAG repeat disease and suggested that increasing expression level or enhancing the function of chaperones will provide an avenue for the treatment of CAG repeat disease.     

CHIP protects from the neurotoxicity of expanded and wild-type ataxin-1 and promotes their ubiquitination and degradation

CHIP (C terminus of Hsc-70 interacting protein) is an E3 ligase that links the protein folding machinery with the ubiquitin-proteasome system and has been implicated in disorders characterized by protein misfolding and aggregation. Here we investigate the role of CHIP in protecting from ataxin-1-induced neurodegeneration. Ataxin-1 is a polyglutamine protein whose expansion causes spinocerebellar ataxia type-1 (SCA1) and triggers the formation of nuclear inclusions (NIs). We find that CHIP and ataxin-1 proteins directly interact and co-localize in NIs both in cell culture and SCA1 postmortem neurons. CHIP promotes ubiquitination of expanded ataxin-1 both in vitro and in cell culture. The Hsp70 chaperone increases CHIP-mediated ubiquitination of ataxin-1 in vitro, and the tetratricopeptide repeat domain, which mediates CHIP interactions with chaperones, is required for ataxin-1 ubitiquination in cell culture. Interestingly, CHIP also interacts with and ubiquitinates unexpanded ataxin-1. Overexpression of CHIP in a Drosophila model of SCA1 decreases the protein steady-state levels of both expanded and unexpanded ataxin-1 and suppresses their toxicity. Finally we investigate the ability of CHIP to protect against toxicity caused by expanded polyglutamine tracts in different protein contexts. We find that CHIP is not effective in suppressing the toxicity caused by a bare 127Q tract with only a short hemagglutinin tag, but it is very efficient in suppressing toxicity caused by a 128Q tract in the context of an N-terminal huntingtin backbone. These data underscore the importance of the protein framework for modulating the effects of polyglutamine-induced neurodegeneration.     

Amyloid in neurodegenerative diseases: friend or foe?

Accumulation of amyloid-like aggregates is a hallmark of numerous neurodegenerative disorders such as Alzheimer's and polyglutamine disease. Yet, whether the amyloid inclusions found in these diseases are toxic or cytoprotective remains unclear. Various studies suggest that the toxic culprit in the amyloid folding pathway is actually a soluble oligomeric species which might interfere with normal cellular function by a multifactorial mechanism including aberrant protein-protein interactions. Molecular chaperones suppress toxicity of amyloidogenic proteins by inhibiting aggregation of non-native disease substrates and targeting them for refolding or degradation. Paradoxically, recent studies also suggest a protective action of chaperones in their promotion of the assembly of large, tightly packed, benign aggregates that sequester toxic protein species.     

Modulation of neurodegeneration by molecular chaperones

Many neurodegenerative disorders are characterized by conformational changes in proteins that result in misfolding, aggregation and intra- or extra-neuronal accumulation of amyloid fibrils. Molecular chaperones provide a first line of defence against misfolded, aggregation-prone proteins and are among the most potent suppressors of neurodegeneration known for animal models of human disease. Recent studies have investigated the role of molecular chaperones in amyotrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease and polyglutamine diseases. We propose that molecular chaperones are neuroprotective because of their ability to modulate the earliest aberrant protein interactions that trigger pathogenic cascades. A detailed understanding of the molecular basis of chaperone-mediated protection against neurodegeneration might lead to the development of therapies for neurodegenerative disorders that are associated with protein misfolding and aggregation.     

Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin-proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14-3-3, and alpha-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin-proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.     

Hosting neurotoxicity in polyglutamine disease

Polyglutamine diseases are caused by an expanded glutamine domain thought to confer a toxic activity onto the respective disease proteins. In this issue, propose that toxicity of the polyglutamine protein Ataxin-1 may not be due to abberant protein interactions mediated by the polyglutamine expansion. Instead, they suggest that toxicity is solely due to interactions of Ataxin-1 with its normal binding partners.     

A compact beta model of huntingtin toxicity

Huntington disease results from an expanded polyglutamine region in the N terminus of the huntingtin protein. HD pathology is characterized by neuronal degeneration and protein inclusions containing N-terminal fragments of mutant huntingtin. Structural information is minimal, though it is believed that mutant huntingtin polyglutamine adopts β structure upon conversion to a toxic form. To this end, we designed mammalian cell expression constructs encoding compact β variants of Htt exon 1 N-terminal fragment and tested their ability to aggregate and induce toxicity in cultured neuronal cells. In parallel, we performed molecular dynamics simulations, which indicate that constructs with expanded polyglutamine β-strands are stabilized by main-chain hydrogen bonding. Finally, we found a correlation between the reactivity to 3B5H10, an expanded polyglutamine antibody that recognizes a compact β rich hairpin structure, and the ability to induce cell toxicity. These data are consistent with an important role for a compact β structure in mutant huntingtin-induced cell toxicity.     

Co-chaperone CHIP associates with expanded polyglutamine protein and promotes their degradation by proteasomes

A major hallmark of the polyglutamine diseases is the formation of neuronal intranuclear inclusions of the disease proteins that are ubiquitinated and often associated with various chaperones and proteasome components. But, how the polyglutamine proteins are ubiquitinated and degraded by the proteasomes are not known. Here, we demonstrate that CHIP (C terminus of Hsp70-interacting protein) co-immunoprecipitates with the polyglutamine-expanded huntingtin or ataxin-3 and associates with their aggregates. Transient overexpression of CHIP increases the ubiquitination and the rate of degradation of polyglutamine-expanded huntingtin or ataxin-3. Finally, we show that overexpression of CHIP suppresses the aggregation and cell death mediated by expanded polyglutamine proteins and the suppressive effect is more prominent when CHIP is overexpressed along with Hsc70.