Polyglutamine Expansion In Protein Research Articles

Valproate Mediated Proteasome Dysfunctions Induce Apoptosis

AbstractSeveral studies suggest Valproate's (VPA) therapeutic use in treating seizures, epilepsy, and bipolar disorder. Valproate is a class I histone deacetylases (HDACs) inhibitor and an attractive chemotherapeutic agent for targeting cancer. Few reports suggest that Valproate can suppress cell growth and cell differentiation and is linked with anti‐tumor activity. However, Valproate‐associated anti‐tumoral function and intracellular signaling cascade‐mediated anti‐cellular proliferation activities still need to be better understood. This current study suggests that Valproate can elevate proteasomal dysfunctions, resulting in misfolded protein accumulation and abnormal mitochondrial functions that successively induce apoptosis. Present findings indicate that treatment of Valproate inhibits Proteasome activities and also aggravates accumulation of expanded polyglutamine proteins and other proteasomal substrates. Overall, the aggregation of aberrant proteins and mitochondrial dysfunctions are observed, such as cytochrome c release, and disturbed mitochondrial membrane potential. Treatment of Valproate induces apoptotic morphological changes and cell death. These observations suggest that Valproate can cause mitochondrial abnormalities associated with apoptosis. These findings can provide new possible insights and suggest molecular approaches for developing better and specific Proteasome inhibitors that can be better useful with other anti‐tumor drugs for treatment of cancer and other complex diseases.

Acetaminophen induces mitochondrial apoptosis through proteasome dysfunctions

Acetaminophen is a known antipyretic and non-opioid analgesic for mild pain and fever. Numerous studies uncover their hidden chemotherapeutics applications, including chronic cancer pain management. Acetaminophen also represents an anti-proliferative effect in some cancer cells. Few studies also suggest that the use of Acetaminophen can trigger apoptosis and impede cellular growth. However, Acetaminophen's molecular potential and precise mechanism against improper cellular proliferation and use as an effective anti-proliferative agent still need to be better understood. Here, our current findings show that Acetaminophen induces proteasomal dysfunctions, resulting in aberrant protein accumulation and mitochondrial abnormalities, and consequently induces cell apoptosis. We observed that the Acetaminophen treatment leads to improper aggregation of ubiquitylated expanded polyglutamine proteins, which may be due to the dysfunctions of proteasome activities. Our in-silico analysis suggests the interaction of Acetaminophen and proteasome. Furthermore, we demonstrated the accumulation of proteasome substrates and the depletion of proteasome activities after treating Acetaminophen in cells. Acetaminophen induces proteasome dysfunctions and mitochondrial abnormalities, leading to pro-apoptotic morphological changes and apoptosis successively. These results suggest that Acetaminophen can induce cell death and may retain a promising anti-proliferative effect. These observations can open new possible molecular strategies in the near future for developing and designing specific and effective proteasome inhibitors, which can be helpful in conjugation with other anti-tumor drugs for their better efficiency.

AAV-Mediated CAG-Targeting Selectively Reduces Polyglutamine-Expanded Protein and Attenuates Disease Phenotypes in a Spinocerebellar Ataxia Mouse Model.

Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.

Polyglutamine diseases.

Polyglutamine diseases are a collection of nine CAG trinucleotide expansion disorders, presenting with a spectrum of neurological and clinical phenotypes. Recent human, mouse and cell studies of Huntington's diseasehave highlighted the role of DNA repair genes in somatic expansion of the CAG repeat region, modifying disease pathogenesis. Incomplete splicing of the HTT gene has also been shown to occur in humans, with the resulting exon 1 fragment most probably contributing to the Huntington's disease phenotype. In the spinocerebellar ataxias, studies have converged on transcriptional dysregulation of ion channels as a key disease modifier. In addition, advances have been made in understanding how increased levels of toxic, polyglutamine-expanded proteins can arise in the spinocerebellar ataxias through post-transcriptional and -translational modifications and autophagic mechanisms. Recent studies in spinal and bulbar muscular atrophyimplicate similar pathogenic pathways to the more common polyglutamine diseases, highlighting autophagy stimulation as a potential therapeutic target. Finally, the therapeutic use of antisense oligonucleotides in several polyglutamine diseases has shown preclinical benefits and serves as potential future therapies in humans.

SynB3 Conjugated QBP1 Passes Blood-Brain Barrier Models and Inhibits PolyQ Protein Aggregation.

Polyglutamine diseases are degenerative diseases in the central nervous system caused by CAG trinucleotide repeat expansion which encodes polyglutamine tracts, leading to the misfolding of pathological proteins. Small peptides can be designed to prevent polyglutamine diseases by inhibiting the polyglutamine protein aggregation, for example, polyglutamine binding peptide 1(QBP1). However, the transportation capability of polyglutamine binding peptide 1 across the blood-brain barrier is less efficient. We hypothesized whether its therapeutic effect could be improved by increasing the rate of membrane penetration. The objective of the study was to explore whether polyglutamine binding peptide 1 conjugated cell-penetrating peptides could pass through the blood-brain barrier and inhibit the aggregation of polyglutamine proteins. In order to investigate the toxic effects, we constructed a novel stable inducible PC12 cells to express Huntington protein that either has 11 glutamine repeats or 63 glutamine repeats to mimic wild type and polyglutamine expand Huntington protein, respectively. Both SynB3 and TAT conjugated polyglutamine binding peptide 1 was synthesized, respectively. We tested their capabilities to pass through a Trans-well system and subsequently studied the counteractive effects on polyglutamine protein aggregation. The conjugation of cell-penetrating peptides to SynB3 and TAT enhanced the transportation of polyglutamine binding peptide 1 across the mono-cell layer and ameliorated polyglutamine-- expanded Huntington protein aggregation; moreover, SynB3 showed better delivery efficiency than TAT. Interestingly, it has been observed that polyglutamine binding peptide 1 specifically inhibited polyglutamine-expanded protein aggregation rather than affected other amyloidosis proteins, for example, β-Amyloid. Our study indicated that SynB3 could be an effective carrier for polyglutamine binding peptide 1 distribution through the blood-brain barrier model and ameliorate the formation of polyglutamine inclusions; thus SynB3 conjugated polyglutamine binding peptide 1 could be considered as a therapeutic candidate for polyglutamine diseases.

Intercellular Propagation and Aggregate Seeding of Mutant Ataxin-1

Intercellular propagation of aggregated protein inclusions along actin-based tunneling nanotubes (TNTs) has been reported as a means of pathogenic spread in Alzheimer’s, Parkinson’s, and Huntington’s diseases. Propagation of oligomeric-structured polyglutamine-expanded ataxin-1 (Atxn1[154Q]) has been reported in the cerebellum of a Spinocerebellar ataxia type 1 (SCA1) knock-in mouse to correlate with disease propagation. In this study, we investigated whether a physiologically relevant polyglutamine-expanded ATXN1 protein (ATXN1[82Q]) could propagate intercellularly. Using a cerebellar-derived live cell model, we observed ATXN1 aggregates form in the nucleus, subsequently form in the cytoplasm, and finally, propagate to neighboring cells along actin-based intercellular connections. Additionally, we observed the facilitation of aggregate-resistant proteins into aggregates given the presence of aggregation-prone proteins within cells. Taken together, our results support a pathogenic role of intercellular propagation of polyglutamine-expanded ATXN1 inclusions.

Parameter inference for a stochastic kinetic model of expanded polyglutamine proteins.

The presence of protein aggregates in cells is a known feature of many human age-related diseases, such as Huntington's disease. Simulations using fixed parameter values in a model of the dynamic evolution of expanded polyglutaime (PolyQ) proteins in cells have been used to gain a better understanding of the biological system. However, there is considerable uncertainty about the values of some of the parameters governing the system. Currently, appropriate values are chosen by ad hoc attempts to tune the parameters so that the model output matches experimental data. The problem is further complicated by the fact that the data only offer a partial insight into the underlying biological process: the data consist only of the proportions of cell death and of cells with inclusion bodies at a few time points, corrupted by measurementerror. Developing inference procedures to estimate the model parameters in this scenario is a significant task. The model probabilities corresponding to the observed proportions cannot be evaluated exactly, and so they are estimated within the inference algorithm by repeatedly simulating realizations from the model. In general such an approach is computationally very expensive, and we therefore construct Gaussian process emulators for the key quantities and reformulate our algorithm around these fast stochastic approximations. We conclude by highlighting appropriate values of the model parameters leading to new insights into the underlying biologicalprocesses.

Selective suppression of polyglutamine-expanded protein by lipid nanoparticle-delivered siRNA targeting CAG expansions in the mouse CNS

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by expansion of cytosine-adenine-guanine (CAG)-trinucleotide repeats in causative genes. These diseases include spinal and bulbar muscular atrophy (SBMA), Huntington’s disease, dentatorubral-pallidoluysian atrophy, and spinocerebellar ataxias. Targeting expanded CAG repeats is a common therapeutic approach to polyQ diseases, but concomitant silencing of genes with normal CAG repeats may lead to toxicity. Previous studies have shown that CAG repeat-targeting small interfering RNA duplexes (CAG-siRNAs) have the potential to selectively suppress mutant proteins in in vitro cell models of polyQ diseases. However, in vivo application of these siRNAs has not yet been investigated. In this study, we demonstrate that an unlocked nucleic acid (UNA)-modified CAG-siRNA shows high selectivity for polyQ-expanded androgen receptor (AR) inhibition in in vitro cell models and that lipid nanoparticle (LNP)-mediated delivery of the CAG-siRNA selectively suppresses mutant AR in the central nervous system of an SBMA mouse model. In addition, a subcutaneous injection of the LNP-delivered CAG-siRNA efficiently suppresses mutant AR in the skeletal muscle of the SBMA mouse model. These results support the therapeutic potential of LNP-delivered UNA-modified CAG-siRNAs for selective suppression of mutant proteins in SBMA and other polyQ diseases.

Targeting Mitochondrial Network Disorganization is Protective in C. elegans Models of Huntington's Disease.

Huntington’s disease (HD) is an adult-onset neurodegenerative disease caused by a trinucleotide CAG repeat expansion in the HTT gene. While the pathogenesis of HD is incompletely understood, mitochondrial dysfunction is thought to be a key contributor. In this work, we used C. elegans models to elucidate the role of mitochondrial dynamics in HD. We found that expression of a disease-length polyglutamine tract in body wall muscle, either with or without exon 1 of huntingtin, results in mitochondrial fragmentation and mitochondrial network disorganization. While mitochondria in young HD worms form elongated tubular networks as in wild-type worms, mitochondrial fragmentation occurs with age as expanded polyglutamine protein forms aggregates. To correct the deficit in mitochondrial morphology, we reduced levels of DRP-1, the GTPase responsible for mitochondrial fission. Surprisingly, we found that disrupting drp-1 can have detrimental effects, which are dependent on how much expression is decreased. To avoid potential negative side effects of disrupting drp-1, we examined whether decreasing mitochondrial fragmentation by targeting other genes could be beneficial. Through this approach, we identified multiple genetic targets that rescue movement deficits in worm models of HD. Three of these genetic targets, pgp-3, F25B5.6 and alh-12, increased movement in the HD worm model and restored mitochondrial morphology to wild-type morphology. This work demonstrates that disrupting the mitochondrial fission gene drp-1 can be detrimental in animal models of HD, but that decreasing mitochondrial fragmentation by targeting other genes can be protective. Overall, this study identifies novel therapeutic targets for HD aimed at improving mitochondrial health.

MR Imaging of SCA3/MJD.

Spinocerebellar ataxia type 3/Machado–Joseph disease (SCA3/MJD) is a progressive autosomal dominantly inherited cerebellar ataxia characterized by the aggregation of polyglutamine-expanded protein within neuronal nuclei in the brain, which can lead to brain damage that precedes the onset of clinical manifestations. Magnetic resonance imaging (MRI) techniques such as morphometric MRI, diffusion tensor imaging (DTI), functional magnetic resonance imaging (fMRI), and magnetic resonance spectroscopy (MRS) have gained increasing attention as non-invasive and quantitative methods for the assessment of structural and functional alterations in clinical SCA3/MJD patients as well as preclinical carriers. Morphometric MRI has demonstrated typical patterns of atrophy or volume loss in the cerebellum and brainstem with extensive lesions in some supratentorial areas. DTI has detected widespread microstructural alterations in brain white matter, which indicate disrupted brain anatomical connectivity. Task-related fMRI has presented unusual brain activation patterns within the cerebellum and some extracerebellar tissue, reflecting the decreased functional connectivity of these brain regions in SCA3/MJD subjects. MRS has revealed abnormal neurochemical profiles, such as the levels or ratios of N-acetyl aspartate, choline, and creatine, in both clinical cases and preclinical cases before the alterations in brain anatomical structure. Moreover, a number of studies have reported correlations of MR imaging alterations with clinical and genetic features. The utility of these MR imaging techniques can help to identify preclinical SCA3/MJD carriers, monitor disease progression, evaluate response to therapeutic interventions, and illustrate the pathophysiological mechanisms underlying the occurrence, development, and prognosis of SCA3/MJD.

Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form

Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington’s disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.

Conformational studies of pathogenic expanded polyglutamine protein deposits from Huntington’s disease

Huntington’s disease, like other neurodegenerative diseases, continues to lack an effective cure. Current treatments that address early symptoms ultimately fail Huntington’s disease patients and their families, with the disease typically being fatal within 10–15 years from onset. Huntington’s disease is an inherited disorder with motor and mental impairment, and is associated with the genetic expansion of a CAG codon repeat encoding a polyglutamine-segment-containing protein called huntingtin. These Huntington’s disease mutations cause misfolding and aggregation of fragments of the mutant huntingtin protein, thereby likely contributing to disease toxicity through a combination of gain-of-toxic-function for the misfolded aggregates and a loss of function from sequestration of huntingtin and other proteins. As with other amyloid diseases, the mutant protein forms non-native fibrillar structures, which in Huntington’s disease are found within patients’ neurons. The intracellular deposits are associated with dysregulation of vital processes, and inter-neuronal transport of aggregates may contribute to disease progression. However, a molecular understanding of these aggregates and their detrimental effects has been frustrated by insufficient structural data on the misfolded protein state. In this review, we examine recent developments in the structural biology of polyglutamine-expanded huntingtin fragments, and especially the contributions enabled by advances in solid-state nuclear magnetic resonance spectroscopy. We summarize and discuss our current structural understanding of the huntingtin deposits and how this information furthers our understanding of the misfolding mechanism and disease toxicity mechanisms.Impact statementMany incurable neurodegenerative disorders are associated with, and potentially caused by, the amyloidogenic misfolding and aggregation of proteins. Usually, complex genetic and behavioral factors dictate disease risk and age of onset. Due to its principally mono-genic origin, which strongly predicts the age-of-onset by the extent of CAG repeat expansion, Huntington’s disease (HD) presents a unique opportunity to dissect the underlying disease-causing processes in molecular detail. Yet, until recently, the mutant huntingtin protein with its expanded polyglutamine domain has resisted structural study at the atomic level. We present here a review of recent developments in HD structural biology, facilitated by breakthrough data from solid-state NMR spectroscopy, electron microscopy, and complementary methods. The misfolded structures of the fibrillar proteins inform our mechanistic understanding of the disease-causing molecular processes in HD, other CAG repeat expansion disorders, and, more generally, protein deposition disease.

Foot-and-mouth disease virus capsid protein VP2 activates the cellular EIF2S1-ATF4 pathway and induces autophagy via HSPB1

ABSTRACTFoot-and-mouth disease virus (FMDV) can result in economical destruction of cloven-hoofed animals. FMDV infection has been reported to induce macroautophagy/autophagy; however, the precise molecular mechanisms of autophagy induction and effect of FMDV capsid protein on autophagy remain unknown. In the present study, we report that FMDV infection induced a complete autophagy process in the natural host cells of FMDV, and inhibition of autophagy significantly decreased FMDV production, suggesting that FMDV-induced autophagy facilitates viral replication. We found that the EIF2S1-ATF4 pathway was activated and the AKT-MTOR signaling pathway was inhibited by FMDV infection. We also observed that ultraviolet (UV)-inactivated FMDV can induce autophagy. Importantly, our work provides the first piece of evidence that expression of FMDV capsid protein VP2 can induce autophagy through the EIF2S1-ATF4-AKT-MTOR cascade, and we found that VP2 interacted with HSPB1 (heat shock protein family B [small] member 1) and activated the EIF2S1-ATF4 pathway, resulting in autophagy and enhanced FMDV replication. In addition, we show that VP2 induced autophagy in a variety of mammalian cell lines and decreased aggregates of a model mutant HTT (huntingtin) polyglutamine expansion protein (HTT103Q). Overall, our results demonstrate that FMDV capsid protein VP2 induces autophagy through interaction with HSPB1 and activation of the EIF2S1-ATF4 pathway.

Polyglutamine expansion affects huntingtin conformation in multiple Huntington\u2019s disease models

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington’s disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.

Proteolytic Cleavage of Polyglutamine Disease-Causing Proteins: Revisiting the Toxic Fragment Hypothesis.

Proteolytic cleavage has been implicated in the pathogenesis of diverse neurodegenerative diseases involving abnormal protein accumulation. Polyglutamine diseases are a group of nine hereditary disorders caused by an abnormal expansion of repeated glutamine tracts contained in otherwise unrelated proteins. When expanded, these proteins display toxic properties and are prone to aggregate, but the mechanisms responsible for the selective neurodegeneration observed in polyglutamine disease patients are still poorly understood. It has been suggested that the neuronal toxicity of polyglutamine-expanded proteins is associated with the production of deleterious protein fragments. This review aims at discussing the involvement of proteolytic cleavage in the six types of spinocerebellar ataxia caused by polyglutamine expansion of proteins. The analysis takes into detailed consideration evidence concerning fragment detection and the mechanisms of fragment toxicity. Current evidence suggests that the proteins involved in spinocerebellar ataxia types 3, 6 and 7 give rise to stable proteolytic fragments. Fragments carrying polyglutamine expansions display increased tendency to aggregate and toxicity, comparing with their non-expanded counterparts or with the correspondent full-length expanded proteins. Data concerning spinocerebellar ataxia types 1, 2 and 17 is still scarce, but available results afford further investigation. Available literature suggests that proteolytic cleavage of expanded polyglutamine-containing proteins enhances toxicity in disease-associated contexts and may constitute an important step in the pathogenic cascade of polyglutamine diseases. Countering protein fragmentation thus presents itself as a promising therapeutic aim.

A combinatorial approach to identify calpain cleavage sites in the Machado-Joseph disease protein ataxin-3.

Ataxin-3, the disease protein in Machado-Joseph disease, is known to be proteolytically modified by various enzymes including two major families of proteases, caspases and calpains. This processing results in the generation of toxic fragments of the polyglutamine-expanded protein. Although various approaches were undertaken to identify cleavage sites within ataxin-3 and to evaluate the impact of fragments on the molecular pathogenesis of Machado-Joseph disease, calpain-mediated cleavage of the disease protein and the localization of cleavage sites remained unclear. Here, we report on the first precise localization of calpain cleavage sites in ataxin-3 and on the characterization of the resulting breakdown products. After confirming the occurrence of calpain-derived fragmentation of ataxin-3 in patient-derived cell lines and post-mortem brain tissue, we combined in silico prediction tools, western blot analysis, mass spectrometry, and peptide overlay assays to identify calpain cleavage sites. We found that ataxin-3 is primarily cleaved at two sites, namely at amino acid positions D208 and S256 and mutating amino acids at both cleavage sites to tryptophan nearly abolished ataxin-3 fragmentation. Furthermore, analysis of calpain cleavage-derived fragments showed distinct aggregation propensities and toxicities of C-terminal polyglutamine-containing breakdown products. Our data elucidate the important role of ataxin-3 proteolysis in the pathogenesis of Machado-Joseph disease and further emphasize the relevance of targeting this disease pathway as a treatment strategy in neurodegenerative disorders.

Ataxin-3 phosphorylation decreases neuronal defects in spinocerebellar ataxia type 3 models.

Different neurodegenerative diseases are caused by aberrant elongation of repeated glutamine sequences normally found in particular human proteins. Although the proteins involved are ubiquitously distributed in human tissues, toxicity targets only defined neuronal populations. Changes caused by an expanded polyglutamine protein are possibly influenced by endogenous cellular mechanisms, which may be harnessed to produce neuroprotection. Here, we show that ataxin-3, the protein involved in spinocerebellar ataxia type 3, also known as Machado-Joseph disease, causes dendritic and synapse loss in cultured neurons when expanded. We report that S12 of ataxin-3 is phosphorylated in neurons and that mutating this residue so as to mimic a constitutive phosphorylated state counters the neuromorphologic defects observed. In rats stereotaxically injected with expanded ataxin-3-encoding lentiviral vectors, mutation of serine 12 reduces aggregation, neuronal loss, and synapse loss. Our results suggest that S12 plays a role in the pathogenic pathways mediated by polyglutamine-expanded ataxin-3 and that phosphorylation of this residue protects against toxicity.

Proteasome-mediated Proteolysis of the Polyglutamine-expanded Androgen Receptor Is a Late Event in Spinal and Bulbar Muscular Atrophy (SBMA) Pathogenesis

Proteolysis of polyglutamine-expanded proteins is thought to be a required step in the pathogenesis of several neurodegenerative diseases. The accepted view for many polyglutamine proteins is that proteolysis of the mutant protein produces a "toxic fragment" that induces neuronal dysfunction and death in a soluble form; toxicity of the fragment is buffered by its incorporation into amyloid-like inclusions. In contrast to this view, we show that, in the polyglutamine disease spinal and bulbar muscular atrophy, proteolysis of the mutant androgen receptor (AR) is a late event. Immunocytochemical and biochemical analyses revealed that the mutant AR aggregates as a full-length protein, becoming proteolyzed to a smaller fragment through a process requiring the proteasome after it is incorporated into intranuclear inclusions. Moreover, the toxicity-predicting conformational antibody 3B5H10 bound to soluble full-length AR species but not to fragment-containing nuclear inclusions. These data suggest that the AR is toxic as a full-length protein, challenging the notion of polyglutamine protein fragment-associated toxicity by redefining the role of AR proteolysis in spinal and bulbar muscular atrophy pathogenesis.

Spinocerebellar ataxia type 6: Lessons from faithful knock‐in mouse models

AbstractSpinocerebellar ataxia type 6 is a dominantly inherited neurodegenerative disorder characterized by slowly progressive ataxia, slurred speech and nystagmus. The molecular pathogenesis of spinocerebellar ataxia type 6 has attracted considerable attention, because it is caused by the small expansion of a CAG trinucleotide repeat encoding a polyglutamine tract within the Cav2.1 voltage‐gated Ca++ channel. To recreate the characteristics of spinocerebellar ataxia type 6, we produced several lines of knock‐in mice carrying normal or mutant CAG repeat tracts in the Cacna1a locus. The mice expressing a hyperexpanded polyglutamine (Sca684Q) developed late‐onset progressive motor impairment and neuronal inclusion formation in the cytoplasm of cerebellar Purkinje cells. The electrophysiological analysis of the Sca684Q Purkinje cells showed that the repeat expansion did not affect the intrinsic properties of the channel. MPI‐118Q knock‐in mice, which expressed a hyperexpanded channel at modestly enhanced levels because of the insertion of a splice‐site mutation, recapitulated many features of spinocerebellar ataxia type 6, including selective Purkinje cell degeneration. An analysis of the cytoplasmic neuronal inclusions showed the lysosomal localization of accumulated mutant Cav2.1 channels. The lack of cathepsin B, a major lysosomal cysteine proteinase, increased the loss of the Purkinje cells and was accompanied by an acceleration of neuronal inclusion formation in this model. These results suggest that the spinocerebellar ataxia type 6 mutation exerts its neurotoxicity through a mechanism associated with age‐dependent accumulation of the expanded polyglutamine protein, and the pathogenesis of spinocerebellar ataxia type 6 involves the endolysosomal degradation pathway.

RNA interference mitigates motor and neuropathological deficits in a cerebellar mouse model of Machado-Joseph disease.

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.