calpain and Machado-Joseph-Disease

Spinocerebellar ataxia type 3 (SCA3) is characterized by the over-repetitive CAG codon in the ataxin-3 gene (

Topics: Animals; Ataxin-3; Autophagy; Calpain; Cell Differentiation; Cells, Cultured; Humans; Induced Pluripotent Stem Cells; Machado-Joseph Disease; Male; Phthalic Anhydrides; Proteasome Endopeptidase Complex; Purkinje Cells; Repressor Proteins

Machado-Joseph disease (MJD) is the most frequent dominantly inherited cerebellar ataxia worldwide. Expansion of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within ataxin-3, which upon proteolysis may lead to MJD. The aim of this work was to understand the in vivo contribution of calpain proteases to the pathogenesis of MJD. Therefore, we investigated (a) the calpain cleavage sites in ataxin-3 protein, (b) the most toxic ataxin-3 fragment generated by calpain cleavage and (c) whether targeting calpain cleavage sites of mutant ataxin-3 could be a therapeutic strategy for MJD.. We generated truncated and calpain-resistant constructs at the predicted cleavage sites of ataxin-3 using inverse PCR mutagenesis. Lentiviral vectors encoding these constructs were transduced in the adult mouse brain prior to western blot and immunohistochemical analysis 5 and 8 weeks later.. We identified the putative calpain cleavage sites for both wild-type and mutant ataxin-3 proteins. The mutation of these sites eliminated the formation of the toxic fragments, namely, the 26-kDa fragment, the major contributor for striatal degeneration. Nonetheless, reducing the formation of both the 26- and 34-kDa fragments was required to preclude the intranuclear localisation of ataxin-3. A neuroprotective effect was observed upon mutagenesis of calpain cleavage sites within mutant ataxin-3 protein.. These findings suggest that the calpain system should be considered a target for MJD therapy. The identified calpain cleavage sites will contribute to the design of targeted drugs and genome editing systems for those specific locations.

Topics: Animals; Ataxin-3; Calpain; Corpus Striatum; Machado-Joseph Disease; Mice; Mutation

Proteolytic fragmentation of polyglutamine-expanded ataxin-3 is a concomitant and modifier of the molecular pathogenesis of Machado-Joseph disease (MJD), the most common autosomal dominant cerebellar ataxia. Calpains, a group of calcium-dependent cysteine proteases, are important mediators of ataxin-3 cleavage and implicated in multiple neurodegenerative conditions. Pharmacologic and genetic approaches lowering calpain activity showed beneficial effects on molecular and behavioural disease characteristics in MJD model organisms. However, specifically targeting one of the calpain isoforms by genetic means has not yet been evaluated as a potential therapeutic strategy. In our study, we tested whether calpains are overactivated in the MJD context and if reduction or ablation of calpain-1 expression ameliorates the disease-associated phenotype in MJD cells and mice. In all analysed MJD models, we detected an elevated calpain activity at baseline. Lowering or removal of calpain-1 in cells or mice counteracted calpain system overactivation and led to reduced cleavage of ataxin-3 without affecting its aggregation. Moreover, calpain-1 knockout in YAC84Q mice alleviated excessive fragmentation of important synaptic proteins. Despite worsening some motor characteristics, YAC84Q mice showed a rescue of body weight loss and extended survival upon calpain-1 knockout. Together, our findings emphasize the general potential of calpains as a therapeutic target in MJD and other neurodegenerative diseases.

Topics: Animals; Ataxin-3; Calcium; Calpain; Disease Models, Animal; Female; Machado-Joseph Disease; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Peptides; Phenotype; Proteolysis

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.

Topics: Ataxin-3; Brain; Calpain; Cells, Cultured; Combinatorial Chemistry Techniques; Computer Simulation; Humans; Induced Pluripotent Stem Cells; Machado-Joseph Disease; Peptide Hydrolases; Protein Aggregation, Pathological; Transfection

The neurodegenerative disease Machado-Joseph disease (MJD), also known as spinocerebellar ataxin-3, affects neurons of the brain and spinal cord, disrupting control of the movement of muscles. We have successfully established the first transgenic zebrafish (

Topics: Animals; Animals, Genetically Modified; Ataxin-3; Autophagy; Calpain; Disease Models, Animal; Female; Glycoproteins; Humans; Machado-Joseph Disease; Male; Repressor Proteins; Zebrafish

Spinocerebellar ataxia type 3 or Machado-Joseph disease (SCA3/MJD) is characterized by the repetition of a CAG codon in the ataxin-3 gene (ATXN3), which leads to the formation of an elongated mutant ATXN3 protein that can neither be denatured nor undergo proteolysis in the normal manner. This abnormal proteolysis leads to the accumulation of cleaved fragments, which have been identified as toxic and further they act as a seed for more aggregate formation, thereby increasing toxicity in neuronal cells. To date, there have been few studies or treatment strategies that have focused on controlling toxic fragment formation. The aim of this study is to develop a potential treatment strategy for addressing the complications of toxic fragment formation and to provide an alternative treatment strategy for SCA3. Our preliminary data on anti-aggregation and toxic fragment formation using an HEK (human embryonic kidney cells) 293T-84Q-eGFP (green fluorescent protein) cell model identified n-butylidenephthalide (n-BP) as a potential drug treatment for SCA3. n-BP decreased toxic fragment formation in both SCA3 cell and animal models. Moreover, results showed that n-BP can improve gait, motor coordination, and activity in SCA3 mice. To comprehend the molecular basis behind the control of toxic fragment formation, we used microarray analysis to identify tryptophan metabolism as a major player in controlling the fate of mutant ATXN3 aggregates. We also demonstrated that n-BP functions by regulating the early part of the kynurenine pathway through the downregulation of tryptophan 2, 3-dioxygenase (TDO2), which decreases the downstream neurotoxic product, quinolinic acid (QA). In addition, through the control of TDO2, n-BP also decreases active calpain levels, an important enzyme involved in the proteolysis of mutant ATXN3, thereby decreasing toxic fragment formation and associated neurotoxicity. Collectively, these findings indicate a correlation between n-BP, TDO2, QA, calpain, and toxic fragment formation. Thus, this study contributes to a better understanding of the molecular interactions involved in SCA3, and provides a novel potential treatment strategy for this neurodegenerative disease.

Topics: Animals; Ataxin-3; Calcium; Calpain; Cerebellum; Disease Models, Animal; HEK293 Cells; Humans; Machado-Joseph Disease; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Neuroprotective Agents; Phthalic Anhydrides; Quinolinic Acid; Ryanodine Receptor Calcium Release Channel; Tryptophan; Tryptophan Oxygenase

Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

Topics: Ataxin-3; Binding Sites; Calpain; Cell Line; DNA Breaks, Double-Stranded; Exons; Humans; Machado-Joseph Disease; Mutant Proteins; Oligonucleotides, Antisense; Polyubiquitin; Proteolysis; Repressor Proteins; RNA Precursors

Machado-Joseph Disease (MJD) is the most prevalent autosomal dominantly inherited cerebellar ataxia. It is caused by an expanded CAG repeat in the ATXN3 gene, which translates into a polyglutamine tract within the ataxin-3 protein. Present treatments are symptomatic and do not prevent disease progression. As calpain overactivation has been shown to contribute to mutant ataxin-3 proteolysis, translocation to the nucleus, inclusions formation and neurodegeneration, we investigated the potential role of calpain inhibition as a therapeutic strategy to alleviate MJD pathology. For this purpose, we administered orally the calpain inhibitor BDA-410 to a lentiviral mouse model of MJD. Western-blot and immunohistochemical analysis revealed the presence of N- and C-terminal mutant ataxin-3 fragments and the colocalization of large inclusions with cleaved caspase-3 in the mice brain. Oral administration of the calpain inhibitor BDA-410 decreased both fragments formation and full-length ataxin-3 levels, reduced aggregation of mutant ataxin-3 and prevented cell injury and striatal and cerebellar degeneration. Importantly, in correlation with the preserved cerebellar morphology, BDA-410 prevented motor behavioural deficits. In conclusion, BDA-410 alleviates Machado-Joseph neuropathology and may therefore be an effective therapeutic option for MJD.

Topics: Animals; Calpain; Disease Models, Animal; Gene Expression Regulation; Humans; Machado-Joseph Disease; Mice; Nerve Tissue Proteins; Sulfonamides

Spinocerebellar ataxia type 3 (SCA3) is pathologically characterized by the formation of intranuclear aggregates which contain ataxin-3, the mutated protein in SCA3, in a specific subtype of neurons. It has been proposed that ataxin-3 is cleaved by proteolytic enzymes, in particular by calpains and caspases, eventually leading to the formation of aggregates. In our study, we examined the ability of calpains to cleave ataxin-3 in vitro and in vivo. We demonstrated in cell culture and mouse brain homogenates that cleavage of overexpressed ataxin-3 by calpains and in particular by calpain-2 occur and that polyglutamine expanded ataxin-3 is more sensitive to calpain degradation. Based on these results, we investigated the influence of calpains on the pathogenesis of SCA3 in vivo. For this purpose, we enhanced calpain activity in a SCA3 transgenic mouse model by knocking out the endogenous calpain inhibitor calpastatin. Double-mutant mice demonstrated an aggravated neurological phenotype with an increased number of nuclear aggregates and accelerated neurodegeneration in the cerebellum. This study confirms the critical importance of calcium-dependent calpain-type proteases in the pathogenesis of SCA3 and suggests that the manipulation of the ataxin-3 cleavage pathway and the regulation of intracellular calcium homeostasis may represent novel targets for therapeutic intervention in SCA3.

Topics: Animals; Ataxin-3; Calcium; Calcium-Binding Proteins; Calpain; Cerebellum; Disease Models, Animal; Gene Deletion; Gene Expression Regulation; Gene Knockout Techniques; Genotype; Glycoproteins; HEK293 Cells; Homeostasis; Humans; Immunohistochemistry; Machado-Joseph Disease; Mice; Mice, Knockout; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Peptides; Phenotype; Repressor Proteins; Transcription Factors

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Topics: Active Transport, Cell Nucleus; Adult; Animals; Ataxin-3; Brain Chemistry; Calcium-Binding Proteins; Calpain; Female; Glycoproteins; Humans; Machado-Joseph Disease; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Mutation; Neuroprotective Agents; Nuclear Proteins; Proteolysis; Transcription Factors

Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

Topics: Ataxin-3; Calcium; Calpain; Cells, Cultured; Excitatory Amino Acids; Glutamic Acid; Humans; Machado-Joseph Disease; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Repressor Proteins

The formation of intraneuronal inclusions is a common feature of neurodegenerative polyglutamine disorders, including Spinocerebellar ataxia type 3. The mechanism that triggers inclusion formation in these typically late onset diseases has remained elusive. However, there is increasing evidence that proteolytic fragments containing the expanded polyglutamine segment are critically required to initiate the aggregation process. We analyzed ataxin-3 proteolysis in neuroblastoma cells and in vitro and show that calcium-dependent calpain proteases generate aggregation-competent ataxin-3 fragments. Co-expression of the highly specific cellular calpain inhibitor calpastatin abrogated fragmentation and the formation of inclusions in cells expressing pathological ataxin-3. These findings suggest a critical role of calpains in the pathogenesis of Spinocerebellar ataxia type 3.

Topics: Animals; Antibodies; Ataxin-3; Calpain; Cell Line, Tumor; Enzyme Inhibitors; Humans; Ionomycin; Ionophores; Kidney; Machado-Joseph Disease; Mice; Nerve Tissue Proteins; Neuroblastoma; Nuclear Proteins; Peptides; Rabbits; Rats; Repressor Proteins; Transcription Factors