sq-23377 and Machado-Joseph-Disease

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study, induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation, expressed comparable levels of several neuronal markers and responded to the neurotransmitters, glutamate/glycine, GABA and acetylcholine. Additionally, all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation, and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity, although not comparable to that of the adult human brain. Interestingly, we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion, we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity, which could potentially be valuable for the study of SCA3 disease mechanisms.

Topics: Antigens, Nuclear; Ataxin-3; Brain; Calcium; Cell Differentiation; Cell Line; Cellular Reprogramming; Fibroblasts; Humans; Induced Pluripotent Stem Cells; Ionomycin; Karyotype; Machado-Joseph Disease; Microscopy, Fluorescence; Nerve Tissue Proteins; Neural Stem Cells; Protein Aggregates; Repressor Proteins; Synapsins; Transcription Factors

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