Molecular characterization of mutant protein kinase C gamma found in spinocerebellar ataxia type 14 and its application to screening of therapeutics

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1. Several causal missense mutations in protein kinase CY (γPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant γPKC found in SCA14 is susceptible to aggregation and induces apoptosis in cultured cell lines In the present study, we examined whether mutant γPKC formed aggregation and how mutant γPKC affect the morphology and survival of cerebellar Purkinje cells (PCs), which are degenerated in SCA14 patients. Wild type (WT) and mutant γPKG-GFP were expressed in PCs of mouse cerebellar primary culture by using adenoviral vectors Aggregates of mutant γPKC-GFP were also formed in PCs and were indispensable for PC apoptosis. However, the long-term time-lapse observation revealed that Its have a protective potential to eliminate the toxic aggregates of mutant γPKG-GFP and this potential appeared prominently after the withdrawal of mutant γPKG-GFP expression. Mutant γPKG-GFP disturbed the … More development of PC dendrites and reduced synapse formation, regardless of presence or absence of its aggregate. FRAP (Fluorescence recovery after photobleaching) analysis revealed the reduced mobility of mutant γPKG-GFP, which may cause the attenuated translocation of mutant γPKG-GFP upon the stimulation of high KC1 These results indicate that mutant γPKC not only forms toxic aggregates but also affects the dendritic development and the synaptic formation in PCs, probably due to the reduced mobility and the insufficient translocation of mutant γPKC. These characters of mutant γPKC in PCs would be involved in the atrophy of cerebellar cortex and subsequently caused cerebellar dysfunction in SCA14 patients.2. Several causal missense mutations in protein kinase Cγ (γPKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant γPKC found in SCA14 is susceptible to two types of aggregation, cytoplasmic dot-like and perinuclear massive aggregation, and causes cell death in Chinese hamster ovary (CHO) cells. Long-term time-lapse imaging revealed that firstly-accumulated dot-Like aggregation of mutant γPKC-GFP gradually formed perinuclear massive aggregations, followed by cell death. However, it remains unclear how aggregate formation of mutant γPKC causes cell death In the present study, we examined whether these mutant aggregations affect the ubiquitin-proteasome system (UPS) and endoplasmic reticular (ER) strum. 'No mutant γPKG-GFPs (S119P and G128D) were strongly ubiquitinated, and dot-like aggregations of these mutants were ubiquitin-positive and colocalized with proteasome 20S. Furthermore, proteasome activity in cells with aggregates, especially massive ones, was significantly decreased. Aggregate formation of mutant γPKG-GFP induced phosphorylation of PERK (PKR-like ER kinase) and nuclear expression of CHOP (C/EBP homologous protein), hallmarks of ER stress and subsequently activated caspase-3. These results indicate that aggregate formation of mutant γPKC found in SCA14 impairs UPS and induces ER stress, leading to apoptotic cell death. Less