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

1.脊髓小脑性共济失调14型（SCA 14）是一种常染色体显性遗传性神经退行性疾病，其蛋白激酶CY（γPKC）基因存在多个错义突变。我们以前发现SCA 14中发现的突变型γPKC在培养的细胞系中易发生聚集并诱导凋亡。本研究检测了突变型γPKC是否形成聚集以及突变型γPKC如何影响SCA 14患者退化的小脑浦肯野细胞（Purkinje cells，PC）的形态和存活。利用腺病毒载体在小鼠小脑原代培养的PC中表达野生型（WT）和突变型γPKG-GFP，在PC中也形成了突变型γPKC-GFP的聚集体，并且是PC凋亡所必需的。然而，长时间的延时观察发现，其具有清除突变体γPKG-GFP毒性聚集体的保护潜力，并且这种潜力在突变体γPKG-GFP表达撤销后表现突出。突变体γPKG-GFP干扰了 关于我们 PC树突的发育和突触形成减少，无论其聚集体的存在与否。FRAP（Fluorescence Recovery after photobleaching）分析表明，突变型γ PKG-GFP的迁移率降低，这可能导致突变型γPKG-GFP在高浓度KC 1刺激下移位减弱。结果表明，突变型γPKC不仅形成毒性聚集体，而且影响PC树突发育和突触形成，可能与突变型γPKC迁移率降低和移位不足有关。结论：1. PC中突变型γPKC的这些特征可能参与了SCA 14患者小脑皮质的萎缩，进而导致小脑功能障碍.脊髓小脑性共济失调14型（SCA 14）是一种常染色体显性遗传性神经退行性疾病，其蛋白激酶Cγ（γPKC）基因存在多个错义突变。我们以前证明，在SCA 14中发现的突变型γPKC对两种类型的聚集敏感，即胞质点状聚集和核周大量聚集，并导致中国仓鼠卵巢（CHO）细胞的细胞死亡。长时程成像显示，突变体γPKC-GFP首先聚集成点状聚集体，逐渐形成核周块状聚集体，随后细胞死亡。然而，目前尚不清楚突变的γPKC聚集体的形成如何导致细胞死亡。本研究检测了这些突变的聚集体是否影响泛素-蛋白酶体系统（UPS）和内质网（ER）结构。没有突变的γPKG-GFPs（S119 P和G128 D）被强烈泛素化，并且这些突变体的点样聚集体是泛素阳性的并且与蛋白酶体20 S共定位。此外，蛋白酶体活性在细胞聚集，特别是大规模的，显着下降。突变体γPKG-GFP的聚集体形成诱导PERK（PKR样ER激酶）的磷酸化和CHOP（C/EBP同源蛋白）的核表达，这是ER应激的标志，随后激活caspase-3。这些结果表明，在SCA 14中发现的突变型γPKC的聚集体形成损害UPS并诱导ER应激，导致凋亡性细胞死亡。少

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