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

1.在常染色体显性遗传性神经退行性疾病脊髓小脑性共济失调14型(SCA14)中发现了蛋白激酶CY(γ)基因的几个原因错义突变。在本研究中，我们研究了在SCA14患者中发现的突变的γ蛋白激酶C对聚集和诱导细胞系凋亡的敏感性。我们研究了突变的γ蛋白激酶C是否形成聚集，以及突变的γ蛋白激酶C如何影响小脑浦肯野细胞的形态和存活。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，并在PC中形成突变型γPKC-GFP聚集体，对PC的凋亡是必不可少的。然而，长期的时间推移观察表明，ITS具有清除突变的γPKG-GFP的毒性聚集体的保护性潜力，并且这种潜力在突变的γPKG-GFP表达取消后显著出现。突变体γpKG-gfp干扰…无论其聚集体的存在与否，PC树突的更多发育和突触形成的减少。FRAP分析显示突变型γPKG-GFP的迁移率降低，这可能导致突变型γPKG-GFP在高Kc1刺激下的移位减弱。结果表明，突变型γPKC不仅形成有毒聚集体，而且影响PC树突状细胞的发育和突触的形成，可能是由于突变型γPKC的迁移率降低和移位不足所致。结论：1.PC中突变的γ蛋白激酶C的这些特征可能参与了SCA14患者小脑皮质的萎缩，进而导致小脑功能障碍。在常染色体显性遗传性神经退行性疾病脊髓小脑性共济失调14型(SCA14)中发现了蛋白激酶C(Protein Kinase C，γ(γ)基因的几个原因错义突变。我们先前的研究表明，在中国仓鼠卵巢(CHO)细胞中发现的突变的γ蛋白激酶C对两种类型的聚集--胞浆点状聚集和核周大量聚集--敏感，并导致细胞死亡。长期时移成像显示，突变体γPKC-GFP首先积累的点状聚集逐渐形成核周大量聚集，随后细胞死亡。然而，目前尚不清楚突变的γ蛋白激酶C的聚集如何导致细胞死亡。在本研究中，我们研究了这些突变聚集是否影响泛素-蛋白酶体系统和内质网(ER)链。没有突变的γPKG-GFP(S119P和G128D)被强烈泛素化，这些突变的点状聚集体是泛素阳性的，并与蛋白酶体20S共定位。此外，有聚集体的细胞中的蛋白酶体活性显著降低。突变的γPKG-GFP聚集体的形成诱导了PERK的磷酸化和CHOP(C/EBP同源蛋白)的核表达，这是ER应激的标志，随后激活了caspase-3。这些结果表明，在SCA14中发现的突变的γPKC聚集体的形成损害了UPS并诱导了内质网应激，导致细胞死亡较少。

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