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

1. 在脊髓小脑性共济失调14型（SCA14）（常染色体显性神经退行性疾病）中发现了蛋白激酶CY （γPKC）基因的几个因果错义突变。我们之前已经证实，在培养的细胞系中，在SCA14中发现的突变型γPKC易聚集并诱导细胞凋亡。在本研究中，我们研究了突变型γPKC是否形成聚集，以及突变型γPKC如何影响sc14患者退行的小脑浦肯野细胞（PCs）的形态和存活。利用腺病毒载体在小鼠小脑原代培养的PC中表达了野生型（WT）和突变型γ - pkg - gfp，在PC中也形成了突变型γ - pkc - gfp的聚集体，这是PC凋亡所必需的。然而，长期延时观察显示，其具有消除突变体γPKG-GFP毒性聚集体的保护潜力，并且这种潜力在突变体γPKG-GFP表达退出后表现突出。突变体γPKG-GFP干扰了PC树突的发育，减少了突触的形成，无论其聚集体是否存在。FRAP（光漂白后荧光恢复）分析显示突变体γPKG-GFP迁移率降低，这可能导致突变体γPKG-GFP在高KC1刺激下易位减弱。这些结果表明，突变体γPKC不仅形成毒性聚集体，而且影响pc树突发育和突触形成，这可能是由于突变体γPKC迁移率降低和易位不足造成的。这些突变γ - pkc的特征可能参与了sc14患者小脑皮质萎缩并导致小脑功能障碍。脊髓小脑性共济失调14型（SCA14）是一种常染色体显性神经退行性疾病，在脊髓小脑性共济失调14型（SCA14）中发现了蛋白激酶Cγ (γPKC)基因的几个因果错义突变。我们之前已经证明，在SCA14中发现的突变γPKC易发生细胞质点状和核周团块聚集两种类型的聚集，并导致中国仓鼠卵巢（CHO）细胞死亡。长时间延时成像显示，突变体γPKC-GFP最初积聚的点状聚集逐渐形成核周块状聚集，随后细胞死亡。然而，目前尚不清楚突变γPKC的聚集形成如何导致细胞死亡。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（UPS）和内质网（ER）谱。没有突变体γ - pkg - gfp （S119P和G128D）被强烈泛素化，这些突变体的点状聚集是泛素阳性的，并与蛋白酶体20S共定位。此外，有聚集体的细胞，尤其是大质量的细胞，蛋白酶体活性显著降低。突变体γPKG-GFP的聚集形成诱导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应激和随后激活的caspase-3的标志。这些结果表明，在SCA14中发现的突变γPKC的聚集形成损害了UPS并诱导内质网应激，导致凋亡细胞死亡。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