The Cell-based Functional Study Of TGM6, A Causative Gene Of Spinocerebellar Ataxia Type35

Autosomal dominant spinocerebellar ataxias constitute a large, heterogeneous group of progressive neurodegenerative diseases with multiple types. Mean age of onset is approximately30to40years old, and its main pathological change is the atrophy of cerebellum and brainstem. It is characterized by progressive cerebellar ataxia, ataxia of gait and dysarthria and sometimes accompanied by ophthalmoplegia, cognitive dysfunction, peripheral neuropathy, pyramidal sign and retinitis pigmentosa.To date, more than30distinct forms of spinocerebellar ataxias have been identified. In our previous study, we have set up a genetic diagnosis platform according to the genetic spectrum and CAG trinucleotide repeat numbers of SCA subtypes in Chinese Han population. We concluded that the most common type of SCAs in Chinese Han population is SCA3, followed by SCA2, SCA1, and SCA7.Whole exome sequencing technology, as the representative of a new generation of sequencing technology with outstanding technical advantages of high-throughput, high-precision, high sensitivity and low running cost, has created a new pathway for studying human genetic diseases in recent years. In2010, we identified a novel spinocerebellar ataxia causative gene TGM6in two Chinese families by using combined strategy of exome sequencing and linkage analysis. Two missense mutations in TGM6(D327G and L517W) were identified. In2012, scholars from Hong Kong identified another new form of missense mutation (D150H) in TGM6gene, which can also lead to the occurrence of the SCA35in a Chinese family, further confirming our previous work. TGM6encodes transglutaminase6protein, which belongs to transglutaminase family. Transglutaminases are a large family of calcium-dependent enzymes, that catalyze an acyl transfer reaction between the y-carboxamide of a peptide-bound glutamine residue and the nucleophilic groups, resulting in an isopeptide bond. TG6was first reported in2001, however, there is no functional studies on TG6until now.To further study the molecular mechanism of SCA35, we established cell models by overexpressing wild-type and mutant TG6proteins, and then performed a series of in vitro functional studies, including detect the expression level by western blot, TG6degradation, transglutaminase activity assay, immunofluorescence assays to study subcellular localization of TG6, GST pull down and co-immunopreciptation assays to detect the interaction between polyQ proteins and TG6, cell apoptosis assays to explore the role of TG6in apoptosis.Conclusions:(1) We constructed wild-type and two mutant form of eukaryotic and prokaryotic TGM6expression plasmids, and established the basis for studying the mechanism of SCA35.(2) Transfecting the wild-type and two mutant eukaryotic TGM6expression plasmids into HEK293cells by using lipofectamine and detecting the expression level by western blot, we found that the expression of three proteins had no significant difference.(3) Chase time experiment concluded that both mutant TG6exhibited a significantly shorter half-life, suggesting that SCA35-causing mutations resulted in destabilization of the TG6protein and tended to be degraded easily. (4) Cell immunofluorescence assays showed that both wild-type and mutant TG6were mainly localized in the cytoplasm and did not co-localize with any of (ER, Golgi, and lysosome) markers.(5) Transglutaminase activity assay showed that the lysates from cells transfected with either of the mutant TG6constructs exhibited significantly reduced enzymatic activity, especially the L517W mutant form.(6) GST pull-down assay and co-immunoprecipitation showed that both wild-type and mutant TG6interacted with PolyQ proteins (ataxin-3-20Q、ataxin-3-70Q、Htt-150Q). Both wild-type and mutant TG6co-localized with PolyQ proteins in HEK293cells and could promoted the formation of polyQ aggregates by converting soluble PolyQ into insoluble PolyQ aggregates.(7) Overexpression of TG6had no effect on the apoptosis rate of HEK293and NIH3T3cells. The cell lines stably expressing mutant TG6exhibited significantly increased levels of apoptosis induced by A23187or STS. TG6mutants sensitized cells to apoptosis by increasing the activities of caspases, which may underlie the pathogenesis of SCA35.