The Relationship Between Phosphorylation Of Ataxin-3 And Autophagy

Spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is a kind of autosomal dominant disease, which is characterized by degeneration of neurons and accumulation of aberrant protein aggregates in affected neurons. The pathogenic gene for SCA3/MJD has been cloned and designated as MJD1. SCA3/MJD is caused by the abnormal expansion of a CAG trinucleotide repeat near the C-terminus of MJD1 encoding sequence. Up to present time, the pathogenesis of SCA3/MJD and other polyglutamine (polyQ) disease is still not fully understood, nonetheless it is generally agreed that a toxic gain-of-function effect of the polyglutamine expansion on the polyQ protein, especially the toxic effect of the truncated small polyQ-containing fragments plays a pivotal role in the pathogenesis of polyQ disease. Autophagy is involved in the degradation of polyQ protein, and pharmacological activation of autophagy has been found to lower the levels of polyQ-expanded polyQ proteins, as well as their neurotoxicity in cellular and animal models. Recently several phosphorylation sites have been identified in ataxin-3, and the phosphorylation of some sites affects the degradation, subcellular localization and formation of aggregation of ataxin-3. It is still unclear whether phosphorylation of ataxin-3 is related to the autophagy-mediated cleavage of ataxin-3, neither is its relation to the generation of toxic fragments.After construction of the eukaryotic expression plasmids of a phospho-dead-mutant(S260A) and a phospho-mimic-mutant (S260D), HEK293 cell was transfected with the phospho-mutant or the wild-type expression plasmids and treated with 3-MA or rapamycin, and then the levels of ataxin-3 was detected by Western blotting to illuminate the relationship between phosphorylation of ataxin-3 its autophagy-mediated cleavage, and also the relationship between phosphorylation of ataxin-3 and the generation of toxic fragments.Objective:To study the influence of phosphorylation of ataxin-3 on the degradation of ataxin-3 through autophagy and its effect on the generation of toxic fragments.Methods:1. Construct the eukaryotic expression plasmids of a phospho-dead-mutant(S260A) and a phospho-mimic-mutant (S260D) of ataxin-32.3-MA was used to inhibit autophagy in transfected cells. Western blotting was applied to examine the protein level of ataxin-3 and to detect the toxic fragment of ataxin-3.3. Rapamycin was used to activate autophagy in transfected cells. Western blotting was applied to examine the protein level of ataxin-3 and to detect the toxic fragment of ataxin-3.Results:1.3-MA treatment lead to a significant increase in the protein levels of both wild-type and polyQ-expanded ataxin-3, whereas rapamycin treatment significantly decrease the protein levels of ataxin-3.2. Autophagy inhibition by 3-MA induces the generation of a C-terminal fragment of ataxin-33. Mutation of S260D promotes the degradation of ataxin-3 only when autophagy is activated by Rapamycin. Mutation of S260A had no effect on the degradation of ataxin-3.Conclusion:1 Autophagy is involved in the degradation of ataxin-3.2 When autophagy is activated by Rapamycin, phosphorylation of 260S is involved in mediating the degradation of ataxin-3 through autophagy.3 Autophagy probably mediates the formation of ataxin-3 toxic fragments.