The Role Of Autophagy On Degradation Of PolyQ-ataxin-3 And On Pathogenesis Of SCA3/MJD Preparation Of Polyclonal Antibody Against Ataxin-3

Background:The hereditary spinocerebellar ataxias (SCAs) are a group of inherited neurodegenerative disorders, which is characterized by accumulation of aberrant protein aggregates in affected neurons. Genetic and transgenic datas suggest that these diseases are caused by codon reiteration mutations, where protein misfolding is mediated by the abnormal expansion of a tract of repeated amino acids. Among them, spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) is the most common subtype. The pathogenic gene for SCA3/MJD has been cloned and designated as MJD1. SCA3/MJD is one of the polyglutamine (polyQ) diseases caused by an expansion of a polyQ stretch near the C-terminus of the MJD1 gene product, ataxin-3. However, the physiological function of ataxin-3 is unknown, and the pathogenesis of the expansion of a polyQ stretch near the C-terminus is still not well illuminated. Studies have found that degeneration of the disease proteins plays a critical role in their physiological function and pathogenesis.Eukaryotic cells have two major protein degradation pathways, the ubiquitin-proteasome pathway and autophagy, which are responsible for cellular homeostasis. Autophagy is a cellular catabolic mechanism mediating the turnover of intracellular long-lived proteins even entire organelles. It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases. Several studies have observed that autophagy is a major route for the degradation of the disease proteins in Alzheimer's disease, Huntington's disease (HD), spinocerebellar ataxia type 1 and Parkinson's disease.Several signalling complexes and pathways are involved in the autophagic route in mammalian cells. And many of these genes remain to be poorly understood. It is widely agreed that the mTOR (mammalian target of rapamycin) signalling pathway negatively regulates autophagy, and LKB1-AMPK pathway plays a role in regulating mTOR. It has found that LKB1, which is the upstream kinase of autophagy, can efficiently phosphorylated AMPK (AMP-activated protein kinase) in vitro specifically at Thr 172 functioning as a upstream regulator of autophagy. The cyclin-dependent kinase inhibitor p27Kipl, is phosphorylated at Thr 198 downstream of the LKB1-AMPK pathway, directly linking sensing of LKB1 and autophagy.In previous work, we have constructed the eukaryotic expression plasmids of wild-type and polyQ-expanded ataxin-3:pCDNA3.1-Myc-His(-)B-ataxin-3-20Q and pCDNA3.1-Myc-His(-)B-ataxin-3-68Q by using recombinant DNA technology.Objective:To research the influence of autophagy on the protein degradation, formation of intracellular protein aggregates, and cell viability of polyQ-expanded ataxin-3; And discuss the role of autophagy on pathogenesis of spinocerebellar ataxia 3/Machado-Josephdisease (SCA3/MJD). Further more, to address the role of LKB1 in degradation of polyQ-expanded ataxin-3, and discuss the mechanism of it.Methods:1. Immunofluorescence-laser cofocalization and western-blot were undertaken to observe the degradation of ataxin-3 influenced by autophagy.2. By Western-blot, we investigated the effect of LKB1 on the protein degradation of polyQ-expanded ataxin-3.3. Immunofluorescence and MTT were used to detect the formation of ataxin-3 aggregates and cell viability modified by autophagy.4. Recombinant DNA technology and Western-blot were undertaken to construct and detect the expression of pGEX-4T-2 prokaryotic expression plasmids of wild-type ataxin-3.5. The expressed proteins were purified from total proteins with Glutathione sepharose 4B agarose column. The New Zealand rabbits were immunized with the purified fusion proteins to prepare polyclonal antiserum.Results:1. We found that ataxin-3-68Q in HEK293T cells expressed in cytoplasm and nucleus, and aggregated in cells which colocalized with GFP-LC3 by Immunofluorescence-laser cofocalization. By western-blot,3-MA or NH4CL treatment led to a significant increase in the levels of ataxin-3-68Q, whereas rapamycin had the opposite effect. These results suggested that the aggregates can be sequestered into autophagic vacuoles. The reduction of autophagy led to the increase of ataxin-3 with expanded polyQ.2. The result from western blot showed that expressing of LKB1 led to a significant increase in the levels of ataxin-3-68Q.3. We discovered by Immunofluorescence-laser that 3-MA treatment resulted in an obvious change in the appearance of the aggregates formed by ataxin-3-68Q constructs transiently transfected into HEK293T cells. In cells with aggregates,3-MA increased their apparent size and number. Treatment with rapamycin after 9 hours of transfection, resulted in a decrease in the proportions of aggregate-containing cells in HEK293T cells expressing ataxin-3-68Q. It indicated that inhibition of autophagy increased aggregate formation in HEK293T cells expressing ataxin-3-68Q, and vice versa. Treatment with rapamycin after 9 hours of transfection, resulted in a decrease in expressing of mutant ataxin-3 in HEK293T cells than that of control cells. But no significant change was observed in cells expressing ataxin-3-68Q treated with rapamycin after 16 hours of transfection. This observation counters the possibility that autophagy can degrade monomeric and oligomeric precursors of aggregates, rather than the large inclusions.4. The MTT assay showed that the cell viability of group of 3-MA obviously decreased in contrast to control group (P<0.05). The cell viability of group of early treatment with rapamycin was higher than that of control group (P<0.05). But the aggregate-containing cells (%) of late treatment with rapamycin group and control group had no significantly different (P>0.05) and the cell viability of which was lower (P<0.01).5. DNA sequencing confirmed that prokaryotic expression plasmids of wild-type ataxin-3-N matched with GenBank standard sequence (S75313). It suggested that prokaryotic expression plasmids constructed successfully when we checked the reading frame of the plasmids without any frameshift after purpose gene order insertion.6. Western-blot and immunoflurescence analysis suggest that the polyclonal antibody raised in the rabbit could recognized ataxin-3.Conclusion:1. We confirm that autophagy is involved in the degradation of mutant ataxin-3.2. We discover that inhibiting or inducing autophagy in early stage after transfection, results in a increase or a decrease in the level of polyglutamine-expanded ataxin-3, respectively.3. For the first time, we find that expressing of LKB1 inhibits the degradation of polyglutamine-expanded ataxin-3.4. We confirm that autophagy induces degradation of mutant ataxin-3, decreases the formation of aggregation and alleviates the protein toxity.5. We prepare anti-ataxin-3 polyclonal antibody which could widely useful in researches of ataxin-3.