Abstract：
Spinocerebellar ataxia type 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of ataxin-3 protein aggregates, neurodegeneration, and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model SCA3. SCA3 SH-SY5Y cells were found to contain high molecular weight ataxin-3 species and detergent-insoluble protein aggregates. Treatment with SB increased the activity of the autophagy protein quality control pathway in the SCA3 cells, decreased the presence of ataxin-3 aggregates and presence of high molecular weight ataxin-3 in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing activity of the autophagy pathway, and decreasing the presence of insoluble ataxin-3 protein species in the transgenic SCA3 zebrafish. Co-treating the SCA3 zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on zebrafish swimming, indicating that the improved swimming performance was autophagy-dependent. To understand the mechanism by which SB induces autophagy we performed proteomic analysis of protein lysates from the SB-treated and untreated SCA3 SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling, with immunoblot analysis confirming that SB treatment had increased levels of AMPK protein and its substrates. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway process and that this has beneficial effects in vitro and in vivo. While our results suggested that this activity may involve activity of a PKA/AMPK-dependent process, this requires further confirmation. We propose that treatment with sodium butyrate warrants further investigation as a potential treatment for neurodegenerative diseases underpinned by mechanisms relating to protein aggregation including SCA3.
摘要：
脊髓小脑共济失调3型（SCA3，也称为MachadoJoseph病）是由ATXN3/MJD基因内的三核苷酸重复区域扩展引起的致命神经退行性疾病。ATXN3的突变导致ataxin-3蛋白聚集体的形成，神经变性,和运动障碍。在这里，我们调查了丁酸钠（SB）的治疗潜力和机制活性，丁酸的钠盐，肠道微生物群自然产生的代谢产物，在培养的SH-SY5Y细胞和表达含有84个谷氨酰胺（Q）残基的人ataxin-3的转基因斑马鱼上建立SCA3模型。发现SCA3SH-SY5Y细胞含有高分子量的ataxin-3物种和洗涤剂不溶性蛋白质聚集体。SB处理增加了SCA3细胞自噬蛋白质量控制途径的活性，以自噬依赖的方式减少了ataxin-3聚集体的存在和高分子量ataxin-3的存在。用SB治疗在体内也是有益的，提高游泳性能，增加自噬途径的活性，并减少转基因SCA3斑马鱼中不溶性共济失调蛋白3的存在。用SB和氯喹共同处理SCA3斑马鱼，自噬抑制剂，防止SB对斑马鱼游泳的有益影响，表明游泳表现的改善是自噬依赖性的。为了了解SB诱导自噬的机制，我们对SB处理和未处理的SCA3SH-SY5Y细胞的蛋白质裂解物进行了蛋白质组学分析。我们发现SB处理增加了蛋白激酶A和AMPK信号的活性,免疫印迹分析证实，SB治疗增加了AMPK蛋白及其底物的水平。我们的发现表明，用SB治疗可以增加自噬途径过程的活性，这在体外和体内都有有益的作用。虽然我们的结果表明这种活性可能涉及PKA/AMPK依赖性过程的活性，这需要进一步确认。我们建议用丁酸钠治疗值得进一步研究，作为神经退行性疾病的潜在治疗方法，其基础是与包括SCA3在内的蛋白质聚集相关的机制。
