Autolysosome biogenesis and developmental senescence are regulated by both Spns1 and v-ATPase

Spns1 (Spinster homolog 1 [Drosophila]) in vertebrates, as well as Spin (Spinster) in Drosophila, is a hypothetical lysosomal H+-carbohydrate transporter, which functions at a late stage of macroautophagy (hereafter autophagy). The Spin/Spns1 defect induces aberrant autolysosome formation that leads to developmental senescence in the embryonic stage and premature aging symptoms in adulthood. However, the molecular mechanism by which loss of Spin/Spns1 leads to the specific pathogenesis remains to be elucidated. Using chemical, genetic and CRISPR/Cas9-mediated genome-editing approaches in zebrafish, we investigated and determined a mechanism that suppresses embryonic senescence as well as autolysosomal impairment mediated by Spns1 deficiency. Unexpectedly, we found that a concurrent disruption of the vacuolar-type H+-ATPase (v-ATPase) subunit gene, atp6v0ca (ATPase, H+ transporting, lysosomal, V0 subunit ca) led to suppression of the senescence induced by the Spns1 defect, whereas the sole loss of Atp6v0ca led to senescent embryos similar to the single spns1 mutation. Moreover, we discovered that the combined stable defect seen in the presence of both the spns1 and atp6v0ca mutant genes still subsequently induced premature autophagosome-lysosome fusion marked by insufficient acidity, while extending developmental life span, compared with the solely mutated spns1 defect. Our data suggest that Spns1 and the v-ATPase orchestrate proper autolysosomal biogenesis with optimal acidification that is critically linked to developmental senescence and survival.脊椎动物中的Spns1（果蝇Spinster同源物1）以及果蝇中的Spin（Spinster）均为假想的溶酶体H+碳水化合物转运蛋白，在巨自噬（后文简称自噬）的晚期阶段发挥功能。Spin/Spns1缺陷会诱导异常自噬溶酶体形成，进而导致胚胎期发育性衰老以及成年个体出现早衰症状。然而，Spin/Spns1缺失引发上述特异性发病机制的分子机理仍有待阐明。本研究在斑马鱼中利用化学、遗传及CRISPR/Cas9介导的基因组编辑技术，探究并明确了一种可抑制Spns1缺陷介导的胚胎衰老及自噬溶酶体功能损伤的机制。出乎意料的是，同时敲除液泡型H+-ATP酶（v-ATPase）亚基基因atp6v0ca（ATPase, H+ transporting, lysosomal, V0 subunit ca）能够抑制Spns1缺陷诱导的衰老，而单独缺失Atp6v0ca则会导致与spns1单基因突变相似的胚胎衰老表型。此外，研究发现相较于单独的spns1突变缺陷，同时存在spns1与atp6v0ca突变基因的联合稳定功能缺陷虽仍会继而引发以酸化不足为特征的自噬体-溶酶体过早融合，但可延长发育寿命。本研究数据表明，Spns1与v-ATPase协同调控具备最优酸化状态的正常自噬溶酶体生物发生，这一过程与发育衰老及个体存活密切相关。