Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, Madrid, Spain.
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain.
J Cell Mol Med. 2023 Jun;27(11):1557-1564. doi: 10.1111/jcmm.17768. Epub 2023 May 10.
VPS13A is a lipid transfer protein localized at different membrane contact sites between organelles, and mutations in the corresponding gene produce a rare neurodegenerative disease called chorea-acanthocytosis (ChAc). Previous studies showed that VPS13A depletion in HeLa cells results in an accumulation of endosomal and lysosomal markers, suggesting a defect in lysosomal degradation capacity leading to partial autophagic dysfunction. Our goal was to determine whether compounds that modulate the endo-lysosomal pathway could be beneficial in the treatment of ChAc. To test this hypothesis, we first generated a KO model using CRISPR/Cas9 to study the consequences of the absence of VPS13A in HeLa cells. We found that inactivation of VPS13A impairs cell growth, which precludes the use of isolated clones due to the undesirable selection of edited clones with residual protein expression. Therefore, we optimized the use of pool cells obtained shortly after transfection with CRISPR/Cas9 components. These cells are a mixture of wild-type and edited cells that allow a comparative analysis of phenotypes and avoids the selection of clones with residual level of VPS13A expression after long-term growth. Consistent with previous observations by siRNA inactivation, VPS13A inactivation by CRISPR/Cas9 resulted in accumulation of the endo-lysosomal markers RAB7A and LAMP1. Notably, we observed that rapamycin partially suppressed the difference in lysosome accumulation between VPS13A KO and WT cells, suggesting that modulation of the autophagic and lysosomal pathway could be a therapeutic target in the treatment of ChAc.
VPS13A 是一种定位于细胞器之间不同膜接触位点的脂质转移蛋白，其相应基因的突变会导致一种罕见的神经退行性疾病，称为舞蹈棘红细胞增多症（ChAc）。先前的研究表明，HeLa 细胞中 VPS13A 的耗竭会导致内体和溶酶体标记物的积累，这表明溶酶体降解能力的缺陷导致部分自噬功能障碍。我们的目标是确定是否可以调节内体溶酶体途径的化合物对 ChAc 的治疗有益。为了验证这一假设，我们首先使用 CRISPR/Cas9 生成 KO 模型，以研究 VPS13A 在 HeLa 细胞中缺失的后果。我们发现 VPS13A 的失活会损害细胞生长，这使得由于残留蛋白表达的编辑克隆的不良选择，无法使用分离的克隆。因此，我们优化了使用 CRISPR/Cas9 成分转染后不久获得的池细胞的使用。这些细胞是野生型和编辑型细胞的混合物，允许对表型进行比较分析，并避免在长期生长后选择残留 VPS13A 表达水平的克隆。与 siRNA 失活的先前观察结果一致，CRISPR/Cas9 对 VPS13A 的失活导致内体 - 溶酶体标记物 RAB7A 和 LAMP1 的积累。值得注意的是，我们观察到雷帕霉素部分抑制了 VPS13A KO 和 WT 细胞之间溶酶体积累的差异，这表明调节自噬和溶酶体途径可能是 ChAc 治疗的一个治疗靶点。
