Department of Neurology, University of Michigan, Ann Arbor, MI, USA.
Ann Arbor Veterans Administration Healthcare, Ann Arbor, MI, USA.
Sci Adv. 2024 Sep 6;10(36):eadn2321. doi: 10.1126/sciadv.adn2321. Epub 2024 Sep 4.
Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recessively inherited neurodegenerative disorder caused by intronic biallelic, nonreference CCCTT/AAGGG repeat expansions within . To investigate how these repeats cause disease, we generated patient induced pluripotent stem cell-derived neurons (iNeurons). CCCTT/AAGGG repeat expansions do not alter neuronal splicing, expression, or DNA repair pathway function. In reporter assays, AAGGG repeats are translated into pentapeptide repeat proteins. However, these proteins and repeat RNA foci were not detected in iNeurons, and overexpression of these repeats failed to induce neuronal toxicity. CANVAS iNeurons exhibit defects in neuronal development and diminished synaptic connectivity that is rescued by CRISPR deletion of a single expanded AAGGG allele. These deficits were neither replicated by knockdown in control iNeurons nor rescued by RFC1 reprovision in CANVAS iNeurons. These findings support a repeat-dependent but RFC1 protein-independent cause of neuronal dysfunction in CANVAS, with implications for therapeutic development in this currently untreatable condition.
小脑共济失调伴神经病和前庭反射消失综合征（CANVAS）是一种隐性遗传性神经退行性疾病，由. 内含子双等位基因、非参考 CCCTT/AAGGG 重复扩展引起。为了研究这些重复如何导致疾病，我们生成了患者诱导多能干细胞衍生的神经元（iNeurons）。CCCTT/AAGGG 重复扩展不会改变神经元剪接、表达或 DNA 修复途径的功能。在报告基因实验中，AAGGG 重复被翻译成五肽重复蛋白。然而，这些蛋白和重复 RNA 焦点在 iNeurons 中未被检测到，并且这些重复的过表达未能诱导神经元毒性。CANVAS iNeurons 表现出神经元发育缺陷和突触连接减少，通过 CRISPR 删除单个扩展的 AAGGG 等位基因可以挽救。在对照 iNeurons 中敲低这些缺陷既没有被复制，也不能通过在 CANVAS iNeurons 中重新提供 RFC1 来挽救。这些发现支持了 CANVAS 中神经元功能障碍的重复依赖性但 RFC1 蛋白非依赖性原因，这对这种目前无法治疗的疾病的治疗开发具有重要意义。
