Al-Azzam Norah, To Jenny H, Gautam Vaishali, Street Lena A, Nguyen Chloe B, Naritomi Jack T, Lam Dylan C, Madrigal Assael A, Lee Benjamin, Jin Wenhao, Avina Anthony, Mizrahi Orel, Mueller Jasmine R, Ford Willard, Schiavon Cara R, Rebollo Elena, Vu Anthony Q, Blue Steven M, Madakamutil Yashwin L, Manor Uri, Rothstein Jeffrey D, Coyne Alyssa N, Jovanovic Marko, Yeo Gene W
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA; Sanford Stem Cell Institute Innovation Center and Stem Cell Program, University of California San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA; Neurosciences Graduate Program, University of California San Diego, San Diego, CA, USA.
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA; Sanford Stem Cell Institute Innovation Center and Stem Cell Program, University of California San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California San Diego, La Jolla, CA, USA.
Neuron. 2024 Dec 18;112(24):4033-4047.e8. doi: 10.1016/j.neuron.2024.10.007. Epub 2024 Oct 31.
Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.
肌萎缩侧索硬化症（ALS）与神经元中某些核孔蛋白的减少有关。参与核孔监测的蛋白——多囊泡体蛋白7（CHMP7）的核定位增加，已被确定为破坏核孔和扰乱运输的关键因素。利用基于CRISPR的微筏技术，随后进行gRNA鉴定（CRaft-ID），我们发现了55种影响CHMP7定位的RNA结合蛋白（RBP），包括运动神经元存活蛋白（SMN）复合体成分SmD1。免疫沉淀-质谱（IP-MS）和增强交联与免疫沉淀（CLIP）分析揭示了CHMP7与运动神经元（MN）中的SmD1、小核RNA和剪接因子mRNA的相互作用。ALS诱导多能干细胞（iPSC）-MN显示SmD1表达降低，抑制SmD1/SMN复合体会增加CHMP7的核定位。至关重要的是，在ALS iPSC-MN中过表达SmD1可恢复CHMP7的细胞质定位并纠正STMN2剪接。我们的研究结果表明，早期ALS发病机制是由SMN复合体失调驱动的。
