Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo and modulates differential splicing. Inhibition of the autophagic protein ULK1 attenuates axonal degeneration in vitro and in vivo and modulates differential splicing

Several traumatic and neurodegenerative disorders of the CNS show axonal degeneration as a key and early pathological feature. Following a focal axonal lesion, an extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, activation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we overexpressed a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons in vitro and in the spinal cord (SC) and optic nerve (ON) in vivo. We found a significant inhibitory effect on autophagy in vitro and in vivo. While no effect on cell survival could be observed, ULK1.DN attenuated AAD after axotomy in microfluidic chambers in vitro. Models of SC injury and ON crush in vivo confirmed an attenuation of axonal degeneration by ULK1.DN. In a translational approach, we employed the selective ULK1 inhibitor SBI-0206965 (SBI) and found an attenuation of AAD after ON crush in vivo mediated by a reduction of autophagy by SBI. Quantitative proteomic profiling after ULK1.DN overexpression in vitro revealed a regulation of proteins associated with translation and splicing. A differential exon expression analysis identified the genes kinesin family member 1B (Kif1b), DNA damage inducible transcript 3 (Ddit3) and WD repeat domain 6 (Wdr6) as targets of ULK1.DN and potential mediators of its degeneration-attenuating effect. These findings reveal ULK1 as an important mediator of axonal degeneration and elucidate its function in splicing, defining it as a putative therapeutic target. Overall design: Analysis of differential exon and gene expression in primary E18 rat cortical neurons (DIV 8) after transduction with AAV.ULK1.DN and AAV.mCherry (control, Genbank ID: KT345943), from 3 independent cultures.

多种中枢神经系统（CNS）创伤性与神经退行性疾病均以轴突退变作为核心且早期的病理特征。局灶性轴突损伤后，数小时内便会发生急性轴突退变（AAD）介导的广泛轴突崩解。研究已证实，急性轴突退变过程中会激活包括Unc-51样自噬激活激酶1（ULK1）在内的自噬相关蛋白，但其具体作用尚未完全阐明。本研究分别在体外将显性负性ULK1突变体（ULK1.DN）过表达于大鼠原代皮层神经元，以及在体内将其递送至脊髓（SC）与视神经（ON）。我们发现该操作在体内外均可显著抑制自噬。尽管未观察到对细胞存活的影响，但ULK1.DN可在体外微流控培养腔模型中减轻轴突切断后的急性轴突退变。脊髓损伤与视神经钳夹伤的体内模型进一步证实，ULK1.DN可缓解轴突退变。在转化研究中，我们使用了选择性ULK1抑制剂SBI-0206965（SBI），发现其可通过抑制自噬减轻体内视神经钳夹伤后的急性轴突退变。体外过表达ULK1.DN后的定量蛋白质组学分析显示，与翻译及RNA剪接相关的蛋白表达发生调控。差异外显子表达分析鉴定出驱动蛋白家族成员1B（Kif1b）、DNA损伤诱导转录蛋白3（Ddit3）及WD重复结构域6（Wdr6）为ULK1.DN的作用靶点，同时也是其减轻轴突退变效应的潜在介导因子。上述研究结果表明ULK1是轴突退变的重要介导因子，并阐明了其在RNA剪接中的功能，将其定为潜在的治疗靶点。整体实验设计：对3批独立培养的胚胎18天（E18）大鼠皮层神经元（体外培养至第8天，DIV 8），分别转导腺相关病毒（AAV）.ULK1.DN与腺相关病毒（AAV）.mCherry（对照组，GenBank登录号：KT345943），并分析其差异外显子与基因表达情况。