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.多种中枢神经系统（central nervous system, CNS）创伤性与神经退行性疾病均以轴突变性作为关键且早期的病理特征。局灶性轴突损伤后，急性轴突变性（acute axonal degeneration, AAD）可在数小时内引发大范围轴突崩解。在急性轴突变性进程中，包括Unc-51样自噬激活激酶1（Unc-51 like autophagy activating kinase 1, ULK1）在内的自噬蛋白已被证实激活，但其具体作用仍未完全明确。本研究通过体外实验，将显性负效突变型ULK1（ULK1.DN）过表达至原代大鼠皮层神经元，并在体内将其递送至脊髓（spinal cord, SC）与视神经（optic nerve, ON）。结果显示，无论体外还是体内环境中，该突变体均可显著抑制自噬活性。尽管未观察到其对细胞存活产生影响，但ULK1.DN可在体外微流控腔室中，减轻轴突切断术后的急性轴突变性。体内脊髓损伤与视神经钳夹模型进一步证实，ULK1.DN可缓解轴突变性进程。在转化研究策略中，本研究使用选择性ULK1抑制剂SBI-0206965（SBI），发现其可通过抑制自噬，在体内减轻视神经钳夹后的急性轴突变性。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显著调控。差异外显子表达分析鉴定出驱动蛋白家族成员1B（kinesin family member 1B, Kif1b）、DNA损伤诱导转录因子3（DNA damage inducible transcript 3, Ddit3）以及WD重复结构域6（WD repeat domain 6, Wdr6）为ULK1.DN的作用靶点，同时也是其缓解轴突变性效应的潜在介导因子。本研究结果揭示ULK1是急性轴突变性的重要调控介质，并阐明了其在RNA剪接中的功能，使其成为潜在的治疗靶点。