ABSTRACT

Macroautophagy/autophagy is essential for maintaining cellular homeostasis through the degradation of organelles and proteins. It also has a prominent role in modulating aging. However, the role of autophagy in the neuronal response to axon injury and axon regeneration, particularly in the context of aging, remains largely unknown. Our candidate genetic screen for axon regeneration regulators has identified genes in the autophagy pathway. Using a reporter that monitors autophagosomes and autolysosomes, we were able to monitor the dynamics of autophagy during axon regeneration. In response to axon injury, there was a significant increase in the number of autophagic vesicles. Injury-triggered autophagy activation and axon regeneration capacity undergo an age-dependent decline, and autophagy-activating agents partially rescued these declines. We found that DLK-1 was both required and sufficient for injury-induced autophagy activation. Autophagic vesicles co-localized with the NOTCH4 ortholog, LIN-12 receptor, a previously identified inhibitor of axon regeneration. Epistasis analyses indicate that LIN-12 might be a target of autophagy in axon regeneration. Together, our data suggest that DLK-mediated injury signaling can activate autophagy, which might limit the level of LIN-12 and NOTCH proteins to promote axon regeneration. Our findings reveal that autophagy activation can promote axon regeneration in neurons that lack maximal regrowth capacity, providing a promising therapeutic strategy for axon injury.

Abbreviations: 3-MA: 3-methyladenine; ALs: autolysosomes; APs: autophagosomes; ARF-6: ADP-Ribosylation Factor related 6; ATG-9: AuTophaGy (yeast Atg homolog) 9; ATG9A: autophagy related 9A; BA1: bafilomycin A₁; BEC-1: BEClin (human autophagy) homolog; BECN1: beclin 1; C. elegans: Caenorhabditis elegans; CEBP-1: C/EBP (CCAAT/enhancer-binding protein) homolog; CNS: central nervous system; DLK-1: Dual-Leucine zipper Kinase; DMSO: dimethyl sulfoxide; DRG: dorsal root ganglion; FOS: Fos proto-oncogene, AP-1 transcription factor subunit; GABA: gamma-aminobutyric acid; GFP: green fluorescent protein; HDA-3: Histone DeAcetylase; IP3: inositol trisphosphate; ITR-1: Inositol Triphosphate Receptor; KLF-2: Kruppel-Like Factor (zinc finger protein) 2; LGG-1: LC3, GABARAP and GATE-16 family; MAK-2: MAP kinase Activated protein Kinase; MAP kinase: mitogen-activated protein kinase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKK-4: mitogen activated protein kinase kinase 4; MTOR: mechanistic target of rapamycin kinase; NGM: nematode growth medium; NICD: Notch intracellular domain; NOTCH: notch receptor; PLM: posterior lateral microtubule; PMK-3: P38 Map kinase family; PNS: peripheral nervous system; SCG10: superior cervical ganglion protein 10; SCI: spinal cord injury; UNC-51: UNCoordinated 51; ULK1: unc-51 like autophagy activating kinase 1; wnd: wallenda

摘要

巨自噬/自噬对于通过降解细胞器和蛋白质来维持细胞稳态至关重要。它在调节衰老方面也有重要作用。然而，自噬在神经元对轴突损伤和轴突再生的反应中的作用，特别是在衰老的背景下，仍然很大程度上是未知的。我们的轴突再生调节剂候选基因筛选已经确定了自噬途径中的基因。使用监测自噬体和自溶酶体的报告器，我们能够监测轴突再生过程中自噬的动态。作为对轴突损伤的反应，自噬囊泡的数量显着增加。损伤触发的自噬激活和轴突再生能力经历年龄依赖性下降，而自噬激活剂部分挽救了这些下降。我们发现 DLK-1 是损伤诱导的自噬激活所必需和充分的。自噬囊泡与 NOTCH4 直系同源物 LIN-12 受体共定位，这是一种先前确定的轴突再生抑制剂。上位性分析表明 LIN-12 可能是轴突再生中自噬的靶标。总之，我们的数据表明 DLK 介导的损伤信号可以激活自噬，这可能会限制 LIN-12 和 NOTCH 蛋白的水平以促进轴突再生。我们的研究结果表明，自噬激活可以促进缺乏最大再生能力的神经元中的轴突再生，为轴突损伤提供有希望的治疗策略。先前确定的轴突再生抑制剂。上位性分析表明 LIN-12 可能是轴突再生中自噬的靶标。总之，我们的数据表明 DLK 介导的损伤信号可以激活自噬，这可能会限制 LIN-12 和 NOTCH 蛋白的水平以促进轴突再生。我们的研究结果表明，自噬激活可以促进缺乏最大再生能力的神经元中的轴突再生，为轴突损伤提供有希望的治疗策略。先前确定的轴突再生抑制剂。上位性分析表明 LIN-12 可能是轴突再生中自噬的靶标。总之，我们的数据表明 DLK 介导的损伤信号可以激活自噬，这可能会限制 LIN-12 和 NOTCH 蛋白的水平以促进轴突再生。我们的研究结果表明，自噬激活可以促进缺乏最大再生能力的神经元中的轴突再生，为轴突损伤提供有希望的治疗策略。

缩写：3-MA：3-甲基腺嘌呤；ALs：自溶酶体；APs：自噬体；ARF-6：ADP-核糖基化因子相关 6；ATG-9：AuTophaGy（酵母 Atg 同源物）9；ATG9A：自噬相关的9A；BA1：巴弗洛霉素A ₁；BEC-1：BEClin（人类自噬）同源物；BECN1：beclin 1；C. elegans：秀丽隐杆线虫; CEBP-1：C/EBP（CCAAT/增强子结合蛋白）同源物；CNS：中枢神经系统；DLK-1：双亮氨酸拉链激酶；DMSO：二甲亚砜；DRG：背根神经节；FOS：Fos原癌基因，AP-1转录因子亚基；GABA：γ-氨基丁酸；GFP：绿色荧光蛋白；HDA-3：组蛋白去乙酰化酶；IP3：三磷酸肌醇；ITR-1：肌醇三磷酸受体；KLF-2：Kruppel-Like Factor（锌指蛋白）2；LGG-1：LC3、GABARAP 和 GATE-16 家族；MAK-2：MAP激酶活化蛋白激酶；MAP激酶：丝裂原活化蛋白激酶；MAP1LC3/LC3：微管相关蛋白1轻链3；MKK-4：丝裂原活化蛋白激酶激酶 4；MTOR：雷帕霉素激酶的机制靶点；NGM：线虫生长培养基；NICD：Notch 胞内结构域；NOTCH：缺口受体；PLM：后外侧微管；PMK-3：P38 映射激酶家族；PNS：周围神经系统；SCG10：颈上神经节蛋白10；SCI：脊髓损伤；UNC-51：联合国协调 51；ULK1：unc-51 样自噬激活激酶 1；wnd：瓦伦达