Disrupted endoplasmic reticulum-mediated autophagosomal biogenesis in a <i>Drosophila</i> model of C9-ALS-FTD
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Macroautophagy/autophagy is a major pathway for the clearance of protein aggregates and damaged organelles, and multiple intracellular organelles participate in the process of autophagy, from autophagosome formation to maturation and degradation. Dysregulation of the autophagy pathway has been implicated in the pathogenesis of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), however the mechanisms underlying autophagy impairment in these diseases are incompletely understood. Since the expansion of GGGGCC (G<sub>4</sub>C<sub>2</sub>) repeats in the first intron of the <i>C9orf72</i> gene is the most common inherited cause of both ALS and FTD (C9-ALS-FTD), we investigated autophagosome dynamics in <i>Drosophila</i> motor neurons expressing 30 G<sub>4</sub>C<sub>2</sub> repeats (30 R). <i>In vivo</i> imaging demonstrates that expression of expanded G<sub>4</sub>C<sub>2</sub> repeats markedly impairs biogenesis of autophagosomes at synaptic termini, whereas trafficking and maturation of axonal autophagosomes are unaffected. Motor neurons expressing 30 R display marked disruption in endoplasmic reticulum (ER) structure and dynamics in the soma, axons, and synapses. Disruption of ER morphology with mutations in Rtnl1 (Reticulon-like 1) or atl (atlastin) also impairs autophagosome formation in motor neurons, suggesting that ER integrity is critical for autophagosome formation. Furthermore, live imaging demonstrates that autophagosomes are generated from dynamic ER tubules at synaptic boutons, and this process fails to occur in a C9-ALS-FTD model. Together, these findings suggest that dynamic ER tubules are required for formation of autophagosomes at the neuromuscular junction, and that this process is disrupted by expanded G<sub>4</sub>C<sub>2</sub> repeats that cause ALS-FTD. 3R: UAS construct expressing 3 G<sub>4</sub>C<sub>2</sub> repeats (used as control); 3WJ: three-way junction; 12R: UAS construct expressing leader sequence and 12 G<sub>4</sub>C<sub>2</sub> repeats; 30R: UAS construct expressing 30 G<sub>4</sub>C<sub>2</sub> repeats; 36R: UAS construct expressing 36 G<sub>4</sub>C<sub>2</sub> repeats; 44R: UAS construct expressing leader sequence and 44 G<sub>4</sub>C<sub>2</sub> repeats; ALS: amyotrophic lateral sclerosis; Atg: autophagy related; atl: atlastin; C9-ALS-FTD: ALS or FTD caused by hexanuleotide repeat expansion in <i>C9orf72</i>; ER: endoplasmic reticulum; FTD: frontotemporal dementia; HRE: GGGGCC hexanucleotide repeat expansion; HSP: hereditary spastic paraplegia; Lamp1: lysosomal associated membrane protein 1; MT: microtubule; NMJ: neuromuscular junction; Rab: Ras-associated binding GTPase; RAN: repeat associated non-AUG (RAN) translation; RO-36: UAS construct expression “RNA-only” version of 36 G<sub>4</sub>C<sub>2</sub> repeats in which stop codons in all six reading frames are inserted.; Rtnl1: Reticulon-like 1; SN: segmental nerve; TFEB/Mitf: transcription factor EB/microphthalmia associated transcription factor (<i>Drosophila</i> ortholog of TFEB); TrpA1: transient receptor potential cation channel A1; VAPB: VAMP associated protein B and C; VNC: ventral nerve cord (spinal cord in <i>Drosophila</i> larvae)
巨自噬（Macroautophagy，又称自噬autophagy）是清除蛋白质聚集物与受损细胞器的主要通路，多种细胞内细胞器参与自噬进程，从自噬体（autophagosome）的形成到成熟与降解均有参与。自噬通路的失调与多种神经退行性疾病的发病机制密切相关，包括肌萎缩侧索硬化（amyotrophic lateral sclerosis, ALS）和额颞叶痴呆（frontotemporal dementia, FTD），但此类疾病中自噬受损的具体分子机制仍未完全阐明。鉴于C9orf72基因第一内含子内的GGGGCC（G₄C₂）重复序列扩增是ALS与FTD最常见的遗传性病因（C9-ALS-FTD），本研究在表达30个G₄C₂重复序列（30 R）的果蝇（Drosophila）运动神经元中探究了自噬体的动态变化。体内（in vivo）成像结果显示，扩增的G₄C₂重复序列的表达会显著损害突触末端的自噬体生物发生过程，而轴突自噬体的运输与成熟则未受影响。表达30 R的运动神经元在胞体、轴突及突触中均表现出内质网（endoplasmic reticulum, ER）的结构与动态变化的显著紊乱。通过Rtnl1（Reticulon-like 1）或atl（atlastin）突变破坏内质网形态，同样会损害运动神经元中的自噬体形成，这表明内质网的完整性对自噬体形成至关重要。此外，活细胞成像证实，自噬体由突触扣结处的动态内质网管状结构生成，而这一过程在C9-ALS-FTD模型中无法正常启动。综上，本研究结果表明，动态内质网管状结构是神经肌肉接头（neuromuscular junction, NMJ）处自噬体形成的必需条件，而致病的扩增G₄C₂重复序列会破坏这一关键过程。 3R：表达3个G₄C₂重复序列的UAS载体（用作对照）； 3WJ：三向连接结构； 12R：包含引导序列与12个G₄C₂重复序列的UAS载体； 30R：表达30个G₄C₂重复序列的UAS载体； 36R：表达36个G₄C₂重复序列的UAS载体； 44R：包含引导序列与44个G₄C₂重复序列的UAS载体； ALS：肌萎缩侧索硬化； Atg：自噬相关（autophagy related）； atl：atlastin； C9-ALS-FTD：由C9orf72基因六核苷酸重复序列扩增导致的ALS或FTD； ER：内质网； FTD：额颞叶痴呆； HRE：GGGGCC六核苷酸重复序列扩增； HSP：遗传性痉挛性截瘫； Lamp1：溶酶体相关膜蛋白1（lysosomal associated membrane protein 1）； MT：微管（microtubule）； NMJ：神经肌肉接头； Rab：Ras相关结合GTP酶（Ras-associated binding GTPase）； RAN：重复序列相关非AUG（RAN）翻译； RO-36：表达36个G₄C₂重复序列的“仅RNA”型UAS载体，该载体在所有六个阅读框中均插入了终止密码子； Rtnl1：Reticulon-like 1； SN：节段神经； TFEB/Mitf：转录因子EB/小眼症相关转录因子（果蝇TFEB的同源物）； TrpA1：瞬时受体电位阳离子通道A1（transient receptor potential cation channel A1）； VAPB：VAMP相关蛋白B和C（VAMP associated protein B and C）； VNC：腹神经索（果蝇幼虫的脊髓）
