Summary statistics for Fig 2C.

Motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) and progressive bulbar palsy, involve loss of muscle control resulting from death of motor neurons. Although the exact pathogenesis of these syndromes remains elusive, many are caused by genetically inherited mutations. Thus, it is valuable to identify additional genes that can impact motor neuron survival and function. In this report, we describe mice that express globally reduced levels of calcium-modulating cyclophilin ligand (CAML) protein. CAML is an essential component in the transmembrane domain recognition complex (TRC) pathway, responsible for inserting C-terminal tail anchored (TA) proteins into the endoplasmic reticulum membrane. The primary phenotype observed in these mice was rapid development of hind limb weakness and paralysis. Spinal cord sections revealed a loss of motor neuron cell bodies. Targeting CAML loss specifically to neurons using SLICK-H-Cre or synapsin-Cre transgenic mice yielded similar phenotypes, indicating that CAML plays a cell autonomous role in this process. We found that intracellular trafficking was perturbed in cells depleted of CAML, with aberrant release of procathepsin D and defective retention of CD222 within the trans-Golgi network, as well as reduced levels and mislocalization of syntaxin 5 (Stx5). Dysfunctional lysosomes and abnormal protein glycosylation were also revealed in CAML deficient cells, further indicating a defect in Golgi trafficking. In addition, we observed an identical phenotype in mice lacking ASNA1 in neurons, suggesting that CAML’s role in sustaining muscle function is related to its involvement in the TRC pathway. Together, these findings implicate motor neuron survival as a key role for the TA protein insertion machinery in mice, which may shed light on the pathogenesis of neuromuscular disease in humans.

运动神经元病（Motor neuron diseases），例如肌萎缩侧索硬化症（amyotrophic lateral sclerosis, ALS）与进行性延髓麻痹（progressive bulbar palsy），均因运动神经元死亡引发肌肉控制能力丧失。尽管此类综合征的确切发病机制仍不明晰，但多数病例由遗传性基因突变所致。因此，挖掘可影响运动神经元存活与功能的新型基因具有重要研究价值。本研究中，我们构建了全局钙调亲环蛋白配体（calcium-modulating cyclophilin ligand, CAML）蛋白表达水平降低的小鼠模型。CAML是跨膜结构域识别复合物（transmembrane domain recognition complex, TRC）通路的关键组成部分，负责将C端锚定（C-terminal tail anchored, TA）蛋白插入内质网膜。该模型小鼠的核心表型为快速出现后肢无力与瘫痪。脊髓组织切片观察显示，小鼠运动神经元胞体出现丢失。利用SLICK-H-Cre或synapsin-Cre转基因小鼠，实现神经元特异性的CAML敲除后，可得到相似的表型，表明CAML在此过程中发挥细胞自主性功能。我们发现，CAML敲低的细胞内体运输过程受到扰动，表现为组织蛋白酶D前体（procathepsin D）异常释放、CD222在反式高尔基体网络（trans-Golgi network）内的滞留功能受损，以及syntaxin 5（Stx5）的表达水平降低与定位异常。CAML缺陷细胞中还观察到溶酶体功能异常与蛋白质糖基化紊乱，进一步证实高尔基体运输存在缺陷。此外，我们在神经元特异性敲除ASNA1的小鼠中观察到了相同的表型，提示CAML维持肌肉功能的作用与其参与TRC通路密切相关。综上，本研究结果表明，TA蛋白插入机器在小鼠运动神经元存活过程中发挥关键作用，这一发现可为阐明人类神经肌肉疾病的发病机制提供新的思路。