Summary statistics for Fig 6A.

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），如肌萎缩侧索硬化（amyotrophic lateral sclerosis, ALS）与进行性延髓麻痹（progressive bulbar palsy），是因运动神经元死亡导致肌肉控制丧失的一类疾病。尽管此类综合征的确切发病机制仍不明朗，但多数病例由遗传性突变引发。因此，鉴定可影响运动神经元存活与功能的新基因具有重要研究价值。本研究报道了一类全身性钙调节亲环蛋白配体（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）通路的必需组分，该通路负责将C端锚定（C-terminal tail anchored, TA）蛋白插入内质网膜。此类小鼠的核心表型为快速出现后肢无力与麻痹。脊髓切片观察显示运动神经元胞体丢失。通过SLICK-H-Cre或突触素-Cre（synapsin-Cre）转基因小鼠将CAML缺失特异性靶向神经元后，可得到相似表型，表明CAML在此过程中发挥细胞自主作用。研究发现，CAML缺失的细胞内运输过程受到干扰，表现为组织蛋白酶D前体（procathepsin D）异常释放、CD222在反式高尔基网络（trans-Golgi network）中的滞留缺陷，以及突触融合蛋白5（syntaxin 5, Stx5）的表达水平降低与定位异常。CAML缺陷细胞中还存在溶酶体功能异常与蛋白质糖基化异常，进一步提示高尔基体内运输存在缺陷。此外，在神经元特异性缺失ASNA1的小鼠中，我们也观察到了相同的表型，这表明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蛋白插入系统在小鼠运动神经元存活过程中发挥关键作用，这可为人类神经肌肉疾病的发病机制研究提供新的线索。