Department of Clinical Genetics, ErasmusMC University Medical Center, 3015 CN, Rotterdam, the Netherlands.
Division of Pediatric Genetics, Department of Pediatrics, Prince Sultan Military Medical City, Riyadh, 12233, Saudi Arabia.
Hum Genet. 2023 Mar;142(3):379-397. doi: 10.1007/s00439-022-02511-3. Epub 2022 Dec 20.
CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development.
CLEC16A 是一种膜相关的 C 型凝集素蛋白，作为 E3-泛素连接酶发挥作用。CLEC16A 调节自噬和线粒体自噬，并据报道定位于晚期内体。GWAS 研究将 CLEC16A 单核苷酸多态性与各种自身免疫和神经退行性疾病相关联，包括多发性硬化症和帕金森病。在小鼠模型中的研究表明 CLEC16A 在神经退行性变中起作用。我们在来自无关家庭的患有严重神经发育障碍的兄弟姐妹中鉴定出双等位 CLEC16A 截断变异，包括小头畸形、脑萎缩、胼胝体发育不良和生长迟缓。为了了解 CLEC16A 在神经发育中的功能，我们使用了体外模型和斑马鱼胚胎。我们观察到 CLEC16A 在 HEK293T 细胞中定位于早期内体。人类 CLEC16A 的质谱分析显示与内体逆行运输复合物亚基和内体泛素连接酶 TRIM27 相互作用。表达导致 C 端截断 CLEC16A 的人变体，消除了其在内体中的定位和与 TRIM27 的相互作用，表明丧失功能效应。CLEC16A 敲低增加了 TRIM27 对早期内体的粘附和内体 F-肌动蛋白的异常积累，这是囊泡分选受损的标志。CRISPR-Cas9 对斑马鱼胚胎中的 clec16a 进行诱变导致神经元和小胶质细胞中酸性/吞噬溶酶体隔间的积累和失调的线粒体自噬。自噬表型通过野生型人 CLEC16A 而不是 C 端截断的 CLEC16A 得到挽救。我们的结果表明，CLEC16A 与逆行运输成分密切相互作用，并通过精细调节内体表面上的 TRIM27 和聚合 F-肌动蛋白的水平来调节内体命运。CLEC16A 介导的内体分选的失调与神经退行性变有关，但它也会导致脑发育过程中自噬体和不健康线粒体的积累。
