Curiel Julian, Rodríguez Bey Guillermo, Takanohashi Asako, Bugiani Marianna, Fu Xiaoqin, Wolf Nicole I, Nmezi Bruce, Schiffmann Raphael, Bugaighis Mona, Pierson Tyler, Helman Guy, Simons Cas, van der Knaap Marjo S, Liu Judy, Padiath Quasar, Vanderver Adeline
Center for Neuroscience Research, Children's National Health System, Children's Research Institute, Washington, DC 20010, USA.
Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Hum Mol Genet. 2017 Nov 15;26(22):4506-4518. doi: 10.1093/hmg/ddx338.
Hypomyelinating leukodystrophies are heritable disorders defined by lack of development of brain myelin, but the cellular mechanisms of hypomyelination are often poorly understood. Mutations in TUBB4A, encoding the tubulin isoform tubulin beta class IVA (Tubb4a), result in the symptom complex of hypomyelination with atrophy of basal ganglia and cerebellum (H-ABC). Additionally, TUBB4A mutations are known to result in a broad phenotypic spectrum, ranging from primary dystonia (DYT4), isolated hypomyelination with spastic quadriplegia, and an infantile onset encephalopathy, suggesting multiple cell types may be involved. We present a study of the cellular effects of TUBB4A mutations responsible for H-ABC (p.Asp249Asn), DYT4 (p.Arg2Gly), a severe combined phenotype with hypomyelination and encephalopathy (p.Asn414Lys), as well as milder phenotypes causing isolated hypomyelination (p.Val255Ile and p.Arg282Pro). We used a combination of histopathological, biochemical and cellular approaches to determine how these different mutations may have variable cellular effects in neurons and/or oligodendrocytes. Our results demonstrate that specific mutations lead to either purely neuronal, combined neuronal and oligodendrocytic or purely oligodendrocytic defects that closely match their respective clinical phenotypes. Thus, the DYT4 mutation that leads to phenotypes attributable to neuronal dysfunction results in altered neuronal morphology, but with unchanged tubulin quantity and polymerization, with normal oligodendrocyte morphology and myelin gene expression. Conversely, mutations associated with isolated hypomyelination (p.Val255Ile and p.Arg282Pro) and the severe combined phenotype (p.Asn414Lys) resulted in normal neuronal morphology but were associated with altered oligodendrocyte morphology, myelin gene expression, and microtubule dysfunction. The H-ABC mutation (p.Asp249Asn) that exhibits a combined neuronal and myelin phenotype had overlapping cellular defects involving both neuronal and oligodendrocyte cell types in vitro. Only mutations causing hypomyelination phenotypes showed altered microtubule dynamics and acted through a dominant toxic gain of function mechanism. The DYT4 mutation had no impact on microtubule dynamics suggesting a distinct mechanism of action. In summary, the different clinical phenotypes associated with TUBB4A reflect the selective and specific cellular effects of the causative mutations. Cellular specificity of disease pathogenesis is relevant to developing targeted treatments for this disabling condition.
低髓鞘形成性脑白质营养不良是一类遗传性疾病，其特征为脑髓鞘发育不全，但低髓鞘形成的细胞机制通常还不太清楚。编码微管蛋白异构体微管蛋白β IVA类（Tubb4a）的TUBB4A基因突变会导致基底神经节和小脑萎缩伴低髓鞘形成的症状复合体（H-ABC）。此外，已知TUBB4A突变会导致广泛的表型谱，从原发性肌张力障碍（DYT4）、伴有痉挛性四肢瘫的孤立性低髓鞘形成，到婴儿期发病的脑病，这表明可能涉及多种细胞类型。我们对导致H-ABC（p.Asp249Asn）、DYT4（p.Arg2Gly）、伴有低髓鞘形成和脑病的严重联合表型（p.Asn414Lys）以及导致孤立性低髓鞘形成的较轻表型（p.Val255Ile和p.Arg282Pro）的TUBB4A突变的细胞效应进行了研究。我们结合组织病理学、生物化学和细胞方法来确定这些不同的突变如何在神经元和/或少突胶质细胞中产生可变的细胞效应。我们的结果表明，特定突变会导致纯粹的神经元缺陷、神经元和少突胶质细胞联合缺陷或纯粹的少突胶质细胞缺陷，这些缺陷与各自的临床表型密切匹配。因此，导致可归因于神经元功能障碍表型的DYT4突变会导致神经元形态改变，但微管蛋白数量和聚合不变，少突胶质细胞形态和髓鞘基因表达正常。相反，与孤立性低髓鞘形成（p.Val255Ile和p.Arg282Pro）和严重联合表型（p.Asn414Lys）相关的突变导致神经元形态正常，但与少突胶质细胞形态改变、髓鞘基因表达和微管功能障碍有关。表现出神经元和髓鞘联合表型的H-ABC突变（p.Asp249Asn）在体外具有涉及神经元和少突胶质细胞类型的重叠细胞缺陷。只有导致低髓鞘形成表型的突变显示微管动力学改变，并通过显性毒性功能获得机制起作用。DYT4突变对微管动力学没有影响，提示其作用机制不同。总之，与TUBB4A相关的不同临床表型反映了致病突变的选择性和特异性细胞效应。疾病发病机制的细胞特异性与开发针对这种致残性疾病的靶向治疗方法相关。
