The role of Lamin B1 in the organization of the nuclear envelope and myelin regulation in development and disease

The nuclear lamina is a structural meshwork composed of intermediate filament proteins known as lamins that maintains nuclear shape and function. Perturbations of lamins lead to diseases, collectively known as laminopathies, which affect a wide variety of organ systems. One such laminopathy is autosomal dominant leukodystrophy (ADLD), a severe and fatal adult-onset demyelinating laminopathy caused by overexpression of LMNB1, one of the lamin proteins that make up the nuclear lamina. My studies aim to elucidate the role of lamin B1 in the organization of the nuclear envelope, its role in myelin regulation during oligodendrocyte maturation, and to understand how the genomic rearrangements involving LMNB1 cause ADLD. Our results suggest a novel concentric organization model of the nuclear lamina, with lamin B1 facing the inner nuclear membrane while lamins A and C together face the nucleoplasm. Lamin B1’s outward-facing localization maintains nuclear shape by restraining the lamin A/C meshwork from protruding outward. To study lamin B1’s function in mature oligodendrocytes, conditional Lmnb1 knockout mice were used to study behavioral and molecular changes in the central nervous system. Knockout mice did not exhibit any overt behavioral phenotypes or myelination defects, but a careful analysis revealed alterations in the number of myelinating oligodendrocyte populations. We conclude that while mature oligodendrocytes do not require lamin B1 for their proper function, it might be important for the regulation of oligodendrocyte cell number. Array CGH studies revealed that deletions upstream of LMNB1 can also lead to ADLD, while large duplications involving LMNB1 and a significant upstream region do not. Real-time PCR analysis demonstrate much higher LMNB1 expression in white matter than in grey matter and fibroblasts. We propose that an oligodendrocyte-specific silencer element lies upstream of LMNB1, explaining ADLD’s central nervous system exclusivity despite a constitutional LMNB1 duplication. As demyelination and white matter injuries are common in disorders affecting a wide age range – from preterm neonates to young adults and the elderly – researching pathways involved in myelination and ways to reverse it could have a significant impact to public health

Genomic deletions upstream of lamin B1 lead to atypical autosomal dominant leukodystrophy

Objective Clinical, radiologic, and molecular analysis of patients with genomic deletions upstream of the LMNB1 gene. Methods Detailed neurologic, MRI examinations, custom array comparative genomic hybridization (aCGH) analysis, and expression analysis were performed in patients at different clinical centers. All procedures were approved by institutional review boards of the respective institutions. Results Five patients from 3 independent families presented at ages ranging from 32 to 52 years with neurologic symptoms that included progressive hypophonia, upper and lower limb weakness and spasticity, and cerebellar dysfunction and MRIs characterized by widespread white matter alterations. Patients had unique nonrecurrent deletions upstream of the LMNB1, varying in size from 250 kb to 670 kb. Deletion junctions were embedded in repetitive elements. Expression analysis revealed increased LMNB1 expression in patient cells. Conclusions Our findings confirmed the association between LMNB1 upstream deletions and leukodystrophy previously reported in a single family, expanding the phenotypic and molecular description of this condition. Although clinical and radiologic features overlapped with those of autosomal dominant leukodystrophy because of LMNB1 duplications, patients with deletions upstream of LMNB1 had an earlier age at symptom onset, lacked early dysautonomia, and appeared to have lesser involvement of the cerebellum and sparing of the spinal cord diameter on MRI. aCGH analysis defined a smaller minimal critical region required for disease causation and revealed that deletions occur at repetitive DNA genomic elements. Search for LMNB1 structural variants (duplications and upstream deletions) should be an integral part of the investigation of patients with autosomal dominant adult-onset leukodystrophy

TUBB4A mutations result in specific neuronal and oligodendrocytic defects that closely match clinically distinct phenotypes

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

Variants in the zinc transporter TMEM163 cause a hypomyelinating leukodystrophy

Hypomyelinating leukodystrophies comprise a subclass of genetic disorders with deficient myelination of the CNS white matter. Here we report four unrelated families with a hypomyelinating leukodystrophy phenotype harbouring variants in TMEM163 (NM_030923.5). The initial clinical presentation resembled Pelizaeus-Merzbacher disease with congenital nystagmus, hypotonia, delayed global development and neuroimaging findings suggestive of significant and diffuse hypomyelination. Genomic testing identified three distinct heterozygous missense variants in TMEM163 with two unrelated individuals sharing the same de novo variant. TMEM163 is highly expressed in the CNS particularly in newly myelinating oligodendrocytes and was recently revealed to function as a zinc efflux transporter. All the variants identified lie in highly conserved residues in the cytoplasmic domain of the protein, and functional in vitro analysis of the mutant protein demonstrated significant impairment in the ability to efflux zinc out of the cell. Expression of the mutant proteins in an oligodendroglial cell line resulted in substantially reduced mRNA expression of key myelin genes, reduced branching and increased cell death. Our findings indicate that variants in TMEM163 cause a hypomyelinating leukodystrophy and uncover a novel role for zinc homeostasis in oligodendrocyte development and myelin formation