Enhanced differentiation of the mouse oli-neu oligodendroglial cell line using optimized culture conditions.

OBJECTIVE: Oligodendrocytes (OL) are the glial cell type in the CNS that are responsible for myelin formation. The ability to culture OLs in vitro has provided critical insights into the mechanisms underlying their function. However, primary OL cultures are tedious to obtain, difficult to propagate and are not easily conducive to genetic manipulation. To overcome these obstacles, researchers have generated immortalized OL like cell lines derived from various species. One such cell line is the mouse Oli-neu line which is thought to recapitulate characteristics of OLs in early stages of maturity. They have been extensively utilized in multiple studies as surrogates for OLs, especially in analyzing epigenetic modifications and regulatory pathways in the OL lineage. RESULTS: In this report we present the development of optimized culture media and growth conditions that greatly facilitate the differentiation of Oli-neu cells. Oli-neu cells differentiated using these new protocols exhibit a higher expression of myelin related genes and increased branching, both of which are defining characteristics of mature OLs, when compared to previous culture protocols. We envision that these new culture conditions will greatly facilitate the use of Oli-neu cells and enhance their ability to recapitulate the salient features of primary OLs.

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.

scMAPA: Identification of cell-type-specific alternative polyadenylation in complex tissues.

BACKGROUND: Alternative polyadenylation (APA) causes shortening or lengthening of the 3'-untranslated region (3'-UTR) of genes (APA genes) in diverse cellular processes such as cell proliferation and differentiation. To identify cell-type-specific APA genes in scRNA-Seq data, current bioinformatic methods have several limitations. First, they assume certain read coverage shapes in the scRNA-Seq data, which can be violated in multiple APA genes. Second, their identification is limited between 2 cell types and not directly applicable to the data of multiple cell types. Third, they do not control undesired source of variance, which potentially introduces noise to the cell-type-specific identification of APA genes. FINDINGS: We developed a combination of a computational change-point algorithm and a statistical model, single-cell Multi-group identification of APA (scMAPA). To avoid the assumptions on the read coverage shape, scMAPA formulates a change-point problem after transforming the 3' biased scRNA-Seq data to represent the full-length 3'-UTR signal. To identify cell-type-specific APA genes while adjusting for undesired source of variation, scMAPA models APA isoforms in consideration of the cell types and the undesired source. In our novel simulation data and data from human peripheral blood mononuclear cells, scMAPA outperforms existing methods in sensitivity, robustness, and stability. In mouse brain data consisting of multiple cell types sampled from multiple regions, scMAPA identifies cell-type-specific APA genes, elucidating novel roles of APA for dividing immune cells and differentiated neuron cells and in multiple brain disorders. CONCLUSIONS: scMAPA elucidates the cell-type-specific function of APA events and sheds novel insights into the functional roles of APA events in complex tissues.

Analysis of LMNB1 duplications in autosomal dominant leukodystrophy provides insights into duplication mechanisms and allele-specific expression.

Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients' fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels.

Autosomal dominant leukodystrophy caused by lamin B1 duplications a clinical and molecular case study of altered nuclear function and disease.

Autosomal dominant leukodystrophy (ADLD) is an adult-onset demyelinating disorder that has recently shown to be caused by duplications of the nuclear lamina gene, lamin B1. This chapter attempts to collate and summarize the current knowledge about the disease and the clinical, pathological, and radiological presentations of the different ADLD families described till date. It also provides an overview of the molecular genetics underlying the disease and the mechanisms that may cause the duplication mutation event. ADLD is the first disease that has ever been linked to lamin B1 mutations and it expands the pathological role of the nuclear lamia to include disorders of the brain. The chapter also speculates on the different mechanisms that may link an important and ubiquitous structure like the nuclear lamina with the complex and cell-specific functions of myelin formation and maintenance. Understanding these mechanisms may not only prove helpful in understanding ADLD pathology but can also help in identifying new pathways that may be involved in myelin biology that can have implications for common demyelinating diseases like multiple sclerosis.

Identification of a novel 45 repeat unstable allele associated with a disease phenotype at the MJD1/SCA3 locus.

We report a three generation Indian pedigree with the proband having 45 repeats at the Machado Joseph Disease (MJD)/spinocerebellar ataxia 3 (SCA3) disease locus. The proband exhibited clinical features of SCA and showed signs of cerebellar and brainstem atrophy on the MRI scan. The 45 repeat allele was unstable upon inter-generational transmission and was associated with a haplotype found in the majority of MJD/SCA3 patients from around the world. This is the smallest unstable allele reported till date at the MJD/SCA3 locus and may greatly reduce the gap between normal and pathological repeat ranges. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299:1/suppmat/index.html.

Glycogen synthase kinase 3beta as a likely target for the action of lithium on circadian clocks.

Although lithium is one of the most commonly used drugs in the prophylaxis and treatment of bipolar disorder, the mechanisms underlying its therapeutic action are still unclear. Together with its mood-stabilizing effects, lithium is also known to influence the circadian clocks of several organisms including man. Circadian rhythms are altered in patients with bipolar disorder, and it is believed that these rhythms may play an important role in disease mechanisms. It is therefore possible that some of the therapeutic actions of lithium may be related to its effect on circadian clocks. Identifying the targets for lithium's action on circadian clocks would therefore be important both for understanding the mechanisms of its therapeutic effect and also in further understanding disease mechanisms in bipolar disorders. Using Drosophila melanogaster as a model system, we show that long-term administration of lithium results in lengthening of the free-running period (tau) of circadian locomotor activity rhythm of flies in constant darkness (DD). This effect occurs at concentrations similar to the plasma levels of lithium used in the treatment of bipolar disorder. The lithium-treated flies also show reduced activity of one of the previously reported targets of lithium action, Glycogen Synthase Kinase 3beta (GSK 3beta). GSK 3beta has been shown to be involved in the regulation of circadian clocks as the down regulation of this protein results in an elongation of tau. The tau elongation resembles the effect seen with lithium administration in a number of organisms including man, and taken together with the earlier observations our results suggest that lithium inhibits the activity of GSK 3beta to produce its effect on circadian clocks.