Hisatsugu KOSHIMIZU

Fukuyama congenital muscular dystrophy (FCMD) is an autosomal recessive disorder caused by fukutin (FKTN) gene mutations. FCMD is the second most common form of childhood muscular dystrophy in Japan, and the most patients possess a homozygous retrotransposal SINE-VNTR-Alu insertion in the 3′-untranslated region of FKTN. A deep-intronic variant (DIV) was previously identified as the second most prevalent loss-of-function mutation in Japanese patients with FCMD. The DIV creates a new splicing donor site in intron 5 that causes aberrant splicing and the formation of a 64-base pair pseudoexon in the mature mRNA, resulting in a truncated nonfunctional protein. Patients with FCMD carrying the DIV present a more severe symptoms, and currently, there is no radical therapy available for this disorder. In the present study, we describe in vitro evaluation of antisense oligonucleotide mediated skipping of pseudoexon inclusion and restoration of functional FKTN protein. A total of 16 19-26-mer antisense oligonucleotide sequences were designed with a 2’-O-methyl backbone and were screened in patient-derived fibroblasts, lymphoblast cells, and minigene splice assays. One antisense oligonucleotide targeting the exonic splice enhancer region significantly induced pseudoexon skipping and increased the expression of normal mRNA. It also rescued FKTN protein production in lymphoblast cells and restored functional O-mannosyl glycosylation of alpha-dystroglycan in patient-derived myotubes. Based on our results, ASO-based splicing correction should be investigated further as a potential treatment for patients with FCMD carrying the DIV.

The striatum, a main component of the basal ganglia, is a critical part of the motor and reward systems of the brain. It consists of GABAergic and cholinergic neurons and receives projections of dopaminergic, glutamatergic, and serotonergic neurons from other brain regions. Brain-derived neurotrophic factor (BDNF) plays multiple roles in the central nervous system, and striatal BDNF has been suggested to be involved in psychiatric and neurodegenerative disorders. However, the transcriptomic impact of BDNF on the striatum remains largely unknown. In the present study, we performed transcriptomic profiling of striatal cells stimulated with BDNF to identify enriched gene sets (GSs) and their novel target genes in vitro. We carried out RNA sequencing (RNA-Seq) of messenger RNA extracted from primary dissociated cultures of rat striatum stimulated with BDNF and conducted Generally Applicable Gene-set Enrichment (GAGE) analysis on 10599 genes. Significant differentially expressed genes (DEGs) were determined by differential expression analysis for sequence count data 2 (DESeq2). GAGE analysis identified significantly enriched GSs that included GSs related to regulation and dysregulation of synaptic functions, such as synaptic vesicle cycle and addiction to nicotine and morphine, respectively. It also detected GSs related to various types of synapses, including not only GABAergic and cholinergic synapses but also dopaminergic and glutamatergic synapses. DESeq2 revealed 72 significant DEGs, among which the highest significance was observed in the apolipoprotein L domain containing 1 (Apold1). The present study indicates that BDNF predominantly regulates the expression of synaptic-function-related genes and that BDNF promotes synaptogenesis in various subtypes of neurons in the developing striatum. Apold1 may represent a unique target gene of BDNF in the striatum.

Chromosome 8 open reading frame 46 (C8orf46), a human protein-coding gene, has recently been named Vexin. A recent study indicated that Vexin is involved in embryonic neurogenesis. Additionally, some transcriptomic studies detected changes in the mRNA levels of patients with psychiatric and neurological diseases. In our previous study, we sought for target genes of brain-derived neurotrophic factor (BDNF) in cultured rat cortical neurons, finding that BDNF potentially leads to the upregulation of Vexin mRNA. However, its underlying mechanisms are unknown. In the present study, we assessed the regulatory mechanisms of the BDNF-induced gene expression of Vexin in vitro. We reanalyzed ChIP-seq data in various human organs provided by the ENCODE project, evaluating acetylation levels of the 27th lysine residue of the histone H3 (H3K27ac) at the Vexin locus. The transcriptomic effects of BDNF on rat Vexin (RGD1561849) were evaluated by real-time quantitative PCR (RT-qPCR) in primary cultures of cerebral cortical neurons, in the presence or absence of inhibitors for signaling molecules activated by BDNF. Results The Vexin locus and its promoter region in the brain angular gyrus show higher acetylation levels of the H3K27 than those in other organs. Stimulation of cultured rat cortical neurons, but not astrocyte, with BDNF, led to marked elevations in the mRNA levels of Vexin, which was inhibited in the presence of K252a and U0126. The upregulated H3K27ac in the brain may be associated with the enriched gene expression of Vexin in the brain. It is indicated that BDNF induces the gene expression of Vexin in the cortical neurons via the TrkB-MEK signaling pathway.

Major depressive disorder (MDD) is a common and disabling psychiatric disorder. A recent mega analysis of genome-wide association studies (GWASs) identified 44 loci associated with MDD, though most of the genetic etiologies of the MDD/psychological distress remain unclear. To further understand the genetic basis of MDD/psychological distress, we conducted a GWAS in East Asia with more than 10,000 participants of Japanese ancestry who had enrolled in a direct-to-consumer genetic test. After quality control on the genotype data, 10,330 subjects with a total of 8,567,708 imputed SNPs were eligible for the analysis. The participants completed a self-administered questionnaire on their past medical history and health conditions that included the 6-item Kessler screening scale (K6 scale) for psychological distress (cut-off point of 5) and past medical history of MDD, resulting in 3981 subjects assigned to “psychologically distressed group” [cases], and the remaining 6349 subjects were assigned to the “non-psychologically distressed group” [controls]. In this GWAS, we found an association with genome-wide significance at rs6073833 (P = 7.60 × 10 −9 ) in 20q13.12. This is, to the best of our knowledge, the first large-scale GWAS for psychological distress using data from direct-to-consumer (DTC) genetic tests in a population of non-European-ancestry, and the present study thus detected a novel locus significantly associated with psychological distress in the Japanese population.