Brain-derived neurotrophic factor predominantly regulates the expression of synapse-related genes in the striatum: Insights from in vitro transcriptomics.

AIM: 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. METHODS: 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). RESULTS: 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). CONCLUSIONS: 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.

Meganuclease-Based Artificial Transcription Factors.

Embedding middle-scale artificial gene networks in live mammalian cells is one of the most important future goals for cell engineering. However, the applications of the highly orthogonal and conventional artificial transcription factors currently available are limited. In this study, we present a scalable pipeline to produce artificial transcription factors based on homing endonucleases, also known as meganucleases. The introduction of mutations at critical sites for nuclease activity renders these homing endonucleases a simple but highly specific DNA binding domain for their specific DNA target. The introduction of inactivated meganucleases linked to transcriptional activator domains strongly induced reporter gene expression, while their fusion to transcriptional repressor domains suppressed them. In addition, we show that inactivated meganuclease-based transcription factors could be embedded in the synthetic membrane receptor synNotch and used to construct synthetic circuits. These results suggest that inactivated meganucleases are useful DNA-binding domains for the construction of synthetic transcription factors in mammalian cells.

Vexin is upregulated in cerebral cortical neurons by brain-derived neurotrophic factor.

AIM: 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. METHODS: 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. CONCLUSION: 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.

Alpha-phenyl-N-tert-butyl nitrone has scavenging activity against singlet oxygen ((1)O(2)) and attenuates (1)O(2)-induced neuronal cell death.

Scavenging activity of alpha-phenyl-N-tert-butyl nitrone (PBN) against singlet oxygen ((1)O(2)) and its effects on (1)O(2)-induced neuronal cell death were examined. PBN at 1 - 4 mM dose-dependently suppressed (1)O(2) released from activated human neutrophils. PBN did not react with hydrogen peroxide or hypochlorite and did not affect myeloperoxidase activity, which are involved in the (1)O(2) formation in neutrophils. PBN also suppressed chemically generated (1)O(2) in a cell-free system. These findings collectively indicated that PBN certainly has scavenging activity against (1)O(2). Furthermore, PBN attenuated (1)O(2)-induced neuronal cell death. The well-known neuroprotective effects of PBN might be attributed to its (1)O(2)-scavenging activity.