Truncated TrkB.T1-Mediated Astrocyte Dysfunction Contributes to Impaired Motor Function and Neuropathic Pain after Spinal Cord Injury.

Following spinal cord injury (SCI), astrocytes demonstrate long-lasting reactive changes, which are associated with the persistence of neuropathic pain and motor dysfunction. We previously demonstrated that upregulation of trkB.T1, a truncated isoform of the brain-derived neurotrophic factor receptor (BDNF), contributes to gliosis after SCI, but little is known about the effects of trkB.T1 on the function of astrocytes. As trkB.T1 is the sole isoform of trkB receptors expressed on astrocytes, we examined the function of trkB.T1-driven astrocytes in vitro and in vivo Immunohistochemistry showed that trkB.T1+ cells were significantly upregulated 7 d after injury, with sustained elevation in white matter through 8 weeks. The latter increase was predominantly found in astrocytes. TrkB.T1 was also highly expressed by neurons and microglia/macrophages at 7 d after injury and declined by 8 weeks. RNA sequencing of cultured astrocytes derived from trkB.T1+/+ (WT) and trkB.T1-/- (KO) mice revealed downregulation of migration and proliferation pathways in KO astrocytes. KO astrocytes also exhibited slower migration/proliferation in vitro in response to FBS or BDNF compared with WT astrocytes. Reduced proliferation of astrocytes was also confirmed after SCI in astrocyte-specific trkB.T1 KO mice; using mechanical allodynia and pain-related measurements on the CatWalk, these animals also showed reduced hyperpathic responses, along with improved motor coordination. Together, our data indicate that trkB.T1 in astrocytes contributes to neuropathic pain and neurological dysfunction following SCI, suggesting that trkB.T1 may provide a novel therapeutic target for SCI.SIGNIFICANCE STATEMENT Neuropathic pain after spinal cord injury (SCI) may in part be caused by upregulation of the brain-derived neurotrophic factor (BDNF) receptor trkB.T1, a truncated isoform of BDNF. TrkB.T1 is the only isoform of tropomyosin-related receptor kinase type B (trkB) receptors expressed on astrocytes. Here, we showed that trkB.T1 is significantly increased in the injured mouse spinal cord, where it is predominantly found in astrocytes. RNA sequencing of cultured astrocytes demonstrated downregulation of migration and proliferation pathways in trkB.T1 KO astrocytes. This was validated in vivo, where deletion of trkB.T1 in astrocytes reduced cell proliferation and migration. After SCI, astrocyte-specific trkB.T1 KO mice showed reduced hyperpathic responses and improved motor coordination. Therefore, the trkB.T1 receptor plays a significant pathophysiological role after SCI, and may provide a novel therapeutic target for SCI.

Hydroquinone increases 5-hydroxymethylcytosine formation through ten eleven translocation 1 (TET1) 5-methylcytosine dioxygenase.

DNA methylation regulates gene expression throughout development and in a wide range of pathologies such as cancer and neurological disorders. Pathways controlling the dynamic levels and targets of methylation are known to be disrupted by chemicals and are therefore of great interest in both prevention and clinical contexts. Benzene and its metabolite hydroquinone have been shown to lead to decreased levels of DNA methylation, although the mechanism is not known. This study employs a cell culture model to investigate the mechanism of hydroquinone-mediated changes in DNA methylation. Exposures that do not affect HEK293 cell viability led to genomic and methylated reporter DNA demethylation. Hydroquinone caused reactivation of a methylated reporter plasmid that was prevented by the addition of N-acetylcysteine. Hydroquinone also caused an increase in Ten Eleven Translocation 1 activity and global levels of 5-hydroxymethylcytosine. 5-Hydroxymethylcytosine was found enriched at LINE-1 prior to a decrease in both 5-hydroxymethylcytosine and 5-methylcytosine. Ten Eleven Translocation-1 knockdown decreased 5-hydroxymethylcytosine formation following hydroquinone exposure as well as the induction of glutamate-cysteine ligase catalytic subunit and 14-3-3σ. Finally, Ten Eleven Translocation 1 knockdown decreased the percentage of cells accumulating in G2+M following hydroquinone exposure, indicating that it may have a role in cell cycle changes in response to toxicants. This work demonstrates that hydroquinone exposure leads to active and functional DNA demethylation in HEK293 cells in a mechanism involving reactive oxygen species and Ten Eleven Translocation 1 5-methylcytosine dioxygenase.

Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Troma-1/cytokeratin 8 and the transcription factor Tead4. A decrease in CpG methylation at three sites in the TEAD4 promoter and the B1 repeated sequence was observed. Knocking down expression of Tead4 with siRNA blocked the increase in Troma-1/cytokeratin 8 and over expression of Tead4 induced the expression of Troma-1/cytokeratin 8. Cells treated for 5days with hydralazine were no longer capable of undergoing retinoic acid-mediated neuronal differentiation. An irreversible loss of the pluripotent transcription factor Oct-4 was observed following hydralazine exposure. In summary, hydralazine induces P19 cells to assume a trophoblast-like phenotype by upregulating Tead4 expression through a mechanism involving DNA demethylation.

Small molecule glutaminase inhibitors block glutamate release from stimulated microglia.

Glutaminase plays a critical role in the generation of glutamate, a key excitatory neurotransmitter in the CNS. Excess glutamate release from activated macrophages and microglia correlates with upregulated glutaminase suggesting a pathogenic role for glutaminase. Both glutaminase siRNA and small molecule inhibitors have been shown to decrease excess glutamate and provide neuroprotection in multiple models of disease, including HIV-associated dementia (HAD), multiple sclerosis and ischemia. Consequently, inhibition of glutaminase could be of interest for treatment of these diseases. Bis-2-(5-phenylacetimido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and 6-diazo-5-oxo-l-norleucine (DON), two most commonly used glutaminase inhibitors, are either poorly soluble or non-specific. Recently, several new BPTES analogs with improved physicochemical properties were reported. To evaluate these new inhibitors, we established a cell-based microglial activation assay measuring glutamate release. Microglia-mediated glutamate levels were significantly augmented by tumor necrosis factor (TNF)-α, phorbol 12-myristate 13-acetate (PMA) and Toll-like receptor (TLR) ligands coincident with increased glutaminase activity. While several potent glutaminase inhibitors abrogated the increase in glutamate, a structurally related analog devoid of glutaminase activity was unable to block the increase. In the absence of glutamine, glutamate levels were significantly attenuated. These data suggest that the in vitro microglia assay may be a useful tool in developing glutaminase inhibitors of therapeutic interest.

Methyl CpG binding protein 2 deficiency enhances expression of inflammatory cytokines by sustaining NF-κB signaling in myeloid derived cells.

Knocking down methyl CpG binding protein 2 (MeCP2) enhances NF-κB activation in human peripheral blood mononuclear cells (PBMC). In this study, we examined whether this caused the expression of cytokines to be elevated. Increased levels of TNFα, IL-6, and IL-3 mRNAs were observed in human PBMC made MeCP2 deficient with a lentiviral shRNA MeCP2 vector and in splenocytes from MeCP2-null mice. TNFα neutralizing antibody attenuated expression of IL-6 and TNFα but did not affect expression of IL-3. Lipopolysaccharide-mediated increases in TNFα, IL-6, and IL-3 mRNAs were also enhanced in MeCP2-deficient PBMC. Two inhibitors of NF-κB blocked the increased levels of IL-6, TNFα, and IL-3 in MeCP2-deficient PBMC treated with lipopolysaccharide. MeCP2 deficiency also enhanced expression of IL-6 and TNFα mRNAs in the THP1 human monocyte cell line, which were also attenuated by the NF-κB inhibitors. In chromatin immunoprecipitation assays, the binding of the NF-κB family member p65 and acetylated H3 to the TNFα promoter was greater after treatment with LPS in MeCP2-deficient THP1 cells. MeCP2 did not bind to the TNFα promoter. In summary, the data indicates that MeCP2 deficiency increases expression of TNFα and other inflammatory cytokines by enhancing NF-κB signaling.

MeCP2 deficiency enhances glutamate release through NF-κB signaling in myeloid derived cells.

The signaling pathway involved in regulating glutamate release was investigated. Glutaminase mRNA levels were higher in microglia isolated from mice treated with lipopolysaccharide. Pam3CSK and lipopolysaccharide stimulated glutamate release in neonatal mouse microglia cultures, which was attenuated by NF-κB inhibitors. Higher levels of glutaminase mRNA and glutamate release were observed in human peripheral blood mononuclear cells made MeCP2 deficient with MeCP2 shRNA, which was blocked by NF-κB inhibitors. NF-κB inhibitors also decreased the higher levels of glutaminase mRNA in MeCP2 deficient THP1 monocyte cell lines. Finally, an inverse relation was observed between MeCP2 levels and NF-κB reporter activity in THP1 cells. We suggest that NF-κB pathway is involved in the release of glutamate in MeCP2 deficient cells from the myeloid lineage.