Human neuroblastoma SH-SY5Y cells are a widely-used human neuronal cell model in the study of neurodegeneration. A recent study shows that, 1-methyl-4-phenylpyridine ion (MPP), which selectively causes dopaminergic neuronal death leading to Parkinson’s disease-like symptoms, can reduce SH-SY5Y cell viability by inducing H₂O₂ generation and subsequent TRPM2 channel activation. MPP-induced cell death is enhanced by increasing the TRPM2 expression. By contrast, increasing the TRPM2 expression has also been reported to support SH-SY5Y cell survival after exposure to H₂O₂, leading to the suggestion of a protective role for the TRPM2 channel. To clarify the role of reactive oxygen species (ROS)-induced TRPM2 channel activation in SH-SY5Y cells, we generated a stable SH-SY5Y cell line overexpressing the human TRPM2 channel and examined cell death and cell viability after exposure to H₂O₂ in the wild-type and TRPM2-overexpressing SH-SY5Y cells. Exposure to H₂O₂ resulted in concentration-dependent cell death and reduction in cell viability in both cell types. TRPM2 overexpression remarkably augmented H₂O₂-induced cell death and reduction in cell viability. Furthermore, H₂O₂-induced cell death in both the wild-type and TRPM2-overexpressing cells was prevented by 2-APB, a TRPM2 inhibitor, and also by PJ34 and DPQ, poly(ADP-ribose) polymerase (PARP) inhibitors. Collectively, our results show that increasing the TRPM2 expression renders SH-SY5Y cells to be more susceptible to ROS-induced cell death and reinforce the notion that the TRPM2 channel plays a critical role in conferring ROS-induced cell death. It is anticipated that SH-SY5Y cells can be useful for better understanding the molecular and signaling mechanisms for ROS-induced TRPM2-mediated neurodegeneration in the pathogenesis of neurodegenerative diseases.

Many mutations in the signal peptide and propeptide of factor IX (FIX) cause hemophilia B. A FIX variants database reports 28 unique missense mutations in these regions that lead to FIX deficiency, but the underlying mechanism is known only for the mutations on R43 that interfere with propeptide cleavage. It remains unclear how other mutations result in FIX deficiency and why patients carrying the same mutation have different bleeding tendencies. Here, we modify a cell-based reporter assay to characterize the missense mutations in the signal peptide and propeptide of FIX. The results show that the level of secreted conformation-specific reporter (SCSR), which has a functional γ-carboxyglutamate (Gla) domain of FIX, decreases significantly in most mutations. The decreased SCSR level is consistent with FIX deficiency in hemophilia B patients. Moreover, we find that the decrease in the SCSR level is caused by several distinct mechanisms, including interfering with cotranslational translocation into the endoplasmic reticulum, protein secretion, γ-carboxylation of the Gla domain, and cleavage of the signal peptide or propeptide. Importantly, our results also show that the SCSR levels of most signal peptide and propeptide mutations increase with vitamin K concentration, suggesting that the heterogeneity of bleeding tendencies may be related to vitamin K levels in the body. Thus, oral administration of vitamin K may alleviate the severity of bleeding tendencies in patients with missense mutations in the FIX signal peptide and propeptide regions.

Introduction: Astrocytes are parenchymal cells widely distributed throughout the brain. Beyond their essential functions in healthy tissue, astrocytes exhibit an evolutionarily conserved response to all forms of brain injury, termed astrocytic reactivity. Nevertheless, conceptual understanding of what astrocytic reactivity encompasses and its functional roles remains incomplete and occasionally contentious. Lipopolysaccharide (LPS) is widely used to induce neuroinflammation. In the current study, Histone deacetylase 7 (HDAC7) has been shown to ameliorate LPS-induced neuroinflammation and mitigate astrocytic reactivity. Methods: We overexpressed HDAC7 using viral vectors and generated primary astrocytes from Hdac7 ^{flox / flox} mice to achieve astrocyte-specific HDAC7 knockout. Subsequently, we assessed astrocytic reactivity and detected the expression of the Interferon regulatory factor 3 (IRF3)/cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. Results: HDAC7 has been implicated in inflammatory regulation, but its role in astrocyte reactivity and the underlying mechanisms remain unclear. Here, we demonstrate that HDAC7 deficiency attenuates LPS-induced astrogliosis by suppressing the cGAS/STING signaling axis. LPS stimulation induced robust upregulation of glial fibrillary acidic protein (GFAP), complement component 3 (C3), and pro-inflammatory cytokines (TNF-α, IL-6) in WT astrocytes, which was significantly blunted in HDAC7 knockout astrocytes. Conversely, lentiviral overexpression of HDAC7 in WT astrocytes exacerbated IRF3/cGAS/STING pathway activation, as validated by Western blot analysis showing upregulated cGAS, STING and IRF3 expression. Pharmacological activation of the STING pathway in astrocytes restored pro-inflammatory cytokine expression and reactive marker levels, indicating pathway dependence. Discussion: Our results delineate a novel HDAC7/IRF3/cGAS/STING signaling axis that governs astrocyte reactivity. This discovery provides a crucial cellular neurophysiological mechanism by which astrocytes integrate inflammatory signals and subsequently modulate the central nervous system microenvironment. Targeting HDAC7, therefore, represents a therapeutic strategy to mitigate neuroinflammation by specifically correcting this aberrant cell-physiological state of astrocytes, ultimately preserving neural circuit function.

Vitamin K epoxide reductases (VKORs) are a large family of integral membrane enzymes found from bacteria to humans. Human VKOR, specific target of warfarin, has both the epoxide and quinone reductase activity to maintain the vitamin K cycle. Bacterial VKOR homologs, however, are insensitive to warfarin inhibition and are quinone reductases incapable of epoxide reduction. What affords the epoxide reductase activity in human VKOR remains unknown. Here, we show that a representative bacterial VKOR homolog can be converted to an epoxide reductase that is also inhibitable by warfarin. To generate this new activity, we first substituted several regions surrounding the active site of bacterial VKOR by those from human VKOR based on comparison of their crystal structures. Subsequent systematic substitutions narrowed down to merely eight residues, with the addition of a membrane anchor domain, that are responsible for the epoxide reductase activity. Substitutions corresponding to N80 and Y139 in human VKOR provide strong hydrogen bonding interactions to facilitate the epoxide reduction. The rest of six substitutions increase the size and change the shape of the substrate‐binding pocket, and the membrane anchor domain stabilizes this pocket while allowing certain flexibility for optimal binding of the epoxide substrate. Overall, our study reveals the structural features of the epoxide reductase activity carried out by a subset of VKOR family in the membrane environment.

Regulating serotonin expression can be used to treat psychotic depression. Mifepristone, a glucocorticoid receptor antagonist, is an effective candidate for psychotic depression treatment. However, the underlying mechanism related to serotonin transporter expression is poorly understood. In this study, we cloned the human brain serotonin transporter into Xenopus oocytes, to establish an in vitro expression system. Two-electrode voltage clamp recordings were used to detect serotonin transporter activity. Our results show that mifepristone attenuates serotonin transporter activity by directly inhibiting the serotonin transporter, and suggests that the serotonin transporter is a pharmacological target of mifepristone for the treatment of psychotic depression.