Lysosomal TFEB-TRPML1 Axis in Astrocytes Modulates Depressive-like Behaviors.

Lysosomes are important cellular structures for human health as centers for recycling, signaling, metabolism and stress adaptation. However, the potential role of lysosomes in stress-related emotions has long been overlooked. Here, it is found that lysosomal morphology in astrocytes is altered in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social defeat stress. A screen of lysosome-related genes revealed that the expression of the mucolipin 1 gene (Mcoln1; protein: mucolipin TRP channel 1) is decreased in susceptible mice and depressed patients. Astrocyte-specific knockout of mucolipin TRP channel 1 (TRPML1) induced depressive-like behaviors by inhibiting lysosomal exocytosis-mediated adenosine 5'-triphosphate (ATP) release. Furthermore, this stress response of astrocytic lysosomes is mediated by the transcription factor EB (TFEB), and overexpression of TRPML1 rescued depressive-like behaviors induced by astrocyte-specific knockout of TFEB. Collectively, these findings reveal a lysosomal stress-sensing signaling pathway contributing to the development of depression and identify the lysosome as a potential target organelle for antidepressants.

Integrating network pharmacology and experimental verification to explore the pharmacological mechanisms of phlorizin against osteoarthritis.

To study the pharmacological effects and mechanisms of phlorizin in the treatment of osteoarthritis (OA) through network pharmacological analysis, molecular docking, and experimental validation. First, we screened out the relevant targets related to phlorizin and OA from the public database. The key targets, biological processes, and signaling pathways of phlorizin against OA were identified by protein-protein interaction (PPI) network, Gene Ontology (GO), and Encyclopedia of Kyoto Genes and Genomes (KEGG) pathway enrichment analysis. Subsequently, molecular docking was performed to predict the binding activity between phlorizin and key targets. Finally, we evaluated the effects of phlorizin on hydrogen peroxide-induced OA in rats and validated its possible mechanism of action based on the findings of the network pharmacology analysis. Network pharmacology revealed a total of 235 cross-targets involved in the treatment of OA. Phlorizin's anti-OA effect was found to be primarily mediated through oxidoreductase activity, with JAK-STAT and NF-κB signaling pathways playing a regulating role, according to pathway enrichment analysis. Phlorizin demonstrated a strong affinity for NF-κB1 targets through molecular docking. Moreover, in vitro experiments demonstrated that phlorizin could enhance intracellular antioxidant enzyme activities with good ROS scavenging ability and significantly reduce the expression of NF-κB1 and inflammatory cytokines. Phlorizin can inhibit the progression of OA. The potential underlying mechanism involves inhibiting the NF-κB pathway to reduce inflammation and promote intracellular antioxidant action.

Astrocytic ALKBH5 in stress response contributes to depressive-like behaviors in mice.

Epigenetic mechanisms bridge genetic and environmental factors that contribute to the pathogenesis of major depression disorder (MDD). However, the cellular specificity and sensitivity of environmental stress on brain epitranscriptomics and its impact on depression remain unclear. Here, we found that ALKBH5, an RNA demethylase of N6-methyladenosine (m6A), was increased in MDD patients' blood and depression models. ALKBH5 in astrocytes was more sensitive to stress than that in neurons and endothelial cells. Selective deletion of ALKBH5 in astrocytes, but not in neurons and endothelial cells, produced antidepressant-like behaviors. Astrocytic ALKBH5 in the mPFC regulated depression-related behaviors bidirectionally. Meanwhile, ALKBH5 modulated glutamate transporter-1 (GLT-1) m6A modification and increased the expression of GLT-1 in astrocytes. ALKBH5 astrocyte-specific knockout preserved stress-induced disruption of glutamatergic synaptic transmission, neuronal atrophy and defective Ca2+ activity. Moreover, enhanced m6A modification with S-adenosylmethionine (SAMe) produced antidepressant-like effects. Our findings indicate that astrocytic epitranscriptomics contribute to depressive-like behaviors and that astrocytic ALKBH5 may be a therapeutic target for depression.

Ultra-Low-Dose UV-C Photo-stimulation Promotes Neural Stem Cells Differentiation via Presenilin 1 Mediated Notch and ß-Catenin Activation.

Light-based photo-stimulation has demonstrated promising effects on stem cell behavior, particularly in optimizing neurogenesis. However, the precise parameters for achieving optimal results, including the wavelengths, light intensity, radiating energy, and underlying mechanisms, remain incompletely understood. In this study, we focused on utilizing ultraviolet-C (UV-C) at a specific wavelength of 254 nm, with an ultra-low dose at intensity of 330 µW/cm2 and a total energy of 594 mJ/cm2 per day over a period of seven days, to stimulate the proliferation and differentiation of mouse neural stem cells (NSCs). The results revealed that the application of ultra-low-dose UV-C yielded the most significant effect in promoting differentiation when compared to mixed ultraviolet (UV) and ultraviolet-A (UV-A) radiation at equivalent exposure levels. The mechanism exploration elucidated the role of Presenilin 1 in mediating the activation of ß-catenin and Notch 1 by the UV-C treatment, both of which are key factors facilitating NSCs proliferation and differentiation. These findings introduce a novel approach employing ultra-low-dose UV-C for specifically enhancing NSC differentiation, as well as the underlying mechanism. It would contribute valuable insights into brain stimulation and neurogenesis modulation for various diseases, offering potential therapeutic avenues for further exploration.

Transcriptomic analysis of rat brain response to alternating current electrical stimulation: unveiling insights via single-nucleus RNA sequencing.

Electrical brain stimulation (EBS) has gained popularity for laboratory and clinical applications. However, comprehensive characterization of cellular diversity and gene expression changes induced by EBS remains limited, particularly with respect to specific brain regions and stimulation sites. Here, we presented the initial single-nucleus RNA sequencing profiles of rat cortex, hippocampus, and thalamus subjected to intracranial alternating current stimulation (iACS) at 40 Hz. The results demonstrated an increased number of neurons in all three regions in response to iACS. Interestingly, less than 0.1% of host gene expression in neurons was significantly altered by iACS. In addition, we identified Rgs9, a known negative regulator of dopaminergic signaling, as a unique downregulated gene in neurons. Unilateral iACS produced a more focused local effect in attenuating the proportion of Rgs9+ neurons in the ipsilateral compared to bilateral iACS treatment. The results suggested that unilateral iACS at 40 Hz was an efficient approach to increase the number of neurons and downregulate Rgs9 gene expression without affecting other cell types or genes in the brain. Our study presented the direct evidence that EBS could boost cerebral neurogenesis and enhance neuronal sensitization to dopaminergic drugs and agonists, through its downregulatory effect on Rgs9 in neurons.

Role of integrin ß1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

Cartilage defects may lead to severe degenerative joint diseases. Tissue engineering based on type I collagen hydrogel that has chondrogenic potential is ideal for cartilage repair. However, the underlying mechanisms of chondrogenic differentiation driven by type I collagen hydrogel have not been fully clarified. Herein, we explored potential collagen receptors and chondrogenic signaling pathways through bioinformatical analysis to investigate the mechanism of collagen-induced chondrogenesis. Results showed that the super enhancer-related genes induced by collagen hydrogel were significantly enriched in the TGF-ß signaling pathway, and integrin-ß1 (ITGB1), a receptor of collagen, was highly expressed in bone marrow mesenchymal stem cells (BMSCs). Further analysis showed genes such as COL2A1 and Tenascin C (TNC) that interacted with ITGB1 were significantly enriched in extracellular matrix (ECM) structural constituents in the chondrogenic induction group. Knockdown of ITGB1 led to the downregulation of cartilage-specific genes (SOX9, ACAN, COL2A1), SMAD2 and TNC, as well as the downregulation of phosphorylation of SMAD2/3. Knockdown of TNC also resulted in the decrease of cartilage markers, ITGB1 and the SMAD2/3 phosphorylation but overexpression of TNC showed the opposite trend. Finally, in vitro and in vivo experiments confirmed the involvement of ITGB1 and TNC in collagen-mediated chondrogenic differentiation and cartilage regeneration. In summary, we demonstrated that ITGB1 was a crucial receptor for chondrogenic differentiation of BMSCs induced by collagen hydrogel. It can activate TGF-SMAD2/3 signaling, followed by impacting TNC expression, which in turn promotes the interaction of ITGB1 and TGF-SMAD2/3 signaling to enhance chondrogenesis. These may provide concernful support for cartilage tissue engineering and biomaterials development.

Identification of autophagy-related genes in osteoarthritis articular cartilage and their roles in immune infiltration.

Background: Autophagy plays a critical role in the progression of osteoarthritis (OA), mainly by regulating inflammatory and immune responses. However, the underlying mechanisms remain unclear. This study aimed to investigate the potential relevance of autophagy-related genes (ARGs) associated with infiltrating immune cells in OA. Methods: GSE114007, GSE169077, and ARGs were obtained from the Gene Expression Omnibus (GEO) database and the Human Autophagy database. R software was used to identify the differentially expressed autophagy-related genes (DEARGs) in OA. Functional enrichment and protein-protein interaction (PPI) analyses were performed to explore the role of DEARGs in OA cartilage, and then Cytoscape was utilized to screen hub ARGs. Single-sample gene set enrichment analysis (ssGSEA) was used to conduct immune infiltration analysis and evaluate the potential correlation of key ARGs and immune cell infiltration. Then, the expression levels of hub ARGs in OA were further verified by the GSE169077 and qRT-PCR. Finally, Western blotting and immunohistochemistry were used to validate the final hub ARGs. Results: A total of 24 downregulated genes and five upregulated genes were identified, and these genes were enriched in autophagy, mitophagy, and inflammation-related pathways. The intersection results identified nine hub genes, namely, CDKN1A, DDIT3, FOS, VEGFA, RELA, MAP1LC3B, MYC, HSPA5, and HSPA8. GSE169077 and qRT-PCR validation results showed that only four genes, CDKN1A, DDT3, MAP1LC3B, and MYC, were consistent with the bioinformatics analysis results. Western blotting and immunohistochemical (IHC) showed that the expression of these four genes was significantly downregulated in the OA group, which is consistent with the qPCR results. Immune infiltration correlation analysis indicated that DDIT3 was negatively correlated with immature dendritic cells in OA, and FOS was positively correlated with eosinophils. Conclusion: CDKN1A, DDIT3, MAP1LC3B, and MYC were identified as ARGs that were closely associated with immune infiltration in OA cartilage. Among them, DDIT3 showed a strong negative correlation with immature dendritic cells. This study found that the interaction between ARGs and immune cell infiltration may play a crucial role in the pathogenesis of OA; however, the specific interaction mechanism needs further research to be clarified. This study provides new insights to further understand the molecular mechanisms of immunity involved in the process of OA by autophagy.

O-GlcNAc transferase in astrocytes modulates depression-related stress susceptibility through glutamatergic synaptic transmission.

Major depressive disorder is a common and devastating psychiatric disease, and the prevalence and burden are substantially increasing worldwide. Multiple studies of depression patients have implicated glucose metabolic dysfunction in the pathophysiology of depression. However, the molecular mechanisms by which glucose and related metabolic pathways modulate depressive-like behaviors are largely uncharacterized. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a donor molecule for O-GlcNAcylation. O-GlcNAc transferase (OGT), a key enzyme in protein O-GlcNAcylation, catalyzes protein posttranslational modification by O-GlcNAc and acts as a stress sensor. Here, we show that Ogt mRNA was increased in depression patients and that astroglial OGT expression was specifically upregulated in the medial prefrontal cortex (mPFC) of susceptible mice after chronic social-defeat stress. The selective deletion of astrocytic OGT resulted in antidepressant-like effects, and moreover, astrocytic OGT in the mPFC bidirectionally regulated vulnerability to social stress. Furthermore, OGT modulated glutamatergic synaptic transmission through O-GlcNAcylation of glutamate transporter-1 (GLT-1) in astrocytes. OGT astrocyte-specific knockout preserved the neuronal morphology atrophy and Ca2+ activity deficits caused by chronic stress and resulted in antidepressant effects. Our study reveals that astrocytic OGT in the mPFC regulates depressive-like behaviors through the O-GlcNAcylation of GLT-1 and could be a potential target for antidepressants.

Autophagy induction promoted by m6A reader YTHDF3 through translation upregulation of FOXO3 mRNA.

Autophagy is crucial for maintaining cellular energy homeostasis and for cells to adapt to nutrient deficiency, and nutrient sensors regulating autophagy have been reported previously. However, the role of eiptranscriptomic modifications such as m6A in the regulation of starvation-induced autophagy is unclear. Here, we show that the m6A reader YTHDF3 is essential for autophagy induction. m6A modification is up-regulated to promote autophagosome formation and lysosomal degradation upon nutrient deficiency. METTL3 depletion leads to a loss of functional m6A modification and inhibits YTHDF3-mediated autophagy flux. YTHDF3 promotes autophagy by recognizing m6A modification sites around the stop codon of FOXO3 mRNA. YTHDF3 also recruits eIF3a and eIF4B to facilitate FOXO3 translation, subsequently initiating autophagy. Overall, our study demonstrates that the epitranscriptome regulator YTHDF3 functions as a nutrient responder, providing a glimpse into the post-transcriptional RNA modifications that regulate metabolic homeostasis.

Proteomic Profiling of Astrocytic O-GlcNAc Transferase-Related Proteins in the Medial Prefrontal Cortex.

The medial prefrontal cortex (mPFC), a key part of the brain networks that are closely related to the regulation of behavior, acts as a key regulator in emotion, social cognition, and decision making. Astrocytes are the majority cell type of glial cells, which play a significant role in a number of processes and establish a suitable environment for the functioning of neurons, including the brain energy metabolism. Astrocyte's dysfunction in the mPFC has been implicated in various neuropsychiatric disorders. Glucose is a major energy source in the brain. In glucose metabolism, part of glucose is used to convert UDP-GlcNAc as a donor molecule for O-GlcNAcylation, which is controlled by a group of enzymes, O-GlcNAc transferase enzyme (OGT), and O-GlcNAcase (OGA). However, the role of O-GlcNAcylation in astrocytes is almost completely unknown. Our research showed that astrocytic OGT could influence the expression of proteins in the mPFC. Most of these altered proteins participate in metabolic processes, transferase activity, and biosynthetic processes. GFAP, an astrocyte maker, was increased after OGT deletion. These results provide a framework for further study on the role of astrocytic OGT/O-GlcNAcylation in the mPFC.

MST4 negatively regulates the EMT, invasion and metastasis of HCC cells by inactivating PI3K/AKT/Snail1 axis.

Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and has a poor prognosis due to the high incidence of invasion and metastasis-related progression. However, the underlying mechanism remains elusive, and valuable biomarkers for predicting invasion, metastasis, and poor prognosis of HCC patients are still lacking. Methods: Immunohistochemistry (IHC) was performed on HCC tissues (n = 325), and the correlations between MST4 expression of the clinical HCC tissues, the clinicopathologic features, and survival were further evaluated. The effects of MST4 on HCC cell migratory and invasive properties in vitro were evaluated by Transwell and Boyden assays. The intrahepatic metastasis mouse model was established to evaluate the HCC metastasis in vivo. The PI3K inhibitor, LY294002, and a specific siRNA against Snail1 were used to investigate the roles of PI3K/AKT pathway and Snail1 in MST4-regulated EMT, migration, and invasion of HCC cells, respectively. Results: In this study, by comprehensively analyzing our clinical data, we discovered that low MST4 expression is highly associated with the advanced progression of HCC and serves as a prognostic biomarker for HCC patients of clinical-stage III-IV. Functional studies indicate that MST4 inactivation induces epithelial-to-mesenchymal transition (EMT) of HCC cells, promotes their migratory and invasive potential in vitro, and facilitates their intrahepatic metastasis in vivo, whereas MST4 overexpression exhibits the opposite phenotypes. Mechanistically, MST4 inactivation elevates the expression and nuclear translocation of Snail1, a key EMT transcription factor (EMT-TF), through the PI3K/AKT signaling pathway, thus inducing the EMT phenotype of HCC cells, and enhancing their invasive and metastatic potential. Moreover, a negative correlation between MST4 and p-AKT, Snail1, and Ki67 and a positive correlation between MST4 and E-cadherin were determined in clinical HCC samples. Conclusions: Our findings indicate that MST4 suppresses EMT, invasion, and metastasis of HCC cells by modulating the PI3K/AKT/Snail1 axis, suggesting that MST4 may be a potential prognostic biomarker for aggressive and metastatic HCC.

MST4 inhibits human hepatocellular carcinoma cell proliferation and induces cell cycle arrest via suppression of PI3K/AKT pathway.

Objective: MST4 has exhibited functions in regulating cell polarity, Golgi apparatus, cell migration, and cancer. Mechanistically, it affects the activity of p-ERK, Hippo-YAP pathway and autophagy. The aim of this study is to further examine the functions of MST4 in hepatocellular carcinoma (HCC) and the underlying mechanism. Methods: The expression level of MST4 in HCC and noncancer adjacent liver tissues was determined by qRT-PCR and immunohistochemistry staining. Wild-type MST4 (MST4) and a dominant-negative mutant of MST4 (dnMST4) were overexpressed in HCC cell lines, respectively. CCK-8 assay, EdU incorporation assay, and soft agar assay were used to determine cell proliferation in vitro. The xenograft mouse model was employed to determine HCC cell growth in vivo. Cell cycle analysis was performed by PI staining and flow cytometry. The expression of key members in PI3K/AKT pathway was detected by Western blot analysis. Results: In our study, we reported new evidence that MST4 was frequently down-regulated in HCC tissues. Gain-of-function and loss-of-function experiments demonstrated that MST4 negatively regulated in vitro HCC cell proliferation. Additionally, MST4 overexpression suppressed Bel-7404 cell tumor growth in nude mice. Further experiments revealed that the growth-inhibitory effect of MST4 overexpression was partly due to a G1-phase cell cycle arrest. Importantly, mechanistic investigations suggested that dnMST4 significantly elevated the phosphorylation levels of key members of PI3K/AKT pathway, and the selective PI3K inhibitor LY294002 can reverse the proliferation-promoting effect of dnMST4. Conclusions: Overall, our results provide a new insight into the clinical significance, functions and molecular mechanism of MST4 in HCC, suggesting that MST4 might have a potential therapeutic value in the HCC clinical treatment.