Dl-3-n-butylphthalide improves stroke outcomes after focal ischemic stroke in mouse model by inhibiting the pyroptosis-regulated cell death and ameliorating neuroinflammation.

Recent studies have highlighted the involvement of pyroptosis-mediated cell death and neuroinflammation in ischemic stroke (IS) pathogenesis. DL-3-n-butylphthalide (NBP), a synthesized compound based on an extract from seeds of Apium graveolens, possesses a broad range of biological effects. However, the efficacy and the underlying mechanisms of NBP in IS remain contentious. Herein, we investigated the therapeutic effects of NBP and elucidated its potential mechanisms in neuronal cell pyroptosis and microglia inflammatory responses. Adult male mice underwent permanent distal middle cerebral artery occlusion (dMCAO), followed by daily oral gavage of NBP (80 mg/kg) for 1, 7, or 21 consecutive days. Gene Expression Omnibus (GEO) dataset of IS patients peripheral blood RNA sequencing was analyzed to identify differentially expressed pyroptosis-related genes (PRGs) during the ischemic process. Our results suggested that NBP treatment effectively alleviated brain ischemic damage, resulting in decreased neurological deficit scores, reduced infarct volume, and improved neurological and behavioral functions. RNA sequence data from human unveiled upregulated PRGs in IS. Subsequently, we observed that NBP downregulated pyroptosis-associated markers at days 7 and 21 post-modeling, at both the protein and mRNA levels. Additionally, NBP suppressed the co-localization of pyroptosis markers with neuronal cells to variable degrees and simultaneously mitigated the accumulation of activated microglia. Overall, our data provide novel evidence that NBP treatment significantly attenuates ischemic brain damage and promotes recovery of neurological function in the early and recovery phases after IS, probably by negatively regulating the pyroptosis cell death of neuronal cells and inhibiting toxic neuroinflammation in the central nervous system.

SARM1 promotes the neuroinflammation and demyelination through IGFBP2/NF-κB pathway in experimental autoimmune encephalomyelitis mice.

AIM: Multiple sclerosis (MS) is an autoimmune disease, and its typical characteristics are neuroinflammation and the demyelination of neurons in the central nervous system (CNS). Sterile alpha and TIR motif containing 1 (SARM1) is an essential factor mediating axonal degeneration and SARM1 deletion reduces the neuroinflammation in spinal cord injury. This study aimed to explore the roles of SARM1 and its underlying mechanisms in MS. METHODS: Experimental autoimmune encephalomyelitis (EAE, a model of MS) model was established. Immunostaining, western blot, electron microscope, and HE staining were used to examine the pathological manifestations such as inflammation, demyelination, and neuronal death in SARM1f/f EAE mice and SARM1Nestin -CKO EAE mice. In addition, RNA-seq, real-time PCR and double-immunostaining were used to examine the underlying mechanism of SARM1 in EAE mice. RESULTS: SARM1 was upregulated in neurons of the spinal cords of EAE mice. SARM1 knockout in CNS ameliorated EAE with less neuroinflammation, demyelination, and dead neurons. Mechanically, SARM1 knockout resulted in the reduction of insulin-like growth factor (IGF)-binding protein 2 (IGFBP2) in neurons of EAE mice, which might inhibit the neuroinflammation through inhibiting NF-κB signaling. Finally, activation of NF-κB partially aggravated the neuroinflammation and demyelination deficits of SARM1Nestin -CKO EAE mice. CONCLUSIONS: These results identified the unknown role of SARM1 in the promotion of neuroinflammation and demyelination and revealed a novel drug target pathway of SARM1/IGFBP2/NF-κB for MS.

Yes-associated protein regulates glutamate homeostasis through promoting the expression of excitatory amino acid transporter-2 in astrocytes via ß-catenin signaling.

The homeostasis of glutamate is mainly regulated by the excitatory amino acid transporters (EAATs), especially by EAAT2 in astrocytes. Excessive glutamate in the synaptic cleft caused by dysfunction or dysregulation of EAAT2 can lead to excitotoxicity, neuronal death and cognitive dysfunction. However, it remains unclear about the detailed regulation mechanism of expression and function of astrocytic EAAT2. In this study, first, we found increased neuronal death and impairment of cognitive function in YAPGFAP -CKO mice (conditionally knock out Yes-associated protein [YAP] in astrocytes), and identified EAAT2 as a downstream target of YAP through RNA sequencing. Second, the expression of EAAT2 was decreased in cultured YAP-/- astrocytes and the hippocampus of YAPGFAP -CKO mice, and glutamate uptake was reduced in YAP-/- astrocytes, but increased in YAP-upregulated astrocytes. Third, further investigation of the mechanism showed that the mRNA and protein levels of ß-catenin were decreased in YAP-/- astrocytes and increased in YAP-upregulated astrocytes. Wnt3a activated YAP signaling and up-regulated EAAT2 through ß-catenin. Furthermore, over-expression or activation of ß-catenin partially restored the downregulation of EAAT2, the impairment of glutamate uptake, neuronal death and cognitive decline that caused by YAP deletion. Finally, activation of EAAT2 also rescued neuronal death and cognitive decline in YAPGFAP -CKO mice. Taken together, our study identifies an unrecognized role of YAP signaling in the regulation of glutamate homeostasis through the ß-catenin/EAAT2 pathway in astrocytes, which may provide novel insights into the pathogenesis of brain diseases that closely related to the dysfunction or dysregulation of EAAT2, and promote the development of clinical strategy.

Astrocytic SARM1 promotes neuroinflammation and axonal demyelination in experimental autoimmune encephalomyelitis through inhibiting GDNF signaling.

Astrocytes are important components of the innate immune response in the central nervous system (CNS), involving in the inflammatory and neurotoxic responses that occur in CNS diseases, such as multiple sclerosis (MS). Recent studies have shown that SARM1 plays a critical role in axonal degeneration and inflammation. However, the detailed role of astrocytic SARM1 in MS remains unclear. Here, we established the MS model of mice - experimental autoimmune encephalomyelitis (EAE) and found that SARM1 was upregulated in astrocytes of the spinal cords of EAE mice. Moreover, conditional knockout of astrocytic SARM1 (SARM1GFAP-CKO mice, SARM1Aldh1L1-CKO mice) delayed EAE with later onset, alleviated the inflammatory infiltration, and inhibited the demyelination and neuronal death. Mechanically, RNA-seq revealed that the expression of glial-derived neurotrophic factor (GDNF) was upregulated in SARM1-/- astrocytes. Western blot and immunostaining further confirmed the upregulation of GDNF in spinal cord astrocytes of SARM1GFAP-CKO EAE mice. Interestingly, the downregulation of GDNF by streptozotocin (STZ, a drug used to downregulate GDNF) treatment worsened the deficits of SARM1GFAP-CKO EAE mice. These findings identify that astrocytic SARM1 promotes neuroinflammation and axonal demyelination in EAE by inhibiting the expression of GDNF, reveal the novel role of SARM1/GDNF signaling in EAE, and provide new therapeutic ideas for the treatment of MS.

Astrocytic YAP prevents the demyelination through promoting expression of cholesterol synthesis genes in experimental autoimmune encephalomyelitis.

Cholesterols are the main components of myelin, and are mainly synthesized in astrocytes and transported to oligodendrocytes and neurons in the adult brain. It has been reported that Hippo/yes-associated protein (YAP) pathways are involved in cholesterol synthesis in the liver, however, it remains unknown whether YAP signaling can prevent the demyelination through promoting cholesterol synthesis in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis characterized by neuroinflammation and demyelination. Here, we found that YAP was upregulated and activated in astrocytes of spinal cords of EAE mice through suppression of the Hippo pathway. YAP deletion in astrocytes aggravated EAE with earlier onset, severer inflammatory infiltration, demyelination, and more loss of neurons. Furthermore, we found that the neuroinflammation was aggravated and the proliferation of astrocytes was decreased in YAPGFAP-CKO EAE mice. Mechanically, RNA-seq revealed that the expression of cholesterol-synthesis pathway genes such as HMGCS1 were decreased in YAP-/- astrocytes. qPCR, western blot, and immunostaining further confirmed the more significant reduction of HMGCS1 in spinal cord astrocytes of YAPGFAP-CKO EAE mice. Interestingly, upregulation of cholesterol-synthesis pathways by diarylpropionitrile (DPN) (an ERß-ligand, to upregulate the expression of HMGCS1) treatment partially rescued the demyelination deficits in YAPGFAP-CKO EAE mice. Finally, activation of YAP by XMU-MP-1 treatment promoted the expression of HMGCS1 in astrocytes and partially rescued the demyelination and inflammatory infiltration deficits in EAE mice. These findings identify unrecognized functions of astrocytic YAP in the prevention of demyelination through promoting cholesterol synthesis in EAE, and reveal a novel pathway of YAP/HMGCS1 for cholesterol synthesis in EAE pathology.

SARM1 promotes neuroinflammation and inhibits neural regeneration after spinal cord injury through NF-κB signaling.

Axonal degeneration is a common pathological feature in many acute and chronic neurological diseases such as spinal cord injury (SCI). SARM1 (sterile alpha and TIR motif-containing 1), the fifth TLR (Toll-like receptor) adaptor, has diverse functions in the immune and nervous systems, and recently has been identified as a key mediator of Wallerian degeneration (WD). However, the detailed functions of SARM1 after SCI still remain unclear. Methods: Modified Allen's method was used to establish a contusion model of SCI in mice. Furthermore, to address the function of SARM1 after SCI, conditional knockout (CKO) mice in the central nervous system (CNS), SARM1Nestin-CKO mice, and SARM1GFAP-CKO mice were successfully generated by Nestin-Cre and GFAP-Cre transgenic mice crossed with SARM1flox/flox mice, respectively. Immunostaining, Hematoxylin-Eosin (HE) staining, Nissl staining and behavioral test assays such as footprint and Basso Mouse Scale (BMS) scoring were used to examine the roles of SARM1 pathway in SCI based on these conditional knockout mice. Drugs such as FK866, an inhibitor of SARM1, and apoptozole, an inhibitor of heat shock protein 70 (HSP70), were used to further explore the molecular mechanism of SARM1 in neural regeneration after SCI. Results: We found that SARM1 was upregulated in neurons and astrocytes at early stage after SCI. SARM1Nestin-CKO and SARM1GFAP-CKO mice displayed normal development of the spinal cords and motor function. Interestingly, conditional deletion of SARM1 in neurons and astrocytes promoted the functional recovery of behavior performance after SCI. Mechanistically, conditional deletion of SARM1 in neurons and astrocytes promoted neuronal regeneration at intermediate phase after SCI, and reduced neuroinflammation at SCI early phase through downregulation of NF-κB signaling after SCI, which may be due to upregulation of HSP70. Finally, FK866, an inhibitor of SARM1, reduced the neuroinflammation and promoted the neuronal regeneration after SCI. Conclusion: Our results indicate that SARM1-mediated prodegenerative pathway and neuroinflammation promotes the pathological progress of SCI and anti-SARM1 therapeutics are viable and promising approaches for preserving neuronal function after SCI.

D-galactose induces senescence of glioblastoma cells through YAP-CDK6 pathway.

Treatment of glioblastoma using radiotherapy and chemotherapy has various outcomes, key among them being cellular senescence. However, the molecular mechanisms of this process remain unclear. In the present study, we tested the ability of D-galactose (D-gal), a reducing sugar, to induce senescence in glioblastoma cells. Following pretreatment with D-gal, glioblastoma cell lines (C6 and U87MG) showed typical characteristics of senescence. These included the reduced cell proliferation, hypertrophic morphology, increased senescence-associated ß-galactosidase activity, downregulation of Lamin B1, and upregulation of several senescence-associated genes such as p16, p53, and NF-κB. Furthermore, our results showed that D-gal was more suitable than etoposide (a DNA-damage drug) in inducing senescence of glioblastoma cells. Mechanistically, D-gal inactivated the YAP-CDK6 signaling pathway, while overexpression of YAP or CDK6 could restore D-gal-induced senescence of C6 cells. Finally, metformin, an anti-aging agent, activated the YAP-CDK6 pathway and suppressed D-gal-induced senescence of C6 cells. Taken together, these findings established a new model for analyzing senescence in glioblastoma cells, which occurred through the YAP-CDK6 pathway. This is expected to provide a basis for development of novel therapies for the treatment of glioblastoma.