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The cell‐to‐cell transfer of α‐synuclein (α‐Syn) greatly contributes to Parkinson's disease (PD) pathogenesis and underlies the spread of α‐Syn pathology. During this process, extracellular α‐Syn can activate microglia and neuroinflammation, which plays an important role in PD. However, the effect of extracellular α‐Syn on microglia autophagy is poorly understood. In the present study, we reported that extracellular α‐Syn inhibited the autophagy initiation, as indicated by LC3‐II reduction and p62 protein elevation in BV2 and cultured primary microglia. The in vitro findings were verified in microglia‐enriched population isolated from α‐Syn‐overexpressing mice induced by adeno‐associated virus (AAV2/9)‐encoded wildtype human α‐Syn injection into the substantia nigra (SN). Mechanistically, α‐Syn led to microglial autophagic impairment through activating toll‐like receptor 4 (Tlr4) and its downstream p38 and Akt‐mTOR signaling because Tlr4 knockout and inhibition of p38, Akt as well as mTOR prevented α‐Syn‐induced autophagy inhibition. Moreover, inhibition of Akt reversed the mTOR activation but failed to affect p38 phosphorylation triggered by α‐Syn. Functionally, the in vivo evidence showed that lysozyme 2 Cre (Lyz2cre)‐mediated depletion of autophagy‐related gene 5 (Atg5) in microglia aggravated the neuroinflammation and dopaminergic neuron losses in the SN and exacerbated the locomotor deficit in α‐Syn‐overexpressing mice. Taken together, the results suggest that extracellular α‐Syn, via Tlr4‐dependent p38 and Akt‐mTOR signaling cascades, disrupts microglial autophagy activity which synergistically contributes to neuroinflammation and PD development. Autophagy‐dependent and independent machinery synergistically contribute to hα‐Syn‐caused neuroinflammation in PD. The basal autophagy activity restricts microglia inflammation. Extracellular hα‐Syn interacts with and activates Tlr4, resulting in inflammatory responses, as well as autophagy suppression in microglia via Tlr4‐dependent p38 and Akt/mTOR signaling cascades. This impairs the inhibitory effect of autophagy on inflammation, and thus aggravating hα‐Syn‐induced inflammatory responses.

Diabetic kidney disease (DKD) is one of the microvascular complications of diabetes mellitus and a major cause of end-stage renal disease with limited treatment options. Wogonin is a flavonoid derived from the root of Scutellaria baicalensis Georgi, which has shown a potent renoprotective effect. But the mechanisms of action in DKD are not fully elucidated. In this study, we investigated the effects of wogonin on glomerular podocytes in DKD using mouse podocyte clone 5 (MPC5) cells and diabetic mice model. MPC5 cells were treated with high glucose (30 mM). We showed that wogonin (4, 8, 16 μM) dose-dependently alleviated high glucose (HG)-induced MPC5 cell damage, accompanied by increased expression of WT-1, nephrin, and podocin proteins, and decreased expression of TNF-α, MCP-1, IL-1β as well as phosphorylated p65. Furthermore, wogonin treatment significantly inhibited HG-induced apoptosis in MPC5 cells. Wogonin reversed HG-suppressed autophagy in MPC5 cells, evidenced by increased ATG7, LC3-II, and Beclin-1 protein, and decreased p62 protein. We demonstrated that wogonin directly bound to Bcl-2 in MPC5 cells. In HG-treated MPC5 cells, knockdown of Bcl-2 abolished the beneficial effects of wogonin, whereas overexpression of Bcl-2 mimicked the protective effects of wogonin. Interestingly, we found that the expression of Bcl-2 was significantly decreased in biopsy renal tissue of diabetic nephropathy patients. In vivo experiments were conducted in STZ-induced diabetic mice, which were administered wogonin (10, 20, 40 mg · kg −1  · d −1 , i.g.) every other day for 12 weeks. We showed that wogonin administration significantly alleviated albuminuria, histopathological lesions, and p65 NF-κB-mediated renal inflammatory response. Wogonin administration dose-dependently inhibited podocyte apoptosis and promoted podocyte autophagy in STZ-induced diabetic mice. This study for the first time demonstrates a novel action of wogonin in mitigating glomerulopathy and podocytes injury by regulating Bcl-2-mediated crosstalk between autophagy and apoptosis. Wogonin may be a potential therapeutic drug against DKD.

Abstract BackgroundSub-anesthetic ketamine has rapid-onset effects for the treatment of major depressive disorder (MDD). However, the mechanism underlying ketamine’s antidepressant properties remains unclear. Recent studies have reported an interrelationship between autophagy and the inflammasome, both of which are involved in the pathophysiology of MDD. In this study, we assess whether ketamine exerts its antidepressant effects via an association with the autophagy-NLRP3 inflammasome pathway.MethodsWe established a depressive-like rat model by treating Wistar Kyoto rats with chronic restraint stress (CRS) for 28 days. Microglial cells from newborn Sprague–Dawley rats were used for in vitro experiments.ResultsWe found sub-anesthetic ketamine treatment reversed depressive-like behavior in CRS rats. Ketamine triggered autophagy in the microglia of prefrontal cortex (PFC) and (hippocampus) HPC, with increased levels of LC3B, decreased levels of p62 protein, and elevated autophagosomes both in vivo and in vitro. Moreover, NLRP3 inflammasome activation was also inhibited by ketamine, with reduced expression of NLRP3-ASC-CASP1 assembly and decreased IL-1β levels in cerebrospinal fluid (CSF) as well as in the serum. Increased BDNF levels and synaptophysin levels were detected in the ketamine-treated group. The rapid anti-depressive effects, elevation of autophagy, reduction in NLRP3, and neuroplasticity-related factors induced by ketamine could be significantly blocked by the autophagy inhibitor Baf A1 (0.1 mg/kg).ConclusionsOur findings demonstrate that sub-anesthetic doses of ketamine exert their antidepressant-like effects by inhibiting inflammation and initiating neuroprotection via autophagy activation. These data might help expand future investigations on the antidepressant properties of ketamine.

Manganese (Mn), the third most abundant transition metal in the earth’s crust, has widespread applications in the emerging field of organometallic catalysis and traditional industries. Excessive Mn exposure causes neurological syndrome resembling Parkinson’s disease (PD). The pathogenesis of PD is thought to involve microglia-mediated neuroinflammatory injury, with mitochondrial dysfunction playing a role in aberrant microglial activation. In the early stages of PD, PINK1/Parkin-mediated mitophagy contributes to the microglial inflammatory response via the cGAS/STING signaling pathway. Suppression of PINK1/Parkin-mediated mitophagy due to excessive Mn exposure exacerbates neuronal injury. Moreover, excessive Mn exposure leads to neuroinflammatory damage via the microglial cGAS-STING pathway. However, the precise role of microglial mitophagy in modulating neuroinflammation in Mn-induced parkinsonism and its underlying molecular mechanism remains unclear. Here, we observed that Mn-exposed mice exhibited neurobehavioral abnormalities and detrimental microglial activation, along with increased apoptosis of nerve cells, proinflammatory cytokines, and intracellular ROS. Furthermore, in vivo and in vitro experiments showed that excessive Mn exposure resulted in microglial mitochondrial dysfunction, manifested by increased mitochondrial ROS, decreased mitochondrial mass, and membrane potential. Additionally, with the escalating Mn dose, PINK1/Parkin-mediated mitophagy changed from activation to suppression. This was evidenced by decreased levels of LC3-II, PINK1, p-Parkin/Parkin, and increased levels of p62 protein expression level, as well as the colocalization between ATPB and LC3B due to excessive Mn exposure. Upregulation of mitophagy by urolithin A could mitigate Mn-induced mitochondrial dysfunction, as indicated by decreased mitochondrial ROS, increased mitochondrial mass, and membrane potential, along with improvements in neurobehavioral deficits and attenuated detrimental microglial activation. Using single-nucleus RNA-sequencing (snRNA-seq) analysis in the Mn-exposed mouse model, we identified the microglial cGAS-STING signaling pathway as a potential mechanism underlying Mn-induced neuroinflammation. This pathway is associated with an increase in cytosolic mtDNA levels, which activate STING signaling. These findings point to the induction of microglial mitophagy as a viable strategy to alleviate Mn-induced neuroinflammation through mtDNA-STING signaling. Graphical Abstract

Autophagy is a cellular mechanism that recycles cellular components to maintain homeostasis. To investigate the clinical implication of autophagy in gastric cancer, the autophagy markers with autophagosome formation, LC3B and selective autophagy substrate p62/SQSTM1 (P62) were validated. LC3B and p62 expression was examined using immunohistochemistry, western blot assays, and reverse-transcription polymerase chain reaction (RT-PCR). The relationship of LC3B and p62 expression in gastric adenocarcinomas with clinicopathological parameters, including patient survival, were analyzed. Normal gastric mucosae exhibit no LC3B and p62 expression, while tubular adenoma and gastric adenocarcinomas exhibit variable nuclear or cytoplasmic p62 expression. High LC3B, high cytoplasmic p62, and low nuclear p62 protein expression in gastric adenocarcinomas is positively correlated with poor prognostic factors including survival. Dynamic LC3B and p62 changes are suggested to be involved in gastric tumorigenesis and cancer progression. LC3B and p62 could be used as prognostic biomarkers and potential therapeutic targets for gastric adenocarcinomas.

The tripartite motif (TRIM) protein family plays important roles in the initiation and progression of various tumors through different mechanisms. However, the biological functions of TRIM39 and its underlying mechanism in hepatocellular carcinoma (HCC) remain largely unknown. In this study, we found that the expression of TRIM39 was increased in HCC tissues compared to adjacent normal tissues and negatively correlated with the prognosis of HCC patients. Functional studies demonstrated that TRIM39 promoted the proliferation of HCC cells in vitro and in vivo. Mechanistically, TRIM39 interacted with p62 and facilitated its ubiquitination, leading to a decrease of p62 protein stability. The downregulation of p62 released KEAP1, which subsequently inhibited NRF2-HO-1 pathway. Moreover, overexpression p62 counteracted the pro-tumor phenotypes induced by TRIM39 in HCC. In conclusion, our study elucidates the oncogenic roles and the relevant mechanisms of TRIM39 in HCC, highlights the TRIM39-p62-Keap1-NRF2 axis in HCC progression. These findings may provide potential therapeutic targets for HCC treatment.

The increased global prevalence of metabolic dysfunction-associated steatohepatitis (MASLD) has been closely associated with chronic disorders of the circadian clock. Herein, we investigate the role of Clock , a core circadian gene, in the pathogenesis of MASLD. Wild-type (WT) and liver-specific Clock knockdown ( Clock -KD) mice were fed a Western diet for 20 weeks to induce MASLD. A cellular MASLD model was established by treating AML12 cells with free fatty acids and the effects of Clock knockdown were examined following transfection with Clock siRNA. Increased lipid deposition and more severe steatohepatitis and fibrosis were observed in the livers of Western diet-fed but not normal chow diet-fed Clock -KD mice after 20 weeks compared to WT mice. Moreover, the Clock gene was found to be significantly downregulated in WT MASLD mice. The Clock gene was shown to regulate the expression of lipophagy-related proteins (LC3B, P62, RAB7, and PLIN2) in vivo and in vitro. Knockdown of Clock was found to inhibit lipophagy resulting in increased accumulation of lipid droplets in the mouse liver and AML12 cells. Interestingly, the CLOCK protein was shown to interact with P62. However, knockdown of the Clock gene did not promote transcription of the P62 gene but suppressed degradation of the P62 protein during lipophagy in AML12 cells. The hepatic Clock gene regulates lipophagy and affects lipid droplet deposition in liver cells, and thus plays a critical role in the development of MASLD induced by a Western diet.

Background Chronic neuropathic pain is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Divanillyl sulfone (DS), a novel structural derivative of 4,4′-dihydroxydibenzyl sulfoxide from a traditional Chinese medicine Gastrodia elata with anti-nociceptive effects, significantly alleviated neuropathic pain following intrathecal injection. Here, we aimed to investigate the underlying mechanisms of DS against neuropathic pain. Methods A chronic constrictive injury (CCI) mouse model of neuropathic pain induced by sciatic nerve ligation was performed to evaluate the effect of DS by measuring the limb withdrawal using Von Frey filament test. Immunofluorescence staining was used to assess the cell localizations and expressions of Iba-1, ASC, NLRP3, and ROS, the formation of autolysosome. The levels of NLRP3-related proteins (caspase-1, NLRP3, and IL-1β), mitophagy-related proteins (LC3, Beclin-1, and p62), and apoptosis-related proteins (Bcl-XL and Bax) were detected by Western blotting. The apoptosis of BV-2 cell and caspase activity were evaluated by flow cytometry. Results DS significantly alleviated the neuropathic pain by increasing the mechanical withdrawal threshold and inhibiting the activation of NLRP3 in CCI-induced model mice. Our findings indicated that DS promoted the mitophagy by increasing the LC3II and Beclin 1 and decreasing the levels of p62 protein in BV-2 cell. This is accompanied by the inhibition of NLRP3 activation, which was shown as inhibited the expression of NLRP3 in lysates as well as the secretion of mature caspase-1 p10 and IL-1β p17 in supernatants in cultured BV-2 microglia. In addition, DS could promote mitophagy-induced improvement of dysfunctional mitochondria by clearing intracellular ROS and restoring mitochondrial membrane potential. Conclusion Together, our findings demonstrated that DS ameliorate chronic neuropathic pain in mice by suppressing NLRP3 inflammasome activation induced by mitophagy in microglia. DS may be a promising therapeutic agent for chronic neuropathic pain.

Paget’s disease of bone (PDB) is a chronic skeletal disorder that can affect one or several bones in individuals older than 55 y of age. PDB-like changes have been reported in archaeological remains as old as Roman, although accurate diagnosis and natural history of the disease is lacking. Six skeletons from a collection of 130 excavated at Norton Priory in the North West of England, which dates to medieval times, show atypical and extensive pathological changes resembling contemporary PDB affecting as many as 75% of individual skeletons. Disease prevalence in the remaining collection is high, at least 16% of adults, with age at death estimations as low as 35 y. Despite these atypical features, paleoproteomic analysis identified sequestosome 1 (SQSTM1) or p62, a protein central to the pathological milieu of PDB, as one of the few noncollagenous human sequences preserved in skeletal samples. Targeted proteomic analysis detected >60% of the ancient p62 primary sequence, with Western blotting indicating p62 abnormalities, including in dentition. Direct sequencing of ancient DNA excluded contemporary PDB-associated SQSTM1 mutations. Our observations indicate that the ancient p62 protein is likely modified within its C-terminal ubiquitin-associated domain. Ancient miRNAs were remarkably preserved in an osteosarcoma from a skeleton with extensive disease, with miR-16 expression consistent with that reported in contemporary PDB-associated bone tumors. Our work displays the use of proteomics to inform diagnosis of ancient diseases such as atypical PDB, which has unusual features presumably potentiated by yet-unidentified environmental or genetic factors.

Cigarette smoke (CS) exposure is known to induce proteostasis imbalance that can initiate accumulation of ubiquitinated proteins. Therefore, the primary goal of this study was to determine if first- and secondhand CS induces localization of ubiquitinated proteins in perinuclear spaces as aggresome bodies. Furthermore, we sought to determine the mechanism by which smoke-induced aggresome formation contributes to chronic obstructive pulmonary disease (COPD)–emphysema pathogenesis. Hence, Beas2b cells were treated with CS extract (CSE) for in vitro experimental analysis of CS-induced aggresome formation by immunoblotting, microscopy, and reporter assays, whereas chronic CS–exposed murine model and human COPD–emphysema lung tissues were used for validation. In preliminary analysis, we observed a significant (P < 0.01) increase in ubiquitinated protein aggregation in the insoluble protein fraction of CSE-treated Beas2b cells. We verified that CS-induced ubiquitin aggregrates are localized in the perinuclear spaces as aggresome bodies. These CS-induced aggresomes (P < 0.001) colocalize with autophagy protein microtubule–associated protein 1 light chain-3B+ autophagy bodies, whereas U.S. Food and Drug Administration–approved autophagy-inducing drug (carbamazepine) significantly (P < 0.01) decreases their colocalization and expression, suggesting CS-impaired autophagy. Moreover, CSE treatment significantly increases valosin-containing protein–p62 protein–protein interaction (P < 0.0005) and p62 expression (aberrant autophagy marker; P < 0.0001), verifying CS-impaired autophagy as an aggresome formation mechanism. We also found that inhibiting protein synthesis by cycloheximide does not deplete CS-induced ubiquitinated protein aggregates, suggesting the role of CS-induced protein synthesis in aggresome formation. Next, we used an emphysema murine model to verify that chronic CS significantly (P < 0.0005) induces aggresome formation. Moreover, we observed that autophagy induction by carbamazepine inhibits CS-induced aggresome formation and alveolar space enlargement (P < 0.001), confirming involvement of aggresome bodies in COPD–emphysema pathogenesis. Finally, significantly higher p62 accumulation in smokers and severe COPD–emphysema lungs (Global Initiative for Chronic Obstructive Lung Disease Stage III/IV) as compared with normal nonsmokers (Global Initiative for Chronic Obstructive Lung Disease Stage 0) substantiates the pathogenic role of autophagy impairment in aggresome formation and COPD–emphysema progression. In conclusion, CS-induced aggresome formation is a novel mechanism involved in COPD–emphysema pathogenesis.