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Reactive astrogliosis and neuronal death are major features of brain tissue damage after transient global cerebral ischemia/reperfusion (I/R). The CA1 subfield in the hippocampus is particularly susceptible to cell death after I/R. Recently, attention has focused on the relationship between the autophagy–lysosomal pathway and cerebral ischemia. Lysosomal‐associated membrane protein type‐2A (LAMP‐2A) is a key protein in chaperone‐mediated autophagy (CMA). However, LAMP‐2A expression in astrocytes of the hippocampus and its influence on brain injury following I/R remain unknown. Here, we show that LAMP‐2A is elevated in astrocytes of the CA1 hippocampal subfield after I/R and in primary cultured astrocytes after transient oxygen–glucose deprivation (OGD). Conditional LAMP‐2A knockdown in CA1 astrocytes inhibited astrocyte activation and prevented neuronal death by inhibiting the mitochondrial pathway of apoptosis after I/R, suggesting that elevated astrocytic LAMP‐2A contributes to regional ischemic vulnerability. Furthermore, astrocytic LAMP‐2A ablation ameliorated the spatial learning and memory deficits caused by I/R. Conditional astrocytic LAMP‐2A knockdown also prevented the loss of hippocampal synapses and dendritic spines, improved the synaptic ultrastructure, and inhibited the reduced expression of synaptic proteins after ischemia. Thus, our findings demonstrate that astrocytic LAMP‐2A expression increases upon I/R and that LAMP‐2A ablation specifically in hippocampal astrocytes contributes to cerebroprotection, suggesting a novel neuroprotective strategy for patients with global ischemia. Hippocampal astrocytic LAMP‐2A knockdown inhibits astrocyte activation, maintains the integrity of neuronal morphology, and ameliorates cognitive deficits after transient global cerebral ischemia.

Autophagy is an intracellular recycling process that maintains cellular homeostasis by degrading excess or defective macromolecules and organelles. Chaperone-mediated autophagy (CMA) is a highly selective form of autophagy in which a substrate containing a KFERQ-like motif is recognized by a chaperone protein, delivered to the lysosomal membrane, and then translocated to the lysosome for degradation with the assistance of lysosomal membrane protein 2A. Normal CMA activity is involved in the regulation of cellular proteostasis, metabolism, differentiation, and survival. CMA dysfunction disturbs cellular homeostasis and directly participates in the pathogenesis of human diseases. Previous investigations on CMA in the central nervous system have primarily focus on neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. Recently, mounting evidence suggested that brain injuries involve a wider range of types and severities, making the involvement of CMA in the bidirectional processes of damage and repair even more crucial. In this review, we summarize the basic processes of CMA and its associated regulatory mechanisms and highlight the critical role of CMA in brain injury such as cerebral ischemia, traumatic brain injury, and other specific brain injuries. We also discuss the potential of CMA as a therapeutic target to treat brain injury and provide valuable insights into clinical strategies.

Autophagy is a central mechanism for maintaining cellular homeostasis in health and disease as it provides the critical energy through the breakdown and recycling of cellular components and molecules within lysosomes. One of the three types of autophagy is chaperone-mediated autophagy (CMA), a degradation pathway selective for soluble cytosolic proteins that contain a targeting motif related to KFERQ in their amino acid sequence. This motif marks them as CMA substrate and is, in the initial step of CMA, recognised by the heat shock protein 70 (Hsc70). The protein complex is then targeted to the lysosomal membrane where the interaction with the splice variant A of the lysosomal-associated membrane protein-2 (LAMP-2A) results in its unfolding and translocation into the lysosome for degradation. Altered levels of CMA have been reported in a wide range of pathologies including many cancer types that upregulate CMA as part of the pro-tumorigenic phenotype, while in aging a decline is observed and associated with a decrease of LAMP-2 expression. The potential of altering CMA to modify a physiological or pathological process has been firmly established through genetic manipulation in animals and chemical interference with this pathway. However, its use for therapeutic purposes has remained limited. Compounds used to target and modify CMA have been applied successfully to gain a better understanding of its cellular mechanisms, but they are mostly not specific, also influence other autophagic pathways and are associated with high levels of toxicity. Here, we will focus on the molecular mechanisms involved in CMA regulation as well as on potential ways to intersect them, describe modulators successfully used, their mechanism of action and therapeutic potential. Furthermore, we will discuss the potential benefits and drawbacks of CMA modulation in diseases such as cancer.

Glioblastoma (GB) is one of the most aggressive and treatment-resistant cancers due to its complex tumor microenvironment (TME). We previously showed that GB progression is dependent on the aberrant induction of chaperone-mediated autophagy (CMA) in pericytes (PCs), which promotes TME immunosuppression through the PC secretome. The secretion of extracellular matrix (ECM) proteins with anti-tumor (Lumican) and pro-tumoral (Osteopontin, OPN) properties was shown to be dependent on the regulation of GB-induced CMA in PCs. As biomarkers are rarely studied in TME, in this work, we aimed to validate Lumican and OPN as prognostic markers in the perivascular areas of the peritumoral niche of a cohort of GB patients. Previously, we had validated their expression in GB xenografted mice presenting GB infiltration (OPN) or GB elimination (Lumican) dependent on competent or deficient CMA PCs, respectively. Then, patient sample classification by GB infiltration into the peritumoral brain parenchyma was related to GB-induced CMA in microvasculature PCs, analyzing the expression of the lysosomal receptor, LAMP-2A. Our results revealed a correlation between GB-induced CMA activity in peritumoral PCs and GB patients’ outcomes, identifying three degrees of severity. The perivascular expression of both immune activation markers, Iba1 and CD68, was related to CMA-dependent PC immune function and determined as useful for efficient GB prognosis. Lumican expression was identified in perivascular areas of patients with less severe outcome and partially co-localizing with PCs presenting low CMA activity, while OPN was primarily found in perivascular areas of patients with poor outcome and partially co-localizing with PCs presenting high CMA activity. Importantly, we found sex differences in the incidence of middle-aged patients, being significantly higher in men but with worse prognosis in women. Our results confirmed that Lumican and OPN in perivascular areas of the GB peritumoral niche are effective predictive biomarkers for evaluating prognosis and monitoring possible therapeutic immune responses dependent on PCs in tumor progression.

The NLRP3 inflammasome/IL-1β-dependent inflammatory response serves as a critical factor and key trigger in exacerbating atherosclerosis (AS), whereas chaperone-mediated autophagy (CMA) recognizes and degrades the NLRP3 inflammasome. Targeting this pathway represents a more nuanced and targeted anti-inflammatory strategy to mitigate AS progression. As a key bioactive component derived from leaves, Ginkgolide C (GC) possesses notable anti-inflammatory effects and confers protection against myocardial and cerebral ischemia-reperfusion injuries. The current research aimed to investigate whether GC could exert protective effects against AS and to elucidate its potential underlying mechanisms. This study established both (high-fat diet/vitamin D3-induced atherosclerotic mouse model) and (LPS/ATP-stimulated RAW264.7 macrophage injury model) systems. evaluations included: H&E and Oil Red O staining for atherosclerotic lesion assessment; biochemical detection for lipid profiles; transmission electron microscopy for autophagic structure observation; immunohistochemistry and immunofluorescence for CMA regulator (LAMP-2A), NLRP3 inflammasome as well as key pro-inflammatory cytokines such as IL-1β, IL-18, and TNF-α. analyses comprised: MTT assay for cell viability; ELISA for quantifying inflammatory cytokine secretion; Western blotting for LAMP-2A, NLRP3 inflammasome, and NF-κB, MAPK signaling pathways molecules. LAMP-2A knockdown was conducted using siRNA to validate the CMA-dependent mechanisms underlying GC's effects. Our results demonstrate that GC potentiated CMA activity in macrophages, leading to promoted degradation of the NLRP3 inflammasome via the lysosomal pathway. This process effectively suppressed the NLRP3 inflammasome/IL-1β-driven inflammatory cascade, ultimately attenuating atherosclerotic progression. GC alleviates AS via a novel LAMP-2A-dependent mechanism that enhances protein clearance and suppresses NLRP3 inflammation, providing a targeted alternative to broad immunosuppression. These results establish GC as a promising therapeutic candidate and prompt further studies on its clinical efficacy and applicability in other chronic inflammatory diseases.

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.

Macroautophagy plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD), but the role of chaperone-mediated autophagy (CMA) has not been investigated. We investigated if and how CMA is involved in the pathogenesis of COPD. We measured the level of lysosome-associated membrane protein-2A (LAMP-2A), which is a critical component of CMA that functions as a receptor for cytosolic substrate proteins, in total lung tissues and primary human bronchial epithelial cells (HBECs) from healthy never smokers, smokers, and COPD patients. We assessed the effects of LAMP-2A knock-down on cigarette smoke extract (CSE)-induced aging, cell cycle arrest, and apoptosis in BEAS-2B cells and the expression levels of apoptosis hallmarks in primary HBECs and lung tissue sections. We found that the protein levels of LAMP-2A in lung homogenates and primary HBECs from smokers and COPD patients were lower than those from never smokers. In addition, its level in primary HBECs was negatively correlated with years of smoking. CSE caused degradation of LAMP-2A protein via the lysosomal pathway by activating macroautophagy. Knock-down of LAMP-2A markedly enhanced CSE-induced expression of senescence markers such as p16, p21, p27, and p53. G2/M cell cycle arrest, up-regulation of cyclin B1, and apoptosis in BEAS-2B cells. Apoptosis was increased in CSE-treated primary HBECs and in lung tissues from smokers and COPD patients. Cigarette smoke-induced down-regulation of LAMP-2A is involved in acceleration of aging and apoptosis of lung epithelial cells, which might at least partially contribute to COPD pathogenesis.

Annexin A1 (ANXA1) is an endogenous protein with potent anti-inflammatory properties in the brain. Although ANXA1 has been predominantly studied for its binding to formyl peptide receptors (FPRs) on plasma membranes, little is known regarding whether this protein has an anti-inflammatory effect in the cytosol. Here, we investigated the mechanism by which the ANXA1 peptide Ac2-26 decreases high TNF-α production and IKKβ activity, which was caused by oxygen glucose deprivation/reperfusion (OGD/R)-induced neuronal conditioned medium (NCM) in microglia. We found that exogenous Ac2-26 crosses into the cytoplasm of microglia and inhibits both gene expression and protein secretion of TNF-α. Ac2-26 also causes a decrease in IKKβ protein but not IKKβ mRNA, and this effect is inverted by lysosome inhibitor NH CL. Furthermore, we demonstrate that Ac2-26 induces IKKβ accumulation in lysosomes and that lysosomal-associated membrane protein 2A (LAMP-2A), not LC-3, is enhanced in microglia exposed to Ac2-26. We hypothesize that Ac2-26 mediates IKKβ degradation in lysosomes through chaperone-mediated autophagy (CMA). Interestingly, ANXA1 in the cytoplasm does not interact with IKKβ but with HSPB1, and Ac2-26 promotes HSPB1 binding to IKKβ. Furthermore, both ANXA1 and HSPB1 can interact with Hsc70 and LAMP-2A, but IKKβ only associates with LAMP-2A. Downregulation of HSPB1 or LAMP-2A reverses the degradation of IKKβ induced by Ac2-26. Taken together, these findings define an essential role of exogenous Ac2-26 in microglia and demonstrate that Ac2-26 is associated with HSPB1 and promotes HSPB1 binding to IKKβ, which is degraded by CMA, thereby reducing TNF-α expression.

Background Lysosome-associated membrane protein type 2A (LAMP-2A) is the key component of chaperone-mediated autophagy (CMA), a cargo-selective lysosomal degradation pathway. Aberrant LAMP-2A expression and CMA activation have been demonstrated in various human malignancies. The study focusing on the intrinsic role of LAMP-2A and CMA in glioblastoma (GBM), and downstream mechanism could provide valuable insight into the pathogenesis and novel therapeutic modality of GBM. Methods The levels of LAMP-2A, nuclear receptor co-repressor (N-CoR), unfolded protein response (UPR) and apoptosis were examined in clinical samples. LAMP-2A siRNA and shRNA were constructed to manipulate CMA activation. The role of CMA and downstream mechanism through degradation of N-CoR and arresting UPR mediated apoptosis were explored in GBM cells and nude mouse xenograft model. Results Elevated LAMP-2A and associated decreased N-CoR expression were observed in GBM as compared with peritumoral region and low-grade glioma. Inhibited UPR and apoptosis were observed in GBM with high LAMP-2A expression. In vitro study demonstrated co-localization and interaction between LAMP-2A and N-CoR. LAMP-2A silencing up-regulated N-CoR and aroused UPR pathway, leading to apoptosis, while N-CoR silencing led to an opposite result. In vivo study further confirmed that LAMP-2A inhibition arrested tumor growth by promoting apoptosis. Conclusions Our results demonstrated the central role of CMA in mediating N-CoR degradation and protecting GBM cells against UPR and apoptosis, and provided evidence of LAMP-2A as potential biomarker. Further research focusing on CMA with other tumorigenic process is needed and selective modulators of LAMP-2A remain to be investigated to provide a novel therapeutic strategy for GBM. Ci7JAW8nJL_a3Xcmr9Ld2G Video Abstract .

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.