Chaperone-mediated autophagy (CMA) is a selective form of autophagy that contributes to the maintenance of cellular homeostasis. CMA activity declines with age in most tissues and systems, including the immune system, due to a reduction in levels of lysosome-associated membrane protein type 2A (LAMP2A), an essential CMA component. In this study, we show that overexpressing a copy of hLAMP2A within T cells since middle-age can prevent some of their age-associated loss of function. Our data support the idea that preserving LAMP2A expression with age through genetic means leads to enhanced proliferative responses, decreased number of regulatory T cell populations, and down-regulated expression of inhibitory receptors by T cells. During aging, elevated numbers of these immunosuppressive T cell populations significantly contribute to the age-associated downregulation of T cell responses. Using comparative proteomics, we confirm that preservation of CMA activity in old mice prevents age-related changes in both the resting and the activated T cell proteome. We also explore the effect of using first-in-class small molecule activators of CMA and demonstrate improved T cell response upon their administration to old mice. We conclude that sustaining CMA activity constitutes a potentially viable therapeutic approach to improving T cell function with age.

In orchestrating cell signaling, facilitating plasma membrane repair, supervising protein secretion, managing waste elimination, and regulating energy consumption, lysosomes are indispensable guardians that play a crucial role in preserving intracellular homeostasis. Neurons are terminally differentiated post-mitotic cells. Neuronal function and waste elimination depend on normal lysosomal function. Converging data suggest that lysosomal dysfunction is a critical event in the etiology of Parkinson\'s disease (PD). Mutations in Glucosylceramidase Beta 1 (GBA1) and leucine-rich repeat kinase 2 (LRRK2) confer an increased risk for the development of parkinsonism. Furthermore, lysosomal dysfunction has been observed in the affected neurons of sporadic PD (sPD) patients. Given that lysosomal hydrolases actively contribute to the breakdown of impaired organelles and misfolded proteins, any compromise in lysosomal integrity could incite abnormal accumulation of proteins, including α-synuclein, the major component of Lewy bodies in PD. Clinical observations have shown that lysosomal protein levels in cerebrospinal fluid may serve as potential biomarkers for PD diagnosis and as signs of lysosomal dysfunction. In this review, we summarize the current evidence regarding lysosomal dysfunction in PD and discuss the intimate relationship between lysosomal dysfunction and pathological α-synuclein. In addition, we discuss therapeutic strategies that target lysosomes to treat PD.

Defects in chaperone-mediated autophagy (CMA) are associated with cellular senescence, but the mechanism remains poorly understood. Here, we found that CMA inhibition induced cellular senescence in a calcium-dependent manner and identified its role in TNF-induced senescence of nucleus pulposus cells (NPC) and intervertebral disc degeneration. Based on structural and functional proteomic screens, PLCG1 (phospholipase C gamma 1) was predicted as a potential substrate for CMA deficiency to affect calcium homeostasis. We further confirmed that PLCG1 was a key mediator of CMA in the regulation of intracellular calcium flux. Aberrant accumulation of PLCG1 caused by CMA blockage resulted in calcium overload, thereby inducing NPC senescence. Immunoassays on human specimens showed that reduced LAMP2A, the rate-limiting protein of CMA, or increased PLCG1 was associated with disc senescence, and the TNF-induced disc degeneration in rats was inhibited by overexpression of Lamp2a or knockdown of Plcg1. Because CMA dysregulation, calcium overload, and cellular senescence are common features of disc degeneration and other age-related degenerative diseases, the discovery of actionable molecular targets that can link these perturbations may have therapeutic value.Abbreviation: ATRA: all-trans-retinoic acid; BrdU: bromodeoxyuridine; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16-INK4A: cyclin dependent kinase inhibitor 2A; CMA: chaperone-mediated autophagy; DHI: disc height index; ER: endoplasmic reticulum; IP: immunoprecipitation; IP3: inositol 1,4,5-trisphosphate; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; IVD: intervertebral disc; IVDD: intervertebral disc degeneration; KD: knockdown; KO: knockout; Leu: leupeptin; MRI: magnetic resonance imaging; MS: mass spectrometry; N/L: NH4Cl and leupeptin; NP: nucleus pulposus; NPC: nucleus pulposus cells; PI: protease inhibitors; PLC: phospholipase C; PLCG1: phospholipase C gamma 1; ROS: reactive oxygen species; RT-qPCR: real-time quantitative reverse transcription PCR; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SASP: senescence-associated secretory phenotype; STV: starvation; TMT: tandem mass tag; TNF: tumor necrosis factor; TP53: tumor protein p53; UPS: ubiquitin-proteasome system.

Introduction: Aromatic (Ar)-turmerone is a bioactive component of turmeric oil obtained from Curcuma longa. We recently identified a novel analog (A2) of ar-turmerone that protects dopaminergic neurons from toxic stimuli by activating nuclear factor erythroid 2-related factor 2 (Nrf2). D-cysteine increases Nrf2, leading to the activation of chaperone-mediated autophagy (CMA), a pathway in the autophagy-lysosome protein degradation system, in primary cultured cerebellar Purkinje cells. In this study, we attempted to identify novel analogs of ar-turmerone that activate Nrf2 more potently and investigated whether these analogs activate CMA. Methods: Four novel analogs (A4-A7) from A2 were synthesized. We investigated the effects of A2 and novel 4 analogs on Nrf2 expression via immunoblotting and CMA activity via fluorescence observation. Results: Although all analogs, including A2, increased Nrf2 expression, only A4 activated CMA in SH-SY5Y cells. Additionally, A4-mediated CMA activation was not reversed by Nrf2 inhibition, indicating that A4 activated CMA via mechanisms other than Nrf2 activation. We focused on p38, which participates in CMA activation. Inhibition of p38 significantly prevented A4-mediated activation of CMA. Although all novel analogs significantly increased the phosphorylation of p38 6 h after drug treatment, only A4 significantly increased phosphorylation 24 h after treatment. Finally, we revealed that A4 protected SH-SY5Y cells from the cytotoxicity of rotenone, and that this protection was reversed by inhibiting p38. Conclusion: These findings suggest that the novel ar-turmerone analog, A4, activates CMA and protects SH-SY5Y cells through the persistent activation of p38.

Heat shock cognate protein 70 (Hsc70/HSPA8) belongs to the Hsp70 family of molecular chaperones. The fundamental functions of Hsp70 family molecular chaperones depend on ATP-dependent allosteric regulation of binding and release of hydrophobic polypeptide substrates. Hsc70 is also involved in various other cellular functions including selective pathways of protein degradation: chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI), in which Hsc70 recruits substrate proteins containing a KFERQ-like pentapeptide motif from the cytosol to lysosomes and late endosomes, respectively. However, whether the interaction between Hsc70 and the pentapeptide motif is direct or mediated by other molecules has remained unknown. In the present study, we introduced a photo-crosslinker near the KFERQ motif in a CMA/eMI model substrate and successfully detected its crosslinking with Hsc70, revealing the direct interaction between Hsc70 and the KFERQ motif for the first time. In addition, we demonstrated that the loss of the Hsc70 ATPase activity by the D10 N mutation appreciably reduced the crosslinking efficiency. Our present results suggested that the ATP allostery of Hsc70 is involved in the direct interaction of Hsc70 with the KFERQ-like pentapeptide.

Autophagy is essential for the adaptive response to exercise and physiological skeletal muscle functionality. However, the mechanisms leading to the activation of macroautophagy and chaperone-mediated autophagy in human skeletal muscle in response to high-intensity exercise remain elusive. Our findings demonstrate that macroautophagy and chaperone-mediated autophagy are stimulated by high-intensity exercise in normoxia (PIO2: 143 mmHg) and severe acute hypoxia (PIO2: 73 mmHg) in healthy humans. High-intensity exercise induces macroautophagy initiation through AMPKα phosphorylation, which phosphorylates and activates ULK1. ULK1 phosphorylates BECN1 at Ser15, eliciting the dissociation of BECN1-BCL2 crucial for phagophore formation. Besides, high-intensity exercise elevates the LC3B-II:LC3B-I ratio, reduces total SQSTM1/p62 levels, and induces p-Ser349 SQSTM1/p62 phosphorylation, suggesting heightened autophagosome degradation. PHAF1/MYTHO, a novel macroautophagy biomarker, is highly upregulated in response to high-intensity exercise. The latter is accompanied by elevated LAMP2A expression, indicating chaperone-mediated autophagy activation regardless of post-exercise HSPA8/HSC70 downregulation. Despite increased glycolytic metabolism, severe acute hypoxia does not exacerbate the autophagy signaling response. Signaling changes revert within 1 min of recovery with free circulation, while the application of immediate post-exercise ischemia impedes recovery. Our study concludes that macroautophagy and chaperone-mediated autophagy pathways are strongly activated by high-intensity exercise, regardless of PO2, and that oxygenation is necessary to revert these signals to pre-exercise values. PHAF1/MYTHO emerges as a pivotal exercise-responsive autophagy marker positively associated with the LC3B-II:LC3B-I ratio.

Objective To elucidate the role of chaperone-mediated autophagy (CMA) in alleviating emotional dysfunction in mice with sepsis-associated encephalopathy (SAE). Methods The SAE mouse model was established by cecal ligation and perforation (CLP). The severity of sepsis was assessed using the sepsis severity score (MSS). Emotional function in SAE mice was assessed by the open-field test and elevated plus-maze. The expression levels of cognitive heat shock cognate protein 70 (HSC70), lysosomal-associated membrane protein 2A (LAMP2A) and high mobility group box 1 protein B1 (HMGB1) were detected using Western blotting. Co-localization of LAMP2A in the hippocampal neurons was observed by immunofluorescence. The release of inflammatory factors interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) was measured using ELISA. Following 12 hours post-CLP, mice were orally administered resveratrol at a dose of 30 mg/kg once daily until day 14. Results The mortality rate of CLP mice was 45.83% 24 days post CLP, and all surviving mice exhibited emotional disturbances. 24 hours after CLP, a significant decrease in HSC70 and LAMP2A expression in hippocampal neurons was observed, indicating impaired CMA activity. Meanwhile, HMGB1 and inflammatory cytokines (IL-6 and TNF-α) levels increased. After resveratrol treatment, an increase of HSC70 and LAMP2A expression, and a decrease of HMGB1 expression and inflammatory cytokine release were observed, suggesting enhanced CMA activity and reduced neuroinflammation. Behavioral tests showed that emotional dysfunction was improved in SAE mice after resveratrol treatment. Conclusion CMA activity of hippocampal neurons in SAE mice is significantly reduced, leading to emotional dysfunction. Resveratrol can alleviate neuroinflammation and emotional dysfunction in SAE mice by promoting CMA and inhibiting the expression of HMGB1 and the release of inflammatory factors.

Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral system. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus with a large economic impact on the swine industry. At present, studies on the escape mechanism of PRRSV in the autophagy process, especially through chaperone-mediated autophagy (CMA), are limited. This study confirmed that PRRSV glycoprotein 5 (GP5) could disrupt the formation of the GFAP-LAMP2A complex by inhibiting the MTORC2/PHLPP1/GFAP pathway, promoting the dissociation of the pGFAP-EF1α complex, and blocking the K63-linked polyubiquitination of LAMP2A to inhibit the activity of CMA. Further research demonstrated that CMA plays an anti-PRRSV role by antagonizing nonstructural protein 11 (NSP11)-mediated inhibition of type I interferon (IFN-I) signaling. Taken together, these results indicate that PRRSV GP5 inhibits the antiviral effect of CMA by targeting LAMP2A. This research provides new insight into the escape mechanism of immunosuppressive viruses in CMA.
OBJECTIVE: Viruses have evolved sophisticated mechanisms to manipulate autophagy to evade degradation and immune responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus that causes enormous economic losses in the swine industry. However, the mechanism by which PRRSV manipulates autophagy to defend against host antiviral effects remains unclear. In this study, we found that PRRSV GP5 interacts with LAMP2A and disrupts the formation of the GFAP-LAMP2A complex, thus inhibiting the activity of CMA and subsequently enhancing the inhibitory effect of the NSP11-mediated IFN-I signaling pathway, ultimately facilitating PRRSV replication. Our study revealed a novel mechanism by which PRRSV escapes host antiviral effects through CMA, providing a potential host target, LAMP2A, for developing antiviral drugs and contributing to understanding the escape mechanism of immunosuppressive viruses.

Recent studies have shown that autophagy is activated in response to nerve damage and occurs simultaneously with the initial stages of Schwann cell-mediated demyelination. Although several studies have reported that macroautophagy is involved in the peripheral nerve, the role of chaperone-mediated autophagy (CMA) has not yet been investigated in peripheral nerve injury. The present study investigates the role of CMA in the sciatic nerve. Using a mouse model of sciatic nerve injury, the authors employed immunofluorescence analysis to observe the expression of LAMP2A, a critical marker for CMA. RNA sequencing was performed to observe the transcriptional profile of Lamp2a in Schwann cells. Bioinformatics analysis was carried out to observe the hub genes associated with Lamp2a . Expression of Lamp2a , a key gene in CMA, increased following sciatic nerve injury, based on an immunofluorescence assay. To identify differentially expressed genes using Lamp2a , RNA sequence analysis was conducted using rat Schwann cells overexpressing Lamp2a . The nine hub genes ( Snrpf, Polr1d, Snip1, Aqr, Polr2h, Ssbp1, Mterf3, Adcy6 , and Sbds ) were identified using the CytoHubba plugin of Cytoscape. Functional analysis revealed that Lamp2a overexpression affected the transcription levels of genes associated with mitotic spindle organization and mRNA splicing via the spliceosome. In addition, Polr1d and Snrpf1 were downregulated throughout postnatal development but elevated following sciatic nerve injury, according to a bioinformatics study. CMA may be an integral pathway in sciatic nerve injury via mRNA splicing.

Breast cancer (BC) is most commonly diagnosed worldwide. Liquiritigenin is a flavonoid found in various species of the Glycyrrhiza genus, showing anti-tumor activity. This article was to explore the influences of liquiritigenin on the biological behaviors of BC cells and its underlying mechanism. BC cells were treated with liquiritigenin alone or transfected with oe-HSP90 before liquiritigenin treatment. RT-qPCR and Western blotting were employed to examine the levels of HSP90, Snail, E-cadherin, HSC70, and LAMP-2A. Cell viability, proliferation, migration, and invasion were evaluated by performing MTT, colony formation, scratch, and Transwell assays, respectively. Liquiritigenin treatment reduced HSP90 and Snail levels and enhanced E-cadherin expression as well as inhibiting the proliferation, migration, and invasion of BC cells. Moreover, liquiritigenin treatment decreased the expression of HSC70 and LAMP-2A, proteins related to chaperone-mediated autophagy (CMA). HSP90 overexpression promoted the CMA, invasion, and migration of BC cells under liquiritigenin treatment. Liquiritigenin inhibits HSP90-mediated CMA, thereby suppressing BC cell growth.