Neuroprotective effects of chaperone-mediated autophagy in neurodegenerative diseases

Chaperone-mediated autophagy is one of three types of autophagy and is characterized by the selective degradation of proteins.Chaperone-mediated autophagy contributes to energy balance and helps maintain cellular homeostasis,while providing nutrients and support for cell survival.Chaperone-mediated autophagy activity can be detected in almost all cells,including neurons.Owing to the extreme sensitivity of neurons to their environmental changes,maintaining neuronal homeostasis is critical for neuronal growth and survival.Chaperone-mediated autophagy dysfunction is closely related to central nervous system diseases.It has been shown that neuronal damage and cell death are accompanied by chaperone-mediated autophagy dysfunction.Under certain conditions,regulation of chaperone-mediated autophagy activity attenuates neurotoxicity.In this paper,we review the changes in chaperone-mediated autophagy in neurodegenerative diseases,brain injury,glioma,and autoimmune diseases.We also summarize the most recent research progress on chaperone-mediated autophagy regulation and discuss the potential of chaperone-mediated autophagy as a therapeutic target for central nervous system diseases.

Chaperone-mediated autophagy targeting chimeras (CMATAC) forthe degradation of ERα in breast cancer

Estrogen receptor alpha(ERα/ESR1)is overexpressed in over half of all breast cancers and is considered a valuable therapeutic target in ERαpositive breast cancer.Here,we designed a membrane-permeant Chaperonemediated Autophagy Targeting Chimeras(CMATAC)peptide to knockdown endogenous ERαprotein through chaperone-mediated autophagy.The peptide contains a cell membrane-penetrating peptide(TAT)that allows the peptide to by-pass the plasma membrane,anαI peptide as a protein-binding peptide(PBD)that binds specifically to ERα,and CMA-targeting peptide(CTM)that targeting chaperone-mediated autophagy.We validated that ERαtargeting peptide was able to target and degrade ERαto reduce the viability of ERαpositive breast cancer cells.Taken together,our studies provided a new method to reduce the level of intracellular ERαprotein via CMATAC,and thus may provide a new strategy for the treatment of ERαpositive breast cancer.

Bcl-xL regulates radiation-induced ferroptosis through chaperone-mediated autophagy of GPX4 in tumor cells

Objective:To investigate the role and the molecular mechanisms of apoptotic signaling in ferroptosis to regulate tumor radiosensitivity.Methods:Reactive oxygen species(ROS)and lipid peroxide levels were detected in Mouse embryonic fibroblasts(MEFs)with Bcl-xL or Mcl-1 deficiency induced by erastin.Colony formation,ROS,lipid peroxidation and the transcription/translation levels of PTGS2 were measured in Bcl-xL knockdown tumor cells induced by 5 Gyγ-rays or co-treated with ferrostatin-1(Ferr-1).The protein levels of LPCAT3,ACSL4 and PEBP1 in Bcl-xL knockout MEF cells were evaluated in Bcl-xL knockout MEF cells post-radiation.Moreover,the interaction of heat shock protein 90(HSP90)with Bcl-xL,GPX4,or LAMP2A was detected by protein mass spectrometry and immunoprecipitation assays.Results:Manipulating Bcl-xL levels facilitated radiation-induced ferroptosis by augmenting the enzymatic oxidation of polyunsaturated fatty acids(PUFAs)and enhancing chaperone-mediated autophagy(CMA)of glutathione peroxidase 4(GPX4)(MEF cell line:t=4.540,P<0.01;A549 cell line:t=56.16,P<0.0001;t=4.885,P<0.01;HCT116 cell line:t=14.75,P<0.01;t=7.363,P<0.05).Downregulating Bcl-xL expression promoted the activity of acyl-CoA synthetase long-chain family member 4(ACSL4),thus increasing the enzymatic oxidation of PUFAs(t=4.258,P<0.01).Moreover,depletion of Bcl-xL expedited the CMA process targeting GPX4 by facilitating the association of GPX4 with heat shock protein 90(HSP90)and LAMP2A following radiation exposure.Subsequent degradation of GPX4 led to the accumulation of lipid peroxides,ultimately triggering ferroptosis.Conclusions:Our study provides initial insights into the regulatory role of Bcl-xL in ferroptosis and underscores the potential of targeting Bcl-xL as a promising therapeutic strategy for cancer by modulating both apoptotic and ferroptotic pathways.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model

Chaperone-mediated autophagy(CMA)is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases.However,the mechanisms that regulate CMA are not fully understood.Here,using unbiased drug screening approaches,we discover Metformin,a drug that is commonly the first medication prescribed for type 2 diabetes,can induce CMA.We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA,Hsc70,and its activation.Notably,we find that amyloid-beta precursor protein(APP)is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner.The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity.Importantly,we find that in the APP/PS1 mouse model of Alzheimer's disease(AD),activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβplaque levels and reverses the molecular and behavioral AD phenotypes.Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases,such as AD,where such therapeutic intervention could be beneficial.

Dihydromyricetin and Salvianolic acid B inhibit alpha-synuclein aggregation and enhance chaperone-mediated autophagy

Background:Progressive accumulation ofα-synuclein is a key step in the pathological development of Parkinson’s disease.Impaired protein degradation and increased levels ofα-synuclein may trigger a pathological aggregation in vitro and in vivo.The chaperone-mediated autophagy(CMA)pathway is involved in the intracellular degradation processes ofα-synuclein.Dysfunction of the CMA pathway impairsα-synuclein degradation and causes cytotoxicity.Results:In the present study,we investigated the effects on the CMA pathway andα-synuclein aggregation using bioactive ingredients(Dihydromyricetin(DHM)and Salvianolic acid B(Sal B))extracted from natural medicinal plants.In both cell-free and cellular models ofα-synuclein aggregation,after administration of DHM and Sal B,we observed significant inhibition ofα-synuclein accumulation and aggregation.Cells were co-transfected with a Cterminal modifiedα-synuclein(SynT)and synphilin-1,and then treated with DHM(10μM)and Sal B(50μM)16 hours after transfection;levels ofα-synuclein aggregation decreased significantly(68%for DHM and 75%for Sal B).Concomitantly,we detected increased levels of LAMP-1(a marker of lysosomal homeostasis)and LAMP-2A(a key marker of CMA).Immunofluorescence analyses showed increased colocalization between LAMP-1 and LAMP-2A withα-synuclein inclusions after treatment with DHM and Sal B.We also found increased levels of LAMP-1 and LAMP-2A both in vitro and in vivo,along with decreased levels ofα-synuclein.Moreover,DHM and Sal B treatments exhibited anti-inflammatory activities,preventing astroglia-and microglia-mediated neuroinflammation in BAC-α-syn-GFP transgenic mice.Conclusions:Our data indicate that DHM and Sal B are effective in modulatingα-synuclein accumulation and aggregate formation and augmenting activation of CMA,holding potential for the treatment of Parkinson’s disease.

Chaperone-mediated autophagy:roles in neuroprotection

Chaperone-mediated autophagy （CMA）, one of the main pathways of lysosomal proteolysis, is characterized by the selective targeting and direct translocation into the lysosomal lumen of substrate proteins containing a targeting motif biochemically related to the pentapeptide KFERQ. Along with the other two lysosomal pathways, macro- and micro-autophagy, CMA is essential for maintaining cellular homeostasis and survival by selectively degrading misfolded, oxidized, or damaged cytosolic proteins. CMA plays an important role in pathologies such as cancer, kidney disorders, and neurodegenerative diseases. Neurons are post-mitotic and highly susceptible to dysfunction of cellular quality-control systems. Maintaining a balance between protein synthesis and degradation is critical for neuronal functions and homeostasis. Recent studies have revealed several new mechanisms by which CMA protects neurons through regulating factors critical for their viability and homeostasis. In the current review, we summarize recent advances in the understanding of the regulation and physiology of CMA with a specific focus on its possible roles in neuroprotection.

Chaperone-mediated autophagy and neurodegeneration:connections,mechanisms,and therapeutic implications

Lysosomes degrade dysfunctional intracellular components via three pathways： macroautophagy, microautophagy, and chaperone-mediated autophagy （CMA）. Unlike the other two, CMA degrades cytosolic proteins with a recognized KFERQ-like motif in lysosomes and is important for cellular homeostasis. CMA activity declines with age and is altered in neurodegenerative diseases. Its impairment leads to the accumulation of aggregated proteins, some of which may be directly tied to the pathogenic processes of neurodegenerative diseases. Its induction may accelerate the clearance of pathogenic proteins and promote cell survival, representing a potential therapeutic approach for the treatment of neurodegenerative diseases. In this review, we summarize the current findings on how CMA is involved in neurodegenerative diseases, especially in Parkinson＇s disease.

Inflammation-induced inhibition of chaperone-mediated autophagy maintains the immunosuppressive function of murine mesenchymal stromal cells

Macroautophagy has been implicated in modulating the therapeutic function of mesenchymal stromal cells(MSCs).However,the biological function of chaperone-mediated autophagy(CMA)in MSCs remains elusive.Here,we found that CMA was inhibited in MSCs in response to the proinflammatory cytokines interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α).In addition,suppression of CMA by knocking down the CMA-related lysosomal receptor lysosomal-associated membrane protein 2(LAMP-2A)in MSCs significantly enhanced the immunosuppressive effect of MSCs on T cell proliferation,and as expected,LAMP-2A overexpression in MSCs exerted the opposite effect on T cell proliferation.This effect of CMA on the immunosuppressive function of MSCs was attributed to its negative regulation of the expression of chemokine C-X-C motif ligand 10(CXCL10),which recruits inflammatory cells,especially T cells,to MSCs,and inducible nitric oxide synthase(iNOS),which leads to the subsequent inhibition of T cell proliferation via nitric oxide(NO).Mechanistically,CMA inhibition dramatically promoted IFN-γplus TNF-α-induced activation of NF-κB and STAT1,leading to the enhanced expression of CXCL10 and iNOS in MSCs.Furthermore,we found that IFN-γplus TNF-α-induced AKT activation contributed to CMA inhibition in MSCs.More interestingly,CMA-deficient MSCs exhibited improved therapeutic efficacy in inflammatory liver injury.Taken together,our findings established CMA inhibition as a critical contributor to the immunosuppressive function of MSCs induced by inflammatory cytokines nd highlighted a previously unknown function of CMA.

Chaperone-mediated autophagy: roles in neurodegeneration

Chaperone-mediated autophagy(CMA)selectively delivers cytosolic proteins with an exposed CMA-targeting motif to lysosomes for degradation and plays an important role in protein quality control and cellular homeostasis.A growing body of evidence supports the hypothesis that CMA dysfunction may be involved in the pathogenic process of neurodegenerative diseases.Both down-regulation and compensatory up-regulation in CMA activities have been observed in association with neurodegenerative conditions.Recent studies have revealed several new mechanisms by which CMA function may be involved in the regulation of factors critical for neuronal viability and homeostasis.Here,we summarize these recent advances in the understanding of the relationship between CMA dysfunction and neurodegeneration and discuss the therapeutic potential of targeting CMA in the treatment of neurodegenerative diseases.

Inhibition of tumor suppressor p73 by nerve growth factor receptor via chaperone-mediated autophagy

The tumor suppressr p73 is a homolog of p53 and is capable of inducing cell cycle arrest and apoptosis.Here,we identify nerve growth factor receptor(NGFR,p75NTR,or CD271)as a novel negative p73 regulator.p73 activates NGFR transcription,which,in turn,promotes p73 degradation in a negative feedback loop.NGFR directly binds to p73 central DNA-binding domain and suppresses p73 transcriptional activity as well as p73-mediated apoptosis in cancer cells.Surprisingly,we uncover a previously unknown mechanism of NGFR-facilitated p73 degradation through the chaperone-mediated autophagy(CMA)pathway.Collectively,our studies demonstrate a new oncogenic function for NGFR in inactivating p73 activity by promoting its degradation through the CMA.

Chaperone-mediated Autophagy Regulates Cell Growth by Targeting SMAD3 in Glioma

Previous studies suggest that the reduction of SMAD3(mothers against decapentaplegic homolog 3)has a great impact on tumor development,but its exact pathological function remains unclear.In this study,we found that the protein level of SMAD3 was greatly reduced in human-grade IV glioblastoma tissues,in which LAMP2A(lysosome-associated membrane protein type 2A)was significantly up-regulated.LAMP2A is a key ratelimiting protein of chaperone-mediated autophagy(CMA),a lysosome pathway of protein degradation that is activated in glioma.We carefully analyzed the amino-acid sequence of SMAD3 and found that it contained a pentapeptide motif biochemically related to KFERQ,which has been proposed to be a targeting sequence for CMA.In vitro,we confirmed that SMAD3 was degraded in either serum-free or KFERQ motif deleted condition,which was regulated by LAMP2A and interacted with HSC70(heat shock cognate 71 kDa protein).Using isolated lysosomes,amino-acid residues 75 and 128 of SMAD3 were found to be of importance for this process,which affected the CMA pathway in which SMAD3 was involved.Similarly,down-regulating SMAD3 or up-regulating LAMP2A in cultured glioma cells enhanced their proliferation and invasion.Taken together,these results suggest that excessive activation of CMA regulates glioma cell growth by promoting the degradation of SMAD3.Therefore,targeting the SMAD3-LAMP2Amediated CMA-lysosome pathway may be a promising approach in anti-cancer therapy.

Autophagy regulation combined with stem cell therapy for treatment of spinal cord injury

Stem cells are a group of cells with unique self-renewal and differentiation abilities that have great prospects in the repair of spinal cord injury. However, stem cell renewal and differentiation require strict control of protein turnover in the stem cells to achieve cell remodeling. As a highly conserved “gatekeeper” of cell homeostasis, autophagy can regulate cell remodeling by precisely controlling protein turnover in cells. Recently, it has been found that the expression of autophagy markers changes in animal models of spinal cord injury. Therefore, understanding whether autophagy can affect the fate of stem cells and promote the repair of spinal cord injury is of considerable clinical value. This review expounds the importance of autophagy homeostasis control for the repair of spinal cord injury from three aspects—pathophysiology of spinal cord injury, autophagy and stem cell function, and autophagy and stem cell function in spinal cord injury—and proposes the synergistic therapeutic effect of autophagy and stem cells in spinal cord injury.

The critical role of the endolysosomal system in cerebral ischemia

Cerebral ischemia is a serious disease that triggers sequential pathological mechanisms, leading to significant morbidity and mortality. Although most studies to date have typically focused on the lysosome, a single organelle, current evidence supports that the function of lysosomes cannot be separated from that of the endolysosomal system as a whole. The associated membrane fusion functions of this system play a crucial role in the biodegradation of cerebral ischemia-related products. Here, we review the regulation of and the changes that occur in the endolysosomal system after cerebral ischemia, focusing on the latest research progress on membrane fusion function. Numerous proteins, including N-ethylmaleimide-sensitive factor and lysosomal potassium channel transmembrane protein 175, regulate the function of this system. However, these proteins are abnormally expressed after cerebral ischemic injury, which disrupts the normal fusion function of membranes within the endolysosomal system and that between autophagosomes and lysosomes. This results in impaired “maturation” of the endolysosomal system and the collapse of energy metabolism balance and protein homeostasis maintained by the autophagy-lysosomal pathway. Autophagy is the final step in the endolysosomal pathway and contributes to maintaining the dynamic balance of the system. The process of autophagosome-lysosome fusion is a necessary part of autophagy and plays a crucial role in maintaining energy homeostasis and clearing aging proteins. We believe that, in cerebral ischemic injury, the endolysosomal system should be considered as a whole rather than focusing on the lysosome. Understanding how this dynamic system is regulated will provide new ideas for the treatment of cerebral ischemia.

Hepatitis B virus replication

Hepadnaviruses, including human hepatitis B virus （HBV）, replicate through reverse transcription of an RNA intermediate, the pregenomic RNA （pgRNA）. Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription： it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, ε, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV （DHBV）, now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cellfree systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the ~ RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

CMA down-regulates p53 expression through degradation of HMGB1 protein to inhibit irradiation-triggered apoptosis in hepatocellular carcinoma

AIM To investigate the mechanism of chaperone-mediated autophagy(CMA)-induced resistance to irradiationtriggered apoptosis through regulation of the p53 protein in hepatocellular carcinoma(HCC). METHODS Firstly, we detected expression of lysosome-associated membrane protein 2a(Lamp-2a), which is the key protein of CMA, by western blot in Hep G2 and SMMC7721 cells after irradiation. We further used sh RNA Lamp-2a HCC cells to verify the radioresistance induced by CMA. Next, we detected the HMGB1 and p53 expression after irradiation by western blot, and we further used RNA interference and ethyl pyruvate(EP), as a HMGB1 inhibitor, to observe changes of p53 expression. Finally, an immunoprecipitation assay was conducted to explore the interaction between Lamp-2a and HMGB1, and the data were analyzed. RESULTS We found the expression of Lamp-2a was increased on irradiation while apoptosis decreased in Hep G2 and SMMC7721 cells. The apoptosis was increased markedly in the sh RNA Lamp-2a Hep G2 and SMMC7721 cells as detected by western blot and colony formation assay. Next, we found p53 expression was gradually reduced on irradiation but obviously increased in sh RNA Lamp-2a cells. Furthermore, p53 increased the cell apoptosis on irradiation in Hep3B(p53-/-) cells. Finally, p53 levels were regulated by HMGB1 as measured through RNA interference and the EP treatment. HMGB1 was able to combine with Lamp-2a as seen by immunoprecipitation assay and was degraded via the CMA pathway. The decreased HMGB1 inhibited p53 expression induced by irradiation and further reduced the apoptosis in HCC cells. CONCLUSION CMA pathway activation appears to down-regulate the susceptibility of HCC to irradiation by degrading HMGB1 with further impact on p53 expression. These findings have clinical relevance for radiotherapy of HCC.

Dysregulation of autophagy and mitochondrial function in Parkinson’s disease

Parkinson’s disease(PD)is the second most common neurodegenerative disease.Increasing evidence supports that dysregulation of autophagy and mitochondrial function are closely related with PD pathogenesis.In this review,we briefly summarized autophagy pathway,which consists of macroautophagy,microautophagy and chaperone-mediated autophagy(CMA).Then,we discussed the involvement of mitochondrial dysfunction in PD pathogenesis.We specifically reviewed the recent developments in the relationship among several PD related genes,autophagy and mitochondrial dysfunction,followed by the therapeutic implications of these pathways.In conclusion,we propose that autophagy activity and mitochondrial homeostasis are of high importance in the pathogenesis of PD.Better understanding of these pathways can shed light on the novel therapeutic methods for PD prevention and amelioration.

Autophagy-lysosome pathway as a source of candidate biomarkers for Parkinson's disease

Parkinson's disease(PD)is a neurodegenerative disorder characterized by progressive motor disturbances and affects more than 1%of the worldwide population.Diagnosis of PD relies on clinical history and physical examination,but misdiagnosis is common in early stages.Despite considerable progress in understanding PD pathophysiology,including genetic and biochemical causes,diagnostic approaches lack accuracy and interventions are restricted to symptomatic treatments.Identification of biomarkers for PD may allow early and more precise diagnosis and monitoring of dopamine replacement strategies and disease-modifying treatments.Increasing evidence suggests that autophagic dysregulation causes the accumulation of abnormal proteins,such as aberrantα-synuclein,a protein critical to PD pathogenesis.Mutations in the GBA gene are a major PD risk factor andβ-glucocerebrosidase(GCase)is also emerging as an important molecule in PD pathogenesis.Consequently,proteins involved in the autophagy-lysosome pathway and GCase protein levels and activity are prime targets for the research and development of new PD biomarkers.The studies so far in PD biological material have yielded some consistent results,particularly regarding the levels of Hsc70,a component of the chaperone-mediated autophagy pathway,and the enzymatic activity of GCase in GBA mutation carriers.In the future,larger longitudinal studies,corroborating previous research on possible biomarker candidates,as well as extending the search for possible candidates for other lysosomal components,may yield more definitive results.