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.

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.

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.

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.

白藜芦醇促进分子伴侣介导的自噬减轻神经炎症改善脓毒症相关性脑病小鼠情感功能障碍

目的 明确分子伴侣介导的自噬(CMA)减轻脓毒症相关性脑病(SAE)小鼠情感功能障碍的作用。方法 通过盲肠结扎穿孔术(CLP)构建SAE小鼠模型,采用小鼠脓毒症严重程度评分(MSS)评估小鼠脓毒症的严重程度,并进行旷场和高架十字迷宫相关行为学实验检测SAE小鼠的情感功能。Western blot法检测热休克蛋白同源蛋白70(HSC70)、溶酶体相关膜蛋白2A(LAMP2A)和高迁移率族蛋白B1(HMGB1)蛋白表达水平,免疫荧光组织化学染色检测LAMP2A与神经元的共定位情况。ELISA检测炎症因子白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)的释放水平。同时按30 mg/kg剂量每天连续给予白藜芦醇灌胃直至第14天。结果 CLP小鼠14 d死亡率为45.83%,而幸存小鼠均存在情感功能障碍。CLP术后24 h海马神经元HSC70和LAMP2A表达水平显著降低,HMGB1表达水平以及炎症因子IL-6和TNF-α释放增加,CMA活性受损,而在灌胃给予白藜芦醇后,HSC70和LAMP2A表达水平增加,HMGB1表达水平降低,炎症因子释放减少,提示CMA活性提高且神经炎症减轻,行为学测试结果表明在给予小鼠白藜芦醇灌胃后小鼠情感功能障碍得到改善。结论 SAE小鼠海马神经元CMA活性显著降低,导致情感功能障碍;白藜芦醇能够促进CMA,从而抑制HMGB1的表达和炎症因子的释放,减轻神经炎症,从而改善SAE小鼠的情感功能障碍。

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.

分子侣伴蛋白介导的自噬障碍在神经退行性病变中的作用

分子偶伴介导的白噬（chaperone—mediatedautophagy，CMA）是单个可溶性蛋白在溶酶体进行选择性降解的一种白噬途径，其特点是所要进行降解的蛋白直接穿过溶酶体膜到达溶酶体内腔进行降解，与其他自噬途径通过睫泡介导的特点完全不同。选择性降解、底物直接转位这两大特点决定丫CMA所参与的各种生理功能以及CMA障碍所引起的不同疾病。最近的研究表明，随着年龄下降的CMA功能是多种疾病的加重因素。本文将简要综述CMA功能相关的分子机制以及通过这一途径进行的溶酶体的选择性降解相关生理的最新研究，也将评述CMA障碍的细胞结局和CMA障碍与神经退行性病变的关系。

分子伴侣介导的自噬在阿尔茨海默病中作用的研究进展

阿尔茨海默病(Alzheimer’s disease,AD)是一种常见的神经系统退行性疾病,其主要病理变化是β淀粉样蛋白(amyloidβ-protein,Aβ)大量堆积形成的老年斑(senile plaque,SP)和细胞内Tau蛋白(Tau protein)过度磷酸化积聚形成的神经原纤维缠结(neurofibrillary tangles,NFTs)。分子伴侣介导的自噬(chaperone-mediated autophagy,CMA)是一种选择性地将带有CMA基序的蛋白传递到溶酶体进行降解的过程。当自噬过程受损时,Aβ与异常磷酸化的Tau蛋白会大量堆积于神经元内,从而破坏细胞的正常功能并加速其死亡。相关研究表明,CMA是早期AD中异常蛋白质降解的重要途径,且该途径的失活可能在AD进展中发挥了重要作用。该文从CMA的概念、生理作用,CMA与AD的病理联系等方面进行综述,并对CMA途径的失活在AD中的作用进行详细阐述,以期为AD发病机制的研究及治疗提供新的思路。

帕金森病细胞模型中分子伴侣介导自噬对α-突触核蛋白低聚体水平的影响

目的·探讨在泛素蛋白酶体系统(ubiquitin proteasome system,UPS)受损的帕金森病(Parkinson's disease,PD)细胞模型中分子伴侣介导自噬(chaperone-mediated autophagy,CMA)对α-突触核蛋白低聚体水平的影响。方法·培养稳转野生型α-突触核蛋白的人神经母细胞瘤SK-N-SH细胞系,加入蛋白酶体抑制剂lactacystin构建PD细胞模型。通过Western blotting检测α-突触核蛋白低聚体、溶酶体相关膜蛋白2A型(lysosome-associated membrane protein type 2A,LAMP2A)和相对分子质量为70 000的热休克同源蛋白(heat-shock cognate protein of 70 kDa,HSC70)水平;使用LAMP2A siRNA抑制CMA功能,检测对α-突触核蛋白低聚体水平及细胞存活率的影响;通过免疫共沉淀观察LAMP2A与α-突触核蛋白低聚体的相互作用。结果·PD细胞模型中α-突触核蛋白低聚体表达水平升高,与CMA功能密切相关的蛋白LAMP2A和HSC70水平应激性增加;抑制LAMP2A表达,α-突触核蛋白低聚体水平进一步升高,细胞存活率降低,且通过免疫共沉淀检测到LAMP2A与α-突触核蛋白低聚体存在相互作用。结论·PD细胞模型中,CMA是调节α-突触核蛋白低聚体水平的途径之一,抑制其功能可进一步增加α-突触核蛋白低聚体水平及细胞毒性作用。

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.

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.

An insight review of autophagy biology and neurodegenerative diseases:machinery,mechanisms and regulation

Autophagy is an intracellular catabolic system in which cytoplasmic proteins or organelles are translocated into lysosomes for degradation.Three different types of autophagy have been identified as macroautophagy,microautophagy,and chaperone-mediated autophagy（CMA）.