Calorie restriction and calorie-restriction mimetics activate chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.

LAMP2A regulates cisplatin resistance in colorectal cancer through mediating autophagy.

BACKGROUND: Drug resistance is an important constraint on clinical outcomes in advanced cancers. LAMP2A is a limiting protein in molecular chaperone-mediated autophagy. This study was aimed to explore LAMP2A function in cisplatin (cis-diamminedichloroplatinum, DDP) resistance colorectal cancer (CRC) to seek new ideas for CRC clinical treatment. METHODS: In this study, LAMP2A expression was analyzed by molecular experimental techniques,such as qRT-PCR and western blot. Then, LAMP2A in cells was interfered by cell transfection experiments. Subsequently, the function of LAMP2A on proliferation, migration, invasion, DDP sensitivity, and autophagy of CRC/DDP cells were further investigated by a series of experiments, such as CCK-8, transwell, and western blot. RESULTS: We revealed that LAMP2A was clearly augmented in DDP-resistant CRC and was related to poor patient prognosis. Functionally, LAMP2A insertion remarkably CRC/DDP proliferation, migration, invasion ability and DDP resistance by strengthen autophagy. In contrast, LAMP2A knockdown limited the proliferation, migration, and invasion while heightened cellular sensitivity to DDP by restraining autophagy in CRC/DDP cells. Furthermore, LAMP2A silencing was able to curb tumor formation and enhance sensitivity to DDP in vivo. CONCLUSION: In summary, LAMP2A boosted malignant progression and DDP resistance in CRC/DDP cells through mediating autophagy. Clarifying LAMP2A function in DDP resistance is promising to seek cancer therapies biomarkers targeting LAMP2A activity.

Chaperone-mediated autophagy protects the bone formation from excessive inflammation through PI3K/AKT/GSK3ß/ß-catenin pathway.

Multiple regulatory mechanisms are in place to ensure the normal processes of bone metabolism, encompassing both bone formation and absorption. This study has identified chaperone-mediated autophagy (CMA) as a critical regulator that safeguards bone formation from the detrimental effects of excessive inflammation. By silencing LAMP2A or HSCA8, we observed a hindrance in the osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. To further elucidate the role of LAMP2A, we generated LAMP2A gene knockdown and overexpression of mouse BMSCs (mBMSCs) using adenovirus. Our results showed that LAMP2A knockdown led to a decrease in osteogenic-specific proteins, while LAMP2A overexpression favored the osteogenesis of mBMSCs. Notably, active-ß-catenin levels were upregulated by LAMP2A overexpression. Furthermore, we found that LAMP2A overexpression effectively protected the osteogenesis of mBMSCs from TNF-α, through the PI3K/AKT/GSK3ß/ß-catenin pathway. Additionally, LAMP2A overexpression significantly inhibited osteoclast hyperactivity induced by TNF-α. Finally, in a murine bone defect model, we demonstrated that controlled release of LAMP2A overexpression adenovirus by alginate sodium capsule efficiently protected bone healing from inflammation, as confirmed by imaging and histological analyses. Collectively, our findings suggest that enhancing CMA has the potential to safeguard bone formation while mitigating hyperactivity in bone absorption.

Sensitivity of substrate translocation in chaperone-mediated autophagy to Alzheimer's disease progression.

Alzheimer's disease (AD) is a progressive brain disorder marked by abnormal protein accumulation and resulting proteotoxicity. This study examines Chaperone-Mediated Autophagy (CMA), particularly substrate translocation into lysosomes, in AD. The study observes: (1) Increased substrate translocation activity into lysosomes, vital for CMA, aligns with AD progression, highlighted by gene upregulation and more efficient substrate delivery. (2) This CMA phase strongly correlates with AD's clinical symptoms; more proteotoxicity links to worse dementia, underscoring the need for active degradation. (3) Proteins like GFAP and LAMP2A, when upregulated, almost certainly indicate AD risk, marking this process as a significant AD biomarker. Based on these observations, this study proposes the following hypothesis: As AD progresses, the aggregation of pathogenic proteins increases, the process of substrate entry into lysosomes via CMA becomes active. The genes associated with this process exhibit heightened sensitivity to AD. This conclusion stems from an analysis of over 10,000 genes and 363 patients using two AI methodologies. These methodologies were instrumental in identifying genes highly sensitive to AD and in mapping the molecular networks that respond to the disease, thereby highlighting the significance of this critical phase of CMA.

Exosomes derived from vMIP-II-Lamp2b gene-modified M2 cells provide neuroprotection by targeting the injured spinal cord, inhibiting chemokine signals and modulating microglia/macrophage polarization in mice.

Inflammation is one of the key injury factors for spinal cord injury (SCI). Exosomes (Exos) derived from M2 macrophages have been shown to inhibit inflammation and be beneficial in SCI animal models. However, lacking targetability restricts their application prospects. Considering that chemokine receptors increase dramatically after SCI, viral macrophage inflammatory protein II (vMIP-II) is a broad-spectrum chemokine receptor binding peptide, and lysosomal associated membrane protein 2b (Lamp2b) is the key membrane component of Exos, we speculated that vMIP-II-Lamp2b gene-modified M2 macrophage-derived Exos (vMIP-II-Lamp2b-M2-Exo) not only have anti-inflammatory properties, but also can target the injured area by vMIP-II. In this study, using a murine contusive SCI model, we revealed that vMIP-II-Lamp2b-M2-Exo could target the chemokine receptors which highly expressed in the injured spinal cords, inhibit some key chemokine receptor signaling pathways (such as MAPK and Akt), further inhibit proinflammatory factors (such as IL-1ß, IL-6, IL-17, IL-18, TNF-α, and iNOS), and promote anti-inflammatory factors (such as IL-4 and Arg1) productions, and the transformation of microglia/macrophages from M1 into M2. Moreover, the improved histological and functional recoveries were also found. Collectively, our results suggest that vMIP-II-Lamp2b-M2-Exo may provide neuroprotection by targeting the injured spinal cord, inhibiting some chemokine signals, reducing proinflammatory factor production and modulating microglia/macrophage polarization.

[Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells]. / åˆ†å­ä¼´ä¾£ä»‹å¯¼çš„è‡ªå™¬å¯¹èƒ†çº¢ç´ è¯±å¯¼çš„å°é¼ å°èƒ¶è´¨ç»†èƒžæŸä¼¤çš„å½±å“.

OBJECTIVES: To investigate the effect of chaperone-mediated autophagy (CMA) on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin (UCB). METHODS: The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide), QX77 group (treated with 20 µmol/L QX77 for 24 hours), UCB group (treated with 40 µmol/L UCB for 24 hours), and UCB+QX77 group (treated with both 20 µmol/L QX77 and 40 µmol/L UCB for 24 hours). (2) For the cell transfection experiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencing control+UCB group (treated with 40 µmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volume of dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 µmol/L UCB for 24 hours). The cell viability was assessed using the modified MTT method. The expression levels of p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence. RESULTS: Compared to the UCB group, the cell viability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as the mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α decreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65 and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2A silencing control+UCB group, the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as increased mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α (P<0.05). CONCLUSIONS: CMA is inhibited in UCB-induced BV2 cell damage, and activating CMA may reduce p65 and NLRP3 protein levels, suppress inflammatory responses, and counteract bilirubin neurotoxicity.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Altered Splicing of LAMP2 in a Multigenerational Family from Latvia Affected by Danon Disease.

Background and Objectives: Danon disease is a multisystemic disorder associated with variants in the LAMP2 gene, mainly affecting the cardiac muscle. Here, we report a multigenerational family from Latvia with two male patients, hemizygous for a novel splice-affecting variant c.928+3A>G. Affected patients exhibit a cardiac phenotype, moderate mental disability, and mild retinal changes. Materials and Methods: Both patients underwent either exome or hypertrophic cardiomyopathy gene panel next-generation sequencing. The pathogenic variant effect was determined using reverse transcription, Sanger sequencing, and high-resolution electrophoresis. Results: Evaluation of the splicing process revealed that approximately 80% of the transcripts exhibited a lack of the entire exon 7. This alteration was predicted to cause a shift of the reading frame, consequently introducing a premature stop codon downstream in the sequence. Conclusions: Based on our data, we propose that c.928+3A>G is a pathogenic variant associated with Danon disease.

International Consensus on Differential Diagnosis and Management of Patients With Danon Disease: JACC State-of-the-Art Review.

Danon disease is a rare X-linked autophagic vacuolar cardioskeletal myopathy associated with severe heart failure that can be accompanied with extracardiac neurologic, skeletal, and ophthalmologic manifestations. It is caused by loss of function variants in the LAMP2 gene and is among the most severe and penetrant of the genetic cardiomyopathies. Most patients with Danon disease will experience symptomatic heart failure. Male individuals generally present earlier than women and die of either heart failure or arrhythmia or receive a heart transplant by the third decade of life. Herein, the authors review the differential diagnosis of Danon disease, diagnostic criteria, natural history, management recommendations, and recent advances in treatment of this increasingly recognized and extremely morbid cardiomyopathy.

Danon Disease: Entire LAMP2 Gene Deletion with Unusual Clinical Presentation-Case Report and Review of the Literature.

Danon disease is a rare x-linked dominant multisystemic disorder with a clinical triad of severe cardiomyopathy, skeletal myopathy, and intellectual disability. It is caused by defects in the lysosome-associated membrane protein-2 (LAMP2) gene. Numerous different mutations in the LAMP2 protein have been described. Danon disease is typically lethal by the mid-twenties in male patients due to cardiomyopathy and heart failure. Female patients usually present with milder and variable symptoms. This report describes a 42-year-old father and his 3-year-old daughter presenting with mild manifestations of the disease. The father has normal intellectual development and normal physical activity. At the age of 13, he was diagnosed with mild ventricular pre-excitation known as Wolf-Parkinson-White syndrome (WPWs), very mild and mostly asymptomatic cardiomyopathy and left ventricular hypertrophy, and at about the age of 25 presented with visual impairment due to cone-rod dystrophy. His daughter showed normal development and very mild asymptomatic electrocardiographic WPWs abnormalities with left mild ventricular hypertrophy. Genetic testing revealed an Xq24 microdeletion encompassing the entire LAMP2 gene. Relevant literature was reviewed as a reference for the etiology, diagnosis, treatment and case management.

LAMP2A, LAMP2B and LAMP2C: similar structures, divergent roles.

LAMP2 (lysosomal associated membrane protein 2) is one of the major protein components of the lysosomal membrane. There currently exist three LAMP2 isoforms, LAMP2A, LAMP2B and LAMP2C, and they vary in distribution and function. LAMP2A serves as a receptor and channel for transporting cytosolic proteins in a process called chaperone-mediated autophagy (CMA). LAMP2B is required for autophagosome-lysosome fusion in cardiomyocytes and is one of the components of exosome membranes. LAMP2C is primarily implicated in a novel type of autophagy in which nucleic acids are taken up into lysosomes for degradation. In this review, the current evidence for the function of each LAMP2 isoform in various pathophysiological processes and human diseases, as well as their possible mechanisms, are comprehensively summarized. We discuss the evolutionary patterns of the three isoforms in vertebrates and provide technical guidance on investigating these isoforms. We are also concerned with the newly arising questions in this particular research area that remain unanswered. Advances in the functions of the three LAMP2 isoforms will uncover new links between lysosomal dysfunction, autophagy and human diseases.Abbreviation: ACSL4: acyl-CoA synthetase long-chain family member 4; AD: Alzheimer disease; Ag: antigens; APP: amyloid beta precursor protein; ATG14: autophagy related 14; AVSF: autophagic vacuoles with unique sarcolemmal features; BBC3/PUMA: BCL2 binding component 3; CCD: C-terminal coiled coil domain; CMA: chaperone-mediated autophagy; CVDs: cardiovascular diseases; DDIT4/REDD1: DNA damage inducible transcript 4; ECs: endothelial cells; ER: endoplasmic reticulum; ESCs: embryonic stem cells; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/ß-glucocerebrosidase: glucosylceramidase beta; GSCs: glioblastoma stem cells; HCC: hepatocellular carcinoma; HD: Huntington disease; HSCs: hematopoietic stem cells; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; IL3: interleukin 3; IR: ischemia-reperfusion; LAMP2: lysosomal associated membrane protein 2; LDs: lipid droplets; LRRK2: leucine rich repeat kinase 2; MA: macroautophagy; MHC: major histocompatibility complex; MST1: macrophage stimulating 1; NAFLD: nonalcoholic fatty liver disease; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NLRP3: NLR family pyrin domain containing 3; PARK7: Parkinsonism associated deglycase; PD: Parkinson disease; PEA15/PED: proliferation and apoptosis adaptor protein 15; PKM/PKM2: pyruvate kinase M1/2; RA: rheumatoid arthritis; RARA: retinoic acid receptor alpha; RCAN1: regulator of calcineurin 1; RCC: renal cell carcinoma; RDA: RNautophagy and DNautophagy; RNAi: RNA interference; RND3: Rho Family GTPase 3; SG-NOS3/eNOS: deleterious glutathionylated NOS3; SLE: systemic lupus erythematosus; TAMs: tumor-associated macrophages; TME: tumor microenvironment; UCHL1: ubiquitin C-terminal hydrolase L1; VAMP8: vesicle associated membrane protein 8.

Clinical features of pediatric Danon disease and the importance of early diagnosis.

Successful therapy in a cohort with early onset Danon disease (DD) highlights the potential importance of earlier disease recognition. We present experience from the largest National Pediatric Center in Russia for cardiomyopathy patients. This report focuses on identification of early clinical features of DD in the pediatric population by detailed pedigree analysis and review of medical records. RESULTS: Nine patients (3 females) were identified with DD at the Russian National Medical Research Center of Children's Health ("National Pediatric Center") aged birth to 16 years. At presentation/evaluation: all patients had left ventricular hypertrophy (LVH), ECG features of Wolff-Parkinson-White (WPW), and an increase in hepatic enzymes (particularly lactate dehydrogenase (LDH)); three had marked increase in NT-proBNP; two had HCM with impaired LV function; one had LVH with LV noncompaction; five had arrhythmia with paroxysmal supraventricular and/or ventricular tachycardia. Two teenagers died at ages 16-17 from refractory heart failure and two underwent heart transplantation. All patients were found to have a pathogenic/likely pathogenic variant in the LAMP2 gene, six patients had no family history and a de novo evolvement was documented in 4/6 of those available for genetic tested. Retrospective review related to family background and earlier clinical evaluations revealed a definitive or highly suspicious family history of DD in 3, early clinical presentation with cardiac abnormalities (ECG, echo) in 3, and cerebral, hepatic and/or neuromuscular symptoms in 5. Abnormalities were detected 9,5 months to 5,8 years, median 3,5 years prior to referral to the National Pediatric Center. CONCLUSION: The earliest clinical manifestations of Danon disease occur in the first 12 years of life with symptoms of skeletal muscle and cerebral disease, raised hepatic enzymes, and evidence of cardiac disease on ECG/echo.

Benzo(a)pyrene regulates chaperone-mediated autophagy via heat shock protein 90.

AIMS: Some studies have shown that the Benzo(a)pyrene (BaP) exposure induced oxidative damage, DNA damage and autophagy, but the molecular mechanism is not clear. Heat shock protein 90 (HSP90) is regarded as an important target in cancer therapy and a key factor in autophagy. Therefore, this study aims to clarify the new mechanism of BaP regulating CMA through HSP90. MAIN METHODS: C57BL mice were fed with BaP at a dose of 25.3 mg/kg. A549 cells were treated with different concerntrations of BaP, and MTT assay was used to observe the effect of BaP on the proliferation of A549 cells. DNA damage was detected by alkaline comet assay. Focus experiment for detection of γ-H2AX by immunofluorescence. The mRNA expression of HSP90, HSC70 and Lamp-2a was detected by qPCR. The protein expressions of HSP90, HSC70 and Lamp-2a were detected by Western blot. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90α shRNA lentivirus transduction in A549 cells. KEY FINDINGS: In these studies, we first found that heat shock protein 90 (HSP90), heat shock cognate 70 (HSC70) and lysosomal-associated membrane protein type 2 receptor (Lamp-2a) expressions of C57BL mice lung tissue and A549 cells exposed to BaP were significant increase, as well as BaP induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by comet assay and γ-H2AX foci analysis in A549 cells. Our results demonstrated BaP induced CMA and caused DNA damage. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90α shRNA lentivirus transduction in A549 cells. HSC70 and Lamp-2a expressions of these cells exposed to BaP were not significant increase, which showed that BaP inducted CMA was mediated by HSP90. Further, HSP90α shRNA prevented BaP induced of BaP which suggested BaP regulated CMA and caused DNA damage by HSP90. Our results elucidated a new mechanism of BaP regulated CMA through HSP90. SIGNIFICANCE: BaP regulated CMA through HSP90. HSP90 is involved in the regulation of gene instability induced by DNA damage by BaP, which promotes CMA. Our study also revealed that BaP regulates CMA through HSP90. This study fills the gap of the effect of BaP on autophagy and its mechanism, which will lead to a more comprehensive understanding of the action mechanism of BaP.

Identification and functional analysis of a novel de novo missense mutation located in the initiation codon of LAMP2 associated with early onset female Danon disease.

BACKGROUND: Danon disease is characterized by the failure of lysosomal biogenesis, maturation, and function due to a deficiency of lysosomal membrane structural protein (LAMP2). METHODS: The current report describes a female patient with a sudden syncope and hypertrophic cardiomyopathy phenotype. We identified the pathogenic mutations in patients by whole-exon sequencing, followed by a series of molecular biology and genetic approaches to identify and functional analysis of the mutations. RESULTS: Suggestive findings by cardiac magnetic resonance (CMR), electrocardiogram (ECG), and laboratory examination suggested Danon disease which was confirmed by genetic testing. The patient carried a novel de novo mutation, LAMP2 c.2T>C located at the initiation codon. The quantitative polymerase chain reaction (qPCR) and Western blot (WB) analysis of peripheral blood leukocytes from the patients revealed evidence of LAMP2 haploinsufficiency. Labeling of the new initiation codon predicted by the software with green fluorescent protein followed by fluorescence microscopy and Western blotting showed that the first ATG downstream from the original initiation codon became the new translational initiation codon. The three-dimensional structure of the mutated protein predicted by alphafold2 revealed that it consisted of only six amino acids and failed to form a functional polypeptide or protein. Overexpression of the mutated LAMP2 c.2T>C showed a loss of function of the protein, as assessed by the dual-fluorescence autophagy indicator system. The mutation was confirmed to be null, AR experiments and sequencing results confirmed that 28% of the mutant X chromosome remained active. CONCLUSION: We propose possible mechanisms of mutations associated with haploinsufficiency of LAMP2: (1) The inactivation X chromosome carrying the mutation was not significantly skewed. However, it decreased in the mRNA level and the expression ratio of the mutant transcripts; (2) The identified mutation is null, and the active mutant transcript fails to translate into the normal LAMP2 proteins. The presence of haploinsufficiency in LAMP2 and the X chromosome inactivation pattern were crucial factors contributing to the early onset of Danon disease in this female patient.

Identification of a novel splicing-altering LAMP2 variant in a Chinese family with Danon disease.

AIMS: This study aimed to identify a novel splicing-altering LAMP2 variant associated with Danon disease. METHODS AND RESULTS: To identify the potential genetic mutation in a Chinese pedigree, whole-exome sequencing was conducted in the proband, and Sanger sequencing was performed on the proband's parents. To verify the impact of the splice-site variant, a minigene splicing assay was applied. The AlphaFold2 analysis was used to analyse the mutant protein structure. A splice-site variant (NM_013995.2:c.864+5G>A) located at intron 6 of the LAMP2 gene was identified as a potential pathogenic variant. The minigene splicing revealed that this variant causes exon 6 to be skipped, resulting in a truncated protein. The AlphaFold2 analysis showed that the mutation caused a protein twist direction change, leading to conformational abnormality. CONCLUSIONS: A novel splice-site variant (NM_013995.2:c.864+5G>A) located at intron 6 of the LAMP2 gene was identified. This discovery may enlarge the LAMP2 variant spectrum, promote accurate genetic counselling, and contribute to the diagnosis of Danon disease.

LncRNA OBFC2A modulated benzene metabolites-induced autophagy and apoptosis by interacting with LAMP2.

Exposure to benzene results in peripheral blood cell reduction, aplastic anemia, and leukemia. We previously observed that the lncRNA OBFC2A was upregulated significantly in benzene-exposed workers and correlated with reduced blood cell counts. However, the role of lncRNA OBFC2A in benzene hematotoxicity remains unclear. In this study, we discovered that lncRNA OBFC2A was regulated by oxidative stress and played roles in cell autophagy and apoptosis caused by the benzene metabolite 1,4-Benzoquinone (1,4-BQ) in vitro. Mechanistically, protein chip, RNA pull-down, and FISH colocalization uncovered that lncRNA OBFC2A directly bound to LAMP2, a regulator of chaperone-mediated autophagy (CMA), and upregulated its expression in 1,4-BQ-treated cells. LncRNA OBFC2A knockdown alleviated LAMP2 overexpression caused by 1,4-BQ, which confirmed their regulatory relationship. In conclusion, we demonstrate that lncRNA OBFC2A mediates 1,4-BQ-induced apoptosis and autophagy by interacting with LAMP2. LncRNA OBFC2A could serve as a biomarker for hematotoxicity caused by benzene.

Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells.

LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A KO cells, and here we assessed the specificity of select commercial LAMP2A antibodies on wild-type and LAMP2A KO human cancer cells. While all tested antibodies were suitable for immunoblotting, the anti-LAMP2A antibody (ab18528) is likely to exhibit an off-target reactivity in immunostaining approaches using human cancer cells, and alternative antibodies, which seem more appropriate, are available.

Thyroid hormone upregulates LAMP2 expression and lysosome activity.

Thyroid hormone (T3)-induced autophagy and its biological significance have been extensively investigated in recent years. However, limited studies to date have focused on the important role of lysosomes in autophagy. In this study, we explored the effects of T3 on lysosomal protein expression and trafficking in detail. Our findings showed that T3 activates rapid lysosomal turnover and expression of numerous lysosomal genes, including TFEB, LAMP2, ARSB, GBA, PSAP, ATP6V0B, ATP6V0D1, ATP6V1E1, CTSB, CTSH, CTSL, and CTSS, in a thyroid hormone receptor-dependent manner. In a murine model, LAMP2 protein was specifically induced in mice with hyperthyroidism. T3-promoted microtubule assembly was significantly disrupted by vinblastine, resulting in accumulation of the lipid droplet marker PLIN2. In the presence of the lysosomal autophagy inhibitors bafilomycin A1, chloroquine and ammonium chloride, we observed substantial accumulation of LAMP2 but not LAMP1 protein. T3 further enhanced the protein levels of ectopically expressed LAMP1 and LAMP2. Upon knockdown of LAMP2, cavities of lysosomes and lipid droplets accumulated in the presence of T3, although the changes in LAMP1 and PLIN2 expression were less pronounced. More specifically, the protective effect of T3 against ER stress-induced death was abolished by knockdown of LAMP2. Our collective results indicate that T3 not only promotes lysosomal gene expression but also LAMP protein stability and microtubule assembly, leading to enhancement of lysosomal activity in digesting any additional autophagosomal burden.

A rare and fatal cause of hypertrophic cardiomyopathy: Danon disease.

Danon disease is a rare and fatal disease caused by a mutation in the lysosome-associated membrane protein 2 gene. Impaired intracellular autophagy causes lysosomal vacuoles to accumulate mainly in myocardial and skeletal muscle cells, leading to hypertrophic cardiomyopathy, skeletal myopathy, and varying degrees of intellectual disability. Two distinct childhood presentations of Danon disease are described in this report.