Patients with liver diseases, such as liver cirrhosis and liver failure, have a high likelihood of developing hepatic encephalopathy (HE). HE is characterised by neurological impairments ranging from mild cognitive dysfunction to severe coma. Hyperammonaemia, metabolic disruption, and inflammation are recognised as key factors in the onset of HE. Recently, impaired autophagy has also been suggested to contribute to the development of HE. Therefore, in this study, we aimed to examine whether the autophagy inducer torin-1 is effective in reducing signs of HE. In this study, C57BL/6 mice were subjected to bile duct ligation (BDL) for 14 days to establish a model of HE. Liver function and structure were assessed using aspartate aminotransferase, alanine aminotransferase, total bilirubin, and haematoxylin and eosin staining, respectively. Neurological function was evaluated through the elevated plus maze, novel object recognition test, marble-burying test, clasping test, and passive avoidance test. To evaluate the effects of torin-1, it was administrated intraperitoneally to mice daily for 14 days. Changes in autophagy, cholesterol metabolism, and the cytokine/chemokine profile in the cerebral cortex were measured with and without torin-1 treatment. We found that although systemic administration of torin-1 could not reduce the induction of jaundice and liver impairment caused by BDL, it appeared to delay the onset of HE. Torin-1 treatment reduced the BDL-induced elevation in serum cholesterol and ammonia levels. Furthermore, abnormal expression of autophagy- and cholesterol-associated molecules in the cerebral cortex was reduced by torin-1 treatment. Cognitive function was improved in mice undergoing BDL with torin-1 treatment. Conclusively, these findings indicate that the induction of autophagy was found to alleviate the adverse effects seen in HE by regulating cholesterol metabolism in the cerebral cortex. The autophagy inducer torin-1 might be a potential approach to alleviate alterations and symptoms in HE.

2025-07-03·Autophagy

Autophagy regulates cellular senescence by mediating the degradation of CDKN1A/p21 and CDKN2A/p16 through SQSTM1/p62-mediated selective autophagy in myxomatous mitral valve degeneration

Article

作者: Tang, Qiyu ; Markby, Greg R ; Tang, Keyi ; Corcoran, Brendan M ; MacRae, Vicky E ; Phadwal, Kanchan ; Parys, Maciej

Myxomatous mitral valve degeneration (MMVD) is one of the most important age-dependent degenerative heart valve disorders in both humans and dogs. It is characterized by the aberrant remodeling of extracellular matrix (ECM), regulated by senescent myofibroblasts (aVICs) transitioning from quiescent valve interstitial cells (qVICs), primarily under TGFB1/TGF-β1 control. In the present study, we found senescent aVICs exhibited impaired macroautophagy/autophagy as evidenced by compromised autophagy flux and immature autophagosomes. MTOR-dependent autophagy induced by rapamycin and torin-1 attenuated cell senescence and decreased the expression of cyclin-dependent kinase inhibitors (CDKIs) CDKN2A/p16^INK4A and CDKN1A/p21^CIP1. Furthermore, induction of autophagy in aVICs by ATG (autophagy related) gene overexpression restored autophagy flux, with a concomitant reduction in CDKN1A and CDKN2A expression and senescence-associated secretory phenotype (SASP). Conversely, autophagy deficiency induced CDKN1A and CDKN2A accumulation and SASP, whereas ATG re-expression alleviated senescent phenotypic transformation. Notably, CDKN1A and CDKN2A localized to autophagosomes and lysosomes following MTOR antagonism or MG132 treatment. SQSTM1/p62 was identified as the autophagy receptor to selectively sequester CDKN1A and CDKN2A cargoes for autophagic degradation. Our findings are the first demonstration that CDKN1A and CDKN2A are degraded through SQSTM1-mediated selective autophagy, independent of the ubiquitin-proteasome pathway. These data will inform development of therapeutic strategies for the treatment of canine and human MMVD, and for the treatment of Alzheimer disease, Parkinson disease and other age-related degenerative disorders.Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle; AKT: AKT serine/threonine kinase; aVICs: activated valve interstitial cells; ATG: autophagy related; baf-A1: bafilomycin A₁; BrdU, bromodeoxyuridine; BSA: bovine serum albumin; CDKIs, cyclin-dependent kinase inhibitors; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; co-IP: co-immunoprecipitation; DMSO: dimethylsulfoxide; ECM, extracellular matrix; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; eGFP: green fluorescent protein; ELISA: enzyme-linked immunosorbent assay; HEK-293T, human embryonic kidney 293T; HRP: horseradish peroxidase; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LIR: MAP1LC3/LC3-interacting region; MFS: Marfan syndrome; MKI67/Ki-67: marker of proliferation Ki-67; MMVD: myxomatous mitral valve degeneration; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; OE: overexpression; PBST, phosphate-buffered saline with 0.1% Tween-20; PCNA: proliferating cell nuclear antigen; PIK3CA/PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha; PLA: proximity ligation assays; PSMA1: proteasome 20S subunit alpha 1; PSMB5: proteasome 20S subunit beta 5; qVICs: quiescent valve interstitial cells; qRT-PCR: quantitative real-time PCR; SA-GLB1/β-gal: SA-senescence-associated GLB1/β-galactosidase; ROS: reactive oxygen species; SASP: senescence-associated secretory phenotype; RPS6KB1/p70 S6K: ribosomal protein S6 kinase B1; SMAD: SMAD family member; SQSTM1/p62: sequestosome 1; STEM: scanning transmission electron microscopy; TGFB: transforming growth factor beta; TGFBR: transforming growth factor beta receptor; TP53/p53: tumor protein p53; UPS: ubiquitin-proteasome system; WT, wild-type.

2025-07-01·AMERICAN JOURNAL OF PATHOLOGY

Constitutive Hepatic mTORC1 Activation Aggravates Alcohol-Induced Liver Injury via Endoplasmic Reticulum Stress–Mediated Ferroptosis

Article

作者: Kang, Xiaohong ; Liu, Boao ; Chen, Zhiguo ; Chang, Tingmin ; Yang, Yang ; Zhao, Yuanyuan ; Xiong, Xiwen ; Xu, Lin ; Song, Shiyi ; Qi, Enbo ; Zhuang, Peili

Alcohol-related liver disease (ALD), a consequence of excessive alcohol use, manifests across a broad spectrum of liver damage, ranging from steatosis to cirrhosis. DEPDC5 (DEP domain-containing protein 5) is a component of the GATOR1 (gap activity towards rags 1) complex, which functions as a repressor of the amino acid-sensing branch of the mammalian target of rapamycin complex 1 (mTORC1) pathway. In this study, hepatocyte-specific Depdc5 knockout mice (Depdc5^△Hep) were generated. Aberrant activation of mTORC1 caused by Depdc5 deletion led to exacerbated endoplasmic reticulum (ER) stress and hepatocyte ferroptosis in the livers of ethanol-fed mice. Torin-1, an ATP-competitive mTOR inhibitor, suppressed the mTORC1 activity and reversed the effects of Depdc5 deletion on ER stress and ferroptosis in ethanol-fed mouse livers. Furthermore, pharmacologic relief of ER stress using tauroursodeoxycholic acid or inhibition of ferroptosis with liproxstatin-1 both alleviated the liver abnormalities induced by Depdc5 ablation in ethanol-fed mice. In addition, ER stress was shown to function as an upstream signal of ferroptosis in the progression of ALD. These findings provide novel in vivo evidence that sustained mTORC1 activation leads to alcoholic liver injury by inducing ER stress and ferroptosis, suggesting that targeting these pathways may represent a potential therapeutic strategy for ALD.

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