1. Academic Validation
  2. MLKL-USP7-UBA52 signaling is indispensable for autophagy in brain through maintaining ubiquitin homeostasis

MLKL-USP7-UBA52 signaling is indispensable for autophagy in brain through maintaining ubiquitin homeostasis

  • Autophagy. 2024 Sep 19:1-23. doi: 10.1080/15548627.2024.2395727.
Zhigang Zhang 1 2 Shuai Chen 1 2 3 Shirui Jun 1 2 Xirong Xu 1 3 Yuchuan Hong 1 3 Xifei Yang 4 Liangyu Zou 5 You-Qiang Song 6 Yu Chen 1 2 3 7 8 Jie Tu 1 2 3 7 9
Affiliations

Affiliations

  • 1 Shenzhen Key Laboratory of Neuroimmunomodulation for Neurological Diseases, Shenzhen-Hong Kong Institute of Brain Science, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
  • 2 Faculty of Life and Health Sciences, Shenzhen University of Advanced Technology, Shenzhen, Guangdong Province, China.
  • 3 University of Chinese of Academy of Sciences, Beijing, China.
  • 4 Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention, Shenzhen, China.
  • 5 Department of Neurology, Shenzhen People's Hospital (The First Affiliated Hospital of Southern University of Science and Technology, The Second Clinical College, Jinan University), Shenzhen, China.
  • 6 School of Biomedical Sciences, The University of Hong Kong, Hong Kong, China.
  • 7 CAS Key Laboratory of Brain Connectome and Manipulation, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.
  • 8 SIAT-HKUST Joint Laboratory for Brain Science, Chinese Academy of Sciences, Shenzhen, China.
  • 9 Guangdong Provincial Key Laboratory of Brain Connectome and Behavior,Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen, Guangdong Province, China.
Abstract

Individuals with genetic elimination of MLKL (Mixed Lineage Kinase domain like pseudokinase) exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer disease (AD). However, the mechanism is not yet fully understood. Here, we observed significant compromise in macroautophagy/Autophagy in the brains of mlkl knockout (KO) mice, as evidenced by the downregulation of BECN1/Beclin1 and ULK1 (unc-51 like Autophagy activating kinase 1). We identified UBA52 (ubiquitin A-52 residue ribosomal protein fusion product 1) as the binding partner of MLKL under physiological conditions. Loss of Mlkl induced a decrease in ubiquitin levels by preventing UBA52 cleavage. Furthermore, we demonstrated that the Deubiquitinase (DUB) USP7 (ubiquitin specific peptidase 7) mediates the processing of UBA52, which is regulated by MLKL. Moreover, our results indicated that the reduction of BECN1 and ULK1 upon Mlkl loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA Sequencing revealed that the loss of Mlkl resulted in the disruption of multiple neurodegenerative disease-related pathways, including those associated with AD. These results were consistent with the observation of cognitive impairment in mlkl KO mice and exacerbation of AD pathologies in an AD mouse model with mlkl deletion. Taken together, our findings demonstrate that MLKL-USP7-UBA52 signaling is required for Autophagy in brain through maintaining ubiquitin homeostasis, and highlight the contribution of Mlkl loss-induced ubiquitin deficits to the development of neurodegeneration. Thus, the maintenance of adequate levels of ubiquitin may provide a novel perspective to protect individuals from multiple neurodegenerative diseases through regulating Autophagy.Abbreviations: 4HB: four-helix bundle; AAV: adeno-associated virus; AD: Alzheimer disease; AIF1: allograft inflammatory factor 1; APOE: Apolipoprotein E; APP: amyloid beta precursor protein; Aβ: amyloid β; BECN1: beclin 1; co-IP: co-immunoprecipitation; DEGs: differentially expressed genes; DLG4: discs large MAGUK scaffold protein 4; DUB: deubiquitinase; EBSS: Earle's balanced salt solution; GFAP: glial fibrillary acidic protein; HRP: horseradish peroxidase; IL1B: interleukin 1 beta; IL6: interleukin 6; IPed: immunoprecipitated; KEGG: Kyoto Encyclopedia of Genes and Genomes; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MLKL: Mixed Lineage Kinase domain like pseudokinase; NSA: necrosulfonamide; OPCs: oligodendrocyte precursor cells; PFA: paraformaldehyde; PsKD: pseudo-kinase domain; SYP: synaptophysin; UB: ubiquitin; UBA52: ubiquitin A-52 residue ribosomal protein fusion product 1; UCHL3: ubiquitin C-terminal hydrolase L3; ULK1: unc-51 like Autophagy activating kinase 1; UMAP: uniform manifold approximation and projection; UPS: ubiquitin-proteasome system; USP7: ubiquitin specific peptidase 7; USP9X: ubiquitin specific peptidase 9 X-linked.

Keywords

Alzheimer disease; BECN1; ULK1; autophagy; cognitive impairment; neurodegeneration.

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