MLKL-USP7-UBA52 signaling is indispensable for autophagy in brain through maintaining ubiquitin homeostasis
Creators: name: Zhang, Zhigang
name: Chen, Shuai
name: Jun, Shirui
name: Xu, Xirong
name: Hong, Yuchuan
name: Yang, Xifei
name: Zou, Liangyu
name: Song, You-Qiang
name: Chen, Yu
name: Tu, Jie
Publisher: name: Taylor & Francis
Year: 2024
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Infectious Diseases
subject: FOS: Health sciences
subject: Plant Biology

description: Individuals with genetic elimination of <i>MLKL</i> (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 <i>mlkl</i> 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 <i>Mlkl</i> 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 <i>Mlkl</i> loss is attributed to a decrease in their lysine 63 (K63)-linked polyubiquitination. Additionally, single-nucleus RNA sequencing revealed that the loss of <i>Mlkl</i> 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 <i>mlkl</i> KO mice and exacerbation of AD pathologies in an AD mouse model with <i>mlkl</i> 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 <i>Mlkl</i> 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.<b>Abbreviations</b>: 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.