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

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; Aß amyloid ß; 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.