MLKL/USP7/UBA52 Signaling Is Indispensable for Autophagy in Brain through Maintaining Ubiquitin Homeostasis

Individuals with genetic elimination of MLKL exhibit an increased susceptibility to neurodegenerative diseases like Alzheimer’s disease (AD). However, the specific mechanism behind this relationship is not yet fully understood. Here, we initially observed significant compromise in autophagy in the brains of MLKL knockout (KO) mice, as evidenced by the down-regulation of autophagy-initiation proteins Beclin1 and ULK1. To elucidate the mechanism by which MLKL, primarily a cytosolic protein, regulates the protein levels of Beclin1 and ULK1, we performed a proteomic screening of MLKL-association proteins and identified UBA52 as the binding partner under physiological conditions. Loss of MLKL induces a decrease in ubiquitin levels by preventing UBA52 from producing ubiquitin and the C-terminal ribosomal protein L40. Furthermore, we demonstrated that in the brain, the deubiquitinase (DUB) USP7 mediates the processing of UBA52, which is regulated by MLKL. Knock down (KD) of USP7 or UBA52 showed similar effects on autophagy and ubiquitin levels as MLKL KD/KO. Moreover, our results indicated that the reduction of Beclin1 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.