Comprehensive analysis of autophagic functions of WIPI family proteins and their implications for the pathogenesis of β-propeller associated neurodegeneration

Abstract

β-propellers that bind polyphosphoinositides (PROPPINs) are an autophagy-related protein family conserved throughout eukaryotes. The PROPPIN family includes Atg18, Atg21, and Hsv2 in yeast and WD-repeat protein interacting with phosphoinositides (WIPI)1-4 in mammals. Mutations in the WIPI genes are associated with human neuronal diseases, including β-propeller associated neurodegeneration (BPAN) caused by mutations in WDR45 (encoding WIPI4). In contrast to yeast PROPPINs, the functions of mammalian WIPI1-WIPI4 have not been systematically investigated. Although the involvement of WIPI2 in autophagy has been clearly shown, the functions of WIPI1, WIPI3, and WIPI4 in autophagy remain poorly understood. In this study, we comprehensively analyzed the roles of WIPI proteins by using WIPI-knockout (single, double, and quadruple knockout) HEK293T cells and recently developed HaloTag-based reporters, which enable us to monitor autophagic flux sensitively and quantitatively. We found that WIPI2 was nearly essential for autophagy and partially redundant with WIPI1. Autophagic flux was unaffected or only slightly reduced by single deletion of WIPI3 (encoded by WDR45B) or WIPI4 but was profoundly reduced by double deletion of WIPI3 and WIPI4. Furthermore, we revealed variable effects of BPAN-related missense mutations on the autophagic activity of WIPI4. BPAN is characterized by neurodevelopmental and neurodegenerative abnormalities, and we found a possible association between the magnitude of the defect of the autophagic activity of WIPI4 mutants and the severity of neurodevelopmental symptoms. However, some of the BPAN-related missense mutations, which produce neurodegenerative signs, showed almost normal autophagic activity, suggesting that non-autophagic functions of WIPI4 may be related to neurodegeneration in BPAN.

AAV-mediated gene transfer of WDR45 corrects neurologic deficits in the mouse model of beta-propeller protein-associated neurodegeneration

“…WIPI4, in response to AMPK stimulation, binds ATG2 and facilitates the elongation of the autophagosomal membrane (18). Unlike WIPI2, the knockdown of WIPI4 alone leads to only a slight reduction of the autophagy, affecting mostly the size of the autophagosomes (20). The interaction of WIPI4 and WIPI2 was reported (18), and a recent study demonstrated the essential role of WIPI2 in supporting the function of WIPI4 in autophagy (20).…”

“…The four mammalian WIPI proteins, WIPI1-4, act as PI3P effectors that decode PI3P signals and consequently enable autophagophore formation, working as scaffolds and attracting their protein partners to the forming autophagosomal membrane. WIPI4 forms a complex with ATG2 that recruits phospholipids from the endoplasmic reticulum to expand the autophagic membrane (18,19,20).…”

“…The four mammalian WIPI proteins, WIPI1-4, act as PI3P effectors that decode PI3P signals and consequently enable autophagophore formation, working as scaffolds and attracting their protein partners to the forming autophagosomal membrane. WIPI4 forms a complex with ATG2 that recruits phospholipids from the endoplasmic reticulum to expand the autophagic membrane (18, 19, 20). Mutations of the ATG2A binding sites in WIPI4 disrupt autophagic flux (21) and have been documented in BPAN patients, where the severity of the disease significantly correlates with the inhibitory effect of these mutations (22).…”

The Organization and Function of the Phagophore-ER Membrane Contact Sites

“…Overall ATG9, ATG2 and/or Atg18/WIPI4 proteins may represent a modular system that eukaryotic cells may use to establish MCSs between specific organelles for lipid transfer. The relevance of this system is also underlined by the fact that mutations in WIPI4 cause ß-propeller protein associated neurodegeneration (BPAN), a lethal neurodevelopmental disorder ( Cong et al, 2021 ; Shimizu et al, 2023 ). Henceforth, the elucidation of the molecular function of the phagophore-ER MCSs could aid to understand the generation of autophagosomes and possibly other organelles, in health and disease.…”

Human WIPI β‐propeller function in autophagy and neurodegeneration

“…We can assume that the PI3P effector and signaling scaffold function of WIPI4, in addition to its role in autophagy initiation, may also extend to later stages in the autophagy process. Furthermore, it may also be reasonable to consider if, analogous to other autophagy proteins, WIPI4 also exerts cellular functions that only indirectly affect autophagy, or do not impact autophagy at all [71,[103][104][105][106]. Clearly, a combinatorial knowledge of WIPI4 functions arising from independent approaches is a prerequisite for the development of future BPAN therapies, be it gene or cell therapy or the use of specific drugs.…”