Our study's objective is to delineate key aspects of WIPI4 function and provide a novel platform to interrogate cell type-specific pathobiology of WDR45 mutations.

BPAN (β-propeller protein-associated neurodegeneration) is a progressive and incurable neurodegenerative disorder that belongs to a subset of NBIA (neurodegeneration with brain iron accumulation). Patients affected by BPAN initially experience cognitive deterioration and epilepsy in childhood, followed by progressive dementia, dystonia, and parkinsonism in adulthood. BPAN is caused by loss-of-function mutations in the gene WDR45, which encodes the protein, WIPI4. WIPI4 plays a critical role in autophagy by serving as a scaffold for recruiting protein complexes necessary for normal maturation of autophagosomes in the degradative pathway of autophagy. Therefore, disease-causing mutations in WDR45 disrupt autophagosome maturation and impair degradation of substrates through autophagy. Although much has been elucidated about how and why WDR45 loss leads to toxic effects in BPAN, the distinct functions of WIPI4 in neurons and its role in BPAN development remain unknown.

We established a novel BPAN model system using induced pluripotent stem cells (iPSCs) capable of rapid differentiation into multiple and isogenic neuronal and non-neuronal cell types. To enable non-invasive characterization of derangements of WIPI4 and autophagy in live cells,  we used CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs) that express fluorescently tagged autophagy markers to introduce two simultaneous genomic edits: (1) BPAN mutations into the native WDR45 locus, and (2) fusion of a HaloTag to WIPI4.

We found a profound reduction in WIPI4 expression and abnormal localization in mutant WIPI4 cells compared to wild-type. In addition, mutant WIPI4 cells showed impaired autophagic activity with abnormal accumulation of dysmorphic autophagosomes and multiple autophagy substrates.