Objective: To examine the impact of mutant WDR45 on autophagy,mitochondrial function,and oxidative stress capacity in patient’s fibroblasts.

Background: Mutations in the WD repeat domain 45 (WDR45) gene were described to cause neurodegeneration with brain iron accumulation (NBIA). WDR45 encodes a protein that serves a putative role in autophagy.Autophagy-related genes are present on mitochondrial membranes suggesting a link between WDR45,autophagosomes,and mitochondria.

Methods: We detected a mutation in WDR45 (c.519+1_519+3delGTG) in a 22-yr-old female patient with NBIA.We collected a skin biopsy from the patient and examined fibroblasts from the patient and healthy controls.To validate our findings in a neuronal cell model, we generated WDR45 knockout (KO) SH-SY5Y cells by using CRISPR/Cas9 gene-editing technology.

Results: The patient presented with mental retardation,autistic features,parkinsonism,and dystonia.MRI showed characteristic iron deposition bilaterally in the globus pallidum and substantia nigra.RT-PCR and sequencing revealed loss of the exon8 splice site in WDR45 leading to aberrant splicing generating a frameshift and premature stop codon.Blood and fibroblast RNA showed skewed X-chromosome inactivation leading to silencing of the WDR45 wild-type allele.The patient’s fibroblasts showed decreased capability to activate autophagy upon treatment with the autophagy activator Torin1 compared to cells from healthy controls.We further observed a reduced mitochondrial membrane potential, as well as a decreased degree of mitochondrial network branching in mutant fibroblasts indicating mitochondrial dysfunction.Accordingly,we determined higher levels of oxidized proteins in the mutant cells under basal conditions and upon treatment with the mitochondrial toxin MPP+ and ferric chloride.This increased susceptibility to high iron levels was seen along with an upregulation of antioxidative defense proteins.All effects were fully rescued by the transduction of the patient’s fibroblasts with wild-type WDR45. Besides the presence of mitochondrial dysfunction and oxidative stress,we detected increased total and phosphorylated tau levels in SH-SY5YWDR45 KO cells supporting the hypothesis of impaired autophagy with tau being a known autophagy substrate.

Conclusions: Further studies are warranted to elucidate the exact molecular mechanism connecting WDR45 mutations with iron deposition,autophagy,and mitochondrial dysfunction.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/mutant-wdr45-links-nbia-to-impaired-autophagy-mitochondrial-dysfunction-and-oxidative-stress/