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Pathological Characteristics and Alterations in Proteomic Profiles of preclinical models Harboring Patient-Derived Point Mutation in the WDR45 Gene

Clinical Genetics and Therapeutics
  • Primary Categories:
    • Basic Research
  • Secondary Categories:
    • Basic Research
Introduction:
WDR45-related neurodevelopmental disorder is a distinct X-linked subtype of neurodegeneration with brain iron accumulation (NBIA), which comprises a heterogeneous group of monogenic disorders. The molecular mechanism underlying the pathogenesis remains unknown. Here we generated induced pluripotent stem cells (iPSCs)-derived midbrain organoids from a female patient with a novel WDR45 point mutation and its homologous mouse model to recapitulate the pathological characteristics and explore the pathogenic mechanism of WDR45 gene mutation.

Methods:
We generated iPSCs from a patient with a novel point mutation and performed pluripotency and functional verification, followed by establishing its natural isogenic control iPSCs based on the X-chromosome inactivation mechanism. We differentiated the iPSCs (mutant group and isogenic control group) into midbrain organoids to recapitulate human physiological and pathological states. We explored their disease phenotypes through immunofluorescence staining and DAB-enhanced Prussian blue iron staining of the induced differentiated midbrain organoids. We also constructed a knock-in mouse model at the homologous site of the patient by using CRISPR/Cas9 technology. We evaluated behavioral changes of the mice through open field tests, balance beam walking tests, and water-maze tests. We explored histological changes through Nissl staining and immunofluorescence staining. We performed transmission electron microscope to evaluated the pathological changes at a subcellular level. And we also assessed the degree of neuronal damage through the expression level of NSE by ELISA. Finally, we performed proteomic analysis on midbrain organoids at D45 to investigate the potential pathogenic mechanisms.

Results:
We identified a patient carrying a novel mutation in WDR45 (c.888G>A/p.W296X, classified as pathogenic according to ACMG guideline). Peripheral blood mononuclear cells (PBMCs) were extracted from the patient and used to establish iPSCs, including mutant clones (MUT) and isogenic control clones (WT). The results revealed a significant reduction in the efficiency of MUT clones in developing into mature dopaminergic neurons in the midbrain, accompanied by a pronounced iron deposition phenotype at late stages of differentiation. In the WDR45-deficient mouse model, hemizygous mutant mice exhibited increased locomotor hyperactivity, motor dysfunction, and cognitive impairments. Histologically, there was a notable decrease in mature dopaminergic neurons in the substantia nigra (SN) of the midbrain, along with a significant increase in the autophagy substrate SQSTM1/p62, aligning with the clinical phenotypes observed in patients. Although no significant iron accumulation was observed at histological level, we observed ferroptosis features at a subcellular level. The expression level of NSE in mutant group showed a significant increase. Proteomic analysis of midbrain organoids differentiated for 45 days revealed that the differentially expressed proteins were primarily enriched in the peroxisomal pathway.

Conclusion:
In this study, we successfully generated midbrain organoids differentiated from iPSCs derived from a patient with a novel point mutation in WDR45 gene, as well as a knock-in mice model at the homologous site. Notably, the midbrain organoid model recapitulated the phenotype of iron deposition in the substantia nigra observed in clinical patients, validating its potential as a reliable preclinical research model. Furthermore, this study unveiled the role of the peroxisomal pathway in NBIA5 at the physiological level of human brain-like organoids, enhancing our understanding of the fundamental molecular mechanisms driving neurodegenerative diseases. Importantly, it may lay the groundwork for the development of mechanism-based therapeutic interventions.

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