Cotfas, Daniel-Marian; (2024) Investigating the pathophysiology of Beta Propeller-protein Associated Neurodegeneration (BPAN). Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Introduction: Beta-propeller protein associated neurodegeneration (BPAN) is an X-linked dominant complex neurological disorder caused by mutations in the WDR45 gene. Patients present with global neurodevelopmental delay and seizures, intellectual issues in childhood with regression in early adulthood, progressive dystonic and rigid parkinsonian symptoms, and dementia. MRI scans of BPAN patients show brain iron deposition in the substantia nigra and globus pallidus, and generalised atrophy of cerebrum and cerebellum. Post-mortem studies reveal pathological iron deposition, neurofibrillary hyperphosphorylated tau (MAPT – microtubule associated protein tau) tangles throughout the cortex, with mixed 3R-4R tau pathology similar to that seen in Alzheimer’s disease (AD). The current views on pathogenesis include abnormal protein clearance and cellular turnover, which have implicated autophagy. Autophagy is a cellular process involved in the turnover of materials and subcellular structures. WDR45 protein has a putative role in autophagy initiation and regulation, and mutations in WDR45 lead to disease. It may act as a regulator of autophagy, possibly behaving as the scaffold for proteins involved in early autophagy to assemble. The function of the WDR45 protein has not been fully elucidated, and it is the focus of our investigations. Methods: Two cell models of BPAN were created. The first using BPAN-patient skin and dural fibroblasts were reprogrammed into induced pluripotent stem (iPS) cells, and differentiated into disease-relevant, cortical neurons to model BPAN in vitro, and recapitulate developmental events that may occur specifically in BPAN patients. Normal control and BPAN-patient fibroblasts, iPS cells, and iPSC-derived neurons were examined for changes in autophagy. Cultured cells were investigated using RT-PCR, western blotting, and immunocytochemistry. Immunohistochemical investigations were also undertaken in paraffin-embedded, formalin-fixed post-mortem human brain using the unique resources at Queen Square Brain Bank. The second cell model utilised tetracycline-inducible shRNA knockdown of WDR45 expression in SH-SY5Y neuroblastoma cells. These cells were further neuralised, and examined using nanostring RNA expression panels, RT-qPCR, and western blotting for changes in neurodegeneration-related and autophagy-related genes. Results: Immunohistochemical investigations in post-mortem BPAN brain yielded region-specific differences in ‘pathway-specific’ (ER-stress, iron metabolism, and autophagy) markers, which were similar in pattern to Alzheimer’s Disease brains. However, BPAN brains showed a high intensity signal for iron metabolism markers, which are a hallmark of NBIAs but not AD. IHC data from post-mortem brains suggest that several pathways (autophagy, iron metabolism, and ER stress) are affected by WDR45 mutation, and that these effects may be specific to microglia and to a lesser extent in neurons. Molecular investigations into WDR45 expression by Western blot and RT-qPCR suggest reduced protein expression as well as reduced autophagic flux as assayed by comparing LC3 protein levels in BPAN-patient derived iPSC, neural stem cells, and iPSC-derived cortical neurons compared to normal controls. This pattern extends into SH-SY5Y neuroblastoma cells, which were engineered to transiently knock-down WDR45 expression in response to doxycycline treatment. Transcriptomics performed on both cell models further extend our understanding of BPAN beyond the pathways already explored, and suggest other cells, pathways, and organelles that could be affected by BPAN, providing the basis for future research.

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