Investigating disease mechanisms in autosomal dominant optic atrophy with retinal ganglion cells derived from induced pluripotent stem cells

Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy in the UK, characterised by the preferential loss of retinal ganglion cells (RGCs) and progressive blindness. 60-70 % of DOA patients harbour mutations in the OPA1 gene, encoding a mitochondrial protein that regulates mitochondrial morphology, bioenergetics and mitochondrial DNA (mtDNA) quality. Currently, DOA has no therapeutic options and the mechanisms driving RGC degeneration are poorly understood. In this study, a biobank of induced pluripotent stem cells iPSCs (iPSCs) encompassing the clinical and genetic DOA spectrum was created using patient-derived OPA1 mutant fibroblast cell lines, and CRISPR/Cas9 gene editing to generate isogenic cell lines. RGC differentiation was optimized and characterised in 2D and 3D in vitro methods, demonstrating expression of RGC-associated genes including BRN3B and ISL1. OPA1 mutant iPSCs showed no differentiation deficit compared to wild-type control cell lines, exhibiting comparable expression of all relevant markers. 2D-RGCs demonstrated enrichment of neuronal associated markers, including ELAVL3 and TAU, when compared to 3D retinal organoids. Phenotypic analysis demonstrated significant deficits in respiration, ATP production and increased mtDNA mutation in fibroblasts, iPSCs and 2D-RGCs compared to isogenic controls. Characterisation of mitochondrial stress through induction of stress associated gene expression demonstrated significant levels of upregulation in iPSCs, however, 3D- and 2D-RGCs exhibited fewer upregulated genes indicating that mitochondrial stress may be a cell type specific response. Importantly, correction of patient-derived iPSCs restored mitochondrial homeostasis, demonstrating that restoration of WT OPA1 expression is able to mitigate mutant associated phenotypes. Thus, an OPA1 mutant iPSC biobank has been established encompassing the clinical disease spectrum, enabling effective in vitro modelling to establish RGC specific disease mechanisms. OPA1 mutant RGCs demonstrate significant reductions in mitochondrial homeostasis, including reduced bioenergetic output and mtDNA quality. This work provides a platform for further investigation of OPA1-mediated disease mechanisms and therapeutic design

VCP: A Gatekeeper for Intracellular Proteopathic Seeding

Protein inclusions such as β-amyloid, tau, α-synuclein, and TDP-43 are considered the pathologic hallmarks of many neurodegenerative diseases. These proteins are prone to misfold, aggregate, and template new aggregates. Accumulating evidence suggests that those proteins in their high-molecular-weight forms can serve as a seed , spread through an interconnected brain network, and induce new inclusions. Therefore, it is essential to understand the mechanism of proteopathic seeding. In this dissertation, we performed a whole genomic CRISPR-Cas9 KO screening to identify gene modifiers of αS seeding. Within the screening, we found several hits of endolysosomal function and trafficking, including VCP. VCP is a versatile protein required for protein homeostasis. Mutations in VCP are associated with type 1 Multisystem proteopathy (MSP1), whose patients can develop multiple neurodegenerative diseases with proteopathic protein inclusions, including Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS), Parkinson and Alzheimer\u27s. We validated that either VCP dysfunction (inhibition or RNAi knockdown) or VCP disease mutations can exacerbate αS seeding in both αS biosensor cells and hippocampal neurons, in which both VCP knockdown and VCP mutation knock-in neurons show increasing αS aggregation following seeding. Additionally, mice carrying a VCP disease mutation exhibit more αS aggregates than C57 control 90 days after intrastriatal injection of αS pre-formed fibrils in vivo. We showed that VCP aggravates endolysosomal membrane damage with the proteopathic seeds. Similarly, seeding activities increased when we co-treated the cells with seeds and LLOMe, a drug specifically permeabilizes the endolysosome. Previously, in our lab, we demonstrated VCP involved in the autophagy-mediated clearance of damaged endolysosome, called lysophagy. Screening different VCP cofactors, we identified that only lysophagy-related cofactor UBXD1 modifies the α-synuclein seeding the same as VCP. We proposed that VCP might regulate αS seeding via UBXD1-dependent lysophagy. This dissertation also developed two novel TDP-43 seeding assays, which phenocopies hyper-phosphorylated TDP-43 aggregations in patients. We observed the same protective effect of VCP on intracellular TDP-43 seeding and tau. Overall, our results support VCP as a gatekeeper for different intracellular proteopathic seeding in a merging pathway