Restriction lift date: 2030-09-30
Optimisation of a GFP-RAB7 hiPSC model to investigate phenotypic differences of point mutations in Charcot-Marie-Tooth Disease type 2B
Loading...
Files
Date
2025
Authors
O'Mahony, Katie
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Charcot-Marie-Tooth disease type 2B (CMT2B) is a debilitating inherited peripheral neuropathy caused by mutations in the RAB7 gene. Despite many clinical trials, effective treatments targeting the underlying mechanisms of CMT2B remain elusive. One potential limitation is the reliance on non-physiological models, such as HeLa cells and mouse models, which fail to capture the complexity of human disease. To address this, the Burk Lab developed isogenic human induced pluripotent stem cell (hiPSC) lines with CRISPR-engineered GFP-RAB7 carrying the CMT2B-associated point mutations. These lines provide a physiologically relevant system to study the functional impact of RAB7 mutations, reducing the variability and enabling systematic comparison across all major missense mutations associated with CMT2B.
This thesis focuses on characterising the Burk Lab’s CMT2B hiPSC lines, optimising their differentiation into induced sensory neurons (iSNs), and validating their use for studying RAB7-associated pathways, including late endosomal trafficking and autophagy. Building on the development of these hiPSC models, initial experiments aimed to validate that the GFP-tag does not affect RAB7 and its known interactors (ORP1L, RILP, WASH1, VPS26, VPS29, and VPS35). Co-immunoprecipitation (co-IP) was performed using lysates from undifferentiated hiPSCs. While the protocol requires further optimisation, initial results are promising as RAB7 can indeed be pulled down with GFP, confirming successful tagging.
To further investigate functional outcomes of RAB7 mutations, neurite length, late endosomal trafficking dynamics, and autophagic flux were assessed. No significant differences were observed between the length of young neurites (DIV1 and DIV2) of wild type (WT) controls and mutant iSNs, This may suggest that neurite length may not be affected in CMT2B. This warrants further investigation at later stages of outgrowth.
Building on these findings, preliminary live cell imaging of RAB7-positive vesicle in V162M mutant iSNs demonstrated increased vesicle movement along neurites compared to WT controls, suggesting altered intracellular trafficking as a potential mechanism underlying CMT2B pathology.
Autophagic flux was assessed in undifferentiated GFP-RAB7 hiPSCs using starvation conditions and bafilomycin A1 treatment, followed by immunoblotting for p62 and LC3. While further optimisation is needed before this protocol can be reliably used, these initial results provide a foundational workflow for future autophagy assessments in this model.
For all experiments, additional differentiations are necessary to conclusively confirm the preliminary results obtained thus far. The continued validation of these models will facilitate the identification of therapeutic targets and pave the way for more effective treatments for CMT2B.
Description
Keywords
CMT2B , Peripheral neuropathy , Receptor signalling , Endosomal trafficking , RAB7 , hiPSC
Citation
O'Mahony, K. 2025. Optimisation of a GFP-RAB7 hiPSC model to investigate phenotypic differences of point mutations in Charcot-Marie-Tooth Disease type 2B. MRes Thesis, University College Cork.