BioSciences Imaging Centre - Masters by Research Theses
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Item Optimisation of a GFP-RAB7 hiPSC model to investigate phenotypic differences of point mutations in Charcot-Marie-Tooth Disease type 2B(University College Cork, 2025) O'Mahony, Katie; Burk, Katja; Lindsay, AndrewCharcot-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.Item The effects of stressors during early life on hippocampal neurogenesis and microglial activation in the male and female brain(University College Cork, 2021-04-05) McGovern, Andrew J.; Nolan, Yvonne M.; O'Leary, OliviaStress during critical periods of brain development and maturation such as adolescence is associated with an increased risk of developing stress-related psychiatric disorders which are more common in women than men. Early life stress such as maternal separation (MS), juvenile stress (JS) and inflammatory insults like lipopolysaccharide (LPS), have been found to induce anxiety and depressive-like behaviours and decrease adult hippocampal neurogenesis in rodents. However, the effects of early life stress on adult hippocampal neurogenesis and associated function have been mostly assessed in male rodents. The impact of early life stress on microglia, which are involved in the regulation of adult hippocampal neurogenesis and dendritic remodelling, has also been predominantly examined in male rodents. Thus, in this study we assessed adult hippocampal neurogenesis and hippocampal microglia following LPS administration in MS juvenile female Sprague-Dawley rats and following JS in male and female Sprague-Dawley rats in adulthood. MS increased the number of newly born hippocampal neurons in the ventral hippocampus, reduced the dendritic complexity of newly born neurons in the whole hippocampus and increased the soma size of microglia, indicating activation. LPS reduced newly born hippocampal dendritic complexity and increased the number of microglia in the dorsal hippocampus. Conversely, LPS administration in MS rats reduced the number of microglia in the dorsal hippocampus and MS attenuated microglial activation in response to LPS. LPS administration in MS increased dendritic complexity in the granule cell layer (GCL) and further reduced dendritic complexity in the ventral but not dorsal hippocampus of juvenile female rats. JS did not affect hippocampal neurogenesis in adult male or female rats but reduced the cell soma size of microglia in the GCL in the dorsal hippocampus of females. We observed significant sex differences in adult rats; females had fewer newly born neurons with less dendritic complexity in the dorsal hippocampus than males. There were also fewer microglia in the molecular layer (ML) of the hippocampus in adult female than male rats. Together the data here shows that the effect of early life stressors differentially affects hippocampal neurogenesis and hippocampal microglia dependent on age, sex and subregion of the hippocampus analysed.