Year: 2020
Type: dissertation
Abstract: Rett syndrome (RTT) is a rare neurodevelopmental disorder affecting 1 in 10,000 females born, leading to early progressive loss of developed language, cognition and motor skills. RTT is primarily caused by heterozygous loss-of-function mutations in the X linked gene encoding the global transcriptional regulator methyl-CpG-binding protein 2 (MECP2). MECP2 undergoes alternative splicing of its exon 2 resulting in the MECP2e1 and MECP2e2 isoforms, whose functions in the brain remain unclear. MECP2 is also alternatively polyadenylated to yield MECP2 short and long 3’-UTR isoforms that can undergo post-transcriptional regulation during neurodevelopment. In this thesis, I modelled RTT using human neurons differentiated from embryonic and induced pluripotent stem cells (ESCs/iPSCs) to study perturbations of MECP2. The overall hypothesis is that MECP2 mutations or changes of its isoform levels in human cortical neurons cause impairments in morphology and electrophysiology. In the first part of this thesis, I studied how modulation of MECP2 level can alter cell size and established a platform for evaluating the functional connectivity of neurons. I demonstrated that iPSC-derived neurons from an RTT patient with an MECP2e1 isoform-specific mutation have soma area defects that were rescued by expression of MECP2e1, while remaining unaffected by MECP2e2. I also showed in control ESC-derived neurons that over-expressed PUM1 consistent with destabilization of the MECP2 long 3’-UTR isoform led to phenocopy of the smaller soma area observed in RTT. Although an automated morphology-based approach for phenotyping neurons was not robust, I established a micro-electrode array (MEA) platform for assessing the network activity of control excitatory neurons. In the latter part of this thesis, I used isogenic gene edited and RTT patient reprogrammed stem cell-derived neurons to evaluate a range of phenotypes associated with different types of MECP2 mutations. I investigated the consequences of a functionally mild MECP2 L124W mutation in RTT patient neurons and used MEAs to identify novel network circuitry impairments in MECP2 null excitatory neuronal cultures. In summary, this work provides insights into RTT using human stem cell-derived neurons to reveal a wide spectrum of severe and core shared morphological and circuitry changes associated with MECP2 dysfunction.
Source: TSpace (University of Toronto)
Author: Rebecca Siu Fung Mok
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3150726449