CYFIP1, an Autism Spectrum Disorder candidate gene, as a potential modulator of IL-6/STAT3 signalling in Chromosome 15q-Duplication Syndrome

Abstract

Neurodevelopmental disorders (NDDs) arise from abnormalities during the development of the nervous system and describe a variety of neurological conditions, a prime example of which is autism spectrum disorder (ASD). ASD is characterised by impaired social interaction, delayed language development and repetitive or restrictive behaviours. Although the aetiology of ASD remains unclear, ASD is known to possess a strong genetic component and is comorbid with several genetic syndromes – these are referred to as syndromic forms of ASD. Two of the most common forms of syndromic ASD are Fragile X syndrome (FXS) and chromosome 15q-duplication syndrome (Dup(15q)). FXS is caused by silencing of the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene and Dup(15q) is the result of a duplication event in the 15q11.2-q13.1 region. Although several genes are encoded within the 15q11.2-q13.1 cytobands, one of these, Cytoplasmic FMRP-Interacting Protein 1 (CYFIP1), is a gene of particular interest as studies have demonstrated that duplications (or deletions) that include CYFIP1 lead to increased severity of neurobehavioural phenotypes. Importantly, CYFIP1 interacts with Fragile X Messenger Ribonucleoprotein (FMRP), the protein product of the FMR1 gene, to regulate local protein synthesis in neurons, in addition to its role in regulating Actin cytoskeleton dynamics as part of the WAVE regulatory complex.

Janus Kinase and Microtubule-Interacting Protein 1 (JAKMIP1) has been identified as an ASD candidate gene due to its common dysregulation in cases of FXS and Dup(15q) with ASD. Furthermore, JAKMIP1 expression has been shown to be reduced following both CYFIP1-overexpression (OE) and FMR1-knockdown (KD). A recent study demonstrated that JAKMIP1 modulates neuronal responses to cytokine signalling, with JAKMIP1-deficient neuroblastoma cells showing reduced expression of Signal Transducer and Activator of Transcription 3 (STAT3), attenuated STAT3 activity following stimulation with Interleukin-6 (IL-6) and impaired IL-6-induced neurite outgrowth. However, it is not yet known whether JAKMIP1 dysregulation in syndromic forms of ASD also leads to impaired IL-6/STAT3 signalling and how this affects neuronal behaviours.

Given that JAKMIP1 expression is dysregulated in CYFIP1-OE cells, I hypothesised that CYFIP1-OE may also lead to changes in IL-6/STAT3 signalling. The overarching aims of this thesis are therefore to investigate whether CYFIP1 plays a role in modulating cytokine signalling via JAKMIP1, and to examine the impact of altered IL-6 signalling on Dup(15q) neuronal morphology. Using both CYFIP1-OE HEK293 and SH-SY5Y neuroblastoma cells, I discovered that CYFIP1-OE cells showed reduced JAKMIP1 and STAT3 expression, but enhanced IL-6-induced STAT3 transcriptional activity and neurite outgrowth. Interestingly, I found that this altered response was not specific to IL-6/STAT3 signalling, with CYFIP1-OE cells showing altered expression of several other cytokine signalling-related transcription factors and enhanced Interferon-γ (IFN-γ)-induced STAT1 transcriptional activity.

To validate these findings in an ASD model, I assessed IL-6/STAT3 signalling in Dup(15q) induced pluripotent stem cells (iPSCs) and iPSC-derived cortical neurons (iNeurons). I observed that Dup(15q) iPSCs and iNeurons with increased CYFIP1 expression again show reduced STAT3 expression, but an enhanced STAT3 transcriptional response following IL-6 stimulation. Furthermore, Dup(15q) iNeurons display increased neuritogenesis and neurite branching complexity, however no change in IL-6-promoted neuritogenesis.

Finally, to elucidate the potential mechanism by which CYFIP1 modulates STAT3 activity, I generated tools using an established CRISPR-Cas9-based gene-editing technique named ORANGE to endogenously tag my proteins of interest with fluorescent proteins. I then created a novel method based on the ORANGE technique, incorporating the Sleeping Beauty transposon system to improve the efficacy of gene editing. I find that this method, named Sleeping ORANGE, improves the gene editing efficiency by approximately four- to six-fold.

Together, this thesis provides the first evidence that the CYFIP1 gene encoded in the chromosome 15q region is a novel regulator of cytokine signalling, capable of modifying key neurodevelopmental processes, such as neurite outgrowth and neurite branching, at the intersection between NDD risk genetics and neuroinflammation. Moreover, this thesis also generates a novel method to increase the efficacy of CRISPR-Cas9-based endogenous epitope tagging, whilst providing insights into the molecular actions of the sleeping beauty transposase.