Simansour, Maud, Thiemeyer, Anke, Klein, Jessica, Stoehr, Natacha, Bernhard, Mario, Fodor, Barna and Galimberti, Ivan (2014) Assessment of neuronal cell type specific transcriptional effects of three pathological sequence variants of the FMR1 gene promoter. Master Thesis.

Abstract

The absence of functional FMR1 gene product (FMRP) causes Fragile X Syndrome; which is the most common inherited form of mental disability. In 1991, Verkerk et al. has discovered abnormalities in dendritic spine morphology in brain sections from Fragile X Syndrome (FXS) patients and synaptic transmission impairments in brain sections from Fmr1 knockout mice, uncovering a key neuronal phenotype associated with this disease. The most common cause of Fragile X Syndrome is the epigenetic silencing of the promoter triggered by a CGG triplet expansion in the 5’UTR of the gene. Additionally, inactivating point mutations in the coding region and deletions of FMR1 were also described to cause FXS.
In 2004, Collins et al., found three novel variants in the promoter region of the FMR1 gene through the use of pooled template massively parallel sequencing. These three novel variants were the first to be identified in conserved defined elements of the FMR1 promoter gene.
Using a luciferase assay, the authors showed that all these three mutations independently reduced the activity of the FMR1 promoter respectively to 36,2%, 29,2% and 5,9% in blood cells. These mutations are altering the transcription factor binding sites which are necessary to initiate FMR1 expression and therefore they are decreasing the FMR1 gene product FMRP.
The decrease in FMR1 expression is preventing the FMR1 gene product FMRP to repress the translation of mRNAs leading to an imbalance of synaptic proteins synthesis such as NMDA (N-Methyl-D-Aspartate) receptors and GABA (γ-aminobutyric acid) receptors. These proteins are required for long lasting forms of synaptic plasticity that underlie consolidation of long term memories such as LTP and LTD. Studies have established that FMRP is important for synaptic plasticity in particular in mGluR-dependent LTD which is exaggerated in FXS leading to impairments in learning and memory and in dendritic spine morphogenesis.
In this study, we created various plasmids containing GFP, mCherry or tdTomato gene reporter under the control of the FMR1 promoter with one of the three variants described in the publication from Collin’s. The plasmids were then transiently transfected in Hela cells, rat cortical neurons and human neuronal precursor neurons. In parallel, mouse hippocampal slice cultures were shot with gold particles coated by the same plasmids (Genegun protocol). The cells and slices were then treated with Bicuculline, TTX and glycine (LTP-Gly) to assess how neuronal activity modulates the FMR1 expression.
We used confocal microscopy to highlight the expression of our constructs under the different experimental paradigms. We found that the mutation located near the first and most important single start transcription was reducing FMR1 expression to 42.08% in human neuronal precursors and to 45.7% in mouse hippocampal slices.
Bicuculline was found to rescue FMR1 expression in brain slices, suggesting that the inhibition of GABA receptors triggers a sufficient increase of excitatory glutamatergic activity to modulate FMR1 expression.

Item Type:	Article
Keywords:	FXS, FMR1, FMRP, synaptic plasticity, transcription factors, neuronal modulators, LTD, NMDAR, AMPAR, glutamatergic receptor.
Date Deposited:	09 May 2016 23:45
Last Modified:	09 May 2016 23:45
URI:	https://oak.novartis.com/id/eprint/25240