Membrane trafficking impairments in fragile x syndrome

Emory University, Atlanta GA

Investigators

Abstract

Fragile x syndrome (FXS), the most common form of inherited intellectual disability and monogenic cause of autism, is caused by loss of FMRP, the fragile x messenger ribonucleoprotein-1. FMRP is a mRNA binding protein known to regulate mRNA translation, including local protein synthesis important for synapse development and function. FMRP often acts to repress translation, which results in elevated expression of many proteins, including cytoskeletal proteins and components of the postsynaptic density. We have previously shown that FMRP represses translation of PSD-95 mRNA at synapses. Loss of FMRP in FXS models also results in dysregulated surface expression of membrane proteins, although underlying mechanisms remain unclear, as several do not appear to be direct FMRP targets. Previously we showed that FMRP depleted neurons have reduced surface expression and enhanced rate of AMPA receptor endocytosis, which is a likely driver of the enhanced mGluR-LTD. Other studies from our lab and others suggest that FMRP may play a broad role to regulate the dynamic trafficking of numerous membrane surface proteins, but underlying mechanisms are not known. A critical gap is lack of understanding of underlying mechanisms for if and how FMRP might directly and/or indirectly regulate membrane protein surface expression to control synapse development and function. We conducted an unbiased mass spectrometry analysis following surface biotinylation and streptavidin pulldown analysis of membrane labelled and associated proteins from control and FMRP depleted cortical neurons (DIV21) from mice. A surprising result was the increased expression of four subunits of the Clathrin-Associated Adaptor Complex Protein-2 (AP2) in FMRP depleted neurons compared to controls. As several data sets of FMRP binding targets have identified Ap2 subunit mRNAs, these results suggest that FMRP may repress translation, resulting in increased levels of AP2 subunits in FXS. We hypothesize that elevated nascent synthesis of AP2 subunits leads to enhanced endocytosis of several membrane proteins to alter synaptic development and function in FXS. Aim 1 will test the hypothesis that FMRP is a negative regulator of the synthesis of AP2 subunits and that elevated levels of AP2 subunits at synapses are responsible for the reduced surface expression of AMPA receptors in FXS. Aim 2 will test the hypothesis that elevated levels of AP2 subunits are also responsible for the reduced surface expression of other membrane proteins identified in our screen that are relevant to FXS and other neurodevelopmental brain disorders. Viral shRNA knockdown and pharmacological strategies will be used to reduce or inhibit the elevated levels of AP-2 subunits in FXS. As an alternative and innovative approach, we will use a new CRISPR-Cas9 TKI method to introduce SEP tags on endogenous GluA1/2 subunits. This research has implications for development of therapeutic strategies that target AP-2 to correct for altered membrane protein surface expression contributing to impairments in synaptic development in FXS.