Membrane trafficking impairments in fragile x syndrome

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.

Grant Number: 1R21NS143271-01A1
NIH Institute/Center: NIH
Principal Investigator: GARY BASSELL