Abstracts

Rationale: Mutations in the FMR1 gene result in loss of function of the Fragile-X Mental Retardation Protein (FMRP) that are closely linked to intellectual disability (ID) and autism in affected individuals with the Fragile X syndrome (FRAX). Outside of FRAX, the role of FMRP function in mediating neuropsychological abnormalities is not clear. We have previously shown that adult rats have loss of CA1 long-term potentiation (LTP) and enhanced long-term depression (LTD) following an early life seizure (ELS) induced by kainate at post-natal day (P) 7. Alterations in LTP and LTD likely underlie the observed behavioral abnormalities at P60+. LTD can be mediated by two distinct mechanisms, NMDA receptor dependent and metabotropic glutamate receptor (mGluR) dependent, differentiated by the effectiveness of different LTD inducing stimulation paradigms. mGluR-LTD is dependent on protein synthesis. In FMR1 knock-outs, mGluR-LTD is enhanced and protein synthesis is dysregulated. Normally, protein phosphatase 2A (PP2A) acts concertedly with FMRP to limit the amount of mGluR-mediated protein synthesis. We hypothesized that alterations in LTD following ELS are mGluR-dependent and due to alterations in FMRP-related signaling.Methods: ELS was induced in rats at P7 with kainate (2 mg/kg, s.c.). Hippocampal slices were prepared at P60+. LTD induction paradigms designed to isolate mGluR-LTD were utilized. Okadaic acid (3 nM) was used to probe the dependence of mGluR-LTD on PP2A signaling. Cycloheximide (50 ?M) and anisomycin (20 ?M) were used to probe the dependence of mGluR-LTD on protein synthesis. Mediators of the observed changes in mGluR dependent LTD were assessed using semi-quantitative western blot expression assays of subcellular fractionated and whole CA1 homogenates. Localization of FMRP was probed with fluorescence immunoctytochemistry in CA1 hippocampal slices.Results: ELS specifically enhanced mGluR-LTD in adult (P60+) CA1 hippocampus measured using field excitatory postsynaptic potentials. PP2A blockade occluded any further enhancement of mGluR-LTD mediated by ELS. mGluR-LTD remained dependent on protein synthesis following ELS. Expression of activated S6K was significantly (p < 0.05) increased. Expression of activated GSK3? and Akt were unchanged. Expression of total S6K, mGluR5, FMRP, GSK3?, Akt, PP2A, ARC and STEP were not significantly altered. Expression of PP2A in subcellular compartments was significantly altered. Localization of FMRP following ELS resulted in a translocation away from dendritic compartments.Conclusions: ELS results in long-term signaling changes causing enhanced mGluR-LTD. Signaling proteins associated with mGluR-LTD were altered in a fashion consistent with altered FMRP function, suggesting possible targets for therapeutic intervention. Thus we establish a critical role for FMRP localization and PP2A in mediating mGluR-LTD. Furthermore, this suggests a role for FMRP in mediating ID and autism outside of FRAX, particularly that associated with ELS.