Neuropeptide Release is Impaired in a Mouse Model of Fragile X Mental Retardation Syndrome.

Fragile X syndrome (FXS) is the most common cause of inherited mental retardation, with an incidence of 1 in 4000 males and 1 in 8000 females (1). The molecular mechanism underlying FXS involves the expansion of an unstable polymorphic CGG triplet repeat to >200 in the 5′ untranslated region of the Fmr1 gene (2,3). This results in hypermethylation of the Fmr1 promoter and silencing of the Fmr1 gene, with consequent absence of the protein product, fragile X mental retardation protein (FMRP) (3). FMRP is an mRNA-binding protein that is enriched at the synapse and known to regulate the transport and localized translation of mRNA in response to mGluR receptor activation (4). Many of the mRNA cargoes associated with FMRP encode proteins crucial for spine maturation and synaptic plasticity (5−8). In the absence of FMRP, there is defective regulation of localized mRNA translation. This absence affects synaptic plasticity in FXS, with abnormalities in long-term potentiation (LTP) and long-term depression (LTD) (9,10) in Fmr1 knockout (KO) mice, which exhibit characteristics of FXS (11,12). The absence of FMRP should lead to dysregulated local protein levels in both axons and dendrites, but previous reports have focused largely on translational regulation deficits at the postsynaptic site in FXS. Studies by Hanson and Madison (13) and Lauterborn et al. (14) recently suggested possible presynaptic effects caused by the loss of FMRP, prompting us to examine neuropeptide release in FXS. The mRNA cargoes of FMRP include presynaptic proteins that participate in the secretory pathway, in particular, vesicle exocytosis (5,6). One such protein is Rab3A, a GTPase that cycles between a soluble Rab3A-GDP form and a vesicle membrane-bound Rab3A-GTP form and is involved in activity-dependent vesicle docking and fusion at the synapse (15,16). Changes in Rab3A levels would be expected to affect activity-dependent release of transmitters and modulators. Using Western blot analyses, we characterized the levels of this protein in wild-type (WT) and Fmr1 KO mice. Next, we used matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to examine synaptoneurosomal preparations and probed the physiology of stimulus-evoked neuropeptide release in Fmr1 KO mice using live brain slices. Our results indicate that these mice are markedly deficient in neuropeptide release. In order to determine whether the neuropeptide release deficit in Fmr1 KO mice is a general deficit in dense-core vesicle (DCV) release, we used electrochemistry to examine the release of biogenic amines. We show that the release deficit is specific to peptides, because there is no significant difference in the release of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) from the striatum of WT and Fmr1 KO mice. Lastly, using electron microscopy to quantify the number of peptide-housing DCVs, we do not observe significant differences between WT and Fmr1 KO mice, again suggesting a specific release deficit in FXS.