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

Abstract

Fragile X syndrome (FXS), an inherited disorder characterized by mental retardation and autismlike behaviors, is caused by the failure to transcribe the gene for fragile X mental retardation protein (FMRP), a translational regulator and transporter of select mRNAs. FXS model mice (Fmr1 KO mice) exhibit impaired neuropeptide release. Release of biogenic amines does not differ between wild-type (WT) and Fmr1 KO mice. Rab3A, an mRNA cargo of FMRP involved in the recruitment of vesicles, is decreased by ∼50% in synaptoneurosomes of Fmr1 KO mice; however, the number of dense-core vesicles (DCVs) does not differ between WT and Fmr1 KO mice. Therefore, deficits associated with FXS may reflect this aberrant vesicle release, specifically involving docking and fusion of peptidergic DCVs, and may lead to defective maturation/maintenance of synaptic connections.

Figure 1

Western blot analysis from P10−14 WT and Fmr1 KO mouse cortical synaptoneurosomes and total cortical homogenates shows a reduction in Rab3A expression. (a) Synaptoneurosomal lysates from WT and Fmr1 KO mouse cortices were run on 12% polyacrylamide gels, blotted to nitrocellulose membranes, and stained with rabbit polyclonal antibody specific for Rab3A, followed by antibody to β-actin to normalize to total protein loaded. (b) Rab3A expression in Fmr1 KO mouse synaptoneurosomes (n = 8) is dramatically reduced compared with that in WT mouse synaptoneurosomes (n = 8). Blots were normalized to β-actin (β-actin, ∗∗, p < 0.01; not shown) and to total protein loaded (∗∗, p < 0.01; error bars, SEM). (c) Rab3A expression in total cortical homogenates is significantly decreased in Fmr1 KO mice, although to a smaller extent than in synaptoneurosomes (n = 4; ∗∗, p < 0.01).

Figure 2

Synaptoneurosomes from Fmr1 KO mice are defective in neuropeptide release. LC−MALDI-MS analysis of releasates collected from stimulated (KCl, 55 mM) and unstimulated (saline) cortical synaptoneurosomes from P10−14 WT and Fmr1 KO mice. Each panel represents a profile in the mass range m/z 1300−5000 of several LC fractions for (a, b) WT and (c, d) Fmr1 KO mice. The mass profiles obtained from the releasates of stimulated WT synaptoneurosomes (a) show a number of high-intensity peaks compared with those from the Fmr1 KO synaptoneurosomes (b). Using MS/MS analysis or accurate mass match, we identified several peaks as peptides derived from (1) PEP-19, (2) cholecystokinin 12, (3) unknown m/z 1776.7, (4) unknown m/z 1923.7, (5) stathmin (6) orexin B, (7) unknown m/z 3387.0, (8) thymosin β4, and (9) thymosin β10. Inset shows an expanded mass region from the fraction containing stathmin peptide at m/z 2348.1.

Figure 3

Defective neuropeptide release from Fmr1 KO brain slice preparations. MALDI-MS analysis of releasates from whole brain slices (400 μm) containing SCN and cortex from P10−15 WT and Fmr1 KO mice. Slices stimulated with a topical application of 55 mM KCl show marked increase in the number and intensity of peptides observed in WT mice compared with Fmr1 KO mice (n = 4, WT; n = 4, Fmr1 KO). (a) Mass profiles obtained from the cortex and (b) from the SCN. Inset in panel a shows relative intensity of neurokinin B and inset in panel b shows arginine vasopressin, for all four conditions.

Figure 4

Biogenic amine (DA, NE, 5-HT) release from WT and Fmr1 KO brain slice preparations is not significantly different: (a) DA release in the CP evoked with a single stimulus pulse; (b) 5-HT release evoked in the SNr by a 20 pulse, 100 Hz stimulation train; (c) NE release in the BNST evoked by a 60 pulse, 60 Hz stimulation (data shown are from WT tissues); (d) identical release concentrations were observed in brain slices from WT and Fmr1 KO (p > 0.05, n = 6 from each genotype for each neurotransmitter).