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. 2013 Nov 22;342(6161):987-91.
doi: 10.1126/science.1245079. Epub 2013 Oct 31.

The Human Language-Associated Gene SRPX2 Regulates Synapse Formation and Vocalization in Mice

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Free PMC article

The Human Language-Associated Gene SRPX2 Regulates Synapse Formation and Vocalization in Mice

G M Sia et al. Science. .
Free PMC article

Abstract

Synapse formation in the developing brain depends on the coordinated activity of synaptogenic proteins, some of which have been implicated in a number of neurodevelopmental disorders. Here, we show that the sushi repeat-containing protein X-linked 2 (SRPX2) gene encodes a protein that promotes synaptogenesis in the cerebral cortex. In humans, SRPX2 is an epilepsy- and language-associated gene that is a target of the foxhead box protein P2 (FoxP2) transcription factor. We also show that FoxP2 modulates synapse formation through regulating SRPX2 levels and that SRPX2 reduction impairs development of ultrasonic vocalization in mice. Our results suggest FoxP2 modulates the development of neural circuits through regulating synaptogenesis and that SRPX2 is a synaptogenic factor that plays a role in the pathogenesis of language disorders.

Figures

Fig. 1
Fig. 1. SRPX2 overexpression increases excitatory synaptic density in cultured neurons
A, Rat cortical neurons were cotransfected with BFP and rat (Rn SRPX2) or human SRPX2 (Hs SRPX2), and stained for excitatory synapse markers VGlut1 (green) and PSD95 (red), as well as for inhibitory synaptic markers VGAT (green) and gephyrin (red). Scale bars, 5 μm. B, Quantification of synapse density shown in (A). (Means ± sem, n = 13–35, *p < 0.05, **p < 0.01). C, Quantitation of spine density shown in (A). (Means ± sem, n = 25–35, **p < 0.01). D, DIV 21 dissociated rat cortical neurons were surface-stained for SRPX2, and then permeabilized and stained for excitatory and inhibitory synaptic markers. Scale bars, 3.5 μm. E, Synaptosome preparation was immunoblotted for SRPX2 and PSD95. Fractions are crude homogenate (Hom), P1 pellet (P1), S1 supernatant (S1), S2 supernatant (S2), P2 pellet (P2), and synaptosomes (Syn). F, Western blot analysis of SRPX2 levels in the cerebral cortex, hippocampus and cerebellum of P7 and P30 mice.
Fig. 2
Fig. 2. Reduction of endogenous SRPX2 and secretion of dominant negative SRPX2 decrease excitatory synaptic density in cultured neurons
A, Rat cortical neurons were cotransfected with BFP and scrambled shRNA, SRPX2 shRNA, or SRPX2 shRNA and rescue construct (SRPX2 rescue), and then stained for excitatory or inhibitory synaptic markers. Scale bar, 5 μm. B, Quantitation of synapse density shown in (A). (Means ± sem, n = 12–35, *p < 0.05, **p < 0.01). C, Quantitation of spine density shown in (A). (Means ± sem, n = 22–35, *p < 0.05). D, Rat cortical neurons were incubated with medium conditioned by HEK293 cells overexpressing GFP, SRPX2, or SRPX2-DN, and immunostained for the dendritic marker MAP2, and excitatory or inhibitory synaptic markers. Scale bar, 5 μm. E, Quantitation of synapse density shown in (D). (Means ± sem, n = 12–21, *p < 0.05, **p < 0.01).
Fig. 3
Fig. 3. FoxP2 regulates excitatory synapse density through SRPX2
A, Western blot analysis of SRPX2 levels in cortical neurons eletroporated with GFP or FoxP2 at plating and cultured for 4 days. B, Quantitation of SRPX2 levels shown in (A). (Means ± sem, n = 6–7, ***p < 0.001. C, ChIP assay of FoxP2 in cortices of P0 C57BL/6J mice. FoxP2 does not bind to the β-actin promoter, but does bind the promoter of the previously identified FoxP2 target gene Slc17a3(32), and the promoter of the SRPX2 gene. D, Rat cortical neurons were cotransfected with GFP and FoxP2, FoxP2 and SRPX2, or FoxP2-R552H, and immunostained for excitatory and inhibitory synaptic markers. Scale bars, 5 μm. E, Quantitation of synapse density shown in (D). (Means ± sem, n = 15–17, *p < 0.05, **p < 0.01). F, Quantitation of spine density shown in (D). (Means ± sem, n = 32–36, **p < 0.01).
Fig. 4
Fig. 4. SRPX2 regulates excitatory synapse density and ultrasonic vocalization in vivo
A, Low magnification micrograph of electroporated layer V/VI neurons in cortices of P21 C57BL/6J mice. B, Representative micrographs of apical dendrites of cortical neurons electroporated in utero with scrambled shRNA, SRPX2 shRNA, SRPX2 shRNA and rescue construct, or SRPX2 dominant negative construct. Scale bar, 10 μm. C, Quantitation of spine density on apical dendrite of layer V/VI neurons electroporated with various constructs. (Means ± sem, n = 16–49, **p < 0.01). D, Representative traces of mEPSC recordings from layer V/VI cortical neurons electroporated with scrambled shRNA, SRPX2 shRNA, SRPX2 shRNA and rescue construct, and SRPX2 dominant negative construct. E, Average frequency and amplitude of mEPSC recordings. (Means ± sem, n = 8–11, **p < 0.01, ***p < 0.001). F, Average number of USVs emitted in 5 minutes by P7 mouse pups electroporated with various SRPX2 constructs. (Means ± sem, n = 22–46, **p < 0.01).

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