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SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins - PubMed

  • ️Wed Jan 01 2020

SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins

Kathlyn J Gan et al. J Neurosci. 2020.

Abstract

Emerging evidence supports roles for secreted extracellular matrix proteins in boosting synaptogenesis, synaptic transmission, and synaptic plasticity. SPARCL1 (also known as Hevin), a secreted non-neuronal protein, was reported to increase synaptogenesis by simultaneously binding to presynaptic neurexin-1α and to postsynaptic neuroligin-1B, thereby catalyzing formation of trans-synaptic neurexin/neuroligin complexes. However, neurexins and neuroligins do not themselves mediate synaptogenesis, raising the question of how SPARCL1 enhances synapse formation by binding to these molecules. Moreover, it remained unclear whether SPARCL1 acts on all synapses containing neurexins and neuroligins or only on a subset of synapses, and whether it enhances synaptic transmission in addition to boosting synaptogenesis or induces silent synapses. To explore these questions, we examined the synaptic effects of SPARCL1 and their dependence on neurexins and neuroligins. Using mixed neuronal and glial cultures from neonatal mouse cortex of both sexes, we show that SPARCL1 selectively increases excitatory but not inhibitory synapse numbers, enhances excitatory but not inhibitory synaptic transmission, and augments NMDAR-mediated synaptic responses more than AMPAR-mediated synaptic responses. None of these effects were mediated by SPARCL1-binding to neurexins or neuroligins. Neurons from triple neurexin-1/2/3 or from quadruple neuroligin-1/2/3/4 conditional KO mice that lacked all neurexins or all neuroligins were fully responsive to SPARCL1. Together, our results reveal that SPARCL1 selectively boosts excitatory but not inhibitory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.SIGNIFICANCE STATEMENT Emerging evidence supports roles for extracellular matrix proteins in boosting synapse formation and function. Previous studies demonstrated that SPARCL1, a secreted non-neuronal protein, promotes synapse formation in rodent and human neurons. However, it remained unclear whether SPARCL1 acts on all or on only a subset of synapses, induces functional or largely inactive synapses, and generates synapses by bridging presynaptic neurexins and postsynaptic neuroligins. Here, we report that SPARCL1 selectively induces excitatory synapses, increases their efficacy, and enhances their NMDAR content. Moreover, using rigorous genetic manipulations, we show that SPARCL1 does not require neurexins and neuroligins for its activity. Thus, SPARCL1 selectively boosts excitatory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.

Keywords: NMDAR; SPARC-like protein 1 (SPARCL1); neurexin; neuroligin; synapse; synapse formation.

Copyright © 2020 the authors.

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Figures

Figure 1.
Figure 1.

Endogenous SPARCL1 is secreted at low levels by cultured mouse glia but is abundant in mouse serum. A, Design of intron-spanning primers for semiquantitative RT-PCR. Forward (F) and reverse (R) primers against SPARCL1 primers against SPARCL1 and β-actin (control) genes were situated in exons to distinguish between gDNA and mRNA amplification (left). Expected amplicon sizes are indicated (right). B, Most cortical SPARCL1 mRNA is expressed by glia. RT-PCR was performed on total RNA isolated from glia cultured alone or from glia cocultured with cortical neurons at DIV14. Amplification of SPARCL1 and β-actin transcripts was optimized to 25 PCR cycles to ensure that quantification was performed before saturation. SPARCL1 and β-actin transcripts from both sources were amplified either individually or in multiplexed format to estimate the relative abundance of SPARCL1 cDNA by normalization against β-actin. Analysis by electrophoresis shows specific amplification of SPARCL1 and β-actin cDNA without gDNA contamination. SPARCL1 mRNA is expressed at similar levels by pure glia and by neurons cocultured with glia, indicating that most cortical SPARCL1 is expressed by glia. SPARCL1 band intensities are normalized to β-actin band intensities within multiplexed RT-PCRs. C, Endogenous SPARCL1 protein is secreted at low levels in primary cortical cultures. Cortical glia were cultured alone (G) or with cortical neurons (N + G) for DIV14. Left, Coomassie stain showing the total protein composition of conditioned media and cell lysates taken from both types of cultures and from mouse serum. Right, Immunoblot of SPARCL1 (green) from conditioned media and cell lysates. Full-length SPARCL1 is secreted and present intracellularly (green arrow). Secreted SPARCL1 is proteolyzed into an array of fragments (light green arrows). Truncated SPARCL1 is present in serum (dark green arrow) but is undetectable in primary cortical cultures. D, Comparison of recombinant and endogenous SPARCL1 protein levels. Equal volumes of HEK293T cell supernatant containing native recombinant SPARCL1 and of conditioned medium harvested from primary cortical cultures before SPARCL1 treatment (DIV13) and 24 h after SPARCL1 treatment were analyzed by SDS-PAGE and immunoblotting. Left, Coomassie stain showing the total protein composition of the HEK cell supernatant and of the conditioned medium before (untreated) and after (treated) SPARCL1 treatment. Asterisks indicate bands corresponding in size to SPARCL1. Right, Immunoblot of recombinant SPARCL1 protein secreted by HEK cells and of SPARCL1 present in the conditioned medium before and after treatment. Dark gray arrows indicate full-length SPARCL1. Light gray arrows indicate proteolyzed SPARCL1. Bar graphs indicate mean ± SEM. Three independent cultures were analyzed. Statistical significance was evaluated by a Student's t test. ***p < 0.001; nonsignificant relations (n.s.) are indicated. For complete statistical analyses, see Extended Data Figure 1-1.

Figure 2.
Figure 2.

Recombinant SPARCL1 selectively boosts excitatory but not inhibitory synapse numbers in primary cultures of cortical neurons and glia. A, Experimental strategy. Cortical neurons and glia were cultured from P0 WT, Nrxn123, or Nlgn1234 cKO mice. At DIV3, neurons were infected with lentiviruses expressing nuclear Cre-recombinase fused to EGFP and driven by the synapsin promoter (Syn-Cre-EGFP), or nonfunctional mutant Cre (Syn-ΔCre-EGFP) as a negative control. At DIV10, neurons were transfected with β-actin-eBFP for morphologic analyses. Neurons were treated with recombinant SPARCL1 at DIV13 and analyzed by electrophysiology, immunocytochemistry, and immunoblotting at DIV14-DIV16. In the experiments described in the present figure analyzing WT neurons, cultures expressing ΔCre were examined. In all subsequent figures describing experiments on conditional neurexin and neuroligin mutants, cultures expressing ΔCre and Cre were investigated. B, Representative images showing that recombinant SPARCL1 treatment increases the excitatory synapse density in WT neurons. Images represent dendritic segments of WT neurons treated at DIV13 with the supernatants of HEK cells expressing either mClover (Control) or recombinant SPARCL1. Neurons were immunostained for vGluT1 (excitatory presynaptic marker), Homer (excitatory postsynaptic marker), and MAP2 (dendritic marker) at DIV14. C, Quantifications showing that recombinant SPARCL1 increases the density (left), but not the size of excitatory synapses (right). D, Representative images showing that recombinant SPARCL1 treatment does not alter inhibitory synapse in WT neurons. Images represent dendritic segments of WT neurons treated as described in B, and immunostained for vGAT (inhibitory presynaptic marker), gephyrin (inhibitory postsynaptic marker), and MAP2 (dendritic marker) at DIV14. E, Quantifications showing that recombinant SPARCL1 does not alter the density (left) or size (right) of inhibitory synapses. F, Representative images showing that recombinant SPARCL1 treatment does not alter the survival or dendritic morphology of WT neurons. Images represent neurons treated as described in B, but additionally sparsely transfected with eBFP to visualize dendritic arborizations of neurons. Neurons were counterstained with MAP2. G–I, Quantifications showing that recombinant SPARCL1 does not alter the neuronal cell density (G), number of primary dendrites, and dendritic branches (H), or soma size of neurons (I). J, Electrophysiological measurements of the capacitance (left) and input resistance (right) of WT neurons treated with SPARCL1 as described in B show that SPARCL1 has no significant effect on these passive electrical membrane properties. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. Statistical significance was evaluated by a Student's t test. **p < 0.01; nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 2-1.

Figure 3.
Figure 3.

Cre-recombinase expression substantially reduces neurexin and neuroligin protein expression in cKO neurons. A, Lentiviral infection of primary cortical neurons. Representative low-magnification images show DIV14 neurons expressing ΔCre-EGFP or Cre-EGFP. Neurons were counterstained for MAP2 to visualize neuronal dendrites, respectively. All neuronal nuclei were positive for ΔCre-EGFP or Cre-EGFP expression, yielding an infection efficiency of 100%. B, Analysis of neurexin protein expression in primary cortical neurons from Nrxn123 cKO mice. Immunoblots were performed on lysates from cortical cultures infected with lentiviral ΔCre or Cre. Left, Representative blots. Right, Summary graph of neurexin protein levels. The pan-neurexin antibody detects full-length (black arrow) and truncated neurexin. A nonspecific band is also observed (red arrow). The neurexin signals were normalized to corresponding β-actin signals for quantification. C, Analysis of neuroligin protein expression in primary cortical neurons from Nlgn1234 cKO mice. Immunoblots were performed on lysates from cortical cultures infected with lentiviral ΔCre or Cre. Left, Representative blots of Nlgn1, Nlgn2, and Nlgn3. Right, Summary graph of neurexin protein levels. The Nlgn3 antibody detects Nlgn3 (black arrow) and two nonspecific bands (red arrows). The neuroligin signals were normalized to corresponding β-actin signals for quantification. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. Statistical significance was evaluated by a Student's t test. ***p < 0.001; nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 3-1.

Figure 4.
Figure 4.

Deletion of neurexins in dissociated cultures of mouse neurons and glia neither decreases excitatory or inhibitory synapse numbers nor impairs the increase in excitatory synapse numbers produced by recombinant SPARCL1. A, Deletion of all neurexins does not significantly decrease synapse numbers and does not block excitatory synaptogenesis induced by recombinant SPARCL1. Representative images show Nrxn123, control (ΔCre), and cKO (Cre) neurons treated at DIV13 with the supernatants of HEK cells expressing either mClover (Control medium) or recombinant SPARCL1. Neurons were immunostained for VGLUT1 (excitatory puncta), VGAT (inhibitory puncta), and MAP2 at DIV14. Higher-magnification images (right) were taken from the boxed areas shown in the corresponding lower-magnification images (left). B, C, Quantifications showing that recombinant SPARCL1 increases the density of excitatory synapses (top summary graphs) but not inhibitory synapses (bottom summary graphs) in control and Nrxn123 cKO neurons. SPARCL1 did not affect the size and staining intensity of excitatory and inhibitory synapses. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. *p < 0.05 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant (n.s.) relations are indicated. For complete statistical analyses, see Extended Data Figure 4-1.

Figure 5.
Figure 5.

Recombinant SPARCL1 increases spontaneous excitatory but not inhibitory synaptic transmission independent of neurexins. A, SPARCL1 increases mEPSC frequency but not amplitude independent of neurexins. Nrxn123, control (ΔCre), and cKO (Cre) neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous excitatory synaptic activity was assessed at DIV14 by mEPSC recordings in the presence of TTX and PTX. Top, Representative traces. Bottom, Summary graphs of the mEPSC frequency and mEPSC amplitude. *p < 0.05 (one-way ANOVA with Tukey's post hoc comparisons). B, SPARCL1 does not affect spontaneous inhibitory synaptic activity. Nrxn123 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous inhibitory synaptic activity was assessed at DIV14 by mIPSC recordings in the presence of TTX, CNQX, and D-AP5. Top, Representative traces. Bottom, Summary graphs of the mIPSC frequency and mIPSC amplitude. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown beside the bars. ***p < 0.001 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 5-1.

Figure 6.
Figure 6.

Deletion of neuroligins does not significantly decrease synapse numbers and does not block excitatory synaptogenesis induced by recombinant SPARCL1. A, Deletion of all neuroligins does not significantly decrease synapse numbers and does not block excitatory synaptogenesis induced by recombinant SPARCL1. Representative images show Nlgn1234, control (ΔCre), and cKO (Cre) neurons treated at DIV13 with the supernatants of HEK cells expressing either mClover (Control medium) or recombinant SPARCL1. Neurons were immunostained for VGLUT1 (excitatory puncta), VGAT (inhibitory puncta), and MAP2 at DIV14. Higher-magnification images (right) were taken from the boxed areas shown in the corresponding lower-magnification images (left). B, C, Quantifications showing that recombinant SPARCL1 increases the density of excitatory synapses (top summary graphs) but not inhibitory synapses (bottom summary graphs) in control and Nlgn1234 cKO neurons. SPARCL1 did not substantially affect the size and staining intensity of excitatory and inhibitory synapses. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within bars. *p < 0.05; **p < 0.01 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 6-1.

Figure 7.
Figure 7.

Recombinant SPARCL1 increases spontaneous excitatory but not inhibitory synaptic transmission independent of neuroligins. A, Deletion of all neuroligins significantly reduces mEPSC frequency and amplitude but does not prevent SPARCL1 from increasing mEPSC frequency. Nlgn1234, control (ΔCre), and cKO (Cre) neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous excitatory synaptic activity was assessed at DIV14 by mEPSC recordings in the presence of TTX and PTX. Top, Representative traces. Bottom, Summary graphs of the mEPSC frequency and mEPSC amplitude. B, Deletion of all neuroligins significantly reduces mIPSC frequency and amplitude, which are unaltered by SPARCL1. Nlgn1234 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Spontaneous inhibitory synaptic activity was assessed at DIV14 by mIPSC recordings in the presence of TTX, CNQX, and D-AP5. Top, Representative traces. Bottom, Summary graphs of the mIPSC frequency and mIPSC amplitude. Bar graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown beside the bars. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey's post hoc comparisons). Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 7-1.

Figure 8.
Figure 8.

SPARCL1 selectively enhances excitatory evoked neurotransmission and increases NMDAR-mediated synaptic responses independent of neuroligins. A, Deletion of all neuroligins reduces the amplitudes of evoked AMPAR-EPSCs and NMDAR-EPSCs. Treatment with recombinant SPARCL1 increases these parameters and particularly enhances the NMDAR/AMPAR ratio independent of neuroligins. Nlgn1234 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Neurons were analyzed at DIV14-DIV16 by recording EPSCs evoked by extracellular stimulation. AMPAR-EPSCs and NMDAR-EPSCs were pharmacologically isolated with PTX and monitored at −70 mV and 40 mV holding potentials, respectively. Representative traces of evoked EPSCs are shown for all conditions. B, Summary graphs of evoked AMPAR-EPSC amplitude, NMDAR-EPSC amplitude, and NMDAR/AMPAR ratio. NMDAR current amplitudes were measured 50 ms after stimulation. C, Deletion of all neuroligins reduces the amplitudes of evoked GABAR-IPSCs, whereas addition of SPARCL1 does not alter these currents. Nlgn1234 control and cKO neurons were treated at DIV13 with control medium or recombinant SPARCL1. Neurons were analyzed at DIV14-DIV16 by recording IPSCs evoked by extracellular stimulation. GABAR-IPSCs were pharmacologically isolated with D-AP5 and CNQX and monitored at −70 mV. Representative traces of evoked IPSCs are shown for all conditions. D, Summary graph of evoked GABAR-IPSC amplitude. Bar and line graphs indicate mean ± SEM. Numbers of cells/independent cultures analyzed are shown within the bars. *p < 0.05; ***p < 0.001; one-way ANOVAs and Tukey's post hoc comparisons. Nonsignificant relations are not indicated. For complete statistical analyses, see Extended Data Figure 8-1.

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