Maternal VPA-treated primary neurons secreted the excitatory presynapse organizer.
Maternal treatment with valproic acid (VPA) has been shown to result in the development of autism spectrum disorder (ASD)-like phenotypes, such as impaired social communication and repetitive behaviors, in mouse models. In these animals, alterations in the excitatory-inhibitory (E/I) ratio have been observed in electrophysiological analyses. However, the effect of maternal VPA administration on synaptogenesis in neurons is not well understood. To investigate this, we examined the protein expression levels of synapse-related molecules in primary cultured neurons derived from VPA-treated pregnant mice at 6 days in vitro (DIV6) (Fig. 1A). We found that prenatal VPA administration resulted in a marked increase in the expression of the excitatory presynaptic marker molecule vesicular glutamate transporter 1 (vGlut1), while the expression of the inhibitory presynaptic marker vesicular GABA transporter (vGat) was unchanged (Fig. 1B and 1C). Immunocytochemical analyses using anti-vGlut1 and vGat antibodies were used to quantify the formation of excitatory and inhibitory synapses, respectively. Consistent with the biochemical analysis, the area of vGlut1-positive puncta was significantly increased, whereas the area of vGat-positive puncta remained unchanged (Figs. 1D and 1E).
To identify the molecule(s) responsible for the VPA-induced increase in excitatory presynapses, we tested the synaptogenic activity of conditioned medium from primary cultured neurons of VPA-treated maternal fetuses (CM-VPA) in primary cultured neurons separately seeded from wild-type mice (Fig. 1F). CM-VPA treatment increased the expression of vGlut1 but did not alter the expression of vGat (Fig. 1G and 1H). Furthermore, the addition of CM-VPA increased the area of vGlut1-positive puncta (Fig. 1I and 1J). Notably, this increase in excitatory synaptic puncta was abolished by boiling CM-VPA, while the percentage of vGat-positive puncta remained unchanged. These results suggest that CM-VPA contains presynaptic organizer protein(s) that enhance the formation of excitatory synapses in primary neurons.
Lingo2 expression level was increased in the VPA maternal administration model.
To identify the VPA-induced synaptic organizer protein, we performed a quantitative secretome analysis using the Secretome Protein Enrichment with Click Sugars (SPECS) method, which overcomes several limitations of secretome analysis, such as the low concentration of secreted proteins and contamination by serum and cytoplasmic proteins in the culture medium [43]. After the metabolic labeling of glycans with azide-labeled sugar in living cells, newly glycosylated proteins were selectively biotinylated by a 2 + 3 cycloaddition reaction. Subsequently, biotin-labeled secreted proteins were specifically detected by the avidin-biotin reaction on the beads. Using the SPECS method, several proteins were identified as substrate proteins for shedding proteases, such as BACE1 and ADAM10 [43, 44]. We used the SPECS method to comprehensively analyze the secretory proteins in CM-VPA. After comparison with the secretome of primary neurons derived from wild-type mice, we found that the levels of three candidate proteins were altered (Fig. 2A and supplementary table). Of these, leucine-rich repeat and immunoglobulin domain-containing protein 2 (Lingo2) was of particular interest. LINGO2 is a gene identified in the human chromosome 9p21.1 region as an ortholog of LINGO1 [46]. The role of LINGO2 in central nervous system functions is not well understood. In addition, copy number variations (CNVs) in the LINGO2 gene have been reported in ASD patients, and genetic analysis using single RNA sequencing data suggested that LINGO2 expression correlates with the clinical severity of ASD [47–49]. We also confirmed increased protein expression of Lingo2 in the cell lysates of primary neurons derived from fetuses exposed to maternal VPA administration (Fig. 2B and 2C), although the mRNA levels of Lingo2 were unaltered (Fig. 2D). These data suggest that the level of Lingo2 is regulated by protein metabolism in VPA-induced model neurons.
Lingo2 was cleaved by ADAM10 to secrete a soluble form of Lingo2.
We identified peptides derived from Lingo2 in CM-VPA, although Lingo2 is a type 1 transmembrane protein. Therefore, we hypothesized that it undergoes proteolytic processing to release the extracellular region into the conditioned medium. This metabolism is observed in another type 1 transmembrane proteins, such as APP and neuroligin [50, 51], which are cleaved at the juxtamembrane region by A disintegrin and metalloproteinase domain-containing proteins (ADAMs), releasing their extracellular domains. To study the protein metabolism of Lingo2, we overexpressed N-terminally HA-tagged Lingo2 in HEK 293A cells. The results showed that full-length HA-Lingo2 was detected as 95 and 110 kDa doublet bands in the cell lysates and a soluble form of HA-Lingo2 (sLingo2) of 80 kDa was found in the conditioned medium (Fig. 4A). The production of sLingo2 was reduced by the administration of GM6001, a broad-spectrum metalloproteinase inhibitor, suggesting that Lingo2 is cleaved by metalloproteinases to secrete sLingo2 (Fig. 3A). We also overexpressed HA-Lingo2 in primary mouse neurons and evaluated the effects of several metalloproteinase inhibitors; a broad-spectrum metalloproteinase inhibitor GM6001, the ADAM10 and ADAM17 inhibitor INCB3619 [51], and the ADAM10 inhibitor GI254023X [52]. sLingo2 production was abolished by all inhibitors (Fig. 3B and 3C), suggesting that Lingo2 is cleaved by ADAM10. To determine which ADAM is responsible for the cleavage of Lingo2, we generated shRNA lentiviral vectors targeting Adam9, Adam10, and Adam17 and confirmed the reduction of the mRNA level of each ADAM protease. The amount of sLingo2 was abolished by Adam10 knockdown, but not by Adam9 or Adam17 (Fig. 3D and 3E). Consistent with this, systematic proteomic analysis of Adam10-deficient neurons using SPECS also revealed that Lingo2 is one of the ADAM10 substrates [44]. Thus, these results suggest that ADAM10 cleaves Lingo2 in primary neurons (Fig. 3F).
ADAM10 cleaves its substrates at the juxtamembrane region [53]. Based on the molecular weight of sLingo2 and the enzymatic character of ADAM10, we hypothesized that Lingo2 is cleaved at the stalk region (i.e., 501–545 aa) located between the immunoglobulin-like domain and the transmembrane domain. To narrow down the cleavage site, we systematically examined the proteolytic processing of HA-Lingo2 with deletion mutations at the stalk region (Fig. 3G) in HEK293A cells. HA-Lingo2 production was significantly reduced in the Δ501–511 and Δ512–522 mutants (Fig. 3H). Expression of the HA-Lingo2 was reduced by the deletion near the transmembrane domain (i.e., Δ534–545). We further generated microdeletion mutants at 501–511 and found that the Δ508–511 mutant failed to release HA-sLingo2 (Fig. 3I). Taken together, 508–522 aa at the stalk region of Lingo2 is critical for sLingo2 generation.
The soluble form of Lingo2 functions as the excitatory presynapses organizer in mouse primary neurons.
To investigate the ability of sLingo2 to induce excitatory synaptogenesis, we tested the synaptogenic activity of recombinant HA-sLingo2-V5His purified from the conditioned medium of HA-sLingo2-V5His overexpressing HEK 293A cells (Fig. 4A). Treatment of primary mouse neurons with 10 nM HA-sLingo2-V5His resulted in a significant increase in the expression level of the excitatory presynaptic marker vGlut1 and no change in the expression level of the inhibitory presynaptic marker vGat (Fig. 4B and 4C). This effect was also confirmed by immunocytochemical analysis, which showed an increase in the area of vGlut1-positive presynaptic puncta and no change in the area of vGat-positive presynaptic puncta (Fig. 4D and 4E). These results indicate that sLingo2 selectively induces the formation of excitatory presynapses in mouse primary neurons.
Because the expression level of endogenous Lingo2 was increased in primary neurons derived from fetuses treated with VPA during pregnancy, we hypothesized that the knockdown of Lingo2 would correct the abnormal excitatory synaptogenesis in the VPA model. To investigate this, we performed Lingo2 knockdown using two Lingo2-targeting shRNAs. Immunoblot analysis showed that maternal administration of VPA significantly increased the expression of vGlut1, and Lingo2 knockdown attenuated this increase (Fig. 5A and 5B). The expression of vGat, however, remained unchanged. To further confirm these results, we analyzed the number of vGlut1-positive puncta, a measure of excitatory presynaptic formation, by immunocytochemistry on Lingo2 knockdown primary neurons expressing GFP. We found that the increased number of vGlut1-positive puncta induced by maternal administration of VPA was attenuated by Lingo2 knockdown (Figs. 5C and 5D). These results suggest that reducing the expression of Lingo2 can normalize excitatory presynaptic formation in the VPA model.
The soluble form of Lingo2 increased excitatory synapses in human iPSC-derived neurons.
To explore the role of sLingo2 in human excitatory neurons, we examined its effects on human iPSC-derived neurons. We used a system that can induce differentiation into excitatory neurons in a short time (2 weeks) by overexpressing Neurogenin-2 [54]. The differentiated excitatory neurons expressed neuronal markers, including β3-tubulin (β3-tub), and excitatory postsynaptic marker proteins, including Homer1. After the iPSCs were fully differentiated into excitatory neurons, we administered purified sLingo2 (Fig. 6A) and confirmed a significant increase in the expression of Homer1, an excitatory postsynaptic marker (Fig. 6B and 6C), similar to that observed in primary mouse neurons. To quantify excitatory synaptogenesis, we stained for puncta co-staining with synapsin-1, a presynaptic marker, and Homer1, a postsynaptic marker, and counted the density of excitatory synapses on the dendritic marker Map2. Our results showed that sLingo2 treatment resulted in a significant increase in excitatory synaptic density (Fig. 6C), indicating that sLingo2 is capable of inducing excitatory synapses in human iPSC-derived excitatory neurons. To determine whether the increase in excitatory synapses induced by recombinant sLingo2 affected synaptic function, we measured miniature excitatory postsynaptic currents (mEPSCs) using the whole-cell patch-clamp method (Fig. 6D). Our results showed that recombinant sLingo2 administration significantly increased the frequency of mEPSCs without affecting the amplitude (Figs. 6E and 6F). Taken together, our data indicate that sLingo2 functions as an excitatory synapse organizer in human neurons.