Many biomaterials have been evaluated using cultured cells. In particular, osteoblast-like cells are often used to evaluate the osteocompatibility, hard-tissue-regeneration, osteoconductive, and osteoinductive characteristics of biomaterials. However, the evaluation of biomaterial osteogenesis-inducing capacity using osteoblast-like cells is not standardized; instead, it is performed under laboratory-specific culture conditions with different culture media. However, the effect of different media conditions on bone formation has not been investigated. Here, we aimed to evaluate the osteogenesis of MC3T3-E1 cells, one of the most commonly used osteoblast-like cell lines for osteogenesis evaluation, and assayed cell proliferation, alkaline phosphatase activity, expression of osteoblast markers, and calcification under varying culture media conditions. Furthermore, the various media conditions were tested in uncoated plates and plates coated with collagen type I and poly-L-lysine, highly biocompatible molecules commonly used as pseudobiomaterials. We found that the type of base medium, the presence or absence of vitamin C, and the freshness of the medium may affect biomaterial regeneration. We posit that an in vitro model that recapitulates in vivo bone formation should be established before evaluating biomaterials.

Genome editing is a powerful technique for studying gene functions. CRISPR/Cas9-mediated gene knock-in has recently been applied to various cells and organisms. Here, we successfully knocked in an EGFP coding sequence at the site immediately after the first ATG codon of the β-actin gene in neurons in the brain by the combined use of the CRISPR/Cas9 system and in utero electroporation technique, resulting in the expression of the EGFP-tagged β-actin protein in cortical layer 2/3 pyramidal neurons. We detected EGFP fluorescence signals in the soma and neurites of EGFP knock-in neurons. These signals were particularly abundant in the head of dendritic spines, corresponding to the localization of the endogenous β-actin protein. EGFP knock-in neurons showed no detectable changes in spine density and basic electrophysiological properties. In contrast, exogenously overexpressed EGFP-β-actin showed increased spine density and EPSC frequency, and changed resting membrane potential. Thus, our technique provides a potential tool to elucidate the localization of various endogenous proteins in neurons by epitope tagging without altering neuronal and synaptic functions. This technique can be also useful for introducing a specific mutation into genes to study the function of proteins and genomic elements in brain neurons.

Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). Here, we report the crystal structure of the Nrxn1β–LRRTM2 complex at 3.4 Å resolution. The Nrxn1β–LRRTM2 interface involves Ca²⁺-mediated interactions and overlaps with the Nrxn–neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn.

Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.

Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain.