The compound heterozygous missense mutations (c.1972C>T:p.R658W and c.2024C>A:p.T675K) was also identified in another sibling with similar clinical features (Charng et al., 2016). We find that this pathogenic GRM7 I154T and R658W/T675K mutations lead to the degradation of the mGlu7 protein. In particular, the GRM7 R658W/T675K mutation results in a lack of surface mGlu7 expression in heterologous cells and cultured neurons isolated from male and female rat embryos. We demonstrate that this expression of mGlu7 variants or exposure to mGlu7 antagonists impairs axon outgrowth through the mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling pathway during early neuronal development, which subsequently leads to a decrease in the number of presynaptic terminals in mature neurons. Treatment with an mGlu7 agonist restores the pathologic phenotypes caused by mGlu7 I154T but not by mGlu7 R658W/T675K because of its lack of neuronal surface expression. These findings provide evidence that stable neuronal surface expression of mGlu7 is essential for neural development and that mGlu7 is usually a promising therapeutic target for NDDs. SIGNIFICANCE STATEMENT Neurodevelopmental disorders (NDDs) affect brain development and function by multiple etiologies. Metabotropic glutamate receptor 7 (mGlu7) is usually a receptor that controls excitatory neurotransmission and synaptic plasticity. Since accumulating evidence indicates that this gene locus is usually associated with NDD risk, we analyzed the functional effects of human variants identified in patients with NDDs. We demonstrate that stable neuronal surface expression of mGlu7 is essential for axon outgrowth and presynaptic terminal development in neurons. We found that mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) signaling and subsequent cytoskeletal dynamics are defective because of the degradation of mGlu7 variants. Finally, we show that the defects caused by mGlu7 I154T can be reversed by agonists, providing the rationale for proposing mGlu7 as a potential therapeutic target for NDDs. as a potential NDD risk locus (Elia et al., 2011; Gai et al., 2012; Park et al., 2013; Yang and Pan, 2013; Liu et al., 2015; Noroozi et al., 2016). These studies have revealed inherited or point mutations or deletions in introns and/or exons in cohorts of ASD or ADHD patients. In addition, a missense mutation (c.1865G>A:p.R622Q) was reported to be associated with ASD on the basis of large-scale whole-exome sequencing (WES) studies in families with ASD (Sanders et al., 2012; Iossifov et al., 2014). A recent WES study on consanguineous families identified as the candidate gene for the highest risk of NDDs, including DD/ID and brain malformations (Charng et al., 2016). This study identified a homozygous missense mutation (c.461T>C:p.I154T) from two affected siblings with DDs/IDs, seizures, hypotonia, and brain atrophy. The compound heterozygous missense mutations (c.1972C>T:p.R658W and c.2024C>A:p.T675K) was also identified in another sibling with similar clinical features (Charng et al., 2016). D-Luciferin In a different set of study, a homozygous nonsense mutation (c.1757G>A:p.W586X, in which X designates a translation termination codon) was identified in families with NDDs (Reuter et al., 2017). In this study, we investigated the mechanism by which human variants carrying mutations in protein-coding sequences lead to the pathologic phenotypes observed in NDD patients. Specifically, we characterized the function of the variants identified from the existing WES literature for NDD patients (Table 1; Sanders et al., 2012; Iossifov et al., 2014; Charng et al., 2016; Reuter et al., 2017). When we expressed human variants in heterologous cells and rat primary cultured neurons, we found a profound reduction in D-Luciferin the protein expression of mGlu7 variants. The instability of mGlu7 variant proteins is usually caused by protein degradation through the proteasomal or autophagosomal-lysosomal degradation pathway. We show that the variants cause a severe impairment in axon outgrowth during Mouse monoclonal antibody to TCF11/NRF1. This gene encodes a protein that homodimerizes and functions as a transcription factor whichactivates the expression of some key metabolic genes regulating cellular growth and nucleargenes required for respiration,heme biosynthesis,and mitochondrial DNA transcription andreplication.The protein has also been associated with the regulation of neuriteoutgrowth.Alternate transcriptional splice variants,which encode the same protein, have beencharacterized.Additional variants encoding different protein isoforms have been described butthey have not been fully characterized.Confusion has occurred in bibliographic databases due tothe shared symbol of NRF1 for this gene and for “”nuclear factor(erythroid-derived 2)-like 1″”which has an official symbol of NFE2L1.[provided by RefSeq, Jul 2008]” early neuronal development, which subsequently leads to a deficit in the number of presynaptic terminals in mature neurons. We discovered that the mitogen-activated protein kinase (MAPK)-cAMP-protein kinase A (PKA) pathway is usually perturbed by the variants. Of particular importance, we found that the deficits in axon outgrowth and presynaptic terminal development induced by mGlu7 I154T were restored by treatment with an mGlu7 agonist during early development. Thus, our study provides mechanistic insight into the development of NDDs by the variants and suggests mGlu7 as a potential therapeutic target for D-Luciferin NDD treatment. Table 1. Pathogenic human variants and clinical features cDNA plasmid from rat WT cDNA (GenBank accession No. “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_000844″,”term_id”:”1519242138″,”term_text”:”NM_000844″NM_000844 and “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_031040″,”term_id”:”13591962″,”term_text”:”NM_031040″NM_031040) by substituting four amino acid mismatches in the coding sequence using site-directed mutagenesis. The following oligonucleotide primers were used: H454N-forward (F), 5-gtatatccgcAatgttaacttcaatgg-3 and H454N-reverse (R), 5-ccattgaagttaacatTgcggatatac-3; T488S-F, 5-caacaaacacaaGcaaccctggtta-3 and T488S-R, 5-taaccagggttgCttgtgtttgttg-3; S520A-F, 5-gagagatcccaGcctctgtgtgtac-3 and S520A-R, 5-gtacacacagaggCtgggatctctc-3; N578D-F, 5-ctggctgtcagGaTatcccaatcatc-3 and N578D-R, 5-gatgattgggatAtCctgacagccag-3 (the mutated nucleotides are indicated by capital letters). Using human WT cDNA as a template, we generated pathogenic mutants by site-directed mutagenesis using the following oligonucleotide primers: I154T-F, 5-tagttggagtgaCtggggcttcggg-3 and I154T-R, D-Luciferin 5-cccgaagccccaGtcactccaacta-3; W586X-F, 5-tcaaactggagtAgcactccccctg-3 and W586X-R, 5-cagggggagtgcTactccagtttga-3; R622Q-F, 5-cacccattgtcCAggcatctgggcg-3 and R622Q-R, 5-cgcccagatgccTGgacaatgggtg-3; R658W-F, 5-tgtgttctttcTgGcgtgtcttctt-3 and R658W-R, 5-aagaagacacgCcAgaaagaacaca-3; T675K-F, 5-ctgcccttttaaAGaagaccaatcg-3 and T675K-R, 5-cgattggtcttCTttaaaagggcag-3. The PCRs were performed using Phusion DNA polymerase (catalog #M0530, New England Biolabs) according to.