Neurexins and neuroligins are emerging as central organizing molecules for excitatory glutamatergic and inhibitory GABAergic synapses in mammalian brain. They function as cell adhesion molecules, bridging the synaptic cleft. Remarkably, each partner can trigger formation of a hemisynapse: neuroligins trigger presynaptic differentiation and neurexins trigger postsynaptic differentiation. Recent protein interaction assays and cell culture studies indicate a selectivity of function conferred by alternative splicing in both partners. An insert at site 4 of β-neurexins selectively promotes GABAergic synaptic function, whereas an insert at site B of neuroligin 1 selectively promotes glutamatergic synaptic function. Initial knockdown and knockout studies indicate that neurexins and neuroligins have an essential role in synaptic transmission, particularly at GABAergic synapses, but further studies are needed to assess the in vivo functions of these complex protein families.

Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) were recently found to instruct presynaptic and mediate postsynaptic glutamatergic differentiation. In a candidate screen, here we identify neurexin-1␤ lacking an insert at splice site 4 (ϪS4) as a ligand for LRRTM2. Neurexins bind LRRTM2 with a similar affinity but distinct code from the code for binding neuroligin-1 (the predominant form of neuroligin-1 at glutamate synapses, containing the B splice site insert). Whereas neuroligin-1 binds to neurexins 1, 2, and 3 ␤ but not ␣ variants, regardless of insert at splice site 4, LRRTM2 binds to neurexins 1, 2, and 3 ␣ and ␤ variants specifically lacking an insert at splice site 4. We further show that this binding code is conserved in LRRTM1, the family member linked to schizophrenia and handedness, and that the code is functional in a coculture hemisynapse formation assay. Mutagenesis of LRRTM2 to prevent binding to neurexins abolishes presynaptic inducing activity of LRRTM2. Remarkably, mutagenesis of neurexins shows that the binding face on neurexin-1␤ (ϪS4) is highly overlapping for the structurally distinct LRRTM2 and neuroligin-1 partners. Finally, we explore here the interplay of neuroligin-1 and LRRTM2 in synapse regulation. In neuron cultures, LRRTM2 is more potent than neuroligin-1 in promoting synaptic differentiation, and, most importantly, these two families of neurexin-binding partners cooperate in an additive or synergistic manner. Thus, we propose a synaptic code hypothesis suggesting that neurexins are master regulators of the cooperative activities of LRRTMs and neuroligins.

Fat mass and obesity-associated gene (FTO) is a member of the Fe (II)- and oxoglutarate-dependent AlkB dioxygenase family and is linked to both obesity and intellectual disability. The role of FTO in neurodevelopment and neurogenesis, however, remains largely unknown. Here we show that FTO is expressed in adult neural stem cells and neurons and displays dynamic expression during postnatal neurodevelopment. The loss of FTO leads to decreased brain size and body weight. We find that FTO deficiency could reduce the proliferation and neuronal differentiation of adult neural stem cells in vivo, which leads to impaired learning and memory. Given the role of FTO as a demethylase of N6-methyladenosine (m6A), we went on to perform genome-wide m6A profiling and observed dynamic m6A modification during postnatal neurodevelopment. The loss of FTO led to the altered expression of several key components of the brain derived neurotrophic factor pathway that were marked by m6A. These results together suggest FTO plays important roles in neurogenesis, as well as in learning and memory.

Recent findings suggest that the neurexin-neuroligin link promotes both GABAergic and glutamatergic synaptogenesis, but the mechanism by which neurexins influence the clustering of appropriate neuroligins and postsynaptic differentiation remains unclear. Previous studies suggested that the presence or absence of alternatively spliced residues at splice site 4 (S4) in the neurexin LNS domain may regulate neurexin function. We demonstrate that addition of the S4 insert selectively reduces the ability of neurexin-1␤ to cluster neuroligin-1/3/4 and glutamatergic postsynaptic proteins, although clustering of neuroligin-2 and GABAergic postsynaptic proteins remain strong. Furthermore, addition of the S4 insert decreases the binding affinity of neurexin-1␤ to neuroligins-1 and -4 but has little effect on binding to neuroligins-2 and -3. Additional structure-function studies reveal the neurexin binding interface mediating synaptogenic activity to be composed primarily of residues in the ␤2␤3, ␤6␤7, and ␤10␤11 loops on one rim of the LNS domain ␤ sandwich. Mutation of two predicted Ca 2ϩ -binding residues disrupts postsynaptic protein clustering and binding to neuroligins, consistent with previous findings that neurexin-neuroligin binding is Ca 2ϩ dependent. Glutamatergic postsynaptic clustering was more readily disrupted by the mutagenesis than GABAergic postsynaptic protein clustering. Perhaps neurexins-neuroligins, or neurexin-1␤ at least, is most important for GABA synapse formation or controlling the balance of GABA and glutamate synapses. These results suggest that differential neurexin-neuroligin binding affinities and splice variations may play an instructive role in postsynaptic differentiation.

Background N6-methyladenosine (m6A) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain. Results We show that m6A modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, m6A shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more m6A sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more m6A sites compared to mice. Differential m6A sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These m6A sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased m6A methylation in a mouse model of Alzheimer’s disease, which is correlated with reduced protein levels. Conclusions Our results suggest that m6A exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, m6A controls protein levels of key genes involved in Alzheimer’s disease-associated pathways, suggesting that m6A plays an important role in aging and neurodegenerative disease.

N6-methyladenosine (m6A) is the most prevalent internal modification of mammalian messenger RNAs (mRNAs) and long non-coding RNAs. The biological functions of this reversible RNA modification can be interpreted by cytoplasmic and nuclear 'm6A reader' proteins to fine-tune gene expression, such as mRNA degradation and translation initiation. Here we profiled transcriptome-wide m6A sites in adult mouse cerebral cortex, underscoring that m6A is a widespread epitranscriptomic modification in brain. Interestingly, the mRNA targets of fragile X mental retardation protein (FMRP), a selective RNA-binding protein, are enriched for m6A marks. Loss of functional FMRP leads to Fragile X syndrome (FXS), the most common inherited form of intellectual disability. Transcriptome-wide gene expression profiling identified 2035 genes differentially expressed in the absence of FMRP in cortex, and 92.5% of 174 downregulated FMRP targets are marked by m6A. Biochemical analyses indicate that FMRP binds to the m6A sites of its mRNA targets and interacts with m6A reader YTHDF2 in an RNA-independent manner. FMRP maintains the stability of its mRNA targets while YTHDF2 promotes the degradation of these mRNAs. These data together suggest that FMRP regulates the stability of its m6A-marked mRNA targets through YTHDF2, which could potentially contribute to the molecular pathogenesis of FXS.

Genetic analyses have linked MicroRNA-137 ( MIR137 ) to neuropsychiatric disorders, including schizophrenia and autism spectrum disorder (ASD). MiR-137 plays important roles in neurogenesis and neuronal maturation, but the impact of miR-137 loss of function in vivo remains unclear. Here we show the complete loss of miR-137 in the mouse germline (gKO) or nervous system (cKO) leads to postnatal lethality, while heterozygous gKO and cKO mice remain viable. Partial loss of miR-137 in heterozygous cKO mice results in dysregulated synaptic plasticity, repetitive behavior, and impaired learning and social behavior. Transcriptomic and proteomic analyses revealed that the miR-137 mRNA target, Phosphodiesterase 10a (Pde10a), is elevated in heterozygous KO mice. Treatment with the PDE10A inhibitor papaverine or knockdown of Pde10a ameliorates the deficits observed in the heterozygous cKO mice. Collectively, our results suggest that MIR137 plays essential roles in postnatal neurodevelopment, and that dysregulation of miR-137 potentially contributes to neuropsychiatric disorders in humans.

␤-Neurexin and neuroligin cell adhesion molecules contribute to synapse development in the brain. The longer ␣-neurexins function at both glutamate and ␥-aminobutyric acid (GABA) synapses in coupling to presynaptic calcium channels. Binding of ␣-neurexins to neuroligins was recently reported, but the role of the ␣-neurexins in synapse development has not been well studied. Here we report that in COS cell neuron coculture assays, all three ␣-neurexins induce clustering of the GABAergic postsynaptic scaffolding protein gephyrin and neuroligin 2 but not of the glutamatergic postsynaptic scaffolding protein PSD-95 or neuroligin 1/3/4. ␣-Neurexins also induce clustering of the GABA A receptor ␥2 subunit. This synapse promoting activity of ␣-neurexins is mediated by the sixth LNS (laminin neurexin sex hormone-binding protein) domain and negatively modulated by upstream sequences. Although inserts at splice site 4 (S4) in ␤-neurexins promote greater clustering activity for GABA than glutamate proteins in coculture assay, ␣-neurexinspecific sequences confer complete specificity for GABA proteins. We further report a developmental increase in the ratio of ؊S4 to ؉S4 forms of neurexins correlating with an increase in glutamate relative to GABA synaptogenesis and activity regulation of splicing at S4. Thus, ؉S4 ␤-neurexins and, even more selectively, ␣-neurexins may be mediators of GABAergic synaptic protein recruitment and stabilization.