Induction of synapse formation by de novo neurotransmitter synthesis.

A vital question in neuroscience is how neurons align their postsynaptic structures with presynaptic release sites. Although synaptic adhesion proteins are known to contribute in this process, the role of neurotransmitters remains unclear. Here we inquire whether de novo biosynthesis and vesicular release of a noncanonical transmitter can facilitate the assembly of its corresponding postsynapses. We demonstrate that, in both stem cell-derived human neurons as well as in vivo mouse neurons of purely glutamatergic identity, ectopic expression of GABA-synthesis enzymes and vesicular transporters is sufficient to both produce GABA from ambient glutamate and transmit it from presynaptic terminals. This enables efficient accumulation and consistent activation of postsynaptic GABAA receptors, and generates fully functional GABAergic synapses that operate in parallel but independently of their glutamatergic counterparts. These findings suggest that presynaptic release of a neurotransmitter itself can signal the organization of relevant postsynaptic apparatus, which could be directly modified to reprogram the synapse identity of neurons.

An Autism-Associated Mutation Impairs Neuroligin-4 Glycosylation and Enhances Excitatory Synaptic Transmission in Human Neurons.

Neuroligins (NLGNs) are a class of postsynaptic cell adhesion molecules that interact with presynaptic neurexins (NRXNs) and regulate synapse function. NLGN4 is a member of the NLGN family and consists of a unique amino acid sequence in humans that is not evolutionarily well conserved in rodents. The human-specific NLGN4 gene has been reported to be mutated in many patients with autism and other neurodevelopmental disorders. However, it remained unclear how these mutations might alter the molecular properties of NLGN4 and affect synaptic transmission in human neurons. Here, we describe a severely autistic male patient carrying a single amino acid substitution (R101Q) in the NLGN4 gene. When expressed in HEK293 cells, the R101Q mutation in NLGN4 did not affect its binding affinity for NRXNs or its capacity to form homodimers. This mutation, however, impaired the maturation of NLGN4 protein by inhibiting N-linked glycosylation at an adjacent residue (N102), which is conserved in all NLGNs. As a result, the R101Q substitution significantly decreased the surface trafficking of NLGN4 and increased its retention in the endoplasmic reticulum and Golgi apparatus. In human neurons derived from male stem cell lines, the R101Q mutation also similarly reduced the synaptic localization of NLGN4, resulting in a loss-of-function phenotype. This mutation-induced trafficking defect substantially diminished the ability of NLGN4 to form excitatory synapses and modulate their functional properties. Viewed together, our findings suggest that the R101Q mutation is pathogenic for NLGN4 and can lead to synaptic dysfunction in autism.

Direct Reprogramming of Human Neurons Identifies MARCKSL1 as a Pathogenic Mediator of Valproic Acid-Induced Teratogenicity.

Human pluripotent stem cells can be rapidly converted into functional neurons by ectopic expression of proneural transcription factors. Here we show that directly reprogrammed neurons, despite their rapid maturation kinetics, can model teratogenic mechanisms that specifically affect early neurodevelopment. We delineated distinct phases of in vitro maturation during reprogramming of human neurons and assessed the cellular phenotypes of valproic acid (VPA), a teratogenic drug. VPA exposure caused chronic impairment of dendritic morphology and functional properties of developing neurons, but not those of mature neurons. These pathogenic effects were associated with VPA-mediated inhibition of the histone deacetylase (HDAC) and glycogen synthase kinase-3 (GSK-3) pathways, which caused transcriptional downregulation of many genes, including MARCKSL1, an actin-stabilizing protein essential for dendritic morphogenesis and synapse maturation during early neurodevelopment. Our findings identify a developmentally restricted pathogenic mechanism of VPA and establish the use of reprogrammed neurons as an effective platform for modeling teratogenic pathways.