Available methods for differentiating human embryonic (ES) and induced pluripotent stem (iPS) cells into neurons are often cumbersome, slow and variable. Alternatively, human fibroblasts can be directly converted into induced neuronal (iN) cells. However, with present techniques conversion is inefficient, synapse formation is limited, and only small amounts of neurons can be generated. Here, we show that human ES and iPS cells can be converted into functional iN cells with nearly 100% yield and purity in less than two weeks by forced expression of a single transcription factor. The resulting ES-iN or iPS-iN cells exhibit quantitatively reproducible properties independent of the cell line of origin, form mature pre- and postsynaptic specializations, and integrate into existing synaptic networks when transplanted into mouse brain. As illustrated by selected examples, our approach enables large-scale studies of human neurons for questions such as analyses of human diseases, examination of human-specific genes, and drug screening.

Heterozygous SHANK3 mutations are associated with idiopathic autism and Phelan-McDermid syndrome. SHANK3 is a ubiquitously expressed scaffolding protein that is enriched in postsynaptic excitatory synapses. Here we used engineered conditional mutations in human neurons to show that heterozygous and homozygous SHANK3 mutations severely and specifically impair Ih-channels. SHANK3 mutations caused alterations in neuronal morphology and synaptic connectivity; chronic pharmacological blockage of Ih-channels reproduced these phenotypes, suggesting they may be secondary to Ih-channel impairment. Moreover, mouse Shank3-deficient neurons also exhibited severe decreases in Ih-currents. SHANK3 protein interacted with hyperpolarization-activated cyclic nucleotide-gated channel proteins (HCN proteins) forming Ih-channels, indicating that SHANK3 functions to organize HCN-channels. Our data suggest SHANK3 mutations predispose to autism, at least partially, by inducing an Ih-channelopathy that may be amenable to pharmacological intervention.

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces.To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices.We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

Fragile X syndrome (FXS) is an X chromosome-linked disease leading to severe intellectual disabilities. FXS is caused by inactivation of the fragile X mental retardation 1 () gene, but how inactivation induces FXS remains unclear. Using human neurons generated from control and FXS patient-derived induced pluripotent stem (iPS) cells or from embryonic stem cells carrying conditional mutations, we show here that loss of function specifically abolished homeostatic synaptic plasticity without affecting basal synaptic transmission. We demonstrated that, in human neurons, homeostatic plasticity induced by synaptic silencing was mediated by retinoic acid, which regulated both excitatory and inhibitory synaptic strength. inactivation impaired homeostatic plasticity by blocking retinoic acid-mediated regulation of synaptic strength. Repairing the genetic mutation in the gene in an FXS patient cell line restored fragile X mental retardation protein (FMRP) expression and fully rescued synaptic retinoic acid signaling. Thus, our study reveals a robust functional impairment caused by mutations that might contribute to neuronal dysfunction in FXS. In addition, our results suggest that FXS patient iPS cell-derived neurons might be useful for studying the mechanisms mediating functional abnormalities in FXS.

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Neuromodulators bind to pre-and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca 2+ levels, and are thought to play important roles in neuropsychiatric and neurodegenerative diseases. Here, we discovered in human neurons an unanticipated presynaptic mechanism that acutely changes synaptic ultrastructure and regulates synaptic communication. Activation of neuromodulator receptors bidirectionally controlled synaptic vesicle numbers within nerve terminals. This control correlated with changes in the levels of cAMP-dependent protein kinase A-mediated phosphorylation of synapsin-1. Using a conditional deletion approach, we reveal that the neuromodulator-induced control of synaptic vesicle numbers was largely dependent on synapsin-1. We propose a mechanism whereby non-phosphorylated synapsin-1 ''latches'' synaptic vesicles to presynaptic clusters at the active zone. cAMP-dependent phosphorylation of synapsin-1 then removes the vesicles. cAMP-independent dephosphorylation of synapsin-1 in turn recruits vesicles. Synapsin-1 thereby bidirectionally regulates synaptic vesicle numbers and modifies presynaptic neurotransmitter release as an effector of neuromodulator signaling in human neurons.

Hundreds of L1CAM gene mutations have been shown to be associated with congenital hydrocephalus, severe intellectual disability, aphasia, and motor symptoms. How such mutations impair neuronal function, however, remains unclear. Here, we generated human embryonic stem (ES) cells carrying a conditional L1CAM loss-of-function mutation and produced precisely matching control and L1CAM-deficient neurons from these ES cells. In analyzing two independent conditionally mutant ES cell clones, we found that deletion of L1CAM dramatically impaired axonal elongation and, to a lesser extent, dendritic arborization. Unexpectedly, we also detected an ∼20–50% and ∼20–30% decrease, respectively, in the levels of ankyrinG and ankyrinB protein, and observed that the size and intensity of ankyrinG staining in the axon initial segment was significantly reduced. Overexpression of wild-type L1CAM, but not of the L1CAM point mutants R1166X and S1224L, rescued the decrease in ankyrin levels. Importantly, we found that the L1CAM mutation selectively decreased activity-dependent Na+-currents, altered neuronal excitability, and caused impairments in action potential (AP) generation. Thus, our results suggest that the clinical presentations of L1CAM mutations in human patients could be accounted for, at least in part, by cell-autonomous changes in the functional development of neurons, such that neurons are unable to develop normal axons and dendrites and to generate normal APs.