Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling.

De novo mutations in CHD8 are strongly associated with autism spectrum disorder, but the basic biology of CHD8 remains poorly understood. Here we report that Chd8 knockdown during cortical development results in defective neural progenitor proliferation and differentiation that ultimately manifests in abnormal neuronal morphology and behaviors in adult mice. Transcriptome analysis revealed that while Chd8 stimulates the transcription of cell cycle genes, it also precludes the induction of neural-specific genes by regulating the expression of PRC2 complex components. Furthermore, knockdown of Chd8 disrupts the expression of key transducers of Wnt signaling, and enhancing Wnt signaling rescues the transcriptional and behavioral deficits caused by Chd8 knockdown. We propose that these roles of Chd8 and the dynamics of Chd8 expression during development help negotiate the fine balance between neural progenitor proliferation and differentiation. Together, these observations provide new insights into the neurodevelopmental role of Chd8.

Evolutionary conservation of complexins: from choanoflagellates to mice.

Complexins are synaptic SNARE complex-binding proteins that cooperate with synaptotagmins in activating Ca(2+)-stimulated, synaptotagmin-dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin-independent synaptic vesicle exocytosis. Here, we show that complexin sequences are conserved in some non-metazoan unicellular organisms and in all metazoans, suggesting that complexins are a universal feature of metazoans that predate metazoan evolution. We show that complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse complexins in activating Ca(2+)-triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of complexins is likely conserved throughout metazoan evolution.

Synaptotagmin-12 phosphorylation by cAMP-dependent protein kinase is essential for hippocampal mossy fiber LTP.

Synaptotagmin-12 (Syt12) is an abundant synaptic vesicle protein that--different from other synaptic vesicle-associated synaptotagmins--does not bind Ca(2+). Syt12 is phosphorylated by cAMP-dependent protein kinase-A at serine-97 in an activity-dependent manner, suggesting a function for Syt12 in cAMP-dependent synaptic plasticity. To test this hypothesis, we here generated (1) Syt12 knock-out mice and (2) Syt12 knockin mice carrying a single amino-acid substitution [the serine-97-to-alanine- (S97A)-substitution]. Both Syt12 knock-out mice and Syt12 S97A-knockin mice were viable and fertile, and exhibited no measurable change in basal synaptic strength or short-term plasticity as analyzed in cultured cortical neurons or in acute hippocampal slices. However, both Syt12 knock-out and Syt12 S97A-knockin mice displayed a major impairment in cAMP-dependent mossy-fiber long-term potentiation (LTP) in the CA3 region of the hippocampus. This impairment was observed using different experimental configurations for inducing and monitoring mossy-fiber LTP. Moreover, although the Syt12 knock-out had no effect on the short-term potentiation of synaptic transmission induced by the adenylate-cyclase activator forskolin in cultured cortical neurons and in the CA1 region of the hippocampus, both the Syt12 knock-out and the Syt12 S97A-knockin impaired the long-term increase in mossy-fiber synaptic transmission induced by forskolin. Thus, Syt12 is essential for cAMP-dependent presynaptic LTP at mossy-fiber synapses, and a single amino-acid substitution that blocks the cAMP-dependent phosphorylation of Syt12 is sufficient to impair the function of Syt12 in mossy-fiber LTP, suggesting that cAMP-dependent phosphorylation of Syt12 on serine-97 contributes to the induction of mossy-fiber LTP.

C-terminal complexin sequence is selectively required for clamping and priming but not for Ca2+ triggering of synaptic exocytosis.

Complexins are small soluble proteins that bind to assembling SNARE complexes during synaptic vesicle exocytosis, which in turn mediates neurotransmitter release. Complexins are required for clamping of spontaneous "mini " release and for the priming and synaptotagmin-dependent Ca(2+) triggering of evoked release. Mammalian genomes encode four complexins that are composed of an N-terminal unstructured sequence that activates synaptic exocytosis, an accessory α-helix that clamps exocytosis, an essential central α-helix that binds to assembling SNARE complexes and is required for all of its functions, and a long, apparently unstructured C-terminal sequence whose function remains unclear. Here, we used cultured mouse neurons to show that the C-terminal sequence of complexin-1 is not required for its synaptotagmin-activating function but is essential for its priming and clamping functions. Wild-type complexin-3 did not clamp exocytosis but nevertheless fully primed and activated exocytosis. Strikingly, exchanging the complexin-1 C terminus for the complexin-3 C terminus abrogated clamping, whereas exchanging the complexin-3 C terminus for the complexin-1 C terminus enabled clamping. Analysis of point mutations in the complexin-1 C terminus identified two single amino-acid substitutions that impaired clamping without altering the activation function of complexin-1. Examination of release induced by stimulus trains revealed that clamping-deficient C-terminal complexin mutants produced a modest relative increase in delayed release. Overall, our results show that the relatively large C-terminal complexin-1 sequence acts in priming and clamping synaptic exocytosis and demonstrate that the clamping function is not conserved in complexin-3, presumably because of its distinct C-terminal sequences.

Postsynaptic complexin controls AMPA receptor exocytosis during LTP.

Long-term potentiation (LTP) is a compelling synaptic correlate of learning and memory. LTP induction requires NMDA receptor (NMDAR) activation, which triggers SNARE-dependent exocytosis of AMPA receptors (AMPARs). However, the molecular mechanisms mediating AMPAR exocytosis induced by NMDAR activation remain largely unknown. Here, we show that complexin, a protein that regulates neurotransmitter release via binding to SNARE complexes, is essential for AMPAR exocytosis during LTP but not for the constitutive AMPAR exocytosis that maintains basal synaptic strength. The regulated postsynaptic AMPAR exocytosis during LTP requires binding of complexin to SNARE complexes. In hippocampal neurons, presynaptic complexin acts together with synaptotagmin-1 to mediate neurotransmitter release. However, postsynaptic synaptotagmin-1 is not required for complexin-dependent AMPAR exocytosis during LTP. These results suggest a complexin-dependent molecular mechanism for regulating AMPAR delivery to synapses, a mechanism that is surprisingly similar to presynaptic exocytosis but controlled by regulators other than synaptotagmin-1.

Complexin clamps asynchronous release by blocking a secondary Ca(2+) sensor via its accessory α helix.

Complexin activates and clamps neurotransmitter release; impairing complexin function decreases synchronous, but increases spontaneous and asynchronous synaptic vesicle exocytosis. Here, we show that complexin-different from the Ca(2+) sensor synaptotagmin-1-activates synchronous exocytosis by promoting synaptic vesicle priming, but clamps spontaneous and asynchronous exocytosis-similar to synaptotagmin-1-by blocking a secondary Ca(2+) sensor. Activation and clamping functions of complexin depend on distinct, autonomously acting sequences, namely its N-terminal region and accessory α helix, respectively. Mutations designed to test whether the accessory α helix of complexin clamps exocytosis by inserting into SNARE-complexes support this hypothesis, suggesting that the accessory α helix blocks completion of trans-SNARE-complex assembly until Ca(2+) binding to synaptotagmin relieves this block. Moreover, a juxtamembranous mutation in the SNARE-protein synaptobrevin-2, which presumably impairs force transfer from nascent trans-SNARE complexes onto fusing membranes, also unclamps spontaneous fusion by disinhibiting a secondary Ca(2+) sensor. Thus, complexin performs mechanistically distinct activation and clamping functions that operate in conjunction with synaptotagmin-1 by controlling trans-SNARE-complex assembly.