Dynamin is primed at endocytic sites for ultrafast endocytosis.

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here, we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane. Single-particle tracking of Dynamin 1xA molecules confirms the liquid-like property of condensates in vivo. When Dynamin 1xA is mutated to disrupt its interaction with Syndapin 1, the condensates do not form, and consequently, ultrafast endocytosis slows down by 100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

Cannabinoid receptor activation acutely increases synaptic vesicle numbers by activating synapsins in human synapses.

Cannabis and cannabinoid drugs are central agents that are used widely recreationally and are employed broadly for treating psychiatric conditions. Cannabinoids primarily act by stimulating presynaptic CB1 receptors (CB1Rs), the most abundant G-protein-coupled receptors in brain. CB1R activation decreases neurotransmitter release by inhibiting presynaptic Ca2+ channels and induces long-term plasticity by decreasing cellular cAMP levels. Here we identified an unanticipated additional mechanism of acute cannabinoid signaling in presynaptic terminals that regulates the size of synaptic vesicle pools available for neurotransmitter release. Specifically, we show that activation of CB1Rs in human and mouse neurons rapidly recruits vesicles to nerve terminals by suppressing the cAMP-dependent phosphorylation of synapsins. We confirmed this unanticipated mechanism using conditional deletion of synapsin-1, the predominant synapsin isoform in human neurons, demonstrating that synapsin-1 significantly contributes to the CB1R-dependent regulation of neurotransmission. Interestingly, acute activation of the Gi-DREADD hM4D mimics the effect of CB1R activation in a synapsin-1-dependent manner, suggesting that the control of synaptic vesicle numbers by synapsin-1 phosphorylation is a general presynaptic mechanism of neuromodulation. Thus, we uncovered a CB1R-dependent presynaptic mechanism that rapidly regulates the organization and neurotransmitter release properties of synapses.

Neuromodulator Signaling Bidirectionally Controls Vesicle Numbers in Human Synapses.

Neuromodulators bind to pre- and postsynaptic G protein-coupled receptors (GPCRs), are able to quickly change intracellular cyclic AMP (cAMP) and Ca2+ 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.

A Single Human Neuron Approach to Synapse Function.

To understand human neuronal function, it is crucial to obtain knowledge of how human synapses operate. New approaches are necessary to define the unique properties of human synapses. Recently, new culturing approaches have been developed to obtain cultures of single human neurons for the first time (Rhee et al., Cell Rep. 2019, Meijer et al., Cell Rep. 2019, and Fenske et al., Sci. Rep. 2019).

Autaptic cultures of human induced neurons as a versatile platform for studying synaptic function and neuronal morphology.

Recently developed technology to differentiate induced pluripotent stem cells (iPSCs) into human induced neurons (iNs) provides an exciting opportunity to study the function of human neurons. However, functional characterisations of iNs have been hampered by the reliance on mass culturing protocols which do not allow assessment of synaptic release characteristics and neuronal morphology at the individual cell level with quantitative precision. Here, we have developed for the first time a protocol to generate autaptic cultures of iPSC-derived iNs. We show that our method efficiently generates mature, autaptic iNs with robust spontaneous and action potential-driven synaptic transmission. The synaptic responses are sensitive to modulation by metabotropic receptor agonists as well as potentiation by acute phorbol ester application. Finally, we demonstrate loss of evoked and spontaneous release by Unc13A knockdown. This culture system provides a versatile platform allowing for quantitative and integrative assessment of morphophysiological and molecular parameters underlying human synaptic transmission.

Synaptojanin and Endophilin Mediate Neck Formation during Ultrafast Endocytosis.

Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis.

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function.

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating-a deficit in the ability to filter out unnecessary information-which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

Transcription factor Yin-Yang 2 alters neuronal outgrowth in vitro.

The Yin-Yang 2 (YY2) protein is the most recently described member of the family of YY transcription factors. Despite its high structural and functional homology with the well-characterized YY1, less is known about its role in biological processes. In previous studies, we have found differential yy2 mRNA expression levels in various cell types of the murine brain. To investigate the functional implication of yy2 in neurons, we have examined the influence of altered cellular yy2 concentrations during neuronal differentiation. Our results indicate that both the up- and down-regulation of yy2 significantly impairs the outgrowth of the major neurite of primary hippocampal neurons and the numbers of neuronal processes in proximate extensions. Moreover, enhanced expression of wild-type yy2 results in increased cell death, whereas elevated expression levels of a yy2 DNA-binding mutant have no effect on cell viability. Therefore, stringent regulation of the cellular yy2 content might be needed to ensure proper neurite outgrowth and cell vitality.