Synaptic transmission relies on coordinated coupling of synaptic vesicle (SV) exocytosis and endocytosis. While much attention has focused on characterizing proteins involved in SV recycling, the roles of membrane lipids and their metabolism remain poorly understood. Diacylglycerol, a major signaling lipid produced at synapses during synaptic transmission, is regulated by diacylglycerol kinase (DGK). Here, we report a role for DGKθ in the mammalian CNS in facilitating recycling of presynaptic vesicles at excitatory synapses. Using synaptophysin- and vGlut1-pHluorin optical reporters, we found that acute and chronic deletion of DGKθ attenuated the recovery of SVs following neuronal stimulation. Rescue of recycling kinetics required DGKθ kinase activity. Our data establish a role for DGK catalytic activity at the presynaptic nerve terminal in SV recycling. Altogether, these data suggest that DGKθ supports synaptic transmission during periods of elevated neuronal activity.

Rheb, a ubiquitous small GTPase, is well known to bind and activate mTOR, which augments protein synthesis. Inhibition of protein synthesis is also physiologically regulated. Thus, with cell stress, the unfolded protein response system leads to phosphorylation of the initiation factor eIF2α and arrest of protein synthesis. We now demonstrate a major role for Rheb in inhibiting protein synthesis by enhancing the phosphorylation of eIF2α by protein kinase-like ER kinase (PERK). Interplay between the stimulatory and inhibitory roles of Rheb may enable cells to modulate protein synthesis in response to varying environmental stresses.

How neuronal signaling affects brain myelination remains poorly understood. We show dysregulated neuronal RHEB-mTORC1-DLK1 axis impairs brain myelination. Neuronal Rheb cKO impairs oligodendrocyte differentiation/myelination, with activated neuronal expression of the imprinted gene Dlk1. Neuronal Dlk1 cKO ameliorates myelination deficit in neuronal Rheb cKO mice, indicating that activated neuronal Dlk1 expression contributes to impaired myelination caused by Rheb cKO. The effect of Rheb cKO on Dlk1 expression is mediated by mTORC1; neuronal mTor cKO and Raptor cKO and pharmacological inhibition of mTORC1 recapitulate elevated neuronal Dlk1 expression. We demonstrate that both a secreted form of DLK1 and a membrane-bound DLK1 inhibit the differentiation of cultured oligodendrocyte precursor cells into oligodendrocytes expressing myelin proteins. Finally, neuronal expression of Dlk1 in transgenic mice reduces the formation of mature oligodendrocytes and myelination. This study identifies Dlk1 as an inhibitor of oligodendrocyte myelination and a mechanism linking altered neuronal signaling with oligodendrocyte dysfunction.

Synapses in the brain exhibit cell-type-specific differences in basal synaptic transmission and plasticity. Here, we evaluated cell-type-specific specializations in the composition of glutamatergic synapses, identifying Btbd11 as an inhibitory interneuron-specific, synapse-enriched protein. Btbd11 is highly conserved across species and binds to core postsynaptic proteins, including Psd-95. Intriguingly, we show that Btbd11 can undergo liquid-liquid phase separation when expressed with Psd-95, supporting the idea that the glutamatergic postsynaptic density in synapses in inhibitory interneurons exists in a phase-separated state. Knockout of Btbd11 decreased glutamatergic signaling onto parvalbumin-positive interneurons. Further, both in vitro and in vivo, Btbd11 knockout disrupts network activity. At the behavioral level, Btbd11 knockout from interneurons alters exploratory behavior, measures of anxiety, and sensitizes mice to pharmacologically induced hyperactivity following NMDA receptor antagonist challenge. Our findings identify a cell-type-specific mechanism that supports glutamatergic synapse function in inhibitory interneurons-with implications for circuit function and animal behavior.

AMPA receptors (AMPARs) have recently been shown to undergo post-translational ubiquitination in mammalian neurons. However, the underlying molecular mechanisms are poorly understood and remain controversial. Here, we report that all four AMPAR subunits (GluA1-4) are rapidly ubiquitinated upon brief application of AMPA or bicuculline in cultured neurons. This process is Ca2+ dependent and requires the activity of L-type voltage-gated Ca2+ channels and Ca2+/calmodulin-dependent kinase II. The ubiquitination of all subunits occurs exclusively on AMPARs located on the plasma membrane post-endocytosis. The sites of ubiquitination were mapped to Lys-868 in GluA1 and Lys-870/Lys-882 in GluA2 C-terminals. Mutation of these lysines did not affect basal surface expression or AMPA-induced internalization of GluA1 and GluA2 subunits. Instead, it reduced the intracellular trafficking of AMPARs to the late endosomes and thus protein degradation. These data indicate that ubiquitination is an important regulatory signal for controlling AMPAR function, which may be crucial for synaptic plasticity.

Mutations of SHANK3 cause Phelan-McDermid syndrome (PMS), and these individuals can exhibit sensitivity to stress, resulting in behavioral deterioration. Here, we examine the interaction of stress with genotype using a mouse model with face validity to PMS. In Shank3ΔC/+ mice, swim stress produces an altered transcriptomic response in pyramidal neurons that impacts genes and pathways involved in synaptic function, signaling, and protein turnover. Homer1a, which is part of the Shank3-mGluR-N-methyl-D-aspartate (NMDA) receptor complex, is super-induced and is implicated in the stress response because stress-induced social deficits in Shank3ΔC/+ mice are mitigated in Shank3ΔC/+;Homer1a-/- mice. Several lines of evidence demonstrate that Shank3 expression is regulated by Homer1a in competition with crosslinking forms of Homer, and consistent with this model, Shank3 expression and function that are reduced in Shank3ΔC/+ mice are rescued in Shank3ΔC/+;Homer1a-/- mice. Studies highlight the interaction between stress and genetics and focus attention on activity-dependent changes that may contribute to pathogenesis.

Perceptual learning improves our ability to interpret sensory stimuli present in our environment through experience. Despite its importance, the underlying mechanisms that enable perceptual learning in our sensory cortices are still not fully understood. In this study, we used in vivo two-photon imaging to investigate the functional and structural changes induced by visual stimulation in the mouse primary visual cortex (V1). Our results demonstrate that repeated stimulation leads to a refinement of V1 circuitry by decreasing the number of responsive neurons while potentiating their response. At the synaptic level, we observe a reduction in the number of dendritic spines and an overall increase in spine AMPA receptor levels in the same subset of neurons. In addition, visual stimulation induces synaptic potentiation in neighboring spines within individual dendrites. These findings provide insights into the mechanisms of synaptic plasticity underlying information processing in the neocortex.