cis-B7:CD28 interactions at invaginated synaptic membranes provide CD28 co-stimulation and promote CD8+ T cell function and anti-tumor immunity.

B7 ligands (CD80 and CD86), expressed by professional antigen-presenting cells (APCs), activate the main co-stimulatory receptor CD28 on T cells in trans. However, in peripheral tissues, APCs expressing B7 ligands are relatively scarce. This raises the questions of whether and how CD28 co-stimulation occurs in peripheral tissues. Here, we report that CD8+ T cells displayed B7 ligands that interacted with CD28 in cis at membrane invaginations of the immunological synapse as a result of membrane remodeling driven by phosphoinositide-3-kinase (PI3K) and sorting-nexin-9 (SNX9). cis-B7:CD28 interactions triggered CD28 signaling through protein kinase C theta (PKCθ) and promoted CD8+ T cell survival, migration, and cytokine production. In mouse tumor models, loss of T cell-intrinsic cis-B7:CD28 interactions decreased intratumoral T cells and accelerated tumor growth. Thus, B7 ligands on CD8+ T cells can evoke cell-autonomous CD28 co-stimulation in cis in peripheral tissues, suggesting cis-signaling as a general mechanism for boosting T cell functionality.

Proteomic Analysis of Unbounded Cellular Compartments: Synaptic Clefts.

Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.

A concerted neuron-astrocyte program declines in ageing and schizophrenia.

Human brains vary across people and over time; such variation is not yet understood in cellular terms. Here we describe a relationship between people's cortical neurons and cortical astrocytes. We used single-nucleus RNA sequencing to analyse the prefrontal cortex of 191 human donors aged 22-97 years, including healthy individuals and people with schizophrenia. Latent-factor analysis of these data revealed that, in people whose cortical neurons more strongly expressed genes encoding synaptic components, cortical astrocytes more strongly expressed distinct genes with synaptic functions and genes for synthesizing cholesterol, an astrocyte-supplied component of synaptic membranes. We call this relationship the synaptic neuron and astrocyte program (SNAP). In schizophrenia and ageing-two conditions that involve declines in cognitive flexibility and plasticity1,2-cells divested from SNAP: astrocytes, glutamatergic (excitatory) neurons and GABAergic (inhibitory) neurons all showed reduced SNAP expression to corresponding degrees. The distinct astrocytic and neuronal components of SNAP both involved genes in which genetic risk factors for schizophrenia were strongly concentrated. SNAP, which varies quantitatively even among healthy people of similar age, may underlie many aspects of normal human interindividual differences and may be an important point of convergence for multiple kinds of pathophysiology.

RIM and RIM-BP Form Presynaptic Active-Zone-like Condensates via Phase Separation.

Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.

Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Priming of synaptic vesicles involves Munc13-catalyzed transition of the Munc18-1/syntaxin-1 complex to the SNARE complex in the presence of SNAP-25 and synaptobrevin-2; Munc13 drives opening of syntaxin-1 via the MUN domain while Munc18-1 primes SNARE assembly via domain 3a. However, the underlying mechanism remains unclear. In this study, we have identified a number of residues in domain 3a of Munc18-1 that are crucial for Munc13 and Munc18-1 actions in SNARE complex assembly and synaptic vesicle priming. Our results showed that two residues (Q301/K308) at the side of domain 3a mediate the interaction between the Munc18-1/syntaxin-1 complex and the MUN domain. This interaction enables the MUN domain to drive the opening of syntaxin-1 linker region, thereby leading to the extension of domain 3a and promoting synaptobrevin-2 binding. In addition, we identified two residues (K332/K333) at the bottom of domain 3a that mediate the interaction between Munc18-1 and the SNARE motif of syntaxin-1. This interaction ensures Munc18-1 to persistently associate with syntaxin-1 during the conformational change of syntaxin-1 from closed to open, which reinforces the role of Munc18-1 in templating SNARE assembly. Taken together, our data suggest a mechanism by which Munc13 activates the Munc18-1/syntaxin-1 complex and enables Munc18-1 to prime SNARE assembly.

Association of a newly identified lncRNA LNC_000280 with the formation of acetylcholine receptor clusters in vitro.

Peripheral nerve injury (PNI) can disintegrate acetylcholine receptor (AChR) clusters in the postsynaptic membrane. In our previous research, lncRNAs that were differentially expressed in the whole transcriptome sequencing of denervated muscle atrophy after PNI were screened. By utilizing Gene Ontology (GO) analysis and protein-protein interaction (PPI) networks, a novel lncRNA LNC_000280 was predicted to be associated with neuromuscular junction (NMJ). The myotubes were used to assess the connection between LNC_000280 and AChR cluster formation in vitro by overexpression and knockdown of LNC_000280 in the C2C12 cell line. Our findings demonstrated that the overexpression of LNC_000280 repressed the gene expression and protein level of AChR subunits in myotubes and further reduced the area of AChR aggregates on the cell membrane. In contrast, the knockdown of LNC_000280 brought about opposite results. In addition, the transcriptional level of Sorbs2 changed inversely with the quantity change of LNC_000280. In conclusion, LNC_000280 may associate with the formation of AChR clusters.

SorCS1-mediated sorting in dendrites maintains neurexin axonal surface polarization required for synaptic function.

The pre- and postsynaptic membranes comprising the synaptic junction differ in protein composition. The membrane trafficking mechanisms by which neurons control surface polarization of synaptic receptors remain poorly understood. The sorting receptor Sortilin-related CNS expressed 1 (SorCS1) is a critical regulator of trafficking of neuronal receptors, including the presynaptic adhesion molecule neurexin (Nrxn), an essential synaptic organizer. Here, we show that SorCS1 maintains a balance between axonal and dendritic Nrxn surface levels in the same neuron. Newly synthesized Nrxn1α traffics to the dendritic surface, where it is endocytosed. Endosomal SorCS1 interacts with the Rab11 GTPase effector Rab11 family-interacting protein 5 (Rab11FIP5)/Rab11 interacting protein (Rip11) to facilitate the transition of internalized Nrxn1α from early to recycling endosomes and bias Nrxn1α surface polarization towards the axon. In the absence of SorCS1, Nrxn1α accumulates in early endosomes and mispolarizes to the dendritic surface, impairing presynaptic differentiation and function. Thus, SorCS1-mediated sorting in dendritic endosomes controls Nrxn axonal surface polarization required for proper synapse development and function.

Depolarization-dependent Induction of Site-specific Changes in Sialylation on N-linked Glycoproteins in Rat Nerve Terminals.

Synaptic transmission leading to release of neurotransmitters in the nervous system is a fast and highly dynamic process. Previously, protein interaction and phosphorylation have been thought to be the main regulators of synaptic transmission. Here we show that sialylation of N-linked glycosylation is a novel potential modulator of neurotransmitter release mechanisms by investigating depolarization-dependent changes of formerly sialylated N-linked glycopeptides. We suggest that negatively charged sialic acids can be modulated, similarly to phosphorylation, by the action of sialyltransferases and sialidases thereby changing local structure and function of membrane glycoproteins. We characterized site-specific alteration in sialylation on N-linked glycoproteins in isolated rat nerve terminals after brief depolarization using quantitative sialiomics. We identified 1965 formerly sialylated N-linked glycosites in synaptic proteins and found that the abundances of 430 glycosites changed after 5 s depolarization. We observed changes on essential synaptic proteins such as synaptic vesicle proteins, ion channels and transporters, neurotransmitter receptors and cell adhesion molecules. This study is to our knowledge the first to describe ultra-fast site-specific modulation of the sialiome after brief stimulation of a biological system.

An overview of the synaptic vesicle lipid composition.

Chemical neurotransmission is the major mechanism of neuronal communication. Neurotransmitters are released from secretory organelles, the synaptic vesicles (SVs) via exocytosis into the synaptic cleft. Fusion of SVs with the presynaptic plasma membrane is balanced by endocytosis, thus maintaining the presynaptic membrane at steady-state levels. The protein machineries responsible for exo- and endocytosis have been extensively investigated. In contrast, less is known about the role of lipids in synaptic transmission and how the lipid composition of SVs is affected by dynamic exo-endocytotic cycling. Here we summarize the current knowledge about the composition, organization, and function of SV membrane lipids. We also cover lipid biogenesis and maintenance during the synaptic vesicle cycle.

Effects of Zinc, Mercury, or Lead on [3H]MK-801 and [3H]Fluorowillardiine Binding to Rat Synaptic Membranes.

Glutamate (Glu) is considered the most important excitatory amino acid neurotransmitter in the mammalian Central Nervous System. Zinc (Zn) is co-released with Glu during synaptic transmission and interacts with Glutamate receptors and transporters. We performed binding experiments using [3H]MK-801 (NMDA), and [3H]Fluorowillardine (AMPA) as ligands to study Zn-Glutamate interactions in rat cortical synaptic membranes. We also examined the effects of mercury and lead on NMDA or AMPA receptors. Zinc at 1 nM, significantly potentiates [3H]MK-801 binding. Lead inhibits [3H]MK-801 binding at micromolar concentrations. At millimolar concentrations, Hg also has a significant inhibitory effect. These effects are not reversed by Zn (1 nM). Zinc displaces the [3H]FW binding curve to the right. Lead (nM) and Hg (µM) inhibit [3H]FW binding. At certain concentrations, Zn reverses the effects of these metals on [3H]FW binding. These specific interactions serve to clarify the role of Zn, Hg, and Pb in physiological and pathological conditions.

Potentiating KCC2 activity is sufficient to limit the onset and severity of seizures.

The type 2 K+/Cl- cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl-, a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl- extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids.

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.

Effect of ethanol on Munc13-1 C1 in Membrane: A Molecular Dynamics Simulation Study.

BACKGROUND: EtOH has a significant effect on synaptic plasticity. Munc13-1 is an essential presynaptic active zone protein involved in priming the synaptic vesicle and releasing neurotransmitter in the brain. It is a peripheral membrane protein and binds to the activator, diacylglycerol (DAG)/phorbol ester at its membrane-targeting C1 domain. Our previous studies identified Glu-582 of C1 domain as the alcohol-binding residue (Das, J. et al, J. Neurochem., 126, 715-726, 2013). METHODS: Here, we describe a 250 ns molecular dynamics (MD) simulation study on the interaction of EtOH and the activator-bound Munc13-1 C1 in the presence of varying concentrations of phosphatidylserine (PS). RESULTS: In this study, Munc13-1 C1 shows higher conformational stability in EtOH than in water. It forms fewer hydrogen bonds with phorbol 13-acetate in the presence of EtOH than in water. EtOH also affected the interaction between the protein and the membrane and between the activator and the membrane. Similar studies in a E582A mutant suggest that these effects of EtOH are mostly mediated through Glu-582. CONCLUSIONS: EtOH forms hydrogen bonds with Glu-582. While occupancy of the EtOH molecules at the vicinity (4Å) of Glu-582 is 34.4%, the occupancy in the E582A mutant is 26.5% of the simulation time. In addition, the amount of PS in the membrane influences the conformational stability of the C1 domain and interactions in the ternary complex. This study is important in providing the structural basis of EtOH's effects on synaptic plasticity.

Morphologies of synaptic protein membrane fusion interfaces.

Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release.

Dynamic Relationship of the SNARE Complex with a Membrane.

Fusion of secretory granules and synaptic vesicles with the plasma membrane is driven by SNARE protein interactions. Intensive investigations in vitro, which include x-ray crystallography, cryoelectron microscopy, and NMR analyses by numerous groups, have elucidated structures relevant to the function of these proteins. Although function depends on the proteins being membrane bound, for experimental reasons, most of the studies have used cytosolic domains, as exemplified by the groundbreaking studies that elucidated the structure of a tetrapeptide helical bundle formed by interaction of the cytosolic domains of syntaxin1A, SNAP25 (two peptides) and synaptobrevin 2. Because the cytosolic fragments were unfettered by membrane attachments, it is likely that the tetrapeptide helical bundle reflects the lowest energy state, such as that found in the "cis" interactions of the SNARE motifs after fusion when they co-localize in the plasma membrane. Much more difficult to study and still poorly understood are critical "trans" interactions between the synaptic vesicle SNARE protein synaptobrevin 2 and the plasma membrane syntaxin1A/SNAP25 complex that initiate the fusion event. In a series of articles from the laboratory of Lukas Tamm, the spontaneous orientation of the SNARE motif of membrane-bound, full-length syntaxin1A with respect to the membrane hosting syntaxin's transmembrane domain was investigated with nanometer precision under a variety of conditions, including those that model aspects of the "trans" configuration. The studies rely on fluorescence interference-contrast microscopy, a technique that utilizes the pattern of constructive and destructive interference arising from incoming and reflected excitation and emission light at the surface of a silicon chip that has been layered with oxidized silicon of varying depths. This Perspective discusses their findings, including the unexpected influence of the degree of lipid unsaturation on the orientation of the SNARE complex.

Lipids and phosphates at odds in synaptic depression.

Long-term depression (LTD) is a reduction in the efficacy of neuronal synapses, but the molecular basis of LTD signaling and how these signals lead to phenotypic outcomes, such as the shrinkage of synaptic regions, is not clear. In a new report, Woolfrey et al use chemically-induced LTD and a multitude of in vitro biochemical assays to provide evidence that synaptic removal of the scaffolding protein AKAP79/150 promotes LTD-induced spine shrinkage. The further identification of CaMKII, a kinase primarily associated with long-term potentiation (LTP), as a requirement for AKAP79/150 removal, uncovers unexpected interplay between different post-translational modifications and points to a new model of LTD.

Reciprocal modulation between amyloid precursor protein and synaptic membrane cholesterol revealed by live cell imaging.

The amyloid precursor protein (APP) has been extensively studied because of its association with Alzheimer's disease (AD). However, APP distribution across different subcellular membrane compartments and its function in neurons remains unclear. We generated an APP fusion protein with a pH-sensitive green fluorescent protein at its ectodomain and a pH-insensitive blue fluorescent protein at its cytosolic domain and used it to measure APP's distribution, subcellular trafficking, and cleavage in live neurons. This reporter, closely resembling endogenous APP, revealed only a limited correlation between synaptic activities and APP trafficking. However, the synaptic surface fraction of APP was increased by a reduction in membrane cholesterol levels, a phenomenon that involves APP's cholesterol-binding motif. Mutations at or near binding sites not only reduced both the surface fraction of APP and membrane cholesterol levels in a dominant negative manner, but also increased synaptic vulnerability to moderate membrane cholesterol reduction. Our results reveal reciprocal modulation of APP and membrane cholesterol levels at synaptic boutons.

Declining levels of functionally specialized synaptic proteins in plasma neuronal exosomes with progression of Alzheimer's disease.

Interactions of the presynaptic proteins, neuronal pentraxin 2 (NPTX2) and neurexin 2α (NRXN2α), with their respective postsynaptic functional partners, GluA4-containing glutamate (AMPA4) receptor and neuroligin 1 (NLGN1), enhance excitatory synaptic activity in some areas of the hippocampus and cerebral cortex. As early damage of such excitatory circuits in the brain tissues of participants with Alzheimer's disease (AD) correlates with cognitive losses, plasma neuron-derived exosome (NDE) levels of these 2 pairs of specialized synaptic proteins were quantified to assess their biomarker characteristics. The NDE contents of all 4 proteins were decreased significantly in AD dementia ( n = 46), and diminished levels of AMPA4 and NLGN1 correlated with the extent of cognitive loss. In a preclinical period, 6-11 yr before the onset of dementia, the NDE levels of all but NPTX2 were significantly lower than those of matched controls, and levels of all proteins declined significantly with the development of dementia. Reductions in NDE levels of these specialized excitatory synaptic proteins may therefore be indicative of the extent of cognitive loss and may reflect progression of the severity of AD.-Goetzl, E. J., Abner, E. L., Jicha, G. A., Kapogiannis, D., Schwartz, J. B. Declining levels of functionally specialized synaptic proteins in plasma neuronal exosomes with progression of Alzheimer's disease.

Variable priming of a docked synaptic vesicle.

The priming of a docked synaptic vesicle determines the probability of its membrane (VM) fusing with the presynaptic membrane (PM) when a nerve impulse arrives. To gain insight into the nature of priming, we searched by electron tomography for structural relationships correlated with fusion probability at active zones of axon terminals at frog neuromuscular junctions. For terminals fixed at rest, the contact area between the VM of docked vesicles and PM varied >10-fold with a normal distribution. There was no merging of the membranes. For terminals fixed during repetitive evoked synaptic transmission, the normal distribution of contact areas was shifted to the left, due in part to a decreased number of large contact areas, and there was a subpopulation of large contact areas where the membranes were hemifused, an intermediate preceding complete fusion. Thus, fusion probability of a docked vesicle is related to the extent of its VM-PM contact area. For terminals fixed 1 h after activity, the distribution of contact areas recovered to that at rest, indicating the extent of a VM-PM contact area is dynamic and in equilibrium. The extent of VM-PM contact areas in resting terminals correlated with eccentricity in vesicle shape caused by force toward the PM and with shortness of active zone material macromolecules linking vesicles to PM components, some thought to include Ca(2+) channels. We propose that priming is a variable continuum of events imposing variable fusion probability on each vesicle and is regulated by force-generating shortening of active zone material macromolecules in dynamic equilibrium.

Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion.

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.