Adenylyl Cyclases 1 and 8 Initiate a Presynaptic Homeostatic Response to Ethanol Treatment

BACKGROUND:Although ethanol exerts widespread action in the brain, only recently has progress been made in understanding the specific events occurring at the synapse during ethanol exposure. Mice deficient in the calcium-stimulated adenylyl cyclases, AC1 and AC8 (DKO), demonstrate increased sedation duration and impaired phosphorylation by protein kinase A (PKA) following acute ethanol treatment. While not direct targets for ethanol, we hypothesize that these cyclases initiate a homeostatic presynaptic response by PKA to reactivate neurons from ethanol-mediated inhibition. METHODOLOGY/PRINCIPAL FINDINGS:Here, we have used phosphoproteomic techniques and identified several presynaptic proteins that are phosphorylated in the brains of wild type mice (WT) after ethanol exposure, including synapsin, a known PKA target. Phosphorylation of synapsins I and II, as well as phosphorylation of non-PKA targets, such as, eukaryotic elongation factor-2 (eEF-2) and dynamin is significantly impaired in the brains of DKO mice. This deficit is primarily driven by AC1, as AC1-deficient, but not AC8-deficient mice also demonstrate significant reductions in phosphorylation of synapsin and eEF-2 in cortical and hippocampal tissues. DKO mice have a reduced pool of functional recycling vesicles and fewer active terminals as measured by FM1-43 uptake compared to WT controls, which may be a contributing factor to the impaired presynaptic response to ethanol treatment. CONCLUSIONS/SIGNIFICANCE:These data demonstrate that calcium-stimulated AC-dependent PKA activation in the presynaptic terminal, primarily driven by AC1, is a critical event in the reactivation of neurons following ethanol-induced activity blockade

VAMP4 directs synaptic vesicles to a pool that selectively maintains asynchronous neurotransmission

Synaptic vesicles in the brain harbor several soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins. With the exception of synaptobrevin2, or VAMP2 (syb2), which is directly involved in vesicle fusion, the role of these SNAREs in neurotransmission is unclear. Here we show that in mice syb2 drives rapid Ca2+-dependent synchronous neurotransmission, whereas the structurally homologous SNARE protein VAMP4 selectively maintains bulk Ca2+-dependent asynchronous release. At inhibitory nerve terminals, up- or downregulation of VAMP4 causes a correlated change in asynchronous release. Biochemically, VAMP4 forms a stable complex with SNAREs syntaxin-1 and SNAP-25 that does not interact with complexins or synaptotagmin-1, proteins essential for synchronous neurotransmission. Optical imaging of individual synapses indicates that trafficking of VAMP4 and syb2 show minimal overlap. Taken together, these findings suggest that VAMP4 and syb2 diverge functionally, traffic independently and support distinct forms of neurotransmission. These results provide molecular insight into how synapses diversify their release properties by taking advantage of distinct synaptic vesicle–associated SNAREs

Neurons are MHC Class I-Dependent Targets for CD8 T Cells upon Neurotropic Viral Infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage

Myogenic tone in mouse mesenteric arteries: evidence for P2Y receptor-mediated, Na+, K+, 2Cl− cotransport-dependent signaling

This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog α,β-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X1 receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y6 receptors. Inhibition of Na+, K+, 2Cl− cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y6-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y6-mediated signaling should be examined further

Bcl-xl regulates metabolic efficiency of neurons through interaction with the mitochondrial F1FoATP synthase

Anti-apoptotic Bcl2 family proteins such as Bcl-xL protect cells from death by sequestering apoptotic molecules, but also contribute to normal neuronal function. We find in hippocampal neurons that Bcl-xL enhances the efficiency of energy metabolism. Our evidence indicates that Bcl-xLinteracts directly with the ?-subunit of the F1FO ATP synthase, decreasing an ion leak within the F1FO ATPase complex and thereby increasing net transport of H+ by F1FO during F1FO ATPase activity. By patch clamping submitochondrial vesicles enriched in F1FO ATP synthase complexes, we find that, in the presence of ATP, pharmacological or genetic inhibition of Bcl-xL activity increases the membrane leak conductance. In addition, recombinant Bcl-xL protein directly increases the level of ATPase activity of purified synthase complexes, and inhibition of endogenous Bcl-xL decreases the level of F1FO enzymatic activity. Our findings indicate that increased mitochondrial efficiency contributes to the enhanced synaptic efficacy found in Bcl-xL-expressing neurons

Properties of glutamatergic synapses in immature layer Vb pyramidal neurons: coupling of pre- and postsynaptic maturational states.

Contains fulltext : 89577.pdf (publisher's version ) (Closed access)Following initial contact formation, glutamatergic synapses in cortical neurons undergo pronounced functional maturation. These maturational events, occurring both pre- and postsynaptically, have been well described in the developing hippocampus. In this paper, we characterized glutamatergic synapses in immature layer Vb pyramidal neurons of the mouse somatosensory cortex during early postnatal development. At postnatal day 7, a significant subpopulation of glutamatergic synapses exhibited a low release probability that was accompanied by strong paired-pulse facilitation of AMPA EPSCs (paired-pulse ratio C > or = 2). Increasing extracellular Ca(2+) concentration increased release probability and led to paired-pulse depression. During further postnatal development, these functionally immature synapses disappeared. As shown pharmacologically,these synapses expressed postsynaptic NMDA receptors containing NR2B subunits, while NMDA receptors with NR2A subunits were lacking. Taken together, a low release probability presynaptically was coupled to postsynaptic NR2B signaling. This subpopulation of neocortical synapses thus differed from the majority of synapses in the developing hippocampus, where high release probability is coupled to NR2B signaling. The novel type of functionally immature glutamatergic synapse described here might play an important role in early developmental synapse elimination and in the activity-dependent refinement of the neocortical synaptic microcircuitry.1 januari 201