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Using a Drosophila whole-genome transgenic RNAi screen for glycogenes regulating synapse function, we have identified two protein α-N-acetylgalactosaminyltransferases (pgant3 and pgant35A) that regulate synaptic O-linked glycosylation (GalNAcα1-O-S/T). Loss of either pgant alone elevates presynaptic/postsynaptic molecular assembly and evoked neurotransmission strength, but synapses appear restored to normal in double mutants. Likewise, activity-dependent facilitation, augmentation, and posttetanic potentiation are all suppressively impaired in pgant mutants. In non-neuronal contexts, pgant function regulates integrin signaling, and we show here that the synaptic Position Specific 2 (αPS2) integrin receptor and transmembrane tenascin ligand are both suppressively downregulated in pgant mutants. Channelrhodopsin-driven activity rapidly (<1 min) drives integrin signaling in wild-type synapses but is suppressively abolished in pgant mutants. Optogenetic stimulation in pgant mutants alters presynaptic vesicle trafficking and postsynaptic pocket size during the perturbed integrin signaling underlying synaptic plasticity defects. Critically, acute blockade of integrin signaling acts synergistically with pgant mutants to eliminate all activity-dependent synaptic plasticity.
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G(i/o)-protein-coupled receptors (GPCRs) ubiquitously inhibit neurotransmission, principally via Gβγ, which acts via a number of possible effectors. GPCR effector specificity has traditionally been attributed to Gα, based on Gα's preferential effector targeting in vitro compared with Gβγ's promiscuous targeting of various effectors. In synapses, however, Gβγ clearly targets unique effectors in a receptor-dependent way to modulate synaptic transmission. It remains unknown whether Gβγ specificity in vivo is due to specific Gβγ isoform-receptor associations or to spatial separation of distinct Gβγ pathways through macromolecular interactions. We thus sought to determine how Gβγ signaling pathways within axons remain distinct from one another. In rat hippocampal CA1 axons, GABA(B) receptors (GABA(B)Rs) inhibit presynaptic Ca(2+) entry, and we have now demonstrated that 5-HT(1B) receptors (5-HT(1B)Rs) liberate Gβγ to interact with SNARE complex C terminals with no effect on Ca(2+) entry. Both GABA(B)Rs and 5-HT(1B)Rs inhibit Ca(2+)-evoked neurotransmitter release, but 5-HT(1B)Rs have no effect on Sr(2+)-evoked release. Sr(2+), unlike Ca(2+), does not cause synaptotagmin to compete with Gβγ binding to SNARE complexes. 5-HT(1B)Rs also fail to inhibit release following cleavage of the C terminus of the SNARE complex protein SNAP-25 with botulinum A toxin. Thus, GABA(B)Rs and 5-HT(1B)Rs both localize to presynaptic terminals, but target distinct effectors. We demonstrate that disruption of SNARE complexes and vesicle priming with botulinum C toxin eliminates this selectivity, allowing 5-HT(1B)R inhibition of Ca(2+) entry. We conclude that receptor-effector specificity requires a microarchitecture provided by the SNARE complex during vesicle priming.
The regulated exocytosis that mediates chemical signaling at synapses requires mechanisms to coordinate the immediate response to stimulation with the recycling needed to sustain release. Two general classes of transporter contribute to release, one located on synaptic vesicles that loads them with transmitter, and a second at the plasma membrane that both terminates signaling and serves to recycle transmitter for subsequent rounds of release. Originally identified as the target of psychoactive drugs, these transport systems have important roles in transmitter release, but we are only beginning to understand their contribution to synaptic transmission, plasticity, behavior, and disease. Recent work has started to provide a structural basis for their activity, to characterize their trafficking and potential for regulation. The results indicate that far from the passive target of psychoactive drugs, neurotransmitter transporters undergo regulation that contributes to synaptic plasticity.
Human cytomegalovirus induces and requires fatty acid synthesis. This suggests an essential role for lipidome remodeling in viral replication. We used mass spectrometry to quantify glycerophospholipids in mock-infected and virus-infected fibroblasts, as well as in virions. Although the lipid composition of mock-infected and virus-infected fibroblasts was similar, virions were markedly different. The virion envelope contained twofold more phosphatidylethanolamines and threefold less phosphatidylserines than the host cell. This indicates that the virus buds from a membrane with a different lipid composition from the host cell as a whole. Compared with published datasets, the virion envelope showed the greatest similarity to the synaptic vesicle lipidome. Synaptosome-associated protein of 25 kDa (SNAP-25) is a component of the complex that mediates exocytosis of synaptic vesicles in neurons; and its homolog, SNAP-23, functions in exocytosis in many other cell types. Infection induced the relocation of SNAP-23 to the cytoplasmic viral assembly zone, and knockdown of SNAP-23 inhibited the production of virus. We propose that cytomegalovirus capsids acquire their envelope by budding into vesicles with a lipid composition similar to that of synaptic vesicles, which subsequently fuse with the plasma membrane to release virions from the cell.
A systematic Drosophila forward genetic screen for photoreceptor synaptic transmission mutants identified no-on-and-no-off transient C (nonC) based on loss of retinal synaptic responses to light stimulation. The cloned gene encodes phosphatidylinositol-3-kinase-like kinase (PIKK) Smg1, a regulatory kinase of the nonsense-mediated decay (NMD) pathway. The Smg proteins act in an mRNA quality control surveillance mechanism to selectively degrade transcripts containing premature stop codons, thereby preventing the translation of truncated proteins with dominant-negative or deleterious gain-of-function activities. At the neuromuscular junction (NMJ) synapse, an extended allelic series of Smg1 mutants show impaired structural architecture, with decreased terminal arbor size, branching and synaptic bouton number. Functionally, loss of Smg1 results in a ~50% reduction in basal neurotransmission strength, as well as progressive transmission fatigue and greatly impaired synaptic vesicle recycling during high-frequency stimulation. Mutation of other NMD pathways genes (Upf2 and Smg6) similarly impairs neurotransmission and synaptic vesicle cycling. These findings suggest that the NMD pathway acts to regulate proper mRNA translation to safeguard synapse morphology and maintain the efficacy of synaptic function.
Vertebrate photoreceptors have a modified cilium composed of a basal body, axoneme and outer segment. The outer segment includes stacked membrane discs, containing opsin and the signal transduction apparatus mediating phototransduction. In photoreceptors, two distinct classes of vesicles are trafficked. Synaptic vesicles are transported down the axon to the synapse, whereas opsin-containing vesicles are transported to the outer segment. The continuous replacement of the outer segments imposes a significant biosynthetic and trafficking burden on the photoreceptors. Here, we show that Ahi1, a gene that when mutated results in the neurodevelopmental disorder, Joubert syndrome (JBTS), is required for photoreceptor sensory cilia formation and the development of photoreceptor outer segments. In mice with a targeted deletion of Ahi1, photoreceptors undergo early degeneration. Whereas synaptic proteins are correctly trafficked, photoreceptor outer segment proteins fail to be transported appropriately or are significantly reduced in their expression levels (i.e., transducin and Rom1) in Ahi1(-/-) mice. We show that vesicular targeting defects in Ahi1(-/-) mice are cilium specific, and our evidence suggests that the defects are caused by a decrease in expression of the small GTPase Rab8a, a protein required for accurate polarized vesicular trafficking. Thus, our results suggest that Ahi1 plays a role in stabilizing the outer segment proteins, transducin and Rom1, and that Ahi1 is an important component of Rab8a-mediated vesicular trafficking in photoreceptors. The retinal degeneration observed in Ahi1(-/-) mice recapitulates aspects of the retinal phenotype observed in patients with JBTS and suggests the importance of Ahi1 in photoreceptor function.
The norepinephrine transporter (NET) is a presynaptic plasma membrane protein that mediates reuptake of synaptically released norepinephrine. NET is also a major target for medications used for the treatment of depression, attention deficit/hyperactivity disorder, narcolepsy, and obesity. NET is regulated by numerous mechanisms, including catalytic activation and membrane trafficking. Amphetamine (AMPH), a psychostimulant and NET substrate, has also been shown to induce NET trafficking. However, neither the molecular basis nor the nature of the relevant membrane compartments of AMPH-modulated NET trafficking has been defined. Indeed, direct visualization of drug-modulated NET trafficking in neurons has yet to be demonstrated. In this study, we used a recently developed NET antibody and the presence of large presynaptic boutons in sympathetic neurons to examine basal and AMPH-modulated NET trafficking. Specifically, we establish a role for Rab11 in AMPH-induced NET trafficking. First, we found that, in cortical slices, AMPH induces a reduction in surface NET. Next, we observed AMPH-induced accumulation and colocalization of NET with Rab11a and Rab4 in presynaptic boutons of cultured neurons. Using tagged proteins, we demonstrated that NET and a truncated Rab11 effector (FIP2DeltaC2) do not redistribute in synchrony, whereas NET and wild-type Rab11a do. Analysis of various Rab11a/b mutants further demonstrates that Rab11 regulates NET trafficking. Expression of the truncated Rab11a effector (FIP2DeltaC2) attenuates endogenous Rab11 function and prevented AMPH-induced NET internalization as does GDP-locked Rab4 S22N. Our data demonstrate that AMPH leads to an increase of NET in endosomes of single boutons and varicosities in a Rab11-dependent manner.
We studied the consequences of expression of wild-type (WT) human NIPA1 and two mutant forms of NIPA1 with known HSP-associated mutations (T45R and G106R) on cultured rat cortical neurons and using equivalent substitutions in the Caenorhabditis elegans NIPA1 homolog CeNIPA. WT NIPA1 localized in transfected neuronal and non-neuronal cells to the Golgi complex, a subset of synaptic vesicles, to a subset of early endosomes, and plasma cell membrane. Mutant NIPA1 accumulated in the endoplasmic reticulum (ER) triggering ER stress and features of apoptotic cell death. Flow cytometric analysis of NIPA1 surface expression demonstrated relatively intact trafficking of mutant forms and only the T45R mutant exhibited modestly reduced patterns of surface expression without evidence for a dominant-negative effect. In vivo pan-neuronal expression of the WT C. elegans NIPA1 homolog (CeNIPA) was well tolerated, with no obvious impact on neuronal morphology or behavior. In striking contrast, expression of CeNIPA bearing HSP-associated mutations caused a progressive neural degeneration and a clear motor phenotype. Neuronal loss in these animals began at day 7 and by day 9 animals were completely paralyzed. These effects appeared to arise from activation of the apoptotic program triggered by unfolded protein response (UPR), as we observed marked modifications of motor and cellular phenotype when mutant NIPA1 was expressed in caspase (ced-3)- and UPR (xbp-1)-deficient backgrounds. We propose that HSP-associated mutations in NIPA1 lead to cellular and functional deficits through a gain-of-function mechanism supporting the ER accumulation of toxic NIPA1 proteins.
The high-affinity choline transporter (CHT) is expressed in cholinergic neurons and efficiently transported to axon terminals where it controls the rate-limiting step in acetylcholine synthesis. Recent studies have shown that the majority of CHT is unexpectedly localized on synaptic vesicles (SV) rather than the presynaptic plasma membrane, establishing vesicular CHT trafficking as a basis for activity-dependent CHT regulation. Here, we analyse the intracellular distribution of CHT in the adaptor protein-3 (AP-3)-deficient mouse model mocha. In the mocha mouse, granular structures in cell bodies are intensely labelled with CHT antibody, indicating possible deficits in CHT trafficking from the cell body to the axon terminal. Western blot analyses reveal that CHT on SV in mocha mice is decreased by 30% compared with wild-type mice. However, no significant difference in synaptosomal choline uptake activity is detected, consistent with the existence of a large reservoir pool for CHT. To further characterize CHT trafficking, we established a PC12D-CHT cell line. In this line, CHT is found associated with a subpopulation of synaptophysin-positive synaptic-like microvesicles (SLMV). The amounts of CHT detected on SLMV are greatly reduced by treating the cell with agents that halt AP-dependent membrane trafficking. These results demonstrate that APs have important functions for CHT trafficking in neuronal cells.
The recent cloning of the human choline transporter (hCHT) has allowed its expression in Xenopus laevis oocytes and the simultaneous measurement of choline transport and choline-induced current under voltage clamp. hCHT currents and choline transport are evident in cRNA-injected oocytes and significantly enhanced by the hCHT trafficking mutant L530A/V531A. The charge/choline ratio of hCHT varies from 10e/choline at -80 mV to 3e/choline at -20 mV, in contrast with the reported fixed stoichiometry of the Na+-coupled glucose transporter in the same gene family. Ion substitution shows that the choline uptake and choline-induced current are Na+ and Cl- dependent; however, the reversal potential of the induced current suggests a Na+-selective mechanism, consigning Cl- to a regulatory role rather than a coupled, cotransported-ion role. The hCHT-specific inhibitor hemicholinium-3 (HC-3) blocks choline uptake and choline-induced current; in addition, HC-3 alone reveals a constitutive, depolarizing leak current through hCHT. We show that external protons reduce hCHT current, transport, and binding with a similar pKa of 7.4, suggesting proton titration of residue(s) that support choline binding and transport. Given the localization of the choline transporter to synaptic vesicles, we propose that proton inactivation of hCHT prevents acetylcholine and proton leakage from the acidic interior of cholinergic synaptic vesicles. This mechanism would allow cholinergic, activity-triggered delivery of silent choline transporters to the plasma membrane, in which normal pH would reactivate the transporters for choline uptake and subsequent acetylcholine synthesis.