Enhancement of Phosphorylation and Transport Activity of the Neuronal Glutamate Transporter Excitatory Amino Acid Transporter 3 by C3bot and a 26mer C3bot Peptide.

In primary murine hippocampal neurons we investigated the regulation of EAAT3-mediated glutamate transport by the Clostridium botulinum C3 transferase C3bot and a 26mer peptide derived from full length protein. Incubation with either enzyme-competent C3bot or enzyme-deficient C3bot156-181 peptide resulted in the upregulation of glutamate uptake by up to 22% compared to untreated cells. A similar enhancement of glutamate transport was also achieved by the classical phorbol-ester-mediated activation of protein kinase C subtypes. Yet comparable, effects elicited by C3 preparations seemed not to rely on PKCα, γ, ε, or ζ activation. Blocking of tyrosine phosphorylation by tyrosine kinase inhibitors prevented the observed effect mediated by C3bot and C3bot 26mer. By using biochemical and molecular biological assays we could rule out that the observed C3bot and C3bot 26mer-mediated effects solely resulted from enhanced transporter expression or translocation to the neuronal surface but was rather mediated by transporter phosphorylation at tyrosine residues that was found to be significantly enhanced following incubation with either full length protein or the 26mer C3 peptide.

Sex and brain region-specific regulation of serotonin transporter activity in synaptosomes in guanine nucleotide-binding protein G(q) alpha knockout mice.

The regulation of the serotonin transporter (SERT) by guanine nucleotide-binding protein alpha (Gα) q was investigated using Gαq knockout mice. In the absence of Gαq, SERT-mediated uptake of 5-hydroxytryptamine (5HT) was enhanced in midbrain and frontal cortex synaptosomes, but only in female mice. The mechanisms underlying this sexual dimorphism were investigated using quantitative western blot analysis revealing brain region-specific differences. In the frontal cortex, SERT protein expression was decreased in male knockout mice, seemingly explaining the sex-dependent variation in SERT activity. The differential expression of Gαi1 in female mice contributes to the sex differences in the midbrain. In fact, Gαi1 levels inversely correlate with 5HT uptake rates across both sexes and genotypes. Likely due to differential SERT regulation as well as sex differences in the expression of tryptophan hydroxylase 2, Gαq knockout mice also displayed sex- and genotype-dependent alterations in total 5HT tissue levels as determined by high-performance liquid chromatography. Gαq inhibitors, YM-254890 and BIM-46187, differentially affected SERT activity in both, synaptosomes and cultured cells. YM-254890 treatment mimicked the effect of Gαq knockout in the frontal cortex. BIM-46187, which promotes the nucleotide-free form of Gα proteins, substantially inhibited 5HT uptake, prompting us to hypothesise that Gαq interacts with SERT similarly as with G-protein-coupled receptors and inhibits SERT activity by modulating transport-associated conformational changes. Taken together, our findings reveal a novel mechanism of SERT regulation and impact our understanding of sex differences in diseases associated with dysregulation of serotonin transmission, such as depression and anxiety.

Vesicular Glutamate Transporters (SLCA17 A6, 7, 8) Control Synaptic Phosphate Levels.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate. VGLUTs were originally identified as sodium-dependent transporters of inorganic phosphate (Pi), but the physiological relevance of this activity remains unclear. Heterologous expression of all three VGLUTs greatly augments intracellular Pi levels. Using neuronal models, we show that translocation of VGLUTs to the plasma membrane during exocytosis results in highly increased Pi uptake. VGLUT-mediated Pi influx is counteracted by Pi efflux. Synaptosomes prepared from perinatal VGLUT2-/- mice that are virtually free of VGLUTs show drastically reduced cytosolic Pi levels and fail to import Pi. Glutamate partially competes with sodium (Na+)/Pi (NaPi)-uptake mediated by VGLUTs but does not appear to be transported. A nanobody that blocks glutamate transport by binding to the cytoplasmic domain of VGLUT1 abolishes Pi transport when co-expressed with VGLUT1. We conclude that VGLUTs have a dual function that is essential for both vesicular glutamate loading and Pi restoration in neurons.

The Higher Sensitivity of GABAergic Compared to Glutamatergic Neurons to Growth-Promoting C3bot Treatment Is Mediated by Vimentin.

The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of ß1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects.

Correction: Autoantibodies to synapsin I sequestrate synapsin I and alter synaptic function.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Autoantibodies to synapsin I sequestrate synapsin I and alter synaptic function.

Synapsin I is a phosphoprotein that coats the cytoplasmic side of synaptic vesicles and regulates their trafficking within nerve terminals. Autoantibodies against Syn I have been described in sera and cerebrospinal fluids of patients with numerous neurological diseases, including limbic encephalitis and clinically isolated syndrome; however, the effects and fate of autoantibodies in neurons are still unexplored. We found that in vitro exposure of primary hippocampal neurons to patient's autoantibodies to SynI decreased the density of excitatory and inhibitory synapses and impaired both glutamatergic and GABAergic synaptic transmission. These effects were reproduced with a purified SynI antibody and completely absent in SynI knockout neurons. Autoantibodies to SynI are internalized by FcγII/III-mediated endocytosis, interact with endogenous SynI, and promote its sequestration and intracellular aggregation. Neurons exposed to human autoantibodies to SynI display a reduced density of SVs, mimicking the SynI loss-of-function phenotype. Our data indicate that autoantibodies to intracellular antigens such as SynI can reach and inactivate their targets and suggest that an antibody-mediated synaptic dysfunction may contribute to the evolution and progression of autoimmune-mediated neurological diseases positive for SynI autoantibodies.

An Alternative Exon of CAPS2 Influences Catecholamine Loading into LDCVs of Chromaffin Cells.

The calcium-dependent activator proteins for secretion (CAPS) are priming factors for synaptic and large dense-core vesicles (LDCVs), promoting their entry into and stabilizing the release-ready state. A modulatory role of CAPS in catecholamine loading of vesicles has been suggested. Although an influence of CAPS on monoamine transporter function and on vesicle acidification has been reported, a role of CAPS in vesicle loading is disputed. Using expression of naturally occurring splice variants of CAPS2 into chromaffin cells from CAPS1/CAPS2 double-deficient mice of both sexes, we show that an alternative exon of 40 aa is responsible for enhanced catecholamine loading of LDCVs in mouse chromaffin cells. The presence of this exon leads to increased activity of both vesicular monoamine transporters. Deletion of CAPS does not alter acidification of vesicles. Our results establish a splice-variant-dependent modulatory effect of CAPS on catecholamine content in LDCVs.SIGNIFICANCE STATEMENT The calcium activator protein for secretion (CAPS) promotes and stabilizes the entry of catecholamine-containing vesicles of the adrenal gland into a release-ready state. Expression of an alternatively spliced exon in CAPS leads to enhanced catecholamine content in chromaffin granules. This exon codes for 40 aa with a high proline content, consistent with an unstructured loop present in the portion of the molecule generally thought to be involved in vesicle priming. CAPS variants containing this exon promote serotonin uptake into Chinese hamster ovary cells expressing either vesicular monoamine transporter. Epigenetic tuning of CAPS variants may allow modulation of endocrine adrenaline and noradrenaline release. This mechanism may extend to monoamine release in central neurons or in the enteric nervous system.

Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons.

Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim-/- mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim-/- astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.

Epitope specificity of anti-synapsin autoantibodies: Differential targeting of synapsin I domains.

OBJECTIVE: To identify the specific domains of the presynaptic protein synapsin targeted by recently described autoantibodies to synapsin. METHODS: Sera of 20 and CSF of two patients with different psychiatric and neurological disorders previously tested positive for immunoglobulin (Ig)G antibodies to full-length synapsin were screened for IgG against synapsin I domains using HEK293 cells transfected with constructs encoding different domains of rat synapsin Ia. Additionally, IgG subclasses were determined using full-length synapsin Ia. Serum and CSF from one patient were also screened for IgA autoantibodies to synapsin I domains. Sera from nine and CSF from two healthy subjects were analyzed as controls. RESULTS: IgG in serum from 12 of 20 IgG synapsin full-length positive patients, but from none of the healthy controls, bound to synapsin domains. Of these 12 sera, six bound to the A domain, five to the D domain, and one to the B- (and possibly A-), D-, and E-domains of synapsin I. IgG antibodies to the D-domain were also detected in one of the CSF samples. Determination of IgG subclasses detected IgG1 in two sera and one CSF, IgG2 in none of the samples, IgG3 in two sera, and IgG4 in eight sera. One patient known to be positive for IgA antibodies to full-length synapsin had IgA antibodies to the D-domain in serum and CSF. CONCLUSIONS: Anti-synapsin autoantibodies preferentially bind to either the A- or the D-domain of synapsin I.

Balance of Go1α and Go2α expression regulates motor function via the striatal dopaminergic system.

The heterotrimeric G-protein Go with its splice variants, Go1α and Go2α, seems to be involved in the regulation of motor function but isoform-specific effects are still unclear. We found that Go1α-/- knockouts performed worse on the rota-rod than Go2α-/- and wild-type (WT) mice. In Go1+2α-/- mice motor function was partially recovered. Furthermore, Go1+2α-/- mice showed an increased spontaneous motor activity. Compared to wild types or Go2α-/- mice, Go1+2α-/- mice developed increased behavioural sensitization following repetitive cocaine treatment, but failed to develop conditioned place preference. Analysis of dopamine concentration and expression of D1 and D2 receptors unravelled splice-variant-specific imbalances in the striatal dopaminergic system: In Go1α-/- mice dopamine concentration and vesicular monoamine uptake were increased compared to wild types. The expression of the D2 receptor was higher in Go1α-/- compared to wild type littermates, but unchanged in Go2α-/- mice. Deletion of both Go1α and Go2α re-established both dopamine and D2 receptor levels comparable to those in the wild-type. Cocaine treatment had no effect on the ratio of D1 receptor to D2 receptor in Go1+2α-/- mutants, but decreased this ratio in Go2α-/- mice. Finally, we observed that the deletion of Go1α led to a threefold higher striatal expression of Go2α. Taken together our data suggest that a balance in the expression of Go1α and Go2α sustains normal motor function. Deletion of either splice variant results in divergent behavioural and molecular alterations in the striatal dopaminergic system. Deletion of both splice variants partially restores the behavioural and molecular changes. Open Data: Materials are available on https://cos.io/our-services/open-science-badges/ https://osf.io/93n6m/.

Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔµH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔµH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons.

VGLUT1 Binding to Endophilin or Intersectin1 and Dynamin Phosphorylation in a Diurnal Context.

Glutamate is concentrated into synaptic vesicles (SV) by the vesicular glutamate transporters (VGLUT) 1 and 2. VGLUTs also harbor a Na+/Pi-transport activity when residing at the plasma membrane. Here we aimed to identify whether the diurnal switches of VGLUT1 parallels interactions with or modification of endocytic proteins. VGLUT1 and dynamin bind to SH3 domains of either endophilin (Enph) or intersectin 1 (ITSN1) harboring one or five SH3 domains A-E, respectively. We followed diurnal variations by pull down experiments using SH3 fusion protein and brains from mice entrained in a strict 24-h light-dark cycle (12-h light Zeitgeber (ZT) 0, 6; 12-h dark ZT 12 and 18). In pull downs with EnphSH3 interaction with VGLUT1 is high during the resting light and reduced during the active dark period while dynamin binding does not vary. This diurnal light/dark pattern depends on a functional period 2 gene and changes when animals are kept in complete darkness. Pull downs using ITSN1SH3 A reveal diurnally varying binding of VGLUT1 with slightly reduced VGLUT1/dynamin ratios at the beginning of the light (ZT 0) or the dark (ZT 12) period. Phosphorylation increases binding of VGLUT1 but not of dynamin to EnphSH3. In contrast binding of dynamin to ITSN1SH3 A decreases under phosphorylating conditions with no changes in VGLUT1 binding. Phosphorylation of dynamin at Ser 774 is high at ZT 6 and ZT 18 when more VGLUT1 is at the plasma membrane but low at ZT 0 and ZT 12 the diurnal peaks of VGLUT1 endocytosis. In conclusion the diurnally varying endocytosis of VGLUT1 involves differential interactions with the SH3 domains of Enph and ITSN1 and correlates with the de-phosphorylation of dynamin1.

The Rho ADP-ribosylating C3 exoenzyme binds cells via an Arg-Gly-Asp motif.

The Rho ADP-ribosylating C3 exoenzyme (C3bot) is a bacterial protein toxin devoid of a cell-binding or -translocation domain. Nevertheless, C3 can efficiently enter intact cells, including neurons, but the mechanism of C3 binding and uptake is not yet understood. Previously, we identified the intermediate filament vimentin as an extracellular membranous interaction partner of C3. However, uptake of C3 into cells still occurs (although reduced) in the absence of vimentin, indicating involvement of an additional host cell receptor. C3 harbors an Arg-Gly-Asp (RGD) motif, which is the major integrin-binding site, present in a variety of integrin ligands. To check whether the RGD motif of C3 is involved in binding to cells, we performed a competition assay with C3 and RGD peptide or with a monoclonal antibody binding to ß1-integrin subunit and binding assays in different cell lines, primary neurons, and synaptosomes with C3-RGD mutants. Here, we report that preincubation of cells with the GRGDNP peptide strongly reduced C3 binding to cells. Moreover, mutation of the RGD motif reduced C3 binding to intact cells and also to recombinant vimentin. Anti-integrin antibodies also lowered the C3 binding to cells. Our results indicate that the RGD motif of C3 is at least one essential C3 motif for binding to host cells and that integrin is an additional receptor for C3 besides vimentin.

Synapsin-antibodies in psychiatric and neurological disorders: Prevalence and clinical findings.

OBJECTIVE: To study the prevalence of autoantibodies to synapsin in patients with psychiatric and neurological disorders and to describe clinical findings in synapsin antibody positive patients. METHODS: Sera of 375 patients with different psychiatric and neurological disorders and sera of 97 healthy controls were screened (dilution 1:320) for anti-synapsin IgG using HEK293 cells transfected with rat synapsin Ia. Positive sera were further analyzed by immunoblots with brain tissue from wild type and synapsin knock out mice and with HEK293 cells transfected with human synapsin Ia and Ib. Binding of synapsin IgG positive sera to primary neurons was studied using murine hippocampal neurons. RESULTS: IgG in serum from 23 (6.1%) of 375 patients, but from none of the 97 healthy controls (p=0.007), bound to rat synapsin Ia transfected cells with a median (range) titer of 1:1000 (1:320-1:100,000). Twelve of the 23 positive sera reacted with a protein of the molecular size of synapsin I in immunoblots of wild type but not of synapsin knock out mouse brain tissue. Out of 19/23 positive sera available for testing, 13 bound to human synapsin Ia and 16 to human synapsin Ib transfected cells. Synapsin IgG positive sera stained fixed and permeabilized murine hippocampal neurons. Synapsin IgG positive patients had various psychiatric and neurological disorders. Tumors were documented in 2 patients (melanoma, small cell lung carcinoma); concomitant anti-neuronal or other autoantibodies were present in 8 patients. CONCLUSIONS: Autoantibodies to human synapsin Ia and Ib are detectable in a proportion of sera from patients with different psychiatric and neurological disorders, warranting further investigation into the potential pathophysiological relevance of these antibodies.

Regulation of the Hippocampal Network by VGLUT3-Positive CCK- GABAergic Basket Cells.

Hippocampal interneurons release the inhibitory transmitter GABA to regulate excitation, rhythm generation and synaptic plasticity. A subpopulation of GABAergic basket cells co-expresses the GABA/glycine vesicular transporters (VIAAT) and the atypical type III vesicular glutamate transporter (VGLUT3); therefore, these cells have the ability to signal with both GABA and glutamate. GABAergic transmission by basket cells has been extensively characterized but nothing is known about the functional implications of VGLUT3-dependent glutamate released by these cells. Here, using VGLUT3-null mice we observed that the loss of VGLUT3 results in a metaplastic shift in synaptic plasticity at Shaeffer's collaterals - CA1 synapses and an altered theta oscillation. These changes were paralleled by the loss of a VGLUT3-dependent inhibition of GABAergic current in CA1 pyramidal layer. Therefore presynaptic type III metabotropic could be activated by glutamate released from VGLUT3-positive interneurons. This putative presynaptic heterologous feedback mechanism inhibits local GABAergic tone and regulates the hippocampal neuronal network.

Serotonin Transporter Associated Protein Complexes Are Enriched in Synaptic Vesicle Proteins and Proteins Involved in Energy Metabolism and Ion Homeostasis.

The serotonin transporter (SERT) mediates Na+-dependent high-affinity serotonin uptake and plays a key role in regulating extracellular serotonin concentration in the brain and periphery. To gain novel insight into SERT regulation, we conducted a comprehensive proteomics screen to identify components of SERT-associated protein complexes in the brain by employing three independent approaches. In vivo SERT complexes were purified from rat brain using an immobilized high-affinity SERT ligand, amino-methyl citalopram. This approach was combined with GST pulldown and yeast two-hybrid screens using N- and C-terminal cytoplasmic transporter domains as bait. Potential SERT associated proteins detected by at least two of the interaction methods were subjected to gene ontology analysis resulting in the identification of functional protein clusters that are enriched in SERT complexes. Prominent clusters include synaptic vesicle proteins, as well as proteins involved in energy metabolism and ion homeostasis. Using subcellular fractionation and electron microscopy we provide further evidence that SERT is indeed associated with synaptic vesicle fractions, and colocalizes with small vesicular structures in axons and axon terminals. We also show that SERT is found in close proximity to mitochondrial membranes in both, hippocampal and neocortical regions. We propose a model of the SERT interactome, in which SERT is distributed between different subcellular compartments through dynamic interactions with site-specific protein complexes. Finally, our protein interaction data suggest novel hypotheses for the regulation of SERT activity and trafficking, which ultimately impact on serotonergic neurotransmission and serotonin dependent brain functions.

The intermediate filament protein vimentin is essential for axonotrophic effects of Clostridium botulinum C3 exoenzyme.

The type III intermediate filament protein vimentin was recently identified to mediate binding and uptake of Clostridium botulinum C3 exoenzyme (C3bot) in two cell lines. Here, we used primary neuronal cultures from vimentin knockout (Vim-/- ) mice to study the impact of vimentin on axonal growth and internalization of C3bot. In contrast to wild type, vimentin knockout neurons were insensitive to C3bot. Application of extracellular vimentin to Vim-/- neurons completely restored the growth-promoting effects of C3bot. In line with this uptake of C3bot into Vim-/- neurons was strongly decreased resulting in reduced ADP-ribosylation of RhoA and B as detected by an antibody recognizing selectively ADP-ribosylated RhoA/B. Again, uptake of C3bot into Vim-/- neurons was rescued by addition of extracellular vimentin. In addition, in purified embryonic stem cell-derived motor neurons that are devoid of glial cells C3bot elicited axonotrophic effects confining neuronal vimentin as a binding partner. Primary neuronal cultures from vimentin knockout (KO) mice were used to study the impact of vimentin on axonal growth and internalization of C3bot. In contrast to wild type, vimentin knockout neurons were insensitive to the axonotrophic effects of C3bot. Application of extracellular vimentin (recombinant vimentin) to vimentin KO neurons completely restored the growth-promoting effects of C3bot. In line with this uptake of C3bot into vimentin KO neurons was strongly decreased resulting in reduced ADP-ribosylation of RhoA and B as detected by an antibody recognizing selectively ADP-ribosylated RhoA/B.

Co-existence of Functionally Different Vesicular Neurotransmitter Transporters.

The vesicular transmitter transporters VGLUT, VGAT, VMAT2 and VAChT, define phenotype and physiological properties of neuronal subtypes. VGLUTs concentrate the excitatory amino acid glutamate, VGAT the inhibitory amino acid GABA, VMAT2 monoamines, and VAChT acetylcholine (ACh) into synaptic vesicle (SV). Following membrane depolarization SV release their content into the synaptic cleft. A strict segregation of vesicular transporters is mandatory for the precise functioning of synaptic communication and of neuronal circuits. In the last years, evidence accumulates that subsets of neurons express more than one of these transporters leading to synaptic co-release of different and functionally opposing transmitters and modulation of synaptic plasticity. Synaptic co-existence of transporters may change during pathological scenarios in order to ameliorate misbalances in neuronal activity. In addition, evidence increases that transporters also co-exist on the same vesicle providing another layer of regulation. Generally, vesicular transmitter loading relies on an electrochemical gradient ΔµH(+) driven by the proton ATPase rendering the lumen of the vesicle with respect to the cytosol positive (Δψ) and acidic (ΔpH). While the activity of VGLUT mainly depends on the Δψ component, VMAT, VGAT and VAChT work best at a high ΔpH. Thus, a vesicular synergy of transporters depending on the combination may increase or decrease the filling of SV with the principal transmitter. We provide an overview on synaptic co-existence of vesicular transmitter transporters including changes in the excitatory/inhibitory balance under pathological conditions. Additionally, we discuss functional aspects of vesicular synergy of transmitter transporters.

An essential role of acetylcholine-glutamate synergy at habenular synapses in nicotine dependence.

A great deal of interest has been focused recently on the habenula and its critical role in aversion, negative-reward and drug dependence. Using a conditional mouse model of the ACh-synthesizing enzyme choline acetyltransferase (Chat), we report that local elimination of acetylcholine (ACh) in medial habenula (MHb) neurons alters glutamate corelease and presynaptic facilitation. Electron microscopy and immuno-isolation analyses revealed colocalization of ACh and glutamate vesicular transporters in synaptic vesicles (SVs) in the central IPN. Glutamate reuptake in SVs prepared from the IPN was increased by ACh, indicating vesicular synergy. Mice lacking CHAT in habenular neurons were insensitive to nicotine-conditioned reward and withdrawal. These data demonstrate that ACh controls the quantal size and release frequency of glutamate at habenular synapses, and suggest that the synergistic functions of ACh and glutamate may be generally important for modulation of cholinergic circuit function and behavior.

Intrathecal immunoglobulin A and G antibodies to synapsin in a patient with limbic encephalitis.

OBJECTIVE: To report on the identification of intrathecally synthesized immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies to synapsin, a synaptic vesicle-associated protein, in a patient with limbic encephalitis. METHODS: Methods included clinical characterization, indirect immunofluorescence, immunoprecipitation, mass spectrometry, immunoblots of wild-type and synapsin I/II/III knockout mice, and cell-based assays with synapsin Ia, Ib, IIa, and IIb plasmids. RESULTS: A 69-year-old man presented with confusion, disorientation, seizures, and left hippocampal hyperintensities on MRI. CSF examinations revealed an intrathecal IgA and IgG synthesis. Except for IgG antibodies to voltage-gated potassium channels in CSF, screening for known neuronal autoantibodies in serum and CSF was negative. However, indirect immunofluorescence using the patient's CSF showed binding of IgA to mouse hippocampus, amygdala, and cerebellum. Immunoprecipitation with CSF IgA followed by mass spectrometry identified synapsin as autoantigenic target. Knockout tissues and cell-based assays confirmed that IgA and IgG in the patient's CSF and serum reacted with synapsin Ia, Ib, and IIa. Calculation of antibody indices proved intrathecal synthesis of anti-synapsin IgA and IgG. The patient responded clinically to immunotherapy but developed left hippocampal atrophy. CSF IgA or IgG of the patient did not bind to live, unfixed, and nonpermeabilized mouse hippocampal neurons, compatible with synapsin being an intracellular antigen. CONCLUSIONS: This report identifies isoforms of the synaptic vesicle-associated protein synapsin as targets of intrathecally produced IgA and IgG antibodies in a patient with limbic encephalitis. Future studies should clarify the prevalence and pathogenic relevance of anti-synapsin antibodies in limbic encephalitis.