Novel SYN1 Variant in Two Brothers with Focal Epilepsy and Their Prompt Response to Valproate.

Synapsins are neuron-specific phosphoproteins that modulate neurotransmitter release, synaptic plasticity, and molecular processes shaping higher brain functions. Pathogenic synapsin-1 (SYN1) variants are associated with epilepsy, intellectual disabilities, and behavioral problems. We detected a novel SYN1 variant [c.477_479delTGG (p.Gly160del)] in brothers with focal epilepsy with secondary generalization. The deleted amino acid was found to be highly conserved among mammalian species. In electroencephalography, the older brother showed a bioelectrical status epilepticus and was also diagnosed with attention deficit hyperactivity disorder. Behavioral abnormalities were seen before or after the seizures. Both patients responded quickly to treatment with valproate. Our case reports are consistent with the clinical heterogeneity of the pathogenic SYN1 variants described in the literature.

Interactions between Membraneless Condensates and Membranous Organelles at the Presynapse: A Phase Separation View of Synaptic Vesicle Cycle.

Action potential-induced neurotransmitter release in presynaptic boutons involves coordinated actions of a large list of proteins that are associated directly or indirectly with membrane structures including synaptic vesicles and plasma membranes. These proteins are often highly abundant in different synaptic bouton sub-compartments, and they rarely act alone. Instead, these proteins interact with each other forming intricate and distinct molecular complexes. Many of these complexes form condensed clusters on membrane surfaces. This review summarizes findings in recent years showing that many of presynaptic protein complex assemblies are formed via phase separation. These protein condensates extensively interact with lipid membranes via distinct modes, forming various mesoscale structures by different mode of organizations between membraneless condensates and membranous organelles. We discuss that such mesoscale interactions could have deep implications on mobilization, exocytosis, and retrieval of synaptic vesicles.

Multivalent electrostatic pi-cation interaction between synaptophysin and synapsin is responsible for the coacervation.

We recently showed that synaptophysin (Syph) and synapsin (Syn) can induce liquid-liquid phase separation (LLPS) to cluster small synaptic-like microvesicles in living cells which are highly reminiscent of SV cluster. However, as there is no physical interaction between them, the underlying mechanism for their coacervation remains unknown. Here, we showed that the coacervation between Syph and Syn is primarily governed by multivalent pi-cation electrostatic interactions among tyrosine residues of Syph C-terminal (Ct) and positively charged Syn. We found that Syph Ct is intrinsically disordered and it alone can form liquid droplets by interactions among themselves at high concentration in a crowding environment in vitro or when assisted by additional interactions by tagging with light-sensitive CRY2PHR or subunits of a multimeric protein in living cells. Syph Ct contains 10 repeated sequences, 9 of them start with tyrosine, and mutating 9 tyrosine to serine (9YS) completely abolished the phase separating property of Syph Ct, indicating tyrosine-mediated pi-interactions are critical. We further found that 9YS mutation failed to coacervate with Syn, and since 9YS retains Syph's negative charge, the results indicate that pi-cation interactions rather than simple charge interactions are responsible for their coacervation. In addition to revealing the underlying mechanism of Syph and Syn coacervation, our results also raise the possibility that physiological regulation of pi-cation interactions between Syph and Syn during synaptic activity may contribute to the dynamics of synaptic vesicle clustering.

Protonation states at different pH, conformational changes and impact of glycosylation in synapsin Ia.

Synapsin I (SynI) is the most abundant brain phosphoprotein present at presynaptic terminals that regulates neurotransmitter release, clustering of synaptic vesicles (SVs) at active zones, and stimulates synaptogenesis and neurite outgrowth. Earlier studies have established that SynI displays pH-dependent tethering of SVs to actin filaments and exhibits a maximum binding around neutral pH, however, the effect of pH shift from acidic to basic on the conformational stability of SynI has not been explored yet. Another important aspect of SynIa is its O-GlcNAcylation (O-GlcNac) at the Thr87 position, which is responsible for the positive regulation of synaptic plasticity linked to learning and memory in mice. Furthermore, reduced levels of O-GlcNAc have been observed in Alzheimer's disease, suggesting a possible link to deficits in synaptic plasticity. In this study, the effect of pH and glycosylation on the structure and functional stability of SynIa is determined through molecular dynamics (MD) simulation approach. The 3D structure of SynIa was established via threading-based homology modeling methods. It was observed that the structure of SynIa adopts extended conformational changes as the pH shifts from acidic to basic, resulting in a compact conformation at pH 8.0. Moreover, the results obtained by comparing the glycosylated and unglycosylated protein indicated that the glycan moiety imparts stability to the protein by forming intramolecular hydrogen bond interactions with the protein residues. The results indicate that although O-GlcNAc moieties do not induce a significant change in SynIa structure they minimize protein dynamics, likely leading to enhanced protein stability.

Synapsin Condensates Recruit alpha-Synuclein.

Neurotransmission relies on the tight spatial and temporal regulation of the synaptic vesicle (SV) cycle. Nerve terminals contain hundreds of SVs that form tight clusters. These clusters represent a distinct liquid phase in which one component of the phase are SVs and the other synapsin 1, a highly abundant synaptic protein. Another major family of disordered proteins at the presynapse includes synucleins, most notably α-synuclein. The precise physiological role of α-synuclein in synaptic physiology remains elusive, albeit its role has been implicated in nearly all steps of the SV cycle. To determine the effect of α-synuclein on the synapsin phase, we employ the reconstitution approach using natively purified SVs from rat brains and the heterologous cell system to generate synapsin condensates. We demonstrate that synapsin condensates recruit α-synuclein, and while enriched into these synapsin condensates, α-synuclein still maintains its high mobility. The presence of SVs enhances the rate of synapsin/α-synuclein condensation, suggesting that SVs act as catalyzers for the formation of synapsin condensates. Notably, at physiological salt and protein concentrations, α-synuclein alone is not able to cluster isolated SVs. Excess of α-synuclein disrupts the kinetics of synapsin/SV condensate formation, indicating that the molar ratio between synapsin and α-synuclein is important in assembling the functional condensates of SVs. Understanding the molecular mechanism of α-synuclein interactions at the nerve terminals is crucial for clarifying the pathogenesis of synucleinopathies, where α-synuclein, synaptic proteins and lipid organelles all accumulate as insoluble intracellular inclusions.

The Role of Low Complexity Regions in Protein Interaction Modes: An Illustration in Huntingtin.

Low complexity regions (LCRs) are very frequent in protein sequences, generally having a lower propensity to form structured domains and tending to be much less evolutionarily conserved than globular domains. Their higher abundance in eukaryotes and in species with more cellular types agrees with a growing number of reports on their function in protein interactions regulated by post-translational modifications. LCRs facilitate the increase of regulatory and network complexity required with the emergence of organisms with more complex tissue distribution and development. Although the low conservation and structural flexibility of LCRs complicate their study, evolutionary studies of proteins across species have been used to evaluate their significance and function. To investigate how to apply this evolutionary approach to the study of LCR function in protein-protein interactions, we performed a detailed analysis for Huntingtin (HTT), a large protein that is a hub for interaction with hundreds of proteins, has a variety of LCRs, and for which partial structural information (in complex with HAP40) is available. We hypothesize that proteins RASA1, SYN2, and KAT2B may compete with HAP40 for their attachment to the core of HTT using similar LCRs. Our results illustrate how evolution might favor the interplay of LCRs with domains, and the possibility of detecting multiple modes of LCR-mediated protein-protein interactions with a large hub such as HTT when enough protein interaction data is available.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery.

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood-brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

Probing Synaptic Signaling with Optogenetic Stimulation and Genetically Encoded Calcium Reporters.

Optogenetics provides a powerful approach for investigating neuronal electrophysiology at the scale required for drug discovery applications. Probing synaptic function with high throughput using optogenetics requires robust tools that enable both precise stimulation of and facile readout of synaptic activity. Here we describe two functional assays to achieve this end: (1) a pre-synaptic calcium assay that utilizes the channelrhodopsin, CheRiff, patterned optogenetic stimulus, and the pre-synaptically targeted calcium reporter jRGECO1a to monitor pre-synaptic changes in calcium influx and (2) a synaptic transmission assay in which CheRiff and cytosolic jRGECO1a are expressed in non-overlapping sets of neurons, enabling pre-synaptic stimulation and post-synaptic readout of activity. This chapter describes the methodology and practical considerations for implementation of these two assays.

Vesicle Clustering in a Living Synapse Depends on a Synapsin Region that Mediates Phase Separation.

Liquid-liquid phase separation is an increasingly recognized mechanism for compartmentalization in cells. Recent in vitro studies suggest that this organizational principle may apply to synaptic vesicle clusters. Here we test this possibility by performing microinjections at the living lamprey giant reticulospinal synapse. Axons are maintained at rest to examine whether reagents introduced into the cytosol enter a putative liquid phase to disrupt critical protein-protein interactions. Compounds that perturb the intrinsically disordered region of synapsin, which is critical for liquid phase organization in vitro, cause dispersion of synaptic vesicles from resting clusters. Reagents that perturb SH3 domain interactions with synapsin are ineffective at rest. Our results indicate that synaptic vesicles at a living central synapse are organized as a distinct liquid phase maintained by interactions via the intrinsically disordered region of synapsin.

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.

Correction of cognitive deficits in mouse models of Down syndrome by a pharmacological inhibitor of DYRK1A.

Growing evidence supports the implication of DYRK1A in the development of cognitive deficits seen in Down syndrome (DS) and Alzheimer's disease (AD). We here demonstrate that pharmacological inhibition of brain DYRK1A is able to correct recognition memory deficits in three DS mouse models with increasing genetic complexity [Tg(Dyrk1a), Ts65Dn, Dp1Yey], all expressing an extra copy of Dyrk1a Overexpressed DYRK1A accumulates in the cytoplasm and at the synapse. Treatment of the three DS models with the pharmacological DYRK1A inhibitor leucettine L41 leads to normalization of DYRK1A activity and corrects the novel object cognitive impairment observed in these models. Brain functional magnetic resonance imaging reveals that this cognitive improvement is paralleled by functional connectivity remodelling of core brain areas involved in learning/memory processes. The impact of Dyrk1a trisomy and L41 treatment on brain phosphoproteins was investigated by a quantitative phosphoproteomics method, revealing the implication of synaptic (synapsin 1) and cytoskeletal components involved in synaptic response and axonal organization. These results encourage the development of DYRK1A inhibitors as drug candidates to treat cognitive deficits associated with DS and AD.

A liquid phase of synapsin and lipid vesicles.

Neurotransmitter-containing synaptic vesicles (SVs) form tight clusters at synapses. These clusters act as a reservoir from which SVs are drawn for exocytosis during sustained activity. Several components associated with SVs that are likely to help form such clusters have been reported, including synapsin. Here we found that synapsin can form a distinct liquid phase in an aqueous environment. Other scaffolding proteins could coassemble into this condensate but were not necessary for its formation. Importantly, the synapsin phase could capture small lipid vesicles. The synapsin phase rapidly disassembled upon phosphorylation by calcium/calmodulin-dependent protein kinase II, mimicking the dispersion of synapsin 1 that occurs at presynaptic sites upon stimulation. Thus, principles of liquid-liquid phase separation may apply to the clustering of SVs at synapses.

FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments.

Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes.

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, ß-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

Serotonin-immunoreactive neurons in the ventral nerve cord of Remipedia (Crustacea): support for a sister group relationship of Remipedia and Hexapoda?

BACKGROUND: Remipedia were initially seen as a primitive taxon within Pancrustacea based on characters considered ancestral, such as the homonomously segmented trunk. Meanwhile, several morphological and molecular studies proposed a more derived position of Remipedia within Pancrustacea, including a sister group relationship to Hexapoda. Because of these conflicting hypotheses, fresh data are crucial to contribute new insights into euarthropod phylogeny. The architecture of individually identifiable serotonin-immunoreactive neurons has successfully been used for phylogenetic considerations in Euarthropoda. Here, we identified neurons in three species of Remipedia with an antiserum against serotonin and compared our findings to reconstructed ground patterns in other euarthropod taxa. Additionally, we traced neurite connectivity and neuropil outlines using antisera against acetylated α-tubulin and synapsin. RESULTS: The ventral nerve cord of Remipedia displays a typical rope-ladder-like arrangement of separate metameric ganglia linked by paired longitudinally projecting connectives. The peripheral projections comprise an intersegmental nerve, consisting of two branches that fuse shortly after exiting the connectives, and the segmental anterior and posterior nerve. The distribution and morphology of serotonin-immunoreactive interneurons in the trunk segments is highly conserved within the remipede species we analyzed, which allows for the reconstruction of a ground pattern: two posterior and one anterior pair of serotonin-immunoreactive neurons that possess a single contralateral projection. Additionally, three pairs of immunoreactive neurons are found in the medial part of each hemiganglion. In one species (Cryptocorynetes haptodiscus), the anterior pair of immunoreactive neurons is missing. CONCLUSIONS: The anatomy of the remipede ventral nerve cord with its separate metameric ganglia mirrors the external morphology of the animal's trunk. The rope-ladder-like structure and principal architecture of the segmental ganglia in Remipedia corresponds closely to that of other Euarthropoda. A comparison of the serotonin-immunoreactive cell arrangement of Remipedia to reconstructed ground patterns of major euarthropod taxa supports a homology of the anterior and posterior neurons in Pancrustacea. These neurons in Remipedia possess unbranched projections across the midline, pointing towards similarities to the hexapod pattern. Our findings are in line with a growing number of phylogenetic investigations proposing Remipedia to be a rather derived crustacean lineage that perhaps has close affinities to Hexapoda.

Epileptogenic Q555X SYN1 mutant triggers imbalances in release dynamics and short-term plasticity.

Synapsin I (SynI) is a synaptic vesicle (SV) phosphoprotein playing multiple roles in synaptic transmission and plasticity by differentially affecting crucial steps of SV trafficking in excitatory and inhibitory synapses. SynI knockout (KO) mice are epileptic, and nonsense and missense mutations in the human SYN1 gene have a causal role in idiopathic epilepsy and autism. To get insights into the mechanisms of epileptogenesis linked to SYN1 mutations, we analyzed the effects of the recently identified Q555X mutation on neurotransmitter release dynamics and short-term plasticity (STP) in excitatory and inhibitory synapses. We used patch-clamp electrophysiology coupled to electron microscopy and multi-electrode arrays to dissect synaptic transmission of primary SynI KO hippocampal neurons in which the human wild-type and mutant SynI were expressed by lentiviral transduction. A parallel decrease in the SV readily releasable pool in inhibitory synapses and in the release probability in excitatory synapses caused a marked reduction in the evoked synchronous release. This effect was accompanied by an increase in asynchronous release that was much more intense in excitatory synapses and associated with an increased total charge transfer. Q555X-hSynI induced larger facilitation and post-tetanic potentiation in excitatory synapses and stronger depression after long trains in inhibitory synapses. These changes were associated with higher network excitability and firing/bursting activity. Our data indicate that imbalances in STP and release dynamics of inhibitory and excitatory synapses trigger network hyperexcitability potentially leading to epilepsy/autism manifestations.

Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice.

Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the ß2-selective antagonist dihydro-ß-erythroidine (DHßE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20min prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHßE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through ß2-containing nicotinic receptors.

Synapsin-1 and tau reciprocal O-GlcNAcylation and phosphorylation sites in mouse brain synaptosomes

O-linked N-acetylglucosamine (O-GlcNAc) represents a key regulatory post-translational modification (PTM) that is reversible and often reciprocal with phosphorylation of serine and threonine at the same or nearby residues. Although recent technical advances in O-GlcNAc site-mapping methods combined with mass spectrometry (MS) techniques have facilitated study of the fundamental roles of O-GlcNAcylation in cellular processes, an efficient technique for examining the dynamic, reciprocal relationships between O-GlcNAcylation and phosphorylation is needed to provide greater insights into the regulatory functions of O-GlcNAcylation. Here, we describe a strategy for selectively identifying both O-GlcNAc- and phospho-modified sites. This strategy involves metal affinity separation of O-GlcNAcylated and phosphorylated peptides, beta-elimination of O-GlcNAcyl or phosphoryl functional groups from the separated peptides followed by dithiothreitol (DTT) conjugation (BEMAD), affinity purification of DTT-conjugated peptides using thiol affinity chromatography, and identification of formerly O-GlcNAcylated or phosphorylated peptides by MS. The combined metal affinity separation and BEMAD approach allows selective enrichment of O-GlcNAcylated peptides over phosphorylated counterparts. Using this approach with mouse brain synaptosomes, we identified the serine residue at 605 of the synapsin-1 peptide, 603QASQAGPGPR612, and the serine residue at 692 of the tau peptide, 688SPVVSGDTSPR698, which were found to be potential reciprocal O-GlcNAcylation and phosphorylation sites. These results demonstrate that our strategy enables mapping of the reciprocal site occupancy of O-GlcNAcylation and phosphorylation of proteins, which permits the assessment of cross-talk between these two PTMs and their regulatory roles.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Synapsin II is involved in the molecular pathway of lithium treatment in bipolar disorder.

Bipolar disorder (BD) is a debilitating psychiatric condition with a prevalence of 1-2% in the general population that is characterized by severe episodic shifts in mood ranging from depressive to manic episodes. One of the most common treatments is lithium (Li), with successful response in 30-60% of patients. Synapsin II (SYN2) is a neuronal phosphoprotein that we have previously identified as a possible candidate gene for the etiology of BD and/or response to Li treatment in a genome-wide linkage study focusing on BD patients characterized for excellent response to Li prophylaxis. In the present study we investigated the role of this gene in BD, particularly as it pertains to Li treatment. We investigated the effect of lithium treatment on the expression of SYN2 in lymphoblastoid cell lines from patients characterized as excellent Li-responders, non-responders, as well as non-psychiatric controls. Finally, we sought to determine if Li has a cell-type-specific effect on gene expression in neuronal-derived cell lines. In both in vitro models, we found SYN2 to be modulated by the presence of Li. By focusing on Li-responsive BD we have identified a potential mechanism for Li response in some patients.