Synapsin IIb Research Articles

Distinct and dementia-related synaptopathy in the hippocampus after military blast exposures.

Explosive shockwaves, and other types of blast exposures, are linked to injuries commonly associated with military service and to an increased risk for the onset of dementia. Neurological complications following a blast injury, including depression, anxiety, and memory problems, often persist even when brain damage is undetectable. Here, hippocampal explants were exposed to the explosive 1,3,5‐trinitro‐1,3,5‐triazinane (RDX) to identify indicators of blast‐induced changes within important neuronal circuitries. Highly controlled detonations of small, 1.7‐gram RDX spherical charges reduced synaptic markers known to be downregulated in cognitive disorders, but without causing overt neuronal loss or astroglial responses. In the absence of neuromorphological alterations, levels of synaptophysin, GluA1, and synapsin IIb were significantly diminished within 24 hr, and these synaptic components exhibited progressive reductions following blast exposure as compared to their stable maintenance in control explants. In contrast, labeling of the synapsin IIa isoform remained unaltered, while neuropilar staining of other markers decreased, including synapsin IIb and neural cell adhesion molecule (NCAM) isoforms, along with evidence of NCAM proteolytic breakdown. NCAM180 displayed a distinct decline after the RDX blasts, whereas NCAM140 and NCAM120 exhibited smaller or no deterioration, respectively. Interestingly, the extent of synaptic marker reduction correlated with AT8‐positive tau levels, with tau pathology stochastically found in CA1 neurons and their dendrites. The decline in synaptic components was also reflected in the size of evoked postsynaptic currents recorded from CA1 pyramidals, which exhibited a severe and selective reduction. The identified indicators of blast‐mediated synaptopathy point to the need for early biomarkers of explosives altering synaptic integrity with links to dementia risk, to advance strategies for both cognitive health and therapeutic monitoring.

A novel pathway regulates social hierarchy via lncRNA AtLAS and postsynaptic synapsin IIb.

Dominance hierarchy is a fundamental phenomenon in grouped animals and human beings, however, the underlying regulatory mechanisms remain elusive. Here, we report that an antisense long non-coding RNA (lncRNA) of synapsin II, named as AtLAS, plays a crucial role in the regulation of social hierarchy. AtLAS is decreased in the prefrontal cortical excitatory pyramidal neurons of dominant mice; consistently, silencing or overexpression of AtLAS increases or decreases the social rank, respectively. Mechanistically, we show that AtLAS regulates alternative polyadenylation of synapsin II gene and increases synapsin 2b (syn2b) expression. Syn2b reduces AMPA receptor (AMPAR)-mediated excitatory synaptic transmission through a direct binding with AMPAR at the postsynaptic sitevia its unique C-terminal sequence. Moreover, a peptide disrupting the binding of syn2b with AMPARs enhances the synaptic strength and social ranks. These findings reveal a novel role for lncRNA AtLAS and its target syn2b in the regulation of social behaviors by controlling postsynaptic AMPAR trafficking.

Reduced expression of synapsin II in a chronic phencyclidine preclinical rat model of schizophrenia.

Schizophrenia is a mental disorder characterized by positive symptoms, negative symptoms, and cognitive dysfunction. Phencyclidine (PCP)-a N-methyl-D-aspartate (NMDA) receptor antagonist-induces symptoms indistinguishable from those of schizophrenia. A reduction of the phosphoprotein synapsin II has also been implicated in schizophrenia and has a well-known role in the maintenance of the presynaptic reserve pool and vesicle mobilization. This study assessed the behavioral and biochemical outcomes of chronic NMDA receptor antagonism in rodents and its implications for the pathophysiology of schizophrenia. Sprague Dawley rats received saline or chronic PCP (5mg/kg/day) for 14days via surgically implanted Alzet® osmotic mini-pumps. Following the treatment period, rats were tested with a series of behavioral paradigms, including locomotor activity, social interaction, and sensorimotor gating. Following behavioral assessment, the medial prefrontal cortex (mPFC) of all rats was isolated for synapsin II protein analysis. Chronic PCP treatment yielded a hyper-locomotive state (p=0.0256), reduced social interaction (p=0.0005), and reduced pre-pulse inhibition (p<0.0001) in comparison to saline-treated controls. Synapsin IIa (p<0.0001) and IIb (p<0.0071) levels in the mPFC of chronically treated PCP rats were reduced in comparison to the saline group. Study results confirm that rats subject to chronic PCP treatment display behavioral phenotypes similar to established preclinical animal models of schizophrenia. Reduction of synapsin II expression in this context implicates the role of this protein in the pathophysiology of schizophrenia and sheds light on the longer-term consequences of NMDA receptor antagonism facilitated by chronic PCP treatment.

Differential Expression of Synapsin I and II upon Treatment by Lithium and Valproic Acid in Various Brain Regions.

IntroductionDue to the heterogeneity of psychiatric illnesses and overlapping mechanisms, patients with psychosis are differentially responsive to pharmaceutical drugs. In addition to having therapeutic effects for schizophrenia and bipolar disorder, antipsychotics and mood stabilizers have many clinical applications and are used unconventionally due to their direct and indirect effects on neurotransmitters. Synapsins, a family of neuronal phosphoproteins, play a key regulatory role in neurotransmitter release at synapses. In this study, we investigated the effects of mood stabilizers, lithium, and valproic acid on synapsin gene expression in the rat brain.MethodsIntraperitoneal injections of saline, lithium, and valproic acid were administered to male Sprague Dawley rats twice daily for 14 d, corresponding to their treatment group. Following decapitation and brain tissue isolation, mRNA was extracted from various brain regions including the hippocampus, striatum, prefrontal cortex, and frontal cortex.ResultsBiochemical analysis revealed that lithium significantly increased gene expression of synapsin I in the striatum, synapsin IIa in the hippocampus and prefrontal cortex, and synapsin IIb in the hippocampus and striatum. Valproic acid significantly increased synapsin IIa in the hippocampus and prefrontal cortex, as well as synapsin IIb in the hippocampus and striatum.ConclusionThese significant changes in synapsin I and II expression may implicate a common transcription factor, early growth response 1, in its mechanistic pathway. Overall, these results elucidate mechanisms through which lithium and valproic acid act on downstream targets compared with antipsychotics and provide deeper insight on the involvement of synaptic proteins in treating neuropsychiatric illnesses.

Inhibitor of Endocannabinoid Deactivation Protects Against In Vitro and In Vivo Neurotoxic Effects of Paraoxon.

The anticholinesterase paraoxon (Pxn) is related to military nerve agents that increase acetylcholine levels, trigger seizures, and cause excitotoxic damage in the brain. In rat hippocampal slice cultures, high-dose Pxn was applied resulting in a presynaptic vulnerability evidenced by a 64% reduction in synapsin IIb (syn IIb) levels, whereas the postsynaptic protein GluR1 was unchanged. Other signs of Pxn-induced cytotoxicity include the oxidative stress-related production of stable 4-hydroxynonenal (4-HNE)-protein adducts. Next, the Pxn toxicity was tested for protective effects by the fatty acid amide hydrolase (FAAH) inhibitor AM5206, a compound linked to enhanced repair signaling through the endocannabinoid pathway. The Pxn-mediated declines in syn IIb and synaptophysin were prevented by AM5206 in the slice cultures. To test if the protective results in the slice model translate to an in vivo model, AM5206 was injected i.p. into rats, followed immediately by subcutaneous Pxn administration. The toxin caused a pathogenic cascade initiated by seizure events, leading to presynaptic marker decline and oxidative changes in the hippocampus and frontal cortex. AM5206 exhibited protective effects including the reduction of seizure severity by 86%, and improving balance and coordination measured 24h post-insult. As observed in hippocampal slices, the FAAH inhibitor also prevented the Pxn-induced loss of syn IIb in vivo. In addition, the AM5206 compound reduced the 4-HNE modifications of proteins and the β1 integrin activation events both in vitro and in vivo. These results indicate that Pxn exposure produces oxidative and synaptic toxicity that leads to the behavioral deficits manifested by the neurotoxin. In contrast, the presence of FAAH inhibitor AM5206 offsets the pathogenic cascade elicited by the Pxn anticholinesterase.

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.

Synapsin IIb as a functional marker of submissive behavior.

Dominance and submissiveness are important functional elements of the social hierarchy. By employing selective breeding based on a social interaction test, we developed mice with strong and stable, inheritable features of dominance and submissiveness. In order to identify candidate genes responsible for dominant and submissive behavior, we applied transcriptomic and proteomic studies supported by molecular, behavioral and pharmacological approaches. We clearly show here that the expression of Synapsin II isoform b (Syn IIb) is constitutively upregulated in the hippocampus and striatum of submissive mice in comparison to their dominant and wild type counterparts. Moreover, the reduction of submissive behavior achieved after mating and delivery was accompanied by a marked reduction of Syn IIb expression. Since submissiveness has been shown to be associated with depressive-like behavior, we applied acute SSRI (Paroxetine) treatment to reduce submissiveness in studied mice. We found that reduction of submissive behavior evoked by Paroxetine was paired with significantly decreased Syn IIb expression. In conclusion, our findings indicate that submissiveness, known to be an important element of depressive-like behavioral abnormalities, is strongly linked with changes in Syn IIb expression.

Sleep fragmentation induces reduction of synapsin II in rat hippocampus

Continuation of sleep is important for learning and memory. Some proteins are differentially expressed in specific brain regions by normal sleep/wake cycle or sleep deprivation, suggesting that specific proteins are regulated by sleep/wake cycle and could play an important role in regulation of normal brain function associated with sleep in the regions. The hippocampus is a major region of the brain accounting for memory formation and organization, and its specific function could be altered by change of synapse-related molecular components. To examine sleep fragmentation-induced alteration of proteins in the hippocampus, two-dimensional gel electrophoresis was performed. Thirty-six male Wistar rats were randomly divided into the home cage group, sleep fragmentation group, and exercise control group. Four day sleep fragmentation was accomplished by a forced walking wheel system with a wheel on/off cycle of 30-s on/90-s off to mimic the arousal that is common in patients with severe sleep apnea. Time interval of exercise control was set at 10 min on/30 min off. Of 58 spots that were differentially expressed among the groups, one protein was consistently reduced in the sleep fragmented group compared to other groups. The protein was identified as synapsin IIa by MALDI-TOF mass spectrometry. Real-time polymerase chain reaction and Western blot analysis confirmed downregulated expression of synapsin IIa and another isoform, synapsin IIb. The results suggest that sleep fragmentation induces molecular changes accounting for neurotransmitter release, integrity of the reserve synaptic vesicle pool, and synaptic plasticity.

Synapsin II gene expression in the dorsolateral prefrontal cortex of brain specimens from patients with schizophrenia and bipolar disorder: effect of lifetime intake of antipsychotic drugs

Synapsins are neuronal phosphoproteins crucial to regulating the processes required for normal neurotransmitter release. Synapsin II, in particular, has been implied as a candidate gene for schizophrenia. This study investigated synapsin II mRNA expression, using Real Time RT-PCR, in coded dorsolateral prefrontal cortical samples provided by the Stanley Foundation Neuropathology Consortium. Synapsin IIa was decreased in patients with schizophrenia when compared to both healthy subjects and patients with bipolar disorder, whereas the synapsin IIb was only significantly reduced in patients with schizophrenia when compared to healthy subjects, but not patients with bipolar disorder. Furthermore, lifetime antipsychotic drug use was positively associated with synapsin IIa expression in patients with schizophrenia. Results suggest that impairment of synaptic transmission by synapsin II reduction may contribute to dysregulated convergent molecular mechanisms which result in aberrant neural circuits that characterize schizophrenia, while implicating involvement of synapsin II in therapeutic mechanisms of currently prescribed antipsychotic drugs.

Synapsin-dependent development of glutamatergic synaptic vesicles and presynaptic plasticity in postnatal mouse brain

Inactivation of the genes encoding the neuronal, synaptic vesicle-associated proteins synapsin I and II leads to severe reductions in the number of synaptic vesicles in the CNS. We here define the postnatal developmental period during which the synapsin I and/or II proteins modulate synaptic vesicle number and function in excitatory glutamatergic synapses in mouse brain. In wild-type mice, brain levels of both synapsin I and synapsin IIb showed developmental increases during synaptogenesis from postnatal days 5–20, while synapsin IIa showed a protracted increase during postnatal days 20–30. The vesicular glutamate transporters (VGLUT) 1 and VGLUT2 showed synapsin-independent development during postnatal days 5–10, following which significant reductions were seen when synapsin-deficient brains were compared with wild-type brains following postnatal day 20. A similar, synapsin-dependent developmental profile of vesicular glutamate uptake occurred during the same age periods. Physiological analysis of the development of excitatory glutamatergic synapses, performed in the CA1 stratum radiatum of the hippocampus from the two genotypes, showed that both the synapsin-dependent part of the frequency facilitation and the synapsin-dependent delayed response enhancement were restricted to the period after postnatal day 10. Our data demonstrate that while both synaptic vesicle number and presynaptic short-term plasticity are essentially independent of synapsin I and II prior to postnatal day 10, maturation and function of excitatory synapses appear to be strongly dependent on synapsin I and II from postnatal day 20.

Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

Glut4 exocytosis in adipocytes uses protein machinery that is shared with other regulated secretory processes. Synapsins are phosphoproteins that regulate a ;reserve pool' of vesicles clustered behind the active zone in neurons. We found that adipocytes (primary cells and the 3T3-L1 cell line) express synapsin IIb mRNA and protein. Synapsin IIb co-localizes with Glut4 in perinuclear vesicle clusters. To test whether synapsin plays a role in Glut4 traffic, a site 1 phosphorylation mutant (S10A synapsin) was expressed in 3T3-L1 adipocytes. Interestingly, expression of S10A synapsin increased basal cell surface Glut4 almost fourfold (50% maximal insulin effect). Insulin caused a further twofold translocation of Glut4 in these cells. Expression of the N-terminus of S10A synapsin (amino acids 1-118) was sufficient to inhibit basal Glut4 retention. Neither wild-type nor S10D synapsin redistributed Glut4. S10A synapsin did not elevate surface levels of the transferrin receptor in adipocytes or Glut4 in fibroblasts. Therefore, S10A synapsin is inhibiting the specialized process of basal intracellular retention of Glut4 in adipocytes, without affecting general endocytic cycling. While mutant forms of many proteins inhibit Glut4 exocytosis in response to insulin, S10A synapsin is one of only a few that specifically inhibits Glut4 retention in basal adipocytes. These data indicate that the synapsins are important regulators of membrane traffic in many cell types.

Synapsin IIb interacts with the C‐terminal SH2 and SH3 domains of PLCγ1 and inhibits its enzymatic activity

To elucidate the function of PLCgamma1, we have investigated the proteins that bind to its SH (Src homology) domain. Immunoscreening was performed with purified antisera specific for SH223 (two SH2 and one SH3)-binding proteins. Several immunoreactive clones were identified as putative binding proteins and one of them was identified as synapsin IIb. We demonstrate a stable association between PLCgamma1 and synapsin IIb, which binds the carboxyl terminal SH2 and SH3 domains of the enzyme and inhibits it.

Upregulation of synapsin IIa and IIb mRNAs in the paraventricular and supraoptic nuclei in chronic salt loaded and lactating rats.

The effects of chronic salt loading (2% saline to drink for 5 and 10 days), gestation, lactation and adrenalectomy on the expression of synapsin IIa and IIb genes were examined in the rat paraventricular (PVN) and supraoptic nuclei (SON), using in situ hybridization histochemistry. In each control, synapsin IIa and IIb genes were moderately expressed in the magnocellular division of the PVN and SON, while few transcripts of synapsin IIa and IIb were observed in the parvocellular division of the PVN. Chronic salt loading, gestation on day 21 and lactation on day 10 caused significant increases in synapsin IIa and IIb transcripts in the magnocellular division of the PVN and SON, compared to each control. Although corticotropin-releasing hormone transcripts in the parvocellular division of the PVN were significantly increased in the adrenalectomized rats, no changes in the transcripts of synapsin IIa and IIb were observed throughout the PVN. These results suggest that physiological stimuli such as osmotic challenge and lactation potently increase synapsin IIa and IIb mRNAs in the magnocellular neurons of the PVN and SON.

Synapsins as major neuronal Ca2+/S100A1-interacting proteins

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

Synapsins as major neuronal Ca2+/S100A1-interacting proteins

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

Interaction of synapsin IIb with calmodulin: identification of a high affinity site.

Interaction of synapsin IIb with calmodulin: identification of a high affinity site.

Differential distribution of synapsin IIa and IIb mRNAs in various brain structures and the effect of chronic morphine administration on the regional expression of these isoforms.

Quantitative reverse transcriptase-polymerase chain reaction and in situ hybridization techniques were used to determine the regional distribution of synapsin IIa and IIb mRNAs in rat central nervous system and to assess the effect of chronic morphine administration on the gene expression of these two isoforms of synapsin II. These isoforms are members of a family of neuron-specific phosphoproteins thought to be involved in the regulation of neurotransmitter release. Our data demonstrate the widespread distribution, yet regionally variable expression, of synapsin IIa and IIb mRNAs throughout the adult rat brain and spinal cord. The ratios of the relative abundance of synapsins IIa and IIb differed by up to 4.5-fold among the various regions studied. Synapsin IIa and IIb mRNAs were shown to be highly concentrated in the thalamus and in the hippocampus, whereas lower concentrations were found in most other central nervous system structures. In this study, we show differential regulation by morphine of synapsins IIa and IIb in various regions of the brain. In the striatum, a 2.4-fold increase was observed in the levels of synapsin IIa mRNA following chronic morphine regime, whereas no change was found for synapsin IIb. On the other hand, mRNA levels of synapsin IIb in spinal cord of chronically treated rats were markedly decreased (by 62%), while no alterations were observed in synapsin IIa. Selective regulation by morphine has also been demonstrated in several other central nervous system structures. The opiate-induced regulation of the gene expression of synapsin II isoforms could be viewed as one of the cellular adaptations to the persistent opiate effects and may be involved in the molecular mechanism underlying opiate tolerance and/or dependence.

Cloning and sequencing analysis of a human synapsin IIb-encoding brain cDNA

Cloning and sequencing of the gene encoding human synapsin IIb, an abundant peripheral membrane protein of synaptic vesicles, is described. The human and rat coding sequences showed 89% nucleotide and 94% amino acid sequence homology.

Changes in the expression of synapsin I and II messenger RNA during postnatal rat brain development.

Synapsin Ia, Ib, IIa, and IIb are neuronal phosphoproteins, which are supposed to play a role in the short-term regulation of neurotransmitter release. Besides a high degree of homology among the four synapsin subtypes, there are structural differences in the 3'end of their coding region. Here we present the first extensive study of the expression of their gene transcripts by using in situ hybridization and northern blot analysis. Our results show regionally and temporally distinct expression patterns of synapsin Ia, Ib, IIa, and IIb, which suggests different functional properties of the four synapsin subtypes. There was no specific messenger RNA (mRNA) expression of synapsin IIb in most brain regions apart from the cerebellum, suggesting a minor functional role of this synapsin subtype. Synapsin Ia, Ib, and IIa mRNA were expressed earlier in ontogenetically older brain regions such as the piriform cortex, the thalamus, and the hippocampus and later in ontogenetically younger areas such as the neocortex and the cerebellum. Owing to the distinct expression pattern of the synapsin subtypes, we suppose that the synapsins might be essential for the underlying molecular mechanism of pattern formation and plasticity in distinct brain regions during different states of rat brain development.

Laminin and neuropeptide Y are increased by synapsin transfection in cultured NG108-15 neuroblastoma/glioma hybrid cells.

We have investigated the presence and expression of laminin and neuropeptide Y (NPY) in several NG108-15 cell lines transfected with synapsin Ib, IIa, or IIb. The content of laminin, a basal membrane glycoprotein that promotes adhesion and induces neurite out-growth and neuronal differentiation, was increased in all transfected cell lines examined. In cells that were chemically differentiated with prostaglandin E1 plus 3-isobutyl-1-methylxanthine, laminin levels were increased even further. The content of NPY, suggested to be a neurotransmitter/neuromodulator in peripheral sympathetic neurons as well as in central neurons, was also increased in all transfected cell lines examined. Immunohistochemical analysis combined with confocal laser microscopy showed that NPY staining was granular and very often enriched in neuritic varicosities. The distribution and the staining pattern of NPY were consistent with storage of NPY in large dense-cored vesicles. The results indicate that, in differentiated neurons, the synapsins increase the levels of a neuropeptide transmitter stored in large dense-cored vesicles and of an extracellular matrix protein associated with neuronal maturation.