Protein Synapsin Research Articles

Herpes Simplex Virus type-1 infection induces synaptic dysfunction in cultured cortical neurons via GSK-3 activation and intraneuronal amyloid-β protein accumulation.

Increasing evidence suggests that recurrent Herpes Simplex Virus type 1 (HSV-1) infection spreading to the CNS is a risk factor for Alzheimer’s Disease (AD) but the underlying mechanisms have not been fully elucidated yet. Here we demonstrate that in cultured mouse cortical neurons HSV-1 induced Ca2+-dependent activation of glycogen synthase kinase (GSK)-3. This event was critical for the HSV-1-dependent phosphorylation of amyloid precursor protein (APP) at Thr668 and the following intraneuronal accumulation of amyloid-β protein (Aβ). HSV-1-infected neurons also exhibited: i) significantly reduced expression of the presynaptic proteins synapsin-1 and synaptophysin; ii) depressed synaptic transmission. These effects depended on GSK-3 activation and intraneuronal accumulation of Aβ. In fact, either the selective GSK-3 inhibitor, SB216763, or a specific antibody recognizing Aβ (4G8) significantly counteracted the effects induced by HSV-1 at the synaptic level. Moreover, in neurons derived from APP KO mice and infected with HSV-1 Aβ accumulation was not found and synaptic protein expression was only slightly reduced when compared to wild-type infected neurons. These data further support our contention that HSV-1 infections spreading to the CNS may contribute to AD phenotype.

Functions of synapsins in corticothalamic facilitation: important roles of synapsin I.

The synaptic vesicle associated proteins synapsin I and synapsin II have important functions in synaptic short-term plasticity. We investigated their functions in cortical facilitatory feedback to neurons in dorsal lateral geniculate nucleus (dLGN), feedback that has important functions in state-dependent regulation of thalamic transmission of visual input to cortex. We compared results from normal wild-type (WT) mice and synapsin knockout (KO) mice in several types of synaptic plasticity, and found clear differences between the responses of neurons in the synapsin I KO and the WT, but no significant differences between the synapsin II KO and the WT. These results are in contrast to the important role of synapsin II previously demonstrated in similar types of synaptic plasticity in other brain regions, indicating that the synapsins can have different roles in similar types of STP in different parts of the brain. The synaptic vesicle associated proteins synapsin I (SynI) and synapsin II (SynII) have important functions in several types of synaptic short-term plasticity in the brain, but their separate functions in different types of synapses are not well known. We investigated possible distinct functions of the two synapsins in synaptic short-term plasticity at corticothalamic synapses on relay neurons in the dorsal lateral geniculate nucleus. These synapses provide excitatory feedback from visual cortex to the relay cells, feedback that can facilitate transmission of signals from retina to cortex. We compared results from normal wild-type (WT), SynI knockout (KO) and SynII KO mice, in three types of synaptic plasticity mainly linked to presynaptic mechanism. In SynI KO mice, paired-pulse stimulation elicited increased facilitation at short interpulse intervals compared to the WT. Pulse-train stimulation elicited weaker facilitation than in the WT, and also post-tetanic potentiation was weaker in SynI KO than in the WT. Between SynII KO and the WT we found no significant differences. Thus, SynI has important functions in these types of synaptic plasticity at corticothalamic synapses. Interestingly, our data are in contrast to the important role of SynII previously shown for sustained synaptic transmission during intense stimulation in excitatory synapses in other parts of the brain, and our results suggest that SynI and SynII may have different roles in similar types of STP in different parts of the brain.

Different protocols of treadmill exercise induce distinct neuroplastic effects in rat brain motor areas

A variety of exercise protocols have been used to promote experimental neuroplasticity. However, the plastic brain responses generated by several aspects of training (types, frequency, regimens, duration) remain undetermined. The aim of this study was to compare the plastic changes in the glutamatergic system and synaptic proteins in motor cortex, striatum and cerebellum promoted by two different treadmill exercise regimens. The present study analyzed by immunohistochemistry and Western blotting the expression of the subunits of AMPA receptors (GluA1 and GluA2/3) and synaptic proteins (synapsin I and synaptophysin) in adult male Wistar rat brains. The animals were divided into animals subjected to two different frequencies of aerobic exercise groups and sedentary animals. The exercise groups were: intermittent treadmill exercise (ITE) – animals that exercised 3 times a week (every other day) during four weeks, and continuous treadmill exercise (CTE) – animals that exercised every day during four weeks. Our results reveal that different protocols of treadmill exercise were able to promote distinct synaptic reorganization processes among the exercised groups. In general, the intermittent exercise regimen induced a higher expression of presynaptic proteins, whereas the continuous exercise regimen increased postsynaptic GluA1 and GluA2/3 receptors.

Sublethal doses of fipronil intensify synapsin immunostaining in Atta sexdens rubropilosa (Hymenoptera: Formicidae) brains.

Although ants are common insects in agricultural ecosystems, few studies have considered how xenobiotics might induce physiological and morphological alterations in these insects. This study aimed to verify the neurotoxic action of sublethal doses of fipronil on the mushroom bodies of brains from the leaf-cutting ant Atta sexdens rubropilosa through immunocytochemistry analysis for the protein synapsin. The LD50 value was established as 1.42 ng ant(-1), and the sublethal doses used were LD50/10 and LD50/100. Synapsin labelling was more evident in the brains extracted from ants exposed to the insecticide, specifically in the regions of glia in the mushroom bodies, compared with the control group. It was possible to measure the intensity of emitted fluorescence in the areas of the mushroom bodies, and a statistical test showed differences between the control group and the treatment group. Thus, it is concluded that sublethal doses of the insecticide fipronil intensified synapsin immunostaining, suggesting an increased release of neurotransmitters, which may be linked to neurotoxicity and overexcitation. These sublethal doses may have two different effects: compromising the operation and maintenance of the colony and leading to the establishment of resistance in insects.

Presynaptic active zones in invertebrates and vertebrates.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

Synapsin determines memory strength after punishment- and relief-learning.

Adverse life events can induce two kinds of memory with opposite valence, dependent on timing: "negative" memories for stimuli preceding them and "positive" memories for stimuli experienced at the moment of "relief." Such punishment memory and relief memory are found in insects, rats, and man. For example, fruit flies (Drosophila melanogaster) avoid an odor after odor-shock training ("forward conditioning" of the odor), whereas after shock-odor training ("backward conditioning" of the odor) they approach it. Do these timing-dependent associative processes share molecular determinants? We focus on the role of Synapsin, a conserved presynaptic phosphoprotein regulating the balance between the reserve pool and the readily releasable pool of synaptic vesicles. We find that a lack of Synapsin leaves task-relevant sensory and motor faculties unaffected. In contrast, both punishment memory and relief memory scores are reduced. These defects reflect a true lessening of associative memory strength, as distortions in nonassociative processing (e.g., susceptibility to handling, adaptation, habituation, sensitization), discrimination ability, and changes in the time course of coincidence detection can be ruled out as alternative explanations. Reductions in punishment- and relief-memory strength are also observed upon an RNAi-mediated knock-down of Synapsin, and are rescued both by acutely restoring Synapsin and by locally restoring it in the mushroom bodies of mutant flies. Thus, both punishment memory and relief memory require the Synapsin protein and in this sense share genetic and molecular determinants. We note that corresponding molecular commonalities between punishment memory and relief memory in humans would constrain pharmacological attempts to selectively interfere with excessive associative punishment memories, e.g., after traumatic experiences.

Brainstem brain-derived neurotrophic factor signaling is required for histone deacetylase inhibitor-induced pain relief.

Our previous study demonstrated that persistent pain can epigenetically suppress the transcription of Gad2 [encoding glutamic acid decarboxylase 65 (GAD65)] and consequently impair the inhibitory function of GABAergic synapses in central pain-modulating neurons. This contributes to the development of persistent pain sensitization. Histone deacetylase (HDAC) inhibitors increased GAD65 activity considerably, restored GABA synaptic function, and rendered sensitized pain behavior less pronounced. However, the molecular mechanisms by which HDAC regulates GABAergic transmission through GAD65 under pain conditions are unknown. This work showed that HDAC inhibitor-induced increases in colocalization of GAD65 and synaptic protein synapsin I on the presynaptic axon terminals of the nucleus raphe magnus (NRM) were blocked by a TrkB receptor antagonist K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], indicating that BDNF-TrkB signaling may be required in GAD65 modulation of GABA synaptic function. At the brain-derived neurotrophic factor (BDNF) promoter, HDAC inhibitors induced significant increases in H3 hyperacetylation, consistent with the increase in BDNF mRNA and total proteins. Although exogenous BDNF facilitated GABA miniature inhibitory postsynaptic currents and GAD65 accumulation in NRM neuronal synapses in normal rats, it failed to do so in animals subjected to persistent inflammation. In addition, blockade of the TrkB receptor with K252a has no effect on miniature inhibitory postsynaptic currents and synaptic GAD65 accumulation under normal conditions. In addition, the analgesic effects of HDAC inhibitors on behavior were blocked by NRM infusion of K252a. These findings suggest that BDNF-TrkB signaling is required for drugs that reverse the epigenetic effects of chronic pain at the gene level, such as HDAC inhibitors.

Antibody against α-gliadin 33-mer peptide: Is the key initiating factor for development of multiple sclerosis during gluten sensitivity?

Abstract Despite great advances in clarifying the pathogenesis of multiple sclerosis (MS), the exact underlying mechanism has not been definitely established. However, the responsibility of cross-reactive antibodies as the initiating factor in MS pathogenesis is a novel idea. Recently, an antibody against-α-gliadin 33-mer peptide which is found in most patients with gluten sensitivity have shown to cross-react significantly with various neural antigens including asialoganglioside, synapsin, and myelin basic protein (MBP). Furthermore, evidence indicates that IL-17, circulating immune complexes and even antibodies produced during gluten sensitivity can contribute to blood–brain barrier (BBB) permeability. Accordingly, extravasation of these anti-α-gliadin antibodies (AGA; especially IgG isotype) through the impaired BBB thought to target asialoganglioside, synapsin, and MBP in neurons. This opsonization may trigger a series of cascade pathways including complement activation, antibody-dependent microglial cytotoxicity against neurons, secretion of inflammatory mediators, myelin sheath damage, chemokine expression, CNS inflammation, BBB disruption and then leukocyte infiltration. The present hypothesis introduces a new antibody-dependent alternative pathway which may lead to multiple sclerosis (MS) during gluten sensitivity.

Change in expression of vesicular protein synapsin II by chronic treatment with D2 allosteric modulator PAOPA

The hallmark symptoms of schizophrenia include profound disturbances in thought, perception, cognition etc., which negatively impacts an individual's quality of life. Current antipsychotic drugs are not effective in treating all symptoms of this disorder, and often cause severe movement and metabolic side effects. Consequently, there remains a strong impetus to develop safer and more efficacious therapeutics for patients, as well as elucidating the etiology of schizophrenia. Previous work in our lab has introduced a novel candidate for the treatment of this disease: the dopamine D2 receptor (D2R) allosteric modulator, 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA). We have previously shown that PAOPA, by selectively modulating D2R, can ameliorate schizophrenia-like symptoms in animal models, although the precise mechanism is presently not understood. Synapsin II is a presynaptic vesicular protein which has been strongly implicated in schizophrenia, as it is reduced in the prefrontal cortex of patients, and knockdown of this protein elicits schizophrenia-like phenotypes in animal models. Given the therapeutic effects of PAOPA and the role of synapsin II in schizophrenia, the objective of this study was to investigate the effect of chronic administration of PAOPA (45 days) on neuronal synapsin II protein expression in rodents. Immunoblot results revealed that the synapsin IIa, but not the IIb isoform, was increased in the dopaminergic regions of the striatum, nucleus accumbens, and medial prefrontal cortex. The results of this study implicate a role for modulation of synapsin II as a possible therapeutic mechanism of action for potential antipsychotic drug PAOPA.

Different effects of bisphenol-A on memory behavior and synaptic modification in intact and estrogen-deprived female mice.

Bisphenol-A (BPA) has the capability of interfering with the effects of estrogens on modulating brain function. The purpose of this study was to investigate the effects of BPA on memory and synaptic modification in the hippocampus of female mice under different levels of cycling estrogen. BPA exposure (40, 400 μg/kg/day) for 8 weeks did not affect spatial memory and passive avoidance task of gonadally intact mice but improved ovariectomy (Ovx)-induced memory impairment, whereas co-exposure of BPA with estradiol benzoate (EB) diminished the rescue effect of EB on memory behavior of Ovx mice. The results of morphometric measurement showed that BPA positively modified the synaptic interface structure and increased the synaptic density of CA1 pyramidal cell in the hippocampus of Ovx females, but inhibited the enhancement of EB on synaptic modification and synaptogenesis of Ovx mice. Furthermore, BPA up-regulated synaptic proteins synapsin I and PSD-95 and NMDA receptor NR2B but inhibited EB-induced increase in PSD-95 and NR2B in the hippocampus of Ovx mice. These results suggest that BPA interfered with normal hormonal regulation in synaptic plasticity and memory of female mice as a potent estrogen mimetic and as a disruptor of estrogen under various concentrations of cycling estrogen.

Designer Self-Assemble Peptides Maximize the Therapeutic Benefits of Neural Stem Cell Transplantation for Alzheimer's Disease via Enhancing Neuron Differentiation and Paracrine Action.

The neuropathological hallmarks of Alzheimer’s disease (AD) include the presence of extracellular amyloid-β peptide (Aβ) in the form of amyloid plaques and neuronal loss. Neural stem cell (NSC) is being scrutinized as a promising cell replacement therapy for various neurodegenerative diseases. However, the unfavorable niche at the site of degenerative disease is hostile to the survival and differentiation of transplanted cells. Here, we undertook in vitro and in vivo works to examine whether a designer self-assemble peptide (DSP), which contains one functional domain Tyr-Ile-Gly-Ser-Arg (YIGSR) derived from laminin, promotes the survival and neuronal differentiation of NSC and behavioral improvement. We found that DSP could undergo spontaneous assembly into well-ordered nanofibers, and it not only facilitated the cell viability in normal culture condition, but also decreased the number of apoptotic cells induced by Aβ in vitro. NSC seeded in DSP showed much more neuronal differentiation than that seeded in self-assemble peptide (SP) or alone. In the AD model, NSC transplantation in DSP-treated AD rats demonstrated much more obvious cognitive rescue with restoration of learning/memory function compared with NSC transplantation in SP, NSC alone, or DSP alone treated ones. Interestingly, DSP enhanced the survival and neuronal differentiation of transplanted NSC. Apoptosis levels in the CA1 region and Aβ level in the hippocampus were significantly decreased in the group of NSC transplantation in DSP. Moreover, synaptic function, indicated by the expression of pre-synaptic protein synapsin-1, was restored and the secretion of anti-inflammatory and neurotrophic factors were increased, such as IL-10, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and insulin-like growth factor 1 (IGF-1), while the expression of pro-inflammatory factors were decreased, such as TNF-α and IL-1β. These data firstly unveiled that the biomaterial DSP can maximize the therapeutic benefits of NSC transplantation for AD through improving the survival and differentiation of transplanted stem cells and promoting the effects of neuroprotection, anti-neuroinflammatory and paracrine action. Our results may have important clinical implications for the design of future NSC-based strategies using the biomaterials for various neurodegenerative diseases including AD.Electronic supplementary materialThe online version of this article (doi:10.1007/s12035-014-9069-y) contains supplementary material, which is available to authorized users.

Chronic copper exposure causes spatial memory impairment, selective loss of hippocampal synaptic proteins, and activation of PKR/eIF2α pathway in mice.

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

A single brain-derived neurotrophic factor infusion into the dorsomedial prefrontal cortex attenuates cocaine self-administration-induced phosphorylation of synapsin in the nucleus accumbens during early withdrawal.

Background:Dysregulation in the prefrontal cortex-nucleus accumbens pathway has been implicated in cocaine addiction. We have previously demonstrated that one intra-dorsomedial prefrontal cortex brain-derived neurotrophic factor (BDNF) infusion immediately following the last cocaine self-administration session caused a long-lasting inhibition of cocaine-seeking and normalized the cocaine-induced disturbance of glutamate transmission in the nucleus accumbens after extinction and a cocaine prime. However, the molecular mechanism mediating the brain-derived neurotrophic factor effect on cocaine-induced alterations in extracellular glutamate levels is unknown.Methods:In the present study, we determined the effects of brain-derived neurotrophic factor on cocaine-induced changes in the phosphorylation of synapsin (p-synapsin), a family of presynaptic proteins that mediate synaptic vesicle mobilization, in the nucleus accumbens during early withdrawal.Results:Two hours after cocaine self-administration, p-synapsin Ser9 and p-synapsin Ser62/67, but not p-synapsin Ser603, were increased in the nucleus accumbens. At 22 hours, only p-synapsin Ser9 was still elevated. Elevations at both time points were attenuated by an intra-dorsomedial prefrontal cortex brain-derived neurotrophic factor infusion immediately after the end of cocaine self-administration. Brain-derived neurotrophic factor also reduced cocaine self-administration withdrawal-induced phosphorylation of the protein phosphatase 2A C-subunit, suggesting that brain-derived neurotrophic factor disinhibits protein phosphatase 2A C-subunit, consistent with p-synapsin Ser9 dephosphorylation. Further, co-immunoprecipitation demonstrated that protein phosphatase 2A C-subunit and synapsin are associated in a protein-protein complex that was reduced after 2 hours of withdrawal from cocaine self-administration and reversed by brain-derived neurotrophic factor.Conclusions:Taken together, these findings demonstrate that brain-derived neurotrophic factor normalizes the cocaine self-administration–induced elevation of p-synapsin in nucleus accumbens that may underlie a disturbance in the probability of neurotransmitter release or represent a compensatory neuroadaptation in response to the hypofunction within the prefrontal cortex-nucleus accumbens pathway during cocaine withdrawal.

Synaptic degeneration in rat brain after prolonged oral exposure to silver nanoparticles

Neurotoxicity of silver nanoparticles has been confirmed in both in vitro and in vivo studies. However, the mechanisms of the toxic action have not been fully clarified. Since nanoparticles are likely to have the ability to enter the brain and significantly accumulate in this organ, it is important to investigate their neurotoxic mechanisms. Here we examine the effect of prolonged exposure of rats to small (10nm) citrate-stabilized silver nanoparticles (as opposed to the ionic silver) on synapse ultrastructure and specific proteins. Administration of both nanosilver and ionic silver over a two-week period resulted in ultrastructural changes including blurred synapse structure and strongly enhanced density of synaptic vesicles clustering in the center of the presynaptic part. Disturbed synaptic membrane leading to liberation of synaptic vesicles into neuropil, which testifies for strong synaptic degeneration, was characteristic feature observed under AgNPs exposure. Also a noteworthy finding was the presence of myelin-like structures derived from fragmented membranes and organelles which are associated with neurodegenerative processes. Additionally, we observed significantly decreased levels of the presynaptic proteins synapsin I and synaptophysin, as well as PSD-95 protein which is an indicator of postsynaptic densities. The present study demonstrates that exposure of adult rats to both forms of silver leads to ultrastructural changes in synapses. However, it seems that small AgNPs lead to more severe synaptic degeneration, mainly in the hippocampal region of brain. The observations may indicate impairment of nerve function and, in the case of hippocampus, may predict impairment of cognitive processes.

Inhibition of ERK1/2 signaling prevents epileptiform behavior in rats prone to audiogenic seizures.

It has recently been proposed that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. We hypothesized that inhibition of ERK1/2 activity could prevent audiogenic seizures by altering GABA and glutamate release mechanisms. Krushinsky-Molodkina rats, genetically prone to audiogenic seizure, were recruited in the experiments. Animals were i.p. injected with an inhibitor of ERK1/2 SL 327 at different doses 60 min before audio stimulation. We demonstrated for the first time that inhibition of ERK1/2 activity by SL 327 injections prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. The obtained data also demonstrated unchanged levels of GABA production, and an increase in the level of vesicular glutamate transporter 2. The study of exocytosis protein expression showed that SL 327 treatment leads to downregulation of vesicle-associated membrane protein 2 and synapsin I, and accumulation of synaptosomal-associated protein 25 (SNAP-25). The obtained data indicate that the inhibition of ERK1/2 blocks seizure behavior presumably by altering the exocytosis machinery, and identifies ERK1/2 as a potential target for the development of new strategies for seizure treatment. Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are one of the factors mediating seizure development. Here we report that inhibition of ERK1/2 by SL 327 prevented seizure behavior and this effect was dose-dependent and correlated with ERK1/2 activity. Accumulation of VGLUT2 was associated with differential changing of synaptic proteins VAMP2, SNAP-25 and synapsin I. The obtained data indicate that the inhibition of ERK1/2 alters neurotransmitter release by changing the exocytosis machinery, thus preventing seizures.

Beta-amyloid oligomers induce early loss of presynaptic proteins in primary neurons by caspase-dependent and proteasome-dependent mechanisms.

Beta-amyloid is a major pathogenic molecule for Alzheimer's disease (AD) and can be aggregated into a soluble oligomer, which is a toxic intermediate, before amyloid fibril formation. Beta-amyloid oligomers are associated closely with early synaptic loss in AD. However, it is still unknown which synaptic proteins are involved in the synaptotoxicity, and a direct comparison among the synaptic proteins should also be addressed. Here, we investigated changes in the expression of several presynaptic and postsynaptic proteins in primary neurons after treatment with a low-molecular weight and a high-molecular weight beta-amyloid oligomer. Both oligomers induced early neuronal dysfunction after 4 h and significantly reduced presynaptic protein (synaptophysin, syntaxin, synapsin, and synaptotagmin) expression. However, the expression of postsynaptic proteins (PSD95, NMDAR2A/B, and GluR2/3), except NMDAR1 was not reduced, and some protein expression levels were increased. Glutamate treatment, which is correlated with postsynaptic activation, showed more postsynaptic-specific protein loss compared with beta-amyloid oligomer treatment. Finally, the caspase inhibitor zVAD and the proteasomal inhibitor MG132 attenuated presynaptic protein loss. Thus, our data showed changes in synaptic proteins by beta-amyloid oligomers, which provides an understanding of early synaptotoxicity and suggests new approaches for AD treatment.

A role for synapsin in FKBP51 modulation of stress responsiveness: Convergent evidence from animal and human studies

Both the molecular co-chaperone FKBP51 and the presynaptic vesicle protein synapsin (alternatively spliced from SYN1-3) are intensively discussed players in the still insufficiently explored pathobiology of psychiatric disorders such as major depression, schizophrenia and posttraumatic stress disorder (PTSD). To address their still unknown interaction, we compared the expression levels of synapsin and five other neurostructural and HPA axis related marker proteins in the prefrontal cortex (PFC) and the hippocampus of restrained-stressed and unstressed Fkbp5 knockout mice and corresponding wild-type littermates. In addition, we compared and correlated the gene expression levels of SYN1, SYN2 and FKBP5 in three different online datasets comprising expression data of human healthy subjects as well as of predominantly medicated patients with different psychiatric disorders. In summary, we found that Fkbp5 deletion, which we previously demonstrated to improve stress-coping behavior in mice, prevents the stress-induced decline in prefrontal cortical (pc), but not in hippocampal synapsin expression. Accordingly, pc, but not hippocampal, synapsin protein levels correlated positively with a more active mouse stress coping behavior. Searching for an underlying mechanism, we found evidence that deletion of Fkbp5 might prevent stress-induced pc synapsin loss, at least in part, through improvement of pc Akt kinase activity. These results, together with our finding that FKBP5 and SYN1 mRNA levels were regulated in opposite directions in the PFC of schizophrenic patients, who are known for exhibiting an altered stress-coping behavior, provide the first evidence of a role for pc synapsin in FKBP51 modulation of stress responsiveness. This role might extend to other tissues, as we found FKBP5 and SYN1 levels to correlate inversely not only in human PFC samples but also in other expression sites. The main limitation of this study is the small number of individuals included in the correlation analyses. Future studies will have to verify the here-postulated role of the FKBP51-Akt kinase-synapsin pathway in stress responsiveness.

BDNF-ERK-CREB signalling mediates the role of miR-132 in the regulation of the effects of oleanolic acid in male mice.

Although previous study has demonstrated that brain-derived neurotrophic factor (BDNF) is involved in the antidepressant-like effect of oleanolic acid, there is little information regarding the details of the molecular mechanism involved in this effect. We used a chronic unpredictable mild stress (CUMS) model to test the antidepressant-like effect of oleanolic acid on depressant-like behaviour, miR-132 expression and synaptic protein expression in the male mouse hippocampus. Furthermore, we explored the possible signalling pathways associated with miR-132 expression that mediate the effect of oleanolic acid on neuronal proliferation. The results demonstrated that a 3-week treatment with oleanolic acid ameliorated CUMS-induced anhedonic and anxiogenic behaviours. Furthermore, we found that oleanolic acid led to the BDNF-related phosphorylation and activation of extracellular signal-regulated kinases (ERK) and cyclic adenosine monophosphate response element binding protein (CREB), which was associated with the upregulation of miR-132 and hippocampal neuronal proliferation. Moreover, experiments with an miR-132 antagomir revealed that targeting miR-132 led to inhibition of neuronal proliferation and the postsynaptic density protein 95, but did not affect presynaptic protein synapsin I. Several other stimuli can also induce CREB phosphorylation in the hippocampus. Thus, regulation of miR-132 may not be restricted to neurotrophic signalling. Our results show that oleanolic acid induces the upregulation of miR-132, which serves as an important regulator of neurotrophic actions, mainly through the activation of the hippocampal BDNF-ERK-CREB signalling pathways.

CHRONIC COPPER EXPOSURE CAUSES SPATIAL MEMORY IMPAIRMENT, SELECTIVE LOSS OF HIPPOCAMPAL SYNAPTIC PROTEINS, AND ACTIVATION OF PKR/EIF2 α PATHWAY IN MICE

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. The effects of chronic copper exposure on spatial memory and the possible mechanisms remain unclear. Inductively coupled plasma mass spectrometry (ICP-MS) was used to measure the concentrations of copper in the serum and hippocampus of mice. Morris water maze test was performed to evaluate spatial learning and memory. Immunohistochemical staining and Western-blot analysis were performed to measure the changes of oxidative stress markers, synaptic proteins. TUNEL staining was performed to measure the change of apoptosis. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was up-regulated and hippocampal neuronal apopotosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway. This work was supported by NSFC (81102154), Medical Scientific Research Foundation of Guangdong Province (B2012322, A2013598) and Shenzhen Scheme of Science and Technology (Medicine and Health) (201202086, 201302148).

Presynaptic Alpha-Synuclein Aggregation in a Mouse Model of Parkinson's Disease

Parkinson's disease and dementia with Lewy bodies are associated with abnormal neuronal aggregation of α-synuclein. However, the mechanisms of aggregation and their relationship to disease are poorly understood. We developed an in vivo multiphoton imaging paradigm to study α-synuclein aggregation in mouse cortex with subcellular resolution. We used a green fluorescent protein-tagged human α-synuclein mouse line that has moderate overexpression levels mimicking human disease. Fluorescence recovery after photobleaching (FRAP) of labeled protein demonstrated that somatic α-synuclein existed primarily in an unbound, soluble pool. In contrast, α-synuclein in presynaptic terminals was in at least three different pools: (1) as unbound, soluble protein; (2) bound to presynaptic vesicles; and (3) as microaggregates. Serial imaging of microaggregates over 1 week demonstrated a heterogeneous population with differing α-synuclein exchange rates. The microaggregate species were resistant to proteinase K, phosphorylated at serine-129, oxidized, and associated with a decrease in the presynaptic vesicle protein synapsin and glutamate immunogold labeling. Multiphoton FRAP provided the specific binding constants for α-synuclein's binding to synaptic vesicles and its effective diffusion coefficient in the soma and axon, setting the stage for future studies targeting synuclein modifications and their effects. Our in vivo results suggest that, under moderate overexpression conditions, α-synuclein aggregates are selectively found in presynaptic terminals.