AP-3-dependent mechanisms control the targeting of a chloride channel (ClC-3) in neuronal and non-neuronal cells

Adaptor protein (AP)-2 and AP-3-dependent mechanisms control the sorting of membrane proteins into synaptic vesicles. Mouse models deficient in AP-3, mocha, develop a neurological phenotype of which the central feature is an alteration of the luminal synaptic vesicle composition. This is caused by a severe reduction of vesicular levels of the zinc transporter 3 (ZnT3). It is presently unknown whether this mocha defect is restricted to ZnT3 or encompasses other synaptic vesicle proteins capable of modifying synaptic vesicle contents, such as transporters or channels. In this study, we identified a chloride channel, ClC-3, whose level in synaptic vesicles and hippocampal mossy fiber terminals was reduced in the context of the mocha AP-3 deficiency. In PC-12 cells, ClC-3 was present in transferrin receptor-positive endosomes, where it was targeted to synaptic-like microvesicles (SLMV) by a mechanism sensitive to brefeldin A, a signature of the AP-3-dependent route of SLMV biogenesis. ClC-3 was packed in SLMV along with the AP-3-targeted synaptic vesicle protein ZnT3. Co-segregation of ClC-3 and ZnT3 to common intracellular compartments was functionally significant as revealed by increased vesicular zinc transport with increased ClC3 expression. Our work has identified a synaptic vesicle protein in which trafficking to synaptic vesicles is regulated by AP-3. In addition, our findings indicate that ClC-3 and ZnT3 reside in a common vesicle population where they functionally interact to determine vesicle luminal composition.     

锌及锌转运蛋白ZnT3在小鼠海马苔藓纤维的一致性分布

目的 研究游离锌离子和锌转运蛋白ZnT3在小鼠海马的定位以及二的分布是否具有一致性。方法 应用锌TSQ荧光技术、锌金属自显影技术检测含锌神经元内的游离锌离子;应用免疫电镜技术检测ZnT3在含锌神经元轴突终末的分布。结果 游离锌离子和ZnT3免疫反应产物的分布在海马苔藓纤维内的分布具有一致性。在齿状回和CA3区的苔藓纤维内,锌和ZnT3蛋白定位于轴突终末的突触小泡。富含锌离子的含锌神经元轴突终末与CA3区锥体细胞的胞体和树突形成突触。尚可见锌离子存在于突触间隙内。结论 ZnT3向突触小泡内转运锌离子使锌离子聚积在含锌神经元轴突终末的突触小泡内,发挥锌离子的神经生物学功能。     

The actions of synaptically released zinc at hippocampal mossy fiber synapses

Zn2+ is present at high concentrations in the synaptic vesicles of hippocampal mossy fibers. We have used Zn2+ chelators and the mocha mutant mouse to address the physiological role of Zn2+ in this pathway. Zn2+ is not involved in the unique presynaptic plasticities observed at mossy fiber synapses but is coreleased with glutamate from these synapses, both spontaneously and with electrical stimulation, where it exerts a strong modulatory effect on the NMDA receptors. Zn2+ tonically occupies the high-affinity binding site of NMDA receptors at mossy fiber synapses, whereas the lower affinity voltage-dependent Zn2+ binding site is occupied during action potential driven-release. We conclude that Zn2+ is a modulatory neurotransmitter released from mossy fiber synapses and plays an important role in shaping the NMDA receptor response at these synapses.     

The maintenance of LTP at hippocampal mossy fiber synapses is independent of sustained presynaptic calcium.

We have examined the role of presynaptic residual calcium in maintaining long-term changes in synaptic efficacy observed at mossy fiber synapses between hippocampal dentate granule cells and CA3 pyramidal cells. Calcium concentrations in individual mossy fiber terminals in hippocampal slice were optically measured with the calcium indicator fura-2 while stimulating the mossy fiber pathway and recording excitatory postsynaptic potentials extracellularly. Short-term synaptic enhancement was accompanied by increased presynaptic residual calcium concentration. A 2-fold enhancement of transmitter release was accompanied by a 10-30 nM increase in residual calcium. Following induction of mossy fiber LTP, transiently elevated presynaptic calcium decayed to prestimulus levels, whereas enhancement of synaptic transmission persisted. Our results demonstrate that, despite an apparent strong sensitivity of synaptic enhancement to presynaptic residual calcium levels, sustained increases in presynaptic residual calcium levels are not responsible for the maintained synaptic enhancement observed during mossy fiber LTP.     

Involvement of the synapse-specific zinc transporter ZnT3 in cadmium-induced hippocampal neurotoxicity

The present study examined the involvement of zinc (Zn)-transporters (ZnT3) in cadmium (Cd)-induced alterations of Zn homeostasis in rat hippocampal neurons. We treated primary rat hippocampal neurons for 24 or 48 hr with various concentrations of CdCl₂ (0, 0.5, 5, 10, 25, or 50 μM) and/or ZnCl ₂ (0, 10, 30, 50, 70, or 90 μM), using normal neuronal medium as control. By The CellTiter 96 ^® Aqueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI) assay and immunohistochemistry for cell death markers, 10 and 25 μM of Cd were found to be noncytotoxic doses, and both 30 and 90 μM of Zn as the best concentrations for cell proliferation. We tested these selected doses. Cd, at concentrations of 10 or 25 μM (and depending on the absence or presence of Zn), decreased the percentage of surviving cells. Cd-induced neuronal death was either apoptotic or necrotic depending on dose, as indicated by 7-AAD and/or annexin V labeling. At the molecular level, Cd exposure induced a decrease in hippocampal brain-derived neurotrophic factor-tropomyosin receptor kinase B (BDNF-TrkB) and Erk1/2 signaling, a significant downregulation of the expression of learning- and memory-related receptors and synaptic proteins such as the NMDAR NR2A subunit and PSD-95, as well as the expression of the synapse-specific vesicular Zn transporter ZnT3 in cultured hippocampal neurons. Zn supplementation, especially at the 30 μM concentration, led to partial or total protection against Cd neurotoxicity both with respect to the number of apoptotic cells and the expression of several genes. Interestingly, after knockdown of ZnT3 by small interfering RNA transfection, we did not find the restoration of the expression of this gene following Zn supplementation at 30 μM concentration. These data indicate the involvement of ZnT3 in the mechanism of Cd-induced hippocampal neurotoxicity.     

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGlu_u that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.

This study uses several high-resolution imaging techniques to investigate the structural correlates of presynaptic potentiation at hippocampal mossy fiber boutons, observing an increase in release sites and in release synchronicity accompanied by synaptic vesicle dispersion in the terminal and accumulation at release sites, but no modulation of the distance between calcium channel and release sites.     

Effect of the zinc chelator N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine (TPEN) on hippocampal mossy fiber calcium signals and on synaptic transmission

An important pool of chelatable zinc is present in the synaptic vesicles of mossy fiber terminals from hippocampal CA3 area, being zinc released following single or repetitive electrical stimulation. Previous studies have suggested different synaptic roles for released mossy fiber zinc, including the inhibition of presynaptic calcium and of postsynaptic N-methyl-D-aspartate (NMDA) and gamma amino-butyric acid (GABAA) receptors. The effect of endogenously released zinc on mossy fiber long-term potentiation (LTP) induction also is not yet established. We have investigated the effect of the permeant zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) on mossy fiber calcium and on synaptic transmission, before and during the application of LTP-inducing stimulation. We have found, using the calcium indicator Fura-2, that single and tetanically-evoked mossy fiber calcium signals are both enhanced in the presence of 20 microM TPEN, while the single field potentials are unaffected. As expected, no effect was observed on the single calcium signals or field potentials obtained at the CA3-CA1 synapses, from the CA1 area, which has a lower concentration of vesicular zinc. These results support the idea that at the hippocampal mossy fiber synapses, released zinc inhibits presynaptic calcium mechanisms. A higher concentration of TPEN (100 microM) significantly reduced mossy fiber synaptic transmission but did not prevent the induction of mossy fiber LTP, suggesting that zinc is not required for the formation of this form of LTP.     

Cytomorphological alterations of mossy fiber boutons of the hippocampus produced by 3-acetylpyridine,methoxypyridoxine, reserpine,and iproniazid

Summary The hippocampal mossy fiber boutons of the rabbit were studied with phase and electron microscopy. The injection of 3-acetylpyridine, methoxypyridoxine, and reserpine diminishes the conspicuous osmiophilic density of the mossy fiber boutons in comparison to similar regions from nontreated animals as observable in phase microscopy. However, electron micrographs of the same samples show little or no diminution in the number of those synaptic vesicles consisting of a clear homogeneous center (Type I). Treatment with monoamine liberator, reserpine, results in the same cytomorphological appearance of the boutons as with convulsant agents. The number of synaptic dense-core vesicles (Type II) is not altered after treatment with the convulsant agents or reserpine.A certain extra-vesicular substance and a certain granular component of the vesicular membranes of Type I vesicles is progressively reduced after treatment with all of these drugs. It is suggested that this accounts for the decreased density by phase microscopy.The monoamine oxidase inhibitor, iproniazid, increases the density of the extra-vesicular substance as well as the particles attached to the vesicular membranes of Type I vesicles.It is suggested that these osmiophilic particles contain the biogenic monoamines (in this instance probably serotonin and/or histamine) and that in acute experiments the liberation of these neurotransmitters is not related to a disappearence of dense-core vesicles concommitant with a depletion of neurotransmitters but is from particles in the extra-vesicular substance and the granular component of the vesicular of the Type I vesicles.Furthermore, the functional role of zinc in the synaptic vesicles of mossy fiber boutons of the hippocampus is discussed in regard to a possible storage mechanism for biogenic monoamines.This study was partly supported by USPHS Grant 5 P10 ESOO159.     

A critical role of a facilitatory presynaptic kainate receptor in mossy fiber LTP.

The mechanisms involved in mossy fiber LTP in the hippocampus are not well established. In the present study, we show that the kainate receptor antagonist LY382884 (10 microM) is selective for presynaptic kainate receptors in the CA3 region of the hippocampus. At a concentration at which it blocks mossy fiber LTP, LY382884 selectively blocks the synaptic activation of a presynaptic kainate receptor that facilitates AMPA receptor-mediated synaptic transmission. Following the induction of mossy fiber LTP, there is a complete loss of the presynaptic kainate receptor-mediated facilitation of synaptic transmission. These results identify a central role for the presynaptic kainate receptor in the induction of mossy fiber LTP. In addition, these results suggest that the pathway by which kainate receptors facilitate glutamate release is utilized for the expression of mossy fiber LTP.     

Vesicular zinc promotes presynaptic and inhibits postsynaptic long-term potentiation of mossy fiber-CA3 synapse

The presence of zinc in glutamatergic synaptic vesicles of excitatory neurons of mammalian cerebral cortex suggests that zinc might regulate plasticity of synapses formed by these neurons. Long-term potentiation (LTP) is a form of synaptic plasticity that may underlie learning and memory. We tested the hypothesis that zinc within vesicles of mossy fibers (mf) contributes to mf-LTP, a classical form of presynaptic LTP. We synthesized an extracellular zinc chelator with selectivity and kinetic properties suitable for study of the large transient of zinc in the synaptic cleft induced by mf stimulation. We found that vesicular zinc is required for presynaptic mf-LTP. Unexpectedly, vesicular zinc also inhibits?a form of postsynaptic mf-LTP. Because the mf-CA3 synapse provides a major source of excitatory input to the hippocampus, regulating its efficacy by these dual actions, vesicular zinc is critical to proper function of hippocampal circuitry in health and disease.     

A comparative morphometric study of synaptic vesicles and other organelles in mossy fiber endings of the pigeon and the rat

The mossy fiber ending organelles of 4 rats and 4 pigeons were studied using ultrastructural morphometric methods. The number of agranular vesicles per square micrometer of synaptic surface, as well as the number of agranular vesicles per synapse, were found to be greater in the rat than in the pigeon, while no significant differences were found in coated and dense core vesicles. A highly positive correlation was found, in both species, between the synaptic surface and the number of agranular vesicles per unit volume of mossy fiber endings, while no correlation was found between the synaptic surface and numerical densities of coated and dense core vesicles.     

Synapsin I deficiency results in the structural change in the presynaptic terminals in the murine nervous system

Synapsin I is one of the major synaptic vesicle-associated proteins. Previous experiments implicated its crucial role in synaptogenesis and transmitter release. To better define the role of synapsin I in vivo, we used gene targeting to disrupt the murine synapsin I gene. Mutant mice lacking synapsin I appeared to develop normally and did not have gross anatomical abnormalities. However, when we examined the presynaptic structure of the hippocampal CA3 field in detail, we found that the sizes of mossy fiber giant terminals were significantly smaller, the number of synaptic vesicles became reduced, and the presynaptic structures altered, although the mossy fiber long-term potentiation remained intact. These results suggest significant contribution of synapsin I to the formation and maintenance of the presynaptic structure.     

Pathway-specific alteration of synaptic plasticity in Tg2576 mice

Various animal models of Alzheimer disease (AD) are characterized by deficits in spatial memory that are causally related to altered synaptic function and impairment of long-term potentiation (LTP) in the hippocampus. In Tg2576 AD mice, we compared LTP in 2 major hippocampal pathways, Schaffer collateral (SC) and mossy fiber (MF) pathways. Whereas LTP was completely abolished in the SC pathway of Tg2576 mice, we found no decrease in LTP induced by stimulation of the MF pathway. In fact, we found that in the MF pathway, LTP was slightly, but significantly, enhanced compared with that in the MF pathway of WT littermates. This pathway-specific impairment of LTP is not attributable to alterations in transmitter release, as indicated by an unaltered paired-pulse ratio. These results suggest that the spatial memory deficits normally seen in AD models arise primarily from LTP impairment at the SC pathway.     

Fine structure of synapses in the hippocampus of the rabbit with special reference to dark presynaptic endings

Summary The stratum radiatum of h ₃ and h ₄ in the hippocampus of the rahbit, where the mossy fiber endings are distributed, was investigated under the electron microscope. These regions contain a certain number of electron dense presynaptic endings. These are characterized by highly dense synaptic vesicles and mitochondrial matrices. The dense endings are not considered as degenerated. Electron dense silver particles, substituted for zinc, occurred on the synaptic vesicles of these dense terminals as well as the mossy fiber endings after the application of Timm's histochemical method modified for electron microscopy. It is concluded that the dark synaptic endings observed might represent mossy fiber terminals in a special functional phase, or might be the result of structural alteration in the course of tissue preparation. The zinc localized in the synaptic vesicles is thought to be associated with the neurotransmitter present in these endings.     

GPER1 Modulates Synaptic Plasticity During the Development of Temporal Lobe Epilepsy in Rats

G-protein coupled estrogen receptor 1 (GPER1) is a novel type of estrogen receptor. Several studies have shown that it has an anti-inflammatory action,which plays an important role in remyelination and cognitive ability adjustment. However, whether it is involved in the development of temporal lobe epilepsy (TLE) is still unknown. The present study established a TLE model by intraperitoneal injection of lithium chloride (3 mmol/kg) and pilocarpine (50 mg/kg) in rats to study the effect of GPER1 in the synaptic plasticity during the development of temporal lobe epilepsy. A microinjection cannula was implanted into the lateral ventricle region of rats via a stereotaxic instrument. G-1 is the specific GPER1 agonist and G15 is the specific GPER1 antagonist. The G1 or G15 and Dimethyl sulfoxide were injected into the rat brains in the intervention groups and control group, respectively. After G1 intervention, the learning and memory abilities and hippocampal neuron damage in epileptic rats were significantly improved, while G15 weakened the neuroprotective effect of GPER1. Meanwhile, G1 controlled the abnormal formation of hippocampal mossy fiber sprouting caused by seizures, and participated in the regulation of synaptic plasticity by reducing the expression of Synapsin I and increasing the expression of gephyrin. Inhibitory synapse gephyrin may play a significant role in synaptic plasticity.

     

IQGAP1 Expression in Spared CA1 Neurons After an Excitotoxic Lesion in the Mouse Hippocampus

Repeated seizures induce permanent alterations in the hippocampal circuits in experimental models with intractable temporal lobe epilepsy. Sprouting and synaptic reorganization induced by seizures has been well-studied in the mossy fiber pathway. However, studies investigating sprouting and synaptic reorganization beyond the mossy fiber pathway are limited. The present study examined the biochemical changes of CA1 pyramidal neurons undergoing morphological changes after excitotoxicity-induced hippocampal CA3 neuronal death. IQ-domain GTPase-activating proteins (IQGAP1), is an effector of Rac1 and Cdc42 and an actin-binding protein, was upregulated in CA1 pyramidal neurons after kainic acid-induced hippocampal CA3 neuronal degeneration. IQGAP1 + cells were colocalized with Nestin, but not in astrocytes or mature neurons. Furthermore, IQGAP1 did not originate from newly divided local precursors or NG2 + cells. IQGAP1 and adenomatous polyposis coli localized in CA1 pyramidal neurons, and Cdc42 activation was followed by IQGAP1 recruitment. These findings suggest that IQGAP1 is upregulated in pre-existed sparing neurons of the CA1 layer undergoing morphological changes after excitoxicity-induced hippocampal CA3 neuronal death. It demonstrates the utility of IQGAP1 as a possible marker for spared pyramidal neurons, which may contribute to structural and functional alternations responsible for the development of epilepsy.     

The Hippocampal Proteomic Analysis of Senescence-Accelerated Mouse: Implications of Uchl3 and Mitofilin in Cognitive Disorder and Mitochondria Dysfunction in SAMP8

Senescence-accelerated mouse prone 8 (SAMP8) is considered as a useful animal model for age-related learning and memory impairments. Hippocampus, a critical brain region associated with cognitive decline during normal aging and various neurodegenerative diseases, appeared a series of abnormalities in SAMP8. To investigate the molecular mechanisms underlying age-related cognitive disorders, we used 2-DE coupled with MALDI TOF/TOF MS to analyze the differential protein expression of the hippocampus of SAMP8 at 6-month-old compared with the age-matched SAM/resistant 1 (SAMR1) which shows normal aging process. Two proteins were found to be markedly changed in SAMP8 as compared to SAMR1: ubiquitin carboxyl-terminal hydrolase L3 (Uchl3), implicating in cytosolic proteolysis of oxidatively damaged proteins, was down-regulated while mitofilin, a vital protein for normal mitochondria function, exhibited four isoforms with a consistent basic shift of isoelectric point among the soluble hippocampal proteins in SAMP8 compared with SAMR1. The alterations were confirmed by Western blotting analysis. The analysis of their expression changes may shed light on the mechanisms of learning and memory deficits and mitochondrial dysfunction as observed in SAMP8.     

Correlated alterations in serotonergic and dopaminergic modulations at the hippocampal mossy fiber synapse in mice lacking dysbindin

Dysbindin-1 (dystrobrevin-binding protein 1, DTNBP1) is one of the promising schizophrenia susceptibility genes. Dysbindin protein is abundantly expressed in synaptic regions of the hippocampus, including the terminal field of the mossy fibers, and this hippocampal expression of dysbindin is strongly reduced in patients with schizophrenia. In the present study, we examined the functional role of dysbindin in hippocampal mossy fiber-CA3 synaptic transmission and its modulation using the sandy mouse, a spontaneous mutant with deletion in the dysbindin gene. Electrophysiological recordings were made in hippocampal slices prepared from adult male sandy mice and their wild-type littermates. Basic properties of the mossy fiber synaptic transmission in the mutant mice were generally normal except for slightly reduced frequency facilitation. Serotonin and dopamine, two major neuromodulators implicated in the pathophysiology of schizophrenia, can potentiate mossy fiber synaptic transmission probably via an increase in cAMP levels. Synaptic potentiation induced by serotonin and dopamine was very variable in magnitude in the mutant mice, with some mice showing prominent enhancement as compared with the wild-type mice. In addition, the magnitude of potentiation induced by these monoamines significantly correlated with each other in the mutant mice, indicating that a subpopulation of sandy mice has marked hypersensitivity to both serotonin and dopamine. While direct activation of the cAMP cascade by forskolin induced robust synaptic potentiation in both wild-type and mutant mice, this forskolin-induced potentaition correlated in magnitude with the serotonin-induced potentiation only in the mutant mice, suggesting a possible change in coupling of receptor activation to downstream signaling. These results suggest that the dysbindin deficiency could be an essential genetic factor that causes synaptic hypersensitivity to dopamine and serotonin. The altered monoaminergic modulation at the mossy fiber synapse could be a candidate pathophysiological basis for impairment of hippocampus-dependent brain functions in schizophrenia.     

Significance of Zn2+ signaling in cognition: Insight from synaptic Zn2+ dyshomeostasis

Zinc is concentrated in the synaptic vesicles via zinc transporter-3 (ZnT3), released from glutamatergic (zincergic) neuron terminals, and serves as a signal factor (Zn²⁺ signal) in the intracellular (cytosol) compartment as well as in the extracellular compartment. Synaptic Zn²⁺ signaling is dynamically linked to neurotransmission via glutamate and is involved in synaptic plasticity such as long-term potentiation (LTP) and cognitive activity. Zinc concentration in the synaptic vesicles is correlated with ZnT3 protein expression and potentially decreased under chronic zinc deficiency. Synaptic vesicle serves as a large pool for Zn²⁺ signaling and other organelles might also serve as a pool for Zn²⁺ signaling. ZnT3KO mice and zinc-deficient animals, which lack or reduce Zn²⁺ release into the extracellular space by action potentials, are able to recognize novel or displaced objects normally. However, the amount of Zn²⁺ functioning as a signal factor increases along with brain development. Exogenous Zn²⁺ lowers the threshold in hippocampal CA1 LTP induction in young rat. Furthermore, ZnT3KO mice lose advanced cognition such as contextual discrimination. It is likely that the optimal range of synaptic Zn²⁺ signaling is involved in cognitive activity. On the basis of the findings on the relationship between dyshomeostasis of synaptic Zn²⁺ and cognition, this paper summarizes the possible involvement of intracellular Zn²⁺ signaling in cognitive ability.     

ZnT3与Aβ在APP/PS1转基因小鼠老年斑内的定位及相关性

目的研究锌转运体-3（zinc transporter 3,ZnT3）在APP／PS1转基因小鼠大脑皮层及海马内的表达,探讨ZnT3与β-淀粉样蛋白（β-amylold,Aβ）在老年斑内的定位分布及相关性。方法应用免疫荧光和共聚焦激光扫描显微镜观察ZnT3在APP／PS1转基因小鼠大脑内的表达及其与Aβ在老年斑内的位置关系。结果ZnT3主要分布于APP／PS1转基因小鼠大脑皮层和海马的老年斑中,海马苔藓纤维也可见ZnT3的阳性反应产物;ZnT3和Aβ双标的共聚焦激光扫描显微镜观察结果证实几乎所有Aβ老年斑中均有不同程度的ZnT3表达,且主要定位在老年斑周围的变性的神经元及其突起内,围绕老年斑的Aβ核心分布。结论ZnT3与Aβ共同表达于APP／PS1转基因小鼠老年斑内,提示ZnT3可能在老年斑内的锌离子的聚集过程中起着重要的调节作用,进而参与了APP／PS1转基因小鼠大脑内Aβ老年斑的形成。