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1.
Proc Natl Acad Sci U S A ; 121(15): e2320505121, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38568977

RESUMO

The presynaptic SNARE-complex regulator complexin (Cplx) enhances the fusogenicity of primed synaptic vesicles (SVs). Consequently, Cplx deletion impairs action potential-evoked transmitter release. Conversely, though, Cplx loss enhances spontaneous and delayed asynchronous release at certain synapse types. Using electrophysiology and kinetic modeling, we show that such seemingly contradictory transmitter release phenotypes seen upon Cplx deletion can be explained by an additional of Cplx in the control of SV priming, where its ablation facilitates the generation of a "faulty" SV fusion apparatus. Supporting this notion, a sequential two-step priming scheme, featuring reduced vesicle fusogenicity and increased transition rates into the faulty primed state, reproduces all aberrations of transmitter release modes and short-term synaptic plasticity seen upon Cplx loss. Accordingly, we propose a dual presynaptic function for the SNARE-complex interactor Cplx, one as a "checkpoint" protein that guarantees the proper assembly of the fusion machinery during vesicle priming, and one in boosting vesicle fusogenicity.


Assuntos
Sinapses , Vesículas Sinápticas , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Potenciais de Ação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia
2.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360929

RESUMO

Complexins (Cplxs) 1 to 4 are components of the presynaptic compartment of chemical synapses where they regulate important steps in synaptic vesicle exocytosis. In the retina, all four Cplxs are present, and while we know a lot about Cplxs 3 and 4, little is known about Cplxs 1 and 2. Here, we performed in situ hybridization experiments and bioinformatics and exploited Cplx 1 and Cplx 2 single-knockout mice combined with immunocytochemistry and light microscopy to characterize in detail the cell type and synapse-specific distribution of Cplx 1 and Cplx 2. We found that Cplx 2 and not Cplx 1 is the main isoform expressed in normal and displaced amacrine cells and ganglion cells in mouse retinae and that amacrine cells seem to operate with a single Cplx isoform at their conventional chemical synapses. Surprising was the finding that retinal function, determined with electroretinographic recordings, was altered in Cplx 1 but not Cplx 2 single-knockout mice. In summary, the results provide an important basis for future studies on the function of Cplxs 1 and 2 in the processing of visual signals in the mammalian retina.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Células Amácrinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras/metabolismo , Células Bipolares da Retina/metabolismo , Células Ganglionares da Retina/metabolismo , Células Horizontais da Retina/metabolismo , Proteínas SNARE/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Células Cultivadas , Biologia Computacional/métodos , Eletrorretinografia/métodos , Feminino , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética
3.
J Neurosci ; 36(25): 6651-67, 2016 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-27335398

RESUMO

UNLABELLED: Complexins (Cplxs) are SNARE complex regulators controlling the speed and Ca(2+) sensitivity of SNARE-mediated synaptic vesicle fusion. We have shown previously that photoreceptor ribbon synapses in mouse retina are equipped with Cplx3 and Cplx4 and that lack of both Cplxs perturbs photoreceptor ribbon synaptic function; however, Cplx3/4 function in photoreceptor synaptic transmission remained elusive. To investigate Cplx3/4 function in photoreceptor ribbon synapses, voltage-clamp recordings from postsynaptic horizontal cells were performed in horizontal slice preparations of Cplx3/4 wild-type (WT) and Cplx3/4 double knock-out (DKO) mice. We measured tonic activity in light and dark, current responses to changes in luminous intensity, and electrically evoked postsynaptic responses. Cplx3/4 decreased the frequency of tonic events and shifted their amplitude distribution to smaller values. Light responses were sustained in the presence of Cplx3/4, but transient in their absence. Finally, Cplx3/4 increased synaptic vesicle release evoked by electrical stimulation. Using electron microscopy, we quantified the number of synaptic vesicles at presynaptic ribbons after light or dark adaptation. In Cplx3/4 WT photoreceptors, the number of synaptic vesicles associated with the ribbon base close to the release site was significantly lower in light than in dark. This is in contrast to Cplx3/4 DKO photoreceptors, in which the number of ribbon-associated synaptic vesicles remained unchanged regardless of the adaptational state. Our results indicate a suppressing and a facilitating action of Cplx3/4 on Ca(2+)-dependent tonic and evoked neurotransmitter release, respectively, and a regulatory role in the adaptation-dependent availability of synaptic vesicles for release at photoreceptor ribbon synapses. SIGNIFICANCE STATEMENT: Synaptic vesicle fusion at active zones of chemical synapses is executed by SNARE complexes. Complexins (Cplxs) are SNARE complex regulators and photoreceptor ribbon synapses are equipped with Cplx3 and Cplx4. The absence of both Cplxs perturbs ribbon synaptic function. Because we lack information on Cplx function in photoreceptor synaptic transmission, we investigated Cplx function using voltage-clamp recordings from postsynaptic horizontal cells of Cplx3/4 wild-type and Cplx3/4 double knock-out mice and quantified synaptic vesicle number at the ribbon after light and dark adaptation using electron microscopy. The findings reveal a suppressing action of Cplx3/4 on tonic neurotransmitter release, a facilitating action on evoked release, and a regulatory role of Cplx3/4 in the adaptation-dependent availability of synaptic vesicles at mouse photoreceptor ribbon synapses.


Assuntos
Proteínas do Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células Fotorreceptoras de Vertebrados/fisiologia , Retina/citologia , Sinapses/fisiologia , Transmissão Sináptica/genética , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Cálcio/metabolismo , Proteínas do Olho/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Células Fotorreceptoras de Vertebrados/ultraestrutura , Proteínas SNARE/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Fatores de Tempo , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo
4.
J Cell Sci ; 128(4): 638-44, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25609709

RESUMO

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.


Assuntos
Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Proteínas de Membrana/genética , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Tomografia com Microscopia Eletrônica , Exocitose/fisiologia , Feminino , Células Ciliadas Auditivas Internas/citologia , Audição/genética , Audição/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp
5.
J Neurosci ; 35(21): 8272-90, 2015 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-26019341

RESUMO

Complexins (Cplxs) are small synaptic proteins that cooperate with SNARE-complexes in the control of synaptic vesicle (SV) fusion. Studies involving genetic mutation, knock-down, or knock-out indicated two key functions of Cplx that are not mutually exclusive but cannot easily be reconciled, one in facilitating SV fusion, and one in "clamping" SVs to prevent premature fusion. Most studies on the role of Cplxs in mammalian synapse function have relied on cultured neurons, heterologous expression systems, or membrane fusion assays in vitro, whereas little is known about the function of Cplxs in native synapses. We therefore studied consequences of genetic ablation of Cplx1 in the mouse calyx of Held synapse, and discovered a developmentally exacerbating phenotype of reduced spontaneous and evoked transmission but excessive asynchronous release after stimulation, compatible with combined facilitating and clamping functions of Cplx1. Because action potential waveforms, Ca(2+) influx, readily releasable SV pool size, and quantal size were unaltered, the reduced synaptic strength in the absence of Cplx1 is most likely a consequence of a decreased release probability, which is caused, in part, by less tight coupling between Ca(2+) channels and docked SV. We found further that the excessive asynchronous release in Cplx1-deficient calyces triggered aberrant action potentials in their target neurons, and slowed-down the recovery of EPSCs after depleting stimuli. The augmented asynchronous release had a delayed onset and lasted hundreds of milliseconds, indicating that it predominantly represents fusion of newly recruited SVs, which remain unstable and prone to premature fusion in the absence of Cplx1.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Tronco Encefálico/metabolismo , Proteínas do Tecido Nervoso/deficiência , Sinapses/metabolismo , Vesículas Sinápticas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/biossíntese , Animais , Tronco Encefálico/citologia , Adesão Celular/fisiologia , Exocitose/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese
6.
EMBO J ; 29(15): 2477-90, 2010 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-20562829

RESUMO

The SNARE-complex consisting of synaptobrevin-2/VAMP-2, SNAP-25 and syntaxin-1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap-25 null mice rescued with mutants challenging the C-terminal, N-terminal and middle domains of the SNARE-bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub-domains of the SNARE-bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C-terminal end of the SNARE-bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C-terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N-terminal end of the SNARE-bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N-terminal deletion delays vesicle priming after a high-frequency train. We propose that the stability of N-terminal two-thirds of the SNARE-bundle has a function for vesicle priming and limiting spontaneous release.


Assuntos
Transmissão Sináptica , Proteína 25 Associada a Sinaptossoma/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Deleção de Genes , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Proteína 25 Associada a Sinaptossoma/química , Proteína 25 Associada a Sinaptossoma/deficiência , Proteína 25 Associada a Sinaptossoma/genética
7.
Front Mol Neurosci ; 17: 1308466, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38481472

RESUMO

Adaptation of photoreceptor sensitivity to varying light intensities is a fundamental requirement for retinal function and vision. Adaptive mechanisms in signal transduction are well described, but little is known about the mechanisms that adapt the photoreceptor synapse to changing light intensities. The SNARE complex regulators Complexin 3 and Complexin 4 have been proposed to be involved in synaptic light adaptation by limiting synaptic vesicle recruitment and fusion. How this Complexin effect is exerted is unknown. Focusing on rod photoreceptors, we established Complexin 4 as the predominant Complexin in the light-dependent regulation of neurotransmitter release. The number of readily releasable synaptic vesicles is significantly smaller in light than in dark at wildtype compared to Complexin 4 deficient rod photoreceptor ribbon synapses. Electrophysiology indicates that Complexin 4 reduces or clamps Ca2+-dependent sustained synaptic vesicle release, thereby enhancing light signaling at the synapse. Complexin 4 deficiency increased synaptic vesicle release and desensitized light signaling. In a quantitative proteomic screen, we identified Transducin as an interactor of the Complexin 4-SNARE complex. Our results provide evidence for a presynaptic interplay of both Complexin 4 and Transducin with the SNARE complex, an interplay that may facilitate the adaptation of synaptic transmission to light at rod photoreceptor ribbon synapses.

8.
J Neurosci ; 32(23): 8040-52, 2012 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-22674279

RESUMO

Munc13 proteins are essential regulators of exocytosis. In hippocampal glutamatergic neurons, the genetic deletion of Munc13s results in the complete loss of primed synaptic vesicles (SVs) in direct contact with the presynaptic active zone membrane, and in a total block of neurotransmitter release. Similarly drastic consequences of Munc13 loss are detectable in hippocampal and striatal GABAergic neurons. We show here that, in the adult mouse retina, the two Munc13-2 splice variants bMunc13-2 and ubMunc13-2 are selectively localized to conventional and ribbon synapses, respectively, and that ubMunc13-2 is the only Munc13 isoform in mature photoreceptor ribbon synapses. Strikingly, the genetic deletion of ubMunc13-2 has little effect on synaptic signaling by photoreceptor ribbon synapses and does not prevent membrane attachment of synaptic vesicles at the photoreceptor ribbon synaptic site. Thus, photoreceptor ribbon synapses and conventional synapses differ fundamentally with regard to their dependence on SV priming proteins of the Munc13 family. Their function is only moderately affected by Munc13 loss, which leads to slight perturbations of signal integration in the retina.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Sinapses/fisiologia , Vesículas Sinápticas/fisiologia , Células Amácrinas/fisiologia , Animais , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Eletrorretinografia , Exocitose/genética , Exocitose/fisiologia , Imunofluorescência , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Isomerismo , Camundongos , Camundongos Knockout , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , RNA/biossíntese , RNA/genética , Retina/citologia , Retina/fisiologia , Retina/ultraestrutura , Sinapses/ultraestrutura , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/ultraestrutura , Transcrição Gênica
9.
J Physiol ; 591(10): 2463-73, 2013 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-23401610

RESUMO

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Canais de Cálcio/fisiologia , Células Cromafins/fisiologia , Exocitose/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Vesículas Secretórias/fisiologia , Animais , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Músculo Esquelético/fisiologia , Junção Neuromuscular/fisiologia , Sinapses/fisiologia
10.
Adv Neurobiol ; 33: 255-285, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37615870

RESUMO

Neurotransmitter release is a spatially and temporally tightly regulated process, which requires assembly and disassembly of SNARE complexes to enable the exocytosis of transmitter-loaded synaptic vesicles (SVs) at presynaptic active zones (AZs). While the requirement for the core SNARE machinery is shared by most membrane fusion processes, SNARE-mediated fusion at AZs is uniquely regulated to allow very rapid Ca2+-triggered SV exocytosis following action potential (AP) arrival. To enable a sub-millisecond time course of AP-triggered SV fusion, synapse-specific accessory SNARE-binding proteins are required in addition to the core fusion machinery. Among the known SNARE regulators specific for Ca2+-triggered SV fusion are complexins, which are almost ubiquitously expressed in neurons. This chapter summarizes the structural features of complexins, models for their molecular interactions with SNAREs, and their roles in SV fusion.


Assuntos
Fusão de Membrana , Vesículas Sinápticas , Humanos , Transmissão Sináptica , Exocitose , Proteínas SNARE
11.
Hum Mol Genet ; 19(17): 3402-12, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20584925

RESUMO

Complexin 2 is a protein modulator of neurotransmitter release that is downregulated in humans suffering from depression, animal models of depression and neurological disorders such as Huntington's disease in which depression is a major symptom. Although complexin 2 knockout (Cplx2-/-) mice are overtly normal, they show significant abnormalities in cognitive function and synaptic plasticity. Here we show that Cplx2-/- mice also have disturbances in emotional behaviours that include abnormal social interactions and depressive-like behaviour. Since neurotransmitter deficiencies are thought to underlie depression, we examined neurotransmitter levels in Cplx2-/- mice and found a significant decrease in levels of noradrenaline and the serotonin metabolite 5-hydroxyindoleacetic acid in the hippocampus. Chronic treatment with clorgyline, an irreversible inhibitor of monoamine oxidase A, restored hippocampal noradrenaline to normal levels (from 60 to 97% of vehicle-treated Cplx2+/+ mice, P<0.001), and reversed the behavioural deficits seen in Cplx2-/- mice. For example, clorgyline-treated Cplx2-/- mice spent significantly more time interacting with a novel visitor mouse compared with vehicle-treated Cplx2-/- mice in the social recognition test (34 compared with 13%, P<0.01). We were also able to reverse the selective deficit seen in mossy fibre-long-term potentiation (MF-LTP) in Cplx2-/- mice using the noradrenergic agonist isoprenaline. Pre-treatment with isoprenaline in vitro increased MF-LTP by 125% (P<0.001), thus restoring it to control levels. Our data strongly support the idea that complexin 2 is a key player in normal neurological function, and that downregulation of complexin 2 could lead to changes in neurotransmitter release sufficient to cause significant behavioural abnormalities such as depression.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Antidepressivos/administração & dosagem , Clorgilina/administração & dosagem , Proteínas do Tecido Nervoso/genética , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/fisiopatologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Norepinefrina/metabolismo
12.
Eur J Neurosci ; 36(4): 2470-81, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22694764

RESUMO

Complexins (Cplxs) regulate the speed and Ca(2+)-sensitivity of synaptic vesicle fusion. It has been shown that all four known Cplxs are present at mouse retinal synapses--at conventional amacrine cell synapses (Cplx 1 to Cplx 3) and at photoreceptor and bipolar cell ribbon synapses (Cplx 3 and Cplx 4) [K. Reim et al. (2005) J. Cell Biol., 169, 669-680]. Electroretinographic recordings in Cplx 3/Cplx 4 double-knockout (DKO) mice showed perturbed transmission in the outer plexiform layer, and possible changes in the inner plexiform layer [K. Reim et al. (2009) J. Cell Sci., 122, 1352-1361]. In the present study, we examined the effects of the absence of Cplx 3 and Cplx 4 on ganglion cell responses. We report that the lack of Cplx 3 and Cplx 4 differentially impacts the ON and OFF pathways. Under photopic conditions, the responses in the cone OFF pathway are largely unaffected, whereas the responses in the cone ON pathway are diminished in Cplx 3/Cplx 4 DKO mice. Under scotopic conditions, both ON and OFF response rates are reduced and high-sensitivity OFF responses are missing in Cplx 3/Cplx 4 DKO mice. The electrophysiological findings are corroborated by new immunocytochemical findings. We now show that rod spherules contain only Cplx 4. However, both Cplx 3 and Cplx 4 co-localize in cone pedicles. In the inner plexiform layer, Cplx 3 is present in rod bipolar cell terminals and in amacrine cell processes. Most importantly, Cplx 3 is localized in the lobular appendages of AII amacrine cells, the sites of signal transmission from the primary rod pathway into the OFF pathway in the inner plexiform layer.


Assuntos
Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios Retinianos/fisiologia , Vias Visuais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Estimulação Luminosa , Neurônios Retinianos/metabolismo
13.
Nat Struct Mol Biol ; 14(10): 949-58, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17828276

RESUMO

Complexins constitute a family of four synaptic high-affinity SNARE complex-binding proteins. They positively regulate a late, post-priming step in Ca2+-triggered synchronous neurotransmitter release, but the underlying molecular mechanisms are unclear. We show here that SNARE complex binding of complexin I (CplxI) via its central alpha-helix is necessary but, unexpectedly, not sufficient for its key function in promoting neurotransmitter release. An accessory alpha-helix on the N-terminal side of the SNARE complex-binding region has an inhibitory effect on fast synaptic exocytosis, whereas sequences N-terminally adjacent to this helix facilitate Ca2+-triggered release even in the absence of the Ca2+ sensor synaptotagmin-1. Our results indicate that distinct functional domains of CplxI differentially regulate synaptic exocytosis and that, through the interplay between these domains, CplxI carries out a crucial role in fine-tuning Ca2+-triggered fast neurotransmitter release.


Assuntos
Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurotransmissores/metabolismo , Estrutura Secundária de Proteína , Proteínas SNARE/metabolismo , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Sequência de Aminoácidos , Animais , Cálcio/metabolismo , Células Cultivadas , Exocitose/fisiologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Alinhamento de Sequência , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo
14.
Cereb Cortex ; 21(10): 2187-203, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21368089

RESUMO

There is currently a debate about the evolutionary origin of the earliest generated cortical preplate neurons and their derivatives (subplate and marginal zone). We examined the subplate with murine markers including nuclear receptor related 1 (Nurr1), monooxygenase Dbh-like 1 (Moxd1), transmembrane protein 163 (Tmem163), and connective tissue growth factor (Ctgf) in developing and adult turtle, chick, opossum, mouse, and rat. Whereas some of these are expressed in dorsal pallium in all species studied (Nurr1, Ctgf, and Tmem163), we observed that the closely related mouse and rat differed in the expression patterns of several others (Dopa decarboxylase, Moxd1, and thyrotropin-releasing hormone). The expression of Ctgf, Moxd1, and Nurr1 in the oppossum suggests a more dispersed subplate population in this marsupial compared with mice and rats. In embryonic and adult chick brains, our selected subplate markers are primarily expressed in the hyperpallium and in the turtle in the main cell dense layer of the dorsal cortex. These observations suggest that some neurons that express these selected markers were present in the common ancestor of sauropsids and mammals.


Assuntos
Córtex Cerebral/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Fatores Etários , Animais , Animais Recém-Nascidos , Córtex Cerebral/crescimento & desenvolvimento , Embrião de Galinha , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Gambás , Ratos , Ratos Wistar , Especificidade da Espécie , Tartarugas
15.
J Neurosci ; 30(24): 8171-9, 2010 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-20554867

RESUMO

Cerebellar feedforward inhibition (FFI) is mediated by two distinct pathways targeting different subcellular compartments of Purkinje cells (PCs). The axon of the granule cell, the parallel fiber, makes excitatory synapses not only onto PCs but also onto two types of interneurons, basket and stellate cells. Basket and stellate cells then send inhibitory signals to the soma and dendrites of Purkinje cells, respectively. Functional differences between somatic and dendritic FFI pathways, however, remain unknown. Here we address this question by examining how basket and stellate cells are recruited dynamically under high-frequency granule cell inputs at mice cerebellum. Short-term plasticity of various synapses within the FFI circuit has been explored. Unexpectedly, the parallel fiber synapse, which was considered to be facilitating during repetitive stimulation, shows depression, when the postsynaptic target is a basket cell. Other factors in the FFI circuit, such as firing properties of interneurons and dynamics of inhibitory synapses, are similar between somatic and dendritic pathways. The target-dependent parallel fiber synaptic plasticity has functional consequences for the two FFI pathways, because we observe that PCs receive transient somatic inhibition during 50 Hz stimulation of granule cells but persistent dendritic inhibition.


Assuntos
Cerebelo/citologia , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Células de Purkinje/fisiologia , Sinapses/fisiologia , Animais , Animais Recém-Nascidos , Biofísica , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/deficiência , Inibição Neural/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Células de Purkinje/efeitos dos fármacos , Piridazinas/farmacologia , Sinapses/efeitos dos fármacos
16.
Proc Natl Acad Sci U S A ; 105(22): 7875-80, 2008 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-18505837

RESUMO

Complexins (Cplxs) are key regulators of synaptic exocytosis, but whether they act as facilitators or inhibitors is currently being disputed controversially. We show that genetic deletion of all Cplxs expressed in the mouse brain causes a reduction in Ca(2+)-triggered and spontaneous neurotransmitter release at both excitatory and inhibitory synapses. Our results demonstrate that at mammalian central nervous system synapses, Cplxs facilitate neurotransmitter release and do not simply act as inhibitory clamps of the synaptic vesicle fusion machinery.


Assuntos
Cálcio/metabolismo , Exocitose , Proteínas do Olho/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurotransmissores/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Animais , Encéfalo/metabolismo , Encéfalo/fisiologia , Cálcio/farmacologia , Potenciais Evocados , Exocitose/genética , Proteínas do Olho/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal , Neurônios/metabolismo , Neurônios/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo
17.
Proc Natl Acad Sci U S A ; 105(49): 19538-43, 2008 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-19033464

RESUMO

SNARE-mediated exocytosis is a multistage process central to synaptic transmission and hormone release. Complexins (CPXs) are small proteins that bind very rapidly and with a high affinity to the SNARE core complex, where they have been proposed recently to inhibit exocytosis by clamping the complex and inhibiting membrane fusion. However, several other studies also suggest that CPXs are positive regulators of neurotransmitter release. Thus, whether CPXs are positive or negative regulators of exocytosis is not known, much less the stage in the vesicle life cycle at which they function. Here, we systematically dissect the vesicle stages leading up to exocytosis using a knockout-rescue strategy in a mammalian model system. We show that adrenal chromaffin cells from CPX II knockout mice exhibit markedly diminished releasable vesicle pools (comprising the readily and slowly releasable pools), while showing no change in the kinetics of fusion pore dilation or morphological vesicle docking. Overexpression of WT CPX II-but not of SNARE-binding-deficient mutants-restores the size of the the releasable pools in knockout cells, and in WT cells it markedly enlarges them. Our results show that CPXs regulate the size of the primed vesicle pools and have a positive role in Ca(2+)-triggered exocytosis.


Assuntos
Cálcio/metabolismo , Células Cromafins/fisiologia , Exocitose/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Células Cromafins/ultraestrutura , Feminino , Masculino , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Mutantes , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Proteínas SNARE/metabolismo , Vesículas Secretórias/ultraestrutura
18.
J Neurosci ; 29(25): 7991-8004, 2009 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-19553439

RESUMO

Complexins (CPXs I-IV) presumably act as regulators of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, but their function in the intact mammalian nervous system is not well established. Here, we explored the role of CPXs in the mouse auditory system. Hearing was impaired in CPX I knock-out mice but normal in knock-out mice for CPXs II, III, IV, and III/IV as measured by auditory brainstem responses. Complexins were not detectable in cochlear hair cells but CPX I was expressed in spiral ganglion neurons (SGNs) that give rise to the auditory nerve. Ca(2+)-dependent exocytosis of inner hair cells and sound encoding by SGNs were unaffected in CPX I knock-out mice. In the absence of CPX I, the resting release probability in the endbulb of Held synapses of the auditory nerve fibers with bushy cells in the cochlear nucleus was reduced. As predicted by computational modeling, bushy cells had decreased spike rates at sound onset as well as longer and more variable first spike latencies explaining the abnormal auditory brainstem responses. In addition, we found synaptic transmission to outlast the stimulus at many endbulb of Held synapses in vitro and in vivo, suggesting impaired synchronization of release to stimulus offset. Although sound encoding in the cochlea proceeds in the absence of complexins, CPX I is required for faithful processing of sound onset and offset in the cochlear nucleus.


Assuntos
Vias Auditivas/fisiologia , Núcleo Coclear/metabolismo , Audição/fisiologia , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Transmissão Sináptica/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Animais , Nervo Coclear/fisiologia , Células Ciliadas Auditivas Internas/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microscopia Confocal , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sinapses/metabolismo
19.
J Cell Biol ; 169(4): 669-80, 2005 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-15911881

RESUMO

Ribbon synapses in retinal sensory neurons maintain large pools of readily releasable synaptic vesicles. This allows them to release several hundreds of vesicles per second at every presynaptic release site. The molecular components that cause this high transmitter release efficiency of ribbon synapses are unknown. In the present study, we identified and characterized two novel vertebrate complexins (CPXs), CPXs III and IV, that are the only CPX isoforms present in retinal ribbon synapses. CPXs III and IV are COOH-terminally farnesylated, and, like CPXs I and II, bind to SNAP receptor complexes. CPXs III and IV can functionally replace CPXs I and II, and their COOH-terminal farnesylation regulates their synaptic targeting and modulatory function in transmitter release. The novel CPXs III and IV may contribute to the unique release efficacy of retinal sensory neurons.


Assuntos
Proteínas do Olho/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Organelas/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Sinapses/metabolismo , Proteínas Adaptadoras de Transporte Vesicular , Animais , Linhagem Celular , Proteínas do Olho/genética , Proteínas do Olho/isolamento & purificação , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Ligação Proteica/fisiologia , Prenilação de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteínas SNARE , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transmissão Sináptica/fisiologia , Proteínas de Transporte Vesicular/metabolismo
20.
Cereb Cortex ; 19(8): 1738-50, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19008461

RESUMO

The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor-related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.


Assuntos
Neurônios/metabolismo , Córtex Somatossensorial/metabolismo , Córtex Visual/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Ácido gama-Aminobutírico/metabolismo
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