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1.
J Neurosci ; 37(17): 4604-4617, 2017 04 26.
Article in English | MEDLINE | ID: mdl-28363983

ABSTRACT

Parvalbumin-expressing inhibitory neurons in the mammalian CNS are specialized for fast transmitter release at their output synapses. However, the Ca2+ sensor(s) used by identified inhibitory synapses, including the output synapses of parvalbumin-expressing inhibitory neurons, have only recently started to be addressed. Here, we investigated the roles of Syt1 and Syt2 at two types of fast-releasing inhibitory connections in the mammalian CNS: the medial nucleus of the trapezoid body to lateral superior olive glycinergic synapse, and the basket/stellate cell-Purkinje GABAergic synapse in the cerebellum. We used conditional and conventional knock-out (KO) mouse lines, with viral expression of Cre-recombinase and a light-activated ion channel for optical stimulation of the transduced fibers, to produce Syt1-Syt2 double KO synapses in vivo Surprisingly, we found that KO of Syt2 alone had only minor effects on evoked transmitter release, despite the clear presence of the protein in inhibitory nerve terminals revealed by immunohistochemistry. We show that Syt1 is weakly coexpressed at these inhibitory synapses and must be genetically inactivated together with Syt2 to achieve a significant reduction and desynchronization of fast release. Thus, our work identifies the functionally relevant Ca2+ sensor(s) at fast-releasing inhibitory synapses and shows that two major Syt isoforms can cooperate to mediate release at a given synaptic connection.SIGNIFICANCE STATEMENT During synaptic transmission, the influx of Ca2+ into the presynaptic nerve terminal activates a Ca2+ sensor for vesicle fusion, a crucial step in the activity-dependent release of neurotransmitter. Synaptotagmin (Syt) proteins, and especially Syt1 and Syt2, have been identified as the Ca2+ sensor at excitatory synapses, but the Ca2+ sensor(s) at inhibitory synapses in native brain tissue are not well known. We found that both Syt1 and Syt2 need to be genetically inactivated to cause a significant reduction of activity-evoked release at two types of fast inhibitory synapses in mouse brain. Thus, we identify Syt2 as a functionally important Ca2+ sensor at fast-releasing inhibitory synapses, and show that Syt1 and Syt2 can redundantly control transmitter release at specific brain synapses.


Subject(s)
Neurons/physiology , Parvalbumins/metabolism , Synaptic Transmission/physiology , Synaptotagmin II/physiology , Synaptotagmin I/physiology , Animals , Cerebellum/metabolism , Glycine/metabolism , Mice , Mice, Knockout , Nerve Fibers/physiology , Neurons/metabolism , Neurotransmitter Agents/metabolism , Photic Stimulation , gamma-Aminobutyric Acid/physiology
2.
Mol Neurodegener ; 10: 31, 2015 Jul 23.
Article in English | MEDLINE | ID: mdl-26202512

ABSTRACT

BACKGROUND: Accumulation of the ß-amyloid peptide (Aß) is a major pathological hallmark of Alzheimer's disease (AD). Recent studies have shown that synaptic Aß toxicity may directly impair synaptic function. However, proteins regulating Aß generation at the synapse have not been characterized. Here, we sought to identify synaptic proteins that interact with the extracellular domain of APP and regulate Aß generation. RESULTS: Affinity purification-coupled mass spectrometry identified members of the Synaptotagmin (Syt) family as novel interacting proteins with the APP ectodomain in mouse brains. Syt-1, -2 and -9 interacted with APP in cells and in mouse brains in vivo. Using a GST pull-down approach, we have further demonstrated that the Syt interaction site lies in the 108 amino acids linker region between the E1 and KPI domains of APP. Stable overexpression of Syt-1 or Syt-9 with APP in CHO and rat pheochromocytoma cells (PC12) significantly increased APP-CTF and sAPP levels, with a 2 to 3 fold increase in secreted Aß levels in PC12 cells. Moreover, using a stable knockdown approach to reduce the expression of endogenous Syt-1 in PC12 cells, we have observed a ~ 50% reduction in secreted Aß generation. APP processing also decreased in these cells, shown by lower CTF levels. Lentiviral-mediated knock down of endogenous Syt-1 in mouse primary neurons also led to a significant reduction in both Aß40 and Aß42 generation. As secreted sAPPß levels were significantly reduced in PC12 cells lacking Syt-1 expression, our results suggest that Syt-1 regulates Aß generation by modulating BACE1-mediated cleavage of APP. CONCLUSION: Altogether, our data identify the synaptic vesicle proteins Syt-1 and 9 as novel APP-interacting proteins that promote Aß generation and thus may play an important role in the pathogenesis of AD.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Synaptotagmins/physiology , Amyloid Precursor Protein Secretases/physiology , Amyloid beta-Peptides/biosynthesis , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/chemistry , Animals , Aspartic Acid Endopeptidases/physiology , CHO Cells , Cricetinae , Cricetulus , Mice , Neurons/metabolism , PC12 Cells , Peptide Fragments/biosynthesis , Peptide Fragments/metabolism , Protein Interaction Mapping , Protein Processing, Post-Translational , Protein Structure, Tertiary , RNA Interference , RNA, Small Interfering/genetics , Rats , Recombinant Fusion Proteins/metabolism , Species Specificity , Synaptotagmin I/deficiency , Synaptotagmin I/genetics , Synaptotagmin I/physiology , Synaptotagmin II/physiology
3.
Open Biol ; 3(11): 130163, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24350389

ABSTRACT

Spontaneous electrical activity generated by developing sensory cells and neurons is crucial for the maturation of neural circuits. The full maturation of mammalian auditory inner hair cells (IHCs) depends on patterns of spontaneous action potentials during a 'critical period' of development. The intrinsic spiking activity of IHCs can be modulated by inhibitory input from cholinergic efferent fibres descending from the brainstem, which transiently innervate immature IHCs. However, it remains unknown whether this transient efferent input to developing IHCs is required for their functional maturation. We used a mouse model that lacks the α9-nicotinic acetylcholine receptor subunit (α9nAChR) in IHCs and another lacking synaptotagmin-2 in the efferent terminals to remove or reduce efferent input to IHCs, respectively. We found that the efferent system is required for the developmental linearization of the Ca(2+)-sensitivity of vesicle fusion at IHC ribbon synapses, without affecting their general cell development. This provides the first direct evidence that the efferent system, by modulating IHC electrical activity, is required for the maturation of the IHC synaptic machinery. The central control of sensory cell development is unique among sensory systems.


Subject(s)
Hair Cells, Auditory, Inner/physiology , Motor Neurons/physiology , Receptors, Nicotinic/physiology , Synapses/physiology , Synaptic Transmission/physiology , Action Potentials , Animals , Cochlea/physiology , Mice , Mice, Knockout , Receptors, Nicotinic/genetics , Stereocilia , Synaptotagmin II/genetics , Synaptotagmin II/physiology
4.
Biochim Biophys Acta ; 1808(10): 2435-9, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21787744

ABSTRACT

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine.


Subject(s)
Exocytosis/physiology , Liposomes , Mast Cells/metabolism , Membrane Fusion/physiology , Synaptotagmin II/physiology , Blotting, Western , Cell Line , Electrophoresis, Polyacrylamide Gel
5.
J Neurosci ; 30(40): 13281-90, 2010 Oct 06.
Article in English | MEDLINE | ID: mdl-20926654

ABSTRACT

In pre-hearing mice, vesicle exocytosis at cochlear inner hair cell (IHC) ribbon synapses is triggered by spontaneous Ca(2+) spikes. At the onset of hearing, IHC exocytosis is then exclusively driven by graded potentials, and is characterized by higher Ca(2+) efficiency and improved synchronization of vesicular release. The molecular players involved in this transition are still unknown. Here we addressed the involvement of synaptotagmins and otoferlin as putative Ca(2+) sensors in IHC exocytosis during postnatal maturation of the cochlea. Using cell capacitance measurements, we showed that Ca(2+)-evoked exocytosis in mouse IHCs switches from an otoferlin-independent to an otoferlin-dependent mechanism at postnatal day 4. During this early exocytotic period, several synaptotagmins (Syts), including Syt1, Syt2 and Syt7, were detected in IHCs. The exocytotic response as well as the release of the readily releasable vesicle pool (RRP) was, however, unchanged in newborn mutant mice lacking Syt1, Syt2 or Syt7 (Syt1(-/-), Syt2(-/-) and Syt7(-/-) mice). We only found a defect in RRP recovery in Syt1(-/-) mice which was apparent as a strongly reduced response to repetitive stimulations. In post-hearing Syt2(-/-) and Syt7(-/-) mutant mice, IHC synaptic exocytosis was unaffected. The transient expression of Syt1 and Syt2, which were no longer detected in IHCs after the onset of hearing, indicates that these two most common Ca(2+)-sensors in CNS synapses are not involved in mature IHCs. We suggest that otoferlin underlies highly efficient Ca(2+)-dependent membrane-membrane fusion, a process likely essential to increase the probability and synchrony of vesicle fusion events at the mature IHC ribbon synapse.


Subject(s)
Cochlea/growth & development , Exocytosis , Hair Cells, Auditory, Inner/physiology , Membrane Proteins/physiology , Synaptotagmin II/physiology , Synaptotagmin I/physiology , Animals , Calcium/physiology , Calcium Signaling/genetics , Cellular Senescence/genetics , Cellular Senescence/physiology , Cochlea/cytology , Electric Capacitance , Exocytosis/genetics , Female , Hair Cells, Auditory, Inner/metabolism , Male , Membrane Proteins/genetics , Mice , Mice, Knockout , Organ Culture Techniques , Patch-Clamp Techniques , Synapses/genetics , Synapses/physiology , Synaptic Transmission/genetics , Synaptotagmin I/genetics , Synaptotagmin II/genetics
6.
Dev Cell ; 19(3): 426-39, 2010 Sep 14.
Article in English | MEDLINE | ID: mdl-20833364

ABSTRACT

Targeting of activated plasma membrane receptors to endocytic pathways is important in determining the outcome of growth factor signaling. However, the molecular mechanisms are still poorly understood. Here, we show that the synaptotagmin-related membrane protein E-Syt2 is essential for rapid endocytosis of the activated FGF receptor and for functional signal transduction during Xenopus development. E-Syt2 depletion prevents an early phase of activated FGF receptor endocytosis that we show is required for ERK activation and the induction of the mesoderm. E-Syt2 interacts selectively with the activated FGF receptor and with Adaptin-2, and is required upstream of Ras activation and of receptor autophosphorylation for ERK activation and the induction of the mesodermal marker Xbra. The data identify E-Syt2 as an endocytic adaptor for the clathrin-mediated pathway whose function is conserved in human and suggest a broader role for the E-Syt subfamily in growth factor signaling.


Subject(s)
Endocytosis/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1/metabolism , Synaptotagmin II/physiology , Xenopus laevis/embryology , Adaptor Protein Complex alpha Subunits/genetics , Adaptor Protein Complex alpha Subunits/metabolism , Animals , Blotting, Western , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Enzyme Activation , Fluorescent Antibody Technique , Humans , Immunoprecipitation , In Situ Hybridization , Mesoderm/cytology , Mesoderm/metabolism , RNA, Messenger/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus laevis/metabolism , ras Proteins/genetics , ras Proteins/metabolism
7.
Neuron ; 63(4): 482-96, 2009 Aug 27.
Article in English | MEDLINE | ID: mdl-19709630

ABSTRACT

A multitude of synaptic proteins interact at the active zones of nerve terminals to achieve the high temporal precision of neurotransmitter release in synchrony with action potentials. Though synaptotagmin has been recognized as the Ca2+ sensor for synchronous release, it may have additional roles of action. We address this question at the calyx of Held, a giant presynaptic terminal, that allows biophysical dissection of multiple roles of molecules in synaptic transmission. Using high-level expression recombinant adenoviruses, in conjunction with a stereotactic surgery in postnatal day 1 rats, we overcame the previous inability to molecular perturb the calyx by overexpression of a mutated synaptotagmin. We report that this mutation leaves intrinsic Ca2+ sensitivity of vesicles intact while it destabilizes the readily releasable pool of vesicles and loosens the tight coupling between Ca2+ influx and release, most likely by interfering with the correct positioning of vesicles with respect to Ca2+ channels.


Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Synaptic Vesicles/metabolism , Synaptotagmin II/physiology , Amino Acid Sequence , Animals , Animals, Newborn , Calcium/physiology , Calcium Signaling/genetics , Cells, Cultured , Molecular Sequence Data , Presynaptic Terminals/metabolism , Rats , Rats, Wistar , Synaptic Vesicles/genetics , Synaptotagmin II/genetics , Time Factors
8.
J Biol Chem ; 284(15): 9781-7, 2009 Apr 10.
Article in English | MEDLINE | ID: mdl-19208631

ABSTRACT

Synaptotagmin 2 (Syt2) functions as a low affinity, fast exocytic Ca(2+) sensor in neurons, where it is activated by Ca(2+) influx through voltage-gated channels. Targeted insertion of lacZ into the mouse syt2 locus reveals expression in mucin-secreting goblet cells of the airways. In these cells, rapid Ca(2+) entry from the extracellular medium does not contribute significantly to stimulated secretion (Davis, C. W., and Dickey, B. F. (2008) Annu. Rev. Physiol. 70, 487-512). Nonetheless, Syt2(-/-) mice show a severe defect in acute agonist-stimulated airway mucin secretion, and Syt2(+/-) mice show a partial defect. In contrast to Munc13-2(-/-) mice (Zhu, Y., Ehre, C., Abdullah, L. H., Sheehan, J. K., Roy, M., Evans, C. M., Dickey, B. F., and Davis, C. W. (2008) J. Physiol. (Lond.) 586, 1977-1992), Syt2(-/-) mice show no spontaneous mucin accumulation, consistent with the inhibitory action of Syt2 at resting cytoplasmic Ca(2+) in neurons. In human airway goblet cells, inositol trisphosphate receptors are found in rough endoplasmic reticulum that closely invests apical mucin granules, consistent with the known dependence of exocytic Ca(2+) signaling on intracellular stores in these cells. Hence, Syt2 can serve as an exocytic sensor for diverse Ca(2+) signaling systems, and its levels are limiting for stimulated secretory function in airway goblet cells.


Subject(s)
Calcium Signaling , Endoplasmic Reticulum/metabolism , Exocytosis , Gene Expression Regulation , Goblet Cells/metabolism , Synaptotagmin II/physiology , Animals , Calcium/metabolism , Cytoplasm/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mucins/metabolism , Neurons/metabolism , Synaptotagmin II/metabolism
9.
J Neurosci ; 26(52): 13493-504, 2006 Dec 27.
Article in English | MEDLINE | ID: mdl-17192432

ABSTRACT

Biochemical and genetic data suggest that synaptotagmin-2 functions as a Ca2+ sensor for fast neurotransmitter release in caudal brain regions, but animals and/or synapses lacking synaptotagmin-2 have not been examined. We have now generated mice in which the 5' end of the synaptotagmin-2 gene was replaced by lacZ. Using beta-galactosidase as a marker, we show that, consistent with previous studies, synaptotagmin-2 is widely expressed in spinal cord, brainstem, and cerebellum, but is additionally present in selected forebrain neurons, including most striatal neurons and some hypothalamic, cortical, and hippocampal neurons. Synaptotagmin-2-deficient mice were indistinguishable from wild-type littermates at birth, but subsequently developed severe motor dysfunction, and perished at approximately 3 weeks of age. Electrophysiological studies in cultured striatal neurons revealed that the synaptotagmin-2 deletion slowed the kinetics of evoked neurotransmitter release without altering the total amount of release. In contrast, synaptotagmin-2-deficient neuromuscular junctions (NMJs) suffered from a large reduction in evoked release and changes in short-term synaptic plasticity. Furthermore, in mutant NMJs, the frequency of spontaneous miniature release events was increased both at rest and during stimulus trains. Viewed together, our results demonstrate that the synaptotagmin-2 deficiency causes a lethal impairment in synaptic transmission in selected synapses. This impairment, however, is less severe than that produced in forebrain neurons by deletion of synaptotagmin-1, presumably because at least in NMJs, synaptotagmin-1 is coexpressed with synaptotagmin-2, and both together mediate fast Ca2+-triggered release. Thus, synaptotagmin-2 is an essential synaptotagmin isoform that functions in concert with other synaptotagmins in the Ca2+ triggering of neurotransmitter release.


Subject(s)
Brain/metabolism , Calcium/physiology , Neuromuscular Junction/metabolism , Neurotransmitter Agents/metabolism , Synapses/metabolism , Synaptotagmin II/physiology , Animals , Animals, Newborn , Brain/drug effects , Cell Survival/genetics , Cells, Cultured , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neuromuscular Junction/genetics , Neurotransmitter Agents/genetics , Protein Isoforms/deficiency , Protein Isoforms/genetics , Protein Isoforms/physiology , Synapses/genetics , Synaptic Transmission/genetics , Synaptotagmin II/deficiency , Synaptotagmin II/genetics
10.
J Neurosci ; 26(2): 632-43, 2006 Jan 11.
Article in English | MEDLINE | ID: mdl-16407561

ABSTRACT

Synaptotagmins comprise a large protein family, of which synaptotagmin 1 (Syt1) is a Ca2+ sensor for fast exocytosis, and its close relative, synaptotagmin 2 (Syt2), is assumed to serve similar functions. Chromaffin cells express Syt1 but not Syt2. We compared secretion from chromaffin cells from Syt1 null mice overexpressing either Syt isoform. High time-resolution capacitance measurement showed that Syt1 null cells lack the exocytotic phase corresponding to the readily-releasable pool (RRP) of vesicles. Comparison with the amperometric signal confirmed that the missing phase of exocytosis consists of catecholamine-containing vesicles. Overexpression of Syt1 rescued the RRP and increased its size above wild-type values, whereas the size of the slowly releasable pool decreased, indicating that the availability of Syt1 regulates the relative size of the two releasable pools. The RRP was also rescued by Syt2 overexpression, but the kinetics of fusion was slightly slower than in cells expressing Syt1. Biochemical experiments showed that Syt2 has a slightly lower Ca2+ affinity for phospholipid binding than Syt1 because of a difference in the C2A domain. These data constitute evidence for the function of Syt1 and Syt2 as alternative, but not identical, calcium-sensors for RRP fusion. By overexpression of Syt1 mutated in the shared PKC/calcium/calmodulin-dependent kinase phosphorylation site, we show that phorbol esters act independently and upstream of Syt1 to regulate the size of the releasable pools. We conclude that exocytosis from mouse chromaffin cells can be modified by the differential expression of Syt isoforms and by Syt abundance but not by phosphorylation of Syt1.


Subject(s)
Chromaffin Cells/physiology , Exocytosis/physiology , Protein Processing, Post-Translational , Synaptotagmin II/physiology , Synaptotagmin I/physiology , Animals , Binding Sites , Calcium/metabolism , Catecholamines/metabolism , Cells, Cultured/physiology , Liposomes/metabolism , Mice , Mice, Knockout , Mutagenesis, Site-Directed , Nerve Tissue Proteins/metabolism , Patch-Clamp Techniques , Phospholipids/metabolism , Phosphorylation , Photolysis , Protein Binding , Protein Kinase C/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/physiology , SNARE Proteins/metabolism , Synaptotagmin I/chemistry , Synaptotagmin I/deficiency , Synaptotagmin I/genetics , Synaptotagmin II/chemistry , Synaptotagmin II/deficiency , Synaptotagmin II/genetics , Tetradecanoylphorbol Acetate/pharmacology , Transfection
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