Structure of synaptogyrin (p29) defines novel synaptic vesicle protein.

Synaptogyrin (p29) is a synaptic vesicle protein that is uniformly distributed in the nervous system (Baumert et al., 1990). We have cloned and sequenced the cDNA encoding synaptogyrin, and the sequence predicts a protein with a molecular mass of 25,900 D with four membrane-spanning domains. The topology of the protein was confirmed by limited proteolysis using domain-specific antibodies. Database searches revealed several cDNA sequences coding polypeptides with sequence identities ranging from 32 to 46%, suggesting that synaptogyrin is a member of a multigene family. When the synaptogyrin cDNA is expressed in COS cells, the generated protein is indistinguishable from native synaptogyrin. To study intracellular sorting, synaptogyrin was expressed in CHO cells that revealed a punctate staining that was very similar to that of synaptophysin and endogenously expressed cellubrevin. Significant overlap with transferrin staining was also observed, suggesting that synaptogyrin is targeted to a recycling compartment involved in membrane traffic to and from the plasma membrane.

Mechanism of action of rab3A in mossy fiber LTP.

In mossy fiber synapses of the hippocampal CA3 region, LTP is induced by cAMP and requires the synaptic vesicle protein rab3A. In contrast, CA1-region synapses do not exhibit this type of LTP. We now show that cAMP enhances glutamate release from CA3 but not CA1 synaptosomes by (1) increasing the readily releasable pool as tested by hypertonic sucrose; (2) potentiating release evoked by KCl depolarization, which opens voltage-gated Ca2+ channels; and (3) by enhancing Ca2+ action on the secretory apparatus as monitored by the Ca2+-ionophore ionomycin. In rab3A-deficient synaptosomes, forskolin still enhances KCl- and sucrose-induced glutamate release but not ionomycin-induced release. Our results show that cAMP has multiple actions in mossy fiber synapses, of which only the direct activation of the secretory apparatus requires rab3A and functions in mfLTP.

SV2A and SV2B function as redundant Ca2+ regulators in neurotransmitter release.

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor isoform (SV2C) that resemble transporter proteins. We now show that SV2B knockout mice are phenotypically normal while SV2A- and SV2A/SV2B double knockout mice exhibit severe seizures and die postnatally. In electrophysiological recordings from cultured hippocampal neurons, SV2A- or SV2B-deficient cells exhibited no detectable abnormalities. Neurons lacking both SV2 isoforms, however, experienced sustained increases in Ca2+-dependent synaptic transmission when two or more action potentials were triggered in succession. These increases could be reversed by EGTA-AM. Our data suggest that without SV2 proteins, presynaptic Ca2+ accumulation during consecutive action potentials causes abnormal increases in neurotransmitter release that destabilize synaptic circuits and induce epilepsy.

A role for cAMP in long-term depression at hippocampal mossy fiber synapses.

Mossy fiber synapses on hippocampal CA3 pyramidal cells, in addition to expressing an NMDA receptor-independent form of long-term potentiation (LTP), have recently been shown to express a novel presynaptic form of long-term depression (LTD). We have studied the mechanisms underlying mossy fiber LTD and present evidence that it is triggered, at least in part, by a metabotropic glutamate receptor-mediated decrease in adenylyl cyclase activity, which leads to a decrease in the activity of the cAMP-dependent protein kinase (PKA) and a reversal of the presynaptic processes responsible for mossy fiber LTP. The bidirectional control of synaptic strength at mossy fiber synapses by activity therefore appears to be due to modulation of the cAMP-PKA signaling pathway in mossy fiber boutons.

Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I.

We have generated mice lacking synaptogyrin I and synaptophysin I to explore the functions of these abundant tyrosine-phosphorylated proteins of synaptic vesicles. Single and double knockout mice were alive and fertile without significant morphological or biochemical changes. Electrophysiological recordings in the hippocampal CA1 region revealed that short-term and long-term synaptic plasticity were severely reduced in the synaptophysin/synaptogyrin double knockout mice. LTP was decreased independent of the induction protocol, suggesting that the defect in LTP was not caused by insufficient induction. Our data show that synaptogyrin I and synaptophysin I perform redundant and essential functions in synaptic plasticity without being required for neurotransmitter release itself.

Rabphilin knock-out mice reveal that rabphilin is not required for rab3 function in regulating neurotransmitter release.

Rab3A and rab3C are GTP-binding proteins of synaptic vesicles that regulate vesicle exocytosis. Rabphilin is a candidate rab3 effector at the synapse because it binds to rab3s in a GTP-dependent manner, it is co-localized with rab3s on synaptic vesicles, and it dissociates with rab3s from the vesicles during exocytosis. Rabphilin contains two C(2) domains, which could function as Ca(2+) sensors in exocytosis and is phosphorylated as a function of stimulation. However, it is unknown what essential function, if any, rabphilin performs. One controversial question regards the respective roles of rab3s and rabphilin in localizing each other to synaptic vesicles: although rabphilin is mislocalized in rab3A knock-out mice, purified synaptic vesicles were shown to require rabphilin for binding of rab3A but not rab3A for binding of rabphilin. To test whether rabphilin is involved in localizing rab3s to synaptic vesicles and to explore the functions of rabphilin in regulating exocytosis, we have now analyzed knock-out mice for rabphilin. Mice that lack rabphilin are viable and fertile without obvious physiological impairments. In rabphilin-deficient mice, rab3A is targeted to synaptic vesicles normally, whereas in rab3A-deficient mice, rabphilin transport to synapses is impaired. These results show that rabphilin binds to vesicles via rab3s, consistent with an effector function of rabphilin for a synaptic rab3-signal. Surprisingly, however, no abnormalities in synaptic transmission or plasticity were observed in rabphilin-deficient mice; synaptic properties that are impaired in rab3A knock-out mice were unchanged in rabphilin knock-out mice. Our data thus demonstrate that rabphilin is endowed with the properties of a rab3 effector but is not essential for the regulatory functions of rab3 in synaptic transmission.

SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family.

We describe here the identification and molecular characterization of a new brain protein that we named SV2C because it is homologous to the synaptic vesicle proteins SV2A and SV2B, and because it is also recognized by the monoclonal SV2 antibody that led to the initial discovery of SV2A and SV2B. SV2C is more closely related to SV2A (62% identity) than to SV2B (57% identity), and contains 12 transmembrane regions similar to these proteins. To characterize SV2C and compare its properties and localization with those of SV2A and SV2B, we raised an SV2C-specific antibody. Using this antibody, we show that SV2C is an N-glycosylated protein that is concentrated on small synaptic vesicles; in addition, it is found on microvesicles in adrenal chromaffin cells. We evaluated the relative localization of the three SV2 isoforms by staining rat brain sections with antibodies specific for SV2A, SV2B and SV2C. Analysis of the resulting staining patterns confirmed previous conclusions that SV2A is ubiquitously expressed in virtually all synapses. SV2B, although more restricted in distribution, was also found in a wide variety of synapses throughout the brain. In striking contrast to this general localization and to similarly wide distributions of other synaptic vesicle proteins, SV2C was observed only in few brain areas. High levels of SV2C were found primarily in phylogenetically old brain regions such as the pallidum, the substantia nigra, the midbrain, the brainstem and the olfactory bulb. SV2C was undetectable in the cerebral cortex and the hippocampus, and found at low levels in the cerebellar cortex. Our data suggest that closely related members of a synaptic vesicle protein family can either have very general (SV2A) or restricted distributions (SV2C), possibly in order to allow specialization in the regulation of the expression or of the function of these abundant synaptic vesicle proteins.

Syntaxin 3B is essential for the exocytosis of synaptic vesicles in ribbon synapses of the retina.

Ribbon synapses of the vertebrate retina are specialized synapses that release neurotransmitter by synaptic vesicle exocytosis in a manner that is proportional to the level of depolarization of the cell. This release property is different from conventional neurons, in which the release of neurotransmitter occurs as a short-lived burst triggered by an action potential. Synaptic vesicle exocytosis is a calcium regulated process that is dependent on a set of interacting synaptic proteins that form the so-called SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) complex. Syntaxin 3B has been identified as a specialized SNARE molecule in ribbon synapses of the rodent retina. However, the best physiologically-characterized neuron that forms ribbon-style synapses is the rod-dominant or Mb1 bipolar cell of the goldfish retina. We report here the molecular characterization of syntaxin 3B from the goldfish retina. Using a combination of reverse transcription (RT) polymerase chain reaction (PCR) and immunostaining with a specific antibody, we show that syntaxin 3B is highly enriched in the plasma membrane of bipolar cell synaptic terminals of the goldfish retina. Using membrane capacitance measurements we demonstrate that a peptide derived from goldfish syntaxin 3B inhibits synaptic vesicle exocytosis. These experiments demonstrate that syntaxin 3B is an important factor for synaptic vesicle exocytosis in ribbon synapses of the vertebrate retina.

Effect of chamber eccentricity on equatorial fiber stress during systole.

A survey was conducted of methods of assessing stress in the left ventricle. Although simultaneous measurements have been made of average wall force and thickness in the dog left ventricle (from which stress can be computed), the experimental procedures are not applicable to man. However, two mathematical models based on ellipsoidal representations of ventricular geometry have been shown to predict average circumferential stress reasonably well. Although both of these models are sensitive to errors in average equatorial wall thickness, they appear to be the most reliable models currently available for estimating stress. One of these models was applied to an analysis of midwall equatorial fiber stress and force in the normal conscious dog left ventricle. It was found that variations in chamber eccentricity during systole were much less important in evaluating this stress than variations in the ratio of equatorial wall thickness to semi-minor radius. A formula was derived that relates fiber stress to fiber force. It was found that fiber stress and force decrease more rapidly during ejection than intraventricular pressure, consistent with previous results.

Estimation of local myocardial stress.

Two formulas are presented for estimating local average circumferential stress in the left ventricle from the cavity pressure and various quantities, available from the angiogram, which characterize the size and shape of the cavity and ventricular wall. The advantages of these formulas are as follows: 1) they are based on thick-wall shell theory; 2) they are intended for application at positions in the ventricular wall other than the base; and 3) they are based on a more general representation of ventricular geometry than a sphere, cylinder, or ellipsoid. Except for one location, both formulas predict average circumferential stresses that agree to within 25% with the corresponding stresses in a finite element model of an aneurysmal ventricle. In addition, at the equator of a thick-wall ellipsoid, the formulas are identical in form to a previously derived formula that has been shown to predict stresses that are in fair to good agreement with measured stresses in the open-chest dog heart.

Characterization of a brain-specific Sp1-like activity interacting with an unusual binding site within the myelin proteolipid protein promoter.

The proteolipid protein (PLP) gene encodes the main integral protein of the myelin membrane of the central nervous system. The expression of the gene is regulated in a cell- and development-specific manner. Comparison of approximately 1.5 kb of the upstream noncoding region from man, mouse, and rat gene revealed an extensive sequence identity of about 95% between -250 and +100 (the most upstream transcription start site is defined as +1) but only about 50% identity further upstream. To define potential cis-acting elements in the promoter of the mouse PLP gene the upstream region was studied by transfection of C6 glioblastoma cells and CHO fibroblasts with various 5' deletion constructs fused to the reporter gene luciferase. We localized a promoter at position -184 to +90, which is active in both cell lines. Analysis of this region by DNase I foot-printing experiments and band shift analysis with nuclear extracts from myelinating brain, liver, C6, and CHO cells shows the binding of several different proteins to the promoter region. One brain-specific and two ubiquitous factors bound to the sequence AAGGGGAGGAG (DR1/2 box). This motif is also present in the upstream region of other myelin-specific genes and in some variants of the glia cell-specific virus JC. The factors bound with similar affinity to a Sp1-binding site. Therefore one of the ubiquitous factors seems to be Sp1 suggesting that Sp1 may play a role in the transcriptional regulation of the PLP gene. It has been shown that the DR1/2 box-binding factors are Zn(2+)-dependent. By Southwestern blotting it has been demonstrated that the DR1/2 box binds a protein of about 66 kDa that is enriched in brain.

Cellugyrin, a novel ubiquitous form of synaptogyrin that is phosphorylated by pp60c-src.

Synaptogyrin is an abundant membrane protein of synaptic vesicles containing four transmembrane regions and a C-terminal cytoplasmic tail that is tyrosine phosphorylated. We have now identified a novel isoform of synaptogyrin called cellugyrin that exhibits 47% sequence identity with synaptogyrin. In rat tissues, cellugyrin and synaptogyrins are expressed in mirror image patterns. Cellugyrin is ubiquitously present in all tissues tested with the lowest levels in brain tissue, whereas synaptogyrin protein is only detectable in brain. Transfection studies in COS cells demonstrated that both cellugyrin and synaptogyrin are tyrosine phosphorylated in vivo by pp60c-src, and experiments with recombinant proteins showed that pp60c-src phosphorylates the cytoplasmic tails of these proteins in vitro. Cellugyrin and synaptogyrin co-localize when transfected into COS cells but are differentially distributed in brain, the only tissue where both proteins are detectable. Our data suggest that the synaptic vesicle protein synaptogyrin is a specialized version of a ubiquitous protein, cellugyrin, with the two proteins sharing structural similarity but differing in localization. This finding supports the emerging concept of synaptic vesicles as the simplified and specialized form of a generic trafficking organelle. The conserved tyrosine phosphorylation of cellugyrin and synaptogyrins suggests a link between tyrosine phosphorylation via pp60c-src and membrane traffic.

Deformation of the diastolic left ventricle. Nonlinear elastic effects.

A linear incremental finite element model is used to analyze the mechanical behavior of the left ventricle. The ventricle is treated as a heterogeneous, non-linearly elastic, isotropic, thick-walled solid of revolution. A new triaxial constitutive relation for the myocardium is presented which exhibits the observed exponential length-passive tension behavior of left ventricular papillary muscle in the limit of uniaxial tension. This triaxial relation contains three parameters: (a) a "small strain" Young's modulus, (b) a Poisson's ratio, and (c) a parameter which characterizes the nonlinear aspect of the elastic behavior of heart muscle. The inner third and outer two-thirds of the ventricular wall are assumed to have small strain Young's moduli of 30 and 60 g/cm(2), respectively. The Poisson's ratio is assumed to be equal to 0.49 throughout the ventricular wall. In general, the results of this study indicate that while a linearly elastic model for the ventricle may be adequate in terms of predicting pressure-volume relationships, a linear model may have serious limitations with regard to predicting fiber elongation within the ventricular wall. For example, volumes and midwall equatorial circumferential strains predicted by the linear and nonlinear models considered in this study differ by approximately 20 and 90%, respectively, at a transmural pressure of 12 cm H(2)O.

Estimation of regional stress in the left ventricular septum and free wall: an echocardiographic study suggesting a mechanism for asymmetric septal hypertrophy.

Although asymmetric septal hypertrophy is noted in a wide variety of cardiac disorders, its cause remains unclear. One possible mechanism is that the septum is subjected to greater systolic stress because of its flatter (more eccentric) contour. This was investigated noninvasively in nine subjects by estimation of regional myocardial stress from measurements of blood pressure by cuff sphygmomanometry and by echocardiographic examinations of left ventricular shape and dimensions. Analysis of left ventricular cavity shape showed that both the free and septal walls were elliptical, but the septum was more eccentric than the free wall. Using a conceptual model to determine changes in regional systolic stress, the theoretical rate of increase in regional stress relative to pressure (delta S/delta P) was significantly greater in the septum compared to the free wall. Increased hypertrophy of the septum to normalize this increased delta S/delta P may be the cause of asymmetric septal hypertrophy in many disorders associated with elevated left ventricular pressure.