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At least 19 recordsLinked to original sources
PubMed1994

Synaptophysin gene expression in human brain: a quantitative in situ hybridization and immunocytochemical study.

Synaptophysin is a presynaptic vesicle protein. Its quantitative detection has become established as a molecular marker of synaptic density. We have studied synaptophysin messenger RNA in the neocortex, hippocampus and cerebellum using in situ hybridization histochemistry to see if the encoding transcript can be detected in post mortem human brain and to investigate factors which might influence its abundance. Synaptophysin was also measured immunocytochemically in the hippocampus. The level of synaptophysin messenger RNA expression was uniform in all neocortical areas examined. Strong correlations were found for the amount of synaptophysin messenger RNA between individual regions and between homologous areas in the two hemispheres. Synaptophysin messenger RNA declined with increasing age and prolonged post mortem interval. Synaptophysin immunoreactivity also reduced with age, as well as with duration of formalin fixation but not post mortem interval. Synaptophysin immunoreactivity correlated with the abundance of the messenger RNA in neurons within, and projecting to, each hippocampal subfield. Significantly greater synaptophysin immunoreactivity was seen in the left than the right CA4 and CA1 regions. These data show that quantitative detection of synaptophysin messenger RNA as well as synaptophysin itself can reliably be carried out in post mortem human brain sections. They are in keeping with other findings that synaptic density is relatively uniform through the neocortex and decreases with age. They also suggest a possible asymmetry of hippocampal synaptophysin expression. The level of synaptophysin messenger RNA paralleled that of synaptophysin immunoreactivity, indicating that changes in gene expression contribute to variations in the latter observed in aging and other situations. Detection of synaptophysin messenger RNA broadens the range of methods by which synaptic protein gene products can be studied and used as markers of synaptic density and synaptic involvement during physiological and pathological processes in human brain.

Adult↗
PubMed1991

Expression of synaptophysin during the prenatal development of the rat spinal cord: correlation with basic differentiation processes of neurons.

The development of the spinal cord involves the proliferation of neurons, their migration to well-defined areas, fiber outgrowth and synapse formation. The present study was designed to correlate the spatiotemporal pattern of expression of synaptophysin, an integral membrane protein of small synaptic vesicles, with these basic processes occurring during the embryonic development of the rat spinal cord. Thoracic segments of spinal cords from embryonic days 12, 14, 16, 18, 20 and of adult spinal cords were studied. S1 nuclease protection assays and immunoblots revealed minute amounts of specific mRNA and synaptophysin at embryonic day 12. There was a steep increase of mRNA between embryonic days 14 and 16, after which levels reached a plateau. A rise in the amount of synaptophysin in the spinal cord occurred between embryonic days 12 and 14, and the levels changed only slightly until the end of embryonic development. Even higher levels of synaptophysin, found in the adult spinal cord, may indicate that its biosynthesis continued after birth. In situ hybridization histochemistry revealed the localization of specific synaptophysin mRNA in the neuroepithelium. However, immunocytochemistry failed to detect synaptophysin in the neuroepithelial cells. Following migration of the neuroblasts, synaptophysins was found in neurons concomitantly with the onset of fiber outgrowth. Thus, already at embryonic day 12, outgrowing fibers of the dorsal root sensory neurons and of motoneurons were synaptophysin positive. From embryonic day 14 throughout the prenatal period, strong synaptophysin immunoreactivity was seen in the ventrolateral and dorsal parts of the marginal layer. Most likely this staining pattern indicates transient functional synaptic contacts because, in the adult spinal cord, the corresponding region, the white matter, exhibited only faint synaptophysin immunoreactivity. In the intermediate layer of the embryonic spinal cord, which corresponds to the gray matter of the adult spinal cord, synaptophysin-positive fibers were observed prior to the formation of functional synapses. The latter are most likely permanent, since synaptophysin in the adult spinal cord is mainly confined to the gray matter. Our data (i) show transcription and translation of synaptophysin within the neurons of the spinal cord and correlate these processes with proliferation, migration, fiber outgrowth and the formation of transient or permanent synapses, and (ii) prove that synaptophysin is a marker for fiber outgrowth in addition to synapse formation.

Aging↗
PubMed1995

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

The synaptic vesicle protein synaptobrevin (VAMP) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion. It interacts with the synaptic membrane proteins syntaxin I and synaptosome-associated protein (SNAP)-25 to form a complex which precedes exocytosis [Söllner et al. (1993b) Cell, 75, 409-418]. Here we demonstrate that the majority of synaptobrevin is bound to the vesicle protein synaptophysin in detergent extracts. No syntaxin I was found in this complex when synaptophysin-specific antibodies were used for immunoprecipitation. Conversely, no synaptophysin was associated with the synaptobrevin-syntaxin I complex when syntaxin-specific antibodies were used for immunoprecipitation. Thus, the synaptobrevin pool bound to synaptophysin is not available for binding to syntaxin I and SNAP-25, and vice versa. Synaptobrevin-synaptophysin binding was also demonstrated by chemical cross-linking in isolated nerve terminals. Furthermore, recombinant synaptobrevin II efficiently bound synaptophysin and its isoform synaptoporin, but not the more distantly related synaptic vesicle protein p29. Recombinant synaptobrevin I bound with similar efficiency, whereas the non-neuronal isoform cellubrevin displayed a lower affinity towards synaptophysin. Treatment with high NaCl concentrations resulted in a dissociation of the synaptobrevin-synaptophysin complex. In addition, the interaction of synaptobrevin with synaptophysin was irreversibly abolished by low amounts of SDS, while the interaction with syntaxin I was enhanced. We conclude that synaptophysin selectively interacts with synaptobrevin in a complex which excludes the t-SNAP receptors syntaxin I and SNAP-25, suggesting a role for synaptophysin in the control of exocytosis.

Amino Acid Sequence↗
PubMed1999

Co-localization of synaptophysin with different neuroendocrine hormones in the human gastrointestinal tract.

Colocalisation of synaptophysin has been studied in different neuroendocrine cell types in histologically normal mucosa from human gastrointestinal tract (corpus, antrum, duodenum, ileum and colon) using double-immunofluorescence stainings. Numerous synaptophysin immunoreactive cells were seen in the antrum, while a smaller number were found in the intestinal tract. Synaptophysin immunoreactivity was strong in the antrum but weak in the intestine. In the intestinal colocalisation studies the synaptophysin immunoreactivity was enhanced by using the tyramide amplification method. Synaptophysin and chromogranin A were colocalised but the latter occurred mainly basally, whereas synaptophysin was found to occur diffusely throughout the cytoplasm. Synaptophysin immunoreactivity occurred in the serotonin cells throughout the gastrointestinal tract, and in the antral gastrin and somatostatin cells. In the intestinal tract only a small fraction of somatostatin, gastrin, cholecystokinin, enteroglucagon, enteroglucagon/ peptide tyrosine tyrosine displayed synaptophysin immunoreactivity. In the gastrointestinal tract (except the antrum), chromogranin A is a better general neuroendocrine marker than synaptophysin. The functional role of synaptophysin is unclear but it may be involved in the intracellular transport and release of hormones. Based on the distribution background of synaptophysin, it seems to be of greater importance in the antrum than in the intestinal tract as a whole.

Adult↗
PubMed1995

Altered synaptophysin expression as a marker of synaptic pathology in schizophrenia.

It has been proposed that synaptic density or synaptic innervation may be altered in schizophrenia as a correlate of the neurodevelopmental pathology of the disease. Synaptophysin is a synaptic vesicle protein whose distribution and abundance provides a synaptic marker which can be reliably measured in post mortem brain. We have used in situ hybridization histochemistry and immunoreactivity to assess the expression of synaptophysin messenger RNA and protein respectively in medial temporal lobe from seven schizophrenics and 13 controls. In the schizophrenic cases, synaptophysin messenger RNA was reduced bilaterally in CA4, CA3, subiculum and parahippocampal gyrus, with a similar trend in dentate gyrus but no change in CA1. It was also decreased in terms of grains per pyramidal neuron in the affected subfields. In parahippocampal gyrus, the loss of synaptophysin messenger RNA per neuron in schizophrenia was greater in deep than superficial laminae. A parallel study in rats showed no effect of haloperidol treatment upon hippocampal synaptophysin messenger RNA, suggesting that neuroleptic treatment does not underlie the reductions found in schizophrenia. In the right medial temporal lobe of schizophrenics, we confirmed the correlation of synaptophysin messenger RNA abundance between ipsilateral subfields seen in both hemispheres of control brains. However, these correlations were not observed in the left medial temporal lobe of the schizophrenic cases. Synaptophysin immunoreactivity in schizophrenia showed no significant differences in any subfield compared to controls. Our data support the broad hypothesis that synaptic pathology occurs in schizophrenia. In so far as synaptophysin expression is a marker for synaptic density, the data suggest that pyramidal neurons within the medial temporal lobe may form fewer synapses. However, the lack of any significant differences in synaptophysin immunoreactivity despite the loss of encoding messenger RNA means that this conclusion must be drawn cautiously. There are several plausible explanations for the preservation of synaptophysin immunoreactivity despite reductions in transcript abundance; one possibility is that the inferrred loss of synapses occurs in extra-hippocampal sites to which the affected pyramidal neurons project. For example, the reduction in synaptophysin messenger RNA in subicular neurons may be accompanied by decreased density of synaptic terminals in the nucleus accumbens. Such differences in the efferent synaptic connectivity of the hippocampus have previously been hypothesized to be an important component of the circuitry underlying schizophrenia.

Animals↗
PubMed1990

The multisubunit structure of synaptophysin. Relationship between disulfide bonding and homo-oligomerization.

Synaptophysin, a major membrane protein of synaptic vesicles, contains four transmembrane regions and two intravesicular loops. Synaptophysin monomers associate into homopolymers that have the potential to form channels in the synaptic vesicle membrane. Here we show that in native synaptophysin, homopolymers are linked by noncovalent forces. The molecule contains unstable intramolecular disulfide bonds that undergo disulfide exchange during solubilization, thereby covalently cross-linking neighboring synaptophysin molecules. The locations of the intramolecular disulfide bonds in synaptophysin were determined, revealing that each of the two intravesicular loops of synaptophysin is circularized by a single disulfide bond. Cross-linking of synaptophysin by disulfide bonds can be triggered in synaptic vesicles and in intact cells by a cycle of reduction and oxidation, suggesting that native synaptophysin is a homomultimer in situ. In addition, chemical cross-linking of native synaptophysin demonstrates that a low molecular weight protein is specifically associated with synaptophysin complexes and is lost upon reduction of the intramolecular disulfide bonds. These data suggest that native synaptophysin forms a noncovalent homomultimeric complex whose structure and interaction with other proteins are dependent on the integrity of its intramolecular disulfide bonds and phospholipid environment.

Adrenal Gland Neoplasms↗
PubMed1994

Development of polyclonal antibodies and evaluation of a sensitive radioimmunoassay for detection and measurement of synaptophysin.

Polyclonal antibodies directed towards synaptophysin were raised against a synthesised peptide corresponding to amino acids 246 to 260 of the human synaptophysin sequence. The antibodies, when applied for immunocytochemical staining, showed a staining pattern identical to that of the commercially available monoclonal antibody SY-38. A radioimmunoassay for measurements of synaptophysin was developed using these antibodies and the peptide as standard and tracer. The radioimmunoassay was used for optimising the conditions for purification of synaptophysin from rat brain. No synaptophysin was detected in blood plasma in humans, not even during an embolisation treatment of tumour metastases in the liver, which induced tumour cell necrosis, in a patient with carcinoid tumours. By radioimmunoassay, synaptophysin was detected in cell homogenate from the PC-12 (160 ng/mg) and LCC-18 (40 ng/mg) cell lines and in the cell culture media. In the LCC-18 cell line the synaptophysin immunoreactivity was found in the plasma membrane, and the presence of synaptophysin was confirmed both by radioimmunoassay measurements and by the Northern blot technique. These data indicate that measurements of synaptophysin using this radioimmunoassay are reliable and that the assay can serve as a useful tool in further explorations of the biological effects of synaptophysin.

Animals↗
PubMed1999

The synaptophysin-synaptobrevin complex is developmentally upregulated in cultivated neurons but is absent in neuroendocrine cells.

Regulated secretion requires the formation of a fusion complex consisting of synaptobrevin, syntaxin and SNAP 25. One of these key proteins, synaptobrevin, also complexes with the vesicle protein synaptophysin. The fusion complex and the synaptophysin-synaptobrevin complex are mutually exclusive. Using a combination of immunoprecipitation and crosslinking experiments we report here that the synaptophysin-synaptobrevin interaction in mouse whole brain and defined brain areas is upregulated during neuronal development as previously reported for rat brain. Furthermore the synaptophysin-synaptobrevin complex is also upregulated within 10-12 days of cultivation in mouse hippocampal neurons in primary culture. Besides being constituents of small synaptic vesicles in neurons synaptophysin and synaptobrevin also occur on small synaptic vesicle analogues of neuroendocrine cells. However, the synaptophysin-synaptobrevin complex was not found in neuroendocrine cell lines and more importantly it was also absent in the adrenal gland, the adenohypophysis and the neurohypophysis although the individual proteins could be clearly detected. In the rat pheochromocytoma cell line PC 12 complex formation between synaptophysin and synaptobrevin could be initiated by adult rat brain cytosol. In conclusion, the synaptophysin-synaptobrevin complex is upregulated in neurons in primary culture but is absent in the neuroendocrine cell lines and tissues tested. The complex may provide a reserve pool of synaptobrevin during periods of high synaptic activity. Such a reserve pool probably is less important for more slowly secreting neuroendocrine cells and neurons. The synaptophysin on small synaptic vesicle analogues in these cells appears to resemble the synaptophysin of embryonic synaptic vesicles since complex formation can be induced by adult brain cytosol.

Adrenal Glands↗
PubMed1991

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

Animals↗
PubMed1990

A comparison of synaptophysin, chromogranin, and L-dopa decarboxylase as markers for neuroendocrine differentiation in lung cancer cell lines.

Synaptophysin is a Mr 38,000 integral membrane glycoprotein expressed by a variety of normal and neoplastic neuroendocrine cells. We studied synaptophysin as an immunocytochemical marker for neuroendocrine differentiation in lung cancer and compared it to the immunocytochemical expression of chromogranin A, a marker for dense core (endocrine) granules, and the biochemical activity of L-dopa decarboxylase (DDC), the key amine-handling enzyme. Of the 250 cell lines available to us, we selected examples representative of the following cell types: bronchial carcinoids (n = 4), small cell lung cancer (SCLC) (n = 7), extrapulmonary small cell carcinomas (n = 4), and non-small cell lung cancers (n = 18) whose neuroendocrine status had been previously determined on the basis of electron microscopy and DDC activity. We demonstrated (a) there was a higher incidence of synaptophysin than chromogranin A immunoreactivity in carcinoid (100 versus 75%), classic SCLC (70 versus 50%), and variant SCLC (57 versus 29%) cell lines; (b) 3 of the 4 (75%) extrapulmonary small cell lung cancer cell lines expressed synaptophysin and chromogranin A; (c) 5 of the 7 (71%) non-small cell lung cancer cell lines previously shown to express multiple neuroendocrine markers were positive for synaptophysin, chromogranin A, and DDC activity; (d) none of the other 11 non-small cell lung cancer cell lines expressed synaptophysin or chromogranin A; and (e) formalin fixation and paraffin embedding reduced synaptophysin immunoreactivity in 11 of 14 (79%) of the cell lines, as compared to freshly prepared specimens fixed in 95% ethanol. Western blot analysis using the synaptophysin antibody (SY38) demonstrated immunoreactive proteins ranging from Mr 43,000 to 45,000 in five representative cell lines. The concordance of expression of all three neuroendocrine markers was statistically significant when values for all cell lines were totalled. Synaptophysin was a more commonly expressed marker for variant SCLC cell lines, which rarely showed DDC activity. We conclude that synaptophysin may be a more sensitive and specific marker for neuroendocrine differentiation, when compared to chromogranin A and DDC in lung cancer cell lines which express only part of the neuroendocrine program.

Aromatic-L-Amino-Acid Decarboxylases↗
PubMed1993

Synaptophysin and synaptoporin expression in the developing rat olfactory system.

The expressions of two closely related synaptic vesicle antigens synaptophysin and synaptoporin were examined in the olfactory system of the adult rat and during pre- and postnatal development. In the adult, immunocytochemistry showed that the continuously regenerating olfactory receptor neurons (primary neurons) produce both synaptophysin and synaptoporin which were localized in the cell bodies of the receptor neurons in the olfactory epithelium, their dendrites, axonal processes in the olfactory nerve and their terminals in the olfactory bulb glomeruli. Furthermore, ultrastructural analysis revealed synaptophysin- and synaptoporin-immunoreactivities associated with synaptic vesicles in most olfactory receptor axonal terminals impinging on dendrites of the mitral and tufted neurons (secondary neurons in the olfactory bulb circuitry) in the olfactory glomeruli. In like manner, tufted neurons, granule and periglomerular neurons (interneurons in the olfactory bulb circuitry) express both synaptophysin and synaptoporin. In contrast, mitral neurons expressed only the synaptophysin antigen which was likewise associated with mitral axonal terminals in their target the olfactory cortex. The patterns of synaptophysin and synaptoporin expressions in mitral neurons (synaptophysin only) and tufted neurons (synaptophysin and synaptoporin) were similar in prenatal, postnatal and adult rats as revealed by immunocytochemistry and in situ hybridization. However, the biosynthesis of synaptophysin and synaptoporin by granule and periglomerular neurons, olfactory bulb interneurons, occurred mainly postnatally.

Animals↗
PubMed1988

Immunoelectron microscopic localization of synaptophysin in a Golgi subcompartment of developing hypothalamic neurons.

Synaptophysin, previously identified as an integral membrane glycoprotein (mol. wt 38,000) characteristic of presynaptic vesicles of mature neurons, provides a molecular marker to study the origin, formation and traffic of synaptic vesicles. Using the monoclonal antibody SY38 against this polypeptide we have localized synaptophysin by immunofluorescence and electron microscope immunoperoxidase methods in cultured mouse hypothalamic neurons taken from 16-day-old fetuses which achieve synaptogenesis after 10-12 days in vitro. We have compared the localization of synaptophysin in perikarya and nerve endings as a function of age (2-19 days in vitro) and of treatment of mature neurons with nocodazole. Using immunofluorescence microscopy, synaptophysin was already detected in neuronal soma at 2 days in vitro, where the initiation of neurite development is observed. At the electron microscope level, virtually all mature synaptic boutons and varicosities showed an extensive synaptophysin labeling of synaptic vesicles at 12-13 days in culture whereas neurites showed only very few labeled vesicles. In neuronal soma taken before synapse formation (6 days in vitro), synaptophysin was selectively localized in membranes of the innermost cisternae of the Golgi zone and in vesicles of variable size and shape in the core of the Golgi zone. In contrast, after synapse formation, synaptophysin labeling was barely detected in the Golgi zone of neurons but a very strong labeling of synaptic vesicles in synaptic boutons was observed. Treatment of mature neurons (12 days in vitro) with nocodazole (10(-5) M) resulted in a conspicuous synaptophysin staining of the innermost trans-Golgi cisternae and numerous vesicles in the cytoplasm. Furthermore, an accumulation of labeled synaptic vesicles on the presynaptic membrane of nerve terminals was found. The data suggest that synaptophysin is released from the Golgi apparatus in a vesicular form, after glycosylation, and is then transported to nerve endings by a mechanism which requires integrity of microtubules.

Animals↗
PubMed1999

Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system.

Synaptophysin is a protein of synaptic vesicles and may be demonstrated in tissue sections of human brain and spinal cord by immunocytochemistry using a monoclonal antibody. Synaptophysin immunoreactivity was studied in paraffin-embedded sections of the central nervous system (CNS) in 14 normal human fetuses and neonates ranging in age from 8 to 41 weeks gestation, and in three brains with heterotopic neurons or malformations. A progressive expression of synaptophysin is seen in axonal terminals within grey matter in various parts of the CNS, beginning in the ventral horns of the spinal cord and brainstem tegmentum at 12-14 weeks. In the cerebellum, the molecular layer shows a band of reactivity from 18 weeks; by term two parallel bands of synaptophysin are seen in the molecular layer and reactivity also is demonstrated in the Purkinje and internal granular layers. In the cerebral neocortex, the molecular zone has weak synaptophysin reactivity as early as 10 weeks, though reactivity is not detected in the deep layers of the cortical plate until 19 weeks and in layers 2-4 until 25 weeks gestation. Synaptophysin reactivity is strong at the surface of neurons but not detected in their somatic cytoplasm; coarsely beaded reactivity within the neuropil probably corresponds to synaptic vesicles in terminal axons. Similar granular synaptophysin reactivity is seen around heterotopic neurons in the subcortical white matter, in dysgenesis of the cerebellar cortex and in the residual anencephalic forebrain. Thermal intensification by heating the incubating solution in a microwave oven often enhances immunoreactivity because of more complete antigen retrieval and is recommended for tissue stored in formalin or in paraffin for long periods. Synaptophysin provides a useful tissue marker of synaptogenesis during normal development and in cerebral dysgeneses, and may provide useful correlations with functional imaging of the brain in living patients. Used in conjunction with other neuronal markers, the expression of synaptophysin in terminal axons of distant neurons, in temporal relation to the maturation of the neurons they innervate, may provide clues to the pathogenesis of epilepsy in early infancy.

Axons↗
PubMed1994

Sorting of synaptophysin into special vesicles in nonneuroendocrine epithelial cells.

Synaptophysin is a major transmembrane glycoprotein of a type of small vesicle with an electron-translucent content (SET vesicles), including the approximately 50-nm presynaptic vesicles in neuronal cells, and of similar, somewhat larger (< or = approximately 90 nm) vesicles (SLMV) in neuroendocrine (NE) cells. When certain epithelial non-NE cells, such as human hepatocellular carcinoma PLC cells, were cDNA transfected to synthesize synaptophysin, the new molecules appeared in specific SET vesicles. As this was in contrast to other reports that only NE cells were able to sort synaptophysin away from other plasma membrane proteins into presynaptic- or SLMV-type vesicles, we have further characterized the vesicles containing synaptophysin in transfected PLC cells. Using fractionation and immunoisolation techniques, we have separated different kinds of vesicles, and we have identified a distinct type of synaptophysin-rich, small (30-90-nm) vesicle that contains little, if any, protein of the constitutive secretory pathway marker hepatitis B surface antigen, of the fluid phase endocytosis marker HRP, and of the plasma membrane recycling endosomal marker transferrin receptor. In addition, we have found variously sized vesicles that contained both synaptophysin and transferrin receptor. A corresponding result was also obtained by direct visualization, using double-label immunofluorescence microscopy for the endocytotic markers and synaptophysin in confocal laser scan microscopy and in double-immunogold label electron microscopy. We conclude that diverse non-NE cells of epithelial nature are able to enrich the "foreign" molecule synaptophysin in a category of SET vesicles that are morphologically indistinguishable from SLMV of NE cells, including one type of vesicle in which synaptophysin is sorted away from endosomal marker proteins. Possible mechanisms of this sorting are discussed.

Biological Transport↗
PubMed1999

The synaptophysin-synaptobrevin complex: a hallmark of synaptic vesicle maturation.

Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, we report here that the synaptophysin-synaptobrevin complex is upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin, a closely related homolog of synaptophysin, to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. We conclude that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, we propose that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity.

Aging↗
PubMed1993

Calcium-dependent serine phosphorylation of synaptophysin.

The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca(2+)-dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K(+)-induced depolarization of slices and synaptosomes prelabeled with 32P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca2+ in the incubation medium. The addition of Ca2+ plus calmodulin to purified synaptic vesicles resulted in a 4-fold increase in serine phosphorylation of synaptophysin, and this phosphorylation was antagonized by a peptide inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II(. Purified rat forebrain CaM kinase II phosphorylated both purified synaptophysin and endogenous, vesicle-associated synaptophysin, and the resulting 2-dimensional chymotryptic phosphopeptide maps were similar to those derived from synaptophysin phosphorylated in cerebrocortical slices. These data demonstrate that Ca(2+)-dependent phosphorylation of synaptophysin, mediated by CaM kinase II, occurs under physiological conditions.

Animals↗
PubMed1995

Mice lacking synaptophysin reproduce and form typical synaptic vesicles.

Synaptophysin is one of the major integral membrane proteins of the small (30-50nm diameter) electron-translucent transmitter-containing vesicles in neurons and of similar vesicles in neuroendocrine cells. Since its expression is tightly linked to the occurrence of these vesicle types, we mutated the X-chromosomally located synaptophysin gene in embryonic stem cells for the generation of synaptophysin-deficient mice in order to study the consequence of synaptophysin ablation for the formation and function of such vesicles in vivo. The behavior and appearance of mice lacking synaptophysin was indistinguishable from that of their litter mates and reproductive capacity was comparable to normal mice. Furthermore, no drastic compensatory changes were noted in the expression of several other neuronal polypeptides or in the mRNA levels of synaptophysin isoforms, the closely related neuronal synaptoporin/synaptophysinII, and the ubiquitous pantophysin. Immunofluorescence microscopy of several neuronal and neuroendocrine tissues showed that overall tissue architecture was maintained in the absence of synaptophysin, and that the distribution of other synaptic vesicle components was not visibly affected. In electron-microscopic preparations, large numbers of vesicles with a diameter of 39.9nm and an electron-translucent interior were seen in synaptic regions of synaptophysin-deficient mice; these vesicles could be labeled by antibodies against synaptic vesicle proteins, such as synaptobrevin 2.

Adrenal Medulla↗
PubMed1991

Synaptophysin immunoreactivity in the mammalian endocrine pancreas.

Synaptophysin, a major membrane glycoprotein of small presynaptic vesicles in neurons, has also been found in microvesicles of endocrine cells, e.g., of the endocrine pancreas. In the present study, the endocrine pancreas in 9 mammalian species (man, dog, mink, bovine, rabbit, guinea pig, rat, mouse, gerbil) has been investigated immunohistochemically for synaptophysin immunoreactivity. Synaptophysin-positive cells have been identified and localized on semithin plastic sections. Our study demonstrates that, in all species examined, all pancreatic endocrine cell types are consistently synaptophysin-positive independent of their location within the tissue, or the conditions of tissue processing. In addition, a few cells that cannot be hormonally identified show synaptophysin immunoreactivity. Hence, synaptophysin appears to be a regular constituent of all pancreatic endocrine cells in mammals. In several species, a subpopulation of endocrine cells, consisting of glucagon-containing and/or pancreatic-polypeptide-containing cells, exhibits a significantly higher degree of synaptophysin immunoreactivity. In the gerbil, this heterogeneity can readily be detected from the day of birth onwards. Our findings indicate that closely related endocrine cell types may differ with respect to the content of synaptophysin.

Animals↗