Interaction with telencephalin and the amyloid precursor protein predicts a ring structure for presenilins.

The carboxyl terminus of presenilin 1 and 2 (PS1 and PS2) binds to the neuron-specific cell adhesion molecule telencephalin (TLN) in the brain. PS1 deficiency results in the abnormal accumulation of TLN in a yet unidentified intracellular compartment. The first transmembrane domain and carboxyl terminus of PS1 form a binding pocket with the transmembrane domain of TLN. Remarkably, APP binds to the same regions via part of its transmembrane domain encompassing the critical residues mutated in familial Alzheimer's disease. Our data surprisingly indicate a spatial dissociation between the binding site and the proposed catalytic site near the critical aspartates in PSs. They provide important experimental evidence to support a ring structure model for PS.

Presenilin 1 controls gamma-secretase processing of amyloid precursor protein in pre-golgi compartments of hippocampal neurons.

Mutations of presenilin 1 (PS1) causing Alzheimer's disease selectively increase the secretion of the amyloidogenic betaA4(1-42), whereas knocking out the gene results in decreased production of both betaA4(1-40) and (1-42) amyloid peptides (De Strooper et al. 1998). Therefore, PS1 function is closely linked to the gamma-secretase processing of the amyloid precursor protein (APP). Given the ongoing controversy on the subcellular localization of PS1, it remains unclear at what level of the secretory and endocytic pathways PS1 exerts its activity on APP and on the APP carboxy-terminal fragments that are the direct substrates for gamma-secretase. Therefore, we have reinvestigated the subcellular localization of endogenously expressed PS1 in neurons in vitro and in vivo using confocal microscopy and fine-tuned subcellular fractionation. We show that uncleaved PS1 holoprotein is recovered in the nuclear envelope fraction, whereas the cleaved PS fragments are found mainly in post-ER membranes including the intermediate compartment (IC). PS1 is concentrated in discrete sec23p- and p58/ERGIC-53-positive patches, suggesting its localization in subdomains involved in ER export. PS1 is not found to significant amounts beyond the cis-Golgi. Surprisingly, we found that APP carboxy-terminal fragments also coenrich in the pre-Golgi membrane fractions, consistent with the idea that these fragments are the real substrates for gamma-secretase. Functional evidence that PS1 exerts its effects on gamma-secretase processing of APP in the ER/IC was obtained using a series of APP trafficking mutants. These mutants were investigated in hippocampal neurons derived from transgenic mice expressing PS1wt or PS1 containing clinical mutations (PS1(M146L) and PS1(L286V)) at physiologically relevant levels. We demonstrate that the APP-London and PS1 mutations have additive effects on the increased secretion of betaA4(1-42) relative to betaA4(1-40), indicating that both mutations operate independently. Overall, our data clearly establish that PS1 controls gamma(42)-secretase activity in pre-Golgi compartments. We discuss models that reconcile this conclusion with the effects of PS1 deficiency on the generation of betaA4(1-40) peptide in the late biosynthetic and endocytic pathways.

Rab3 is present on endosomes from bovine chromaffin cells in primary culture.

Rab3a, a small GTP-binding protein, is believed to mediate Ca2+-dependent exocytosis. Consistent with such a role was the previously reported specific association of Rab3a with synaptic vesicles in neurons and secretory granules in adrenal chromaffin cells. Secretory vesicles are believed to be the final point of Rab3a membrane association, as it was shown by several groups that Rab3a dissociates from the secretory vesicle membrane during stimulated exocytosis. In chromaffin cells, Rab3a is not exclusively localized on secretory granules since a fraction is present on a previously unidentified subcellular compartment equilibrating at light sucrose density. This 'light' membraneous structure could be the starting point for reassociation of Rab3a with membranes involved in granule formation, or it could be a structure unrelated to granules. The present study used several subcellular fractionation techniques and immunomicroscopy to unravel the nature of the 'light' Rab3a-containing structures from bovine chromaffin cells in primary culture. After stimulation, amounts of both Rab3a-d and the granule marker dopamine-beta-hydroxylase (DbetaH) increase transiently in sucrose gradient fractions enriched in endosomal markers. A diaminobenzidine-induced density shift of endosomes alters the distribution of DbetaH and Rab3a-d. At the ultrastructural level, subplasmalemmal pleiomorphic organelles were detected by Rab3a-d-immunogold labelling. Taken together our data provide for the first time evidence that internalised secretory granule membranes go through an endosomal stage where Rab3a is present, resembling the neuronal synaptic vesicle cycle. This indicates that the endosome is an important trafficking route in the biogenesis/recycling of secretory vesicles in chromaffin cells, in which Rab3a could have an as yet unknown regulatory function, and could point to the existence of alternative recycling pathways for the chromaffin granule membrane.

Rab3 dissociation and clathrin-mediated endocytosis, two key steps in the exo-endocytotic pathway of large dense-cored vesicles in primary cultures of superior cervical ganglia.

In this study we have used primary cultures of porcine superior cervical ganglia as a model system to study exo-endocytosis in sympathetic neurons. Pure neuronal cultures with a defined noradrenergic phenotype can be obtained when antimitotics are included in the culture medium, and the high yield from prenatal piglets allows a biochemical approach in addition to morphological studies. Release of large dense-cored vesicles (LDCVs) was visualized by exposing stimulated neurons to anti-dopamine-beta-hydroxylase (D beta H) prior to fixation. Double immunofluorescent staining revealed that synaptotagmin was colocalized with anti-D beta H labeled exocytotic spots, but not the small GTP-binding protein rab3. Density gradient centrifugation of a postmitochondrial supernatant of cultured cells confirmed the dissociation of rab3 from a population of mature LDCVs. As expected, rab3 did not reassociate with the lighter D beta H-positive membrane fraction in the gradient representing internalized LDCV membranes. The fluid phase marker horseradish peroxidase colocalized with retrieved LDCV membranes. Indirect immunofluorescence demonstrated the colocalization of clathrin with D beta H-positive exocytotic spots on the plasma membrane. At the ultrastructural level, depolarization of neurons resulted in the abrupt loss of LDCVs and concomitant appearance of clathrin-coated vesicles and coated omega profiles. The size distribution of coated structures overlapped strongly with that of LDCVs. Taken together, these data clearly suggest that two key regulatory events in the process of exo-endocytosis, i.e. rab3 dissociation and clathrin-mediated internalization, are not only reminiscent of small synaptic vesicles, but also are a feature of the LDCV pathway at the presynaptic plasma membrane of sympathetic neurons.

Export of cellubrevin from the endoplasmic reticulum is controlled by BAP31.

Cellubrevin is a ubiquitously expressed membrane protein that is localized to endosomes throughout the endocytotic pathway and functions in constitutive exocytosis. We report that cellubrevin binds with high specificity to BAP31, a representative of a highly conserved family of integral membrane proteins that has recently been discovered to be binding proteins of membrane immunoglobulins. The interaction between BAP31 and cellubrevin is sensitive to high ionic strength and appears to require the transmembrane regions of both proteins. No other proteins of liver membrane extracts copurified with BAP31 on immobilized recombinant cellubrevin, demonstrating that the interaction is specific. Synaptobrevin I bound to BAP31 with comparable affinity, whereas only weak binding was detectable with synaptobrevin II. Furthermore, a fraction of BAP31 and cellubrevin was complexed when each of them was quantitatively immunoprecipitated from detergent extracts of fibroblasts (BHK 21 cells). During purification of clathrin-coated vesicles or early endosomes, BAP31 did not cofractionate with cellubrevin. Rather, the protein was enriched in ER-containing fractions. When BHK cells were analyzed by immunocytochemistry, BAP31 did not overlap with cellubrevin, but rather colocalized with resident proteins of the ER. In addition, immunoreactive vesicles were clustered in a paranuclear region close to the microtubule organizing center, but different from the Golgi apparatus. When microtubules were depolymerized with nocodazole, this accumulation disappeared and BAP31 was confined to the ER. Truncation of the cytoplasmic tail of BAP31 prevented export of cellubrevin, but not of the transferrin receptor from the ER. We conclude that BAP31 represents a novel class of sorting proteins that controls anterograde transport of certain membrane proteins from the ER to the Golgi complex.

Alzheimer amyloid protein precursor is localized in nerve terminal preparations to Rab5-containing vesicular organelles distinct from those implicated in the synaptic vesicle pathway.

In order to localize amyloid protein precursor (APP) in nerve terminals, we have immunoisolated vesicular organelles from nerve terminal preparations using antibodies to Rab5 and synaptophysin. These immunoisolates were then analyzed by electron microscopy and by immunoblotting. The synaptophysin immunoisolates represented a nearly homogeneous population of small synaptic vesicles, with less than 10% contamination by other organelles, and very little APP. In contrast, Rab5 immunoisolates contained, in addition to small synaptic vesicles, substantial numbers of large uni- and bilamellar vesicles and high levels of APP. Thus, it appears that nerve terminal APP is contained predominantly in large vesicular organelles, distinct from synaptic vesicles and from the synaptic vesicle recycling pathway.

Ultrastructural localization of B-50/growth-associated protein-43 to anterogradely transported synaptophysin-positive and calcitonin gene-related peptide-negative vesicles in the regenerating rat sciatic nerve.

The growth-associated protein-43/B-50 (B-50/GAP-43) is conveyed from the neuronal soma into the axon by fast axonal transport and moved to the nerve terminal. To visualize and determine the type of vesicles by which B-50/GAP-43 is anterogradely transported in the regenerating rat sciatic nerve, we have investigated Lowicryl HM20 embedded nerve pieces dissected from the proximal side of a collection ligature. Ultrastructurally, numerous vesicular profiles of various sizes, tubules and mitochondria were seen to accumulate proximal to the collection ligature. Both, in unmyelinated and myelinated axons, B-50/GAP-43 immunoreactivity was associated with vesicular profiles which had a diameter of 50 nm. A fraction of the B-50/GAP-43 label co-localized with the small vesicle marker synaptophysin. Co-localization of B-50/GAP-43 was not detected with the large dense-core vesicle marker calcitonin gene-related peptide. These results indicate that, in rat sciatic nerve axons, B-50/GAP-43 is anterogradely transported in small 50 nm vesicles of the constitutive pathway. These transport vesicles were distinguished in two types. We suggest that one type carrying, both, B-50 GAP-43 and synaptophysin has as destination the nerve terminal, whereas the second type, which only contains B-50/GAP-43 and no synaptophysin, may be primarily targeted to the axolemma for local membrane fusion.

Subcellular localization of synaptophysin in noradrenergic nerve terminals: a biochemical and morphological study.

The subcellular localization of synaptophysin was investigated in noradrenergic nerve terminals of bovine vas deferens and dog spleen and compared with membrane-bound and soluble markers of noradrenergic storage vesicles. At the light microscopical level chromogranin A- and cytochrome b561-immunoreactivity revealed an identical and very dense innervation of the entire vas deferens. In the case of synaptophysin, most immunoreactivity was found only in the outmost varicosities closest to the lumen, which were also positive for chromogranin A. Small dense-core vesicles of dog spleen were purified using a combination of velocity gradient centrifugation and size exclusion chromatography. Small dense-core vesicles were enriched 64 times as measured by the noradrenaline content. Enrichments for dopamine-beta-hydroxylase were in a similar range. Synaptophysin-containing vesicles were smaller in size and they did not contain the typical noradrenergic markers dopamine-beta-hydroxylase, cytochrome b561, and noradrenaline. Instead, they might store adenosine triphosphate (ATP). A greater part of synaptophysin immunoreactivity was consistently found at high sucrose densities at the position of large dense-core vesicles. We conclude that in the noradrenergic nerve terminal: (1) small dense-core vesicles have a membrane composition similar to large dense-core vesicles, indicating that the former are derived from the latter, and (2) synaptophysin seems not to be present on small dense-core vesicles. We suggest the possibility that synaptophysin-containing vesicles form a residual population whose role in neurotransmission has been taken over by large and small dense-core vesicles following noradrenergic differentiation.

Co-storage in large 'dense-core' vesicles of dopamine and cholecystokinin in rat striatum.

The subcellular localization of cholecystokinin in the striatum--an area where a high density of cholecystokinin containing terminals has been demonstrated--was studied using biochemical techniques. Cholecystokinin containing vesicles were partially purified using iso-osmotic Ficoll gradients. As judged from their size and their buoyant density in isopycnic gradients, cholecystokinin containing vesicles represent large 'dense-core' vesicles. Negative staining and subsequent immunolabelling for synaptophysin at the electron microscopical level, showed labelled vesicles of 50-70 nm. binding of dihydrotetrabenazine was detected in the cholecystokinin containing fractions. The results suggest that dopamine is co-stored with cholecystokinin in large dense vesicles in rat striatum.

Differences in the distribution of cytochrome b561 and synaptophysin in dog splenic nerve: a biochemical and immunocytochemical study.

Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotransmitter-containing vesicles, i.e., large (LDVs) and small dense-core vesicles, coexist with the axonal reticulum (AR) and the well-characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b561, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b561 coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b561 immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b561, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data clearly illustrate that synaptophysin is absent from LDVs and AR elements in adrenergic axons.

Catecholamines are present in a synaptic-like microvesicle-enriched fraction from bovine adrenal medulla.

"Synaptic-like microvesicles" are present in all neuroendocrine cells and cell lines. Despite their resemblance to small synaptic vesicles of the CNS, a thorough biochemical characterization is lacking. Moreover, the subcellular distribution of synaptophysin, the most abundant integral membrane protein of small synaptic vesicles, in adrenal medulla is still controversial. Using gradient centrifugation, we were able to compare the distribution of several markers for small synaptic vesicles and chromaffin granules. Synaptophysin was found at a high density (1.16 g/ml), purifying away from dopamine beta-hydroxylase and cytochrome b561. Both noradrenaline and adrenaline showed a parallel distribution with synaptophysin, suggesting their presence in synaptic-like microvesicles. Experiments in the presence of tetrabenazine did not influence the catecholamine content. Additionally, tetrabenazine binding showed a consistent shoulder in the region of synaptophysin. [3H]Noradrenaline uptake was blocked by tetrabenazine, but not by desipramine. Also chromogranin A parallels the distribution of synaptophysin; however, a localization in the Golgi cannot be ruled out. Synaptophysin was shown to undergo very fast phosphorylation, together with another triplet protein of approximately 18 kDa. In contrast, the latter showed a rather bimodal distribution coinciding with synaptophysin and dopamine beta-hydroxylase. Immunoelectron microscopy of synaptic-like microvesicle fractions showed an intense labeling for synaptophysin on 60-90-nm organelles. Whereas abundant gold labeling for cytochrome b561 was found over the entire surface of chromaffin granules, synaptophysin labeling was encountered mostly on vesicles adsorbed to granules. We conclude that catecholamines might be stored in synaptic-like microvesicles of the chromaffin cell.

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.

Simultaneous purification of the neuroproteins synapsin I and synaptophysin.

A procedure for the simultaneous purification of synapsin I and synaptophysin from calf brain was developed. Demyelinated membranes were extracted with 2% Triton X-100 and 2 M KCl. The extracted proteins were separated by weak cation-exchange chromatography on carboxymethyl-Sepharose Fast Flow. Synaptophysin was finally purified by preparative sodium dodecyl sulphate-polyacrylamine gel electrophoresis and synapsin I by affinity chromatography using a calmodulin-Sepharose column. The recovery obtained was 40 micrograms/g in brain for synaptophysin and 25 micrograms/g in brain for synapsin I.

Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry.

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.

Presence and subcellular localization of alpha 2-adrenoceptors in dog splenic nerve.

Two weeks after denervation of the dog spleen, alpha 2-adrenoceptors labelled by [3H]rauwolscine were decreased to 44% of control. In splenic nerve, high-affinity, specific, stereoselective [3H]rauwolscine binding was observed, with a Bmax of 211 +/- 49.8 fmol/mg protein and a Kd of 4.33 +/- 0.87 nM. The [3H]rauwolscine binding sites in the splenic nerve were not co-localized with noradrenaline (NA) in the NA-storage vesicles, as revealed by sucrose density gradients. Moreover, unlike NA and its storage vesicles, no accumulation of alpha 2-adrenoceptors against a ligature could be observed. The results demonstrate the presence of alpha 2-receptors in the splenic nerve, and their localization in the axon, distinct from the large dense-cored vesicles.