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M Matteoli

Publications and source records attributed to M Matteoli.

At least 37 records · Page 2Linked to original sources
PubMed1995

Calcium-dependent glutamate release during neuronal development and synaptogenesis: different involvement of omega-agatoxin IVA- and omega-conotoxin GVIA-sensitive channels.

Hippocampal neurons maintained in primary culture recycle synaptic vesicles and express functional glutamate receptors since early stages of neuronal development. By analyzing glutamate-induced cytosolic calcium changes to sense presynaptically released neurotransmitter, we demonstrate that the ability of neurons to release glutamate in the extracellular space is temporally coincident with the property of synaptic vesicles to undergo exocytotic-endocytotic recycling. Neuronal differentiation and maturation of synaptic contacts coincide with a change in the subtype of calcium channels primarily involved in controlling neurosecretion. Whereas omega-agatoxin IVA-sensitive channels play a role in controlling neurotransmitter secretion at all stages of neuronal differentiation, omega-conotoxin GVIA-sensitive channels are primarily involved in mediating glutamate release at early developmental stages only.

2-Amino-5-phosphonovalerate↗
PubMed1995

Mechanisms of synaptogenesis in hippocampal neurons in primary culture.

To improve our understanding of the mechanisms which regulate the formation and the functional maturation of synaptic contacts between neurons, we used hippocampal neurons maintained in primary cultures as experimental system. In this model, which offers several advantages for the study of neuronal development and synaptogenesis, we investigated some of the cellular mechanisms underlying the formation of presynaptic and postsynaptic compartments.

Animals↗
PubMed1995

Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin.

Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse.

Animals↗
PubMed1995

A radioimmunoassay to monitor synaptic activity in hippocampal neurons in vitro.

Exocytosis of synaptic vesicles (SV) results in the surface exposure of lumenal epitopes of SV proteins. We have recently described the use of antibodies directed against the lumenal N-terminus of synaptotagmin I (Sytlum-Abs) to morphologically monitor exo-endocytic recycling of SVs. We report here that a radioimmunoassay based on these antibodies can be used to quantify levels of synaptic activity in primary neuronal cultures. High density cultures of hippocampal neurons grown in the absence of glia were used for these studies. A significant cell surface pool of synaptotagmin I immunoreactivity was detectable by Sytlum-Abs at steady state. The increase in the amount of Sytlum-Abs which became cell bound during a 3 min incubation at 37 degrees C over the Ab bound to this cell surface pool, was substantially higher in depolarizing media containing extracellular Ca2+ than in Ca(2+)-free media. Incubation of the cultures with Sytlum-Abs for longer time periods indicated a sustained increase in the rate of SV exocytosis in depolarizing media which lasted for at least 1 h. This increase was completely abolished by pretreating the neurons with tetanus toxin and this block correlated with a disappearance of synaptobrevin immunoreactivity. This radioimmunoassay therefore offers a new way to monitor SV exocytosis of neuronal populations in vitro irrespective of the type of neurotransmitter secreted and of postsynaptic effects.

Animals↗
PubMed1994

Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis.

Calcium imaging techniques were used to obtain a clear although indirect evidence about the distribution of functional glutamate receptors of NMDA and non-NMDA type in cultured hippocampal neurons during establishment of polarity and synaptogenesis. Glutamate receptors were expressed and were already functional as early as one day after plating. At this stage NMDA and non-NMDA receptors were distributed in all plasmalemmal areas. During the establishment of neuronal polarity, responses to either types of glutamate receptors became restricted to the soma and dendrites. Compartmentalization of glutamate receptors occurred at stages of development when synaptic vesicles were already fully segregated to the axon. Formation of synapses was accompanied by a further redistribution of receptors, which segregated to synapse-enriched portions of dendrites. Receptor compartmentalization and dendritic redistribution as well as accumulation of synaptic vesicles at synaptic sites occurred also in neurons cultured in the presence of either the sodium channel blocker tetrodotoxin or glutamate receptor antagonists. These results indicate that signals generated by neuronal electrical activity or receptor activation are not involved in the establishment of neuronal polarity and synaptogenesis.

Animals↗
PubMed1994

Morphologic and biochemical analysis of the intracellular trafficking of the Alzheimer beta/A4 amyloid precursor protein.

Abnormal metabolic processing of the beta/A4 amyloid precursor protein (APP) has been implicated in the pathogenesis of Alzheimer disease. Several aspects of normal APP processing have been elucidated, but the precise cellular trafficking of APP remains unclear. To investigate APP trafficking pathways further, we have examined the subcellular distribution of APP in rat brain tissue and a variety of cultured cell types, and correlated this distribution with the biochemical processing of APP. In immunofluorescence microscopy of rat brain sections, APP immunoreactivity was concentrated in the Golgi complex and in proximal axon segments. In addition, a lower level of punctate fluorescence was visible throughout the neuropil. By immunoelectron microscopy of rat brain tissue fragments, APP was found associated with Golgi elements and with medium-sized, invaginated vesicles in both axons and dendrites. Prominent localization of APP to the Golgi complex was also found in primary cultures of rat hippocampal neurons and in non-neuronal cell lines. When cultured cells were treated with brefeldin A (BFA), APP immunoreactivity changed from a Golgi-like to an endoplasmic reticulum-like distribution. No APP was detected in the BFA-induced reticulum identified by the transferrin receptor, indicating that concentration of APP in the Golgi does not reflect recycling between the trans-Golgi network and early endosomal system. In immunoblots of BFA-treated cells, there was an accumulation of full-length APP and inhibition of APP secretory processing. Treatment with phorbol ester resulted in a marked elevation of APP secretion, but no obvious redistribution of APP immunoreactivity was apparent at the light microscope level. The lysosomotropic drug chloroquine induced accumulation of APP in cell lysates, as seen by immunoblotting. Immunofluorescence microscopy of chloroquine-treated cells demonstrated a colocalization of APP with the lysosomal marker Igp 120, whereas no colocalization was seen in untreated cells. Taken together, these results support a scheme in which APP is concentrated in the Golgi complex as it travels through the central vacuolar system en route to the plasma membrane for secretion of its amino-terminal domain and/or to lysosomes for degradation.

Alzheimer Disease↗
PubMed1993

Synaptic vesicle proteins and early endosomes in cultured hippocampal neurons: differential effects of Brefeldin A in axon and dendrites.

The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal-dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagmin, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase- or Lucifer yellow-labeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.

Amino Acid Sequence↗
PubMed1992

Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.

The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.

Animals↗
PubMed1992

Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons.

In mature neurons synaptic vesicles (SVs) undergo cycles of exo-endocytosis at synapses. It is currently unknown whether SV exocytosis and recycling occurs also in developing axons prior to synapse formation. To address this question, we have developed an immunocytochemical assay to reveal SV exo-endocytosis in hippocampal neurons developing in culture. In this assay antibodies directed against the lumenal domain of synaptotagmin I (Syt I), an intrinsic membrane protein of SVs, are used to reveal exposure of SV membranes at the cell surface. Addition of antibodies to the culture medium of living neurons for 1 hr at 37 degrees C resulted in their rapid and specific internalization by all neuronal processes and, particularly, by axons. Double immunofluorescence and electron microscopy immunocytochemistry indicated that the antibodies were retained within SVs in cell processes and underwent cycles of exo-endocytosis in parallel with SV membranes. In contrast, another endocytotic marker, wheat germ agglutinin, was rapidly cleared from the processes and transported to the cell body. Antibody-labeled SVs were still present in axons several days after antibody loading and became clustered at presynaptic sites in parallel with synaptogenesis. These results demonstrate that SVs undergo multiple cycles of exo-endocytosis in developing neuronal processes irrespective of the presence of synaptic contacts.

Calcium-Binding Proteins↗
PubMed1992

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains.

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

GABA and pancreatic beta-cells: colocalization of glutamic acid decarboxylase (GAD) and GABA with synaptic-like microvesicles suggests their role in GABA storage and secretion.

GABA, a major inhibitory neurotransmitter of the brain, is also present at high concentration in pancreatic islets. Current evidence suggests that within islets GABA is secreted from beta-cells and regulates the function of mantle cells (alpha- and delta-cells). In the nervous system GABA is stored in, and secreted from, synaptic vesicles. The mechanism of GABA secretion from beta-cells remains to be elucidated. Recently the existence of synaptic-like microvesicles has been demonstrated in some peptide-secreting endocrine cells. The function of these vesicles is so far unknown. The proposed paracrine action of GABA in pancreatic islets makes beta-cells a useful model system to explore the possibility that synaptic-like microvesicles, like synaptic vesicles, are involved in the storage and release of non-peptide neurotransmitters. We report here the presence of synaptic-like microvesicles in beta-cells and in beta-cells. Some beta-cells in culture were found to extend neurite-like processes. When these were present, synaptic-like microvesicles were particularly concentrated in their distal portions. The GABA synthesizing enzyme, glutamic acid decarboxylase (GAD), was found to be localized around synaptic-like microvesicles. This was similar to the localization of GAD around synaptic vesicles in GABA-secreting neurons. GABA immunoreactivity was found to be concentrated in regions of beta-cells which were enriched in synaptic-like microvesicles. These findings suggest that in beta-cells synaptic-like microvesicles are storage organelles for GABA and support the hypothesis that storage of non-peptide signal molecules destined for secretion might be a general feature of synaptic-like microvesicles of endocrine cells.

Animals↗
PubMed1991

Molecular mechanisms in neurotransmitter release.

The vesicle hypothesis of neurotransmitter release was first formulated in the 1950s, but only recently have the molecular mechanisms involved in neurotransmitter release begun to be elucidated. This short review summarizes current concepts on neurosecretion and the available information on synaptic vesicle exocytosis.

Animals↗
PubMed1991

Association of Rab3A with synaptic vesicles at late stages of the secretory pathway.

Rab3A is a small GTP-binding protein highly concentrated on synaptic vesicles. Like other small GTP-binding proteins it is thought to cycle between a soluble and a membrane-associated state. To determine at which stage of the life cycle of synaptic vesicles rab3A is associated with their membranes, the localization of the protein in neurons and neuroendocrine cells at different developmental and functional stages was investigated. In all cases, rab3A was colocalized with synaptic vesicle markers at the cell periphery, but was absent from the Golgi area, suggesting that rab3A associates with vesicles distally to the Golgi complex and dissociates from vesicle membranes before they recycle to this region. Immunofluorescence experiments carried out on frog motor end plates demonstrated that massive exocytosis of synaptic vesicles is accompanied by a translocation of rab3A to the cell surface. The selective localization of rab3A on synaptic vesicles at stages preceding their fusion with the plasmalemma suggests that the protein is part of a regulatory machinery that is assembled onto the vesicles in preparation for exocytosis.

Animals↗
PubMed1990

Developmentally regulated expression of calcitonin gene-related peptide at mammalian neuromuscular junction.

Using indirect immunofluorescence techniques, we have found that calcitonin gene-related peptide-like immunoreactivity is present in the neuromuscular junctions of somatic muscles as well as in almost all motor neurons of the lumbar enlargement of 1-week-old rats. It gradually decreases in both motor neurons and motor nerve endings as the animal grows up and completely disappears from the neuromuscular junctions in adult rats, persisting only in the motor nerve endings on the intrafusal fibers. In situ hybridization experiments have shown that the down-regulation of calcitonin gene-related peptide-like immunoreactivity is strictly related to a reduction in CGRP mRNA levels in the spinal motor neurons. These results indicate that the expression of CGRP is developmentally regulated in spinal cord alpha motor neurons. They also suggest that the peptide may play an important role at the immature neuromuscular junction.

Aging↗
PubMed1990

Substance P-like immunoreactivity at the frog neuromuscular junction.

It has been recently shown that frog motoneurons and their nerve terminals contain calcitonin gene-related peptide-like immunoreactivity in large dense-core vesicles (Matteoli et al., Proc. natn. Acad. Sci. U.S.A. 85, 7366-7370, 1988). We report here by an immunofluorescence approach that the same neurons and nerve terminals also contain substance P-like immunoreactivity. The demonstration of substance P-like immunoreactivity in the frog motor nerve endings supports previous data suggesting a physiological role for this peptide in the modulation of cholinergic transmission.

Acetylcholine↗
PubMed1990

Neurofilament proteins are co-expressed with desmin in heart conduction system myocytes.

We have recently shown that specialized myocytes of the rabbit heart express a cytoskeletal protein similar to the M subunit of neurofilaments (NF). Since this result was obtained using a single anti-NF-M monoclonal antibody, we tested on conduction myocytes a panel of five anti-NF antibodies, specific for each of the three NF subunits and for phosphorylated and non-phosphorylated epitopes. Two antibodies, one specific for the L subunit and one for phosphorylated M subunit of NF, reacted with specialized myocytes in immunohistochemistry. In immunoblots on conduction tissue homogenates the two antibodies recognized two polypeptides with electrophoretic mobility and solubility properties identical to those of NF-L and NF-M in the sciatic nerve. The subcellular distribution of NF immunoreactivity in specialized myocytes was very similar to desmin localization; namely, it was distributed on large filamentous bundles and on fine filaments localized transversely at the level of the Z line. At the ultrastructural level, immunoreactive filaments were localized in the intermyofibrillar space and connected myofibrils with mitochondria. Co-expression of NF proteins and desmin was also observed in vitro in a minor population of cardiac myocytes cultured from embryonic rabbit heart. In most cases NF immunoreactivity co-localized with desmin, especially where filaments were well organized, but in some cells anti-NF and anti-desmin antibodies labelled different filamentous structures. These results indicate that NF proteins are structural components of the cytoskeleton of specialized myocytes and show a subcellular distribution very similar to desmin. Such a composition of intermediate filaments indicates that in these cardiac cells muscle differentiation is compatible with the expression of neuronal proteins.

Animals↗
PubMed1988

Differential effect of alpha-latrotoxin on exocytosis from small synaptic vesicles and from large dense-core vesicles containing calcitonin gene-related peptide at the frog neuromuscular junction.

The regulatory peptide called calcitonin gene-related peptide (CGRP) was detected by immunofluorescence in frog motor neurons and motor nerve terminals. In motor nerve terminals, CGRP-like immunoreactivity was found to be segregated within large dense-core vesicles. To determine whether exocytosis from acetylcholine-containing small synaptic vesicles and from CGRP-containing large dense-core vesicles can be independently stimulated, nerve-muscle preparations were exposed to alpha-latrotoxin. This toxin induced complete depletion of acetylcholine-containing small synaptic vesicles but did not induce a parallel depletion of CGRP-like immunoreactivity and of large dense-core vesicles. These effects were independent of the presence of extracellular Ca2+ and occurred both at room temperature and at low temperature (1-3 degrees C). These findings suggest that exocytosis from the two vesicle populations is mediated by distinct biochemical mechanisms, which might be differentially regulated by physiological stimuli.

Animals↗