GRP78 clustering at the cell surface of neurons transduces the action of exogenous alpha-synuclein.

Mutation or multiplication of the alpha-synuclein (Syn)-encoding gene is frequent cause of early onset Parkinson's disease (PD). Recent evidences point to the pathogenic role of excess Syn also in sporadic PD. Syn is a cytosolic protein, which has been shown to be released from neurons. Here we provide evidence that extracellular Syn induces an increase in surface-exposed glucose-related protein of 78 kDa (GRP78), which becomes clustered in microdomains of the neuronal plasma membrane. Upon interacting with Syn, GRP78 activates a signaling cascade leading to cofilin 1 inactivation and stabilization of microfilaments, thus affecting morphology and dynamics of actin cytoskeleton in cultured neurons. Downregulation of GRP78 abolishes the activity of exogenous Syn, indicating that it is the primary target of Syn. Inactivation of cofilin 1 and stabilization of actin cytoskeleton are present also in fibroblasts derived from genetic PD patients, which show a dramatic increase in stress fibers. Similar changes are displayed by control cells incubated with the medium of PD fibroblasts, only when Syn is present. The accumulation of Syn in the extracellular milieu, its interaction with the plasma membrane and Syn-driven clustering of GRP78 appear, therefore, responsible for the dysregulation of actin turnover, leading to early deficits in synaptic function that precede neurodegeneration.

Cortico-hippocampal hyperexcitability in synapsin I/II/III knockout mice: age-dependency and response to the antiepileptic drug levetiracetam.

Synapsins (SynI, SynII, SynIII) are a multigene family of synaptic vesicle (SV) phosphoproteins implicated in the regulation of synaptic transmission and plasticity. Synapsin I, II, I/II and I/II/III knockout mice are epileptic and SYN1/2 genes have been identified as major epilepsy susceptibility genes in humans. We analyzed cortico-hippocampal epileptiform activity induced by 4-aminopyridine (4AP) in acute slices from presymptomatic (3-weeks-old) and symptomatic (1-year-old) Syn I/II/III triple knockout (TKO) mice and aged-matched triple wild type (TWT) controls and assessed the effect of the SV-targeted antiepileptic drug (AED) levetiracetam (LEV) in reverting the epileptic phenotype. Both fast and slow interictal (I-IC) and ictal (IC) events were observed in both genotypes. The incidence of fast I-IC events was higher in presymptomatic TKO slices, while frequency and latency of I-IC events were similar in both genotypes. The major age and genotype effects were observed in IC activity, that was much more pronounced in 3-weeks-old TKO and persisted with age, while it disappeared from 1-year-old TWT slices. LEV virtually suppressed fast I-IC and IC discharges from 3-weeks-old TWT slices, while it only increased the latency of fast I-IC and IC activity in TKO slices. Analysis of I-IC events in patch-clamped CA1 pyramidal neurons revealed that LEV increased the inhibitory/excitatory ratio of I-IC activity in both genotypes. The lower LEV potency in TKO slices of both ages was associated with a decreased expression of SV2A, a SV protein acting as LEV receptor, in cortex and hippocampus. The results demonstrate that deletion of Syn genes is associated with a higher propensity to 4AP-induced epileptic paroxysms that precedes the onset of epilepsy and consolidates with age. LEV ameliorates such hyper excitability by enhancing the inhibition/excitation ratio, although the effect is hindered in TKO slices which exhibit a concomitant decrease in the levels of the LEV receptor SV2A.

The synapsins: key actors of synapse function and plasticity.

The synapsins are a family of neuronal phosphoproteins evolutionarily conserved in invertebrate and vertebrate organisms. Their best-characterised function is to modulate neurotransmitter release at the pre-synaptic terminal, by reversibly tethering synaptic vesicles (SVs) to the actin cytoskeleton. However, many recent data have suggested novel functions for synapsins in other aspects of the pre-synaptic physiology, such as SV docking, fusion and recycling. Synapsin activity is tightly regulated by several protein kinases and phosphatases, which modulate the association of synapsins to SVs as well as their interaction with actin filaments and other synaptic proteins. In this context, synapsins act as a link between extracellular stimuli and the intracellular signalling events activated upon neuronal stimulation. Genetic manipulation of synapsins in various in vivo models has revealed that, although not essential for the basic development and functioning of neuronal networks, these proteins are extremely important in the fine-tuning of neuronal plasticity, as shown by the epileptic phenotype and behavioural abnormalities characterising mouse lines lacking one or more synapsin isoforms. In this review, we summarise the current knowledge about how the various members of the synapsin family are involved in the modulation of the pre-synaptic physiology. We give a comprehensive description of the molecular basis of synapsin function, as well as an overview of the more recent evidence linking mutations in the synapsin proteins to the onset of severe central nervous system diseases such as epilepsy and schizophrenia.

Synaptophysin is an autoantigen in paraneoplastic neuropathy.

Paraneoplastic neurological syndromes (PNS) are often associated with antineuronal autoantibodies and many of them could be identified in the recent years. However, there are still new antineuronal binding patterns with yet unidentified autoantigens. We here describe a new autoantibody associated with paraneoplastic sensorimotor and autonomic neuropathy in a patient with small cell lung cancer. In indirect immunofluorescence test, the patient's serum colocalised with the synaptic protein synaptophysin in the cerebellum and myenteric plexus of the gut. Immunoblotting showed a 38 kDa reactivity, which is also the molecular weight of synaptophysin. Therefore a Western Blot with recombinant synaptophysin has been used and revealed reactivity of the serum against synaptophysin. In patients with non-paraneoplastic neuropathies or healthy controls, anti-synaptophysin autoantibodies were not detectable. In 20 SCLC patients without neurological syndromes, two patients had low-titer anti-synaptophysin autoantibodies. The patient's serum and IgG fraction showed cytotoxicity to primary cultured myenteric plexus neurons. We conclude that synaptophysin is an autoantigen in paraneoplastic neurological syndromes.

Synaptic vesicles: is kissing a matter of competence?

The "kiss-and-run" model of exocytosis and endocytosis predicts that synaptic vesicles can undergo fast and efficient recycling, after fusion with the plasmalemma, without intermixing of membranes. Evidence is mounting from several new experimental approaches that kiss-and-run occurs at synapses. Distinct vesicle pools, which initially were identified in morphological terms, are now being characterized in biochemical and functional terms. In addition, at least two functional recycling pathways, operating on different time scales (from milliseconds to tens of seconds), have been shown to coexist in the same synaptic system, and the two pathways appear to be differentially regulated. Taken together, these data suggest that kiss-and-run operates in parallel with the classical, coated-vesicle recycling. Here, we review recent evidence for kiss-and-run recycling and discuss whether it is a distinct process, dependent on the molecular organization of the fusing vesicle. We propose that vesicles undergo a process of "competence maturation". According to this view, the specific molecular make-up of the vesicles, their location and their interactions with nerve terminal proteins might determine not only the differential availability of the vesicles for fusion and neurotransmitter release but also the recycling path that they will follow.

Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals.

We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.

Specificity of the binding of synapsin I to Src homology 3 domains.

Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.

Neurite extension occurs in the absence of regulated exocytosis in PC12 subclones.

We have investigated the process leading to differentiation of PC12 cells. This process is known to include extension of neurites and changes in the expression of subsets of proteins involved in cytoskeletal rearrangements or in neurosecretion. To this aim, we have studied a PC12 clone (trk-PC12) stably transfected with the nerve growth factor receptor TrkA. These cells are able to undergo both spontaneous and neurotrophin-induced morphological differentiation. However, both undifferentiated and nerve growth factor-differentiated trk-PC12 cells appear to be completely defective in the expression of proteins of the secretory apparatus, including proteins of synaptic vesicles and large dense-core granules, neurotransmitter transporters, and neurotransmitter-synthesizing enzymes. These results indicate that neurite extension can occur independently of the presence of the neurosecretory machinery, including the proteins that constitute the fusion machine, suggesting the existence of differential activation pathways for the two processes during neuronal differentiation. These findings have been confirmed in independent clones obtained from PC12-27, a previously characterized PC12 variant clone globally incompetent for regulated secretion. In contrast, the integrity of the Rab cycle appears to be necessary for neurite extension, because antisense oligonucleotides against the neurospecific isoform of Rab-guanosine diphosphate-dissociation inhibitor significantly interfere with process formation.

Molecular mechanisms of neurite extension.

The extension of neurites is a major task of developing neurons, requiring a significant metabolic effort to sustain the increase in molecular synthesis necessary for plasma membrane expansion. In addition, neurite extension involves changes in the subsets of expressed proteins and reorganization of the cytomatrix. These phenomena are driven by environmental cues which activate signal transduction processes as well as by the intrinsic genetic program of the cell. The present review summarizes some of the most recent progress made in the elucidation of the molecular mechanisms underlying these processes.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

Downregulation of MAP kinase activity signalled by HIV-1-gp120 coat protein in granular neurons and glial cells from rat cerebellum.

We have studied the effect of gp120 coat protein from HIV-1 on tyrosine phosphorylation processes in primary cultures of granular neurons or glial cells from the cerebellum of neonatal rats. The extracellular application of recombinant gp120 (200 pM) was able to reduce the phosphotyrosine content and the immunoreactivity for active form-specific antibodies of MAP kinase. Whereas in neurons MAP kinase appeared to be the only protein whose phosphotyrosine content was decreased, in glial cultures the inhibitory effect of gp120 on tyrosine phosphorylation processes appeared to be more widespread. In neuronal cultures, the effect of the viral protein was prevented by the concomitant treatment with depolarizing agents.

Kinetic analysis of the phosphorylation-dependent interactions of synapsin I with rat brain synaptic vesicles.

1. Synapsin I, a major synaptic vesicle (SV)-associated phosphoprotein, is involved in the regulation of neurotransmitter release and synapse formation. By binding to both phospholipid and protein components of SV with high affinity and in a phosphorylation-dependent fashion, synapsin I is believed to cluster SV and to attach them to the actin-based cytoskeleton of the nerve terminal. 2. In the present study we have investigated the kinetic aspects of synapsin I-SV interactions and the mechanisms of their modulation by ionic strength and site-specific phosphorylation, using fluorescence resonance energy transfer between suitable fluorophores linked to synapsin I and to the membrane bilayer. 3. The binding of synapsin I to the phospholipid and protein components of SV has fast kinetics: mean time constants ranged between 1 and 4 s for association and 9 and 11's for ionic strength-induced dissociation at 20 degrees C. The interaction with the phospholipid component consists predominantly of a hydrophobic binding with the core of the membrane which may account for the membrane stabilizing effect of synapsin I. 4. Phosphorylation of synapsin I by either SV-associated or purified exogenous Ca2+/calmodulin-dependent protein kinase II (CaMPKII) inhibited the association rate and the binding to SV at steady state by acting on the ionic strength-sensitive component of the binding. When dephosphorylated synapsin I was previously bound to SV, exposure of SV to Ca2+/calmodulin in the presence of ATP triggered a prompt dissociation of synapsin I with a time constant similar to that of ionic strength-induced dissociation. 5. In conclusion, the reversible interactions between synapsin I and SV are highly regulated by site-specific phosphorylation and have kinetics of the same order of magnitude as the kinetics of SV recycling determined in mammalian neurons under comparable temperature conditions. These findings are consistent with the hypothesis that synapsin I associates with, and dissociates from, SV during the exo-endocytotic cycle. The on-vesicle phosphorylation of synapsin I by the SV-associated CaMPKII, and the subsequent dissociation of the protein from the vesicle membrane, though not involved in mediating exocytosis of primed vesicles evoked by a single stimulus, may represent a prompt and efficient mechanism for the modulation of neurotransmitter release and presynaptic plasticity.

Alteration of the actin-based cytoskeleton by rabies virus.

We investigated the effect of rabies virus infection on the actin cytoskeleton using various techniques. Confocal microscopic examination of rabies virus-infected neuroblastoma cells at late stages of infection revealed a dramatic decrease in F-actin staining. The results of a fluorimetric assay with pyrenylated actin indicated that purified rabies virus nucleocapsid has no direct action on the kinetics of actin polymerization and only a weak effect on the final extent of polymerization. Video-microscopy experiments with purified components showed that rabies virus nucleocapsid inhibits the actin-bundling effect induced by dephospho-synapsin I, a neuron-specific protein which is known to exert a control on the actin-based cytoskeleton. Thus, the observed decrease in F-actin staining in infected cells might be ascribed to an indirect action of rabies nucleocapsid on the effects of actin-binding proteins such as synapsin I.

Synapsin I interacts with c-Src and stimulates its tyrosine kinase activity.

Synapsin I is a synaptic vesicle-associated phosphoprotein that has been implicated in the formation of presynaptic specializations and in the regulation of neurotransmitter release. The nonreceptor tyrosine kinase c-Src is enriched on synaptic vesicles, where it accounts for most of the vesicle-associated tyrosine kinase activity. Using overlay, affinity chromatography, and coprecipitation assays, we have now shown that synapsin I is the major binding protein for the Src homology 3 (SH3) domain of c-Src in highly purified synaptic vesicle preparations. The interaction was mediated by the proline-rich domain D of synapsin I and was not significantly affected by stoichiometric phosphorylation of synapsin I at any of the known regulatory sites. The interaction of purified c-Src and synapsin I resulted in a severalfold stimulation of tyrosine kinase activity and was antagonized by the purified c-Src-SH3 domain. Depletion of synapsin I from purified synaptic vesicles resulted in a decrease of endogenous tyrosine kinase activity. Portions of the total cellular pools of synapsin I and Src were coprecipitated from detergent extracts of rat brain synaptosomal fractions using antibodies to either protein species. The interaction between synapsin I and c-Src, as well as the synapsin I-induced stimulation of tyrosine kinase activity, may be physiologically important in signal transduction and in the modulation of the function of axon terminals, both during synaptogenesis and at mature synapses.

Tat protein from HIV-1 activates MAP kinase in granular neurons and glial cells from rat cerebellum.

We have investigated the effect of extracellularly applied Tat protein of the human immunodeficiency virus type 1 (HIV-1) on tyrosine phosphorylation processes, which represent a major signal transduction pathway of cells of the central nervous system. Primary cultures of rat cerebellar astrocytes or granule cells were incubated with synthetic Tat (10 ng/ml) for various periods of time and analyzed for their phosphotyrosine content by Western blotting. In both types of cultures Tat was able to induce the phosphorylation of mitogen-activated protein kinase (MAP kinase) on tyrosine residues, although with different kinetics and isoform specificity. In addition, in neuronal cells, but not in astrocytes, Tat increased the phosphotyrosine content of Shc, a protein involved in signal transduction downstream of receptor tyrosine kinase activation. This study shows that Tat applied extracellularly is able to induce the generation of intracellular signals in neuronal as well as glial cells.

Anti-synapsin monoclonal antibodies: epitope mapping and inhibitory effects on phosphorylation and Grb2 binding.

The synapsins are a family of major neuron-specific synaptic vesicle-associated phosphoproteins which play important roles in synaptic function. In an effort to identify molecular tools which can be used to perturb the activity of the synapsins in in vitro as well as in vivo experiments, we have localized the epitopes of a panel of monoclonal antibodies (mAbs) raised against synapsins I and II and have characterized their ability to interfere with the interactions of the synapsins with protein kinases, actin and Src homology-3 (SH3) domains. The epitopes of the six mAbs were found to be concentrated in the N-terminal region within domains A and B for the synapsin II-reactive mAbs 19.4, 19.11, 19.51 and 19.21, and in two C-terminal clusters in the proline-rich domains D for synapsin I (mAbs 10.22, 19.51, 19.11 and 19.8) and G for synapsin II (mAb 19.8). The synapsin II-specific mAbs 19.4 and 19.21, whose overlapping epitopes are adjacent to phosphorylation site 1, specifically inhibited synapsin II phosphorylation by endogenous or exogenous cAMP-dependent protein kinase. While all the anti-synapsin I mAbs were unable to affect the interactions of synapsin I both with Ca2+/calmodulin-dependent protein kinase II and with actin monomers and filaments, mAbs 19.8 and 19.51 were found to inhibit the binding of Grb2 SH3 domains to the proline-rich C-terminal region of synapsin I.

Phosphorylation-dependent effects of synapsin IIa on actin polymerization and network formation.

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphorylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.

Effects of synaptic vesicles on actin polymerization.

We have analyzed the effects of synaptic vesicles on actin polymerization by using a time-resolved spectrofluorometric assay. We have found that synaptic vesicles have complex effects on the kinetics of actin polymerization, which vary depending on whether the synaptic vesicle-specific phosphoprotein synapsin I is absent or present on their membrane. Synapsin I bound either to synaptic vesicles or to pure phospholipid vesicles exhibits phosphorylation-dependent actin-nucleating activity. Synaptic vesicles depleted of endogenous synapsin I decrease the rate and the final extent of actin polymerization, an effect which is not observed with pure phospholipid vesicles. Thus, the state of association of synapsin I with synaptic vesicles, which is modulated by its state of phosphorylation, may affect actin assembly and the physico-chemical characteristics of the synaptic vesicle microenvironment.