Franco Onofri

The Inhibitory Effects of Interleukin-6 on Synaptic Plasticity in the Rat Hippocampus Are Associated with an Inhibition of Mitogen-Activated Protein Kinase ERK

Several cytokines have short‐term effects on synaptic transmission and plasticity that are thought to be mediated by the activation of intracellular protein kinases. We have studied the effects of interleukin‐6 (IL‐6) on the expression of paired pulse facilitation (PPF), posttetanic potentiation (PTP), and long‐term potentiation (LTP) in the CA1 region of the hippocampus as well as on the activation of the signal transducer and activator of transcription‐3 (STAT3), the mitogen‐activated protein kinase ERK (MAPK/ERK), and the stress‐activated protein kinase/c‐Jun NH2‐terminal kinase (SAPK/JNK). IL‐6 induced a marked and dose‐dependent decrease in the expression of PTP and LTP that could be counteracted by the simultaneous treatment with the tyrosine kinase inhibitor lavendustin A (LavA) but did not significantly affect PPF. The IL‐6‐induced inhibition of PTP and LTP was accompanied by a simulation of STAT3 tyrosine phosphorylation and an inhibition of MAPK/ERK dual phosphorylation, in the absence of changes in the state of activation of SAPK/JNK. Both effects of IL‐6 on STAT3 and MAPK/ERK activation were effectively counteracted by LavA treatment. The results indicate the tyrosine kinases and MAPK/ERK are involved in hippocampal synaptic plasticity and may represent preferential intracellular targets for the actions of IL‐6 in the adult nervous system.

SYN1 loss-of-function mutations in autism and partial epilepsy cause impaired synaptic function

Several genes predisposing to autism spectrum disorders (ASDs) with or without epilepsy have been identified, many of which are implicated in synaptic function. Here we report a Q555X mutation in synapsin 1 (SYN1), an X-linked gene encoding for a neuron-specific phosphoprotein implicated in the regulation of neurotransmitter release and synaptogenesis. This nonsense mutation was found in all affected individuals from a large French-Canadian family segregating epilepsy and ASDs. Additional mutations in SYN1 (A51G, A550T and T567A) were found in 1.0 and 3.5% of French-Canadian individuals with autism and epilepsy, respectively. The majority of these SYN1 mutations were clustered in the proline-rich D-domain which is substrate of multiple protein kinases. When expressed in synapsin I (SynI) knockout (KO) neurons, all the D-domain mutants failed in rescuing the impairment in the size and trafficking of synaptic vesicle pools, whereas the wild-type human SynI fully reverted the KO phenotype. Moreover, the nonsense Q555X mutation had a dramatic impact on phosphorylation by MAPK/Erk and neurite outgrowth, whereas the missense A550T and T567A mutants displayed impaired targeting to nerve terminals. These results demonstrate that SYN1 is a novel predisposing gene to ASDs, in addition to epilepsy, and strengthen the hypothesis that a disturbance of synaptic homeostasis underlies the pathogenesis of both diseases.

Phosphatidylinositol 4-kinase type IIα is responsible for the phosphatidylinositol 4-kinase activity associated with synaptic vesicles

Phosphorylation of inositol phospholipids plays a key role in cellular regulation via the generation of intracellular second messengers. In addition, it represents a mechanism to regulate interactions of the lipid bilayer with proteins and protein scaffolds involved in vesicle budding, cytoskeletal organization, and signaling. Generation of phosphatidylinositol 4-phosphate [PI(4)P] from phosphatidylinositol (PI) is an important step in this metabolic pathway because PI(4)P is a precursor of other important phosphoinositides and has protein binding properties of its own. We report here that a PI 4-kinase (PI4K) activity previously reported on synaptic vesicles is accounted for by the α isoform of the recently characterized type II PI4K (PI4KII) family. PI4KIIα, which also accounts for the bulk of PI4K activity in brain extracts, is concentrated at synapses and in the region of the Golgi complex in neuronal perikarya. Our results provide new evidence for the occurrence of a cycle of phosphoinositide synthesis and hydrolysis nested within the exo–endocytic cycle of synaptic vesicles and point to PI4KIIα as a critical player in this cycle.

Phosphorylation of VAMP/synaptobrevin in synaptic vesicles by endogenous protein kinases.

Abstract: VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca2+/calmodulin‐dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.

Interleukin‐6 inhibits neurotransmitter release and the spread of excitation in the rat cerebral cortex

Cytokines are extracellular mediators that have been reported to affect neurotransmitter release and synaptic plasticity phenomena when applied in vitro. Most of these effects occur rapidly after the application of the cytokines and are presumably mediated through the activation of protein phosphorylation processes. While many cytokines have an inflammatory action, interleukin‐6 (IL‐6) has been found to have a neuroprotective effect against ischaemia lesions and glutamate excitotoxicity, and to increase neuronal survival in a variety of experimental conditions. In this paper, the functional effects of IL‐6 on the spread of excitation visualized by dark‐field/infrared videomicroscopy in rat cortical slices and on glutamate release from cortical synaptosomes were analysed and correlated with the activation of the STAT3, mitogen‐activated protein kinase ERK (MAPK/ERK) and stress‐activated protein kinase/cJun NH2‐terminal kinase (SAPK/JNK) pathways. We have found that IL‐6 depresses the spread of excitation and evoked glutamate release in the cerebral cortex, and that these effects are accompanied by a stimulation of STAT3 tyrosine phosphorylation, an inhibition of MAPK/ERK activity, a decreased phosphorylation of the presynaptic MAPK/ERK substrate synapsin I and no detectable effects on SAPK/JNK. The effects of IL‐6 were effectively counteracted by treatment of the cortical slices with the tyrosine kinase inhibitor lavendustin A. The inhibitory effects of IL‐6 on glutamate release and on the spread of excitation in the rat cerebral cortex indicate that the protective effect of IL‐6 on neuronal survival could be mediated by a downregulation of neuronal activity, release of excitatory neurotransmitters and MAPK/ERK activity.

Synapsin-I- and synapsin-II-null mice display an increased age-dependent cognitive impairment

Synapsin I (SynI) and synapsin II (SynII) are major synaptic vesicle (SV) proteins that function in the regulation of the availability of SVs for release in mature neurons. SynI and SynII show a high level of sequence similarity and share many functions in vivo, although distinct physiological roles for the two proteins have been proposed. Both SynI–/– and SynII–/– mice have a normal lifespan, but exhibit a decreased number of SVs and synaptic depression upon high-frequency stimulation. Because of the role of the synapsin proteins in synaptic organization and plasticity, we studied the long-lasting effects of synapsin deletion on the phenotype of SynI–/– and SynII–/– mice during aging. Both SynI–/– and SynII–/– mice displayed behavioural defects that emerged during aging and involved emotional memory in both mutants, and spatial memory in SynII–/– mice. These abnormalities, which were more pronounced in SynII–/– mice, were associated with neuronal loss and gliosis in the cerebral cortex and hippocampus. The data indicate that SynI and SynII have specific and non-redundant functions, and that synaptic dysfunctions associated with synapsin mutations negatively modulate cognitive performances and neuronal survival during senescence.

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 11s for ionic strength‐induced dissociation at 20°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.

Tyrosine phosphorylation of synapsin I by Src regulates synaptic-vesicle trafficking

Synapsins are synaptic vesicle (SV)-associated phosphoproteins involved in the regulation of neurotransmitter release. Synapsins reversibly tether SVs to the cytoskeleton and their phosphorylation by serine/threonine kinases increases SV availability for exocytosis by impairing their association with SVs and/or actin. We recently showed that synapsin I, through SH3- or SH2-mediated interactions, activates Src and is phosphorylated by the same kinase at Tyr301. Here, we demonstrate that, in contrast to serine phosphorylation, Src-mediated tyrosine phosphorylation of synapsin I increases its binding to SVs and actin, and increases the formation of synapsin dimers, which are both potentially involved in SV clustering. Synapsin I phosphorylation by Src affected SV dynamics and was physiologically regulated in brain slices in response to depolarization. Expression of the non-phosphorylatable (Y301F) synapsin I mutant in synapsin-I-knockout neurons increased the sizes of the readily releasable and recycling pools of SVs with respect to the wild-type form, which is consistent with an increased availability of recycled SVs for exocytosis. The data provide a mechanism for the effects of Src on SV trafficking and indicate that tyrosine phosphorylation of synapsins, unlike serine phosphorylation, stimulates the reclustering of recycled SVs and their recruitment to the reserve pool.

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-γ, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH2-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, α-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 Ca2+/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and α-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.

Binding of Protein Kinase Inhibitors to Synapsin I Inferred from Pair-Wise Binding Site Similarity Measurements

Predicting off-targets by computational methods is getting increasing importance in early drug discovery stages. We herewith present a computational method based on binding site three-dimensional comparisons, which prompted us to investigate the cross-reaction of protein kinase inhibitors with synapsin I, an ATP-binding protein regulating neurotransmitter release in the synapse. Systematic pair-wise comparison of the staurosporine-binding site of the proto-oncogene Pim-1 kinase with 6,412 druggable protein-ligand binding sites suggested that the ATP-binding site of synapsin I may recognize the pan-kinase inhibitor staurosporine. Biochemical validation of this hypothesis was realized by competition experiments of staurosporine with ATP-γ35S for binding to synapsin I. Staurosporine, as well as three other inhibitors of protein kinases (cdk2, Pim-1 and casein kinase type 2), effectively bound to synapsin I with nanomolar affinities and promoted synapsin-induced F-actin bundling. The selective Pim-1 kinase inhibitor quercetagetin was shown to be the most potent synapsin I binder (IC50  = 0.15 µM), in agreement with the predicted binding site similarities between synapsin I and various protein kinases. Other protein kinase inhibitors (protein kinase A and chk1 inhibitor), kinase inhibitors (diacylglycerolkinase inhibitor) and various other ATP-competitors (DNA topoisomerase II and HSP-90α inhibitors) did not bind to synapsin I, as predicted from a lower similarity of their respective ATP-binding sites to that of synapsin I. The present data suggest that the observed downregulation of neurotransmitter release by some but not all protein kinase inhibitors may also be contributed by a direct binding to synapsin I and phosphorylation-independent perturbation of synapsin I function. More generally, the data also demonstrate that cross-reactivity with various targets may be detected by systematic pair-wise similarity measurement of ligand-annotated binding sites.