Silvia Giovedì

Recruitment of Endophilin to Clathrin-Coated Pit Necks Is Required for Efficient Vesicle Uncoating after Fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

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.

Synaptojanin 1-linked phosphoinositide dyshomeostasis and cognitive deficits in mouse models of Down's syndrome

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal PtdIns(4,5)P2 balance is required for proper neurotransmission, the phosphoinositide phosphatase synaptojanin 1 is a key regulator of its metabolism. The underlying gene, SYNJ1, maps to human chromosome 21 and is thus a candidate for involvement in Downs syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that PtdIns(4,5)P2 metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring Synj1 to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing Synj1 from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that PtdIns(4,5)P2 dyshomeostasis caused by gene dosage imbalance for Synj1 may contribute to brain dysfunction and cognitive disabilities in DS.

Overlapping Role of Dynamin Isoforms in Synaptic Vesicle Endocytosis

The existence of neuron-specific endocytic protein isoforms raises questions about their importance for specialized neuronal functions. Dynamin, a GTPase implicated in the fission reaction of endocytosis, is encoded by three genes, two of which, dynamin 1 and 3, are highly expressed in neurons. We show that dynamin 3, thought to play a predominantly postsynaptic role, has a major presynaptic function. Although lack of dynamin 3 does not produce an overt phenotype in mice, it worsens the dynamin 1 KO phenotype, leading to perinatal lethality and a more severe defect in activity-dependent synaptic vesicle endocytosis. Thus, dynamin 1 and 3, which together account for the overwhelming majority of brain dynamin, cooperate in supporting optimal rates of synaptic vesicle endocytosis. Persistence of synaptic transmission in their absence indicates that if dynamin plays essential functions in neurons, such functions can be achieved by the very low levels of dynamin 2.

Role of dynamin, synaptojanin, and endophilin in podocyte foot processes

Podocytes are specialized cells that play an integral role in the renal glomerular filtration barrier via their foot processes. The foot processes form a highly organized structure, the disruption of which causes nephrotic syndrome. Interestingly, several similarities have been observed between mechanisms that govern podocyte organization and mechanisms that mediate neuronal synapse development. Dynamin, synaptojanin, and endophilin are functional partners in synaptic vesicle recycling via interconnected actions in clathrin-mediated endocytosis and actin dynamics in neurons. A role of dynamin in the maintenance of the kidney filtration barrier via an action on the actin cytoskeleton of podocytes was suggested. Here we used a conditional double-KO of dynamin 1 (Dnm1) and Dnm2 in mouse podocytes to confirm dynamins role in podocyte foot process maintenance. In addition, we demonstrated that while synaptojanin 1 (Synj1) KO mice and endophilin 1 (Sh3gl2), endophilin 2 (Sh3gl1), and endophilin 3 (Sh3gl3) triple-KO mice had grossly normal embryonic development, these mutants failed to establish a normal filtration barrier and exhibited severe proteinuria due to abnormal podocyte foot process formation. These results strongly implicate a protein network that functions at the interface between endocytosis and actin at neuronal synapses in the formation and maintenance of the kidney glomerular filtration barrier.

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.

Heterogeneity of glutamatergic and GABAergic release machinery in cerebral cortex

We investigated whether cortical glutamatergic and GABAergic release machineries can be differentiated on the basis of the proteins they express, by studying the degree of co-localization of synapsin (SYN) I and II, synaptophysin (SYP) I and II, synaptosomal-associated protein (SNAP)-25 and SNAP-23 in vesicular glutamate transporter (VGLUT) 1-, VGLUT2- and vesicular GABA transporter (VGAT)-positive (+) puncta in the rat cerebral cortex. Co-localization studies showed that SYNI and II were expressed in approximately 90% of VGLUT1+, approximately 30% of VGLUT2+ and 30-50% of VGAT+ puncta; SYPI was expressed in approximately 95% of VGLUT1+, 30% of VGLUT2+, and 45% of VGAT+ puncta; SYPII in approximately 7% of VGLUT1+, 3% of VGLUT2+, and 20% of VGAT+ puncta; SNAP-25 in approximately 94% of VGLUT1+, 5% of VGLUT2+, and 1% of VGAT+ puncta, and SNAP-23 in approximately 3% of VGLUT1+, 86% of VGLUT2+, and 22% of VGAT+ puncta. Since SYPI, which is considered ubiquitous, was expressed in about half of GABAergic axon terminals, we studied its localization electron microscopically and in immunoisolated synaptic vesicles: these studies showed that approximately 30% of axon terminals forming symmetric synapses were SYPI-negative, and that immunoisolated VGAT-positive synaptic vesicles were relatively depleted of SYPI as compared with VGLUT1+ vesicles. Overall, the present investigation shows that in the cerebral cortex of rats distinct presynaptic proteins involved in neurotransmitter release are differentially expressed in GABAergic and in the two major types of glutamatergic axon terminals in the cerebral cortex of rats.

Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles II. FUNCTIONAL EFFECTS OF THE Rab3A-SYNAPSIN I INTERACTION

Synapsins, a family of neuron-specific phosphoproteins that play an important role in the regulation of synaptic vesicle trafficking and neurotransmitter release, were recently demonstrated to interact with the synaptic vesicle-associated small G protein Rab3A within nerve terminals (Giovedì, S., Vaccaro, P., Valtorta, F., Darchen, F., Greengard, P., Cesareni, G., and Benfenati, F. (2004) J. Biol. Chem. 279, 43760–43768). We have analyzed the functional consequences of this interaction on the biological activities of both proteins and on their subcellular distribution within nerve terminals. The presence of synapsin I stimulated GTP binding and GTPase activity of both purified and endogenous synaptic vesicle-associated Rab3A. Conversely, Rab3A inhibited synapsin I binding to F-actin, as well as synapsin-induced actin bundling and vesicle clustering. Moreover, the amount of Rab3A associated with synaptic vesicles was decreased in synapsin knockout mice, and the presence of synapsin I prevented RabGDI-induced Rab3A dissociation from synaptic vesicles. The results indicate that an interaction between synapsin I and Rab3A exists on synaptic vesicles that modulates the functional properties of both proteins. Given the well recognized importance of both synapsins and Rab3A in synaptic vesicles exocytosis, this interaction is likely to play a major role in the modulation of neurotransmitter release.

Phosphorylation by cAMP-dependent protein kinase is essential for synapsin-induced enhancement of neurotransmitter release in invertebrate neurons.

Synapsins are synaptic vesicle-associated phosphoproteins involved in the regulation of neurotransmitter release and synapse formation; they are substrates for multiple protein kinases that phosphorylate them on distinct sites. We have previously found that injection of synapsin into Helix snail neurons cultured under low-release conditions increases the efficiency of neurotransmitter release. In order to investigate the role of phosphorylation in this modulatory action of synapsins, we examined the substrate properties of the snail synapsin orthologue recently cloned in Aplysia (apSyn) for various protein kinases and compared the effects of the intracellular injection of wild-type apSyn with those of its phosphorylation site mutants. ApSyn was found to be an excellent in vitro substrate for cAMP-dependent protein kinase, which phosphorylated it at high stoichiometry on a single site (Ser-9) in the highly conserved domain A, unlike the other kinases reported to phosphorylate mammalian synapsins, which phosphorylated apSyn to a much lesser extent. The functional effect of apSyn phosphorylation by cAMP-dependent protein kinase on neurotransmitter release was studied by injecting wild-type or Ser-9 mutated apSyn into the soma of Helix serotonergic C1 neurons cultured under low-release conditions, i.e. in contact with the non-physiological target neuron C3. In this model of impaired neurotransmitter release, the injection of wild-type apSyn induced a significant enhancement of release. This enhancement was virtually absent after injection of the non-phosphorylatable mutant (Ser-9→Ala), but it was maintained after injection of the pseudophosphorylated mutant (Ser-9→Asp). These functional effects of apSyn injection were paralleled by marked ultrastructural changes in the C1 neuron, with the formation of extensive interdigitations of neurite-like processes containing an increased complement of C1 dense core vesicles at the sites of cell-to-cell contact. This structural rearrangement was virtually absent in mock-injected C1 neurons or after injection of the non-phosphorylatable apSyn mutant. These data indicate that phosphorylation of synapsin domain A is essential for the synapsin-induced enhancement of neurotransmitter release and suggest that endogenous kinases phosphorylating this domain play a central role in the regulation of the efficiency of the exocytotic machinery.

Synapsin Is a Novel Rab3 Effector Protein on Small Synaptic Vesicles I. IDENTIFICATION AND CHARACTERIZATION OF THE SYNAPSIN I-Rab3 INTERACTIONS IN VITRO AND IN INTACT NERVE TERMINALS

Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.