Showing papers by "Fabio Benfenati published in 2007"

Lack of Synapsin I Reduces the Readily Releasable Pool of Synaptic Vesicles at Central Inhibitory Synapses

Abstract: Synapsins (Syns) are synaptic vesicle (SV) phosphoproteins that play a role in neurotransmitter release and synaptic plasticity by acting at multiple steps of exocytosis. Mutation of SYN genes results in an epileptic phenotype in mouse and man suggesting a role of Syns in the control of network excitability. We have studied the effects of the genetic ablation of the SYN1 gene on inhibitory synaptic transmission in primary hippocampal neurons. Inhibitory neurons lacking SynI showed reduced amplitude of IPSCs evoked by isolated action potentials. The impairment in inhibitory transmission was caused by a decrease in the size of the SV readily releasable pool, rather than by changes in release probability or quantal size. The reduction of the readily releasable pool was caused by a decrease in the number of SVs released by single synaptic boutons in response to the action potential, in the absence of variations in the number of synaptic contacts between couples of monosynaptically connected neurons. The deletion of SYN1 did not affect paired-pulse depression or post-tetanic potentiation, but was associated with a moderate increase of synaptic depression evoked by trains of action potentials, which became apparent at high stimulation frequencies and was accompanied by a slow down of recovery from depression. The decreased size of the SV readily releasable pool, coupled with a decreased SV recycling rate and refilling by the SV reserve pool, may contribute to the epileptic phenotype of SynI knock-out mice.

The synapsin cycle: A view from the synaptic endocytic zone

Abstract: Although the synapsin phosphoproteins were discovered more than 30 years ago and are known to play important roles in neurotransmitter release and synaptogenesis, a complete picture of their functions within the nerve terminal is lacking. It has been shown that these proteins play an important role in the clustering of synaptic vesicles (SVs) at active zones and function as modulators of synaptic strength by acting at both pre- and postdocking levels. Recent studies have demonstrated that synapsins migrate to the endocytic zone of central synapses during neurotransmitter release, which suggests that there are additional functions for these proteins in SV recycling.

Phosphorylation of synapsin domain A is required for post-tetanic potentiation.

Abstract: Post-tetanic potentiation (PTP) is a form of homosynaptic plasticity important for information processing and short-term memory in the nervous system. The synapsins, a family of synaptic vesicle (SV)-associated phosphoproteins, have been implicated in PTP. Although several synapsin functions are known to be regulated by phosphorylation by multiple protein kinases, the role of individual phosphorylation sites in synaptic plasticity is poorly understood. All the synapsins share a phosphorylation site in the N-terminal domain A (site 1) that regulates neurite elongation and SV mobilization. Here, we have examined the role of phosphorylation of synapsin domain A in PTP and other forms of short-term synaptic enhancement (STE) at synapses between cultured Helix pomatia neurons. To this aim, we cloned H. pomatia synapsin (helSyn) and overexpressed GFP-tagged wild-type helSyn or site-1-mutant helSyn mutated in the presynaptic compartment of C1-B2 synapses. We found that PTP at these synapses depends both on Ca2+/calmodulin-dependent and cAMP-dependent protein kinases, and that overexpression of the non-phosphorylatable helSyn mutant, but not wild-type helSyn, specifically impairs PTP, while not altering facilitation and augmentation. Our findings show that phosphorylation of site 1 has a prominent role in the expression of PTP, thus defining a novel role for phosphorylation of synapsin domain A in short-term homosynaptic plasticity.

Synaptophysin I selectively specifies the exocytic pathway of synaptobrevin 2/VAMP2

Abstract: Biogenesis and recycling of synaptic vesicles are accompanied by sorting processes that preserve the molecular composition of the compartments involved. In the present study, we have addressed the targeting of synaptobrevin 2/VAMP2 (vesicle-associated membrane protein 2), a critical component of the synaptic vesicle­-fusion machinery, in a heterotypic context where its sorting is not confounded by the presence of other neuron-specific molecules. Ectopically expressed synaptophysin I interacts with VAMP2 and alters its default surface targeting to a prominent vesicular distribution, with no effect on the targeting of other membrane proteins. Protein–protein interaction is not sufficient for the control of VAMP2 sorting, which is mediated by the C-terminal domain of synaptophysin I. Synaptophysin I directs the sorting of VAMP2 to vesicles before surface delivery, without influencing VAMP2 endocytosis. Consistent with this, dynamin and α-SNAP (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein) mutants which block trafficking at the plasma membrane do not abrogate the effect of synaptophysin I on VAMP2 sorting. These results indicate that the sorting determinants of synaptic vesicle proteins can operate independently of a neuronal context and implicate the association of VAMP2 with synaptophysin I in the specification of the pathway of synaptic vesicle biogenesis.

Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization.

Abstract: The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH3 cells that express voltage-dependent calcium channels and are subjected to Ca2+ oscillations, we found that treatment with high K+-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH3 cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH3 cells and rat brain synaptosomes showed that K+-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH3 cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.

Fast changes in the functional status of release sites during short-term plasticity: involvement of a frequency-dependent bypass of Rac at Aplysia synapses.

Abstract: Synaptic transmission can be described as a stochastic quantal process defined by three main parameters: N, the number of functional release sites; P, the release probability; and Q, the quantum of response. Many changes in synaptic strength that are observed during expression of short term plasticity rely on modifications in P. Regulation of N has been also suggested. We have investigated at identified cholinergic inhibitory Aplysia synapses the cellular mechanism of post-tetanic potentiation (PTP) expressed under control conditions or after N has been depressed by applying lethal toxin (LT) from Clostridium sordellii or tetanus toxin (TeNT). The analysis of the Ca2+ dependency, paired-pulse ratio and variance to mean amplitude relationship of the postsynaptic responses elicited at distinct extracellular [Ca2+]/[Mg2+] elicited during control post-tetanic potentiation (PTPcont) indicated that PTPcont is mainly driven by an increase in release probability, P. The PTP expressed at TeNT-treated synapses (PTPTeNT) was found to be similar to PTPcont, but scaled to the extent of reduction in N produced by TeNT. Despite LT inducing a decrease in N as TeNT does, the PTP expressed at LT-treated synapses (PTPLT) was characterized by exceptionally large amplitude and bi-exponential time course, as compared to PTPcont or the PTPTeNT. Analysis of the Ca2+ dependency of PTPLT, paired-pulse ratio and fluctuations in amplitude of the postsynaptic responses elicited during PTPLT or the variance to mean amplitude relationship of time-locked postsynaptic responses in a series of subsequent PTPLT indicated that an N-driven change is involved in the early phase (1 s time scale) of PTPLT, while at a later stage PTPLT is composed of both N and P increases. Our results suggest that fast switching on of the functional status of the release sites occurs also during the early events of PTPcont. The early N-driven phase of PTPLT is likely to be a functional recovery of the release sites silenced by Rac inactivation. This effect did not appear to result from reversion of LT inhibitory action but from bypassing the step regulated by Rac. Altogether the data suggest that Rac and the intracellular pathway which allows the bypassing of Rac are key players in new forms of short-term plasticity that rely on fast, activity-dependent changes in the functional status of the release sites.