Intrinsic calcium dynamics control botulinum toxin A susceptibility in distinct neuronal populations.

SNAP-25 is a SNARE protein implicated in exocytosis and in the negative modulation of voltage-gated calcium channels. We have previously shown that GABAergic synapses, which express SNAP-25 at much lower levels relative to glutamatergic ones, are characterized by a higher calcium responsiveness to depolarization and are largely resistant to botulinum toxin A. We show here that silencing of SNAP-25 in glutamatergic neurons, a procedure which increases KCl-induced calcium elevations, confers these synapses with toxin resistance. Since it is known that calcium reverts the efficacy of botulinum A, we investigated whether the lower effectiveness of the toxin in inhibiting GABAergic vesicle cycling might be attributable to higher evoked calcium transients of inhibitory neurons. We demonstrate that either expression of SNAP-25(1-197) or BAPTA/AM treatment, both inhibiting calcium dynamics, facilitate block of GABAergic vesicle exocytosis upon toxin treatment. These data indicate that intrinsic calcium dynamics control botulinum A susceptibility in distinct neuronal populations.

The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair.

Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as "danger signals" to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD(4)), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD(4) promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a "sensor" that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.

Calpain activity contributes to the control of SNAP-25 levels in neurons.

Calpains are a family of calcium-dependent proteases with abundant expression in the CNS, and potent in cleaving some synaptic components. Assessment of calpain activity by its fluorescent substrate, Boc-Leu-Met-CMAC, revealed that cultured neurons display a significant level of constitutive enzyme activity. Notably, calpain activity differs in distinct neuronal populations, with a significantly higher level of activity in GABAergic cells. Using selectively-enriched cultures of fast-spiking GABAergic interneurons, we show that calpain activity partially contributes to the post-translational down regulation of SNAP-25, a calpain substrate, in differentiated GABA cells. In addition, we demonstrate that SNAP-25 is cleaved by calpain in response to acute seizures induced by intraperitoneal kainate injection in vivo. These data indicate that calpains in neurons are active even at physiological calcium concentrations and that different levels of calpain activation in selected neuron subtypes may contribute to the pattern of synaptic protein expression.

Activity-dependent phosphorylation of Ser187 is required for SNAP-25-negative modulation of neuronal voltage-gated calcium channels.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a SNARE protein that regulates neurotransmission by the formation of a complex with syntaxin 1 and synaptobrevin/VAMP2. SNAP-25 also reduces neuronal calcium responses to stimuli, but neither the functional relevance nor the molecular mechanisms of this modulation have been clarified. In this study, we demonstrate that hippocampal slices from Snap25(+/-) mice display a significantly larger facilitation and that higher calcium peaks are reached after depolarization by Snap25(-/-) and Snap25(+/-) cultured neurons compared with wild type. We also show that SNAP-25b modulates calcium dynamics by inhibiting voltage-gated calcium channels (VGCCs) and that PKC phosphorylation of SNAP-25 at ser187 is essential for this process, as indicated by the use of phosphomimetic (S187E) or nonphosphorylated (S187A) mutants. Neuronal activity is the trigger that induces the transient phosphorylation of SNAP-25 at ser187. Indeed, enhancement of network activity increases the levels of phosphorylated SNAP-25, whereas network inhibition reduces the extent of protein phosphorylation. A transient peak of SNAP-25 phosphorylation also is detectable in rat hippocampus in vivo after i.p. injection with kainate to induce seizures. These findings demonstrate that differences in the expression levels of SNAP-25 impact on calcium dynamics and neuronal plasticity, and that SNAP-25 phosphorylation, by promoting inhibition of VGCCs, may mediate a negative feedback modulation of neuronal activity during intense activation.

Traffic of botulinum toxins A and E in excitatory and inhibitory neurons.

Botulinum neurotoxins (BoNTs), proteases specific for the SNARE proteins, are used to study the molecular machinery supporting exocytosis and are used to treat human diseases characterized by cholinergic hyperactivity. The recent extension of the use of BoNTs to central nervous system (CNS) pathologies prompted the study of their traffic in central neurons. We used fluorescent BoNT/A and BoNT/E to study the penetration, the translocation and the catalytic action of these toxins in excitatory and inhibitory neurons. We show that BoNT/A and BoNT/E, besides preferentially inhibiting synaptic vesicle recycling at glutamatergic relative to Gamma-aminobutyric acid (GABA)-ergic neurons, are more efficient in impairing the release of excitatory than inhibitory neurotransmitter from brain synaptosomes. This differential effect does not result from a defective penetration of the toxin in line with the presence of the BoNT/A receptor, synaptic vesicle protein 2 (SV2), in both types of neurons. Interestingly, exogenous expression of SNAP-25 in GABAergic neurons confers sensitivity to BoNT/A. These results indicate that the expression of the toxin substrate, and not the toxin penetration, most likely accounts for the distinct effects of the two neurotoxins at the two types of terminals and support the use of BoNTs for the therapy of CNS diseases caused by the altered activity of selected neuronal populations.

Entering neurons: botulinum toxins and synaptic vesicle recycling.

Botulinum toxins are metalloproteases that act inside nerve terminals and block neurotransmitter release through their cleavage of components of the exocytosis machinery. These toxins are used to treat human diseases that are characterized by hyperfunction of cholinergic terminals. Recently, evidence has accumulated that gangliosides and synaptic vesicle proteins cooperate to mediate toxin binding to the presynaptic terminal. The differential distribution of synaptic vesicle protein receptors, gangliosides and toxin substrates in distinct neuronal populations opens up the possibility of using different serotypes of botulinum toxins for the treatment of central nervous system diseases caused by altered activity of selected neuronal populations.

Internalization and mechanism of action of clostridial toxins in neurons.

Botulinum toxins are metalloproteases that act inside nerve terminals and block neurotransmitter release via their activity directed specifically on SNARE proteins. This review summarizes data on botulinum toxin modes of binding, sites of action, and biochemical activities. Their use in cell biology and neuroscience is considered, as well as their therapeutic utilization in human diseases characterized by hyperfunction of cholinergic terminals.