Giambattista Bonanno

Chronic Antidepressants Reduce Depolarization-Evoked Glutamate Release and Protein Interactions Favoring Formation of SNARE Complex in Hippocampus

Glutamate neurotransmission was recently implicated in the action of stress and in antidepressant mechanisms. We report that chronic (not acute) treatment with three antidepressants with different primary mechanisms (fluoxetine, reboxetine, and desipramine) markedly reduced depolarization-evoked release of glutamate, stimulated by 15 or 25 mm KCl, but not release of GABA. Endogenous glutamate and GABA release was measured in superfused synaptosomes, freshly prepared from hippocampus of drug-treated rats. Interestingly, treatment with the three drugs only barely changed the release of glutamate (and of GABA) induced by ionomycin. In synaptic membranes of chronically treated rats we found a marked reduction in the protein-protein interaction between syntaxin 1 and Thr286-phosphorylated αCaM kinase II (α-calcium/calmodulin-dependent protein kinase II) (an interaction previously proposed to promote neurotransmitter release) and a marked increase in the interaction between syntaxin 1 and Munc-18 (an interaction proposed to reduce neurotransmitter release). Furthermore, we found a selective reduction in the expression level of the three proteins forming the core SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex. These findings suggest that antidepressants work by stabilizing glutamate neurotransmission in the hippocampus and that they may represent a useful tool for the study of relationship between functional and molecular processes in nerve terminals.

Acute Stress Increases Depolarization-Evoked Glutamate Release in the Rat Prefrontal/Frontal Cortex: The Dampening Action of Antidepressants

Background Behavioral stress is recognized as a main risk factor for neuropsychiatric diseases. Converging evidence suggested that acute stress is associated with increase of excitatory transmission in certain forebrain areas. Aim of this work was to investigate the mechanism whereby acute stress increases glutamate release, and if therapeutic drugs prevent the effect of stress on glutamate release. Methodology/Findings Rats were chronically treated with vehicle or drugs employed for therapy of mood/anxiety disorders (fluoxetine, desipramine, venlafaxine, agomelatine) and then subjected to unpredictable footshock stress. Acute stress induced marked increase in depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex in superfusion, and the chronic drug treatments prevented the increase of glutamate release. Stress induced rapid increase in the circulating levels of corticosterone in all rats (both vehicle- and drug-treated), and glutamate release increase was blocked by previous administration of selective antagonist of glucocorticoid receptor (RU 486). On the molecular level, stress induced accumulation of presynaptic SNARE complexes in synaptic membranes (both in vehicle- and drug-treated rats). Patch-clamp recordings of pyramidal neurons in the prefrontal cortex revealed that stress increased glutamatergic transmission through both pre- and postsynaptic mechanisms, and that antidepressants may normalize it by reducing release probability. Conclusions/Significance Acute footshock stress up-regulated depolarization-evoked release of glutamate from synaptosomes of prefrontal/frontal cortex. Stress-induced increase of glutamate release was dependent on stimulation of glucocorticoid receptor by corticosterone. Because all drugs employed did not block either elevation of corticosterone or accumulation of SNARE complexes, the dampening action of the drugs on glutamate release must be downstream of these processes. This novel effect of antidepressants on the response to stress, shown here for the first time, could be related to the therapeutic action of these drugs.

Nerve growth factor and brain-derived neurotrophic factor increase neurotransmitter release in the rat visual cortex.

A number of experiments have shown that neurotrophins are involved in the development and plasticity of the visual cortex (Bonhoeffer, T., Curr. Op. Neurobiol., 6, 119 1996). A possible mechanism underlying these effects is the neurotrophin modulation of synaptic transmission. We investigated whether nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF) can modulate the release of neurotransmitter in the rat visual cortex at the peak of the critical period for plasticity (P23). The release of glutamate, acetylcholine and gamma‐aminobutyric acid (GABA) from visual cortical synaptosomes was analysed in continuous perfusion conditions. We found that NGF enhances the depolarization‐evoked release of glutamate (≈ 90%) and acetylcholine (≈ 35%) but not that of GABA. By contrast, BDNF enhances the depolarization‐evoked release of all three neurotransmitters investigated (≈ 30%). BDNF and NGF were ineffective on basal release of neurotransmitters. The effect of NGF was not blocked by cholinergic antagonists atropine and mecamylamine. NGF and BDNF potentiation of transmitter release was strongly but not completely blocked by K252a, a tyrosine kinase inhibitor. The role of TrkA and p75NTR receptors was investigated in NGF‐induced potentiation of glutamate release. Block of NGF binding to p75NTR using specific blocking antibodies (REX‐IgG) slightly but significantly reduced the effect of NGF. Activation of TrkA in isolation by RTA‐IgG, an antibody that specifically activates TrkA, was less effective than activation of both receptors by NGF. These results show that neurotrophin action on neurotransmitter release was mostly mediated by Trk receptors with p75NTR having a little but significant positive role. Antigen blot analysis showed the presence of TrkA, TrkB and p75NTR receptors in the visual cortex.

Glia re-sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re‐sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia‐specific proteins glial fibrillary acidic protein (GFAP) and S‐100, but not the neuronal proteins 95‐kDa postsynaptic density protein (PSD‐95), microtubule‐associated protein 2 (MAP‐2) and β‐tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin‐αM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1–5 µm) efficiently stimulated the release of tritium from gliosomes pre‐labelled with [3H]d‐aspartate and of endogenous glutamate in a Ca2+‐dependent and bafilomycin A1‐sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca2+‐dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d‐aspartate release in a concentration‐ (0.1–3 mm) and Ca2+‐dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin‐1, vesicular‐associated membrane protein type 2 (VAMP‐2), 23‐kDa synaptosome‐associated protein (SNAP‐23) and 25‐kDa synaptosome‐associated protein (SNAP‐25)] co‐existing with GFAP immunoreactivity. Moreover, GFAP or VAMP‐2 co‐expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several ∼30‐nm non‐clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate‐accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.

Intravenous mesenchymal stem cells improve survival and motor function in experimental amyotrophic lateral sclerosis.

Despite some advances in the understanding of amyotrophic lateral sclerosis (ALS) pathogenesis, significant achievements in treating this disease are still lacking. Mesenchymal stromal (stem) cells (MSCs) have been shown to be effective in several models of neurological disease. To determine the effects of the intravenous injection of MSCs in an ALS mouse model during the symptomatic stage of disease, MSCs (1 × 106) were intravenously injected in mice expressing human superoxide dismutase 1 (SOD1) carrying the G93A mutation (SOD1/G93A) presenting with experimental ALS. Survival, motor abilities, histology, oxidative stress markers and [3H]d-aspartate release in the spinal cord were investigated. MSC injection in SOD1/G93A mice improved survival and motor functions compared with saline-injected controls. Injected MSCs scantly home to the central nervous system and poorly engraft. We observed a reduced accumulation of ubiquitin agglomerates and of activated astrocytes and microglia in the spinal cord of MSC-treated SOD1/G93A mice, with no changes in the number of choline acetyltransferase- and glutamate transporter type 1-positive cells. MSC administration turned around the upregulation of metallothionein mRNA expression and of the activity of the antioxidant enzyme glutathione S-transferase, both associated with disease progression. Last, we observed that MSCs reverted both spontaneous and stimulus-evoked neuronal release of (3H)d-aspartate, a marker of endogenous glutamate, which is upregulated in SOD1/G93A mice. These findings suggest that intravenous administration of MSCs significantly improves the clinical outcome and pathological scores of mutant SOD1/G93A mice, thus providing the rationale for their exploitation for the treatment of ALS.

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.

CGP 52432 : a novel potent and selective GABAB autoreceptor antagonist in rat cerebral cortex

As previously reported GABAB receptors are heterogeneous. Three pharmacologically distinct receptor subtypes mediating inhibition of gamma-aminobutyric acid (GABA), glutamate or somatostatin release, respectively, exist on axon terminals of rat cerebral cortex. We investigated the novel GABAB receptor antagonist, [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxy-methyl) phosphinic acid (CGP 52432), on the above receptor subtypes. The effects of (-)-baclofen on the K(+)-evoked release of GABA, glutamate or somatostatin from rat cortical synaptosomes were antagonized by CGP 52432. The IC50 of the drug at GABA autoreceptors (0.085 microM) was 35- and 100-fold lower than at the receptors regulating somatostatin and glutamate overflow, respectively. At the autoreceptor the calculated pA2 for CGP 52432 amounted to 7.70, which makes the drug about 1000-fold more potent than phaclofen at this receptor. The potency and selectivity characteristics of CGP 52432 indicate that the drug is by far the most appropriate tool to investigate the terminal GABAB autoreceptors of the rat cerebral cortex.

Stimulation of excitatory amino acid release from adult mouse brain glia subcellular particles by high mobility group box 1 protein

The multifunctional protein high mobility group box 1 (HMGB1) is expressed in hippocampus and cerebellum of adult mouse brain. Our aim was to determine whether HMGB1 affects glutamatergic transmission by monitoring neurotransmitter release from glial (gliosomes) and neuronal (synaptosomes) re‐sealed subcellular particles isolated from cerebellum and hippocampus. HMGB1 induced release of the glutamate analogue [3H]d‐aspartate form gliosomes in a concentration‐dependent manner, whereas nerve terminals were insensitive to the protein. The HMGB1‐evoked release of [3H]d‐aspartate was independent of modifications of cytosolic Ca2+ , but it was blocked by dl‐threo‐β‐benzyloxyaspartate (dl‐TBOA), an inhibitor of glutamate transporters. HMGB1 also stimulated the release of endogenous glutamate in a Ca2+‐independent and dl‐TBOA‐sensitive manner. These findings suggest the involvement of carrier‐mediated release. Moreover, dihydrokainic acid, a selective inhibitor of glutamate transporter 1 (GLT1), does not block the effect of HMGB1, indicating a role for the glial glutamate‐aspartate transporter (GLAST) subtype in this response. We also demonstrate that HMGB1/glial particles association is promoted by Ca2+. Furthermore, although HMGB1 can physically interact with GLAST and the receptor for advanced glycation end products (RAGE), only its binding with RAGE is promoted by Ca2+. These results suggest that the HMGB1 cytokine could act as a modulator of glutamate homeostasis in adult mammal brain.

Desmethylclomipramine induces the accumulation of autophagy markers by blocking autophagic flux

Alterations in the autophagic pathway are associated with the onset and progression of various diseases. However, despite the therapeutic potential for pharmacological modulators of autophagic flux, few such compounds have been characterised. Here we show that clomipramine, an FDA-approved drug long used for the treatment of psychiatric disorders, and its active metabolite desmethylclomipramine (DCMI) interfere with autophagic flux. Treating cells with DCMI caused a significant and specific increase in autophagosomal markers and a concomitant blockage of the degradation of autophagic cargo. This observation might be relevant in therapy in which malignant cells exploit autophagy to survive stress conditions, rendering them more susceptible to the action of cytotoxic agents. In accordance, DCMI-mediated obstruction of autophagic flux increased the cytotoxic effect of chemotherapeutic agents. Collectively, our studies describe a new function of DCMI that can be exploited for the treatment of pathological conditions in which manipulation of autophagic flux is thought to be beneficial.

Coexistence and function of different neurotransmitter transporters in the plasma membrane of CNS neurons

Transporters able to recapture released neurotransmitters into neurons can no longer be considered as cell-specific neuronal markers. In fact, colocalization on one nerve terminal of transporters able to selectively recapture the released endogenously synthesized transmitter (homotransporters) and of transporters that can selectively take up transmitters/modulators originating from neighboring structures (heterotransporters) has been demonstrated to occur on several families of nerve terminals. Activation of heterotransporters often increases the release of the transmitter stored in the terminals on which the heterotransporters are localized. The release caused by heterotransporter activation takes place through multiple mechanisms including exocytosis, either dependent on external Ca(2+) or on Ca(2+) mobilized from intraterminal stores, and homotransporter reversal. Homocarrier-mediated release elicited by heterocarrier activation represents a clear case of transporter-transporter interaction. Although the functional significance of transporter coexpression on one nerve terminal remains to be established, it may in some instances reflect cotransmission. In other cases, heterotransporters may mediate modulation of basal transmitter release in addition to the modulation of the evoked release brought about by presynaptic heteroreceptors. Heterotransporters are also increasingly reported to exist on neuronal soma/dendrites. With the exception of EAAT4, the glutamate transporter/chloride channel situated on GABAergic Purkinje cells in the cerebellum, the functions of somatodendritic heterocarriers is not understood.