João O. Malva

Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures.

The inflammatory cytokines interleukin-1β and tumor necrosis factor-α (TNF-α) have been identified as mediators of several forms of neurodegeneration in the brain. However, they can produce either deleterious or beneficial effects on neuronal function. We investigated the effects of these cytokines on neuronal death caused by exposure of mouse organotypic hippocampal slice cultures to toxic concentrations of AMPA. Either potentiation of excitotoxicity or neuroprotection was observed, depending on the concentration of the cytokines and the timing of exposure. A relatively high concentration of mouse recombinant TNF-α (10 ng/ml) enhanced excitotoxicity when the cultures were simultaneously exposed to AMPA and to this cytokine. Decreasing the concentration of TNF-α to 1 ng/ml resulted in neuroprotection against AMPA-induced neuronal death independently on the application protocol. By using TNF-α receptor (TNFR) knock-out mice, we demonstrated that the potentiation of AMPA-induced toxicity by TNF-α involves TNF receptor-1, whereas the neuroprotective effect is mediated by TNF receptor-2. AMPA exposure was associated with activation and proliferation of microglia as assessed by macrophage antigen-1 and bromodeoxyuridine immunohistochemistry, suggesting a functional recruitment of cytokine-producing cells at sites of neurodegeneration. Together, these findings are relevant for understanding the role of proinflammatory cytokines and microglia activation in acute and chronic excitotoxic conditions.

Tumor Necrosis Factor‐α Modulates Survival, Proliferation, and Neuronal Differentiation in Neonatal Subventricular Zone Cell Cultures

Tumor necrosis factor (TNF)‐α has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF‐α strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1–3 C57BL/6 mice. By using single‐cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF‐α resulted in increased differentiation of cells displaying a neuronal‐like profile of [Ca2+]i responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF‐α receptor, we found that the proneurogenic effect of 1 ng/ml TNF‐α is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein‐positive neurons was increased following exposure to mouse TNF‐α. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF‐α induced cell proliferation, whereas 10 and 100 ng/ml TNF‐α induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF‐α for 15 minutes or 6 hours caused an increase in the phospho‐stress‐activated protein kinase/c‐Jun N‐terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF‐α induces neurogenesis in neonatal SVZ cell cultures of mice. TNF‐α, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.

Inactivation of Caspase-1 in Rodent Brain : A Novel Anticonvulsive Strategy

Summary:  Purpose: Cytokines and related inflammatory mediators are rapidly synthesized in the brain during seizures. We previously found that intracerebral administration of interleukin‐1 (IL‐1)‐β has proconvulsant effects, whereas its endogenous receptor antagonist (IL‐1Ra) mediates potent anticonvulsant actions in various models of limbic seizures. In this study, we investigated whether seizures can be effectively inhibited by blocking the brain production of IL‐1β, by using selective inhibitors of interleukin‐converting enzyme (ICE/caspase‐1) or through caspase‐1 gene deletion.

Subcellular localization of adenosine A1 receptors in nerve terminals and synapses of the rat hippocampus

Adenosine is a neuromodulator in the CNS that mainly acts through pre- and postsynaptic A(1) receptors to inhibit the release of excitatory neurotransmitters and NMDA receptor function. This might result from a highly localized distribution of A(1) receptors in the active zone and postsynaptic density of CNS synapses that we now investigated in the rat hippocampus. The binding density of the selective A(1) receptor antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX), was enriched in membranes from Percoll-purified nerve terminals (B(max)=1839+/-52 fM/mg protein) compared to total membranes from the hippocampus (B(max)=984+/-31 fM/mg protein), the same occurring with A(1) receptor immunoreactivity. [3H]DPCPX binding occurred mainly to the plasma membrane rather than to intracellular sites, since the binding of the membrane permeable A(1) receptor ligand [3H]DPCPX to intact hippocampal nerve terminals (B(max)=1901+/-192 fM/mg protein) was markedly reduced (B(max)=321+/-30 fM/mg protein) by the membrane impermeable adenosine receptor antagonist, 8-sulfophenyltheophilline (25 microM). Further subcellular fractionation of hippocampal nerve terminals revealed that A(1) receptor immunoreactivity was strategically located in the active zone of presynaptic nerve terminals, as expected to understand the efficiency of A(1) receptors to depress neurotransmitter release. A(1) Receptors were also present in nerve terminals outside the active zone in accordance with the existence of a presynaptic A(1) receptor reserve. Finally, A(1) receptor immunoreactivity was evident in the postsynaptic density together with NMDA receptor subunits 1, 2A and 2B and with N-and P/Q-type calcium channel immunoreactivity, emphasizing the importance of A(1) receptors in the control of dendritic integration.

GluR7 is an essential subunit of presynaptic kainate autoreceptors at hippocampal mossy fiber synapses

Presynaptic ionotropic glutamate receptors are emerging as key players in the regulation of synaptic transmission. Here we identify GluR7, a kainate receptor (KAR) subunit with no known function in the brain, as an essential subunit of presynaptic autoreceptors that facilitate hippocampal mossy fiber synaptic transmission. GluR7−/− mice display markedly reduced short- and long-term synaptic potentiation. Our data suggest that presynaptic KARs are GluR6/GluR7 heteromers that coassemble and are localized within synapses. We show that recombinant GluR6/GluR7 KARs exhibit low sensitivity to glutamate, and we provide evidence that presynaptic KARs at mossy fiber synapses are likely activated by high concentrations of glutamate. Overall, from our data, we propose a model whereby presynaptic KARs are localized in the presynaptic active zone close to release sites, display low affinity for glutamate, are likely Ca2+-permeable, are activated by single release events, and operate within a short time window to facilitate the subsequent release of glutamate.

Neuropeptide Y Promotes Neurogenesis in Murine Subventricular Zone

Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 μM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal‐regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c‐Jun‐NH2‐terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell‐based brain therapy.

Methamphetamine‐induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin

Methamphetamine (METH) causes irreversible damage to brain cells leading to neurological and psychiatric abnormalities. However, the mechanisms underlying life‐threatening effects of acute METH intoxication remain unclear. Indeed, most of the hypotheses focused on intra‐neuronal events, such as dopamine oxidation, oxidative stress and excitotoxicity. Yet, recent reports suggested that glia may contribute to METH‐induced neuropathology. In the present study, we investigated the hippocampal dysfunction induced by an acute high dose of METH (30 mg/kg; intraperitoneal injection), focusing on the inflammatory process and changes in several neuronal structural proteins. For that, 3‐month‐old male wild‐type C57BL/6J mice were killed at different time‐points post‐METH. We observed that METH caused an inflammatory response characterized by astrocytic and microglia reactivity, and tumor necrosis factor (TNF) system alterations. Indeed, glial fibrillary acidic protein (GFAP) and CD11b immunoreactivity were upregulated, likewise TNF‐α and TNF receptor 1 protein levels. Furthermore, the effect of METH on hippocampal neurons was also investigated, and we observed a downregulation in beta III tubulin expression. To clarify the possible neuronal dysfunction induced by METH, several neuronal proteins were analysed. Syntaxin‐1, calbindin D28k and tau protein levels were downregulated, whereas synaptophysin was upregulated. We also evaluated whether an anti‐inflammatory drug could prevent or diminish METH‐induced neuroinflammation, and we concluded that indomethacin (10 mg/kg; i.p.) prevented METH‐induced glia activation and both TNF system and beta III tubulin alterations. In conclusion, we demonstrated that METH triggers an inflammatory process and leads to neuronal dysfunction in the hippocampus, which can be prevented by an anti‐inflammatory treatment.

Activation of neuropeptide Y receptors is neuroprotective against excitotoxicity in organotypic hippocampal slice cultures

Glutamate and NPY have been implicated in hippocampal neuropathology in temporal lobe epilepsy. Thus, we investigated the involvement of NPY receptors in mediating neuroprotection against excitotoxic insults in organotypic cultures of rat hippocampal slices. Exposure of hippocampal slice cultures to 2 μM AMPA (α‐amino‐3‐hydroxy‐5‐methyl‐isoxazole‐4‐propionate) induced neuronal degeneration, monitored by propidium iodide uptake, of granule cells and CA1 pyramidal cells. For dentate granule cells, selective activation of Y1, Y2, or Y5 receptors with 1 μM [Leu31,Pro34]NPY, 300 nM NPY13–36 or 1 μM 500 nM NPY(19–23)‐(Gly1,Ser3,Gln4,Thr6,Ala31,Aib32,Gln34)‐PP, respectively, had a neuroprotective effect against AMPA, whereas only the activation of Y2 receptors was effective for CA1 pyramidal cells. When the slice cultures were exposed to 6 μM kainate, the CA3 pyramidal cells displayed significant degeneration, and in this case the activation of Y1, Y2, and Y5 receptors was neuroprotective. For the kainic acid‐induced degeneration of CA1 pyramidal cells, it was again found that only the Y2 receptor activation was effective. Based on the present findings, it was concluded that Y1, Y2, and Y5 receptors effectively can modify glutamate receptor‐mediated neurodegeneration in the hippocampus.

Methamphetamine‐Induced Early Increase of IL‐6 and TNF‐α mRNA Expression in the Mouse Brain

The mechanisms by which methamphetamine (METH) causes neurotoxicity are not well understood. Recent studies have suggested that METH‐induced neuropathology may result from a multicellular response in which glial cells play a prominent role, and so it is plausible to suggest that cytokines may participate in the toxic effects of METH. Therefore, in the present work we evaluated the effect of an acute administration of METH (30 mg/kg in a single intraperitoneal injection) on the interleukin (IL)‐1β, IL‐6, and tumor necrosis factor (TNF)‐α mRNA expression levels in the hippocampus, frontal cortex, and striatum of mice. We observed that METH did not induce changes in the IL‐1β mRNA expression levels in both hippocampus and striatum, with immeasurable levels in the frontal cortex. Regarding IL‐6, METH induced an increase in the expression levels of this cytokine in the hippocampus and striatum, 1 h and 30 min post injection, respectively. In the frontal cortex, the increase in IL‐6 mRNA levels was more significant and remained high even after 2 h. Moreover, the expression levels of TNF‐α were increased in both hippocampus and frontal cortex 30 min post METH administration, with immeasurable levels in the striatum. We conclude that the pro‐inflammatory cytokines IL‐6 and TNF‐α rapidly increase after METH administration, providing a new insight for understanding the effect of this drug of abuse in the brain.

Susceptibility of hippocampal neurons to Aβ peptide toxicity is associated with perturbation of Ca2+ homeostasis

Neuritic dystrophy, loss of synapses and neuronal death in the cerebral cortex and hippocampus are hallmarks of Alzheimers disease. The aim of the present study was to investigate the differential susceptibility of cortical and hippocampal neurons to amyloid-beta (Abeta)-induced toxicity. For that, we have used primary neuronal cultures prepared from rat brain cortex and hippocampus which were treated with the synthetic peptides Abeta25-35 or Abeta1-40. Abeta-induced apoptotic cell death was analyzed by determining caspase-3-like activity. Neuritic dystrophy was evaluated by cobalt staining and MAP2 immunoreactivity. Perturbation of Ca(2+) homeostasis caused by exposure to Abeta was evaluated by determining basal cytosolic calcium levels in the whole neuronal population and by single cell calcium imaging under basal and KCl-depolarization conditions. Finally, levels of GluR2 subunit of glutamate AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate) receptors were quantified by western blotting. Our results demonstrated that hippocampal neurons in culture are more susceptible than cortical neurons to Abeta-induced apoptosis and also that this mechanism involves the perturbation of Ca(2+) homeostasis. Accordingly, the exposure of hippocampal neurons to Abeta peptides decreases the protein levels of the GluR2 subunit of glutamate AMPA receptors that may be associated with a significant rise of cytosolic Ca(2+) concentration, leading to dendritic dystrophy and activation of apoptotic neuronal death.