The chemokine CX3CL1 and its receptor CX3CR1 are constitutively expressed in the nervous system. In this study, we usedin vivomurine models of permanent middle cerebral artery occlusion (pMCAO) to investigate the protective potential of CX3CL1. We report that exogenous CX3CL1 reduced ischemia-induced cerebral infarct size, neurological deficits, and caspase-3 activation. CX3CL1-induced neuroprotective effects were long lasting, being observed up to 50 d after pMCAO in rats. The neuroprotective action of CX3CL1 in different models of brain injuries is mediated by its inhibitory activity on microglia and,in vitro, requires the activation of adenosine receptor 1 (A₁R). We show that, in the presence of the A₁R antagonist 1,3-dipropyl-8-cyclopentylxanthine and inA₁R^−/−mice, the neuroprotective effect of CX3CL1 on pMCAO was abolished, indicating the critical importance of the adenosine system in CX3CL1 protection alsoin vivo. In apparent contrast with the above reported data but in agreement with previous findings,cx3cl1^−/−andcx3cr1^GFP/GFPmice, respectively, deficient in CX3CL1 or CX3CR1, had less severe brain injury on pMCAO, and the administration of exogenous CX3CL1 increased brain damage incx3cl1^−/−ischemic mice. We also report that CX3CL1 induced a different phagocytic activity in wild type andcx3cl1^−/−microgliain vitroduring cotreatment with the medium conditioned by neurons damaged by oxygen-glucose deprivation. Together, these data suggest that acute administration of CX3CL1 reduces ischemic damage via an adenosine-dependent mechanism and that the absence of constitutive CX3CL1–CX3CR1 signaling changes the outcome of microglia-mediated effects during CX3CL1 administration to ischemic brain.

Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca²⁺-dependent K⁺ channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.

Purpose: Several factors contribute to epileptogenesis in patients with brain tumors, including reduced γ-aminobutyric acid (GABA)ergic inhibition. In particular, changes in Cl⁻ homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl⁻ homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl⁻ concentration ([Cl⁻]_i) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl⁻]_i. Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors. Methods: Membranes from peritumoral cortical tissues of epileptic patients afflicted by gliomas (from II to IV WHO grade) and from cortical tissues of nonepileptic patients were injected into Xenopus oocytes leading to the incorporation of functional GABA_A receptors. The GABA-evoked currents were recorded using standard two-microelectrode voltage-clamp technique. In addition, immunoblot analysis and immunohistochemical staining were carried out on membranes and tissues from the same patients. Key Findings: We found that in oocytes injected with epileptic peritumoral cerebral cortex, the GABA-evoked currents had a more depolarized reversal potential (E_GABA) compared to those from nonepileptic healthy cortex. This difference of E_GABA was abolished by the NKCC1 blocker bumetanide or unblocking of KCC2 with the Zn²⁺ chelator TPEN. Moreover, Western blot analysis revealed an increased expression of NKCC1, and more modestly, of KCC2 transporters in epileptic peritumoral tissues compared to nonepileptic control tissues. In addition, NKCC1 immunoreactivity was strongly increased in peritumoral cortex with respect to nonepileptic cortex, with a prominent expression in neuronal cells. Significance: We report that the positive shift of E_GABA in epileptic peritumoral human cortex is due to an altered expression of NKCC1 and KCC2, perturbing Cl⁻ homeostasis, which might lead to a consequent reduction in GABAergic inhibition. These findings point to a key role of Cl⁻ transporters KCC2 and NKCC1 in tumor-related epilepsy, suggesting a more specific drug therapy and surgical approaches for the epileptic patients afflicted by brain tumors.

The chemokine fractalkine (CX₃CL1) is constitutively expressed by central neurons, regulating microglial responses including chemotaxis, activation, and toxicity. Through the activation of its own specific receptor, CX₃CR1, CX₃CL1 exerts both neuroprotection against glutamate (Glu) toxicity and neuromodulation of the glutamatergic synaptic transmission in hippocampal neurons. Using cultured hippocampal neuronal cell preparations, obtained from CX₃CR1^−/− (CX₃CR1^GFP/GFP) mice, we report that these same effects are mimicked by exposing neurons to a medium conditioned with CX₃CL1-treated mouse microglial cell line BV2 (BV2-st medium). Furthermore, CX₃CL1-induced neuroprotection from Glu toxicity is mediated through the adenosine receptor 1 (AR₁), being blocked by neuronal cell preparations treatment with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a specific inhibitor of AR₁, and mimicked by both adenosine and the specific AR₁ agonist 2-chloro-N⁶-cyclopentyladenosine. Similarly, experiments from whole-cell patch-clamped hippocampal neurons in culture, obtained from CX₃CR1^+/+ mice, show that CX₃CL1-induced depression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- (AMPA-) type Glu receptor-mediated current (AMPA-current), is associated with AR₁ activity being blocked by DPCPX and mimicked by adenosine. Furthermore, BV2-st medium induced a similar AMPA-current depression in CX₃CR1^GFP/GFP hippocampal neurons and this depression was again blocked by DPCPX. We also report that CX₃CL1 induced a significant release of adenosine from microglial BV2 cells, as measured by HPLC analysis. We demonstrate that (i) CX₃CL1, along with AR₁, are critical players for counteracting Glu-mediated neurotoxicity in the brain and (ii) AR₁ mediates neuromodulatory action of CX₃CL1 on hippocampal neurons.

FTY720 (fingolimod, Gilenya™), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P1 receptors (S1P1R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection. Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 μg/2 μl) in Sprague–Dawley rats. FTY720 was applied icv (1 μg/2 μl), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs). FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation. These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.