Activity of Adenosine Receptors Type 1 Is Required for CX3CL1-Mediated Neuroprotection and Neuromodulation in Hippocampal Neurons

Abstract

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−/− (CX3CR1GFP/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 CX3CR1GFP/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.

Adenosine A1 Receptors and Microglial Cells Mediate CX3CL1-Induced Protection of Hippocampal Neurons Against Glu-Induced Death

“…This corresponded to about 70% of total neuronal loss on Glu treatment, as assessed by immunofluorescence analysis with b-tubulin III staining (data not shown). We confirm, in this manuscript, that CX3CL1 protects hippocampal neurons from Glu-induced excitotoxicity ( Figure 1a) similarly to what already shown in neuronal preparations, which contain different ratios of neurons:astrocytes:microglia (Limatola et al, 2005;Lauro et al, 2008). Given that CX3CR1 are predominantly expressed in microglial cells, it is likely that microglial cells mediate the neurotrophic effect of CX3CL1.…”

“…However, as immortalized cell lines may differ from primary cells, we wanted to investigate (i) whether primary microglia also release adenosine on CX3CL1 treatment and (ii) whether the reduction of extracellular adenosine levels, by treating cultured cells with ADA (the enzyme that degrades adenosine to inosine), is sufficient to prevent CX3CL1 neuroprotection against Glu-induced excitotoxicity. Supplementary Figure S3 shows that CX3CL1 treatment of primary cultures of murine microglia induces adenosine release, as previously shown with BV2 cells (Lauro et al, 2008). Results in Figure 3a show that ADA treatment (1 U/ml, 1 h, 371C) of Glu-treated hippocampal cultures completely Figure 2 Endogenous levels of CX3CL1 are not sufficient to protect neurons by excitotoxicity.…”

“…We have previously shown that CX3CL1 induces the release of adenosine from the murine microglial cell line BV2 and from mixed hippocampal cultures (Lauro et al, 2008). However, as immortalized cell lines may differ from primary cells, we wanted to investigate (i) whether primary microglia also release adenosine on CX3CL1 treatment and (ii) whether the reduction of extracellular adenosine levels, by treating cultured cells with ADA (the enzyme that degrades adenosine to inosine), is sufficient to prevent CX3CL1 neuroprotection against Glu-induced excitotoxicity.…”

“…In these protocols, ADA was present till the end of the experiments. To evaluate neuron viability, cells were then treated with detergent-containing buffer (0.05% ethyl hexadecyl dimethylammonium bromide, 0.028% acetic acid, 0.05% Triton X-100, 0.3 mM NaCl, 0.2 mM MgCl 2 , in PBS pH 7.4) and counted in a hemacytometer as already described (Lauro et al, 2008). Alternatively, cell viability was analyzed by the MTT assay: in detail, 5 mg/ml MTT was added 1 : 10 to the cell medium and incubated for 2 h; the medium was aspired, cells were treated with DMSO, and incubated at 371C for 10 min.…”

“…The neurotrophic activity of exogenously administered CX3CL1 has been related to the simultaneous production of protective factors from microglial cells and in particular with the activation of adenosine receptors (ARs, Lauro et al, 2008). Adenosine is a cellular metabolite whose intracellular and extracellular levels can be rapidly modulated by variation of cellular metabolic state (see Fredholm, 2007).…”