Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Our work aimed to provide a topographical analysis of all known ionotropic P2X1–7 and metabotropic P2Y1,2,4,6,11–14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and substantia nigra, P2X6,7 and P2Y1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X1,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease

P2Y12 receptor protein in cortical gray matter lesions in multiple sclerosis

Although Multiple Sclerosis (MS) is regarded as a white matter disease, the incidence of demyelination and axonal injury is prominent also in gray matter. In MS, extracellular adenosine triphosphate (ATP) is an important mediator of central nervous system pathology via its ability to cause oligodendrocyte excitotoxicity. We have analyzed the distribution pattern of all ionotropic P2X and metabotropic P2Y receptors for ATP in postmortem samples of the cerebral cortex from healthy human subjects as well as MS patients. We focus particularly on the P2Y(12) subtype that is highly enriched in oligodendrocytes. We correlate the expression of this receptor to the extent of gray matter demyelination and pathological alterations occurring during secondary progressive MS. Using triple immunofluorescence and confocal analysis, we show that in sections of cerebral cortex from postmortem MS brains, the P2Y(12) protein is present in myelin and interlaminar astrocytes but absent from protoplasmic astrocytes residing in the deeper cortical layers, from microglia/macrophages, and from intact demyelinated axons. We report that a decreased P2Y(12) receptor immunoreactivity in proximity to the lesions is directly correlated with the extent of demyelination found in all types of gray matter cortical plaques (I-III) and subcortical white matter. Our study provides further insights into the pathogenetic features of MS and suggests that the loss of purinergic P2Y(12) receptors might be detrimental to tissue integrity

P2Y12 Receptor on the Verge of a Neuroinflammatory Breakdown

In the CNS, neuroinflammation occurring during pathologies as amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) is the consequence of an intricate interplay orchestrated by various cell phenotypes. Among the molecular cues having a role in this process, extracellular nucleotides are responsible for intercellular communication and propagation of inflammatory stimuli. This occurs by binding to several receptor subtypes, defined P2X/P2Y, which are widespread in different tissues and simultaneously localized on multiple cells. For instance, the metabotropic P2Y12 subtype is found in the CNS on microglia, affecting activation and chemotaxis, on oligodendrocytes, possessing a hypothesized role in myelination, and on astrocytes. By comparative analysis, we have established here that P2Y12 receptor immunolabelled by antibodies against C-terminus or second intracellular loop, is, respectively, distributed and modulated under neuroinflammatory conditions on ramified microglia or myelinated fibers, in primary organotypic cerebellar cultures, tissue slices from rat striatum and cerebellum, spinal cord sections from symptomatic/end stage SOD1-G93A ALS mice, and finally autoptic cortical tissue from progressive MS donors. We suggest that modulation of P2Y12 expression might play a dual role as analytic marker of branched/surveillant microglia and demyelinating lesions, thus potentially acquiring a predictive value under neuroinflammatory conditions as those found in ALS and MS

Synthetic cannabinoid ajulemic acid exerts potent antifibrotic effects in experimental models of systemic sclerosis

Background Cannabinoids modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol that can bind the peroxisome proliferator-activated receptor-γ (PPAR-γ). Recent evidence suggests a key role for PPAR-γ in fibrogenesis. Objective To determine whether AjA can modulate fibrogenesis in murine models of scleroderma. Material and methods Bleomycin-induced experimental fibrosis was used to assess the antifibrotic effects of AjA in vivo. In addition, the efficacy of AjA in pre-established fibrosis was analysed in a modified model of bleomycin-induced dermal fibrosis and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor I. Skin fibrosis was evaluated by quantification of skin thickness and hydroxyproline content. As a marker of fibroblast activation, α-smooth muscle actin was examined. To study the direct effect of AjA in collagen neosynthesis, skin fibroblasts from patients with scleroderma were treated with increasing concentrations of AjA. Protein expression of PPAR-γ, and its endogenous ligand 15d-PGJ2, and TGFβ were assessed before and after AjA treatment. Results AjA significantly prevented experimental bleomycin-induced dermal fibrosis and modestly reduced its progression when started 3 weeks into the disease. AjA strongly reduced collagen neosynthesis by scleroderma fibroblasts in vitro, an action which was reversed completely by co-treatment with a selective PPAR-γ antagonist. Conclusions AjA prevents progression of fibrosis in vivo and inhibits fibrogenesis in vitro by stimulating PPAR-γ signalling. Since therapeutic doses of AjA are well tolerated in humans, it is suggested that AjA as an interesting molecule targeting fibrosis in patients with scleroderma

Synthetic cannabinoid ajulemic acid exerts potent antifibrotic effects in experimental models of systemic sclerosis

Background: Cannabinoids modulate fibrogenesis in scleroderma. Ajulemic acid (AjA) is a non-psychoactive synthetic analogue of tetrahydrocannabinol that can bind the peroxisome proliferator-activated receptor-γ (PPAR-γ). Recent evidence suggests a key role for PPAR-γ in fibrogenesis. Objective: To determine whether AjA can modulate fibrogenesis in murine models of scleroderma. Material and methods: Bleomycin-induced experimental fibrosis was used to assess the antifibrotic effects of AjA in vivo. In addition, the efficacy of AjA in pre-established fibrosis was analysed in a modified model of bleomycin-induced dermal fibrosis and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor I. Skin fibrosis was evaluated by quantification of skin thickness and hydroxyproline content. As a marker of fibroblast activation, α-smooth muscle actin was examined. To study the direct effect of AjA in collagen neosynthesis, skin fibroblasts from patients with scleroderma were treated with increasing concentrations of AjA. Protein expression of PPAR-γ, and its endogenous ligand 15d-PGJ2, and TGFβ were assessed before and after AjA treatment. Results: AjA significantly prevented experimental bleomycin-induced dermal fibrosis and modestly reduced its progression when started 3 weeks into the disease. AjA strongly reduced collagen neosynthesis by scleroderma fibroblasts in vitro, an action which was reversed completely by co-treatment with a selective PPAR-γ antagonist. Conclusions: AjA prevents progression of fibrosis in vivo and inhibits fibrogenesis in vitro by stimulating PPAR-γ signalling. Since therapeutic doses of AjA are well tolerated in humans, it is suggested that AjA as an interesting molecule targeting fibrosis in patients with scleroderma