L. Facci
Doctorate School in Molecular and Cellular Pharmacology
Department of Pharmacology and Anesthesiology, Largo “E. Meneghetti” 2, University of Padua, Padua 35131, Italy.
Inflammation-derived reactive oxygen and nitrogen species and pro-inflammatory cytokines may contribute to neuropathologies such as stroke, traumatic brain injury, and progressive neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis. ATP is commonly released from central nervous system neurons during tissue damage and inflammation, and its actions on glia-mediated toxicity are especially pertinent. ATP can act as a neurotransmitter, and activation of the purinergic receptor subclass P2X7 (P2X7R) has dramatic cytotoxic properties which, together with its ability to regulate cytokine production and release, propose that it can act as an important regulator of cell death in response to pathological insults in nervous system tissues (1). Within the central nervous system, functional P2X7Rs are localized on microglia and astrocytes. P2X7Rs may affect neuronal cell death through their ability to regulate the processing and release of interleukin-1b (IL-1b), a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Extracellular ATP concentrations increase markedly under inflammatory conditions in vivo (2) and in response to tissue trauma. Delivery of a P2X7R antagonist after acute spinal cord impact injury in the rat improved functional recovery and diminished cell death in the peritraumatic zone (3). P2X7R-like immunoreactivity was up-regulated around amyloid b-peptide plaques in a mouse transgenic Alzheimer’s disease model and was regionally localized with activated microglia and astrocytes (4). Intra-hippocampal injection of amyloid b-peptide caused a large accumulation of IL-1b in wild-type but not P2X7R-deficient mice (5).
The aim of this project is to examine the comparative behaviors of P2X7R on microglia and astrocytes cultured from newborn rat cortex and spinal cord. These glial cell populations were prepared following established procedures (6). Upon reaching confluency, the loosely adhering microglia were dislodged from the underlying astrocyte monolayer by shaking (200 cycles/min) for 1 h, followed by purification over plastic dishes. Microglia (³99%) were removed by mechanically scraping and seeded in 96 well culture plates. The enriched (³95%) astrocyte population was detached by mild trypsinization and seeded in 96 well plates. Microglia and astrocytes from both cortex and spinal cord expressed P2X7R mRNA. ATP is an efficient stimulus for IL-1b secretion only after the cells have undergone a short priming with the endotoxin lipopolysaccharide (LPS), a process referred to as ‘priming’. A 2-h pre-treatment of all cell populations with LPS (1 mg/ml) or 1 h with 5 mM ATP led to little or no IL-1b release into the culture medium, as measured by ELISA. However, when LPS pre-treatment was followed by ATP challenge (in the continued presence of LPS) significantly larger amounts of IL-1b were detected. This response was inhibited by a panel of P2X7R antagonists in a concentration-dependent manner. Unexpectedly, benzoylATP, which is also acts as a P2X7R agonist was ineffective.
Future studies will investigate the action of pharmacological agents (e.g. analgesics, antidepressants) on the priming response, as well as the potential for interactions between microglia and astrocytes.
L. Facci is supported by Fondazione CARIPARO Progetto Dottorati di Ricerca" Anno 2009.
(1) Skaper et al FASEB J (doi:10.1096/fj.09-138883)
(2) Lazarowski et al (2000) J Biol Chem 275: 31061-31068
(3) Wang et al (2004) Nat Med 10: 821-827
(4) Parvathenani et al (2003) J Biol Chem 278:13309-13317
(5) Sanz et al (2009) J Immunol 182: 4378-4385
(6) Skaper et al (2006) Glia 54: 234-242