title: Loss of microglial ramification in microglia-astrocyte cocultures: involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems.
abstract: Reduction in microglial branching is a common feature in brain pathology and culminates in the transformation into small, rounded, microglia-derived phagocytes in the presence of neural debris. The molecular factors responsible for this transformation are unknown. Here we explored the effect of different classes of intra- and extracellular stimuli in vitro on the morphology of ramified microglia cultured on a confluent astrocyte substrate. These studies showed a strong dose-dependent effect for the Ca(2+) ionophore calcimycine/A21837 (50 microM) and for dibutyryl-cAMP (1 mM), with a loss of microglial ramification. Direct activation of the adenylate cyclase with forskolin (0.1 mM) also led to the disappearance of microglial branching. Okadaic acid (70 nM), the inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), and pertussis toxin (12.5 microg/ml), a G(i)-protein inhibitor, also showed similar effects. No effect was observed for dibutyryl-cGMP or for UTP; addition of ATP had a moderate effect, but only at very high, probably nonphysiological concentrations (100 mM). Extracellular matrix components such as keratatan-sulfate, integrin receptor blockers, the disintegrins kistrin, echistatin, and flavoridin, or the serine protease thrombin all had no effect. Addition of prostaglandin D(2) (PGD(2)), a molecule produced by activated microglial cells, had a transforming effect, but at concentrations two orders of magnitude higher than that of established PGD(2) receptors. In summary, addition of agents causing intracellular elevation of Ca(2+) and cAMP or inhibition of G(i)-proteins and phosphatases to ramified microglia cultured on top of confluent astrocytes leads to a rapid loss of microglial branching. Signaling cascades controlled by these molecules may play an important role in the regulation of this common physiological process in the injured brain.
authors.affiliation: Department of Neuromorphology, Max Planck Institute of Neurobiology, Martinsried, Germany.
authors.forename: Roger
authors.initials: R
authors.lastname: Kalla
authors.forename: Marion
authors.initials: M
authors.lastname: Bohatschek
authors.forename: Christian U A
authors.initials: CU
authors.lastname: Kloss
authors.forename: Janna
authors.initials: J
authors.lastname: Krol
authors.forename: Xenia
authors.initials: X
authors.lastname: Von Maltzan
authors.forename: Gennadij
authors.initials: G
authors.lastname: Raivich
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D005576:Colforsin / Q000494:pharmacology; D003995:Dibutyryl Cyclic GMP / Q000494:pharmacology; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D007476:Ionophores / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D015230:Prostaglandin D2 / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014544:Uridine Triphosphate / Q000494:pharmacology
chemical_list: D007476:Ionophores; D010455:Peptides; D005576:Colforsin; D019319:Okadaic Acid; D003995:Dibutyryl Cyclic GMP; D003994:Bucladesine; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D010744:Phosphoric Monoester Hydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D015230:Prostaglandin D2; D002118:Calcium; D014544:Uridine Triphosphate