Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death.

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.

Development of polycystic kidney disease in juvenile cystic kidney mice: insights into pathogenesis, ciliary abnormalities, and common features with human disease.

Significant progress in understanding the molecular mechanisms of polycystic kidney disease (PKD) has been made in recent years. Translating this understanding into effective therapeutics will require testing in animal models that closely resemble human PKD by multiple parameters. Similar to autosomal dominant PKD, juvenile cystic kidney (jck) mice develop cysts in multiple nephron segments, including cortical collecting ducts, distal tubules, and loop of Henle. The jck mice display gender dimorphism in kidney disease progression with more aggressive disease in male mice. Gonadectomy experiments show that testosterone aggravates the severity of the disease in jck male mice, while female gonadal hormones have protective effects. EGF receptor is overexpressed and mislocalized in jck cystic epithelia, a hallmark of human disease. Increased cAMP levels in jck kidneys and activation of the B-Raf/extracellular signal-regulated kinase pathway are demonstrated. The effect of jck mutation on the expression of Nek8, a NIMA-related (never in mitosis A) kinase, and polycystins in jck cilia is shown for the first time. Nek8 overexpression and loss of ciliary localization in jck epithelia are accompanied by enhanced expression of polycystins along the cilia. The primary cilia in jck kidneys are significantly more lengthened than the cilia in wild-type mice, suggesting a role for Nek8 in controlling ciliary length. Collectively, these data demonstrate that the jck mice should be useful for testing potential therapies and for studying the molecular mechanisms that link ciliary structure/function and cystogenesis.

Signaling cascades activated upon antibody cross-linking of myelin oligodendrocyte glycoprotein: potential implications for multiple sclerosis.

Antibody-induced demyelination is an important component of pathology in multiple sclerosis. In particular, antibodies to myelin oligodendrocyte glycoprotein (MOG) are elevated in multiple sclerosis patients, and they have been implicated as mediators of demyelination. We have shown previously that antibody cross-linking of MOG in oligodendrocytes results in the repartitioning of MOG into glycosphingolipid-cholesterol membrane microdomains ("lipid rafts"), followed by changes in the phosphorylation of specific proteins, including dephosphorylation of beta-tubulin and the beta subunit of the trimeric G protein and culminating in rapid and dramatic morphological alterations. In order to further elucidate the mechanism of anti-MOG-mediated demyelination, we have carried out a proteomic analysis to identify the set of proteins for which the phosphorylation states or expression levels are altered upon anti-MOG treatment. We demonstrate that treatment of oligodendrocytes with anti-MOG alone leads to an increase in calcium influx and activation of the MAPK/Akt pathways that is independent of MOG repartitioning. However, further cross-linking of anti-MOG.MOG complexes with a secondary anti-IgG results in the lipid raft-dependent phosphorylation of specific proteins related to cellular stress response and cytoskeletal stability. Oligodendrocyte survival is not compromised by these treatments. We discuss the possible significance of the anti-MOG-induced signaling cascade in relation to the initial steps of MOG-mediated demyelination.

Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons.

Brain cells in situ contain low concentrations of free polyunsaturated fatty acids such as arachidonic acid (AA) that are released following pathological insults. As a large rise in extracellular [K(+)] accompanies cerebral ischemia, we explored whether this was a stimulus for cellular AA release employing a murine mixed cortical cell culture preparation radiolabeled with AA. Elevating the [K(+)](o) from 5 to 52 mm induced a time-dependent increase in [(3)H]AA release, which reached a plateau after 15 min. Removal of [Ca(2+)](o) or addition of CdCl(2) (100 microm) diminished the net high K(+)-induced AA release, as did treatment of the cultures with tetanus toxin (300 ng/ml) to block endogenous neurotransmitter release. Pharmacological antagonism of both ionotropic and metabotropic glutamate receptors completely prevented high K(+)-evoked AA release, indicating that glutamate was the neurotransmitter in question. Addition of exogenous glutamate mimicked precisely the characteristics of AA release that followed increases in [K(+)](o). Finally, glutamate and AA were released solely from neurons as tetanus toxin did not cleave astrocytic synaptobrevin-2, nor was AA released from pure astrocyte cultures using the same stimuli that were effective in mixed cultures. Taken in toto, our data are consistent with the following scenario: high [K(+)](o) depolarizes neurons, causing an influx of Ca(2+) via voltage-gated Ca(2+) channels. This Ca(2+) influx stimulates the release of glutamate into the synaptic cleft, where it activates postsynaptic glutamate receptors. Events likely converge on the activation of a phospholipase A(2) family member and possibly the enzymes diacylglycerol and monoacylglycerol lipases to yield free AA.