Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory.

Non-fibrillar soluble oligomeric forms of amyloid-ß peptide (oAß) and tau proteins are likely to play a major role in Alzheimer's disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAß initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aß, eventually leading to memory loss. Here we show that a brief exposure to extracellular recombinant human tau oligomers (oTau), but not monomers, produces an impairment of long-term potentiation (LTP) and memory, independent of the presence of high oAß levels. The impairment is immediate as it raises as soon as 20 min after exposure to the oligomers. These effects are reproduced either by oTau extracted from AD human specimens, or naturally produced in mice overexpressing human tau. Finally, we found that oTau could also act in combination with oAß to produce these effects, as sub-toxic doses of the two peptides combined lead to LTP and memory impairment. These findings provide a novel view of the effects of tau and Aß on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aß and tau pathology.

Interleukin-1beta stimulates cyclic GMP efflux in brain astrocytes.

In rat brain astroglia-enriched cultures long-term treatment with interleukin-1beta induces NO release and stimulation of soluble guanylyl cyclase. The cGMP formed is recovered in the extracellular medium but not in the cell monolayer. The interleukin-1beta effect is mediated by type I receptor and potentiated by interferon-gamma. In cells treated with bacterial endotoxin a larger NO-dependent cGMP accumulation occurs only intracellularly, however a significant cGMP egression is observed when cells are co-treated with interleukin-1beta. The non-selective anion transport inhibitors probenecid and verapamil block cGMP efflux, indicating that interleukin-1beta stimulates a cGMP transporter.

Nitric oxide-independent down-regulation of soluble guanylyl cyclase by bacterial endotoxin in astroglial cells.

Induction of nitric oxide (NO) synthase (NOS) type 2 (NOS-2) in glial cells after exposure to bacterial endotoxin [lipopolysaccharide (LPS)] or inflammatory cytokines has been repeatedly demonstrated both in vitro and in vivo. However, little is known about effects of these agents on NO-dependent cyclic GMP (cGMP) formation. In this work, we show that treatment of rat cerebellar astrocyte-enriched primary cultures with LPS decreases NO donor-stimulated cGMP formation with a similar initial time course (up to 9-12 h) and concentration dependency (0.1-1 ng/ml) as for induction of NOS-2. This effect appears to be due to a down-regulation of soluble guanylyl cyclase (sGC) because LPS treatment decreases sGC activity and sGC beta1 subunit levels. In contrast, cGMP phosphodiesterase activity and stimulation of the particulate guanylyl cyclase by atrial natriuretic peptide are not affected. Incubation of astroglial cultures with a transcription inhibitor (actinomycin D) or a protein synthesis inhibitor (cycloheximide) for 18-20 h does not decrease sGC activity but totally prevents LPS-induced desensitization of sGC. Inhibition of NOS-2 activity with N(G)-monomethyl-L-arginine or inhibition of NOS-2 induction with the synthetic glucocorticoid dexamethasone failed to prevent the inhibitory effect of LPS on sGC, indicating that NO production is not involved. Moreover, after removal of LPS the time for recovery of sGC responsiveness is much longer than that for NOS-2 return to basal levels. LPS impairment of cGMP formation also occurs in cortical astrocytes but not in cerebellar granule neurons. The decreased responsiveness of sGC to NO stimulation following LPS challenge may prevent inappropriate astroglial cGMP signaling caused by excess production of NO by adjacent activated glial cells. Key Words: Astroglia-Neurons-Nitric oxide-Soluble guanylyl cyclase-Lipopolysaccharide.

Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture.

Ca2+ entry induced by N-methyl-D-aspartate (NMDA) in neurons and by noradrenaline (NA) in astrocytes is known to increase intracellular cyclic GMP (cGMP) levels through stimulation of the Ca2+-dependent nitric oxide synthase type I (NOS-I). The possibility that Ca2+ entry could also down-regulate intracellular cGMP by activating a Ca2+/calmodulin-dependent phosphodiesterase (CaM-PDE) has been investigated here in primary cultures enriched in granule neurons or in astroglia from rat cerebellum. We show that the same agonists that stimulate nitric oxide (NO) formation (NMDA and NA at 100 microM) and the Ca2+ ionophore A23187 (10 microM) decrease cGMP generated in response to direct stimulation of soluble guanylyl cyclase (sGC) by NO donors in both cell types. This effect requires extracellular Ca2+ and is prevented by the calmodulin inhibitor W7 (100 microM). Membrane depolarization, manipulations of the Na+ gradient, and intracellular Ca2+ mobilization also decrease NO donor-induced cGMP formation in granule cells. In astroglia Ca2+ entry additionally down-regulates cGMP generated by stimulation of the particulate GC by atrial natriuretic peptide (ANF). Decreases in cGMP produced by A23187 were more pronounced in the absence than in the presence of the PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 mM), indicating that a CaM-PDE was involved. We also show that astroglial cells can accumulate similar amounts of cGMP than neurons in response to NO donors when IBMX is present but much lower levels in its absence. This may result from a lower ratio of sGC to PDE activities in astroglia.

AMPA receptors are coupled to the nitric oxide/cyclic GMP pathway in cerebellar astroglial cells.

In cultured rat cerebellar astroglia kainate induces cGMP formation with low potency (EC50 310 microM). In the presence of cyclothiazide, a blocker of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor desensitization, the effect of kainate was potentiated and glutamate and AMPA elicited large increases (> 100-fold) in cGMP levels. The response to all three agonists was abolished by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine and required extracellular calcium. Uptake of Co2+ was induced by AMPA in a limited population of astroglial cells and this effect was potentiated by cyclothiazide. These results indicate that calcium-permeable AMPA receptors mediate stimulation of nitric oxide formation in cerebellar astroglia. This effect may be relevant for glutamate-dependent synaptic plasticity processes in the cerebellum.

Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes.

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.

Calcium-dependent nitric oxide formation in glial cells.

We have previously demonstrated nitric oxide (NO)-dependent cyclic GMP (cGMP) formation in response to noradrenaline (NA) and glutamate (GLU) in astrocyte-enriched cultures from rat cerebrum. In the present work we show heterogeneity in agonist responses in astrocyte cultures from cerebellum, hippocampus and cortex. The response to NA was higher in cells from cerebellum, intermediate in cultures from hippocampus and low in cortical astrocytes. GLU had no significant effect in cortical and cerebellar cultures and presented lower effects than NA in cells from hippocampus. The NO donor sodium nitroprusside (SNP) produced much higher cGMP levels than agonists and the order of efficacies was cerebellum > cortex > hippocampus. Responses to NA and SNP in cerebellar astrocytes were sensitive to culture conditions decreasing when cells were seeded at low density or subcultured. Microglial cells were the main contaminants of the cerebellar astrocyte cultures but did not contribute to the NA or the SNP responses. No soluble guanylyl cyclase or calcium-dependent NO synthase (cNOS) activities were detected in microglial cultures. The effect of NA in cerebellar astrocytes was blocked by L-arginine analogues and by the alpha 1-adrenoceptor antagonist prazosin. The calcium ionophore A23187 mimicked the effect of NA and omission of calcium from the medium prevented both responses. NA did not elicit cGMP formation in granule cell cultures. These results support an astroglial location of the alpha 1-adrenoceptors and the cNOS that mediate NA stimulation of cGMP formation in cerebellum.

Dexamethasone up-regulates a constitutive nitric oxide synthase in cerebellar astrocytes but not in granule cells in culture.

Treatment of rat cerebellar astrocyte-enriched primary cultures with dexamethasone enhances the nitric oxide-dependent cyclic GMP formation induced by noradrenaline in a time-(> 6 h) and concentration-dependent manner (half-maximal effect at 1 nM). Stimulation of cyclic GMP formation by the calcium ionophore A23187 is similarly enhanced. In contrast, cyclic GMP accumulation in cells treated with lipopolysaccharide is inhibited by dexamethasone. The potentiating effect of dexamethasone is prevented by the protein synthesis inhibitor cycloheximide and is not due to increased soluble guanylate cyclase activity. Agonist stimulation of [3H]arginine to [3H]citrulline conversion is enhanced by dexamethasone in astrocytes but not in cerebellar granule cells. These results indicate that glucocorticoids may up-regulate astroglial calcium-dependent nitric oxide synthase while preventing expression of inducible nitric oxide synthase and are the first report of a differential long-term regulation of the expression of neuronal and astroglial constitutive nitric oxide synthase activities.