María Antonia Baltrons

Extracellular Tau Oligomers Produce An Immediate Impairment of LTP and Memory

Non-fibrillar soluble oligomeric forms of amyloid-β peptide (oAβ) and tau proteins are likely to play a major role in Alzheimer’s disease (AD). The prevailing hypothesis on the disease etiopathogenesis is that oAβ initiates tau pathology that slowly spreads throughout the medial temporal cortex and neocortices independently of Aβ, 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 oAβ 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 oAβ 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 Aβ on memory loss, offering new therapeutic opportunities in the therapy of AD and other neurodegenerative diseases associated with Aβ and tau pathology.

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.

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 astrocyte‐enriched 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 100,000g, 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.

Sildenafil (Viagra) ameliorates clinical symptoms and neuropathology in a mouse model of multiple sclerosis

Cyclic GMP (cGMP)-mediated pathways regulate inflammatory responses in immune and CNS cells. Recently, cGMP phosphodiesterase inhibitors such as sildenafil, commonly used to treat sexual dysfunction in humans including multiple sclerosis (MS) patients, have been reported to be neuroprotective in animal models of stroke, Alzheimer’s disease, and focal brain lesion. In this work, we have examined if sildenafil ameliorates myelin oligodendrocyte glycoprotein peptide (MOG35–55)-induced experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. We show for the first time that treatment with sildenafil after disease onset markedly reduces the clinical signs of EAE by preventing axonal loss and promoting remyelination. Furthermore, sildenafil decreases CD3+ leukocyte infiltration and microglial/macrophage activation in the spinal cord, while increasing forkhead box transcription factor 3-expressing T regulatory cells (Foxp3 Tregs). However, sildenafil treatment did not significantly affect MOG35–55-stimulated proliferation or release of Th1/Th2 cytokines in splenocytes but decreased ICAM-1 in spinal cord infiltrated cells. The presence of reactive astrocytes forming scar-like structures around infiltrates was enhanced by sildenafil suggesting a possible mechanism for restriction of leukocyte spread into healthy parenchyma. These results highlight novel actions of sildenafil that may contribute to its beneficial effects in EAE and suggest that treatment with this widely used and well-tolerated drug may be a useful therapeutic intervention to ameliorate MS neuropathology.

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 μM) and the Ca2+ ionophore A23187 (10 μM) 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 μM). 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. J. Neurosci. Res. 49:333–341, 1997.

β-Amyloid Peptides Decrease Soluble Guanylyl Cyclase Expression in Astroglial Cells

In astroglial cells beta-amyloid peptides (betaA) induce a reactive phenotype and increase expression of NO synthase. Here we show that treatment of rat brain astrocytes with betaA decreases their capacity to accumulate cyclic GMP (cGMP) in response to NO as a result of a decreased expression of soluble guanylyl cyclase (sGC) at the protein and mRNA levels. Potentiation of betaA-induced NO formation by interferon-gamma did not result in a larger decrease in cGMP formation and inhibition of NO synthase failed to reverse down-regulation of sGC, indicating that NO is not involved. The betaA effect was prevented by the protein synthesis inhibitor cycloheximide. Intracerebral betaA injection also decreased sGC beta1 subunit mRNA levels in adult rat hippocampus and cerebellum. A loss of sGC in reactive astrocytes surrounding beta-amyloid plaques could be a mechanism to prevent excess signalling via cGMP at sites of high NO production.

Altered Distribution of RhoA in Alzheimer's Disease and AβPP Overexpressing Mice

RhoGTPases control cytoskeleton dynamics thereby modulating synaptic plasticity. Because Alzheimers disease (AD) is characterized by synaptic dysfunction, we sought to determine whether the expression, activity, or localization of the GTPases RhoA, Rac1 and Cdc42, as well as p21-PAK, a downstream target of Rac1/Cdc42, were altered in 18-month-old AbetaPP Tg2576 mice (Swedish mutation) or in brains from patients with AD and, for comparison in the case of RhoA, Picks disease (PiD), a neurodegenerative disorder characterized by hyper-phosphorylated tau accumulation. Immunohistochemical analyses revealed a distinct localization of each RhoGTPase in synapses, dendrite shafts, neuronal bodies, or astrocytes. The association of RhoA with synapses and dendritic microtubules was confirmed by electron microscopy. In AbetaPP mice, RhoA expression decreased in synapses and increased in dystrophic neurites, suggesting altered subcellular targeting of RhoA. In AD, RhoA immunostaining decreased in the neuropil and markedly increased in neurons, co-localizing with hyperphosphorylated tau inclusions, as though RhoA were sequestered by neurofibrillary tangles. Additionally, total RhoA protein was lower in the AD brain hippocampus, reflecting loss of the membrane bound, presumably active, GTPase. RhoA colocalized with hyperphosphorylated tau in PiD, again suggesting that altered subcellular targeting of RhoA is related to neurodegeneration. No major immunohistochemical changes were observed for Rac1, Cdc42, or p21-PAK, thus identifying RhoA among RhoGTPases as a possible therapeutic target in AD.

Reduced expression of NO-sensitive guanylyl cyclase in reactive astrocytes of Alzheimer disease, Creutzfeldt–Jakob disease, and multiple sclerosis brains

In Alzheimers disease (AD) brains increased NO synthase (NOS) expression is found in reactive astrocytes surrounding amyloid plaques. We have recently shown that treatment with beta-amyloid peptides or IL-1beta down-regulates NO-sensitive soluble guanylyl cyclase (sGC) in cultured astrocytes and in adult rat brain. In this work, we have examined sGC activity and expression in postmortem brain tissue of AD patients and matched controls. No significant alteration was observed in basal or NO-stimulated sGC activity, nor in sGC beta1 and alpha1 subunit levels in cortical extracts of AD brains. Immunohistochemistry showed intense and widespread labeling of sGC beta1 in cortical and hippocampal neurons and white matter fibrillar astrocytes, while grey matter astrocytes were faintly stained. In AD, expression of sGC in neurons and fibrillar astrocytes is not altered but is markedly reduced in reactive astrocytes surrounding amyloid plaques. Immunostaining for sGC beta1 was also lacking in reactive astrocytes in cortex and subcortical white matter in Creutzfeldt-Jakob disease brains and in subacute and chronic plaques in multiple sclerosis (MS) brains. Thus, induction of astrocyte reactivity is associated with decreased capacity to generate cGMP in response to NO both in vitro and in vivo. This effect may be related to the development of the astroglial inflammatory response.

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 μM). In the presence of cyclothiazide, a blocker of α‐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 NG‐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.

Interleukin-1β and lipopolysaccharide decrease soluble guanylyl cyclase in brain cells: NO-independent destabilization of protein and NO-dependent decrease of mRNA

We previously showed that soluble guanylyl cyclase (sGC) is down-regulated in astroglial cells after exposure to LPS. Here, we show that this effect is not mediated by released IL-1beta but that this cytokine is also able to decrease NO-dependent cGMP accumulation in a time- and concentration-dependent manner. The effect of IL-1beta is receptor-mediated, mimicked by tumor necrosis factor-alpha and involves a decrease in sGC activity and protein. IL-1beta and LPS decrease the half-life of the sGC beta1 subunit by a NO-independent but transcription- and translation-dependent mechanism. Additionally, both agents induce a NO-dependent decrease of sGC subunit mRNA. Decreased sGC subunit protein and mRNA levels are also observed in adult rat brain after focal injection of IL-1beta or LPS.