Alicia Gonzalo-Ruiz

Oligomers of beta-amyloid protein (Aβ1-42) induce the activation of cyclooxygenase-2 in astrocytes via an interaction with interleukin-1beta, tumour necrosis factor-alpha, and a nuclear factor kappa-B mechanism in the rat brain

Despite growing evidence indicating the effects of cytokines, including interleukin-1beta (IL-1β) and tumour necrosis factor-α (TNFα), and the enzyme cyclooxygenase-2 (COX-2) in Alzheimers diseases, little is known about the signalling mechanisms that mediate its activation in response to beta-amyloid protein (Aβ). The aim of this study was first to investigate whether Aβ1-42 peptide induced the up-regulation of COX-2. We then examined the expression of COX-2 and cytokines, such as IL-1β and TNFα, in reactive astrocytes. Finally, we analyzed the role of nuclear factor kappa-B (NF-κB) as a signalling pathway in early stages of Aβ-toxicity. In Wistar rats anaesthetised with equitesine, a single microinjection of Aβ1-42 oligomers was made in the left retrosplenial cortex. Control animals were injected with Aβ42-1 peptide into the corresponding region of the cerebral cortex. By COX-2 immunoblotting, we detected two immunopositive protein bands, at 70 and 50 kDa molecular mass. In the Aβ1-42-injected animals the 50 kDa fragment showed a significant increase at 3 and 14 days, as compared with that seen in control animals. The 70 kDa fragment showed a maximal increase at 14 days. In the Aβ1-42-injected animals immunoblot staining of NF-κB detected an active protein band at 50 kDa molecular mass, showing a maximal increase at the 72 h time point. Confocal analysis revealed that COX-2 protein co-localized with Aβ-IR material at the injection site and in endothelial blood vessels, increasing at 72 h. In the Aβ oligomer-treated animals, COX-2, IL-1β, and TNFα proteins were expressed in reactive astrocytes surrounding the injection site and blood vessels at early stages of Aβ toxicity. Double-labelling immunofluorescence studies also revealed that GFAP and COX-2 proteins co-localized with NF-κB-positive material at early time-points. In conclusion, our results suggest that in reactive astrocytes and in COX-2 positive cells NF-κB may mediate pro-, and/or inflammatory gene expression and that, develop strategies that target the GFAP/NF-κB and COX-2/NF-κB pathways might contribute to reducing Aβ-induced toxicity.

In vitro anti-inflammatory activity of phenolic rich extracts from white and red common beans.

According to epidemiological evidence, diets rich in fruits and vegetables can reduce the incidence of several chronic diseases that share an inflammatory component. These protective effects are attributed, in part, to the occurrence of different antioxidant components, mainly phenolic compounds. Our aim was to characterise phenolic composition, and to determine antioxidant and anti-inflammatory activities of phenolic rich extracts obtained from two kinds of common beans, white kidney beans (WKB) and round purple beans (RPB). Phenolic acids were the predominant component in WKB extracts, whereas RPB extracts presented higher concentrations of phenolic compounds, mainly catechin derivatives, proanthocyanidins and catechin glucoside. In addition, RPB extracts showed higher antioxidant capacity and higher anti-inflammatory activity by the reduction of NO production and cytokine mRNA expression of LPS stimulated macrophages. These results suggest that common bean extracts may be used as a source of anti-inflammatory agents as well as a dietary complement for health promotion.

Effects of β-amyloid protein on serotoninergic, noradrenergic, and cholinergic markers in neurons of the pontomesencephalic tegmentum in the rat

The effects on serotoninergic, noradrenergic and cholinergic markers on neurons of the pontomesencephalic tegmentum nuclei were studied in rats following local administration of fibrillar beta-amyloid peptide (Abeta1-40) into the left retrosplenial cortex. Focal deposition of Abeta in the retrosplenial cortex resulted in a loss of serotoninergic neurons in the dorsal and median raphe nuclei. The dorsal raphe nucleus showed a statistically significant reduction of 31.7% in the number of serotoninergic neurons and a decrease (up to 17.38%) in neuronal density in comparison with the same parameters in uninjected controls. A statistically significant reduction of 50.3%, together with a significant decrease of 53.94% in the density of serotoninergic neurons, was also observed in the median raphe nucleus as compared with control animals. Furthermore, a significant reduction of 35.07% in the number of noradrenergic neurons as well as a statistically significant decrease of 56.55% in the density of dopamine-beta-hydroxylase-immunoreactive neurons were also found in the locus coeruleus as compared with the corresponding hemisphere in uninjected controls. By contrast, a reduction of 24.37% in the number of choline acetyltransferase-positive neurons and a slight decrease (up to 22.28%) in the density of cholinergic neurons, which were not statistically significant, was observed in the laterodorsal tegmental nucleus in comparison with the same parameters in control animals. These results show that three different neurochemically defined populations of neurons in the pontomesencephalic tegmentum are affected by the neurotoxicity of Abeta in vivo and that Abeta might indirectly affect serotoninergic, noradrenergic and cholinergic innervation in the retrosplenial cortex.

Soluble oligomeric forms of beta-amyloid (Aβ) peptide stimulate Aβ production via astrogliosis in the rat brain

The aim of this study was to investigate the interaction between beta-amyloid (Abeta) peptide and astrogliosis in early stages of Abeta toxicity. In Wistar rats, anaesthetised with equitesine, a single microinjection of Abeta1-42 oligomers was placed into the retrosplenial cortex. Control animals were injected with Abeta42-1 peptide into the corresponding regions of cerebral cortex. Immunocytochemical analysis revealed an intense Abeta immunoreactivity (IR) at the level of Abeta1-42 injection site, increasing from the first 24 h to later (72 h) time point. Control injection showed a light staining surrounding the injection site. In Abeta oligomers-treated animals, Abeta-immunopositive product also accumulates in cortical cells, particularly in frontal and temporal cortices at an early (24 h) time point. Abeta-IR structures-like diffuse aggregates forms were also observed in hippocampus and in several cortical areas, increasing from the first 24 h to later (72 h) time point. In control animals no specific staining was seen neither in cortical cells nor in structures-like diffuse aggregates forms. Injections of Abeta oligomers also induce activation of astrocytes surrounding and infiltrating the injection site. Astrocyte activation is evidenced by morphological changes and upregulation of glial fibrillary acidic protein (GFAP). By GFAP immunoblotting we detected two immunopositive protein bands, at 50 and 48 kDa molecular mass. Confocal analysis also showed that GFAP co-localized with Abeta-IR material in a time-dependent manner. In conclusion, our results indicate that astrocyte activation might have a critical role in the mechanisms of Abeta-induced neurodegeneration, and that should be further studied as possible targets for therapeutic intervention in AD.

Neuronal and axonal loss are selectively linked to fibrillar amyloid-β within plaques of the aged primate cerebral cortex

The amyloid-beta peptide (Abeta) deposited in plaques in Alzheimers disease has been shown to cause degeneration of neurons in experimental paradigms in vivo and in vitro. However, it has been difficult to convincingly demonstrate toxicity of native amyloid deposits in the aged and Alzheimer brains. Here we provide evidence that the fibrillar conformation of Abeta (fAbeta) deposited in compact plaques is associated with the pathologies observed in Alzheimer brains. fAbeta containing compact but not diffuse plaques in the aged rhesus cortex contained activated microglia and clusters of phosphorylated tau-positive swollen neurites. Scholls quantitative analysis revealed that the area adjacent to fAbeta, containing compact but not diffuse plaques in aged rhesus, aged human, and Alzheimers disease cortex, displays significant loss of neurons and small but statistically significant reduction in the density of cholinergic axons. These observations suggest that fAbeta toxicity may not be restricted to cultured cells and animal injection models. Rather, fAbeta deposited in native compact plaques in aged and AD brains may exert selective toxic effects on its surrounding neural environment.

Effects of lipids and aging on the neurotoxicity and neuronal loss caused by intracerebral injections of the amyloid-β peptide in the rat

The influence of diet and age on the area of lesion and on the neuronal density in the cerebral cortex was studied in rats following local injections of the amyloid-beta peptide (Abeta1-40) in PBS vehicle into the left frontal and cingulate cortices and compared with effects of injections of PBS alone into the corresponding regions of the right hemisphere The experiments were carried out in two groups of animals: one group of young adult rats and a second group of aged rats. Each group of animals, depending on the diet received, was divided into high-cholesterol, high-fat, and a control group. In order to evaluate the interaction of Abeta/PBS-cholesterol and of Abeta/PBS-fat, animals without dietary manipulation receiving Abeta and PBS injection were used as controls. The results showed that the greatest area of lesion was at Abeta injection sites in the high-cholesterol fed group of aged animals. The results also revealed a significant variance in the neuronal density by group and by injection type. Thus, high-cholesterol fed animals showed a greater reduction in neuronal density at Abeta and PBS-injected sites than that seen in the high-fat or control groups. The results also indicate that the loss of neurons at the Abeta injection site exceeds that seen in the PBS-injected area. The greatest reduction in the neuronal density was found at Abeta-injected site in the high-cholesterol fed group of aged animals. In conclusion, our findings indicate an interaction between lipids, age, and Abeta neurotoxicity, and might provide insights into the basic mechanisms involved in a short-term (acute-to-subchronic) response to Abeta peptide.

Amyloid beta peptide-induced cholinergic fibres loss in the cerebral cortex of the rat is modified by diet high in lipids and by age

The influence of diet and age on the effects of intracerebral injection of beta-amyloid peptide (Abeta1-40) in vehicle phosphate-buffered saline (PBS) and on the effects of vehicle alone on cholinergic fibres of the cerebral cortex was studied in rats. The experiments were carried in two groups of animals: one group of young adult rats and a second group of aged rats. Each group of animals, depending on the diet received, was divided into high-cholesterol, high-fat, and a control diet group. In order to evaluate the interaction of Abeta/PBS-cholesterol and of Abeta/PBS-fat, animals without dietary manipulation receiving Abeta and PBS injection were used as controls. High-cholesterol fed animals showed a statistically significant reduction of 49.62% in the number of cholinergic fibres at the Abeta injection site as compared with that at PBS injection site, while the high-fat and control animals showed a significant reduction of 28.13 and 26.81%, respectively. In all diet groups, the loss of cholinergic fibres caused by Abeta as compared to that caused by PBS injection was significantly greater in aged rats in comparison with that observed in the young animals. Furthermore, the results of a multivariate linear regression model revealed that the greatest reduction in cholinergic fibres was in the high-cholesterol fed animals (35 fibres/mm) as compared with that seen in the high-fat and control animals. A significantly greater reduction was also observed at Abeta injection site (28 fibres/mm) as compared with that caused by PBS injection, and a reduction of 16 cholinergic fibres per mm was found in aged animals as compared to that seen in young adult rats. These results show that high-cholesterol diet enhances the toxicity of Abeta peptide and that this is also age-dependent. Therefore, this study increases the evidences of the role of cholesterol in the pathology of Alzheimers disease (AD).

Effects of β‐amyloid peptide on the density of M2 muscarinic acetylcholine receptor protein in the hippocampus of the rat: relationship with GABA‐, calcium‐binding protein and somatostatin‐containing cells

Aims: The deposition of amyloid peptides (Aβ) in the cortex and hippocampus is the primary trigger of Alzheimers disease (AD). Recent studies also indicated that the M2 subtype of muscarinic acetylcholine receptors (M2mAChR) may be a key molecule involved in cognitive dysfunction. Thus, the purpose of this study was to determine the effects of extracellular deposition of Aβ on the density of M2mAChR in the hippocampus of the rat by M2mAChR‐immunohistochemistry. Methods: Special attention was paid to discerning any interaction between Aβ and M2mAChR in GABA‐, and calcium‐binding protein containing cells by double‐labelling immunohistochemistry. Densitometric analysis of M2mAChR‐immunoreactivity was performed using Scion Image Beta Software. Quantitative analysis of GABA‐, and calcium‐binding protein interneurones containing M2mAChR protein was performed using a NeuroLucida morphometric system. Results: Injections of Aβ into the retrosplenial cortex resulted in a significant reduction in M2mAChR‐immunoreactivity in the CA1 ipsilateral to the Aβ‐injected side as compared with the corresponding hemisphere of non‐treated control animals and with that in the corresponding region of the CA1 in the phosphate‐buffered saline‐injected side. Co‐localization studies showed that the M2mAChR is localized in a subset of GABA‐positive cells of the hippocampus, in cells that contain calcium‐binding proteins, and in a subpopulation of cells that contain the neuropeptide somatostatin. Conclusions: Our findings suggest that Aβ induces a significant reduction in M2mAChR‐immunoreactivity in the CA1 of the hippocampus and a reduction in GABAergic interneurones containing M2mAChR, which may contribute to impairment of GABAergic synaptic transmission in area CA1 of hippocampus.

Effects of β-amyloid protein on M1 and M2 subtypes of muscarinic acetylcholine receptors in the medial septum-diagonal band complex of the rat: relationship with cholinergic, GABAergic, and calcium-binding protein perikarya

Cortical cholinergic dysfunction has been correlated with the expression and processing of β-amyloid precursor protein. However, it remains unclear as to how cholinergic dysfunction and beta-amyloid (Aβ) formation and deposition might be related to one another. Since the M1- and M2 subtypes of muscarinic acetylcholine receptors (mAChRs) are considered key molecules that transduce the cholinergic message, the purpose of the present study was to assess the effects of the injected Aβ peptide on the number of M1mAchR- and M2mAChR-immunoreactive cells in the medial septum-diagonal band (MS-nDBB) complex of the rat. Injections of Aβ protein into the retrosplenial cortex resulted in a decrease in M1mAChR and M2mAChR immunoreactivity in the MS-nDBB complex. Quantitative analysis revealed a significant reduction in the number of M1mAChR- and M2mAChR-immunoreactive cells in the medial septum nucleus (MS) and in the horizontal nucleus of the diagonal band of Broca (HDB) as compared to the corresponding hemisphere in control animals and with that seen in the contralateral hemisphere, which corresponds to the PBS-injected side. Co-localization studies showed that the M1mAChR protein is localized in GABA-immunoreactive cells of the MS-nDBB complex, in particular those of the MS nucleus, while M2mAChR protein is localized in both the cholinergic and GABAergic cells. Moreover, GABAergic cells containing M2mAChR are mainly localized in the MS nucleus, while cholinergic cells containing M2mAChR are localized in the MS and the HDB nuclei. Our findings suggest that Aβ induces a reduction in M1mAChR- and M2mAChR-containing cells, which may contribute to impairments of cholinergic and GABAergic transmission in the MS-nDBB complex.

A new procedure for amyloid β oligomers preparation enables the unambiguous testing of their effects on cytosolic and mitochondrial Ca(2+) entry and cell death in primary neurons.

Oligomers of the amyloid β peptide (Aβo) are becoming the most likely neurotoxin in Alzheimers disease. Controversy remains on the mechanisms involved in neurotoxicity induced by Aβo and the targets involved. We have reported that Aβo promote Ca(2+) entry, mitochondrial Ca(2+) overload and apoptosis in cultured cerebellar neurons. However, recent evidence suggests that some of these effects could be induced by glutamate receptor agonists solved in F12, the media in which Aβo are prepared. Here we have tested the effects of different media on Aβo formation and on cytosolic Ca(2+) concentration ([Ca(2+)]cyt) in rat cerebellar and hippocampal cell cultures. We found that Aβo prepared according to previous protocols but solved in alternative media including saline, MEM and DMEM do not allow oligomer formation and fail to increase [Ca(2+)]cyt. Changes in the oligomerization protocol and supplementation of media with selected salts reported to favor oligomer formation enable Aβo formation. Aβo prepared by the new procedure and containing small molecular weight oligomers increased [Ca(2+)]cyt, promoted mitochondrial Ca(2+) overload and cell death in cerebellar granule cells and hippocampal neurons. These results foster a role for Ca(2+) entry in neurotoxicity induced by Aβo and provide a reliable procedure for investigating the Ca(2+) entry pathway promoted by Aβo.