Stéphane Bastianetto

Alzheimer’s disease and the basal forebrain cholinergic system: relations to β-amyloid peptides, cognition, and treatment strategies

Abstract Alzheimer’s disease (AD) is the most common form of degenerative dementia and is characterized by progressive impairment in cognitive function during mid- to late-adult life. Brains from AD patients show several distinct neuropathological features, including extracellular β-amyloid-containing plaques, intracellular neurofibrillary tangles composed of abnormally phosphorylated τ, and degeneration of cholinergic neurons of the basal forebrain. In this review, we will present evidence implicating involvement of the basal forebrain cholinergic system in AD pathogenesis and its accompanying cognitive deficits. We will initially discuss recent results indicating a link between cholinergic mechanisms and the pathogenic events that characterize AD, notably amyloid-β peptides. Following this, animal models of dementia will be discussed in light of the relationship between basal forebrain cholinergic hypofunction and cognitive impairments in AD. Finally, past, present, and future treatment strategies aimed at alleviating the cognitive symptomatology of AD by improving basal forebrain cholinergic function will be addressed.

The ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by β-amyloid

Substantial evidence suggests that the accumulation of β‐amyloid (Aβ)‐derived peptides, and to a lesser extent free radicals, may contribute to the aetiology and/or progression of Alzheimers disease (AD). Ginkgo biloba extract (EGb 761) is a well‐defined plant extract containing two major groups of constituents, i.e. flavonoids and terpenoids. It is viewed as a polyvalent agent with a possible therapeutic use in the treatment of neurodegenerative diseases of multifactorial origin, e.g. AD. We have investigated here the potential effectiveness of EGb 761 against toxicity induced by (Aβ)‐derived peptides (Aβ25−35, Aβ1−40 and Aβ1−42) on hippocampal primary cultured cells, this area being severely affected in AD. A co‐treatment with EGb 761 concentration‐dependently (10–100 μg/mL) protected hippocampal neurons against toxicity induced by Aβ fragments, with a maximal and complete protection at the highest concentration tested. Similar, albeit less potent protective effects were seen with the flavonoid fraction of the extract (CP 205), while the terpenes were ineffective. Most interestingly, EGb 761 (100 μg/mL) was even able to protect (up to 8 h) hippocampal cells from a pre‐exposure to Aβ25−35 and Aβ1−40. EGb 761 was also able to both protect and rescue hippocampal cells from toxicity induced by H2O2 (50–150 μm), a major peroxide possibly involved in mediating Aβ toxicity. Moreover, EGb 761 (10–100 μg/mL), and to a lesser extent CP 205 (10–50 μg/mL), completely blocked Aβ‐induced events, e.g. reactive oxygen species accumulation and apoptosis. These results suggest that the neuroprotective effects of EGb 761 are partly associated with its antioxidant properties and highlight its possible effectiveness in neurodegenerative diseases, e.g. AD via the inhibition of Aβ‐induced toxicity and cell death.

Neuroprotective abilities of resveratrol and other red wine constituents against nitric oxide-related toxicity in cultured hippocampal neurons

Animal and epidemiological studies suggest that polyphenol constituents of red wine possess antioxidant activities that favour protection against cardiovascular disease – the so‐called. ‘French paradox’ – and possibly, central nervous system disorders such as Alzheimers disease (AD) and ischaemia. In the present study, the potential of three major red wine derived‐polyphenols to protect against toxicity induced by the nitric oxide free radical donors sodium nitroprusside (SNP) and 3‐morpholinosydnonimine (SIN‐1) was examined in cultured rat hippocampal cells. Both co‐ and post‐treatments with either the stilbene resveratrol (5–25 μM) or the flavonoids quercetin (5–25 μM) and (+)‐catechin (1–10 μM) were capable of attenuating hippocampal cell death and intracellular reactive oxygen species accumulation produced by SNP (100 μM and 1 mM, respectively). However, among the phenolic compounds tested, only the flavonoids afforded significant protection against 5 mM SIN‐1‐induced toxicity. The effects of phenolic constituents were shared by Trolox (100 μM), a vitamin E analogue, but not by selective inhibitors of cyclo‐oxygenases (COX) and lipoxygenases (LOX). Among the phenolic compounds tested, only quercetin (10 μM) inhibited 100 μM SNP‐stimulated protein kinase C (PKC) activation, whereas none of them were able to attenuate nitrite accumulation caused by SNP (100 μM). Taken together, these data suggest that the neuroprotective abilities of quercetin, resveratrol, and (+)‐catechin result from their antioxidant properties rather than their purported inhibitory effects on intracellular enzymes such as COX, LOX, or nitric oxide synthase. Quercetin, however, may also act via PKC to produce its protective effects.

Neuroprotective effects of resveratrol against β-amyloid-induced neurotoxicity in rat hippocampal neurons: involvement of protein kinase C

Resveratrol, an active ingredient of red wine extracts, has been shown to exhibit neuroprotective effects in several experimental models. The present study evaluated the neuroprotective effects of resveratrol against amyloid β(Aβ)‐induced toxicity in cultured rat hippocampal cells and examined the role of the protein kinase C (PKC) pathway in this effect. Pre‐, co‐ and post‐treatment with resveratrol significantly attenuated Aβ‐induced cell death in a concentration‐dependent manner, with a concentration of 25 μM being maximally effective. Pretreatment (1 h) of hippocampal cells with phorbol‐12‐myristate‐13‐acetate, a PKC activator, at increasing concentrations (1–100 ng ml−1), resulted in a dose‐dependent reduction in Aβ‐induced toxicity, whereas the inactive 4α‐phorbol had no effect. Pretreatment (30 min) of hippocampal cells with GF 109203X (1 μM), a general PKC inhibitor, significantly attenuated the neuroprotective effect of resveratrol against Aβ‐induced cell death. Treatment of hippocampal cells with resveratrol (20 μM) also induced the phosphorylation of various isoforms of PKC leading to activation. Taken together, the present results indicate that PKC is involved in the neuroprotective action of resveratrol against Aβ‐induced toxicity.

Dehydroepiandrosterone (DHEA) protects hippocampal cells from oxidative stress-induced damage.

It has been postulated that decreases in plasma levels of dehydroepiandrosterone (DHEA) may contribute to the development of some age-related disorders. Along with neuroprotective and memory enhancing effects, DHEA has been shown to display antioxidant properties. Moreover, oxidative stress is known to cause lipid peroxidation and degenerative changes in the hippocampus, an area involved in memory processes and especially afflicted in Alzheimers disease (AD). Accordingly, we investigated the antioxidant effects of DHEA in models of oxidative stress using rat primary hippocampal cells and human hippocampal tissue from AD patients and age-matched controls. A pre-treatment of rat primary mixed hippocampal cell cultures with DHEA (10-100 microM) protected against the toxicity induced by H2O2 and sodium nitroprusside. Moreover, DHEA (10-100 microM) was also able to prevent H2O2/FeSO4-stimulated lipid oxidation in both control and AD hippocampal tissues. Taken together, these data suggest that DHEA may be useful in treating age-related central nervous system diseases based on its protective effects in the hippocampus.

Oxidative damage and protection by antioxidants in the frontal cortex of Alzheimer’s disease is related to the apolipoprotein E genotype

A great number of epidemiological studies have demonstrated that the frequency of the epsilon4 allele of the apolipoprotein E gene (APOE) is markedly higher in sporadic and in familial late onset Alzheimer disease (AD). In the frontal cortex of AD patients, oxidative damage is elevated. We address the hypothesis that the APOE genotype and reactive oxygen-mediated damage are linked in the frontal cortex of AD patients. We have related the APOE genotype to the levels of lipid oxidation (LPO) and to the antioxidant status, in frontal cortex tissues from age-matched control and AD cases with different APOE genotypes. LPO levels were significantly elevated in tissues from Alzheimers cases which are homozygous for the epsilon4 allele of APOE, compared to AD epsilon3/epsilon3 cases and controls. Activities of enzymatic antioxidants, such as catalase and glutathione peroxidase (GSH-PX), were also higher in AD cases with at least one epsilon4 allele of APOE, while superoxide dismutase (SOD) activity was unchanged. In the frontal cortex, the concentration of apoE protein was not different between controls and AD cases, and was genotype independent. The Ginkgo biloba extract (EGb 761), the neurosteroid dehydroepiandrosterone (DHEA) and human recombinant apoE3 (hapoE3rec) were able to protect control, AD epsilon3/epsilon3 and epsilon3/epsilon4 cases against hydrogen peroxide/iron-induced LPO, while hapoE4rec was completely ineffective. Moreover, EGb 761 and DHEA had no effect in homozygous epsilon4 cases. These results demonstrate that oxidative stress-induced injury and protection by antioxidants in the frontal cortex of AD cases are related to the APOE genotype.

Neuroprotective effects of green and black teas and their catechin gallate esters against β‐amyloid‐induced toxicity

Teas represent a large family of plants containing high amounts of polyphenols that may confer health benefits in various diseases. Recently, it has been hypothesized that tea consumption may also reduce the risk of age‐related neurodegenerative pathologies. Considering the deleterious role of β‐amyloid (Aβ) in the aetiology of Alzheimers disease (AD), we investigated green and black tea extracts and flavan‐3‐ols (present as monomers and dimers in green and black forms, respectively) against toxicity induced by Aβ‐derived peptides using primary cultures of rat hippocampal cells as model. Both green and black tea extracts (5–25 µg/mL) displayed neuroprotective action against Aβ toxicity. These effects were shared by gallic acid (1–20 µm), epicatechin gallate (ECG; 1–20 µm) and epigallocatechin gallate (EGCG; 1–10 µm), the former being the most potent flavan‐3‐ol. In contrast, epicatechin and epigallocatechin were ineffective in the same range of concentrations. Moreover, only tea flavan‐3‐ol gallate esters (i.e. ECG, EGCG) and gallic acid inhibited apoptotic events induced by Aβ25‐35. Interestingly, EGCG and gallic acid inhibited Aβ aggregation and/or the formation of Aβ‐derived diffusible neurotoxin ligands. Taken together, these results indicate that the catechin gallates (through the galloyl moiety) contribute to the neuroprotective effects of both green and black teas. Moreover, the protective effect of EGCG is likely to be associated, at least in part, with its inhibitory action on Aβ fibrils/oligomers formation. These data also support the hypothesis that not only green but also black teas may reduce age‐related neurodegenerative diseases, such as AD.

Amyloid β peptide induces tau phosphorylation and loss of cholinergic neurons in rat primary septal cultures

The neuropathological features associated with Alzheimers disease (AD) brain include the presence of extracellular neuritic plaques composed of amyloid beta protein (Abeta), intracellular neurofibrillary tangles containing phosphorylated tau protein and the loss of basal forebrain cholinergic neurons which innervate regions such as the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Abeta accumulation in vivo may initiate phosphorylation of tau protein, which by disrupting neuronal network may trigger the process of neurodegeneration observed in AD brains. However, the underlying cause of degeneration of the basal forebrain cholinergic neurons and their association, if any, to Abeta peptides or phosphorylated tau remains mostly unknown. In the present study, using rat primary septal cultures, we have shown that aggregated Abeta peptides, in a time (18-96 h)- and concentration (0.7-60 microM)-dependent manner, induce toxicity and decrease choline acetyltransferase enzyme activity in cultured neurons. Using immunocytochemistry and immunoblotting, we have also demonstrated that Abeta treatment can significantly increase the phosphorylation of tau protein in septal cultures. At the cellular level, hyperphosphorylated tau is mostly apparent in the somatodendritic compartment of the neurons. Abeta peptide (10 microM), in addition to tau phosphorylation, also activates mitogen-activated protein kinase and glycogen synthase kinase-3beta, the two kinases which are known to be involved in the formation of hyperphosphorylated tau in the AD brain. Exposure to specific inhibitors of the mitogen-activated protein kinase (i.e. PD98059) or glycogen synthase kinase-3beta (i.e. LiCl) attenuated the hyperphosphorylation of the tau protein in cultured neurons. Given the evidence that tau phosphorylation can induce cell loss by disrupting neuronal cytoskeleton, it is likely that aggregated Abeta peptide triggers degeneration of septal neurons, including those expressing the cholinergic phenotype, by phosphorylation of the tau protein activated by mitogen-activated protein kinase and glycogen synthase kinase-3beta. These results, taken together, suggest that cultured septal cholinergic neurons are vulnerable to Abeta-mediated toxicity and tau phosphorylation may play an important role in Abeta-induced neurodegeneration.

The Ginkgo biloba extract (EGb 761) protects and rescues hippocampal cells against nitric oxide-induced toxicity: involvement of its flavonoid constituents and protein kinase C.

Abstract: An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 μM) or 3‐morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP‐induced events were blocked by either EGb 761 (10‐100 μg/ml) or its flavonoid fraction CP 205 (25 μg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L‐type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3‐[(4‐octadecyl)benzoyl]acrylic acid (OBAA)] and C (U‐73122), failed to display any significant effects. Moreover, EGb 761 (50 μg/ml), CP 205 (25 μg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 μg/ml) was shown to block the activation of PKC induced by SNP (100 μM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO‐stimulated PKC activity.

Natural extracts as possible protective agents of brain aging

A growing number of studies suggest that natural extracts and phytochemicals have a positive impact on brain aging. We examined the potential of the Ginkgo biloba extract EGb 761 and red wine-derived constituents on cell death produced by beta-amyloid (Abeta) peptides and oxidative stress, with respect to their possible deleterious role in age-related neurological disorders. We found that EGb 761, possibly through the antioxidant properties of its flavonoids, was able to protect hippocampal cells against toxic effects induced by Abeta peptides. Moreover, we showed that an exposure of rat hippocampal cells to the nitric oxide (NO) donor sodium nitroprusside (SNP) resulted in a decrease in cell survival and increase in reactive oxygen species (ROS) accumulation. However, EGb 761 and red wine-derived polyphenols protected against these events, due to their antioxidant activities, and their ability to block SNP-stimulated activity of protein kinase C (PKC). Taken together, these results support the hypothesis that dietary intake of natural substances may be beneficial in normal aging of the brain.