Rosa Resende

Brain oxidative stress in a triple-transgenic mouse model of Alzheimer disease

Alzheimer disease (AD) is a neurodegenerative disease which is characterized by the presence of extracellular senile plaques mainly composed of amyloid-beta peptide (Abeta), intracellular neurofibrillary tangles, and selective synaptic and neuronal loss. AD brains revealed elevated levels of oxidative stress markers which have been implicated in Abeta-induced toxicity. In the present work we addressed the hypothesis that oxidative stress occurs early in the development of AD and evaluated the extension of the oxidative stress and the levels of antioxidants in an in vivo model of AD, the triple-transgenic mouse, which develops plaques, tangles, and cognitive impairments and thus mimics AD progression in humans. We have shown that in this model, levels of antioxidants, namely, reduced glutathione and vitamin E, are decreased and the extent of lipid peroxidation is increased. We have also observed increased activity of the antioxidant enzymes glutathione peroxidase and superoxide dismutase. These alterations are evident during the Abeta oligomerization period, before the appearance of Abeta plaques and neurofibrillary tangles, supporting the view that oxidative stress occurs early in the development of the disease.

An endoplasmic-reticulum-specific apoptotic pathway is involved in prion and amyloid-beta peptides neurotoxicity

Prion (PrP) and amyloid-beta (Abeta) peptides are involved in the neuronal loss that occurs in Prion disorders (PrD) and Alzheimers disease (AD), respectively, partially due to Ca(2+) dysregulation. Besides, the endoplasmic reticulum (ER) stress has an active role in the neurotoxic mechanisms that lead to these pathologies. Here, we analyzed whether the ER-mediated apoptotic pathway is involved in the toxic effect of synthetic PrP and Abeta peptides. In PrP106-126- and Abeta1-40-treated cortical neurons, the release of Ca(2+) through ER ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP(3)R) receptors induces ER stress and leads to increased cytosolic Ca(2+) and reactive oxygen species (ROS) levels and subsequently to apoptotic death involving mitochondrial cytochrome c release and caspases activation. These results demonstrate that the early PrP- and Abeta-induced perturbation of ER Ca(2+) homeostasis is a death message that leads to neuronal loss, suggesting that the regulation of ER Ca(2+) levels may be a potential therapeutical target for PrD and AD.

Neurotoxic effect of oligomeric and fibrillar species of amyloid-beta peptide 1-42: involvement of endoplasmic reticulum calcium release in oligomer-induced cell death.

The nature of the toxic form of amyloid-beta peptide (Abeta) involved in early Alzheimers disease (AD) pathology and whether it is the fibrillar or the oligomeric peptide that is the most deleterious to neurons remain controversial. This work aimed to compare the neurotoxicity of different amyloid-beta peptide 1-42 (Abeta1-42) assemblies, using fresh and aged samples enriched in oligomeric and fibrillar species, respectively, and also isolated oligomers and fibrils. The results obtained with fresh and aged Abeta1-42 preparations suggested that oligomeric species are more toxic to cortical neurons in culture than fibrillar forms, which was confirmed by using isolated oligomers and fibrils. In order to further elucidate the mechanisms involved in soluble Abeta toxicity, the involvement of endoplasmic reticulum (ER) calcium (Ca(2+)) release in oligomer-induced apoptosis was evaluated. We observed that oligomeric Abeta1-42 depletes ER Ca(2+) levels leading to intracellular Ca(2+) dyshomeostasis involving phospholipase C activation. Moreover, in the presence of dantrolene, an inhibitor of ER Ca(2+) release through ryanodine receptors, the oligomer-induced apoptosis was prevented demonstrating the involvement of ER Ca(2+) release.

ER stress is involved in Aβ-induced GSK-3β activation and tau phosphorylation

Intracellular neurofibrillary tangles, one of the characteristic hallmarks of Alzheimers disease (AD), are mainly composed of hyperphosphorylated tau. The abnormal tau phosphorylation seems to be related to altered activity of kinases such as glycogen synthase kinase‐3β (GSK‐3β). Tau pathology is thought to be a later event during the progression of the disease, and it seems to occur as a consequence of amyloid‐beta (Aβ) peptide accumulation. The aim of this work was to investigate whether soluble Aβ1–42, particularly oligomers that correspond to the neurotoxic species involved early in the development of AD, triggers tau phosphorylation by a mechanism involving activation of tau‐kinase GSK‐3β. Several studies suggest that GSK‐3β plays a central role in signaling the downstream effects of endoplasmic reticulum (ER) stress. Therefore, the involvement of ER Ca2+ release in GSK‐3β activation and tau phosphorylation induced by Aβ1–42 oligomers was evaluated using dantrolene, an inhibitor of Ca2+ release through channels associated with ER ryanodine receptors. We observed that Aβ1–42 oligomers increase tau phosphorylation and compromises cell survival through a mechanism mediated by GSK‐3β activation. We also demonstrated that oligomeric Aβ1–42 induces ER stress and that ER Ca2+ release is involved in oligomer‐induced GSK‐3β activation and tau phosphorylation. This work suggests that GSK‐3β can be a promising target for therapeutic intervention in AD.

Cholesterol and statins in Alzheimer's disease: current controversies.

Alzheimers disease (AD) is the principal cause of dementia in older people, and accumulation of amyloid-beta (Abeta) peptide is a crucial event in AD pathogenesis. Despite opposite results found in literature, increased evidence posits that high cholesterol levels enhance the risk to develop AD. In fact, cholesterol metabolism and catabolism are affected in this neurodegenerative disorder. Since amyloid precursor protein (APP) processing and subsequent Abeta production are dependent on membrane cholesterol content and on levels of isoprenoid intermediates in the cholesterol biosynthesis pathway, changes in cholesterol might have different consequences on Abeta formation. These pieces of evidence support that inhibitors of cholesterol synthesis, like statins, could have a therapeutic role in AD. Many studies about the effect of statins use in AD show conflicting results; however, some authors explain it by the differences in administrated doses. Recent studies demonstrate that statins can efficiently decrease Abeta formation from APP and be neuroprotective against the peptide toxicity. Because of the high number of pleiotropic effects of statins, novel molecular mechanisms that account for the beneficial effect of these drugs on AD might be discovered in a near future.

Endoplasmic reticulum stress occurs downstream of GluN2B subunit of N-methyl-D-aspartate receptor in mature hippocampal cultures treated with amyloid-β oligomers

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting both the hippocampus and the cerebral cortex. Reduced synaptic density that occurs early in the disease process seems to be partially due to the overactivation of N‐methyl‐d‐aspartate receptors (NMDARs) leading to excitotoxicity. Recently, we demonstrated that amyloid‐beta oligomers (AβO), the species implicated in synaptic loss during the initial disease stages, induce endoplasmic reticulum (ER) stress in cultured neurons. Here, we investigated whether AβO trigger ER stress by an NMDAR‐dependent mechanism leading to neuronal dysfunction and analyzed the contribution of GluN2A and GluN2B subunits of this glutamate receptor. Our data revealed that AβO induce ER stress in mature hippocampal cultures, activating ER stress‐associated sensors and increasing the levels of the ER chaperone GRP78. We also showed that AβO induce NADPH oxidase (NOX)‐mediated superoxide production downstream of GluN2B and impairs ER and cytosolic Ca2+ homeostasis. These events precede changes in cell viability and activation of the ER stress‐mediated apoptotic pathway, which was associated with translocation of the transcription factor GADD153 / CHOP to the nucleus and occurred by a caspase‐12‐independent mechanism. Significantly, ER stress took place after AβO interaction with GluN2B subunits. In addition, AβO‐induced ER stress and hippocampal dysfunction were prevented by ifenprodil, an antagonist of GluN2B subunits, while the GluN2A antagonist NVP‐AAM077 only slightly attenuated AβO‐induced neurotoxicity. Taken together, our results highlight the role of GluN2B subunit of NMDARs on ER stress‐mediated hippocampal dysfunction caused by AβO suggesting that it might be a potential therapeutic target during the early stages of AD.

Protective effect of leptin and ghrelin against toxicity induced by amyloid-β oligomers in a hypothalamic cell line.

In addition to cognitive decline, Alzheimers disease (AD) patients also exhibit an unexplained weight loss that correlates with disease progression. In young and middle‐aged AD patients, large amounts of amyloid‐β (Aβ) deposits were observed in the hypothalamus, a brain region involved in the control of feeding and body weight through the action of peripheral metabolic peptides, which have recently been shown to have neuroprotective effects. Moreover, levels of peripheral metabolic peptides, such as leptin and ghrelin, are changed in AD patients. The present study aimed to investigate the role of Aβ peptide in the survival of hypothalamic cells and to explore the receptor‐mediated protective effect of leptin and ghrelin against Aβ‐induced toxicity in these cells. Using the mHypoE‐N42 cell line, we demonstrated for the first time that oligomeric Aβ is toxic to hypothalamic cells, leading to cell death. It was also demonstrated that leptin and ghrelin protect these cells against AβO‐induced cell death through the activation of the leptin and ghrelin receptors, respectively. Furthermore, ghrelin and leptin prevented superoxide production, calcium rise and mitochondrial dysfunction triggered by AβO. Taken together, these results suggest that peripheral metabolic peptides, in particular leptin and ghrelin, might be considered as preventive strategies for ameliorating hypothalamic alterations in AD.

Leptin and ghrelin prevent hippocampal dysfunction induced by Aβ oligomers.

It was recently established that the stomach-derived ghrelin and the adipokine leptin promote learning and memory through actions within the hippocampus. Changes in the peripheral or brain levels of these peptides were described in Alzheimers disease (AD) patients and were shown to correlate with the severity of cognitive decline. Furthermore, in vivo and in vitro studies demonstrated that leptin or ghrelin can ameliorate amyloid and tau pathologies as well as cognitive deficits. However, the exact role of these peptides in AD is far from being elucidated. To fill this gap, our working hypothesis was that leptin and ghrelin can exert a neuroprotective role in AD suppressing hippocampal dysfunction triggered by synapto- and neurotoxic amyloid-β oligomers (AβO). Using primary cultured hippocampal neurons, we demonstrated that both peptides reduce AβO-induced production of superoxide and mitochondrial membrane depolarization, improving cell survival, and inhibit cell death through a receptor-dependent mechanism. Furthermore, it was shown that in AβO-treated neurons both leptin and ghrelin prevent glycogen synthase kinase 3β activation. Therefore, the evidence gathered in this study revealed that leptin and ghrelin can act as neuroprotective agents able to rescue hippocampal neurons from AβO toxicity, thus highlighting their potential therapeutic role in AD.

Neuroprotective effects of statins in an in vitro model of Alzheimer's disease.

Statins, used as cholesterol-lowering drugs, were reported to reduce the progression of Alzheimers disease (AD). However, the molecular mechanisms underlying these findings remain to be clarified and it is not well understood whether this beneficial effect is due to simply lowering cholesterol levels. This study was aimed to investigate the neuroprotective effect of simvastatin and lovastatin, lipophilic statins that can transverse the blood brain barrier, against the toxicity triggered by the AD-associated amyloid-beta (Abeta) peptides and to analyze if such protection is cholesterol-independent. Using primary cultures of cortical neurons treated with Abeta1-40 peptide, we have demonstrated that pre-incubation with statins prevents the rise in cytosolic Ca2+ concentration and the accumulation of reactive oxygen species induced by Abeta through mechanisms independent of cholesterol reduction. The neuroprotective actions of statins were rather attributable to their ability to reduce isoprenyl intermediates levels in the cholesterol biosynthetic pathway since their effect was reversed by geranyl pyrophosphate while cholesterol addition was ineffective. Consequently, statins were shown to rescue cortical neurons from Abeta-40-induced caspase-3-dependent apoptosis. Moreover, our results revealed that simvastatin, at neuroprotective concentrations against Abeta-induced toxicity, is not able to activate Akt or ERK2, two signaling kinases with neuroprotective roles against apoptosis.

Dose-Dependent Inhibition of BACE-1 by the Monoterpenoid 2,3,4,4-Tetramethyl-5-methylenecyclopent-2-enone in Cellular and Mouse Models of Alzheimer’s Disease

BACE-1 is an aspartic protease involved in the conversion of amyloid precursor protein (APP) to amyloid-β (Aβ) in vivo, which is one of the key steps in the development and progression of Alzheimers disease. In a previous screening procedure for inhibitors of BACE-1 activity, the oil of Lavandula luisieri was identified as the most potent among several essential oils. The inhibitory effect of this essential oil on Aβ production was also demonstrated in a cellular assay. The composition of the volatile oil and the isolation of the compound responsible for the inhibitory activity were also reported. The present work focused on the characterization of the inhibition of BACE-1 by this active compound, a monoterpene necrodane ketone, 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1), with assessment of its Ki value and the type of inhibition. The dose-related effects of the compound were also evaluated using two different cell lines, with determinations of the respective EC50 values. The entire oil and the 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1) were tested on a triple transgenic mouse model of Alzheimers disease. The overall results showed that compound 1 displayed a dose-dependent inhibition of BACE-1 in cellular and mouse models of Alzheimers disease and is therefore capable of passing through cellular membranes and the blood-brain barrier.