Elisabete Ferreiro

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.

The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway

In this study, we analyzed whether ER Ca2+ release, induced by amyloid-beta (Abeta) and prion (PrP) peptides activates the mitochondrial-mediated apoptotic pathway. In cortical neurons, addition of the synthetic Abeta1-40 or PrP106-126 peptides depletes ER Ca2+ content, leading to cytosolic Ca2+ overload. The Ca2+ released through ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP3R) receptors was shown to be involved in the loss of mitochondrial membrane potential, Bax translocation to mitochondria and apoptotic death. Our data further demonstrate that Ca2+ released from the ER leads to the depletion of endogenous GSH levels and accumulation of reactive oxygen species, which were also involved in the depolarization of the mitochondrial membrane. These results illustrate that the early Abeta- and PrP -induced perturbation of ER Ca2+ homeostasis affects mitochondrial function, activating the mitochondrial-mediated apoptotic pathway and help to clarify the mechanism implicated in neuronal death that occurs in AD and PrD.

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide

Studies with in‐vitro‐cultured neurons treated with amyloid‐β (Aβ) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca2+ homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)‐specific apoptotic pathway mediated by caspase‐12, which is activated upon the perturbation of ER Ca2+ homeostasis, may contribute to Aβ toxicity. To elucidate the involvement of deregulation of ER Ca2+ homeostasis in neuronal death induced by Aβ peptides, we have performed a comparative study using the synthetic peptides Aβ25–35 or Aβ1–40 and thapsigargin, a selective inhibitor of Ca2+ uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 μM) increased the intracellular Ca2+ levels and activated caspase‐3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the Aβ peptides (Aβ25–35, 25 μM; Aβ1–40, 0.5 μM), we observed a significant increase in [Ca2+]i, in caspase‐3‐like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca2+ release through ryanodine receptors (RyR) or inositol 1,4,5‐trisphosphate receptors (IP3R) in Aβ neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca2+ release through RyR or IP3R, were able to prevent the increase in [Ca2+]i and the activation of caspase‐3 and to protect partially against apoptosis induced by treatment with Aβ25–35 or Aβ1–40. In conclusion, our results demonstrate that the release of Ca2+ from the ER, mediated by both RyR and IP3R, is involved in Aβ toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to Aβ‐induced neuronal death. This study suggests that Aβ accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca2+ homeostasis.

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.

Mitochondrial- and Endoplasmic Reticulum-Associated Oxidative Stress in Alzheimer's Disease: From Pathogenesis to Biomarkers

Alzheimers disease (AD) is the most common cause of dementia in the elderly, affecting several million of people worldwide. Pathological changes in the AD brain include the presence of amyloid plaques, neurofibrillary tangles, loss of neurons and synapses, and oxidative damage. These changes strongly associate with mitochondrial dysfunction and stress of the endoplasmic reticulum (ER). Mitochondrial dysfunction is intimately linked to the production of reactive oxygen species (ROS) and mitochondrial-driven apoptosis, which appear to be aggravated in the brain of AD patients. Concomitantly, mitochondria are closely associated with ER, and the deleterious crosstalk between both organelles has been shown to be involved in neuronal degeneration in AD. Stimuli that enhance expression of normal and/or folding-defective proteins activate an adaptive unfolded protein response (UPR) that, if unresolved, can cause apoptotic cell death. ER stress also induces the generation of ROS that, together with mitochondrial ROS and decreased activity of several antioxidant defenses, promotes chronic oxidative stress. In this paper we discuss the critical role of mitochondrial and ER dysfunction in oxidative injury in AD cellular and animal models, as well as in biological fluids from AD patients. Progress in developing peripheral and cerebrospinal fluid biomarkers related to oxidative stress will also be summarized.

Cell degeneration induced by amyloid-β peptides

Extracellular accumulation of amyloid-beta (Abeta) peptide and death of neurons in brain regions involved in learning and memory, particularly the cortex and the hippocampus, are central features of Alzheimers disease (AD). Neuronal Ca2+ overload and apoptosis are known to occur in AD. Abeta might play a role in disrupting Ca2+ homeostasis, and this AD-associated amyloidogenic peptide has been reported to induce apoptotic death in cultured cells. However, the specific intracellular signaling pathways by which Abeta triggers cell death are not yet well defined. This article provides evidence for the involvement of mitochondrial dysfunction in Abeta-induced toxicity and for the role of mitochondria in apoptosis triggered by Abeta. In addition, the endoplasmic reticulum (ER) seems to play a role in Abeta-induced apoptotic neuronal death, the ER stress being mediated by the perturbation of ER Ca2+ homeostasis. It is likely that a better understanding of how Abeta induces neuronal apoptosis will lead to the identification of potential molecular targets for the development of therapies for AD.Extracellular accumulation of amyloid-β (Aβ) peptide and death of neurons in brain regions involved in learning and memory, particularly the cortex and the hippocampus, are central features of Alzheimer’s disease (AD). Neuronal Ca2+ overload and apoptosis are known to occur in AD. Aβ might play a role in disrupting Ca2+ homeostasis, and this AD-associated amyloidogenic peptide has been reported to induce apoptotic death in cultured cells. However, the specific intracellular signaling pathways by which Aβ triggers cell death are not yet well defined. This article provides evidence for the involvement of mitochondrial dysfunction in Aβ-induced toxicity and for the role of mitochondria in apoptotsis triggered by Aβ. In addition, the endoplasmic reticulum (ER) seems to play a role in Aβ-induced apoptotic neuronal death, the ER stress being mediated by the perturbation of ER Ca2+ homeostasis. It is likely that a better understanding of how Aβ induces neuronal apoptosis will lead to the identification of potential molecular targets for the development of therapies for AD.

Epigenetic regulation of BACE1 in Alzheimer’s disease patients and in transgenic mice

In Alzheimers disease (AD) the complex interplay between environment and genetics has hampered the identification of effective therapeutics. However, epigenetic mechanisms could underlie this complexity. Here, we explored the potential role of epigenetic alterations in AD by investigating gene expression levels and chromatin remodeling in selected AD-related genes. Analysis was performed in the brain of the triple transgenic animal model of AD (3xTg-AD) and in peripheral blood mononuclear cells (PBMCs) from patients diagnosed with AD or Mild Cognitive Impairment (MCI). BACE1 mRNA levels were increased in aged 3xTg-AD mice as well as in AD PBMCs along with an increase in promoter accessibility and histone H3 acetylation, while the BACE1 promoter region was less accessible in PBMCs from MCI individuals. Ncstn was downregulated in aged 3xTg-AD brains with a condensation of chromatin and Sirt1 mRNA levels were decreased in these animals despite alterations in histone H3 acetylation. Neither gene was altered in AD PBMCs. The ADORA2A gene was not altered in patients or in the 3xTg-AD mice. Overall, our results suggest that chromatin remodeling plays a role in mRNA alterations in AD, prompting for broader and more detailed studies of chromatin and other epigenetic alterations and their potential use as biomarkers in AD.

Involvement of mitochondria in endoplasmic reticulum stress‐induced apoptotic cell death pathway triggered by the prion peptide PrP106–126

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrPSc. Recently, we have shown that PrP106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress‐mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP106–126 induces ER stress in both cell lines, given that ER Ca2+ content is low, glucose‐regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP106–126‐activated caspase 9 and 3, induced poly (ADP‐ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP106–126‐induced cell death, demonstrating the involvement of Ca2+ release through ER ryanodine receptors. However, in PrP106–126‐treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.

ER Stress-Mediated Apoptotic Pathway Induced by Aβ Peptide Requires the Presence of Functional Mitochondria

Amyloid-beta (Abeta) peptide plays a significant role in the pathogenesis of Alzheimers disease (AD). Previously we found that Abeta induces both mitochondrial and endoplasmic reticulum (ER) dysfunction leading to apoptosis, and now we address the relevance of ER-mitochondria crosstalk in apoptotic cell death triggered by Abeta peptide. Using mitochondrial DNA-depleted rho0 cells derived from the human NT2 teratocarcinoma cell line, characterized by the absence of functional mitochondria, and the parental rho+ cells, we report here that treatment with the synthetic Abeta1-40 peptide, or the classical ER stressors thapsigargin or brefeldin A, increases GRP78 expression levels and caspase activity, two ER stress markers, and also depletes ER calcium stores. Significantly, we show that the presence of functional mitochondria is required for ER stress-mediated apoptotic cell death triggered by toxic insults such as Abeta. We found that the increase in the levels of the pro-apoptotic transcription factor GADD153/CHOP, which mediates ER stress-induced cell death, as well as caspase-9 and -3 activation and increased number of TUNEL-positive cells, occurs in treated parental rho+ cells but is abolished in rho0 cells. Our results strongly support the close communication between ER and mitochondria during apoptotic cell death induced by the Abeta peptide and provide insights into the molecular cascade of cell death in AD.