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Abnormal accumulation of amyloid-β (Aβ) peptide in the brain is a pathological hallmark of Alzheimer's disease (AD). In addition to neurotoxic effects, Aβ also damages brain endothelial cells (ECs) and may thus contribute to the degeneration of cerebral vasculature, which has been proposed as an early pathogenic event in the course of AD and is able to trigger and/or potentiate the neurodegenerative process and cognitive decline. However, the mechanisms underlying Aβ-induced endothelial dysfunction are not completely understood. Here we hypothesized that Aβ impairs protein quality control mechanisms both in the secretory pathway and in the cytosol in brain ECs, leading cells to death. In rat brain RBE4 cells, we demonstrated that Aβ1-40 induces the failure of the ER stress-adaptive unfolded protein response (UPR), deregulates the ubiquitin-proteasome system (UPS) decreasing overall proteasome activity with accumulation of ubiquitinated proteins and impairs the autophagic protein degradation pathway due to failure in the autophagic flux, which culminates in cell demise. In conclusion, Aβ deregulates proteostasis in brain ECs and, as a consequence, these cells die by apoptosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Previously we reported that amyloid-β (Aβ) leads to endoplasmic reticulum (ER) stress in cultured cortical neurons and that ER-mitochondria Ca(2+) transfer is involved in Aβ-induced apoptotic neuronal cell death. In cybrid cells which recreate the defect in mitochondrial cytochrome c oxidase (COX) activity observed in platelets from Alzheimer's disease (AD) patients, we have shown that mitochondrial dysfunction affects the ER stress response triggered by Aβ. Here, we further investigated the impact of COX inhibition on Aβ-induced ER dysfunction using a neuronal model. Primary cultures of cortical neurons were challenged with toxic concentrations of Aβ upon chemical inhibition of COX with potassium cyanide (KCN). ER Ca(2+) homeostasis was evaluated under these conditions, together with the levels of ER stress markers, namely the chaperone GRP78 and XBP-1, a mediator of the ER unfolded protein response (UPR). We demonstrated that COX inhibition potentiates the Aβ-induced depletion of ER Ca(2+) content. KCN pre-treatment was also shown to enhance the rise of cytosolic Ca(2+) levels triggered by Aβ and thapsigargin, a widely used ER stressor. This effect was reverted in the presence of dantrolene, an inhibitor of ER Ca(2+) release through ryanodine receptors. Similarly, the increase in GRP78 and XBP-1 protein levels was shown to be higher in neurons treated with Aβ or thapsigargin in the presence of KCN in comparison with levels determined in neurons treated with the neurotoxins alone. Although the decrease in cell survival, the activation of caspase-9- and -3-mediated apoptotic cell death observed in Aβ- and thapsigargin-treated neurons were also potentiated by KCN, this effect is less pronounced than that observed in Ca(2+) signalling and UPR. Furthermore, in neurons treated with Aβ, the potentiating effect of the COX inhibitor in cell survival and death was not prevented by dantrolene. These results show that inhibition of mitochondrial COX activity potentiates Aβ-induced ER dysfunction and, to a less extent, neuronal cell death. Furthermore, data supports that the effect of impaired COX on Aβ-induced cell death occurs independently of Ca(2+) release through ER ryanodine receptors. Together, our data demonstrate that mitochondria dysfunction in AD enhances the neuronal susceptibility to toxic insults, namely to Aβ-induced ER stress, and strongly suggest that the close communication between ER and mitochondria can be a valuable future therapeutic target in AD., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We investigated the involvement of mitochondrial-dependent apoptosis in Huntington's disease (HD) vs. control (CTR) cybrids, obtained from the fusion of human platelets with mitochondrial DNA-depleted NT2 cells, and further exposed to 3-nitropropionic acid (3-NP) or staurosporine (STS). Untreated HD cybrids did not exhibit significant modifications in the activity of mitochondrial respiratory chain complexes I-IV or in mtDNA sequence variations suggestive of a primary role in mitochondrial susceptibility in the subpopulation of HD carriers studied. However, a slight decrease in mitochondrial membrane potential and increased formation of intracellular hydroperoxides was observed in HD cybrids under basal conditions. Furthermore, apoptotic nuclei morphology and a moderate increase in caspase-3 activation, as well as increased levels of superoxide ions and hydroperoxides were observed in HD cybrids upon 3-NP or STS treatment. 3-NP-evoked apoptosis in HD cybrids involved cytochrome c and AIF release from mitochondria, which was associated with mitochondrial Bax translocation. CTR cybrids subjected to 3-NP showed increased mitochondrial Bax and Bim levels and the release of AIF, but not cytochrome c, suggesting a different mode of cell death, linked to the loss of membrane integrity. Additionally, increased mitochondrial Bim and Bak levels, and a slight release of cytochrome c in untreated HD cybrids may help to explain their moderate susceptibility to mitochondrial-dependent apoptosis., (Copyright 2010 Elsevier Inc. All rights reserved.)

Amyloid-beta (Abeta) peptide plays a significant role in the pathogenesis of Alzheimer's 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.

3-Nitropropionic acid (3-NP) is an irreversible inhibitor of succinate dehydrogenase that has been used to explore the primary mechanisms of cell death associated with mitochondrial dysfunction and neurodegeneration in Huntington's disease. In this study we investigated the ability of brain-derived neurotrophic factor (BDNF) to suppress mitochondrial-dependent cell death induced by 3-NP in primary cortical neurons. This neurotrophin prevented 3-NP-induced release of cytochrome c and Smac/Diablo, caspase-3-like activity and nuclear condensation/fragmentation. Furthermore, it greatly increased phosphorylation of Akt and MAPK, suggesting the involvement of these signalling pathways in BDNF neuroprotection. Interestingly, BDNF decreased the levels of the pro-apoptotic protein Bim in mitochondrial and total cell lysates through the activation of the MEK1/2 pathway. This effect was due to an increase in the degradation rates of Bim. Our data support an important role for BDNF, in protecting cortical neurons against apoptotic cell death caused by inhibition of mitochondrial complex II.

Sporadic Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by a loss of dopaminergic neurons in the substantia nigra pars compacta. Many cellular mechanisms are thought to be involved in the death of these specific neurons in PD, including oxidative stress, changes of intracellular calcium homeostasis, and mitochondrial dysfunction. Since recent studies have revealed that also endoplasmic reticulum (ER) stress in conjunction with abnormal protein degradation can contribute to the PD pathophysiology, we investigated here the molecular mechanisms underlying the interplay between ER and mitochondria and its relevance in the control of neuronal cell death in PD. We observed that MPP+ induced changes in the mitochondrial function, affecting mitochondrial membrane potential and electron transport chain function. Likewise, it was also evident the unfolded protein response activation by an overexpression of GRP78 protein. Moreover, stress stimuli caused the release of Ca2+ from the ER that consistently induced mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Besides, Ca2+ release inhibition prevented MPP+ mediated mitochondria-dependent caspases activation. Our findings show that ER and mitochondria are in a close communication, establishing a dynamic ER-Ca2+-mitochondria interconnection that can play a prominent role in the neuronal cell death induction under particular stressful circumstances of PD pathology.

The mechanisms by which neurons die in Huntington's disease (HD) are uncertain, however, mitochondrial dysfunction and apoptosis have been implicated. Because peripheral abnormalities may reflect similar consequences of mutant huntingtin in the brain, we evaluated markers of apoptotic cell death and mitochondrial function in peripheral blood cells of 10 HD patients and 16 age- and gender-matched controls. We found increased Bax expression in B and T lymphocytes, and monocytes from HD patients, but no alterations in Bcl-2 expression levels. B lymphocytes also showed decreased mitochondrial membrane potential. However, HD peripheral blood cells showed no differences in reactive oxygen species (ROS) levels when compared to controls. Our results suggest that peripheral blood cells, in particularly B lymphocytes may reflect changes observed in HD brain.

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.

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP(Sc). Recently, we have shown that PrP(106-126) 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 PrP(106-126) on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 rho0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 rho+ cells. In this study, we show that PrP(106-126) 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 rho+ cells, PrP(106-126)-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 rho+ from PrP(106-126)-induced cell death, demonstrating the involvement of Ca2+ release through ER ryanodine receptors. However, in PrP(106-126)-treated NT2 rho0 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.

Since adenosine A(2A) receptor (A(2A)Rs) blockade protects against noxious brain insults involving apoptosis, we directly tested if A(2A)R blockade prevents apoptosis induced by staurosporine (STS). Exposure of rat hippocampal neurons to STS (30 nM, 24 h) decreased neuronal viability while increasing the number apoptotic-like neurons and de-localizing mitochondria and cytochrome c immunoreactivities. This was prevented by the selective A(2A)R antagonists, SCH58261 and ZM241385 (50 nM). Shorter incubation periods (6 h) with STS caused no neuronal loss but decreased synaptophysin and MAP-2 immunoreactivities, which was prevented by SCH58261. Furthermore, STS (100 nM) decreased MTT reduction and increased caspase-3 activity in rat hippocampal nerve terminals, which was prevented by SCH58261. These results show that A(2A)R blockade inhibits STS-induced apoptotic-like neuronal cell death. This begins with an apoptotic-like synaptotoxicity, which later evolved into an overt neurotoxicity, and A(2A)Rs effectively control this initial synaptotoxicity, in agreement with their predominant synaptic localization in the hippocampus.

Prion (PrP) and amyloid-beta (Abeta) peptides are involved in the neuronal loss that occurs in Prion disorders (PrD) and Alzheimer's 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.

Drug abuse is associated with brain dysfunction and neurodegeneration, and various recreational drugs induce apoptotic cell death. This study examined the role of the mitochondrial apoptotic pathway in psychostimulant-induced neuronal dysfunction. Using primary neuronal cultures, we observed that amphetamine (IC50=1.40 mM) was more potent than cocaine (IC50=4.30 mM) in inducing cell toxicity. Apoptotic cell death was further evaluated using cocaine and amphetamine concentrations that moderately decreased cell reduction capacity but did not affect plasma membrane integrity. Compared to cocaine, amphetamine highly decreased the mitochondrial membrane potential, as determined using the fluorescent probe rhodamine-123, whereas both drugs decreased mitochondrial cytochrome c. In contrast to amphetamine, cocaine cytotoxicity was partly mediated through effects on the electron transport chain, since cocaine toxicity was ameliorated in mitochondrial DNA-depleted cells lacking mitochondrially encoded electron transport chain subunits. Cocaine and amphetamine induced activation of caspases-2, -3 and -9 but did not affect activity of caspases-6 or -8. In addition, amphetamine, but not cocaine, was associated with the appearance of evident nuclear apoptotic morphology. These events were not accompanied by differences in the release of the apoptosis-inducing factor (AIF) from mitochondria. Our results demonstrate that although both amphetamine and cocaine activate the mitochondrial apoptotic pathway in cortical neurons, amphetamine is more likely to promote apoptosis.

Mitochondria play a critical role in cell death by releasing apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), from the intermembrane space into the cytoplasm. Because mitochondrial dysfunction has been shown to be involved in several neurodegenerative diseases, mitochondrial toxins are largely used to model these disorders. These include 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, which has been used to model Huntington's disease and was previously reported by us to induce apoptotic cell death through caspase activation. In the present study, we evaluated the involvement of caspase-independent neuronal cell death induced by 3-NP (1 mM) and the effect of z-VDVAD-fmk, an inhibitor of caspase-2, using cortical neurons in culture. Our results highly suggest that 3-NP induces both caspase-dependent and -independent cell death. We showed that z-VDVAD-fmk prevented both caspase-2 and -3-like activities evoked by 3-NP, but only partly prevented chromatin fragmentation/condensation. However, z-VDVAD-fmk did not avoid 3-NP-induced release of cytochrome c or AIF from mitochondria nor did it affect the levels of mitochondrial Bax. Furthermore, 3-NP-mediated decrease in plasma membrane integrity was not affected by z-VDVAD-fmk. Under these conditions, the inhibitor prevented the caspase-dependent cell death.

Amyloid beta-peptide (Abeta) is widely held to be associated with Alzheimer's disease. It was previously demonstrated by our group that Abeta induces cell death by an apoptotic process. We report here that activation of the caspase-3 apoptotic cascade is regulated by calcineurin-mediated BAD dephosphorylation. Calcineurin inhibitors were also proven to be effective by preventing the loss of mitochondrial membrane potential (Delta Psim) induced by Abeta, not allowing cytochrome c release from mitochondria and subsequently caspase-3 activation. Considering the results presented, we argue that calcineurin activation and BAD dephosphorylation are upstream in premitochondrial signaling events leading to caspase-3 activation in Abeta-peptide-treated cells.

The mitochondrial toxin 3-nitropropionic acid (3-NP) has been largely used to study neurodegenerative disorders in which bioenergetic defects are implicated. In the present study, we analyzed the molecular pathways involved in FK506 neuroprotection against cell death induced by 3-NP, using cultured cortical neurons. 3-NP induced cytochrome c release and increased caspases -2, -3, -8, and -9-like activities, although, calpain activity was not significantly affected. FK506 decreased cytochrome c release and caspase-3-like activity induced by 3-NP, without changing the activities of other caspases. FK-506 also decreased the number of apoptotic neurons, determined by Hoechst. Under these conditions, FK506 alone significantly reduced calcineurin activity by about 50%. Our results also showed a decrease in mitochondrial Bax and an increase in mitochondrial Bcl-2 levels upon exposure to FK506 and 3-NP. However, no significant changes occurred in total Bcl-2 and Bax levels. Altogether, the results suggest that FK506 neuroprotection against 3-NP-induced apoptosis is associated with the redistribution of Bcl-2 and Bax in the mitochondrial membrane.

Oxidative stress has been implicated in several pathologies associated with degenerative processes. Mitochondria are involved in cell death by necrosis or apoptosis due to a large load of Ca2+, the formation of reactive oxygen species (ROS), mitochondrial depolarization and the release of cytochrome c that initiates the caspase cascade. Nevertheless, the role of mitochondria in cell death processes induced by hydrogen peroxide (H2O2) has not been fully established. In this study, we analyzed the cytotoxic effect of H2O2 on rho+ human teratocarcinoma (NT2) cells and on mitochondria-DNA depleted rho0 NT2 cells, lacking functional mitochondria. The cells were exposed to H2O2 for 24 h and cell viability was dose-dependently decreased in both cell lines upon H2O2 exposure, although cell susceptibility was higher in rho0 NT2 cells. Moreover a decrease in mitochondrial membrane potential (Deltapsi(m)), mitochondrial cytochrome c release, caspases activation and DNA fragmentation were largely induced by H2O2 and occurred in both cell lines. Nevertheless, increased cell toxicity in rho0 cells upon H2O2 exposure was accompanied by a higher activation of the effector caspases-3 and -6. The data support that, in general, no differences were observed in cells containing functional (rho+) or non-functional (rho0) mitochondria upon H2O2-induced apoptotic cell death.

In this study, we analyzed the effect of Bcl-2 overexpression, an important anti-apoptotic protein, by using two hypothalamic cell lines, GT1-7puro or GT1-7bcl-2. 3-Nitropropionic acid (3-NP) mediated a dose-dependent decrease in cell viability in GT1-7puro cells, as determined by following the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, which was significantly prevented in GT1-7bcl-2 cells. In addition, activation of caspases-2, -3, and -6 induced by 3-NP was prevented by Bcl-2 overexpression. The data suggest that irreversible inhibition of mitochondrial complex II induces apoptotic features of cell death in a process prevented by Bcl-2.

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF(3)), a phospholipase A(2) (PLA(2)) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.

In recent years we have witnessed a major interest in the study of the role of mitochondria, not only as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca2+ homeostasis and endogenous producers of reactive oxygen species (ROS). Interestingly, the mitochondria have been also implicated as central executioners of cell death. Increased mitochondrial Ca2+ overload as a result of excitotoxicity has been associated with the generation of superoxide and may induce the release of proapoptotic mitochondrial proteins, proceeding through DNA fragmentation/condensation and culminating in cell demise by apoptosis and/or necrosis. In addition, these processes have been implicated in the pathogenesis of many neurodegenerative diseases, which share several features of cell death: selective brain areas undergo neurodegeneration, involving mitochondrial dysfunction (mitochondrial complexes are affected), loss of intracellular Ca2+ homeostasis, excitotoxicity, and the extracellular or intracellular accumulation of insoluble protein aggregates in the brain.

In the present work we investigated the effect of selective stimulation of non-desensitizing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), an AMPA-preferring receptor antagonist. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non-desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK-801, significantly decreased the number of Co2+-positive neurones, used as a cytochemical marker of Ca2+-permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA-mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases-1 and -3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn- superoxide dismutase (SOD) was observed on selective activation of non-desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor-mediated excitotoxicity in cultured rat hippocampal neurones.

Accumulating data suggest a central role for mitochondria and oxidative stress in neurodegenerative apoptosis. We previously demonstrated that amyloid-beta peptide 25-35 (Abeta 25-35) toxicity in cultured cells is mediated by its effects on functioning mitochondria. In this study, we further explored the hypothesis that Abeta 25-35 might induce apoptotic cell death by altering mitochondrial physiology. Mitochondria in Ntera2 (NT2 rho+) human teratocarcinoma cells exposed to either staurosporine (STS) or Abeta 25-35 were found to release cytochrome c, with subsequent activation of caspases 9 and 3. However, NT2 cells depleted of mitochondrial DNA (rho0 cells), which maintain a normal mitochondrial membrane potential (Deltapsi(m)) despite the absence of a functional electron transport chain (ETC), demonstrated cytochrome c release and caspase activation only with STS. We further observed increased reactive oxygen species (ROS) production and decreased reduced glutathione (GSH) levels in rho+ and rho0 cells treated with STS, but only in rho+ cells treated with Abeta 25-35. We conclude that under in vitro conditions, Abeta can induce oxidative stress and apoptosis only when a functional mitochondrial ETC is present.

Statins, used as cholesterol-lowering drugs, were reported to reduce the progression of Alzheimer's 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-β (Aβ) peptides and to analyze if such protection is cholesterol-independent. Using primary cultures of cortical neurons treated with Aβ_{1-40} peptide, we have demonstrated that pre-incubation with statins prevents the rise in cytosolic Ca^{2+} concentration and the accumulation of reactive oxygen species induced by Aβ 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 Aβ_{1-40}-induced caspase-3-dependent apoptosis. Moreover, our results revealed that simvastatin, at neuroprotective concentrations against Aβ-induced toxicity, is not able to activate Akt or ERK2, two signaling kinases with neuroprotective roles against apoptosis. [ABSTRACT FROM AUTHOR]

MPP+ (1-methyl-4-phenylpyridium ion), a complex I - inhibiting metabolite of 1-methyl-4-phenyl-1,2,3,6 -tetrahydropyridine (MPTP), causes anatomic-specific neurodegeneration. To evaluate the broader role of mitochondria in MPP+ -induced cell death, we exposed neuron-like NT2 human teratocarcinoma cells with mtDNA (rho+) and without mtDNA (rho0) to MPP+. MPP+ minimized the ability of both rho+ and rho0 cells to reduce MTT. Only rho+ cells, though, initiated intrinsic pathway-mediated apoptosis. MPP+ also activated calpains in rho0 cell lines. The calpain inhibitor MDL 28710 was able to prevent the MPP+-related MTT reduction change in rho0 but not rho+ cells. We conclude that 1) MPP+-induced apoptosis requires functional mitochondria, 2) MPP+ activates calpains independent of respiratory chain inhibition, and 3) calpain activation mediates some aspects of MPP+ toxicity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this study we analysed the effect of Bcl-2 on the cytotoxicity induced by the amyloid-β (Aβ_{25-35}) and prion (PrP_{106-126}) peptides by using GT1-7 puro and GT1-7 bcl-2 (overexpressing the anti-apoptotic protein Bcl-2) neural cells. Exposure to Aβ _{25-35} (1–5 μM) and PrP_{106-126} (25 μM) caused a decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data were correlated with Aβ_{25-35} and PrP_{106-126}-induced activation of caspase-9, which is linked to the mitochondrial death pathway, and the activation of the effector caspase-3, suggesting cell death by apoptosis. Furthermore, Bcl-2 overexpression protected from loss of cell viability and caspase-9 and -3 activation induced by Aβ_{25-35} and PrP_{106-126}, showing that Bcl-2 is neuroprotective against apoptotic cell death caused by amyloidogenic peptides. [ABSTRACT FROM AUTHOR]

In this study, we analyzed the effect of Bcl-2 overexpression, an important anti-apoptotic protein, by using two hypothalamic cell lines, GT1-7puro or GT1-7bcl-2. 3-Nitropropionic acid (3-NP) mediated a dose-dependent decrease in cell viability in GT1-7puro cells, as determined by following the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, which was significantly prevented in GT1-7bcl-2 cells. In addition, activation of caspases-2, -3, and -6 induced by 3-NP was prevented by Bcl-2 overexpression. The data suggest that irreversible inhibition of mitochondrial complex II induces apoptotic features of cell death in a process prevented by Bcl-2. [ABSTRACT FROM AUTHOR]

Abstract: Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1–40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca2+ homeostasis due to the release of Ca2+ from this intracellular store. Finally, Aβ1–40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1–40 concomitantly with caspase-12 activation. Furthermore, Aβ1–40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1–40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. [Copyright &y& Elsevier]

Abstract: Previously we demonstrated that insulin protects against neuronal oxidative stress by restoring antioxidants and energy metabolism. In this study, we analysed how insulin influences insulin- (IR) and insulin growth factor-1 receptor (IGF-1R) intracellular signaling pathways after oxidative stress caused by ascorbate/Fe2+ in rat cortical neurons. Insulin prevented oxidative stress-induced decrease in tyrosine phosphorylation of IR and IGF-1R and Akt inactivation. Insulin also decreased the active form of glycogen synthase kinase-3β (GSK-3β) upon oxidation. Since phosphatidylinositol 3-kinase (PI-3K)/Akt-mediated inhibition of GSK-3β may stimulate protein synthesis and decrease apoptosis, we analysed mRNA and protein expression of “candidate” proteins involved in antioxidant defense, glucose metabolism and apoptosis. Insulin prevented oxidative stress-induced increase in glutathione peroxidase-1 and decrease in hexokinase-II expression, supporting previous findings of changes in glutathione redox cycle and glycolysis. Moreover, insulin precluded Bcl-2 decrease and caspase-3 increased expression. Concordantly, insulin abolished caspase-3 activity and DNA fragmentation caused by oxidative stress. Thus, insulin-mediated activation of IR/IGF-1R stimulates PI-3K/Akt and inhibits GSK-3β signaling pathways, modifying neuronal antioxidant defense-, glucose metabolism- and anti-apoptotic-associated protein synthesis. These and previous data implicate insulin as a promising neuroprotective agent against oxidative stress associated with neurodegenerative diseases. [Copyright &y& Elsevier]

Abstract: Substance abuse and addiction are the most costly of all the neuropsychiatric disorders. In the last decades, much progress has been achieved in understanding the effects of the drugs of abuse in the brain. However, efficient treatments that prevent relapse have not been developed. Drug addiction is now considered a brain disease, because the abuse of drugs affects several brain functions. Neurological impairments observed in drug addicts may reflect drug-induced neuronal dysfunction and neurotoxicity. The drugs of abuse directly or indirectly affect neurotransmitter systems, particularly dopaminergic and glutamatergic neurons. This review explores the literature reporting cellular and molecular alterations reflecting the cytotoxicity induced by amphetamines, cocaine and opiates in neuronal systems. The neurotoxic effects of drugs of abuse are often associated with oxidative stress, mitochondrial dysfunction, apoptosis and inhibition of neurogenesis, among other mechanisms. Understanding the mechanisms that underlie brain dysfunction observed in drug-addicted individuals may contribute to improve the treatment of drug addiction, which may have social and economic consequences. [Copyright &y& Elsevier]

Abstract: Huntington’s disease (HD) is a genetic neurodegenerative disorder characterized by striatal neurodegeneration, involving apoptosis. FK506, an inhibitor of calcineurin (or protein phosphatase 3, formerly known as protein phosphatase 2B), has shown neuroprotective effects in several cellular and animal models of HD. In the present study, we show the protective effects of FK506 in two striatal HD models, primary rat striatal neurons treated with 3-nitropropionic acid (3-NP) and immortalized striatal STHdh cells derived from HD knock-in mice expressing normal (STHdh 7/7) or full-length mutant huntingtin (FL-mHtt) with 111 glutamines (STHdh 111/111), under basal conditions and after exposure to 3-NP or staurosporine (STS). In rat striatal neurons, FK506 abolished 3-NP-induced increase in caspase-3 activation, DNA fragmentation/condensation and necrosis. Nevertheless, in STHdh 111/111 cells under basal conditions, FK506 did not prevent, in a significant manner, the release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, or alter Bax/Bcl-2 ratio, but significantly reverted caspase-3 activation. In STHdh 111/111 cells treated with 0.3mM 3-NP or 25 nM STS, linked to high necrosis, exposure to FK506 exerted no significant effects on caspase-3 activation. However, treatment of STHdh 111/111 cells exposed to 10nM STS with FK506 effectively prevented cell death by apoptosis and moderate necrosis. The results suggest that FK506 may be neuroprotective against apoptosis and necrosis under mild cell death stimulus in the presence of FLmHtt. [Copyright &y& Elsevier]

Abstract: Cocaine and heroin are frequently co-abused by humans, in a combination known as speedball. Recently, chemical interactions between heroin (Her) or its metabolite morphine (Mor) and cocaine (Coc) were described, resulting in the formation of strong adducts. In this work, we evaluated whether combinations of Coc and Her affect the neurotoxicity of these drugs, using rat cortical neurons incubated with Coc, Her, Her followed by Coc (Her+Coc) and Her plus Coc (Her:Coc, 1:1). Neurons exposed to Her, Her+Coc and Her:Coc exhibited a decrease in cell viability, which was more pronounced in neurons exposed to Her and Her+Coc, in comparison with neurons exposed to the mixture (Her:Coc). Cells exposed to the mixture showed increased intracellular calcium and mitochondrial dysfunction, as determined by a decrease in intracellular ATP levels and in mitochondrial membrane potential, displaying both apoptotic and necrotic characteristics. Conversely, a major increase in cytochrome c release, caspase 3-dependent apoptosis, and decreased metabolic neuronal viability were observed upon sequential exposure to Her and Coc. The data show that drug combinations potentiate cortical neurotoxicity and that the mode of co-exposure changes cellular death pathways activated by the drugs, strongly suggesting that chemical interactions occurring in Her:Coc, such as adduct formation, shift cell death mechanisms towards necrosis. Since impairment of the prefrontal cortex is involved in the loss of impulse control observed in drug addicts, the data presented here may contribute to explain the increase in treatment failure observed in speedball abusers. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria likely play a role in Parkinson’s disease (PD) neurodegeneration. We modelled PD by creating cytoplasmic hybrid (cybrid) cell lines in which endogenous mitochondrial DNA (mtDNA) from PD or control subject platelets was expressed within human teratocarcinoma (NT2) cells previously depleted of endogenous mtDNA. Complex I activity was reduced in both PD cybrid lines and in the platelet mitochondria used to generate them. Under basal conditions PD cybrids had less ATP, more LDH release, depolarized mitochondria, less mitochondrial cytochrome c, and higher caspase 3 activity. Equivalent MPP+ exposures are more likely to trigger programmed cell death in PD cybrid cells than in control cybrid cells. Our data support a relatively upstream role for mitochondrial dysfunction in idiopathic PD. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Since adenosine A2A receptor (A2ARs) blockade protects against noxious brain insults involving apoptosis, we directly tested if A2AR blockade prevents apoptosis induced by staurosporine (STS). Exposure of rat hippocampal neurons to STS (30 nM, 24 h) decreased neuronal viability while increasing the number apoptotic-like neurons and de-localizing mitochondria and cytochrome c immunoreactivities. This was prevented by the selective A2AR antagonists, SCH58261 and ZM241385 (50 nM). Shorter incubation periods (6 h) with STS caused no neuronal loss but decreased synaptophysin and MAP-2 immunoreactivities, which was prevented by SCH58261. Furthermore, STS (100 nM) decreased MTT reduction and increased caspase-3 activity in rat hippocampal nerve terminals, which was prevented by SCH58261. These results show that A2AR blockade inhibits STS-induced apoptotic-like neuronal cell death. This begins with an apoptotic-like synaptotoxicity, which later evolved into an overt neurotoxicity, and A2ARs effectively control this initial synaptotoxicity, in agreement with their predominant synaptic localization in the hippocampus. [Copyright &y& Elsevier]

Abstract: In the hippocampus, impaired neurophysiology, compromised neurogenesis, and eventually apoptosis accompany cognitive deficits in insulinopenic (type-1) diabetes (T1D). The underlying pathological mechanisms remain to be defined. The hippocampus has a high density of the cannabinoid type 1 receptor (CB1R), which has been shown to control several brain functions affected by diabetes, such as synaptic plasticity, glucose utilisation, memory consolidation and cognition, and maturation and survival of hippocampal neurons. However, the role of this receptor has not been investigated yet in diabetic encephalopathy. We report now that in the streptozotocin animal model of T1D, the hippocampal densities of CB1R protein and of specific CB1R binding sites are significantly increased both in the nerve terminals and in total membranes (changes between 13% and 38%), whereas CB1R mRNA expression is decreased by 25%, suggesting that CB1Rs might play a role in diabetic encephalopathy. [Copyright &y& Elsevier]

Abstract: Previously we reported that endoplasmic reticulum (ER)-mitochondria crosstalk is involved in amyloid-β (Aβ)-induced apoptosis. Now we show that mitochondrial dysfunction affects the ER stress response triggered by Aβ using cybrids that recreate the defect in mitochondrial cytochrome c oxidase (COX) activity detected in platelets from Alzheimer''s disease (AD) patients. AD and control cybrids were treated with Aβ or classical ER stressors and the ER stress-mediated apoptotic cell death pathway was accessed. Upon treatment, we found increased glucose-regulated protein 78 (GRP78) levels and caspase-4 activation (ER stress markers) which were more pronounced in AD cybrids. Treated AD cybrids also exhibited decreased cell survival as well as increased caspase-3-like activity, poli-ADP-ribose-polymerase (PARP) levels and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells. Finally, we showed that Aβ-induced caspase-3 activation in both cybrid cell lines was prevented by dantrolene, thus implicating ER Ca2+ release in ER stress-mediated apoptosis. Our results demonstrate that mitochondrial dysfunction occurring in AD patients due to COX inhibition potentiates cell susceptibility to Aβ-induced ER stress. This study further supports the close communication between ER and mitochondria during apoptosis in AD. [Copyright &y& Elsevier]