Ildete L. Ferreira

Amyloid beta peptide 1-42 disturbs intracellular calcium homeostasis through activation of GluN2B-containing N-methyl-d-aspartate receptors in cortical cultures.

Alzheimers disease (AD) is a progressive neurodegenerative disorder that leads to debilitating cognitive deficits. Recent evidence demonstrates that glutamate receptors are dysregulated by amyloid beta peptide (Aβ) oligomers, resulting in disruption of glutamatergic synaptic transmission which parallels early cognitive deficits. Although it is well accepted that neuronal death in AD is related to disturbed intracellular Ca(2+) (Ca(2+)(i)) homeostasis, little is known about the contribution of NMDARs containing GluN2A or GluN2B subunits on Aβ-induced Ca(2+)(i) rise and neuronal dysfunction. Thus, the main goal of this work was to evaluate the role of NMDAR subunits in dysregulation of Ca(2+)(i) homeostasis induced by Aβ 1-42 preparation containing both oligomers (in higher percentage) and monomers in rat cerebral cortical neurons. The involvement of NMDARs was evaluated by pharmacological inhibition with MK-801 or the selective GluN2A and GLUN2B subunit antagonists NVP-AAM077 and ifenprodil, respectively. We show that Aβ, like NMDA, increase Ca(2+)(i) levels mainly through activation of NMDARs containing GluN2B subunits. Conversely, GluN2A-NMDARs antagonism potentiates Ca(2+)(i) rise induced by a high concentration of Aβ (1μM), suggesting that GluN2A and GluN2B subunits have opposite roles in regulating Ca(2+)(i) homeostasis. Moreover, Aβ modulate NMDA-induced responses and vice versa. Indeed, pre-exposure to Aβ (1μM) decrease NMDA-evoked Ca(2+)(I) rise and pre-exposure to NMDA decrease Aβ response. Interestingly, simultaneous addition of Aβ and NMDA potentiate Ca(2+)(I) levels, this effect being regulated by GluN2A and GluN2B subunits in opposite manners. This study contributes to the understanding of the molecular basis of early AD pathogenesis, by exploring the role of GluN2A and GluN2B subunits in the mechanism of Aβ toxicity 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.

Mitochondrial function in Parkinson’s disease cybrids containing an nt2 neuron-like nuclear background

Mitochondria likely play a role in Parkinsons 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.

Ca2+ influx through glutamate receptor-associated channels in retina cells correlates with neuronal cell death

We studied the effect of glutamate, N-methyl-D-aspartate (NMDA), kainate or K+ depolarization, on neurotoxicity in cultured chick retinal cells, under conditions in which we could discriminate between Ca2+ entering through ionotropic glutamate receptors and voltage-sensitive Ca2+ channels (VSCCs). When neurons were challenged with NMDA, kainate or glutamate, in Na(+)-containing medium, a decrease in cell survival was observed, whereas K+ depolarization did not affect the viability of the cells. The Mg2+ ion completely prevented the toxic effect mediated by the NMDA receptor, and had a small but significant protective effect at the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate (AMPA/kainate) receptor-induced cell death. We observed that, in a Na(+)-free N-methyl-D-glucamine (NMG) medium, to avoid the activation of VSCCs indirectly by the glutamate receptor agonists, stimulation of the glutamate receptors causes Ca2+ influx only through NMDA and AMPA/kainate receptor-associated channels, and that Ca2+ entry correlates well with subsequent cell death. These results show that the activation of NMDA or AMPA/kainate receptors can cause excitotoxicity in retinal neurons by mechanisms not involving Na+ influx, but rather depending on the permeation of Ca2+ through glutamate receptor-associated channels. For small Ca2+ loads the entry of Ca2+ through the NMDA receptor-associated channel was more efficient in triggering cell death than the influx of Ca2+ through the AMPA/kainate receptor.

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.

Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer's disease

Oxidative stress and endoplasmic reticulum (ER) stress have been associated with Alzheimers disease (AD) progression. In this study we analyzed whether oxidative stress involving changes in Nrf2 and ER stress may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased phosphorylated Nrf2 (p(Ser40)Nrf2) were observed in human peripheral blood mononuclear cells (PBMCs) isolated from individuals with mild cognitive impairment (MCI). Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of early oxidative stress defense mechanisms in AD was substantiated by increased p(Ser40)Nrf2 in 3month-old 3xTg-AD male mice PBMCs, and also with increased nuclear Nrf2 levels in brain cortex. However, SOD1 protein levels were decreased in human MCI PBMCs and in 3xTg-AD mice brain cortex; the latter further correlated with reduced SOD1 mRNA levels. Increased ER stress was also detected in the brain cortex of young female and old male 3xTg-AD mice. We demonstrate oxidative stress and early Nrf2 activation in AD human and mouse models, which fails to regulate some of its targets, leading to repressed expression of antioxidant defenses (e.g., SOD-1), and extending to ER stress. Results suggest markers of prodromal AD linked to oxidative stress associated with Nrf2 activation and ER stress that may be followed in human peripheral blood mononuclear cells.

Ca2+-dependent release of [3H]GABA in cultured chick retina cells

Depolarization by K+ (50 mM) of cultured chick retina cells released 1.14 +/- 0.28% of the accumulated [3H] gamma-aminobutyric acid (GABA) in the absence of Ca2+, but when 1.0 mM Ca2+ was present, the internal free calcium ion concentration [Ca2+]i rose by about 750 nM and the [3H]GABA release about doubled to a value of 2.22 +/- 0.2% of the total [3H]GABA. Nitrendipine (0.1 microM), a blocker of the L-type Ca2+ channels, blocked the [Ca2+]i response to K+ depolarization by about 65%, and the omega-Conotoxin GVIA (omega-CgTx) (0.5 microM), a blocker of the N-type of Ca2+ channels, inhibited by 27% the [Ca2+]i rise due to K+ depolarization. Parallel experiments showed that nitrendipine inhibits [3H]GABA release to the level observed in the absence of Ca2+, whereas omega-CgTx did not inhibit significantly the release of [3H]GABA. The results also show that the release of [3H]GABA due to K(+)-depolarization in the absence of Ca2+ can be totally blocked by 1-(2-(((Diphenylmethylene) amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711), an inhibitor of the GABA carrier. However, in the presence of Ca2+, NNC-711 blocks the release only by about 66%, corresponding to the Ca(2+)-independent release. Thus, it is concluded that [3H]GABA is released in chick retina cells by the exocytotic mechanism, which is Ca(2+)-dependent, and by reversal of the carrier, which is Ca(2+)-independent, in much the same way as has been found for other GABAergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial-dependent apoptosis in Huntington's disease human cybrids

We investigated the involvement of mitochondrial-dependent apoptosis in Huntingtons 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.

Glutamate increases the [Ca2+]i but stimulates Ca2+-independent release of [3H]GABA in cultured chick retina cells

The effect of glutamate on [Ca2+]i and on [3H] gamma-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 microM) increases the [Ca2+]i by Ca2+ influx through Ca2+ channels sensitive to nitrendipine, but not to omega-conotoxin GVIA (omega-Cg Tx) (50%), and by other channels insensitive to either Ca2+ channel blocker. Mobilization of Ca2+ by glutamate required the presence of external Na+, suggesting that Na+ mobilization through the ionotropic glutamate receptors is necessary for the Ca2+ channels to open. The increase in [Ca2+]i was not related to the release of [3H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca2+ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [3H]GABA was significantly depressed by Ca(2+)o, but it was dependent on Na(+)o, just as was observed for the [3H]GABA release induced by veratridine (50 microM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [3H]GABA release. Both veratridine- and glutamate-induced [3H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-py ridine- carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [3H]GABA evoked by glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)

Relation of [Ca2+]i to dopamine release in striatal synaptosomes: role of Ca2+ channels

We compared the effects of KCl and 4-aminopyridine (4-AP) stimulation on the coupling of Ca2+ channel activation to [3H]dopamine ([3H]DA) release in rat striatal synaptosomes and used specific Ca2+ channel blockers to discriminate between the different VSCCs activated by the two stimulatory agents. We found that whereas [3H]DA release is strictly Ca(2+)-dependent in the case of KCl depolarization, 4-AP, at concentrations above 100 microM, progressively causes a large Ca(2+)-independent release of [3H]DA. Thus, at 1 to 3 mM 4-AP, as much as 80-95% of the [3H]DA release is Ca(2+)-independent and can be partially blocked by nomifensine, indicating that some [3H]DA release is occurring through reversal of the DA carrier. Therefore, in the studies relating [Ca2+]i to [3H]DA release we selected 4-AP concentrations lower than 100 microM and corrected for the Ca(2+)-independent release. Under these conditions, we determined that: (1) Ca2+ entry through N-type VSCCs is involved in [3H]DA release both in the case of KCl depolarization (35% inhibition by omega-CgTx) and in 4-AP stimulation (23% inhibition by omega-CgTx); (2) Ca2+ entering through P-type and/or Q-type VSCCs is also involved in [3H]DA release due to 4-AP stimulation (26% inhibition by 200 nM omega-Aga IVA); (3) Neomycin (0.35 mM) inhibited the [3H]DA release due to 4-AP stimulation by about 20% and decreased the KCl induced [3H]DA release by 55%; the effects of neomycin (0.35 mM) and omega-CgTx were additive in both cases, indicating that, at this concentration, the antibiotic does not affect significantly N-type Ca2+ channels; (4) When applied together, omega-CgTx and omega-Aga IVA inhibited the 4-AP stimulated [3H]DA release by about 40-50%, suggesting that the remaining large fraction of the VSCCs activated by 4-AP stimulation are non-N, non-P VSCCs and are coupled to Ca(2+)-dependent [3H]DA release; (5) The contribution of L-type VSCCs is uncertain, since there seemed to be a small contribution in the case of KCl depolarization, but not in the case of 4-AP stimulation. On the whole, the results suggest that the release of [3H]DA in the rat striatal nerve terminals depends on Ca2+ entry through N-, P-, possibly Q-, and other non-N-, non-P-type VSCCs when either KCl or 4-AP stimulation is utilized.