Ana Cristina Rego

Mechanisms of neurodegeneration in Huntington’s disease

Huntington’s disease (HD) is caused by an expansion of cytosine–adenine–guanine (CAG) repeats in the huntingtin gene, which leads to neuronal loss in the striatum and cortex and to the appearance of neuronal intranuclear inclusions of mutant huntingtin. Huntingtin plays a role in protein trafficking, vesicle transport, postsynaptic signaling, transcriptional regulation, and apoptosis. Thus, a loss of function of the normal protein and a toxic gain of function of the mutant huntingtin contribute to the disruption of multiple intracellular pathways. Furthermore, excitotoxicity, dopamine toxicity, metabolic impairment, mitochondrial dysfunction, oxidative stress, apoptosis, and autophagy have been implicated in the progressive degeneration observed in HD. Nevertheless, despite the efforts of a multidisciplinary scientific community, there is no cure for this devastating neurodegenerative disorder. This review presents an overview of the mechanisms that may contribute for HD pathogenesis. Ultimately, a better understanding of these mechanisms will lead to the development of more effective therapeutic targets.

Cellular and molecular mechanisms involved in the neurotoxicity of opioid and psychostimulant drugs

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.

Oxidative Stress, Hypoxia, and Ischemia‐Like Conditions Increase the Release of Endogenous Amino Acids by Distinct Mechanisms in Cultured Retinal Cells

Abstract: The aim of this study was to elucidate the mechanisms by which retinal cells release endogenous amino acids in response to ascorbate/Fe2+‐induced oxidative stress, as compared with chemical hypoxia or ischemia. In the absence of stimulation, oxidative stress increased the release of aspartate, glutamate, taurine, and GABA only when Ca2+ was present. Under hypoxia or ischemia, the release of aspartate, glutamate, glycine, alanine, taurine, and GABA increased mainly by a Ca2+‐independent mechanism. The increased release observed in N‐methyl‐d‐glucamine+ medium suggested the reversal of the Na+‐dependent amino acid transporters. Upon oxidative stress, the release of aspartate, glutamate, and GABA, occurring through the reversal of the Na+‐dependent transporters, was reduced by about 30%, although the release of taurine was enhanced. An increased release of [3H]arachidonic acid and free radicals seems to affect the Na+‐dependent transporters for glutamate and GABA in oxidized cells. All cell treatments increased [Ca2+]i (1.5 to twofold), although no differences were observed in membrane depolarization. The energy charge of cells submitted to hypoxia or oxidative stress was not changed. However, ischemia highly potentiated the reduction of the energy charge, as compared with hypoglycemia or hypoxia alone. The present work is important for understanding the mechanisms of amino acid release that occur in vivo upon oxidative stress, hypoxia, or ischemia, frequently associated with the impairment of energy metabolism.

Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease--resemblance to the effect of amphetamine drugs of abuse.

Parkinson disease (PD) is a chronic and progressive neurological disease associated with a loss of dopaminergic neurons. In most cases the disease is sporadic but genetically inherited cases also exist. One of the major pathological features of PD is the presence of aggregates that localize in neuronal cytoplasm as Lewy bodies, mainly composed of α-synuclein (α-syn) and ubiquitin. The selective degeneration of dopaminergic neurons suggests that dopamine itself may contribute to the neurodegenerative process in PD. Furthermore, mitochondrial dysfunction and oxidative stress constitute key pathogenic events of this disorder. Thus, in this review we give an actual perspective to classical pathways involving these two mechanisms of neurodegeneration, including the role of dopamine in sporadic and familial PD, as well as in the case of abuse of amphetamine-type drugs. Mutations in genes related to familial PD causing autosomal dominant or recessive forms may also have crucial effects on mitochondrial morphology, function, and oxidative stress. Environmental factors, such as MPTP and rotenone, have been reported to induce selective degeneration of the nigrostriatal pathways leading to α-syn-positive inclusions, possibly by inhibiting mitochondrial complex I of the respiratory chain and subsequently increasing oxidative stress. Recently, increased risk for PD was found in amphetamine users. Amphetamine drugs have effects similar to those of other environmental factors for PD, because long-term exposure to these drugs leads to dopamine depletion. Moreover, amphetamine neurotoxicity involves α-syn aggregation, mitochondrial dysfunction, and oxidative stress. Therefore, dopamine and related oxidative stress, as well as mitochondrial dysfunction, seem to be common links between PD and amphetamine neurotoxicity.

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.

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.

Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress

A role for the antioxidants vitamin E and idebenone in decreasing retinal cell injury, after metabolic inhibition induced by chemical ischemia and hypoglycemia, was investigated and compared with oxidative stress conditions. Preincubation of the antioxidants, vitamin E (20 microM) and idebenone (10 microM), effectively protected from retinal cell injury after oxidative stress or hypoglycemia, whereas the protection afforded after postincubation of both antioxidants was decreased. Delayed retinal cell damage, mediated by chemical ischemia, was attenuated at 10 or 12 h postischemia, only after exposure to the antioxidants during all the experimental procedure. An antagonist of the N-methyl-D-aspartate (NMDA) receptors, an inhibitor of nitric oxide synthase (NOS) or a blocker of L-type Ca2+ channels were ineffective in reducing cell injury induced by chemical ischemia, hypoglycemia or oxidative stress. Oxidative stress and hypoglycemia increased (about 1.2-fold) significantly the fluorescence of the probe DCFH2-DA, that is indicative of intracellular ROS formation. Free radical generation detected with the probe dihydrorhodamine 123 (DHR 123) was enhanced after oxidative stress, chemical ischemia or hypoglycemia (about 2-fold). Nevertheless, the antioxidants vitamin E or idebenone were ineffective against intracellular ROS generation. Cellular energy charge decreased greatly after chemical ischemia, was moderately affected after hypoglycemia, but no significant changes were observed after oxidative stress. Preincubation with vitamin E prevented the changes in energy charge upon 6 h posthypoglycemia. We can conclude that irreversible changes occurring during chemical ischemia mainly reflect the alterations taking place at the ischemic core, whereas hypoglycemia situations may reflect changes occurring at the penumbra area, whereby vitamin E or idebenone may help to increase cell survival, exerting a beneficial neuroprotective effect.

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.

Toxicity of β-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits

Neurotoxicity induced by beta-amyloid peptide (Abeta) involves glutamate toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Abeta(1-40) toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(1-40) decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Abeta(1-40). Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Abeta(1-40) through changes in calcium homeostasis.

Adenosine Triphosphate Degradation Products After Oxidative Stress and Metabolic Dysfunction in Cultured Retinal Cells

Abstract: The alteration in energy metabolic products was analyzed in cultured retinal cells submitted to oxidative stress, hypoxia, glucopenia, or ischemia‐like conditions. Ischemia highly reduced cellular ATP and increased AMP formation, without significant changes in ADP. Ischemia induced a significant increase in extracellular adenosine (ADO) and hypoxanthine (HYP), and to a lesser extent inosine (INO). Glucopenia reduced cellular ATP by about two‐ to threefold, which was not compensated for by AMP formation. Under glucopenia, extracellular ADO and HYP were significantly increased, although a major increase in extracellular INO was observed. 5‐(4‐Nitrobenzyl)‐6‐thioinosine (10 µM) reduced extracellular ADO during glucopenia or ischemia by ∼80%, indicating that ADO accumulation occurs mainly via the transporter. Intracellular ATP, ADP, or AMP and extracellular ADO, INO, or HYP were not apparently changed after oxidative stress or hypoxia. Nevertheless, in the presence of 10 µM erythro‐9‐(2‐hydroxy‐3‐nonyl)adenosine, oxidative stress was shown to increase significantly the accumulation of ADO, which was reduced in the presence of 200 µMα,β‐methyleneadenosine 5′‐diphosphate, suggesting that ADO accumulation after oxidative stress may result from extracellular degradation of adenine nucleotides. The increase in ADO accumulation resulting from the depletion of cellular ATP was directly related to the release of endogenous glutamate occurring through a Ca2+‐independent pathway after ischemia. Increased metabolic products derived from ATP are suggested to exert a modulating effect against excitotoxic neuronal death.