No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Involvement of Oxidative Stress on the Impairment of Energy Metabolism Induced by A?? Peptides on PC12 Cells: Protection by Antioxidants
Abstract
Alzheimer's disease is widely held to be associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta) aggregates. In this study, the involvement of oxidative stress on A beta-induced energy metabolism dysfunction was evaluated on PC12 cells. It was shown that A beta peptides (A beta25-35 and A beta1-40) induce a concentration-dependent accumulation of reactive oxygen species (ROS), decrease the cellular redox activity, and lead to the depletion of ATP levels. The observed inhibition by A beta of mitochondrial function and of glycolysis is blocked by the antioxidants vitamin E, idebenone, and GSH ethyl ester. Taken together, these data suggest that exposure of PC12 cells to A beta results in an impairment of energy metabolism, leading to a deficit in ATP levels and to the compromise of cellular viability. Furthermore, the generation of ROS seems to be a crucial event responsible for the energetic metabolic dysfunction induced by A beta.
... Using the method given by Pereira et al. (1999) [20] ROS values were generated. PA-1 cells were added in 6 well plates (2 x 10 6 cells/well) for 24 h before exposing them to different concentrations of the betanin (40 cells/well) for 24 h prior to exposure and different concentrations of the betanin (40 µg/ml) and untreated cells were maintained at 37°C (5% CO 2 ). ...
... Using the method given by Pereira et al. (1999) [20] ROS values were generated. PA-1 cells were added in 6 well plates (2 x 10 6 cells/well) for 24 h before exposing them to different concentrations of the betanin (40 cells/well) for 24 h prior to exposure and different concentrations of the betanin (40 µg/ml) and untreated cells were maintained at 37°C (5% CO 2 ). ...
... DCFH-DA is cell permeable, lipophilic compound. DCFH-DA is deacetylayed to DCF in the cytoplasm by cellular esterases (Pereira et al.,1999). A uorescent molecule is obtained by oxidation of DCF by various radicals such as hydroxyl, alkoxyl, peroxyl, carbonate and nitrate (emission 485nm, excitation 530nm). ...
Ovarian carcinoma has a cure rate of 30% which makes it deadlier than any other disease. There are a number of genetic and epigenetic changes that lead to ovarian carcinoma cell transformation. Chemoprevention of cancer through application of natural compounds is the need of present generation as other methods are rigorous and have many side effects. Betanin, a compound from Beta vulgaris extract is used in present study to check its potential for inhibition of (PA-1) cancer cell proliferation. Determination of IC50 values through MTT assay was carried out, in addition measurement of Mitochondrial Membrane Potential (MMP), effect of Reactive Oxygen Species (ROS) generation and induction of Apoptosis in ovarian cancer cells through betanin was also observed. Results have shown betanin as a potential candidate for inhibition of ovarian cancer cell proliferation and it can be taken up as a serious compound for further studies for its application in cancer cure.
... Using the method given by Pereira et al. (1999) [26], ROS values were generated. PA-1 cells were added in 6-well plates (2 × 10 6 cells/well) for 24 h before exposing them to betanin (40 μg/well), and untreated cells were maintained at 37 °C (5% CO 2 ). ...
... Using the method given by Pereira et al. (1999) [26], ROS values were generated. PA-1 cells were added in 6-well plates (2 × 10 6 cells/well) for 24 h before exposing them to betanin (40 μg/well), and untreated cells were maintained at 37 °C (5% CO 2 ). ...
Ovarian carcinoma has a cure rate of 30% which makes it deadlier than any other disease. There are a number of genetic and epigenetic changes that lead to ovarian carcinoma cell transformation. Chemoprevention of cancer through application of natural compounds is the need of present generation as other methods are rigorous and have many side effects. Betanin, a compound from Beta vulgaris extract is used in present study to check its potential for inhibition of (PA-1) cancer cell proliferation. Determination of IC50 values through MTT assay was carried out, in addition measurement of mitochondrial membrane potential (MMP), effect of reactive oxygen species (ROS) generation, and induction of apoptosis in ovarian cancer cells through betanin was also observed. Results have shown betanin as a potential candidate for inhibition of ovarian cancer cell proliferation and it can be taken up as a serious compound for further studies for its application in cancer cure.
... Aβ-induced mitochondrial dysfunction has been demonstrated on several occasions [26]. PC12 cell from rat adrenal medulla cells treated with varied concentrations of Aβ peptides showed improper function in electron transport through complexes in the ETC [35]. Additionally, these cells showed an impairment in glycolysis and thus a reduction in ATP generation [35]. ...
... PC12 cell from rat adrenal medulla cells treated with varied concentrations of Aβ peptides showed improper function in electron transport through complexes in the ETC [35]. Additionally, these cells showed an impairment in glycolysis and thus a reduction in ATP generation [35]. ...
Mitochondria are the primary source for energy generation in the cell, which manifests itself in the form of the adenosine triphosphate (ATP). Nicotinamide dinucleotide (NADH) molecules are the first to enter the so-called electron transport chain or ETC of the mitochondria. The ETC represents a chain of reducing agents organized into four major protein-metal complexes (I-IV) that utilize the flow of electrons to drive the production of ATP. An additional integral protein that is related to oxidative phosphorylation is ATP synthase, referred to as complex V. Complex V carries out ATP synthesis as a result of the electron flow through the ETC. The coupling of electron flow from NADH to molecular oxygen to the production of ATP represents a process known as oxidative phosphorylation. In this review, we describe mainly the bioenergetic properties of mitochondria, such as those found in the ETC that may be altered in Alzheimer's disease (AD). Increasing evidence points to several mitochondrial functions that are affected in AD. Furthermore, it is becoming apparent that mitochondria are a potential target for treatment in early-stage AD. With growing interest in the mitochondria as a target for AD, it has been hypothesized that deficit in this organelle may be at the heart of the progression of AD itself. The role of mitochondria in AD may be significant and is emerging as a main area of AD research.
... Os produtos obtidos pela redução dos hidroperóxidos do ácido linoleico (ácidos 9-hidroxi-10,12-octadecadienóico e 13hidroxi-9,11-octadecadienóico), por exemplo, são susceptíveis de ser detectados em grandes quantidades (Spiteller e Spiteller, 1997). Os aldeídos são os produtos produzidos em maior quantidade, especialmente o 4-hidroxinonenal (4-HNE), que deriva do ácido araquidónico e é altamente reactivo, sendo responsável pela maioria dos efeitos citotóxicos verificados em situação de stresse oxidativo (Markesbery, 1997;Pereira et al., 1999). No entanto, a medição do malonildialdeído é o método mais utilizado para quantificar a peroxidação lipídica. ...
... Fe 3+ + ascorbato Fe 2+ + ascorbato • (reacção 5) O Fe 2+ resultante desta reacção é depois canalizado para a reacção de Fenton, levando à formação de radicais hidroxilo (Shapiro e Saliou, 2001;Halliwell e Gutteridge, 1999 Na verdade, os fibroblastos são células que, pela sua natureza indiferenciada, são difíceis de oxidar. As células PC12, por seu turno, têm sido bons modelos para estudos deste tipo (Kruman et al., 1998;Pereira et al., 1999 /" 5" 0/ Os resultados já apresentados e outros previamente obtidos (Silva et al., 2003) revelaram que os novos compostos não são tóxicos e poderão ser bons protectores de fenómenos de peroxidação lipídica. Com este trabalho pretendeu-se também determinar a sua capacidade para actuarem intracelularmente e desse modo exercerem protecção a nível dos fenómenos apoptóticos. ...
... Aβinduced metabolic dysfunction and accompanied oxidative stress has been largely demonstrated. In PC12 cells treated with increasing concentrations of Aβ peptides resulted in mitochondrial dysfunction where dysfunction of complex I, II, III and IV was evident (Pereira et al., 1999). Indeed it was verified an impairment of glycolysis resulting in ATP depletion where ROS play a major role in the process (Pereira et al., 1999). ...
... In PC12 cells treated with increasing concentrations of Aβ peptides resulted in mitochondrial dysfunction where dysfunction of complex I, II, III and IV was evident (Pereira et al., 1999). Indeed it was verified an impairment of glycolysis resulting in ATP depletion where ROS play a major role in the process (Pereira et al., 1999). Although the exact mechanism by which Aβ induces cell death is not yet fully clarified, there is evidence that this peptide, added extracelullary, can act intracelullary, increasing mitochondrial dysfunction (Hansson Petersen et al., 2008), lowering antioxidant enzymatic activity, and increasing oxidative stress as well. ...
Mitochondria are the power house of living cells and are exceptional regulators of cell survival, since they are the main ATP and reactive oxygen species (ROS) producers and regulate intracellular calcium homeostasis. Many evidences have been raised implicating mitochondria defects as key players in the pathogenesis of several neurodegenerative diseases, as well as in aging. However, in order to understand the key role of mitochondria in disease we will discuss mitochondria normal physiology and its relevant involvement in brain metabolism. We will highlight some of the findings that provide evidence for the role of mitochondria in neurodegeneration associated with Alzheimer's and Parkinson's disease, the most frequent neurodegenerative disorders. Ultimately, we propose to use these insights to better understand mitochondrial dependent mechanisms involved in disease to develop potential mitochondrial therapeutic targets.
... GAPDH was also previously shown to undergo increased disulfide bonding in detergent-insoluble extracts from AD patient and transgenic AD mouse brain tissue, compared with age-matched controls, and in Aβtreated primary cortical neurons, which was associated to a reduction in GAPDH enzymatic activity thus affecting neuronal glycolytic metabolism [34]. Previously, we have shown that Aβ-induced toxicity is linked with reduced production of pyruvate through glycolysis that can be prevented by several antioxidants [35]. In AD there is also an increase in oxidatively modified glycolytic enzymes, as identified by proteomic studies, including enolase (catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate in glycolysis) which is also up-regulated in MCI, early-onset AD, and AD patients [36]. ...
... In isolated brain mitochondria, it was shown that Aβ impairs the respiratory chain, uncouples OXPHOS, decreases the energetic levels and exacerbates the susceptibility to PTP opening [77][78][79]. Previous data from our laboratory showed that Aβ inhibits the respiratory chain complexes and reduces ATP levels in PC12 cells [35,80]. By using human wild-type APP stably transfected neuroblastoma SH-SY5Y cells, a decrease in COX and an increase in complex III activity was recently shown [81]. ...
Alzheimer's disease (AD) is the most common form of dementia in old age. Cognitive impairment in AD may be partially due to overall hypometabolism. Indeed, AD is characterized by an early region-specific decline in glucose utilization and by mitochondrial dysfunction, which have deleterious consequences for neurons through increased production of reactive oxygen species (ROS), ATP depletion and activation of cell death processes. In this article, we provide an overview of the alterations on energetic metabolism occurring in AD. First, we resume the evidences that link the 'metabolic syndrome' with increased risk for developing AD and revisit the major changes occurring on both extra-mitochondrial and mitochondrial metabolic pathways, as revealed by imaging studies and biochemical analysis of brain and peripheral samples obtained from AD patients. We also cover the recent findings on cellular and animal models that highlight mitochondrial dysfunction as a fundamental mechanism in AD pathogenesis. Recent evidence posits that mitochondrial abnormalities in this neurodegenerative disorder are associated with changes in mitochondrial dynamics and can be induced by amyloid-beta (Aβ) that progressively accumulates within this organelle, acting as a direct toxin. Furthermore, Aβ induces activation of glutamate N-methyl-D-aspartate receptors (NMDARs) and/or excessive release of calcium from endoplasmic reticulum (ER) that may underlie mitochondrial calcium dyshomeostasis thereby disturbing organelle functioning and, ultimately, damaging neurons. Throughout the review, we further discuss several therapeutic strategies aimed to restore neuronal metabolic function in cellular and animal models of AD, some of which have reached the stage of clinical trials.
... The ROS level of cells was determined using the dichloro-fluorescein diacetate (DCFH-DA) test. DCFH-DA is a molecule that is lipophilic in nature and has the ability of cell-permeable which is oxidized by ROS into dichlorofluorescein (DCF) inside the cell 13 . Radicals like hydroxyl, nitrate, peroxyl, carbonate and alkoxyl oxidized DCF to produce a luminous molecule (excitation 530 nm, emission 485 nm). ...
The use of natural substances in cancer treatment is the current need because alternative approaches are intensive and have numerous negative side effects. In this current study, the natural product Prunus dulcis (Almond) was screened for its ability to inhibit the proliferation of (PA-1) cancer cells. In the MTT assay, IC50 values obtained are 220 μg/ml. Reactive Oxygen Species (ROS), Mitochondrial Membrane Potential (MMP) and Apoptosis induction in cancer cells of the ovary are noticed and this assay shows the anti-cancerous activity of Prunus dulcis. Catechin has emerged as the major bioactive compound of Prunus dulcis. Anti-bacterial activity of Prunus dulcis at various concentrations of 50, 80, 90 and 100μl was tested. 90μl of Prunus dulcis extract showed a good antibacterial effect against Saccharomyces cerevisiae. This research shows that the almond bioactive molecules inhibit the proliferation of ovarian cancer cell lines. The identified promising active pharmaceutical ingredient catechin needs focused research.
... Mitochondrial alterations have been reported in several neurodegenerative diseases [16][17][18] associated mostly with oxidative damage [19]. Oxidative stress is mostly associated with amyloid β (Aβ) accumulation in the neocortex [20,21], playing, therefore, an important role in the pathogenetic mechanisms of Alzheimer's disease [22], since it is not only involved in damage to the proteins of NFT and the formation of senile plaques but also involves extensive damage to the cytoplasm of neuronal populations vulnerable to death during AD [23]. ...
"Alzheimer’s disease (AD) is a devastating neurodegenerative disorder characterized by cognitive impairment, affecting memory and associated with behavioral and mood changes. The pathophysiology of Alzheimer’s disease involves a number of cellular and biochemical mechanisms. In the present study we aimed to describe the morphological alterations of dendrites, dendritic spines, synapses and mitochondria in Alzheimer’s disease. For the purpose of the study we examined different brain areas in twenty brains from Alzheimer’s disease patients, and twenty age-matched individuals who died accidentally. We found significant loss of dendritic branches and decrease of spinal density, changes of the synapses and severe alterations of the mitochondria. The findings of the present study constitute the pathological background for the cognitive decline seen in Alzheimer’s disease, and are in favour of a significant role of mitochondria in early stages of the pathogenesis of the disease."
... Measurement of reactive oxygen species: Intracellular ROS level was determined by a previously described method using 2,7-dichloro-dihydrofluorescein diacetate (DCFH-DA) assay [47]. Approximately 2 × 10 6 cells/well (PA-1 cells) were seeded in 6 well plate and allowed to attach for 24 h. ...
An enhanced anticancer properties of Ageratina adenophora mediated silver nanoparticles (AgNPs) were evaluated in the present study. The biogenic AgNPs effectively inhibited the viability and proliferation of ovarian teratocarcinoma cells (PA-1) by significantly increasing the initiation of apoptosis. The AgNPs were synthesized with the phytochemicals present in aqueous extract of Ageratina adenophora leaves which performed the dual function of reducing silver ions as well as the capping the nanoparticles. Characterization of nanoparticles with UV-vis spectroscopy revealed surface plasmon resonance peak at 470 nm. Fourier transform infrared spectrum showed the presence of functional groups such as alcohol, aldehyde, alkane, aromatic amines and phenolic compounds. X-Ray diffraction (XRD) analysis revealed the crystalline nature and face centred cubic structure of AgNPs. The size, morphology and distribution of AgNPs were confirmed with scanning electron microscope (SEM). Further, dynamic light scattering (DLS) and zeta potential measurements also confirmed the size and charge of the synthesized nanoparticles. The AgNPs significantly increased the cytotoxicity and inhibited the proliferation of PA-1 cells. Apoptosis of PA-1 cells were increased with treatment of AgNPs. The increased cytotoxicity of PA-1 cells was due to the synergistic activity of phytochemicals and AgNPs. Hence, the current research on the silver nanoparticles mediated through A. adenophora leaf extract could be an effective alternative in treatment of ovarian cancer.
... The intracellular level of the ROS in the HCT-116 cells was measured by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay [29,30]. The HCT-116 cells were seeded in 6-well plates (2 × 10 6 cells/well) and then treated with varying concentrations of CuO NPs (20 and 25 µg/mL) for 24 h at 37°C. ...
In the present study, pumpkin seed extract was used to synthesize copper oxide nanoparticles (CuO NPs) along with evaluating its anticancer activity using different molecular biology tools in the human colorectal cancer cell line (HCT-116). Morphological and structural properties of the biogenically synthesized CuO NPs were characterized by UV-visible spectrophotometry (UV-vis), energy-dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). For estimating the anticancer efficacy, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity, morphological alteration, reactive oxygen species (ROS) formation, and alterations in the mitochondrial membrane potential (MMP) were determined. SEM and TEM data revealed the formation of spherical nanoparticles possessing an average size of 20 nm. The CuO NPs showed 50% inhibitory concentration (IC50) at 25 µg/mL against the HCT-116 cell line. The treatment with IC50 concentration of CuO NPs showed significant shrinking, detachment, membrane blebbing, and shape distortion of cancer cells. Similarly, the IC50 dose of CuO NPs showed significantly early apoptosis in cancer cells compared to late apoptosis. The cancer cell line also showed a dose-dependent increase and decrease in ROS formation and MMP, respectively. The results obtained through various assays indicated significant anticancer efficacy of biogenically synthesized CuO NPs. Thus, further studies are recommended to validate our results using ex vivo and in vivo models.
... Another randomized, double-blind, placebo-controlled, multicenter study, 300 patients with Alzheimer-type dementia were treated for 2 years with 30 or 90 mg/day idebenone or placebo [53]. There was no significant improvement in a small trial in HD [54]. These results are controversial since another large trial showed no benefits [55]. ...
Alzheimer’s Disease (AD) is the most frequent cause of dementia in
aged population. Till now, there is no effective treatment for AD. Now various
studies have kinked AD to mitochondrial dysfunction, due to accumulation of
Aβaggregates, neurofibrillary tangles, cholinergic transmission, oxidative stress
and neuroinflammation causing neurodegeneration and cognitive decline
seen in AD. Here in this article, how the mitochondrial dysfunction/dynamics
are involved in the pathogenesis of cognitive decline seen in cases of AD and
various therapeutics targeted against mitochondrial dysfunctions are described,
thereby causing a ray of hope for preventing progression of AD.
... Another possibility is that of abnormal mitochondrial energy metabolism. Studies have shown that PC-12 cells exposed to Aβ demonstrate reduced ATP generation [57], mediated via its interaction with the mitochondrial membrane [58]. Furthermore, deficiencies in complex IV of the electron transport chain have been shown in vitro, in vivo and in post-mortem human brain to be an inherent feature of AD [59][60][61][62]. ...
We have previously shown that the expression of nicotinamide N-methyltransferase (NNMT) is significantly increased in the brains of patients who have died of Parkinson’s disease (PD). In this study, we have compared the expression of NNMT in post-mortem medial temporal lobe, hippocampus and cerebellum of 10 Alzheimer’s disease (AD) and 9 non-disease control subjects using a combination of quantitative Western blotting, immunohistochemistry and dual-label confocal microscopy coupled with quantitative analysis of colocalisation. NNMT was detected as a single protein of 29 kDa in both AD and non-disease control brains, which was significantly increased in AD medial temporal lobe compared to non-disease controls (7.5-fold, P < 0.026). There was no significant difference in expression in the cerebellum (P = 0.91). NNMT expression in AD medial temporal lobe and hippocampus was present in cholinergic neurones with no glial localisation. Cell-type expression was identical in both non-disease control and AD tissues. These results are the first to show, in a proof-of-concept study using a small patient cohort, that NNMT protein expression is increased in the AD brain and is present in neurones which degenerate in AD. These results suggest that the elevation of NNMT may be a common feature of many neurodegenerative diseases. Confirmation of this overexpression using a larger AD patient cohort will drive the future development of NNMT-targetting therapeutics which may slow or stop the disease pathogenesis, in contrast to current therapies which solely address AD symptoms.
... MTT assay was performed to measure the cytoxic efficacy of M.t-ZnONPs on viability of Hep-2 cells by the method of Mosmann [16]. 1 × 10 5 Hep-2 cells (200 μl/well) were plated in microtiter plates (96-well) and incubated with various concentrations of M.t-ZnONPs (1-10 μg/ml) at 37°C for 24 h in a CO 2 incubator. After incubation, to each well 20 μl of MTT solution was added and then incubated for 4 h in dark. ...
Biosynthesis of Zinc oxide nanoparticles (ZnONPs) from natural plants stands as a promising nanodrug delivery system in cancer therapeutics. Marsdenia tenacissima (M.t), a Chinese medicinal plant has been extensively used as clinical remedy for treating several types of cancer. In this present study, ZnONPs were synthesized from Marsdenia tenacissima and its anti cancer potency was assessed against in vitro laryngeal cancer cell line Hep-2. The biosynthesized Marsdenia tenacissima Zinc Oxide Nanoparticles [M.t-ZnONPs] was characterized using UV-visible Spec, SEM, TEM and EDAX analysis. The cytotoxic and apoptotic inducing potential of M.t-ZnONPs was assessed by MTT assay and staining such as DCFH-DA, AO/EtBr, Rhodamine 123, DAPI and comet assay. The anticancer potential of M.t-ZnONPs was analysed by Real time PCR analysis of proapoptotic, antiapoptotic and caspases proteins. Our present findings showed characteristic and morphological representation of synthesized M.t-ZnONPs by UV-visible Spec, SEM, TEM and EDAX analysis. M.t-ZnONPs exhibits its cytotoxicity by inhibiting the viability of Hep-2 cells and IC50 value was obtained by MTT assay. The results of apoptotic staining techniques in M.t-ZnONPs treated Hep-2 cells confirm with excess ROS generation, disruption of mitochondrial membrane potential and nuclear damage. The apoptotic inducing potential of M.t-ZnONPs was also evidenced by upregulation of proapoptotic proteins Bax, Caspase 3 & 9 and downregultion of antiapoptotic protein Bcl-2 by RT-PCR analysis. Finally, these results suggested that biosynthesized M.t-ZnONPs is an effective anticancer agent which induces apoptosis in Hep-2 laryngeal cell line and thus conclude that M.t-ZnONPs, a valid anticancer strategy in treating various cancer.
... Firstly, neurons and myocytes produce energy using the same enzyme activities. Secondly, the pathogenic β-AP, which is present in skeletal muscle [6], also exerts its toxicity on the production of pyruvate through glycolysis [7]. ...
... In addition, a glycolytic shift in response to UCP4 expression is supported by the increased cellular lactate levels in cells with high-UCP4 levels and the increased vulnerability of UCP4 cells to iodoacetate, an inhibitor of glycolysis. Although glycolysis is more prominent in astrocytes, it also occurs in neurons and PC12 cells (Pereira et al., 1999;Gjedde and Marrett, 2001). Cells with a high glycolytic capability exhibit increased resistance to oxidative stress (Almeida et al., 2001;Brown and Bonitaite, 2001), consistent with a role for the glycolytic shift induced Fig. 10. ...
Data S1 Immunoblotting
... It has been demonstrated, that Aβ added to the culture medium inhibited PDHC and the key enzymes of TCA cycle, in primary and clonal neuronal and glial cells [36,46,71,135,136]. It resulted in depletion of acetyl-CoA, yielding suppression of respiratory chain and ATP levels in affected neuronal cells (Fig. 1) [46,135,137]. These alterations could be aggravated by Aβ-evoked disruption of endogenous metal homeostasis, including calcium, iron, zinc and copper [78,83]. ...
There are several systemic and intracerebral pathologic conditions, which limit provision and utilization of energy precursor metabolites in neuronal cells. Energy deficits cause excessive depolarization of neuronal cells triggering glutamate-zinc evoked excitotoxic cascade. The intracellular zinc excess hits several intraneuronal targets yielding collapse of energy balance and impairment functional and structural impairments cholinergic neurons. Disturbances in metabolism of acetyl-CoA, which is a direct precursor for energy, acetylcholine, N-acetyl-L-aspartate and acetylated proteins synthesis, play an important role in these pathomechanisms. Disruption of brain homeostasis activates slow accumulation of amyloid-β 1-42 , which extra and intracellular oligomeric deposits disrupt diverse transporting and signaling processes in all membrane structures of the cell. Both neurotoxic signals may combine aggravating detrimental effects on neuronal cell. Different neuroglial and neuronal cell types may display differential susceptibility to similar pathogenic insults depending on specific features of their energy and functional parameters. This review, basing on findings gained from cellular and animal models of Alzheimer's disease, discusses putative energy/acetyl-CoA dependent mechanism in early and late stages of neurodegeneration.
... This could also explain the highest incidence of the disease in women compared to age-matched men (31)(32)(33)(34), where the decline in SHs is more subtle and progressive. A major AD pathophysiologic feature is Ab accumulation, consensually recognised as a source of oxidative stress and mitochondrial dysfunction, and often leading to apoptosis of neuronal cells (35)(36)(37)(38). Indeed, Ab is seen as a toxin that might have beneficial effects at low doses, including quenching of oxidative damage, but becomes noxious at high doses (39,40). ...
The choroid plexus (CP) epithelium is a unique structure in the brain that forms the interface between the peripheral blood on the basal side and the cerebrospinal fluid (CSF) on the apical side. It is a relevant source of many polypeptides secreted to the CSF with neuroprotective functions and participates in the elimination and detoxification of brain metabolites, such as β-amyloid (Aβ) removal from the CSF through transporter-mediated influx. The CP is also a target tissue for sex hormones (SHs) which have recognized neuroprotective effects against a variety of insults, including Aβ toxicity and oxidative stress in the central nervous system. This study aimed at understanding how SHs modulate Aβ-induced oxidative stress in a CP cell line (Z310 cell line) by analysing the effects of Aβ1-42 on oxidative stress, mitochondrial function and apoptosis, and assessing how 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) modulated these effects and the cellular uptake of Aβ1-42 by CP cells. Our findings show that E2 and DHT treatment reduce Aβ1-42 -induced oxidative stress and the internalization of Aβ1-42 by CP epithelial cells, highlighting the importance of considering the SHs background and therefore sex-related differences in the Aβ metabolism and clearance by CP cells. This article is protected by copyright. All rights reserved.
... APH-M seemed more potent than APH at the same concentration, possibly owing to the presence of some highly antioxidative compounds generated during the Maillard reactions including polymers (such as melanoidins), volatile compounds (nitrogen heterocyclics) and peptide-sugar complexes (Liu et al., 2012;Martins, Jongen, & Boekel, 2000). The generation of reactive oxygen species is a crucial event that is responsible for the energetic metabolic dysfunction induced by βamyloid peptides in PC12 cells (Pereira, Santos, & Oliveira, 1999). Some antioxidants could play an important role in preventing neuronal cell death and resisting neurodegenerative disorder (Dong et al., 2014). ...
Anchovy (Coilia mystus) protein hydrolysate (APH) and its Maillard reaction product (APH-M) with d-ribose were subjected to in vivo mouse behavioral trials and associated with biochemical analyses, in vitro H2O2-stressed PC12 cell assay and inhibition of acetylcholin esterase (AchE) to explore the mechanism of the behavioral trial findings. Results revealed that both APH and APH-M could alleviate undesired alterations on hippocampus ultrastructure of mice, protect PC12 from H2O2-induced oxidative stress, and inhibit AchE activity in vivo and in vitro, suggesting the well-being potential for combating memory-impairment in mice. Moreover, APH-M exhibited much stronger abilities above than APH. The test of acetylcholinergic system indicated that APH-M would improve memory mainly through regulating the AchE activity, while APH through controlling the mRNA expression of ChAT and AchE activity. Therefore, protein hydrolysates from anchovy could possess therapeutic potential for memory deficits, and Maillard reaction might further promote their memory-improving capacity.
... In some reports it is mentioned that one of the insulin degrading enzymes isoform, a well established regulator of Aβ dynamicity targets mitochondria and interfere with its normal functioning [213] . Aβ is also found to be a good inhibitor of respiratory chain complex and thus leads to marked decrease in cellular ATP levels [214,215] . Importantly, Aβ 40 and Aβ 2535 contribute in uncoupling of oxidative phosphorylation and impair respiratory chain as well as MPT opening [204,210,211] . ...
Diabetes mellitus (DM), a metabolic disorder is a major orchestra influencing brain and behavioral responses via direct or indirect mechanisms. Many lines of evidence suggest that diabetic patients apparently face severe brain complications, but the story is far from being fully understood. Type 2 diabetes, an ever increasing epidemic and its chronic brain complications are implicated in the development of Alzheimer's disease (AD). Evidences from clinical and experimental studies suggest that insulin draws a clear trajectory from the peripheral system to the central nervous system. This review is a spot light on striking pathological, bio chemical, molecular and behavioral commonalities of AD and DM. Incidence of cognitive decline in diabetic patients and diabetic symptoms in AD patients has brought the concept of brain diabetes to attention. Brain diabetes reflects insulin resistant brain state with oxidative stress, cognitive impairment, activation of various inflammatory cascade and mitochondrial vulnerability as a shared footprint of AD and DM. It has become extremely important for the investigators to understand the pathophysiology of brain complications in diabetes and put intensive pursuits for therapeutic interventions. Although, decades of research have yielded a range of molecules with potential beneficial effects, but they are yet to meet the expectations.
... In some reports it is mentioned that one of the insulin degrading enzymes isoform, a well established regulator of Aβ dynamicity targets mitochondria and interfere with its normal functioning [213] . Aβ is also found to be a good inhibitor of respiratory chain complex and thus leads to marked decrease in cellular ATP levels [214,215] . Importantly, Aβ 40 and Aβ 2535 contribute in uncoupling of oxidative phosphorylation and impair respiratory chain as well as MPT opening [204,210,211] . ...
... In some reports it is mentioned that one of the insulin degrading enzymes isoform, a well established regulator of Aβ dynamicity targets mitochondria and interfere with its normal functioning [213] . Aβ is also found to be a good inhibitor of respiratory chain complex and thus leads to marked decrease in cellular ATP levels [214,215] . Importantly, Aβ 40 and Aβ 2535 contribute in uncoupling of oxidative phosphorylation and impair respiratory chain as well as MPT opening [204,210,211] . ...
Diabetes mellitus (DM), a metabolic disorder is a major orchestra influencing brain and behavioral responses via direct or indirect mechanisms. Many lines of evidence suggest that diabetic patients apparently face severe brain complications, but the story is far from being fully understood. Type 2 diabetes, an ever increasing epidemic and its chronic brain complications are implicated in the development of Alzheimer's disease (AD). Evidences from clinical and experimental studies suggest that insulin draws a clear trajectory from the peripheral system to the central nervous system. This review is a spot light on striking pathological, bio chemical, molecular and behavioral commonalities of AD and DM. Incidence of cognitive decline in diabetic patients and diabetic symptoms in AD patients has brought the concept of brain diabetes to attention. Brain diabetes reflects insulin resistant brain state with oxidative stress, cognitive impairment, activation of various inflammatory cascade and mitochondrial vulnerability as a shared footprint of AD and DM. It has become extremely important for the investigators to understand the pathophysiology of brain complications in diabetes and put intensive pursuits for therapeutic interventions. Although, decades of research have yielded a range of molecules with potential beneficial effects, but they are yet to meet the expectations.
... Besides being a target for the oxidative stress that occurs in the AD brain [60], mitochondria themselves may be a source of ROS that amplify the mitochondrial dysfunction occurring in this disease [61][62]. The generation of ROS may also be critically involved in the impairment of energy metabolism induced by A which has been associated with decreases in complex III core protein 1, decreases in complex V βchain in brain mitochondria [63], and also with morphological changes in the cristae, resulting in smaller and poorly functional mitochondria [64]. ...
... MTT assay, according to the method of Mosmann, (1983), was utilized to find out the efficacy of apigenin on Hep-2 cell viability. The effect of apigenin on the generation of intracellular ROS was assessed according to the method of Pereira, et al., (1999) using a non fluorescent probe 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The apoptosis inducing potential of apigenin in Hep-2 cells was measured using DAPI staining according to the method of Arung, et al., (2009) as well as by acridine orange/ethidium bromide dual staining according to the method of Baskic, et al., (2006). ...
The aim of the present study is to explore the cytotoxic potential of apigenin in Hep-2 cells. The mechanistic pathway for its cytotoxic potential was analyzed using MTT assay (cytotoxicity), DNA fragmentation (agarose gel electrophoresis), Nuclear damage (DAPI Staining), ROS generation ability (DCFH-DA method), apoptosis inducing potential (dual staining) and expression of apoptosis and angiogenic proteins (Western blotting). The overall findings of the present study suggest that apigenin might have reduced the cell viability of Hep-2 cells through its apoptotic and anti-angiogenic potential.
... It is important to underline that mitochondrial cytochrome c oxidase may be inhibited by a dimeric conformer of Aβ42, a mechanism which is copper dependent [72][73][74]. Oxidative stress, is reasonably associated with amyloid β (Αβ) accumulation in the neocortex, [75,76], a fact which plays a crucial role in the pathogenesis of Alzheimer's disease, inducing alterations to the cytoplasm of sensitive cells [77] by increasing mitochondrial reactive oxygen species (ROS) production [78,79]. This would cause further mitochondrial dysfunction [80], since the lack of histones in mitochondrial DNA makes them very sensitive to oxidative stress [81,82]. ...
Alzheimer’s disease (AD) is a neurodegenerative disorder of the brain, inducing
progressive severe presenile and senile cognitive decline, resulting in vegetative stage
eventually. From the etiological point of view the main causative factor, remains unknown,
in spite of the steady augmentation of the research efforts. Golgi staining revealed the
substantial alterations of the dendritic branches and the tremendous loss of spines even in
the initial stages of the disease. Electron microscopy reveals morphological changes of the
mitochondria in neurons and astrocytes associated with fragmentation of cisternae of Golgi
complex and pathological alteration of the dendritic spines, even in areas of the brain,
which demonstrate minimal tau pathology and few amyloid β deposits. It is attempted to
describe the ultrastructural alterations of the cerebellar cortex in early cases of AD,
focusing the study mostly on mitochondria, Golgi apparatus, dendritic branches, dendritic
spines and synapses in the cerebellar hemispheres and the vermis. Mitochondria
demonstrated an impressive polymorphism in the soma, the axonal and dendritic profiles of
Purkinje cells, the climbing fibers, the mossy fibers and the synapses. Electron microscopy
revealed also marked fragmentation of cisternae of Golgi complex in large number of
Purkinje cells, granule and stellate cells in the vermis and the cerebellar hemispheres. The
fragmentation of the Golgi complex and the poverty in vesicles in cis- and trans-Golgi
network in the soma of Purkinje cells in Alzheimer’s brains coincide with the synaptic loss,
the shortage of the dendritic arborization and the pathological alterations of the spines.
Numerous spines included large multivesicular bodies, altered spine apparatus, and unusual
mitochondria. Giant elongated spines were seen in a substantial number of Purkinje cells.
In many presynaptic terminals of parallel and mossy fibers, electron microscopy revealed a
dramatic loss of the synaptic vesicles associated with marked polymorphism. On the basis
of the mitochondrial and Golgi complex pathology, new therapeutic strategies protecting
those organelles might be proposed for the treatment of early cases of AD.
... Several studies have demonstrated that compounds with antioxidant properties can have a protective effect in different situations of cellular dysfunction. For example, IDB was shown to decrease retinal cell injury induced by oxidative stress and hypoglycemia [3], and to protect synaptosomes from oxidative damage induced by ascorbate/iron [34]. On the other side, GSH/EE, were also shown to promote the mitochondrial and glycolytic metabolism in PC12 cells exposed to β-amyloid [7], whereas in sympathetic neurons deprived from nerve growth factor, GSH/EE also prevented from cytochrome c release [30]. ...
Recently we showed that staurosporine uses the extracellular calcium ions to affect on neurite outgrowth in PC12 cells. However, these mechanisms during staurosporine-induced cell death are still unclear. In this study, we investigated the relation between of reactive oxygen species (ROS) and extracellular calcium entry during staurosporine-induced cell death in PC12 cells. The results showed that decrease or increase of extracellular calcium ions resulted in correspondingly significant increase in intracellular calcium concentration ([Ca2+]i). At high extracellular Ca2+ concentration (0.5-0.7 mM) increase in intracellular calcium concentration ([Ca2+]i), cells which was not prevented in the presence of IDB and GSH/EE, indicating its independence on ROS generation in treated cells. Another side, at low extracellular Ca2+ (0.-0.3 mM) was prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation in treated cells. Thus, endogenous ROS generation and the rise in intracellular calcium by reducing extracellular calcium entry are important inter-players in staurosporine-induced cell death.
... When taken in high doses, it becomes a potent hepatotoxin and can cause fatal hepatic necrosis 24 and is employed as an experimental hepatotoxic agent. Oxidative stress caused by APAP results in the release of LDH, a marker of cell damage 25 . GSH is the major nonenzymatic antioxidant and regulator of intracellular redox homeostasis, ubiquitously present in all cell types. ...
ABSTRACT: Drug-induced hepatotoxicity represents a major clinical
problem and an impediment to new medicine development. In vitro
evaluation of hepatotoxicity is an essential stage in the research and
development of new pharmaceuticals as the liver is one of the most
commonly impacted organs during preclinical toxicity studies. The effect of
an aqueous leaf extract of Tridax procumbens was evaluated against
acetaminophen-induced free radical reaction and liver cell necrosis in mouse
primary hepatocyte culture. The liver was excised from the male albino
mouse and cells were isolated and cultured. After monolayer developed, cells
were treated with acetaminophen and different concentrations of Tridax
procumbens aqueous extract. After the treatment for 3 to 24 h, free radical
reaction, mitochondrial and extramitochondrial dehydrogenase activity,
lactate dehydrogenase release, glutathione level, trypan blue uptake and
haptocyte morphology were determined. Increased free radical reactions,
LDH release, trypan blue uptake, liver cell necrosis and decreased levels of
cellular glutathione, mitochondrial and extramitochondrial dehydrogenase
activity were detected in acetaminophen-treated groups. Pretreatment of
hepatocytes with Tridax procumbens extract caused attenuation of
acetaminophen induced toxicity in various extents of oxidative stress,
increased cell viability, glutathione level and mitochondrial and
extramitochondrial dehydrogenase in a dose dependant manner.
... The intracellular ROS level was determined according to the method of Pereira et al. [17] using a non fluorescent probe 2', 7'dichlorodihydrofluorescein diacetate (DCFH-DA). This non fluorescent probe can diffuse into the intracellular matrix of cells, where it is acted upon by intracellular esterase to form polar 2', 7'dichlorodihydrofluorescein (DCFH), which gets trapped inside the cells and oxidised by intracellular oxidants to a highly fluorescent 2', 7'-Dichlorofluorescein (DCF). ...
... γ-Secretase cleavage of C99 results in the release of the Aβ peptides, Aβ 1-40 (amino acid residues 672-711) and Aβ 1-42 (amino acid residues 688-713), major components of amyloid plaques, and of cytotoxic γ-CTF(57) and γ-CTF(59), respectively. Recent findings of different effects of specific cysteine protease inhibitor E-64d on production of Aβ 1-40 and Aβ 1-42 in APP-transfected cell cultures suggested that the two Aβs may be generated by the action of two different proteases termed γ 40 -and γ 42 -secretase, respectively (Figueiredo- Pereira et al., 1999). Besides, cytotoxic γ-CTF(50) fragment is released at third γ-secretase cleavage site, Leu 720 -Val 721 . ...
Amyloid-β peptide (Aβ) is a small protein that accumulates in Alzheimer's disease brains. It aggregates to form the core of Alzheimer disease-associated amyloid plaques that are neurotoxic. No satisfactory hypothesis has yet been proposed to explain the mechanism of Aβ aggregation and toxicity. To throw light on the problem, many investigators dealed with Aβ 25-35 or other short fragments of amyloid protein precursor, and some authors dissected such investigations as futile undertakings. For the last years, we and many others observed that Aβ 25-35 was more effective than Aβ 1-40 in inducing oxidative stress and metabolic disturbances in brain neurones. We here review biochemical responses to short Aβs, in particular our recent explorations of the mechanisms of Aβ 25-35 toxicity in rat and human cells, and discuss physiological significance and role of Aβ 25-35 in the progression of amyloidosis in animals. We are trying to answer the question whether Aβ 25-35 does good for amyloidosis knowledge.
... Cell viability was evaluated using the MTT assay, which measures the ability of metabolic active cells to form formazan through cleavage of the tetrazolium ring of MTT [41]. Briefly, cells were washed in normal sodium medium (in mM: 132 NaCl, 4 KCl, 1.2 Na 2 HPO 4 , 1.4 MgCl 2 , 6 glucose, 10 HEPES, and 1 CaCl 2 , pH 7.4) and incubated with 0.5 mg/ml MTT for 2 h at 37°C. ...
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 Ca(2+) homeostasis due to the release of Ca(2+) 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.
... ROS was measured by using a non-fluorescent probe, 2 7 diacetyl dichlorofluorescein (DCFH-DH) that can penetrate in to the intracellular matrix of cells and becomes oxidized by ROS to fluorescent dichlorofluorescein (DCF), as per the protocol [26] . Briefly, an aliquot of the above mentioned isolated cells (100 µl) were made up to a final volume of 2 ml in normal phosphate buffered saline (pH 7.4). ...
Recent reports indicate that β-amyloid peptide (Aβ) vaccine based therapy for Alzheimer’s disease (AD) may be on the horizon. There are however, concerns about the safety of this approach. Immunization with Aβ has several disadvantages, because it crosses the blood brain barrier and cause inflammation and neurotoxicity. The present work is aimed to study the protective effective of α-lipoic acid (LA) in the oxidative vulnerability of β-amyloid in plasma, liver, spleen and brain, when Aβ fibrils are given intraperitoneally in inflammation induced mice. Result shows that reactive oxygen species (ROS) in the astrocytes of inflammation induced mice along with Aβ (IA) has shown 2.5-fold increase when compared with LA treated mice. The increased level of lipid peroxidase (LPO) (p < 0.05) and decreased antioxidant status (p < 0.05) were observed in the plasma, liver, spleen and brain of IA induced mice when compared with LA treated mice.
Data shows that there were no significant changes observed between the control and LA treated mice. Our biochemical and histological results highlight that significant oxidative vulnerability was observed in IA treated mice, which was prevented by LA therapy. Our findings suggest that the antioxidant effect of LA when induced with Aβ may serve as a potent therapeutic tool for inflammatory AD models. (Mol Cell Biochem 270: 29–37, 2005)
Bacterial secondary metabolites are the current focus of investigation as a potential solution to various drug resistance and other problems. Palladium nanoparticles produced by Bacillus sp are considered to be green, eco-friendly, cost-effective, and they also have antibacterial and anticancer capabilities which could be used in pharmaceuticals. Agar well screening has proven that the secondary metabolites of Bacillus sp (TAP 4) have antibacterial properties. Ultraviolet–visible (UV–Vis) spectroscopy was used to determine the synthesis of palladium nanoparticles. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and X ray diffraction (XRD) were used to examine the morphological appearances, stability, and crystalline structure of palladium nanoparticles from the secondary metabolites of Bacillus sp. Using DCFH-DA, we determined the impact of secondary metabolites of synthesized nanoparticles on ROS levels and mitochondrial membrane potential (MMP) by two-fold serial dilution method. On A549 lung cancer cells, the cytotoxicity of synthesized secondary metabolites mediated palladium nanoparticles was measured using MTT assay to determine their anticancer activities. Our findings indicated that their secondary metabolites have a promising role as a first-line cancer drug. However, further research is required to entirely understand their mode of mechanistic action.
Alzheimer’s disease remains the main cause of dementia in advanced age worldwide. Among the etiopathological background of the disease mitochondrial alterations may play a crucial role, given that they are closely related to metabolic and energy deficiency in neurons, glia, and endothelial cells in Alzheimer’s disease and other neurodegenerative disorders. In a series of morphological and morphometric studies of mitochondria in the cerebrum and the cerebellar cortex in Alzheimer’s disease, by electron microscopy, we described marked morphological and morphometric alterations. The most frequent ultrastructural alterations of the mitochondria consist of disruption of the cristae, accumulation of osmiophilic material, and marked changes of shape and size in comparison with the normal controls. Mitochondrial alterations were particularly prominent in dendritic profiles and dendritic spines. The ultrastructural study of a substantial number of neurons in the cerebellum revealed that mitochondrial alterations do not coexist, as a rule, with the typical Alzheimer’s pathology, such as cytoskeletal alterations, amyloid deposits, and tau pathology, though they are frequently observed coexisting with alterations of the cisternae of the Golgi apparatus. Therapeutical regimes targeting mitochondria may be beneficial in early cases of Alzheimer’s disease.
Alzheimer’s disease (AD) is the most frequent cause of age-related neurodegeneration and cognitive impairment, and there are currently no broadly effective therapies. The underlying pathogenesis is complex, but a growing body of evidence implicates mitochondrial dysfunction as a common pathomechanism involved in many of the hallmark features of the AD brain, such as formation of amyloid-beta (Aβ) aggregates (amyloid plaques), neurofibrillary tangles, cholinergic system dysfunction, impaired synaptic transmission and plasticity, oxidative stress, and neuroin-flammation, that lead to neurodegeneration and cognitive dysfunction. Indeed, mitochondrial dysfunction concomitant with progressive accumulation of mitochondrial Aβ is an early event in AD pathogenesis. Healthy mitochondria are critical for providing sufficient energy to maintain en-dogenous neuroprotective and reparative mechanisms, while disturbances in mitochondrial function, motility, fission, and fusion lead to neuronal malfunction and degeneration associated with excess free radical production and reduced intracellular calcium buffering. In addition, mitochondrial dysfunction can contribute to amyloid-β precursor protein (APP) expression and misprocessing to produce pathogenic fragments (e.g., Aβ1-40). Given this background, we present an overview of the importance of mitochondria for maintenance of neuronal function and how mitochondrial dysfunction acts as a driver of cognitive impairment in AD. Additionally, we provide a brief summary of possible treatments targeting mitochondrial dysfunction as therapeutic approaches for AD.
Neuritic plaques composed of [beta]-amyloid peptide and compromised cerebral energy metabolism are two characteristic features of Alzheimer's disease. Synthetic [beta]-amyloid peptide is toxic to cultured neurons. The mechanism underlying [beta]-amyloid neurotoxicity is not well defined, but is thought to involve induction of oxidative stress. Recent studies have shown that [beta]-amyloid inhibits mitochondrial enzymes in a neural cell line and in isolated mitochondria. The present study shows that [beta]-amyloid causes mitochondrial dysfunction in primary neuronal cultures, with a much smaller effect on astrocyte cultures. [beta]-Amyloid was found to decrease the number of mitochondria and induced abnormal mitochondrial swelling in neurons. [beta]-Amyloid caused a decrease in cellular ATP of 42% in neurons and 29% in astrocytes in 24 hours. Reduced glutathione levels were also lowered by [beta]-amyloid in both astrocytes (down from 25.2 to 14.9 nmol.mg protein-1) and neurons (down from 5.1 to 2.9 nmol.mg protein-1). [beta]-Amyloid directly inhibited cytochrome oxidase, [beta]-ketoglutarate dehydrogenase and pyruvate dehydrogenase in purified form and in isolated brain mitochondria. The inhibition of these essential mitochondrial enzyme complexes was reflected in an inhibition of integrated mitochondrial respiration by [beta]-amyloid. There is considerable evidence implicating elevated nitric oxide levels in the pathogenesis of Alzheimer's disease. In this study, the deleterious effects of [beta]-amyloid were found to be exacerbated by nitric oxide in cultured astrocytes and neurons and also in isolated mitochondria. Stimulation of endogenous nitric oxide production in astrocytes with cytokines, together with [beta]-amyloid treatment caused a marked increase in cell death and impaired respiration. Treatment of neuronal cultures with the nitric oxide donor DETA-NO exacerbated the toxicity of [beta]-amyloid, while authentic nitric oxide was found to significantly enhance inhibition of respiration caused by [beta]-amyloid in isolated mitochondria. The results of this study suggest that [beta]-amyloid may contribute to the impaired cerebral energy metabolism observed in Alzheimer's disease. In addition, these results suggest that neuronal degeneration due to [beta]-amyloid in Alzheimer's disease may be accelerated by any insult that stimulates excessive NO production such as infection, ischaemic episodes and head trauma.
To understand the molecular mechanisms underlying the beneficial effects of sildenafil in animal models of neurological disorders, we investigated the effects of sildenafil on the mitochondrial toxicity induced by β-amyloid (Aβ) peptide. Treatment of HT-22 hippocampal neuronal cells with Aβ25∼35 results in increased mitochondrial Ca2+ load, which is subsequently suppressed by sildenafil as well as by diazoxide, a selective opener of the ATP-sensitive K+ channels (KATP). However, the suppressive effects of sildenafil and diazoxide are significantly attenuated by 5-hydroxydecanoic acid (5-HD), a KATP inhibitor. The increased mitochondrial Ca2+ overload is accompanied by decrease in the intracellular ATP concentration, increase in intracellular ROS generation, occurrence of mitochondrial permeability transition, and activation of caspase-9 and cell death. Exposure to sildenafil inhibited the mitochondria-associated changes and cell death induced by Aβ. However, the inhibitory effects of sildenafil are abolished or weakened in the presence of 5-HD, suggesting that opening of the mitochondrial KATP is required for sildenafil to exert these effects. Taken together, these results indicate that at the mitochondrial levels, sildenafil plays a protective role towards neuronal cell in an environment rich in Aβ, and exerts its effects via the mitochondrial KATP channels-dependent mechanisms.
Reactive oxygen species (ROS) are by-products of cellular metabolism or of xenobiotic exposure. Depending on their level, ROS can be detrimental leading to oxidative modifications in cellular lipids, proteins, or DNA or can be beneficial participating in intracellular signaling or cell regulation. Mitochondria are a quantitatively relevant intracellular source of ROS which are mainly produced as a result of monoelectron reduction of oxygen at the level of the respiratory chain complexes. Additionally, mitochondrial redox systems, such us glutathione, thioredoxin, and pyridine nucleotide redox couples, are involved in the antioxidant defense participating in modulation of mitochondrial functions including apoptotic cell death. Imbalance between ROS and the antioxidant defense leads to oxidative stress and oxidative changes to cellular biomolecules. Apoptosis or programmed cell death is initiated either at the plasma membrane (extrinsic) receptor-mediated apoptosis or at the mitochondria (intrinsic)-mediated apoptosis, respectively. High levels of mitochondrial ROS (mtROS) can initiate intrinsic apoptosis leading to the release of mitochondrial apoptogenic factors like cytochrome c, apoptosis-inducing factor, into the cytosol. Moreover, mtROS can oxidize mitochondrial glutathione (GSH) causing the loss of intramitochondrial redox homeostasis and irreversible oxidative modifications to mitochondrial macromolecules including mitochondrial DNA. In addition to glutathione, thioredoxin and thioredoxin-dependent enzymes are involved in the removal of hydrogen peroxide and in the redox regulation of mitochondrial protein functions. The reductant for these two redox systems is NADPH and mitochondrial sources, including nicotinamide nucleotide transhydrogenase, isocitrate dehydrogenase, and malic enzyme. A better understanding of the redox control of apoptosis initiation and execution could stem the development of novel therapeutic interventions for oxidative stress-associated pathologies.
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 AD 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 rho(0) 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.
To explore the pathogenesis of rheumatoid arthritis (RA) from the perspective of metabolomics, gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) technology was used to observe changes in the metabolic profiles of urine output from rats with adjuvant-induced arthritis (AA). Spague-Dawley (SD) rats were randomly divided into control group (NC) and experimental group, with 8 in each. Rats in experimental group were induced by intracutaneous innoculation of 0.1mL Freund's complete adjuvant (FCA) to right paws. On day 20 after immunization, the metabolic profiles between rat control and experimental groups were compared by combining GC-TOF/MS technology with multivariate statistical approaches, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and orthogonal projections to latent structures-discriminant analysis (OPLS-DA). Nine potential biomarkers were identified, including 2,2-dimethylsuccinic acid, tartronic acid, dehydroshikimic acid, hippuric acid, adenine, phenaceturic acid, L-dopa, 1,4-dihydroxy-2-naphthoic acid and melibiose. The findings indicate that the rats with AA are disturbed in metabolism of purine, amino acid, fat and energy. This study also demonstrates that the dysfunction in a range of biosynthetic and catabolic pathways, which leads to increased oxygen free radicals and inflammation, could cause underlying pathogenesis of RA. This article is protected by copyright. All rights reserved.
The aim of the present study is to explore the cytotoxic potential of apigenin in Hep-2 cells. The mechanistic pathway for its cytotoxic potential was analyzed using MTT assay (cytotoxicity), DNA fragmentation (agarose gel electrophoresis), Nuclear damage (DAPI Staining), ROS generation ability (DCFH-DA method), apoptosis inducing potential (dual staining) and expression of apoptosis and angiogenic proteins (Western blotting). The overall findings of the present study suggest that apigenin might have reduced the cell viability of Hep-2 cells through its apoptotic and anti-angiogenic potential.
More than twenty different human proteins can fold abnormally resulting in the formation of pathological deposits and several feared degenerative diseases. These proteins lack primary sequence homology, yet they can self-assemble into fibrils with a characteristic cross β-sheet structure. Among them, β-amyloid of Alzheimer's disease is the best characterized. β-amyloid is the principal protein component of senile plaques seen in the brains of individuals diagnosed with Alzheimer's disease and is implicated in the neurotoxicity associated with the disease. Although Alzheimer's disease has been the center of intense research, much remains to be learned concerning the role of β-amyloid and the toxicity mechanisms that mediate its biological responses. In the current review, the general background information on Alzheimer's disease and β-amyloid are first presented. Next, attention is focused on the relevant research works regarding structural aspect and molecular features of Aβ species as well as β-amyloid fribrillogenesis/ aggregation process. Importantly, an emerging issue that an oligomeric intermediate is the primary toxic β-amyloid species is also discussed. Lastly, several β-amyloid-induced toxicity mechanisms are proposed and reviewed. It is our belief that the advances in basic understanding of the conformational changes as well as biological functions of β-amyloid and β-amyloid-elicited toxicity mechanisms will shed light on the development and/or design of potential interfering agents against amyloid formation associated with Alzheimer's disease.
Physical activity prevents numerous disorders and improves many pathophysiological disease features. Exercise has been classically associated to muscular and metabolic benefits. However, the effects of exercise training on brain function, specialty known as “Neurobiology of exercise”, have recently received much attention.
The beneficial effects of exercise have been clearly established in several pathologies such as Alzheimer´s disease, Parkinson´s disease, amyotrophic lateral sclerosis, schizophrenia, bipolar disorder and depressive disorder, among others. These findings have lead to use exercise training as a therapeutic coadjutant strategy not only in research but also in the clinical practice. According to a large amount of evidence, physical exercise not only restores the altered physiology of several neuropsychiatric diseases but also improves the brain function, cognition and psychological condition in healthy people.
Exercise exerts its action on the brain through many molecular pathways and physiological mechanisms. Some of them include the prevention of oxidative damage, release of endorphins, restoration of dopamine signaling and those actions mediated by the neurotrophic factors. Neurotrophic factors are growth factors with different sources and pathways of action. Although there are several factors included in this family of proteins, we would like to highlight the insulin-like growth factor 1 (IGF-1), the vascular endothelial growth factor (VEGF), the brain-derived neurotrophic factor (BDNF), the nerve growth factor (NGF), and the neurotrophins 3 and 4/5 (NT-3 and NT-4/5 respectively). The main actions mediated by these factors include the hippocampal neurogenesis, neuron repair, axogenesis, dendrogenesis, synaptic transmission modulation, synaptogenesis, and thereby brain plasticity. The functional consequences of their modulation include long term potentiation, improvement of learning and memory, anxiolytic and antidepressant effects.
BDNF is one of the most important neurotrophic factors since it mediates pleiotropic trophic effects in the brain. In addition, it is also one of the main neurotrophic factors induced by both chronic and acute exercise. High levels of BDNF have been found in brains of exercised persons, through post mortem studies and jugular blood in vivo analysis, as well as in trained animals. The brain modulation is produced mainly in the hippocampus and limbic system and thereby it has been associated with several cognitive and psychological improvements.
There is yet no consensus about the adequate blood processing conditions to standardize peripheral BDNF assessment in exercise studies. Serum, plasma, whole blood, and platelets-rich plasma with several methodological processing conditions have been indistinctly used in the literature. This leads to inconsistencies in the studies.
This Doctoral Thesis aims to clarify the effects of acute exercise and training in human blood levels of neurotrophic factors as well as the appropriate methodological protocol for the BDNF analysis. We also aim to determine the molecular pathways involved in the beneficial effects of exercise training in two mice models of Alzheimer´s disease. Finally, we aim to test the possible synergistic beneficial effect of exercise training and a BDNF pharmacologic mimetic on brain function in rats.
In our first experimental model, healthy adolescents were divided into two groups according to their exercise habits. The trained group included members of an elite cyclist team, and thereby highly trained. The control group included sedentary matched controls. We compared the IGF-1 and BDNF blood levels of both groups in the pre-season and post-competition period. We evaluated the possible effect of the circulating BDNF on the cAMP response element-binding (CREB) activation in peripheral blood mononuclear cells. All participants were also evaluated through anthropometric, hematological and acelerometric analysis.
We found that the BDNF and IGF-1 blood levels were increased in the trained adolescents compared with the sedentary controls when we analyzed it during the pre-season, characterized by a moderate physical demand. This increment did not affect to the CREB. Moreover, the differences between both groups disappeared when we compared the neurotrophic factor levels in the post-competition period, characterized by a maximum physical performance requirement.
In our second experimental model we determined the effect of an acute bout of exercise on BDNF blood levels in healthy adults under different blood processing conditions in a time-course analysis (at baseline, immediately after exercise, at 30 and 60 minutes of recovery). The blood samples that we analyzed included serum coagulated 10 minutes and 24 hours; plasma with EDTA, with and without platelets; and whole blood.
We found an increment in BDNF levels after the acute exercise in the serum coagulated during 24 hours and in whole blood samples. These changes were not evident when analyzed in the serum coagulated during 10 minutes, total plasma and platelet-free plasma samples. The interference of the anticoagulants used for the plasma and the irregular platelet activation in the serum coagulated during 10 minutes led to a high variability in the BDNF levels. We have also found that the processing temperature of the samples and the hemoconcentration are relevant factors to take into account in these studies.
In our third experimental model we used non transgenic and double transgenic mice (2xTg) for Alzheimer´s disease. We divided the animals into two groups: sedentary and exercised. At 10 months of age, trained groups were subjected to 12 weeks of a training combination of forced and voluntary exercise. Different psychological and physical tests were performed to the animals. We also evaluated brain glucose uptake by positron emission tomography and biochemical markers related to amyloid-β (Aβ) (1-42) levels and its modulation through the low density lipoprotein receptor-related protein 1 (LRP1), BDNF pathway (tyrosine kinase type B (TrkB) levels and CREB levels and activation), oxidative damage (malondialdehyde, glutathione, protein carbonylation), antioxidant defense levels (superoxide dismutase dependent of copper/zinc and manganese (Cu/Zn-SOD and Mn-SOD), glutathione peroxidase (GPx) and catalase (CAT)), and mitochondrial content (cytochrome-C) and biogenesis (peroxisome proliferator-activated receptor-gamma coactivator 1α, PGC-1α).
The exercise training improved the behavior of the 2xTg mice as well as their physical performance. These improvements were accompanied by a hippocampal Aβ (1-42) reduction in the 2xTg mice. The cerebral and systemic oxidative damage, LRP1 and hippocampal BDNF levels were reduced in the 2xTg mice and the exercise did not affect it. Nevertheless, the brain glucose uptake was higher in the transgenic mice and the antioxidant defense, determined by the CAT, increased in the 2xTg mice after exercise.
Our forth experimental model included a triple transgenic mouse model (3xTg) of Alzheimer´s disease and non transgenic mice as control. We studied the protective effect of exercise in ovariectomized mice. The exercise protocol included 12 weeks of spontaneous wheel-running. The animals were divided into eight experimental groups which included an Alzheimer´s disease model and/or artificial climacteric and/or exercise treatment. We sacrificed animals and analyzed several brain biomarkers which included the Aβ and hiperphosphorylated tau levels, the amyloidogenic pathway (C99/APP), BDNF levels and its pathway through TrkB and CREB, PGC-1α, and the expression the antioxidant enzymes GPx, Mn-SOD and CAT.
The exercise training performed by the 3xTg mice and the ovariectomy did not affect to the brain Aβ and the tau hyperphosphorylated levels. Nevertheless, the exercise partially prevented activation of the amyloidogenic pathway in all cases. In addition, training incremented the hippocampal BDNF levels of the 3xTg, ovariectomized and sham. We obtained a CREB activation increment in the non transgenic and 3xTg non ovariectomized mice subjected to exercise. Moreover, the hippocampal expression of CAT increased in the 3xTg mice, trained and sedentary, whereas the ovariectomy reduced it. This was reverted through physical exercise.
Finally, in the fifth experimental model we aimed to evaluate the possible synergic effects of 6 weeks of exercise training and a pharmacological BDNF mimetic, 7,8-dihydroxyflavone (7,8-DHF), in healthy young rats. Thus, we used four experimental groups which included the trained group, the group with 7,8-DHF, the group subjected to a combination of exercise and 7,8-DHF and a control group. We evaluated the cognition of the animals thought the object recognition test and their behavioral condition by the open field test.
The treatment with 7,8-DHF and/or forced exercise training did not show a synergic effect on the psychological parameters analyzed. The learning and memory did not improve, whereas the exploratory behavior incremented with all treatments but especially with the exercise training.
Taking together our findings we can conclude that acute and chronic exercise increases BDNF peripheral levels in humans whereas exercise training improves the pathophysiological and functional features of Alzheimer´s disease in two different transgenic models. Exercise seems to contribute to brain benefits by a wide range of physiological mechanisms including the BDNF pathway.
Amyloid peptides (A) aggregation is considered as a crucial pathological biomarker of Alzheimer's disease (AD). It was found that A and heme can form A-heme complex, which results in increase of heme pseudo-peroxidase activity. Recently, we found that the increase of pseudo-peroxidase activity would induce an elevated tyrosine nitration on A in the presence of nitrite and hydrogen peroxide. However, the nature of tyrosine nitration of A and its physiologic significance are still unknown. In this study, we revealed that A1-40 can be nitrated in vitro by binding to heme with the presence of nitrite and hydrogen peroxide. Moreover, we found that tyrosine nitration had little effect on A1-40's binding activity with heme. TMB assay also revealed that the peroxidase activity of heme-A1-40Y10(3N)T (tyrosine 10 was replaced with 3-nitrtotyrosine in A1-40) complex was moderately increased compared with that of heme-A1-40 complex. Furthermore, Thioflavin T fluorescence and transmission electron microscopic characterization indicated that tyrosine nitration significantly decreased the aggregation of A1-40. In addition, cytotoxicity test verified that the cytotoxicity of wild type A1-40 was more toxic than A1-40Y10(3N)T. These results suggested that nitration of A1-40 might be an A detoxicant process and a compensatory reaction to nitrative stress. Our findings may lead to a detailed understanding of the function of Aβ1-40 and would be helpful in preventing and curing AD.
Biological ageing is characterised by prolonged cellular damage over the lifespan, which results from oxidative stress processes that are implicated in both the initiation and progression of age-related disorders such as Alzheimer's disease (AD). Recently, the role of the plasma membrane redox system, which consists of at least three major components: the lipophilic antioxidants (Coenzyme Q (CoQ) and α-tocopherol), the intracellular cytosolic electron donor (NAD(P)H) and membrane-associated quinone reductases (cytochrome b5 reductase and NADH-quinone), in protecting against oxidative stress has come into focus. Research shows that this redox system plays a protective role during mitochondrial dysfunction by aiding in the alternative glycolytic ATP production pathway and reducing oxidative stress. The different aspects of the plasma membrane operate in tandem to protect the membrane from lipid peroxidation, preventing the formation of semiquinone free radicals and reactive oxygen species, and to ultimately limit oxidative stress while still maintaining cellular levels of each component. Past studies have made a link between the plasma membrane redox system and AD and have revealed that each aspect is affected during the disease. Moreover, research has revealed that manipulating the system components can protect cells from amyloid-β toxicity, suggesting a key role in cell survival. To date, the relationship of the plasma membrane redox system to the brain changes seen in AD has yet to be fully elucidated, and we will review the current literature here.
Segregation in the use of the water column by two congeneric species of Characidae, Hyphessobrycon bifasciatus and H. luetkenii, was investigated through underwater observations in the Cabiúnas coastal lagoon in northeastern Rio de Janeiro, Brazil. The use of the water column by the two species differed significantly. Hyphessobrycon luetkenii occupied mainly the uppermost stratum, with 79% of the observations within 20 cm of the surface; whereas H. bifasciatus was more common between 20 cm and 40 cm below the surface (55% of the observations). Predation pressure, macrophyte cover and nutrient distribution may influence this vertical segregation.
Based on a fish survey and preliminary underwater observations, 17 "morphotypes" were identified that characterize the morphological diversity found within 27 nektonic fish species sampled at São Sebastião Channel. Such "morphotypes" were studied using an ecomorphological approach, with the intention to investigate similarities and differences in shape and habits. Underwater field observations were also performed, to verify if the lifestyle of these species, such as vertical occupation of the water column and the habitat use, are in accordance with their distribution in the morphospace. The results, complemented with data from scientific literature on the taxonomy and phylogenies of these species, allowed discussing some of the typical cases of convergent and divergent evolution. Some of the ecomorphological clusters had no phylogenetic support although this is probably due to the environmental conditions in which theirs members have evolved. The body shape and fins positions of a fish clearly influence its ecological performance and habitat use, corroborating the ecomorphological hypothesis on the intimate link between phenotype and ecology.
Insoluble aggregates of the amyloid beta-peptide (A beta) are a major constituent of senile plaques found in brains of Alzheimer's disease patients. The beta-amyloid fragment A beta(1-40) is toxic to rat pheochromocytoma PC12 cells, leading to a concentration-dependent decrease in the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The detrimental effects of A beta(1-40) are enhanced in the presence of 1 m M zinc, whereas 50 mu M zinc exerts a protective effect against A beta(1-40)-induced toxicity. Exposure of PC12 cells to low zinc concentrations (50 mu M) affords a decrease (1.4-fold) in the extent of lipid peroxidation, a decrement in protein oxidation (1.1-fold), and an increase in ATP levels (1.2-fold), although the differences were not statistically significant. However, treatment of cells with high concentrations of zinc (1 m M) led to significant increases in lipid peroxidation (3.7-fold) and protein oxidation (1.5-fold) and to depletion of the ATP pool (21-fold). These data suggest that zinc has a concentration-dependent dual effect, protective and toxic, thus playing an important role in the pathogenesis of Alzheimer's disease. Antiox. Redox Signal. 2, 317-325.
In Alzheimer's disease (AD), abnormal accumulations of beta-amyloid are present in the brain and degenerating neurons exhibit cytoskeletal aberrations (neurofibrillary tangles). Roles for beta-amyloid in the neuronal degeneration of AD have been suggested based on recent data obtained in rodent studies demonstrating neurotoxic actions of beta- amyloid. However, the cellular mechanism of action of beta-amyloid is unknown, and there is no direct information concerning the biological activity of beta-amyloid in human neurons. We now report on experiments in human cerebral cortical cell cultures that tested the hypothesis that beta-amyloid can destabilize neuronal calcium regulation and render neurons more vulnerable to environmental stimuli that elevate intracellular calcium levels. Synthetic beta-amyloid peptides (beta APs) corresponding to amino acids 1–38 or 25–35 of the beta-amyloid protein enhanced glutamate neurotoxicity in cortical cultures, while a peptide with a scrambled sequence was without effect. beta APs alone had no effect on neuronal survival during a 4 d exposure period. beta APs enhanced both kainate and NMDA neurotoxicity, indicating that the effect was not specific for a particular subtype of glutamate receptor. The effects of beta APs on excitatory amino acid (EAA)-induced neuronal degeneration were concentration dependent and required prolonged (days) exposures. The beta APs also rendered neurons more vulnerable to calcium ionophore neurotoxicity, indicating that beta APs compromised the ability of the neurons to reduce intracellular calcium levels to normal limits. Direct measurements of intracellular calcium levels demonstrated that beta APs elevated rest levels of calcium and enhanced calcium responses to EAAs and calcium ionophore. The neurotoxicity caused by EAAs and potentiated by beta APs was dependent upon calcium influx since it did not occur in calcium-deficient culture medium. Finally, the beta APs made neurons more vulnerable to neurofibrillary tangle-like antigenic changes induced by EAAs or calcium ionophore (i.e., increased staining with tau and ubiquitin antibodies). Taken together, these data suggest that beta-amyloid destabilizes neuronal calcium homeostasis and thereby renders neurons more vulnerable to environmental insults.
Hydrogen peroxide (H2O2) is suspected to be involved in numerous brain pathologies such as neurodegenerative diseases or in acute injury such as ischemia or trauma. In this study, we examined the ability of pyruvate to improve the survival of cultured striatal neurons exposed for 30 min to H2O2, as estimated 24 hr later by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. Pyruvate strongly protected neurons against both H2O2 added to the external medium and H2O2 endogenously produced through the redox cycling of the experimental quinone menadione. The neuroprotective effect of pyruvate appeared to result rather from the ability of alpha-ketoacids to undergo nonenzymatic decarboxylation in the presence of H2O2 than from an improvement of energy metabolism. Indeed, several other alpha-ketoacids, including alpha-ketobutyrate, which is not an energy substrate, reproduced the neuroprotective effect of pyruvate. In contrast, lactate, a neuronal energy substrate, did not protect neurons from H2O2. Optimal neuroprotection was achieved with relatively low concentrations of pyruvate (</=1 mM), whereas at high concentration (10 mM) pyruvate was ineffective. This paradox could result from the cytosolic acidification induced by the cotransport of pyruvate and protons into neurons. Indeed, cytosolic acidification both enhanced the H2O2-induced neurotoxicity and decreased the rate of pyruvate decarboxylation by H2O2. Together, these results indicate that pyruvate efficiently protects neurons against both exogenous and endogenous H2O2. Its low toxicity and its capacity to cross the blood-brain barrier open a new therapeutic perspective in brain pathologies in which H2O2 is involved.
The amyloid beta-peptide (A beta) that accumulates as insoluble plaques in the brain in Alzheimer's disease can be directly neurotoxic and can increase neuronal vulnerability to excitotoxic insults. The mechanism of A beta toxicity is unclear but is believed to involve generation of reactive oxygen species (ROS) and loss of calcium homeostasis. We now report that exposure of cultured rat hippocampal neurons to A beta 1-40 or A beta 25-35 causes a selective reduction in Na+/K(+)-ATPase activity which precedes loss of calcium homeostasis and cell degeneration. Na+/K(+)-ATPase activity was reduced within 30 min of exposure to A beta 25-35 and declined to less than 40% of basal level by 3 hr. A beta did not impair other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. Experiments with ouabain, a specific inhibitor of the Na+/K(+)-ATPase, demonstrated that impairment of this enzyme was sufficient to induce an elevation of [Ca2+]i and neuronal injury. Impairment of Na+/K(+)-ATPase activity appeared to be causally involved in the elevation of [Ca2+]i and neurotoxicity since suppression of Na+ influx significantly reduced A beta- and ouabain-induced [Ca2+]i elevation and neuronal death. Neuronal degeneration induced by ouabain appeared to be of an apoptotic form as indicated by nuclear condensation and DNA fragmentation. The antioxidant free radical scavengers vitamin E and propylgallate significantly attenuated A beta-induced impairment of Na+/K(+)-ATPase activity, elevation of [Ca2+]i and neurotoxicity, suggesting a role for ROS. Finally, exposure of synaptosomes from postmortem human hippocampus to A beta resulted in a significant and specific reduction in Na+/K(+)-ATPase and Ca(2+)-ATPase activities, without affecting other Mg(2+)-dependent ATPase activities or Na+/Ca2+ exchange. These data suggest that impairment of ion-motive ATPases may play a role in the pathogenesis of neuronal injury in Alzheimer's disease.
Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.
A single cell clonal line which responds reversibly to nerve growth factor (NGF) has been established from a transplantable rat adrenal pheochromocytoma. This line, designated PC12, has a homogeneous and near-diploid chromosome number of 40. By 1 week's exposure to NGF, PC12 cells cease to multiply and begin to extend branching varicose processes similar to those produced by sympathetic neurons in primary cell culture. By several weeks of exposure to NGF, the PC12 processes reach 500-1000 mum in length. Removal of NGF is followed by degeneration of processes within 24 hr and by resumption of cell multiplication within 72 hr. PC12 cells grown with or without NGF contain dense core chromaffin-like granules up to 350 nm in diameter. The NGF-treated cells also contain small vesicles which accumulate in process varicosities and endings. PC12 cells synthesize and store the catecholamine neurotransmitters dopamine and norepinephrine. The levels (per mg of protein) of catecholamines and of the their synthetic enzymes in PC12 cells are comparable to or higher than those found in rat adrenals. NGF-treatment of PC12 cells results in no change in the levels of catecholamines or of their synthetic enzymes when expressed on a per cell basis, but does result in a 4- to 6-fold decrease in levels when expressed on a per mg of protein basis. PC12 cells do not synthesize epinephrine and cannot be induced to do so by treatment with dexamethasone. The PC12 cell line should be a useful model system for neurobiological and neurochemical studies.
Previously we have shown that the COOH-terminal 100 residues (A4CT) of the amyloid protein precursor (APP), which carry the sequence of the amyloid beta A4 protein of Alzheimer's disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte lysate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Müller-Hill, B., Masters, C. L., and Beyreuther, K. (1988) EMBO J. 7, 949-957). Here we report that aggregation of this COOH-terminal APP fragment A4CT and also of beta A4 itself depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT and beta A4 in the wheat germ expression system resulted in only monomeric forms. We have identified the factors which are capable of transforming both soluble A4CT and beta A4 into insoluble and aggregating molecules. Monomeric A4CT or beta A4 expressed in the wheat germ lysate could be transformed into aggregating molecules by the addition of metal-catalyzed oxidation systems. The addition of radical scavengers such as ascorbic acid, trolox, and amino acids prevented the aggregation process induced by the radical initiators. Thus, the aggregation of amyloidogenic APP fragments if analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis requires amino acid oxidation and protein cross-linking induced by radical generation systems.
The relationship between Alzheimer disease (AD) and aging is not currently known. In this study, postmortem frontal- and occipital-pole brain samples were obtained from 16 subjects with AD, 8 age-matched controls, and 5 young controls. These samples were analyzed both for protein oxidation products (carbonyl) and the activities of two enzymes vulnerable to mixed-function oxidation, glutamine synthetase and creatine kinase. Glutamine synthetase is more sensitive to mixed-function oxidation than creatine kinase. Carbonyl content rises exponentially with age, at double the rate in the frontal pole compared with the occipital pole. Compared with young controls, both aged groups (AD and age-matched controls) have increased carbonyl content and decreased glutamine synthetase and creatine kinase activities, which are more marked in the frontal than occipital pole in all instances. We conclude that protein oxidation products accumulate in the brain and that oxidation-vulnerable enzyme activities decrease with aging in the same regional pattern (frontal more affected than occipital). However, only glutamine synthetase activity distinguishes AD from age-matched controls: Because glutamine synthetase activity is differentially reduced in the frontal pole in AD, we suggest that AD may represent a specific brain vulnerability to age-related oxidation.
Dynamic positron emission tomography with [18F]fluorodeoxyglucose was used in six patients with Alzheimer's disease (AD) and seven healthy age-matched control subjects to estimate the kinetic parameters K1*, k2*, and k3* that describe glucose transport and phosphorylation. A high-resolution tomograph was used to acquire brain uptake data in one tomographic plane, and a radial artery catheter connected to a plastic scintillator was used to acquire arterial input data. A nonlinear iterative least-squares fitting procedure that included terms for the vascular fraction and time delay to the peripheral sampling site was used to fit a three-compartment model to the brain data. Regions studied included frontal, temporal, occipital, and the entire cortex and subcortical white matter. The values obtained for the individual rate constants and regional CMRglc (rCMRglc; calculated using regional values of the rate constants) were higher than those reported previously. A significant (p less than 0.05) decrease was found in K1* in frontal and temporal cortex in the AD patients compared with the controls, with values of 0.157 and 0.161 ml/g/min in frontal and temporal cortex, respectively, of controls and 0.127 and 0.126 ml/g/min in frontal and temporal cortex of the AD patients. rCMRglc was also significantly (p less than 0.02) lower in the AD patients than controls in all cortical brain regions. Lower values of k3* were found in all brain regions in the AD patients, although these were not statistically significant. These findings provide evidence of an in vivo abnormality of forward glucose transport in AD.(ABSTRACT TRUNCATED AT 250 WORDS)
The amyloid beta protein is deposited in the brains of patients with Alzheimer's disease but its pathogenic role is unknown. In culture, the amyloid beta protein was neurotrophic to undifferentiated hippocampal neurons at low concentrations and neurotoxic to mature neurons at higher concentrations. In differentiated neurons, amyloid beta protein caused dendritic and axonal retraction followed by neuronal death. A portion of the amyloid beta protein (amino acids 25 to 35) mediated both the trophic and toxic effects and was homologous to the tachykinin neuropeptide family. The effects of the amyloid beta protein were mimicked by tachykinin antagonists and completely reversed by specific tachykinin agonists. Thus, the amyloid beta protein could function as a neurotrophic factor for differentiating neurons, but at high concentrations in mature neurons, as in Alzheimer's disease, could cause neuronal degeneration.
The effect of nerve growth factor (NGF) on the utilization and fate of uniformly labeled 14C glucose and on the content of several pyridine and purine nucleotides has been tested in the clonal cell line PC12. After incubation for 72 h with NGF, PC12 cells exhibit a 2.7-fold increase in glucose utilization and a 4.7-fold increase in CO2 release. During the same incubation period, all the nucleotides tested (NAD+, AMP, GMP, UDP-glucose, UDP-galactose, UDP, ADP, GDP, UTP, CTP, ATP, and GTP) underwent significant increments, varying from a minimum of 27% for ADP to a maximum of 90-120% for AMP, GMP, UDP-glucose, and UDP-galactose. These findings are discussed in connection with the trophic and differentiative effects of NGF in PC12 cells, which, in the presence of this factor, shifted from a neoplastic to a neuronal-like cell population.
We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.
beta-Amyloid protein (A beta) is a member of a small group of proteins that accumulate as amyloid deposits in various tissues. It has recently been demonstrated that the toxicity of A beta toward some neural cells is caused by oxidative damage. Since all of the amyloid diseases are characterized by protein deposited in the antiparallel beta-sheet conformation, it was asked whether there is a common toxic mechanism. It is shown here that the protein components of other human amyloidoses, including amylin, calcitonin, and atrial natriuretic peptide, are all toxic to clonal and primary cells. The toxicity is mediated via a free radical pathway indistinguishable from that of A beta. Experiments with synthetic peptides suggest that it is the amphiphilic nature of the peptides generated by their beta structure rather than their beta structure per se that causes toxicity. These results tend to rule out the alternative that amyloid toxicity is exclusively mediated via specific cell surface receptors.
beta-Amyloid is a 39- to 43-amino-acid neurotoxic peptide that aggregates to form the core of Alzheimer disease-associated senile (amyloid) plaques. No satisfactory hypothesis has yet been proposed to explain the mechanism of beta-amyloid aggregation and toxicity. We present mass spectrometric and electron paramagnetic resonance spin trapping evidence that beta-amyloid, in aqueous solution, fragments and generates free radical peptides. beta-Amyloid fragments, at concentrations that previously have been shown to be neurotoxic to cultured neurons, can inactivate oxidation-sensitive glutamine synthetase and creatine kinase enzymes. Also, salicylate hydroxylation assays indicate that reactive oxygen species are generated by the beta-amyloid-(25-35) fragment during cell-free incubation. These results are formulated into a free radical-based unifying hypothesis for neurotoxicity of beta-amyloid and are discussed with reference to membrane molecular alterations in Alzheimer disease.
The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.
In isolated mitochondria, t-butylhydroperoxide (t-BuOOH) and other pro-oxidants cause a permeability transition characterized by increased permeability to small ions, swelling and loss of membrane potential. Cyclosporin A and trifluoperazine inhibit this permeability transition. Here, we investigated the role of the mitochondrial permeability transition in lethal cellular injury from t-BuOOH. Hepatocytes from fasted rats were isolated by collagenase perfusion, and cell viability was assessed by propidium iodide fluorescence. t-BuOOH caused dose- and time-dependent cell killing. Fructose, a substrate for glycolytic ATP formation, protected at lower (< or = 100 microM), but not at higher concentrations of t-BuOOH. In fructose-treated cells, oligomycin (10 micrograms/ml) delayed cell killing after 100 to 300 microM t-BuOOH, whereas cyclosporin A (0.5 microM) plus trifluoperazine (5 microM) even more potently reduced lethal injury. In hepatocyte suspensions, 100 microM t-BuOOH caused mitochondrial depolarization as determined by release of rhodamine 123. Cyclosporin A plus trifluoperazine in the presence of fructose substantially reduced release of rhodamine 123. Similarly, in single cultured hepatocytes viewed by laser scanning confocal microscopy, t-BuOOH caused leakage of rhodamine 123 from mitochondria, an event which preceded cell death and which was delayed by fructose in combination with cyclosporin A plus trifluoperazine. At 1 mM, t-BuOOH inhibited glycolysis, and fructose in combination with either oligomycin or cyclosporin A plus trifluoperazine had only a short-lived protective effect. In conclusion, t-BuOOH toxicity was progressive with increasing dosages. At low t-BuOOH (< or = 50 microM), mitochondrial ATP synthetic capacity was inhibited, but not uncoupled.(ABSTRACT TRUNCATED AT 250 WORDS)
Elucidation of cellular and molecular mechanisms of excitotoxic neuronal injury have been facilitated by the development of several cell biological technologies—namely, (1) neural cell culture, (2) quantitative assays of neuronal injury and death, (3) fluorescence imaging of intracellular free calcium levels ([Ca2+]i), (4) evaluation of mitochondrial function, (5) quantification of reactive oxygen species, (6) assessment of cytoskeletal alterations elicited by excitotoxic/metabolic insults, and (7) detection of “stress response” proteins. This chapter presents protocols for each of these technical approaches, as applied to embryonic rat and human brain neurons in dissociated cell culture. One strategy employed to study neuronal death is to elucidate the mechanisms that normally protect neurons from adverse environmental conditions. These studies have shown that many cellular signaling mechanisms exist that are designed to protect neurons against adverse environmental conditions such as metabolic and excitotoxic insults. Many of the neuroprotective strategies acquired during evolution involve systems that control calcium and free radical metabolism.
THE aim of the present study was to determine whether amyloid beta-peptide (A beta) induces mitochondrial dysfunction. Mitochondrial function was reported to be affected following A beta exposure, as demonstrated by depolarization of the mitochondrial membrane, decrease of oxygen consumption and by the inhibition of complexes I, III and IV of the mitochondrial respiratory chain. A beta 25-35 and A beta 1-40 peptides also inhibited MTT reduction in a dose-dependent manner in undifferentiated and differentiated PC12 cells. Several antioxidants prevented this inhibitory response, suggesting that oxidative stress is involved in A beta-induced cytotoxicity. These data suggest that mitochondrial dysfunction contributes to amyloid beta-protein cytotoxicity and may play a major role in the abnormalities of energy metabolism observed in Alzheimer's disease. (C) 1998 Rapid Science Ltd.
This chapter describes the preparation and properties of succinie–cytochrome c reductase (complex II - III). Assays of enzymatic activity are carried out at 38° in a Beckman Model DU spectrophotometer, equipped with a photomultiplier. The final reaction mixture contains ten micromoles phosphate buffer (0.10 ml); one micromole NaN3 (0.01 ml); 0.2 micromole EDTA (0.02 ml); five mg BSA (0.05 ml); 10 micromoles potassium succinate, pH 7.0 (0.10 ml); and water to a volume of 0.9 ml. The enzyme preparation to be assayed is diluted to a concentration of 100-200 μg protein per milliliter in a solution of 0.88 M sucrose 0.005 M in succinatc. The enzyme-catalyzed reduction of cytochrome c by succinate proceeds at a linear rate under standard conditions over the first minute. Then specific activity is calculated. The resulting increase in absorbancy at 550 mμ is followed during this linear phase. In the method of preparation as described in the chapter, beef heart mitochondria are isolated in 0.25 M sucrose and are frozen at –40° for a period of 1-7 days. The yield and increase of activity during the purification of the succinic-cytochrome c reductase is also illustrated.
Astrocytes possess plasma membrane glutamate transporters that rapidly remove glutamate from the extracellular milieu and thereby prevent excitotoxic injury to neurons. Cellular oxidative stress is increased in neural tissues in a variety of acute and chronic neurodegenerative conditions. Recent findings suggest that oxidative stress increases neuronal vulnerability to excitotoxicity and that membrane lipid peroxidation plays a key role in this process. We now report that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, impairs glutamate transport in cultured cortical astrocytes. Impairment of glutamate transport occurred within 1–3 h of exposure to HNE; FeSO4, an inducer of membrane lipid peroxidation, also impaired glutamate transport. Vitamin E prevented impairment of glutamate transport induced by FeSO4, but not that induced by HNE, consistent with HNE acting as an effector of lipid peroxidation-induced impairment of glutamate transport. Glutathione, which binds and thereby detoxifies HNE, prevented HNE from impairing glutamate transport. Western blot, immunoprecipitation, and immunocytochemical analyses using an antibody against HNE-protein conjugates provided evidence that HNE covalently binds to many different astrocytic proteins including the glutamate transporter GLT-1. Data further suggest that HNE promotes intermolecular cross-linking of GLT-1 monomers to form dimers. HNE also induced mitochondrial dysfunction and accumulation of peroxides in astrocytes. Impairment of glutamate transport and mitochondrial function occurred with sublethal concentrations of HNE, concentrations known to be generated in cells exposed to various oxidative insults. Collectively, our data suggest that HNE may be an important mediator of oxidative stress-induced impairment of astrocytic glutamate transport and may thereby play a role in promoting neuronal excitotoxicity. GLIA 22:149–160, 1998. © 1998 Wiley-Liss, Inc.
Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid β-peptide (Aβ). Aβ-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2-trans-nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of Aβ and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6(2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1–10 µM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and Aβ-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 µM). The results obtained are discussed with relevance to the hypothesis of Aβ-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.
The pathological presentation of Alzheimer's disease, the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intracellular and extracellular filamentous proteins aggregates that form lesions termed neurofibrillary tangles and senile plaques, respectively. Several independent parameters have been suggested as the primary factor that is responsible for this pathogenesis, including apolipoprotein ϵ genotype, hyperphosphorylation of cytoskeletal proteins, or metabolism of amyloid β. However, at present, no one theory explains adequately the host of complex biochemical and pathological facets of the disease. Recent findings suggest that age-related increases in oxidative stress and protein glycation either individually, or more probably in a synergistic manner, could, exclusive of the other theories or in concert with them, account for all aspects of Alzheimer's disease.
An assay for proteins in solution is described that depends on the conversion of Coomassie brilliant blue G250 in dilute acid from a brownish-orange to an intense blue color. As soon as the dye solution is mixed with a protein sample, the absorbance of the mixture can be measured. The absorbance of the solution is stable for 60–90 min at room temperature. The test can be used with a variety of proteins and polypeptides with molecular weights greater than 3000. The assay has high reproducibility and can detect less than 1.0 μg of albumin. The single reagent required is very stable, and free amino acids and several chemicals that interfere with the Lowry protein assay cause no interference. The test is adaptable to a micromethod, which is also described.
This chapter discusses the preparation and activity of native and chemically modified cytochromes c. Cytochrome c is extracted from ground and homogenized tissue with a dilute solution of aluminum sulfate at pH 4.5, the trivalent cations effectively displacing the protein even at low ionic strength. Aluminum ions are precipitated as the hydroxide at slightly alkaline pH, and each is replaced in solution by three monovalent ammonium ions. The cytochrome c is purified by (NH4)2SO4 fractionation and cation-exchange chromatography. This method of extraction is suited to vertebrate and invertebrate tissues, but for plant materials, protists, or fungi, special cytolysis procedures are often required before use of a similar scheme. Although both these aspects of cytochrome c function are interesting, it seems that the biologically significant evolutionary variations of cytochrome c structure predominantly affect the mechanics of protein–protein interaction with its physiological oxidants and reductants, rather than the mechanism of electron transfer.
The amyloid beta protein (ABP) is a 40 to 42 amino acid peptide which accumulates in Alzheimer's disease plaques. It has been demonstrated that this peptide and a fragment derived from it are cytotoxic for cultured cortical nerve cells. It is shown here that ABP and an internal fragment encompassing residues 25 to 35 (beta 25-35) are cytotoxic to a clone of PC12 cells at concentrations above 1 x 10(-9)M and to several other cell lines at higher concentrations. Between 10(-9) and 10(-11) M beta 25-35 protects PC12 cells from glutamate toxicity. The antioxidant and free radical scavenger vitamin E inhibits ABP induced cell death. These results have implications regarding the prevention and treatment of Alzheimer's disease.
In normoglycemic patients with either incipient early-onset or incipient late-onset dementia of the Alzheimer type, the predominant disturbance consisted of a significant reduction in cerebral glucose utilization. Alterations in cerebral blood flow and oxygen consumption first occurred in late-onset dementia types. In advanced late-onset dementia, these parameters had decreased most severely. The calculated ATP production rate from glucose indicated a drastic loss of energy in all patients studied. As not all oxygen consumed by the brain was used for glucose oxidation, oxidation of substrates other than glucose (endogenous amino acids and free fatty acids) is assumed to minimize the energy loss from glucose. The possibility that the abnormalities in oxidative and energy metabolism in dementias of the Alzheimer's type are due to metabolic abnormalities in glycolytic glucose breakdown and pyruvate oxidation, rather than to an uncoupling of oxidative phosphorylation, is discussed.
Excitatory amino acids (EAA) such as glutamate and aspartate are major transmitters of the cerebral cortex and hippocampus, and EAA mechanisms appear to play a role in learning and memory. Anatomical and biochemical evidence suggests that there is both pre- and postsynaptic disruption of EAA pathways in Alzheimer's disease. Dysfunction of EAA pathways could play a role in the clinical manifestations of Alzheimer's disease, such as memory loss and signs of cortical disconnection. Furthermore, EAA might be involved in the pathogenesis of Alzheimer's disease, by virtue of their neurotoxic (excitotoxic) properties. Circumstantial evidence raises the possibility that the EAA system may partially determine the distribution of pathology in Alzheimer's disease and may be important in producing the neurofibrillary tangles, RNA reductions and dendritic changes which characterize this devastating disorder. In this article, we will review the evidence suggesting a role for EAA in the clinical manifestations and pathogenesis of Alzheimer's disease.
We studied the hexose transporter protein of the frontal and temporal neocortex, hippocampus, putamen, cerebellum, and cerebral microvessels (which constitute the blood-brain barrier) in Alzheimer disease and control subjects by reversible and covalent binding with [3H]cytochalasin B and by immunological reactivity. In Alzheimer disease subjects, we found a marked decrease in the hexose transporter in brain microvessels and in the cerebral neocortex and hippocampus, regions that are most affected in Alzheimer disease, but there were no abnormalities in the putamen or cerebellum. Hexose transporter reduction in cerebral microvessels of Alzheimer subjects is relatively specific because other enzyme markers of brain endothelium were not significantly altered. The low density of the hexose transporter at the blood-brain barrier and in the cerebral cortex in Alzheimer disease may be related to decreased in vivo measurements of cerebral oxidative metabolism.
Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.
A simple and rapid method for the determination of ATP, ADP, AMP, NADP+, NAD+, NADPH, and NADH in human erythrocytes is described. A single-step extraction procedure employing alkaline medium and CF 50A Amicon ultrafiltration membranes allows a simultaneous and total recovery of the compounds of interest. Analysis is performed by reverse-phase high-performance liquid chromatography on a 5-micron Supelcosil LC-18 column and uv detection. Extraction and analysis require about 30 min. Levels of adenine and pyridine nucleotides in normal adults are also presented.
A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis.
A highly sensitive fluorometric method for the quantitation of cholesterol, lipid, and other hydroperoxides at the picomole level is described. The method is based on the oxidation of dichlorofluoroscin to the fluorescent dichlorofluoroscein by hydroperoxide and hematin under mild conditions. A 1:1 stoichiometry is observed between the hydroperoxide added and the dichlorofluoroscein produced. Since endoperoxides (e.g., PGH2) do not react in the assay, they do not interfere in the determination of lipid hydroperoxides.
Aging is a major risk factor for several common neurodegenerative diseases, including Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Huntington's disease (HD). Recent studies have implicated mitochondrial dysfunction and oxidative stress in the aging process and also in the pathogenesis of neurodegenerative diseases. In brain and other tissues, aging is associated with progressive impairment of mitochondrial function and increased oxidative damage. In PD, several studies have demonstrated decreased complex I activity, increased oxidative damage, and altered activities of antioxidant defense systems. Some cases of familial ALS are associated with mutations in the gene for Cu, Zn superoxide dismutase (Cu, Zn SOD) and decreased Cu, Zn SOD activity, while in sporadic ALS oxidative damage may be increased. Defects in energy metabolism and increased cortical lactate levels have been detected in HD patients. Studies of AD patients have identified decreased complex IV activity, and some patients with AD and PD have mitochondrial DNA mutations. The age-related onset and progressive course of these neurodegenerative diseases may be due to a cycling process between impaired energy metabolism and oxidative stress.
Recent findings link altered processing of beta-amyloid precursor protein (beta APP) to disruption of neuronal Ca2+ homeostasis and an excitotoxic mechanism of cell death in Alzheimer's disease. A major pathway of beta APP metabolism results in the release of secreted forms of beta APP, APPss. These secreted forms are released in response to electrical activity and can modulate neuronal responses to glutamate, suggesting roles in developmental and synaptic plasticity. beta APP is upregulated in response to neural injury and APPss can protect neurons against excitotoxic or ischemic insults by stabilizing the intracellular Ca2+ concentration [Ca2+]i. An alternative beta APP processing pathway liberates intact beta-amyloid peptide, which can form aggregates that disrupt Ca2+ homeostasis and render neurons vulnerable to metabolic or excitotoxic insults. Genetic abnormalities (e.g. certain beta APP mutations or Down syndrome) and age-related changes in brain metabolism (e.g. reduced energy availability or increased oxidative stress) may favor accumulation of [Ca2+]i-destabilizing beta-amyloid peptide and diminish the release of [Ca2+]i-stabilizing, neuroprotective APPss.
There is increasing evidence for genetic heterogeneity in Alzheimer disease. A longitudinal clinical and imaging study had been established in order to determine whether specific phenotypic profiles are present in aetiologically distinct familial Alzheimer disease (FAD) pedigrees. [18F]fluorodeoxyglucose positron emission tomography has been used in conjunction with statistical parametric mapping to determine the relative distribution of hypometabolism. A parietotemporal deficit has been observed in individuals from both amyloid precursor protein mutation and chromosome 14 linked FAD families. Preliminary data from asymptomatic individuals at risk of FAD shows similar, although a less extensive pattern of deficit.
The aging brain undergoes a process of enhanced peroxidative stress, as shown by reports of altered membrane lipids, oxidized proteins, and damaged DNA. The aims of this review are to examine: (1) the possible contribution of mitochondrial processes to the formation and release of reactive oxygen species (ROS) in the aging brain; and (2) the age-related changes of antioxidant defenses, both enzymatic and nonenzymatic. It will focus on studies investigating the role of the electron transfer chain as the site of ROS formation in brain aging and the alterations of the glutathione system, also in relation to the effects of exogenous pro-oxidant agents. The possible role of peroxidative stress in age-related neurodegenerative diseases will also be discussed.
We determined levels of thiobarbituric acid-reactive substances (TBARS), a measure of lipid peroxidation, and the activity of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), and catalase (CAT) in the amygdala, hippocampus, pyriform cortex, superior and middle temporal gyri, inferior parietal lobule, middle frontal gyrus, occipital pole, and cerebellum of 13 Alzheimer's disease (AD) and 10 control brains. Levels of TBARS were elevated in all AD brain regions except the middle frontal gyrus, and elevation levels reached statistical significance in the hippocampus and pyriform cortex and marginal significance in the amygdala of AD subjects compared with age-matched controls. Significant elevation of GSH-Px activity was present in AD hippocampus compared with control. Moderate but statistically insignificant elevations of GSH-Px activity also were present in the amygdala and pyriform cortex in AD. GSSG-R activity was significantly elevated in the amygdala and hippocampus in AD subjects compared with controls. CAT activity was significantly elevated in AD hippocampus and superior and middle temporal gyri. SOD levels were elevated in all brain regions in AD patients compared with controls, although none of these elevations reached statistical significance. Antioxidant enzyme activities were significantly elevated where lipid peroxidation was most pronounced, suggesting a compensatory rise in antioxidant activity in response to increased free radical formation. This study supports the concept that the brain in AD is under increased oxidative stress and demonstrates that the oxidative changes are most pronounced in the medial temporal lobe, where histopathologic alterations are most severe.
In order to establish whether positron emission tomography (PET) can identify metabolic changes in Alzheimer's disease at a presymptomatic stage, we have examined 24 asymptomatic at risk individuals from families with Alzheimer's disease. A significant reduction in global cerebral metabolic rate for glucose was found when compared with 16 age-matched controls. There was also a focal, parieto-temporal deficit similar to, although less extensive than, that found in 18 symptomatic individuals from familial Alzheimer's disease (FAD) pedigrees. Follow up of this cohort will establish whether these metabolic changes relate to a presymptomatic stage of the disease.
In vitro cell culture model systems for investigating the biochemical mechanisms involved in the neurodegenerative actions of beta-amyloid peptide (beta-AP) have been established. Using rat pheochromocytoma PC12 or human epitheloid HeLa cell lines, submicromolar concentrations of the beta-AP fragments beta 1-40, beta 1-39, and beta 25-35, but not beta 1-28, were found to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In both cell lines, the beta-AP-sensitive component represented approximately 70% of total cellular MTT reduction. When the reduction of a series of structurally related dyes was compared with that of MTT, the reduction of 3 alpha-naphthyl-2-phenyl-5-(4-nitrophenyl)-2H-tetrazolium chloride (NTV) was also found to be sensitive to beta 25-35, but that of seven other redox dyes was not. A property common to MTT and NTV is that they are both readily taken up into PC12 and HeLa cells and do not require an artificial electron coupling agent to be reduced. Microscopic analysis of MTT-formazan product formation in PC12 and HeLa cells following beta 25-35 treatment revealed that it was the intracellular component of the reduction of this dye that was abolished. These results support the hypothesis that the cellular reduction of MTT represents a specific indicator of the initial events underlying the mechanism of beta-AP toxicity.
Age-related damage to the mitochondrial membrane, including decreased membrane fluidity, has been attributed to free radical reactions. Our previous studies point to lipid peroxidation as a primary cause in age-related changes in membrane fluidity. This report offers new evidence that lipid peroxidation-modulated decreases in membrane fluidity are mediated through two aldehydic lipid peroxidation products, 4-hydroxynonenal (HNE) and malondialdehyde (MDA). Hepatic mitochondria were isolated from both ad libitum fed (AL) and dietary restricted (DR) rats of different ages. Introduction of the aldehydes was found to decrease mitochondrial membrane fluidity, although the fluidity decrease induced by HNE was more pronounced than that induced by MDA. It seems likely that HNE modifies membrane fluidity by direct interaction with membrane phospholipids, as shown by the generation of a fluorescent complex between HNE and membrane phospholipids. Finally, HNE and MDA were isolated and quantitated in mitochondria. Their levels clearly differentiated between animals of different age and dietary groups. These data indicate that the reactive products of lipid peroxidation, especially HNE, may play an important role in mediating the decreased mitochondrial membrane fluidity observed in aging animals.
It has previously been reported that isolated rat hepatocytes rapidly and completely metabolize high concentrations of 4-hydroxy-2,3-(E)-nonenal (4-HNE). However, until this report, the degree to which oxidative-reductive and nonoxidative metabolic pathways function in the depletion of 4-HNE by isolated rat hepatocytes has been speculative. The objective of the present study was to quantitate the extent to which cellular aldehyde dehydrogenases (ALDH; EC 1.2.1.3.), alcohol dehydrogenase (ADH; EC 1.1.1.1.), and glutathione S-transferases (GST; EC 2.5.1.18) function simultaneously during hepatocellular metabolism of 4-HNE. Hepatocytes were incubated with varying concentrations of 4-HNE (50, 100, 250 microM) and reversed-phase HPLC was used to quantitate 4-HNE and the oxidative and reductive metabolites, 4-hydroxy-2-nonenoic acid and 1,4-dihydroxy-2-nonene, respectively. Conjugative metabolism of 4-HNE was determined from the depletion of cellular reduced glutathione (GSH) and concomitant formation of a GSH-4-HNE adduct detected as 2,4-dinitrofluorobenzene derivatives measured by reversed-phase HPLC. Hepatocellular elimination of 4-HNE was estimated at rates of 1.666, 0.902, and 0.219 nmol min-1 10(6) hepatocytes-1 for 50, 100, and 250 microM aldehyde, respectively. At aldehyde concentrations of 50, 100, and 250 microM the maximal concentrations of oxidative (acid) metabolites formed were 5.9, 12.7, and 28.9 nmoles 10(6) hepatocytes-1, whereas the concentrations of the reductive (diol) metabolite were 0.4, 12.6, and 42.3 nmoles 10(6) hepatocytes-1, respectively. The presence of 4-methylpyrazole or cyanamide abolished formation of the reductive metabolite 1,4-dihydroxy-2-nonene or the oxidative metabolite 4-hydroxy-2-nonenoic acid in hepatocyte suspensions. At all 4-HNE concentrations evaluated, hepatocellular glutathione was not completely depleted by the aldehyde and the depletion of cellular reduced GSH corresponded to the production of the GSH-4-HNE conjugate. Metabolism by the alcohol/aldehyde dehydrogenase pathways accounted for approximately 10% of the 4-HNE elimination, while bioconversion by GST represent 50-60% of the total 4-HNE removal by hepatocytes. The enzymatic pathways responsible for the remaining 40% of 4-HNE metabolism remain to be identified. Taken together these results describe the quantitative and dynamic importance of oxidative, reductive, and nonoxidative routes in the metabolism and detoxification of 4-HNE.
Amyloid beta protein (A beta) is a 40-43 amino acid peptide that is associated with plaques in the brains of Alzheimer's patients and is cytotoxic to cultured neurons. Using both primary central nervous system cultures and clonal cell lines, it is shown that a number of anti-oxidants protect cells from A beta toxicity, suggesting that at least one pathway to A beta cytotoxicity results in free radical damage. A beta causes increased levels of H2O2 and lipid peroxides to accumulate in cells. The H2O2-degrading enzyme catalase protects cells from A beta toxicity. Clonal cell lines selected for their resistance to A beta toxicity also become resistant to the cytolytic action of H2O2. In addition, A beta induces the activity of NF-kappa B, a transcription factor thought to be regulated by oxidative stress. Finally, A beta-induced H2O2 production and A beta toxicity are blocked by reagents that inhibit flavin oxidases, suggesting that A beta activates a member of this class of enzymes. These results show that the cytotoxic action of A beta on neurons results from free radical damage to susceptible cells.
Alternative processing of the beta-amyloid precursor protein (beta APP) can result in liberation of either secreted forms of beta APP (APPSs), which may play roles in neuronal plasticity and survival, or amyloid beta-peptide (A beta), which can be neurotoxic. In rat hippocampal cell cultures A beta 1-40 caused a time- and concentration-dependent reduction in neuronal survival. APPS695 and APPS751 significantly reduced A beta-induced injury in a concentration-dependent manner. A beta caused an elevation of intracellular calcium levels ([Ca2+]i) which was significantly attenuated by APPSs. A beta also caused induction of reactive oxygen species (measured using the oxidation-sensitive fluorescent dye 2,7-dichlorofluorescein) which was also attenuated by APPSs. A beta-induced neurotoxicity and elevations of [Ca2+]i were attenuated by vitamin E, suggesting the involvement of free radicals in A beta-induced loss of calcium homeostasis and neuronal injury. The APPSs protected neurons against oxidative injury caused by exposure to iron. Taken together, the data indicate that A beta kills neurons by causing free radical production and increased [Ca2+]i. APPSs can protect neurons against such free radical- and Ca(2+)-mediated injury. These findings support the hypothesis that altered processing of beta APP contributes to neuronal injury in Alzheimer's disease.
We have previously reported (Hensley et al., Proc. Natl. Acad. Sci. USA (1994) in press) that beta-amyloid peptide fragments in aqueous media, in a metal-independent reaction, produce reactive peptide free radicals and reactive oxygen species. In contrast to the hours or days necessary to produce neurotoxicity and a detectable free radical for beta-amyloid, the extremely neurotoxic A beta(25-35) fragment of beta-amyloid peptide produces a detectable radical in minutes. We now report that A beta(25-35) is a potent lipoperoxidation initiator, as inferred from peptide-mediated reduction of nitroxyl stearate spin labels bound to rodent neocortical synaptosomal membranes. A beta(25-35) rapidly quenches the paramagnetism of membrane-bound 12-nitroxyl stearate spin probe deep within the lipid bilayer, but reacts poorly with the 5-nitroxyl isomer whose paramagnetic center is near the lipid/water interface. A beta(35-25), the non-neurotoxic reverse sequence of A beta(25-35), shows little proclivity to reduce either spin label. These findings are formulated into a "molecular shrapnel" model of neuronal membrane damage in Alzheimer's disease.
During aging long-lived proteins accumulate specific post-translational modifications. One family of modifications, termed Maillard reaction products, are initiated by the condensation between amino groups of proteins and reducing sugars. Protein modification by the Maillard reaction is associated with crosslink formation, decreased protein solubility, and increased protease resistance. Here, we present evidence that the characteristic pathological structures associated with Alzheimer disease contain modifications typical of advanced Maillard reaction end products. Specifically, antibodies against two Maillard end products, pyrraline and pentosidine, immunocytochemically label neurofibrillary tangles and senile plaques in brain tissue from patients with Alzheimer disease. In contrast, little or no staining is observed in apparently healthy neurons of the same brain. The Maillard-reaction-related modifications described herein could account for the biochemical and insolubility properties of the lesions of Alzheimer disease through the formation of protein crosslinks.