Mubeen A. Ansari

Oxidative Stress in the Progression of Alzheimer Disease in the Frontal Cortex

We investigated oxidative stress in human postmortem frontal cortexfrom individuals characterized as mild cognitive impairment (n= 8), mild/moderate Alzheimer disease (n = 4), and late-stage Alzheimer disease (n = 9). Samples from subjects with no cognitive impairment (n = 10) that were age- and postmortem interval-matched with these cases were used as controls. The short postmortem intervalbrain samples were processed for postmitochondrial supernatant, nonsynaptic mitochondria, and synaptosome fractions. Samples were analyzed for several antioxidants (glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, superoxide dismutase, catalase) and the oxidative marker, thiobarbituric acid reactive substances. The tissue was also analyzed for possible changes in protein damage using neurochemical markers for protein carbonyls, 3-nitrotyrosine, 4-hydroxynonenal, andacrolein. All 3 neuropil fractions (postmitochondrial supernatant, mitochondrial, and synaptosomal) demonstrated significant disease-dependent increases in oxidative markers. The highest changes were observed in the synaptosomal fraction. Both mitochondrial and synaptosomal fractions had significant declines in antioxidants (glutathione, glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase). Levels of oxidative markers significantly correlated with Mini-Mental Status Examination scores. Oxidative stress was more localized to the synapses, with levels increasing in a disease-dependent fashion. These correlations implicate an involvement of oxidative stress in Alzheimer disease-related synaptic loss.

Protective effect of quercetin in primary neurons against Aβ(1–42): relevance to Alzheimer's disease

Quercetin, a flavonoid found in various foodstuffs, has antioxidant properties and increases glutathione (GSH) levels and antioxidant enzyme function. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimers disease (AD). Amyloid beta-peptide [Abeta(1-42)], elevated in AD brain, is associated with oxidative stress and neurotoxicity. We aimed to investigate the protective effects of quercetin on Abeta(1-42)-induced oxidative cell toxicity in cultured neurons in the present study. Decreased cell survival in neuronal cultures treated with Abeta(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (protein-bound 4-hydroxy-2-nonenal). Pretreatment of primary hippocampal cultures with quercetin significantly attenuated Abeta(1-42)-induced cytotoxicity, protein oxidation, lipid peroxidation and apoptosis. A dose-response study suggested that quercetin showed protective effects against Abeta(1-42) toxicity by modulating oxidative stress at lower doses, but higher doses were not only non-neuroprotective but also toxic. These findings provide motivation to test the hypothesis that quercetin may provide a promising approach for the treatment of AD and other oxidative-stress-related neurodegenerative diseases.

Oxidative stress and modification of synaptic proteins in hippocampus after traumatic brain injury

Oxidative stress, an imbalance between oxidants and antioxidants, contributes to the pathogenesis of traumatic brain injury (TBI). Oxidative neurodegeneration is a key mediator of exacerbated morphological responses and deficits in behavioral recoveries. The present study assessed early hippocampal sequential imbalance to possibly enhance antioxidant therapy. Young adult male Sprague-Dawley rats were subjected to a unilateral moderate cortical contusion. At various times post-TBI, animals were killed and the hippocampus was analyzed for antioxidants (GSH, GSSG, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, glucose-6-phosphate dehydrogenase, superoxide dismutase, and catalase) and oxidants (acrolein, 4-hydroxynonenal, protein carbonyl, and 3-nitrotyrosine). Synaptic markers (synapsin I, postsynaptic density protein 95, synapse-associated protein 97, growth-associated protein 43) were also analyzed. All values were compared with those for sham-operated animals. Significant time-dependent changes in antioxidants were observed as early as 3 h posttrauma and paralleled increases in oxidants (4-hydroxynonenal, acrolein, and protein carbonyl), with peak values obtained at 24-48 h. Time-dependent changes in synaptic proteins (synapsin I, postsynaptic density protein 95, and synapse-associated protein 97) occurred well after levels of oxidants peaked. These results indicate that depletion of antioxidant systems following trauma could adversely affect synaptic function and plasticity. Early onset of oxidative stress suggests that the initial therapeutic window following TBI appears to be relatively short, and it may be necessary to stagger selective types of antioxidant therapy to target specific oxidative components.

A Time Course of Contusion-Induced Oxidative Stress and Synaptic Proteins in Cortex in a Rat Model of TBI

An imbalance between oxidants and antioxidants has been postulated to lead to oxidative damage in traumatic brain injury (TBI). Oxidative neurodegeneration is a key mediator of exacerbated morphological responses and deficits in behavioral recoveries. The present study was designed to delineate the early temporal sequence of this imbalance in order to enhance possible antioxidant therapy. Young adult male Sprague-Dawley rats were subjected to a unilateral moderate cortical contusion. At various times post-trauma (3, 6, 12, 24, 48, 72, and 96 h), animals were killed and the cortex analyzed for enzymatic and non-enzymatic oxidative stress markers. Fresh tissues were prepared for biochemical analysis of several antioxidants (glutathione [GSH], glutathione peroxidase [GPx], glutathione reductase [GR], glutathione-S-transferase [GST], and thiobarbituric acid reactive substances [TBARS]). Synaptic markers Synapsin-I, PSD-95, SAP-97 and GAP-43 were analyzed by Western blot with antibodies directed against them. All activity levels were compared to sham-operated animals. Activity of antioxidant enzymes and GSH clearly demonstrate a significant time-dependent increase in oxidative stress. Changes in pre- and post-synaptic proteins (Synapsin-I and PSD-95) occur early (24 h), whereas SAP-97 levels demonstrate a protracted reduction. These results indicate that depletion of antioxidant systems following trauma could adversely affect synaptic function and plasticity. Because of the observed differences in the time-course of various markers, it may be necessary to stagger selective types of anti-oxidant therapy to target specific oxidative components. The initial therapeutic window following TBI appears relatively short since oxidative damage occurs as early as 3 h.

Pin1 in Alzheimer's disease

Proteolytic processing and phosphorylation of amyloid precursor protein (APP), and hyperphosphorylation of tau protein, have been shown to be increased in Alzheimers disease (AD) brains, leading to increased production of β‐amyloid (Aβ) peptides and neurofibrillary tangles, respectively. These observations suggest that phosphorylation events are critical to the understanding of the pathogenesis and treatment of this devastating disease. Pin‐1, one of the peptidyl‐prolyl isomerases (PPIase), catalyzes the isomerization of the peptide bond between pSer/Thr‐Pro in proteins, thereby regulating their biological functions which include protein assembly, folding, intracellular transport, intracellular signaling, transcription, cell cycle progression and apoptosis. A number of previous studies have shown that Pin1 is co‐localized with phosphorylated tau in AD brain, and shows an inverse relationship to the expression of tau. Pin1 protects neurons under in vitro conditions. Moreover, recent studies demonstrate that APP is a target for Pin1 and thus, in Aβ production. Furthermore, Pin1 was found to be oxidatively modified and to have reduced activity in the hippocampus in mild cognitive impairment (MCI) and AD. Because of the diverse functions of Pin1, and the discovery that this protein is one of the oxidized proteins common to both MCI and AD brain, the question arises as to whether Pin1 is one of the driving forces for the initiation or progression of AD pathogenesis, finally leading to neurodegeneration and neuronal apoptosis. In the present review, we discuss the role of Pin1 with respect to Alzheimers disease.

REVIEW: Pin1 in Alzheimer's disease

Proteolytic processing and phosphorylation of amyloid precursor protein (APP), and hyperphosphorylation of tau protein, have been shown to be increased in Alzheimers disease (AD) brains, leading to increased production of β‐amyloid (Aβ) peptides and neurofibrillary tangles, respectively. These observations suggest that phosphorylation events are critical to the understanding of the pathogenesis and treatment of this devastating disease. Pin‐1, one of the peptidyl‐prolyl isomerases (PPIase), catalyzes the isomerization of the peptide bond between pSer/Thr‐Pro in proteins, thereby regulating their biological functions which include protein assembly, folding, intracellular transport, intracellular signaling, transcription, cell cycle progression and apoptosis. A number of previous studies have shown that Pin1 is co‐localized with phosphorylated tau in AD brain, and shows an inverse relationship to the expression of tau. Pin1 protects neurons under in vitro conditions. Moreover, recent studies demonstrate that APP is a target for Pin1 and thus, in Aβ production. Furthermore, Pin1 was found to be oxidatively modified and to have reduced activity in the hippocampus in mild cognitive impairment (MCI) and AD. Because of the diverse functions of Pin1, and the discovery that this protein is one of the oxidized proteins common to both MCI and AD brain, the question arises as to whether Pin1 is one of the driving forces for the initiation or progression of AD pathogenesis, finally leading to neurodegeneration and neuronal apoptosis. In the present review, we discuss the role of Pin1 with respect to Alzheimers disease.

NADPH-oxidase activation and cognition in Alzheimer disease progression.

Superoxide production via NADPH-oxidase (NOX) has been shown to play a role in a variety of neurological disorders, including Alzheimer disease (AD). To improve our understanding of the NOX system and cognitive impairment, we studied the various protein components of the phagocytic isoform (gp91(phox), or NOX2) in the frontal and temporal cortex of age- and postmortem-matched samples. Individuals underwent antemortem cognitive testing and postmortem histopathologic assessment to determine disease progression and assignment to one of the following groups: no cognitive impairment (NCI), preclinical AD, mild cognitive impairment (MCI), early AD, and mild-to-moderate AD. Biochemical methods were used to determine overall NOX activity as well as levels of the various subunits (gp91(phox), p67(phox), p47(phox), p40(phox), and p22(phox)). Overall enzyme activity was significantly elevated in the MCI cohort in both cortical regions compared to the NCI cohort. This activity level remained elevated in the AD groups. Only the NOX cytosolic subunit proteins (p67(phox), p47(phox), and p40(phox) ) were significantly elevated with disease progression; the membrane-bound subunits (gp91(phox) and p22(phox)) remained stable. In addition, there was a robust correlation between NOX activity and the individuals cognitive status such that as the enzyme activity increased, cognitive performance decreased. Collectively, these data show that upregulated NADPH-oxidase in frontal and temporal cortex suggests that increases in NOX-associated redox pathways might participate in early pathogenesis and contribute to AD progression.

Age-related changes in mitochondrial respiration and oxidative damage in the cerebral cortex of the Fischer 344 rat.

This study probed possible age-related changes in mitochondrial bioenergetics in naïve Fischer 344 rats. Synaptic and extrasynaptic mitochondria were isolated from the cortex of one hemisphere of young (3-5 months), middle (12-14 months), or aged (22-24 months) rats. Respiration parameters were obtained using a Clarke-type electrode. Aged rats displayed no significant alterations in respiration, indicating mitochondria must be more resilient to the aging process than previously thought. Synaptic mitochondria displayed lower respiration capacities than the extrasynaptic fraction. Aged F344 rats appear capable of normal electron transport chain function without declines in ability to produce ATP. Markers of cortical oxidative damage (3-nitrotyrosine [3-NT], 4-hydroxynonenal [4-HNE], and protein carbonyls [PC]) were collected from the post-mitochondrial supernatant (PMS) from the contralateral hemisphere, and from mitochondrial samples following respiration analysis. Age-related increases in PC and 3-NT levels were found in synaptic mitochondria, whereas significant extrasynaptic elevations were only found in middle aged rats. These findings support an age-related increase in oxidative damage in the cortex, while proposing the two fractions of mitochondria are differentially affected by the aging process. Levels of oxidative damage that accumulates in the cortex with age does not appear to significantly impair cortical mitochondrial respiration of F344 rats.

Protective effect of Pycnogenol in human neuroblastoma SH-SY5Y cells following acrolein-induced cytotoxicity.

Oxidative stress is one of the hypotheses involved in the etiology of Alzheimers disease (AD). Considerable attention has been focused on increasing the intracellular glutathione (GSH) levels in many neurodegenerative diseases, including AD. Pycnogenol (PYC) has antioxidant properties and stabilizes intracellular antioxidant defense systems including glutathione levels. The present study investigated the protective effects of PYC on acrolein-induced oxidative cell toxicity in cultured SH-SY5Y neuroblastoma cells. Decreased cell survival in SH-SY5Y cultures treated with acrolein correlated with oxidative stress, increased NADPH oxidase activity, free radical production, protein oxidation/nitration (protein carbonyl, 3-nitrotyrosine), and lipid peroxidation (4-hydroxy-2-nonenal). Pretreatment with PYC significantly attenuated acrolein-induced cytotoxicity, protein damage, lipid peroxidation, and cell death. A dose-response study suggested that PYC showed protective effects against acrolein toxicity by modulating oxidative stress and increasing GSH. These findings provide support that PYC may provide a promising approach for the treatment of oxidative stress-related neurodegenerative diseases such as AD.

Synaptic Change in the Posterior Cingulate Gyrus in the Progression of Alzheimer's Disease

Mild cognitive impairment (MCI) is considered to be an early stage in the progression of Alzheimers disease (AD) providing an opportunity to investigate brain pathogenesis prior to the onset of dementia. Neuroimaging studies have identified the posterior cingulate gyrus (PostC) as a cortical region affected early in the onset of AD. This association cortex is involved in a variety of different cognitive tasks and is intimately connected with the hippocampal/entorhinal cortex region, a component of the medial temporal memory circuit that displays early AD pathology. We quantified the total number of synapses in lamina 3 of the PostC using unbiased stereology coupled with electron microscopy from short postmortem autopsy tissue harvested from cases at different stage of AD progression. Individuals in the early stages of AD showed a significant decline in synaptic numbers compared to individuals with no cognitive impairment (NCI). Subjects with MCI exhibited synaptic numbers that were between the AD and NCI cohorts. Adjacent tissue was evaluated for changes in both pre and postsynaptic proteins levels. Individuals with MCI demonstrated a significant loss in presynaptic markers synapsin-1 and synaptophysin and postsynaptic markers PSD-95 and SAP-97. Levels of [3H]PiB binding was significantly increased in MCI and AD and correlated strongly with levels of synaptic proteins. All synaptic markers showed a significant association with Mini-Mental Status Examination scores. These results support the idea that the PostC synaptic function is affected during the prodromal stage of the disease and may underlie some of the early clinical sequelae associated with AD.