Hafiz Mohmmad Abdul

Cognitive Decline in Alzheimer's Disease Is Associated with Selective Changes in Calcineurin/NFAT Signaling

Upon activation by calcineurin, the nuclear factor of activated T-cells (NFAT) translocates to the nucleus and guides the transcription of numerous molecules involved in inflammation and Ca2+ dysregulation, both of which are prominent features of Alzheimers disease (AD). However, NFAT signaling in AD remains relatively uninvestigated. Using isolated cytosolic and nuclear fractions prepared from rapid-autopsy postmortem human brain tissue, we show that NFATs 1 and 3 shifted to nuclear compartments in the hippocampus at different stages of neuropathology and cognitive decline, whereas NFAT2 remained unchanged. NFAT1 exhibited greater association with isolated nuclear fractions in subjects with mild cognitive impairment (MCI), whereas NFAT3 showed a strong nuclear bias in subjects with severe dementia and AD. Similar to NFAT1, calcineurin-Aα also exhibited a nuclear bias in the early stages of cognitive decline. But, unlike NFAT1 and similar to NFAT3, the nuclear bias for calcineurin became more pronounced as cognition worsened. Changes in calcineurin/NFAT3 were directly correlated to soluble amyloid-β (Aβ(1-42)) levels in postmortem hippocampus, and oligomeric Aβ, in particular, robustly stimulated NFAT activation in primary rat astrocyte cultures. Oligomeric Aβ also caused a significant reduction in excitatory amino acid transporter 2 (EAAT2) protein levels in astrocyte cultures, which was blocked by NFAT inhibition. Moreover, inhibition of astrocytic NFAT activity in mixed cultures ameliorated Aβ-dependent elevations in glutamate and neuronal death. The results suggest that NFAT signaling is selectively altered in AD and may play an important role in driving Aβ-mediated neurodegeneration.

Acetyl-L-carnitine-induced up-regulation of heat shock proteins protects cortical neurons against amyloid-beta peptide 1–42-mediated oxidative stress and neurotoxicity: Implications for Alzheimer's disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and cognition and by senile plaques and neurofibrillary tangles in brain. Amyloid‐beta peptide, particularly the 42‐amino‐acid peptide (Aβ1–42), is a principal component of senile plaques and is thought to be central to the pathogenesis of the disease. The AD brain is under significant oxidative stress, and Aβ1–42 peptide is known to cause oxidative stress in vitro and in vivo. Acetyl‐L‐carnitine (ALCAR) is an endogenous mitochondrial membrane compound that helps to maintain mitochondrial bioenergetics and lowers the increased oxidative stress associated with aging. Glutathione (GSH) is an important endogenous antioxidant, and its levels have been shown to decrease with aging. Administration of ALCAR increases cellular levels of GSH in rat astrocytes. In the current study, we investigated whether ALCAR plays a protective role in cortical neuronal cells against Aβ1–42‐mediated oxidative stress and neurotoxicity. Decreased cell survival in neuronal cultures treated with Aβ1–42 correlated with an increase in protein oxidation (protein carbonyl, 3‐nitrotyrosine) and lipid peroxidation (4‐hydroxy‐2‐nonenal) formation. Pretreatment of primary cortical neuronal cultures with ALCAR significantly attenuated Aβ1–42‐induced cytotoxicity, protein oxidation, lipid peroxidation, and apoptosis in a dose‐dependent manner. Addition of ALCAR to neurons also led to an elevated cellular GSH and heat shock proteins (HSPs) levels compared with untreated control cells. Our results suggest that ALCAR exerts protective effects against Aβ1–42 toxicity and oxidative stress in part by up‐regulating the levels of GSH and HSPs. This evidence supports the pharmacological potential of acetyl carnitine in the management of Aβ1–42‐induced oxidative stress and neurotoxicity. Therefore, ALCAR may be useful as a possible therapeutic strategy for patients with AD.

A neuronal model of Alzheimer's disease: an insight into the mechanisms of oxidative stress-mediated mitochondrial injury.

Alzheimers disease (AD) is associated with beta-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and amyloid precursor protein (APP) (NLh/NLh) and presenilin 1 (PS1) (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared with WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to beta-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared with mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP(5+) (SOD mimetic) protected against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against beta-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.

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.

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid β-peptide (1–42), H2O2 and kainic acid: implications for Alzheimer's disease

Oxidative stress is observed in Alzheimers disease (AD) brain, including protein oxidation and lipid peroxidation. One of the major pathological hallmarks of AD is the brain deposition of amyloid beta‐peptide (Aβ). This 42‐mer peptide is derived from the β‐amyloid precursor protein (APP) and is associated with oxidative stress in vitro and in vivo. Mutations in the PS‐1 and APP genes, which increase production of the highly amyloidogenic amyloid β‐peptide (Aβ42), are the major causes of early onset familial AD. Several lines of evidence suggest that enhanced oxidative stress, inflammation, and apoptosis play important roles in the pathogenesis of AD. In the present study, primary neuronal cultures from knock‐in mice expressing mutant human PS‐1 and APP were compared with those from wild‐type mice, in the presence or absence of various oxidizing agents, viz, Aβ(1–42), H2O2 and kainic acid (KA). APP/PS‐1 double mutant neurons displayed a significant basal increase in oxidative stress as measured by protein oxidation, lipid peroxidation, and 3‐nitrotyrosine when compared with the wild‐type neurons (p < 0.0005). Elevated levels of human APP, PS‐1 and Aβ(1–42) were found in APP/PS‐1 cultures compared with wild‐type neurons. APP/PS‐1 double mutant neuron cultures exhibited increased vulnerability to oxidative stress, mitochondrial dysfunction and apoptosis induced by Aβ(1–42), H2O2 and KA compared with wild‐type neuronal cultures. The results are consonant with the hypothesis that Aβ(1–42)‐associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.

Interleukin-1β-dependent Signaling between Astrocytes and Neurons Depends Critically on Astrocytic Calcineurin/NFAT Activity

Interleukin-1β (IL-1β) and the Ca2+/calmodulin-dependent protein phosphatase, calcineurin, have each been shown to play an important role in neuroinflammation. However, whether these signaling molecules interact to coordinate immune/inflammatory processes and neurodegeneration has not been investigated. Here, we show that exogenous application of IL-1β (10 ng/ml) recruited calcineurin/NFAT (nuclear factor of activated T cells) activation in primary astrocyte-enriched cultures within minutes, through a pathway involving IL-1 receptors and L-type Ca2+ channels. Adenovirus-mediated delivery of the NFAT inhibitor, VIVIT, suppressed the IL-1β-dependent induction of several inflammatory mediators and/or markers of astrocyte activation, including tumor necrosis factor α, granulocyte/macrophage colony-stimulating factor, and vimentin. Expression of an activated form of calcineurin in one set of astrocyte cultures also triggered the release of factors that, in turn, stimulated NFAT activity in a second set of “naive” astrocytes. This effect was prevented when calcineurin-expressing cultures co-expressed VIVIT, suggesting that the calcineurin/NFAT pathway coordinates positive feedback signaling between astrocytes. In the presence of astrocytes and neurons, 48-h delivery of IL-1β was associated with several excitotoxic effects, including NMDA receptor-dependent neuronal death, elevated extracellular glutamate, and hyperexcitable synaptic activity. Each of these effects were reversed or ameliorated by targeted delivery of VIVIT to astrocytes. IL-1β also caused an NFAT-dependent reduction in excitatory amino acid transporter levels, indicating a possible mechanism for IL-1β-mediated excitotoxicity. Taken together, the results have potentially important implications for the propagation and maintenance of neuroinflammatory signaling processes associated with many neurodegenerative conditions and diseases.

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease.

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age

Oxidative stress is strongly implicated in the progressive decline of cognition associated with aging and neurodegenerative disorders. In the brain, free radical-mediated oxidative stress plays a critical role in the age-related decline of cellular function as a result of the oxidation of proteins, lipids, and nucleic acids. A number of studies indicate that an increase in protein oxidation and lipid peroxidation is associated with age-related neurodegenerative diseases and cellular dysfunction observed in aging brains. Oxidative stress is one of the important factors contributing to Alzheimers disease (AD), one of whose major hallmarks includes brain depositions of amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP). Mutation in APP and PS-1 genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. In the present study, protein oxidation and lipid peroxidation in the brain from knock-in mice expressing human mutant APP and PS-1 were compared with brain from wild type, as a function of age. The results suggest that there is an increased oxidative stress in the brain of wild-type mice as a function of age. In APP/PS-1 mouse brain, there is a basal increase (at 1 month) in oxidative stress compared to the wild type (1 month), as measured by protein oxidation and lipid peroxidation. In addition, age-related elevation of oxidative damage was observed in APP/PS-1 mice brain compared to that of wild-type mice brain. These results are discussed with reference to the importance of Abeta42-associated oxidative stress in the pathogenesis of AD.

APP and PS-1 mutations induce brain oxidative stress independent of dietary cholesterol: implications for Alzheimer's disease

Epidemiological and biochemical studies strongly implicate a role for cholesterol in the pathogenesis of Alzheimers disease (AD). Mutation in the PS-1 and APP genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. The AD brain is under significant oxidative stress, including protein oxidation and lipid peroxidation. In the present study, protein oxidation and lipid peroxidation were compared in the brain homogenates from knock-in mice expressing mutant human PS-1 and APP in relation to the intake of dietary cholesterol. The APP and PS-1 mice displayed increased oxidative stress as measured by protein oxidation and lipid peroxidation, independent of dietary cholesterol. These results are discussed with reference to proposed therapeutic strategies of AD.