Blockade of Astrocytic Calcineurin/NFAT Signaling Helps to Normalize Hippocampal Synaptic Function and Plasticity in a Rat Model of Traumatic Brain Injury.

Increasing evidence suggests that the calcineurin (CN)-dependent transcription factor NFAT (Nuclear Factor of Activated T cells) mediates deleterious effects of astrocytes in progressive neurodegenerative conditions. However, the impact of astrocytic CN/NFAT signaling on neural function/recovery after acute injury has not been investigated extensively. Using a controlled cortical impact (CCI) procedure in rats, we show that traumatic brain injury is associated with an increase in the activities of NFATs 1 and 4 in the hippocampus at 7 d after injury. NFAT4, but not NFAT1, exhibited extensive labeling in astrocytes and was found throughout the axon/dendrite layers of CA1 and the dentate gyrus. Blockade of the astrocytic CN/NFAT pathway in rats using adeno-associated virus (AAV) vectors expressing the astrocyte-specific promoter Gfa2 and the NFAT-inhibitory peptide VIVIT prevented the injury-related loss of basal CA1 synaptic strength and key synaptic proteins and reduced the susceptibility to induction of long-term depression. In conjunction with these seemingly beneficial effects, VIVIT treatment elicited a marked increase in the expression of the prosynaptogenic factor SPARCL1 (hevin), especially in hippocampal tissue ipsilateral to the CCI injury. However, in contrast to previous work on Alzheimer's mouse models, AAV-Gfa2-VIVIT had no effects on the levels of GFAP and Iba1, suggesting that synaptic benefits of VIVIT were not attributable to a reduction in glial activation per se. Together, the results implicate the astrocytic CN/NFAT4 pathway as a key mechanism for disrupting synaptic remodeling and homeostasis in the hippocampus after acute injury. SIGNIFICANCE STATEMENT: Similar to microglia, astrocytes become strongly "activated" with neural damage and exhibit numerous morphologic/biochemical changes, including an increase in the expression/activity of the protein phosphatase calcineurin. Using adeno-associated virus (AAV) to inhibit the calcineurin-dependent activation of the transcription factor NFAT (Nuclear Factor of Activated T cells) selectively, we have shown that activated astrocytes contribute to neural dysfunction in animal models characterized by progressive/chronic neuropathology. Here, we show that the suppression of astrocytic calcineurin/NFATs helps to protect synaptic function and plasticity in an animal model in which pathology arises from a single traumatic brain injury. The findings suggest that at least some astrocyte functions impair recovery after trauma and may provide druggable targets for treating victims of acute nervous system injury.

Inhibition of soluble tumor necrosis factor ameliorates synaptic alterations and Ca2+ dysregulation in aged rats.

The role of tumor necrosis factor α (TNF) in neural function has been investigated extensively in several neurodegenerative conditions, but rarely in brain aging, where cognitive and physiologic changes are milder and more variable. Here, we show that protein levels for TNF receptor 1 (TNFR1) are significantly elevated in the hippocampus relative to TNF receptor 2 (TNFR2) in aged (22 months) but not young adult (6 months) Fischer 344 rats. To determine if altered TNF/TNFR1 interactions contribute to key brain aging biomarkers, aged rats received chronic (4-6 week) intracranial infusions of XPro1595: a soluble dominant negative TNF that preferentially inhibits TNFR1 signaling. Aged rats treated with XPro1595 showed improved Morris Water Maze performance, reduced microglial activation, reduced susceptibility to hippocampal long-term depression, increased protein levels for the GluR1 type glutamate receptor, and lower L-type voltage sensitive Ca(2+) channel (VSCC) activity in hippocampal CA1 neurons. The results suggest that diverse functional changes associated with brain aging may arise, in part, from selective alterations in TNF signaling.

Proteolysis of calcineurin is increased in human hippocampus during mild cognitive impairment and is stimulated by oligomeric Abeta in primary cell culture.

Recent reports demonstrate that the activation and interaction of the protease calpain (CP) and the protein phosphatase calcineurin (CN) are elevated in the late stages of Alzheimer's disease (AD). However, the extent to which CPs and CN interact during earlier stages of disease progression remains unknown. Here, we investigated CP and CN protein levels in cytosolic, nuclear, and membrane fractions prepared from human postmortem hippocampal tissue from aged non-demented subjects, and subjects diagnosed with mild cognitive impairment (MCI). The results revealed a parallel increase in CP I and the 48 kDa CN-Aα (ΔCN-Aα48) proteolytic fragment in cytosolic fractions during MCI. In primary rat hippocampal cultures, CP-dependent proteolysis and activation of CN was stimulated by application of oligomeric Aß((1-42)) peptides. Deleterious effects of Aß on neuronal morphology were reduced by blockade of either CP or CN. NMDA-type glutamate receptors, which help regulate cognition and neuronal viability, and are modulated by CPs and CN, were also investigated in human hippocampus. Relative to controls, MCI subjects showed significantly greater proteolytic levels of the NR2B subunit. Within subjects, the extent of NR2B proteolysis was strongly correlated with the generation of ΔCN-Aα48 in the cytosol. A similar proteolytic pattern for NR2B was also observed in primary rat hippocampal cultures treated with oligomeric Aß and prevented by inhibition of CP or CN. Together, the results demonstrate that the activation and interaction of CPs and CN are increased early in cognitive decline associated with AD and may help drive other pathologic processes during disease progression.

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 beta-peptide (1-42), HO and kainic acid: implications for Alzheimer's disease.

Oxidative stress is observed in Alzheimer's 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 (Abeta). This 42-mer peptide is derived from the beta-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 beta-peptide (Abeta42), 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, Abeta(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 Abeta(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 Abeta(1-42), H2O2 and KA compared with wild-type neuronal cultures. The results are consonant with the hypothesis that Abeta(1-42)-associated oxidative stress and increased vulnerability to oxidative stress may contribute significantly to neuronal apoptosis and death in familial early onset AD.

Protection against amyloid beta-peptide (1-42)-induced loss of phospholipid asymmetry in synaptosomal membranes by tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester: implications for Alzheimer's disease.

Amyloid-beta (1-42) [Abeta (1-42)] deposition in the brain is a hallmark of Alzheimer's disease (AD) and has been shown to induce apoptosis and disrupt cellular ion homeostasis. Abeta (1-42) induces membrane lipid peroxidation, and 4-hydroxynonenal (HNE) and 2-propenal (acrolein) are the two reactive products of lipid peroxidation, which structurally modify proteins by covalent interaction and inhibit enzyme function. Phosphatidylserine (PS), an aminophospholipid, is sequestered in the inner leaflet of the plasma membrane in nonstimulated cells. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine in the outer leaflet of the membrane. The ATP-requiring enzyme, flippase, maintains phospholipid asymmetry of PS. Here, we have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by Abeta (1-42). Flippase activity depends on a critical cysteine residue, a putative site of covalent modification by the Abeta (1-42)-induced lipid peroxidation products, HNE or acrolein. The present study is aimed to investigate the protective effects of tricyclodecan-9-xanthogenate (D609) and ferulic acid ethyl ester (FAEE) on Abeta (1-42) induced modulation in phospholipid asymmetry in the synaptosomal membranes. Pretreatment of synaptosomes with D609 and FAEE significantly protected Abeta (1-42)-induced loss of phospholipid asymmetry in synaptosomal membranes. Our results suggest that D609 and FAEE exert protective effects against Abeta (1-42) induced apoptosis. The increase in intracellular Ca(2+) might not be the sole cause for the loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in the modulation of phospholipid asymmetry. The results of this study are discussed with relevance to neuronal loss in the AD brain.

HIV-dementia, Tat-induced oxidative stress, and antioxidant therapeutic considerations.

Oxidative stress is thought to play a role in the onset of dementia. HIV-dementia has recently been demonstrated to be associated with oxidative stress as indexed by increased protein and lipid peroxidation in the brain and cerebrospinal fluid compared to HIV non-demented patients. The HIV protein Tat induces neurotoxicity, and, more recently, Tat was found to induce oxidative stress directly and indirectly. The role of Tat in HIV-dementia and possible therapeutic strategies involving endogenous and exogenous antioxidants are discussed.

Neurotoxicity and oxidative stress in D1M-substituted Alzheimer's A beta(1-42): relevance to N-terminal methionine chemistry in small model peptides.

Small model peptides containing N-terminal methionine are reported to form sulfur-centered-free radicals that are stabilized by the terminal N atom. To test whether a similar chemistry would apply to a disease-relevant longer peptide, Alzheimer's disease (AD)-associated amyloid beta-peptide 1-42 was employed. Methionine at residue 35 of this 42-mer has been shown to be a key amino acid residue involved in amyloid beta-peptide 1-42 [A beta1-42]-mediated toxicity and therefore, the pathogenesis of AD. Previous studies have shown that mutation of the methionine residue to norleucine abrogates the oxidative stress and neurotoxic properties of A beta(1-42). In the current study, we examined if the position of methionine at residue 35 is a criterion for toxicity. In doing so, we tested the effects of moving methionine to the N-terminus of the peptide in a synthetic peptide, A beta(1-42)D1M, in which methionine was substituted for aspartic acid at the N-terminus of the peptide and all subsequent residues from D1 to L34 were shifted one position towards the carboxy-terminus. A beta(1-42)D1M exhibited oxidative stress and neurotoxicity properties similar to those of the native peptide, A beta(1-42), all of which are inhibited by the free radical scavenger Vitamin E, suggesting that reactive oxygen species may play a role in the A beta-mediated toxicity. Additionally, substitution of methionine at the N-terminus by norleucine, A beta(1-42)D1Nle, completely abrogated the oxidative stress and neurotoxicity associated with the A beta(1-42)D1M peptide. The results of this study validate the chemistry reported for short peptides with N-terminal methionines in a disease-relevant peptide.

Ferulic acid ethyl ester protects neurons against amyloid beta- peptide(1-42)-induced oxidative stress and neurotoxicity: relationship to antioxidant activity.

Alzheimer's disease (AD) is neuropathologically characterized by depositions of extracellular amyloid and intracellular neurofibrillary tangles, associated with loss of neurons in the brain. Amyloid beta-peptide (Abeta) is the major component of senile plaques and is considered to have a causal role in the development and progress of AD. Several lines of evidence suggest that enhanced oxidative stress and inflammation play important roles in the pathogenesis or progression of AD. The present study aimed to investigate the protective effects of ethyl-4-hydroxy-3-methoxycinnamic acid (FAEE), a phenolic compound which shows antioxidant and anti-inflammatory activity, on Abeta(1-42)-induced oxidative stress and neurotoxicity. We hypothesized that the structure of FAEE would facilitate radical scavenging and may induce protective proteins. Abeta(1-42) decreases cell viability, which was correlated with increased free radical formation, protein oxidation (protein carbonyl, 3-nitrotyrosine), lipid peroxidation (4-hydroxy-2-trans-nonenal) and inducible nitric oxide synthase. Pre-treatment of primary hippocampal cultures with FAEE significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular reactive oxygen species accumulation, protein oxidation, lipid peroxidation and induction of inducible nitric oxide synthase. Treatment of neurons with Abeta(1-42) increases levels of heme oxygenase-1 and heat shock protein 72. Consistent with a cellular stress response to the Abeta(1-42)-induced oxidative stress, FAEE treatment increases the levels of heme oxygenase-1 and heat shock protein 72, which may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells against Abeta(1-42)-induced toxicity. These results suggest that FAEE exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress directly and by inducing protective genes. These findings suggest that FAEE could potentially be of importance for the treatment of AD and other oxidative stress-related diseases.

Gamma-glutamylcysteine ethyl ester protection of proteins from Abeta(1-42)-mediated oxidative stress in neuronal cell culture: a proteomics approach.

Protein oxidation mediated by amyloid beta-peptide (1-42) (Abeta[1-42]) has been proposed to play a central role in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disorder associated with aging and the loss of cognitive function. The specific mechanism by which Abeta(1-42), the primary component of the senile plaque and a pathologic hallmark of AD, contributes to the oxidative damage evident in AD brain is unknown. Moreover, the specific proteins that are vulnerable to oxidative damage induced by Abeta(1-42) are unknown. Identification of such proteins could contribute to our understanding of not only the role of Abeta(1-42) in the pathogenesis of AD, but also provide insight into the mechanisms of neurodegeneration at the protein level in AD. We report the proteomic identification of two proteins found to be oxidized significantly in neuronal cultures treated with Abeta(1-42): 14-3-3zeta and glyceraldehyde-3-phosphate dehydrogenase. We also report that pretreatment of neuronal cultures with gamma-glutamylcysteine ethyl ester, a compound that supplies the limiting substrate for the synthesis of glutathione and results in the upregulation of glutathione in neuronal cultures, protects both proteins against Abeta(1-42)-mediated protein oxidation.

Role of phenylalanine 20 in Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity.

Senile plaques are a hallmark of Alzheimer's disease (AD), a neurodegenerative disease associated with cognitive decline and aging. Abeta(1-42) is the primary component of the senile plaque in AD brain and has been shown to induce protein oxidation in vitro and in vivo. Oxidative stress is extensive in AD brain. As a result, Abeta(1-42) has been proposed to play a central role in the pathogenesis of AD; however, the specific mechanism of neurotoxicity remains unknown. Recently, it has been proposed that long distance electron transfer from methionine 35 to the Cu(II) bound at the N terminus of Abeta(1-42) occurs via phenylalanine 20. Additionally, it was proposed that substitution of phenylalanine 20 of Abeta(1-42) by alanine [Abeta(1-42)F20A] would lessen the neurotoxicity induced by Abeta(1-42). In this study, we evaluate the predictions of this theoretical study by determining the oxidative stress and neurotoxic properties of Abeta(1-42)F20A relative to Abeta(1-42) in primary neuronal cell culture. Abeta(1-42)F20A induced protein oxidation and lipid peroxidation similar to Abeta(1-42) but to a lesser extent and in a manner inhibited by pretreatment of neurons with vitamin E. Additionally, Abeta(1-42)F20A affected mitochondrial function similar to Abeta(1-42), albeit to a lesser extent. Furthermore, the mutation does not appear to abolish the ability of the native peptide to reduce Cu(II). Abeta(1-42)F20A did not compromise neuronal morphology at 24 h incubation with neurons, but did so after 48 h incubation. Taken together, these results suggest that long distance electron transfer from methionine 35 through phenylalanine 20 may not play a pivotal role in Abeta(1-42)-mediated oxidative stress and neurotoxicity.

Rodent Abeta(1-42) exhibits oxidative stress properties similar to those of human Abeta(1-42): Implications for proposed mechanisms of toxicity.

Alzheimer's disease is a neurodegenerative disorder associated with aging and cognitive decline. Amyloid beta peptide (1-42) [Abeta(1-42)] is a primary constituent of senile plaques - a hallmark of Alzheimer's disease - and has been implicated in the pathogenesis of the disease. Previous studies have shown that methionine residue 35 of beta(1-42) may play a critical role in Abeta(1-42)-mediated oxidative stress and neurotoxicity. Several additional mechanisms of neurotoxicity have been proposed, including the role of Cu(II) binding and reduction to produce hydrogen peroxide and the role of peptide aggregation. It has been reported that rodent Abeta is less likely to form larger beta-sheet structures, and consequently, large aggregates. As a consequence of the lack of deposition of the peptide in rodent brain, rodent Abeta has been proposed to be non-toxic. Additionally, the sequence of the rodent variety of Abeta(1-42) contains three amino acid substitutions compared to the human sequence. These substitutions include the shift of arginine 5, trysosine 10, and histidine 13 to glycine, phenylalanine, and arginine, respectively. This shift in sequence within the Cu(II) binding region of the peptide results in a decrease in the ability of the rodent Abeta peptide to reduce Cu(II) to Cu(I) compared to the human Abeta peptide. As a result of the effect of the amino acid variations on the ability of the rodent peptide to reduce Cu(II) to Cu(I) compared to the human peptide, the rodent beta has been proposed to lack oxidative stress properties. In this study, the oxidative stress and neurotoxic properties of rodent beta(1-42) [Abeta(1-42)Rat] were evaluated and compared to those of human Abeta(1-42). Both human Abeta(1-42) and beta(1-42)Rat were found to have a significant effect on neuronal DNA fragmentation, loss of neuritic networks, and cell viability. beta(1-42) Rat was found to cause a significant increase in both protein oxidation and lipid peroxidation, similar to Abeta(1-42), both of which were inhibited by the lipid-soluble, chain breaking antioxidant vitamin E, suggesting that reactive oxygen species play a role in the Abeta-mediated toxicity. Taken together, these results suggest that Cu(II) reduction may not play a critical role inbeta(1-42)Rat-induced oxidative stress, and that the oxidative stress exhibited by this peptide may be a consequence of the presence of methionine 35, similar to the findings associated with the native human beta(1-42) peptide.

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 Alzheimer's 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.

Protective effect of the xanthate, D609, on Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress in primary neuronal cells.

Tricyclodecan-9-yl-xanthogenate (D609) is an inhibitor of phosphatidylcholine-specific phospholipase C, and this agent also has been reported to protect rodents against oxidative damage induced by ionizing radiation. Previously, we showed that D609 mimics glutathione (GSH) functions and that a disulfide is formed upon oxidation of D609 and the resulting dixanthate is a substrate for GSH reductase, regenerating D609. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid beta-peptide [Abeta(1-42)], elevated in AD brain, is associated with oxidative stress and toxicity. The present study aimed to investigate the protective effects of D609 on Abeta(1-42)-induced oxidative cell toxicity in cultured neurons. 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 (4-hydroxy-2-nonenal) formation. Pretreatment of primary hippocampal cultures with D609 significantly attenuated Abeta(1-42)-induced cytotoxicity, intracellular ROS accumulation, protein oxidation, lipid peroxidation and apoptosis. Methylated D609, with the thiol functionality no longer able to form the disulfide upon oxidation, did not protect neuronal cells against Abeta(1-42)-induced oxidative stress. Our results suggest that D609 exerts protective effects against Abeta(1-42) toxicity by modulating oxidative stress. These results may be of importance for the treatment of AD and other oxidative stress-related diseases.

Modulation of phospholipid asymmetry in synaptosomal membranes by the lipid peroxidation products, 4-hydroxynonenal and acrolein: implications for Alzheimer's disease.

Membrane lipid bilayer asymmetry is maintained by the ATP-dependent enzyme flippase. An early signal of synaptosomal apoptosis is the loss of phospholipid asymmetry and the appearance of phosphatidylserine (PS) in the outer leaflet of the membrane. Two highly reactive products of lipid peroxidation, 4-hydroxynonenal (HNE) and acrolein, both elevated in Alzheimer's disease (AD) brain, have been shown to induce apoptosis and disrupt cellular ion homeostasis. These reactive aldehydes can structurally modify proteins by covalent interaction and inhibit enzyme function. Phospholipid asymmetry of PS is maintained by the ATP-requiring enzyme flippase. We have investigated the inactivation of the transmembrane enzyme aminophospholipid-translocase (or flippase) by HNE and acrolein. Flippase activity depends on a critical cysteine residue, a possible site of covalent modification by HNE or acrolein. The present study demonstrates that these alkenals induce the appearance of PS on the outer bilayer lamellae and suggests that increases in intracellular Ca(2+) might not be the sole cause for loss of flippase activity. Rather, other mechanisms that could modulate the function of flippase might be important in phospholipid asymmetry disruption. These results are discussed with potential relevance to neuronal loss in Alzheimer's disease brain.