Manuela Aragno

Oxidative stress activates a positive feedback between the γ‐ and β‐secretase cleavages of the β‐amyloid precursor protein

Sequential cleavages of the β‐amyloid precursor protein cleaving enzyme 1 (BACE1) by β‐secretase and γ‐secretase generate the amyloid β‐peptides, believed to be responsible of synaptic dysfunction and neuronal cell death in Alzheimer’s disease (AD). Levels of BACE1 are increased in vulnerable regions of the AD brain, but the underlying mechanism is unknown. Here we show that oxidative stress (OS) stimulates BACE1 expression by a mechanism requiring γ‐secretase activity involving the c‐jun N‐terminal kinase (JNK)/c‐jun pathway. BACE1 levels are increased in response to OS in normal cells, but not in cells lacking presenilins or amyloid precursor protein. Moreover, BACE1 is induced in association with OS in the brains of mice subjected to cerebral ischaemia/reperfusion. The OS‐induced BACE1 expression correlates with an activation of JNK and c‐jun, but is absent in cultured cells or mice lacking JNK. Our findings suggest a mechanism by which OS induces BACE1 transcription, thereby promoting production of pathological levels of amyloid β in AD.

H2o2 and 4-hydroxynonenal mediate amyloid β-induced neuronal apoptosis by activating jnks and p38mapk

Amyloid beta peptides (Abeta) may be neurotoxic during the progression of Alzheimers disease by eliciting oxidative stress. Exposure of neuronally differentiated SK-N-BE cells to Abeta(25-35) fragment as well as to full-length Abeta(1-40) and Abeta(1-42) induces early and time-dependent generation of oxidative stress that has been evaluated by carefully monitoring generation of hydrogen peroxide (H(2)O(2)), 4-hydroxynonenal (HNE), thiobarbituric acid reactive substances (TBARS), and fluorescent chromolipids. Abeta treatment also results in the activation of c-Jun aminoterminal kinases (JNKs) and p38(MAPK) and is followed by characteristic nuclear changes of apoptosis as evaluated by DAPI staining and TUNEL technique. To reproduce the relationships between oxidative stress and Abeta apoptosis we found that only the simultaneous administration of HNE and H(2)O(2), at concentrations similar to those generated within the first 3 h of Abeta exposure, can fully mimic Abeta-dependent activation of JNKs and p38(MAPK) and occurrence of apoptosis. Antioxidants such as alpha-tocopherol and N-acetylcysteine prevent completely either neuronal apoptosis or activation of JNKs and p38(MAPK) elicited by Abeta or by simultaneous HNE and H(2)O(2) addition. Finally, direct evidence that activation of these kinases is required for cell death induced by Abeta has been obtained by pretreating cell with specific inhibitors of JNKs and p38(MAPK). These results suggest the existence of a sequence of events in Abeta-induced apoptosis involving simultaneous generation of HNE and H(2)O(2) and oxidative stress-dependent activation of JNKs and p38(MAPK).

The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1α

While it is well established that stroke and cerebral hypoperfusion are both significant risk factors for Alzheimer’s disease, the molecular link between ischemia and amyloid precursor protein processing has only been recently established. Specifically, hypoxia significantly increases β‐site APP cleaving enzyme (BACE1) gene transcription through the over‐expression of hypoxia inducible factor 1α, resulting in increased BACE1 secretase activity and amyloid‐β production. In this study, we significantly extend these findings both in vitro, in differentiated SK‐N‐BE neuroblastoma cells, and in vivo, in rats subjected to cerebral ischemia, showing that hypoxia up‐regulates BACE1 expression through a biphasic mechanism. The early post‐hypoxic up‐regulation of BACE1 depends on the production of reactive oxygen species mediated by the sudden interruption of the mitochondrial electron transport chain, while the later expression of BACE1 is caused by hypoxia inducible factor 1α activation. The involvement of reactive oxygen species released by mitochondria in the BACE1 up‐regulation was confirmed by the complete protection exerted by complex I inhibitors such as rotenone and diphenyl‐phenylen iodonium. Moreover, the oxidative stress‐mediated up‐regulation of BACE1 is mediated by c‐jun N terminal kinase pathway as demonstrated by the protection exerted by the silencing of c‐jun N‐terminal kinase isoforms 1 and 2. Our study strengthens the hypothesis that oxidative stress is a basic common mechanism of amyloid‐β accumulation.

Dehydroepiandrosterone protects tissues of streptozotocin-treated rats against oxidative stress

Chronic hyperglycemia in diabetes determines the overproduction of free radicals, and evidence is increasing that these contribute to the development of diabetic complications. It has recently been reported that dehydroepiandrosterone possesses antioxidant properties; this study evaluates whether, administered daily for three weeks per os, it may provide antioxidant protection in tissues of rats with streptozotocin-induced diabetes. Lipid peroxidation was evaluated on liver, brain and kidney homogenates from diabetic animals, measuring both steady-state concentrations of thiobarbituric acid reactive substances and fluorescent chromolipids. Hyperglycemic rats had higher thiobarbituric acid reactive substances formation and fluorescent chromolipids levels than controls. Dehydroepiandrosterone-treatment (4 mg/day for 3 weeks) protected tissues against lipid peroxidation: liver, kidney and brain homogenates from dehydroepiandrosterone-treated animals showed a significant decrease of both thiobarbituric acid reactive substances and fluorescent chromolipids formation. The effect of dehydroepiandrosterone on the cellular antioxidant defenses was also investigated, as impaired antioxidant enzyme activities were considered proof of oxygen-dependent toxicity. In kidney and liver homogenates, dehydroepiandrosterone treatment restored to near-control values the cytosolic level of reduced glutathione, as well as the enzymatic activities of superoxide-dismutase, glutathione-peroxidase, catalase. In the brain, only an increase of catalase activity was evident (p < .05), which reverted with dehydroepiandrosterone treatment. The results demonstrate that DHEA treatment clearly reduces oxidative stress products in the tissues of streptozotocin-treated rats.

AGEs/RAGE complex upregulates BACE1 via NF-κB pathway activation.

Although the pathogenesis of sporadic Alzheimer disease (AD) is not clearly understood, it is likely dependent on several age-related factors. Diabetes is a risk factor for AD, and multiple mechanisms connecting the 2 diseases have been proposed. Hyperglycemia enhances the formation of advanced glycation end products (AGEs) that result from the auto-oxidation of glucose and fructose. The interaction of AGEs with their receptor, named RAGE, elicits the formation of reactive oxygen species that are also believed to be an early event in AD pathology. To investigate a functional link between the disorders diabetes and AD, the effect of 2 AGEs, pentosidine and glyceraldehydes-derived pyridinium (GLAP), was studied on BACE1 expression both in vivo, in streptozotocin treated rats, and in vitro in differentiated neuroblastoma cells. We showed that pentosidine and GLAP were able to upregulate BACE1 expression through their binding with RAGE and the consequent activation of NF-κB. In addition, both pentosidine and GLAP were found to be increased in the brain in sporadic AD patients. Our findings demonstrate that activation of the AGEs/RAGE axis, by upregulating the key enzyme for amyloid-β production, provides a pathologic link between diabetes mellitus and AD.

PROTECTIVE EFFECT OF DEHYDROEPIANDROSTERONE AGAINST COPPER-INDUCED LIPID PEROXIDATION IN THE RAT

This study investigates the effectiveness and multitargeted activity of dehydroepiandrosterone (DHEA) as antioxidant in vivo. A single dose of DHEA was given IP to male rats. Liver and brain microsomes, and plasma low density lipoprotein (LDL), were isolated from rats sacrificed 17 h later. Liver and brain microsomes were challenged with CuSO(4) and, as index of lipid peroxidation, the production of thiobarbituric acid reactive substances (TBARS) was measaured. Also, plasma low-density lipoprotein (LDL) were challenged with copper and the time course of lipid peroxidation was evaluated following the formation of conjugated dienes. The onset of TBARS generation induced by copper was marked delayed in both liver and brain microsomes from DHEA-treated animals. Also, the resistance of LDL to oxidation, expressed by the duration of the lag-phase of the kinetic curve, was significantly enhanced in DHEA-treated rats. Results indicate that in vivo DHEA supplementation makes subcellular fractions isolated from different tissues and plasma constituents (LDL) more resistant to lipid peroxidation triggered by copper. The antioxidant effect on plasma LDL might be of special relevance to the proposed antiatherogenic activity of DHEA. Moreover, multitargeted antioxidant activity of DHEA might protect tissues from oxygen radicals damage.

Oxidative derangement in rat synaptosomes induced by hyperglycaemia : Restorative effect of dehydroepiandrosterone treatment

Central nervous system damage in diabetes is caused by both cerebral atherosclerosis and the detrimental effect of chronic hyperglycaemia on nervous tissue. Hyperglycaemia is the primer of a series of cascade reactions causing overproduction of free radicals. There is increasing evidence that these reactive molecules contribute to neuronal tissue damage. Dehydroepiandrosterone (DHEA) has been reported to possess antioxidant properties. This study evaluates the oxidative status in the synaptosomal fraction isolated from the brain of streptozotocin-treated rats and the antioxidant effect of DHEA treatment on diabetic rats. Hydroxyl radical generation, hydrogen peroxide content, and the level of the reactive oxygen species was increased (P<0.05) in synaptosomes isolated from streptozotocin-treated rats. The derangement of the oxidative status was confirmed by a low level of reduced glutathione and alpha-tocopherol. DHEA treatment (4 mg per day for 3 weeks, per os) protected the synaptosomes against oxidative damage: synaptosomes from diabetic DHEA-treated rats showed a significant decrease in reactive species (P<0.05) and in the formation of end products of lipid peroxidation, evaluated in terms of fluorescent chromolipid (P<0.01). Moreover, DHEA treatment restored the unsaturated fatty acid content of the membrane and the reduced glutathione and alpha-tocopherol levels to normal levels and restored membrane NaK-ATPase activity close to control levels. The results demonstrate that DHEA supplementation greatly reduces oxidative damage in synaptosomes isolated from diabetic rats and suggest that this neurosteroid may participate in protecting the integrity of synaptic membranes against hyperglycaemia-induced damage.

SREBP-1c in nonalcoholic fatty liver disease induced by Western-type high-fat diet plus fructose in rats.

This study concentrated on the initial events triggering the development of nonalcoholic fatty liver disease induced by a high-fat plus fructose (HF-F) diet and on the possibility of delaying nonalcoholic fatty liver disease progression by adding dehydroepiandrosterone (DHEA) to the diet. Sterol regulatory element binding protein-1c (SREBP-1c) activation plays a crucial role in the progression of nonalcoholic fatty liver disease induced by an HF-F diet. This study investigated the protective effects of DHEA, a compound of physiological origin with multitargeted antioxidant properties, against the induction of SREBP-1c and on liver insulin resistance in rats fed an HF-F diet, which mimics a typical unhealthy Western diet. An HF-F diet, fortified or not with DHEA (0.01%, w/w), was administered for 15 weeks to male Wistar rats. After HF-F the liver showed unbalanced oxidative status, fatty infiltration, hepatic insulin resistance, and inflammation. The addition of DHEA to the diet reduced both activation of oxidative-stress-dependent pathways and expression of SREBP-1c and partially restored the expression of liver X-activated receptor-alpha and insulin receptor substrate-2 genes. DHEA supplementation of the HF-F diet reduced de novo lipogenesis and delayed progression of nonalcoholic fatty liver disease, demonstrating a relationship between oxidative stress and nonalcoholic fatty liver disease via SREBP-1c.

Pioglitazone improves lipid and insulin levels in overweight rats on a high cholesterol and fructose diet by decreasing hepatic inflammation

Background and purpose:  Nutrient overload leads to obesity and insulin resistance. Pioglitazone, a selective peroxisome proliferator‐activated receptor (PPAR)γ agonist, is currently used to manage insulin resistance, but the specific molecular mechanisms activated by PPARγ are not yet fully understood. Recent studies suggest the involvement of suppressor of cytokine signalling (SOCS)‐3 in the pathogenesis of insulin resistance. This study aimed to investigate the hepatic signalling pathway activated by PPARγ activation in a non‐genetic insulin‐resistant animal model.

Insulin Reduces Cerebral Ischemia/Reperfusion Injury in the Hippocampus of Diabetic Rats: A Role for Glycogen Synthase Kinase-3β

OBJECTIVE—There is evidence that insulin reduces brain injury evoked by ischemia/reperfusion (I/R). However, the molecular mechanisms underlying the protective effects of insulin remain unknown. Insulin is a well-known inhibitor of glycogen synthase kinase-3β (GSK-3β). Here, we investigate the role of GSK-3β inhibition on I/R-induced cerebral injury in a rat model of insulinopenic diabetes. RESEARCH DESIGN AND METHODS—Rats with streptozotocin-induced diabetes were subjected to 30-min occlusion of common carotid arteries followed by 1 or 24 h of reperfusion. Insulin (2–12 IU/kg i.v.) or the selective GSK-3β inhibitor TDZD-8 (0.2–3 mg/kg i.v.) was administered during reperfusion. RESULTS—Insulin or TDZD-8 dramatically reduced infarct volume and levels of S100B protein, a marker of cerebral injury. Both drugs induced phosphorylation of the Ser9 residue, thereby inactivating GSK-3β in the rat hippocampus. Insulin, but not TDZD-8, lowered blood glucose. The hippocampi of the drug-treated animals displayed reduced oxidative stress at 1 h of reperfusion as shown by the decreased generation of reactive oxygen species and lipid peroxidation. I/R-induced activation of nuclear factor-κB was attenuated by both drug treatments. At 24 h of reperfusion, TDZD-8 and insulin significantly reduced plasma levels of tumor necrosis factor-α; neutrophil infiltration, measured as myeloperoxidase activity and intercellular-adhesion-molecule-1 expression; and cyclooxygenase-2 and inducible-NO-synthase expression. CONCLUSIONS—Acute administration of insulin or TDZD-8 reduced cerebral I/R injury in diabetic rats. We propose that the inhibitory effect on the activity of GSK-3β contributes to the protective effect of insulin independently of any effects on blood glucose.