Cláudia P. Figueiredo

Connecting TNF-α Signaling Pathways to iNOS Expression in a Mouse Model of Alzheimer's Disease: Relevance for the Behavioral and Synaptic Deficits Induced by Amyloid β Protein

Increased brain deposition of amyloid β protein (Aβ) and cognitive deficits are classical signals of Alzheimers disease (AD) that have been highly associated with inflammatory alterations. The present work was designed to determine the correlation between tumor necrosis factor-α (TNF-α)-related signaling pathways and inducible nitric oxide synthase (iNOS) expression in a mouse model of AD, by means of both in vivo and in vitro approaches. The intracerebroventricular injection of Aβ1–40 in mice resulted in marked deficits of learning and memory, according to assessment in the water maze paradigm. This cognition impairment seems to be related to synapse dysfunction and glial cell activation. The pharmacological blockage of either TNF-α or iNOS reduced the cognitive deficit evoked by Aβ1–40 in mice. Similar results were obtained in TNF-α receptor 1 and iNOS knock-out mice. Aβ1–40 administration induced an increase in TNF-α expression and oxidative alterations in prefrontal cortex and hippocampus. Likewise, Aβ1–40 led to activation of both JNK (c-Jun-NH2-terminal kinase)/c-Jun and nuclear factor-κB, resulting in iNOS upregulation in both brain structures. The anti-TNF-α antibody reduced all of the molecular and biochemical alterations promoted by Aβ1–40. These results provide new insights in mouse models of AD, revealing TNF-α and iNOS as central mediators of Aβ action. These pathways might be targeted for AD drug development.

Atorvastatin prevents hippocampal cell death, neuroinflammation and oxidative stress following amyloid-β1–40 administration in mice: Evidence for dissociation between cognitive deficits and neuronal damage

The accumulation of amyloid-beta (Aβ) peptides in the brain of human and rodents has been associated with the activation of glial cells, neuroinflammatory and oxidative responses, and cognitive deficits. These oxidative changes leave glutamate transporters more vulnerable and may result in reduction of their functions, resulting in excitotoxic damage. Herein, we evaluated the effects of atorvastatin, a HMG-CoA reductase inhibitor, in molecular and behavioral alterations induced by a single intracerebroventricular injection of aggregated Aβ(1-40) (400 pmol) in mice. An increased glial fibrillar acidic protein (GFAP) expression and cyclooxygenase-2 (COX-2) levels, as well as increased lipid peroxidation and impairment in the glutathione antioxidant system and cell degeneration was found in the hippocampus of Aβ(1-40)-treated mice. Aβ(1-40) also induced a marked decrease in glutamatergic transporters (GLAST and GLT-1) expression and in l-[³H] glutamate uptake in mice hippocampus, in addition to spatial learning and memory deficits. Atorvastatin (10 mg/kg/day v.o.) was administered after Aβ(1-40) injection and through seven consecutive days. Atorvastatin treatment was neuroprotective against cell degeneration induced by Aβ(1-40), reducing inflammatory and oxidative responses and increasing the expression of glutamatergic transporters. On the other hand, atorvastatin did not reverse the cognitive impairments and failed to alter the hippocampal glutamate uptake in Aβ(1-40)-treated mice. These results reinforce and extend the notion of the potential neuroprotective action of atorvastatin against the neuronal toxicity induced by Aβ(1-40). In addition, the present findings suggest that the spatial learning and memory deficits induced by Aβ peptides in rodents may not be entirely related to neuronal damage.

Effects of Traumatic Brain Injury of Different Severities on Emotional, Cognitive, and Oxidative Stress-Related Parameters in Mice

Cognitive deficits and psychiatric disorders are significant sequelae of traumatic brain injury (TBI). Animal models have been widely employed in TBI research, but few studies have addressed the effects of experimental TBI of different severities on emotional and cognitive parameters. In this study, mice were subjected to weight-drop TBI to induce mild, intermediate, or severe TBI. After neurological assessment, the mice recovered for 10 days, and were then subjected to a battery of behavioral tests, which included open-field, elevated plus-maze, forced swimming, tail suspension, and step-down inhibitory avoidance tests. Oxidative stress-related parameters (nonprotein thiols [NPSH], glutathione peroxidase [GPx], glutathione reductase [GR], and thiobarbituric acid reactive species [TBARS]) were quantified in the cortex and hippocampus at 2 and 24 h and 14 days after TBI, and histopathological analysis was performed 15 days after TBI. Mice subjected to mild TBI showed increased anxiety and depressive-like behaviors, while intermediate and severe TBI induced robust memory deficits. The severe TBI group also displayed increased locomotor activity. Intermediate and severe TBI caused extensive macroscopic and microscopic brain damage, while mild TBI typically had no histological abnormalities. Moreover, a significant increase in TBARS in the ipsilateral cortex and GPx in the ipsilateral hippocampus was observed at 24 h and 14 days, respectively, following intermediate TBI. The current experimental TBI model induced emotional and cognitive changes comparable to sequelae seen in human TBI, and it might therefore represent a useful approach to the study of mechanisms of and new treatments for TBI and related disorders.

Differential susceptibility following β-amyloid peptide-(1–40) administration in C57BL/6 and Swiss albino mice: Evidence for a dissociation between cognitive deficits and the glutathione system response

Considerable evidence supports the role of oxidative stress in the pathogenesis of Alzheimers disease (AD). Previous studies suggest that the central nervous system (CNS) administration of beta-amyloid peptide, the major constituent of senile plaque in AD, induces oxidative stress in rodents which may contribute to the learning and memory deficits verified in the beta-amyloid model of AD. In the present study, we compared the effects of a single intracerebroventricular (i.c.v.) injection of aggregated beta-amyloid peptide-(1-40) (Abeta(1-40)) (400pmol/mouse) on spatial learning and memory performance, synaptic density and the glutathione (GSH)-dependent antioxidant status in adult male C57BL/6 and Swiss albino mice. Seven days after Abeta(1-40) administration, C57BL/6 and Swiss mice presented similar spatial learning and memory impairments, as evaluated in the water maze task, although these impairments were not found in Abeta(40-1)-treated mice. Moreover, a similar decline of synaptophysin levels was observed in the hippocampus (HC) and prefrontal cortex (PFC) of both Swiss and C57BL/6 mice treated with Abeta(1-40), which suggests synaptic loss. C57BL/6 mice presented lower levels of glutathione-related antioxidant defences (total glutathione (GSH-t) levels, glutathione peroxidase (GPx) and glutathione reductase (GR) activity) in the HC and PFC in comparison to Swiss mice. Despite the reduced basal GSH-dependent antioxidant defences observed in C57BL/6 mice, Abeta(1-40) administration induced significant alterations in the brain antioxidant parameters only in Swiss mice, decreasing GSH-t levels and increasing GPx and GR activity in the HC and PFC 24h after treatment. These results indicate strain differences in the susceptibility to Abeta(1-40)-induced changes in the GSH-dependent antioxidant defences in mice, which should be taken into account in further studies using the Abeta model of AD in mice. In addition, the present findings suggest that the spatial learning and memory deficits induced by beta-amyloid peptides in rodents may not be entirely related to glutathione-dependent antioxidant response.

The role of TNF-α signaling pathway on COX-2 upregulation and cognitive decline induced by β-amyloid peptide

Alzheimers disease (AD), a chronic degenerative and inflammatory brain disorder characterized by neuronal dysfunction and loss, is linked to accumulation of beta-amyloid (Abeta) peptide. Tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase-2 (COX-2) are proteins that have key roles in immune cell activation, inflammation and cognitive function in the brain. Here, we evaluated the link between TNF-alpha and COX-2 on the acute responses elicited by Abeta. Behavioral and molecular analyses were performed in mice after an intracerebroventricular (i.c.v.) injection of Abeta(1-40). Genetic and/or pharmacological approaches were used to inhibit TNF-alpha and COX-2. I.c.v. Abeta(1-40) injection in mice activates TNF-alpha signaling pathway resulting in COX-2 upregulation, synaptic loss and cognitive decline. Pharmacological studies revealed that COX-2 is involved in the cognitive impairment mediated by TNF-alpha. However, COX-2 inhibition failed in reducing the synaptophysin loss induced by Abeta(1-40). The COX-2 upregulation induced by Abeta(1-40) was attributed to activation of different protein kinases and transcriptional factors that are greatly regulated by TNF-alpha. Together, these results indicate that Abeta(1-40) induces the activation of several TNF-alpha-dependent intracellular signaling pathways that play a key role in the control of COX-2 upregulation and activation, synaptic loss and cognitive decline in mice. Therefore, selective TNF-alpha inhibitors may be potentially interesting tools for AD drug development.

Alzheimer‐associated Aβ oligomers impact the central nervous system to induce peripheral metabolic deregulation

Alzheimers disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD‐associated Aβ oligomers (AβOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AβOs failed to induce glucose intolerance, suggesting AβOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AβOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α‐P). AβOs further induced eIF2α‐P and activated pro‐inflammatory IKKβ/NF‐κB signaling in the hypothalamus of mice and macaques. AβOs failed to trigger peripheral glucose intolerance in tumor necrosis factor‐α (TNF‐α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AβOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AβOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.

Memantine Rescues Transient Cognitive Impairment Caused by High-Molecular-Weight Aβ Oligomers But Not the Persistent Impairment Induced by Low-Molecular-Weight Oligomers

Brain accumulation of soluble amyloid-β oligomers (AβOs) has been implicated in synapse failure and cognitive impairment in Alzheimers disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) Aβ oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW AβOs (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW AβOs (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different Aβ oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.

Amyloid-β oligomers link depressive-like behavior and cognitive deficits in mice

Depression is one of the most common psychiatric symptoms in Alzheimers disease (AD), and considerable evidence indicates that major depressive disorder increases the risk of AD.1, 2, 3 To date, however, the molecular mechanisms underlying the clinical association between depression and AD have remained elusive. Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of AD patients and are increasingly recognized as the proximal neurotoxins responsible for synapse failure and memory deficits in AD.4, 5 We have hypothesized that AβOs might be mechanistically linked to behavioral changes in AD. In order to test this hypothesis, mice were given a single intracerebroventricular (i.c.v.) injection of 10 pmol AβOs and were subsequently evaluated in the Porsolt forced swim test (FST) for assessment of depressive-like behavior. Compared with vehicle-injected control mice, AβO-injected mice exhibited a significant increase in immobility in the FST, both 24 h and 8 days after AβO injections (Figure 1a). Similar results were obtained when animals were assessed in the tail suspension test, another classical task to evaluate depressive-like behavior in rodents (Supplementary Figure S1a). AβO-induced immobility in the FST was blocked by anti-depressant (fluoxetine) treatment (Figure 1a). An important feature of depressive disorder is anhedonic behavior, including decreased interest for pleasurable sensorial experiences. Whereas vehicle-injected mice exhibited an expected preference for sucrose solution over plain water, AβO-injected mice did not exhibit such preference, indicating that AβOs instigate anhedonic behavior (Figure 1b). Figure 1 Amyloid-β oligomers (AβOs) induce depressive-like behavior, memory deficits and hippocampal recruitment of microglia and astrocytes in mice. Three-month-old Swiss mice received a single intracerebroventricular (i.c.v.) injection of AβOs ... As memory deficit is the main clinical symptom of AD, we investigated the impact of AβOs on mice memory using the novel object recognition (OR) task. Results showed that 24 h after i.c.v. injection, AβO-treated mice spent equal amounts of time exploring both old (familiar) and new (novel) objects, indicating a deficit in declarative recognition memory, whereas vehicle-injected animals exhibited a significant preference for the novel object (Figure 1c). Treatment with fluoxetine prevented the memory deficit induced by AβOs (Figure 1c). Control measurements showed no changes in spontaneous exploratory or locomotor activities of fluoxetine-, saline-, vehicle- or AβO-injected animals during the training phase of the OR test (Supplementary Figure S1c–e). As the hippocampus is a key anatomical structure for OR memory, we sought to determine whether AβOs injected i.c.v. reached the hippocampus. Indeed, robust AβO immunoreactivity was verified using an anti-oligomer monoclonal antibody (NU4)6 in hippocampi from AβO-injected mice, but not in hippocampi from control vehicle-injected animals (Supplementary Figure S1b). Together, these results indicate that AβOs have an acute impact on memory, learning and mood in mice, and that fluoxetine treatment prevented both cognitive impairment and depressive-like behavior induced by AβOs. The beneficial actions of fluoxetine have been partly ascribed to its anti-inflammatory effect. This led us to ask whether AβOs triggered an inflammatory response in the mouse brain. The brains of mice used in the tests described above were analyzed for levels of pro-inflammatory cytokines after i.c.v. injection of AβOs or vehicle. AβO-injected animals showed significantly elevated brain levels of interleukin-1β and tumor necrosis factor-α compared with vehicle-injected animals (Figure 1d, e). Sections from the hippocampus and cortex of AβO- or vehicle-injected mice were further immunostained for the presence of microglia (anti-Iba-1 antibody) and astrocytes (anti-GFAP antibody). Compared with vehicle-injected animals, AβO-injected mice showed markedly increased immunoreactivities for both Iba-1 and GFAP in the hippocampus and cortex 24 h after injection (Figure 1f–o, and Supplementary Figure 1f–o). The increase in glial cell numbers instigated by AβOs was blocked by fluoxetine treatment of the animals before AβO injection (Figure 1f–o, and Supplementary Figure 1f–o). The current findings establish that AβOs link memory impairment and depressive-like behavior in mice, providing molecular mechanistic support to clinical evidence connecting AD and depressive disorder. The impact of AβOs on mood, learning and memory, and its prevention by fluoxetine, can likely be attributed to the activation of inflammatory pathways (as shown here) and, possibly, to the deregulation of the serotonergic axis. The latter possibility is in line with the observation that pro-inflammatory cytokines impact serotonin metabolism7, 8 and that increased serotonin levels are associated with lower brain Aβ levels in transgenic mouse models of AD and in humans.9 Moreover, 5-HT1A and 5-HT2A receptors have been reported to be reduced in post-mortem AD brain,10 and 5-HT1A receptors have been found to be reduced in vivo in AD.11 Brain disturbances that place a person at risk for developing depression and AD are still largely unknown. By revealing that AβOs underlie both cognitive and depressive-like symptoms in mice, our results suggest a mechanism by which elevated brain levels of AβOs may be linked to changes in cognition and mood in AD.

Therapeutic action and underlying mechanisms of a combination of two pentacyclic triterpenes, α‐ and β‐amyrin, in a mouse model of colitis

Background and purpose:  α‐ and β‐amyrin are pentacyclic triterpenes found in plants and are known to exhibit pronounced anti‐inflammatory effects. Here, we evaluated the effects of a 1:1 mixture of α‐ and β‐amyrin (α,β‐amyrin) on an experimental model of colitis in mice.

A study of the relative importance of the peroxiredoxin-, catalase-, and glutathione-dependent systems in neural peroxide metabolism

Cells are endowed with several overlapping peroxide-degrading systems whose relative importance is a matter of debate. In this study, three different sources of neural cells (rat hippocampal slices, rat C6 glioma cells, and mouse N2a neuroblastoma cells) were used as models to understand the relative contributions of individual peroxide-degrading systems. After a pretreatment (30 min) with specific inhibitors, each system was challenged with either H₂O₂ or cumene hydroperoxide (CuOOH), both at 100 μM. Hippocampal slices, C6 cells, and N2a cells showed a decrease in the H₂O₂ decomposition rate (23-28%) by a pretreatment with the catalase inhibitor aminotriazole. The inhibition of glutathione reductase (GR) by BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) significantly decreased H₂O₂ and CuOOH decomposition rates (31-77%). Inhibition of catalase was not as effective as BCNU at decreasing cell viability (MTT assay) and cell permeability or at increasing DNA damage (comet test). Impairing the thioredoxin (Trx)-dependent peroxiredoxin (Prx) recycling by thioredoxin reductase (TrxR) inhibition with auranofin neither potentiated peroxide toxicity nor decreased the peroxide-decomposition rate. The results indicate that neural peroxidatic systems depending on Trx/TrxR for recycling are not as important as those depending on GSH/GR. Dimer formation, which leads to Prx2 inactivation, was observed in hippocampal slices and N2a cells treated with H₂O₂, but not in C6 cells. However, Prx-SO₃ formation, another form of Prx inactivation, was observed in all neural cell types tested, indicating that redox-mediated signaling pathways can be modulated in neural cells. These differences in Prx2 dimerization suggest specific redox regulation mechanisms in glia-derived (C6) compared to neuron-derived (N2a) cells and hippocampal slices.