Cellular senescence promotes progenitor cell expansion during axolotl limb regeneration.

Axolotl limb regeneration is accompanied by the transient induction of cellular senescence within the blastema, the structure that nucleates regeneration. The precise role of this blastemal senescent cell (bSC) population, however, remains unknown. Here, through a combination of gain- and loss-of-function assays, we elucidate the functions and molecular features of cellular senescence in vivo. We demonstrate that cellular senescence plays a positive role during axolotl regeneration by creating a pro-proliferative niche that supports progenitor cell expansion and blastema outgrowth. Senescent cells impact their microenvironment via Wnt pathway modulation. Further, we identify a link between Wnt signaling and senescence induction and propose that bSC-derived Wnt signals facilitate the proliferation of neighboring cells in part by preventing their induction into senescence. This work defines the roles of cellular senescence in the regeneration of complex structures.

Baculovirus Production and Infection in Axolotls.

Salamanders have served as an excellent model for developmental and tissue regeneration studies. While transgenic approaches are available for various salamander species, their long generation time and expensive maintenance have driven the development of alternative gene delivery methods for functional studies. We have previously developed pseudotyped baculovirus (BV) as a tool for gene delivery in the axolotl (Oliveira et al. Dev Biol 433(2):262-275, 2018). Since its initial conception, we have refined our protocol of BV production and usage in salamander models. In this chapter, we describe a detailed and versatile protocol for BV-mediated transduction in urodeles.

Tig1 regulates proximo-distal identity during salamander limb regeneration.

Salamander limb regeneration is an accurate process which gives rise exclusively to the missing structures, irrespective of the amputation level. This suggests that cells in the stump have an awareness of their spatial location, a property termed positional identity. Little is known about how positional identity is encoded, in salamanders or other biological systems. Through single-cell RNAseq analysis, we identified Tig1/Rarres1 as a potential determinant of proximal identity. Tig1 encodes a conserved cell surface molecule, is regulated by retinoic acid and exhibits a graded expression along the proximo-distal axis of the limb. Its overexpression leads to regeneration defects in the distal elements and elicits proximal displacement of blastema cells, while its neutralisation blocks proximo-distal cell surface interactions. Critically, Tig1 reprogrammes distal cells to a proximal identity, upregulating Prod1 and inhibiting Hoxa13 and distal transcriptional networks. Thus, Tig1 is a central cell surface determinant of proximal identity in the salamander limb.

Liraglutide Protects Against Brain Amyloid-ß1-42 Accumulation in Female Mice with Early Alzheimer's Disease-Like Pathology by Partially Rescuing Oxidative/Nitrosative Stress and Inflammation.

Alzheimer's disease (AD) is the most common form of dementia worldwide, being characterized by the deposition of senile plaques, neurofibrillary tangles (enriched in the amyloid beta (Aß) peptide and hyperphosphorylated tau (p-tau), respectively) and memory loss. Aging, type 2 diabetes (T2D) and female sex (especially after menopause) are risk factors for AD, but their crosslinking mechanisms remain unclear. Most clinical trials targeting AD neuropathology failed and it remains incurable. However, evidence suggests that effective anti-T2D drugs, such as the GLP-1 mimetic and neuroprotector liraglutide, can be also efficient against AD. Thus, we aimed to study the benefits of a peripheral liraglutide treatment in AD female mice. We used blood and brain cortical lysates from 10-month-old 3xTg-AD female mice, treated for 28 days with liraglutide (0.2 mg/kg, once/day) to evaluate parameters affected in AD (e.g., Aß and p-tau, motor and cognitive function, glucose metabolism, inflammation and oxidative/nitrosative stress). Despite the limited signs of cognitive changes in mature female mice, liraglutide only reduced their cortical Aß1-42 levels. Liraglutide partially attenuated brain estradiol and GLP-1 and activated PKA levels, oxidative/nitrosative stress and inflammation in these AD female mice. Our results support the earlier use of liraglutide as a potential preventive/therapeutic agent against the accumulation of the first neuropathological features of AD in females.

Dual Therapy with Liraglutide and Ghrelin Promotes Brain and Peripheral Energy Metabolism in the R6/2 Mouse Model of Huntington's Disease.

Neuronal loss alongside altered energy metabolism, are key features of Huntington's disease (HD) pathology. The orexigenic gut-peptide hormone ghrelin is known to stimulate appetite and affect whole body energy metabolism. Liraglutide is an efficient anti-type 2 diabetes incretin drug, with neuroprotective effects alongside anorectic properties. Combining liraglutide with the orexigenic peptide ghrelin may potentially promote brain/cognitive function in HD. The R6/2 mouse model of HD exhibits progressive central pathology, weight loss, deranged glucose metabolism, skeletal muscle atrophy and altered body composition. In this study, we targeted energy metabolism in R6/2 mice using a co-administration of liraglutide and ghrelin. We investigated their effect on brain cortical hormone-mediated intracellular signalling pathways, metabolic and apoptotic markers, and the impact on motor function in HD. We here demonstrate that liraglutide, alone or together with ghrelin (subcutaneous daily injections of 150 µg/kg (ghrelin) and 0.2 mg/kg (liraglutide), for 2 weeks), normalized glucose homeostatic features in the R6/2 mouse, without substantially affecting body weight or body composition. Liraglutide alone decreased brain cortical active GLP-1 and IGF-1 levels in R6/2 mice, alongside higher ADP levels. Liraglutide plus ghrelin decreased brain insulin, lactate, AMP and cholesterol levels in R6/2 mice. Taken together, our findings encourage further studies targeting energy metabolism in HD.

Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration.

Axolotls can regenerate complex structures through recruitment and remodeling of cells within mature tissues. Accessing the underlying mechanisms at a molecular resolution is crucial to understand how injury triggers regeneration and how it proceeds. However, gene transformation in adult tissues can be challenging. Here we characterize the use of pseudotyped baculovirus (BV) as an effective gene transfer method both for cells within mature limb tissue and within the blastema. These cells remain competent to participate in regeneration after transduction. We further characterize the effectiveness of BV for gene overexpression studies by overexpressing Shh in the blastema, which yields a high penetrance of classic polydactyly phenotypes. Overall, our work establishes BV as a powerful tool to access gene function in axolotl limb regeneration.

Instruments for measuring undergraduate nursing students' knowledge, attitudes and skills in evidence-based practice: a systematic review protocol.

REVIEW QUESTION/OBJECTIVE: The objective of this systematic review is to identify and assess the properties of instruments for measuring undergraduate nursing students' knowledge, attitudes and skills in evidence-based practice (EBP).More specifically, the review questions are as follows.

Phosphatase 2A Inhibition Affects Endoplasmic Reticulum and Mitochondria Homeostasis Via Cytoskeletal Alterations in Brain Endothelial Cells.

The loss of endothelial cells (ECs) homeostasis is a trigger for cerebrovascular dysfunction that is a common event in several neurodegenerative disorders such as Alzheimer's disease (AD). The present work addressed the role of phosphatase 2A (PP2A) in cytoskeleton rearrangement, endoplasmic reticulum (ER) homeostasis, ER-mitochondria communication and mitochondrial dynamics in brain ECs. For this purpose, rat brain endothelial (RBE4) cells were exposed to okadaic acid, a well-known inhibitor of PP2A activity. An increase in the levels of tau phosphorylated on Ser396 and Thr181 residues was observed upon PP2A inhibition, concomitantly with the rearrangement of microtubules and actin cytoskeleton. Under these conditions, an increase in the levels of ER stress markers, namely GRP78, XBP1, p-eIF2αSer51, and ERO1α, was observed. Moreover, PP2A inhibition upregulated the Sigma-1 receptor, an ER chaperone located at the ER-mitochondria interface, and enhanced inter-organelle Ca2+ transfer, culminating in mitochondrial Ca2+ overload and activation of mitochondria-dependent apoptosis. The inhibition of PP2A activity also promoted an alteration of the structural and spatial mitochondria network due to upregulation of mitochondrial fission (Drp1 and Fis1) and fusion (Mfn1, Mfn2 and OPA1) proteins, suggesting detrimental changes in mitochondrial dynamics. In accordance with our in vitro observations, brain vessels from 3xTg-AD mice showed a significant decrease in PP2A protein levels accompanied by an increase in tau phosphorylated on Ser396 and GRP78 protein levels. Collectively, these results suggest that the loss of cerebrovascular homeostasis that occurs in AD might be a downstream event of the compromised activity and/or expression of PP2A, which is observed in the brain of individuals affected with this devastating neurodegenerative disorder.

Comparative Mitochondrial-Based Protective Effects of Resveratrol and Nicotinamide in Huntington's Disease Models.

Sirtuin 1 (SIRT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylase that regulates longevity and enhances mitochondrial metabolism. Both activation and inhibition of SIRT1 were previously shown to ameliorate neuropathological mechanisms in Huntington's disease (HD), a neurodegenerative disease that selectively affects the striatum and cortex and is commonly linked to mitochondrial dysfunction. Thus, in this study, we tested the influence of resveratrol (RESV, a SIRT1 activator) versus nicotinamide (NAM, a SIRT1 inhibitor) in counteracting mitochondrial dysfunction in HD models, namely striatal and cortical neurons isolated from YAC128 transgenic mice embryos, HD human lymphoblasts, and an in vivo HD model. HD cell models displayed a deregulation in mitochondrial membrane potential and respiration, implicating a decline in mitochondrial function. Further studies revealed decreased PGC-1α and TFAM protein levels, linked to mitochondrial DNA loss in HD lymphoblasts. Remarkably, RESV completely restored these parameters, while NAM increased NAD+ levels, providing a positive add on mitochondrial function in in vitro HD models. In general, RESV decreased while NAM increased H3 acetylation at lysine 9. In agreement with in vitro data, continuous RESV treatment for 28 days significantly improved motor coordination and learning and enhanced expression of mitochondrial-encoded electron transport chain genes in YAC128 mice. In contrast, high concentrations of NAM blocked mitochondrial-related transcription, worsening motor phenotype. Overall, data indicate that activation of deacetylase activity by RESV improved gene transcription associated to mitochondrial function in HD, which may partially control HD-related motor disturbances.

FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration.

In salamanders, grafting of a left limb blastema onto a right limb stump yields regeneration of three limbs, the normal limb and two 'supernumerary' limbs. This experiment and other research have shown that the juxtaposition of anterior and posterior limb tissue plus innervation are necessary and sufficient to induce complete limb regeneration in salamanders. However, the cellular and molecular basis of the requirement for anterior-posterior tissue interactions were unknown. Here we have clarified the molecular basis of the requirement for both anterior and posterior tissue during limb regeneration and supernumerary limb formation in axolotls (Ambystoma mexicanum). We show that the two tissues provide complementary cross-inductive signals that are required for limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a limb, but activation of hedgehog (HH) signalling was sufficient to drive regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signalling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signalling was not sufficient to drive regeneration; however, ectopic expression of FGF8 together with endogenous HH signalling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on sonic hedgehog (SHH) signalling from posterior tissue. Together, our findings identify key anteriorly and posteriorly localized signals that promote limb regeneration and show that these single factors are sufficient to drive non-regenerating blastemas to complete regeneration with full elaboration of skeletal elements.

Assessment of fight outcome is needed to activate socially driven transcriptional changes in the zebrafish brain.

Group living animals must be able to express different behavior profiles depending on their social status. Therefore, the same genotype may translate into different behavioral phenotypes through socially driven differential gene expression. However, how social information is translated into a neurogenomic response and what are the specific cues in a social interaction that signal a change in social status are questions that have remained unanswered. Here, we show for the first time, to our knowledge, that the switch between status-specific neurogenomic states relies on the assessment of fight outcome rather than just on self- or opponent-only assessment of fighting ability. For this purpose, we manipulated the perception of fight outcome in male zebrafish and measured its impact on the brain transcriptome using a zebrafish whole genome gene chip. Males fought either a real opponent, and a winner and a loser were identified, or their own image on a mirror, in which case, despite expressing aggressive behavior, males did not experience either a victory or a defeat. Massive changes in the brain transcriptome were observed in real opponent fighters, with losers displaying both a higher number of differentially expressed genes and of coexpressed gene modules than winners. In contrast, mirror fighters expressed a neurogenomic state similar to that of noninteracting fish. The genes that responded to fight outcome included immediate early genes and genes involved in neuroplasticity and epigenetic modifications. These results indicate that, even in cognitively simple organisms such as zebrafish, neurogenomic responses underlying changes in social status rely on mutual assessment of fighting ability.

The Association of Amyloid-ß Protein Precursor With α- and ß-Secretases in Mouse Cerebral Cortex Synapses Is Altered in Early Alzheimer's Disease.

Amyloid-ß peptides (Aß), the proposed triggers of synaptic dysfunction in early Alzheimer's disease (AD), derive from the endoproteolytic cleavage of amyloid-ß precursor protein (APP) by ß-secretases (BACE1), whereas APP cleavage by α-secretases (ADAM10) abrogates Aß formation. We now mapped the synaptic localization of APP, ADAM10, and BACE1 in the mouse cerebral cortex. All three proteins were present in cortical synapses and subsynaptic fractionation revealed that APP was located mainly in the pre-synaptic active zone (53 %) and in the post-synaptic density (37 %), whereas ADAM10 was enriched in the post-synaptic density (61 %) and BACE1 was concentrated in extra-synaptic regions (72 %). Immunocytochemistry analysis further showed that APP and BACE1 were co-localized in about 30 % of both glutamatergic and GABAergic terminals, whereas few terminals were endowed with ADAM10. This distribution is modified in a mouse model of early AD based on Aß1-42-intracerebroventricular injection, where the synaptic levels of APP and ADAM10 increased by 30 %, whereas BACE1 levels were reduced. This suggests that, in early AD, there are compensatory mechanisms to avoid Aß overload in cortical synapses favoring the non-amyloidogenic processing of APP.

Alzheimer's disease and type 2 diabetes-related alterations in brain mitochondria, autophagy and synaptic markers.

We aimed to investigate mitochondrial function, biogenesis and autophagy in the brain of type 2 diabetes (T2D) and Alzheimer's disease (AD) mice. Isolated brain mitochondria and homogenates from cerebral cortex and hippocampus of wild-type (WT), triple transgenic AD (3xTg-AD) and T2D mice were used to evaluate mitochondrial functional parameters and protein levels of mitochondrial biogenesis, autophagy and synaptic integrity markers, respectively. A significant decrease in mitochondrial respiration, membrane potential and energy levels was observed in T2D and 3xTg-AD mice. Also, a significant decrease in the levels of autophagy-related protein 7 (ATG7) and glycosylated lysosomal membrane protein 1 (LAMP1) was observed in cerebral cortex and hippocampus of T2D and 3xTg-AD mice. Moreover, both brain regions of 3xTg-AD mice present lower levels of nuclear respiratory factor (NRF) 1 while the levels of NRF2 are lower in both brain regions of T2D and 3xTg-AD mice. A decrease in mitochondrial encoded, nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) was also observed in T2D and 3xTg-AD mice although only statistically significant in T2D cortex. Furthermore, a decrease in the levels of postsynaptic density protein 95 (PSD95) in the cerebral cortex of 3xTg-AD mice and in hippocampus of T2D and 3xTg-AD mice and a decrease in the levels of synaptosomal-associated protein 25 (SNAP 25) in the hippocampus of T2D and 3xTg-AD mice were observed suggesting synaptic integrity loss. These results support the idea that alterations in mitochondrial function, biogenesis and autophagy cause synaptic damage in AD and T2D.

Modulation of endoplasmic reticulum stress: an opportunity to prevent neurodegeneration?

Neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and prion-related diseases) have in common the presence of protein aggregates in specific brain areas where significant neuronal loss is detected. In these pathologies, accumulating evidence supports a close correlation between neurodegeneration and endoplasmic reticulum (ER) stress, a condition that arises from ER lumen overload with misfolded proteins. Under these conditions, ER stress sensors initiate the unfolded protein response to restore normal ER function. If stress is too prolonged, or adaptive responses fail, apoptotic cell death ensues. Therefore, it was recently suggested that the manipulation of the ER unfolded protein response could be an effective strategy to avoid neuronal loss in neurodegenerative disorders. We will review the mechanisms underlying ER stress-associated neurodegeneration and discuss the possibility of ER as a therapeutic target.

Oxidative stress involving changes in Nrf2 and ER stress in early stages of Alzheimer's disease.

Oxidative stress and endoplasmic reticulum (ER) stress have been associated with Alzheimer's disease (AD) progression. In this study we analyzed whether oxidative stress involving changes in Nrf2 and ER stress may constitute early events in AD pathogenesis by using human peripheral blood cells and an AD transgenic mouse model at different disease stages. Increased oxidative stress and increased phosphorylated Nrf2 (p(Ser40)Nrf2) were observed in human peripheral blood mononuclear cells (PBMCs) isolated from individuals with mild cognitive impairment (MCI). Moreover, we observed impaired ER Ca2+ homeostasis and increased ER stress markers in PBMCs from MCI individuals and mild AD patients. Evidence of early oxidative stress defense mechanisms in AD was substantiated by increased p(Ser40)Nrf2 in 3month-old 3xTg-AD male mice PBMCs, and also with increased nuclear Nrf2 levels in brain cortex. However, SOD1 protein levels were decreased in human MCI PBMCs and in 3xTg-AD mice brain cortex; the latter further correlated with reduced SOD1 mRNA levels. Increased ER stress was also detected in the brain cortex of young female and old male 3xTg-AD mice. We demonstrate oxidative stress and early Nrf2 activation in AD human and mouse models, which fails to regulate some of its targets, leading to repressed expression of antioxidant defenses (e.g., SOD-1), and extending to ER stress. Results suggest markers of prodromal AD linked to oxidative stress associated with Nrf2 activation and ER stress that may be followed in human peripheral blood mononuclear cells.

Localization and Trafficking of Amyloid-ß Protein Precursor and Secretases: Impact on Alzheimer's Disease.

Alzheimer's disease (AD) affects almost 35 million people worldwide. One of the neuropathological features of AD is the presence of extracellular amyloid plaques, which are mainly composed of amyloid-ß (Aß) peptides. These peptides derive from the amyloidogenic proteolytic processing of the amyloid-ß protein precursor (AßPP), through the sequential action of ß- and γ-secretases. However, AßPP can also be cleaved by a non-amyloidogenic pathway, involving an α-secretase, and in this case the Aß formation is precluded. The production of Aß and of other AßPP catabolites depends on the spatial and temporal co-localization of AßPP with α- or ß-secretases and γ-secretase, which traffic through the secretory pathway in a highly regulated manner. Disturbances on AßPP and secretases intracellular trafficking and, consequently, in their localization may affect dynamic interactions between these proteins with consequences in the AD pathogenesis. In this article, we critically review the recent knowledge about the trafficking and co-localization of AßPP and related secretases in the brain under physiological and AD conditions. A particular focus is given to data concerning the distribution of AßPP and secretases in different types of synapses relatively to other neuronal or glial localizations. Furthermore, we discuss some possible signals that govern the dynamic encounter of AßPP with each group of secretases, such as AßPP mutations, estrogen deprivation, chronic stress, metabolic impairment, and alterations in sleep pattern-associated with aging. The knowledge of key signals that are responsible for the shifting of AßPP processing away from α-secretases and toward the ß-secretases might be useful to develop AD therapeutic strategies.

Aß and NMDAR activation cause mitochondrial dysfunction involving ER calcium release.

Early cognitive deficits in Alzheimer's disease (AD) seem to be correlated to dysregulation of glutamate receptors evoked by amyloid-beta (Aß) peptide. Aß interference with the activity of N-methyl-d-aspartate receptors (NMDARs) may be a relevant factor for Aß-induced mitochondrial toxicity and neuronal dysfunction. To evaluate the role of mitochondria in NMDARs activation mediated by Aß, we followed in situ single-cell simultaneous measurement of cytosolic free Ca(2+)(Cai(2+)) and mitochondrial membrane potential in primary cortical neurons. Our results show that direct exposure to Aß + NMDA largely increased Cai(2+) and induced immediate mitochondrial depolarization, compared with Aß or NMDA alone. Mitochondrial depolarization induced by rotenone strongly inhibited the rise in Cai(2+) evoked by Aß or NMDA, suggesting that mitochondria control Ca(2+) entry through NMDARs. However, incubation with rotenone did not preclude mitochondrial Ca(2+) (mitCa(2+)) retention in cells treated with Aß. Aß-induced Cai(2+) and mitCa(2+) rise were inhibited by ifenprodil, an antagonist of GluN2B-containing NMDARs. Exposure to Aß + NMDA further evoked a higher mitCa(2+) retention, which was ameliorated in GluN2B(-/-) cortical neurons, largely implicating the involvement of this NMDAR subunit. Moreover, pharmacologic inhibition of endoplasmic reticulum (ER) inositol-1,4,5-triphosphate receptor (IP3R) and mitCa(2+) uniporter (MCU) evidenced that Aß + NMDA-induced mitCa(2+) rise involves ER Ca(2+) release through IP3R and mitochondrial entry by the MCU. Altogether, data highlight mitCa(2+) dyshomeostasis and subsequent dysfunction as mechanisms relevant for early neuronal dysfunction in AD linked to Aß-mediated GluN2B-composed NMDARs activation.

Activation of IGF-1 and insulin signaling pathways ameliorate mitochondrial function and energy metabolism in Huntington's Disease human lymphoblasts.

Huntington's disease (HD) is an inherited neurodegenerative disease caused by a polyglutamine repeat expansion in the huntingtin protein. Mitochondrial dysfunction associated with energy failure plays an important role in this untreated pathology. In the present work, we used lymphoblasts obtained from HD patients or unaffected parentally related individuals to study the protective role of insulin-like growth factor 1 (IGF-1) versus insulin (at low nM) on signaling and metabolic and mitochondrial functions. Deregulation of intracellular signaling pathways linked to activation of insulin and IGF-1 receptors (IR,IGF-1R), Akt, and ERK was largely restored by IGF-1 and, at a less extent, by insulin in HD human lymphoblasts. Importantly, both neurotrophic factors stimulated huntingtin phosphorylation at Ser421 in HD cells. IGF-1 and insulin also rescued energy levels in HD peripheral cells, as evaluated by increased ATP and phosphocreatine, and decreased lactate levels. Moreover, IGF-1 effectively ameliorated O2 consumption and mitochondrial membrane potential (Δψm) in HD lymphoblasts, which occurred concomitantly with increased levels of cytochrome c. Indeed, constitutive phosphorylation of huntingtin was able to restore the Δψm in lymphoblasts expressing an abnormal expansion of polyglutamines. HD lymphoblasts further exhibited increased intracellular Ca(2+) levels before and after exposure to hydrogen peroxide (H2O2), and decreased mitochondrial Ca(2+) accumulation, being the later recovered by IGF-1 and insulin in HD lymphoblasts pre-exposed to H2O2. In summary, the data support an important role for IR/IGF-1R mediated activation of signaling pathways and improved mitochondrial and metabolic function in HD human lymphoblasts.

Amyloid-beta disrupts calcium and redox homeostasis in brain endothelial cells.

In Alzheimer's disease, the accumulation of amyloid-beta (Aß) in the brain occurs in the parenchyma and cerebrovasculature. Several evidences support that the neuronal demise is potentiated by vascular alterations in the early stages of the disease, but the mechanisms responsible for the dysfunction of brain endothelial cells that underlie these cerebrovascular changes are unknown. Using rat brain microvascular endothelial cells, we found that short-term treatment with a toxic dose of Aß1-40 inhibits the Ca(2+) refill and retention ability of the endoplasmic reticulum and enhances the mitochondrial and cytosolic response to adenosine triphosphate (ATP)-stimulated endoplasmic reticulum Ca(2+) release. Upon prolonged Aß1-40 exposure, Ca(2+) homeostasis was restored concomitantly with a decrease in the levels of proteins involved in its regulation operating at the plasma membrane, endoplasmic reticulum, and mitochondria. Along with perturbations in Ca(2+) regulation, an early increase in the levels of oxidants and a decrease in the ratio between reduced and oxidized glutathione were observed in Aß1-40-treated endothelial cells. Under these conditions, the nuclear levels of oxidative stress-related transcription factors, namely, hypoxia-inducible factor 1α and nuclear factor (erythroid-derived 2)-related factor 2, were enhanced as well as the protein levels of target genes. In conclusion, Aß1-40 affects several mechanisms involved in Ca(2+) homeostasis and impairs the redox homeostasis simultaneously with stimulation of protective stress responses in brain endothelial cells. However, the imbalance between cell death and survival pathways leads to endothelial dysfunction that in turn contributes to cerebrovascular impairment in Alzheimer's disease.