Roles of Erythrocytes in Human Health and Disease 2.0.

This Special Issue titled "Roles of Erythrocytes in Human Health and Disease 2 [...].

Sphingosine-1-phosphate Decreases Erythrocyte Dysfunction Induced by ß-Amyloid.

Amyloid beta peptides (Aß) have been identified as the main pathogenic agents in Alzheimer's disease (AD). Soluble Aß oligomers, rather than monomer or insoluble amyloid fibrils, show red blood cell (RBC) membrane-binding capacity and trigger several morphological and functional alterations in RBCs that can result in impaired oxygen transport and delivery. Since bioactive lipids have been recently proposed as potent protective agents against Aß toxicity, we investigated the role of sphingosine-1-phosphate (S1P) in signaling pathways involved in the mechanism underlying ATP release in Ab-treated RBCs. In RBCs following different treatments, the ATP, 2,3 DPG and cAMP levels and caspase 3 activity were determined by spectrophotometric and immunoassay. S1P rescued the inhibition of ATP release from RBCs triggered by Ab, through a mechanism involving caspase-3 and restoring 2,3 DPG and cAMP levels within the cell. These findings reveal the molecular basis of S1P protection against Aß in RBCs and suggest new therapeutic avenues in AD.

The Nutraceuticals as Modern Key to Achieve Erythrocyte Oxidative Stress Fighting in Osteoarthritis.

Osteoarthritis (OA), the most common joint disease, shows an increasing prevalence in the aging population in industrialized countries. OA is characterized by low-grade chronic inflammation, which causes degeneration of all joint tissues, such as articular cartilage, subchondral bone, and synovial membrane, leading to pain and loss of functionality. Erythrocytes, the most abundant blood cells, have as their primary function oxygen transport, which induces reactive oxygen species (ROS) production. For this reason, the erythrocytes have several mechanisms to counteract ROS injuries, which cause damage to lipids and proteins of the cell membrane. Oxidative stress and inflammation are highly correlated and are both causes of joint disorders. In the synovial fluid and blood of osteoarthritis patients, erythrocyte antioxidant enzyme expression is decreased. To date, OA is a non-curable disease, treated mainly with non-steroidal anti-inflammatory drugs and corticosteroids for a prolonged period of time, which cause several side effects; thus, the search for natural remedies with anti-inflammatory and antioxidant activities is always ongoing. In this review, we analyze several manuscripts describing the effect of traditional remedies, such as Harpagophytum procumbens, Curcumin longa, and Boswellia serrata extracts, in the treatments of OA for their anti-inflammatory, analgesic, and antioxidant activity. The effects of such remedies have been studied both in in vitro and in vivo models, considering both joint cells and erythrocytes.

The Neuroprotective Potentiality of Flavonoids on Alzheimer's Disease.

Alzheimer's disease (AD), due to its spread, has become a global health priority, and is characterized by senile dementia and progressive disability. The main cause of AD and other neurodegenerations (Huntington, Parkinson, Amyotrophic Lateral Sclerosis) are aggregated protein accumulation and oxidative damage. Recent research on secondary metabolites of plants such as polyphenols demonstrated that they may slow the progression of AD. The flavonoids' mechanism of action in AD involved the inhibition of acetylcholinesterase, butyrylcholinesterase, Tau protein aggregation, ß-secretase, oxidative stress, inflammation, and apoptosis through modulation of signaling pathways which are implicated in cognitive and neuroprotective functions, such as ERK, PI3-kinase/Akt, NFKB, MAPKs, and endogenous antioxidant enzymatic systems. This review focuses on flavonoids and their role in AD, in terms of therapeutic potentiality for human health, antioxidant potential, and specific AD molecular targets.

Methionine 35 sulphoxide reduces toxicity of Aß in red blood cell.

BACKGROUND: The oxidation of methionine residue in position 35 of Ab to sulphoxide (Ab-sulphoxide) has the ability to deeply modify wild-type Ab 1-42 (Ab) neurotoxic action. Our previous studies suggest that in nucleated cells, lower toxicity of Ab-sulphoxide might result not from structural alteration, but from elevation of methionine sulphoxide reductase A (MsrA) activity and mRNA levels. DESIGN: On this basis, we hypothesised that red blood cell (RBC), a cell devoid almost completely of MsrA activity, shares with nucleated cells an antioxidant system induced by methionine 35 sulphoxide, responsible for the lower toxicity of Ab-sulphoxide in RBC. (Results) Supporting this hypothesis, we found that the low toxicity of Ab-sulphoxide in RBC correlated with pentose phosphate pathway (PPP) flux increase, and this event was associated with a low level of methionine oxidation in total proteins. None of these effects were observed when cells were exposed to Ab native. DISCUSSION: These results outline the importance of the redox state of methionine 35 in the modulation of Ab-mediated events and suggest an important protective role for PPP in RBC of patients affected by Alzheimer's disease.

Amyloid beta peptide (1-42)-mediated antioxidant imbalance is associated with activation of protein kinase C in red blood cells.

Glycolysis and pentose phosphate pathway (PPP) in red blood cell (RBC) are modulated by the cell oxygenation state. This metabolic modulation is connected to variations in intracellular nicotinamide adenine dinucleotide phosphate-reduced form (NADPH) and adenosine triphosphate (ATP) levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. In the present study, we analysed the effects of amyloid beta peptide (1-42) (Abeta) on RBC metabolism and its relationship with the activity of protein kinase C (PKC). Our results showed that metabolic response to Abeta depended on the degree of cell oxygenation. In particular, under high O2 pressure, in Abeta-treated RBC, glucose metabolized through PPP approached that metabolized by RBC under low O2 pressure, differently to that observed in untreated cells. The effect of Abeta on RBC metabolism was paralleled by increase in PKC enzyme activity, but cytosolic Ca2+ concentration does not seem to be involved in this mechanism. Incubation of Abeta-treated RBC with a specific inhibitor of PKC partially restores PPP flux. A possible rationalization of the different metabolic behaviours shown by RBC following Abeta treatment is proposed. It takes into account the known post-translational modifications to cytoskeleton proteins induced by PKC. The reduction in PPP flux may lead to a weakened defence system of antioxidant reserve in RBC, becoming a source of reactive species, and, consequently, its typical, structural and functional features are lost. Therefore, oxidative stress may outflow from the RBC and trigger damage events in adjacent cells and tissue, thus contributing to vascular damage.

Enhancing nutritional knowledge and self-regulation among adolescents: efficacy of a multifaceted food literacy intervention.

This health literacy intervention study, conducted on adolescent students, aimed to evaluate the impact of a comprehensive educational program on promoting healthy eating habits. The intervention sought to enhance adolescents' knowledge about nutrition, foster self-regulation skills, and ultimately improve their overall health, including their body mass index (BMI). Through a multi-component approach that combined theoretical learning with practical activities and the integration of digital tools such as the MyFitnessPal app, the study targeted improvements in food literacy, which encompasses nutrition knowledge, food label interpretation skills, and cooking abilities. These elements are critical in influencing adolescents' food choices and eating behaviors, with a focus on increasing the consumption of fruits and vegetables while reducing the intake of fast food and processed snacks. The intervention was structured around a series of educational workshops and interactive sessions, facilitated by professionals experienced in nutrition. These sessions covered topics such as food composition, the importance of a balanced diet, and strategies for avoiding excessive consumption of processed and high-energy-density foods. A distinctive feature of the program was its use of digital tools to enhance engagement and allow for the practical application of learned concepts through food tracking and activity monitoring. Preliminary results indicate positive outcomes in terms of increased food knowledge and improved dietary habits among participants. Notably, there was a significant improvement in adolescents' BMI, highlighting the potential of food literacy interventions to contribute to better physical health outcomes. The study underscores the importance of adopting multidisciplinary and technology-enhanced approaches in educational programs aimed at promoting healthy eating among adolescents. The study's findings suggest that well-structured food education programs, tailored to address the specific needs of adolescents, can facilitate positive changes in eating behavior. This highlights the critical role of food literacy in adolescents' health and wellbeing and points to the need for continued investment in research and development to optimize intervention strategies. The integration of digital technologies and a focus on self-regulation strategies are identified as promising avenues for future research and educational practice, reinforcing the call for innovative investments in food education and health promotion among the youth.

ß-amyloid decreases detectable endothelial nitric oxide synthase in human erythrocytes: a role for membrane acetylcholinesterase.

Until few years ago, many studies of Alzheimer's disease investigated the effects of this syndrome in the central nervous system. Only recently, the detection of amyloid beta peptide (Aß) in the blood has evidenced the necessity to extend studies on extraneuronal cells, particularly on erythrocytes. Aß is also present in brain capillaries, where it interacts with the erythrocytes, inducing several metabolic and functional alterations. Recently, functionally active endothelial type nitric oxide synthase (eNOS) was discovered in human erythrocytes. The goal of the present study was to evidence the effect of Aß on erythrocyte eNOS. We found that Aß following to 24-h exposure causes a decrease in the immune staining of erythrocyte eNOS. Concurrently, Aß alters erythrocyte cell morphology, decreases nitrites and nitrates levels, and affects membrane acetylcholinesterase activity. Propidium, an acetylcholinesterase inhibitor, was able to reverse the effects elicited by Aß. These events could contribute to the vascular alterations associated with Alzheimer's disease disease.

ATP release from erythrocytes: A role of adenosine1.

BACKGROUND: The oxygen required to meet metabolic needs of all tissues is delivered by the red blood cell (RBC), a small, flexible cell which, in mammals, is devoid of a nucleus and mitochondria. Despite its simple appearance, this cell has an important role in its own distribution, enabling the delivery of oxygen to precisely meet localized metabolic need. When red blood cells enter in hypoxic area, a signalling pathway is activated within the cell, resulting in the release of ATP in amounts adequate to activate purinergic receptors on vascular endothelium, which trigger secretion of nitric oxide and other factors resulting in vasodilatation. OBJECTIVE: The present study investigates the effect of adenosine exposure on this molecular mechanism. METHODS AND RESULTS: We report that RBC in the presence of adenosine in low oxygen conditions, ATP release increase after 24 h exposure. Adenosine induced-ATP release in deoxygenated red blood cell show data similar to that of RBC in high oxygen conditions: (1) RBC after band 3 modification by 4,4'- diisothio-cyanatostilbene- 2,2'-disulphonic acid; (2) CO-treated RBC. In the presence of Sphingosine kinase (SphK1) inhibitor, adenosine mediated effects on ATP release were abolished. Activity of adenylate cyclase increase following to adenosine exposure, on the contrary red cell phosphofructokinase is not modified within the RBC in the presence of adenosine. CONCLUSION: Our data support involvement of band 3/deoxyHb binding and adenylate cyclase in the pathway responsible for ATP release from RBC following exposure to adenosine.

Intention to Screen for Hepatitis C Among University Students: Influence of Different Communicative Scenarios.

This study aimed to evaluate the influence of different narrative scenarios regarding students' intentions to undergo diagnostic screening for hepatitis C, and whether gender identification with the characters of the scenario could influence the students' intentions to undergo a medical test. A sample of 600 participants was administered three narrative scenarios with different frames (positive, negative, and ambivalent), including two gender options (male and female) for the main character of the story. A statistically significant three-way interaction between scenario, gender identification, and time resulted. There were significant simple main effects on the intention to have a diagnostic test for hepatitis C for the scenarios with the protagonist of the same gender as the participant and after the administration of the negative scenario. The use of a negative scenario with the same gender character was always more effective than the use of a positive framed scenario, even though there was a high level of knowledge regarding the disease. Personal diagnostic testing was not directly associated with knowledge regarding the infection. The findings of this study can ultimately help policymakers develop communication campaigns adapted to target populations such as college students, in order to raise awareness of the risk, promote prevention and behavioral change, and encourage medical screening.

Psychometric properties and measurement invariance across gender of the Italian version of the tempest self-regulation questionnaire for eating adapted for young adults.

The prevalence of overweight and obesity in young adults has increased dramatically in recent decades. The unhealthy eating habits that develop at this time can often lead to negative health consequences in the future. It is therefore important to learn about self-regulation and self-control strategies and help young adults to have healthy eating behaviours. This study aims to present an Italian version of the Tempest Self-Regulation Questionnaire for Eating (TESQ-E) adapted for young adults. The instrument assesses self-regulation and self-control strategies to counteract the desire and temptation to eat unhealthy food and to choose healthy foods. A total of 645 students (271 males and 374 females with an average age of 24.82 and SD = 4.34) took part in the study. The results of the confirmatory factor analysis supported the six first-order factors model concerning specific self-regulation strategies and a higher-order structure of the TESQ-E [χ2 (152) = 336.480, p < 0.001; CFI = 0.95; RMSEA = 0.04; SRMR = 0.04]: three correlated second-order factors (addressing the temptation directly, addressing the psychological meaning of temptation, and addressing the goal directly) and one-third-order factor (self-regulatory competence). The results also confirmed the strong measurement invariance of the TESQ-E across gender. To have reliable comparisons across males and females, the latent mean differences tests were performed on the six specific self-regulation strategies. The analysis showed that females appear to prefer strategies that directly address the goal by expressing explicit intentions or plans to eat in a healthy way. Convergence validity was tested through correlations with Eating-Self-Efficacy Brief Scale (ESEBS), Emotional Eating Scale (EES), Scale of Attitudes towards Healthy Eating (SAHE), and Body Mass Index (BMI). In conclusion, the TESQ-E appears to be a psychometrically sound questionnaire that can be effectively used with young adults to measure self-regulation strategies in eating in order to plan personalised interventions for the prevention and control of the metabolic syndrome, and to reduce a wide range of diet-related diseases.

Derangement of erythrocytic AE1 in beta-thalassemia by caspase 3: pathogenic mechanisms and implications in red blood cell senescence.

Considering its complex molecular pathophysiology, beta-thalassemia could be a good in vivo model to study some aspects related to erythrocyte functions with potential therapeutic implications not only within the frame of this particular hemoglobinopathy but also with respect to conditions in which the cellular milieu, altered by a deranged anion exchanger, could display a significant pathogenetic role (i.e., erythrocyte senescence, complications of red cell storage, renal tubular acidosis and some abnormal protein thesaurismosis). This work evaluates the anionic influx across band 3 protein in normal and beta-thalassemic red blood cells (RBCs) and ghosts. Since redox-mediated injury is an important pathway in the destruction of beta-thalassemic RBCs, we studied the anion transport and the activity of caspase 3 in the absence and presence of t-butylhydroperoxide in order to evaluate the effect of an increase of cellular oxidative stress. Interestingly, beta-thalassemic erythrocytes show a faster rate of anion exchange than normal RBCs and absence of any modulation mechanism of anion influx. These findings led us to formulate a hypothesis about the metabolic characteristics of beta-thalassemic erythrocytes, outlining that one of the main targets of caspase 3 in RBCs is the cytoplasmic domain of band 3 protein.

Fibroblast apoptosis in a patient affected by lamellar ichthyosis.

BACKGROUND: Lamellar ichthyosis (LI) is a congenital recessive skin disorder characterized by generalized scaling and hyperkeratosis. The pathology may be caused by mutations in transglutaminase 1 (TGM1) gene that encodes an enzyme critical for terminally differentiating keratinocytes. Because of evidences that transglutaminase enzymes are involved in programmed cell death, we investigated morphological and biochemical apoptotic parameters in cultured skin fibroblasts from a patient with a severe LI and homozygous for the TGM1 R142H mutation. METHOD: The principle apoptotic signals (mitochondrial membrane potential, analysis of oxygen consumption, DNA fragmentation and Bax/Bcl-2 gene expression) were analyzed in cultured fibroblasts from a LI patient, his mother (TGM1 mutation carrier) and a control subject. RESULTS: LI fibroblasts showing a reduction of fibronectin expression evidenced a strong inhibition of oxygen consumption, a dramatic drop in the mitochondrial membrane potential (Delta psi(m)), and a higher apoptotic index. CONCLUSION: The present results suggest a possible connection between the alterations in the keratinization process leading to LI and the observed increased fibroblast apoptosis.

Amyloid ß peptide affects erythrocyte morphology: Role of intracellular signaling pathways.

BACKGROUND: Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. OBJECTIVE: Understanding the role of Aß in the cross talk between cell signalling pathways and modulation of the cell structural and biomechanical properties occurring in RBCs during aging. METHODS: The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. RESULTS: We show that treatment with Aß accelerates the occurrence of morphological and biochemical aging markers in human RBC and influences the cell metabolism. Biochemical data demonstrate that contemporaneously to morphological alterations, Aß triggers: (i) metabolic alterations and (ii) a complex signaling pathway involving caspase 3, protein kinase C and nitric oxide derived metabolites. CONCLUSIONS: our study provides a comprehensive picture in which Aß treatment of RBC induces changes in specific cell signalling events and/or metabolic pathways, in turns affecting the membrane-cytoskeleton interaction and the membrane integrity.

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study.

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1-42 (Aß) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aß boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aß-induced effects. Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aß and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites. As a whole, the collected data revealed that, the damaging path induced by Aß in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.

Role of methionine 35 in the intracellular Ca2+ homeostasis dysregulation and Ca2+-dependent apoptosis induced by amyloid beta-peptide in human neuroblastoma IMR32 cells.

Amyloid beta-peptide (Abeta) plays a fundamental role in the pathogenesis of Alzheimer's disease. We recently reported that the redox state of the methionine residue in position 35 of amyloid beta-peptide (Abeta) 1-42 (Met35) strongly affects the peptide's ability to trigger apoptosis and is thus a major determinant of its neurotoxicity. Dysregulation of intracellular Ca(2+) homeostasis resulting in the activation of pro-apoptotic pathways has been proposed as a mechanism underlying Abeta toxicity. Therefore, we investigated correlations between the Met35 redox state, Abeta toxicity, and altered intracellular Ca(2+) signaling in human neuroblastoma IMR32 cells. Cells incubated for 6-24 h with 10 microM Abeta1-42 exhibited significantly increased KCl-induced Ca(2+) transient amplitudes and resting free Ca(2+) concentrations. Nifedipine-sensitive Ca(2+) current densities and Ca(v)1 channel expression were markedly enhanced by Abeta1-42. None of these effects were observed when cells were exposed to Abeta containing oxidized Met35 (Abeta1-42(Met35-Ox)). Cell pre-treatment with the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (1 microM) or the Ca(v)1 channel blocker nifedipine (5 microM) significantly attenuated Abeta1-42-induced apoptosis but had no effect on Abeta1-42(Met35-Ox) toxicity. Collectively, these data suggest that reduced Met35 plays a critical role in Abeta1-42 toxicity by rendering the peptide capable of disrupting intracellular Ca(2+) homeostasis and thereby provoking apoptotic cell death.

Mitochondrial oxygen consumption inhibition importance for TMT-dependent cell death in undifferentiated PC12 cells.

The evolving role of mitochondria as a target for different death-inducing noxae prompted us to investigate trimethyltin (TMT)-dependent effects on mitochondrial functionality. For this purpose, we used a homogeneous cell culture model represented by undifferentiated PC12 cells. Mitochondria isolated from PC12 cells treated with TMT for 6, 12 and 24h, showed a time-dependent inhibition of ADP-stimulated oxygen consumption using succinate or glutamate/malate as substrate. Using a fluorescent assay, the effect of TMT on mitochondrial membrane potential (delta Psi) in PC12 cells was also determined. After 24h in culture, a strong loss of mitochondrial membrane potential (delta Psi) was observed in TMT-treated cells. Collapse of mitochondrial membrane potential correlated with an increased expression of bax/bcl-2 ratio, as evaluated by polymerase chain reaction. Western blotting and spectrophotometric analysis showed that cytochrome c release and activation of caspase 3 were concurrently induced. Our findings suggest that inhibition of mitochondrial respiration represents the early toxic event for cell death in PC12 due to trimethyltin.

Amyloid-beta peptide affects the oxygen dependence of erythrocyte metabolism: a role for caspase 3.

Human erythrocyte metabolism is modulated by the cell oxygenation state. Among other mechanisms, competition of deoxyhemoglobin and some glycolytic enzymes for the cytoplasmic domain of band 3 is probably involved in modulation. This metabolic modulation is connected to variations in intracellular NADPH and ATP levels as a function of the oxygenation state of the cell, and, consequently, it should have physiologic relevance. The present study investigates the effect of amyloid-beta peptide exposure on this metabolic modulation and its relationship with the activity of erythrocyte caspase 3. Metabolic differences between erythrocytes incubated at high and low oxygen saturation disappear following to 24 h exposure to amyloid-beta peptide. Western blotting analysis shows that caspase 3 is concurrently activated. Pre-incubation of amyloid-beta peptide-treated erythrocytes with a specific inhibitor of caspase 3, partially restores the oxygen-dependent modulation. This finding suggests that human erythrocytes following to exposure to amyloid-beta peptide show a complete loss of the oxygen-dependent metabolic modulation, which is partially restored by caspase 3 inhibitor-treatment.

Erythrocytes as Potential Link between Diabetes and Alzheimer's Disease.

Many studies support the existence of an association between type 2 diabetes (T2DM) and Alzheimer's disease (AD). In AD, in addition to brain, a number of peripheral tissues and cells are affected, including red blood cell (RBC) and because there are currently no reliable diagnostic biomarkers of AD in the blood, a gradually increasing attention has been given to the study of RBC's alterations. Recently it has been evidenced in diabetes, RBC alterations superimposable to the ones occurring in AD RBC. Furthermore, growing evidence suggests that oxidative stress plays a pivotal role in the development of RBC's alterations and vice versa. Once again this represents a further evidence of a shared pathway between AD and T2DM. The present review summarizes the two disorders, highlighting the role of RBC in the postulated common biochemical links, and suggests RBC as a possible target for clinical trials.

Fragment 31-35 of beta-amyloid peptide induces neurodegeneration in rat cerebellar granule cells via bax gene expression and caspase-3 activation. A crucial role for the redox state of methionine-35 residue.

The amyloid beta-peptide (AbetaP) is the major protein component of brain senile plaques in Alzheimer's disease. The redox state of methionine-35 residue plays a critical role in peptide neurotoxic actions. We used the fragment 31-35 of AbetaP [AbetaP(31-35)], containing a single methionine-35 residue (Met-35), to investigate the relationship between the oxidative state of Met-35 and neurotoxic and pro-apoptotic actions induced by the peptide; in rat cerebellar granule cells (CGC), we compared the effects of AbetaP(31-35), in which the Met-35 is present in the reduced state, with those of a modified peptide with oxidized Met-35 [AbetaP(31-35)Met-35(OX)](,) as well as an AbetaP-derivative with Met-35 substituted by norleucine [AbetaP(31-35)Nle-35]. AbetaP(31-35) induced a time-dependent decrease in cell viability. AbetaP(31-35)Met-35(OX) was significantly less potent, but still induced a significant decrease in cell viability compared to control. No toxic effects were observed after treatment with AbetaP(31-35)Nle-35. AbetaP(31-35) induced a 2-fold increase in bax mRNA levels after 4h, whereas AbetaP(31-35)Met-35(OX) raised bax mRNA levels by 41% and AbetaP(31-35)Nle-35 had no effect. Finally, AbetaP(31-35) caused a 43% increase in caspase-3 activity after 24h; AbetaP(31-35)Met-35(OX) caused only a 18% increase, and AbetaP(31-35)Nle-35 had no effect. These findings suggest that AbetaP(31-35)-induced neurodegeneration in CGC is mediated by a selective early increase in bax mRNA levels followed by delayed caspase-3 activation; the redox state of the single Met-35 residue is crucial in the occurrence and extent of the above phenomena.