Soluble amyloid β-peptide oligomers (AβOs), increasingly recognized as causative agents of Alzheimer's disease (AD), disrupt neuronal Ca(2+) homeostasis and synaptic function. Here, we report that AβOs at sublethal concentrations generate prolonged Ca(2+) signals in primary hippocampal neurons; incubation in Ca(2+)-free solutions, inhibition of ryanodine receptors (RyRs) or N-methyl-d-aspartate receptors (NMDARs), or preincubation with N-acetyl-l-cysteine abolished these signals. AβOs decreased (6 h) RyR2 and RyR3 mRNA and RyR2 protein, and promoted mitochondrial fragmentation after 24 h. NMDAR inhibition abolished the RyR2 decrease, whereas RyR inhibition prevented significantly the RyR2 protein decrease and mitochondrial fragmentation induced by AβOs. Incubation with AβOs (6 h) eliminated the RyR2 increase induced by brain-derived nerve factor (BDNF) and the dendritic spine remodeling induced within minutes by BDNF or the RyR agonist caffeine. Addition of BDNF to neurons incubated with AβOs for 24 h, which had RyR2 similar to and slightly higher RyR3 protein content than those of controls, induced dendritic spine growth but at slower rates than in controls. These combined effects of sublethal AβOs concentrations (which include redox-sensitive stimulation of RyR-mediated Ca(2+) release, decreased RyR2 protein expression, mitochondrial fragmentation, and prevention of RyR-mediated spine remodeling) may contribute to impairing the synaptic plasticity in AD.

Background: Soluble amyloid-F peptide oligomers (AFOs), which are centrally involved in the pathogenesis of Alzheimer’s disease, trigger Ca²⁺ influx through N-methyl-D-aspartate receptors and stimulate reactive oxygen species generation in primary hippocampal neurons. We have previously reported that AFOs promote Ca²⁺ release mediated by ryanodine receptors (RyR), which in turn triggers mitochondrial fragmentation. We have also reported that the antioxidant N-acetylcysteine (NAC) prevents AFOs-induced Ca²⁺ signal generation. Objectives: To determine if RyR-mediated Ca²⁺ release activated by the specific agonist 4-chloro-m-cresol (4-CMC) induces fragmentation of the mitochondrial network, and to ascertain if NAC prevents the mitochondrial fragmentation induced by AFOs and/or 4-CMC. Methods: Mature primary rat hippocampal neurons were incubated for 24 h with sublethal concentrations of AFOs (500 nM) or for 1–3 h with 4-CMC (0.5–1 mM), w10 mM NAC. Mitochondrial morphology was assessed by confocal microscopy of fixed neurons stained with anti-mHsp70. Intracellular Ca²⁺ levels were determined by time series microscopy of neurons preloaded with Fluo-4 AM. Results: Preincubation of neurons for 30 min with NAC prevented the mitochondrial fragmentation induced by AFOs or 4-CMC. In addition, we confirmed that preincubation with NAC abolished the stimulation of RyR-mediated Ca²⁺ release induced by AFOs or 4-CMC. Conclusion: The present results strongly suggest that the general antioxidant NAC prevents AFO-induced mitochondrial fragmentation by preventing RyR-mediated Ca²⁺-induced Ca²⁺ release. Copyright i 2012 S. Karger AG, Basel

Amyloid β peptide oligomers (AβOs), toxic aggregates with pivotal roles in Alzheimer’s disease, trigger persistent and low magnitude Ca2+ signals in neurons. We reported previously that these Ca2+ signals, which arise from Ca2+ entry and subsequent amplification by Ca2+ release through ryanodine receptor (RyR) channels, promote mitochondrial network fragmentation and reduce RyR2 expression. Here, we examined if AβOs, by inducing redox sensitive RyR-mediated Ca2+ release, stimulate mitochondrial Ca2+-uptake, ROS generation and mitochondrial fragmentation, and also investigated the effects of the antioxidant N-acetyl cysteine (NAC) and the mitochondrial antioxidant EUK-134 on AβOs-induced mitochondrial dysfunction. In addition, we studied the contribution of the RyR2 isoform to AβOs-induced Ca2+ release, mitochondrial Ca2+ uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca2+ signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca2+ uptake and increased mitochondrial superoxide and hydrogen peroxide levels; ryanodine, at concentrations that suppress RyR activity, prevented these responses. The antioxidants NAC and EUK-134 impeded the mitochondrial ROS increase induced by AβOs. Additionally, EUK-134 prevented the mitochondrial fragmentation induced by AβOs, as previously reported for NAC and ryanodine. These findings show that both antioxidants, NAC and EUK-134, prevented the Ca2+-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca2+ release mediated by the RyR2 isoform causes the deleterious effects of AβOs on mitochondrial function. Knockdown of RyR2 with antisense oligonucleotides reduced by about 50% RyR2 mRNA and protein levels in primary hippocampal neurons, decreased by 40% Ca2+ release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced the cytoplasmic and mitochondrial Ca2+ signals and the mitochondrial fragmentation induced by AβOs. Based on our results, we propose that AβOs-induced Ca2+ entry and ROS generation jointly stimulate RyR2 activity, causing mitochondrial Ca2+ overload and fragmentation in a feed forward injurious cycle. The present novel findings highlight the specific participation of RyR2-mediated Ca2+ release on AβOs-induced mitochondrial malfunction.

We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimer's disease (AD) patients when exposed to hydrogen peroxide (H 2 O 2 )-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimer's disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) © 2014 Bentham Science Publishers CONFLICT OF INTERESTThe authors confirm that this article content has no conflict of interest. DISCLOSURE STATEMENTNone of the authors have conflicts of interest related to this study. AUTHORS' CONTRIBUTIONSDaniela P Ponce: Performed research, designed experiments, collected data, analyzed data, revised paper. Felipe Salech: Performed research, designed experiments, collected data, analyzed data, revised paper. Mónica Silva: Performed research, collected data. Chengjie Xiong: Analyzed data, revised paper. Catherine M Roe: Analyzed data, revised paper. Mauricio Henriquez: Designed experiments, analyzed data, revised paper. Andrew FG Quest: Designed experiments, analyzed data, contributed important reagents, and revised paper. María Isabel Behrens: Directed the project, designed experiments, contributed important reagents, analyzed data, wrote paper. and mild-to-moderate dementia (CDR 1-2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H 2 O 2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H 2 O 2 -induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H 2 O 2 -induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H 2 O 2 -induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/ necrosis ratio. HHS Public Access

Mild cognitive impairment (MCI) is a clinically detectable initial stage of cognitive deterioration with a high conversion rate to dementia. There is increasing evidence that some of the cerebral alterations present in Alzheimer type dementia can be found in peripheral tissues. We have previously shown that lymphocytes from Alzheimer’s disease (AD) patients have increased susceptibility to hydrogen peroxide (H2O2)-induced death that depends on dementia severity. We here investigated whether lymphocytes from MCI patients show increased vulnerability to death, and explored the involvement of Poly [ADP-ribose] polymerase (PARP-1) and p53 in the regulation of this process. Lymphocytes from 16 MCI and 10 AD patients, and 15 healthy controls (HCs) were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibitors (3-aminobenzamide (3-ABA) or Nicotinamide (NAM)), or the p53 inhibitor (nutlin-3) or stabilizer (pifithrin-α). PARP-1 and p53 mRNA levels were determined by quantitative PCR (qPCR). Lymphocytes from MCI patients showed increased susceptibility to death, attaining intermediate values between AD and controls. PARP inhibitors -3-ABA and NAM- markedly protected from H2O2-induced death, making the difference between MCI and controls disappear, but not the difference between AD and controls. PARP-1 mRNA expression was increased in MCI lymphocytes. Modulation of p53 with Nutlin-3 or pifithrin-α did not modify the H2O2-induced death of lymphocytes from MCI or AD patients, but augmented the death in control lymphocytes attaining levels similar to MCI and AD. Accordingly, p53 mRNA expression was increased in AD and MCI lymphocytes compared to controls. In all, these results show that increased oxidative death is present in lymphocytes at the MCI stage. PARP-1 has a preponderant role, with complete death protection achieved with PARP inhibition in MCI lymphocytes, but not in AD, suggesting that PARP-1 might have a protective role. In addition, deregulations of the p53 pathway seem to contribute to the H2O2-induced death in MCI and AD lymphocytes, which show increased p53 expression. The results showing a prominent protective role of PARP inhibitors opens the door to study the use of these agents to prevent oxidative death in MCI patients.

Mounting evidence indicates that iron accumulation impairs brain function. We have reported previously that addition of sub-lethal concentrations of iron to primary hippocampal neurons produces Ca2+ signals and promotes cytoplasmic generation of reactive oxygen species. These Ca2+ signals, which emerge within seconds after iron addition, arise mostly from Ca2+ release through the redox-sensitive ryanodine receptor (RyR) channels present in the endoplasmic reticulum. We have reported also that addition of synaptotoxic amyloid-β oligomers to primary hippocampal neurons stimulates RyR-mediated Ca2+ release, generating long-lasting Ca2+ signals that activate Ca2+-sensitive cellular effectors and promote the disruption of the mitochondrial network. Here, we describe that 24 h incubation of primary hippocampal neurons with iron enhanced agonist-induced RyR-mediated Ca2+ release and promoted mitochondrial network fragmentation in 43% of neurons, a response significantly prevented by RyR inhibition and by the antioxidant agent N-acetyl-L-cysteine. Stimulation of RyR-mediated Ca2+ release by a RyR agonist promoted mitochondrial Ca2+ uptake in control neurons and in iron-treated neurons that displayed non-fragmented mitochondria, but not in neurons with fragmented mitochondria. Yet, the global cytoplasmic Ca2+ increase induced by the Ca2+ ionophore ionomycin prompted significant mitochondrial Ca2+ uptake in neurons with fragmented mitochondria, indicating that fragmentation did not prevent mitochondrial Ca2+ uptake but presumably decreased the functional coupling between RyR-mediated Ca2+ release and the mitochondrial Ca2+ uniporter. Taken together, our results indicate that stimulation of redox-sensitive RyR-mediated Ca2+ release by iron causes significant neuronal mitochondrial fragmentation, which presumably contributes to the impairment of neuronal function produced by iron accumulation.

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Recent studies suggest that cellular senescence plays a role in Alzheimer’s Disease (AD) pathogenesis. We hypothesize that cellular senescence markers might be tracked in the peripheral tissues of AD patients. Senescence hallmarks, including altered metabolism, cell-cycle arrest, DNA damage response (DDR) and senescence secretory associated phenotype (SASP), were measured in peripheral blood mononuclear cells (PBMCs) of healthy controls (HC), amnestic mild cognitive impairment (aMCI) and AD patients. Senescence-associated βeta-galactosidase (SA-β-Gal) activity, G0-G1 phase cell-cycle arrest, p16 and p53 were analyzed by flow cytometry, while IL-6 and IL-8 mRNA were analyzed by qPCR, and phosphorylated H2A histone family member X (γH2AX) was analyzed by immunofluorescence. Senescent cells in the brain tissue were determined with lipofuscin staining. An increase in the number of senescent cells was observed in the frontal cortex and hippocampus of advanced AD patients. PBMCs of aMCI patients, but not in AD, showed increased SA-β-Gal compared with HCs. aMCI PBMCs also had increased IL-6 and IL8 mRNA expression and number of cells arrested at G0-G1, which were absent in AD. Instead, AD PBMCs had significantly increased p16 and p53 expression and decreased γH2Ax activity compared with HC. This study reports that several markers of cellular senescence can be measured in PBMCs of aMCI and AD patients.