Serotype b of Aggregatibacter actinomycetemcomitans triggers pro-inflammatory responses and amyloid beta secretion in hippocampal cells: a novel link between periodontitis and Alzheimer´s disease?

Introduction: Previous reports have proposed that Periodontal disease (PDis) predisposes to Alzheimer's disease (AD), both highly prevalent pathologies among the elderly. The bacteria Aggregatibacter actinomycetemcomitans (Aa), associated with the most aggressive forms of PDis, are classified in different serotypes with distinct virulence according to the antigenicity of their lipopolysaccharide (LPS). Methods: Here, we determined the effects of purified LPS, from serotypes a, b or c of Aa, on primary cultures of microglia or mixed hippocampal cells. Results: We found that both culture types exhibited higher levels of inflammatory cytokines (IL-1ß, IL-6 and TNFα) when treated with serotype b-LPS, compared with controls, as quantified by qPCR and/or ELISA. Also, cultures treated with serotype a-LPS displayed increased mRNA levels of the modulatory cytokines IL-4 and IL-10. Mixed hippocampal cultures treated with serotype b-LPS exhibited severe neuronal morphological changes and displayed increased levels of secreted Aß1-42 peptide. These results indicate that LPS from different Aa serotypes triggers discriminatory immune responses, which differentially affect primary hippocampal cells. Conclusion: Altogether, our results show that treatment with serotype b-LPS triggers the secretion of proinflammatory cytokines by microglia, induces neurite shrinking, and increases the extracellular Aß1-42 levels, all features strongly associated with the etiology of AD.

H2 O2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and Ca(2+) signals in human periodontal fibroblasts.

BACKGROUND: The mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown. OBJECTIVE: To determine the effect of H2 O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFκB) and Ca(2+) -mediated signals in human periodontal ligament fibroblasts. MATERIAL AND METHODS: Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5-10 µm) of H2 O2 or control media. NFκB involvement was evaluated through immunofluorescence of p65 subunit, using the NFκB blocking peptide SN50 and catalase. Ca(2+) signals were analyzed by loading the cells with Fluo4-AM and recording the fluorescence changes in a confocal microscope before and after the addition of H2 O2 . 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl was used to chelate intracellular Ca(2+) . The activity and levels of MMP-2 and MMP-9 were analyzed by gelatin zymogram and densitometric scanning, and enzyme-linked immunosorbent assay, respectively. Statistical analysis was performed with stata V11.1 software using the ANOVA test. RESULTS: H2 O2 at concentrations of 2.5-5 µm induced Ca(2+) signaling and NFκB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2 O2 at 2.5-5 µm significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls. CONCLUSION: Sublethal H2 O2 induces Ca(2+) -dependent NFκB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.

Sub-lethal levels of amyloid ß-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons.

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aß) peptide. Current hypotheses regard soluble amyloid beta oligomers (AßOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aß fibrils and AßOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AßOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AßOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AßOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AßOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AßOs did not potentiate iron-induced cell death while iron chelation did not decrease AßOs-induced cell death. The lack of synergism between iron and AßOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.

The antioxidant N-acetylcysteine prevents the mitochondrial fragmentation induced by soluble amyloid-ß peptide oligomers.

BACKGROUND: Soluble amyloid-ß peptide oligomers (AßOs), which are centrally involved in the pathogenesis of Alzheimer's disease, trigger Ca(2+) influx through N-methyl-D-aspartate receptors and stimulate reactive oxygen species generation in primary hippocampal neurons. We have previously reported that AßOs promote Ca(2+) release mediated by ryanodine receptors (RyR), which in turn triggers mitochondrial fragmentation. We have also reported that the antioxidant N-acetylcysteine (NAC) prevents AßOs-induced Ca(2+) signal generation. OBJECTIVES: To determine if RyR-mediated Ca(2+) 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 AßOs and/or 4-CMC. METHODS: Mature primary rat hippocampal neurons were incubated for 24 h with sublethal concentrations of AßOs (500 nM) or for 1-3 h with 4-CMC (0.5-1 mM), ± 10 mM NAC. Mitochondrial morphology was assessed by confocal microscopy of fixed neurons stained with anti-mHsp70. Intracellular Ca(2+) 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 AßOs or 4-CMC. In addition, we confirmed that preincubation with NAC abolished the stimulation of RyR-mediated Ca(2+) release induced by AßOs or 4-CMC. CONCLUSION: The present results strongly suggest that the general antioxidant NAC prevents AßO-induced mitochondrial fragmentation by preventing RyR-mediated Ca(2+)-induced Ca(2+) release.

Aß oligomers induce glutamate release from hippocampal neurons.

Soluble oligomers of the amyloid-ß peptide (AßOs) accumulate in Alzheimer's disease (AD) brain and have been implicated in mechanisms of pathogenesis. The neurotoxicity of AßOs appears to be, at least in part, due to dysregulation of glutamate signaling. Here, we show that AßOs promote extracellular accumulation of glutamate and d-serine, a co-agonist at glutamate receptors of the N-methyl-d-aspartate subtype (NMDARs), in hippocampal neuronal cultures. The increase in extracellular glutamate levels induced by AßOs was blocked by the sodium channel blocker tetrodotoxin (TTX), by the NMDAR blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) and by removal of Ca(2+) from the extracellular medium, indicating dependence on excitatory neuronal activity. AßOs enhanced both the release of pre-synaptic vesicles labeled by FM1-43 and spontaneous post-synaptic activity measured by whole-cell patch-clamp. Activation of inhibitory GABA(A) receptors by taurine blocked the increase in extracellular glutamate levels, suggesting that selective pharmacological inhibition of neuronal activity can counteract the impact of AbOs on glutamate dyshomeostasis. Results reveal a novel mechanism by which Ab oligomers promote abnormal release of glutamate in hippocampal neurons, which may contribute to dysregulation of excitatory signaling in the brain.

Dual role of glutamatergic neurotransmission on amyloid beta(1-42) aggregation and neurotoxicity in embryonic avian retina.

The effects of glutamate receptor antagonists on the toxicity of the beta-amyloid peptide (Abeta(1-42)) in embryonic chick retina were investigated. When used alone or in combination, the N-methyl-D-asparate antagonist, MK-801, the (+/-)-alphaamino-3-hydroxyl-5-methylisoxazole-4-propionic acid/kainate antagonist, DNQX, and the metabotropic receptor antagonist, (RS)-1-aminoindan-1,5-dicarboxylic acid, blocked the neurotoxicity of Abeta(1-42). Aggregation of Abeta(1-42) was significantly increased in the presence of acidic glutamate solutions, but not in the presence of other neurotransmitters. These results point to a dual role of glutamatergic transmission in Alzheimer's disease (AD): (i) Abeta neurotoxicity requires activation of glutamate receptors and its blockade prevents cell death; (ii) high concentrations of glutamate in the synaptic cleft indirectly enhance Abeta aggregation through acidification of the medium, resulting in increased amounts of neurotoxic amyloid fibrils. These results suggest that glutamatergic neurotransmission may represent a novel target for therapeutic approaches in AD.