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.

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We have previously reported that primary hippocampal neurons exposed to synaptotoxic amyloid beta oligomers (AβOs), which are likely causative agents of Alzheimer’s disease (AD), exhibit abnormal Ca2+ signals, mitochondrial dysfunction and defective structural plasticity. Additionally, AβOs-exposed neurons exhibit a decrease in the protein content of type-2 ryanodine receptor (RyR2) Ca2+ channels, which exert critical roles in hippocampal synaptic plasticity and spatial memory processes. The antioxidant N-acetylcysteine (NAC) prevents these deleterious effects of AβOs in vitro. The main contribution of the present work is to show that AβOs injections directly into the hippocampus, by engaging oxidation-mediated reversible pathways significantly decreased RyR2 protein content but increased single RyR2 channel activation by Ca2+ and caused considerable spatial memory deficits. AβOs injections into the CA3 hippocampal region impaired rat performance in the Oasis maze spatial memory task, decreased hippocampal glutathione levels and overall content of plasticity-related proteins (c-Fos, Arc, and RyR2) and increased ERK1/2 phosphorylation. In contrast, in hippocampus-derived mitochondria-associated membranes (MAM) AβOs injections increased RyR2 levels. Rats fed with NAC for 3-weeks prior to AβOs injections displayed comparable redox potential, RyR2 and Arc protein contents, similar ERK1/2 phosphorylation and RyR2 single channel activation by Ca2+ as saline-injected (control) rats. NAC-fed rats subsequently injected with AβOs displayed the same behavior in the spatial memory task as control rats. Based on the present in vivo results, we propose that redox-sensitive neuronal RyR2 channels partake in the mechanism underlying AβOs-induced memory disruption in rodents.

AimTo determine the methylation pattern of TLR2 gene promoter and its association with the transcriptional regulation of periapical inflammatory and angiogenic responses in symptomatic and asymptomatic forms of apical periodontitis.MethodologyIn this cross‐sectional study, apical lesions were obtained from volunteers with asymptomatic apical periodontitis (AAP) (n = 17) and symptomatic apical periodontitis (SAP) (n = 17) scheduled for tooth extraction, and both total RNA and DNA were extracted. DNA was bisulfite‐treated, a region of CpG island within the TLR2 gene was amplified by qPCR and the products were sequenced. Additionally, the mRNA expression of TLR2, TLR4, IL‐6, IL‐12, TNFalpha, IL‐23, IL‐10, TGFbeta, VEGFA and CDH5 was analysed by qPCR. The data were analysed with chi‐square tests, Mann–Whitney or unpaired t‐tests, and Spearman´s correlation; variable adjustments were performed using multiple linear regression (P < 0.05).ResultsTLR2 depicted a hypomethylated DNA profile at the CpG island in SAP when compared with AAP, along with upregulated expression of TLR2, with pro‐inflammatory cytokines IL‐6 and IL‐23, and the angiogenesis marker CDH5 (P < 0.05). TLR2 methylation percentage negatively correlated with mRNA levels of IL‐23 and CDH5 in apical periodontitis. Lower methylation frequencies of single CpG dinucleotides −8 and −10 localized in close proximity to nuclear factor κB (NFκB) binding within the TLR2 promoter were identified in SAP versus AAP (P < 0.05). Finally, unmethylated −10 and −8 single sites demonstrated up‐regulation of IL‐23, IL‐10 and CDH5 transcripts compared to their methylated counterparts (P < 0.05).ConclusionsTLR2 gene promoter hypomethylation was linked to transcriptional activity of pro‐inflammatory cytokines and angiogenic markers in exacerbated periapical inflammation. Moreover, unmethylated single sites in close proximity to NFκB binding were involved in active transcription of IL‐23, IL‐10 and CDH5.

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IL-6 trans-signalling induced Th1 and Th17-related cytokines and represents an extra-hepatic mechanism for PCR synthesis in human periodontal ligament fibroblasts, contributing to explain the bone-destructive phenotype of apical lesions and eventually its systemic complications.

Apical Lesions of Endodontic Origin (ALEO) are initiated by polymicrobial endodontic canal infection. Porphyromonas gingivalis (Pg) and Porphyromonas endodontalis (Pe) lipopolysaccharides (LPS) can induce a pro-inflammatory macrophage response through their recognition by TLR2 and TLR4. However, polarization responses induced by Pg and/or Pe LPS in macrophages are not fully understood. We aimed to characterize the polarization profiles of macrophages differentiated from THP-1 cells following Pg and/or Pe LPS stimulation from reference strain and clinical isolates. A modified LPS purification protocol was implemented and the electrophoretic LPS profiles were characterized. THP-1 human monocytes differentiated to macrophages were stimulated with Pg and Pe LPS. Polarization profiles were characterized through cell surface markers and secreted cytokines levels after 24 h of stimulation. TLR2 and TLR4 cell surfaces and transcriptional levels were determined after 24 or 2 h of LPS stimulation, respectively. LPS from Pg induced a predominant M1 profile in macrophages evidenced by changes in the expression of the surface marker CD64 and pro-inflammatory cytokine profiles, TNF-α, IL-1β, IL-6, and IL-12. Pe LPS was unable to induce a significant response. TLR2 and TLR4 expressions were neither modified by Pg or Pe LPS. Pg LPS, but not Pe LPS, induced a macrophage M1 Profile.