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.

Ryanodine receptors (RyR) amplify activity-dependent calcium influx via calcium-induced calcium release. Calcium signals trigger postsynaptic pathways in hippocampal neurons that underlie synaptic plasticity, learning, and memory. Recent evidence supports a role of the RyR2 and RyR3 isoforms in these processes. Along with calcium signals, brain-derived neurotrophic factor (BDNF) is a key signaling molecule for hippocampal synaptic plasticity and spatial memory. Upon binding to specific TrkB receptors, BDNF initiates complex signaling pathways that modify synaptic structure and function. Here, we show that BDNF-induced remodeling of hippocampal dendritic spines required functional RyR. Additionally, incubation with BDNF enhanced the expression of RyR2, RyR3, and PKMζ, an atypical protein kinase C isoform with key roles in hippocampal memory consolidation. Consistent with their increased RyR protein content, BDNF-treated neurons generated larger RyR-mediated calcium signals than controls. Selective inhibition of RyR-mediated calcium release with inhibitory ryanodine concentrations prevented the PKMζ, RyR2, and RyR3 protein content enhancement induced by BDNF. Intrahippocampal injection of BDNF or training rats in a spatial memory task enhanced PKMζ, RyR2, RyR3, and BDNF hippocampal protein content, while injection of ryanodine at concentrations that stimulate RyR-mediated calcium release improved spatial memory learning and enhanced memory consolidation. We propose that RyR-generated calcium signals are key features of the complex neuronal plasticity processes induced by BDNF, which include increased expression of RyR2, RyR3, and PKMζ and the spine remodeling required for spatial memory formation.dendritic spine remodeling | Morris water maze | endoplasmic reticulum | protein expression

Based on these combined results, we propose (i) that BDNF-induced RyR2-mediated Ca release and ROS generation via NOS/NOX2 are strictly required for the dendritic spine remodeling and the RyR2 upregulation induced by BDNF, and (ii) that RyR2 channel expression is crucial for spatial memory processes. Antioxid. Redox Signal. 29, 1125-1146.

Additional studies should examine the precise molecular mechanisms by which Ca2+ release channels contribute to hippocampal synaptic plasticity and spatial memory formation/consolidation. Future studies should test whether redox-modified Ca2+ release channels contribute toward generating the intracellular Ca2+ signals required for sustained synaptic plasticity and hippocampal spatial memory, and whether loss of redox balance and oxidative stress, by altering Ca2+ release channel function, presumably contribute to the abnormal memory processes that occur during aging and AD.

Recognition memory comprises recollection judgment and familiarity, two different processes that engage the hippocampus and the perirhinal cortex, respectively. Previous studies have shown that aged rodents display defective recognition memory and alterations in hippocampal synaptic plasticity. We report here that young rats efficiently performed at short-term (5 min) and long-term (24 h) hippocampus-associated object-location tasks and perirhinal cortex-related novel-object recognition tasks. In contrast, aged rats successfully performed the object-location and the novel-object recognition tasks only at short-term. In addition, aged rats displayed defective long-term potentiation (LTP) and enhanced long-term depression (LTD). Successful long-term performance of object-location but not of novel-object recognition tasks increased the protein levels of ryanodine receptor types-2/3 (RyR2/RyR3) and of IP3R1 in young rat hippocampus. Likewise, sustained LTP induction (1 h) significantly increased RyR2, RyR3 and IP3R1 protein levels in hippocampal slices from young rats. In contrast, LTD induction (1 h) did not modify the levels of these three proteins. Naïve (untrained) aged rats displayed higher RyR2/RyR3 hippocampal protein levels but similar IP3R1 protein content relative to young rats; these levels did not change following exposure to either memory recognition task or after LTP or LTD induction. The perirhinal cortex from young or aged rats did not display changes in the protein contents of RyR2, RyR3, and IP3R1 after exposure at long-term (24 h) to the object-location or the novel-object recognition tasks. Naïve aged rats displayed higher RyR2 channel oxidation levels in the hippocampus compared to naïve young rats. The RyR2/RyR3 up-regulation and the increased RyR2 oxidation levels exhibited by aged rat hippocampus are likely to generate anomalous calcium signals, which may contribute to the well-known impairments in hippocampal LTP and spatial memory that take place during aging.

Increased reactive oxygen species (ROS) generation and the ensuing oxidative stress contribute to Alzheimer's disease pathology. We reported previously that amyloid-β peptide oligomers (AβOs) produce aberrant Ca2+ signals at sublethal concentrations and decrease the expression of type-2 ryanodine receptors (RyR2), which are crucial for hippocampal synaptic plasticity and memory. Here, we investigated whether the antioxidant agent astaxanthin (ATX) protects neurons from AβOs-induced excessive mitochondrial ROS generation, NFATc4 activation, and RyR2 mRNA downregulation. To determine mitochondrial H2O2 production or NFATc4 nuclear translocation, neurons were transfected with plasmids coding for HyperMito or NFATc4-eGFP, respectively. Primary hippocampal cultures were incubated with 0.1 μM ATX for 1.5 h prior to AβOs addition (500 nM). We found that incubation with ATX (≤10 μM) for ≤24 h was nontoxic to neurons, evaluated by the live/dead assay. Preincubation with 0.1 μM ATX also prevented the neuronal mitochondrial H2O2 generation induced within minutes of AβOs addition. Longer exposures to AβOs (6 h) promoted NFATc4-eGFP nuclear translocation and decreased RyR2 mRNA levels, evaluated by detection of the eGFP-tagged fluorescent plasmid and qPCR, respectively. Preincubation with 0.1 μM ATX prevented both effects. These results indicate that ATX protects neurons from the noxious effects of AβOs on mitochondrial ROS production, NFATc4 activation, and RyR2 gene expression downregulation.

Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.

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