BackgroundToll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer’s disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence.MethodsCa2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4.ResultsLPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death.ConclusionsAging and amyloid β oligomers, the neurotoxin involved in Alzheimer’s disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0802-0) contains supplementary material, which is available to authorized users.
Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca 2+ homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated
The most important risk factor for Alzheimer's disease (AD) is aging. Neurotoxicity in AD has been linked to dyshomeostasis of intracellular Ca2+ induced by small aggregates of the amyloid-β peptide 1-42 (Aβ42 oligomers). However, how aging influences susceptibility to neurotoxicity induced by Aβ42 oligomers is unknown. In this study, we used long-term cultures of rat hippocampal neurons, a model of neuronal in vitro aging, to investigate the contribution of aging to Ca2+ dishomeostasis and neuron cell death induced by Aβ42 oligomers. In addition, we tested whether non-steroidal anti-inflammatory drugs (NSAIDs) and R-flurbiprofen prevent apoptosis acting on subcellular Ca2+ in aged neurons. We found that Aβ42 oligomers have no effect on young hippocampal neurons cultured for 2 days in vitro (2 DIV). However, they promoted apoptosis modestly in mature neurons (8 DIV) and these effects increased dramatically after 13 DIV, when neurons display many hallmarks of in vivo aging. Consistently, cytosolic and mitochondrial Ca2+ responses induced by Aβ42 oligomers increased dramatically with culture age. At low concentrations, NSAIDs and the enantiomer R-flurbiprofen lacking anti-inflammatory activity prevent Ca2+ overload and neuron cell death induced by Aβ42 oligomers in aged neurons. However, at high concentrations R-flurbiprofen induces apoptosis. Thus, Aβ42 oligomers promote Ca2+ overload and neuron cell death only in aged rat hippocampal neurons. These effects are prevented by low concentrations of NSAIDs and R-flurbiprofen acting on mitochondrial Ca2+ overload.
Objectives: Epidemiological data suggest that non-steroidal anti-inflammatory drugs
(NSAIDs) may protect against Alzheimer's disease (AD). Unfortunately, recent trials have failed in providing
compelling evidence of neuroprotection. Discussion as to why NSAIDs effectivity is uncertain is
ongoing. Possible explanations include the view that NSAIDs and other possible disease-modifying
drugs should be provided before the patients develop symptoms of AD or cognitive decline. In addition,
NSAID targets for neuroprotection are unclear. Both COX-dependent and independent mechanisms
have been proposed, including γ-secretase that cleaves the amyloid precursor protein (APP) and yields
amyloid β peptide (Aβ).
Methods:
We have proposed a neuroprotection mechanism for NSAIDs based on inhibition of mitochondrial
Ca2+ overload. Aβ oligomers promote Ca2+ influx and mitochondrial Ca2+ overload leading to
neuron cell death. Several non-specific NSAIDs including ibuprofen, sulindac, indomethacin and Rflurbiprofen
depolarize mitochondria in the low µM range and prevent mitochondrial Ca2+ overload induced
by Aβ oligomers and/or N-methyl-D-aspartate (NMDA). However, at larger concentrations,
NSAIDs may collapse mitochondrial potential (ΔΨ) leading to cell death.
Results:
Accordingly, this mechanism may explain neuroprotection at low concentrations and damage at
larger doses, thus providing clues on the failure of promising trials. Perhaps lower NSAID concentrations
and/or alternative compounds with larger dynamic ranges should be considered for future trials to
provide definitive evidence of neuroprotection against AD.
Alzheimer's disease (AD), the most prevalent dementia linked to aging, involves neurotoxic effects of amyloid β species and dishomeostasis of intracellular Ca. To investigate mechanisms of AD, the effects of soluble species of amyloid β oligomers (Aβo) prepared in medium devoid of glutamate receptor agonists can be tested on intracellular Ca in long-term cultures of rat hippocampal neurons that reflect aging neurons. Furthermore, changes in expression of proteins involved in oligomer responses and AD can be tested in the same neurons using quantitative immunofluorescence. Detailed procedures for the preparation of Aβ species in defined medium, long-term culture of rat hippocampal neurons mimicking aged neurons, calcium imaging and quantitative immunofluorescence in these cultures are described in this chapter.
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