In vitro aging promotes endoplasmic reticulum (ER)-mitochondria Ca2+ cross talk and loss of store-operated Ca2+ entry (SOCE) in rat hippocampal neurons.

Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca2+ homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated Ca2+ entry (SOCE). In addition, Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria sustains energy production but mitochondrial Ca2+ overload promotes apoptosis. We have addressed whether SOCE and ER-mitochondria Ca2+ transfer are influenced by culture time in long-term cultures of rat hippocampal neurons, a model of neuronal aging. We found that short-term cultured neurons show large SOCE, low Ca2+ store content and no functional coupling between ER and mitochondria. In contrast, in long-term cultures reflecting aging neurons, SOCE is essentially lost, Stim1 and Orai1 are downregulated, Ca2+ stores become overloaded, Ca2+ release is enhanced, expression of the mitochondrial Ca2+ uniporter (MCU) increases and most Ca2+ released from the ER is transferred to mitochondria. These results suggest that neuronal aging is associated to increased ER-mitochondrial cross talking and loss of SOCE. This subcellular Ca2+ remodeling might contribute to cognitive decline and susceptibility to neuron cell death in the elderly.

Aging and amyloid ß oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons.

BACKGROUND: Toll-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 ß peptide (Aßo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aßo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. METHODS: Ca2+ 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 Aßo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4. RESULTS: LPS 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 Aßo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death. CONCLUSIONS: Aging and amyloid ß 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.

Antagonism of P2X7 receptors enhances lorazepam action in delaying seizure onset in an in vitro model of status epilepticus.

Approximately 30% of patients with status epilepticus (SE) become refractory to two or more antiseizure medications (ASMs). There is thus a real need to identify novel targets against which to develop new ASMs for treating this clinical emergency. Among purinergic receptors, the ionotropic ATP-gated P2X7 receptor (P2X7R) has received attention as a potential ASM target. This study evaluated the effect of the selective P2X7R antagonist A740003 on acute seizures in the dentate gyrus (DG) of hippocampal brain slices, where P2X7Rs are highly expressed, with a view to establishing the potential of P2X7R antagonists as a therapy or adjunct with lorazepam (LZP) in refractory SE. Extracellular electrophysiological recordings were made from the DG of male mouse hippocampal slices. Spontaneous seizure-like events (SLEs) were induced by removing extracellular Mg2+ and sequentially adding the K+ channel blocker 4-aminopyridine and the adenosine A1 receptor antagonist 8-cyclopentyltheophylline, during which the early and late application of A740003 and/or lorazepam was evaluated. Our study revealed that, in the absence of changes in mRNA for P2X7Rs or inflammatory markers, P2X7R antagonism did not reduce the frequency of SLEs. However, A740003 in conjunction with LZP delayed the onset of seizures. Furthermore, our results support the need for employing LZP before seizures become refractory during SE as delayed application of LZP increased seizure frequency. These studies reveal possible sites of intervention that could have a positive impact in patients with high risk of suffering SE.

Neurotoxic Ca2+ Signaling Induced by Amyloid-ß Oligomers in Aged Hippocampal Neurons In Vitro.

Alzheimer's disease (AD), the most prevalent dementia linked to aging, involves neurotoxic effects of amyloid ß species and dishomeostasis of intracellular Ca2+. To investigate mechanisms of AD, the effects of soluble species of amyloid ß oligomers (Aßo) prepared in medium devoid of glutamate receptor agonists can be tested on intracellular Ca2+ 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 Aß 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.

Is it All Said for NSAIDs in Alzheimer's Disease? Role of Mitochondrial Calcium Uptake.

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 ß peptide (Aß). METHODS: We have proposed a neuroprotection mechanism for NSAIDs based on inhibition of mitochondrial Ca2+ overload. Aß 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 Aß 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.

Aging Enables Ca2+ Overload and Apoptosis Induced by Amyloid-ß Oligomers in Rat Hippocampal Neurons: Neuroprotection by Non-Steroidal Anti-Inflammatory Drugs and R-Flurbiprofen in Aging Neurons.

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-ß peptide 1-42 (Aß42 oligomers). However, how aging influences susceptibility to neurotoxicity induced by Aß42 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 Aß42 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 Aß42 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 Aß42 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 Aß42 oligomers in aged neurons. However, at high concentrations R-flurbiprofen induces apoptosis. Thus, Aß42 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.