Research Article: Mitochondrial Ca2+ Overload Underlies Aβ Oligomers Neurotoxicity Providing an Unexpected Mechanism of Neuroprotection by NSAIDs

Date Published: July 23, 2008

Publisher: Public Library of Science

Author(s): Sara Sanz-Blasco, Ruth A. Valero, Ignacio Rodríguez-Crespo, Carlos Villalobos, Lucía Núñez, Ernest Greene. http://doi.org/10.1371/journal.pone.0002718

Abstract: Dysregulation of intracellular Ca2+ homeostasis may underlie amyloid β peptide (Aβ) toxicity in Alzheimer’s Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Aβ1–42 oligomers, the assembly state correlating best with cognitive decline in AD, but not Aβ fibrils, induce a massive entry of Ca2+ in neurons and promote mitochondrial Ca2+ overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Aβ oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca2+ overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca2+ overload, cytochrome c release and cell death induced by Aβ oligomers. Our results indicate that i) mitochondrial Ca2+ overload underlies the neurotoxicity induced by Aβ oligomers and ii) inhibition of mitochondrial Ca2+ overload provides a novel mechanism of neuroprotection by NSAIDs against Aβ oligomers and AD.

Partial Text: Alzheimer’s Disease (AD) is a devastating neurodegenerative disorder due to a massive neuron dysfunction and loss related to development of senile plaques that are made of amyloid β peptide (Aβ), a cleavage product of the amyloid precursor protein. Early affected areas in the brain are the cortex and hippocampus. However, neuropathological studies show frequent and varied cerebellar changes in the late stages of the disease [1]. In fact, the cerebellum has been shown to be a unique organ in terms of AD manifestations because it is virtually free of neurofibrillary pathology but there is an strong correlation between cerebellar atrophy -with large cell death in the granular layer- and duration and stage of AD [1]. Although many in vitro studies have been carried out using cortical and hippocampal neurons, cerebellar granule cells have been also used for studies of Aβ neurotoxicity [2]–[4]. Several mechanisms have been envisioned. First, the “inflammatory hypothesis” proposes that Aβ may promote a damaging inflammation reaction. This view is supported among other evidence by the neuroprotection afforded by NSAIDs [5]. Second, Aβ promotes mitochondrial dysfunction and apoptosis and this toxicity contributes to AD [6] but the mechanism is unclear. Aβ may associate with mitochondrial membranes in mutant mice and patients with AD and mitochondria from mutant mice show lower levels of oxygen consumption and reduced respiratory complex-associated enzymatic activity suggesting that mitochondria-bound Aβ may impact on mitochondrial activity 7–9. Finally, AD has been also related to a general dyshomeostasis of intracellular Ca2+, a key second messenger involved in multiple neuronal functions. This view is supported by reports on dysregulation of intracellular Ca2+ promoted by Aβ and mutant presenilins [10]. Aβ may promote Ca2+ entry into neurons but results are controversial [11], [12]. Part of the controversy may relate to the fact that Aβ toxicity depends on its assembly state that varies from monomers to small, soluble oligomers and fibrils [13]. Small assemblies (oligomers) of unmodified Aβ are becoming the proximate neurotoxin in AD [13], [14], but most studies used fibrils. Intracellular Ca2+ levels are important for AD since overexpression of calbindin28k, an endogenous Ca2+ buffer, prevents neuron death in AD models [15]. However the link between putative changes in intracellular Ca2+ and cell damage is unknown. A rise in mitochondrial Ca2+ concentration ([Ca2+]mit) might contribute to neurotoxicity but monitoring [Ca2+]mit in individual neurons has been challenging. We have addressed the effects of Aβ assembly state on Ca2+ influx and mitochondrial Ca2+ uptake using photon counting imaging of neurons expressing targeted aequorin [16]. We found that only oligomers, but not fibrils, increased cytosolic and mitochondrial Ca2+ concentrations. Accordingly we asked for the role of mitochondrial Ca2+ uptake on neurotoxicity induced by Aβ oligomers. Finally, we tested whether NSAIDs may protect against Aβ toxicity acting on subcellular Ca2+ fluxes. For these studies we have used mainly cerebellar granule cells although some experiments have been also carried out in cortical and hippocampal neurons.

We show here that Aβ1–42 oligomers, the assembly state that correlates best with brain damage and cognitive deficits in AD [13], [14], but not Aβ fibrils, promote massive entry of Ca2+ into GT1 neural cells and cerebellar granule neurons but not glia. This view is supported by the finding that cells responding to Aβ1–42 oligomers also respond to NMDA and by immunocytochemical identification of responsive cells. Aβ1–42 oligomers, but not fibrils, also induced large increases in [Ca2+]cyt in cortical and hippocampal neurons. These results agree with those recently reported [34] showing that Aβ1–42 oligomers but not monomers or fibrils increased [Ca2+]cyt in a human neuroblastoma cell line. The results suggest that the mechanism of neurotoxicity by fibrils and oligomers may be different as previously proposed [21]. The effects of Aβ1–42 oligomers are quite well reproduced by the toxic fragment Aβ25–35 although at 40-fold larger concentrations. The increases in [Ca2+]cyt induced by Aβ1–42 oligomers and the toxic fragment Aβ25–35 can be attributed to enhanced entry of extracellular Ca2+ through the plasma membrane rather than release from intracellular Ca2+ stores as they were entirely prevented by removal of extracellular Ca2+. The route for this enhanced Ca2+ influx is not solved yet but candidate mechanisms may include plasma membrane permeabilization [34], formation of the so-called amyloid channels [35], [36] and/or activation of NMDA receptors [37]. Neither Aβ25–35 or Aβ1–42 oligomers induced parallel decreases in fura2 fluorescence excited at 340 and 380 nm (data not shown) suggesting that the rises in [Ca2+]cyt are not due to membrane permeabilization. It has been shown previously that the increases in [Ca2+]cyt induced by Aβ species can be prevented by the NMDA receptor open channel blocker memantine [37] and specific amyloid channel blockers [36] suggesting that both NMDA receptors and amyloid channels might be involved. In any case, the massive entry of Ca2+ induced by Aβ1–42 oligomers promotes mitochondrial Ca2+ overload as shown directly by bioluminescence imaging of neurons expressing a low-affinity aequorin targeted to mitochondria. This probe was originally developed to monitor the high [Ca2+] inside the endoplasmic reticulum that reach the mM range in resting conditions [24]. Using this probe, we and others have shown previously that [Ca2+]mit may reach several hundred µM upon stimulation of voltage-gated Ca2+ entry and Ca2+ release from intracellular stores [16], [23], [24]. However, the above mentioned increases in [Ca2+]mit were transient and restricted to a pool of mitochondria close to sites of Ca2+ entry or release [23], [24]. The recent introduction of low-affinity, targeted chameleons [38] has confirmed that [Ca2+]mit may reach values above 200 µM, the sensitivity limit of these probes, stressing that [Ca2+]mit levels actually rise substantially and the requirement of low-affinity probes for actual measurements of [Ca2+]mit, particularly when a mitochondrial Ca2+ overload is to be measured. Using the low-affinity, mitochondria-targeted aequorin, we find that Aβ25–35 and Aβ1–42 oligomers, but not fibrils, induce a massive mitochondrial Ca2+ overload that reaches values close to the mM level. As aequorin is consumed by the high Ca2+ level achieved, the results suggest that, at variance with stimulation with high K+, most mitochondria take up Ca2+ when cells are stimulated by Aβ1–42 oligomers. As mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter requires high [Ca2+]cyt levels, these results suggest that Aβ1–42 oligomers promote a massive entry of Ca2+, large and sustained enough to activate the mitochondrial Ca2+ uniporter of most mitochondria.

Source:

http://doi.org/10.1371/journal.pone.0002718