The release of calcium from the endoplasmic reticulum induced by amyloid-beta and prion peptides activates the mitochondrial apoptotic pathway

In this study, we analyzed whether ER Ca²⁺ release, induced by amyloid-β (Aβ) and prion (PrP) peptides activates the mitochondrial-mediated apoptotic pathway. In cortical neurons, addition of the synthetic Aβ1–40 or PrP106–126 peptides depletes ER Ca²⁺ content, leading to cytosolic Ca²⁺ overload. The Ca²⁺ released through ryanodine (RyR) and inositol 1,4,5-trisphosphate (IP₃R) receptors was shown to be involved in the loss of mitochondrial membrane potential, Bax translocation to mitochondria and apoptotic death. Our data further demonstrate that Ca²⁺ released from the ER leads to the depletion of endogenous GSH levels and accumulation of reactive oxygen species, which were also involved in the depolarization of the mitochondrial membrane. These results illustrate that the early Aβ- and PrP -induced perturbation of ER Ca²⁺ homeostasis affects mitochondrial function, activating the mitochondrial-mediated apoptotic pathway and help to clarify the mechanism implicated in neuronal death that occurs in AD and PrD.     

Involvement of endoplasmic reticulum Ca2+ release through ryanodine and inositol 1,4,5-triphosphate receptors in the neurotoxic effects induced by the amyloid-beta peptide

Studies with in-vitro-cultured neurons treated with amyloid-beta (A beta) peptides demonstrated neuronal loss by apoptosis that is due, at least in part, to the perturbation of intracellular Ca(2+) homeostasis. In addition, it was shown that an endoplasmic reticulum (ER)-specific apoptotic pathway mediated by caspase-12, which is activated upon the perturbation of ER Ca(2+) homeostasis, may contribute to A beta toxicity. To elucidate the involvement of deregulation of ER Ca(2+) homeostasis in neuronal death induced by A beta peptides, we have performed a comparative study using the synthetic peptides A beta(25-35) or A beta(1-40) and thapsigargin, a selective inhibitor of Ca(2+) uptake into the ER. Incubation of cortical neurons with thapsigargin (2.5 microM) increased the intracellular Ca(2+) levels and activated caspase-3, leading to a significant increase in the number of apoptotic cells. Similarly, upon incubation of cortical cultures with the A beta peptides (A beta(25-35), 25 microM; A beta(1-40), 0.5 microM), we observed a significant increase in [Ca(2+)](i), in caspase-3-like activity, and in number of neurons exhibiting apoptotic morphology. The role of ER Ca(2+) release through ryanodine receptors (RyR) or inositol 1,4,5-trisphosphate receptors (IP(3)R) in A beta neurotoxicity has been also investigated. Dantrolene and xestospongin C, inhibitors of ER Ca(2+) release through RyR or IP(3)R, were able to prevent the increase in [Ca(2+)](i) and the activation of caspase-3 and to protect partially against apoptosis induced by treatment with A beta(25-35) or A beta(1-40). In conclusion, our results demonstrate that the release of Ca(2+) from the ER, mediated by both RyR and IP(3)R, is involved in A beta toxicity and can contribute, together with the activation of other intracellular neurotoxic mechanisms, to A beta-induced neuronal death. This study suggests that A beta accumulation may have a key role in the pathogenesis of AD as a result of deregulation of ER Ca(2+) homeostasis.     

Cell degeneration induced by amyloid-β peptides

Extracellular accumulation of amyloid-beta (Abeta) peptide and death of neurons in brain regions involved in learning and memory, particularly the cortex and the hippocampus, are central features of Alzheimer's disease (AD). Neuronal Ca2+ overload and apoptosis are known to occur in AD. Abeta might play a role in disrupting Ca2+ homeostasis, and this AD-associated amyloidogenic peptide has been reported to induce apoptotic death in cultured cells. However, the specific intracellular signaling pathways by which Abeta triggers cell death are not yet well defined. This article provides evidence for the involvement of mitochondrial dysfunction in Abeta-induced toxicity and for the role of mitochondria in apoptosis triggered by Abeta. In addition, the endoplasmic reticulum (ER) seems to play a role in Abeta-induced apoptotic neuronal death, the ER stress being mediated by the perturbation of ER Ca2+ homeostasis. It is likely that a better understanding of how Abeta induces neuronal apoptosis will lead to the identification of potential molecular targets for the development of therapies for AD.     

ER stress is involved in Abeta-induced GSK-3beta activation and tau phosphorylation

Intracellular neurofibrillary tangles, one of the characteristic hallmarks of Alzheimer's disease (AD), are mainly composed of hyperphosphorylated tau. The abnormal tau phosphorylation seems to be related to altered activity of kinases such as glycogen synthase kinase-3beta (GSK-3beta). Tau pathology is thought to be a later event during the progression of the disease, and it seems to occur as a consequence of amyloid-beta (Abeta) peptide accumulation. The aim of this work was to investigate whether soluble Abeta1-42, particularly oligomers that correspond to the neurotoxic species involved early in the development of AD, triggers tau phosphorylation by a mechanism involving activation of tau-kinase GSK-3beta. Several studies suggest that GSK-3beta plays a central role in signaling the downstream effects of endoplasmic reticulum (ER) stress. Therefore, the involvement of ER Ca(2+) release in GSK-3beta activation and tau phosphorylation induced by Abeta1-42 oligomers was evaluated using dantrolene, an inhibitor of Ca(2+) release through channels associated with ER ryanodine receptors. We observed that Abeta1-42 oligomers increase tau phosphorylation and compromises cell survival through a mechanism mediated by GSK-3beta activation. We also demonstrated that oligomeric Abeta1-42 induces ER stress and that ER Ca(2+) release is involved in oligomer-induced GSK-3beta activation and tau phosphorylation. This work suggests that GSK-3beta can be a promising target for therapeutic intervention in AD.     

Adult human microglia secrete cytokines when exposed to neurotoxic prion protein peptide: no intermediary role for prostaglandin E2

Prion diseases are characterized by accumulation of protease resistant isoforms of prion protein (termed PrP(SC)), glial activation and neurodegeneration. The time course of PrP deposition, appearance of activated microglia, and of neuronal apoptosis in experimentally-induced prion disease suggests that microglial activation precedes the process of neuronal loss. Activated microglia and inflammatory mediators, including cytokines and prostaglandin E2 (PGE2) co-localize with PrP deposits. In vitro, mouse microglia secrete neurotoxic agents and interleukins (IL)-1 and IL-6, when exposed to synthetic peptides representing the neurotoxic fragment of PrP. In this study, adult human microglia were found to secrete IL-6 and TNF-alpha upon exposure to synthetic fibrillar PrP105-132, the putative transmembrane domain of PrP. Little cytokine release occurred following exposure of microglia to C-terminally amidated, nonfibrillar PrP105-132, suggesting that the degree of fibrillarity of PrP peptides affects their biological properties. Non-steroidal anti-inflammatory drugs (NSAIDs) are thought to exert beneficial effects in neurodegenerative disorders through suppressive effects on microglial activation and on cyclooxygenase (COX) activity. Since microglial COX-2 expression and PGE(2) synthesis are increased in human and experimental prion diseases, we investigated the effects of the NSAIDs indomethacin and BF389, an experimental COX-2 selective inhibitor, on the PrP105-132-induced microglial IL-6 and TNF-alpha synthesis in vitro. No inhibitory effects of the NSAIDs were observed. Furthermore, PrP105-132 did not stimulate microglial PGE(2) synthesis. We conclude that, unlike IL-1beta-induced IL-6 synthesis in astrocytes, the PrP-induced IL-6 synthesis in human adult microglia is not PGE2 mediated.     

Prostaglandin E2 rescues cortical neurons from amyloid beta protein-induced apoptosis

Cerebrospinal fluid prostaglandin E(2) (PGE(2)) levels are elevated in patients with Alzheimer's disease (AD), suggesting an involvement of PGE(2) in the neurodegeneration. AD is characterized by deposits of amyloid beta protein (Abeta) in various regions of the brain, e.g. the cerebral cortex. In the present study, we investigated the effects of PGE(2) on neuronal survival in primary cultures of rat cortical neurons. PGE(2) had no effect on neuronal cell viability or its morphology. Therefore, we examined the synergistic effects of PGE(2) with Abeta, a neurotoxin. Abeta caused neuronal cell death via apoptosis. PGE(2) significantly suppressed Abeta neurotoxicity, but did not promote the neurotoxicity. Furthermore, PGE(2) ameliorated Abeta-induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA. Abeta increased the influx of Ca(2+) into neurons before cell death. Nimodipine, an inhibitor of the L-type voltage-sensitive calcium channel (L-VSCC), significantly reduced Abeta-potentiated Ca(2+) uptake. On the other hand, there was no effect on the Abeta-induced Ca(2+) influx by an N-VSCC blocker or P/Q-VSCC blockers. Moreover, the inhibitor of L-VSCC suppressed Abeta-induced neuronal cell death, whereas neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of Abeta. PGE(2) also suppressed the Abeta-induced Ca(2+) influx in a concentration-dependent manner. This study demonstrated that PGE(2) rescues cortical neurons from Abeta-induced apoptosis by reducing Ca(2+) influx in the primary culture. Furthermore, the present study suggested that the inhibition of L-VSCC contributes to the neuroprotective effect of PGE(2).     

Comparative study of microglia activation induced by amyloid-beta and prion peptides: role in neurodegeneration

The inflammatory responses in Alzheimer's disease (AD) and prion-related encephalopathies (PRE) are dominated by microglia activation. Several studies have reported that the amyloid-beta (Abeta) peptides, which are associated with AD, and the pathogenic isoform of prion protein (PrPSc) have a crucial role in neuronal death and gliosis that occur in both of these disorders. In this study, we investigate whether Abeta and PrPSc cause microglia activation per se and whether these amyloidogenic peptides differentially affect these immunoeffector cells. In addition, we also determined whether substances released by Abeta- and PrP-activated microglia induce neuronal death. Cultures of rat brain microglia cells were treated with the synthetic peptides Abeta1-40, Abeta1-42 and PrP106-126 for different time periods. The lipopolysaccharide was used as a positive control of microglia activation. Our results show that Abeta1-40 and PrP106-126 caused similar morphological changes in microglia and increased the production of nitric oxide and hydroperoxides. An increase on inducible nitric oxide synthase expression was also observed in microglia treated with Abeta1-40 or PrP106. However, these peptides affected in a different manner the secretion of interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) secretion. In cocultures of microglia-neurons, it was observed that microglia treated with Abeta1-40 or PrP106-126 induced a comparable extent of neuronal death. The neutralizing antibody for IL-6 significantly reduced the neuronal death induced by Abeta- or PrP-activated microglia. Taken together, the data indicate that Abeta and PrP peptides caused microglia activation and differentially affected cytokine secretion. The IL-6 released by reactive microglia caused neuronal injury.     

Sublethal concentrations of prion peptide PrP106-126 or the amyloid beta peptide of Alzheimer's disease activates expression of proapoptotic markers in primary cortical neurons

Neurodegenerative disorders such as prion diseases and Alzheimer's disease (AD) are characterized by neuronal dysfunction and accumulation of amyloidogenic protein. In vitro studies have demonstrated that these amyloidogenic proteins can induce cellular oxidative stress and therefore may contribute to the neuronal dysfunction observed in these illnesses. Although the neurotoxic pathways are not fully elucidated, recent studies in AD have demonstrated up-regulation of caspases in neurons treated with amyloid beta (Abeta) peptide, suggesting involvement of apoptotic processes. To examine the role of proapoptotic pathways in prion diseases we treated primary mouse cortical neurons with the toxic prion protein peptide PrP106-126 and measured caspase activation and annexin V binding. We found that PrP106-126 induced a rapid and marked elevation in caspase 3, 6, and 8-like activity in neuronal cultures. Increased annexin V binding was observed predominantly on cortical cell neurites in peptide-treated cultures. Interestingly, these effects were induced by sublethal (5-50 microM) or lethal (100-200 microM) concentrations of PrP106-126. Sublethal concentrations of PrP106-126 maintained elevated caspase activation for at least 10 days with no loss of cell viability. Abeta1-40 also up-regulated caspase 3 activity and annexin V binding at both sublethal (5 microM) and lethal (25 microM) concentrations. There were no changes to proapoptotic marker expression in cultures treated with scrambled PrP106-126 (200 microM) or Abeta1-28 (25 microM) peptides. These studies demonstrate that amyloidogenic peptides can induce prolonged activation of proapoptotic marker expression in cultured neurons even at sublethal concentrations. These effects could contribute to chronic neuronal dysfunction and increase susceptibility to additional metabolic insults in neurodegenerative disorders. If so, targeting of therapeutic strategies against neuronal caspase activation early in the disease course could be beneficial in AD and prion diseases.     

Involvement of calcineurin in the neurotoxic effects induced by amyloid-beta and prion peptides

It is usually accepted that prion and amyloid-beta (A beta) peptides induce apoptotic cell death. However, the mechanisms that trigger neuronal death, induced by these amyloidogenic peptides, remain to be clarified. In the present study we analysed the neurotoxic effects of the synthetic prion and A beta peptides, PrP106-126 and A beta 25-35, in primary cultures of rat brain cortical cells. PrP106-126 and A beta 25-35 incubated at a concentration of 25 micro m for 24 h, did not affect cell membrane integrity, but decreased the metabolic capacity of the cells. The intracellular free Ca2+ concentration and reactive oxygen species levels increased significantly after 24 h treatment with PrP106-126 and A beta 25-35. Furthermore, these peptides (after 24 h exposure) also induced cytochrome c release from mitochondria and increased caspase-3-like activity. FK506, an inhibitor of the Ca2+/calmodulin-dependent phosphatase, calcineurin, was able to prevent cytochrome c release, caspase-3 activation and cell death induced by A beta 25-35 or PrP106-126 peptides. Taken together these data suggest that calcineurin is involved in A beta 25-35 and PrP106-126 neurotoxicity.     

Evaluation of beta-amyloid peptide 25-35 on calcium homeostasis in cultured rat dorsal root ganglion neurons

Accumulation of beta-amyloid (Abeta) protein in brain is an important characteristic for the etiology of Alzheimer's disease. Of all the possible processes generating the neurotoxic effects by Abeta, disruption of intracellular Ca(2+) homeostasis is the primary event. In this process, various intracellular Ca(2+) regulatory mechanisms are reported to be involved. Using patch-clamp techniques, both low and high voltage activated Ca(2+) channel currents were recorded in the cultured dorsal root ganglion (DRG) neurons. Application of Abeta protein fragment, Abeta(25-35) (2 microM), for 30 s increased the amplitude in both currents. The Abeta-triggered facilitation effect of Ca(2+) channel was found in all the depolarized potentials tested, as shown in the current-voltage relationship. Furthermore, after applying single cell Ca(2+) microfluorometric method, it was found that Abeta(25-35) alone could trigger elevations of intracellular Ca(2+) concentration ([Ca(2+)](i)) level in 90% of the cells tested. The elevation diminished completely by cumulatively adding CdCl(2), NiCl(2), thapsigargin (TG), FCCP and Zn(2+) in the normal bath solution. Combining pharmacological approaches, we found that voltage-dependent Ca(2+) channels, Ca(2+) stores and a putative Zn(2+)-sensitive extracellular Ca(2+) entry, respectively, makes 61.0, 25.1, and 13.9% contribution to the [Ca(2+)](i) increase caused by Abeta. When tested in a Ca(2+)-free buffer, mitochondria was found to contribute 41.3% of Abeta produced [Ca(2+)](i) elevation and the remaining 58.7% was attributed to endoplasmic reticulum (ER) release.     

Modulation of Ca channel currents in primary cultures of rat cortical neurones by amyloid β protein (1–40) is dependent on solubility status

The Alzheimer's disease peptide amyloid beta protein (Abeta) can exist in soluble and fibrillar, aggregated forms. Abeta in the aggregated form is thought to be pro-apoptotic, causing cell death when applied to cultured neurones by disrupting Ca(2+) homeostasis. This process may involve changes in Ca(2+) influx across the plasma membrane. The aim of this study was to quantify this effect by applying both the aggregated and unaggregated forms of Abeta to cultured rat cortical neurones. Unaggregated Abeta(1-40) (24-h pretreatment, 1 microM) stimulated an increase in voltage-dependent Ca(2+) channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Abeta(1-40) pre-treatment reduced Ca(2+) channel current density in cortical neurones via an action on N-type Ca(2+) current. This failure of aggregated Abeta(1-40) to increase the Ca(2+) channel current was confirmed on cerebellar granule neurone Ca(2+) currents which normally undergo an increase in activity following soluble Abeta application. Using the MTT and TUNEL assays, aggregated Abeta(1-40) was found to promote apoptotic cell death in cortical neurones confirming that Abeta exhibited the expected biological activity. Unaggregated Abeta had no neurotoxic effect. These data indicate that the unaggregated, non-pathological form of Abeta(1-40), and not the aggregated form, cause changes in neuronal Ca(2+) channel activity. This may reflect a normal functional role for amyloid peptides in the central nervous system.     

Modulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid beta protein (1-40) is dependent on solubility status

The Alzheimer's disease peptide amyloid beta protein (Abeta) can exist in soluble and fibrillar, aggregated forms. Abeta in the aggregated form is thought to be pro-apoptotic, causing cell death when applied to cultured neurones by disrupting Ca(2+) homeostasis. This process may involve changes in Ca(2+) influx across the plasma membrane. The aim of this study was to quantify this effect by applying both the aggregated and unaggregated forms of Abeta to cultured rat cortical neurones. Unaggregated Abeta(1-40) (24-h pretreatment, 1 microM) stimulated an increase in voltage-dependent Ca(2+) channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Abeta(1-40) pre-treatment reduced Ca(2+) channel current density in cortical neurones via an action on N-type Ca(2+) current. This failure of aggregated Abeta(1-40) to increase the Ca(2+) channel current was confirmed on cerebellar granule neurone Ca(2+) currents which normally undergo an increase in activity following soluble Abeta application. Using the MTT and TUNEL assays, aggregated Abeta(1-40) was found to promote apoptotic cell death in cortical neurones confirming that Abeta exhibited the expected biological activity. Unaggregated Abeta had no neurotoxic effect. These data indicate that the unaggregated, non-pathological form of Abeta(1-40), and not the aggregated form, cause changes in neuronal Ca(2+) channel activity. This may reflect a normal functional role for amyloid peptides in the central nervous system.     

The cellular prion protein (PrPC) prevents apoptotic neuronal cell death and mitochondrial dysfunction induced by serum deprivation

Prion diseases are transmissible neurodegenerative disorders that are invariably fatal in humans and animals. Although the nature of the infectious agent and pathogenic mechanisms of prion diseases are not clear, it has been reported that prion diseases may be associated with aberrant metabolism of cellular prion protein (PrP(C)). In various reports, it has been postulated that PrP(C) may be involved in one or more of the following: neurotransmitter metabolism, cell adhesion, signal transduction, copper metabolism, antioxidant activity or programmed cell death. Despite suggestive results supporting each of these mechanisms, the physiological function(s) of PrP(C) is not known. To investigate whether PrP(C) can prevent apoptotic cell death in prion diseases, we established the cell lines stably expressing PrP(C) from PrP knockout (PrP(-/-)) neuronal cells and examined the role of PrP(C) under apoptosis and/or serum-deprived condition. We found that PrP(-/-) cells were vulnerable to apoptotic cell death and that this vulnerability was rescued by the expression of PrP(C). The expression levels of apoptosis-related proteins including p53, Bax, caspase-3, poly(ADP-ribose) polymerase (PARP) and cytochrome c were significantly increased in PrP(-/-) cells. In addition, Ca(2+) levels of mitochondria were increased, whereas mitochondrial membrane potentials were decreased in PrP(-/-) cells. These results strongly suggest that PrP(C) may play a central role as an effective anti-apoptotic protein through caspase-dependent apoptotic pathways in mitochondria, supporting the concept that disruption of PrP(C) and consequent reduction of anti-apoptotic capacity of PrP(C) may be one of the pathogenic mechanisms of prion diseases.     

Role of potassium channels in Abeta(1-40)-activated apoptotic pathway in cultured cortical neurons

Potassium channel dysfunction has been implicated in Alzheimer's disease (AD). In the present study, by using potassium channel blocker tetraethylammonium (TEA), we investigated the relationship between the enhancement of potassium currents and the alteration of apoptotic cascade in the neuronal apoptotic model induced by beta-amyloid peptide 1-40(Abeta(1-40)). Cortical neurons exposed to Abeta(1-40) 5 muM developed a specific increase in the delayed rectifier potassium current (I(K)), but not the transient outward potassium currents (I(A)), before the appearance of neuronal apoptosis. Abeta(1-40) induced various apoptotic features such as chromatin condensation, a decrease in the amount of Bcl-2 protein, an increase in the amount of Bax protein, cytochrome c release from mitochondria, and caspase-3 activation. Potassium channel blocker 5 mM TEA attenuated Abeta(1-40)-induced neuronal death and prevented the alterations of all above mentioned apoptotic indicators. The study indicates that I(K) enhancement might play an important role in certain form of programmed cell death induced by beta-amyloid peptide (Abeta). Increased potassium channel activity might trigger the activation of apoptosis cascade in Abeta(1-40)-treated rat cortical neurons.     

Bcl-2 overexpression protects against amyloid-beta and prion toxicity in GT1-7 neural cells

In this study we analysed the effect of Bcl-2 on the cytotoxicity induced by the amyloid-beta (Abeta(25-35)) and prion (PrP(106-126)) peptides by using GT1-7puro and GT1-7bcl-2 (overexpressing the anti-apoptotic protein Bcl-2) neural cells. Exposure to Abeta(25-35) (1-5 microM) and PrP(106-126) (25 microM) caused a decrease in cell viability, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. These data were correlated with Abeta(25-35) and PrP(106-126)-induced activation of caspase-9, which is linked to the mitochondrial death pathway, and the activation of the effector caspase-3, suggesting cell death by apoptosis. Furthermore, Bcl-2 overexpression protected from loss of cell viability and caspase-9 and -3 activation induced by Abeta(25-35) and PrP(106-126), showing that Bcl-2 is neuroprotective against apoptotic cell death caused by amyloidogenic peptides.     

Apoptotic cell death and impairment of L-type voltage-sensitive calcium channel activity in rat cerebellar granule cells treated with the prion protein fragment 106-126

Prion diseases are neurodegenerative pathologies characterized by the accumulation, in the brain, of altered forms of the prion protein (PrP), named PrP(Sc). A synthetic peptide homologous to residues 106-126 of PrP (PrP106-126) was reported to maintain the neurodegenerative characteristics of PrP(Sc). We investigated the intracellular mechanisms involved in PrP106-126-dependent degeneration of primary cultures of cerebellar granule neurons. Prolonged exposure of such neurons to PrP106-126 induced apoptotic cell death. The L-type voltage-sensitive calcium channel blocker nicardipine reproduced this effect, suggesting that blockade of Ca(2+) entry through this class of calcium channels may be responsible for the granule cell degeneration. Microfluorometric analysis showed that PrP106-126 caused a reduction in cytosolic calcium levels, elicited by depolarizing K(+) concentrations in these neurons. Electrophysiological studies demonstrated that PrP106-126 and nicardipine selectively reduce the L-type calcium channel current. These data demonstrate that PrP106-126 alters the activity of L-type voltage-sensitive calcium channels in rat cerebellar granule cells and suggest that this phenomenon is related to the cell death induced by the peptide.     

A three amino acid peptide, Gly-Pro-Arg, protects and rescues cell death induced by amyloid beta-peptide

Amyloid beta-peptide (Abeta) contributes to the pathogenesis of Alzheimer's disease (AD), causing neuronal death through apoptosis. In this study, the neuroprotective role of small peptides, Gly-Pro-Glu (GPE), Gly-Glu (GE), Gly-Pro-Asp (GPD), and Gly-Pro-Arg (GPR) were examined against Abeta-induced toxicity in cultured rat hippocampal neurons. We report here that GPR (10-100 microM) prevented Abeta-mediated increase in lactate dehydrogenase (LDH) release and Abeta inhibition of MTT reduction, even in neurons that were pre-exposed to Abeta for 24 or 48 h. Since GPR prevented Abeta inhibition of MTT reduction, the anti-apoptotic effect of GPR was studied by examining activation of caspase-3 and expression of p53 protein. Caspase-3 was significantly activated by 20 microM Abeta25-35 and 5 microM Abeta1-40, but GPR effectively prevented the Abeta-mediated activation of caspase-3. Similarly, Abeta increased numbers of p53-positive cells, but GPR prevented this Abeta effect. Our findings suggest that GPR can rescue cultured rat hippocampal neurons from Abeta-induced neuronal death by inhibiting caspase-3/p53-dependent apoptosis.     

Calcium-regulated signaling pathways

Amyloid beta (Abeta) peptides have been shown to impair synaptic function, especially long-term synaptic plasticity, in transgenic mouse models of Alzheimer's disease (AD) and in acute hippocampal preparations. In the transgenic mice overexpressing mutant forms of human amyloid precursor protein (APP), the deficits in hippocampal long-term potentiation (LTP) occur prior to synaptic loss and cell death, suggesting early functional changes at these synapses. Recent studies demonstrate that Abeta-induced synaptic dysfunction is linked with altered Ca2+ signaling in hippocampal neurons. While reducing Ca2+ influx through NMDA receptors, Abeta peptides elevate intracellular Ca2+ concentration by enhancing Ca2+ influx from voltage-gated Ca2+ channels or nonselective cation channels, or by stimulating Ca2+ release from intracellular stores. Interestingly, acute application of Abeta or APP overexpression inhibits activity-dependent regulation of several protein kinase pathways that require Ca2+ influx via NMDA receptors for activation, including Ca2+/calmodulin-dependent protein kinase II, protein kinase A, and extracellular regulated kinases (Erk). On the other hand, activation of Ca2+-dependent protein phosphatase 2B (calcineurin) is implicated in Abeta inhibition of LTP. Thus, multiple Ca2+-regulated signaling pathways are involved in the synaptic action of Abeta, and malfunction of these pathways may underlie the synaptic dysfunction in early AD.     

Presenilin-1 mutations sensitize neurons to DNA damage-induced death by a mechanism involving perturbed calcium homeostasis and activation of calpains and caspase-12

Mutations in presenilin-1 (PS1) can cause early onset familial Alzheimer's disease (AD). Studies of cultured cells and mice expressing mutant PS1 suggest that PS1 mutations may promote neuronal dysfunction and degeneration by altering cellular calcium homeostasis. On the other hand, it has been suggested that age-related damage to DNA in neurons may be an important early event in the pathogenesis of AD. We now report that PC12 cells and primary hippocampal neurons expressing mutant PS1 exhibit increased sensitivity to death induced by DNA damage. The hypersensitivity to DNA damage is correlated with increased intracellular Ca(2+) levels, induction of p53, upregulation of the Ca(2+)-dependent protease m-calpain, and mitochondrial membrane depolarization. Moreover, activation of caspase-12, an endoplasmic reticulum (ER)-associated caspase, is greatly increased in cells expressing mutant PS1. DNA damage-induced death of cells expressing mutant PS1 was attenuated by inhibitors of calpains I and II, by an intracellular Ca(2+) chelator, by the protein synthesis inhibitor cycloheximide, and by a broad-spectrum caspase inhibitor, but not by an inhibitor of caspase-1. Agents that release Ca(2+) from the ER increased the vulnerability of cells expressing mutant PS1 to DNA damage. By promoting ER-mediated apoptotic proteolytic cascades, PS1 mutations may sensitize neurons to DNA damage.     

Neuronal calcium mishandling and the pathogenesis of Alzheimer's disease

Perturbed neuronal Ca(2+) homeostasis is implicated in age-related cognitive impairment and Alzheimer's disease (AD). With advancing age, neurons encounter increased oxidative stress and impaired energy metabolism, which compromise the function of proteins that control membrane excitability and subcellular Ca(2+) dynamics. Toxic forms of amyloid beta-peptide (Abeta) can induce Ca(2+) influx into neurons by inducing membrane-associated oxidative stress or by forming an oligomeric pore in the membrane, thereby rendering neurons vulnerable to excitotoxicity and apoptosis. AD-causing mutations in the beta-amyloid precursor protein and presenilins can compromise these normal proteins in the plasma membrane and endoplasmic reticulum, respectively. Emerging knowledge of the actions of Ca(2+) upstream and downstream of Abeta provides opportunities to develop novel preventative and therapeutic interventions for AD.