RAGE regulates BACE1 and Abeta generation via NFAT1 activation in Alzheimer's disease animal model.

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor, and amyloid beta peptide (Abeta) is one of the ligands for RAGE. Because RAGE is a transporter of Abeta from the blood to the brain, RAGE is believed to play an important role in Alzheimer's disease (AD) pathogenesis. In the present study, the role of RAGE in Abeta production was examined in the brain tissue of an AD animal model, Tg2576 mice, as well as cultured cells. Because beta-site APP-cleaving enzyme 1 (BACE1), an essential protease for Abeta production, is up-regulated in cells overexpressing RAGE and in RAGE-injected brains of Tg2576 mice, the molecular mechanisms underlying RAGE, BACE1 expression, and Abeta production were examined. Because RAGE stimulates intracellular calcium, nuclear factor of activated T-cells 1 (NFAT1) was examined. NFAT1 was activated following RAGE-induced BACE1 expression followed by Abeta generation. Injection of soluble RAGE (sRAGE), which acts as a competitor with full-length RAGE (fRAGE), into aged Tg2576 mouse brains reduced the levels of plaques, Abeta, BACE1, and the active form of NFAT1 compared with fRAGE-injected Tg2576 mice. Taken together, RAGE stimulates functional BACE1 expression through NFAT1 activation, resulting in more Abeta production and deposition in the brain.

DNA damage-inducing agents elicit gamma-secretase activation mediated by oxidative stress.

According to the amyloid cascade hypothesis, Alzheimer's disease is the consequence of neuronal cell death induced by beta-amyloid (Abeta), which accumulates by abnormal clearance or production. On the other hand, recent studies have shown cell death-induced alteration in amyloid precursor protein (APP) processing, suggesting potential mutual interactions between APP processing and cell death. We have shown previously that the cell death caused by DNA damage-inducing agents (DDIAs) facilitated gamma-secretase activity and Abeta generation in a Bax/Bcl-2-dependent, but caspase-independent manner. Here, we attempted to elucidate the downstream mechanism that modulates gamma-secretase activity in DDIA-treated cells. N-acetyl cysteine, a potent antioxidant, attenuated DDIA-induced enhancement of gamma-secretase activity but failed to rescue cell death. Overexpression of heat shock protein 70, which blocks cytochrome c release from mitochondria, also reduced gamma-secretase activity. Moreover, glutathione depletion significantly facilitated gamma-secretase activity and Abeta generation by enhancing the formation of higher molecular weight gamma-secretase complex before signs of cell death developed. Finally, Abeta treatment, a known inducer of oxidative stress, also increased gamma-secretase activity. Taken together, these results indicate that DDIA-induced gamma-secretase activation is dependent on augmented oxidative stress, and that Abeta and gamma-secretase may activate each other. On the basis of these results, we propose a feed-back loop between oxidative stress and Abeta generation mediated by gamma-secretase activation.

Negative regulation of the SAPK/JNK signaling pathway by presenilin 1.

Presenilin 1 (PS1) plays a pivotal role in Notch signaling and the intracellular metabolism of the amyloid beta-protein. To understand intracellular signaling events downstream of PS1, we investigated in this study the action of PS1 on mitogen-activated protein kinase pathways. Overexpressed PS1 suppressed the stress-induced stimulation of stress-activated protein kinase (SAPK)/c-Jun NH(2)-terminal kinase (JNK) in human embryonic kidney 293 cells. Interestingly, two functionally inactive PS1 mutants, PS1(D257A) and PS1(D385A), failed to inhibit UV-stimulated SAPK/JNK. Furthermore, H(2)O(2-) or UV-stimulated SAPK activity was higher in mouse embryonic fibroblast (MEF) cells from PS1-null mice than in MEF cells from PS(+/+) mice. MEF(PS1(-/-)) cells were more sensitive to the H(2)O(2)-induced apoptosis than MEF(PS1(+/+)) cells. Ectopic expression of PS1 in MEF(PS1(-/-)) cells suppressed H(2)O(2)-stimulated SAPK/JNK activity and apoptotic cell death. Together, our data suggest that PS1 inhibits the stress-activated signaling by suppressing the SAPK/JNK pathway.

Dynamics of population code for working memory in the prefrontal cortex.

Some neurons (delay cells) in the prefrontal cortex elevate their activities throughout the time period during which the animal is required to remember past events and prepare future behavior, suggesting that working memory is mediated by continuous neural activity. It is unknown, however, how working memory is represented within a population of prefrontal cortical neurons. We recorded from neuronal ensembles in the prefrontal cortex as rats learned a new delayed alternation task. Ensemble activities changed in parallel with behavioral learning so that they increasingly allowed correct decoding of previous and future goal choices. In well-trained rats, considerable decoding was possible based on only a few neurons and after removing continuously active delay cells. These results show that neural activity in the prefrontal cortex changes dynamically during new task learning so that working memory is robustly represented and that working memory can be mediated by sequential activation of different neural populations.

Serum anti-amyloid-beta antibodies and Alzheimer's disease in elderly Korean patients.

Alzheimer's disease (AD) is characterized by the deposition of senile plaques and neurofibrillary tangles in the brain. The presence of the amyloid-beta (Abeta) peptide in senile plaques seems to play a central role in the neuropathology of AD. Diagnosis of AD involves neuropsychological examinations or magnetic resonance imaging and, to date, a specific diagnostic marker indicating AD has not been found. This study analysed anti-Abeta antibodies from the serum of 153 patients with AD using an enzyme-linked immunosorbent assay method. The levels of anti-Abeta antibody from patients in the control group (n=193) were compared with those of patients with AD. Our results showed a significantly lower anti-Abeta antibody level in patients with AD than in the control group. These results showed that the anti-Abeta antibody level in serum could potentially be used to diagnose the presence of AD.

Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice.

Endogenous metals such as zinc may contribute to beta-amyloid (Abeta) aggregation and hence the plaque formation. In the present study, we examined brains of four Swedish mutant amyloid precursor protein (APP) transgenic mice at 12 months of age for histochemically reactive zinc in the plaques. Here, we report that all the Congo red (+) mature plaques contained chelatable zinc, as demonstrated by staining with the zinc-specific fluorescent dye 6-methoxy-8-quinolyl-para-toluenesulfonamide (TSQ). On the other hand, Congo red (-) preamyloid Abeta deposits were not stained with TSQ. Interestingly, although cerebellum contained similar degree of preamyloid Abeta deposits as cerebral cortex, it was completely devoid of Congo red- or TSQ-stained mature plaques. Although zinc from plaques was only slowly and partially removed by a specific zinc remover, dithizone, treatment of brain sections with heparinase-III, which degrades heparan sulfate proteoglycan (HSPG), another major constituent of plaques, greatly fastened the zinc removal with dithizone. The present study has demonstrated the presence of histochemically reactive zinc in plaques, but not preamyloid Abeta deposits, of the Swedish mutant APP transgenic mice. Because preamyloid Abeta deposits fail to develop into congophilic plaques in cerebellum where synaptic vesicle zinc is deficient, the synaptic zinc may be a necessary element in the plaque formation. In holding zinc inside plaques, HSPG may contribute in addition to Abeta.

CR6-interacting factor 1 is a key regulator in Aß-induced mitochondrial disruption and pathogenesis of Alzheimer's disease.

Mitochondrial dysfunction, often characterized by massive fission and other morphological abnormalities, is a well-known risk factor for Alzheimer's disease (AD). One causative mechanism underlying AD-associated mitochondrial dysfunction is thought to be amyloid-ß (Aß), yet the pathways between Aß and mitochondrial dysfunction remain elusive. In this study, we report that CR6-interacting factor 1 (Crif1), a mitochondrial inner membrane protein, is a key player in Aß-induced mitochondrial dysfunction. Specifically, we found that Crif1 levels were downregulated in the pathological regions of Tg6799 mice brains, wherein overexpressed Aß undergoes self-aggregation. Downregulation of Crif1 was similarly observed in human AD brains as well as in SH-SY5Y cells treated with Aß. In addition, knockdown of Crif1, using RNA interference, induced mitochondrial dysfunction with phenotypes similar to those observed in Aß-treated cells. Conversely, Crif1 overexpression prevented Aß-induced mitochondrial dysfunction and cell death. Finally, we show that Aß-induced downregulation of Crif1 is mediated by enhanced reactive oxygen species (ROS) and ROS-dependent sumoylation of the transcription factor specificity protein 1 (Sp1). These results identify the ROS-Sp1-Crif1 pathway to be a new mechanism underlying Aß-induced mitochondrial dysfunction and suggest that ROS-mediated downregulation of Crif1 is a crucial event in AD pathology. We propose that Crif1 may serve as a novel therapeutic target in the treatment of AD.

Cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus of the rat.

Effects of cholinergic agents on synaptic transmission and plasticity were examined in entorhinal cortex and hippocampus. Bath application of carbachol (0.25-0.75 microM) induced transient depression of field potential responses in all cases tested (24/24 in layer III of medial entorhinal cortex slices and 24/24 in CA1 of hippocampal slices; 11.0+/-1.9% and 7.8+/-2.5%, respectively) and long-lasting potentiation in some cases (4/24 in entorhinal cortex and 12/24 in hippocampus; 33.7+/-3.7% and 32.1+/-9.9%, respectively, in successful cases). Carbachol (0.5 microM) induced transient depression, but not long-lasting potentiation, of N-methyl-D-aspartate receptor-mediated responses in entorhinal cortex. At 5 microM, carbachol induced transient depression only (55. 9+/-4.7% in entorhinal cortex and 41.4+/-2.9% in hippocampus), which was blocked by atropine. Paired-pulse facilitation was not altered during carbachol-induced potentiation but enhanced during carbachol-induced depression. These results suggest that the underlying mechanisms of carbachol-induced depression and potentiation are decreased transmitter release and selective enhancement of non-N-methyl-D-aspartate receptor-mediated responses, respectively. Long-term potentiation could be induced in the presence of 10 microM atropine by theta burst stimulation. The magnitude was significantly lower (15.2+/-5.2%, n=9) compared with control (37.2+/-6.1%, n=8) in entorhinal cortex, however. These results demonstrate similar, but not identical, cholinergic modulation of synaptic transmission and plasticity in entorhinal cortex and hippocampus.

Long-term potentiation in visual cortical projections to the medial prefrontal cortex of the rat.

In order to investigate neural mechanisms by which the prefrontal cortex adaptively modifies its activities based on past experience, we examined whether or not sensory cortical projections to the medial prefrontal cortex support long-term potentiation (LTP) in rats. Monosynaptic projections from the secondary visual cortex, mediomedial area (V2MM) to the infralimbic cortex were confirmed by orthodromic as well as antidromic activation of single units. High-frequency stimulation (50 Hz, 2 s) induced LTP (approximately 45% increase over the baseline) in the V2MM projection to the infralimbic cortex. LTP induction in this pathway was completely blocked by an injection (i.p.) of CPP, an N-methyl-D-aspartate receptor antagonist. LTP was also induced in the ventral hippocampal projection to the infralimbic cortex by the same high-frequency stimulation. The present results suggest that modification of synaptic weights of afferent sensory cortical projections is one mechanism underlying learning-induced changes in prefrontal cortical neural activities.

Is understanding the biological function of APP important in understanding Alzheimer's disease?

The presence of mutations around the A beta sequence in APP provides strong argument for the involvement of APP, and A beta in particular, in pathogenesis of Alzheimer's disease (AD). In vitro studies demonstrated that A beta may cause neuronal death, supporting the hypothetical involvement of A beta in neurodegeneration in AD. However, concentrations of A beta required for neuronal death are nonphysiologically high. Nevertheless, the predominant idea in the field is that it is sufficient to postulate A beta as a major culprit in AD development. The question we pose is whether the potentially important involvement of A beta precludes the etiological (primary) involvement (not pathological, i.e., secondary) of APP functions. We do not have an adequate answer to this question. Current knowledge about APP functions indicates that APP is critically required for the maintenance of neuronal and synaptic structure and function. Because AD is a disease of neuronal and synaptic deterioration, APP may be involved during the course of AD pathogenesis, perhaps secondarily. To ponder the question whether APP may be etiologically involved in AD, much needs to be learned about APP functions. This article is intended to provide a foundation for this challenging task.

Amyloid precursor protein activates phosphotyrosine signaling pathway.

Amyloid precursor protein (APP) is known to have neurotrophic effects but little information is available on the signaling pathways activated by APP. Since neurotrophic factors activate tyrosine phosphorylation signaling pathway in general, we investigated whether or not APP activates tyrosine phosphorylation pathway. Alpha-secretase derived APP (sAPP alpha) increased the number of neurites per cell and enhanced tyrosine phosphorylation levels on distinct 125 and 200 kDa protein bands. The APP3 19-335 17-mer peptide, which has been reported to be responsible for the neurotrophic effect of sAPP alpha [Jin, L.-W., Ninomiya, H., Roch, J.-M., Schubert, D., Masliah, E., Otero, D.A.C. and Saitoh, T., J. Neurosci., 14 (1994) 5461-5470], increased neurite extension as well as tyrosine phosphorylation on 125 and 200 kDa proteins in a similar manner to sAPP alpha. Both effects were blocked by an antagonist peptide to 17-mer ERMSQ (APP329-333). These results indicate that the 17-mer domain of APP induces tyrosine phosphorylation on distinct proteins under the condition that induces neurite extension.

Presenilin 1 mediates protein kinase C dependent alpha-secretase derived amyloid precursor protein secretion and mitogen-activated protein kinase activation in presenilin 1 transfected human embryonic kidney 293 cell.

We investigated the role of presenilin 1 (PS1) in the secretion of alpha-secretase derived amyloid precursor protein (sAPP alpha) and associated intracellular signaling pathways. Human embryonic kidney (HEK) 293 cells were transfected with exon 9 deletion (deltaE9) mutant PS1 cDNA in an ecdyson-inducible system. sAPPalpha secretion was lower in the mutant PS1 expressing cells compared with non-expressing cells. When activated by PDBu, secretion of sAPPalpha and the level of phosphorylated mitogen activated protein kinase (MAPK) were greatly enhanced in deltaE9 PS1 uninduced cells, but not in the mutant PS1 induced cells. PD98059, a MAPK inhibitor, blocked PDBu induced sAPPalpha secretion from deltaE9 PS1 uninduced cells but had no effect on the mutant PS1 induced cells. These data indicate that PS1 mediates PDBu-induced sAPPalpha secretion and MAPK activation.

Estrogen blocks neurotoxic effects of beta-amyloid (1-42) and induces neurite extension on B103 cells.

Clinical studies have shown that estrogen replacement therapy is associated with reduced risk of Alzheimer's disease (AD). We tested whether or not estrogen blocks neurotoxic effects of beta-amyloid (1-42) (A beta1-42) on cultured B103 cells. A beta1-42 (1 microM) induced typical necrotic cell death, as revealed by light and electron microscopic examinations. Co-administration of estrogen not only blocked A beta1-42 toxicity to a large degree, but also enhanced neurite extension. Pretreatment with estrogen was even more effective in blocking A beta1-42 toxicity. When added 18 h after the beginning of A beta1-42 treatment, estrogen was still effective in halting the progress of cell death and enhancing neurite extension. The protection against A beta1-42-induced neuronal death by estrogen was unlikely due to a blockade of lipid peroxidation injury, since estrogen completely failed to attenuate ferrous chloride-induced cell death. These results demonstrate that estrogen blocks A beta1-42-induced neurotoxicity and enhances neurite extension on B103 cells, both of which may well be underlying mechanisms of beneficial effects of estrogen in AD.

Haloperidol and clozapine increase neural activity in the rat prefrontal cortex.

Haloperidol and clozapine have been widely used to alleviate schizophrenic symptoms, but their physiological effects in the prefrontal cortex (PFC) are not known. Effects of haloperidol and clozapine on single unit activity were investigated in the medial PFC of anesthetized rats. Injection (intraperitoneal) of haloperidol (1 mg/kg) or clozapine (20 mg/kg) significantly elevated discharge rates of PFC neurons. Considering that hypofrontality is one characteristic of schizophrenic symptoms, these results raise the possibility that enhancement of PFC neural activity contributes to therapeutic effects of haloperidol and clozapine.

Neuroprotective effects of estrogen against beta-amyloid toxicity are mediated by estrogen receptors in cultured neuronal cells.

Although estrogen is known to exert beneficial effects on Alzheimer's disease, its underlying cellular mechanisms have not been clear. In this study we investigated whether or not neuroprotective effects of estrogen are mediated by estrogen receptors (ERs). Treatment of estrogen (1.8 nM) reduced beta-amyloid (Abeta)-induced death of ER-expressing W4 cells. This effect of estrogen was blocked by a specific ER blocker ICI 182,780. When estrogen was treated to HT22 cells, which lack functional ERs, Abeta-induced cell death was not affected. Transfection of HT22 cells with human ERalpha, but not ERbeta, restored protective action of estrogen against Abeta. Hoechst staining revealed that estrogen protected ERalpha-expressing cells by blocking Abeta-induced apoptosis. These results indicate that estrogen blocks Abeta-induced cell death via ERalpha-dependent pathways.

Aß1-42 reduces P-glycoprotein in the blood-brain barrier through RAGE-NF-κB signaling.

The reduced clearance of amyloid-ß peptide (Aß) from the brain partly accounts for the neurotoxic accumulation of Aß in Alzheimer's disease (AD). Recently, it has been suggested that P-glycoprotein (P-gp), which is an efflux transporter expressed on the luminal membrane of the brain capillary endothelium, is capable of transporting Aß out of the brain. Although evidence has shown that restoring P-gp reduces brain Aß in a mouse model of AD, the molecular mechanisms underlying the decrease in P-gp expression in AD is largely unknown. We found that Aß1-42 reduced P-gp expression in the murine brain endothelial cell line bEnd.3, which was consistent with our in vivo data that P-gp expression was significantly reduced, especially near amyloid plaques in the brains of five familial AD mutations (5XFAD) mice that are used as an animal model for AD. A neutralizing antibody against the receptor for advanced glycation end products (RAGE) and an inhibitor of nuclear factor-kappa B (NF-κB) signaling prevented the decrease in Aß1-42-induced P-gp expression, suggesting that Aß reduced P-gp expression through NF-κB signaling by interacting with RAGE. In addition, we observed that the P-gp reduction by Aß was rescued in bEnd.3 cells receiving inductive signals or factors from astrocytes making contacts with endothelial cells (ECs). These results support that alterations of astrocyte-EC contacts were closely associated with P-gp expression. This suggestion was further supported by the observation of a loss of astrocyte polarity in the brains of 5XFAD mice. Taken together, we found that P-gp downregulation by Aß was mediated through RAGE-NF-κB signaling pathway in ECs and that the contact between astrocytes and ECs was an important factor in the regulation of P-gp expression.

Reduced IRE1α mediates apoptotic cell death by disrupting calcium homeostasis via the InsP3 receptor.

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca(2+). Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca(2+) homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca(2+) dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca(2+) concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca(2+) through the InsP3 receptor (InsP3R). The Ca(2+) efflux in IRE1α-deficient cells correlates with dissociation of the Ca(2+)-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α-TRAF2-ASK1 complex. The increased cytosolic concentration of Ca(2+) induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca(2+) dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca(2+) influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca(2+) homeostasis and cell survival during ER stress and reveal a previously unknown Ca(2+)-mediated cell death signaling between the IRE1α-InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

High-dose of vitamin C supplementation reduces amyloid plaque burden and ameliorates pathological changes in the brain of 5XFAD mice.

Blood-brain barrier (BBB) breakdown and mitochondrial dysfunction have been implicated in the pathogenesis of Alzheimer's disease (AD), a neurodegenerative disease characterized by cognitive deficits and neuronal loss. Besides vitamin C being as one of the important antioxidants, recently, it has also been reported as a modulator of BBB integrity and mitochondria morphology. Plasma levels of vitamin C are decreased in AD patients, which can affect disease progression. However, investigation using animal models on the role of vitamin C in the AD pathogenesis has been hampered because rodents produce with no dependence on external supply. Therefore, to identify the pathogenic importance of vitamin C in an AD mouse model, we cross-bred 5 familial Alzheimer's disease mutation (5XFAD) mice (AD mouse model) with ι-gulono-γ-lactone oxidase (Gulo) knockout (KO) mice, which are unable to synthesize their own vitamin C, and produced Gulo KO mice with 5XFAD mice background (KO-Tg). These mice were maintained on either low (0.66 g/l) or high (3.3 g/l) supplementation of vitamin C. We found that the higher supplementation of vitamin C had reduced amyloid plaque burden in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration. These results suggest that intake of a larger amount of vitamin C could be protective against AD-like pathologies.

Crucial role of calbindin-D28k in the pathogenesis of Alzheimer's disease mouse model.

Calbindin-D28k (CB), one of the major calcium-binding and buffering proteins, has a critical role in preventing a neuronal death as well as maintaining calcium homeostasis. Although marked reductions of CB expression have been observed in the brains of mice and humans with Alzheimer disease (AD), it is unknown whether these changes contribute to AD-related dysfunction. To determine the pathogenic importance of CB depletions in AD models, we crossed 5 familial AD mutations (5XFAD; Tg) mice with CB knock-out (CBKO) mice and generated a novel line CBKO·5XFAD (CBKOTg) mice. We first identified the change of signaling pathways and differentially expressed proteins globally by removing CB in Tg mice using mass spectrometry and antibody microarray. Immunohistochemistry showed that CBKOTg mice had significant neuronal loss in the subiculum area without changing the magnitude (number) of amyloid ß-peptide (Aß) plaques deposition and elicited significant apoptotic features and mitochondrial dysfunction compared with Tg mice. Moreover, CBKOTg mice reduced levels of phosphorylated mitogen-activated protein kinase (extracellular signal-regulated kinase) 1/2 and cAMP response element-binding protein at Ser-133 and synaptic molecules such as N-methyl-D-aspartate receptor 1 (NMDA receptor 1), NMDA receptor 2A, PSD-95 and synaptophysin in the subiculum compared with Tg mice. Importantly, this is the first experimental evidence that removal of CB from amyloid precursor protein/presenilin transgenic mice aggravates AD pathogenesis, suggesting that CB has a critical role in AD pathogenesis.