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.

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.

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.

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.

Critical role of presenilin-dependent γ-secretase activity in DNA damage-induced promyelocytic leukemia protein expression and apoptosis.

Promyelocytic leukemia (PML) is a major component of macromolecular multiprotein complexes called PML nuclear-bodies (PML-NBs). These PML-NBs recruit numerous proteins including CBP, p53 and HIPK2 in response to DNA damage, senescence and apoptosis. In this study, we investigated the effect of presenilin (PS), the main component of the γ-secretase complex, in PML/p53 expression and downstream consequences during DNA damage-induced cell death using camptothecin (CPT). We found that the loss of PS in PS knockout (KO) MEFs (mouse embryonic fibroblasts) results in severely blunted PML expression and attenuated cell death upon CPT exposure, a phenotype that is fully reversed by re-expression of PS1 in PS KO cells and recapitulated by γ-secretase inhibitors in hPS1 MEFs. Interestingly, the γ-secretase cleavage product, APP intracellular domain (AICD), together with Fe65-induced PML expression at the protein and transcriptional levels in PS KO cells. PML and p53 reciprocally positively regulated each other during CPT-induced DNA damage, both of which were dependent on PS. Finally, elevated levels of PML-NB, PML protein and PML mRNA were detected in the brain tissues from Alzheimer's disease (AD) patients, where γ-secretase activity is essential for pathogenesis. Our data provide for the first time, a critical role of the PS/AICD-PML/p53 pathway in DNA damage-induced apoptosis, and implicate this pathway in AD pathogenesis.

Vitamin D-binding protein interacts with Aß and suppresses Aß-mediated pathology.

The level of vitamin D-binding protein (DBP) is increased in the cerebrospinal fluid of patients with Alzheimer's disease (AD), suggesting a relationship with its pathogenesis. In this study, we investigated whether and how DBP is related to AD using several different approaches. A pull-down assay and a surface plasmon resonance binding assay indicated direct interactions between purified DBP and amyloid beta (Aß), which was confirmed in the brain of AD patients and transgenic AD model mice by immunoprecipitation assay and immunohistochemical double-staining method. Moreover, atomic force microscopic examination revealed that DBP reduced Aß aggregation in vitro. DBP also prevented Aß-mediated death in cultured mouse hippocampal HT22 cell line. Finally, DBP decreased Aß-induced synaptic loss in the hippocampus and rescued memory deficits in mice after injection of Aß into the lateral ventricle. These results provide converging evidence that DBP attenuates the harmful effects of Aß by a direct interaction, and suggest that DBP is a promising therapeutic agent for the treatment of AD.

Pivotal role of the RanBP9-cofilin pathway in Aß-induced apoptosis and neurodegeneration.

Neurodegeneration associated with amyloid ß (Aß) peptide accumulation, synaptic loss, neuroinflammation, tauopathy, and memory impairments encompass the pathophysiological features of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9, which is overall increased in brains of AD patients, simultaneously promotes Aß generation and focal adhesion disruption by accelerating the endocytosis of amyloid precursor protein (APP) and ß1-integrin, respectively. Here, we show that RanBP9 protein levels are increased by fourfold in FAD mutant APP transgenic mice. Accordingly, RanBP9 transgenic mice demonstrate significantly increased synapse loss, neurodegeneration, gliosis, and spatial memory deficits. RanBP9 overexpression promotes apoptosis and potentiates Aß-induced neurotoxicity independent of its capacity to promote Aß generation. Conversely, RanBP9 reduction by siRNA or gene dosage mitigates Aß-induced neurotoxicity. Importantly, RanBP9 activates/dephosphorylates cofilin, a key regulator of actin dynamics and mitochondria-mediated apoptosis, and siRNA knockdown of cofilin abolishes both Aß and RanBP9-induced apoptosis. These findings implicate the RanBP9-cofilin pathway as critical therapeutic targets not only for stemming Aß generation but also antagonizing Aß-induced neurotoxicity.

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.

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.

ER stress-induced caspase-12 activation is inhibited by PKC in neuronal cells.

Caspase-12 is activated when the cells are exposed to excess levels of various stimuli, which cause endoplasmic reticulum (ER) stress. Protein kinase C (PKC) plays an important role in many signaling pathways in cells, and the activation of PKC has multiple actions in the signaling function of the ER. This study examined whether or not phorbol 12, 13-dibutyrate (PDBu)-induced PKC activation modulates caspase-12 cleavage and it's processing, using a wild type caspase-12 overexpressing neuronal cell line, known as Cas-12 cells. The thapsigargin treatment induced caspase-12 fragmentation in the Cas-12 cells. This was inhibited by PKC, which had previously been stimulated by PDBu. The PDBu treatment attenuated the ER stress-induced translocation of caspase-12 from the ER to the cytoplasm. The caspase-3 specific inhibitor blocked caspase-12 fragmentation, and purified caspase-12 was cleaved by the active caspase-3 in vitro, suggesting that caspase-12 might be a substrate for caspase-3. In addition, the PDBu treatment influenced the decrease of active caspase-3 fragment. These results suggest that an ER stress induces the activation of caspase-12 via caspase-3, and that PKC regulates both caspase-12 and caspase-3 activations in Cas-12 cells.

Circulating beta amyloid protein is elevated in patients with acute ischemic stroke.

Recent clinical and experimental studies suggest that ischemic strokes may play an important role in the pathogenesis of Alzheimer's disease (AD). Beta amyloid (Abeta), a major component of senile plaque in AD, is known to be derived from ischemic brain or activated platelets. We prospectively enrolled 62 patients with acute ischemic stroke and 27 age-matched controls. The serum Abeta and P-selectin levels were determined using the Sandwich-ELISA. We divided ischemic strokes into subgroups according to the clinical syndrome, pathogenesis, and infarct size, and compared the Abeta level between each subgroup. The Abeta1-40 level was markedly elevated in ischemic stroke patients, as compared to controls (140.2 +/- 54.0 vs 88.44 +/- 34.96 pg/ml, p<0.001). Cardioembolic and larger artery atherosclerotic infarcts had higher Abeta1-40 level than small vessel disease (p = 0.001). Both infarct size and the initial NIHSS score had significantly positive correlations with the serum level of Abeta1-40 (r = 0.539, p<0.001 and r = 0.425, p = 0.001, respectively). However, the P-selectin level was not significantly correlated with serum Abeta1-40. Our data suggest that elevated circulating Abeta1-40 in ischemic stroke patients may be derived from brain as a consequence of ischemic insults.

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.

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.

Interferon gamma stimulates beta-secretase expression and sAPPbeta production in astrocytes.

Neurons, but not astrocytes, are known as the major source of Abeta, because astrocytes express low levels of putative beta-secretase (BACE). Astrocytes near senile plaque cores show enhanced levels of BACE protein expression, however, suggesting that astrocytes can contribute to Abeta production under pathological conditions. To investigate factors that stimulate BACE protein expression in astrocytes, we tested the effects of interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) on BACE protein expression in U373MG astrocytoma cells and primary astrocyte cultures from Tg2576 mouse brains. BACE protein expression and sAPPbeta production were dramatically increased, without changes in holo APP levels, following IFN-gamma treatment in both cell types. AG490, which is a blocker of IFN-gamma-induced STAT signaling, decreased IFN-gamma-induced BACE protein expression and sAPPbeta production in a dose-dependent manner. These results show that astrocytes are capable of expressing BACE and producing sAPPbeta in response to certain stimulating factors, and IFN-gamma is one such factor.

Cholinergic modulation of synaptic physiology in deep layer entorhinal cortex of the rat.

We have recently shown that cholinergic effects on synaptic transmission and plasticity in the superficial (II/III) layers of the rat medial entorhinal cortex (EC) are similar, but not identical, to those in the hippocampus (Yun et al. [2000] Neuroscience 97:671-676). Because the superficial and deep layers of the EC preferentially convey afferent and efferent hippocampal projections, respectively, it is of interest to compare cholinergic effects between the two regions. We therefore investigated the physiological effects of cholinergic agents in the layer V of medial EC slices under experimental conditions identical to those in the previous study. Bath application of carbachol (0.5 microM) induced transient depression of field potential responses in all cases tested (30 of 30; 18.5% +/- 2.3%) and rarely induced long-lasting potentiation (only 3 of 30; 20.4% +/- 3.2% in successful cases). At 5 microM, carbachol induced transient depression only (20 of 20, 48.9% +/- 2.8%), which was blocked by atropine (10 microM). Paired-pulse facilitation was enhanced during carbachol-induced depression, suggesting presynaptic action of carbachol. Long-term potentiation (LTP) could be induced in the presence of 10 microM atropine by theta burst stimulation, but its magnitude was significantly lower (9.1% +/- 4.7%, n = 15) compared to LTP in control slices (22.4% +/- 3.9%, n = 20). These results, combined with our previous findings, demonstrate remarkably similar cholinergic modulation of synaptic transmission and plasticity across the superficial and deep layers of EC.

Zinc enhances synthesis of presenilin 1 in mouse primary cortical culture.

Whether zinc interacts with presenilin 1 (PS1), one of the causative genes of familial Alzheimer's disease (AD), is not known. Here we report that zinc modulates the synthesis of PS1. Exogenous zinc enhanced the amount of C-terminal fragments of PS1 (PS1-CTF) in neonatal mouse cortical cultures in a dose-dependent manner. Zinc also induced cell death in a dose-dependent manner. These effects of zinc were not mimicked by calcium, copper, or iron, and were blocked by a zinc-specific chelator, TPEN. Experiments using metabolic labeling and cycloheximide treatment revealed that zinc increased PS1-CTF by elevating the de novo synthesis of PS1. Time course experiments revealed that cell death commenced sooner (0.5-1 h) than enhancement of PS1-CTF (1-2 h) following zinc treatment. However, the amount of PS1-CTF remained unchanged during etoposide- or H(2)O(2)-induced cell death, suggesting that enhancement of PS1 synthesis is specifically correlated with zinc-induced cell death.

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.