Dopamine D1 receptor signaling system regulates ryanodine receptor expression after intermittent exposure to methamphetamine in primary cultures of midbrain and cerebral cortical neurons

Regulatory mechanisms of ryanodine receptor (RyR) expression are not well known, although methamphetamine (METH) has been reported to up-regulate RyRs in mouse brain. This study investigate regulatory mechanisms of RyR expression by dopaminergic system using the midbrain and cerebral cortical neurons in primary culture intermittently exposed to METH and dopamine receptor (DR) agonists (1 h/day, for 3 days). Intermittent METH (10 μM) exposure enhanced RyR-1 and -2 proteins and their mRNA, but not RyR-3 expression in the both types of the neurons. These METH-induced increases of RyR proteins and their mRNA were dose-dependently blocked by SCH23390 (a selective D(1) DR antagonist), but not a D(2)DR antagonist sulpiride, suggesting a regulatory role of D(1)DRs in RyR expression by METH in these neurons. In cerebral cortical neurons, intermittent SKF82958 (a selective D(1)DR agonist) exposure increased RyR-1 and -2 proteins and their mRNA, whereas quinpirole (a selective D(2)DR agonist) showed no effects. KT5720, a protein kinase A inhibitor, dose-dependently attenuated the METH-stimulated RyR-1 and -2 expressions in cerebral cortical neurons. METH significantly increased phosphorylation of cAMP-response element-binding protein, which was completely suppressed by SCH23390. These results indicate that RyR-1 and -2 expressions are regulated by D(1)DRs via the signal transduction linked to D(1)DRs.     

Tg2576 cortical neurons that express human Ab are susceptible to extracellular Aβ-induced, K+ efflux dependent neurodegeneration

Background

One of the key pathological features of AD is the formation of insoluble amyloid plaques. The major constituent of these extracellular plaques is the beta-amyloid peptide (Aβ), although Aβ is also found to accumulate intraneuronally in AD. Due to the slowly progressive nature of the disease, it is likely that neurons are exposed to sublethal concentrations of both intracellular and extracellular Aβ for extended periods of time.

Results

In this study, we report that daily exposure to a sublethal concentration of Aβ_1-40 (1 µM) for six days induces substantial apoptosis of cortical neurons cultured from Tg2576 mice (which express substantial but sublethal levels of intracellular Aβ). Notably, untreated Tg2576 neurons of similar age did not display any signs of apoptosis, indicating that the level of intracellular Aβ present in these neurons was not the cause of toxicity. Furthermore, wildtype neurons did not become apoptotic under the same chronic Aβ_1-40 treatment. We found that this apoptosis was linked to Tg2576 neurons being unable to maintain K⁺ homeostasis following Aβ treatment. Furthermore, blocking K⁺ efflux protected Tg2576 neurons from Aβ-induced neurotoxicity. Interestingly, chronic exposure to 1 µM Aβ_1-40 caused the generation of axonal swellings in Tg2576 neurons that contained dense concentrations of hyperphosphorylated tau. These were not observed in wildtype neurons under the same treatment conditions.

Conclusions

Our data suggest that when neurons are chronically exposed to sublethal levels of both intra- and extra-cellular Aβ, this causes a K⁺-dependent neurodegeneration that has pathological characteristics similar to AD.     

Increased intraneuronal resting [Ca2+] in adult Alzheimer's disease mice

Neurodegeneration in Alzheimer's disease (AD) has been linked to intracellular accumulation of misfolded proteins and dysregulation of intracellular Ca2+. In the current work, we determined the contribution of specific Ca2+ pathways to an alteration in Ca2+ homeostasis in primary cortical neurons from an adult triple transgenic (3xTg-AD) mouse model of AD that exhibits intraneuronal accumulation of beta-amyloid proteins. Resting free Ca2+ concentration ([Ca2+](i)), as measured with Ca2+-selective microelectrodes, was greatly elevated in neurons from 3xTg-AD and APP(SWE) mouse strains when compared with their respective non-transgenic neurons, while there was no alteration in the resting membrane potential. In the absence of the extracellular Ca2+, the [Ca2+](i) returned to near normal levels in 3xTg-AD neurons, demonstrating that extracellular Ca2+contributed to elevated [Ca2+](i). Application of nifedipine, or a non-L-type channel blocker, SKF-96365, partially reduced [Ca2+](i). Blocking the ryanodine receptors, with ryanodine or FLA-365 had no effect, suggesting that these channels do not contribute to the elevated [Ca2+](i). Conversely, inhibition of inositol trisphosphate receptors with xestospongin C produced a partial reduction in [Ca2+](i). These results demonstrate that an elevation in resting [Ca2+](i), contributed by aberrant Ca2+entry and release pathways, should be considered a major component of the abnormal Ca2+ homeostasis associated with AD.     

Internalization of beta-amyloid peptide by primary neurons in the absence of apolipoprotein E

Extracellular accumulation of beta-amyloid peptide (Abeta) has been linked to the development of Alzheimer disease. The importance of intraneuronal Abeta has been recognized more recently. Although considerable evidence indicates that extracellular Abeta contributes to the intracellular pool of Abeta, the mechanisms involved in Abeta uptake by neurons are poorly understood. We examined the molecular mechanisms involved in Abeta-(1-42) internalization by primary neurons in the absence of apolipoprotein E. We demonstrated that Abeta-(1-42) is more efficiently internalized by axons than by cell bodies of sympathetic neurons, suggesting that Abeta-(1-42) uptake might be mediated by proteins enriched in the axons. Although the acetylcholine receptor alpha7nAChR, previously suggested to be involved in Abeta internalization, is enriched in axons, our results indicate that it does not mediate Abeta-(1-42) internalization. Moreover, receptors of the low density lipoprotein receptor family are not essential for Abeta-(1-42) uptake in the absence of apolipoprotein E because receptor-associated protein had no effect on Abeta uptake. By expressing the inactive dynamin mutant dynK44A and the clathrin hub we found that Abeta-(1-42) internalization is independent of clathrin but dependent on dynamin, which suggests an endocytic pathway involving caveolae/lipid rafts. Confocal microscopy studies showing that Abeta did not co-localize with the early endosome marker EEA1 further support a clathrin-independent mechanism. The lack of co-localization of Abeta with caveolin in intracellular vesicles and the normal uptake of Abeta by neurons that do not express caveolin indicate that Abeta does not require caveolin either. Instead partial co-localization of Abeta-(1-42) with cholera toxin subunit B and sensitivity to reduction of cellular cholesterol and sphingolipid levels suggest a caveolae-independent, raft-mediated mechanism. Understanding the molecular events involved in neuronal Abeta internalization might identify potential therapeutic targets for Alzheimer disease.     

Calpain cleavage and inactivation of the sodium calcium exchanger‐3 occur downstream of Aβ in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β‐amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase‐3, activity is a prominent feature of AD brain. In addition, we observe increased calpain‐mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain‐dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.     

Age-Dependent Neuroplasticity Mechanisms in Alzheimer Tg2576 Mice Following Modulation of Brain Amyloid-β Levels

The objective of this study was to investigate the effects of modulating brain amyloid-β (Aβ) levels at different stages of amyloid pathology on synaptic function, inflammatory cell changes and hippocampal neurogenesis, i.e. processes perturbed in Alzheimer’s disease (AD). Young (4- to 6-month-old) and older (15- to 18-month-old) APP_SWE transgenic (Tg2576) mice were treated with the AD candidate drug (+)-phenserine for 16 consecutive days. We found significant reductions in insoluble Aβ1-42 levels in the cortices of both young and older transgenic mice, while significant reductions in soluble Aβ1-42 levels and insoluble Aβ1-40 levels were only found in animals aged 15–18 months. Autoradiography binding with the amyloid ligand Pittsburgh Compound B (³H-PIB) revealed a trend for reduced fibrillar Aβ deposition in the brains of older phenserine-treated Tg2576 mice. Phenserine treatment increased cortical synaptophysin levels in younger mice, while decreased interleukin-1β and increased monocyte chemoattractant protein-1 and tumor necrosis factor-alpha levels were detected in the cortices of older mice. The reduction in Aβ1-42 levels was associated with an increased number of bromodeoxyuridine-positive proliferating cells in the hippocampi of both young and older Tg2576 mice. To determine whether the increased cell proliferation was accompanied by increased neuronal production, the endogenous early neuronal marker doublecortin (DCX) was examined in the dentate gyrus (DG) using immunohistochemical detection. Although no changes in the total number of DCX⁺-expressing neurons were detected in the DG in Tg2576 mice at either age following (+)-phenserine treatment, dendritic arborization was increased in differentiating neurons in young Tg2576 mice. Collectively, these findings indicate that reducing Aβ1-42 levels in Tg2576 mice at an early pathological stage affects synaptic function by modulating the maturation and plasticity of newborn neurons in the brain. In contrast, lowering Aβ levels in Tg2576 mice when Aβ plaque pathology is prominent mainly alters the levels of proinflammatory cytokines and chemokines.     

Enhanced caffeine-induced Ca2+ release in the 3xTg-AD mouse model of Alzheimer's disease

Alzheimer's disease (AD) is the most prevalent form of dementia among the elderly and is a complex disorder that involves altered proteolysis, oxidative stress and disruption of ion homeostasis. Animal models have proven useful in studying the impact of mutant AD-related genes on other cellular signaling pathways, such as Ca2+ signaling. Along these lines, disturbances of intracellular Ca2+ ([Ca2+]i) homeostasis are an early event in the pathogenesis of AD. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate disturbances in Ca2+ homeostasis in primary cortical neurons from a triple transgenic mouse model of Alzheimer's disease (3xTg-AD). Application of caffeine to mutant presenilin-1 knock-in neurons (PS1KI) and 3xTg-AD neurons evoked a peak rise of [Ca2+]i that was significantly greater than those observed in non-transgenic neurons, although all groups had similar decay rates of their Ca2+ transient. This finding suggests that Ca2+ stores are greater in both PS1KI and 3xTg-AD neurons as calculated by the integral of the caffeine-induced Ca2+ transient signal. Western blot analysis failed to identify changes in the levels of several Ca2+ binding proteins (SERCA-2B, calbindin, calsenilin and calreticulin) implicated in the pathogenesis of AD. However, ryanodine receptor expression in both PS1KI and 3xTg-AD cortex was significantly increased. Our results suggest that the enhanced Ca2+ response to caffeine observed in both PS1KI and 3xTg-AD neurons may not be attributable to an alteration of endoplasmic reticulum store size, but to the increased steady-state levels of the ryanodine receptor.     

Profile for Amyloid-β and Tau Expression in Primary Cortical Cultures from 3xTg-AD Mice

Advances in transgenic technology as well as in the genetics of Alzheimer disease (AD) have allowed the establishment of animal models that reproduce amyloid-beta plaques and neurofibrillary tangles, the main pathological hallmarks of AD. Among these models, 3xTg-AD mice harboring PS1 _M146V, APP _Swe and tau _P301L human transgenes provided the model that most closely mimics human AD features. Although cortical cultures from 3xTg-AD mice have been shown to present disturbances in intracellular [Ca²⁺] homeostasis, the development of AD pathology in vitro has not been previously evaluated. In the current work, we determined the temporal profile for amyloid precursor protein, amyloid-β and tau expression in primary cortical cultures from 3xTg-AD mice. Immunocytochemistry and Western blot analysis showed an increased expression of these proteins as well as several phosphorylated tau isoforms with time in culture. Alterations in calcium homeostasis and cholinergic and glutamatergic responses were also observed early in vitro. Thus, 3x-TgAD cortical neurons in vitro provide an exceptional tool to investigate pharmacological approaches as well as the cellular basis for AD and related diseases.     

Attenuated hippocampus-dependent learning and memory decline in transgenic TgAPPswe Fischer-344 rats

Alzheimer's disease (AD) is characterized by increased beta amyloid (Abeta) levels, extracellular Abeta deposits in senile plaques, neurofibrillary tangles, and neuronal loss. However, the physiological role of normal levels of Abeta and its parent protein, the amyloid precursor protein (APP) are unknown. Here we report that low-level transgenic (Tg) expression of the Swedish APP mutant gene (APPswe) in Fischer-344 rats results in attenuated age-dependent cognitive performance decline in 2 hippocampus-dependent learning and memory tasks compared with age-matched nontransgenic Fischer-344 controls. TgAPPswe rats exhibit mild increases in brain APP mRNA (56.8%), Abeta-42 (21%), and Abeta-40 (6.1%) peptide levels at 12 mo of age, with no extracellular Abeta deposits or senile plaques at 6, 12, and 18 mo of age, whereas 3- to 6-fold increases in Abeta levels are detected in plaque-positive human AD patients and transgenic mouse models. The data support the hypothesis that a threshold paradigm underlies Abeta-related pathology, below which APP expression may play a physiological role in specific hippocampus-dependent tasks, most likely related to its neurotrophic role.     

Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons

Beta-amyloid ((A)beta) is a peptide of 39-42 amino acids that is the primary component of plaques in Alzheimer's disease (AD). The mechanism by which (A)beta expresses its neurotoxic effects may involve induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels. Cultured cortical cells were utilized to study the alterations in calcium homeostasis underlying the neurotoxic effect of (A)beta. Serum supplement B27 and vitamin E were effective in preventing neuronal death as assessed by lactate dehydrogenase (LDH) release, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and number of apoptotic nuclei. In addition, (A)beta-induced cytosolic free calcium ([Ca2+]i) was blocked by antioxidants vitamin E and U83836E, but not by N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801, or by voltage-gated calcium channel blocker nimodipine. Taken together, the results suggest that NMDA receptor and voltage-gated calcium channels are not involved in (A)beta-induced [Ca2+]i increase. This increase appeared to be the result of extracellular calcium influx by some unknown mechanisms. In addition, antioxidants such as B27 were effective in protecting cultured cortical neurons against (A)beta, and correlated with (A)beta attenuation of early calcium response.     

Neuronal and glial calcium signaling in Alzheimer's disease

Cognitive impairment and emotional disturbances in Alzheimer's disease (AD) result from the degeneration of synapses and death of neurons in the limbic system and associated regions of the cerebral cortex. An alteration in the proteolytic processing of the amyloid precursor protein (APP) results in increased production and accumulation of amyloid beta-peptide (Abeta) in the brain. Abeta has been shown to cause synaptic dysfunction and can render neurons vulnerable to excitotoxicity and apoptosis by a mechanism involving disruption of cellular calcium homeostasis. By inducing membrane lipid peroxidation and generation of the aldehyde 4-hydroxynonenal, Abeta impairs the function of membrane ion-motive ATPases and glucose and glutamate transporters, and can enhance calcium influx through voltage-dependent and ligand-gated calcium channels. Reduced levels of a secreted form of APP which normally regulates synaptic plasticity and cell survival may also promote disruption of synaptic calcium homeostasis in AD. Some cases of inherited AD are caused by mutations in presenilins 1 and 2 which perturb endoplasmic reticulum (ER) calcium homeostasis such that greater amounts of calcium are released upon stimulation, possibly as the result of alterations in IP(3) and ryanodine receptor channels, Ca(2+)-ATPases and the ER stress protein Herp. Abnormalities in calcium regulation in astrocytes, oligodendrocytes, and microglia have also been documented in studies of experimental models of AD, suggesting contributions of these alterations to neuronal dysfunction and cell death in AD. Collectively, the available data show that perturbed cellular calcium homeostasis plays a prominent role in the pathogenesis of AD, suggesting potential benefits of preventative and therapeutic strategies that stabilize cellular calcium homeostasis.     

Cholesterol distribution in the Golgi complex of DITNC1 astrocytes is differentially altered by fresh and aged amyloid beta-peptide-(1-42)

The Golgi complex plays an important role in cholesterol trafficking in cells, and amyloid beta-peptides (Abetas) alter cholesterol trafficking. The hypothesis was tested that fresh and aged Abeta-(1-42) would differentially modify Golgi cholesterol content in DINTC1 astrocytes and that the effects of Abeta-(1-42) would be associated with the region of the Golgi complex. Two different methods were used to determine the effects of Abeta-(1-42) on Golgi complex cholesterol. Confocal microscopy showed that fresh Abeta-(1-42) significantly increased cholesterol and that aged Abeta-(1-42) significantly reduced cholesterol content in the Golgi complex. Isolation of the Golgi complex into two fractions using density gradient centrifugation showed effects of aged Abeta-(1-42) similar to those observed with confocal microscopy but revealed the novel finding that fresh Abeta-(1-42) had opposite effects on the two Golgi fractions suggesting a specificity of Abeta-(1-42) perturbation of the Golgi complex. Phosphatidylcholine-phospholipase D activity, cell membrane cholesterol, and apolipoprotein E levels were associated with effects of fresh Abeta-(1-42) on cholesterol distribution but not with effects of aged Abeta-(1-42), arguing against a common mechanism. Extracellular Abeta-(1-42) targets the Golgi complex and disrupts cell cholesterol homeostasis, and this action of Abeta-(1-42) could alter cell functions requiring optimal levels of cholesterol.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

Embryonic lethality and abnormal cardiac myocytes in mice lacking ryanodine receptor type 2.

The ryanodine receptor type 2 (RyR-2) functions as a Ca2+-induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR-2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage-gated Ca2+ channel to constitute the cardiac type of excitation-contraction (E-C) coupling. Here we report on mutant mice lacking RyR-2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5-11.5. These results suggest no essential contribution of the RyR-2 to E-C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR-2 is not in E-C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.     

Comparison of Ca(2+) sparks produced independently by two ryanodine receptor isoforms (type 1 or type 3)

The molecular determinants of a Ca(2+) spark, those events that determine the sudden opening and closing of a small number of ryanodine receptor (RyR) channels limiting Ca(2+) release to a few milliseconds, are unknown. As a first step we investigated which of two RyR isoforms present in mammalian embryonic skeletal muscle, RyR type 1(RyR-1) or RyR type 3 (RyR-3) has the ability to generate Ca(2+) sparks. Their separate contributions were investigated in intercostal muscle cells of RyR-1 null and RyR-3 null mouse embryos. A comparison of Ca(2+) spark parameters of RyR-1 null versus RyR-3 null cells measured at rest with fluo-3 showed that neither the peak fluorescence intensity (DeltaF/F(o) = 1.25 +/- 0.7 vs. 1.55 +/- 0.6), spatial width at half-max intensity (FWHM = 2.7 +/- 1.2 vs. 2.6 +/- 0.6 microm), nor the duration at half-max intensity (FTHM = 45 +/- 49 vs. 43 +/- 25 ms) was significantly different. Sensitivity to caffeine (0.1 mM) was remarkably different, with sparks in RyR-1 null myotubes becoming brighter and longer in duration, whereas those in RyR-3 null cells remained unchanged. Controls performed in double RyR-1/RyR-3 null cells obtained by mice breeding showed that sparks were not observed in the absence of both isoforms in >150 cells imaged. In conclusion, 1) RyR-1 and RyR-3 appear to be the only intracellular Ca(2+) channels that participate in Ca(2+) spark activity in embryonic skeletal muscle; 2) except in their responsiveness to caffeine, both isoforms have the ability to produce Ca(2+) sparks with nearly identical properties, so it is rather unlikely that a single RyR isoform, when others are also present, would be responsible for Ca(2+) sparks; and 3) because RyR-1 null cells are excitation-contraction (EC) uncoupled and RyR-3 null cells exhibit a normal phenotype, Ca(2+) sparks result from the inherent activity of small clusters of RyRs regardless of the participation of these RyRs in EC coupling.     

Carboxyl-terminal fragment of Alzheimer's APP destabilizes calcium homeostasis and renders neuronal cells vulnerable to excitotoxicity.

Numerous lines of evidence indicate that some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the amyloid precursor protein (APP). Most research has focused on the amyloid beta peptide (Abeta). However, the possible role of other cleaved products of APP is less clear. We have previously shown that a recombinant carboxy-terminal 105 amino acid fragment (CT 105) of APP induced strong nonselective inward currents in XENOPUS: oocyte; it also revealed neurotoxicity in PC12 cells and primary cortical neurons, blocked later phase of long-term potentiation in rat hippocampus in vivo, and induced memory deficits and neuropathological changes in mice. We report here that the pretreatment with CT 105 for 24 h at a 10 microM concentration increases intracellular calcium concentration by about twofold in SK-N-SH and PC 12 cells, but not in U251 cells, originated from human glioblastoma. In addition, the calcium increase and toxicity induced by CT 105 were reduced by cholesterol and MK 801 in SK-N-SH and PC 12 cells, whereas the toxicity of Abeta(1-42) was attenuated by nifedipine and verapamil. CT 105 rendered SK-N-SH cells and rat primary cortical neurons more vulnerable to glutamate-induced excitotoxicity. Also, conformational studies using circular dichroism experiments showed that CT 105 has approximately 15% of beta-sheet content in phosphate buffer and aqueous 2,2, 2-trifluoroethanol solutions. However, the content of beta-sheet conformation in dodecyl phosphocholine micelle or in the negatively charged vesicles, is increased to 22%-23%. The results of this study showed that CT 105 disrupts calcium homeostasis and renders neuronal cells more vulnerable to glutamate-induced excitotoxicity, and that some portion of CT 105 has partial beta-sheet conformation in various environments, which may be related to the self-aggregation and toxicity. This may be significantly possibly involved in inducing the neurotoxicity characteristic of AD.     

Early Changes in Behavior Deficits, Amyloid β-42 Deposits and MAPK Activation in Doubly Transgenic Mice Co-expressing NSE-Controlled Human Mutant PS2 and APPsw

1. Doubly transgenic mice were some differences in the period proceeding of the development of Abeta-42 deposits and behavioral deficits. It was not characterized human mutant PS2 (hPS2) with APPsw in the brains of double transgenic mice. The aim of this study was to examine whether doubly transgenic mice co-expressing NSE-controlled APPsw and hPS2m develop AD-like phenotypes much earlier than singly APPsw or hPS2m alone. 2. We produced doubly transgenic mice from a cross between our previously created NSE-controlled hPS2m and an APPsw transgenic line. This doubly transgenic line was quantitatively produced by cross with age-matched control mice, and the produced mice were separated into 5, 6, 7 and 8-month old age groups. At the age of 8 months, the four groups of mice were tested for behavioral function, levels of Abeta-42 deposition, and potential signaling events. 3. It was shown that all the AD-like phenotypes, including behavior deficits, Abeta-42 levels, MAPK activation and ER expressions in doubly transgenic mice develop much earlier in the early time of AD development than their singly transgenic and non-transgenic littermates. 4. The results suggest that elevated Abeta-42 levels, and MAPK activation in doubly transgenic mice are model for early diagnosis and treatment of AD with therapeutic drug.