Brains Of AD Patients Research Articles

Genetic suppression of β2-adrenergic receptors ameliorates tau pathology in a mouse model of tauopathies

Accumulation of the microtubule-binding protein tau is a key event in several neurodegenerative disorders referred to as tauopathies, which include Alzheimer's disease, frontotemporal lobar degeneration, Pick's disease, progressive supranuclear palsy and corticobasal degeneration. Thus, understanding the molecular pathways leading to tau accumulation will have a major impact across multiple neurodegenerative disorders. To elucidate the pathways involved in tau pathology, we removed the gene encoding the beta-2 adrenergic receptors (β2ARs) from a mouse model overexpressing mutant human tau. Notably, the number of β2ARs is increased in brains of AD patients and epidemiological studies show that the use of beta-blockers decreases the incidence of AD. The mechanisms underlying these observations, however, are not clear. We show that the tau transgenic mice lacking the β2AR gene had a reduced mortality rate compared with the parental tau transgenic mice. Removing the gene encoding the β2ARs from the tau transgenic mice also significantly improved motor deficits. Neuropathologically, the improvement in lifespan and motor function was associated with a reduction in brain tau immunoreactivity and phosphorylation. Mechanistically, we provide compelling evidence that the β2AR-mediated changes in tau were linked to a reduction in the activity of GSK3β and CDK5, two of the major tau kinases. These studies provide a mechanistic link between β2ARs and tau and suggest the molecular basis linking the use of beta-blockers to a reduced incidence of AD. Furthermore, these data suggest that a detailed pharmacological modulation of β2ARs could be exploited to develop better therapeutic strategies for AD and other tauopathies.

Permeability transition pore-mediated mitochondrial superoxide flashes mediate an early inhibitory effect of amyloid beta1−42 on neural progenitor cell proliferation

Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study, we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6–24 hours of exposure to aggregating amyloid β-peptide (Aβ1–42), indicating an increase in transient mPTP opening. Subsequently, the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1–42, during which time global cellular reactive oxygen species increased, mitochondrial membrane potential decreased, cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1–42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1–42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism, then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ.

Nature of the neurotoxic membrane actions of amyloid-β on hippocampal neurons in Alzheimer's disease

The mechanism by which amyloid-β (Aβ) produces brain dysfunction in patients with Alzheimer's disease is largely unknown. According to previous studies, Aβ might share perforating properties with gramicidin, a well-accepted membrane-disrupting peptide. Therefore, we hypothesize that the key steps leading to synaptotoxicity by Aβ and gramicidin involve peptide aggregation, pore formation, and calcium dysregulation. Here, we show that Aβ and gramicidin form aggregates enriched in β-sheet structures using electron microscopy, and Thioflavin and Congo Red staining techniques. Also, we found that Aβ and gramicidin display fairly similar actions in hippocampal cell membranes, i.e. inducing Ca2+ entry and synaptoxicity characterized by the loss of synaptic proteins and a decrease in neuronal viability. These effects were not observed in a Ca2+ free solution, indicating that both Aβ and gramicidin induce neurotoxicity by a Ca2+-dependent mechanism. Using combined perforated patch clamp and imaging recordings, we found that only Aβ produced a perforation that progressed from a small (Cl−-selective pore) to a larger perforation that allowed the entry of fluorescent molecules. Therefore, based on these results, we propose that the perforation at the plasma membrane by Aβ is a dynamic process that is critical in producing neurotoxicity similar to that found in the brains of AD patients.

β-arrestin 2 regulates Aβ generation and γ-secretase activity in Alzheimer’s disease

Deficits in several neurotransmitter systems are characteristic features of the brains of AD patients. The majority of neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs) or 7-transmembrane receptors (7TMRs). It has recently been appreciated that a small family of multifunctional GPCR regulatory known as the β-arrestins which play an almost universal role in facilitating traditional GPCR desensitization, are also capable of initiating distinct signals in their own right, conveying receptor subtype-specific signaling events. These signals are often both spatially and temporally distinct, and result in unique cellular and physiological or pathophysiological consequences. As mediators of GPCR desensitization, trafficking and cell signaling, the β-arrestins provide a putative basis to understand GPCR dysfunction in AD. Here, we report that β-arrestin 2 levels are elevated in two independent cohorts of patients with AD. Genetic deletion of Arrb2 (β-arrestin 2) reduces accumulation of the amyloid-β (Aβ) peptide in an AD mouse model. Consistent with these observations, endogenous murine Aβ generation is also reduced in Arrb2-/- mice. Elucidation of the mechanism of the β-arrestin 2-mediated effect on Aβ levels indicates that recruitment of β-arrestin 2 to two GPCRs implicated in the pathogenesis of AD, GPR3 and the β2-adrenergic receptor (β2-AR), is required for the promotion of Aβ release. Collectively, these studies identify β-arrestin 2 as a novel avenue for targeting amyloid pathology and GPCR dysfunction in AD.

Structural analysis of the pyroglutamate‐modified isoform of the Alzheimer's disease‐related amyloid‐β using NMR spectroscopy

The aggregation of the Aβ plays a fundamental role in the pathology of AD. Recently, N-terminally modified Aβ species, pE-Aβ, have been described as major constituents of Aβ deposits in the brains of AD patients. pE-Aβ has an increased aggregation propensity and shows increased toxicity compared with Aβ1-40 and Aβ1-42. In the present work, high-resolution NMR spectroscopy was performed to study pE-Aβ3-40 in aqueous TFE-containing solution. Two-dimensional TOCSY and NOESY experiments were performed. On the basis of NOE and chemical shift data, pE-Aβ3-40 was shown to contain two helical regions formed by residues 14-22 and 30-36. This is similar as previously described for Aβ1-40. However, the secondary chemical shift data indicate decreased helical propensity in pE-Aβ3-40 when compared with Aβ1-40 under exactly the same conditions. This is in agreement with the observation that pE-Aβ3-40 shows a drastically increased tendency to form β-sheet-rich structures under more physiologic conditions. Structural studies of pE-Aβ are crucial for better understanding the structural basis of amyloid fibril formation in the brain during development of AD, especially because an increasing number of reports indicate a decisive role of pE-Aβ for the pathogenesis of AD.

The physiological effect of ingested β-N-methylamino-L-alanine on a glutamatergic synapse in an in vivo preparation

The neurotoxin, BMAA (β-N-methylamino-L-alanine), may be a risk factor for amyotrophic lateral sclerosis (ALS), Parkinson's (PD) and Alzheimer's (AD) disease. In vivo experiments have demonstrated that BMAA can cause a number of motor dysfunctions if ingested or injected, and in vitro experiments show that this toxin binds to glutamate receptors with deleterious results. Also, BMAA exists in the human food chain worldwide, and has been detected in the brains of ALS and AD patients. This paper offers the first demonstration by intracellular recording of the effect of ingested BMAA on the postsynaptic response of an identified glutamatergic cell in a living, undissected organism (Drosophila melanogaster), and correlates these observations with the specific motor dysfunctions that result from ingestion. The results suggest that BMAA acts as a glutamate agonist, causing NMDA receptor channels to remain open for prolonged periods of time, thereby damaging the cell by excitotoxicity. The effect on the postsynaptic response became apparent days before the function of the postsynaptic cell (wing beat) became affected. Severely depolarized cells were able to fully recover with the removal of BMAA from the food source, suggesting that blocking BMAA binding in the brain might be a good treatment strategy.

P1‐301: Characterization of APPsi transgenic rats for cognitive deficits and brain pathology

Alzheimer's disease is one of the most devastating neurodegenerative diseases of the 21st century. Pathological aggregation of the amyloid precursor protein (APP) in the brain of AD patients is thought to be one of the main causes for the observed progressive cognitive decline in affected people. So far, mostly transgenic mouse models are used to mimic the pathology of AD. Some o these models reflect the pathology of AD strikingly well but harbor the disadvantages of mice models in general. We therefore characterized the transgenic rat model by Claudio Cuello and his team (Leon et al. 2010) that expresses the human APP751 cDNA with Swedish (K679N/N671L) and Indiana (V717F) mutations under the regulatory control of the rat Thy-1 promoter. High-APP expressing animals of both sexes were tested over age for behavioral deficits like general health, activity and memory in the open field test, forced swim test, elevated plus maze, Morris water maze, new object recognition and passive avoidance test. Brain, CSF and plasma amyloid β38, 40 and 42 levels were analyzed using the immunosorbent assay MSD-ISA. Brain pathology was assessed immunohistologically for amyloid and amyloid depositions and for brain inflammation i.e. astrogliosis and activated microglia. Our results show that these rats have altered activity levels and memory deficits as measured by different cognitive tests already in early age. Furthermore, amyloid β38, 40 and 42 levels in the cerebrospinal fluid and in the brain of rats increase over age and this can be supported by brain amyloid immunohistochemical results. Accordingly, brain inflammation, which is altered already in the youngest age group, is elevated in all age groups. This transgenic rat model thus not only provides AD specific behavioral deficits but also corresponding brain pathology that starts earl y and increases over age. Since rats are more suitable to perform most behavioral tests compared to mice the APPsi transgenic rat model presents an appropriate alternative to well established transgenic APP mouse models for AD research as well as novel compound tests against AD.

P4‐299: Gamma‐secretase/notch signaling in insulin resistance

Type 2 diabetes (T2D) is a risk factor for Alzheimer's disease, most likely linked to an impairment of insulin signaling in the brain. Insulin receptors (IR) are desensitized in the brains of AD patients. The impairment of insulin signaling in the brain could play a role in the development of neurodegenerative disorders. Insulin regulates glucose intake from the blood to the liver, muscle, and brain through IR-mediated signal transduction. When insulin signaling is disturbed by insulin resistance, the secretion of insulin into the blood is increased in order to transmit insulin signals to nuclei, resulting in hyperinsulinemia. Soluble Aβ oligomer interferes with insulin-induced autophosphorylation through its binding to IR. The expression of insulin-degrading enzyme is stimulated by insulin signaling in order to degrade Aβ. Insulin also regulates Aβ levels by modulating Aβ production via their actions on BACE1 and/or γ-secretase, as well as by modulating Aβ degradation via Aβ-degrading enzymes, such as insulin-degrading enzyme. In this study we will determine whether gamma-secretase/notch signaling regulates insulin resistance and/or secretion. Glucose and insulin tolerance tests Insulin tolerance tests Induce insulin resistance Insulin-sensitizing effects of γ-secretase inhibitors(GSI) Metabolic characterization of Notch antisense mice and wild type mice NAS and WT mice show similar effects in glucose tolerance test and the insulin tolerance test Four month old NAS and WT mice show different effects in glucose tolerance test and the insulin tolerance test Seven month old NAS and WT mice show different effects in glucose tolerance test and the insulin tolerance test than four month Fasting effects on plasma level of diabetes markers This study shows that in a db/db mice and high fat diet model of insulin resistance, Notch expression was increased in liver and muscle of db/db mice. And also NAS mice increased glucose tolerance and insulin sensitivity.

P4‐062: Neprilysin/ABC‐transporter‐deficient mice are a new physiologic model of Alzheimer's disease

In Alzheimer's disease the accumulation of the APP cleavage product Aβ is thought to be one initial trigger of the pathogenesis leading to a progressive loss of memory. Several studies led to the recognition that the accumulation of soluble A β species, and not fibrils or plaques, is the most devastating factor in AD. However, the trigger for the enhanced accumulation of Aβ in the brain of AD patients has only recently become obvious. Majuwenge et al. (2011) could show that it is the clearance of A β peptides from the brain which is impaired in AD patients. We have extensively studied the contribution of these ABC transporters in APP/PS1 and APPdt transgenic mouse models and demonstrated that, besides ABCB1, ABCC1 is a before unrecognized A β -transporting protein with extraordinary impact on Aβ brain content. However, transgenic AD models are (i) early depositing, (ii) models for overproduction of A β and (iii) have up to 10times higher amounts of insoluble over soluble Aβ. Here we use neprilysin knockout mice (NEP-/-) backcrossed to ABCB1-/- or ABCC1-/- mice, respectively. NEP-/- mice are not a model of β-amyloidosis per se, but a model of slightly elevated, physiological murine Aβ (mAβ) levels prior to deposition. We analyzed spine morphology, long term potentiation, immunohistochemistry and used behavioral tests to characterize these mouse models. We found that NEP-/- mice deficient for an ABC transporter showed a lower probability of survival, reduced LTPs and altered spine morphology that were due to significant mAβ accumulations within the brain. Additionally ABC-transporter deficient NEP-/- mice displayed disturbances of the sleep wake cycle and severe reactive gliosis was detectable especially in NEP-/-/ABCC1-/- mice. The work presented here introduces a mouse model that displays functional deficits common to (early) Alzheimer's disease without overexpression of a transgene or plaque deposition, thereby strengthening the view that soluble Aβ species are a main scourge in the progression of AD. Moreover, the results support evidences from former studies that ABC transporters, especially ABCC1, are of considerable importance for Aβ proteostasis of the brain.

Metabolic and immune risk factors for dementia and their modification by flavonoids: New targets for the prevention of cognitive impairment?

A number of contributory factors have been implicated in the pathogenesis of Alzheimer's disease. One of these factors is chronic inflammation, with the over expression of pro-inflammatory cytokines and acute phase reactants consistently observed in the post mortem brain and plasma of AD patients. Furthermore, cardiovascular risk factors, such as hypertension, impaired vascular function and elevated LDL cholesterol, also appear to be predictive of increased dementia risk. Although classically associated with cardiovascular disease risk, both vascular and immune mediators may have direct deleterious effects on the brain, which contribute to the development of vascular dementia and Alzheimer's disease, as well as impairments in memory and neuro-cognitive function. Dietary agents previously noted for their ability to modulate these cardiovascular risk factors leading to reductions in chronic, low-grade inflammation and/or vascular dysfunction, may also possess an ability to moderate the progression of dementia. Flavonoid-rich foods such as tea, berries and cocoa have been reported to attenuate age-related deficits in memory and cognition, although the precise mechanisms of their action are unclear. As these flavonoid rich-foods/beverages also appear to mediate inflammatory processes, attenuate endothelial dysfunction and reduce hypertension, such actions may contribute to their efficacy in the brain. This review will explore these concepts with the view to further unravelling the actions of flavonoids and flavonoid-rich foods against brain disease and to highlight the importance measuring such factors in future clinical studies.

BACE1 inhibitory activity of fungal endophytic extracts from Malaysian medicinal plants.

BackgroundBACE1 was found to be the major β-secretase in neurons and its appearance and activity were found to be elevated in the brains of AD patients. Fungal endophytic extracts for BACE1 inhibitory activity and cytotoxicity against PC-12 (a rat pheochromocytoma with neuronal properties) and WRL68 (a non-tumorigenic human hepatic) were investigated.MethodsEndophytes were isolated from plants collected from Kuala Pilah, Negeri Sembilan and the National Park, Pahang and the extracts were tested for BACE1 inhibition. For investigation of biological activity, the pure endophytic cultures were cultivated for 14 days on PDA plates at 28°C and underwent semipolar extraction with ethyl acetate.ResultsOf 212 endophytic extracts (1000 μg/ml), 29 exhibited more than 90% inhibition of BACE1 in the preliminary screening. Four extracts from isolates HAB16R13, HAB16R14, HAB16R18 and HAB8R24 identified as Cytospora rhizophorae were the most active with IC50(BACE1) values of less than 3.0 μg/ml. The most active extract HAB16R13 was shown to non-competitively inhibit BACE1 with Ki value of 10.0 μg/ml. HAB16R13 was considered non-potent against PC-12 and WRL68 (IC50(CT) of 60.0 and 40.0 μg/ml, respectively).ConclusionsThis first report on endophytic fungal extract with good BACE1 inhibitory activity demonstrates that more extensive study is required to uncover the potential of endophytes.

P4‐445: Early and chronic treatment with dantrolene blocked later learning and memory deficits in older Alzheimer's triple transgenic mice

Background: The major neuropathological hallmarks in the brains of AD patients are senile plaques formed by beta amyloid (As) aggregates and neurofibrillary tangles, containing hyperphosphorylated tau, both causing progressive neuronal dysfunction. These pathological alterations are likely causally involved in eventual neuronal death, particularly in brain regions related to memory and cognition. The first attempt to develop an active vaccine to As, AN1793, failed in clinical trials due to a Th1 type T cell immune response, attributed to the adjuvant used, QS21 and polysorbate 80, provoking a meningoencephalitis-like effect and death in 6% of the patients. To overcome this failure, we designed a novel As1-42 active vaccine incorporated and delivered in a liposomal adjuvant that evoked aTh2 T cell reaction. Methods: As was incorporated and delivered in a liposomal matrix composed of phosphatidylcholine: phosphatidylglycerol: cholesterol:sphingosine-1-phosphate (EB101) and administered intraperitoneally for seven months to double transgenic mice (B6C3-Tg (APPswe,PSEN1dE9)), before or after the AD pathology was developed (In 7-week and 37-week old mice respectively). As plaques and neurofibrillary tangles were quantified by ELISAs and brain histology using specific antibodies. Basal immunological interaction between the T-cells in the affected hippocampalarea and other immune activation markers, including glial fibrillary acidicprotein (GFAP) (astroglia) and CD-45 (B-cells) were also studied using immunoreactive-specific staining. T-cell reaction type was assessed by cytokine type production. Results: Both preventive and therapeutic vaccination with EB101 resulted in a marked inhibition of As deposits (60 to 80%), a reduction of neurofibrillary tangles (70 to 90%) and almost completely suppression of reactive gliosis as measured by GFAP immunoreactivity and cytokine profile, consistent with a marked decrease in amyloidosis-induced inflammation. No external neurological deficits were observed as a result of EB101 immunization (limb paralysis or brain atrophy). Psychomotor and cognitive tests performed in a rota-rod in these animals also showed a dramatic improvement in learning and psychomotor activity in the EB101-treated group. Conclusions: The present findings unequivocally indicate that immunization with EB101 not only prevents and reverses AD neuropathology and underlined inflammation, but also improves cognitive symptoms, thus warranting further studies.

Changes in cerebral blood flow following administration of memantine hydrochloride in Alzheimer's Dementia (DAT) patients - comparative study of spect scan findings using SPM8

conclusion of the trial showed no greater brain tissue loss than they had at the end of the 1-year study, and autopsied brains of several persons dying after receiving AN1792 contained no amyloid deposits. Can the AN1792 observations and the expectation of decreased atrophy with successful disease-modifying AD treatment be reconciled? Methods: We identified several competing factors which might affect rates of brain volume changewith anti-amyloid treatment, including 1) normal rates of age-associated brain volume loss; 2) the additional disease-related contribution to brain volume loss; 3) estimated volume of the brain occupied by amyloid deposits and its regional distribution; 4) rate of accumulation of amyloid in the brain; 5) the contribution of inflammation to brain volume and 6) the rate of brain amyloid removal or decreased rate of brain amyloid accumulation. A mathematical model was constructed, using literature-derived values for as many of the variables as possible. Results: The model was used to predict changes in brain volume under modeled clinical trial conditions. Results will be presented. Conclusions: Several factors contribute to the steady-state levels of amyoid deposits in the brains of AD patients. These factors, as well as the mechanism of action of the drug under study, may affect observed changes in brain volume during clinical trials.

Reduced Sodium Channel Nav1.1 Levels in BACE1-null Mice

The Alzheimer BACE1 enzyme cleaves numerous substrates, with largely unknown physiological consequences. We have previously identified the contribution of elevated BACE1 activity to voltage-gated sodium channel Na(v)1.1 density and neuronal function. Here, we analyzed physiological changes in sodium channel metabolism in BACE1-null mice. Mechanistically, we first confirmed that endogenous BACE1 requires its substrate, the β-subunit Na(v)β(2), to regulate levels of the pore-forming α-subunit Na(v)1.1 in cultured primary neurons. Next, we analyzed sodium channel α-subunit levels in brains of BACE1-null mice at 1 and 3 months of age. At both ages, we found that Na(v)1.1 protein levels were significantly decreased in BACE1-null versus wild-type mouse brains, remaining unchanged in BACE1-heterozygous mouse brains. Interestingly, levels of Na(v)1.2 and Na(v)1.6 α-subunits also decreased in 1-month-old BACE1-null mice. In the hippocampus of BACE1-null mice, we found a robust 57% decrease of Na(v)1.1 levels. Next, we performed surface biotinylation studies in acutely dissociated hippocampal slices from BACE1-null mice. Hippocampal surface Na(v)1.1 levels were significantly decreased, but Na(v)1.2 surface levels were increased in BACE1-null mice perhaps as a compensatory mechanism for reduced surface Na(v)1.1. We also found that Na(v)β(2) processing and Na(v)1.1 mRNA levels were significantly decreased in brains of BACE1-null mice. This suggests a mechanism consistent with BACE1 activity regulating mRNA levels of the α-subunit Na(v)1.1 via cleavage of cell-surface Na(v)β(2). Together, our data show that endogenous BACE1 activity regulates total and surface levels of voltage-gated sodium channels in mouse brains. Both decreased Na(v)1.1 and elevated surface Na(v)1.2 may result in a seizure phenotype. Our data caution that therapeutic BACE1 activity inhibition in Alzheimer disease patients may affect Na(v)1 metabolism and alter neuronal membrane excitability in Alzheimer disease patients.

Synthesis and biological evaluation of indole-chalcone derivatives as β-amyloid imaging probe

A series of chaclone derivatives containing an indole moiety were evaluated in competitive binding assays with Aβ(1-42) aggregates versus [(125)I]IMPY. The affinity of these compounds ranged from 4.46 to >1008 nM, depending on the substitution on the phenyl ring. Fluorescent staining in vitro showed that one compound with a N,N-dimethylamino group intensely stained Aβ plaques within brain sections of AD transgenic mice. The radioiodinated probe [(125)I]-(E)-3-(1H-indol-5-yl)-1-(4-iodophenyl)prop-2-en-1-one, [(125)I]4, was prepared and autoradiography in sections of brain tissue from an animal model of AD showed that it labeled Aβ plaques specifically. However, experiments with normal mice indicated that [(125)I]4 exhibited a low uptake into the brain in vivo (0.41% ID/g at 2 min). Additional chemical modifications of this indole-chalcone structure may lead to more useful imaging agents for detecting β-amyloid plaques in the brains of AD patients.

Advanced glycation end products enhance amyloid precursor protein expression by inducing reactive oxygen species

Accumulation of senile plaques comprising Aβ and neurofibrillary tangles is a major hallmark in the brain of AD patients. Ample evidence has shown that inflammation and reactive oxygen species (ROS) increase in the AD patient's brain. However, the pathogenesis of AD is unclear. Some risk factors of AD include age, sex, genetics, and chronic disease. Epidemiological studies have suggested that the levels of advanced glycation end products (AGEs) are enhanced in patients with diabetes mellitus and during the aging process. At the same time, evidence indicates a moderately increased risk of development of AD in diabetic patients. AGEs may play a role in the pathogenesis of AD. In this study, we proposed that amyloid precursor protein (APP) expression was regulated by AGEs. Our data showed that APP was up-regulated by AGEs in vitro and in vivo, and pretreatment of cells with an ROS inhibitor ( N-acetyl-L-cysteine) blocked the effects of AGEs. In conditioned medium, the level of Aβ 1-42 increased after AGE treatment. Furthermore, the combination of AGEs and aggregated Aβ 1-42 increased ROS production and decreased cell viability. We suggest that AGEs participate in AD development and are an important risk factor in the pathogenesis of AD.

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for β-amyloid plaques in Alzheimer’s disease

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential β-amyloid probes. In vitro binding studies using Aβ aggregates showed that all of them demonstrated high binding affinities with Ki values ranged from 0.11 to 4.64nM. In vitro fluorescent staining results showed that these compounds can intensely stained Aβ plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [125I]-2-(5′-iodo-2,2′-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [125I]10 and [125I]-2-(2,2′-bithiophen-5-yl)-6-iodobenzo[d]thiazole [125I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [125I]10 and [125I]13 labeled the Aβ plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [125I]13 exhibited high brain uptake (3.42% ID/g at 2min) and rapid clearance from the brain (0.53% ID/g at 60min), while [125I]10 showed lower brain uptake (0.87% ID/g at 2min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [125I]13 may be a candidate as an in vivo imaging agent for detecting β-amyloid plaques in the brain of AD patients.

N-Acetylcysteine Prevents 4-Hydroxynonenal- and Amyloid-β-Induced Modification and Inactivation of Neprilysin in SH-SY5Y Cells

As one of the dominant amyloid-beta peptide (Abeta) proteases, neprilysin (NEP) plays a crucial role in maintaining a physiologic balance between Abeta production and catabolism. We have previously shown that NEP is modified by 4-hydroxynonenal (HNE) adducts, resulting in decreased activity in the brain of AD patients and cultured cells. In order to determine whether antioxidants can rescue NEP, SH-SY5Y cells were treated with HNE or Abeta, together with N-acetylcysteine for 24 hours, prior to analysis of NEP protein levels, activity, and oxidative modifications. Intracellular NEP developed HNE adducts after 24 hours of HNE or Abeta treatment as determined by immunoprecipitation, immunoblotting, and double immunofluorescence staining. N-acetylcysteine at 10 to 100 microM alleviated HNE adduction after HNE or Abeta treatment. In keeping with previous reports, HNE-modified NEP showed decreased catalytic activity. The present study demonstrates that antioxidants can be used to spare NEP from oxidative modification, suggesting a potential mechanism underlying the neuroprotective effects of antioxidants in aging or Alzheimer's disease.

Regulation of pyroglutamyl amyloid beta peptide formation by neprilysin

Amyloid beta peptide (Abeta) starting with pyroglutamate (pE) at position 3 is a dominant species that accumulates in the brains of AD patients, whereas APP Tg mice mainly accumulate Abeta1-40/42. Pyroglutamyl Abeta is more hydrophobic and metabolically more stable than others, and thus may play a specific role in AD pathogenesis. We first aimed to quantify N-truncated Abeta species in familial and sporadic AD cases and then to examine the relationship between neprilysin activity and pyroglutamyl Abeta formation. We first quantified N-terminally truncated species in AD brains using a series of endospecific antibodies. We then examined the effect of neprilysin deficiency on the neuropathology of APP-transgenic mice using immunohistochemical and mass spectrometric methods. Abeta3(pE)-42 was consistently the predominant Abeta species in all AD brains examined. In APP-Tg mice, neprilysin deficiency augmented pathological accumulation of pyroglutamyl Abeta in a distinct manner. We also observed upregulation of glutaminyl cyclase, which is responsible for cyclization of glutamate residue of Abeta. Aging-associated neprilysin deficiency accounts not only for elevation of the steady-state levels of Abeta in the brain but also for pyroglutamyl Abeta formation.

Activated PKR levels correlates with PACT levels in the brains of AD patients and in APP/PS1 KI transgenic mice

Activated PKR levels correlates with PACT levels in the brains of AD patients and in APP/PS1 KI transgenic mice