Characterization and use of human brain microvascular endothelial cells to examine β-amyloid exchange in the blood-brain barrier. Bachmeier C, Mullan M, Paris D.

Alzheimer’s disease (AD) is characterized by excessive cerebrovascular deposition of the β-amyloid peptide (Aβ). The investigation of Aβ transport across the blood-brain barrier (BBB) has been hindered by inherent limitations in the cellular systems currently used to model the BBB, such as insufficient barrier properties and poor reproducibility. In addition, many of the existing models are not of human or brain origin and are often arduous to establish and maintain. Thus, we characterized an in vitro model of the BBB employing human brain microvascular endothelial cells (HBMEC) and evaluated its utility to investigate Aβ exchange at the blood-brain interface. Our HBMEC model offers an ease of culture compared with primary isolated or coculture BBB models and is more representative of the human brain endothelium than many of the cell lines currently used to study the BBB. In our studies, the HBMEC model exhibited barrier properties comparable to existing BBB models as evidenced by the restricted permeability of a known paracellular marker. In addition, using a simple and rapid fluormetric assay, we showed that antagonism of key Aβ transport proteins significantly altered the bi-directional transcytosis of fluorescein-Aβ (1-42) across the HBMEC model. Moreover, the magnitude of these effects was consistent with reports in the literature using the same ligands in existing in vitro models of the BBB. These studies establish the HBMEC as a representative in vitro model of the BBB and offer a rapid fluorometric method of assessing Aβ exchange between the periphery and the brain.

or more information on the Roskamp Institute and Alzheimer’s please visit:

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Depletion of CXCR2 inhibits γ-secretase activity and amyloid-β production in a murine model of Alzheimer’s disease.

Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to progressive cognitive decline. Recent studies from our group and others have suggested that certain G-protein coupled receptors (GPCRs) can influence the processing of the amyloid precursor protein (APP). Earlier, we demonstrated that stimulation of a chemokine receptor, CXCR2, results in enhanced γ-secretase activity and in increased amyloid-beta (Aβ) production. Taken together, results obtained from in vitro studies indicate that therapeutic targeting of CXCR2 might aid in lowering Aβ levels in the AD brain. To better understand the precise function and to predict the consequences of CXCR2 depletion in the AD brain, we have crossed CXCR2 knockout mice with mice expressing presenilin (PS1 M146L) and APPsw mutations (PSAPP). Our present study confirms that CXCR2 depletion results in reduction of Aβ with concurrent increases of γ-secretase substrates. At the mechanistic level, the effect of CXCR2 on γ-secretase was not found to occur via their direct interaction. Furthermore, we provide evidence that Aβ promotes endocytosis of CXCR2 via increasing levels of CXCR2 ligands. In conclusion, our current study confirms the regulatory role of CXCR2 in APP processing, and poses it as a potential target for developing novel therapeutics for intervention in AD.

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Induction of drug efflux protein expression by venlafaxine but not desvenlafaxine.

Venlafaxine and its metabolite desvenlafaxine are serotonin-norepinephrine reuptake inhibitors currently prescribed for the treatment of depression. Previously, it was reported that venlafaxine is an inducer of MDR1, the gene responsible for P-glycoprotein (P-gp). The present study expanded upon these findings by examining the effect of venlafaxine and desvenlafaxine on the expression of both P-gp and the breast cancer resistance protein (BCRP) in human brain endothelial cells (HBMEC), an in vitro model of the blood-brain barrier (BBB). The HBMEC were treated for 1 h with various concentrations (500 nM to 50 µM) of venlafaxine and desvenlafaxine. Western blot analysis revealed treatment with venlafaxine significantly induced the expression of P-gp (2-fold) and BCRP (1.75-fold) in a dose-dependent manner, while treatment with desvenlafaxine had no effect on drug efflux transporter expression. To determine the functional significance of this effect, the permeability of a known drug efflux probe, rhodamine 123, across the BBB model and Caco-2 cells, a model of intestinal absorption, were examined. Treatment with venlafaxine (1-50 µM) for 1 h significantly reduced the apical-to-basolateral permeability of R123 across the BBB model (30%) and Caco-2 cell monolayers (25%), indicative of increased drug efflux transporter expression at the apical membrane. Conversely, desvenlafaxine had no effect on R123 permeability in either cellular model. These studies indicate that venlafaxine, but not desvenlafaxine is an inducer of drug efflux transporter expression, which consequently increases the potential for clinical drug-drug interactions. Therefore, based on these preliminary results, caution should be taken when prescribing venlafaxine with other P-gp substrates.

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Selective dihydropyiridine compounds facilitate the clearance of β-amyloid across the blood-brain barrier.

Increasing evidence suggests that the soluble form of the β-amyloid peptide (Aβ) plays a critical role in the pathogenesis of Alzheimer’s disease. Previously, we reported that treatment with certain antihypertensive dihydropyridine (DHP) compounds can mitigate Aβ production in whole cells and reduce brain Aβ burden in a mouse model of Alzheimer’s disease. As Aβ clearance across the blood-brain barrier (BBB) is a key regulatory step in the deposition of Aβ in the brain, we examined the effect of DHP treatment on Aβ brain clearance. Treatment with certain DHP compounds significantly increased Aβ(1-42) transcytosis across the BBB in an in vitro model. The rank order of these compounds was nitrendipine>nicardipine=cilnidipine=lercanidipine>nimodipine>azelnidipine=nilvadipine. Conversely, amlodipine, felodipine, isradipine, and nifedipine had no effect on Aβ(1-42) BBB transcytosis. In an in vivo paradigm of Aβ clearance across the BBB, peripheral administration of nitrendipine, cilnidipine, and nilvadipine to wild-type animals facilitated the brain clearance of centrally administered exogenous Aβ(1-42), whereas with amlodipine, there was no effect. We also observed improved cognitive function in mice treated with nilvadipine following central Aβ(1-42) insult. Thus, in addition to the effect of certain DHP compounds on Aβ production, we demonstrate that certain DHP compounds also facilitate the clearance of Aβ across the BBB. This dual mechanism of action may be particularly effective in attenuating Aβ brain burden in Alzheimer’s disease and could open the door to a new class of therapies for the treatment of this disease.

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Feasibility of Predicting MCI/AD Using Neuropsychological Tests and Serum β-Amyloid.

We examined the usefulness of brief neuropsychological tests and serum Aβ as a predictive test for detecting MCI/AD in older adults. Serum Aβ levels were measured from 208 subjects who were cognitively normal at enrollment and blood draw. Twenty-eight of the subjects subsequently developed MCI (n = 18) or AD (n = 10) over the follow-up period. Baseline measures of global cognition, memory, language fluency, and serum Aβ(1-42) and the ratio of serum Aβ(1-42)/Aβ(1-40) were significant predictors for future MCI/AD using Cox regression with demographic variables, APOE ε4, vascular risk factors, and specific medication as covariates. An optimal sensitivity of 85.2% and specificity of 86.5% for predicting MCI/AD was achieved using ROC analyses. Brief neuropsychological tests and measurements of Aβ(1-42) obtained via blood warrants further study as a practical and cost effective method for wide-scale screening for identifying older adults who may be at-risk for pathological cognitive decline.

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Identification of Plasma Biomarkers of TBI Outcome Using Proteomic Approaches in an APOE Mouse Model.

Abstract The current lack of diagnostic and prognostic biomarkers for traumatic brain injury (TBI) confounds treatment and management of patients and is of increasing concern as the TBI population grows. We have generated plasma proteomic profiles from mice receiving TBI by controlled cortical impact at either 1.3 mm or 1.8 mm depth, comparing these against those of sham injured-animals to identify plasma biomarkers specific to mild or severe TBI at 24 hours, 1 month, or 3 months post-injury. To identify possible prognostic biomarkers, we used apolipoprotein E (APOE)3 and APOE4 transgenic mice, which demonstrate relatively favorable and unfavorable outcomes respectively, following TBI. Using a quantitative proteomics approach (isobaric tagging for relative and absolute quantitation – iTRAQ) we have identified proteins that are significantly modulated as a function of TBI and also in response to the TBI*APOE genotype interaction, the latter representing potential prognostic biomarkers. These preliminary data clearly demonstrate plasma protein changes that are not only injury dependent but also interaction dependent. Importantly, these results demonstrate the presence of TBI-dependent and interaction-dependent plasma proteins at a 3-month time point, which is a considerable time post-injury in the mouse model, and will potentially be of significance for combat veterans receiving assessment at extended periods post-injury. Furthermore, our identification of clusters of functionally related proteins indicates disturbance of particular biological modules, which potentially increases their value beyond that of solitary biomarkers.

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Anatabine lowers Alzheimer’s Aβ production in vitro and in vivo

Brain Aβ accumulation represents a key pathological hallmark in Alzheimer’s disease. In this study, we investigated the impact of anatabine, a minor alkaloid present in plants of the Solanacea family on Aβ production in vitro using a cell line overexpressing the human amyloid precursor protein (APP) and in vivo using a transgenic mouse model of Alzheimer’s disease. In vitro, anatabine lowers Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in a dose dependent manner and reduces sAPPβ production without impacting sAPPα levels suggesting that anatabine lowers Aβ production by mainly impacting the β-cleavage of APP. Additionally, we show that anatabine lowers NFκB activation at doses that inhibit Aβ production in vitro. Since NFκB is known to regulate BACE-1 expression (the rate limiting enzyme responsible for Aβ production), we determined the impact of anatabine on BACE-1 transcription. We show that anatabine inhibits BACE-1 transcription and reduces BACE-1 protein levels in human neuronal like SHSY-5Y cells suggesting that the Aβ lowering properties of anatabine are mediated via a regulation of BACE-1 expression. In vivo, we show that an acute treatment with anatabine for four days significantly lowers brain soluble Aβ₁₋₄₀ and Aβ₁₋₄₂ levels in a transgenic mouse model of Alzheimer’s disease. Altogether our data suggest that anatabine may represent an interesting compound for regulating brain Aβ accumulation.

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