David Beaulieu-Abdelahad

Reduction of β-amyloid pathology by celastrol in a transgenic mouse model of Alzheimer's disease

BackgroundAβ deposits represent a neuropathological hallmark of Alzheimers disease (AD). Both soluble and insoluble Aβ species are considered to be responsible for initiating the pathological cascade that eventually leads to AD. Therefore, the identification of therapeutic approaches that can lower Aβ production or accumulation remains a priority. NFκB has been shown to regulate BACE-1 expression level, the rate limiting enzyme responsible for the production of Aβ. We therefore explored whether the known NFκB inhibitor celastrol could represent a suitable compound for decreasing Aβ production and accumulation in vivo.MethodsThe effect of celastrol on amyloid precursor protein (APP) processing, Aβ production and NFκB activation was investigated by western blotting and ELISAs using a cell line overexpressing APP. The impact of celastrol on brain Aβ accumulation was tested in a transgenic mouse model of AD overexpressing the human APP695sw mutation and the presenilin-1 mutation M146L (Tg PS1/APPsw) by immunostaining and ELISAs. An acute treatment with celastrol was investigated by administering celastrol intraperitoneally at a dosage of 1 mg/Kg in 35 week-old Tg PS1/APPsw for 4 consecutive days. In addition, a chronic treatment (32 days) with celastrol was tested using a matrix-driven delivery pellet system implanted subcutaneously in 5 month-old Tg PS1/APPsw to ensure a continuous daily release of 2.5 mg/Kg of celastrol.ResultsIn vitro, celastrol dose dependently prevented NFκB activation and inhibited BACE-1 expression. Celastrol potently inhibited Aβ1-40 and Aβ1-42 production by reducing the β-cleavage of APP, leading to decreased levels of APP-CTFβ and APPsβ. In vivo, celastrol appeared to reduce the levels of both soluble and insoluble Aβ1-38, Aβ1-40 and Aβ1-42. In addition, a reduction in Aβ plaque burden and microglial activation was observed in the brains of Tg PS1/APPsw following a chronic administration of celastrol.ConclusionsOverall our data suggest that celastrol is a potent Aβ lowering compound that acts as an indirect BACE-1 inhibitor possibly by regulating BACE-1 expression level via an NFκB dependent mechanism. Additional work is required to determine whether chronic administration of celastrol can be safely achieved with cognitive benefits in a transgenic mouse model of AD.

Alzheimer’s β‐amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR‐2

J. Neurochem. (2010) 112, 66–76.

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 (500u2009nM to 50u2009µM) 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 (1–50u2009µM) for 1u2009h 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. Copyright

Stimulation of the Retinoid X Receptor Facilitates Beta-Amyloid Clearance Across the Blood–Brain Barrier

Alzheimer’s disease (AD) is a neurodegenerative process characterized, in part, by the accumulation of beta-amyloid proteins (Aβ) in the brain. Evidence now suggests that the excessive Aβ accumulation is the result of impaired clearance from the brain. Recent studies have indicated that retinoid X receptor (RXR) activation stimulates the metabolic clearance of Aβ and rapidly reverses Aβ-induced behavioral deficits, doing so in an apoE-dependent manner. Previously, we reported that soluble apoE (i.e., not bound to Aβ) facilitated Aβ transit across the blood–brain barrier (BBB). As Aβ clearance from the brain involves both metabolic and BBB-mediated processes, the current studies investigated the impact of RXR stimulation on Aβ clearance across the BBB. Treatment with RXR agonists increased Aβ clearance across the BBB both in vitro and in vivo. Moreover, this processes appeared to involve apoE as RXR agonism did not stimulate Aβ BBB clearance when apoE was absent. Thus, RXR activation could mitigate Aβ brain burden by promoting both the metabolic and BBB clearance of Aβ, offering a novel approach to the treatment of AD.

Anatabine lowers Alzheimer's Aβ production in vitro and in vivo.

Brain Aβ accumulation represents a key pathological hallmark in Alzheimers 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 Alzheimers 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 Alzheimers disease. Altogether our data suggest that anatabine may represent an interesting compound for regulating brain Aβ accumulation.

A Multifaceted Role for apoE in the Clearance of Beta-Amyloid across the Blood-Brain Barrier

Background: While apolipoprotein E4 (apoE4) is highly correlated with the development of Alzheimer’s disease (AD), its role in AD pathology and, in particular, beta-amyloid (Aβ) removal from the brain, is not clearly defined. Objective: To elucidate the influence of apoE on the clearance of Aβ across the blood-brain barrier (BBB). Methods: Aβ(1–42) was intracerebrally administered to transgenic mice expressing human apoE isoforms and examined in the periphery. Results: apoE3 and apoE4 mice had 5 times and 2 times, respectively, more Aβ(1–42) appearing in the plasma than wild-type or apoE knockout mice, indicating an enhanced clearance of Aβ from the brain to the periphery. In vitro, unbound basolateral apoE3 (i.e., not bound to Aβ), and to a lesser extent unbound apoE4, at concentrations ≤10 nm facilitated basolateral-to-apical fluorescein-Aβ(1–42) transcytosis across a BBB model, while apoE isoforms bound to Aβ significantly disrupted Aβ transcytosis. Additionally, following apical exposure to the BBB model, we found that apoE4 bound to Aβ is able to penetrate the BBB more readily than apoE3 bound to Aβ and does so via the RAGE (receptor for advanced glycation end products) transporter. Conclusion: These studies indicate a multifaceted, isoform-dependent role for apoE in the exchange of Aβ across the BBB and may partially explain the association of apoE4 and Aβ brain accumulation in AD.

Anti-inflammatory activity of anatabine via inhibition of STAT3 phosphorylation

Previous investigations have demonstrated the anti-inflammatory effects of cholinergic agonists, such as nicotine. In the present study, we investigated the potential anti-inflammatory activity of anatabine, a minor tobacco alkaloid also present in plants of the Solanacea family which displays a chemical structural similarity with nicotine. Our data show that anatabine prevents STAT3 and NFκB phosphorylation induced by lipopolysaccharide (LPS) or TNF-α in SH-SY5Y, HEK293, human microglia and human blood mononuclear cells. Using human whole blood, we found that anatabine prevents IL-1β production induced by LPS. We assessed anatabines anti-inflammatory activity in vivo using an acute model of inflammation by challenging wild-type mice with LPS. We observed that anatabine reduces pro-inflammatory cytokine production (IL-6, IL-1β and TNF-α) in the plasma, kidney and spleen of the animals following the injection of LPS and concomitantly opposes STAT3 phosphorylation induced by LPS in the spleen and kidney. We also investigated the impact of anatabine on neuroinflammation using a transgenic mouse model of Alzheimers disease (Tg APPsw) that displays elevated cytokine levels in the brain. Following a chronic oral treatment with anatabine, a reduction in brain TNF-α and IL-6 levels compared to untreated Tg APPsw mice was observed. Moreover, an increased STAT3 phosphorylation was detected in the brains of Tg APPsw mice compared to wild-type littermates and was inhibited by anatabine treatment. Overall our data show that the anti-inflammatory activity of anatabine in vitro and in vivo is mediated in part via an inhibition of STAT3 phosphorylation.

Role of the cannabinoid system in the transit of beta-amyloid across the blood–brain barrier

Emerging evidence suggests beta-amyloid (Aβ) deposition in the Alzheimers disease (AD) brain is the result of impaired clearance, due in part to diminished Aβ transport across the blood-brain barrier (BBB). Recently, modulation of the cannabinoid system was shown to reduce Aβ brain levels and improve cognitive behavior in AD animal models. The purpose of the current studies was to investigate the role of the cannabinoid system in the clearance of Aβ across the BBB. Using in vitro and in vivo models of BBB clearance, Aβ transit across the BBB was examined in the presence of cannabinoid receptor agonists and inhibitors. In addition, expression levels of the Aβ transport protein, lipoprotein receptor-related protein1 (LRP1), were determined in the brain and plasma of mice following cannabinoid treatment. Cannabinoid receptor agonism or inhibition of endocannabinoid-degrading enzymes significantly enhanced Aβ clearance across the BBB (2-fold). Moreover, cannabinoid receptor inhibition negated the stimulatory influence of cannabinoid treatment on Aβ BBB clearance. Additionally, LRP1 levels in the brain and plasma were elevated following cannabinoid treatment (1.5-fold), providing rationale for the observed increase in Aβ transit from the brain to the periphery. The current studies demonstrate, for the first time, a role for the cannabinoid system in the transit of Aβ across the BBB. These findings provide insight into the mechanism by which cannabinoid treatment reduces Aβ burden in the AD brain and offer additional evidence on the utility of this pathway as a treatment for AD.

Amelioration of Experimental Autoimmune Encephalomyelitis by Anatabine

Anatabine, a naturally occurring alkaloid, is becoming a commonly used human food supplement, taken for its claimed anti-inflammatory properties although this has not yet been reported in human clinical trials. We have previously shown that anatabine does display certain anti-inflammatory properties and readily crosses the blood-brain barrier suggesting it could represent an important compound for mitigating neuro-inflammatory conditions. The present study was designed to determine whether anatabine had beneficial effects on the development of experimental autoimmune encephalomyelitis (EAE) in mice and to precisely determine its underlying mechanism of action in this mouse model of multiple sclerosis (MS). We found that orally administered anatabine markedly suppressed neurological deficits associated with EAE. Analyses of cytokine production in the periphery of the animals revealed that anatabine significantly reduced Th1 and Th17 cytokines known to contribute to the development of EAE. Anatabine appears to significantly suppress STAT3 and p65 NFκB phosphorylation in the spleen and the brain of EAE mice. These two transcription factors regulate a large array of inflammatory genes including cytokines suggesting a mechanism by which anatabine antagonizes pro-inflammatory cytokine production. Additionally, we found that anatabine alleviated the infiltration of macrophages/microglia and astrogliosis and significantly prevented demyelination in the spinal cord of EAE mice. Altogether our data suggest that anatabine may be effective in the treatment of MS and should be piloted in clinical trials.

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 Alzheimers 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 Alzheimers 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 Alzheimers disease and could open the door to a new class of therapies for the treatment of this disease.