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

<p>Abstract</p> <p>Background</p> <p>Aβ deposits represent a neuropathological hallmark of Alzheimer's 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 <it>in vivo</it>.</p> <p>Methods</p> <p>The 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.</p> <p>Results</p> <p><it>In vitro</it>, celastrol dose dependently prevented NFκB activation and inhibited BACE-1 expression. Celastrol potently inhibited Aβ_1-40and Aβ_1-42production by reducing the β-cleavage of APP, leading to decreased levels of APP-CTFβ and APPsβ. <it>In vivo</it>, celastrol appeared to reduce the levels of both soluble and insoluble Aβ_1-38, Aβ_1-40and Aβ_1-42. 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.</p> <p>Conclusions</p> <p>Overall 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.</p

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

Chronic Anatabine Treatment Reduces Alzheimer’s Disease (AD)-Like Pathology and Improves Socio-Behavioral Deficits in a Transgenic Mouse Model of AD

<div><p>Anatabine is a minor tobacco alkaloid, which is also found in plants of the Solanaceae family and displays a chemical structure similarity with nicotine. We have shown previously that anatabine displays some anti-inflammatory properties and reduces microgliosis and tau phosphorylation in a pure mouse model of tauopathy. We therefore investigated the effects of a chronic oral treatment with anatabine in a transgenic mouse model (Tg PS1/APPswe) of Alzheimer’s disease (AD) which displays pathological Aβ deposits, neuroinflammation and behavioral deficits. In the elevated plus maze, Tg PS1/APPswe mice exhibited hyperactivity and disinhibition compared to wild-type mice. Six and a half months of chronic oral anatabine treatment, suppressed hyperactivity and disinhibition in Tg PS1/APPswe mice compared to Tg PS1/APPswe receiving regular drinking water. Tg PS1/APPswe mice also elicited profound social interaction and social memory deficits, which were both alleviated by the anatabine treatment. We found that anatabine reduces the activation of STAT3 and NFκB in the vicinity of Aβ deposits in Tg PS1/APPswe mice resulting in a reduction of the expression of some of their target genes including <i>Bace1</i>, <i>iNOS</i> and <i>Cox-2</i>. In addition, a significant reduction in microgliosis and pathological deposition of Aβ was observed in the brain of Tg PS1/APPswe mice treated with anatabine. This is the first study to investigate the impact of chronic anatabine treatment on AD-like pathology and behavior in a transgenic mouse model of AD. Overall, our data show that anatabine reduces β-amyloidosis, neuroinflammation and alleviates some behavioral deficits in Tg PS1/APPswe, supporting further exploration of anatabine as a possible disease modifying agent for the treatment of AD.</p></div

Anatabine reduces the amount of phosphorylated p65 NFκB immunopositive cells associated with β-amyloid deposits in the cortex of Tg PS1/APPswe mice.

<p>A) Representative 40X microscopic field revealing the presence of phosphorylated p65 NFκB immunopositive cells around Congo red stained β-amyloid deposits in the cortex of Tg PS1/APPswe mice receiving regular drinking water (placebo) or anatabine at a dosage of 10 and 20 mg/Kg/Day in their drinking water. Data is presented as mean±SEM. B) The histogram represents the average amount of phospho p65 NFκB burden (expressed as a percentage of the brain area examined) associated with β-amyloid deposits in the cortex Tg PS1/APPswe receiving regular drinking water (placebo) and the anatabine treatment at a dosage of 10 and 20 mg/Kg/Day. ANOVA shows a statistically significant main effect of the anatabine treatment on phosphorylated p65 NFκB immunoreactive cells associated with Aβ deposits (P<0.001). Post-hoc comparisons reveal statistically significant differences between Tg PS1/APPswe receiving regular drinking water (placebo) and anatabine at a dosage of 10 mg/Kg/Day (P<0.001) and 20 mg/Kg/Day (P<0.001) for the amount of p65 NFκB phosphorylation associated with β-amyloid burden.</p

Timeline of anatabine treatment and behavior tests performed in Tg PS1/APPswe mice and their control wild-type littermate.

<p>Oral anatabine treatment with 10mg/Kg/day or 20mg/Kg/Day was started in 10 months old Tg PS1/APPswe mice and control wild-type littermates and lasted for 6 and half months. During the course of treatment, a battery of behavior tasks was performed at the time points indicated above. Possible effects of the anatabine treatment on anxiety in mice were evaluated using the elevated plus maze; 0.5 month and 2.5 months after the initiation of the anatabine treatment (age of mice 10.5 and 12.5 months respectively). To evaluate the effects of anatabine on social interaction and social memory, the 3-chamber test was used 6 months after the treatment initiation (age of mice 16 months). At the end of study mice were humanely euthanized at the age of 16.5 months for pathological evaluations.</p

Anatabine reduces <i>Bace</i>1 mRNA expression in brain of Tg PS1/APPswe mice.

<p>The histogram represents quantitative results from RT-qPCR analysis calculated using the Delta-Delta Ct Method. The means ± SEM <i>Bace1</i> mRNA levels are expressed as a relative fold change compared to control (wild-type placebo). ANOVA shows a significant main effect of the genotype and treatments (P<0.009 and P<0.001) on <i>Bace1</i> transcription. Post-hoc analyses reveal a significant difference in <i>Bace1</i> mRNA levels between wild-type and Tg PS1/APPswe placebo mice (P<0.009) showing an upregulation of <i>Bace1</i> mRNA in Tg PS1/APPswe mice compared to wild-type littermates. A significant reduction in <i>Bace1</i> transcription was observed in Tg PS1/APPswe treated with 20 mg/Kg/Day of anatabine in their drinking water (P<0.001).</p

Anatabine reduces β-amyloidosis in the hippocampus and the cortex of Tg PS1/APPswe mice.

<p>A) Representative 20X microscopic fields showing β-amyloid deposits (4G8 immunostaining) in the cortex and hippocampus of Tg PS1/APPswe receiving regular drinking water (placebo) and anatabine at a dosage of 10 and 20 mg/Kg/Day dissolved in their drinking water are shown. Data is presented as mean±SEM. B) The histogram represents the average amount of 4G8 burden quantified in the hippocampus and cortex of Tg PS1/APPswe mice. ANOVA shows a statistically significant main effect of the anatabine treatment (P<0.001) on β-amyloid plaque burden. Post-hoc analyses reveal statistically significant differences in β-amyloid burden in the hippocampus (P<0.002 and P<0.001) and cortex (P<0.001 and P<0.001) between Tg PS1/APPswe mice receiving regular drinking water (placebo) and Tg PS1/APPswe receiving anatabine at a dosage of 10 and 20 mg/Kg/Day dissolved in their drinking water.</p