RNA-sequencing reveals transcriptional up-regulation of Trem2 in response to bexarotene treatment.
ABSTRACT: We have recently demonstrated that short term bexarotene treatment of APP/PS1 mice significantly improves their cognitive performance. While there were no changes in plaque load, or insoluble A? levels in brain, biochemical analysis strongly suggested improved clearance of soluble A?, including A? oligomers. To get further insight into molecular mechanisms underlying this therapeutic effect, we explored genome-wide differential gene expression in brain of bexarotene and control treated APP/PS1 mice. We performed high throughput massively parallel sequencing on mRNA libraries generated from cortices of bexarotene or vehicle treated APP/PS1 mice and compared the expression profiles for differential gene expression. Gene Ontology (GO) Biological Process categories with the highest fold enrichment and lowest False Discovery Rate (FDR) are clustered in GO terms immune response, inflammatory response, oxidation-reduction and immunoglobulin mediated immune response. Chromatin immunoprecipitation (ChIP) followed by ChIP-QPCR, and RT-QPCR expression assays were used to validate select genes, including Trem2, Tyrobp, Apoe and Ttr, differentially expressed in response to Retinoid X Receptor (RXR) activation. We found that bexarotene significantly increased the phagocytosis of soluble and insoluble A? in BV2 cells. The results of our study demonstrate that in AD model mice expressing human APP, gene networks up-regulated in response to RXR activation by the specific, small molecule, ligand bexarotene may influence diverse regulatory pathways that are considered critical for cognitive performance, inflammatory response and A? clearance, and may provide an explanation of the bexarotene therapeutic effect at the molecular level. This study also confirms that unbiased massive parallel sequencing approaches are useful and highly informative for revealing brain molecular and cellular mechanisms underlying responses to activated nuclear hormone receptors in AD animal models.
Project description:Bexarotene, a selective agonist for Retinoid X receptors (RXR) improves cognitive deficits and amyloid-β (Aβ) clearance in mice. Here we examine if the effect of bexarotene on RXR cistrome and transcriptomes depend on APOE isoform and Aβ deposition. We found bexarotene increased RXR binding to promoter regions in cortex of APOE3 mice. Rho family GTPases and Wnt signaling pathway were highly enriched in ChIP-seq and RNA-seq datasets and members of those pathways - Lrp1, Lrp5, Sfrp5 and Sema3f were validated. The effect of APOE isoform was compared in APOE3 and APOE4 mice and we found significant overlapping in affected pathways. ChIP-seq using mouse embryonic stem cells and enrichment levels of histone marks H3K4me3 and H3K27me3 revealed that, bexarotene induced epigenetic changes, consistent with increased neuronal differentiation and in correlation with changes in transcription. Comparison of transcriptome in APOE3 and APP/APOE3 mice revealed that amyloid deposition significantly affects the response to bexarotene. In primary neurons, bexarotene ameliorated the damaged dendrite complexity and loss of neurites caused by Aβ42. Finally, we show that the disruption of actin cytoskeleton induced by Aβ42 in vitro was inhibited by bexarotene treatment. Our results suggest a mechanism to establish RXR therapeutic targets with significance in neurodegeneration.
Project description:Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimer's disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that bexarotene-liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. To further validate the significance of RXR for these functions, we used mouse embryonic stem (ES) cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene. In vitro data from ES cells confirmed that bexarotene-activated RXR affected neuronal development at different levels, including proliferation of neural progenitors and neuronal differentiation, and stimulated neurite outgrowth. This effect was validated in vivo by demonstrating an increased number of neuronal progenitors after bexarotene treatment in the dentate gyrus of APOE3 and APOE4 mice. In primary neurons, bexarotene enhanced the dendritic complexity characterized by increased branching, intersections, and bifurcations. This effect was confirmed by in vivo studies demonstrating that bexarotene significantly improved the compromised dendritic structure in the hippocampus of APOE4 mice. We conclude that bexarotene-activated RXRs promote genetic programs involved in the neurogenesis and development of neuronal projections and these results have significance for the improvement of cognitive deficits.Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimer's disease mouse models, including mice expressing human apolipoprotein E (APOE) isoforms. The goal of this study was to gain further insight into molecular mechanisms whereby ligand-activated RXR can affect or restore cognitive functions. We used an unbiased approach to discover genome-wide changes in RXR cistrome (ChIP-Seq) and gene expression profile (RNA-Seq) in response to bexarotene in the cortex of APOE4 mice. Functional categories enriched in both datasets revealed that liganded RXR affected signaling pathways associated with neurogenesis and neuron projection development. The significance of RXR for these functions was validated in mouse embryonic stem cells, primary neurons, and APOE3 and APOE4 mice treated with bexarotene.
Project description:Though the precise cause(s) of Alzheimer's disease (AD) remain unknown, there is strong evidence that decreased clearance of ?-amyloid (A?) from the brain can contribute to the disease. Therapeutic strategies to promote natural A? clearance mechanisms, such as the protein apolipoprotein-E (APOE), hold promise for the treatment of AD. The amount of APOE in the brain is regulated by nuclear receptors including retinoid X receptors (RXRs). Drugs that activate RXRs, including bexarotene, can increase APOE and ABCA1 production, and have been shown to decrease the A? burden and improve cognition in mouse models of A? amyloidosis. Although recent bexarotene studies failed to replicate the rapid clearance of A? from brains, behavioral and cognitive effects of this compound remain controversial.In efforts to clarify these behavioral findings, mutant APP/PS1 mice were acutely dosed with bexarotene. While ABCA1 was upregulated in mutant APP/PS1 mice treated with bexarotene, this drug failed to attenuate A? plaques or cognitive deficits in these mice.We recommend rigorous preclinical study to evaluate the mechanism and utility of such a compound for AD therapy.
Project description:A hallmark of Alzheimer disease (AD) is the deposition of amyloid ? (A?) in brain parenchyma and cerebral blood vessels, accompanied by cognitive decline. Previously, we showed that human apolipoprotein A-I (apoA-I) decreases A?(40) aggregation and toxicity. Here we demonstrate that apoA-I in lipidated or non-lipidated form prevents the formation of high molecular weight aggregates of A?(42) and decreases A?(42) toxicity in primary brain cells. To determine the effects of apoA-I on AD phenotype in vivo, we crossed APP/PS1?E9 to apoA-I(KO) mice. Using a Morris water maze, we demonstrate that the deletion of mouse Apoa-I exacerbates memory deficits in APP/PS1?E9 mice. Further characterization of APP/PS1?E9/apoA-I(KO) mice showed that apoA-I deficiency did not affect amyloid precursor protein processing, soluble A? oligomer levels, A? plaque load, or levels of insoluble A? in brain parenchyma. To examine the effect of Apoa-I deletion on cerebral amyloid angiopathy, we measured insoluble A? isolated from cerebral blood vessels. Our data show that in APP/PS1?E9/apoA-I(KO) mice, insoluble A?(40) is increased more than 10-fold, and A?(42) is increased 1.5-fold. The increased levels of deposited amyloid in the vessels of cortices and hippocampi of APP/PS1?E9/apoA-I(KO) mice, measured by X-34 staining, confirmed the results. Finally, we demonstrate that lipidated and non-lipidated apoA-I significantly decreased A? toxicity against brain vascular smooth muscle cells. We conclude that lack of apoA-I aggravates the memory deficits in APP/PS1?E9 mice in parallel to significantly increased cerebral amyloid angiopathy.
Project description:The presence of senile plaques is one of the major pathologic hallmarks of the brain with Alzheimer's disease (AD). The plaques predominantly contain insoluble amyloid ?-peptide, a cleavage product of the larger amyloid precursor protein (APP). Two enzymes, named ? and ? secretase, generate the neurotoxic amyloid-? peptide from APP. Mature APP is also turned over endogenously by autophagy, more specifically by the endosomal-lysosomal pathway. A defective lysosomal system is known to be pathogenic in AD. Modulation of NF-E2 related factor 2 (Nrf2) has been shown in several neurodegenerative disorders, and Nrf2 has become a potential therapeutic target for various neurodegenerative disorders, including AD, Parkinson's disease, and amyotrophic lateral sclerosis. In the current study, we explored the effect of genetic ablation of Nrf2 on APP/A? processing and/or aggregation as well as changes in autophagic dysfunction in APP/PS1 mice. There was a significant increase in inflammatory response in APP/PS1 mice lacking Nrf2. This was accompanied by increased intracellular levels of APP, A? (1-42), and A? (1-40), without a change total full-length APP. There was a shift of APP and A? into the insoluble fraction, as well as increased poly-ubiquitin conjugated proteins in mice lacking Nrf2. APP/PS1-mediated autophagic dysfunction is also enhanced in Nrf2-deficient mice. Finally, neurons in the APP/PS1/Nrf2-/- mice had increased accumulation of multivesicular bodies, endosomes, and lysosomes. These outcomes provide a better understanding of the role of Nrf2 in modulating autophagy in an AD mouse model and may help design better Nrf2 targeted therapeutics that could be efficacious in the treatment of AD.
Project description:The amyloid beta (A?) peptide is associated with the neurodegenerative and inflammatory changes in brains affected by Alzheimer's disease (AD). We hypothesized that the enteric nervous system also produces A? in an intestinal component of disease. To test this idea, we compared C57BL/6 wild-type (WT) male and female mice to two models of Alzheimer's disease, amyloid precursor protein (APP)/presenilin 1 (PS1) mice and amyloid precursor protein NL-G-F (AppNL-G-F) mice, at 3, 6, and 12 months of age. Brain A? plaque deposition in AppNL-G-F mice preceded that in the APP/PS1 mice, observable by 3 months. Three-month-old female AppNL-G-F mice had decreased intestinal motility compared with WT and APP/PS1 mice. However, 3-month-old female APP/PS1 mice demonstrated increased intestinal permeability compared with WT and AppNL-G-F mice. Both sexes of APP/PS1 and AppNL-G-F mice demonstrated increased colon lipocalin 2 mRNA and insoluble A? 1-42 levels at 3 months. These data demonstrate an unrecognized enteric aspect of disease in 2 different mouse models correlating with the earliest brain changes.
Project description:The amyloid hypothesis of Alzheimer's disease (AD) postulates that amyloid-beta (Abeta) deposition and neurotoxicity play a causative role in AD; oxidative injury is thought to be central in the pathogenesis. An endogenous defense system against oxidative stress is induced by binding of the transcription factor nuclear factor E2-related factor 2 (Nrf2) to the antioxidant response element (ARE) enhancer sequence. The Nrf2-ARE pathway is activated in response to reactive oxygen species to trigger the simultaneous expression of numerous protective enzymes and scavengers. To exploit the Nrf2-ARE pathway therapeutically, we delivered Nrf2 bilaterally into the hippocampus of 9-month-old transgenic AD mice (APP/PS1 mice) using a lentiviral vector encoding human Nrf2. The data indicate that significant reductions in spatial learning deficits of aged APP/PS1 mice in a Morris Water Maze can be achieved by modulating levels of Nrf2 in the brain. Memory improvement in APP/PS1 mice after Nrf2 transduction shifts the balance between soluble and insoluble Abeta toward an insoluble Abeta pool without concomitant change in total brain Abeta burden. Nrf2 gene transfer is associated with a robust reduction in astrocytic but not microglial activation and induction of Nrf2 target gene heme oxygenase 1, indicating overall activation of the Nrf2-ARE pathway in hippocampal neurons 6 months after injection. Results warrant further exploration of the Nrf2-ARE pathway for treatment of AD and suggest that the Nrf2-ARE pathway may represent a potential therapeutic strategy to pursue in AD in humans, particularly in view of the multiple mechanisms by which Nrf2 can exert its protective effects.
Project description:High throughput massively parallel sequencing on mRNA libraries generated from cortices of bexarotene or vehicle treated APP/PS1 Read counts analyzed for differential gene expression using edgeR
Project description:Carrying the ?4 allele of the APOE gene encoding apolipoprotein E (APOE4) markedly increases the risk for late-onset Alzheimer's disease (AD), in which APOE4 exacerbates the brain accumulation and subsequent deposition of amyloid-? (A?) peptides. While the LDL receptor-related protein 1 (LRP1) is a major apoE receptor in the brain, we found that its levels are associated with those of insoluble A? depending on APOE genotype status in postmortem AD brains. Thus, to determine the functional interaction of apoE4 and LRP1 in brain A? metabolism, we crossed neuronal LRP1-knockout mice with amyloid model APP/PS1 mice and APOE3-targeted replacement (APO3-TR) or APOE4-TR mice. Consistent with previous findings, mice expressing apoE4 had increased A? deposition and insoluble amounts of A?40 and A?42 in the hippocampus of APP/PS1 mice compared with those expressing apoE3. Intriguingly, such effects were reversed in the absence of neuronal LRP1. Neuronal LRP1 deficiency also increased detergent-soluble apoE4 levels, which may contribute to the inhibition of A? deposition. Together, our results suggest that apoE4 exacerbates A? pathology through a mechanism that depends on neuronal LRP1. A better understanding of apoE isoform-specific interaction with their metabolic receptor LRP1 on A? metabolism is crucial for defining APOE4-related risk for AD.
Project description:Dysfunction of the serotonergic (5-HTergic) system has been implicated in the cognitive and behavioural symptoms of Alzheimer's disease (AD). Accumulation of toxic amyloid-? (A?) species is a hallmark of AD and an instigator of pathology. Serotonin (5-HT) augmentation therapy by treatment with selective serotonin reuptake inhibitors (SSRIs) in patients with AD has had mixed success in improving cognitive function, whereas SSRI administration to mice with AD-like disease has been shown to reduce A? pathology. The objective of this study was to investigate whether an increase in extracellular levels of 5-HT induced by chronic SSRI treatment reduces A? pathology and whether 5-HTergic deafferentation of the cerebral cortex could worsen A? pathology in the APPswe/PS1?E9 (APP/PS1) mouse model of AD.We administered a therapeutic dose of the SSRI escitalopram (5 mg/kg/day) in the drinking water of 3-month-old APP/PS1 mice to increase levels of 5-HT, and we performed intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT) to remove 5-HTergic afferents. We validated the effectiveness of these interventions by serotonin transporter autoradiography (neocortex 79.7?±?7.6%) and by high-performance liquid chromatography for 5-HT (neocortex 64% reduction). After 6 months of escitalopram treatment or housing after DHT-induced lesion, we evaluated brain tissue by mesoscale multiplex analysis and sections by IHC analysis.Amyloid-?-containing plaques had formed in the neocortex and hippocampus of 9-month-old APP/PS1 mice after 6 months of escitalopram treatment and 5-HTergic deafferentation. Unexpectedly, levels of insoluble A?42 were unaffected in the neocortex and hippocampus after both types of interventions. Levels of insoluble A?40 increased in the neocortex of SSRI-treated mice compared with those treated with vehicle control, but they were unaffected in the hippocampus. 5-HTergic deafferentation was without effect on the levels of insoluble/soluble A?42 and A?40 in both the neocortex and hippocampus. However, levels of soluble amyloid precursor protein ? were reduced in the neocortex after 5-HTergic deafferentation.Because this study shows that modulation of the 5-HTergic system has either no effect or increases levels of insoluble/soluble A?42 and A?40 in the cerebral cortex of APP/PS1 mice, our observations do not support 5-HT augmentation therapy as a preventive strategy for reducing A? pathology.