Cody M. Wolfe

Bexarotene-Activated Retinoid X Receptors Regulate Neuronal Differentiation and Dendritic Complexity

Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimers 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. SIGNIFICANCE STATEMENT Bexarotene-activated retinoid X receptors (RXRs) ameliorate memory deficits in Alzheimers 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.

RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers

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.

Effect of high fat diet on phenotype, brain transcriptome and lipidome in Alzheimer’s model mice

We examined the effect of chronic high fat diet (HFD) on amyloid deposition and cognition of 12-months old APP23 mice, and correlated the phenotype to brain transcriptome and lipidome. HFD significantly increased amyloid plaques and worsened cognitive performance compared to mice on normal diet (ND). RNA-seq results revealed that in HFD mice there was an increased expression of genes related to immune response, such as Trem2 and Tyrobp. We found a significant increase of TREM2 immunoreactivity in the cortex in response to HFD, most pronounced in female mice that correlated to the amyloid pathology. Down-regulated by HFD were genes related to neuron projections and synaptic transmission in agreement to the significantly deteriorated neurite morphology and cognition in these mice. To examine the effect of the diet on the brain lipidome, we performed Shotgun Lipidomics. While there was no difference in the total amounts of phospholipids of each class, we revealed that the levels of 24 lipid sub-species in the brain were significantly modulated by HFD. Network visualization of correlated lipids demonstrated overall imbalance with most prominent effect on cardiolipin molecular sub-species. This integrative approach demonstrates that HFD elicits a complex response at molecular, cellular and system levels in the CNS.

Gene co-expression networks identify Trem2 and Tyrobp as major hubs in human APOE expressing mice following traumatic brain injury

Traumatic brain injury (TBI) is strongly linked to an increased risk of developing dementia, including chronic traumatic encephalopathy and possibly Alzheimers disease (AD). APOEε4 allele of human Apolipoprotein E (APOE) gene is the major genetic risk factor for late onset AD and has been associated with chronic traumatic encephalopathy and unfavorable outcome following TBI. To determine if there is an APOE isoform-specific response to TBI we performed controlled cortical impact on 3-month-old mice expressing human APOE3 or APOE4 isoforms. Following injury, we used several behavior paradigms to test for anxiety and learning and found that APOE3 and APOE4 targeted replacement mice demonstrate cognitive impairments following moderate TBI. Transcriptional profiling 14days following injury revealed a significant effect of TBI, which was similar in both genotypes. Significantly upregulated by injury in both genotypes were mRNA expression and protein level of ABCA1 transporter and APOJ, but not APOE. To identify gene-networks correlated to injury and APOE isoform, we performed Weighted Gene Co-expression Network Analysis. We determined that the network mostly correlated to TBI in animals expressing both isoforms is immune response with major hub genes including Trem2, Tyrobp, Clec7a and Cd68. We also found a significant increase of TREM2, IBA-1 and GFAP protein levels in the brains of injured mice. We identified a network representing myelination that correlated significantly with APOE isoform in both injury groups. This network was significantly enriched in oligodendrocyte signature genes, such as Mbp and Plp1. Our results demonstrate unique and distinct gene networks at this acute time point for injury and APOE isoform, as well as a network driven by APOE isoform across TBI groups.

Heavy Chronic Intermittent Ethanol Exposure Alters Small Noncoding RNAs in Mouse Sperm and Epididymosomes

While the risks of maternal alcohol abuse during pregnancy are well-established, several preclinical studies suggest that chronic preconception alcohol consumption by either parent may also have significance consequences for offspring health and development. Notably, since isogenic male mice used in these studies are not involved in gestation or rearing of offspring, the cross-generational effects of paternal alcohol exposure suggest a germline-based epigenetic mechanism. Many recent studies have demonstrated that the effects of paternal environmental exposures such as stress or malnutrition can be transmitted to the next generation via alterations to small noncoding RNAs in sperm. Therefore, we used high throughput sequencing to examine the effect of preconception ethanol on small noncoding RNAs in sperm. We found that chronic intermittent ethanol exposure altered several small noncoding RNAs from three of the major small RNA classes in sperm, tRNA-derived small RNA (tDR), mitochondrial small RNA, and microRNA. Six of the ethanol-responsive small noncoding RNAs were evaluated with RT-qPCR on a separate cohort of mice and five of the six were confirmed to be altered by chronic ethanol exposure, supporting the validity of the sequencing results. In addition to altered sperm RNA abundance, chronic ethanol exposure affected post-transcriptional modifications to sperm small noncoding RNAs, increasing two nucleoside modifications previously identified in mitochondrial tRNA. Furthermore, we found that chronic ethanol reduced epididymal expression of a tRNA methyltransferase, Nsun2, known to directly regulate tDR biogenesis. Finally, ethanol-responsive sperm tDR are similarly altered in extracellular vesicles of the epididymis (i.e., epididymosomes), supporting the hypothesis that alterations to sperm tDR emerge in the epididymis and that epididymosomes are the primary source of small noncoding RNAs in sperm. These results add chronic ethanol to the growing list of paternal exposures that can affect small noncoding RNA abundance and nucleoside modifications in sperm. As small noncoding RNAs in sperm have been shown to causally induce heritable phenotypes in offspring, additional research is warranted to understand the potential effects of ethanol-responsive sperm small noncoding RNAs on offspring health and development.

Liver X receptor agonist treatment significantly affects phenotype and transcriptome of APOE3 and APOE4 Abca1 haplo-deficient mice

ATP-binding cassette transporter A1 (ABCA1) controls cholesterol and phospholipid efflux to lipid-poor apolipoprotein E (APOE) and is transcriptionally controlled by Liver X receptors (LXRs) and Retinoic X Receptors (RXRs). In APP transgenic mice, lack of Abca1 increased Aβ deposition and cognitive deficits. Abca1 haplo-deficiency in mice expressing human APOE isoforms, increased level of Aβ oligomers and worsened memory deficits, preferentially in APOE4 mice. In contrast upregulation of Abca1 by LXR/RXR agonists significantly ameliorated pathological phenotype of those mice. The goal of this study was to examine the effect of LXR agonist T0901317 (T0) on the phenotype and brain transcriptome of APP/E3 and APP/E4 Abca1 haplo-deficient (APP/E3/Abca1+/- and APP/E4/Abca1+/-) mice. Our data demonstrate that activated LXRs/RXR ameliorated APOE4-driven pathological phenotype and significantly affected brain transcriptome. We show that in mice expressing either APOE isoform, T0 treatment increased mRNA level of genes known to affect brain APOE lipidation such as Abca1 and Abcg1. In both APP/E3/Abca1+/- and APP/E4/Abca1+/- mice, the application of LXR agonist significantly increased ABCA1 protein level accompanied by an increased APOE lipidation, and was associated with restoration of APOE4 cognitive deficits, reduced levels of Aβ oligomers, but unchanged amyloid load. Finally, using Gene set enrichment analysis we show a significant APOE isoform specific response to LXR agonist treatment: Gene Ontology categories “Microtubule Based Process” and “Synapse Organization” were differentially affected in T0-treated APP/E4/Abca1+/- mice. Altogether, the results are suggesting that treatment of APP/E4/Abca1+/- mice with LXR agonist T0 ameliorates APOE4-induced AD-like pathology and therefore targeting the LXR-ABCA1-APOE regulatory axis could be effective as a potential therapeutic approach in AD patients, carriers of APOEε4.

Integrated approach reveals diet, APOE genotype and sex affect immune response in APP mice

Alzheimers disease (AD) is a multifactorial neurodegenerative disorder that is influenced by genetic and environmental risk factors, such as inheritance of ε4 allele of APOE (APOE4), sex and diet. Here, we examined the effect of high fat diet (HFD) on amyloid pathology and expression profile in brains of AD model mice expressing human APOE isoforms (APP/E3 and APP/E4 mice). APP/E3 and APP/E4 mice were fed HFD or Normal diet for 3months. We found that HFD significantly increased amyloid plaques in male and female APP/E4, but not in APP/E3 mice. To identify differentially expressed genes and gene-networks correlated to diet, APOE isoform and sex, we performed RNA sequencing and applied Weighted Gene Co-expression Network Analysis. We determined that the immune response network with major hubs Tyrobp/DAP12, Csf1r, Tlr2, C1qc and Laptm5 correlated significantly and positively to the phenotype of female APP/E4-HFD mice. Correspondingly, we found that in female APP/E4-HFD mice, microglia coverage around plaques, particularly of larger size, was significantly reduced. This suggests altered containment of the plaque growth and sex-dependent vulnerability in response to diet. The results of our study show concurrent impact of diet, APOE isoform and sex on the brain transcriptome and AD-like phenotype.

MULTI-OMICS PROFILING IDENTIFIES APOE-ALLELE-ASSOCIATED LIPID AND GENE EXPRESSION PATTERNS IN ALZHEIMER'S DISEASE

Age (y) 35.393 9.21 64.5 11.64 31.941 11.46 73.667 12.89 83.714 6.1 ANOVA p<0.001 Pm Delay (h) 48.336 18.3 57.661 27.13 34.118 19.95 56.172 25.32 42.875 29.49 Kwallis p1⁄40.045 Cause of death Suicide 28 30 0 0 0 Accident 0 0 8 9 0 Natural causes 0 0 9 33 28 DSM Axis 1 Diagnosis Nil 0 0 17 42 0 Major depressive disorder 22 23 0 0 0 Depressive Disorder NOS 6 7 0 0 0 Dementia/possible dementia 0 0 0 0 28 DSM Axis 1 dependence 0 0 0 Nil 21 27 17 42 28 Substance 6 3 0 0 0 Alcohol 1 0 0 0 0 Psychotropic medications in last 3 months 15 9 17 20 0 Nil 0 6 0 2 0 Antidepressant 0 0 0 1 0 Antipsychotic 1 4 0 2 1 Benzodiazepine 1 1 0 0 0 Mood stabiliser 11 10 0 2 0 Antidepressant + another class Unknown 15 27 Braak stage 1.714 1.14 3.773 0.43 OFC Ab40 (ng/ml) 14.876 19.64 22.168 32.64 8.549 19.63 80.295 250.01 230.563 202.98 Kwallis p<0.001 OFC Ab42 (ng/ml) 42.777 63.46 265.668 692.21 21.251 25.32 520.9 892.79 3481.984 2382.29 Kwallis p<0.001 OFC Hb (g/dl) 0.293 0.3 0.299 0.3 0.375 0.44 0.314 0.24 0.216 0.16 Kwallis X1⁄43.23, p1⁄40.52 OFC MAG (ng/ml) 114.865 42.27 126.282 47.02 115.905 46.64 120.424 25.93 126.443 31.3 MAG:PLP ratio 16.21 3.61 13.80 3.40 13.32 2.95 15.59 3.74 15.49 4.23 ANOVA p1⁄40.036 OFC PLP (ng/ml) 7.252 3.02 9.582 4.92 10.468 10.54 8.235 2.78 8.491 2.29 OFC VEGF (ng/ml) 0.66 0.38 0.719 0.49 0.412 0.22 1.002 0.66 0.948 0.72 ANOVA p1⁄40.114 Poster Presentations: Wednesday, July 25, 2018 P1543

ABCA1 haplodeficiency affects the brain transcriptome following traumatic brain injury in mice expressing human APOE isoforms

Expression of human Apolipoprotein E (APOE) modulates the inflammatory response in an isoform specific manner, with APOE4 isoform eliciting a stronger pro-inflammatory response, suggesting a possible mechanism for worse outcome following traumatic brain injury (TBI). APOE lipidation and stability is modulated by ATP-binding cassette transporter A1 (ABCA1), a transmembrane protein that transports lipids and cholesterol onto APOE. We examined the impact of Abca1 deficiency and APOE isoform expression on the response to TBI using 3-months-old, human APOE3+/+ (E3/Abca1+/+) and APOE4+/+ (E4/Abca1+/+) targeted replacement mice, and APOE3+/+ and APOE4+/+ mice with only one functional copy of the Abca1 gene (E3/Abca1+/−; E4/Abca1+/−). TBI-treated mice received a craniotomy followed by a controlled cortical impact (CCI) brain injury in the left hemisphere; sham-treated mice received the same surgical procedure without the impact. We performed RNA-seq using samples from cortices and hippocampi followed by genome-wide differential gene expression analysis. We found that TBI significantly impacted unique transcripts within each group, however, the proportion of unique transcripts was highest in E4/Abca1+/− mice. Additionally, we found that Abca1 haplodeficiency increased the expression of microglia sensome genes among only APOE4 injured mice, a response not seen in injured APOE3 mice, nor in either group of sham-treated mice. To identify gene networks, or modules, correlated to TBI, APOE isoform and Abca1 haplodeficiency, we used weighted gene co-expression network analysis (WGCNA). The module that positively correlated to TBI groups was associated with immune response and featured hub genes that were microglia-specific, including Trem2, Tyrobp, Cd68 and Hexb. The modules positively correlated with APOE4 isoform and negatively to Abca1 haplodeficient mice represented “protein translation” and “oxidation-reduction process”, respectively. Our results reveal E4/Abca1+/− TBI mice have a distinct response to injury, and unique gene networks are associated with APOE isoform, Abca1 insufficiency and injury.

SHOTGUN LIPIDOMICS AND RNA-SEQ REVEAL EXTENSIVE CHANGES IN CARDIOLIPIN AND PHOSPHOLIPID PATHWAYS IN BRAINS OF APOEε3/3, APOEε3/4 AND APOEε4/4 ALZHEIMER'S DISEASE PATIENTS

Methods: Our subjects are from Religious Orders Study and the Rush Memory and Aging Project (ROS-MAP), two communitybased longitudinal cohort studies of aging and dementia that were designed for combined analyses. We assessed whether APOE ε4 allele count was associated with more advanced TDP-43 stages (stage 0 1⁄4 no inclusions; stage 1 1⁄4 inclusions in amygdala only; stage 2 1⁄4 stage 1 + entorhinal cortex or hippocampus CA1; stage 31⁄4 stage 2 + neocortex), using an ordinal logistic regression model adjusting for age, sex, and study cohort. Stratified analysis was done in subjects with and without pathologic diagnosis of AD. We used logistic regression models to examine the extent to which the effect of APOEε4 on AD dementia was attributable to AD (bamyloid, paired helical filament (PHF) tau) and TDP-43 pathologies. Results: In 1,084 deceased subjects from ROS-MAP, APOE ε4 was strongly associated with more advanced TDP-43 stage (odds ratio (OR) 1.9, 95% confidence interval (CI) 1.5–2.4, p1⁄44.4310). In stratified analyses, the association of APOE ε4 with TDP-43 stage was observed only in the subgroup with pathologic diagnosis of AD (OR1⁄41.7, 95% CI 1.3–2.3, p1⁄45.8310 (n1⁄4677); OR1⁄41.3, p1⁄40.32 for people without the diagnosis (n1⁄4387)). Of note, APOE ε4 count was marginally associated with higher likelihood of AD dementia, even after controlling for beta-amyloid load and PHF-tau tangle density (OR1⁄41.4, 95% CI 1.0–1.9, p1⁄40.049 (n1⁄4917)). However, this association was no longer significant when TDP-43 stage was included in the model (p1⁄40.13 (n1⁄4917)), suggesting that previously reported effect of APOEε4 in AD dementia is partially mediated by the increased burden of TDP-43 pathology. Conclusions:APOE ε4 is a risk factor of more advanced stages of TDP-43 proteinopathy, especially in the presence of AD pathology, and higher TDP-43 proteinopathy burden contributes to worse clinical outcome associated with APOE ε4.