Alzheimer’s disease (AD), the most common form of dementia, is recognized as a heterogeneous disease with diverse pathophysiologic mechanisms. In this study, we interrogate the molecular heterogeneity of AD by analyzing 1543 transcriptomes across five brain regions in two AD cohorts using an integrative network approach. We identify three major molecular subtypes of AD corresponding to different combinations of multiple dysregulated pathways, such as susceptibility to tau-mediated neurodegeneration, amyloid-β neuroinflammation, synaptic signaling, immune activity, mitochondria organization, and myelination. Multiscale network analysis reveals subtype-specific drivers such as GABRB2, LRP10, MSN, PLP1, and ATP6V1A. We further demonstrate that variations between existing AD mouse models recapitulate a certain degree of subtype heterogeneity, which may partially explain why a vast majority of drugs that succeeded in specific mouse models do not align with generalized human trials across all AD subtypes. Therefore, subtyping patients with AD is a critical step toward precision medicine for this devastating disease.

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.

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 bexaroteneactivated 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.

Early growth response gene 1 (Egr1) is a member of the immediate early gene (IEG) family of transcription factors and plays a role in memory formation. To identify EGR1 target genes in brain of Alzheimer's disease (AD) model mice - APP23, we applied chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq). Functional annotation of genes associated with EGR1 binding revealed a set of related networks including synaptic vesicle transport, clathrin-mediated endocytosis (CME), intracellular membrane fusion and transmission of signals elicited by Ca2+ influx. EGR1 binding is associated with significant enrichment of activating chromatin marks and appears enriched near genes that are up-regulated in the brains of APP23 mice. Among the putative EGR1 targets identified and validated in this study are genes related to synaptic plasticity and transport of proteins, such as Arc, Grin1, Syn2, Vamp2 and Stx6, and genes implicated in AD such as Picalm, Psen2 and App. We also demonstrate a potential regulatory link between EGR1 and its newly identified targets in vivo, since conditions that up-regulate Egr1 levels in brain, such as a spatial memory test, also lead to increased expression of the targets. On the other hand, protein levels of EGR1 and ARC, SYN2, STX6 and PICALM are significantly lower in the brain of adult APP mice than in age-matched wild type animals. The results of this study suggest that EGR1 regulates the expression of genes involved in CME, vesicular transport and synaptic transmission that may be critical for AD pathogenesis.

Traumatic brain injury (TBI) is strongly linked to an increased risk of developing dementia, including chronic traumatic encephalopathy and possibly Alzheimer’s 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 14 days 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.