Emilie L. Castranio

Genome-wide approaches reveal EGR1-controlled regulatory networks associated with neurodegeneration

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 Alzheimers 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 Ca(2+) 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.

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.

Improvement of Memory Deficits and Amyloid-β Clearance in Aged APP23 Mice Treated with a Combination of Anti-Amyloid-β Antibody and LXR Agonist

Passive amyloid-β (Aβ) vaccination has shown significant effects on amyloid pathology in pre-depositing amyloid-β protein precursor (AβPP) mice but the results in older mice are inconsistent. A therapeutic effect of LXR and RXR agonists consisting of improved memory deficits and Aβ pathology has been demonstrated in different Alzheimers disease (AD) mouse models. Here, we report the effect of a combination of N-terminal Aβ antibody and synthetic LXR agonist T0901317 (T0) on AD-like phenotype of APP23 mice. To examine the therapeutic potential of this combination, the treatment of mice started at 11 months of age, when amyloid phenotype in this model is fully developed, and continued for 50 days. We show that Aβ immunization with or without LXR agonist restored the performance of APP23 transgenic mice in two behavior paradigms without affecting the existing amyloid plaques. Importantly, we did not observe an increase of brain microhemorrhage which is considered a significant side effect of Aβ vaccination. Target engagement was confirmed by increased Abca1 and ApoE protein level as well as increased ApoE lipidation in soluble brain extract. In interstitial fluid obtained by microdialysis, we demonstrate that immunization and T0 significantly reduced Aβ levels, indicating an increased Aβ clearance. We found no interaction between the immunotherapy and T0, suggesting no synergism, at least with these doses. The results of our study demonstrate that anti-Aβ treatments can ameliorate cognitive deficits in AβPP mice with advanced AD-like phenotype in conjunction with a decrease of Aβ in brain interstitium and increase of ApoE lipidation without affecting the existing amyloid plaques.

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.

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.

ABCA1 Deficiency Affects Basal Cognitive Deficits and Dendritic Density in Mice

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-free apolipoproteins and regulates the generation of high density lipoproteins. Previously, we have shown that lack of Abca1 significantly increases amyloid deposition and cognitive deficits in Alzheimer’s disease model mice expressing human amyloid-β protein precursor (APP). The goal of this study was to determine if ABCA1 plays a role in memory deficits caused by amyloid-β (Aβ) oligomers and examine neurite architecture of pyramidal hippocampal neurons. Our results confirm previous findings that Abca1 deficiency significantly impairs spatial memory acquisition and retention in the Morris water maze and long-term memory in novel object recognition of APP transgenic mice at a stage of early amyloid pathology. Neither test demonstrated a significant difference between Abca1ko and wild-type (WT) mice. We also examined the effect of intra-hippocampal infused Aβ oligomers on cognitive performance of Abca1ko mice, compared to control infusion of scrambled Aβ peptide. Age-matched WT mice undergoing the same infusions were also used as controls. In this model system, we found a statistically significant difference between WT and Abca1ko mice infused with scrambled Aβ, suggesting that Abca1ko mice are vulnerable to the effect of mild stresses. Moreover, examination of neurite architecture in the hippocampi revealed a significant decrease in neurite length, number of neurite segments, and branches in Abca1ko mice when compared to WT mice. We conclude that mice lacking ABCA1 have basal cognitive deficits that prevent them from coping with additional stressors, which is in part due to impairment of neurite morphology in the hippocampus.

EFFECTS OF APOLIPOPROTEIN E ON COGNITIVE PERFORMANCE AND AMYLOID OLIGOMERIZATION IN A MOUSE MODEL

medium (Gibco-Invitrogen, Carlsbad, CA) for 7 days. Cells were transiently transfected with 8 m g of neurutin subcloned in flag-IRES-EGFP vector, which was a generous gift from Dr. Nedivi at Massachusetts Institute of Technology, 9 m l CaCl 2, 58 m l DW and 75 m l 2 X Hank’s buffered solution (Invitrogen, Carlsbad, CA) in 1ml of growing medium according to the manufacturer’s instruction. At 48h post-transfection, the efficiencies of the transient transfections of plasmids were confirmed by the expression of GFP fluorescence. Fluorescence microscopic observation was performed using Olympus IX50 (Tokyo, Japan). Western blotting Protein was separated in SDS/PAGE gel, electrophoresed at 20w100 m g of protein/lane, and transferred onto a nitrocellulose membrane (Amersham Pharmacia, Buckinghamshire, UK). The protein blot was probed with anti-synaptophysin (Chemicon international, Temecula, CA) detected using horseradish peroxidase-conjugated secondary antibody (GE Healthcare AB, Sweden). Immunoreactive bands were visualized using an enhanced chemiluminescence system (ECL plus ; GE Healthcare AB, Sweden). BrdU staining BrdU (50mg/kg/day, Sigma) was injected intraperitoneally for 6 days into the SD rats with viral-mediated expression of EGFP or neuritin. H ippocampal regions were dissected from the brains isolated from the rats and processed for immunohistochemistry. Immunohistochemistry Fixed brains in 10% neutral buffered formalin for 48 h were dehydrated and embedded in paraffin. Before immunostaining, slides were deparaffinized by oven heating and immersion in xylene. After dehydration through graded alcohols to water, a primary antibody was revealed by incubating the cells for overnight with Cy3-conjugated secondary antibody (Molecular Probes, CA, USA). After three washes with permeabilization buffer and one wash with TBS, slices were mounted on microscope slides in mounting medium (DAKO, CA, USA). Frozen virus-injected wt, Tg2576 or SD rat brain block were sectioned 30 mm and placed into vial for free floating immunohistochemistry. Brain section inactivated endogenous peroxidase and incubated 2N HCl for 30 min at 37 C. They were blocked with 5% bovine serum albumin (BSA) in TBS with TritonX-100 and incubated primary and secondary antibody. Then they washed 3 times with TBS and placed onto glass slide to mount. Confocal microscopic observation was performed using Zeiss LSM510 (Jena, Germany). Fetal neural stem cell culture Fetal neural stem cell culture was performed as previously described [17]. Cells were isolated by mechanical dissociation in MEM (minimum essential medium) supplemented with 1M HEPES and 1% penicillin/streptomysin (pH 7.2), and 10 7 cells were cultured in a T-75 flask. The generation of neural spheres derived from fetal neural stem cells was performed as previously described [18] for immunocytochemistry analysis, we dissociated the neurospheres, and subcultured the fetal neural stem cells (5 x 10 4 cells/well) in 24-well plates that contained 12 mm glass coverslips (Marienfeld, Lauda-Konigshofen, Germany) pre-coated with laminin (Roche, Mannheim, Germany) at a concentration of 20 mg/ml in Minimum Essential Medium with 1M HEPES and penicillin/streptomycin. The cells were incubated in the differentiation medium without growth factors (EGF and bFGF) for 5 days and analyzed with immunocytochemistry Immunocytochemistry Cells were fixed in 4% paraformaldehyde for 30 min at room temperature and were them washed with phosphate buffered saline (PBS), and with PBS containing 0.05% Tween 20 for permeabilization. Next, cells were blocked wit blocking buffer (3% normal goat serum, 2% BSA and 3% Triton-X 100 in PBS) for 1h at room temperature. After washing, cells were incubated in PBS containing antibodies against mouse anti-Tuj-1 (1:100) and rabbit GFAP (1:200) for 2 h at room temperature. After the 2 h incubation, cells were washed with PBS, and then cells were incubated in PBS containing Alexa Flour 488 or 546conjugates secondary antibodies and 4’.6-diamidino-2-phenylindole (DAPI,1 m M) 1h at room temperature. Cells were then observed under a confocal microscope (Olympus FluoView 1000) Statistical Analysis Data are expressed as the means6 SEM values. One-way ANOVA and student’s t -test (SPSS, IL, USA) were used to determine statistical significancewhere p<0.05 was considered to be significant. Results: 1)Neutin is down-regulated in the brains of AD patients 2)Neutin attenuates learning and memory impairments in Tg2576 mice 3)Neuritin regulates neurogenesis in the dentate gyrus 4)Neuritin increases the neuronal population during the differentiation of neural stem cells Conclusions: Here we demonstrate that the expression of neuritin was significantly down-regulated in the hippocampus and cerebral cortex of AD patients compared to age-matched control subjects. We also demonstrate that viral-mediated expression of neuritin in the dentate gyrus of Tg2576 mice harboring the Swedish double mutated human APP695 gene attenuated the deficits in learning and memory.It was also found that the expression of neuritin up-regulates protein level of synaptophysin, a presynaptic marker, in primary hippocampal neuron cultures. In addition, NeuN/BrdU staining was up-regulated in the hippocampal region of the Sprawue-Dawley (SD) rats expressing neuritin. Furthermore, neuritin increased the neural differentiation in fetal neural stem cells. Taken together, our results suggest that neuritin possesses a therapeutic potential for AD by upregulating synaptogenesis and neurogenesis in brains.

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.

IMPACT OF APOE POLYMORPHISMS ON AMYLOID-BETA OLIGOMERIZATION AND ASSOCIATED COGNITIVE DECLINE

this C-terminal cleavage of APP in vivo by mutating the cleavage site, many mouse AD phenotypes were ameliorated. This work describes our progress toward discovering small molecule inhibitors of this destructive process. Methods: Using the D664 (APP695) cleavage site-specific antibody (APP delta C31, Enzo), we measured the level of N-terminal fragment resulting from this intracellular APP proteolysis. We developed an AlphaLISA assay for library screening purposes and a commercial ELISA for validation and research purposes for this C-terminal cleavage. For our library screening, the cell model system used was CHO cells stably transfected with APP770 (CHO-7W). We stimulated cleavage at the D739 caspase cleavage site (APP770) by treating these cells with cerevastatin and were able to reduce this cleavage and rescue cell death with the pan-caspase inhibitor Q-VD-OPh. An ex vivo I5 (hAPPwt) mouse hippocampal slice culture model was also utilized to validate active compounds. Results: Several classes and individual compounds were found to be effective at reducing C-terminal APP caspase cleavage and rescuing cell death in a dose-dependent manner. The most effective include SERCA inhibitors, inhibitors of Wnt signaling and calcium channel antagonists. Conclusions: We have identified and validated individual and classes of compounds that inhibit C-terminal APP caspase cleavage and rescue cell death in a dose–dependent manner. The resulting N-terminal APP fragment could be an important AD biomarker and blocking this cleavage could prove to be an important AD therapeutic approach.