Florent Letronne

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.

ADAM30 Downregulates APP-Linked Defects Through Cathepsin D Activation in Alzheimer's Disease

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimers disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimers disease 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.

Involvement of IL-33 in Alzheimer's disease

tides (Ab) and biometals in the brain represent a major pathogenic pathway in Alzheimer’s disease (AD) and provide the basis for clinical effective disease-modifying therapy (Duce and Bush, 2010; Lannfelt, 2008). Mapping the micro anatomical distribution of essential and trace elements and isotopes in the brain is essential for understanding AD pathobiology and designing new disease-modifying therapies. To date, mapping the brain metallome has been limited by technical barriers. Here we developed a new technique, High-Resolution Metallomic Imaging Mass Spectrometry (HRMIMS), to perform the first high-resolution multi-elemental and isotopic distribution maps of the brain metallome in the Tg2576 transgenic AD mouse model compared to wild-type control mice. Methods: Mice were procured from the Boston University Alzheimer’s Disease Center Transgenic Mouse Facility. Mouse brains were flash frozen and analyzed by metallomic imaging mass spectrometry (MIMS mapping) using a custom cryogenic cell coupled to nanosecond ultraviolet laser ablation (NULA) and hyphenated high-resolution magnetic sector field ICP-MS at the Boston University Center for Biometals & Metallomics, Boston, MA. Laser wavelength, 213 nm; rate, 5-50 mms-1; spot size: 10 micron (high resolution) to 100 micron (scanning resolution). MIMS analysis was conducted on surround to establish elemental background and calibrated with NIST standards. Results: High resolution metallomic maps generated from Tg2576 (Tg) and wild-type (Wt) mouse brain demonstrated unique elemental and isotopic distribution patterns. Zinc (Zn) brain maps revealed a distinctive distribution pattern marked by cortical lamination, prominent allocortical (hippocampus, amygdala) deposition, and isotopic (67Zn, 70Zn). This pattern was progressively disrupted in Tg mice as a function of age with abnormal Zn accumulation co-localizing with amyloid plaque and Timm’s staining. Simultaneous metallomic maps of the same Wt and Tg brains revealed distinctive elemental and isotopic distribution patterns for other important biometals, including copper (Cu), iron (Fe), selenium (Se), molybdenum (Mo), manganese (Mn), and others. Conclusions: We deployed HR-MIMS analysis to generate detailed quantitative high-resolution spatial distribution maps of essential and trace elements and isotopes in Tg2576 transgenic and wild-type mouse brain at 10-100 micron spatial resolution. This study strongly supports a role for zinc in AD-linked brain pathology.