The Zebrafish equivalent of Alzheimer's disease-associated PRESENILIN Isoform PS2V regulates inflammatory and other responses to hypoxic stress

Dominant mutations in the PRESENILIN genes PSEN1 and PSEN2 cause familial Alzheimer's disease (fAD) that usually shows onset before 65 years of age. In contrast, genetic variation at the PSEN1 and PSEN2 loci does not appear to contribute to risk for the sporadic, late onset form of the disease (sAD), leading to doubts that these genes play a role in the majority of AD cases. However, a truncated isoform of PSEN2, PS2V, is upregulated in sAD brains and is induced by hypoxia and high cholesterol intake. PS2V can increase γ-secretase activity and suppress the unfolded protein response (UPR), but detailed analysis of its function has been hindered by lack of a suitable, genetically manipulable animal model since mice and rats lack this PRESENILIN isoform. We recently showed that zebrafish possess an isoform, PS1IV, that is cognate to human PS2V. Using an antisense morpholino oligonucleotide, we can block specifically the induction of PS1IV that normally occurs under hypoxia. Here, we exploit this ability to identify gene regulatory networks that are modulated by PS1IV. When PS1IV is absent under hypoxia-like conditions, we observe changes in expression of genes controlling inflammation (particularly sAD-associated IL1B and CCR5), vascular development, the UPR, protein synthesis, calcium homeostasis, catecholamine biosynthesis, TOR signaling, and cell proliferation. Our results imply an important role for PS2V in sAD as a component of a pathological mechanism that includes hypoxia/oxidative stress and support investigation of the role of PS2V in other diseases, including schizophrenia, when these are implicated in the pathology.Esmaeil Ebrahimie, Seyyed Hani Moussavi Nik, Morgan Newman, Mark Van Der Hoek and Michael Lardell

Identification and expression analysis of the zebrafish orthologues of the mammalian MAP1LC3 gene family

Autophagy is the principle pathway within cells involved in clearing damaged proteins and organelles. Therefore autophagy is necessary to maintain the turnover balance of peptides and homoeostasis. Autophagy occurs at basal levels under normal conditions but can be upregulated by chemical inducers or stress conditions. The zebrafish (Danio rerio) serves as a versatile tool to understand the functions of genes implicated in autophagy. We report the identification of the zebrafish orthologues of mammalian genes MAP1LC3A (map1lc3a) and MAP1LC3B (map1lc3b) by phylogenetic and conserved synteny analysis and we examine their expression during embryonic development. The zebrafish map1lc3a and map1lc3b genes both show maternally contributed transcripts in early embryogenesis. However, levels of map1lc3a transcript steadily increase until at least 120h post-fertilisation while the levels of map1lc3b show a more variable pattern across developmental time. We have also validated the LC3I ratio/LC3I immunoblot autophagy assay in the presence of chloroquine (a lysosomal proteolysis inhibitor). We found that the LC3II/LC3I ratio is significantly increased in the presence of sodium azide with chloroquine supporting that hypoxia induces autophagy in zebrafish. This was supported by our qPCR assay that showed increased map1lc3a transcript levels in the presence of sodium azide. In contrast, levels of map1lc3b transcripts were reduced in the presence of rapamycin but the decrease in the presence of sodium azide did not reach statistical significance. Our study supports the use of zebrafish for analysing the interplay between hypoxia, development and autophagy.Swamynathan Ganesan, Seyyed Hani Moussavi Nik, Morgan Newman, Michael Lardell

Gene Ontology-based analysis of zebrafish’ omics data using the web tool Comparative Gene Ontology

Gene Ontology (GO) analysis is a powerful tool in systems biology, which uses a defined nomenclature to annotate genes/proteins within three categories: ‘‘Molecular Function,’’ ‘‘Biological Process,’’ and ‘‘Cellular Component.’’ GOanalysis can assist in revealing functionalmechanisms underlying observed patterns in transcriptomic, genomic, and proteomic data. The already extensive and increasing use of zebrafish for modeling genetic and other diseases highlights the need to develop a GO analytical tool for this organism. The web tool Comparative GO was originally developed for GO analysis of bacterial data in 2013 (www.comparativego.com). We have now upgraded and elaborated this web tool for analysis of zebrafish genetic data using GOs and annotations from the Gene Ontology Consortium.Esmaeil Ebrahimie, Mario Fruzangohar, Seyyed Hani Moussavi-Nik, and Morgan Newma

Accelerated brain aging towards transcriptional inversion in a zebrafish model of the K115fs mutation of human PSEN2

BACKGROUND:The molecular changes involved in Alzheimer's disease (AD) progression remain unclear since we cannot easily access antemortem human brains. Some non-mammalian vertebrates such as the zebrafish preserve AD-relevant transcript isoforms of the PRESENILIN genes lost from mice and rats. One example is PS2V, the alternative transcript isoform of the PSEN2 gene. PS2V is induced by hypoxia/oxidative stress and shows increased expression in late onset, sporadic AD brains. A unique, early onset familial AD mutation of PSEN2, K115fs, mimics the PS2V coding sequence suggesting that forced, early expression of PS2V-like isoforms may contribute to AD pathogenesis. Here we use zebrafish to model the K115fs mutation to investigate the effects of forced PS2V-like expression on the transcriptomes of young adult and aged adult brains. METHODS:We edited the zebrafish genome to model the K115fs mutation. To explore its effects at the molecular level, we analysed the brain transcriptome and proteome of young (6-month-old) and aged (24-month-old) wild type and heterozygous mutant female sibling zebrafish. Finally, we used gene co-expression network analysis (WGCNA) to compare molecular changes in the brains of these fish to human AD. RESULTS:Young heterozygous mutant fish show transcriptional changes suggesting accelerated brain aging and increased glucocorticoid signalling. These early changes precede a transcriptional 'inversion' that leads to glucocorticoid resistance and other likely pathological changes in aged heterozygous mutant fish. Notably, microglia-associated immune responses regulated by the ETS transcription factor family are altered in both our zebrafish mutant model and in human AD. The molecular changes we observe in aged heterozygous mutant fish occur without obvious histopathology and possibly in the absence of Aβ. CONCLUSIONS:Our results suggest that forced expression of a PS2V-like isoform contributes to immune and stress responses favouring AD pathogenesis. This highlights the value of our zebrafish genetic model for exploring molecular mechanisms involved in AD pathogenesis.Nhi Hin, Morgan Newman, Jan Kaslin, Alon M. Douek, Amanda Lumsden, Seyed Hani Moussavi Nik ... et al

The Guinea Pig as a model for sporadic Alzheimer's Disease (AD): the impact of cholesterol intake on expression of AD-related genes

Extent: 12p.We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes.Mathew J. Sharman, Seyyed H. Moussavi Nik, Mengqi M. Chen, Daniel Ong, Linda Wijaya, Simon M. Laws, Kevin Taddei, Morgan Newman, Michael Lardelli, Ralph N. Martins, Giuseppe Verdil

Identification of additional risk loci for stroke and small vessel disease: a meta-analysis of genome-wide association studies

BACKGROUND: Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. METHODS: For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10(-6)) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10(-8)), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. FINDINGS: We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05-1·12, p=1·48 × 10(-8); minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity-a marker of cerebral small vessel disease-in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2-32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b(-/-) cerebral vessels show decreased smooth muscle cell and pericyte coverage. INTERPRETATION: We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms

The response of HMGA1 to changes in oxygen availability is evolutionarily conserved

Zebrafish embryos have evolved to cope with hypoxia during development. This includes the ability to completely suspend embryo development for extended periods until normoxia is restored. However, only a limited number of studies have examined the gene regulatory responses of zebrafish embryos to hypoxia. The High Mobility Group A1 protein encoded by the mammalian gene HMGA1 is widely expressed during embryo development but not in adults. Its expression can be induced in adult neurons by hypoxia/oxidative stress and it is commonly reactivated in many types of cancer. We report the identification by phylogenetic and conserved synteny analyses of an HMGA1 orthologue in zebrafish, hmga1 (hmg-i/y) and analysis of sodium azide as a chemical agent for inducing hypoxia-like responses in zebrafish embryos including temporary suspension of development ("suspended animation"). Evidence was only found for the existence of the "a" isoform of HMGA1 in fish. The "b" and "c" isoforms were not detected. We show that zebrafish hmga1 is expressed in a manner similar to in mammals including its induction by hypoxia during hatching stage and in adult zebrafish brain. However, earlier during development, hypoxia causes a decrease in hmga1 transcript levels. By analysis of conservation of the HMGA1a isoform binding site in zebrafish psen2 gene transcripts, we predict that a zebrafish equivalent of the PS2V isoform of human PSEN2 is not formed and we support this by RT-PCR analyses. Thus, analysis of hmga1 function in zebrafish embryogenesis may be valuable for understanding its wider role in vertebrate development, cancer and cellular responses to hypoxia but not for analysis of the action of HMGA1 in PS2V formation.Seyyed Hani Moussavi Nik, Morgan Newman, Michael Lardell

Analysis of nicastrin gene phylogeny and expression in zebrafish

NICASTRIN is a component of the aspartyl protease γ-secretase complex which is involved in intramembranous cleavage of type I transmembrane proteins, notably the Notch receptor proteins and the AMYLOID BETA A4 PRECURSOR PROTEIN (APP). This study aimed to characterize the orthologue of the human NICASTRIN (NCSTN) gene in zebrafish, an advantageous model organism for the study of human disease. Zebrafish Nicastrin protein was predicted to possess the conserved glutamate 333 residue and DYIGS motif of human NCSTN that are important for substrate recognition processing in γ-secretase. Quantitative real-time RT-PCR revealed the profile of relative zebrafish nicastrin (ncstn) transcript levels in embryos at different times during development and in adult tissues. The analysis of synteny conservation revealed local rearrangements of ncstn and another gene, mpz, relative to copa, and pex19. In situ hybridization showed higher relative levels of ncstn transcripts in the developing brain and otic vesicles of embryos at 24 and 48 h post fertilization, respectively. Our observations are consistent with a role for Ncstn protein in Notch signaling within the proliferative ventricular zone of the developing central nervous system.Anne Lim, Seyyed Hani Moussavi Nik, Esmaeil Ebrahimie, Michael Lardell