The Use of Zebrafish in Transcriptome Analysis of the Early Effects of Mutations Causing Early Onset Familial Alzheimer's Disease and Other Inherited Neurodegenerative Conditions.

The degree to which non-human animals can be used to model Alzheimer's disease is a contentious issue, particularly as there is still widespread disagreement regarding the pathogenesis of this neurodegenerative dementia. The currently popular transgenic models are based on artificial expression of genes mutated in early onset forms of familial Alzheimer's disease (EOfAD). Uncertainty regarding the veracity of these models led us to focus on heterozygous, single mutations of endogenous genes (knock-in models) as these most closely resemble the genetic state of humans with EOfAD, and so incorporate the fewest assumptions regarding pathological mechanism. We have generated a number of lines of zebrafish bearing EOfAD-like and non-EOfAD-like mutations in genes equivalent to human PSEN1, PSEN2, and SORL1. To analyze the young adult brain transcriptomes of these mutants, we exploited the ability of zebrafish to produce very large families of simultaneous siblings composed of a variety of genotypes and raised in a uniform environment. This "intra-family" analysis strategy greatly reduced genetic and environmental "noise" thereby allowing detection of subtle changes in gene sets after bulk RNA sequencing of entire brains. Changes to oxidative phosphorylation were predicted for all EOfAD-like mutations in the three genes studied. Here we describe some of the analytical lessons learned in our program combining zebrafish genome editing with transcriptomics to understand the molecular pathologies of neurodegenerative disease.

Mitochondrial supplementation of Sus scrofa metaphase II oocytes alters DNA methylation and gene expression profiles of blastocysts.

BACKGROUND: Mitochondrial DNA (mtDNA) copy number in oocytes correlates with oocyte quality and fertilisation outcome. The introduction of additional copies of mtDNA through mitochondrial supplementation of mtDNA-deficient Sus scrofa oocytes resulted in: (1) improved rates of fertilisation; (2) increased mtDNA copy number in the 2-cell stage embryo; and (3) improved development of the embryo to the blastocyst stage. Furthermore, a subset of genes showed changes in gene expression. However, it is still unknown if mitochondrial supplementation alters global and local DNA methylation patterns during early development. RESULTS: We generated a series of embryos in a model animal, Sus scrofa, by intracytoplasmic sperm injection (ICSI) and mitochondrial supplementation in combination with ICSI (mICSI). The DNA methylation status of ICSI- and mICSI-derived blastocysts was analysed by whole genome bisulfite sequencing. At a global level, the additional copies of mtDNA did not affect nuclear DNA methylation profiles of blastocysts, though over 2000 local genomic regions exhibited differential levels of DNA methylation. In terms of the imprinted genes, DNA methylation patterns were conserved in putative imprint control regions; and the gene expression profile of these genes and genes involved in embryonic genome activation were not affected by mitochondrial supplementation. However, 52 genes showed significant differences in expression as demonstrated by RNAseq analysis. The affected gene networks involved haematological system development and function, tissue morphology and cell cycle. Furthermore, seven mtDNA-encoded t-RNAs were downregulated in mICSI-derived blastocysts suggesting that extra copies of mtDNA affected tRNA processing and/or turnover, hence protein synthesis in blastocysts. We also showed a potential association between differentially methylated regions and changes in expression for 55 genes due to mitochondrial supplementation. CONCLUSIONS: The addition of just an extra ~ 800 copies of mtDNA into oocytes can have a significant impact on both gene expression and DNA methylation profiles in Sus scrofa blastocysts by altering the epigenetic programming established during oogenesis. Some of these changes may affect specific tissue-types later in life. Consequently, it is important to determine the longitudinal effect of these molecular changes on growth and development before considering human clinical practice.

Iron Responsive Element-Mediated Responses to Iron Dyshomeostasis in Alzheimer's Disease.

BACKGROUND: Iron trafficking and accumulation is associated with Alzheimer's disease (AD) pathogenesis. However, the role of iron dyshomeostasis in early disease stages is uncertain. Currently, gene expression changes indicative of iron dyshomeostasis are not well characterized, making it difficult to explore these in existing datasets. OBJECTIVE: To identify sets of genes predicted to contain iron responsive elements (IREs) and use these to explore possible iron dyshomeostasis-associated gene expression responses in AD. METHODS: Comprehensive sets of genes containing predicted IRE or IRE-like motifs in their 3' or 5' untranslated regions (UTRs) were identified in human, mouse, and zebrafish reference transcriptomes. Further analyses focusing on these genes were applied to a range of cultured cell, human, mouse, and zebrafish gene expression datasets. RESULTS: IRE gene sets are sufficiently sensitive to distinguish not only between iron overload and deficiency in cultured cells, but also between AD and other pathological brain conditions. Notably, changes in IRE transcript abundance are among the earliest observable changes in zebrafish familial AD (fAD)-like brains, preceding other AD-typical pathologies such as inflammatory changes. Unexpectedly, while some IREs in the 3' untranslated regions of transcripts show significantly increased stability under iron deficiency in line with current assumptions, many such transcripts instead display decreased stability, indicating that this is not a generalizable paradigm. CONCLUSION: Our results reveal IRE gene expression changes as early markers of the pathogenic process in fAD and are consistent with iron dyshomeostasis as an important driver of this disease. Our work demonstrates how differences in the stability of IRE-containing transcripts can be used to explore and compare iron dyshomeostasis-associated gene expression responses across different species, tissues, and conditions.

Transcriptome analyses of 7-day-old zebrafish larvae possessing a familial Alzheimer's disease-like mutation in psen1 indicate effects on oxidative phosphorylation, ECM and MCM functions, and iron homeostasis.

BACKGROUND: Early-onset familial Alzheimer's disease (EOfAD) is promoted by dominant mutations, enabling the study of Alzheimer's disease (AD) pathogenic mechanisms through generation of EOfAD-like mutations in animal models. In a previous study, we generated an EOfAD-like mutation, psen1Q96_K97del, in zebrafish and performed transcriptome analysis comparing entire brains from 6-month-old wild type and heterozygous mutant fish. We identified predicted effects on mitochondrial function and endolysosomal acidification. Here we aimed to determine whether similar effects occur in 7 day post fertilization (dpf) zebrafish larvae that might be exploited in screening of chemical libraries to find ameliorative drugs. RESULTS: We generated clutches of wild type and heterozygous psen1Q96_K97del 7 dpf larvae using a paired-mating strategy to reduce extraneous genetic variation before performing a comparative transcriptome analysis. We identified 228 differentially expressed genes and performed various bioinformatics analyses to predict cellular functions. CONCLUSIONS: Our analyses predicted a significant effect on oxidative phosphorylation, consistent with our earlier observations of predicted effects on ATP synthesis in adult heterozygous psen1Q96_K97del brains. The dysregulation of minichromosome maintenance protein complex (MCM) genes strongly contributed to predicted effects on DNA replication and the cell cycle and may explain earlier observations of genome instability due to PSEN1 mutation. The upregulation of crystallin gene expression may be a response to defective activity of mutant Psen1 protein in endolysosomal acidification. Genes related to extracellular matrix (ECM) were downregulated, consistent with previous studies of EOfAD mutant iPSC neurons and postmortem late onset AD brains. Also, changes in expression of genes controlling iron ion transport were observed without identifiable changes in the prevalence of transcripts containing iron responsive elements (IREs) in their 3' untranslated regions (UTRs). These changes may, therefore, predispose to the apparent iron dyshomeostasis previously observed in 6-month-old heterozygous psen1Q96_K97del EOfAD-like mutant brains.

Accelerated loss of hypoxia response in zebrafish with familial Alzheimer's disease-like mutation of presenilin 1.

Ageing is the major risk factor for Alzheimer's disease (AD), a condition involving brain hypoxia. The majority of early-onset familial AD (EOfAD) cases involve dominant mutations in the gene PSEN1. PSEN1 null mutations do not cause EOfAD. We exploited putative hypomorphic and EOfAD-like mutations in the zebrafish psen1 gene to explore the effects of age and genotype on brain responses to acute hypoxia. Both mutations accelerate age-dependent changes in hypoxia-sensitive gene expression supporting that ageing is necessary, but insufficient, for AD occurrence. Curiously, the responses to acute hypoxia become inverted in extremely aged fish. This is associated with an apparent inability to upregulate glycolysis. Wild-type PSEN1 allele expression is reduced in post-mortem brains of human EOfAD mutation carriers (and extremely aged fish), possibly contributing to EOfAD pathogenesis. We also observed that age-dependent loss of HIF1 stabilization under hypoxia is a phenomenon conserved across vertebrate classes.

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 Aß. 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.

Brain transcriptome analysis of a familial Alzheimer's disease-like mutation in the zebrafish presenilin 1 gene implies effects on energy production.

To prevent or ameliorate Alzheimer's disease (AD) we must understand its molecular basis. AD develops over decades but detailed molecular analysis of AD brains is limited to postmortem tissue where the stresses initiating the disease may be obscured by compensatory responses and neurodegenerative processes. Rare, dominant mutations in a small number of genes, but particularly the gene PRESENILIN 1 (PSEN1), drive early onset of familial AD (EOfAD). Numerous transgenic models of AD have been constructed in mouse and other organisms, but transcriptomic analysis of these models has raised serious doubts regarding their representation of the disease state. Since we lack clarity regarding the molecular mechanism(s) underlying AD, we posit that the most valid approach is to model the human EOfAD genetic state as closely as possible. Therefore, we sought to analyse brains from zebrafish heterozygous for a single, EOfAD-like mutation in their PSEN1-orthologous gene, psen1. We previously introduced an EOfAD-like mutation (Q96_K97del) into the endogenous psen1 gene of zebrafish. Here, we analysed transcriptomes of young adult (6-month-old) entire brains from a family of heterozygous mutant and wild type sibling fish. Gene ontology (GO) analysis implies effects on mitochondria, particularly ATP synthesis, and on ATP-dependent processes including vacuolar acidification.