Karissa Barthelson

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when the effects of homozygosity for recessive mutations are studied in non-isogenic backgrounds, genes located proximal to the mutation on the same chromosome often appear over-represented among those genes identified as differentially expressed (DE). One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect functional responses to the mutation but, instead, result from an unequal distribution of expression quantitative trait loci (eQTLs) between sample groups of mutant or wild-type genotypes. This is problematic because eQTL expression differences are difficult to distinguish from genes that are DE due to functional responses to a mutation. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) are also observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between those sample groups subjected to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting-based approaches. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. Our method for DAR analysis requires only RNA-sequencing data, facilitating its application across new and existing datasets.

Background: The endo‐lysosomal pathway (ELP) of cells is central to their importation of nutrients and for recycling of cellular components. Much genetic evidence supports the importance of dysfunction of the ELP in Alzheimer’s disease (AD). However, AD is a complex disorder and how AD‐associated genetic variation affects ELP function is unclear. Sanfilippo syndrome childhood dementia is a consequence of extreme ELP dysfunction, where inherited recessive mutations result in the ELP’s inability to properly catabolise a particular polysaccharide. Unlike AD, Sanfilippo is genetically simple. All the causative genes have been identified and the biochemical consequences of their mutations are understood. Animal models of Sanfilippo are considered excellent models of the human disease. Using zebrafish as a model organism, we are dissecting shared disease mechanisms between these two dementia subtypes.Method: Zebrafish models of both (early‐onset familial) AD and Sanfilippo syndrome were generated using CRISPR‐Cas9‐based approaches. The brain transcriptomes (RNA‐seq) and metabolomes (metabolomics/fluxomics) of both models are compared within single zebrafish families. Spatial working memory in both models is assessed using a Y‐maze.Result: Preliminary brain transcriptome analyses have identified changes‐in‐common to both oxidative phosphorylation and ribosome gene expression in both AD and Sanfilippo model zebrafish. Metabolomics and behavioural analyses of these models are ongoing.Conclusion: Neurodegeneration in Sanfilippo syndrome and Alzheimer’s disease may share common mechanisms that are amenable to common therapeutic approaches.

Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII) causes childhood dementia, while Alzheimer’s disease is the most common type of adult-onset dementia. There is no cure for either of these diseases, and therapeutic options are extremely limited. Increasing evidence suggests commonalities in the pathogenesis of these diseases. However, a direct molecular-level comparison of these diseases has never been performed. Here, we exploited the power of zebrafish reproduction (large families of siblings from single mating events raised together in consistent environments) to conduct sensitive, internally controlled, comparative transcriptome and proteome analyses of zebrafish models of early-onset familial Alzheimer’s disease (EOfAD, psen1 ^Q96_K97del/+ ) and MPSIIIB ( naglu ^{A603fs/A603fs} ) within single families. We examined larval zebrafish (7 days post fertilisation), representing early disease stages. We also examined the brains of 6-month-old zebrafish, which are approximately equivalent to young adults in humans. We identified substantially more differentially expressed genes and pathways in MPS III zebrafish than in EOfAD-like zebrafish. This is consistent with MPS III being a rapidly progressing and earlier onset form of dementia. Similar changes in expression were detected between the two disease models in gene sets representing extracellular matrix receptor interactions in larvae, and the ribosome and lysosome pathways in 6-month-old adult brains. Cell type-specific changes were detected in MPSIIIB brains at 6 months of age, likely reflecting significant disturbances of oligodendrocyte, neural stem cell, and inflammatory cell functions and/or numbers. Our ‘omics analyses have illuminated similar disease pathways between EOfAD and MPS III indicating where efforts to find mutually effective therapeutic strategies can be targeted.

Sanfilippo syndrome childhood dementia, also known as mucopolysaccharidosis type III (MPS III), is a rare inherited lysosomal storage disorder. Subtypes of MPS III are caused by deficiencies in one of four enzymes required for degradation of the glycosaminoglycan heparan sulfate (HS). An inability to degrade HS leads to progressive neurodegeneration and death in the second or third decades of life. Knowledge of MPS III pathogenesis is incomplete, and no effective therapies exist. We generated the hypomorphic mutations sgsh^S387Lfs, naglu^A603Efs and hgsnat^G577Sfsin the endogenous zebrafish genes orthologous to human SGSH, NAGLU, and HGSNAT that are loci for mutations causing MPS III subtypes MPS IIIA, B and C respectively. Our models display the primary MPS III disease signature of significant brain accumulation of HS, while behavioural analyses support hyperactivity phenotypes. Brain transcriptome analysis revealed changes related to lysosomal, glycosaminoglycan, immune system and iron homeostasis biology in all three models but also distinct differences in brain transcriptome state between models. The transcriptome analysis also indicated marked disturbance of the oligodendrocyte cell state in the brains of MPS IIIA, B and C zebrafish, supporting that effects on this cell type are an early and consistent characteristic of MPS III. Overall, our zebrafish models recapture key characteristics of the human disease and phenotypes seen in mouse models. Our models will allow exploitation of the zebrafish’s extreme fecundity and accessible anatomy to dissect the pathological mechanisms both common and divergent between the MPS IIIA, B, and C subtypes.

In comparisons between mutant and wild-type genotypes, transcriptome analysis can reveal the direct impacts of a mutation, together with the homeostatic responses of the biological system. Recent studies have highlighted that, when homozygous mutations are studied in non-isogenic backgrounds, genes from the same chromosome as a mutation often appear over-represented among differentially expressed (DE) genes. One hypothesis suggests that DE genes chromosomally linked to a mutation may not reflect true biological responses to the mutation but, instead, result from differences in representation of expression quantitative trait loci (eQTLs) between sample groups selected on the basis of mutant or wild-type genotype. This is problematic when inclusion of spurious DE genes in a functional enrichment study results in incorrect inferences of mutation effect. Here we show that chromosomally co-located differentially expressed genes (CC-DEGs) can also be observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-sequencing data, localised differential allelic representation (DAR) between groups of samples subject to differential expression analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting of gene-level rankings. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs that are likely to be functionally related to a mutant phenotype. This supports a long-standing prediction that selection for advantageous linkage disequilibrium influences chromosome evolution. By comparing the genomes of zebrafish ( Danio rerio ) and medaka ( Oryzias latipes ), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. The DAR metric provides a solid foundation for addressing the eQTL issue in new and existing datasets because it relies solely on RNA-sequencing data.

Energy production is the most fundamentally important cellular activity supporting all other functions, particularly in highly active organs, such as brains. Here, we summarise transcriptome analyses of young adult (pre-disease) brains from a collection of 11 early-onset familial Alzheimer's disease (EOFAD)-like and non-EOFAD-like mutations in three zebrafish genes. The one cellular activity consistently predicted as affected by only the EOFAD-like mutations is oxidative phosphorylation, which produces most of the energy of the brain. All the mutations were predicted to affect protein synthesis. We extended our analysis to knock-in mouse models of APOE alleles and found the same effect for the late onset Alzheimer's disease risk allele ε4. Our results support a common molecular basis for the initiation of the pathological processes leading to both early and late onset forms of Alzheimer's disease, and illustrate the utility of zebrafish and knock-in single EOFAD mutation models for understanding the causes of this disease.

Mutation of the gene PARK7 (DJ1) causes monogenic autosomal recessive Parkinson’s disease (PD) in humans. Subsequent alterations of PARK7 protein function lead to mitochondrial dysfunction, a major element in PD pathology. Homozygous mutants for the PARK7-orthologous genes in zebrafish, park7, show changes to gene expression in the oxidative phosphorylation pathway, supporting that disruption of energy production is a key feature of neurodegeneration in PD. Iron is critical for normal mitochondrial function, and we have previously used bioinformatic analysis of IRE-bearing transcripts in brain transcriptomes to find evidence supporting the existence of iron dyshomeostasis in Alzheimer’s disease. Here, we analysed IRE-bearing transcripts in the transcriptome data from homozygous park7^−/− mutant zebrafish brains. We found that the set of genes with “high quality” IREs in their 5′ untranslated regions (UTRs, the HQ5′IRE gene set) was significantly altered in these 4-month-old park7^−/− brains. However, sets of genes with IREs in their 3′ UTRs appeared unaffected. The effects on HQ5′IRE genes are possibly driven by iron dyshomeostasis and/or oxidative stress, but illuminate the existence of currently unknown mechanisms with differential overall effects on 5′ and 3′ IREs.

Mutation of the gene PARK7 ( DJ1 ) causes monogenic autosomal recessive Parkinson’s disease (PD) in humans. Subsequent alterations of PARK7 protein function lead to mitochondrial dysfunction, a major element in PD pathology. Homozygous mutants for the PARK7 -orthologous genes in zebrafish, park7 , show changes to gene expression in the oxidative phosphorylation pathway, supporting that disruption of energy production is a key feature of neurodegeneration in PD. Iron is critical for normal mitochondrial function, and we have previously used bioinformatic analysis of IRE-bearing transcripts in brain transcriptomes to find evidence supporting the existence of iron dyshomeostasis in Alzheimer’s disease. Here, we analysed IRE-bearing transcripts in the transcriptome data from homozygous park7 ^−/− mutant zebrafish brains. We found that the set of genes with “high quality” IREs in their 5’ untranslated regions (UTRs, the HQ5’IRE gene set) was significantly altered in these 4-month-old park7 ^−/− brains. However, sets of genes with IREs in their 3’ UTRs appeared unaffected. The effects on HQ5’IRE genes are possibly driven by iron dyshomeostasis and/or oxidative stress, but illuminate the existence of currently unknown mechanisms with differential overall effects on 5’ and 3’ IREs.

Background: Mutations in PRESENILIN 2 ( PSEN2) cause early onset familial Alzheimer’s disease (EOfAD) but their mode of action remains elusive. One consistent observation for all PRESENILIN gene mutations causing EOfAD is that a transcript is produced with a reading frame terminated by the normal stop codon—the “reading frame preservation rule”. Mutations that do not obey this rule do not cause the disease. The reasons for this are debated. Objective: To predict cellular functions affected by heterozygosity for a frameshift, or a reading frame-preserving mutation in zebrafish psen2 using bioinformatic techniques. Methods: A frameshift mutation ( psen2 ^{N140 fs} ) and a reading frame-preserving (in-frame) mutation ( psen2 ^{T141 _  L142 delinsMISLISV} ) were previously isolated during genome editing directed at the N140 codon of zebrafish psen2 (equivalent to N141 of human PSEN2). We mated a pair of fish heterozygous for each mutation to generate a family of siblings including wild type and heterozygous mutant genotypes. Transcriptomes from young adult (6 months) brains of these genotypes were analyzed. Results: The in-frame mutation uniquely caused subtle, but statistically significant, changes to expression of genes involved in oxidative phosphorylation, long-term potentiation and the cell cycle. The frameshift mutation uniquely affected genes involved in Notch and MAPK signaling, extracellular matrix receptor interactions and focal adhesion. Both mutations affected ribosomal protein gene expression but in opposite directions. Conclusion: A frameshift and an in-frame mutation at the same position in zebrafish psen2 cause discrete effects. Changes in oxidative phosphorylation, long-term potentiation and the cell cycle may promote EOfAD pathogenesis in humans.

Summary: Energy production is the most fundamentally important cellular activity supporting all other functions, particularly in highly active organs such as brains. Here, we summarise transcriptome analyses of young adult (pre-disease) brains from a collection of eleven early-onset familial Alzheimer’s disease (EOfAD)-like and non-EOfAD-like mutations in three zebrafish genes. The one cellular activity consistently predicted as affected by only the EOfAD-like mutations is oxidative phosphorylation that produces most of the brain’s energy. All the mutations were predicted to affect protein synthesis. We extended our analysis to knock-in mouse models of APOE alleles and found the same effect for the late onset Alzheimer’s disease risk allele ɛ4. Our results support a common molecular basis for initiation of the pathological processes leading to both early and late onset forms of Alzheimer’s disease and illustrate the utility of both zebrafish and knock-in, single EOfAD mutation models for understanding the causes of this disease.