Biology subjects

Barthelson, K.

Publications and source records attributed to Barthelson, K..

10 recordsLinked to original sources

bioRxiv2023

Transcriptome analysis of early-onset familial Alzheimer's disease and Sanfilippo syndrome zebrafish models reveals commonalities in disease mechanisms

Sanfilippo syndrome (mucopolysaccharidosis type III, MPSIII) causes childhood dementia, while Alzheimers 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 Alzheimers disease (EOfAD, psen1Q96_K97del/+) and MPSIIIB (nagluA603fs/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.

genetics↗

bioRxiv2023

Zebrafish models of Mucopolysaccharidosis types IIIA, B, & C show hyperactivity and changes in oligodendrocyte state

Sanfilippo syndrome, also known as mucopolysaccharidosis type III (MPS III), is a group of rare inherited lysosomal storage disorders which causes childhood dementia. 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, often in the second or third decades of life in the classical course of the disease. Knowledge of MPS III pathogenesis is incomplete, and no effective therapies are yet approved for human use. We generated hypomorphic mutations in the endogenous zebrafish genes orthologous to those associated with MPS IIIA, MPS IIIB and MPS IIIC. Our models display the primary MPS III disease signature of significant brain accumulation of HS, and behavioural analyses revealed hyperactivity phenotypes in MPS IIIA and MPS IIIB zebrafish. Brain transcriptome analysis revealed changes related to lysosomal, glycosaminoglycan and immune system biology in all three models but also distinct differences in brain transcriptome state between models, which may be mutation dependent. The transcriptome analysis also indicated marked disturbance of oligodendrocyte-related genes in the brains of MPS III zebrafish, supporting that effects on this cell type are a 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 zebrafishs extreme fecundity and accessible anatomy to dissect the pathological mechanisms in MPS IIIA, IIIB, and IIIC.

genetics↗

bioRxiv2023

Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis

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.

genetics↗

bioRxiv2022

pHAPE: a plasmid for production of DNA size marker ladders for gel electrophoresis

DNA size markers (also known as "molecular weight markers" or "DNA ladders") are an essential tool when using gel electrophoresis to identify and purify nucleic acids. However, the cost of these DNA ladders is not insignificant and, over time, impinges on the funds available for research and training in molecular biology. Here, we describe a method for the generation of "pHAPE", a plasmid from which a variety of DNA ladders can be generated via simple restriction enzyme digestions. The pHAPE plasmid can be generated by mutagenesis of the commonly used pBluescript II SK+ phagemid followed by insertion of a custom 7,141 bp sequence (made up of three smaller fragments). Our use of pHAPE allows us some small relief from the ever-rising costs of performing molecular biology experiments ("Dont worry, pHAPE").

synthetic biology↗

bioRxiv2021

Differential effects of loss of park7 activity on Iron Responsive Element (IRE) gene sets: Implications for the role of iron dyshomeostasis in the pathophysiology of Parkinson's disease

Mutation of the gene PARK7 (DJ1) causes monogenic autosomal recessive Parkinsons 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 Alzheimers 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 HQ5IRE 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 HQ5IRE 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.

genetics↗

bioRxiv2021

Comparative analysis of Alzheimer's disease knock-in model brain transcriptomes implies changes to energy metabolism as a causative pathogenic stress

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 Alzheimers 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 brains 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 Alzheimers disease risk allele {varepsilon}4. Our results support a common molecular basis for initiation of the pathological processes leading to both early and late onset forms of Alzheimers disease and illustrate the utility of both zebrafish and knock-in, single EOfAD mutation models for understanding the causes of this disease.

neuroscience↗

bioRxiv2021

PRESENILIN 1 mutations causing early-onset familial Alzheimer's disease or familial acne inversa differ in their effects on genes facilitating energy metabolism and signal transduction

BackgroundThe most common cause of early-onset familial Alzheimers disease (EOfAD) is mutations in PRESENILIN 1 (PSEN1) allowing production of mRNAs encoding full-length, but mutant, proteins. In contrast, a single known frameshift mutation in PSEN1 causes familial acne inversa (fAI) without EOfAD. The molecular consequences of heterozygosity for these mutation types, and how they cause completely different diseases, remains largely unexplored. ObjectiveTo analyse brain transcriptomes of young adult zebrafish to identify similarities and differences in the effects of heterozygosity for psen1 mutations causing EOfAD or fAI. MethodsRNA sequencing was performed on mRNA isolated from the brains of a single family of 6-month-old zebrafish siblings either wild type or possessing a single, heterozygous EOfAD-like or fAI-like mutation in their endogenous psen1 gene. ResultsBoth mutations downregulate genes encoding ribosomal subunits, and upregulate genes involved in inflammation. Genes involved in energy metabolism appeared significantly affected only by the EOfAD-like mutation, while genes involved in Notch, Wnt and neurotrophin signalling pathways appeared significantly affected only by the fAI-like mutation. However, investigation of direct transcriptional targets of Notch signalling revealed possible increases in {gamma}-secretase activity due to heterozygosity for either psen1 mutation. Transcriptional adaptation due to the fAI-like frameshift mutation was evident. ConclusionWe observed both similar and contrasting effects on brain transcriptomes of the heterozygous EOfAD-like and fAI-like mutations. The contrasting effects may illuminate how these mutation types cause distinct diseases.

genetics↗

bioRxiv2020

Frameshift and frame-preserving mutations in zebrafish presenilin 2 affect different cellular functions in young adult brains

BackgroundMutations in PRESENILIN 2 (PSEN2) cause early disease onset familial Alzheimers 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. MethodsA frameshift mutation (psen2N140fs) and a reading frame-preserving mutation (psen2T141_L142delinsMISLISV) 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 analysed. Bioinformatics techniques were used to predict cellular functions affected by heterozygosity for each mutation. ResultsThe reading frame preserving 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 signalling, extracellular matrix receptor interactions and focal adhesion. Both mutations affected ribosomal protein gene expression but in opposite directions. ConclusionA frameshift and frame-preserving 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.

bioinformatics↗

bioRxiv2020

Transcriptome analysis of a protein-truncating mutation in sortilin-related receptor 1 associated with early-onset familial Alzheimer's disease indicates effects on mitochondrial and ribosome function in young-adult zebrafish brains

The early cellular stresses which eventually lead to Alzheimers disease (AD) remain poorly understood because we cannot access living, asymptomatic human AD brains for detailed molecular analyses. Sortilin-related receptor 1 (SORL1) encodes a multi-domain receptor protein genetically associated with both rare, early-onset familial AD (EOfAD) and common, sporadic late-onset AD (LOAD). SORL1 has been shown to play a role in the trafficking of the amyloid {beta} A4 precursor protein (APP) which is cleaved proteolytically to form one of the pathological hallmarks of AD, amyloid {beta} (A{beta}) peptide. However, the other functions of SORL1 are less well understood. Here, we employed a reverse genetics approach to characterise the effect of an EOfAD mutation in SORL1 using zebrafish as a model organism. We performed targeted mutagenesis to generate an EOfAD-like mutation in the zebrafish orthologue of SORL1, and performed RNA-sequencing on mRNA isolated from a family of fish either heterozygous for the EOfAD-like mutation or their wild type siblings and identified subtle effects on the expression of genes which likely indicate changes in mitochondrial and ribosomal function. These changes appear to be independent of changes to expression of APP-related proteins in zebrafish, and mitochondrial content.

neuroscience↗

bioRxiv2020

Brain transcriptome analysis reveals subtle effects on mitochondrial function and iron homeostasis of mutations in the SORL1 gene implicated in early onset familial Alzheimers disease

BackgroundTo prevent or delay the onset of Alzheimers disease (AD), we must understand its molecular basis. The great majority of AD cases arise sporadically with a late onset after 65 years of age (LOAD). However, rare familial cases of AD can occur due to dominant mutations in a small number of genes that cause an early onset prior to 65 years of age (EOfAD). As EOfAD and LOAD share similar pathologies and disease progression, analysis of EOfAD genetic models may give insight into both subtypes of AD. Sortilin-related receptor 1 (SORL1) is genetically associated with both EOfAD and LOAD and provides a unique opportunity to investigate the relationships between both forms of AD. Currently, the role of SORL1 mutations in AD pathogenesis is unclear. MethodsTo understand the molecular consequences of SORL1 mutation, we performed targeted mutagenesis of the orthologous gene in zebrafish. We generated an EOfAD-like mutation, V1482Afs, and a putatively null mutation, to investigate whether EOfAD-like mutations in sorl1 display haploinsufficiency by acting through loss-of-function mechanisms. We performed mRNA-sequencing on whole brains comparing normal (wild type) fish with their siblings heterozygous for EOfAD-like or complete loss-of-function mutations in sorl1 or transheterozygous for these mutations. Differential gene expression and gene set enrichment analyses identified, respectively, changes in young adult zebrafish brain transcriptomes, and putative effects on neural subcellular functions. ResultsWe identified subtle effects on expression of genes involved in energy production, mRNA translation and mTORC1 signalling in both the EOfAD-like and null mutant brains, implying that these effects are due to sorl1 haploinsufficiency. Surprisingly, we also observed changes to expression of genes occurring only in the EOfAD-mutation carrier brains, suggesting gain-of-function effects. Transheterozygosity for the EOfAD-like and null mutations (i.e. lacking wild type sorl1), caused apparent effects on iron homeostasis and other transcriptome changes distinct from the single-mutation heterozygous fish. ConclusionsOur results provide insight into the possible early brain molecular effects of an EOfAD mutation in human SORL1. Differential effects of heterozygosity and complete loss of normal SORL1 expression are revealed.

neuroscience↗