RESUMO
X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift toward monounsaturated fatty acids relieved toxicity. In human ALD fibroblasts, chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts, whereas monounsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated toward monounsaturated VLCFA and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to monounsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.
Assuntos
Adrenoleucodistrofia/enzimologia , Cloroquina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Receptores X do Fígado/agonistas , Estearoil-CoA Dessaturase/biossíntese , Proteínas de Peixe-Zebra/metabolismo , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adrenoleucodistrofia/tratamento farmacológico , Adrenoleucodistrofia/genética , Animais , Linhagem Celular , Ácidos Graxos/metabolismo , Humanos , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Camundongos , Camundongos Knockout , Mutação , Estearoil-CoA Dessaturase/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed "ZEG" (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising.
