Sideroblastic anemias are acquired or inherited anemias that result in a decreased ability to synthesize hemoglobin in red blood cells and result in the presence of iron deposits in the mitochondria ...of red blood cell precursors. A common subtype of congenital sideroblastic anemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatment for SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupled with iron chelation. The function of SLC25A38 is not known. Here we report that the SLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transporters required for the initiation of heme synthesis. To do so, we took advantage of the fact that mitochondrial glycine has several roles beyond the synthesis of heme, including the synthesis of folate derivatives through the glycine cleavage system. The data were consistent with Hem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25 family member Ymc1, as a potential secondary mitochondrial glycine importer. Based on these findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid (5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able to restore heme levels to normal in yeast cells lacking Hem25 function. While neither glycine nor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafish model, we determined that the addition of folate with glycine was able to restore hemoglobin levels. This difference is likely due to the fact that yeast can synthesize folate, whereas in zebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerability of glycine and folate in humans, this study points to a potential novel treatment for SLC25A38 congenital sideroblastic anemia.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (
) to generate ...frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in
,
and
genes. An insertion of seven nucleotides in
resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in
, suggesting activity of alternative translation sites in the other two mutants even though
exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.
Abstract
We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded ...oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.
While adult zebrafish, Danio rerio, possess ammonia and urea transporters (Rh and UT proteins, respectively) in a number of tissues, they are most heavily concentrated within the gills. UT has a ...diffuse expression pattern within Na+-K+-ATPase (NKA)-type mitochondrion-rich cells and Rh proteins form a network similar to the arrangement seen in pufferfish gills (Nakada et al., 2007b). Rhag expression appeared to be limited to the pillar cells lining the blood spaces of the lamellae while Rhbg was localized to the outer layer of both the lamellae and the filament, upon the pavement cells. Exposure to high external ammonia (HEA) or phloretin increased tissue levels of ammonia and urea, respectively, in adult and juvenile zebrafish; however, the responses to these stressors were age dependent. HEA increased mRNA levels for a number of Rh proteins in embryos and larvae but did not elicit similar effects in adult gills, which appear to compensate for the unfavourable ammonia excretory gradient by increasing expression of V-type H+-ATPase. Phloretin exposure increased UT mRNA levels in embryos and larvae but was without effect in adult gill tissue. Surprisingly, in both adults and juveniles, HEA increased the mRNA expression of UT and phloretin increased the mRNA expression of Rh proteins. These results imply that, in zebrafish, there may be a tighter link between ammonia and urea excretion than is thought to occur in most teleosts.
Cryptococcal meningoencephalitis (CM) is a devastating fungal disease with high morbidity and mortality. The current regimen that is standard-of-care involves a combination of three different drugs ...administered for up to one year. There is a critical need for new therapies due to both toxicity and inadequate fungicidal activity of the currently available antifungal drugs. ATI-2307 is a novel aryl amidine that disrupts the mitochondrial membrane potential and inhibits the respiratory chain complexes of fungi-it thus represents a new mechanism for direct antifungal action. Furthermore, ATI-2307 selectively targets fungal mitochondria via a fungal-specific transporter that is not present in mammalian cells. It has very potent
anticryptococcal activity. In this study, the efficacy of ATI-2307 was tested in a rabbit model of CM. ATI-2307 demonstrated significant fungicidal activity at dosages between 1 and 2 mg/kg/d, and these results were superior to fluconazole and similar to amphotericin B treatment. When ATI-2307 was combined with fluconazole, the antifungal effect was greater than either therapy alone. While ATI-2307 has potent anticryptococcal activity in the subarachnoid space, its ability to reduce yeasts in the brain parenchyma was relatively less over the same study period. This new drug, with its unique mechanism of fungicidal action and ability to positively interact with an azole, has demonstrated sufficient anticryptococcal potential in this experimental setting to be further evaluated in clinical studies.
Dose-limiting toxicities for cisplatin administration, including ototoxicity and nephrotoxicity, impact the clinical utility of this effective chemotherapy agent and lead to lifelong complications, ...particularly in pediatric cancer survivors. Using a two-pronged drug screen employing the zebrafish lateral line as an in vivo readout for ototoxicity and kidney cell-based nephrotoxicity assay, we screened 1280 compounds and identified 22 that were both oto- and nephroprotective. Of these, dopamine and L-mimosine, a plant-based amino acid active in the dopamine pathway, were further investigated. Dopamine and L-mimosine protected the hair cells in the zebrafish otic vesicle from cisplatin-induced damage and preserved zebrafish larval glomerular filtration. Importantly, these compounds did not abrogate the cytotoxic effects of cisplatin on human cancer cells. This study provides insights into the mechanisms underlying cisplatin-induced oto- and nephrotoxicity and compelling preclinical evidence for the potential utility of dopamine and L-mimosine in the safer administration of cisplatin.
CHARGE syndrome typically results from mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (
). CHD7 is involved in regulating neural crest development, which gives rise to ...tissues of the skull/face and the autonomic nervous system (ANS). Individuals with CHARGE syndrome are frequently born with anomalies requiring multiple surgeries and often experience adverse events post-anesthesia, including oxygen desaturations, decreased respiratory rates, and heart rate abnormalities. Central congenital hypoventilation syndrome (CCHS) affects ANS components that regulate breathing. Its hallmark feature is hypoventilation during sleep, clinically resembling observations in anesthetized CHARGE patients. Loss of
(paired-like homeobox 2b) underlies CCHS. Employing a
-null zebrafish model, we investigated physiologic responses to anesthesia and compared these to loss of
. Heart rates were lower in
mutants compared to the wild-type. Exposure to tricaine, a zebrafish anesthetic/muscle relaxant, revealed that
mutants took longer to become anesthetized, with higher respiratory rates during recovery.
mutant larvae demonstrated unique
expression patterns. The knockdown of
reduced larval heart rates similar to
mutants.
mutant fish are a valuable preclinical model to investigate anesthesia in CHARGE syndrome and reveal a novel functional link between CHARGE syndrome and CCHS.
Animal models for studying human disease are essential to the continuing evolution of medicine. Rodent models are attractive for the obvious similarities in development and genetic makeup compared ...with humans, but have cost and technical limitations. The zebrafish ( Danio rerio ) represents an ideal alternative vertebrate model of human disease because of its high conservation of genetic information and physiological processes, inexpensive maintenance, and optical clarity facilitating direct observation. This review highlights recent advances in understanding genetic disease states associated with the dynamic organelle, the mitochondrion, using the zebrafish. Mitochondrial diseases that have been replicated in the zebrafish include those affecting the nervous and cardiovascular systems, as well as red blood cell function. Gene silencing techniques, including morpholino knockdown and transcription activator-like (TAL)-effector endonucleases, have been exploited to demonstrate how loss of function can induce human disease-like states in zebrafish. Moreover, modeling mitochondrial diseases has been facilitated greatly by the creation of transgenic fish with fluorescently labeled mitochondria for in vivo visualization of these structures. In addition, behavioral assays have been developed to examine changes in motor activity and sensory responses, particularly in larval stages. Zebrafish are poised to advance our understanding of the pathogenesis of human mitochondrial diseases beyond the current state of knowledge and provide a key tool in the development of novel therapeutic approaches to treat these conditions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
CHARGE syndrome is linked to autosomal‐dominant mutations in the CHD7 gene and results in a number of physiological and structural abnormalities, including heart defects, hearing and vision loss, and ...gastrointestinal (GI) problems. Of these challenges, GI problems have a profound impact throughout an individual's life, resulting in increased morbidity and mortality. A homolog of CHD7 has been identified in the zebrafish, the loss of which recapitulates many of the features of the human disease. Using a morpholino chd7 knockdown model complemented by a chd7 null mutant zebrafish line, we examined GI structure, innervation, and motility in larval zebrafish. Loss of chd7 resulted in physically smaller GI tracts with normal epithelial and muscular histology, but decreased and disorganized vagal projections, particularly in the foregut. chd7 morphant larvae had significantly less ability to empty their GI tract of gavaged fluorescent beads, and this condition was only minimally improved by the prokinetic agents, domperidone and erythromycin, in keeping with mixed responses to these agents in patients with CHARGE syndrome. The conserved genetics and transparency of the zebrafish have provided new insights into the consequences of chd7 gene dysfunction on the GI system and cranial nerve patterning. These findings highlight the opportunity of the zebrafish to serve as a preclinical model for studying compounds that may improve GI motility in individuals with CHARGE syndrome.
A larval zebrafish model of CHARGE syndrome was gavage‐fed fluorescent green beads and the passage of the beads through the intestine was monitored for 24 h. Compared to normal larvae, these CHARGE fish were less able to pass the beads from their intestines. This suggests that intestinal motility is impaired in these fish and likely in individuals with CHARGE.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
β-Adrenergic receptors (βARs) are crucial for maintaining the rate and force of cardiac muscle contraction in vertebrates. Zebrafish (Danio rerio) have one β1AR gene and two β2AR genes (β2aAR and ...β2bAR). We examined the roles of these receptors in larval zebrafish in vivo by assessing the impact of translational gene knockdown on cardiac function. Zebrafish larvae lacking β1AR expression by morpholino knockdown displayed lower heart rates than control fish, whereas larvae deficient in both β2aAR and β2bAR expression exhibited significantly higher heart rates than controls. These results suggested a potential inhibitory role for one or both β2AR genes. By using cultured HEK293 cells transfected with zebrafish βARs, we demonstrated that stimulation with adrenaline or procaterol (a β2AR agonist) resulted in an increase in intracellular cAMP levels in cells expressing any of the three zebrafish βARs. In comparison with its human βAR counterpart, zebrafish β2aAR expressed in HEK293 cells appeared to exhibit a unique binding affinity profile for adrenergic ligands. Specifically, zebrafish β2aAR had a high binding affinity for phenylephrine, a classical α-adrenergic receptor agonist. The zebrafish receptors also had distinct ligand binding affinities for adrenergic agonists when compared with human βARs in culture, with zebrafish β2aAR being distinct from human β2AR and zebrafish β2bAR. Overall, this study provides insight into the function and evolution of both fish and mammalian β-adrenergic receptors.
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