Congenital anaemia associated with loss-of-function variants in DNA polymerase epsilon 1.

DNA polymerase epsilon (Pol ε), a component of the core replisome, is involved in DNA replication. Although genetic defects of Pol ε have been reported to cause immunodeficiency syndromes, its role in haematopoiesis remains unknown. Here, we identified compound heterozygous variants (p.[Asp1131fs];[Thr1891del]) in POLE, encoding Pol ε catalytic subunit A (POLE1), in siblings with a syndromic form of severe congenital transfusion-dependent anaemia. In contrast to Diamond-Blackfan anaemia, marked reticulocytopenia or marked erythroid hypoplasia was not found. Their bone marrow aspirates during infancy revealed erythroid dysplasia with strongly positive TP53 in immunostaining. Repetitive examinations demonstrated trilineage myelodysplasia within 2 years from birth. They had short stature and facial dysmorphism. HEK293 cell-based expression experiments and analyses of patient-derived induced pluripotent stem cells (iPSCs) disclosed a reduced mRNA level of Asp1131fs-POLE1 and defective nuclear translocation of Thr1891del-POLE1. Analysis of iPSCs showed compensatory mRNA upregulation of the other replisome components and increase of the TP53 protein, both suggesting dysfunction of the replisome. We created Pole-knockout medaka fish and found that heterozygous fishes were viable, but with decreased RBCs. Our observations expand the phenotypic spectrum of the Pol ε defect in humans, additionally providing unique evidence linking Pol ε to haematopoiesis.

LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition.

During embryonic development, primitive and definitive waves of hematopoiesis take place to provide proper blood cells for each developmental stage, with the possible involvement of epigenetic factors. We previously found that lysine-specific demethylase 1 (LSD1/KDM1A) promotes primitive hematopoietic differentiation by shutting down the gene expression program of hemangioblasts in an Etv2/Etsrp-dependent manner. In the present study, we demonstrated that zebrafish LSD1 also plays important roles in definitive hematopoiesis in the development of hematopoietic stem and progenitor cells. A combination of genetic approaches and imaging analyses allowed us to show that LSD1 promotes the egress of hematopoietic stem and progenitor cells into the bloodstream during the endothelial-to-hematopoietic transition. Analysis of compound mutant lines with Etv2/Etsrp mutant zebrafish revealed that, unlike in primitive hematopoiesis, this function of LSD1 was independent of Etv2/Etsrp. The phenotype of LSD1 mutant zebrafish during the endothelial-to-hematopoietic transition was similar to that of previously reported compound knockout mice of Gfi1/Gfi1b, which forms a complex with LSD1 and represses endothelial genes. Moreover, co-knockdown of zebrafish Gfi1/Gfi1b genes inhibited the development of hematopoietic stem and progenitor cells. We therefore hypothesize that the shutdown of the Gfi1/Gfi1b-target genes during the endothelial-to-hematopoietic transition is one of the key evolutionarily conserved functions of LSD1 in definitive hematopoiesis.

Design of a new self-assembling antioxidant nanomedicine to ameliorate oxidative stress in zebrafish embryos.

Oxidative stress, which is a persistent state of elevated reactive oxygen species (ROS), is implicated in the pathogeneses of several diseases, making antioxidant-based therapeutics the aptest intervention. Nevertheless, the clinical failure of conventional low-molecular-weight (LMW) antioxidants in oxidative stress-related diseases to yield favorable therapeutic outcomes and an increased mortality rate attributable to their poor pharmacokinetic characteristics, necessitates the development of alternative therapeutics. In light of this, we designed and synthesized a new amphiphilic polymer functionalized with a clinically safe base polymer of poly(styrene-co-maleic anhydride) copolymer conjugated with the LMW pleiotropic antioxidant TEMPO (a potent antioxidant) and biocompatible poly(ethylene glycol) (TEMPO-installed PSMA-g-PEG), which self-assembles into nano-sized micelles (SMAPoTN) under physiological conditions. We investigated its safety and antioxidant ability using zebrafish models. Common LMW antioxidants, such as 4-hydroxy-TEMPO (TEMPOL), vitamin C, N-acetyl-L-cysteine, and edaravone exposure induced phenotypic distortions, a manifestation of developmental toxicity, and resulted in high lethality in zebrafish larvae. LMW TEMPOL also adversely affected embryo hatchability, induced arrhythmia and cardiac edema, and failed to protect against oxidative stress. In contrast, exposure of zebrafish embryos to SMAPoTN increased the hatchability, protected embryos against various inducers of oxidative stress, and did not induce any phenotypic alterations or discernible toxicity. Taken together, we conclude that SMAPoTN surpasses LMW TEMPOL in terms of the ability to protect zebrafish, attributable to efficient ROS scavenging without perturbing normal redox homeostasis. These results imply that SMAPoTN can be used as a therapeutic intervention against various oxidative stress-induced diseases. STATEMENT OF SIGNIFICANCE: Failure of low molecular weight (LMW) antioxidants to improve therapeutic index in various oxidative stress-related pathogenesis, attributable to their poor pharmacokinetic characteristics, greatly limits their clinical translation. To overcome this limitation, we developed a self-assembling antioxidant nanoparticle (SMAPoTN) comprised of amphiphilic polymer; poly(styrene-co-maleic anhydride) conjugated with TEMPO as an antioxidant and biocompatible poly(ethylene glycol). Preliminary studies carried out in the in vivo models of zebrafish embryos confirmed that exposure of LMW antioxidant resulted in acute developmental toxicity, high lethality, and failure to rescue embryos against oxidative stress inducers. In contrast, SMAPoTN did not exert discernible toxicity and significantly improved their survival under oxidative stress. Our finding establishes antioxidant nanoparticles as more suitable therapeutic intervention for oxidative stress-induced diseases than LMW antioxidants.

Longitudinal investigation of a xenograft tumor zebrafish model using polarization-sensitive optical coherence tomography.

Breast cancer is a leading cause of death in female patients worldwide. Further research is needed to get a deeper insight into the mechanisms involved in the development of this devastating disease and to find new therapy strategies. The zebrafish is an established animal model, especially in the field of oncology, which has shown to be a promising candidate for pre-clinical research and precision-based medicine. To investigate cancer growth in vivo in zebrafish, one approach is to explore xenograft tumor models. In this article, we present the investigation of a juvenile xenograft zebrafish model using a Jones matrix optical coherence tomography (JM-OCT) prototype. Immunosuppressed wild-type fish at 1-month post-fertilization were injected with human breast cancer cells and control animals with phosphate buffered saline in the tail musculature. In a longitudinal study, the scatter, polarization, and vasculature changes over time were investigated and quantified in control versus tumor injected animals. A significant decrease in birefringence and an increase in scattering signal was detected in tumor injected zebrafish in comparison to the control once. This work shows the potential of JM-OCT as a non-invasive, label-free, three-dimensional, high-resolution, and tissue-specific imaging tool in pre-clinical cancer research based on juvenile zebrafish models.

Pathogenic variants in the survival of motor neurons complex gene GEMIN5 cause cerebellar atrophy.

Cerebellar ataxia is a genetically heterogeneous disorder. GEMIN5 encoding an RNA-binding protein of the survival of motor neuron complex, is essential for small nuclear ribonucleoprotein biogenesis, and it was recently reported that biallelic loss-of-function variants cause neurodevelopmental delay, hypotonia, and cerebellar ataxia. Here, whole-exome analysis revealed compound heterozygous GEMIN5 variants in two individuals from our cohort of 162 patients with cerebellar atrophy/hypoplasia. Three novel truncating variants and one previously reported missense variant were identified: c.2196dupA, p.(Arg733Thrfs*6) and c.1831G > A, p.(Val611Met) in individual 1, and c.3913delG, p.(Ala1305Leufs*14) and c.4496dupA, p.(Tyr1499*) in individual 2. Western blotting analysis using lymphoblastoid cell lines derived from both affected individuals showed significantly reduced levels of GEMIN5 protein. Zebrafish model for null variants p.(Arg733Thrfs*6) and p.(Ala1305Leufs*14) exhibited complete lethality at 2 weeks and recapitulated a distinct dysplastic phenotype. The phenotypes of affected individuals and the zebrafish mutant models strongly suggest that biallelic loss-of-function variants in GEMIN5 cause cerebellar atrophy/hypoplasia.

Splicing- and demethylase-independent functions of LSD1 in zebrafish primitive hematopoiesis.

LSD1/KDM1A is a widely conserved lysine-specific demethylase that removes methyl groups from methylated proteins, mainly histone H3. We previously isolated the zebrafish LSD1 gene and demonstrated that it is required for primitive hematopoiesis. Recently, a neuron-specific splicing variant of LSD1 was found in mammals and its specific functions and substrate specificities were reported. To our surprise, zebrafish LSD1 cDNA, which we previously analyzed, was corresponded to the neuron-specific variant in mammals. In this study, we investigated the structures and expression of LSD1 splicing variants in zebrafish and found all 4 types of LSD1 isoforms: LSD1, LSD1+2al, LSD1+8al and LSD1+2al8al. Interestingly, LSD1+8al/LSD1+2al8al, which correspond to mammalian neuron-specific variants, expressed ubiquitously in zebrafish. We also performed phenotypic rescue experiments of a zebrafish LSD1 mutant (kdm1ait627) using human and zebrafish LSD1 variants to identify which variant is involved in primitive hematopoiesis. Unexpectedly, the overexpression of all types of human and zebrafish variants was able to rescue the hematopoietic phenotypes in LSD1 mutants. Furthermore, enzymatic-deficient LSD1K661A (human) and K638A (zebrafish) were also able to rescue the mutant phenotypes. These results suggest that the LSD1 functions in zebrafish primitive hematopoiesis are free from any splicing-dependent regulation or demethylation reaction.