Dopaminergic Neuronal Loss and Dopamine-Dependent Locomotor Defects in Fbxo7-Deficient Zebrafish

Recessive mutations in the F-box only protein 7 gene (FBXO7) cause PARK15, a Mendelian form of early-onset, levodopa-responsive parkinsonism with severe loss of nigrostriatal dopaminergic neurons. However, the function of the protein encoded by FBXO7, and the pathogenesis of PARK15 remain unknown. No animal models of this disease exist. Here, we report the generation of a vertebrate model of PARK15 in zebrafish. We first show that the zebrafish Fbxo7 homolog protein (zFbxo7) is expressed abundantly in the normal zebrafish brain. Next, we used two zFbxo7-specific morpholinos (targeting protein translation and mRNA splicing, respectively), to knock down the zFbxo7 expression. The injection of either of these zFbxo7-specific morpholinos in the fish embryos induced a marked decrease in the zFbxo7 protein expression, and a range of developmental defects. Furthermore, whole-mount in situ mRNA hybridization showed abnormal patterning and significant decrease in the number of diencephalic tyrosine hydroxylase-expressing neurons, corresponding to the human nigrostriatal or ventral tegmental dopaminergic neurons. Of note, the number of the dopamine transporter-expressing neurons was much more severely depleted, suggesting dopaminergic dysfunctions earlier and larger than those due to neuronal loss. Last, the zFbxo7 morphants displayed severe locomotor disturbances (bradykinesia), which were dramatically improved by the dopaminergic agonist apomorphine. The severity of these morphological and behavioral abnormalities correlated with the severity of zFbxo7 protein deficiency. Moreover, the effects of the co-injection of zFbxo7- and p53-specific morpholinos were similar to those obtained with zFbxo7-specific morpholinos alone, supporting further the contention that the observed phenotypes were specifically due to the knock down of zFbxo7. In conclusion, this novel vertebrate model reproduces pathologic and behavioral hallmarks of human parkinsonism (dopaminergic neuronal loss and dopamine-dependent bradykinesia), representing therefore a valid tool for investigating the mechanisms of selective dopaminergic neuronal death, and screening for modifier genes and therapeutic compounds

Idh1-mutated transgenic zebrafish lines: An in-vivo model for drug screening and functional analysis

Introduction The gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants. Materials and methods IDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage. Results In total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncome-tabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity. Conclusions We have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation

Exome sequencing and functional analyses suggest that SIX6 is a gene involved in an altered proliferation-differentiation balance early in life and optic nerve degeneration at old age

Primary open-angle glaucoma (POAG) is a hereditary neurodegenerative disease, characterized by optic nerve changes including increased excavation, notching and optic disc hemorrhages. The excavation can be described by the vertical cup-disc ratio (VCDR). Previously, genome-wide significant evidence for the association of rs10483727 in SIX1-SIX6 locus with VCDR and subsequent POAG was found. Using 1000 genomes-based imputation of four independent population-based cohorts in the Netherlands, we identified a missense variant rs33912345 (His141Asn) in SIX6 associated with VCDR (Pmeta = 7.74 × 10-7, n = 11 473) and POAG (Pmeta = 6.09 × 10-3, n = 292). Exome sequencing analysis revealed another missense variant rs146737847 (Glu129Lys) also in SIX6 associated with VCDR (P = 5.09 × 10-3, n = 1208). These two findings point to SIX6 as the responsible gene for the previously reported association signal. Functional characterization of SIX6 in zebrafish revealed that knockdown of six6b led to a small eye phenotype. Histological analysis showed retinal lamination, implying an apparent normal development of the eye, but an underdeveloped lens, and reduced optic nerve diameter. Expression analysis of morphants at 3 dpf showed a 5.5-fold up-regulation of cdkn2b, a cyclin-dependent kinase inhibitor, involved in cell cycle regulation and previously associated with VCDR and POAG in genome-wide association studies (GWASs). Since both six6b and cdkn2b play a key role in cell proliferation, we assessed the proliferative activity in the eye of morphants and found an alteration in the proliferative pattern of retinal cells. Our findings in humans and zebrafish suggest a functional involvement of six6b in early eye development, and open new insights into the genetic architecture of POAG

Effect of Treatment in a Specialized Pediatric Hemato-Oncology Setting on 5-Year Survival in Acute Lymphoblastic Leukemia:A Quasi-Experimental Study

Survival rates of adolescents and young adults (AYAs) with acute lymphoblastic leukemia (ALL) are inferior to those of pediatric ALL patients. In part, this may be caused by differences in treatment setting. Generally, children are treated in specialized pediatric hemato-oncology settings, whereas AYAs are treated in adult hemato-oncology settings. Since 2005, adult treatment protocols have included pediatric-inspired chemotherapy, which has been the standard of care for AYAs from 2008 onwards. This study aims to assess whether, despite protocols in both settings having become more similar, there remains an effect of treatment in specialized pediatric hemato-oncology settings on 5-year survival for ALL patients in the Netherlands. We used nationwide registry data (2004–2013) on 472 ALL patients aged between 10 and 30 years old. A fuzzy regression discontinuity design was applied to estimate the treatment effect using two-stage least squares regression with the treatment threshold at 17 years and 7 months of age, adjusting for sex, age at diagnosis, and immunophenotype. We found a risk difference of 0.419 (p = 0.092; 95% CI = −0.0686; 0.907), meaning a 41.9 percentage point greater probability of surviving five years after diagnosis for ALL patients treated in specialized pediatric hemato-oncology settings. Our results suggest that ALL patients around the threshold could benefit from increased collaboration between pediatric and adult hemato-oncology in terms of survival.</p

Reduction of oxidative stress suppresses poly-GR-mediated toxicity in zebrafish embryos

The hexanucleotide (G4C2)-repeat expansion in the C9ORF72 gene is the most common pathogenic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This repeat expansion can be translated into dipeptide repeat proteins (DPRs), and distribution of the poly-GR DPR correlates with neurodegeneration in postmortem C9FTD/ALS brains. Here, we assessed poly-GR toxicity in zebrafish embryos, using an annexin A5-based fluorescent transgenic line (secA5) that allows for detection and quantification of apoptosis in vivo. Microinjection of RNA encoding poly-GR into fertilized oocytes evoked apoptosis in the brain and abnormal motor neuron morphology in the trunk of 1-4-days postfertilization embryos. Poly-GR can be specifically detected in protein homogenates from injected zebrafish and in the frontal cortexes of C9FTD/ALS cases. Poly-GR expression further elevated MitoSOX levels in zebrafish embryos, indicating oxidative stress. Inhibition of reactive oxygen species using Trolox showed full suppression of poly-GR toxicity. Our study indicates that poly-GR can exert its toxicity via oxidative stress. This zebrafish model can be used to find suppressors of poly-GR toxicity and identify its molecular targets underlying neurodegeneration observed in C9FTD/ALS.</p