The Joubert Syndrome Gene arl13b is Critical for Early Cerebellar Development in Zebrafish.

Joubert syndrome is characterized by unique malformation of the cerebellar vermis. More than thirty Joubert syndrome genes have been identified, including ARL13B. However, its role in cerebellar development remains unexplored. We found that knockdown or knockout of arl13b impaired balance and locomotion in zebrafish larvae. Granule cells were selectively reduced in the corpus cerebelli, a structure homologous to the mammalian vermis. Purkinje cell progenitors were also selectively disturbed dorsomedially. The expression of atoh1 and ptf1, proneural genes of granule and Purkinje cells, respectively, were selectively down-regulated along the dorsal midline of the cerebellum. Moreover, wnt1, which is transiently expressed early in cerebellar development, was selectively reduced. Intriguingly, activating Wnt signaling partially rescued the granule cell defects in arl13b mutants. These findings suggested that Arl13b is necessary for the early development of cerebellar granule and Purkinje cells. The arl13b-deficient zebrafish can serve as a model organism for studying Joubert syndrome.

Establishment of an adult zebrafish model of retinal neurodegeneration induced by NMDA.

AIM: To establish a model of retinal neurodegeneration induced by N-Methyl-D-aspartic acid (NMDA) in adult zebrafish. METHODS: We compared the effects of three different NMDA delivery methods on retinal neurodegeneration in adult zebrafish: immersion (I.M.), intravitreal injection (I.V.), and intraperitoneal injection (I.P.), and examined retinal pathology and degeneration by hematoxylin and eosin and TUNEL staining in the treated zebrafish. Effects of the NMDA receptor antagonist MK-801 and the natural product resveratrol on NMDA-induced retinal neurodegeneration were also assessed. RESULTS: The thickened inner retina was seen in histology with 100 µmol/L NMDA by I.M. administration. Significant apoptosis in the retinal ganglion cell layer and retinal thickness reduction occurred in 0.5 mol/L NMDA I.P. administration group.Seizure-like behavioral changes, but no retinal histological alteration occurred in 16 mg/kg NMDA I.P. administration group. Resveratrol and MK-801 prevented NMDA-induced retinal neurodegeneration in the zebrafish. CONCLUSION: Among the three drug administration methods, I.V. injection of NMDA is the most suitable for establishment of an acute retinal damage model in zebrafish. I.M. with NMDA is likely the best for use as a chronic retinal damage model. I.P. treatment with NMDA causes brain damage. Resveratrol and MK801 may be a clinically valuable treatment for retinal neurodegeneration.

Exploration of age-related mitochondrial dysfunction and the anti-aging effects of resveratrol in zebrafish retina.

It is currently believed that aging is closely linked with mitochondrial dysfunction, and that resveratrol exhibits anti-aging and neuroprotective effects by improving mitochondrial function, even though the mechanisms are not well defined. This study explored mitochondrial quality (mitochondrial DNA integrity and copy number), mitochondrial function (fusion/fission, mitophagy/autophagy), antioxidant system and activity of the Akt/mTOR and Ampk/Sirt1/Pgc1α pathways, and inflammation in aging zebrafish retinas to identify the probable mechanisms of resveratrol's anti-aging and neuroprotective effects. mtDNA integrity, mtDNA copy number, mitochondrial fusion regulators, mitophagy, and antioxidant-related genes were all decreased whereas Akt/mTOR activity and inflammation was increased upon aging in zebrafish retinas. Resveratrol was shown to not only increase mitochondrial quality and function, but also to suppress Akt/mTOR activity in zebrafish retinas. These results support the notion that mitochondrial dysfunction and increased Akt/mTOR activity are major players in age-related retinal neuropathy in zebrafish, and demonstrate a trend towards mitochondrial fragmentation in the aging retina. Importantly, resveratrol promoted mitochondrial function, up-regulating Ampk/Sirt1/Pgc1α, and down-regulated Akt/mTOR pathway activity in zebrafish retinas, suggesting that it may be able to prevent age-related oculopathy.

Effect of Resveratrol on Sirtuins, OPA1, and Fis1 Expression in Adult Zebrafish Retina.

Purpose: We determined whether sirtuins (SIRT1-SIRT7) are expressed in the zebrafish retina, evaluated the modulatory effect of resveratrol in the normal retina, and examined N-Methyl-D-aspartic acid (NMDA)-induced zebrafish retinal damage associated with mitochondrial sirtuins and mitochondrial fusion and fission mediators, OPA1 and Fis1. Methods: Sirtuins, OPA1, and Fis1 mRNA expression was analyzed by RT-PCR and quantitative real time PCR (qPCR) in adult zebrafish (AB type) retina and liver. qPCR showed an effect of resveratrol on SIRTs (SIRT1, 3, 4, 5) and OPA1 and Fis1 in low and high concentrations (5 and 50 mg/L) at different time points (0, 1, 24, and 48 hours) in the retina. Western blots were performed to examine the expression of SIRTs and OPA1 proteins under high concentrations of resveratrol for 24 hours. Hematoxylin and eosin staining, qPCR and mitochondrial copy number, and DNA damage assays then were used to confirm the protective effects of resveratrol on NMDA-induced retinal damage. Results: The seven sirtuins and OPA1 were highly expressed in zebrafish retina compared to the liver. Treatment with resveratrol promoted SIRT1, mitochondrial sirtuins, and OPA1 gene and protein expression, and improved mitochondrial DNA repair in adult zebrafish retina. Interestingly, the effect of resveratrol on SIRT4 gene and protein expression was significantly higher in the zebrafish retina. Importantly, resveratrol offered protection against NMDA-induced retinal damage by activating the SIRT1 gene and subsequent protein expression. Mitochondrial sirtuins and OPA1 genes likely had a role in regulating mitochondrial dynamics. Conclusions: To our knowledge, our study is the first composite analysis of sirtuins in adult zebrafish retina and provides sufficient evidence that resveratrol, as an activator of SIRT1, protects NMDA-induced zebrafish retinal damage by potentially mediating mitochondrial sirtuins and OPA1 genes.

Chd7 Is Critical for Early T-Cell Development and Thymus Organogenesis in Zebrafish.

Coloboma, heart defect, atresia choanae, retarded growth and development, genital hypoplasia, ear anomalies/deafness (CHARGE) syndrome is a congenital disorder affecting multiple organs and mainly caused by mutations in CHD7, a gene encoding a chromatin-remodeling protein. Immunodeficiency and reduced T cells have been noted in CHARGE syndrome. However, the mechanisms underlying T lymphopenia are largely unexplored. Herein, we observed dramatic decrease of T cells in both chd7knockdown and knockout zebrafish embryos. Unexpectedly, hematopoietic stem and progenitor cells and, particularly, lymphoid progenitor cells were increased peripherally in nonthymic areas in chd7-deficient embryos, unlikely to contribute to the T-cell decrease. Further analysis demonstrated that both the organogenesis and homing function of the thymus were seriously impaired. Chd7 might regulate thymus organogenesis through modulating the development of both neural crest cell-derived mesenchyme and pharyngeal endoderm-derived thymic epithelial cells. The expression of foxn1, a central regulator of thymic epithelium, was remarkably down-regulated in the pharyngeal region in chd7-deficient embryos. Moreover, the T-cell reduction in chd7-deficient embryos was partially rescued by overexpressing foxn1, suggesting that restoring thymic epithelium may be a potential therapeutic strategy for treating immunodeficiency in CHARGE syndrome. Collectively, the results indicated that chd7 was critical for thymic development and T-lymphopenia in CHARGE syndrome may be mainly attributed to the defects of thymic organogenesis. The current finding may benefit the diagnosis and therapy of T lymphopenia and immunodeficiency in CHARGE syndrome.