CERKL regulates autophagy via the NAD-dependent deacetylase SIRT1

<p>Macroautophagy/autophagy is an important intracellular mechanism for the maintenance of cellular homeostasis. Here we show that the <i>CERKL</i> (ceramide kinase like) gene, a retinal degeneration (RD) pathogenic gene, plays a critical role in regulating autophagy by stabilizing SIRT1. <i>In vitro</i> and <i>in vivo</i>, suppressing CERKL results in impaired autophagy. SIRT1 is one of the main regulators of acetylation/deacetylation in autophagy. In CERKL-depleted retinas and cells, SIRT1 is downregulated. ATG5 and ATG7, 2 essential components of autophagy, show a higher degree of acetylation in CERKL-depleted cells. Overexpression of SIRT1 rescues autophagy in CERKL-depleted cells, whereas CERKL loses its function of regulating autophagy in SIRT1-depleted cells, and overexpression of CERKL upregulates SIRT1. Finally, we show that CERKL directly interacts with SIRT1, and may regulate its phosphorylation at Ser27 to stabilize SIRT1. These results show that CERKL is an important regulator of autophagy and it plays this role by stabilizing the deacetylase SIRT1.</p

Tulp1 deficiency causes early-onset retinal degeneration through affecting ciliogenesis and activating ferroptosis in zebrafish

Mutations in TUB-like protein 1 (TULP1) are associated with severe early-onset retinal degeneration in humans. However, the pathogenesis remains largely unknown. There are two homologous genes of TULP1 in zebrafish, namely tulp1a and tulp1b. Here, we generated the single knockout (tulp1a(−/−) and tulp1b(−/−)) and double knockout (tulp1-dKO) models in zebrafish. Knockout of tulp1a resulted in the mislocalization of UV cone opsins and the degeneration of UV cones specifically, while knockout of tulp1b resulted in mislocalization of rod opsins and rod-cone degeneration. In the tulp1-dKO zebrafish, mislocalization of opsins was present in all types of photoreceptors, and severe degeneration was observed at a very early age, mimicking the clinical manifestations of TULP1 patients. Photoreceptor cilium length was significantly reduced in the tulp1-dKO retinas. RNA-seq analysis showed that the expression of tektin2 (tekt2), a ciliary and flagellar microtubule structural component, was downregulated in the tulp1-dKO zebrafish. Dual-luciferase reporter assay suggested that Tulp1a and Tulp1b transcriptionally activate the promoter of tekt2. In addition, ferroptosis might be activated in the tulp1-dKO zebrafish, as suggested by the up-regulation of genes related to the ferroptosis pathway, the shrinkage of mitochondria, reduction or disappearance of mitochondria cristae, and the iron and lipid droplet deposition in the retina of tulp1-dKO zebrafish. In conclusion, our study establishes an appropriate zebrafish model for TULP1-associated retinal degeneration and proposes that loss of TULP1 causes defects in cilia structure and opsin trafficking through the downregulation of tekt2, which further increases the death of photoreceptors via ferroptosis. These findings offer insight into the pathogenesis and clinical treatment of early-onset retinal degeneration

Expression of ZnT and ZIP Zinc Transporters in the Human RPE and Their Regulation by Neurotrophic Factors

PURPOSE. Zinc is an essential cofactor for normal cell function. Altered expression and function of zinc transporters may contribute to the pathogenesis of neurodegenerative disorders including macular degeneration. The expression and regulation of zinc transporters in the RPE and the toxicity of zinc to these cells were examined. METHODS. Zinc transporters were identified in a human RPE cell line, ARPE19, using a 28K human array, and their expression was confirmed by PCR, immunocytochemistry, and Western blot analysis in primary human RPE cultures and ARPE19. Zinc toxicity to ARPE19 was determined using monotetrazolium, propidium iodide, and TUNEL assays, and Zn 2ϩ uptake was visualized with Zinquin ethyl ester. The effect of various growth factors on zinc transporter expression also was examined. RESULTS. Transcripts for 20 of 23 zinc transporters are expressed in fetal human RPE, 16 of 23 in adult human RPE, and 21 of 23 in ARPE19. Zn transporter proteins were also detected in ARPE19. ZnT5 expression was not observed, whereas ZnT6, ZIP1, and ZIP13 were the most abundantly expressed in all RPE samples. The addition of low concentrations of Zn 2ϩ to cultures resulted in a dose-dependent increase in intracellular Zn 2ϩ content in ARPE19, and Ͼ30 nM Zn 2ϩ induced necrosis with an LC 50 of 117.4 nM. Brain-derived neurotrophic factor, ciliary neurotrophic factor, glial-derived neurotrophic factor (GDNF), and pigment epithelial-derived neurotrophic factor (PEDF) increased ZIP2 expression, GDNF and PEDF increased ZnT2 expression, and PEDF increased ZnT3 and ZnT8 expression. These neurotrophic factors also promoted Zn 2ϩ uptake in the RPE. CONCLUSIONS. The array of zinc transporters expressed by the RPE may play a key role in zinc homeostasis in the retina and in ocular health and diseases. (Invest Ophthalmol Vis Sci

Rod genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor composition and progressive retinal degeneration

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish

The splicing factor DHX38 enables retinal development through safeguarding genome integrity

DEAH-Box Helicase 38 (DHX38) is a pre-mRNA splicing factor and also a disease-causing gene of autosomal recessive retinitis pigmentosa (arRP). The role of DHX38 in the development and maintenance of the retina remains largely unknown. In this study, by using the dhx38 knockout zebrafish model, wedemonstrated that Dhx38 deficiency causes severe differentiation defects and apoptosis of retinal progenitor cells (RPCs) through disrupted mitosis and increased DNA damage. Furthermore, we found a significant accumulation of R-loops in the dhx38-deficient RPCs and human cell lines. Finally, we found that DNA replication stress is the prerequisite for R-loop-induced DNA damage in the DHX38 knockdown cells. Taken together, our study demonstrates a necessary role of DHX38 in the development of retina and reveals a DHX38/R-loop/replication stress/DNA damage regulatory axis that is relatively independent of the known functions of DHX38 in mitosis control

Retinal degeneration in rpgra mutant zebrafish

Introduction: Pathogenic mutations in RPGRORF15, one of two major human RPGR isoforms, were responsible for most X-linked retinitis pigmentosa cases. Previous studies have shown that RPGR plays a critical role in ciliary protein transport. However, the precise mechanisms of disease triggered by RPGRORF15 mutations have yet to be clearly defined. There are two homologous genes in zebrafish, rpgra and rpgrb. Zebrafish rpgra has a single transcript homologous to human RPGRORF15; rpgrb has two major transcripts: rpgrbex1-17 and rpgrbORF15, similar to human RPGRex1-19 and RPGRORF15, respectively. rpgrb knockdown in zebrafish resulted in both abnormal development and increased cell death in the dysplastic retina. However, the impact of knocking down rpgra in zebrafish remains undetermined. Here, we constructed a rpgra mutant zebrafish model to investigate the retina defect and related molecular mechanism.Methods: we utilized transcription activator-like effector nuclease (TALEN) to generate a rpgra mutant zebrafish. Western blot was used to determine protein expression. RT-PCR was used to quantify gene transcription levels. The visual function of embryonic zebrafish was detected by electroretinography. Immunohistochemistry was used to observe the pathological changes in the retina of mutant zebrafish and transmission electron microscope was employed to view subcellular structure of photoreceptor cells.Results: A homozygous rpgra mutant zebrafish with c.1675_1678delins21 mutation was successfully constructed. Despite the normal morphological development of the retina at 5 days post-fertilization, visual dysfunction was observed in the mutant zebrafish. Further histological and immunofluorescence assays indicated that rpgra mutant zebrafish retina photoreceptors progressively began to degenerate at 3-6 months. Additionally, the mislocalization of cone outer segment proteins (Opn1lw and Gnb3) and the accumulation of vacuole-like structures around the connecting cilium below the OSs were observed in mutant zebrafish. Furthermore, Rab8a, a key regulator of opsin-carrier vesicle trafficking, exhibited decreased expression and evident mislocalization in mutant zebrafish.Discussion: This study generated a novel rpgra mutant zebrafish model, which showed retinal degeneration. our data suggested Rpgra is necessary for the ciliary transport of cone-associated proteins, and further investigation is required to determine its function in rods. The rpgra mutant zebrafish constructed in this study may help us gain a better understanding of the molecular mechanism of retinal degeneration caused by RPGRORF15 mutation and find some useful treatment in the future