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The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants.

ABSTRACT: Photoreceptor degeneration can lead to blindness and represents the most common form of neural degenerative disease worldwide. Although many genes involved in photoreceptor degeneration have been identified, the underlying mechanisms remain to be elucidated. Here we examined photoreceptor development in zebrafish kif3a and kif3b mutants, which affect two subunits of the kinesin-2 complex. In both mutants, rods degenerated quickly, whereas cones underwent a slow degeneration process. Notably, the photoreceptor defects were considerably more severe in kif3a mutants than in kif3b mutants. In the cone photoreceptors of kif3a mutants, opsin proteins accumulated in the apical region and formed abnormal membrane structures. In contrast, rhodopsins were enriched in the rod cell body membrane and represented the primary reason for rapid rod degeneration in these mutants. Moreover, removal of the cytoplasmic tail of rhodopsin to reduce its function, but not decreasing rhodopsin expression levels, prevented rod degeneration in both kif3a and kif3b mutants. Of note, overexpression of full-length rhodopsin or its cytoplasmic tail domain, but not of rhodopsin lacking the cytoplasmic tail, exacerbated rod degeneration in kif3a mutants, implying an important role of the cytoplasmic tail in rod degeneration. Finally, we showed that the cytoplasmic tail of rhodopsin might trigger rod degeneration through activating the downstream calcium signaling pathway, as drug treatment with inhibitors of intracellular calcium release prevented rod degeneration in kif3a mutants. Our results demonstrate a previously unknown function of the rhodopsin cytoplasmic domain during opsin-triggered photoreceptor degeneration and may open up new avenues for managing this disease.

SUBMITTER: Feng D

PROVIDER: S-EPMC5655514 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants.

Feng Dong D Chen Zhe Z Yang Kuang K Miao Shanshan S Xu Bolin B Kang Yunsi Y Xie Haibo H Zhao Chengtian C

The Journal of biological chemistry 20170830 42

Photoreceptor degeneration can lead to blindness and represents the most common form of neural degenerative disease worldwide. Although many genes involved in photoreceptor degeneration have been identified, the underlying mechanisms remain to be elucidated. Here we examined photoreceptor development in zebrafish <i>kif3a</i> and <i>kif3b</i> mutants, which affect two subunits of the kinesin-2 complex. In both mutants, rods degenerated quickly, whereas cones underwent a slow degeneration process ...[more]

PMID: 28855254

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Project description:PurposeRetinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1-1.MethodsOne- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes.ResultsUsing gRNAs that targeted highly conserved regions of rh1-1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5'-coding sequence of rh1-1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3'-coding region of rh1-1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane.ConclusionsThe efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.

Dataset Information

The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants.

Publications

The cytoplasmic tail of rhodopsin triggers rapid rod degeneration in kinesin-2 mutants.

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