@article {2051, title = {Cilia-associated wound repair mediated by IFT88 in retinal pigment epithelium.}, journal = {Sci Rep}, volume = {13}, year = {2023}, month = {2023 May 21}, pages = {8205}, abstract = {

Primary cilia are conserved organelles that integrate extracellular cues into intracellular signals and are critical for diverse processes, including cellular development and repair responses. Deficits in ciliary function cause multisystemic human diseases known as ciliopathies. In the eye, atrophy of the retinal pigment epithelium (RPE) is a common feature of many ciliopathies. However, the roles of RPE cilia in vivo remain poorly understood. In this study, we first found that mouse RPE cells only transiently form primary cilia. We then examined the RPE in the mouse model of Bardet-Biedl Syndrome 4 (BBS4), a ciliopathy associated with retinal degeneration in humans, and found that ciliation in BBS4 mutant RPE cells is disrupted early during development. Next, using a laser-induced injury model in vivo, we found that primary cilia in RPE reassemble in response to laser injury during RPE wound healing and then rapidly disassemble after the repair is completed. Finally, we demonstrated that RPE-specific depletion of primary cilia in a conditional mouse model of cilia loss promoted wound healing and enhanced cell proliferation. In summary, our data suggest that RPE cilia contribute to both retinal development and repair and provide insights into potential therapeutic targets for more common RPE degenerative diseases.

}, keywords = {Animals, Cilia, Ciliopathies, Disease Models, Animal, Humans, Mice, Microtubule-Associated Proteins, Retinal Degeneration, Retinal Pigment Epithelium, Tumor Suppressor Proteins}, issn = {2045-2322}, doi = {10.1038/s41598-023-35099-3}, author = {Ning, Ke and Bhuckory, Mohajeet B and Lo, Chien-Hui and Sendayen, Brent E and Kowal, Tia J and Chen, Ming and Bansal, Ruchi and Chang, Kun-Che and Vollrath, Douglas and Berbari, Nicolas F and Mahajan, Vinit B and Hu, Yang and Sun, Yang} } @article {655, title = {CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa.}, journal = {Mol Ther}, volume = {24}, year = {2016}, month = {2016 08}, pages = {1388-94}, abstract = {

Massive parallel sequencing enables identification of numerous genetic variants in mutant organisms, but determining pathogenicity of any one mutation can be daunting. The most commonly studied preclinical model of retinitis pigmentosa called the "rodless" (rd1) mouse is homozygous for two mutations: a nonsense point mutation (Y347X) and an intronic insertion of a leukemia virus (Xmv-28). Distinguishing which mutation causes retinal degeneration is still under debate nearly a century after the discovery of this model organism. Here, we performed gene editing using the CRISPR/Cas9 system and demonstrated that the Y347X mutation is the causative variant of disease. Genome editing in the first generation produced animals that were mosaic for the corrected allele but still showed neurofunction preservation despite low repair frequencies. Furthermore, second-generation CRISPR-repaired mice showed an even more robust rescue and amelioration of the disease. This predicts excellent outcomes for gene editing in diseased human tissue, as Pde6b, the mutated gene in rd1 mice, has an orthologous intron-exon relationship comparable with the human PDE6B gene. Not only do these findings resolve the debate surrounding the source of neurodegeneration in the rd1 model, but they also provide the first example of homology-directed recombination-mediated gene correction in the visual system.

}, keywords = {Animals, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, Cyclic Nucleotide Phosphodiesterases, Type 6, Disease Models, Animal, DNA Repair, Electroretinography, Exons, Gene Editing, Genetic Loci, Homologous Recombination, Mice, Mice, Transgenic, Mutation, Photoreceptor Cells, Vertebrate, Retinal Degeneration, Retinitis Pigmentosa, RNA, Guide}, issn = {1525-0024}, doi = {10.1038/mt.2016.107}, author = {Wu, Wen-Hsuan and Tsai, Yi-Ting and Justus, Sally and Lee, Ting-Ting and Zhang, Lijuan and Lin, Chyuan-Sheng and Bassuk, Alexander G and Mahajan, Vinit B and Tsang, Stephen H} } @article {637, title = {Reprogramming metabolism by targeting sirtuin 6 attenuates retinal degeneration.}, journal = {J Clin Invest}, volume = {126}, year = {2016}, month = {2016 Dec 01}, pages = {4659-4673}, abstract = {

Retinitis pigmentosa (RP) encompasses a diverse group of Mendelian disorders leading to progressive degeneration of rods and then cones. For reasons that remain unclear, diseased RP photoreceptors begin to deteriorate, eventually leading to cell death and, consequently, loss of vision. Here, we have hypothesized that RP associated with mutations in phosphodiesterase-6 (PDE6) provokes a metabolic aberration in rod cells that promotes the pathological consequences of elevated cGMP and Ca2+, which are induced by the Pde6 mutation. Inhibition of sirtuin 6 (SIRT6), a histone deacetylase repressor of glycolytic flux, reprogrammed rods into perpetual glycolysis, thereby driving the accumulation of biosynthetic intermediates, improving outer segment (OS) length, enhancing photoreceptor survival, and preserving vision. In mouse retinae lacking Sirt6, effectors of glycolytic flux were dramatically increased, leading to upregulation of key intermediates in glycolysis, TCA cycle, and glutaminolysis. Both transgenic and AAV2/8 gene therapy-mediated ablation of Sirt6 in rods provided electrophysiological and anatomic rescue of both rod and cone photoreceptors in a preclinical model of RP. Due to the extensive network of downstream effectors of Sirt6, this study motivates further research into the role that these pathways play in retinal degeneration. Because reprogramming metabolism by enhancing glycolysis is not gene specific, this strategy may be applicable to a wide range of neurodegenerative disorders.

}, keywords = {Animals, Cellular Reprogramming, Cellular Reprogramming Techniques, Citric Acid Cycle, Cyclic GMP, Cyclic Nucleotide Phosphodiesterases, Type 6, Dependovirus, Disease Models, Animal, Eye Proteins, Genetic Therapy, Glycolysis, Mice, Mice, Mutant Strains, Mutation, Retinal Rod Photoreceptor Cells, Retinitis Pigmentosa, Sirtuins, Transduction, Genetic}, issn = {1558-8238}, doi = {10.1172/JCI86905}, author = {Zhang, Lijuan and Du, Jianhai and Justus, Sally and Hsu, Chun-Wei and Bonet-Ponce, Luis and Wu, Wen-Hsuan and Tsai, Yi-Ting and Wu, Wei-Pu and Jia, Yading and Duong, Jimmy K and Mahajan, Vinit B and Lin, Chyuan-Sheng and Wang, Shuang and Hurley, James B and Tsang, Stephen H} } @article {641, title = {Reprogramming towards anabolism impedes degeneration in a preclinical model of retinitis pigmentosa.}, journal = {Hum Mol Genet}, volume = {25}, year = {2016}, month = {2016 10 01}, pages = {4244-4255}, abstract = {

Retinitis pigmentosa (RP) is an incurable neurodegenerative condition featuring photoreceptor death that leads to blindness. Currently, there is no approved therapeutic for photoreceptor degenerative conditions like RP and atrophic age-related macular degeneration (AMD). Although there are promising results in human gene therapy, RP is a genetically diverse disorder, such that gene-specific therapies would be practical in a small fraction of patients with RP. Here, we explore a non-gene-specific strategy that entails reprogramming photoreceptors towards anabolism by upregulating the mechanistic target of rapamycin (mTOR) pathway. We conditionally ablated the tuberous sclerosis complex 1 (Tsc1) gene, an mTOR inhibitor, in the rods of the Pde6bpreclinical RP mouse model and observed, functionally and morphologically, an improvement in the survival of rods and cones at early and late disease stages. These results elucidate the ability of reprogramming the metabolome to slow photoreceptor degeneration. This strategy may also be applicable to a wider range of neurodegenerative diseases, as enhancement of nutrient uptake is not gene-specific and is implicated in multiple pathologies. Enhancing anabolism promoted neuronal survival and function and could potentially benefit a number of photoreceptor and other degenerative conditions.

}, keywords = {Animals, Cell Death, Cellular Reprogramming, Cone-Rod Dystrophies, Disease Models, Animal, Humans, Macular Degeneration, Metabolism, Mice, Photoreceptor Cells, Retinal Cone Photoreceptor Cells, Retinal Rod Photoreceptor Cells, Retinitis Pigmentosa, TOR Serine-Threonine Kinases, Tumor Suppressor Proteins}, issn = {1460-2083}, doi = {10.1093/hmg/ddw256}, author = {Zhang, Lijuan and Justus, Sally and Xu, Yu and Pluchenik, Tamara and Hsu, Chun-Wei and Yang, Jin and Duong, Jimmy K and Lin, Chyuan-Sheng and Jia, Yading and Bassuk, Alexander G and Mahajan, Vinit B and Tsang, Stephen H} } @article {707, title = {Silencing of tuberin enhances photoreceptor survival and function in a preclinical model of retinitis pigmentosa (an american ophthalmological society thesis).}, journal = {Trans Am Ophthalmol Soc}, volume = {112}, year = {2014}, month = {2014 Jul}, pages = {103-15}, abstract = {

PURPOSE: To assess the functional consequences of silencing of tuberin, an inhibitor of the mTOR signaling pathway, in a preclinical model of retinitis pigmentosa (RP) in order to test the hypothesis that insufficient induction of the protein kinase B (PKB)-regulated tuberin/mTOR self-survival pathway initiates apoptosis.

METHODS: In an unbiased genome-scale approach, kinase peptide substrate arrays were used to analyze self-survival pathways at the onset of photoreceptor degeneration. The mutant Pde6b(H620Q)/Pde6b(H620Q) at P14 and P18 photoreceptor outer segment (OS) lysates were labeled with P-ATP and hybridized to an array of 1,164 different synthetic peptide substrates. At this stage, OS of Pde6b(H620Q)/Pde6b(H620Q) rods are morphologically normal. In vitro kinase assays and immunohistochemistry were used to validate phosphorylation. Short hairpin RNA (shRNA) gene silencing was used to validate tuberin{\textquoteright}s role in regulating survival.

RESULTS: At the onset of degeneration, 162 peptides were differentially phosphorylated. Protein kinases A, G, C (AGC kinases), and B exhibited increased activity in both peptide array and in vitro kinase assays. Immunohistochemical data confirmed altered phosphorylation patterns for phosphoinositide-dependent kinase-1 (PDK1), ribosomal protein S6 (RPS6), and tuberin. Tuberin gene silencing rescued photoreceptors from degeneration.

CONCLUSIONS: Phosphorylation of tuberin and RPS6 is due to the upregulated activity of PKB. PKB/tuberin cell growth/survival signaling is activated before the onset of degeneration. Substrates of the AGC kinases in the PKB/tuberin pathway are phosphorylated to promote cell survival. Knockdown of tuberin, the inhibitor of the mTOR pathway, increased photoreceptor survival and function in a preclinical model of RP.

}, keywords = {Animals, Apoptosis, Disease Models, Animal, Gene Silencing, Mice, Mice, Mutant Strains, Phosphorylation, Phosphotransferases, Proto-Oncogene Proteins c-akt, Retinitis Pigmentosa, Ribosomal Protein S6, Signal Transduction, Tumor Suppressor Proteins}, issn = {1545-6110}, author = {Tsang, Stephen H and Chan, Lawrence and Tsai, Yi-Ting and Wu, Wen-Hsuan and Hsu, Chun-Wei and Yang, Jin and Tosi, Joaquin and Wert, Katherine J and Davis, Richard J and Mahajan, Vinit B} } @article {97, title = {Genome-wide generation and systematic phenotyping of knockout mice reveals new roles for many genes.}, journal = {Cell}, volume = {154}, year = {2013}, month = {2013 Jul 18}, pages = {452-64}, abstract = {

Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PAPERCLIP:

}, keywords = {Animals, Disease, Disease Models, Animal, Female, Genes, Essential, Genetic Techniques, Genome-Wide Association Study, Male, Mice, Mice, Knockout, Phenotype}, author = {White, Jacqueline K and Gerdin, Anna-Karin and Karp, Natasha A and Ryder, Ed and Buljan, Marija and Bussell, James N and Salisbury, Jennifer and Clare, Simon and Ingham, Neil J and Podrini, Christine and Houghton, Richard and Estabel, Jeanne and Bottomley, Joanna R and Melvin, David G and Sunter, David and Adams, Niels C and Tannahill, David and Logan, Darren W and Macarthur, Daniel G and Flint, Jonathan and Mahajan, Vinit B and Tsang, Stephen H and Smyth, Ian and Watt, Fiona M and Skarnes, William C and Dougan, Gordon and Adams, David J and Ramirez-Solis, Ramiro and Bradley, Allan and Steel, Karen P} } @article {177, title = {Transgenic mice carrying the H258N mutation in the gene encoding the beta-subunit of phosphodiesterase-6 (PDE6B) provide a model for human congenital stationary night blindness.}, journal = {Human mutation}, volume = {28}, year = {2007}, month = {2007 Mar}, pages = {243-54}, abstract = {

Mutations in the beta-subunit of cGMP-phosphodiesterase (PDE6beta) can lead to either progressive retinal disease, such as human retinitis pigmentosa (RP), or stationary disease, such as congenital stationary night blindness (CSNB). Individuals with CSNB in the Rambusch pedigree were found to carry the H258N allele of PDE6B (MIM$\#$ 180072); a similar mutation was not found in RP patients. This report describes an individual carrying the H258N allele, who presented with generalized retinal dysfunction affecting the rod system and a locus of dysfunction at the rod-bipolar interface. Also described are preclinical studies in which transgenic mice with the H258N allele were generated to study the pathophysiological mechanisms of CSNB. While Pde6b(rd1)/Pde6b(rd1) mice have severe photoreceptor degeneration, as in human RP, the H258N transgene rescued these cells. The cGMP-PDE6 activity of dark-adapted H258N mice showed an approximate three-fold increase in the rate of retinal cGMP hydrolysis: from 130.1 nmol x min(-1) x nmol(-1) rhodopsin in wild-type controls to 319.2 nmol x min(-1) x nmol(-1) rhodopsin in mutants, consistent with the hypothesis that inhibition of the PDE6beta activity by the regulatory PDE6gamma subunit is blocked by this mutation. In the albino (B6CBA x FVB) F2 hybrid background, electroretinograms (ERG) from H258N mice were similar to those obtained from affected Rambusch family members, as well as humans with the most common form of CSNB (X-linked), demonstrating a selective loss of the b-wave with relatively normal a-waves. When the H258N allele was introduced into the DBA background, there was no evidence of selective reduction in b-wave amplitudes; rather a- and b-wave amplitudes were both reduced. Thus, factors other than the PDE6B mutation itself could contribute to the variance of an electrophysiological response. Therefore, caution is advisable when interpreting physiological phenotypes associated with the same allele on different genetic backgrounds. Nevertheless, such animals should be of considerable value in further studies of the molecular pathology of CSNB.

}, keywords = {Adult, Animals, Cyclic Nucleotide Phosphodiesterases, Type 6, Disease Models, Animal, DNA Mutational Analysis, Electroretinography, Female, Humans, Male, Mice, Mice, Inbred DBA, Mice, Transgenic, Mutation, Night Blindness, Phosphoric Diester Hydrolases, Retinal Degeneration, Transgenes}, author = {Tsang, Stephen H and Woodruff, Michael L and Jun, Lin and Mahajan, Vinit and Yamashita, Clyde K and Pedersen, Robert and Lin, Chyuan-Sheng and Goff, Stephen P and Rosenberg, Thomas and Larsen, Michael and Farber, Debora B and Nusinowitz, Steven} }