@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 {2042, title = {Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression.}, journal = {Nat Methods}, volume = {20}, year = {2023}, month = {2023 Jul}, pages = {1070-1081}, abstract = {

The development of transgenic mouse models that express genes of interest in specific cell types has transformed our understanding of basic biology and disease. However, generating these models is time- and resource-intensive. Here we describe a model system, SELective Expression and Controlled Transduction In Vivo (SELECTIV), that enables efficient and specific expression of transgenes by coupling adeno-associated virus (AAV) vectors with Cre-inducible overexpression of the multi-serotype AAV receptor, AAVR. We demonstrate that transgenic AAVR overexpression greatly increases the efficiency of transduction of many diverse cell types, including muscle stem cells, which are normally refractory to AAV transduction. Superior specificity is achieved by combining Cre-mediated AAVR overexpression with whole-body knockout of endogenous Aavr, which is demonstrated in heart cardiomyocytes, liver hepatocytes and cholinergic neurons. The enhanced efficacy and exquisite specificity of SELECTIV has broad utility in development of new mouse model systems and expands the use of AAV for gene delivery in vivo.

}, keywords = {Animals, Dependovirus, Gene Transfer Techniques, Genetic Therapy, Genetic Vectors, Mice, Mice, Transgenic, Transduction, Genetic, Transgenes}, issn = {1548-7105}, doi = {10.1038/s41592-023-01896-x}, author = {Zengel, James and Wang, Yu Xin and Seo, Jai Woong and Ning, Ke and Hamilton, James N and Wu, Bo and Raie, Marina and Holbrook, Colin and Su, Shiqi and Clements, Derek R and Pillay, Sirika and Puschnik, Andreas S and Winslow, Monte M and Idoyaga, Juliana and Nagamine, Claude M and Sun, Yang and Mahajan, Vinit B and Ferrara, Katherine W and Blau, Helen M and Carette, Jan E} } @article {1896, title = {Investigation of Cas9 antibodies in the human eye.}, journal = {Nat Commun}, volume = {13}, year = {2022}, month = {2022 02 25}, pages = {1053}, abstract = {

Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.

}, keywords = {Animals, Antibodies, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Humans, Mice, Streptococcus pyogenes, T-Lymphocytes}, issn = {2041-1723}, doi = {10.1038/s41467-022-28674-1}, author = {Toral, Marcus A and Charlesworth, Carsten T and Ng, Benjamin and Chemudupati, Teja and Homma, Shota and Nakauchi, Hiromitsu and Bassuk, Alexander G and Porteus, Matthew H and Mahajan, Vinit B} } @article {1901, title = {A protocol to inject ocular drug implants into mouse eyes.}, journal = {STAR Protoc}, volume = {3}, year = {2022}, month = {2022 03 18}, pages = {101143}, abstract = {

Ocular drug implants (ODIs) are beneficial for treating ocular diseases. However, the lack of a robust injection approach for small-eyed model organisms has been a major technical limitation in developing ODIs. Here, we present a cost-effective, minimally invasive protocol to deliver ODIs into the mouse vitreous called Mouse Implant Intravitreal Injection (MI3). MI3 provides two alternative surgical approaches (air-pressure or plunger) to deliver micro-scaled ODIs into milli-scaled eyes, and expands the preclinical platforms to determine ODIs\&$\#$39; efficacy, toxicity, and pharmacokinetics. For complete details on the use and execution of this protocol, please refer to Sun et\ al. (2021).

}, keywords = {Animals, Drug Implants, Intravitreal Injections, Mice, Vitreous Body}, issn = {2666-1667}, doi = {10.1016/j.xpro.2022.101143}, author = {Lin, Cheng-Hui and Sun, Young Joo and Lee, Soo Hyeon and Mujica, Elena M and Kunchur, Caitlin R and Wu, Man-Ru and Yang, Jing and Jung, Youn Soo and Chiang, Bryce and Wang, Sui and Mahajan, Vinit B} } @article {635, title = {Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO).}, journal = {PLoS One}, volume = {12}, year = {2017}, month = {2017}, pages = {e0169687}, abstract = {

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.

}, keywords = {Amino Acid Sequence, Animals, Cell Adhesion Molecules, Cell Line, Tumor, Child, Cytoskeletal Proteins, Female, Genes, Recessive, Humans, Interleukin-10, Mice, Mutation, Osteomyelitis, Promoter Regions, Genetic, Sequence Homology, Amino Acid}, issn = {1932-6203}, doi = {10.1371/journal.pone.0169687}, author = {Cox, Allison J and Darbro, Benjamin W and Laxer, Ronald M and Velez, Gabriel and Bing, Xinyu and Finer, Alexis L and Erives, Albert and Mahajan, Vinit B and Bassuk, Alexander G and Ferguson, Polly J} } @article {663, title = {Calpain-5 Expression in the Retina Localizes to Photoreceptor Synapses.}, journal = {Invest Ophthalmol Vis Sci}, volume = {57}, year = {2016}, month = {2016 05 01}, pages = {2509-21}, abstract = {

PURPOSE: We characterize calpain-5 (CAPN5) expression in retinal and neuronal subcellular compartments.

METHODS: CAPN5 gene variants were classified using the exome variant server, and RNA-sequencing was used to compare expression of CAPN5 mRNA in the mouse and human retina and in retinoblastoma cells. Expression of CAPN5 protein was ascertained in humans and mice in silico, in mouse retina by immunohistochemistry, and in neuronal cancer cell lines and fractionated central nervous system tissue extracts by Western analysis with eight antibodies targeting different CAPN5 regions.

RESULTS: Most CAPN5 genetic variation occurs outside its protease core; and searches of cancer and epilepsy/autism genetic databases found no variants similar to hyperactivating retinal disease alleles. The mouse retina expressed one transcript for CAPN5 plus those of nine other calpains, similar to the human retina. In Y79 retinoblastoma cells, the level of CAPN5 transcript was very low. Immunohistochemistry detected CAPN5 expression in the inner and outer nuclear layers and at synapses in the outer plexiform layer. Western analysis of fractionated retinal extracts confirmed CAPN5 synapse localization. Western blots of fractionated brain neuronal extracts revealed distinct subcellular patterns and the potential presence of autoproteolytic CAPN5 domains.

CONCLUSIONS: CAPN5 is moderately expressed in the retina and, despite higher expression in other tissues, hyperactive disease mutants of CAPN5 only manifest as eye disease. At the cellular level, CAPN5 is expressed in several different functional compartments. CAPN5 localization at the photoreceptor synapse and with mitochondria explains the neural circuitry phenotype in human CAPN5 disease alleles.

}, keywords = {Animals, Blotting, Western, Calpain, Cattle, Female, Gene Expression Regulation, Neoplastic, Humans, Immunohistochemistry, Male, Mice, Neoplasms, Experimental, Photoreceptor Cells, Retina, Retinal Neoplasms, Retinoblastoma, RNA, Neoplasm, Synapses, Tumor Cells, Cultured}, issn = {1552-5783}, doi = {10.1167/iovs.15-18680}, author = {Schaefer, Kellie A and Toral, Marcus A and Velez, Gabriel and Cox, Allison J and Baker, Sheila A and Borcherding, Nicholas C and Colgan, Diana F and Bondada, Vimala and Mashburn, Charles B and Yu, Chen-Guang and Geddes, James W and Tsang, Stephen H and Bassuk, Alexander G and Mahajan, Vinit B} } @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 {715, title = {Spinster homolog 2 (spns2) deficiency causes early onset progressive hearing loss.}, journal = {PLoS Genet}, volume = {10}, year = {2014}, month = {2014 Oct}, pages = {e1004688}, abstract = {

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

}, keywords = {Age of Onset, Animals, Anion Transport Proteins, Anterior Eye Segment, Cochlea, Connexins, Ear, Inner, Hair Cells, Auditory, Hearing Loss, Lysophospholipids, Mice, Organogenesis, Sphingosine, Stria Vascularis, Zebrafish}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1004688}, author = {Chen, Jing and Ingham, Neil and Kelly, John and Jadeja, Shalini and Goulding, David and Pass, Johanna and Mahajan, Vinit B and Tsang, Stephen H and Nijnik, Anastasia and Jackson, Ian J and White, Jacqueline K and Forge, Andrew and Jagger, Daniel and Steel, Karen P} } @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 {117, title = {Disruption of mouse Cenpj, a regulator of centriole biogenesis, phenocopies Seckel syndrome.}, journal = {PLoS Genet}, volume = {8}, year = {2012}, month = {2012}, pages = {e1003022}, abstract = {

Disruption of the centromere protein J gene, CENPJ (CPAP, MCPH6, SCKL4), which is a highly conserved and ubiquitiously expressed centrosomal protein, has been associated with primary microcephaly and the microcephalic primordial dwarfism disorder Seckel syndrome. The mechanism by which disruption of CENPJ causes the proportionate, primordial growth failure that is characteristic of Seckel syndrome is unknown. By generating a hypomorphic allele of Cenpj, we have developed a mouse (Cenpj(tm/tm)) that recapitulates many of the clinical features of Seckel syndrome, including intrauterine dwarfism, microcephaly with memory impairment, ossification defects, and ocular and skeletal abnormalities, thus providing clear confirmation that specific mutations of CENPJ can cause Seckel syndrome. Immunohistochemistry revealed increased levels of DNA damage and apoptosis throughout Cenpj(tm/tm) embryos and adult mice showed an elevated frequency of micronucleus induction, suggesting that Cenpj-deficiency results in genomic instability. Notably, however, genomic instability was not the result of defective ATR-dependent DNA damage signaling, as is the case for the majority of genes associated with Seckel syndrome. Instead, Cenpj(tm/tm) embryonic fibroblasts exhibited irregular centriole and centrosome numbers and mono- and multipolar spindles, and many were near-tetraploid with numerical and structural chromosomal abnormalities when compared to passage-matched wild-type cells. Increased cell death due to mitotic failure during embryonic development is likely to contribute to the proportionate dwarfism that is associated with CENPJ-Seckel syndrome.

}, keywords = {Animals, Apoptosis, Centrioles, DNA Damage, Dwarfism, Embryonic Development, Genomic Instability, Mice, Mice, Transgenic, Microcephaly, Microtubule-Associated Proteins, Mitosis, Mitotic Spindle Apparatus, Mutation, Signal Transduction}, author = {McIntyre, Rebecca E and Lakshminarasimhan Chavali, Pavithra and Ismail, Ozama and Carragher, Damian M and Sanchez-Andrade, Gabriela and Forment, Josep V and Fu, Beiyuan and Del Castillo Velasco-Herrera, Martin and Edwards, Andrew and van der Weyden, Louise and Yang, Fengtang and Ramirez-Solis, Ramiro and Estabel, Jeanne and Gallagher, Ferdia A and Logan, Darren W and Arends, Mark J and Tsang, Stephen H and Mahajan, Vinit B and Scudamore, Cheryl L and White, Jacqueline K and Jackson, Stephen P and Gergely, Fanni and Adams, David J} } @article {141, title = {Evisceration of mouse vitreous and retina for proteomic analyses.}, journal = {Journal of visualized experiments : JoVE}, year = {2011}, month = {2011}, abstract = {

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.(1-3) Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.(1,4) The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75\% of its eye.(5) This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

}, keywords = {Animals, Eye Evisceration, Eye Proteins, Mice, Models, Animal, Proteomics, Retina, Vitreous Body}, author = {Skeie, Jessica M and Tsang, Stephen H and Mahajan, Vinit B} } @article {147, title = {Mouse eye enucleation for remote high-throughput phenotyping.}, journal = {Journal of visualized experiments : JoVE}, year = {2011}, month = {2011}, abstract = {

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.(1-5) Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. (6-9) An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

}, keywords = {Animals, Eye Enucleation, High-Throughput Screening Assays, Mice, Mice, Transgenic, Phenotype, Tissue Fixation}, author = {Mahajan, Vinit B and Skeie, Jessica M and Assefnia, Amir H and Mahajan, Maryann and Tsang, Stephen H} } @article {149, title = {Mutations in prickle orthologs cause seizures in flies, mice, and humans.}, journal = {American journal of human genetics}, volume = {88}, year = {2011}, month = {2011 Feb 11}, pages = {138-49}, abstract = {

Epilepsy is heritable, yet few causative gene mutations have been identified, and thus far no human epilepsy gene mutations have been found to produce seizures in invertebrates. Here we show that mutations in prickle genes are associated with seizures in humans, mice, and flies. We identified human epilepsy patients with heterozygous mutations in either PRICKLE1 or PRICKLE2. In overexpression assays in zebrafish, prickle mutations resulted in aberrant prickle function. A seizure phenotype was present in the Prickle1-null mutant mouse, two Prickle1 point mutant (missense and nonsense) mice, and a Prickle2-null mutant mouse. Drosophila with prickle mutations displayed seizures that were responsive to anti-epileptic medication, and homozygous mutant embryos showed neuronal defects. These results suggest that prickle mutations have caused seizures throughout evolution.

}, keywords = {Animals, Blotting, Western, Brain, Calcium, Carrier Proteins, DNA-Binding Proteins, Drosophila melanogaster, Drosophila Proteins, Embryo, Nonmammalian, Epilepsies, Myoclonic, Female, Heterozygote, Humans, Immunoenzyme Techniques, In Situ Hybridization, Male, Mice, Mice, Knockout, Mutation, Nerve Tissue Proteins, Phenotype, Reverse Transcriptase Polymerase Chain Reaction, RNA, Messenger, Seizures, Tumor Suppressor Proteins, Zebrafish, Zebrafish Proteins}, author = {Tao, Hirotaka and Manak, J Robert and Sowers, Levi and Mei, Xue and Kiyonari, Hiroshi and Abe, Takaya and Dahdaleh, Nader S and Yang, Tian and Wu, Shu and Chen, Shan and Fox, Mark H and Gurnett, Christina and Montine, Thomas and Bird, Thomas and Shaffer, Lisa G and Rosenfeld, Jill A and McConnell, Juliann and Madan-Khetarpal, Suneeta and Berry-Kravis, Elizabeth and Griesbach, Hilary and Saneto, Russell P and Scott, Matthew P and Antic, Dragana and Reed, Jordan and Boland, Riley and Ehaideb, Salleh N and El-Shanti, Hatem and Mahajan, Vinit B and Ferguson, Polly J and Axelrod, Jeffrey D and Lehesjoki, Anna-Elina and Fritzsch, Bernd and Slusarski, Diane C and Wemmie, John and Ueno, Naoto and Bassuk, Alexander G} } @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} }