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1.  The Role of Vldlr in Intraretinal Angiogenesis in Mice 
The study further demonstrates the essential role of Vldlr in the regulation of intraretinal angiogenesis in mice and suggests that it probably mediates an antiangiogenic signal to prevent the migration of vascular endothelial cells into the photoreceptor cell layer.
Purpose.
To identify and characterize the r26 mouse line, which displays depigmented patches in the retina, and to determine the causative gene mutation and study the underlying mechanism.
Methods.
Fundus examination, fluorescein angiography, histology, and immunostaining were used to determine the retinal phenotypes. Genome-wide linkage analysis, DNA sequencing, and an allelic test were used to identify the causative gene mutation. Wild-type and mutant gene products were examined by Western blot and transient transfection.
Results.
Homozygous r26/r26 mice displayed depigmented patches in the fundus that overlapped the hyperfluorescent spots in the angiogram. Histology showed overgrown retinal vessels in the subretinal space. Immunostaining verified the presence of endothelial cells in the photoreceptor layer. Chromosome mapping and DNA sequencing revealed a point mutation, c.2239C>T, in the very-low-density lipoprotein receptor (Vldlr) gene. An allelic test in Vldlr knockout (−/−) mice confirmed that r26/− mice display a phenotype similar to that of r26/r26 mice. The Vldlr protein was predominantly localized at the plasma membrane of transfected cells, whereas the truncated Vldlr was diffusely expressed in the cell cytosol. The r26 truncated Vldlr was undetectable in mutant retinas by Western blot.
Conclusions.
The r26 is a recessive mutant caused by a missense mutation in the Vldlr gene. This results in a truncated Vldlr protein that lacks the C-terminal 127 amino acid residues including the single transmembrane domain and fails to localize at the plasma membrane. Thus, the r26 is a loss-of-function Vldlr mutation. Vldlr on the cell surface probably mediates an antiangiogenic signal to prevent retinal endothelial cells from migrating into the photoreceptor cell layer.
doi:10.1167/iovs.10-7082
PMCID: PMC3176028  PMID: 21757581
2.  Deletion of LRP5 in VLDLR Knockout Mice Inhibits Retinal Neovascularization 
PLoS ONE  2013;8(9):e75186.
The development and maintenance of retinal vasculature require a precise balance between pro-angiogenic and anti-angiogenic factors. However, mechanisms underlying normal homeostasis of retinal vasculature and pathological changes of disrupted retinal vessel development are not fully understood. Recent studies of the low-density lipoprotein receptor-related protein 5 (LRP5) and the very low-density lipoprotein receptor (VLDLR) mutant mice indicate that LRP5 mediates a pro-angiogenic signal while VLDLR mediates an anti-angiogenic signal in retinal vasculature. Mice with a loss of LRP5 display underdeveloped intraretinal vasculature associated with endothelial cell (EC) clustering and failed EC migration into deep retinal layers. In contrast, VLDLR knockout mice show overgrown intraretinal vasculature and subretinal neovascularization. To understand the mechanisms for the opposite retinal vascular abnormalities between LRP5 and VLDLR mutant mice and to test how a loss of LRP5 perturbs subretinal neovascularization caused by a loss of VLDLR, we have generated and characterized the retinal vasculature in LRP5/VLDLR double knockout (DKO) mice. Our data show that DKO mice develop substantial EC clustering without subretinal neovascularization. The absence of subretinal neovascularization in DKO mice is associated with inhibited migration of ECs into the photoreceptor cell layer. In addition, the transcription level of Slc38a5, which encodes a Müller cell specific glutamine transporter, is significantly reduced in DKO mice, similar to previously reported changes in LRP5 single knockout mice. Thus, LRP5 signaling is a prerequisite for neovascularization in VLDLR knockout mice. LRP5 may be an effective target for inhibiting intraretinal neovascularization.
doi:10.1371/journal.pone.0075186
PMCID: PMC3772893  PMID: 24058663
3.  Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles 
Cell  2011;144(4):551-565.
SUMMARY
Kinesin and dynein are opposite-polarity microtubule motors that drive the tightly regulated transport of a variety of cargoes. Both motors can bind to cargo but their overall composition on axonal vesicles and whether this composition directly modulates transport activity, is unknown. Here we characterize the intracellular transport and steady state motor subunit composition of mammalian prion protein (PrPC) vesicles. We identify Kinesin-1 and cytoplasmic dynein as major PrPC vesicle motor complexes, and show that their activities are tightly coupled. Regulation of normal retrograde transport by Kinesin-1 is independent of dynein-vesicle attachment, and requires the vesicle association of a complete Kinesin-1 heavy and light chain holoenzyme. Furthermore, motor subunits remain stably associated with stationary as well as with moving vesicles. Our data suggest a coordination model where PrPC vesicles maintain a stable population of associated motors whose activity is modulated by regulatory factors instead of by structural changes to motor-cargo associations.
doi:10.1016/j.cell.2011.01.021
PMCID: PMC3576050  PMID: 21335237
4.  Cataracts and Microphthalmia Caused by a Gja8 Mutation in Extracellular Loop 2 
PLoS ONE  2012;7(12):e52894.
The mouse semi-dominant Nm2249 mutation displays variable cataracts in heterozygous mice and smaller lenses with severe cataracts in homozygous mice. This mutation is caused by a Gja8R205G point mutation in the second extracellular loop of the Cx50 (or α8 connexin) protein. Immunohistological data reveal that Cx50-R205G mutant proteins and endogenous wild-type Cx46 (or α3 connexin) proteins form diffuse tiny spots rather than typical punctate signals of normal gap junctions in the lens. The level of phosphorylated Cx46 proteins is decreased in Gja8R205G/R205G mutant lenses. Genetic analysis reveals that the Cx50-R205G mutation needs the presence of wild-type Cx46 to disrupt lens peripheral fibers and epithelial cells. Electrophysiological data in Xenopus oocytes reveal that Cx50-R205G mutant proteins block channel function of gap junctions composed of wild-type Cx50, but only affect the gating of wild-type Cx46 channels. Both genetic and electrophysiological results suggest that Cx50-R205G mutant proteins alone are unable to form functional channels. These findings imply that the Gja8R205G mutation differentially impairs the functions of Cx50 and Cx46 to cause cataracts, small lenses and microphthalmia. The Gja8R205G mutation occurs at the same conserved residue as the human GJA8R198W mutation. This work provides molecular insights to understand the cataract and microphthalmia/microcornea phenotype caused by Gja8 mutations in mice and humans.
doi:10.1371/journal.pone.0052894
PMCID: PMC3530494  PMID: 23300808
5.  Mechanism of Cataract Formation in αA-crystallin Y118D Mutation 
Purpose
The aim of this study was to elucidate the molecular mechanisms that lead to a dominant nuclear cataract in a mouse harboring the Y118D mutation in the αA-crystallin gene.
Methods
The physicochemical properties of α-crystallin obtained from mouse lenses with the Y118D mutation as well as a recombinant Y118D αA-crystallin were studied using gel filtration, two-dimensional (2D) gel electrophoresis, multi-angle light scattering, circular dichroism, fluorescence, and chaperone activities.
Results
Both native α-crystallin from mutant lens and recombinant αA-Y118D displayed higher molecular mass distribution than the wild-type. Circular dichroism spectra indicated changes in the secondary structures of αA-Y118D. The αA-Y118D protein prevented nonspecific protein aggregation more effectively than wild-type αA-crystallin. The gel filtration and 2D gel electrophoresis analysis showed a significant reduction of Y118D mutant protein in comparison with wild-type αA protein of heterozygous mutant lenses. Quantitative RT-PCR results confirmed a decrease in αA and αB transcripts in the homozygous mutant α A(Y118D/Y118D) lenses.
Conclusions
The αA-Y118D mutant protein itself displays an increased chaperone-like activity. However, the dominant nuclear cataract is associated with a significant decrease in the amount of αA-crystallin, leading to a reduction in total chaperone capacity needed for maintaining lens transparency.
doi:10.1167/iovs.08-3070
PMCID: PMC3001329  PMID: 19151380
6.  Connexin Mediated Cataract Prevention in Mice 
PLoS ONE  2010;5(9):e12624.
Cataracts, named for any opacity in the ocular lens, remain the leading cause of vision loss in the world. Non-surgical methods for cataract prevention are still elusive. We have genetically tested whether enhanced lens gap junction communication, provided by increased α3 connexin (Cx46) proteins expressed from α8(Kiα3) knock-in alleles in Gja8tm1(Gja3)Tww mice, could prevent nuclear cataracts caused by the γB-crystallin S11R mutation in CrygbS11R/S11R mice. Remarkably, homozygous knock-in α8(Kiα3/Kiα3) mice fully prevented nuclear cataracts, while single knock-in α8(Kiα3/−) allele mice showed variable suppression of nuclear opacities in CrygbS11R/S11R mutant mice. Cataract prevention was correlated with the suppression of many pathological processes, including crystallin degradation and fiber cell degeneration, as well as preservation of normal calcium levels and stable actin filaments in the lens. This work demonstrates that enhanced intercellular gap junction communication can effectively prevent or delay nuclear cataract formation and suggests that small metabolites transported through gap junction channels protect the stability of crystallin proteins and the cytoskeletal structures in the lens core. Thus, the use of an array of small molecules to promote lens homeostasis may become a feasible non-surgical approach for nuclear cataract prevention in the future.
doi:10.1371/journal.pone.0012624
PMCID: PMC2936561  PMID: 20844585
7.  Severe Retinal Degeneration Caused by a Novel Rhodopsin Mutation 
A point mutation of the rhodopsin gene results in a mutant Rho-C185R protein and causes dominant retinal degeneration in mice.
Purpose.
To identify a new mouse mutation developing early-onset dominant retinal degeneration, to determine the causative gene mutation, and to investigate the underlying mechanism.
Methods.
Retinal phenotype was examined by indirect ophthalmoscopy, histology, transmission electron microscopy, immunohistochemistry, Western blot analysis, and electroretinography. Causative gene mutation was determined by genomewide linkage analysis and DNA sequencing. Structural modeling was used to predict the impact of the mutation on protein structure.
Results.
An ENU-mutagenized mouse line (R3), displaying attenuated retinal vessels and pigmented patches, was identified by fundus examination. Homozygous R3/R3 mice lost photoreceptors rapidly, leaving only a single row of photoreceptor nuclei at postnatal day 18. The a- and b-waves of ERG were flat in R3/R3 mice, whereas heterozygous R3/+ mice showed reduced amplitude of a- and b-waves. The R3/+ mice had a slower rate of photoreceptor cell loss than compound heterozygous R3/− mice with a null mutant allele. The R3 mutation was mapped and verified to be a rhodopsin point mutation, a c.553T>C for a p.C185R substitution. The side chain of Arg185 impacted on the extracellular loop of the protein. Mutant rhodopsin-C185R protein accumulated in the photoreceptor inner segments, cellular bodies, or both.
Conclusions.
Rhodopsin C185R mutation leads to severe retinal degeneration in R3 mutant mice. A dosage-dependent accumulation of misfolded mutant proteins likely triggers or stimulates the death of rod photoreceptors. The presence of a wild-type rhodopsin allele can delay the loss of photoreceptor cells in R3/+ mice.
doi:10.1167/iovs.09-3585
PMCID: PMC2868463  PMID: 19741247
8.  LRP5 Is Required for Vascular Development in Deeper Layers of the Retina 
PLoS ONE  2010;5(7):e11676.
Background
The low-density lipoprotein receptor-related protein 5 (LRP5) plays an important role in the development of retinal vasculature. LRP5 loss-of-function mutations cause incomplete development of retinal vessel network in humans as well as in mice. To understand the underlying mechanism for how LRP5 mutations lead to retinal vascular abnormalities, we have determined the retinal cell types that express LRP5 and investigated specific molecular and cellular functions that may be regulated by LRP5 signaling in the retina.
Methods and Findings
We characterized the development of retinal vasculature in LRP5 mutant mice using specific retinal cell makers and a GFP transgene expressed in retinal endothelial cells. Our data revealed that retinal vascular endothelial cells predominantly formed cell clusters in the inner-plexiform layer of LRP5 mutant retina rather than sprouting out or migrating into deeper layers to form normal vascular network in the retina. The IRES-β-galactosidase (LacZ) report gene under the control of the endogenous LRP5 promoter was highly expressed in Müller cells and was also weakly detected in endothelial cells of the retinal surface vasculature. Moreover, the LRP5 mutant mice had a reduction of a Müller cell-specific glutamine transporter, Slc38a5, and showed a decrease in b-wave amplitude of electroretinogram.
Conclusions
LRP5 is not only essential for vascular endothelial cells to sprout, migrate and/or anastomose in the deeper plexus during retinal vasculature development but is also important for the functions of Müller cells and retinal interneurons. Müller cells may utilize LRP5-mediated signaling pathway to regulate vascular development in deeper layers and to maintain the function of retinal interneurons.
doi:10.1371/journal.pone.0011676
PMCID: PMC2907392  PMID: 20652025
9.  Papillorenal Syndrome-Causing Missense Mutations in PAX2/Pax2 Result in Hypomorphic Alleles in Mouse and Human 
PLoS Genetics  2010;6(3):e1000870.
Papillorenal syndrome (PRS, also known as renal-coloboma syndrome) is an autosomal dominant disease characterized by potentially-blinding congenital optic nerve excavation and congenital kidney abnormalities. Many patients with PRS have mutations in the paired box transcription factor gene, PAX2. Although most mutations in PAX2 are predicted to result in complete loss of one allele's function, three missense mutations have been reported, raising the possibility that more subtle alterations in PAX2 function may be disease-causing. To date, the molecular behaviors of these mutations have not been explored. We describe a novel mouse model of PRS due to a missense mutation in a highly-conserved threonine residue in the paired domain of Pax2 (p.T74A) that recapitulates the ocular and kidney findings of patients. This mutation is in the Pax2 paired domain at the same location as two human missense mutations. We show that all three missense mutations disrupt potentially critical hydrogen bonds in atomic models and result in reduced Pax2 transactivation, but do not affect nuclear localization, steady state mRNA levels, or the ability of Pax2 to bind its DNA consensus sequence. Moreover, these mutations show reduced steady-state levels of Pax2 protein in vitro and (for p.T74A) in vivo, likely by reducing protein stability. These results suggest that hypomorphic alleles of PAX2/Pax2 can lead to significant disease in humans and mice.
Author Summary
Congenital ocular malformations affecting the optic nerve are an important cause of childhood blindness. The papillorenal syndrome (PRS) is an autosomal dominant disorder that causes congenital optic nerve and kidney abnormalities, which may result in legal blindness and renal failure, respectively. Many cases of PRS are caused by mutations in the paired-box transcription factor PAX2. In this paper, we describe a novel mouse model of this human disease caused by a missense mutation in the Pax2 gene at the same position of one of the few disease-causing missense mutations in humans. We characterize the ocular and non-ocular phenotypes of this mouse and model the effect that murine and human Pax2/PAX2 mutations have on protein structure. We also experimentally test the effect these missense mutations have on protein localization, transactivation, and DNA binding, concluding that all three reduce steady-state levels of protein in vitro and (in p.T74A) in vivo by reducing protein stability. This work will help us better understand the pathophysiology of PRS and to dissect the molecular interactions important in normal PAX2 function.
doi:10.1371/journal.pgen.1000870
PMCID: PMC2832668  PMID: 20221250
10.  Gap junction communication influences intercellular protein distribution in the lens 
Experimental eye research  2008;86(6):966-974.
Lens transparency and high refractive index presumably depend on the appropriate arrangement and distribution of lens proteins among lens fiber cells. Intercellular gap junction channels formed by α3 and α8 connexins are known to transport small molecules, ions and water, but not proteins, in the lens. Mosaic expression of green fluorescent protein (GFP) in the lens is a useful marker for monitoring macromolecule distribution between fiber cells and for constructing 3-dimensional images of living lens cells. In α3(−/−) α8(−/−) double knockout (DKO) lenses, three-dimensional images of GFP-positive cells demonstrate the changes of epithelial cell surfaces and insufficient elongation of inner fiber cells. Uniform distribution of GFP between inner lens fiber cells is observed in both wild-type and α3(−/−) lenses. In contrast, uniform GFP distribution is slightly delayed in α8(−/−) lenses and is abolished in DKO lenses. Without endogenous wild-type α3 and α8 connexins, knock-in α3 connexin (expressed under the α8 gene promoter) restores the uniform distribution of GFP protein in the lens. Thus, the presence of either α3 or α8 connexins seems sufficient to support the uniform distribution of GFP between differentiated lens fiber cells. Although the mechanism that drives GFP transport between fiber cells remains unknown, this work reveals that gap junction communication plays a novel role in the regulation of intercellular protein distribution in the lens.
doi:10.1016/j.exer.2008.03.015
PMCID: PMC2528023  PMID: 18462719
Connexin; Gap junction; Cell-cell communication
11.  A model for familial exudative vitreoretinopathy caused by LPR5 mutations 
Human Molecular Genetics  2008;17(11):1605-1612.
We have identified a mouse recessive mutation that leads to attenuated and hyperpermeable retinal vessels, recapitulating some pathological features of familial exudative vitreoretinopathy (FEVR) in human patients. DNA sequencing reveals a single nucleotide insertion in the gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5), causing a frame shift and resulting in the replacement of the C-terminal 39 amino acid residues by 20 new amino acids. This change eliminates the last three PPP(S/T)P repeats in the LRP5 cytoplasmic domain that are important for mediating Wnt/β-catenin signaling. Thus, mutant LRP5 protein is probably unable to mediate its downstream signaling. Immunostaining and three-dimensional reconstructions of retinal vasculature confirm attenuated retinal vessels. Ultrastructural data further reveal that some capillaries lack lumen structure in the mutant retina. We have also verified that LRP5 null mice develop similar alterations in the retinal vasculature. This study provides direct evidence that LRP5 is essential for the development of retinal vasculature, and suggests a novel role played by LRP5 in capillary maturation. LRP5 mutant mice can be a useful model to explore the clinical manifestations of FEVR.
doi:10.1093/hmg/ddn047
PMCID: PMC2902293  PMID: 18263894
12.  Abnormal neurofilament transport caused by targeted disruption of neuronal kinesin heavy chain KIF5A 
The Journal of Cell Biology  2003;161(1):55-66.
To test the hypothesis that fast anterograde molecular motor proteins power the slow axonal transport of neurofilaments (NFs), we used homologous recombination to generate mice lacking the neuronal-specific conventional kinesin heavy chain, KIF5A. Because null KIF5A mutants die immediately after birth, a synapsin-promoted Cre-recombinase transgene was used to direct inactivation of KIF5A in neurons postnatally. Three fourths of such mutant mice exhibited seizures and death at around 3 wk of age; the remaining animals survived to 3 mo or longer. In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber. Older animals also developed age-dependent sensory neuron degeneration, an accumulation of NF subunits in cell bodies and a reduction in axons, loss of large caliber axons, and hind limb paralysis. These data support the hypothesis that a conventional kinesin plays a role in the microtubule-dependent slow axonal transport of at least one cargo, the NF proteins.
doi:10.1083/jcb.200301026
PMCID: PMC2172877  PMID: 12682084
slow axonal transport; neuronal kinesin heavy chain KIF5A; neurofilament; axonal caliber; DRG sensory neuron
13.  Molecular Cloning and Functional Analysis of Mouse C-Terminal Kinesin Motor KifC3 
Molecular and Cellular Biology  2001;21(3):765-770.
Proteins of the kinesin superfamily define a class of microtubule-dependent motors that play crucial roles in cell division and intracellular transport. To study the molecular mechanism of intracellular transport involving microtubule-dependent motors, a cDNA encoding a new kinesin-like protein called KifC3 was cloned from a mouse brain cDNA library. Sequence and secondary structure analysis revealed that KifC3 is a member of the C-terminal motor family. In contrast to other mouse C-terminal motors, KifC3 is apparently ubiquitous and may have a general role in intracellular transport. To understand the in vivo function of the KifC3 gene, we used homologous recombination in embryonic stem cells to construct knockout mouse strains for the KifC3 gene. Homozygous mutants of the KifC3 gene are viable, reproduce normally, and apparently develop normally. These results suggest that KifC3 is dispensable for normal development and reproduction in the mouse.
doi:10.1128/MCB.21.3.765-770.2001
PMCID: PMC86668  PMID: 11154264

Results 1-13 (13)