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1.  Endothelial SRF/MRTF ablation causes vascular disease phenotypes in murine retinae 
The Journal of Clinical Investigation  2013;123(5):2193-2206.
Retinal vessel homeostasis ensures normal ocular functions. Consequently, retinal hypovascularization and neovascularization, causing a lack and an excess of vessels, respectively, are hallmarks of human retinal pathology. We provide evidence that EC-specific genetic ablation of either the transcription factor SRF or its cofactors MRTF-A and MRTF-B, but not the SRF cofactors ELK1 or ELK4, cause retinal hypovascularization in the postnatal mouse eye. Inducible, EC-specific deficiency of SRF or MRTF-A/MRTF-B during postnatal angiogenesis impaired endothelial tip cell filopodia protrusion, resulting in incomplete formation of the retinal primary vascular plexus, absence of the deep plexi, and persistence of hyaloid vessels. All of these features are typical of human hypovascularization-related vitreoretinopathies, such as familial exudative vitreoretinopathies including Norrie disease. In contrast, conditional EC deletion of Srf in adult murine vessels elicited intraretinal neovascularization that was reminiscent of the age-related human pathologies retinal angiomatous proliferation and macular telangiectasia. These results indicate that angiogenic homeostasis is ensured by differential stage-specific functions of SRF target gene products in the developing versus the mature retinal vasculature and suggest that the actin-directed MRTF-SRF signaling axis could serve as a therapeutic target in the treatment of human vascular retinal diseases.
doi:10.1172/JCI64201
PMCID: PMC3635718  PMID: 23563308
2.  PGC-1α Determines Light Damage Susceptibility of the Murine Retina 
PLoS ONE  2012;7(2):e31272.
The peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) proteins are key regulators of cellular bioenergetics and are accordingly expressed in tissues with a high energetic demand. For example, PGC-1α and PGC-1β control organ function of brown adipose tissue, heart, brain, liver and skeletal muscle. Surprisingly, despite their prominent role in the control of mitochondrial biogenesis and oxidative metabolism, expression and function of the PGC-1 coactivators in the retina, an organ with one of the highest energy demands per tissue weight, are completely unknown. Moreover, the molecular mechanisms that coordinate energy production with repair processes in the damaged retina remain enigmatic. In the present study, we thus investigated the expression and function of the PGC-1 coactivators in the healthy and the damaged retina. We show that PGC-1α and PGC-1β are found at high levels in different structures of the mouse retina, most prominently in the photoreceptors. Furthermore, PGC-1α knockout mice suffer from a striking deterioration in retinal morphology and function upon detrimental light exposure. Gene expression studies revealed dysregulation of all major pathways involved in retinal damage and apoptosis, repair and renewal in the PGC-1α knockouts. The light-induced increase in apoptosis in vivo in the absence of PGC-1α was substantiated in vitro, where overexpression of PGC-1α evoked strong anti-apoptotic effects. Finally, we found that retinal levels of PGC-1 expression are reduced in different mouse models for retinitis pigmentosa. We demonstrate that PGC-1α is a central coordinator of energy production and, importantly, all of the major processes involved in retinal damage and subsequent repair. Together with the observed dysregulation of PGC-1α and PGC-1β in retinitis pigmentosa mouse models, these findings thus imply that PGC-1α might be an attractive target for therapeutic approaches aimed at retinal degeneration diseases.
doi:10.1371/journal.pone.0031272
PMCID: PMC3278422  PMID: 22348062
4.  Spectral Domain Optical Coherence Tomography in Mouse Models of Retinal Degeneration 
Purpose
Spectral domain optical coherence tomography (SD-OCT) allows cross-sectional visualization of retinal structures in vivo. Here, we report the efficacy of a commercially available SD-OCT device to study mouse models of retinal degeneration.
Methods
C57BL/6 and BALB/c wild type mice and three different mouse models of hereditary retinal degeneration (Rho-/-, rd1, RPE65-/-) were investigated using confocal scanning laser ophthalmoscopy (cSLO) for en face visualization and SD-OCT for cross-sectional imaging of retinal structures. Histology was performed to correlate structural findings in SD-OCT with light microscopic data.
Results
In C57BL/6 and BALB/c mice, cSLO and SD-OCT imaging provided structural details of frequently used control animals (central retinal thickness, CRTC57BL/6 = 237±2μm and CRTBALB/c = 211±10μm). RPE65-/- mice at 11 months of age showed a significant reduction of retinal thickness (CRTRPE65 = 193±2μm) with thinning of the outer nuclear layer. Rho-/- mice at P28 demonstrated degenerative changes mainly in the outer retinal layers (CRTRho = 193±2μm). Examining rd1 animals before and after the onset of retinal degeneration allowed to monitor disease progression (CRTrd1 P11 = 246±4μm, CRTrd1 P28 = 143±4μm). Correlation of CRT assessed by histology and SD-OCT was high (r2 = 0.897).
Conclusion
We demonstrated cross sectional visualization of retinal structures in wild type mice and mouse models for retinal degeneration in vivo using a commercially available SD-OCT device. This method will help to reduce numbers of animals needed per study by allowing longitudinal study designs and facilitate characterization of disease dynamics and evaluation of putative therapeutic effects following experimental interventions.
doi:10.1167/iovs.09-3724
PMCID: PMC2800101  PMID: 19661229
optical coherence tomography; retinal degeneration; imaging; mouse models
5.  Novel Rodent Models for Macular Research 
PLoS ONE  2010;5(10):e13403.
Background
Many disabling human retinal disorders involve the central retina, particularly the macula. However, the commonly used rodent models in research, mouse and rat, do not possess a macula. The purpose of this study was to identify small laboratory rodents with a significant central region as potential new models for macular research.
Methodology/Principal Findings
Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli, laboratory rodents less commonly used in retinal research, were subjected to confocal scanning laser ophthalmoscopy (cSLO), fluorescein and indocyanine green angiography, and spectral-domain optical coherence tomography (SD-OCT) using standard equipment (Heidelberg Engineering HRA1 and Spectralis™) adapted to small rodent eyes. The existence of a visual streak-like pattern was assessed on the basis of vascular topography, retinal thickness, and the topography of retinal ganglion cells and cone photoreceptors. All three species examined showed evidence of a significant horizontal streak-like specialization. cSLO angiography and retinal wholemounts revealed that superficial retinal blood vessels typically ramify and narrow into a sparse capillary net at the border of the respective area located dorsal to the optic nerve. Similar to the macular region, there was an absence of larger blood vessels in the streak region. Furthermore, the thickness of the photoreceptor layer and the population density of neurons in the ganglion cell layer were markedly increased in the visual streak region.
Conclusions/Significance
The retinal specializations of Gerbillus perpallidus, Meriones unguiculatus and Phodopus campbelli resemble features of the primate macula. Hence, the rodents reported here may serve to study aspects of macular development and diseases like age-related macular degeneration and diabetic macular edema, and the preclinical assessment of therapeutic strategies.
doi:10.1371/journal.pone.0013403
PMCID: PMC2955520  PMID: 20976212
6.  Retinal Degenerative and Hypoxic Ischemic Disease 
A broad spectrum of retinal diseases affects both the retinal vasculature and the neural retina, including photoreceptor and postreceptor layers. The accepted clinical hallmarks of acute retinopathy of prematurity (ROP) are dilation and tortuosity of the retinal vasculature. Additionally, significant early and persistent effects on photoreceptor and postreceptor neural structures and function are demonstrated in ROP. In this paper, we focus on the results of longitudinal studies of electroretinographic (ERG) and vascular features in rats with induced retinopathies that model the gamut of human ROP, mild to severe. Two potential targets for pharmaceutical interventions emerge from the observations. The first target is immature photoreceptors because the status of the photoreceptors at an early age predicts later vascular outcome; this approach is appealing as it holds promise to prevent ROP. The second target is the interplay of the neural and vascular retinal networks, which develop cooperatively. Beneficial pharmaceutical interventions may be measured in improved visual outcome as well as lessening of the vascular abnormalities.
doi:10.1007/s10633-008-9127-8
PMCID: PMC2629502  PMID: 18483822
7.  Phosducin influences sympathetic activity and prevents stress-induced hypertension in humans and mice 
The Journal of Clinical Investigation  2009;119(12):3597-3612.
Hypertension and its complications represent leading causes of morbidity and mortality. Although the cause of hypertension is unknown in most patients, genetic factors are recognized as contributing significantly to an individual’s lifetime risk of developing the condition. Here, we investigated the role of the G protein regulator phosducin (Pdc) in hypertension. Mice with a targeted deletion of the gene encoding Pdc (Pdc–/– mice) had increased blood pressure despite normal cardiac function and vascular reactivity, and displayed elevated catecholamine turnover in the peripheral sympathetic system. Isolated postganglionic sympathetic neurons from Pdc–/– mice showed prolonged action potential firing after stimulation with acetylcholine and increased firing frequencies during membrane depolarization. Furthermore, Pdc–/– mice displayed exaggerated increases in blood pressure in response to post-operative stress. Candidate gene–based association studies in 2 different human populations revealed several SNPs in the PDC gene to be associated with stress-dependent blood pressure phenotypes. Individuals homozygous for the G allele of an intronic PDC SNP (rs12402521) had 12–15 mmHg higher blood pressure than those carrying the A allele. These findings demonstrate that PDC is an important modulator of sympathetic activity and blood pressure and may thus represent a promising target for treatment of stress-dependent hypertension.
doi:10.1172/JCI38433
PMCID: PMC2786789  PMID: 19959875
9.  Vasoregression Linked to Neuronal Damage in the Rat with Defect of Polycystin-2 
PLoS ONE  2009;4(10):e7328.
Background
Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.
Methods
Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.
Findings
At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.
Interpretation
Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.
doi:10.1371/journal.pone.0007328
PMCID: PMC2752170  PMID: 19806208
10.  Rb-Mediated Neuronal Differentiation through Cell-Cycle–Independent Regulation of E2f3a 
PLoS Biology  2007;5(7):e179.
It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle–independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors.
Author Summary
The retinoblastoma protein (Rb), an important tumor suppressor, blocks division and death by inhibiting the E2f transcription factor family. In contrast, Rb is thought to promote differentiation by potentiating tissue-specific transcription factors, although differentiation defects in Rb null cells could be an indirect consequence of E2f-driven division and death. Here, we resolve different mechanisms by which Rb controls division, death, and differentiation in the retina. Removing E2f1 rescues aberrant division of differentiating Rb-deficient retinal neurons, as well as death in cells prone to apoptosis, and restores both normal differentiation and function of major cell types, such as photoreceptors. However, Rb-deficient starburst amacrine neurons differentiate abnormally even when E2f1 is removed, providing an unequivocal example of a direct role for Rb in neuronal differentiation. Rather than potentiating a cell-specific factor, Rb promotes starburst cell differentiation by inhibiting another E2f, E2f3a. This cell-cycle–independent activity broadens the importance of the Rb–E2f pathway, and suggests we should reassess its role in the differentiation of other cell types.
The retinoblastoma protein (Rb), a tumor suppressor, promotes the differentiation of starburst amacrine cells in the retina by inhibiting the transcription factor E2f3a, whereas it suppresses retinal cell division and death by inhibiting E2f1.
doi:10.1371/journal.pbio.0050179
PMCID: PMC1914394  PMID: 17608565
11.  Lentiviral Gene Transfer of Rpe65 Rescues Survival and Function of Cones in a Mouse Model of Leber Congenital Amaurosis 
PLoS Medicine  2006;3(10):e347.
Background
RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice.
Methods and Findings
Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease.
Conclusions
By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65−/− mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose.
In theRpe65-/- mouse model of Leber congenital amaurosis, injection of a lentiviral vector expressing the Rpe65 mouse cDNA was able to prevent cone degeneration and restore cone function.
Editors' Summary
Background.
Leber congenital amaurosis (LCA) is the name of a group of hereditary diseases that cause blindness in infants and children. Changes in any one of a number of different genes can cause the blindness, which affects vision starting at birth or soon after. The condition was first described by a German doctor, Theodore Leber, in the 19th century, hence the first part of the name; “amaurosis” is another word for blindness. Mutations in one gene called retinal pigment epithelium-specific protein, 65 kDa (RPE65)—so called because it is expressed in the pigment epithelium, a cell layer adjacent to the light-sensitive cells, and is 65 kilodaltons in size—cause about 10% of cases of LCA. The product of this gene is essential for the recycling of a substance called 11-cis-retinal, which is necessary for the light-sensitive rods and cones of the retina to capture light. If the gene is abnormal, the sensitivity of the retina to light is drastically reduced, but it also leads to damage to the light-sensitive cells themselves.
Why Was This Study Done?
Potentially, eyes diseases such as this one could be treated by gene therapy, which works by replacing a defective gene with a normal functional one, usually by putting a copy of the normal gene into a harmless virus and injecting it into the affected tissue—in this case, the eye. The researchers here wanted to see whether expressing wild-type RPE65 using a particular type of gene vector that can carry large pieces of DNA transcript—a lentiviral vector—could prevent degeneration of cone cells and restore cone function in a mouse model of this type of LCA—mice who had had this Rpe65 gene genetically removed.
What Did the Researchers Do and Find?
Injection of the normal gene into the retina of Rpe65-deficient mice led to sustained expression of the protein RPE65 in the retinal pigment epithelium. Electrical recordings of the activity of the eyes in these mice showed that Rpe65 gene transfer restored retinal function to a near-normal level. In addition, Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mice.
What Do These Findings Mean?
These findings suggest that it is theoretically possible to treat this type of blindness by gene therapy. However, because this study was done in mice, many other steps need to be taken before it will be clear whether the treatment could work in humans. These steps include a demonstration that the virus is safe in humans, and experiments to determine what dose of virus would be needed and how long the effects of the treatment would last. Another question is whether it would be necessary (or even possible) to treat affected children during early childhood or when children start losing vision.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030347.
The Foundation for Retinal Research has detailed information on Leber's congenital amaurosis
Contact a Family is a UK organization that aims to put families of children with illnesses in touch with each other
The Foundation for Fighting Blindness funds research into, and provides information about many types of blindness, including Leber's congenital amaurosis
This Web site provides information on gene therapy clinical trials, including those dedicated to cure eye diseases
This foundation provides information on diseases leading to blindness, including Leber's congenital amaurosis
doi:10.1371/journal.pmed.0030347
PMCID: PMC1592340  PMID: 17032058
12.  Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia 
Journal of Clinical Investigation  2006;116(2):386-394.
Choroideremia (CHM) is an X-linked degeneration of the retinal pigment epithelium (RPE), photoreceptors, and choroid, caused by loss of function of the CHM/REP1 gene. REP1 is involved in lipid modification (prenylation) of Rab GTPases, key regulators of intracellular vesicular transport and organelle dynamics. To study the pathogenesis of CHM and to develop a model for assessing gene therapy, we have created a conditional mouse knockout of the Chm gene. Heterozygous-null females exhibit characteristic hallmarks of CHM: progressive degeneration of the photoreceptors, patchy depigmentation of the RPE, and Rab prenylation defects. Using tamoxifen-inducible and tissue-specific Cre expression in combination with floxed Chm alleles, we show that CHM pathogenesis involves independently triggered degeneration of photoreceptors and the RPE, associated with different subsets of defective Rabs.
doi:10.1172/JCI26617
PMCID: PMC1326146  PMID: 16410831
13.  The Role of Rab27a in the Regulation of Melanosome Distribution within Retinal Pigment Epithelial Cells 
Molecular Biology of the Cell  2004;15(5):2264-2275.
Melanosomes within the retinal pigment epithelium (RPE) of mammals have long been thought to exhibit no movement in response to light, unlike fish and amphibian RPE. Here we show that the distribution of melanosomes within the mouse RPE undergoes modest but significant changes with the light cycle. Two hours after light onset, there is a threefold increase in the number of melanosomes in the apical processes that surround adjacent photoreceptors. In skin melanocytes, melanosomes are motile and evenly distributed throughout the cell periphery. This distribution is due to the interaction with the cortical actin cytoskeleton mediated by a tripartite complex of Rab27a, melanophilin, and myosin Va. In ashen (Rab27a null) mice RPE, melanosomes are unable to move beyond the adherens junction axis and do not enter apical processes, suggesting that Rab27a regulates melanosome distribution in the RPE. Unlike skin melanocytes, the effects of Rab27a are mediated through myosin VIIa in the RPE, as evidenced by the similar melanosome distribution phenotype observed in shaker-1 mice, defective in myosin VIIa. Rab27a and myosin VIIa are likely to be required for association with and movement through the apical actin cytoskeleton, which is a prerequisite for entry into the apical processes.
doi:10.1091/mbc.E03-10-0772
PMCID: PMC404021  PMID: 14978221
14.  Rapid degradation of dominant-negative Rab27 proteins in vivo precludes their use in transgenic mouse models 
BMC Cell Biology  2002;3:26.
Background
Transgenic mice have proven to be a powerful system to study normal and pathological gene functions. Here we describe an attempt to generate a transgenic mouse model for choroideremia (CHM), a slow-onset X-linked retinal degeneration caused by mutations in the Rab Escort Protein-1 (REP1) gene. REP1 is part of the Rab geranylgeranylation machinery, a modification that is essential for Rab function in membrane traffic. The loss of REP1 in CHM patients may trigger retinal degeneration through its effects on Rab proteins. We have previously reported that Rab27a is the Rab most affected in CHM lymphoblasts and hypothesised that the selective dysfunction of Rab27a (and possibly a few other Rab GTPases) plays an essential role in the retinal degenerative process.
Results
To investigate this hypothesis, we generated several lines of dominant-negative, constitutively-active and wild-type Rab27a (and Rab27b) transgenic mice whose expression was driven either by the pigment cell-specific tyrosinase promoter or the ubiquitous β-actin promoter. High levels of mRNA and protein were observed in transgenic lines expressing wild-type or constitutively active Rab27a and Rab27b. However, only modest levels of transgenic protein were expressed. Pulse-chase experiments suggest that the dominant-negative proteins, but not the constitutively-active or wild type proteins, are rapidly degraded. Consistently, no significant phenotype was observed in our transgenic lines. Coat-colour was normal, indicating normal Rab27a activity. Retinal function as determined by fundoscopy, angiography, electroretinography and histology was also normal.
Conclusions
We suggest that the instability of the dominant-negative mutant Rab27 proteins in vivo precludes the use of this approach to generate mouse models of disease caused by Rab27 GTPases.
doi:10.1186/1471-2121-3-26
PMCID: PMC137576  PMID: 12401133
15.  Targeted Ablation of Crb1 and Crb2 in Retinal Progenitor Cells Mimics Leber Congenital Amaurosis 
PLoS Genetics  2013;9(12):e1003976.
Development in the central nervous system is highly dependent on the regulation of the switch from progenitor cell proliferation to differentiation, but the molecular and cellular events controlling this process remain poorly understood. Here, we report that ablation of Crb1 and Crb2 genes results in severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. We show that the levels of CRB1 and CRB2 proteins are crucial for mouse retinal development, as they restrain the proliferation of retinal progenitor cells. The lack of these apical proteins results in altered cell cycle progression and increased number of mitotic cells leading to an increased number of late-born cell types such as rod photoreceptors, bipolar and Müller glia cells in postmitotic retinas. Loss of CRB1 and CRB2 in the retina results in dysregulation of target genes for the Notch1 and YAP/Hippo signaling pathways and increased levels of P120-catenin. Loss of CRB1 and CRB2 result in altered progenitor cell cycle distribution with a decrease in number of late progenitors in G1 and an increase in S and G2/M phase. These findings suggest that CRB1 and CRB2 suppress late progenitor pool expansion by regulating multiple proliferative signaling pathways.
Author Summary
Mutations in the human CRB1 gene lead to one of the most severe forms of retinal dystrophies, called Leber congenital amaurosis. Here, we report that ablation of CRB1 and the second family member CRB2 are crucial for proper retinal development. These mice display severe impairment of retinal function, abnormal lamination and thickening of the retina mimicking human Leber congenital amaurosis due to loss of CRB1 function. The thickening of the retina is due to increased cell proliferation during late retinal development leading to an increased number of late-born retinal cells. We describe in these CRB1 Leber congenital amaurosis mouse models the molecular and cellular events involving CRB proteins during the development of the retina.
doi:10.1371/journal.pgen.1003976
PMCID: PMC3854796  PMID: 24339791
16.  Myosin7a Deficiency Results in Reduced Retinal Activity Which Is Improved by Gene Therapy 
PLoS ONE  2013;8(8):e72027.
Mutations in MYO7A cause autosomal recessive Usher syndrome type IB (USH1B), one of the most frequent conditions that combine severe congenital hearing impairment and retinitis pigmentosa. A promising therapeutic strategy for retinitis pigmentosa is gene therapy, however its pre-clinical development is limited by the mild retinal phenotype of the shaker1 (sh1−/−) murine model of USH1B which lacks both retinal functional abnormalities and degeneration. Here we report a significant, early-onset delay of sh1−/− photoreceptor ability to recover from light desensitization as well as a progressive reduction of both b-wave electroretinogram amplitude and light sensitivity, in the absence of significant loss of photoreceptors up to 12 months of age. We additionally show that subretinal delivery to the sh1−/− retina of AAV vectors encoding the large MYO7A protein results in significant improvement of sh1−/− photoreceptor and retinal pigment epithelium ultrastructural anomalies which is associated with improvement of recovery from light desensitization. These findings provide new tools to evaluate the efficacy of experimental therapies for USH1B. In addition, although AAV vectors expressing large genes might have limited clinical applications due to their genome heterogeneity, our data show that AAV-mediated MYO7A gene transfer to the sh1−/− retina is effective.
doi:10.1371/journal.pone.0072027
PMCID: PMC3753344  PMID: 23991031
17.  Successful Subretinal Delivery and Monitoring of MicroBeads in Mice 
PLoS ONE  2013;8(1):e55173.
Background
To monitor viability of implanted genetically engineered and microencapsulated human stem cells (MicroBeads) in the mouse eye, and to study the impact of the beads and/or xenogenic cells on retinal integrity.
Methodology/Principal Findings
MicroBeads were implanted into the subretinal space of SV126 wild type mice using an ab externo approach. Viability of microencapsulated cells was monitored by noninvasive retinal imaging (Spectralis™ HRA+OCT). Retinal integrity was also assessed with retinal imaging and upon the end of the study by light and electron microscopy. The implanted GFP-marked cells encapsulated in subretinal MicroBeads remained viable over a period of up to 4 months. Retinal integrity and viability appeared unaltered apart from the focal damage due to the surgical implantation, GFAP upregulation, and opsin mistargeting in the immediate surrounding tissue.
Conclusions/Significance
The accessibility for routine surgery and its immune privileged state make the eye an ideal target for release system implants for therapeutic substances, including neurotrophic and anti-angiogenic compounds or protein based biosimilars. Microencapsulated human stem cells (MicroBeads) promise to overcome limitations inherent with single factor release systems, as they are able to produce physiologic combinations of bioactive compounds.
doi:10.1371/journal.pone.0055173
PMCID: PMC3557268  PMID: 23383096
18.  Long-Term In Vivo Imaging of Fibrillar Tau in the Retina of P301S Transgenic Mice 
PLoS ONE  2012;7(12):e53547.
Tauopathies are widespread neurodegenerative disorders characterised by the intracellular accumulation of hyperphosphorylated tau. Especially in Alzheimer's disease, pathological alterations in the retina are discussed as potential biomarkers to improve early diagnosis of the disease. Using mice expressing human mutant P301S tau, we demonstrate for the first time a straightforward optical approach for the in vivo detection of fibrillar tau in the retina. Longitudinal examinations of individual animals revealed the fate of single cells containing fibrillar tau and the progression of tau pathology over several months. This technique is most suitable to monitor therapeutic interventions aimed at reducing the accumulation of fibrillar tau. In order to evaluate if this approach can be translated to human diagnosis, we tried to detect fibrillar protein aggregates in the post-mortem retinas of patients that had suffered from Alzheimer's disease or Progressive Supranuclear Palsy. Even though we could detect hyperphosphorylated tau, we did not observe any fibrillar tau or Aß aggregates. In contradiction to previous studies, our observations do not support the notion that Aβ or tau in the retina are of diagnostic value in Alzheimer's disease.
doi:10.1371/journal.pone.0053547
PMCID: PMC3534024  PMID: 23300938
19.  Lack of the Sodium-Driven Chloride Bicarbonate Exchanger NCBE Impairs Visual Function in the Mouse Retina 
PLoS ONE  2012;7(10):e46155.
Regulation of ion and pH homeostasis is essential for normal neuronal function. The sodium-driven chloride bicarbonate exchanger NCBE (Slc4a10), a member of the SLC4 family of bicarbonate transporters, uses the transmembrane gradient of sodium to drive cellular net uptake of bicarbonate and to extrude chloride, thereby modulating both intracellular pH (pHi) and chloride concentration ([Cl−]i) in neurons. Here we show that NCBE is strongly expressed in the retina. As GABAA receptors conduct both chloride and bicarbonate, we hypothesized that NCBE may be relevant for GABAergic transmission in the retina. Importantly, we found a differential expression of NCBE in bipolar cells: whereas NCBE was expressed on ON and OFF bipolar cell axon terminals, it only localized to dendrites of OFF bipolar cells. On these compartments, NCBE colocalized with the main neuronal chloride extruder KCC2, which renders GABA hyperpolarizing. NCBE was also expressed in starburst amacrine cells, but was absent from neurons known to depolarize in response to GABA, like horizontal cells. Mice lacking NCBE showed decreased visual acuity and contrast sensitivity in behavioral experiments and smaller b-wave amplitudes and longer latencies in electroretinograms. Ganglion cells from NCBE-deficient mice also showed altered temporal response properties. In summary, our data suggest that NCBE may serve to maintain intracellular chloride and bicarbonate concentration in retinal neurons. Consequently, lack of NCBE in the retina may result in changes in pHi regulation and chloride-dependent inhibition, leading to altered signal transmission and impaired visual function.
doi:10.1371/journal.pone.0046155
PMCID: PMC3467262  PMID: 23056253
20.  Noninvasive, In Vivo Assessment of Mouse Retinal Structure Using Optical Coherence Tomography 
PLoS ONE  2009;4(10):e7507.
Background
Optical coherence tomography (OCT) is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration.
Methodology/Principal Findings
We achieved to adapt a commercial 3rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis), retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE) due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified.
Conclusions/Significance
We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical assessment of therapeutic strategies.
doi:10.1371/journal.pone.0007507
PMCID: PMC2759518  PMID: 19838301

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