Sainte-Foy-lès-Lyon, France
Sainte-Foy-lès-Lyon, France

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Prabakaran T.,University of Aarhus | Nielsen R.,University of Aarhus | Larsen J.V.,University of Aarhus | Sorensen S.S.,Rigshospitalet | And 5 more authors.
PLoS ONE | Year: 2011

Injury to the glomerular podocyte is a key mechanism in human glomerular disease and podocyte repair is an important therapeutic target. In Fabry disease, podocyte injury is caused by the intracellular accumulation of globotriaosylceramide. This study identifies in the human podocyte three endocytic receptors, mannose 6-phosphate/insulin-like growth II receptor, megalin, and sortilin and demonstrates their drug delivery capabilities for enzyme replacement therapy. Sortilin, a novel α-galactosidase A binding protein, reveals a predominant intracellular expression but also surface expression in the podocyte. The present study provides the rationale for the renal effect of treatment with α-galactosidase A and identifies potential pathways for future non-carbohydrate based drug delivery to the kidney podocyte and other potential affected organs. © 2011 Prabakaran et al.


Galy A.,Institute Of La Vision | Schenck A.,Radboud University Nijmegen | Sahin H.B.,Bogazici University | Qurashi A.,Emory University | And 4 more authors.
Developmental Biology | Year: 2011

Cell rearrangements shape organs and organisms using molecular pathways and cellular processes that are still poorly understood. Here we investigate the role of the Actin cytoskeleton in the formation of the Drosophila compound eye, which requires extensive remodeling and coordination between different cell types. We show that CYFIP/Sra-1, a member of the WAVE/SCAR complex and regulator of Actin remodeling, controls specific aspects of eye architecture: rhabdomere extension, rhabdomere terminal web organization, adherens junctions, retina depth and basement membrane integrity. We demonstrate that some phenotypes manifest independently, due to defects in different cell types. Mutations in WAVE/SCAR and in ARP2/3 complex subunits but not in WASP, another major regulator of Actin nucleation, phenocopy CYFIP defects. Thus, the CYFIP-SCAR-ARP2/3 pathway orchestrates specific tissue remodeling processes. © 2011 Elsevier Inc.


Oliver V.F.,Johns Hopkins University | Wan J.,Johns Hopkins University | Agarwal S.,Ludwig Institute for Cancer Research | Agarwal S.,University of California at San Diego | And 4 more authors.
Epigenetics and Chromatin | Year: 2013

Background: Growing evidence suggests that DNA methylation plays a role in tissue-specific differentiation. Current approaches to methylome analysis using enrichment with the methyl-binding domain protein (MBD) are restricted to large (≥1 μg) DNA samples, limiting the analysis of small tissue samples. Here we present a technique that enables characterization of genome-wide tissue-specific methylation patterns from nanogram quantities of DNA. Results: We have developed a methodology utilizing MBD2b/MBD3L1 enrichment for methylated DNA, kinase pre-treated ligation-mediated PCR amplification (MeKL) and hybridization to the comprehensive high-throughput array for relative methylation (CHARM) customized tiling arrays, which we termed MeKL-chip. Kinase modification in combination with the addition of PEG has increased ligation-mediated PCR amplification over 20-fold, enabling >400-fold amplification of starting DNA. We have shown that MeKL-chip can be applied to as little as 20 ng of DNA, enabling comprehensive analysis of small DNA samples. Applying MeKL-chip to the mouse retina (a limited tissue source) and brain, 2,498 tissue-specific differentially methylated regions (T-DMRs) were characterized. The top five T-DMRs (Rgs20, Hes2, Nfic, Cckbr and Six3os1) were validated by pyrosequencing. Conclusions: MeKL-chip enables genome-wide methylation analysis of nanogram quantities of DNA with a wide range of observed-to-expected CpG ratios due to the binding properties of the MBD2b/MBD3L1 protein complex. This methodology enabled the first analysis of genome-wide methylation in the mouse retina, characterizing novel T-DMRs. © 2013 Oliver et al.; licensee BioMed Central Ltd.


Bhise N.S.,Institute for Nanobiotechnology | Wahlin K.J.,Johns Hopkins University | Zack D.J.,Johns Hopkins University | Zack D.J.,Institute Of La Vision | And 2 more authors.
International Journal of Nanomedicine | Year: 2013

Background: Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. Methods: A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. Results: 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacrylate-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE®HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. Conclusion: This study shows that certain nonviral reprogramming methods may not necessarily be safer than viral approaches and that maximizing exogenous gene expression of reprogramming factors is not sufficient to ensure successful reprogramming. © 2013 Bhise et al.


Wahlin K.J.,Johns Hopkins University | Maruotti J.,Johns Hopkins University | Zack D.J.,Johns Hopkins University | Zack D.J.,Institute Of La Vision
Advances in Experimental Medicine and Biology | Year: 2014

Retinal degenerative disease involving photoreceptor (PR) cell loss results in permanent vision loss and often blindness. Generation of induced pluripotent stem cell (iPSC)-derived retinal cells and tissues from individuals with retinal dystrophies is a relatively new and promising method for studying retinal degeneration mechanisms in vitro. Recent advancements in strategies to differentiate human iPSCs (hiPSCs) into 3D retinal eyecups with a strong resemblance to the mature retina raise the possibility that this system could offer a reliable model for translational drug studies. However, despite the potential benefits, there are challenges that remain to be overcome before stem-cell-derived retinal eyecups can be routinely used to model human retinal diseases. This chapter will discuss both the potential of these 3D eyecup approaches and the nature of some of the challenges that remain. © Springer Science+Business Media, LLC 2014.


Wan J.,Johns Hopkins University | Oliver V.F.,Johns Hopkins University | Zhu H.,Johns Hopkins University | Zack D.J.,Johns Hopkins University | And 3 more authors.
Nucleic Acids Research | Year: 2013

The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation. © 2013 The Author(s). Published by Oxford University Press.


Ranganathan V.,Wilmer Eye Institute | Wahlin K.,Wilmer Eye Institute | Maruotti J.,Wilmer Eye Institute | Zack D.J.,Wilmer Eye Institute | And 2 more authors.
Nature Communications | Year: 2014

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription, thus constraining genomic-targeting sites to GN 19 NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs, which can be used to target both AN 19 NGG and GN 19 NGG genomic sites. AN 19 NGG sites occur ∼15% more frequently than GN 19 NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together, our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species. © 2014 Macmillan Publishers Limited.


Fauser S.,University of Cologne | Lambrou G.N.,Institute Of La Vision
Survey of Ophthalmology | Year: 2015

Anti-vascular endothelial growth factor (anti-VEGF) therapies for neovascular age-related macular degeneration (nAMD) have proven efficacy at a study-population level, although individual patient responses vary, with most of the patients responding well to anti-VEGF therapies, while a few respond poorly. The pathogenesis of AMD is known to have a genetic component, but it is unclear if any particular genotype can predict response to anti-VEGF therapy. With the advent of less expensive genotyping technology, there have been numerous studies within this area. Here we analyze potential biomarker candidates identified that could be used in a clinical setting to predict response to anti-VEGF treatment of nAMD. We analyze single nucleotide polymorphisms (SNPs) identified from 39 publications. The SNPs that appeared to be of most importance fell into two main groups: those previously associated with AMD pathogenesis and those within the signaling pathway targeted by anti-VEGF therapies. A number of small studies found evidence supporting an association between anti-VEGF treatment response and two SNPs, CFH rs1061170 and VEGFA rs699947, but results from randomized controlled trials found no such association. It is possible that, in the future, the cumulative effect of several high-risk SNPs may prove useful in a clinical setting and that other genetic biomarkers may emerge. © 2015 Elsevier Inc.


Lorach H.,Stanford University | Lorach H.,Institute Of La Vision | Goetz G.,Stanford University | Mandel Y.,Bar - Ilan University | And 7 more authors.
Vision Research | Year: 2015

Loss of photoreceptors during retinal degeneration leads to blindness, but information can be reintroduced into the visual system using electrical stimulation of the remaining retinal neurons. Subretinal photovoltaic arrays convert pulsed illumination into pulsed electric current to stimulate the inner retinal neurons. Since required irradiance exceeds the natural luminance levels, an invisible near-infrared (915 nm) light is used to avoid photophobic effects. We characterized the thresholds and dynamic range of cortical responses to prosthetic stimulation with arrays of various pixel sizes and with different number of photodiodes. Stimulation thresholds for devices with 140. μm pixels were approximately half those of 70. μm pixels, and with both pixel sizes, thresholds were lower with 2 diodes than with 3 diodes per pixel. In all cases these thresholds were more than two orders of magnitude below the ocular safety limit. At high stimulation frequencies (>20 Hz), the cortical response exhibited flicker fusion. Over one order of magnitude of dynamic range could be achieved by varying either pulse duration or irradiance. However, contrast sensitivity was very limited. Cortical responses could be detected even with only a few illuminated pixels. Finally, we demonstrate that recording of the corneal electric potential in response to patterned illumination of the subretinal arrays allows monitoring the current produced by each pixel, and thereby assessing the changes in the implant performance over time. © 2014 Elsevier B.V.


Nasser H.,French Institute for Research in Computer Science and Automation | Marre O.,Institute Of La Vision | Cessac B.,French Institute for Research in Computer Science and Automation
Journal of Statistical Mechanics: Theory and Experiment | Year: 2013

Understanding the dynamics of neural networks is a major challenge in experimental neuroscience. For that purpose, a modelling of the recorded activity that reproduces the main statistics of the data is required. In the first part, we present a review on recent results dealing with spike train statistics analysis using maximum entropy models (MaxEnt). Most of these studies have focused on modelling synchronous spike patterns, leaving aside the temporal dynamics of the neural activity. However, the maximum entropy principle can be generalized to the temporal case, leading to Markovian models where memory effects and time correlations in the dynamics are properly taken into account. In the second part, we present a new method based on Monte Carlo sampling which is suited for the fitting of large-scale spatio-temporal MaxEnt models. The formalism and the tools presented here will be essential to fit MaxEnt spatio-temporal models to large neural ensembles. © 2013 IOP Publishing Ltd and SISSA Medialab srl.

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