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1.
EMBO Rep ; 22(11): e53732, 2021 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-34494703

RESUMO

Neuronal communication is typically mediated via synapses and gap junctions. New forms of intercellular communication, including nanotubes (NTs) and extracellular vesicles (EVs), have been described for non-neuronal cells, but their role in neuronal communication is not known. Recently, transfer of cytoplasmic material between donor and host neurons ("material transfer") was shown to occur after photoreceptor transplantation. The cellular mechanism(s) underlying this surprising finding are unknown. Here, using transplantation, primary neuronal cultures and the generation of chimeric retinae, we show for the first time that mammalian photoreceptor neurons can form open-end NT-like processes. These processes permit the transfer of cytoplasmic and membrane-bound molecules in culture and after transplantation and can mediate gain-of-function in the acceptor cells. Rarely, organelles were also observed to transfer. Strikingly, use of chimeric retinae revealed that material transfer can occur between photoreceptors in the intact adult retina. Conversely, while photoreceptors are capable of releasing EVs, at least in culture, these are taken up by glia and not by retinal neurons. Our findings provide the first evidence of functional NT-like processes forming between sensory neurons in culture and in vivo.

2.
Cell Rep ; 36(5): 109461, 2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34348137

RESUMO

In development, almost all stratified neurons must migrate from their birthplace to the appropriate neural layer. Photoreceptors reside in the most apical layer of the retina, near their place of birth. Whether photoreceptors require migratory events for fine-positioning and/or retention within this layer is not well understood. Here, we show that photoreceptor nuclei of the developing mouse retina cyclically exhibit rapid, dynein-1-dependent translocation toward the apical surface, before moving more slowly in the basal direction, likely due to passive displacement by neighboring retinal nuclei. Attenuating dynein 1 function in rod photoreceptors results in their ectopic basal displacement into the outer plexiform layer and inner nuclear layer. Synapse formation is also compromised in these displaced cells. We propose that repeated, apically directed nuclear translocation events are necessary to ensure retention of post-mitotic photoreceptors within the emerging outer nuclear layer during retinogenesis, which is critical for correct neuronal lamination.

3.
Glia ; 69(9): 2272-2290, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34029407

RESUMO

Gliosis is a complex process comprising upregulation of intermediate filament (IF) proteins, particularly glial fibrillary acidic protein (GFAP) and vimentin, changes in glial cell morphology (hypertrophy) and increased deposition of inhibitory extracellular matrix molecules. Gliosis is common to numerous pathologies and can have deleterious effects on tissue function and regeneration. The role of IFs in gliosis is controversial, but a key hypothesized function is the stabilization of glial cell hypertrophy. Here, we developed RNAi approaches to examine the role of GFAP and vimentin in vivo in a murine model of inherited retinal degeneration, the Rhodopsin knockout (Rho-/- ) mouse. Specifically, we sought to examine the role of these IFs in the establishment of Müller glial hypertrophy during progressive degeneration, as opposed to (more commonly assessed) acute injury. Prevention of Gfap upregulation had a significant effect on the morphology of reactive Müller glia cells in vivo and, more strikingly, the reduction of Vimentin expression almost completely prevented these cells from undergoing degeneration-associated hypertrophy. Moreover, and in contrast to studies in knockout mice, simultaneous suppression of both GFAP and vimentin expression led to severe changes in the cytoarchitecture of the retina, in both diseased and wild-type eyes. These data demonstrate a crucial role for Vimentin, as well as GFAP, in the establishment of glial hypertrophy and support the further exploration of RNAi-mediated knockdown of vimentin as a potential therapeutic approach for modulating scar formation in the degenerating retina.

4.
Cell Rep ; 35(3): 109022, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33882303

RESUMO

Age-related macular degeneration and other macular diseases result in the loss of light-sensing cone photoreceptors, causing irreversible sight impairment. Photoreceptor replacement may restore vision by transplanting healthy cells, which must form new synaptic connections with the recipient retina. Despite recent advances, convincing evidence of functional connectivity arising from transplanted human cone photoreceptors in advanced retinal degeneration is lacking. Here, we show restoration of visual function after transplantation of purified human pluripotent stem cell-derived cones into a mouse model of advanced degeneration. Transplanted human cones elaborate nascent outer segments and make putative synapses with recipient murine bipolar cells (BCs), which themselves undergo significant remodeling. Electrophysiological and behavioral assessments demonstrate restoration of surprisingly complex light-evoked retinal ganglion cell responses and improved light-evoked behaviors in treated animals. Stringent controls exclude alternative explanations, including material transfer and neuroprotection. These data provide crucial validation for photoreceptor replacement therapy and for the potential to rescue cone-mediated vision.

5.
Aging (Albany NY) ; 13(8): 10866-10890, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33872219

RESUMO

Dry age-related macular degeneration (AMD) is marked by the accumulation of extracellular and intracellular lipid-rich deposits within and around the retinal pigment epithelium (RPE). Inducing autophagy, a conserved, intracellular degradative pathway, is a potential treatment strategy to prevent disease by clearing these deposits. However, mTOR inhibition, the major mechanism for inducing autophagy, disrupts core RPE functions. Here, we screened autophagy inducers that do not directly inhibit mTOR for their potential as an AMD therapeutic in primary human RPE culture. Only two out of more than thirty autophagy inducers tested reliably increased autophagy flux in RPE, emphasizing that autophagy induction mechanistically differs across distinct tissues. In contrast to mTOR inhibitors, these compounds preserved RPE health, and one inducer, the FDA-approved compound flubendazole (FLBZ), reduced the secretion of apolipoprotein that contributes to extracellular deposits termed drusen. Simultaneously, FLBZ increased production of the lipid-degradation product ß-hydroxybutyrate, which is used by photoreceptor cells as an energy source. FLBZ also reduced the accumulation of intracellular deposits, termed lipofuscin, and alleviated lipofuscin-induced cellular senescence and tight-junction disruption. FLBZ triggered compaction of lipofuscin-like granules into a potentially less toxic form. Thus, induction of RPE autophagy without direct mTOR inhibition is a promising therapeutic approach for dry AMD.


Assuntos
Autofagia/efeitos dos fármacos , Atrofia Geográfica/tratamento farmacológico , Mebendazol/análogos & derivados , Feto Abortado , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Atrofia Geográfica/patologia , Humanos , Lipofuscina/metabolismo , Mebendazol/farmacologia , Mebendazol/uso terapêutico , Cultura Primária de Células , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/patologia , Serina-Treonina Quinases TOR/metabolismo
6.
Cells ; 10(1)2021 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-33435495

RESUMO

The Rpe65-deficient dog has been important for development of translational therapies of Leber congenital amaurosis type 2 (LCA2). The purpose of this study was to provide a comprehensive report of the natural history of retinal changes in this dog model. Rpe65-deficient dogs from 2 months to 10 years of age were assessed by fundus imaging, electroretinography (ERG) and vision testing (VT). Changes in retinal layer thickness were assessed by optical coherence tomography and on plastic retinal sections. ERG showed marked loss of retinal sensitivity, with amplitudes declining with age. Retinal thinning initially developed in the area centralis, with a slower thinning of the outer retina in other areas starting with the inferior retina. VT showed that dogs of all ages performed well in bright light, while at lower light levels they were blind. Retinal pigment epithelial (RPE) inclusions developed and in younger dogs and increased in size with age. The loss of photoreceptors was mirrored by a decline in ERG amplitudes. The slow degeneration meant that sufficient photoreceptors, albeit very desensitized, remained to allow for residual bright light vision in older dogs. This study shows the natural history of the Rpe65-deficient dog model of LCA2.


Assuntos
Retina/enzimologia , Retina/patologia , cis-trans-Isomerases/deficiência , Adaptação Ocular/efeitos da radiação , Envelhecimento/patologia , Animais , Cães , Eletrorretinografia , Fundo de Olho , Luz , Fenótipo , Retina/diagnóstico por imagem , Retina/fisiopatologia , Epitélio Pigmentado da Retina/patologia , Epitélio Pigmentado da Retina/fisiopatologia , Tomografia de Coerência Óptica , Visão Ocular , cis-trans-Isomerases/metabolismo
7.
Hum Mol Genet ; 29(22): 3706-3716, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33355362

RESUMO

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGRex1-19 and RPGRORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGRORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGRORF15 mRNA also exhibited significantly higher levels of RPGRex1-19 mRNA. Four samples with unaltered RPGRex1-19 levels carried mutations in RPGRORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGRex1-19/RPGRORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGRex1-19 (mimicking the increase in RPGRex1-19 to RPGRORF15 isoform ratio) or RPGRORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGRORF15 protein and to the higher levels of the RPGRex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGRex1-9 to RPGRORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGRex1-19/RPGRORF15 ratio may lead to better therapeutic outcomes.


Assuntos
Proteínas do Olho/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Retinite Pigmentosa/genética , Processamento Alternativo/genética , Proteínas de Transporte/genética , Cílios/genética , Cílios/patologia , Éxons/genética , Feminino , Fibroblastos , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Masculino , Mutação/genética , Isoformas de Proteínas/genética , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/patologia
8.
Transl Vis Sci Technol ; 9(10): 5, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32953245

RESUMO

Purpose: To validate a vision-guided mobility assessment for individuals affected by RPE65-associated retinal dystrophy (RPE65-RD). Methods: In this comparative cross-sectional study, 29 subjects, comprising 19 subjects with RPE65-RD and 10 normally-sighted subjects undertook three assessments of mobility: following a straight line, navigating a simple maze, and stepping over a sidewalk "kerb." Performance was quantified as the time taken to complete each assessment, number of errors made, walking speed, and percent preferred walking speed, for each assessment. Subjects also undertook assessments of visual acuity, contrast sensitivity, full-field static perimetry, and age-appropriate quality of life questionnaires. To identify the most relevant metric to quantify vision-guided mobility, we investigated repeatability, as well as convergent, discriminant, and criterion validity. We also measured the effect of illumination on mobility. Results: Walking speed through the maze assessment best discriminated between RPE65-RD and normally-sighted subjects, with both convergent and discriminant validity. Walking speed also approached statistical significance when assessed for criterion validity (P = 0.052). Subjects with RPE65-RD had quantifiably poorer mobility at lower illumination levels. A relatively small mean difference (-0.09 m/s) was identified in comparison to a relatively large repeatability coefficient (1.10 m/s). Conclusions: We describe a novel, quantifiable, repeatable, and valid assessment of mobility designed specifically for subjects with RPE65-RD. The assessment is sensitive to the visual impairment of individuals with RPE65-RD in low illumination, identifies the known phenotypic heterogeneity and will furthermore provide an important outcome measure for RPE65-RD. Translational Relevance: This assessment of vision-guided mobility, validated in a dedicated cohort of subjects with RPE65-RD, is a relevant and quantifiable outcome measure for RPE65-RD.


Assuntos
Qualidade de Vida , Distrofias Retinianas , Estudos Transversais , Humanos , Distrofias Retinianas/diagnóstico , Caminhada , cis-trans-Isomerases
9.
Development ; 147(18)2020 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-32878923

RESUMO

Multipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated 'The people behind the papers' interview.


Assuntos
Proteínas do Olho/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Fator 2 de Transcrição de Octâmero/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Drosophila/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células-Tronco/metabolismo
10.
Transl Vis Sci Technol ; 9(7): 2, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32832209

RESUMO

Major advances in the study of inherited retinal diseases (IRDs) have placed efforts to develop treatments for these blinding conditions at the forefront of the emerging field of precision medicine. As a result, the growth of clinical trials for IRDs has increased rapidly over the past decade and is expected to further accelerate as more therapeutic possibilities emerge and qualified participants are identified. Although guided by established principles, these specialized trials, requiring analysis of novel outcome measures and endpoints in small patient populations, present multiple challenges relative to study design and ethical considerations. This position paper reviews recent accomplishments and existing challenges in clinical trials for IRDs and presents a set of recommendations aimed at rapidly advancing future progress. The goal is to stimulate discussions among researchers, funding agencies, industry, and policy makers that will further the design, conduct, and analysis of clinical trials needed to accelerate the approval of effective treatments for IRDs, while promoting advocacy and ensuring patient safety.


Assuntos
Doenças Retinianas , Humanos , Medicina de Precisão , Retina , Doenças Retinianas/tratamento farmacológico
12.
Hum Gene Ther ; 31(13-14): 709-718, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32578444

RESUMO

The neuronal ceroid lipofuscinoses (NCLs), often referred to as Batten disease, are inherited lysosomal storage disorders that represent the most common neurodegeneration during childhood. Symptoms include seizures, vision loss, motor and cognitive decline, and premature death. The development of brain-directed treatments for NCLs has made noteworthy progress in recent years. Clinical trials are currently ongoing or planned for different forms of the disease. Despite these promising advances, it is unlikely that therapeutic interventions targeting the brain will prevent loss of vision in patients as retinal cells remain untreated and will continue to degenerate. Here, we demonstrate that Cln3Δex7/8 mice, a mouse model of CLN3 Batten disease with juvenile onset, suffer from a decline in inner retinal function resulting from the death of rod bipolar cells, interneurons vital for signal transmission from photoreceptors to ganglion cells in the retina. We also show that this ocular phenotype can be treated by adeno-associated virus (AAV)-mediated expression of CLN3 in cells of the inner retina, leading to significant survival of bipolar cells and preserved retinal function. In contrast, the treatment of photoreceptors, which are lost in patients at late disease stages, was not therapeutic in Cln3Δex7/8 mice, underlining the notion that CLN3 disease is primarily a disease of the inner retina with secondary changes in the outer retina. These data indicate that bipolar cells play a central role in this disease and identify this cell type as an important target for ocular AAV-based gene therapies for CLN3 disease.


Assuntos
Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética/métodos , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Lipofuscinoses Ceroides Neuronais/complicações , Células Fotorreceptoras/metabolismo , Doenças Retinianas/terapia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Doenças Retinianas/etiologia , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia
13.
Biochim Biophys Acta Mol Basis Dis ; 1866(9): 165772, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32220628

RESUMO

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of rare monogenic neurodegenerative diseases predominantly affecting children. All NCLs are lethal and incurable and only one has an approved treatment available. To date, 13 NCL subtypes (CLN1-8, CLN10-14) have been identified, based on the particular disease-causing defective gene. The exact functions of NCL proteins and the pathological mechanisms underlying the diseases are still unclear. However, gene therapy has emerged as an attractive therapeutic strategy for this group of conditions. Here we provide a short review discussing updates on the current gene therapy studies for the NCLs.


Assuntos
Terapia Genética , Lipofuscinoses Ceroides Neuronais/terapia , Animais , Humanos , Lipofuscinoses Ceroides Neuronais/genética
14.
Hum Mol Genet ; 29(8): 1310-1318, 2020 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-32196553

RESUMO

Rhodopsin misfolding caused by the P23H mutation is a major cause of autosomal dominant retinitis pigmentosa (adRP). To date, there are no effective treatments for adRP. The BiP co-chaperone and reductase ERdj5 (DNAJC10) is part of the endoplasmic reticulum (ER) quality control machinery, and previous studies have shown that overexpression of ERdj5 in vitro enhanced the degradation of P23H rhodopsin, whereas knockdown of ERdj5 increased P23H rhodopsin ER retention and aggregation. Here, we investigated the role of ERdj5 in photoreceptor homeostasis in vivo by using an Erdj5 knockout mouse crossed with the P23H knock-in mouse and by adeno-associated viral (AAV) vector-mediated gene augmentation of ERdj5 in P23H-3 rats. Electroretinogram (ERG) and optical coherence tomography of Erdj5-/- and P23H+/-:Erdj5-/- mice showed no effect of ERdj5 ablation on retinal function or photoreceptor survival. Rhodopsin levels and localization were similar to those of control animals at a range of time points. By contrast, when AAV2/8-ERdj5-HA was subretinally injected into P23H-3 rats, analysis of the full-field ERG suggested that overexpression of ERdj5 reduced visual function loss 10 weeks post-injection (PI). This correlated with a significant preservation of photoreceptor cells at 4 and 10 weeks PI. Assessment of the outer nuclear layer (ONL) morphology showed preserved ONL thickness and reduced rhodopsin retention in the ONL in the injected superior retina. Overall, these data suggest that manipulation of the ER quality control and ER-associated degradation factors to promote mutant protein degradation could be beneficial for the treatment of adRP caused by mutant rhodopsin.


Assuntos
Proteínas de Choque Térmico HSP40/genética , Chaperonas Moleculares/genética , Retinite Pigmentosa/genética , Rodopsina/genética , Animais , Modelos Animais de Doenças , Eletrorretinografia , Retículo Endoplasmático/genética , Técnicas de Introdução de Genes , Camundongos , Camundongos Knockout , Mutação/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Ratos , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/patologia , Rodopsina/metabolismo , Transfecção
15.
Mol Ther ; 28(3): 820-829, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-31981492

RESUMO

Glaucoma is a common cause of blindness, yet current therapeutic options are imperfect. Clinical trials have invariably shown that reduction in intraocular pressure (IOP) regardless of disease subtype prevents visual loss. Reducing ciliary body aqueous humor production can lower IOP, and the adeno-associated virus ShH10 serotype was identified as able to transduce mouse ciliary body epithelium following intravitreal injection. Using ShH10 to deliver a single vector CRISPR-Cas9 system disrupting Aquaporin 1 resulted in reduced IOP in treated eyes (10.4 ± 2.4 mmHg) compared with control (13.2 ± 2.0 mmHg) or non-injected eyes (13.1 ± 2.8 mmHg; p < 0.001; n = 12). Editing in the aquaporin 1 gene could be detected in ciliary body, and no off-target increases in corneal or retinal thickness were identified. In experimental mouse models of corticosteroid and microbead-induced ocular hypertension, IOP could be reduced to prevent ganglion cell loss (32 ± 4 /mm2) compared with untreated eyes (25 ± 5/mm2; p < 0.01). ShH10 could transduce human ciliary body from post-mortem donor eyes in ex vivo culture with indel formation detectable in the Aquaporin 1 locus. Clinical translation of this approach to patients with glaucoma may permit long-term reduction of IOP following a single injection.


Assuntos
Aquaporina 1/genética , Corpo Ciliar/metabolismo , Edição de Genes , Terapia Genética , Glaucoma/genética , Glaucoma/terapia , Animais , Aquaporina 1/metabolismo , Sequência de Bases , Sistemas CRISPR-Cas , Dependovirus/genética , Expressão Gênica , Marcação de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Glaucoma/diagnóstico , Glaucoma/fisiopatologia , Camundongos , Retina/metabolismo , Retina/patologia , Transdução Genética , Transgenes
16.
Angiogenesis ; 23(2): 83-90, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31583505

RESUMO

The retinal vasculature is tightly organized in a structure that provides for the high metabolic demand of neurons while minimizing interference with incident light. The adverse impact of retinal vascular insufficiency is mitigated by adaptive vascular regeneration but exacerbated by pathological neovascularization. Aberrant growth of neovessels in the retina is responsible for impairment of sight in common blinding disorders including retinopathy of prematurity, proliferative diabetic retinopathy, and age-related macular degeneration. Myeloid cells are key players in this process, with diverse roles that can either promote or protect against ocular neovascularization. We have previously demonstrated that myeloid-derived VEGF, HIF1, and HIF2 are not essential for pathological retinal neovascularization. Here, however, we show by cell-specific depletion of Vhl in a mouse model of retinal ischemia (oxygen-induced retinopathy, OIR) that myeloid-derived HIFs promote VEGF and bFGF expression and enhance vascular regeneration in association with improved density and organization of the astrocytic network.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Isquemia/genética , Células Mieloides/metabolismo , Regeneração/genética , Vasos Retinianos/fisiologia , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular/genética , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Isquemia/patologia , Camundongos , Camundongos Transgênicos , Retina/patologia , Doenças Retinianas/genética , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-31818854

RESUMO

Degenerative retinal disease is the major cause of sight loss in the developed world and currently there is a lack of effective treatments. As the loss of vision is directly the result of the loss of retinal cells, effective cell replacement through stem-cell-based therapies may have the potential to treat a great number of retinal diseases whatever their underlying etiology. The eye is an ideal organ to develop cell therapies as it is immune privileged, and modern surgical techniques enable precise delivery of cells to the retina. Furthermore, a range of noninvasive diagnostic tests and high-resolution imaging techniques facilitate the evaluation of any therapeutic intervention. In this review, we evaluate the progress to date of current cell therapy strategies for retinal repair, focusing on transplantation of pluripotent stem-cell-derived retinal pigment epithelium (RPE) and photoreceptor cells.


Assuntos
Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/metabolismo , Transplante de Células-Tronco/métodos , Transplante de Células-Tronco/tendências , Células-Tronco/citologia , Animais , Pesquisa Biomédica/tendências , Ensaios Clínicos como Assunto , Células-Tronco Embrionárias/citologia , Feminino , Humanos , Masculino , Camundongos , Células-Tronco Pluripotentes/citologia , Regeneração , Retina/patologia , Doadores de Tecidos
18.
Hum Mol Genet ; 28(23): 3867-3879, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31807779

RESUMO

The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and pre-clinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.


Assuntos
Vetores Genéticos/administração & dosagem , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/terapia , Animais , Animais Recém-Nascidos , Encéfalo/crescimento & desenvolvimento , Dependovirus/genética , Modelos Animais de Doenças , Terapia Genética , Humanos , Injeções Intraventriculares , Proteínas de Membrana/metabolismo , Camundongos , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Resultado do Tratamento
19.
Invest Ophthalmol Vis Sci ; 60(10): 3468-3479, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408109

RESUMO

Purpose: The accumulation of undigestible autofluorescent material (UAM), termed lipofuscin in vivo, is a hallmark of aged RPE. Lipofuscin derives, in part, from the incomplete degradation of phagocytized photoreceptor outer segments (OS). Whether this accumulated waste is toxic is unclear. We therefore investigated the effects of UAM in highly differentiated human fetal RPE (hfRPE) cultures. Methods: Unmodified and photo-oxidized OS were fed daily to confluent cultures of ARPE-19 RPE or hfRPE. The emission spectrum, composition, and morphology of resulting UAM were measured and compared to in vivo lipofuscin. Effects of UAM on multiple RPE phenotypes were assessed. Results: Compared to ARPE-19, hfRPE were markedly less susceptible to UAM buildup. Accumulated UAM in hfRPE initially resembled the morphology of lipofuscin from AMD eyes, but compacted and shifted spectrum over time to resemble lipofuscin from healthy aged human RPE. UAM accumulation mildly reduced transepithelial electrical resistance, ketogenesis, certain RPE differentiation markers, and phagocytosis efficiency, while inducing senescence and rare, focal pockets of epithelial-mesenchymal transition. However, it had no effects on mitochondrial oxygen consumption rate, certain other RPE differentiation markers, secretion of drusen components or polarity markers, nor cell death. Conclusions: hfRPE demonstrates a remarkable resistance to UAM accumulation, suggesting mechanisms for efficient OS processing that may be lost in other RPE culture models. Furthermore, while UAM alters hfRPE phenotype, the effects are modest, consistent with conflicting reports in the literature on the toxicity of lipofuscin. Our results suggest that healthy RPE may adequately adapt to and tolerate lipofuscin accumulation.


Assuntos
Diferenciação Celular/fisiologia , Lipofuscina/metabolismo , Lipofuscina/toxicidade , Epitélio Pigmentado da Retina/citologia , Células Cultivadas , Humanos , Imagem Óptica , Fagocitose/fisiologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Segmento Externo da Célula Bastonete/fisiologia
20.
Invest Ophthalmol Vis Sci ; 60(8): 3170-3177, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31335954

RESUMO

Purpose: Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer. Methods: HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment. Results: LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD. Conclusions: ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.


Assuntos
Transplante de Córnea , Endotélio Corneano/metabolismo , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , RNA Mensageiro/genética , Proteína da Zônula de Oclusão-1/genética , Contagem de Células , Células Cultivadas , Inibição de Contato , Doenças da Córnea/genética , Doenças da Córnea/patologia , Doenças da Córnea/cirurgia , Endotélio Corneano/patologia , Humanos , Transdução de Sinais , Proteína da Zônula de Oclusão-1/biossíntese
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