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1.
Nat Immunol ; 24(4): 700-713, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36807640

RESUMO

Non-neuronal cells are key to the complex cellular interplay that follows central nervous system insult. To understand this interplay, we generated a single-cell atlas of immune, glial and retinal pigment epithelial cells from adult mouse retina before and at multiple time points after axonal transection. We identified rare subsets in naive retina, including interferon (IFN)-response glia and border-associated macrophages, and delineated injury-induced changes in cell composition, expression programs and interactions. Computational analysis charted a three-phase multicellular inflammatory cascade after injury. In the early phase, retinal macroglia and microglia were reactivated, providing chemotactic signals concurrent with infiltration of CCR2+ monocytes from the circulation. These cells differentiated into macrophages in the intermediate phase, while an IFN-response program, likely driven by microglia-derived type I IFN, was activated across resident glia. The late phase indicated inflammatory resolution. Our findings provide a framework to decipher cellular circuitry, spatial relationships and molecular interactions following tissue injury.


Assuntos
Macrófagos , Retina , Animais , Camundongos , Retina/lesões , Retina/metabolismo , Microglia , Sistema Nervoso Central , Monócitos
2.
Cell ; 166(5): 1308-1323.e30, 2016 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-27565351

RESUMO

Patterns of gene expression can be used to characterize and classify neuronal types. It is challenging, however, to generate taxonomies that fulfill the essential criteria of being comprehensive, harmonizing with conventional classification schemes, and lacking superfluous subdivisions of genuine types. To address these challenges, we used massively parallel single-cell RNA profiling and optimized computational methods on a heterogeneous class of neurons, mouse retinal bipolar cells (BCs). From a population of ∼25,000 BCs, we derived a molecular classification that identified 15 types, including all types observed previously and two novel types, one of which has a non-canonical morphology and position. We validated the classification scheme and identified dozens of novel markers using methods that match molecular expression to cell morphology. This work provides a systematic methodology for achieving comprehensive molecular classification of neurons, identifies novel neuronal types, and uncovers transcriptional differences that distinguish types within a class.


Assuntos
Células Bipolares da Retina/classificação , Transcriptoma , Células Amácrinas/citologia , Animais , Análise por Conglomerados , Feminino , Marcadores Genéticos , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Células Bipolares da Retina/citologia , Células Bipolares da Retina/metabolismo , Análise de Sequência de RNA , Análise de Célula Única/métodos , Transcrição Gênica
3.
J Neurosci ; 40(27): 5177-5195, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32457074

RESUMO

Amacrine cells (ACs) are a diverse class of interneurons that modulate input from photoreceptors to retinal ganglion cells (RGCs), rendering each RGC type selectively sensitive to particular visual features, which are then relayed to the brain. While many AC types have been identified morphologically and physiologically, they have not been comprehensively classified or molecularly characterized. We used high-throughput single-cell RNA sequencing to profile >32,000 ACs from mice of both sexes and applied computational methods to identify 63 AC types. We identified molecular markers for each type and used them to characterize the morphology of multiple types. We show that they include nearly all previously known AC types as well as many that had not been described. Consistent with previous studies, most of the AC types expressed markers for the canonical inhibitory neurotransmitters GABA or glycine, but several expressed neither or both. In addition, many expressed one or more neuropeptides, and two expressed glutamatergic markers. We also explored transcriptomic relationships among AC types and identified transcription factors expressed by individual or multiple closely related types. Noteworthy among these were Meis2 and Tcf4, expressed by most GABAergic and most glycinergic types, respectively. Together, these results provide a foundation for developmental and functional studies of ACs, as well as means for genetically accessing them. Along with previous molecular, physiological, and morphologic analyses, they establish the existence of at least 130 neuronal types and nearly 140 cell types in the mouse retina.SIGNIFICANCE STATEMENT The mouse retina is a leading model for analyzing the development, structure, function, and pathology of neural circuits. A complete molecular atlas of retinal cell types provides an important foundation for these studies. We used high-throughput single-cell RNA sequencing to characterize the most heterogeneous class of retinal interneurons, amacrine cells, identifying 63 distinct types. The atlas includes types identified previously as well as many novel types. We provide evidence for the use of multiple neurotransmitters and neuropeptides, and identify transcription factors expressed by groups of closely related types. Combining these results with those obtained previously, we proposed that the mouse retina contains ∼130 neuronal types and is therefore comparable in complexity to other regions of the brain.


Assuntos
Células Amácrinas/classificação , Retina/citologia , Células Amácrinas/metabolismo , Células Amácrinas/ultraestrutura , Animais , Feminino , Glicina/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Receptores de Neurotransmissores/classificação , Receptores de Neurotransmissores/metabolismo , Retina/ultraestrutura , Fator de Transcrição 4/metabolismo , Fatores de Transcrição/genética , Ácido gama-Aminobutírico/metabolismo
4.
PLoS Genet ; 10(2): e1004111, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24516401

RESUMO

Cone-rod homeobox (CRX) protein is a "paired-like" homeodomain transcription factor that is essential for regulating rod and cone photoreceptor transcription. Mutations in human CRX are associated with the dominant retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD) and Leber Congenital Amaurosis (LCA), with variable severity. Heterozygous Crx Knock-Out (KO) mice ("+/-") have normal vision as adults and fail to model the dominant human disease. To investigate how different mutant CRX proteins produce distinct disease pathologies, we generated two Crx Knock-IN (K-IN) mouse models: Crx(E168d2) ("E168d2") and Crx(R90W) ("R90W"). E168d2 mice carry a frameshift mutation in the CRX activation domain, Glu168del2, which is associated with severe dominant CoRD or LCA in humans. R90W mice carry a substitution mutation in the CRX homeodomain, Arg90Trp, which is associated with dominant mild late-onset CoRD and recessive LCA. As seen in human patients, heterozygous E168d2 ("E168d2/+") but not R90W ("R90W/+") mice show severely impaired retinal function, while mice homozygous for either mutation are blind and undergo rapid photoreceptor degeneration. E168d2/+ mice also display abnormal rod/cone morphology, greater impairment of CRX target gene expression than R90W/+ or +/- mice, and undergo progressive photoreceptor degeneration. Surprisingly, E168d2/+ mice express more mutant CRX protein than wild-type CRX. E168d2neo/+, a subline of E168d2 with reduced mutant allele expression, displays a much milder retinal phenotype, demonstrating the impact of Crx expression level on disease severity. Both CRX([E168d2]) and CRX([R90W]) proteins fail to activate transcription in vitro, but CRX([E168d2]) interferes more strongly with the function of wild type (WT) CRX, supporting an antimorphic mechanism. E168d2 and R90W are mechanistically distinct mouse models for CRX-associated disease that will allow the elucidation of molecular mechanisms and testing of novel therapeutic approaches for different forms of CRX-associated disease.


Assuntos
Proteínas de Homeodomínio/genética , Amaurose Congênita de Leber/genética , Retinose Pigmentar/genética , Transativadores/genética , Animais , Modelos Animais de Doenças , Mutação da Fase de Leitura , Homozigoto , Humanos , Amaurose Congênita de Leber/patologia , Camundongos , Fenótipo , Células Fotorreceptoras Retinianas Cones , Degeneração Retiniana/genética , Retinose Pigmentar/patologia
5.
Dev Dyn ; 243(10): 1153-66, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24888636

RESUMO

BACKGROUND: The homeodomain transcription factor CRX is a crucial regulator of mammalian photoreceptor gene expression. Mutations in the human CRX gene are associated with dominant inherited retinopathies Retinitis Pigmentosa (RP), Cone-Rod Dystrophy (CoRD), and Leber Congenital Amaurosis (LCA), of varying severity. In vitro and in vivo assessment of mutant CRX proteins have revealed pathogenic mechanisms for several mutations, but no comprehensive mutation-disease correlation has yet been reported. RESULTS: Here we describe four different classes of disease-causing CRX mutations, characterized by mutation type, pathogenetic mechanism, and the molecular activity of the mutant protein: (1) hypomorphic missense mutations with reduced DNA binding, (2) antimorphic missense mutations with variable DNA binding, (3) antimorphic frameshift/nonsense mutations with intact DNA binding, and (4) antimorphic frameshift mutations with reduced DNA binding. Mammalian models representing three of these classes have been characterized. CONCLUSIONS: Models carrying Class I mutations display a mild dominant retinal phenotype and recessive LCA, while models carrying Class III and IV mutations display characteristically distinct dominant LCA phenotypes. These animal models also reveal unexpected pathogenic mechanisms underlying CRX-associated retinopathies. The complexity of genotype-phenotype correlation for CRX-associated diseases highlights the value of developing comprehensive "true-to-disease" animal models for understanding pathologic mechanisms and testing novel therapeutic approaches.


Assuntos
Cegueira/genética , Modelos Animais de Doenças , Proteínas de Homeodomínio/genética , Doenças Retinianas/genética , Transativadores/genética , Animais , Cegueira/patologia , Redes Reguladoras de Genes , Humanos , Camundongos , Glândula Pineal/metabolismo , Glândula Pineal/patologia , Retina/metabolismo , Retina/patologia , Doenças Retinianas/patologia
6.
bioRxiv ; 2024 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-38328114

RESUMO

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity at the single-cell resolution by classifying and characterizing cell types in multiple tissues and species. While several mouse retinal scRNA-seq reference datasets have been published, each dataset either has a relatively small number of cells or is focused on specific cell classes, and thus is suboptimal for assessing gene expression patterns across all retina types at the same time. To establish a unified and comprehensive reference for the mouse retina, we first generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse whole retinas. This dataset was generated through the targeted enrichment of rare population cells via antibody-based magnetic cell sorting. By integrating this new dataset with public datasets, we conducted an integrated analysis to construct the Mouse Retina Cell Atlas (MRCA) for wild-type mice, which encompasses over 330,000 single cells. The MRCA characterizes 12 major classes and 138 cell types. It captured consensus cell type characterization from public datasets and identified additional new cell types. To facilitate the public use of the MRCA, we have deposited it in CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal for visualization and gene expression exploration. The comprehensive MRCA serves as an easy-to-use, one-stop data resource for the mouse retina communities.

7.
bioRxiv ; 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38712294

RESUMO

Rare cell populations can be challenging to characterize using microfluidic single-cell RNA sequencing (scRNA-seq) platforms. Typically, the population of interest must be enriched and pooled from multiple biological specimens for efficient collection. However, these practices preclude the resolution of sample origin together with phenotypic data and are problematic in experiments in which biological or technical variation is expected to be high (e.g., disease models, genetic perturbation screens, or human samples). One solution is sample multiplexing whereby each sample is tagged with a unique sequence barcode that is resolved bioinformatically. We have established a scRNA-seq sample multiplexing pipeline for mouse retinal ganglion cells using cholesterol-modified-oligos and utilized the enhanced precision to investigate cell type distribution and transcriptomic variance across retinal samples. As single cell transcriptomics are becoming more widely used to research development and disease, sample multiplexing represents a useful method to enhance the precision of scRNA-seq analysis.

8.
iScience ; 27(6): 109916, 2024 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-38812536

RESUMO

Single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular heterogeneity by characterizing cell types across tissues and species. While several mouse retinal scRNA-seq datasets exist, each dataset is either limited in cell numbers or focused on specific cell classes, thereby hindering comprehensive gene expression analysis across all retina types. To fill the gap, we generated the largest retinal scRNA-seq dataset to date, comprising approximately 190,000 single cells from C57BL/6J mouse retinas, enriched for rare population cells via antibody-based magnetic cell sorting. Integrating this dataset with public datasets, we constructed the Mouse Retina Cell Atlas (MRCA) for wild-type mice, encompassing over 330,000 cells, characterizing 12 major classes and 138 cell types. The MRCA consolidates existing knowledge, identifies new cell types, and is publicly accessible via CELLxGENE, UCSC Cell Browser, and the Broad Single Cell Portal, providing a user-friendly resource for the mouse retina research community.

9.
Methods Mol Biol ; 2636: 1-18, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36881292

RESUMO

A prevalent feature among neurodegenerative conditions, including axonal injury, is that certain neuronal types are disproportionately affected, while others are more resilient. Identifying molecular features that separate resilient from susceptible populations could reveal potential targets for neuroprotection and axon regeneration. A powerful approach to resolve molecular differences across cell types is single-cell RNA-sequencing (scRNA-seq). scRNA-seq is a robustly scalable approach that enables the parallel sampling of gene expression across many individual cells. Here we present a systematic framework to apply scRNA-seq to track neuronal survival and gene expression changes following axonal injury. Our methods utilize the mouse retina because it is an experimentally accessible central nervous system tissue and its cell types have been comprehensively characterized by scRNA-seq. This chapter will focus on preparing retinal ganglion cells (RGCs) for scRNA-seq and pre-processing of sequencing results.


Assuntos
Axônios , Neuroproteção , Animais , Camundongos , Neuroproteção/genética , Regeneração Nervosa/genética , Células Ganglionares da Retina , Análise de Sequência de RNA
10.
Transl Vis Sci Technol ; 12(6): 15, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37351895

RESUMO

Purpose: Mutations in the CRX transcription factor are associated with dominant retinopathies often with more severe macular changes. The CRX-mutant cat (Rdy-A182d2) is the only animal model with the equivalent of the critical retinal region for high-acuity vision, the macula. Heterozygous cats (CRXRdy/+) have a severe phenotype modeling Leber congenital amaurosis. This study reports the distinct ocular phenotype of homozygous cats (CRXRdy/Rdy). Methods: Gene expression changes were assessed at both mRNA and protein levels. Changes in globe morphology and retinal structure were analyzed. Results: CRXRdy/Rdy cats had high levels of mutant CRX mRNA and protein. The expression of photoreceptor target genes was severely impaired although there were variable effects on the expression of other transcription factors. The photoreceptor cells remained immature and failed to elaborate outer segments consistent with the lack of retinal function. The retinal layers displayed a progressive remodeling with cell loss but maintained overall retinal thickness due to gliosis. Rapid photoreceptor loss largely occurred in the macula-equivalent retinal region. The homozygous cats developed markedly increased ocular globe length. Conclusions: The phenotype of CRXRdy/Rdy cats was more severe compared to CRXRdy/+ cats by several metrics. Translational Relevance: The CRX-mutant cat is the only model for CRX-retinopathies with a macula-equivalent region. A prominent feature of the CRXRdy/Rdy cat phenotype not detectable in homozygous mouse models was the rapid degeneration of the macula-equivalent retinal region highlighting the value of this large animal model and its future importance in the testing of translational therapies aiming to restore vision.


Assuntos
Doenças Retinianas , Transativadores , Animais , Camundongos , Transativadores/genética , Proteínas de Homeodomínio/genética , Mutação , Fenótipo , RNA Mensageiro
11.
bioRxiv ; 2023 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-37034690

RESUMO

Previously we showed that neurodegeneration initiated by axonal insults depends in part on the stress-responsive kinase Perk (Larhammar et al., 2017). Here we show that Perk acts primarily through Activating Transcription Factor-4 (Atf4) to stimulate not only pro-apoptotic but also pro-regenerative responses following optic nerve injury. Using conditional knockout mice, we find an extensive Perk/Atf4-dependent transcriptional response that includes canonical Atf4 target genes and modest contributions by C/ebp homologous protein (Chop). Overlap with c-Jun-dependent transcription suggests interplay with a parallel stress pathway that couples regenerative and apoptotic responses. Accordingly, neuronal knockout of Atf4 recapitulates the neuroprotection afforded by Perk deficiency, and Perk or Atf4 knockout impairs optic axon regeneration enabled by disrupting the tumor suppressor Pten. These findings contrast with the transcriptional and functional consequences reported for CRISPR targeting of Atf4 or Chop and reveal an integral role for Perk/Atf4 in coordinating neurodegenerative and regenerative responses to CNS axon injury.

12.
Res Sq ; 2023 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-38014002

RESUMO

Single-cell sequencing has revolutionized the scale and resolution of molecular profiling of tissues and organs. Here, we present an integrated multimodal reference atlas of the most accessible portion of the mammalian central nervous system, the retina. We compiled around 2.4 million cells from 55 donors, including 1.4 million unpublished data points, to create a comprehensive human retina cell atlas (HRCA) of transcriptome and chromatin accessibility, unveiling over 110 types. Engaging the retina community, we annotated each cluster, refined the Cell Ontology for the retina, identified distinct marker genes, and characterized cis-regulatory elements and gene regulatory networks (GRNs) for these cell types. Our analysis uncovered intriguing differences in transcriptome, chromatin, and GRNs across cell types. In addition, we modeled changes in gene expression and chromatin openness across gender and age. This integrated atlas also enabled the fine-mapping of GWAS and eQTL variants. Accessible through interactive browsers, this multimodal cross-donor and cross-lab HRCA, can facilitate a better understanding of retinal function and pathology.

13.
J Biol Chem ; 286(42): 36921-31, 2011 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-21865162

RESUMO

The transcription factor neural retina leucine zipper (Nrl) is a critical determinant of rod photoreceptor cell fate and a key regulator of rod differentiation. Nrl(-/-) rod precursors fail to turn on rod genes and instead differentiate as cones. Furthermore, NRL mutations in humans cause retinitis pigmentosa. Despite the developmental and clinical significance of this gene, little is known about the transcriptional regulation of Nrl itself. In this study, we sought to define the cis- and trans-acting factors responsible for initiation and maintenance of Nrl transcription in the mouse retina. Utilizing a quantitative mouse retinal explant electroporation assay, we discovered a phylogenetically conserved, 30-base pair region immediately upstream of the transcription start site that is required for Nrl promoter activity. This region contains binding sites for the retinal transcription factors CRX, OTX2, and RORß, and point mutations in these sites completely abolish promoter activity in living retinas. Gel-shift experiments show that CRX, OTX2, and RORß can bind to the critical region in vitro, whereas ChIP experiments demonstrate binding of CRX and OTX2 to the critical region in vivo. Thus, our results indicate that CRX, OTX2, and RORß directly regulate Nrl transcription by binding to critical sites within the Nrl promoter. We propose a model in which Nrl expression is primarily initiated by OTX2 and RORß and later maintained at high levels by CRX and RORß.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/biossíntese , Proteínas do Olho/metabolismo , Modelos Biológicos , Elementos de Resposta/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transcrição Gênica/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/fisiologia , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Mutação Puntual , Células Fotorreceptoras Retinianas Bastonetes/citologia
14.
Neuron ; 110(16): 2625-2645.e7, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35767994

RESUMO

Injured neurons in the adult mammalian central nervous system often die and seldom regenerate axons. To uncover transcriptional pathways that could ameliorate these disappointing responses, we analyzed three interventions that increase survival and regeneration of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC) injury, albeit not to a clinically useful extent. We assessed gene expression in each of 46 RGC types by single-cell transcriptomics following ONC and treatment. We also compared RGCs that regenerated with those that survived but did not regenerate. Each intervention enhanced survival of most RGC types, but type-independent axon regeneration required manipulation of multiple pathways. Distinct computational methods converged on separate sets of genes selectively expressed by RGCs likely to be dying, surviving, or regenerating. Overexpression of genes associated with the regeneration program enhanced both survival and axon regeneration in vivo, indicating that mechanistic analysis can be used to identify novel therapeutic strategies.


Assuntos
Traumatismos do Nervo Óptico , Células Ganglionares da Retina , Animais , Axônios/metabolismo , Sobrevivência Celular/genética , Mamíferos , Camundongos , Regeneração Nervosa/genética , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/metabolismo , Células Ganglionares da Retina/fisiologia
15.
Cell Rep ; 34(9): 108777, 2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33657370

RESUMO

Adult mammalian central nervous system (CNS) trauma interrupts neural networks and, because axonal regeneration is minimal, neurological deficits persist. Repair via axonal growth is limited by extracellular inhibitors and cell-autonomous factors. Based on results from a screen in vitro, we evaluate nearly 400 genes through a large-scale in vivo regeneration screen. Suppression of 40 genes using viral-driven short hairpin RNAs (shRNAs) promotes retinal ganglion cell (RGC) axon regeneration after optic nerve crush (ONC), and most are validated by separate CRISPR-Cas9 editing experiments. Expression of these axon-regeneration-suppressing genes is not significantly altered by axotomy. Among regeneration-limiting genes, loss of the interleukin 22 (IL-22) cytokine allows an early, yet transient, inflammatory response in the retina after injury. Reduced IL-22 drives concurrent activation of signal transducer and activator of transcription 3 (Stat3) and dual leucine zipper kinase (DLK) pathways and upregulation of multiple neuron-intrinsic regeneration-associated genes (RAGs). Including IL-22, our screen identifies dozens of genes that limit CNS regeneration. Suppression of these genes in the context of axonal damage could support improved neural repair.


Assuntos
Regeneração Nervosa/genética , Neurogênese/genética , Traumatismos do Nervo Óptico/genética , Nervo Óptico/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Sistemas CRISPR-Cas , Dependovirus/genética , Feminino , Edição de Genes , Regulação da Expressão Gênica , Estudos de Associação Genética , Células HEK293 , Humanos , Interleucinas/genética , Interleucinas/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nervo Óptico/patologia , Nervo Óptico/fisiopatologia , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Traumatismos do Nervo Óptico/fisiopatologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Interleucina 22
17.
Neuron ; 104(6): 1039-1055.e12, 2019 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-31784286

RESUMO

Neuronal types in the central nervous system differ dramatically in their resilience to injury or other insults. Here we studied the selective resilience of mouse retinal ganglion cells (RGCs) following optic nerve crush (ONC), which severs their axons and leads to death of ∼80% of RGCs within 2 weeks. To identify expression programs associated with differential resilience, we first used single-cell RNA-seq (scRNA-seq) to generate a comprehensive molecular atlas of 46 RGC types in adult retina. We then tracked their survival after ONC; characterized transcriptomic, physiological, and morphological changes that preceded degeneration; and identified genes selectively expressed by each type. Finally, using loss- and gain-of-function assays in vivo, we showed that manipulating some of these genes improved neuronal survival and axon regeneration following ONC. This study provides a systematic framework for parsing type-specific responses to injury and demonstrates that differential gene expression can be used to reveal molecular targets for intervention.


Assuntos
Sobrevivência Celular/genética , Regeneração Nervosa/genética , Neuroproteção/genética , Células Ganglionares da Retina/fisiologia , Animais , Feminino , Masculino , Camundongos , Compressão Nervosa
18.
Neuron ; 95(4): 869-883.e6, 2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28781169

RESUMO

The size and shape of dendritic arbors are prime determinants of neuronal connectivity and function. We asked how ON-OFF direction-selective ganglion cells (ooDSGCs) in mouse retina acquire their bistratified dendrites, in which responses to light onset and light offset are segregated to distinct strata. We found that the transcriptional regulator Satb1 is selectively expressed by ooDSGCs. In Satb1 mutant mice, ooDSGC dendrites lack ON arbors, and the cells selectively lose ON responses. Satb1 regulates expression of a homophilic adhesion molecule, Contactin 5 (Cntn5). Both Cntn5 and its co-receptor Caspr4 are expressed not only by ooDSGCs, but also by interneurons that form a scaffold on which ooDSGC ON dendrites fasciculate. Removing Cntn5 from either ooDSGCs or interneurons partially phenocopies Satb1 mutants, demonstrating that Satb1-dependent Cntn5 expression in ooDSGCs leads to branch-specific homophilic interactions with interneurons. Thus, Satb1 directs formation of a morphologically and functionally specialized compartment within a complex dendritic arbor.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Contactinas/metabolismo , Dendritos/metabolismo , Retina/citologia , Células Ganglionares da Retina/citologia , Animais , Animais Recém-Nascidos , Caderinas/metabolismo , Moléculas de Adesão Celular Neuronais/genética , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células HEK293 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Técnicas In Vitro , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Receptores de Dopamina D4/genética , Receptores de Dopamina D4/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
19.
Neuron ; 94(6): 1112-1120.e4, 2017 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-28641110

RESUMO

At least 30 types of retinal ganglion cells (RGCs) send distinct messages through the optic nerve to the brain. Available strategies of promoting axon regeneration act on only some of these types. Here we tested the hypothesis that overexpressing developmentally important transcription factors in adult RGCs could reprogram them to a "youthful" growth-competent state and promote regeneration of other types. From a screen of transcription factors, we identified Sox11 as one that could induce substantial axon regeneration. Transcriptome profiling indicated that Sox11 activates genes involved in cytoskeletal remodeling and axon growth. Remarkably, α-RGCs, which preferentially regenerate following treatments such as Pten deletion, were killed by Sox11 overexpression. Thus, Sox11 promotes regeneration of non-α-RGCs, which are refractory to Pten deletion-induced regeneration. We conclude that Sox11 can reprogram adult RGCs to a growth-competent state, suggesting that different growth-promoting interventions promote regeneration in distinct neuronal types.


Assuntos
Axônios/metabolismo , Regeneração Nervosa/genética , Crescimento Neuronal/genética , Traumatismos do Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismo , Fatores de Transcrição SOXC/genética , Animais , Sobrevivência Celular , Perfilação da Expressão Gênica , Camundongos , Microscopia de Fluorescência , Traumatismos do Nervo Óptico/patologia , PTEN Fosfo-Hidrolase/genética , Regeneração/genética , Retina/metabolismo , Retina/patologia , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/patologia , Fatores de Transcrição SOXC/metabolismo
20.
Invest Ophthalmol Vis Sci ; 57(8): 3780-92, 2016 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-27427859

RESUMO

PURPOSE: Mutations in the retinal transcription factor cone-rod homeobox (CRX) gene result in severe dominant retinopathies. A large animal model, the Rdy cat, carrying a spontaneous frameshift mutation in Crx, was reported previously. The present study aimed to further understand pathogenesis in this model by thoroughly characterizing the Rdy retina. METHODS: Structural and functional changes were found in a comparison between the retinas of CrxRdy/+ kittens and those of wild-type littermates and were determined at various ages by fundus examination, electroretinography (ERG), optical coherence tomography, and histologic analyses. RNA and protein expression changes of Crx and key target genes were analyzed using quantitative reverse-transcribed PCR, Western blot analysis, and immunohistochemistry. Transcription activity of the mutant Crx was measured by a dual-luciferase transactivation assay. RESULTS: CrxRdy/+ kittens had no recordable cone ERGs. Rod responses were delayed in development and markedly reduced at young ages and lost by 20 weeks. Photoreceptor outer segment development was incomplete and was followed by progressive outer retinal thinning starting in the cone-rich area centralis. Expression of cone and rod Crx target genes was significantly down-regulated. The mutant Crx allele was overexpressed, leading to high levels of the mutant protein lacking transactivation activity. CONCLUSIONS: The CrxRdy mutation exerts a dominant negative effect on wild-type Crx by overexpressing mutant protein. These findings, consistent with those of studies in a mouse model, support a conserved pathogenic mechanism for CRX frameshift mutations. The similarities between the feline eye and the human eye with the presence of a central region of high cone density makes the CrxRdy/+ cat a valuable model for preclinical testing of therapies for dominant CRX diseases.


Assuntos
Mutação da Fase de Leitura/genética , Proteínas de Homeodomínio/genética , Amaurose Congênita de Leber/genética , Transativadores/genética , Animais , Gatos , Adaptação à Escuridão/fisiologia , Modelos Animais de Doenças , Amaurose Congênita de Leber/patologia , Amaurose Congênita de Leber/fisiopatologia , Fenótipo , Retina/metabolismo , Retina/patologia , Retina/fisiopatologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Limiar Sensorial/fisiologia , Transcrição Gênica
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