RESUMEN
Systemic lupus erythematosus is an autoimmune disease that results in immune-mediated damage to kidneys and other organs. We investigated the role of response gene to complement-32 (RGC-32), a proinflammatory and profibrotic mediator induced by TGFß and C5b-9, in nephrotoxic nephritis (NTN), an experimental model that mimics human lupus nephritis. Proteinuria, loss of renal function and kidney histopathology were attenuated in RGC-32 KO NTN mice. RGC-32 KO NTN mice displayed downregulation of the CCL20/CCR6 and CXCL9/CXCR3 ligand/receptor pairs resulting in decreased renal recruitment of IL-17+ and IFNγ+ cells and subsequent decrease in the influx of innate immune cells. RGC-32 deficiency attenuated renal fibrosis as demonstrated by decreased deposition of collagen I, III and fibronectin. Thus, RGC-32 is a unique mediator shared by the Th17 and Th1 dependent proinflammatory and profibrotic pathways and a potential novel therapeutic target in the treatment of immune complex mediated glomerulonephritis such as lupus nephritis.
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Riñón , Animales , Humanos , Ratones , Modelos Animales de Enfermedad , Fibrosis , Inflamación/inmunología , Riñón/patología , Riñón/inmunología , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares , Células TH1/inmunología , Células Th17/inmunologíaRESUMEN
More than 40 retinal ganglion cell (RGC) subtypes have been categorized in mouse based on their morphologies, functions, and molecular features. Among these diverse subtypes, orientation-selective Jam2-expressing RGCs (J-RGCs) has two unique morphologic characteristics: the ventral-facing dendritic arbor and the OFF-sublaminae stratified terminal dendrites in the inner plexiform layer. Previously, we have discovered that T-box transcription factor T-brain 1 (Tbr1) is expressed in J-RGCs. We further found that Tbr1 is essential for the expression of Jam2, and Tbr1 regulates the formation and the dendritic morphogenesis of J-RGCs. However, Tbr1 begins to express in terminally differentiated RGCs around perinatal stage, suggesting that it is unlikely involved in the initial fate determination for J-RGC and other upstream transcription factors must control Tbr1 expression and J-RGC formation. Using the Cleavage Under Targets and Tagmentation technique, we discovered that Pou4f1 binds to Tbr1 on the evolutionary conserved exon 6 and an intergenic region downstream of the 3'UTR, and on a region flanking the promoter and the first exon of Jam2. We showed that Pou4f1 is required for the expression of Tbr1 and Jam2, indicating Pou4f1 as a direct upstream regulator of Tbr1 and Jam2. Most interestingly, the Pou4f1-bound element in exon 6 of Tbr1 possesses high-level enhancer activity, capable of directing reporter gene expression in J-RGCs. Together, these data revealed a Pou4f1-Tbr1-Jam2 genetic hierarchy as a critical pathway in the formation of J-RGC subtype.
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Purpose: Uveal coloboma is a congenital eye malformation caused by failure of the optic fissure to close in early human development. Despite significant progress in identifying genes whose regulation is important for executing this closure, mutations are detected in a minority of cases using known gene panels, implying additional genetic complexity. We have previously shown knockdown of znf503 (the ortholog of mouse Zfp503) in zebrafish causes coloboma. Here we characterize Zfp503 knockout (KO) mice and evaluate transcriptomic profiling of mutant versus wild-type (WT) retinal pigment epithelium (RPE)/choroid. Methods: Zfp503 KO mice were generated by gene targeting using homologous recombination. Embryos were characterized grossly and histologically. Patterns and level of developmentally relevant proteins/genes were examined with immunostaining/in situ hybridization. The transcriptomic profile of E11.5 KO RPE/choroid was compared to that of WT. Results: Zfp503 is dynamically expressed in developing mouse eyes, and loss of its expression results in uveal coloboma. KO embryos exhibit altered mRNA levels and expression patterns of several key transcription factors involved in eye development, including Otx2, Mitf, Pax6, Pax2, Vax1, and Vax2, resulting in a failure to maintain the presumptive RPE, as evidenced by reduced melanin pigmentation and its differentiation into a neural retina-like lineage. Comparison of RNA sequencing data from WT and KO E11.5 embryos demonstrated reduced expression of melanin-related genes and significant overlap with genes known to be dynamically regulated at the optic fissure. Conclusions: These results demonstrate a critical role of Zfp503 in maintaining RPE fate and optic fissure closure.
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Coloboma , Neuropéptidos , Animales , Humanos , Ratones , Coloboma/genética , Coloboma/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melaninas/metabolismo , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuropéptidos/genética , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Pez Cebra/genéticaRESUMEN
Recent advances in understanding the pathogenesis of multiple sclerosis (MS) have brought into the spotlight the major role played by reactive astrocytes in this condition. Response Gene to Complement (RGC)-32 is a gene induced by complement activation, growth factors, and cytokines, notably transforming growth factor ß, that is involved in the modulation of processes such as angiogenesis, fibrosis, cell migration, and cell differentiation. Studies have uncovered the crucial role that RGC-32 plays in promoting the differentiation of Th17 cells, a subtype of CD4+ T lymphocytes with an important role in MS and its murine model, experimental autoimmune encephalomyelitis. The latest data have also shown that RGC-32 is involved in regulating major transcriptomic changes in astrocytes and in favoring the synthesis and secretion of extracellular matrix components, growth factors, axonal growth molecules, and pro-astrogliogenic molecules. These results suggest that RGC-32 plays a major role in driving reactive astrocytosis and the generation of astrocytes from radial glia precursors. In this review, we summarize recent advances in understanding how RGC-32 regulates the behavior of Th17 cells and astrocytes in neuroinflammation, providing insight into its role as a potential new biomarker and therapeutic target.
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Proteínas de Ciclo Celular , Esclerosis Múltiple , Proteínas Musculares , Proteínas del Tejido Nervioso , Animales , Biomarcadores , Proteínas de Ciclo Celular/genética , Proteínas del Sistema Complemento , Citocinas , Humanos , Ratones , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Enfermedades Neuroinflamatorias , Proteínas Nucleares/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The mouse retina encodes diverse visual features in the spike trains of >40 retinal ganglion cell (RGC) types. Each RGC type innervates a specific subset of the >50 retinorecipient brain areas. Our catalog of RGC types and feature representations is nearing completion. Yet, we know little about where specific RGC types send their information. Furthermore, the developmental strategies by which RGC axons choose their targets and pattern their terminal arbors remain obscure. Here, we identify a genetic intersection (Cck-Cre and Brn3cCKOAP ) that selectively labels transient Suppressed-by-Contrast (tSbC) RGCs, a member of an evolutionarily conserved functionally mysterious RGC subclass. We find that tSbC RGCs selectively innervate the dorsolateral geniculate nucleus (dLGN) and ventrolateral geniculate nucleus (vLGN) of the thalamus, the superior colliculus (SC), and the nucleus of the optic tract (NOT) in mice of either sex. They binocularly innervate dLGN and vLGN but project only contralaterally to SC and NOT. In each target, tSbC RGC axons occupy a specific sublayer, suggesting that they restrict their input to specific circuits. The tSbC RGC axons span the length of the optic tract by birth and remain poised there until they simultaneously innervate their four targets around postnatal day 3. The tSbC RGC axons choose the right targets and establish mature stratification patterns from the outset. This precision is maintained in the absence of Brn3c. Our results provide the first map of SbC inputs to the brain, revealing a narrow target set, unexpected laminar organization, target-specific binocularity, and developmental precision.SIGNIFICANCE STATEMENTIn recent years, we have learned a lot about the visual features encoded by RGCs, the output neurons of the eye. In contrast, we know little about where RGCs send their information and how RGC axons, which carry this information, target specific brain areas during development. Here, we develop an intersectional strategy to label a unique RGC type, the tSbC RGC, and map its projections. We find that tSbC RGC axons are highly selective. They innervate few retinal targets and restrict their arbors to specific sublayers within these targets. The selective tSbC RGC projection patterns develop synchronously and without trial and error, suggesting molecular determinism and coordination.
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Proliferation of endothelial cells (EC) and smooth muscle cells (SMC) is a critical process in atherosclerosis. Here, we investigated the involvement of sublytic C5b-9 effector Response Gene to Complement 32 (RGC-32) in cell cycle activation, phenotypic switch, and production of extracellular matrix (ECM) in SMC. Overexpression of RGC-32 augmented C5b-9-induced cell cycle activation and proliferation of SMC in an ERK1-dependent manner and silencing of RGC-32 inhibited C5b-9-induced cell cycle activation. C5b-9-induced cell cycle activation also required phosphorylation of RGC-32 at threonine 91. We found that ECM components fibronectin and collagens I-V were expressed by SMC in human aortic atherosclerotic tissue. Silencing of RGC-32 in cultured SMC was followed by a significant reduction in TGF-ß-induced expression of SMC differentiation markers myocardin, SM22 and α-SMA, and that of collagens I, IV and V. These data suggest that RGC-32 participates in both sublytic C5b-9-induced cell cycle activation and TGF-ß-induced ECM production.
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Aterosclerosis , Proteínas de Ciclo Celular , Complejo de Ataque a Membrana del Sistema Complemento , Proteínas Musculares , Proteínas del Tejido Nervioso , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Proteínas del Sistema Complemento , Células Endoteliales , Humanos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Factor de Crecimiento Transformador betaRESUMEN
Some previous studies in tissue fibrosis have suggested a profibrotic contribution from elevated expression of a protein termed either RGCC (regulator of cell cycle) or RGC-32 (response gene to complement 32 protein). Our analysis of public gene expression datasets, by contrast, revealed a consistent decrease in RGCC mRNA levels in association with pulmonary fibrosis. Consistent with this observation, we found that stimulating primary adult human lung fibroblasts with transforming growth factor (TGF)-ß in cell cultures elevated collagen expression and simultaneously attenuated RGCC mRNA and protein levels. Moreover, overexpression of RGCC in cultured lung fibroblasts attenuated the stimulating effect of TGF-ß on collagen levels. Similar to humans with pulmonary fibrosis, the levels of RGCC were also decreased in vivo in lung tissues of wild-type mice challenged with bleomycin in both acute and chronic models. Mice with constitutive RGCC gene deletion accumulated more collagen in their lungs in response to chronic bleomycin challenge than did wild-type mice. RNA-Seq analyses of lung fibroblasts revealed that RGCC overexpression alone had a modest transcriptomic effect, but in combination with TGF-ß stimulation, induced notable transcriptomic changes that negated the effects of TGF-ß, including on extracellular matrix-related genes. At the level of intracellular signaling, RGCC overexpression delayed early TGF-ß-induced Smad2/3 phosphorylation, elevated the expression of total and phosphorylated antifibrotic mediator STAT1, and attenuated the expression of a profibrotic mediator STAT3. We conclude that RGCC plays a protective role in pulmonary fibrosis and that its decline permits collagen accumulation. Restoration of RGCC expression may have therapeutic potential in pulmonary fibrosis.
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Fibroblastos/metabolismo , Pulmón/metabolismo , Proteínas Nucleares/fisiología , Fibrosis Pulmonar/prevención & control , Proteína Smad2/metabolismo , Factor de Crecimiento Transformador beta3/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Femenino , Fibroblastos/patología , Humanos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Fosforilación , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Proteína Smad2/genética , Transcriptoma , Factor de Crecimiento Transformador beta3/genéticaRESUMEN
The molecular mechanisms underlying morphological diversity in retinal cell types are poorly understood. We have previously reported that several members of the Copine family of Ca-dependent membrane adaptors are expressed in Retinal Ganglion Cells and transcriptionally regulated by Brn3 transcription factors. Several Copines are enriched in the retina and their over-expression leads to morphological changes -formation of elongated processes-, reminiscent of neurites, in HEK293 cells. However, the role of Copines in the retina is largely unknown. We now investigate Cpne4, a Copine whose expression is restricted to Retinal Ganglion Cells. Over-expression of Cpne4 in RGCs in vivo led to formation of large varicosities on the dendrites but did not otherwise visibly affect dendrite or axon formation. Protein interactions studies using yeast two hybrid analysis from whole retina cDNA revealed two Cpne4 interacting proteins-Host Cell Factor 1 and Morn2. Mass Spectrometry analysis of retina lysate pulled down using Cpne4 or its vonWillebrand A domain showed 207 interacting proteins. A Gene Ontology analysis of the discovered proteins suggests that Cpne4 is involved in several metabolic and signaling pathways in the retina.
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Axones/metabolismo , Proteínas de Unión al Calcio/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Animales , Proteínas de Unión al Calcio/genética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Neuritas/metabolismo , TransfecciónRESUMEN
The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin.
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Encéfalo/metabolismo , Proteína de Unión a CREB/genética , Proteínas del Citoesqueleto/metabolismo , Proteína p300 Asociada a E1A/genética , Células Madre Embrionarias/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Factores de Transcripción/metabolismo , Animales , Diferenciación Celular , Cromatina/química , Femenino , Genómica , Células HEK293 , Heterocigoto , Humanos , Masculino , Ratones , Neuronas/metabolismo , Unión Proteica , Dominios Proteicos , Proteómica , Activación TranscripcionalRESUMEN
Response Gene to Complement 32 (RGC-32) is an important mediator of the TGF-ß signaling pathway, and an increasing amount of evidence implicates this protein in regulating astrocyte biology. We showed recently that spinal cord astrocytes in mice lacking RGC-32 display an immature phenotype reminiscent of progenitors and radial glia, with an overall elongated morphology, increased proliferative capacity, and increased expression of progenitor markers when compared to their wild-type (WT) counterparts that make them incapable of undergoing reactive changes during the acute phase of experimental autoimmune encephalomyelitis (EAE). Here, in order to decipher the molecular networks underlying RGC-32's ability to regulate astrocytic maturation and reactivity, we performed next-generation sequencing of RNA from WT and RGC-32 knockout (KO) neonatal mouse brain astrocytes, either unstimulated or stimulated with the pleiotropic cytokine TGF-ß. Pathway enrichment analysis showed that RGC-32 is critical for the TGF-ß-induced up-regulation of transcripts encoding proteins involved in brain development and tissue remodeling, such as axonal guidance molecules, transcription factors, extracellular matrix (ECM)-related proteins, and proteoglycans. Our next-generation sequencing of RNA analysis also demonstrated that a lack of RGC-32 results in a significant induction of WD repeat and FYVE domain-containing protein 1 (Wdfy1) and stanniocalcin-1 (Stc1). Immunohistochemical analysis of spinal cords isolated from normal adult mice and mice with EAE at the peak of disease showed that RGC-32 is necessary for the in vivo expression of ephrin receptor type A7 in reactive astrocytes, and that the lack of RGC-32 results in a higher number of homeodomain-only protein homeobox (HOPX)+ and CD133+ radial glia cells. Collectively, these findings suggest that RGC-32 plays a major role in modulating the transcriptomic changes in astrocytes that ultimately lead to molecular programs involved in astrocytic differentiation and reactive changes during neuroinflammation.
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Astrocitos/metabolismo , Gliosis/genética , Enfermedades Neuroinflamatorias/genética , Proteínas Nucleares/fisiología , Transcriptoma , Animales , Orientación del Axón/genética , Encéfalo/patología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Regulación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Gliosis/etiología , Gliosis/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Células-Madre Neurales/metabolismo , Neurogénesis , Enfermedades Neuroinflamatorias/metabolismo , Proteínas Nucleares/deficiencia , Organismos Libres de Patógenos Específicos , Médula Espinal/patologíaRESUMEN
The mammalian retina contains more than 40 retinal ganglion cell (RGC) subtypes based on their unique morphologies, functions, and molecular profiles. Among them, intrinsically photosensitive RGCs (ipRGCs) are the first specified RGC type emerging from a common retinal progenitor pool during development. Previous work has shown that T-box transcription factor T-brain 2 (Tbr2) is essential for the formation and maintenance of ipRGCs, and that Tbr2-expressing RGCs activate Opn4 expression upon native ipRGC ablation, suggesting that Tbr2+ RGCs contain a reservoir for ipRGCs. However, the identity of Tbr2+ RGCs has not been fully vetted. Here, using genetic sparse labeling and single cell recording, we showed that Tbr2-expressing retinal neurons include RGCs and a subset of GABAergic displaced amacrine cells (dACs). Most Tbr2+ RGCs are intrinsically photosensitive and morphologically resemble native ipRGCs with identical retinofugal projections. Tbr2+ RGCs also include a unique and rare Pou4f1-expressing OFF RGC subtype. Using a loss-of-function strategy, we have further demonstrated that Tbr2 is essential for the survival of these RGCs and dACs, as well as maintaining the expression of Opn4. These data set a strong foundation to study how Tbr2 regulates ipRGC development and survival, as well as the expression of molecular machinery regulating intrinsic photosensitivity.
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Células Ganglionares de la Retina/metabolismo , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/genética , Animales , Dendritas/química , Dendritas/metabolismo , Femenino , Expresión Génica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Ganglionares de la Retina/química , Proteínas de Dominio T Box/análisisRESUMEN
Astrocytes are increasingly recognized as critical contributors to multiple sclerosis pathogenesis. We have previously shown that lack of Response Gene to Complement 32 (RGC-32) alters astrocyte morphology in the spinal cord at the peak of experimental autoimmune encephalomyelitis (EAE), suggesting a role for RGC-32 in astrocyte differentiation. In this study, we analyzed the expression and distribution of astrocytes and astrocyte progenitors by immunohistochemistry in spinal cords of wild-type (WT) and RGC-32-knockout (KO) mice with EAE and of normal adult mice. Our analysis showed that during acute EAE, WT astrocytes had a reactive morphology and increased GFAP expression, whereas RGC-32 KO astrocytes had a morphology similar to that of radial glia and an increased expression of progenitor markers such as vimentin and fatty acid binding protein 7 (FABP7). In control mice, GFAP expression and astrocyte density were also significantly higher in the WT group, whereas the number of vimentin and FABP7-positive radial glia was significantly higher in the RGC-32 KO group. In vitro studies on cultured neonatal astrocytes from WT and RGC-32 KO mice showed that RGC-32 regulates a complex array of molecular networks pertaining to signal transduction, growth factor expression and secretion, and extracellular matrix (ECM) remodeling. Among the most differentially expressed factors were insulin-like growth factor 1 (IGF1), insulin-like growth factor binding proteins (IGFBPs), and connective tissue growth factor (CTGF); their expression was downregulated in RGC-32-depleted astrocytes. The nuclear translocation of STAT3, a transcription factor critical for astrogliogenesis and driving glial scar formation, was also impaired after RGC-32 silencing. Taken together, these data suggest that RGC-32 is an important regulator of astrocyte differentiation during EAE and that in the absence of RGC-32, astrocytes are unable to fully mature and become reactive astrocytes.
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Astrocitos/metabolismo , Proliferación Celular , Encefalomielitis Autoinmune Experimental/metabolismo , Proteínas Nucleares/metabolismo , Médula Espinal/metabolismo , Animales , Astrocitos/patología , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Femenino , Proteína Ácida Fibrilar de la Glía/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Nucleares/genética , Fenotipo , Ratas Sprague-Dawley , Transducción de Señal , Médula Espinal/patología , Vimentina/metabolismoRESUMEN
In the mouse retina, more than 30 retinal ganglion cell (RGC) subtypes have been classified based on a combined metric of morphological and functional characteristics. RGCs arise from a common pool of retinal progenitor cells during embryonic stages and differentiate into mature subtypes in adult retinas. However, the cellular and molecular mechanisms controlling formation and maturation of such remarkable cellular diversity remain unknown. Here, we demonstrate that T-box transcription factor T-brain 1 (Tbr1) is expressed in two groups of morphologically and functionally distinct RGCs: the orientation-selective J-RGCs and a group of OFF-sustained RGCs with symmetrical dendritic arbors. When Tbr1 is genetically ablated during retinal development, these two RGC groups cannot develop. Ectopically expressing Tbr1 in M4 ipRGCs during development alters dendritic branching and density but not the inner plexiform layer stratification level. Our data indicate that Tbr1 plays critical roles in regulating the formation and dendritic morphogenesis of specific RGC types.
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Células Ganglionares de la Retina/metabolismo , Proteínas de Dominio T Box/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Axones/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Toxina del Cólera/toxicidad , Dendritas/fisiología , Embrión de Mamíferos/metabolismo , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Potasio/farmacología , Retina/crecimiento & desarrollo , Retina/metabolismo , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Proteínas de Dominio T Box/genéticaRESUMEN
Combinatorial expression of Brn3 transcription factors is required for the development of cell-specific morphologies in retinal ganglion cells (RGCs). The molecular mechanisms by which Brn3s regulate RGC type specific features are largely unexplored. We previously identified several members of the Copine (Cpne) family of molecules as potential targets of Brn3 transcription factors in the retina. We now use in situ hybridization and immunohistochemistry to characterize Copine expression in the postnatal and adult mouse retina. We find that Cpne5, 6, and 9 are expressed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in both amacrine cells and RGCs. Cpne4 expression is restricted to one amacrine cell population of the INL, but is specifically expressed in RGCs in the GCL. Cpne4 expression in RGCs is regulated by Brn3b both cell autonomously (in Brn3b+ RGCs) and cell nonautonomously (in Brn3b- RGCs). Copines exhibit a variety of subcellular distributions when overexpressed in tissue culture cells (HEK293), and can induce the formation of elongated processes reminiscent of neurites in these non-neuronal cells. Our results suggest that Copines might be involved in a combinatorial fashion in Brn3b-dependent specification of RGC types. Given their expression profile and previously proven role as Ca2+ sensors, they may participate in the morphogenetic processes that shape RGC dendrite and axon formation at early postnatal ages.
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Proteínas de la Membrana/análisis , Proteínas de la Membrana/biosíntesis , Retina/química , Retina/metabolismo , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Secuencia de Aminoácidos , Animales , Expresión Génica , Técnicas de Sustitución del Gen/métodos , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Células Ganglionares de la Retina/química , Células Ganglionares de la Retina/metabolismoRESUMEN
Extracellular matrix (ECM) deposition in active demyelinating multiple sclerosis (MS) lesions may impede axonal regeneration and can modify immune reactions. Response gene to complement (RGC)-32 plays an important role in the mediation of TGF-ß downstream effects, but its role in gliosis has not been investigated. To gain more insight into the role played by RGC-32 in gliosis, we investigated its involvement in TGF-ß-induced ECM expression and the upregulation of the reactive astrocyte markers α-smooth muscle actin (α-SMA) and nestin. In cultured neonatal rat astrocytes, collagens I, IV, and V, fibronectin, α-SMA, and nestin were significantly induced by TGF-ß stimulation, and RGC-32 silencing resulted in a significant reduction in their expression. Using astrocytes isolated from RGC-32 knock-out (KO) mice, we found that the expression of TGF-ß-induced collagens I, IV, and V, fibronectin, and α-SMA was significantly reduced in RGC-32 KO mice when compared with wild-type (WT) mice. SIS3 inhibition of Smad3 phosphorylation was also associated with a significant reduction in RGC-32 nuclear translocation and TGF-ß-induced collagen I expression. In addition, during experimental autoimmune encephalomyelitis (EAE), RGC-32 KO mouse astrocytes displayed an elongated, bipolar phenotype, resembling immature astrocytes and glial progenitors whereas those from WT mice had a reactive, hypertrophied phenotype. Taken together, our data demonstrate that RGC-32 plays an important role in mediating TGF-ß-induced reactive astrogliosis in EAE. Therefore, RGC-32 may represent a new target for therapeutic intervention in MS.
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Astrocitos/fisiología , Encefalomielitis Autoinmune Experimental/metabolismo , Gliosis/metabolismo , Esclerosis Múltiple/metabolismo , Proteínas Nucleares/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Colágeno/metabolismo , Modelos Animales de Enfermedad , Matriz Extracelular/metabolismo , Femenino , Colágenos Asociados a Fibrillas , Humanos , Ratones , Ratones Noqueados , Nestina/metabolismo , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Ratas , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
An important goal in visual neuroscience is to understand how neuronal population coding in vertebrate retina mediates the broad range of visual functions. Microelectrode arrays interface on isolated retina registers a collective measure of the spiking dynamics of retinal ganglion cells (RGCs) by probing them simultaneously and in large numbers. The recorded data stream is then processed to identify spike trains of individual RGCs by efficient and scalable spike detection and sorting routines. Most spike sorting software packages, available either commercially or as freeware, combine automated steps with judgment calls by the investigator to verify the quality of sorted spikes. This work focused on sorting spikes of RGCs into clusters using an integrated analytical platform for the data recorded during visual stimulation of wild-type mice retinas with whole field stimuli. After spike train detection, we projected each spike onto two feature spaces: a parametric space and a principal components space. We then applied clustering algorithms to sort spikes into separate clusters. To eliminate the need for human intervention, the initial clustering results were submitted to diagnostic tests that evaluated the results to detect the sources of failure in cluster assignment. This failure diagnosis formed a decision logic for diagnosable electrodes to enhance the clustering quality iteratively through rerunning the clustering algorithms. The new clustering results showed that the spike sorting accuracy was improved. Subsequently, the number of active RGCs during each whole field stimulation was found, and the light responsiveness of each RGC was identified. Our approach led to error-resilient spike sorting in both feature extraction methods; however, using parametric features led to less erroneous spike sorting compared to principal components, particularly for low signal-to-noise ratios. As our approach is reliable for retinal signal processing in response to simple visual stimuli, it could be applied to the evaluation of disrupted physiological signaling in retinal neurodegenerative diseases.
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Potenciales de Acción , Reconocimiento de Normas Patrones Automatizadas/métodos , Células Ganglionares de la Retina/fisiología , Visión Ocular/fisiología , Algoritmos , Animales , Análisis por Conglomerados , Ratones , Microelectrodos , Estimulación Luminosa , Análisis de Componente Principal , Procesamiento de Señales Asistido por ComputadorRESUMEN
Th17 cells play a critical role in autoimmune diseases, including multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Response gene to complement (RGC)-32 is a cell cycle regulator and a downstream target of TGF-ß that mediates its profibrotic activity. In this study, we report that RGC-32 is preferentially upregulated during Th17 cell differentiation. RGC-32-/- mice have normal Th1, Th2, and regulatory T cell differentiation but show defective Th17 differentiation in vitro. The impaired Th17 differentiation is associated with defects in IFN regulatory factor 4, B cell-activating transcription factor, retinoic acid-related orphan receptor γt, and SMAD2 activation. In vivo, RGC-32-/- mice display an attenuated experimental autoimmune encephalomyelitis phenotype accompanied by decreased CNS inflammation and reduced frequency of IL-17- and GM-CSF-producing CD4+ T cells. Collectively, our results identify RGC-32 as a novel regulator of Th17 cell differentiation in vitro and in vivo and suggest that RGC-32 is a potential therapeutic target in multiple sclerosis and other Th17-mediated autoimmune diseases.
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Diferenciación Celular/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Células Th17/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/fisiopatología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Factores Reguladores del Interferón/genética , Factores Reguladores del Interferón/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares/deficiencia , Proteínas Nucleares/farmacología , Receptores de Ácido Retinoico/genética , Receptores de Ácido Retinoico/metabolismo , Células TH1/inmunología , Células Th17/inmunología , Células Th17/patologíaRESUMEN
Photoreceptor degeneration is a cause of irreversible vision loss in incurable blinding retinal diseases including retinitis pigmentosa (RP) and atrophic age-related macular degeneration. We found in two separate mouse models of photoreceptor degeneration that tamoxifen, a selective estrogen receptor modulator and a drug previously linked with retinal toxicity, paradoxically provided potent neuroprotective effects. In a light-induced degeneration model, tamoxifen prevented onset of photoreceptor apoptosis and atrophy and maintained near-normal levels of electroretinographic responses. Rescue effects were correlated with decreased microglial activation and inflammatory cytokine production in the retina in vivo and a reduction of microglia-mediated toxicity to photoreceptors in vitro, indicating a microglia-mediated mechanism of rescue. Tamoxifen also rescued degeneration in a genetic (Pde6brd10) model of RP, significantly improving retinal structure, electrophysiological responses, and visual behavior. These prominent neuroprotective effects warrant the consideration of tamoxifen as a drug suitable for being repurposed to treat photoreceptor degenerative disease.SIGNIFICANCE STATEMENT Photoreceptor degeneration is a cause of irreversible blindness in a number of retinal diseases such as retinitis pigmentosa (RP) and atrophic age-related macular degeneration. Tamoxifen, a selective estrogen receptor modulator approved for the treatment of breast cancer and previously linked to a low incidence of retinal toxicity, was unexpectedly found to exert marked protective effects against photoreceptor degeneration. Structural and functional protective effects were found for an acute model of light-induced photoreceptor injury and for a genetic model for RP. The mechanism of protection involved the modulation of microglial activation and the production of inflammatory cytokines, highlighting the role of inflammatory mechanisms in photoreceptor degeneration. Tamoxifen may be suitable for clinical study as a potential treatment for diseases involving photoreceptor degeneration.
Asunto(s)
Regeneración Nerviosa/fisiología , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/patología , Degeneración Retiniana/tratamiento farmacológico , Degeneración Retiniana/fisiopatología , Tamoxifeno/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Regeneración Nerviosa/efectos de la radiación , Fármacos Neuroprotectores/administración & dosificación , Células Fotorreceptoras de Vertebrados/fisiología , Recuperación de la Función/efectos de los fármacos , Degeneración Retiniana/patología , Resultado del TratamientoRESUMEN
Rapid and stable control of pupil size in response to light is critical for vision, but the neural coding mechanisms remain unclear. Here, we investigated the neural basis of pupil control by monitoring pupil size across time while manipulating each photoreceptor input or neurotransmitter output of intrinsically photosensitive retinal ganglion cells (ipRGCs), a critical relay in the control of pupil size. We show that transient and sustained pupil responses are mediated by distinct photoreceptors and neurotransmitters. Transient responses utilize input from rod photoreceptors and output by the classical neurotransmitter glutamate, but adapt within minutes. In contrast, sustained responses are dominated by non-conventional signaling mechanisms: melanopsin phototransduction in ipRGCs and output by the neuropeptide PACAP, which provide stable pupil maintenance across the day. These results highlight a temporal switch in the coding mechanisms of a neural circuit to support proper behavioral dynamics.