RESUMO
In daylight, demand for visual chromophore (11-cis-retinal) exceeds supply by the classical visual cycle. This shortfall is compensated, in part, by the retinal G-protein-coupled receptor (RGR) photoisomerase, which is expressed in both the retinal pigment epithelium (RPE) and in Müller cells. The relative contributions of these two cellular pools of RGR to the maintenance of photoreceptor light responses are not known. Here, we use a cell-specific gene reactivation approach to elucidate the kinetics of RGR-mediated recovery of photoreceptor responses following light exposure. Electroretinographic measurements in mice with RGR expression limited to either cell type reveal that the RPE and a specialized subset of Müller glia contribute both to scotopic and photopic function. We demonstrate that 11-cis-retinal formed through photoisomerization is rapidly hydrolyzed, consistent with its role in a rapid visual pigment regeneration process. Our study shows that RGR provides a pan-retinal sink for all-trans-retinal released under sustained light conditions and supports rapid chromophore regeneration through the photic visual cycle.
Assuntos
Epitélio Pigmentado da Retina , Retinaldeído , Animais , Camundongos , Epitélio Pigmentado da Retina/metabolismo , Retinaldeído/metabolismo , Pigmentos da Retina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Neuroglia/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismoRESUMO
Background: Inspired by research indicating that exposure to humanities correlates with reduced burnout, the Nebraska Medical Orchestra was founded in 2018 as a collaboration between the University of Nebraska Medical Center and the University of Nebraska at Omaha School of Music. Methods: Semistructured interviews about orchestra participants' experiences were conducted with 9 musicians and recorded and transcribed. Transcripts were analyzed using the constant comparative method. Results: The interviews suggested that participants are drawn to the orchestra to pursue a love of music, to be part of an ensemble, and to connect with others in an environment that provides a lighthearted, nonjudgmental, noncompetitive forum in which to create and enjoy music for its own sake. Conclusions: This study has implications for designing arts-based wellness activities for clinicians and scaling them nationwide.
Assuntos
Empatia , Música , Criatividade , Ciências Humanas , Humanos , Nebraska , Inquéritos e QuestionáriosRESUMO
Stress and burnout afflict medical students and professionals at alarming rates, which has led institutions to invest in counseling services and other traditional wellness programming. However, the stigma of utilizing these services permeates the medical community. This narrative explores the founding of the Nebraska Medical Orchestra-an orchestra created as a nontraditional antidote to reduce stress and burnout among health care students and professionals-and also examines the concept of wellness through interactions between the orchestra's director and health care-related musicians.
Assuntos
Esgotamento Profissional , Música , Estudantes de Medicina , Esgotamento Profissional/prevenção & controle , Humanos , Nebraska , Estudantes de Medicina/psicologiaRESUMO
The basic motif-leucine zipper (bZIP) transcription factor neural retina leucine zipper (NRL) determines rod photoreceptor cell fate during retinal development, and its loss leads to cone-only retina in mice. NRL works synergistically with homeodomain protein Cone-Rod Homeobox and other regulatory factors to control the transcription of most genes associated with rod morphogenesis and functional maturation, which span over a period of several weeks in the mammalian retina. We predicted that NRL gradually establishes rod cell identity and function by temporal and dynamic regulation of stage-specific transcriptional targets. Therefore, we mapped the genomic occupancy of NRL at four stages of mouse photoreceptor differentiation by CUT&RUN analysis. Dynamics of NRL binding revealed concordance with the corresponding changes in transcriptome of the developing rods. Notably, we identified c-Jun proto-oncogene as one of the targets of NRL, which could bind to specific cis-elements in the c-Jun promoter and modulate its activity in HEK293 cells. Coimmunoprecipitation studies showed the association of NRL with c-Jun, also a bZIP protein, in transfected cells as well as in developing mouse retina. Additionally, shRNA-mediated knockdown of c-Jun in the mouse retina in vivo resulted in altered expression of almost 1000 genes, with reduced expression of phototransduction genes and many direct targets of NRL in rod photoreceptors. We propose that c-Jun-NRL heterodimers prime the NRL-directed transcriptional program in neonatal rod photoreceptors before high NRL expression suppresses c-Jun at later stages. Our study highlights a broader cooperation among cell-type restricted and widely expressed bZIP proteins, such as c-Jun, in specific spatiotemporal contexts during cellular differentiation.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Células Fotorreceptoras Retinianas Bastonetes , Animais , Humanos , Camundongos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Diferenciação Celular/genética , Proteínas do Olho/genética , Células HEK293 , Mamíferos/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismoRESUMO
Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.
Assuntos
Degeneração Retiniana , Retinose Pigmentar , Animais , Morte Celular/genética , Modelos Animais de Doenças , Camundongos , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/metabolismo , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Retinose Pigmentar/patologiaRESUMO
Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.
Assuntos
Diferenciação Celular/fisiologia , Neocórtex/embriologia , Neocórtex/fisiologia , Neurogênese/fisiologia , Neurônios/fisiologia , Animais , Células Cultivadas , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Neocórtex/citologia , Gravidez , Imagem com Lapso de Tempo/métodosRESUMO
To understand RS1 gene interaction networks in the X-linked retinoschisis (XLRS) mouse retina (Rs1-/y), we analyzed the transcriptome by RNA sequencing before and after in vivo expression of exogenous retinoschisin (RS1) gene delivered by AAV8. RS1 is a secreted cell adhesion protein that is critical for maintaining structural lamination and synaptic integrity of the neural retina. RS1 loss-of-function mutations cause XLRS disease in young boys and men, with splitting ("schisis") of retinal layers and synaptic dysfunction that cause progressive vision loss with age. Analysis of differential gene expression profiles and pathway enrichment analysis of Rs1-KO (Rs1-/y) retina identified cell surface receptor signaling and positive regulation of cell adhesion as potential RS1 gene interaction networks. Most importantly, it also showed massive dysregulation of immune response genes at early age, with characteristics of a microglia-driven proinflammatory state. Delivery of AAV8-RS1 primed the Rs1-KO retina toward structural and functional recovery. The disease transcriptome transitioned toward a recovery phase with upregulation of genes implicated in wound healing, anatomical structure (camera type eye) development, metabolic pathways, and collagen IV networks that provide mechanical stability to basement membrane. AAV8-RS1 expression also attenuated the microglia gene signatures to low levels toward immune quiescence. This study is among the first to identify RS1 gene interaction networks that underlie retinal structural and functional recovery after RS1 gene therapy. Significantly, it also shows that providing wild-type RS1 gene function caused the retina immune status to transition from a degenerative inflammatory phenotype toward immune quiescence, even though the transgene is not directly linked to microglia function. This study indicates that inhibition of microglial proinflammatory responses is an integral part of therapeutic rescue in XLRS gene therapy, and gene therapy might realize its full potential if delivered before microglia activation and photoreceptor cell death. Clinical Trials. gov Identifier NTC 02317887.
Assuntos
Retinosquise , Animais , Eletrorretinografia , Proteínas do Olho/genética , Redes Reguladoras de Genes , Terapia Genética , Vetores Genéticos/genética , Camundongos , Microglia , Retina , Retinosquise/genética , Retinosquise/terapiaRESUMO
Retinal pigment epithelium (RPE) differentiated from human induced pluripotent stem cells, called induced retinal pigment epithelium (iRPE), is being explored as a cell-based therapy for the treatment of retinal degenerative diseases, especially age-related macular degeneration. The success of RPE implantation is linked to the use of biomimetic scaffolds that simulate Bruch's membrane and promote RPE maturation and integration as a functional tissue. Due to difficulties associated with animal protein-derived scaffolds, including sterility and pro-inflammatory responses, current practices favor the use of synthetic polymers, such as polycaprolactone (PCL), for generating nanofibrous scaffolds. In this study, we tested the hypothesis that plant protein-derived fibrous scaffolds can provide favorable conditions permissive for the maturation of RPE tissue sheets in vitro. Our natural, soy protein-derived nanofibrous scaffolds exhibited a J-shaped stress-strain curve that more closely resembled the mechanical properties of native tissues than PCL with significantly higher hydrophilicity of the natural scaffolds, favoring in vivo implantation. We then demonstrate that iRPE sheets growing on these soy protein scaffolds are equivalent to iRPE monolayers cultured on synthetic PCL nanofibrous scaffolds. Immunohistochemistry demonstrated RPE-like morphology and functionality with appropriate localization of RPE markers RPE65, PMEL17, Ezrin, and ZO1 and with anticipated histotypic polarization of vascular endothelial growth factor and pigment epithelium-derived growth factor as indicated by enzyme-linked immunosorbent assay. Scanning electron microscopy revealed dense microvilli on the cell surface and homogeneous tight junctional contacts between the cells. Finally, comparative transcriptome analysis in conjunction with principal component analysis demonstrated that iRPE on nanofibrous scaffolds, either natural or synthetic, matured more consistently than on nonfibrous substrates. Taken together, our studies suggest that the maturation of cultured iRPE sheets for subsequent clinical applications might benefit from the use of nanofibrous scaffolds generated from natural proteins. Impact statement Induced retinal pigment epithelium (iRPE) from patient-derived induced pluripotent stem cells (iPSCs) may yield powerful treatments of retinal diseases, including age-related macular degeneration. Recent studies, including early human clinical trials, demonstrate the importance of selecting appropriate biomaterial scaffolds to support tissue-engineered iRPE sheets during implantation. Electrospun scaffolds show particular promise due to their similarity to the structure of the native Bruch's membrane. In this study, we describe the use of electroprocessed nanofibrous soy protein scaffolds to generate polarized sheets of human iPSC-derived iRPE sheets. Our evaluation, including RNA-seq transcriptomics, indicates that these scaffolds are viable alternatives to scaffolds electrospun from synthetic polymers.
Assuntos
Diferenciação Celular , Células-Tronco Pluripotentes Induzidas/citologia , Nanofibras/química , Epitélio Pigmentado da Retina/citologia , Proteínas de Soja/química , Alicerces Teciduais/química , Linhagem Celular , Módulo de Elasticidade , Perfilação da Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Nanofibras/ultraestrutura , Poliésteres/química , Epitélio Pigmentado da Retina/ultraestrutura , Proteínas de Soja/ultraestruturaRESUMO
Cigarette smoking, a powerful mixture of chemical oxidants, is the strongest environmental risk factor for developing age-related macular degeneration (AMD), the most common cause of blindness among the elderly in western societies. Despite intensive study, the full impact of smoking on the retinal pigment epithelium (RPE), a central cell type involved in AMD pathobiology, remains unknown. The relative contribution of the known dysfunctional pathways to AMD, at what stage they are most pathogenic, or whether other processes are relevant, is poorly understood, and furthermore, whether smoking activates them, is unknown. We performed global RNA-sequencing of the RPE from C57BL/6J mice exposed to chronic cigarette smoke for 6 months to identify potential pathogenic and cytoprotective pathways. The RPE transcriptome induced by chronic cigarette smoking exhibited a mixed response of marked suppression of the innate immune response including type I and II interferons and upregulation of cell differentiation and morphogenic gene clusters, suggesting an attempt by the RPE to maintain its differentiated state despite smoke-induced injury. Given that mice exposed to chronic smoke develop early features of AMD, these novel findings are potentially relevant to the transition from aging to AMD.
Assuntos
Epitélio Pigmentado da Retina , Fumar , Animais , Diferenciação Celular , Perfilação da Expressão Gênica , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , Fumar/efeitos adversosRESUMO
Aging-associated functional decline is accompanied by alterations in the epigenome. To explore DNA modifications that could influence visual function with age, we perform whole-genome bisulfite sequencing of purified mouse rod photoreceptors at four ages and identify 2,054 differentially methylated regions (DMRs). We detect many DMRs during early stages of aging and in rod regulatory regions, and some of these cluster at chromosomal hotspots, especially on chromosome 10, which includes a longevity interactome. Integration of methylome to age-related transcriptome changes, chromatin signatures, and first-order protein-protein interactions uncover an enrichment of DMRs in altered pathways that are associated with rod function, aging, and energy metabolism. In concordance, we detect reduced basal mitochondrial respiration and increased fatty acid dependency with retinal age in ex vivo assays. Our study reveals age-dependent genomic and chromatin features susceptible to DNA methylation changes in rod photoreceptors and identifies a link between DNA methylation and energy metabolism in aging.
Assuntos
Envelhecimento/genética , Metilação de DNA/genética , Metabolismo Energético/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , CamundongosRESUMO
Purpose: Genes involved in the development and differentiation of the mammalian retina are also associated with inherited retinal dystrophies (IRDs) and age-related macular degeneration. Transcriptional regulation of retinal cell differentiation has been addressed by genetic and transcriptomic studies. Much less is known about the posttranslational regulation of key regulatory proteins, although mutations in some genes involved in ubiquitination and proteostasis-E3 ligases and deubiquitinating enzymes (DUBs)-cause IRDs. This study intends to provide new data on DUB gene expression during different developmental stages of mouse and human fetal retinas. Methods: We performed a comprehensive transcriptomic analysis of all the annotated human and mouse DUBs (87) in the developing mouse retina at several embryonic and postnatal time points compared with the transcriptome of the fetal human retina. An integrated comparison of data from transcriptomics, reported chromatin immunoprecipitation sequencing (ChIP-seq) of CRX and NRL transcription factors, and the phenotypic retinal alterations in different animal models is presented. Results: Several DUB genes are differentially expressed during the development of the mouse and human retinas in relation to proliferation or differentiation stages. Some DUB genes appear to be distinctly expressed during the differentiation stages of rod and cone photoreceptor cells, and their expression is altered in mouse knockout models of relevant photoreceptor transcription factors. We complemented this RNA-sequencing (RNA-seq) analysis with other reported expression and phenotypic data to underscore the involvement of DUBs in cell fate decision and photoreceptor differentiation. Conclusions: The present results highlight a short list of potential DUB candidates for retinal disorders, which require further study.
Assuntos
Enzimas Desubiquitinantes/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Mamíferos/embriologia , Mamíferos/genética , Retina/embriologia , Retina/enzimologia , Animais , Enzimas Desubiquitinantes/metabolismo , Feto/metabolismo , Humanos , Camundongos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Transcriptoma/genéticaRESUMO
PURPOSE: Retinal organoids generated from human pluripotent stem cells exhibit considerable variability during differentiation. Our goals are to assess developmental maturity of the neural retina in vitro and design improved protocols based on objective criteria. METHODS: We performed transcriptome analyses of developing retinal organoids from human embryonic and induced pluripotent stem cell lines and utilized multiple bioinformatic tools for comparative analysis. Immunohistochemistry, immunoblotting and electron microscopy were employed for validation. RESULTS: We show that the developmental variability in organoids was reflected in gene expression profiles and could be evaluated by molecular staging with the human fetal and adult retinal transcriptome data. We also demonstrate that the addition of 9-cis retinal, instead of the widely used all-trans retinoic acid, accelerated rod photoreceptor differentiation in organoid cultures, with higher rhodopsin expression and more mature mitochondrial morphology evident by day 120. CONCLUSION: Our studies provide an objective transcriptome-based modality for determining the differentiation state of retinal organoids and for comparisons across different stem cell lines and platforms, which should facilitate disease modeling and evaluation of therapies in vitro.
Assuntos
Diferenciação Celular , Diterpenos/farmacologia , Células-Tronco Embrionárias Humanas/citologia , Organoides/citologia , Retina/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Retinaldeído/farmacologia , Transcriptoma/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Transcriptoma/efeitos dos fármacosRESUMO
Stem cell-derived retinal organoids recapitulate many landmarks of in vivo differentiation but lack functional maturation of distinct cell types, especially photoreceptors. Using comprehensive temporal transcriptome analyses, we show that transcriptome shift from postnatal day 6 (P6) to P10, associated with morphogenesis and synapse formation during mouse retina development, was not evident in organoids, and co-expression clusters with similar patterns included different sets of genes. Furthermore, network analysis identified divergent regulatory dynamics between developing retina in vivo and in organoids, with temporal dysregulation of specific signaling pathways and delayed or reduced expression of genes involved in photoreceptor function(s) and survival. Accordingly, addition of docosahexaenoic acid and fibroblast growth factor 1 to organoid cultures specifically promoted the maturation of photoreceptors, including cones. Our study thus identifies regulatory signals deficient in developing retinal organoids and provides experimental validation by producing a more mature retina in vitro, thereby facilitating investigations in disease modeling and therapies.
Assuntos
Organoides/citologia , Retina/citologia , Transcriptoma , Animais , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Camundongos , Camundongos Endogâmicos C57BL , Organoides/crescimento & desenvolvimento , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Transdução de SinaisRESUMO
Reloading of atrophied muscles after hindlimb suspension (HLS) can induce muscle injury and prolong recovery after disuse in old rats, especially in fast contracting muscles. Less is known about the responses in mice and whether fast and slow muscles from geriatric mice will respond in a similar fashion to HLS unloading and recovery (HLSâ¯+â¯R). Furthermore, while slow muscles undergo atrophy with disuse, they typically are more resistant to sarcopenia than fast contracting muscles. Geriatric (28â¯mo. of age) male C57BL/6 mice were randomly placed into 3 groups. These included HLS for 14â¯days nâ¯=â¯9, and HLS followed by 14â¯days of reloading recovery (HLSâ¯+â¯R; nâ¯=â¯9), or normal ambulatory cage controls (nâ¯=â¯9). Control mice were not exposed to unloading. Electrically evoked maximal muscle function was assessed in vivo in anesthetized mice at baseline, after 14â¯days of HLS or HLSâ¯+â¯R. As expected, HLS significantly reduced body weight, wet weight of gastrocnemius and soleus muscles and in vivo maximal force. There were no differences in vivo fatigability of the plantar flexor muscles and overall fiber size. There were only minor fiber type distribution and frequency distribution of fiber sizes that differ between HLSâ¯+â¯R and control gastrocnemius and soleus muscles. Soleus muscle wet weight had recovered to control levels after reloading, but type I/IIA fibers in the soleus muscles were significantly smaller after HLSâ¯+â¯R than control muscles. In contrast, gastrocnemius muscle wet weight did not recover to control levels after reloading. Plantar flexion muscle force (primarily influenced by the gastrocnemius muscles) did not recover in HLSâ¯+â¯R conditions as compared to HLS conditions and both were lower than control force production signaling for apoptosis, autophagy and anabolic markers were not different between control and HLSâ¯+â¯R gastrocnemius and soleus muscles in geriatric mice. These results suggest that molecular signaling does not explain attenuated ability to regain muscle wet weight, fiber size or muscle force production after HLS in geriatric mice. It is possible that fluid shifts, reduced blood flow, or shortened muscle fibers which failed to regain control lengths contributed to the attenuation of muscle wet weight after HLS and reloading and this affected force production. Further work is needed to determine if altered/loss of neural activity contributed to the inability of geriatric mice to regain gastrocnemius muscle weight and function after HLS and reloading.
Assuntos
Envelhecimento/fisiologia , Elevação dos Membros Posteriores , Músculo Esquelético/fisiologia , Atrofia Muscular/patologia , Animais , Contração Isométrica/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/patologia , Tamanho do ÓrgãoRESUMO
Mucopolysaccharidosis type I (MPS I) is caused by deficiency of α-l-iduronidase (IDUA), a lysosomal enzyme involved in the breakdown and recycling of glycosaminoglycans (GAGs). Although enzyme replacement therapy is available, the efficacy of the treatment for neuropathic manifestations is limited. To facilitate drug discovery and model disease pathophysiology, we generated neural stem cells (NSCs) from MPS I patient-derived induced pluripotent stem cells (iPSCs). The NSCs exhibited characteristic disease phenotypes with deficiency of IDUA, accumulation of GAGs and enlargement of lysosomes, in agreement with the severity of clinical subgroups of MPS I. Transcriptome profiling of NSCs revealed 429 genes that demonstrated a more extensive change in expression in the most severe Hurler syndrome subgroup compared to the intermediate Hurler-Scheie or the least severe Scheie syndrome subgroups. Clustering and pathway analysis revealed high concordance of the severity of neurological defects with marked dysregulation of GAG biosynthesis, GAG degradation, lysosomal function and autophagy. Gene ontology (GO) analysis identified a dramatic upregulation of the autophagy pathway, especially in the Hurler syndrome subgroup. We conclude that GAG accumulation in the patient-derived cells disrupts lysosomal homeostasis, affecting multiple related cellular pathways in response to IDUA deficiency. These dysregulated processes likely lead to enhanced autophagy and progressively severe disease states. Our study provides potentially useful targets for clinical biomarker development, disease diagnosis and prognosis, and drug discovery.
Assuntos
Iduronidase/genética , Mucopolissacaridose I/enzimologia , Células-Tronco Neurais , Fenótipo , Linhagem Celular , Criança , Pré-Escolar , Perfilação da Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Iduronidase/metabolismo , Células-Tronco Pluripotentes Induzidas , Lisossomos , Masculino , Mucopolissacaridose I/genética , Mucopolissacaridose I/metabolismo , MutaçãoRESUMO
Reloading of atrophied muscles after hindlimb suspension unloading (HSU) can induce injury and prolong recovery. Low-impact exercise, such as voluntary wheel running, has been identified as a nondamaging rehabilitation therapy in rodents, but its effects on muscle function, morphology, and satellite cell activity after HSU are unclear. This study tested the hypothesis that low-impact wheel running would increase satellite cell proliferation and improve recovery of muscle structure and function after HSU in mice. Young adult male and female C57BL/6 mice ( n = 6/group) were randomly placed into five groups. These included HSU without recovery (HSU), normal ambulatory recovery for 14 days after HSU (HSU+NoWR), and voluntary wheel running recovery for 14 days after HSU (HSU+WR). Two control groups were used: nonsuspended mouse cage controls (Control) and voluntary wheel running controls (ControlWR). Satellite cell activation was evaluated by providing mice 5-bromo-2'-deoxyuridine (BrdU) in their drinking water. As expected, HSU significantly reduced in vivo maximal force, decreased in vivo fatigability, and decreased type I and IIa myosin heavy chain (MHC) abundance in plantarflexor muscles. HSU+WR mice significantly improved plantarflexor fatigue resistance, increased type I and IIa MHC abundance, increased fiber cross-sectional area, and increased the percentage of type I and IIA muscle fibers in the gastrocnemius muscle. HSU+WR mice also had a significantly greater percentage of BrdU-positive and Pax 7-positive nuclei inside muscle fibers and a greater MyoD-to-Pax 7 protein ratio compared with HSU+NoWR mice. The mechanotransduction protein Yes-associated protein (YAP) was elevated with reloading after HSU, but HSU+WR mice had lower levels of the inactive phosphorylated YAPserine127, which may have contributed to increased satellite cell activation with reloading after HSU. These results indicate that voluntary wheel running increased YAP signaling and satellite cell activity after HSU and this was associated with improved recovery. NEW & NOTEWORTHY Although satellite cell involvement in muscle remodeling has been challenged, the data in this study suggest that voluntary wheel running increased satellite cell activity and suppressed Yes-associated protein (YAP) protein relative to no wheel running and this was associated with improved muscle recovery of force, fatigue resistance, expression of type I myosin heavy chain, and greater fiber cross-sectional area after disuse.
Assuntos
Músculo Esquelético/citologia , Recuperação de Função Fisiológica , Corrida/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Feminino , Elevação dos Membros Posteriores , Via de Sinalização Hippo , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiologia , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Distribuição Aleatória , Proteínas de Sinalização YAPRESUMO
Pluripotent stem cells can be differentiated into 3D retinal organoids, with major cell types self-patterning into a polarized, laminated architecture. In static cultures, organoid development may be hindered by limitations in diffusion of oxygen and nutrients. Herein, we report a bioprocess using rotating-wall vessel (RWV) bioreactors to culture retinal organoids derived from mouse pluripotent stem cells. Organoids in RWV demonstrate enhanced proliferation, with well-defined morphology and improved differentiation of neurons including ganglion cells and S-cone photoreceptors. Furthermore, RWV organoids at day 25 (D25) reveal similar maturation and transcriptome profile as those at D32 in static culture, closely recapitulating spatiotemporal development of postnatal day 6 mouse retina in vivo. Interestingly, however, retinal organoids do not differentiate further under any in vitro condition tested here, suggesting additional requirements for functional maturation. Our studies demonstrate that bioreactors can accelerate and improve organoid growth and differentiation for modeling retinal disease and evaluation of therapies.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Organoides/metabolismo , Células-Tronco Pluripotentes/metabolismo , Retina/metabolismo , Animais , Camundongos , Camundongos Transgênicos , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Retina/citologiaRESUMO
Clinical and genetic heterogeneity associated with retinal diseases makes stem-cell-based therapies an attractive strategy for personalized medicine. However, we have limited understanding of the timing of key events in the developing human retina, and in particular the factors critical for generating the unique architecture of the fovea and surrounding macula. Here we define three key epochs in the transcriptome dynamics of human retina from fetal day (D) 52 to 136. Coincident histological analyses confirmed the cellular basis of transcriptional changes and highlighted the dramatic acceleration of development in the fovea compared with peripheral retina. Human and mouse retinal transcriptomes show remarkable similarity in developmental stages, although morphogenesis was greatly expanded in humans. Integration of DNA accessibility data allowed us to reconstruct transcriptional networks controlling photoreceptor differentiation. Our studies provide insights into human retinal development and serve as a resource for molecular staging of human stem-cell-derived retinal organoids.
Assuntos
Neurogênese/fisiologia , Retina/citologia , Retina/fisiologia , Animais , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Fóvea Central/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Humanos , Macula Lutea/embriologia , Camundongos , Morfogênese , Neurogênese/genética , Neurônios/metabolismo , Retina/embriologia , Retina/crescimento & desenvolvimento , Análise de Sequência de RNA/métodos , TranscriptomaRESUMO
AIM: A systematic review and meta-analysis, evaluating ischemic conditioning during percutaneous coronary intervention (PCI). METHODS & RESULTS: A database search of randomized trials of ischemic conditioning in PCI created three subgroups for meta-analysis: mortality in elective PCI with remote ischemic preconditioning (RIPreC; subgroup 1a, n = 3) - no outcome difference between RIPreC and control (odds ratio: 0.34; 95% CI: 0.08-1.56), myocardial salvage index in ST-elevation myocardial infarction (STEMI) with RIPreC (subgroup 1b, n = 2) - favored RIPreC (mean difference: 0.13; 95% CI: 0.07-0.19), and infarct size in STEMI with local ischemic postconditioning (LIPostC) (subgroup 4b, n = 12) - favored LIPostC (mean difference: -4.13 g.m-2; 95% CI: -7.36 to -0.90 g.m-2). CONCLUSION: RIPreC and LIPostC improve myocardial salvage index and myocardial infarct size respectively in PCI for STEMI. No mortality benefit detected with RIPreC in elective PCI.