RESUMEN
Primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide, disproportionately affects individuals of African ancestry. We conducted a genome-wide association study (GWAS) for POAG in 11,275 individuals of African ancestry (6,003 cases; 5,272 controls). We detected 46 risk loci associated with POAG at genome-wide significance. Replication and post-GWAS analyses, including functionally informed fine-mapping, multiple trait co-localization, and in silico validation, implicated two previously undescribed variants (rs1666698 mapping to DBF4P2; rs34957764 mapping to ROCK1P1) and one previously associated variant (rs11824032 mapping to ARHGEF12) as likely causal. For individuals of African ancestry, a polygenic risk score (PRS) for POAG from our mega-analysis (African ancestry individuals) outperformed a PRS from summary statistics of a much larger GWAS derived from European ancestry individuals. This study quantifies the genetic architecture similarities and differences between African and non-African ancestry populations for this blinding disease.
Asunto(s)
Estudio de Asociación del Genoma Completo , Glaucoma de Ángulo Abierto , Humanos , Predisposición Genética a la Enfermedad , Glaucoma de Ángulo Abierto/genética , Población Negra/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. METHODS: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. RESULTS: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2ß from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. CONCLUSION: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.
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Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Osteogénesis/efectos de los fármacos , Fragmentos de Péptidos/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Línea Celular , Femenino , Sistema de Señalización de MAP Quinasas , Ratones , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoporosis/fisiopatología , Fragmentos de Péptidos/farmacología , Transducción de Señal , Proteínas Smad Reguladas por Receptores/metabolismoRESUMEN
Piezo channels are associated with neuropathology in diseases like traumatic brain injury and glaucoma, but pathways linking tissue stretch to aberrant neural signaling remain unclear. The present study demonstrates that Piezo1 activation increases action potential frequency in response to light and the spontaneous dark signal from mouse retinal explants. Piezo1 stimulation was sufficient to increase cytoplasmic Ca 2+ in soma and neurites, while stretch increased spiking activity in current clamp recordings from of isolated retinal ganglion cells (RGCs). Axon-marker beta-tubulin III colocalized with both Piezo1 and Piezo2 protein in the mouse optic nerve head, while RGC nuclear marker BRN3A colocalized with Piezo channels in the soma. Piezo1 was also present on GFAP-positive regions in the optic nerve head and colocalized with glutamine synthetase in the nerve fiber layer, suggesting expression in optic nerve head astrocytes and Müller glia end feet, respectively. Human RGCs from induced pluripotent stem cells also expressed Piezo1 and Piezo2 in soma and axons, while staining patterns in rats resembled those in mice. mRNA message for Piezo1 was greatest in the RPE/choroid tissue, while Piezo2 levels were highest in the optic nerve, with both channels also expressed in the retina. Increased expression of Piezo1 and Piezo2 occurred both 1 and 10 days after a single stretch in vivo; this increase suggests a potential role in rising sensitivity to repeated nerve stretch. In summary, Piezo1 and Piezo2 were detected in the soma and axons of RGCs, and stimulation affected the light-dependent output of RGCs. The rise in RGCs excitability induced by Piezo stimulation may have parallels to the early disease progression in models of glaucoma and other retinal degenerations. Highlights: Activation of Piezo1 excites retinal ganglion cells, paralleling the early neurodegenerative progression in glaucoma mouse models and retinal degeneration.Piezo1 and Piezo2 were expressed in axons and soma of retinal ganglion cells in mice, rats, and human iPSC-RGCs.Functional assays confirmed Piezo1 in soma and neurites of neurons. Sustained elevation of Piezo1 and Piezo2 occurred after a single transient stretch may enhance damage from repeated traumatic nerve injury.
RESUMEN
Glaucoma is an optic neuropathy characterized by permanent visual field loss caused by the death of retinal ganglion cells (RGCs) and it is the leading cause of irreversible blindness worldwide. Consequently, there is an unmet need for the development of new strategies for its treatment. We investigated RGC replacement therapy as a treatment for ganglion cell loss. Human-induced pluripotent stem cells (hiPSCs) were differentiated into mature, functional RGCs in vitro, labeled with AAV2.7m8-SNCG-eGFP, and transplanted intravitreally in wild-type 4-month-old C57BL/6J mice. Survival of the transplanted hiPSC-RGCs was assessed by color fundus photography and histological studies confirmed the localization of the transplanted hiPSC-RGCs within the retina. Two-photon live imaging of retinal explants and electrophysiological studies confirmed that the morphology and function of the transplanted hiPSC-RGCs were similar to native RGCs. These experiments will provide key strategies to enhance the efficiency of stem cell replacement therapy for neurodegenerative diseases, including glaucoma.
RESUMEN
We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3' V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.
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Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Células Ganglionares de la Retina/citología , Análisis de la Célula Individual/métodos , Diferenciación Celular , Células Cultivadas , Mapeo Cromosómico , Citometría de Flujo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Madre Pluripotentes Inducidas/química , Células Madre Pluripotentes Inducidas/citología , Células Ganglionares de la Retina/química , Análisis de Secuencia de ARN/métodosRESUMEN
Quantum Dot®s (QDot®s) are novel, semi-conductive nanostructures that emit a certain fluorescence when excited by specific wavelengths. QDot®s are more photostable, brighter, and photobleach less than other fluorescent dyes. These characteristics give them the potential to be used in many biological applications. The shells of QDot®s are coated with functional groups, such as carboxylate and organic groups, allowing them to couple to peptides/proteins and be used for real-time imaging and high-resolution microscopy. Here, we utilize Quantum Dot®s and Bone Morphogenetic Protein-2 (BMP-2) to create a BMP-2-QDot®s conjugate. BMP-2 is a growth factor that drives many processes such as cardiogenesis, neural growth, and osteogenesis. Despite its numerous roles, the trafficking and uptake of BMP-2 into cells is not well-established, especially during progression of diseases. The results presented here demonstrate for the first time a fluorescent BMP-2 analog that binds to the BMP-receptors (BMPRs), remains biologically active, and is stable for long time periods. Previous attempts to develop a biological BMP-2 analog with Fluorescein isothiocyanate (FITC) or nanodiamonds lacked data on the analog's stability. Furthermore, these analogs did not address whether they can signal within the cell by binding to the BMPRs or were mediated by non-stable conjugates.
RESUMEN
Glaucoma is a group of progressive optic neuropathies that share common biological and clinical characteristics including irreversible changes to the optic nerve and visual field loss caused by the death of retinal ganglion cells (RGCs). The loss of RGCs manifests as characteristic cupping or optic nerve degeneration, resulting in visual field loss in patients with Glaucoma. Published studies on in vitro RGC differentiation from stem cells utilized classical RGC signaling pathways mimicking retinal development in vivo. Although many strategies allowed for the generation of RGCs, increased variability between experiments and lower yield hampered the cross comparison between individual lines and between experiments. To address this critical need, we developed a reproducible chemically defined in vitro methodology for generating retinal progenitor cell (RPC) populations from iPSCs, that are efficiently directed towards RGC lineage. Using this method, we reproducibly differentiated iPSCs into RGCs with greater than 80% purity, without any genetic modifications. We used small molecules and peptide modulators to inhibit BMP, TGF-ß (SMAD), and canonical Wnt pathways that reduced variability between iPSC lines and yielded functional and mature iPSC-RGCs. Using CD90.2 antibody and Magnetic Activated Cell Sorter (MACS) technique, we successfully purified Thy-1 positive RGCs with nearly 95% purity.
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Diferenciación Celular/efectos de los fármacos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Ganglionares de la Retina/citología , Células Ganglionares de la Retina/metabolismo , Proteínas Smad/antagonistas & inhibidores , Proteínas Wnt/antagonistas & inhibidores , Biología Computacional , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Inmunofenotipificación , Neurogénesis , Retina/citología , Transducción de SeñalRESUMEN
Osteoporosis is a debilitating skeletal disorder that is characterized by loss of bone densityover time. It affects one in two women and one in four men, age 50 and older. New treatmentsthat specifically drive bone formation are desperately needed. We developed a peptide, CK2.3, thatacts downstream of the bone morphogenetic protein receptor type Ia and it induces osteogenesisin-vitro and in-vivo. However, its mechanism of action, especially its mode of uptake by cellsremains unknown. To demonstrate CK2.3 internalization within a cell, we conjugated CK2.3to Quantum Dot®s (Qdot®s), semiconductor nanoparticles. We purified CK2.3-Qdot®s by sizeexclusion chromatography and verified the conjugation and stability using UV/VIS and Fouriertransform infrared spectroscopy. Our results show that CK2.3 was conjugated to the Qdot®s andthe conjugate was stable for at least 4 days at 37 °C. Moreover, CK2.3-Qdot®s exerted biologicalresponse similar to CK2.3. Addition of CK2.3-Qdot®s to cells followed by confocal imaging revealedthat CK2.3-Qdot®s were internalized at 6 h post stimulation. Furthermore, using pharmacologicalinhibitors against endocytic pathways, we demonstrated that CK2.3-Qdot®s were internalized bycaveolae. These results show for the first time that the novel peptide CK2.3 is taken up by the cellthrough caveolae mediated endocytosis.