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The Journal of Clinical Investigation Jun 2022Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases....
Once human photoreceptors die, they do not regenerate, thus, photoreceptor transplantation has emerged as a potential treatment approach for blinding diseases. Improvements in transplant organization, donor cell maturation, and synaptic connectivity to the host will be critical in advancing this technology for use in clinical practice. Unlike the unstructured grafts of prior cell-suspension transplantations into end-stage degeneration models, we describe the extensive incorporation of induced pluripotent stem cell (iPSC) retinal organoid-derived human photoreceptors into mice with cone dysfunction. This incorporative phenotype was validated in both cone-only as well as pan-photoreceptor transplantations. Rather than forming a glial barrier, Müller cells extended throughout the graft, even forming a series of adherens junctions between mouse and human cells, reminiscent of an outer limiting membrane. Donor-host interaction appeared to promote polarization as well as the development of morphological features critical for light detection, namely the formation of inner and well-stacked outer segments oriented toward the retinal pigment epithelium. Putative synapse formation and graft function were evident at both structural and electrophysiological levels. Overall, these results show that human photoreceptors interacted readily with a partially degenerated retina. Moreover, incorporation into the host retina appeared to be beneficial to graft maturation, polarization, and function.
Topics: Animals; Ependymoglial Cells; Humans; Induced Pluripotent Stem Cells; Mice; Photoreceptor Cells, Vertebrate; Retina; Retinal Cone Photoreceptor Cells; Retinal Degeneration
PubMed: 35482419
DOI: 10.1172/JCI154619 -
Investigative Ophthalmology & Visual... Nov 2022Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development...
PURPOSE
Cone and rod photoreceptors in the retina convert light to electrical signals which are transmitted to the visual cortex of the brain. Abnormal photoreceptor development and degeneration results in blindness. So far, the mechanism that controls photoreceptor specification and its subsequent fate bifurcation remain elusive.
METHODS
To trace and enrich the human photoreceptor lineage, we first engineered H9 human embryonic stem cell (hESC) reporter line by fusing EGFP to endogenous BLIMP1 using CRISPR/CAS9 gene-editing technology, and then used the cell line to generate 3D retinal organoids. Following EGFP-based cell sorting, single-cell RNA-sequencing was conducted via 10x Genomics Chromium system, and the data were analyzed using Seurat. Immunofluorescence combined with lentivirus-mediated knockdown and overexpression experiments were used as validation approaches.
RESULTS
Single-cell transcriptomic profiling revealed that retinal progenitor cells were temporally programmed to differentiate to cone and rod sequentially. We identified PHLDA1 as a novel regulator of photoreceptor specification. PHLDA1 mediated the effects of IGF1 through IGF1R, and inhibited AKT phosphorylation during photoreceptor development.
CONCLUSIONS
Our data established a transcriptomic cell atlas of the human photoreceptor lineage, and identified IGF1-PHLDA1 axis to regulate human photoreceptor development.
Topics: Humans; Organoids; Retinal Rod Photoreceptor Cells; Transcriptome; Cell Differentiation; Retina; Retinal Cone Photoreceptor Cells; Embryonic Stem Cells; Transcription Factors; Insulin-Like Growth Factor I
PubMed: 36331259
DOI: 10.1167/iovs.63.12.9 -
The Journal of Experimental Biology Apr 2022Retinal bipolar cells receive direct input from rod and cone photoreceptors and send axons into the inner retina, synapsing onto amacrine and ganglion cells. Bipolar...
Retinal bipolar cells receive direct input from rod and cone photoreceptors and send axons into the inner retina, synapsing onto amacrine and ganglion cells. Bipolar cell responses can be either depolarizing (ON) or hyperpolarizing (OFF); in lower vertebrates, bipolar cells receive mixed rod and cone input, whereas in mammals, input is mostly segregated into 14 classes of cone ON and OFF cells and a single rod ON bipolar cell. We show that lamprey, like mammals, have rod bipolar cells with little or no cone input, but these cells are OFF rather than ON. They have a characteristic morphology and a spectral sensitivity nearly indistinguishable from that of rod photoreceptors. In background light known to saturate rods, rod bipolar cells are also saturated and cannot respond to increment flashes. Our results suggest that early vertebrate progenitors of both agnathans and gnathostomes may have had a more fluid retinal organization than previously thought.
Topics: Animals; Mammals; Petromyzon; Retina; Retinal Bipolar Cells; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Vertebrates
PubMed: 35319772
DOI: 10.1242/jeb.243949 -
Biochimica Et Biophysica Acta.... Aug 2022Reversible phosphorylation of phosphatidylinositol by phosphoinositide (PI) kinases and phosphatases generates seven distinct phosphoinositide phosphates, called...
UNLABELLED
Reversible phosphorylation of phosphatidylinositol by phosphoinositide (PI) kinases and phosphatases generates seven distinct phosphoinositide phosphates, called phosphoinositides or PIPs. All seven PIPs are formed in the retina and photoreceptor cells. Around 50 genes in the mammalian genome encode PI kinases and PI phosphatases. There are no studies available on the distribution of these enzymes in the retina and photoreceptors.
AIM
To employ Ribosomal Targeting Strategy and Nuclear Labeling to Analyze Phosphoinositide Signatures in rod-photoreceptor cells.
METHODS
HA-tagging of ribosomal protein Rpl22 was induced with Cre-recombinase under the control of the rhodopsin promoter. Actively translating mRNAs associated with polyribosomes were isolated by immunoprecipitation with HA antibody, followed by RNA isolation and gene identification. We also isolated biotinylated-rod nuclei from NuTRAP mice under the control of the rhodopsin-Cre promoter and analyzed nuclear phosphoinositides.
RESULTS
Our results indicate that the expression of class I and class III PI 3-kinase, PI4K IIIβ, PI 5-kinase, PIKfyve, PI3-phosphatases, MTMR2, 4, 6, 7, 14, PI4-phosphatase, TMEM55A, PI 5-phosphatases, SYNJI, INPP5B, INPP5E, INPP5F, SKIP and other phosphatases with dual substrate specificity, PTPMT1, SCAM1, and FIG4 are highly enriched in rod photoreceptor cells compared with the retina and cone-like retina. Our analysis identified the presence of PI(4)P, PI(3,4)P, PI(3,5)P, and PI(4,5)P in the rod nuclei.
CONCLUSIONS
Our studies for the first time demonstrate the expression of PI kinases, PI phosphatases, and nuclear PIPs in rod photoreceptor cells. The NuTRAP mice may be useful not only for epigenetic and transcriptomic studies but also for in vivo cell-specific lipidomics research.
Topics: 1-Phosphatidylinositol 4-Kinase; Animals; Flavoproteins; Mice; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphoinositide Phosphatases; Phosphoric Monoester Hydrolases; Photoreceptor Cells; Protein Tyrosine Phosphatases, Non-Receptor; Rhodopsin; Ribosomes
PubMed: 35427794
DOI: 10.1016/j.bbalip.2022.159161 -
Development (Cambridge, England) Dec 2019As with all glial cells, the major role of retinal Müller glia (MG) is to provide essential neuronal support. However, the MG of some non-mammalian species have the... (Review)
Review
As with all glial cells, the major role of retinal Müller glia (MG) is to provide essential neuronal support. However, the MG of some non-mammalian species have the additional ability to generate new retinal neurons capable of sight restoration. Unfortunately, mammalian MG do not possess this ability. However, if we could understand the reasons why, we may be able to devise strategies to confer regenerative potential. The recent discovery that the Hippo signaling pathway acts as an intrinsic block to mammalian MG proliferation, along with reports of adeno-associated virus (AAV)-based MG reprogramming and functional photoreceptor differentiation, may indicate a watershed moment in the field of mammalian retinal regeneration. However, as researchers delve deeper into the cellular and molecular mechanisms, and further refine MG reprogramming strategies, we should recall past misinterpretations of data in this field and proceed with caution. Here, we provide a summary of these emerging data and a discussion of technical concerns specific to AAV-mediated reprogramming experiments that must be addressed in order for the field to move forward.
Topics: Animals; Cell Proliferation; Cellular Reprogramming; Cellular Reprogramming Techniques; Dependovirus; Ependymoglial Cells; Genetic Vectors; Humans; Photoreceptor Cells, Vertebrate; Regeneration
PubMed: 31792065
DOI: 10.1242/dev.182642 -
Acta Pharmacologica Sinica Nov 2020Photoreceptors, as polarised sensory neurons, are essential for light sensation and phototransduction, which are highly dependent on the photoreceptor cilium. Structural... (Review)
Review
Photoreceptors, as polarised sensory neurons, are essential for light sensation and phototransduction, which are highly dependent on the photoreceptor cilium. Structural defects and/or dysfunction of the photoreceptor cilium caused by mutations in photoreceptor-specific genes or common ciliary genes can lead to retinal diseases, including syndromic and nonsyndromic diseases. In this review, we describe the structure and function of the photoreceptor cilium. We also discuss recent findings that underscore the dysregulation of the photoreceptor cilium in various retinal diseases and the therapeutic potential of targeting ciliary genes in these diseases.
Topics: Animals; Cilia; Ciliopathies; Eye Proteins; Genetic Therapy; Humans; Mutation; Photoreceptor Cells, Vertebrate; Retinal Diseases; Stem Cell Transplantation
PubMed: 32753732
DOI: 10.1038/s41401-020-0486-3 -
Scientific Reports May 2022NR2E3 is an orphan nuclear receptor whose loss-of-function causes abnormal retinal photoreceptor development and degeneration. However, despite that many nuclear...
NR2E3 is an orphan nuclear receptor whose loss-of-function causes abnormal retinal photoreceptor development and degeneration. However, despite that many nuclear receptors are regulated by binding of small molecule ligands, biological small molecule ligands regulating NR2E3 have not been identified. Identification of an endogenous NR2E3 ligand might reveal a previously unrecognized component contributing to retinal development and maintenance. Here we report that biliverdin, a conserved green pigment from heme catabolism, regulates NR2E3 and is necessary for zebrafish retinal photoreceptor development. Biliverdin from retinal extracts specifically bound to NR2E3's ligand-binding domain and induced NR2E3-dependent reporter gene expression. Inhibition of biliverdin synthesis decreased photoreceptor cell populations in zebrafish larvae, and this phenotype was alleviated by exogenously supplied biliverdin. Thus, biliverdin is an endogenous small molecule ligand for NR2E3 and a component necessary for the proper development of photoreceptor cells. This result suggests a possible role of heme metabolism in the regulation of retinal photoreceptor cell development.
Topics: Animals; Biliverdine; Heme; Ligands; Orphan Nuclear Receptors; Photoreceptor Cells; Photoreceptor Cells, Vertebrate; Receptors, Cytoplasmic and Nuclear; Retinal Degeneration; Zebrafish; Zebrafish Proteins
PubMed: 35508617
DOI: 10.1038/s41598-022-11502-3 -
European Review For Medical and... Nov 2021Cytoplasmic dynein is a multi-subunit complex that includes cytoplasmic dynein-1 (dynein1) and cytoplasmic dynein-2 (dynein2). It participates in various basic cellular... (Review)
Review
Cytoplasmic dynein is a multi-subunit complex that includes cytoplasmic dynein-1 (dynein1) and cytoplasmic dynein-2 (dynein2). It participates in various basic cellular processes, including nuclear migration, mitotic spindle organization, chromosome separation during mitosis, and the location and function of numerous intracellular organelles. Retinal photoreceptor cells are terminally differentiated neurons that cannot regenerate and cannot be replaced once lost. It is thus crucial to study their development to facilitate the generation and improvement of photoreceptor disease treatments. The outer segment (OS) of photoreceptor cells is a specific sensory cilium. An increasing number of studies have shown that cytoplasmic dynein plays an essential role in the development of retinal photoreceptor cells. To date, people have done a lot of studies on the various functions of dynein in cells and have a very detailed understanding. However, the role of dynein in retinal photoreceptor cells has not been summarized in detail. This article summarizes the currently available knowledge relating to the effects and mechanisms of cytoplasmic dynein on the development and functional maintenance of retinal photoreceptor cells.
Topics: Animals; Cytoplasmic Dyneins; Humans; Photoreceptor Cells, Vertebrate
PubMed: 34787856
DOI: 10.26355/eurrev_202111_27096 -
Stem Cells Translational Medicine Apr 2024Age-related macular degeneration and retinitis pigmentosa are degenerative retinal diseases that cause severe vision loss. Early clinical trials involving... (Review)
Review
Age-related macular degeneration and retinitis pigmentosa are degenerative retinal diseases that cause severe vision loss. Early clinical trials involving transplantation of photoreceptors as treatment for these conditions are underway. In this review, we summarize recent progress in the field of photoreceptor transplantation, including some pertinent results regarding photoreceptor manufacture, photoreceptor transplantation, mechanisms of donor-host cell integration such as material transfer and photoreceptor transplant immunology. We conclude by proposing several approaches that may provide a rational basis for selecting a vision restoration strategy (eg, donor-host synapse formation vs donor-host nanotube formation) and improved transplant efficiency.
Topics: Humans; Retinal Degeneration; Retina; Macular Degeneration; Photoreceptor Cells; Cell- and Tissue-Based Therapy; Stem Cell Transplantation
PubMed: 38417110
DOI: 10.1093/stcltm/szae005 -
Nature Communications Sep 2020Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish an alternative layer of control over these processes, we...
Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish an alternative layer of control over these processes, we generated chimeric regulatory RNAs that interact reversibly and light-dependently with the light-oxygen-voltage photoreceptor PAL. By harnessing this interaction, the function of micro RNAs (miRs) and short hairpin (sh) RNAs in mammalian cells can be regulated in a spatiotemporally precise manner. The underlying strategy is generic and can be adapted to near-arbitrary target sequences. Owing to full genetic encodability, it establishes optoribogenetic control of cell state and physiology. The method stands to facilitate the non-invasive, reversible and spatiotemporally resolved study of regulatory RNAs and protein function in cellular and organismal environments.
Topics: Animals; Gene Expression; HEK293 Cells; Humans; MicroRNAs; Photoreceptor Cells; RNA; RNA, Small Interfering
PubMed: 32973178
DOI: 10.1038/s41467-020-18673-5