Dynamic in vivo quantification of rod photoreceptor degeneration using fluorescent reporter mouse models of retinitis pigmentosa

AbstractRetinitis pigmentosa (RP) is an inherited retinal disease, affecting >20 million people worldwide. Loss of daylight vision typically occurs due to the dysfunction/loss of cone photoreceptors, the cell type that initiates our color and high acuity vision. Currently, there is no effective treatment for RP, other than gene therapy for a limited number of specific disease genes. To develop a gene-agnostic therapy, we screened ≈20 genes for their ability to prolong cone photoreceptor survival in vivo. Here, we report an adeno-associated virus (AAV) vector expressing Txnip, which prolongs the survival of cone photoreceptors and improves visual acuity in RP mouse models. A Txnip allele, C247S, which blocks the association of Txnip with thioredoxin, provides an even greater benefit. Additionally, the rescue effect of Txnip depends on lactate dehydrogenase b (Ldhb), and correlates with the presence of healthier mitochondria, suggesting that Txnip saves RP cones by enhancing their lactate catabolism.

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Retinitis Pigmentosa Results in Neurodegeneration Concomitant With Neuroinflammation, Extracellular Matrix Disorganization and the Upregulation of Neuroprotective Pathways in Glial Cells and Neurons

10.21203/rs.3.rs-101559/v1 ◽

2020 ◽

Author(s):

Christina B. Bielmeier ◽

Saskia Roth ◽

Sabrina I. Schmitt ◽

Stefaniya K. Boneva ◽

Anja Schlecht ◽

...

Keyword(s):

Extracellular Matrix ◽

Retinitis Pigmentosa ◽

Retinal Degeneration ◽

Molecular Mechanisms ◽

Genetic Model ◽

Immunofluorescent Staining ◽

System Response ◽

Photoreceptor Degeneration ◽

The Impact

Abstract BackgroundHereditary retinal degenerations like retinitis pigmentosa (RP) are amongst the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. Here, we in-depth characterized the transgenic VPP mouse model, a genetic model for autosomal dominant RP. MethodsWe examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescent staining and 3D reconstruction. ResultsOne month-old VPP mice showed a significantly higher number of apoptotic photoreceptor cells that resulted in a significantly thinner ONL in three months-old VPP mice, concomitant with an increase in reactivity of microglia and Müller cells. By RNASeq analysis we identified 9,256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, amongst others, dysregulation of TGF-β regulated extracellular matrix organization, factors of the (ocular) immune system/response and apoptosis. ConclusionThe predominant effect pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated and VEGF signaling that were significantly associated with the VPP transgene-induced photoreceptor degeneration. Thus, modulation of these processes might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.

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Evaluation of BMP-2 Mediated Bone Formation Using Enzymatically Crosslinkable Injectable Hydrogels: An In Vivo Study Using Transgenic Fluorescent Reporter Mouse Model

Healthcare Engineering ◽

10.1007/978-981-10-3111-3_8 ◽

2016 ◽

pp. 49-52

Author(s):

Shalini V. Gohil ◽

Lakshmi S. Nair

Keyword(s):

Mouse Model ◽

Bone Formation ◽

In Vivo Study ◽

Injectable Hydrogels ◽

Fluorescent Reporter ◽

Reporter Mouse

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In Vivo CRISPR/Cas9-Mediated Genome Editing Mitigates Photoreceptor Degeneration in a Mouse Model of X-Linked Retinitis Pigmentosa

Investigative Opthalmology & Visual Science ◽

10.1167/iovs.61.4.31 ◽

2020 ◽

Vol 61 (4) ◽

pp. 31 ◽

Cited By ~ 1

Author(s):

Shuang Hu ◽

Juan Du ◽

Ningning Chen ◽

Ruixuan Jia ◽

Jinlu Zhang ◽

...

Keyword(s):

Mouse Model ◽

Retinitis Pigmentosa ◽

Genome Editing ◽

Photoreceptor Degeneration

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New In Vitro Cellular Model for Molecular Studies of Retinitis Pigmentosa

International Journal of Molecular Sciences ◽

10.3390/ijms22126440 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6440

Author(s):

Li Huang ◽

Meltem Kutluer ◽

Elisa Adani ◽

Antonella Comitato ◽

Valeria Marigo

Keyword(s):

Cell Death ◽

Retinitis Pigmentosa ◽

Leucine Zipper ◽

Target Genes ◽

Cyclic Guanosine Monophosphate ◽

Guanosine Monophosphate ◽

Rod Photoreceptor ◽

Photoreceptor Degeneration ◽

Molecular Studies

Retinitis pigmentosa (RP) is an inherited form of retinal degeneration characterized by primary rod photoreceptor cell death followed by cone loss. Mutations in several genes linked to the disease cause increased levels of cyclic guanosine monophosphate (cGMP) and calcium ion influxes. The purpose of this project was to develop a new in vitro photoreceptor degeneration model for molecular studies of RP. 661W cells were genetically modified to stably express the neural retina leucine zipper (NRL) transcription factor. One clone (661W-A11) was selected based on the expression of Nrl target genes. 661W-A11 showed a significant increase in expression of rod-specific genes but not of cone-specific genes, compared with 661W cells. Zaprinast was used to inhibit phosphodiesterase 6 (PDE6) activity to mimic photoreceptor degeneration in vitro. The activation of cell death pathways resulting from PDE6 inhibition was confirmed by detection of decreased viability and increased intracellular cGMP and calcium, as well as activation of protein kinase G (PKG) and calpains. In this new in vitro system, we validated the effects of previously published neuroprotective drugs. The 661W-A11 cells may serve as a new model for molecular studies of RP and for high-throughput drug screening.

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Complement C3- and CR3-dependent microglial clearance protects photoreceptors in retinitis pigmentosa

10.1101/504647 ◽

2018 ◽

Author(s):

Sean M. Silverman ◽

Wenxin Ma ◽

Xu Wang ◽

Lian Zhao ◽

Wai T. Wong

Keyword(s):

Retinitis Pigmentosa ◽

Complement Activation ◽

Complement C3 ◽

Photoreceptor Degeneration ◽

Therapeutic Approaches ◽

Complement Components ◽

Genetic Ablation ◽

Microglial Phagocytosis

AbstractComplement activation has been implicated as an inflammatory driver of neurodegeneration in retinal and brain pathologies. However, its involvement and influence of photoreceptor degeneration in retinitis pigmentosa (RP), an inherited, largely incurable blinding disease, is unclear. We discover that markedly upregulated retinal expression of multiple complement components coincided spatiotemporally with photoreceptor degeneration in both the rd10 mouse model and in human specimens of RP, with increased complement C3 expression and activation localizing to infiltrating microglia near photoreceptors. Genetic ablation of C3 in the rd10 background resulted in accelerated structural and functional photoreceptor degeneration and altered retinal expression of inflammatory genes. These effects were phenocopied by the genetic deletion of CR3, a microglia-expressed receptor for the C3 activation product C3b, implicating an adaptive microglial-mediation mechanism involving C3-CR3 interaction. Deficiency of either C3 or CR3 resulted in deficient microglial phagocytosis of apoptotic photoreceptors in vivo, as well as increased microglial neurotoxicity to photoreceptors in vitro. These findings demonstrate a novel adaptive role for complement activation in RP that facilitates microglial clearance of apoptotic photoreceptors, without which increased proinflammatory microglial neurotoxicity ensues. These positive contributions of complement via microglial-mediated mechanisms are important in the design of immunomodulatory therapeutic approaches to neurodegeneration.One Sentence SummaryComplement activation mediates adaptive neuroprotection for photoreceptors by facilitating C3-CR3 dependent microglial clearance of apoptotic cells.

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