Independent degeneration of photoreceptors and retinal pigment epithelium in conditional knockout mouse models of choroideremia.
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ABSTRACT:
SUBMITTER: Tolmachova T
PROVIDER: S-EPMC1326146 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Retinal degenerative diseases are generally characterized by a permanent loss of light-sensitive retinal neurons known as photoreceptors, or their support cells, the retinal pigmented epithelium (RPE). Metabolic dysfunction has been implicated as a common mechanism of degeneration. In this study, we used the drug metformin in a gain-of-function approach to activate adenosine monophosphate-activated protein kinase (AMPK). We found that treatment protected photoreceptors and the RPE from acute injury and delayed inherited retinal degeneration. Protection was associated with decreased oxidative stress, decreased DNA damage, and increased mitochondrial energy production. To determine whether protection was a local or a systemic effect of metformin, we used AMPK retinal knockout mice and found that local expression of AMPK catalytic subunit ?2 was required for metformin-induced protection. Our data demonstrate that increasing the activity of AMPK in retinal neurons or glia can delay or prevent degeneration of photoreceptors and the RPE from multiple types of cell-death triggers.
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Project description:The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.
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Project description:The retina is the light sensing tissue of the eye which contains multiple layers of cells required for the detection and transmission of a visual signal. Loss of the light-sensing photoreceptors leads to defects in visual function and blindness. Previously, we found that mosaic deletion of Kcnj13, and subsequent loss of the potassium channel Kir7.1, in mice leads to photoreceptor degeneration and recapitulates the human retinal disease phenotype (Zhong et al., 2015). Kcnj13 expression in the retinal pigment epithelium (RPE) is essential for normal retinal electrophysiology, function, and survival. Mice with homozygous loss of Kcnj13 die at postnatal day 1 (P1), requiring a tissue-specific approach to study retinal degeneration phenotypes in adult mice. We used the CRISPR-Cas9 system to generate a floxed, conditional loss-of-function (cKO) Kcnj13flox allele to study the pathogenesis of Kcnj13 deficiency in the retina. To investigate if the Kcnj13 is required in the RPE for photoreceptor function and survival, we used Best1-cre, which is specifically expressed in the RPE. We observed complete loss of Kcnj13 expression in Cre-positive RPE cells. Furthermore, our findings show that widespread loss of Kcnj13 in the RPE leads to severe and progressive thinning of the outer nuclear layer and a reduced response to light. Finally, to detect Best1-cre expression in the RPE of live animals without sacrificing the animal for histology, we generated a Cre-reporter-containing Kcnj13 cKO mouse line (cKOR: Kcnj13flox/flox; Best1-cre; Ai9) which can be rapidly screened using retinal fluorescence microscopy. These findings provide new tools for studying the roles of Kcnj13 in retinal homeostasis.
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Project description:BackgroundAge-related macular degeneration (AMD) is a result of degeneration/damage of the retinal pigment epithelium (RPE) while retinitis pigmentosa (RP), an inherited early-onset disease, results from premature loss of photoreceptors. A promising therapeutic approach for both is the replacement of lost/damaged cells with human induced pluripotent stem cell (hiPSC)-derived retinal cells.MethodsThe aim of this study was to investigate the in vivo functionality of RPE and photoreceptor progenitor (PRP) cells derived from a clinical-grade hiPSC line through a unified protocol. De novo-generated RPE and PRP were characterized extensively to validate their identity, purity, and potency.ResultsRPE expressed tight junction proteins, showed pigmentation and ciliation, and secreted polarization-related factors vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PRP expressed neural retina proteins and cone and rod markers, and responded to KCl-induced polarization. Transcriptomic analysis demonstrated an increase in the expression of mature retinal tissue-specific genes coupled with concomitant downregulation of genes from undesired lineages. RPE transplantation rescued visual function in RCS rats shown via optokinetic tracking and photoreceptor rescue. PRP transplantation improved light perception in NOD.SCID-rd1 mice, and positive electroretinography signals indicated functional photoreceptor activity in the host's outer nuclear layer. Graft survival and integration were confirmed using immunohistochemistry, and no animals showed teratoma formation or any kind of ectopic growth in the eye.ConclusionsTo our knowledge, this is the first demonstration of a unified, scalable, and GMP-adaptable protocol indicating strong animal efficacy and safety data with hiPSC-derived RPE and PRP cells. These findings provide robust proof-of-principle results for IND-enabling studies to test these potential regenerative cell therapies in patients.
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Project description:Antibody-mediated rejection is characterized by donor-specific antibody produced by B cells. However, to our knowledge, B cell invasion and antibody in the inflamed retina after transplantation of retinal pigment epithelial (RPE) cells has not been reported. To determine if RPE transplantation could be performed using allografts, we established in vivo immune rejection models with induced pluripotent stem cell (iPSC)-RPE allografts and determined whether RPE-specific antibody could be detected in these models. We detected alloantibodies in the serum from recipient monkeys that had immune attacks in the retina in an immunofluorescent assay using the transplanted iPSC-RPE cells as the antigen. In addition to T cell and antigen-presenting cell immunity, peripheral blood cells and lymph nodes in animal models with allogeneic iPSC-RPE cells also had activated B cells, which were probably secreting alloantibodies. Using serum and transplanted cells, alloreactive antibody can be detected for the diagnosis of immune rejection after transplantation.
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Project description:Retinal pigment epithelial (RPE) degeneration is potentially involved in the pathogenesis of several retinal degenerative diseases. mTORC1 signaling is shown as a crucial regulator of many biological processes and disease progression. In this study, we aimed at investigating the role of mTORC1 signaling in RPE degeneration. Methods: Western blots were conducted to detect mTORC1 expression pattern during RPE degeneration. Cre-loxP system was used to generate RPE-specific mTORC1 activation mice. Fundus, immunofluorescence staining, transmission electron microscopy, and targeted metabolomic analysis were conducted to determine the effects of mTORC1 activation on RPE degeneration in vivo. Electroretinography, spectral-domain optical coherence tomography, and histological experiments were conducted to determine the effects of mTORC1 activation on choroidal and retinal function in vivo. Results: RPE-specific activation of mTORC1 led to RPE degeneration as shown by the loss of RPE-specific marker, compromised cell junction integrity, and intracellular accumulation of lipid droplets. RPE degeneration further led to abnormal choroidal and retinal function. The inhibition of mTORC1 signaling with rapamycin could partially reverse RPE degeneration. Targeted metabolomics analysis further revealed that mTORC1 activation affected the metabolism of purine, carboxylic acid, and niacin in RPE. Conclusion: This study revealed that abnormal activation of mTORC1 signaling leads to RPE degeneration, which could provide a promising target for the treatment of RPE dysfunction-related diseases.
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Project description:The mammalian retina consists of multiple cell layers including photoreceptor cells, which are light sensing neurons that play essential functions in the visual process. Previously, we identified mutations in SPATA7, encoding spermatogenesis associated protein 7, in families with Leber Congenital Amaurosis (LCA) and juvenile Retinitis Pigmentosa (RP), and showed that Spata7 null mice recapitulate the human disease phenotype of retinal degeneration. SPATA7 is expressed in the connecting cilium of photoreceptor (PR) cells in the mouse retina, as well as in retinal pigment epithelium (RPE) cells, but the functional role of Spata7 in the RPE remains unknown. To investigate whether Spata7 is required in PRs, the RPE, or both, we conditionally knocked out Spata7 in photoreceptors and RPE cells using Crx-Cre and Best1-Cre transgenic mouse lines, respectively. In Spata7 photoreceptor-specific conditional (cKO) mice, both rod and cone photoreceptor dysfunction and degeneration is observed, characterized by progressive thinning of the outer nuclear layer and reduced response to light; however, RPE-specific deletion of Spata7 does not impair retinal function or cell survival. Furthermore, our findings show that both Rhodopsin and RPGRIP1 are mislocalized in the Spata7Flox/-; Crx-Cre cKO mice, suggesting that loss of Spata7 in photoreceptors alone can result in altered trafficking of these proteins in the connecting cilium. Together, our findings suggest that loss of Spata7 in photoreceptors alone is sufficient to cause photoreceptor degeneration, but its function in the RPE is not required for photoreceptor survival; therefore, loss of Spata7 in photoreceptors alters both rod and cone function and survival, consistent with the clinical phenotypes observed in LCA and RP patients with mutations in SPATA7.
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Project description:Age-related macular degeneration (AMD), featured with dysfunction and loss of retinal pigment epithelium (RPE), is lacking efficient therapeutic approaches. According to our previous studies, human amniotic epithelial stem cells (hAESCs) may serve as a potential seed cell source of RPE cells for therapy because they have no ethical concerns, no tumorigenicity, and little immunogenicity. Herein, trichostatin A and nicotinamide can direct hAESCs differentiation into RPE like cells. The differentiated cells display the morphology, marker expression and cellular function of the native RPE cells, and noticeably express little MHC class II antigens and high level of HLA-G. Moreover, visual function and retinal structure of Royal College of Surgeon (RCS) rats, a classical animal model of retinal degeneration, were rescued after subretinal transplantation with the hAESCs-derived RPE like cells. Our study possibly makes some contribution to the resource of functional RPE cells for cell therapy. Subretinal transplantation of hAESCs-RPE could be an optional therapeutic strategy for retinal degeneration diseases.
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Project description:Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented Abca4-/- mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs. These enlarged endosomes internalize the complement protein C3 into the RPE, resulting in the intracellular generation of C3a fragments. Increased C3a in turn activates the mechanistic target of rapamycin (mTOR), a regulator of critical metabolic processes such as autophagy. The antidepressant desipramine, which decreases ceramide levels by inhibiting acid sphingomyelinase, corrects EE defects in the RPE of Abca4-/- mice. This prevents C3 internalization and limits the formation of C3a fragments within the RPE. Although uncontrolled complement activation is associated with macular degenerations, how complement contributes to pathology in a progressive disease is not well understood. Our studies link expansion of the EE compartment with intracellular complement generation and aberrant mTOR activation, which could set the stage for chronic metabolic reprogramming in the RPE as a prelude to disease. The pivotal role of ceramide in driving EE biogenesis and fusion in the Abca4-/- mice RPE suggests that therapeutic targeting of ceramide could be effective in Stargardt disease and other macular degenerations.
| S-EPMC6130344 | biostudies-literature
Project description:Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1?) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.
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