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Retinal pigment epithelial cell necroptosis in response to sodium iodate

View Article: PubMed Central - PubMed

ABSTRACT

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly in developed countries. The late stage of dry AMD, or geographic atrophy (GA), is characterized by extensive retinal pigment epithelium (RPE) degeneration. The underlying molecular mechanism for RPE cell death in GA remains unclear. Our previous study has established that RPE cells die predominantly from necroptosis in response to oxidative stress in vitro. Here, we extend our study and aim to characterize the nature of RPE cell death in response to sodium iodate (NaIO3) in vitro and in a NaIO3-induced retina degeneration mouse model. We found that NaIO3 induces RPE necroptosis in vitro by using a combination of molecular hallmarks. By using TUNEL assays, active caspase-3 and HMGB1 immunostaining, we confirmed that photoreceptor cells die mainly from apoptosis and RPE cells die mainly from necroptosis in response to NaIO3in vivo. RPE necroptosis in this model is also supported by use of the RIPK1 inhibitor, Necrostatin-1. Furthermore, using novel RIPK3-GFP transgenic mouse lines, we detected RIPK3 aggregation, a hallmark of necroptosis, in the RPE cells in vivo after NaIO3 injection. Our findings suggest the necessity of re-evaluating RPE cell death mechanism in AMD models and have the potential to influence therapeutic development for dry AMD, especially GA.

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Related in: MedlinePlus

Molecular hallmarks of RPE degeneration in C57BL/6J mice RPE after retro-orbital NaIO3 administration. (a–d) Tight junctions in the central retina were visualized by ZO-1 staining. At 48 h post 20 mg/kg NaIO3 administration, compromise of tight junctions was visible indicating impairment of the blood–retina barrier (n=6 each). (e–h) Flatmount DAPI and PI staining highlighted necrotic RPE cells as early as 24 h post NaIO3 administration. At 72 h, areas with diffused PI signal were seen (n=6 each). (i–l) TUNEL staining was used to highlight dying cells in the retina. Both RPE cells and photoreceptors were TUNEL-positive as early as 24 h post 20 mg/kg NaIO3 administration (n=6 each). (m–p) Active caspase-3 staining was observed only in the photoreceptor layer at all analyzed time points (n=5 each). The scale bar is 25 μM.
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fig3: Molecular hallmarks of RPE degeneration in C57BL/6J mice RPE after retro-orbital NaIO3 administration. (a–d) Tight junctions in the central retina were visualized by ZO-1 staining. At 48 h post 20 mg/kg NaIO3 administration, compromise of tight junctions was visible indicating impairment of the blood–retina barrier (n=6 each). (e–h) Flatmount DAPI and PI staining highlighted necrotic RPE cells as early as 24 h post NaIO3 administration. At 72 h, areas with diffused PI signal were seen (n=6 each). (i–l) TUNEL staining was used to highlight dying cells in the retina. Both RPE cells and photoreceptors were TUNEL-positive as early as 24 h post 20 mg/kg NaIO3 administration (n=6 each). (m–p) Active caspase-3 staining was observed only in the photoreceptor layer at all analyzed time points (n=5 each). The scale bar is 25 μM.

Mentions: To dissect the molecular changes in the retina after retro-orbital NaIO3 administration, tight junctions were visualized by immunostaining with antibody to ZO-1, a tight junction-associated protein. NaIO3 administration resulted in the loss of RPE monolayer properties as visualized by ZO-1 staining, at 48 h after NaIO3 injection, indicating a compromise of the blood–retina barrier (Figure 3a–d).


Retinal pigment epithelial cell necroptosis in response to sodium iodate
Molecular hallmarks of RPE degeneration in C57BL/6J mice RPE after retro-orbital NaIO3 administration. (a–d) Tight junctions in the central retina were visualized by ZO-1 staining. At 48 h post 20 mg/kg NaIO3 administration, compromise of tight junctions was visible indicating impairment of the blood–retina barrier (n=6 each). (e–h) Flatmount DAPI and PI staining highlighted necrotic RPE cells as early as 24 h post NaIO3 administration. At 72 h, areas with diffused PI signal were seen (n=6 each). (i–l) TUNEL staining was used to highlight dying cells in the retina. Both RPE cells and photoreceptors were TUNEL-positive as early as 24 h post 20 mg/kg NaIO3 administration (n=6 each). (m–p) Active caspase-3 staining was observed only in the photoreceptor layer at all analyzed time points (n=5 each). The scale bar is 25 μM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4979458&req=5

fig3: Molecular hallmarks of RPE degeneration in C57BL/6J mice RPE after retro-orbital NaIO3 administration. (a–d) Tight junctions in the central retina were visualized by ZO-1 staining. At 48 h post 20 mg/kg NaIO3 administration, compromise of tight junctions was visible indicating impairment of the blood–retina barrier (n=6 each). (e–h) Flatmount DAPI and PI staining highlighted necrotic RPE cells as early as 24 h post NaIO3 administration. At 72 h, areas with diffused PI signal were seen (n=6 each). (i–l) TUNEL staining was used to highlight dying cells in the retina. Both RPE cells and photoreceptors were TUNEL-positive as early as 24 h post 20 mg/kg NaIO3 administration (n=6 each). (m–p) Active caspase-3 staining was observed only in the photoreceptor layer at all analyzed time points (n=5 each). The scale bar is 25 μM.
Mentions: To dissect the molecular changes in the retina after retro-orbital NaIO3 administration, tight junctions were visualized by immunostaining with antibody to ZO-1, a tight junction-associated protein. NaIO3 administration resulted in the loss of RPE monolayer properties as visualized by ZO-1 staining, at 48 h after NaIO3 injection, indicating a compromise of the blood–retina barrier (Figure 3a–d).

View Article: PubMed Central - PubMed

ABSTRACT

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly in developed countries. The late stage of dry AMD, or geographic atrophy (GA), is characterized by extensive retinal pigment epithelium (RPE) degeneration. The underlying molecular mechanism for RPE cell death in GA remains unclear. Our previous study has established that RPE cells die predominantly from necroptosis in response to oxidative stress in vitro. Here, we extend our study and aim to characterize the nature of RPE cell death in response to sodium iodate (NaIO3) in vitro and in a NaIO3-induced retina degeneration mouse model. We found that NaIO3 induces RPE necroptosis in vitro by using a combination of molecular hallmarks. By using TUNEL assays, active caspase-3 and HMGB1 immunostaining, we confirmed that photoreceptor cells die mainly from apoptosis and RPE cells die mainly from necroptosis in response to NaIO3in vivo. RPE necroptosis in this model is also supported by use of the RIPK1 inhibitor, Necrostatin-1. Furthermore, using novel RIPK3-GFP transgenic mouse lines, we detected RIPK3 aggregation, a hallmark of necroptosis, in the RPE cells in vivo after NaIO3 injection. Our findings suggest the necessity of re-evaluating RPE cell death mechanism in AMD models and have the potential to influence therapeutic development for dry AMD, especially GA.

No MeSH data available.


Related in: MedlinePlus