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Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

ABSTRACT: Photoreceptor death contributes to 50% of irreversible vision loss in the western world. Pro23His (P23H) transgenic albino rat strains are widely used models for the most common rhodopsin gene mutation associated with the autosomal dominant form of retinitis pigmentosa. However, the mechanism(s) by which photoreceptor death occurs are not well understood and were the principal aim of this study. We first used electroretinogram recording and optical coherence tomography to confirm the time course of functional and structural loss. Electroretinogram analyses revealed significantly decreased rod photoreceptor (a-wave), bipolar cell (b-wave) and amacrine cell responses (oscillatory potentials) from P30 onward. The cone-mediated b-wave was also decreased from P30. TUNEL analysis showed extensive cell death at P18, with continued labeling detected until P30. Focused gene expression arrays indicated activation of, apoptosis, autophagy and necroptosis in whole retina from P14-18. However, analysis of mitochondrial permeability changes (??m) using JC-1 dye, combined with immunofluorescence markers for caspase-dependent (cleaved caspase-3) and caspase-independent (AIF) cell death pathways, indicated mitochondrial-mediated cell death was not a major contributor to photoreceptor death. By contrast, reverse-phase protein array data combined with RIPK3 and phospho-MLKL immunofluorescence indicated widespread necroptosis as the predominant mechanism of photoreceptor death. These findings highlight the complexity of mechanisms involved in photoreceptor death in the Pro23His rat model of degeneration and suggest therapies that target necroptosis should be considered for their potential to reduce photoreceptor death.

SUBMITTER: Kakavand K

PROVIDER: S-EPMC7670078 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

Kakavand Kiana K Jobling Andrew I AI Greferath Ursula U Vessey Kirstan A KA de Iongh Robb U RU Fletcher Erica L EL

Frontiers in neuroscience 20201103

Photoreceptor death contributes to 50% of irreversible vision loss in the western world. Pro23His (P23H) transgenic albino rat strains are widely used models for the most common rhodopsin gene mutation associated with the autosomal dominant form of retinitis pigmentosa. However, the mechanism(s) by which photoreceptor death occurs are not well understood and were the principal aim of this study. We first used electroretinogram recording and optical coherence tomography to confirm the time course ...[more]

PMID: 33224023

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Project description:To characterize the optical coherence tomography (OCT) appearances of photoreceptor degeneration in the rhodopsin P23H transgenic rat (line 2) in relation to the histological, ultrastructural, and electroretinography (ERG) findings.Homozygous rhodopsin P23H transgenic albino rats (line 2, very-slow degeneration model) were employed. Using OCT (Micron IV®; Phoenix Research Labs, Pleasanton, CA, USA), the natural course of photoreceptor degeneration was recorded from postnatal day (P) 15 to P 287. The OCT images were qualitatively observed by comparing them to histological and ultrastructural findings at P 62 and P 169. In addition, each retinal layer was quantitatively analyzed longitudinally during degeneration, compared it to that observed in wild type Sprague-Dawley (SD) rats. The relationships between the ERG (full-field combined rod-cone response, 3.0 cds/m2 stimulation) findings and OCT images were also analyzed.In the qualitative study, the two layers presumably corresponding to the photoreceptor inner segment ellipsoid zone (EZ) and interdigitation zone (IZ) were identified in the P23H rat until PN day 32. However, the photoreceptor inner and outer segment (IS/OS) layer became diffusely hyperreflective on OCT after P 46, and the EZ and IZ zones could no longer be identified on OCT. In contrast, in the SD rats, the EZ and IZ were clearly distinguished until at least P 247. The ultrastructural study showed partial disarrangements of the photoreceptor outer segment discs in the P23H rats at P 62, although a light-microscopic histological study detected almost no abnormality in the outer segment. In the quantitative study, the outer retinal layer including the outer plexiform layer (OPL) and the outer nuclear layer (ONL) became significantly thinner in the P23H rats than in the SD rats after P 71. The thickness of the IS/OS layer was maintained in the P23H rats until P 130, and it became statistically thinner than in the SD rats at P 237. The longitudinal attenuation in the amplitude of the a- and b-waves of ERG was significantly correlated with the thickness of the combined OPL and ONL but not with that of the IS/OS layer.OCT showed the degenerated photoreceptor IS/OS layer in rhodopsin P23H transgenic rats (line 2) as a diffuse hyperreflective zone, even in the early stage, with the partially disarranged and destabilized OS discs recognizable by ultrastructural assessment but not by a histological study. The amplitude of the a- and b-waves mainly depends on the thickness of the OPL and ONL layer rather than the thickness of the photoreceptor IS/OS layer in P23H rats.

Project description:BackgroundRetinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2.MethodsAge-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution.FindingsMüller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators.InterpretationCellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.

Project description:The pathophysiology of most neurodegenerative diseases includes aberrant accumulation of protein aggregates. Recent evidence highlights the role of protein degradation pathways in neurodegeneration. Concurrently, genetic tools have been generated to enable zebrafish, Danio rerio, to be used as an animal model to study neurodegenerative processes. In addition to optical clarity and fast ex utero development, the zebrafish brain is relatively small and has conserved structures with its mammalian counterparts. To take advantage of this model organism and to aid further studies on autophagy and neurodegeneration, we created a stable transgenic zebrafish line that expresses eGFP-Map1lc3b specifically in post-mitotic neurons under the elavl3 promoter. This line is useful for indirectly monitoring autophagic activity in neurons in vivo and screening for macroautophagy/autophagy-modulating compounds. We determined the applicability of this transgenic line by modulating and quantifying the number of autophagosomes via treatment with a known autophagy inducer (rapamycin) and inhibitors (3-methyladenine, protease inhibitors). Additionally, we proposed an in vivo method for quantifying rates of autophagosome accumulation, which can be used to infer occurrence of autophagic flux. Last, we tested two FDA-approved drugs currently undergoing clinical studies for Parkinson disease, isradipine and nilotinib, and found that isradipine did not modulate autophagy, whereas nilotinib induced both autophagosome number and autophagic flux. It is hoped that others will find this line useful as an in vivo vertebrate model to find or validate autophagy modulators that might be used to halt the progression of neurodegenerative diseases. Abbreviations: 3MA: 3-methyladenine; BafA: bafilomycin A1; dd: dorsal diencephalon; dpf: days post fertilization; e: eye; eGFP: enhanced green fluorescent protein; Elavl3: ELAV like neuron-specific RNA binding protein 3; FDA: Food and Drug Administration; hb: habenula; hpt, hours post treatment; Map1lc3b: microtubule-associated protein 1 light chain 3 beta; nt: neural tube; ot, optic tectum; P/E: pepstatin A and E64d; PD: Parkinson disease; PMTs: photomultiplier tubes; PTU: 1-phenyl-2-thiourea; Ta: annealing temperature; Tel, telencephalon.

Dataset Information

Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

Publications

Photoreceptor Degeneration in Pro23His Transgenic Rats (Line 3) Involves Autophagic and Necroptotic Mechanisms.

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