Immunomodulation-accelerated neuronal regeneration following selective rod photoreceptor cell ablation in the zebrafish retina

Abstract

Müller glia (MG) function as inducible retinal stem cells in zebrafish, completely repairing the eye after damage. The innate immune system has recently been shown to promote tissue regeneration in which classic wound-healing responses predominate. However, regulatory roles for leukocytes during cellular regeneration-i.e., selective cell-loss paradigms akin to degenerative disease-are less well defined. To investigate possible roles innate immune cells play during retinal cell regeneration, we used intravital microscopy to visualize neutrophil, macrophage, and retinal microglia responses to induced rod photoreceptor apoptosis. Neutrophils displayed no reactivity to rod cell loss. Peripheral macrophage cells responded to rod cell loss, as evidenced by morphological transitions and increased migration, but did not enter the retina. Retinal microglia displayed multiple hallmarks of immune cell activation: increased migration, translocation to the photoreceptor cell layer, proliferation, and phagocytosis of dying cells. To test function during rod cell regeneration, we coablated microglia and rod cells or applied immune suppression and quantified the kinetics of (i) rod cell clearance, (ii) MG/progenitor cell proliferation, and (iii) rod cell replacement. Coablation and immune suppressants applied before cell loss caused delays in MG/progenitor proliferation rates and slowed the rate of rod cell replacement. Conversely, immune suppressants applied after cell loss had been initiated led to accelerated photoreceptor regeneration kinetics, possibly by promoting rapid resolution of an acute immune response. Our findings suggest that microglia control MG responsiveness to photoreceptor loss and support the development of immune-targeted therapeutic strategies for reversing cell loss associated with degenerative retinal conditions. retina | microglia | glucocorticoid | regeneration | macrophage N eurodegenerative diseases are caused by the loss of discrete neuronal cell types. The goal of regenerative medicine is to reverse this process. One strategy for doing so involves harnessing the regenerative potential of endogenous neural stem cells. Unfortunately, although adult neural stem cells exist in the mammalian CNS, innate potentials for neuronal repair remain dormant in the absence of exogenous stimulation. Conversely, many other species are endowed with remarkable capacities for neuronal regeneration. For instance, in the zebrafish eye, retinal Müller glia (MG) cells can dedifferentiate to a stem cell-like state and give rise to progenitors that replace lost neurons and restore visual function (1, 2). Intriguingly, human MG cells are able to give rise to new neurons in culture (3) that can restore visual function when transplanted into mammalian disease models (4), suggesting that human MG retain a stem cell-like capacity for mediating retinal repair. Mechanisms controlling the regenerative potential of MG are therefore of great interest. We and others study zebrafish to learn more about how retinal regeneration is cont...

Large-scale Phenotypic Drug Screen Identifies Neuroprotectants in Zebrafish and Mouse Models of Retinitis Pigmentosa

“…The ARQiv-HTS platform was used to screen compounds for neuroprotective effects in a transgenic zebrafish model of RP, Tg(rho:YFP-Eco.NfsB)gmc500, hereafter, rho:YFP-NTR (Walker et al, 2012;White et al, 2017). In this line, a 3.7 kb rhodopsin (rho) promoter fragment (Hamaoka et al, 2002) drives transgene expression exclusively in rod photoreceptor cells.…”

“…Having extensive MOA data is a key advantage afforded by testing human-approved compounds which we leveraged in a previous large-scale zebrafish PDD screen (G. . Similarly here, as studies have suggested inflammation plays a key role in retinal degeneration and regeneration (Hollyfield et al, 2008;Mitchell et al, 2018;White et al, 2017;Yoshida et al, 2013), hits implicated as modulators of inflammatory signaling were included. In addition, several compounds that did not produce concentrationdependent effects were selected to test whether this criterion was useful in predicting reproducibility.…”

“…Three contiguous image slices were stacked into a single maximal intensity projection image using Image J. As a means of assessing rod photoreceptor survival, intravital confocal retinal images were collected of YFP-expressing rod cells per condition and processed for volumetric quantification of YFP signal volume using automated image analysis software (Imaris 3.9.1, see White et al, 2017). Briefly, YFP-expressing rod cells volumes were automatically rendered using the same parameters across all treatment groups with the sum of all volumes used to estimate rod photoreceptor numbers per each retina.…”

“…Here, to identify neuroprotective compounds promoting rod photoreceptor survival, ARQiv-HTS was used to perform a large-scale chemical screen in an inducible zebrafish model of RP (Walker et al, 2012;White et al, 2017). Close to 3,000 largely human-approved drugs were tested across six concentrations (i.e., using qHTS principles, Inglese et al, 2006) in more than 350,000 zebrafish larvae.…”

miR‐30a‐5p inhibition promotes interaction of Fas+endothelial cells and FasL+microglia to decrease pathological neovascularization and promote physiological angiogenesis

“…An overwhelming amount of literature supports that microglia contribute to pathological changes through secretion of proinflammatory cytokines leading to cytotoxicity (Chen & Xu, 2015;Madeira, Boia, Santos, Ambrosio, & Santiago, 2015;McVicar et al, 2015;Murinello et al, 2014;Perry & Holmes, 2014;Zhang et al, 2012). However, few recent studies have shown that in certain instances, microglia activation can be protective (Chuang et al, 2016;Holtman et al, 2017;Le, Wu, & Tang, 2016;White et al, 2017). With regard to ischemic injury of the CNS, microglia depletion has been shown to induce more severe ischemic neuronal damage (Szalay et al, 2016).…”