Photoreceptor ablation following ATP induced injury triggers Müller glia driven regeneration in zebrafish

https://doi.org/10.1016/j.exer.2021.108569Get rights and content

Highlights

  • Intravitreal injection of adenosine triphosphate (ATP) caused photoreceptor and ganglion cell death in fish.

  • ATP induced Muller glia proliferated three and four days after injection.

  • Regeneration of rod photoreceptors observed 14 days after injection.

Abstract

Retinal regeneration research offers hope to people affected by visual impairment due to disease and injury. Ongoing research has explored many avenues towards retinal regeneration, including those that utilizes implantation of devices, cells or targeted viral-mediated gene therapy. These results have so far been limited, as gene therapy only has applications for rare single-gene mutations and implantations are invasive and in the case of cell transplantation donor cells often fail to integrate with adult neurons. An alternative mode of retinal regeneration utilizes a stem cell population unique to vertebrate retina – Müller glia (MG). Endogenous MG can readily regenerate lost neurons spontaneously in zebrafish and to a very limited extent in mammalian retina. The use of adenosine triphosphate (ATP) has been shown to induce retinal degeneration and activation of the MG in mammals, but whether this is conserved to other vertebrate species including those with higher regenerative capacity remains unknown. In our study, we injected a single dose of ATP intravitreal in zebrafish to characterize the cell death and MG induced regeneration. We used TUNEL labelling on retinal sections to show that ATP caused localised death of photoreceptors and ganglion cells within 24 h. Histology of GFP-transgenic zebrafish and BrdU injected fish demonstrated that MG proliferation peaked at days 3 and 4 post-ATP injection. Using BrdU labelling and photoreceptor markers (Zpr1) we observed regeneration of lost rod photoreceptors at day 14. This study has been undertaken to allow for comparative studies between mammals and zebrafish that use the same specific induction method of injury, i.e. ATP induced injury to allow for direct comparison of across species to narrow down resulting differences that might reflect the differing regenerative capacity. The ultimate aim of this work is to recapitulate pro-neurogenesis Müller glia signaling in mammals to produce new neurons that integrate with the existing retinal circuit to restore vision.

Introduction

Photoreceptors are the light sensing neurons in the eye that initiate our ability to see the world, and without which blindness and vision impairment results. Photoreceptors can be lost due to traumatic injury or diseases such as retinitis pigmentosa or more commonly age-related macular degeneration which is the leading cause of visual impairment in industrialized countries (Bourne et al., 2018). Currently, there are no treatments to cure blindness. There has been however significant progress made towards restoring some sight such as with the use of electronic implants commonly known as the bionic eye (Brandli et al., 2016), viral-mediated gene therapy (Cepko, 2012; Fortuny and Flannery, 2018), artificial nanowire arrays (Tang et al., 2018), exosome delivery to reduce inflammation (Bian et al., 2020) and photoreceptor or, photoreceptor-like cell transplantation (Decembrini et al., 2014; Kruczek et al., 2017; Mahato et al., 2020; Pearson et al., 2012; Singh et al., 2013). Implant and cell transplantation methods particularly require invasive surgery and implantation of foreign substances to the eye or even the brain. Additionally, whilst visual function improvements have been observed after cell transplantation, the integration efficiency varies (Barber et al., 2012) and rather than integrating into existing neural circuits, transplanted cells may transfer proteins and cytoplasmic content to host photoreceptors (Pearson et al., 2016; Santos-Ferreira et al., 2016; Singh et al., 2016). There is an alternative strategy that is being pursued, one that aims to utilize regenerative stem cells found in the vertebrate eye to restore vision.

The resident regenerative stem cell source in retinas are Müller glia. Müller glia are found in all vertebrate species where they contribute to retinal homeostasis, such as recycling neurotransmitters, retinoids and buffering ions (Bringmann et al., 2006). In response to loss of neurons, Müller glia can re-enter the cell cycle to proliferate and produce multipotent progenitors, albeit with varying levels of proficiency across species (Goldman, 2014). In zebrafish Müller glia generously proliferate and produce multipotent progenitors that migrate to the area of damage, differentiate into mature functional neurons, and integrate into visual circuits, thereby repopulating the lost neurons and restoring function (reviewed in Kei et al. (2014), reviewed in (Angueyra and Kindt, 2018). In mammals the Müller glia population faces two barriers to regeneration as they rarely proliferate and rarely become neurogenic (i.e. produce progenitors) (Goldman, 2014). There have been many studies undertaken in zebrafish to identify proliferative and neurogenic associated genes in Müller glia after injury, that regulate the formation of progenitors such as wingless-related integration site (WNT), transforming growth factor beta (TGFb), fibroblast growth factor (FGF) and the hippo pathway (Gallina et al., 2016; Lenkowski et al., 2013; Qin et al., 2011; Rueda et al., 2019). Some of these genes are also found to be upregulated in mammalian Müller glia, demonstrating an underlying and evolutionary conserved regeneration transcriptome in vertebrates (Martin and Poche, 2019). By studying Müller glia in model systems such as zebrafish it is hoped that the underlying genetic blueprint of regeneration could be discovered and then applied to mammals including humans to improve endogenous vision restoration, such as has been demonstrated for key genes (Jorstad et al., 2017).

In the pursuit of understanding the regeneration transcriptomes (and also the epigenomes) of zebrafish and mammals a number of regenerative paradigms have been established in the retina. These include some that cause loss of different neuron types indiscriminately, such as mechanical injury, or those that target specific neural subpopulations either genetically, or chemically (reviewed in Ng et al., 2014). All of these result in efficient neurogenic regeneration, though the neural type specific differentiation of individual cells is driven by distinct genetic programs mirroring, but not necessarily recapitulating developmental processes (D'Orazi et al., 2020; Fraser et al., 2013; Kei et al., 2017; McGinn et al., 2019). There is a need for cross-species and cross-model injury (and regenerative) paradigms that aids the identification of genetic factors that are particularly relevant for the differential neurogenic response observed in regenerative vertebrates. There are two approaches for cross-species comparisons, either compare multiple injury paradigms as ambitiously undertaken by Hoang et al. to identify a unifying regeneration pathway or attempt a single injury applicable across established vertebrate animal models, which is the aim of our study (Hoang et al., 2019).

Of the many models of photoreceptor damage in mammals, extracellular ATP induced neuron loss can initiate a relatively high proliferative response in mammalian resident Müller glia population as demonstrated in rats (Puthussery and Fletcher, 2009; Vessey et al., 2014), and cats (Aplin et al., 2014). An excess of ATP may be released by dying or damaged cells and has been shown to activate the P2X7 purinoceptors in rodent photoreceptors, which in turn can lead to an influx in calcium and contribute to neural degeneration (Notomi et al., 2011; Puthussery and Fletcher, 2009). Similarly, ATP has also been shown to induce neural loss in other central nervous system neural populations, including spinal cord (Browne, 2013), and cultured hippocampus and cerebellum (Amadio et al., 2002; Cavaliere et al., 2002), which can be ameliorated to some extent with P2X antagonists (Wang et al., 2004). Our study aims to test whether extracellular ATP selectively damages photoreceptors in zebrafish as seen in mammals to establish a cross-species, single-injury model that is not confounded by differences in injury induction or neural population targeted.

Section snippets

Zebrafish strains

We used zebrafish (5–11 months old) of either sex from various strains including wild type, Tg (gfap:EGFPmi 2001) (Bernardos and Raymond, 2006) transgenic lines. All experiments were conducted in accordance with Monash University and University of Melbourne animal care guidelines and approved by the relevant local animal ethics committees.

Retinal injury model

Adult zebrafish were anaesthetised in buffered 0.033% tricaine methanesulfonate (MS-222) in fish tank water until respiratory movements of the operculum

Adenosine triphosphate injections induce preferential cell death in subpopulations of neurons in zebrafish retina

Intravitreal injections of ATP into various mammalian species has been shown to specifically cause cell death of photoreceptors (Vessey et al., 2014). Whilst photoreceptors are also ablated in genetic disease models, such as those with mutations that cause retinitis pigmentosa in humans, the ATP induced photoreceptor injury model is unique as it also shows a particularly robust proliferative response in resident mammalian Müller glia. Hence, the ATP photoreceptor injury model will enable us to

Discussion

In this study, we were able to establish and characterize a comparative vertebrate model of ATP induced retinal photoreceptor death and subsequent Müller cell driven regeneration of photoreceptors in zebrafish. The ATP induced neural cell death occurred relatively quickly comparable to that observed in other injury models of zebrafish retinal neurons including mechanical needle stick injury and genetic-chemical injury using exposure to metronidazole to cause specific cell death in neural

Conclusion

Our study showed ATP damaged photoreceptors and ganglion cells, which are closest to the injection site and may of been exposed to higher concentrations of ATP. Since the photoreceptors are furthest away and affected more than inner retinal neuron, their damage is indicative of a specific response to the ATP presumably via purinergic receptor activation. The resulting cellular death shows the same hallmarks of rapid neural damage within the retinas of a variety of mammalian and non-mammalian

Acknowledgements

We thank Dr. Ophelia Ehrlich for experimental assistance. The Australian Regenerative Medicine Institute is supported by funds from the State Government of Victoria and the Australian Federal Government. We thank FishCore facility staff (Monash University) and Walter and Eliza Hall zebrafish facility staff for housing and taking care of our animals. We thank Biological Optical Microscopy Platform (BOMP) for use of microscopes.

This work was supported by a Kaye Merlin Brutton bequest (University

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