Stimulation of Retinal Pigment Epithelium With an α7 nAChR Agonist Leads to Müller Glia Dependent Neurogenesis in the Adult Mammalian Retina

Abstract

PurposeThe adult mammalian retina is typically incapable of regeneration when damaged by disease or trauma. Restoration of function would require generation of new adult neurons, something that until recently, mammals were thought to be incapable of doing. However, previous studies from this laboratory have shown that the α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, PNU-282987, induces cell cycle reentry of Müller glia and generation of mature retinal neurons in adult rats, in the absence of detectible injury. This study analyzes how PNU-282987 treatment in RPE leads to robust BrdU incorporation in Müller glia in adult mice and leads to generation of Müller-derived retinal progenitors and neuronal differentiation.MethodsRetinal BrdU incorporation was examined after eye drop application of PNU-282987 in adult wild-type and transgenic mice that contain tamoxifen-inducible tdTomato Müller glia, or after intraocular injection of conditioned medium from PNU-282987–treated cultured RPE cells.ResultsPNU-282987 induced robust incorporation of BrdU in all layers of the adult mouse retina. The α7 nAChR agonist was found to stimulate cell cycle reentry of Müller glia and their generation of new retinal progenitors indirectly, via the RPE, in an α7 nAChR-dependent fashion.ConclusionsThe results from this study point to RPE as a contributor to Müller glial neurogenic responses. The manipulation of the RPE to stimulate retinal neurogenesis offers a new direction for developing novel and potentially transformative treatments to reverse the loss of neurons associated with neurodegenerative disease, traumatic injury, or aging.

Inherited Eye Diseases with Retinal Manifestations through the Eyes of Homeobox Genes

“…For example, this strategy involves the use of purinergic P2X receptor antagonists and A3R adenosine receptor agonists as neuroprotectors and regulators of retinal repair [383]. Stimulation of RPE cells by a nicotinic acetylcholine receptor agonist also contributes to the activation of Muller glia proliferation and neural differentiation in adult mice [384].…”

Involvement of HB-EGF/Ascl1/Lin28a Genes in Dedifferentiation of Adult Mammalian Müller Glia

“…Previous studies from this lab found that supernatant collected from RPE cells treated with PNU-282987 (activated RPE) was sufficient to produce cell cycle reentry of the adult mammalian Müller glia in the retina when the supernatant was injected into the vitreal chamber of adult rodents in vivo. These same studies demonstrated that BrdU positive Müller glia dedifferentiate into MDPCs (Webster et al, 2017(Webster et al, , 2019. To identify changes in Müller glia gene expression after exposure to activated RPE supernatant, RNA-seq was performed (GeneWiz Inc., NJ, United States) from total RNA extracted from Müller glia.…”

“…Of the cell types within the neural retina which contain α7 nAChRs, namely bipolar, some amacrine cells and retinal ganglion cells (Webster et al, 2017;Hall et al, 2019), acetylcholine (ACh) released from the starburst amacrine cells does not induce proliferation or interkinetic nuclear migration of Müller glia under physiological conditions. Instead, evidence was provided that PNU-282987 acts on the retinal pigment epithelium (RPE) when applied as eye drops (Webster et al, 2019). RPE cells contain α7 nACh receptors, are known to secret at least 20 different compounds under normal physiological conditions including growth factors and hormones, and are separated from ACh stimulation under normal physiological conditions by the outer limiting membrane (Grierson et al, 1994;Yoshii et al, 2007;Maneu et al, 2010).…”

“…RPE cells contain α7 nACh receptors, are known to secret at least 20 different compounds under normal physiological conditions including growth factors and hormones, and are separated from ACh stimulation under normal physiological conditions by the outer limiting membrane (Grierson et al, 1994;Yoshii et al, 2007;Maneu et al, 2010). When PNU-282987 is administered as eyedrops into the eyes of adult mice and rats, it is hypothesized that PNU-282987 binds to alpha7 nAChRs on RPE directly outside the neural retina (Webster et al, 2019) to release signaling molecules onto the end feet of Müller glia, that then asymmetrically divide to produce Müller-derived progenitor cells (MDPCs). Evidence to support this hypothesis was provided by experiments in which RPE-J cell lines in culture were treated with 100 nM PNU-282987 and the resulting intraocular injection of supernatant was sufficient to produce cell cycle reentry of Müller glia in adult rodents in vivo (Webster et al, 2019).…”

“…When PNU-282987 is administered as eyedrops into the eyes of adult mice and rats, it is hypothesized that PNU-282987 binds to alpha7 nAChRs on RPE directly outside the neural retina (Webster et al, 2019) to release signaling molecules onto the end feet of Müller glia, that then asymmetrically divide to produce Müller-derived progenitor cells (MDPCs). Evidence to support this hypothesis was provided by experiments in which RPE-J cell lines in culture were treated with 100 nM PNU-282987 and the resulting intraocular injection of supernatant was sufficient to produce cell cycle reentry of Müller glia in adult rodents in vivo (Webster et al, 2019).…”