α7 Nicotinic Acetylcholine Receptor Activation in Retinal Pigment Epithelium promotes Müller glia Proliferation and Progenitor-like States

Description

Adult mammalian Müller glia (MG) cells typically undergo a gliotic response following retinal injury, limiting endogenous neuronal regeneration. Previous studies demonstrated that the potent and selective α7 nicotinic acetylcholine receptor (nAChR) agonist, PNU-282987, induces a neurogenic response in adult mammalian MG through signaling initiated in retinal pigment epithelial (RPE) cells. In the present study, we further investigate the cellular and transcriptomic responses underlying α7 nAChR-mediated MG reprogramming using primary mouse MG and RPE cell cultures. Primary MG cells from adult mice were co-cultured with RPE cells treated with either PNU-282987 or DMSO, and phenotypic changes associated with proliferation and progenitor formation were evaluated using immunocytochemistry. PNU-282987-treated primary MG cultures demonstrated a significant increase in Ki67-positive and Pax6-positive cells compared to DMSO-control primary MG cultures. To further characterize the responses, RNA sequencing was performed to characterize transcriptomic effects associated with α7 nAChR activation. Differential expression analyses identified several candidate regulators associated with glial plasticity, progenitor formation, and developmental signaling. These results provide insight into receptor-mediated responses that can enhance the neurogenic potential of central nervous system glia. Moreover, these studies are foundational for future experiments examining the precise mechanism of how PNU-282987 elicits changes in adult mammalian MG cells.

Steps to reproduce

For ICC analysis, MG cells were seeded on laminin-coated glass coverslips in 24-well plates during co-culture with control or PNU-282987-treated RPE cells. Following treatment, the MG cells were fixed with 4% paraformaldehyde for 10 minutes. Cells were treated with 0.5% Triton™ X-100 in Tris-buffered saline (TBSTr) to promote permeabilization with 10% goat or donkey serum to minimize non-specific binding for 3 hours. Primary antibodies were diluted in 0.05% TBSTr with 1% goat or donkey serum and incubated at 4℃ overnight. Supplemental Table 2 provides a list of primary antibodies used, along with their dilutions. Primary antibodies were removed and replaced with fluorescent dye-labeled secondary antibodies (Supplemental Table 2) for 2 hours in the dark. Hoechst (1:10,000) was used as a counterstain, and the coverslips were mounted on microscope slides for viewing on a Nikon C2+ scanning confocal microscope. Primary MG cultures were independently isolated from 10 mice and expanded prior to experimental plating. Each biological replicate consisted of cells derived from an independent mouse, with cells from each mouse distributed across the DMSO and PNU-282987 treatment conditions. Cell counts were obtained from one coverslip for each biological replicate within each culture condition, representing one biological replicate. These biological replicate values (n = 5–10, as indicated for each experiment) were used for all statistical analyses. Cell counts were performed in Fiji (Schindelin et al., 2012) by an investigator blinded to the experimental conditions.

Institutions

• Western Michigan University

  Michigan, Kalamazoo

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