Neuroprotection of rat retinal ganglion cells mediated through alpha7 nicotinic acetylcholine receptors

Abstract

Glutamate-induced excitotoxicity is thought to play an important role in several neurodegenerative diseases in the central nervous system (CNS). In this study, neuroprotection against glutamate-induced excitotoxicity was analyzed using acetylcholine (ACh), nicotine and the α7 specific nicotinic acetylcholine receptor (α7 nAChR) agonist, N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987), in cultured adult rat retinal neurons. Adult Long Evans rat retinas were dissociated and retinal ganglion cells (RGCs) were isolated from all other retinal tissue using a two-step panning technique. Once isolated, RGCs were cultured under various pharmacological conditions to demonstrate excitotoxicity and neuroprotection against excitotoxicity. After 3 days, RGCs were immunostained with antibodies against the glycoprotein, Thy 1.1, counted and cell survival was assessed relative to control untreated conditions. 500 μM glutamate induced excitotoxicity in large and small RGCs in an adult rat dissociated culture. After 3 days in culture with glutamate, the cell survival of large RGCs decreased by an average of 48.16% while the cell survival of small RGCs decreased by an average of 42.03%. Using specific glutamate receptor agonists and antagonists, we provide evidence that the excitotoxic response was mediated through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid (KA) and N-methyl-d-aspartate (NMDA) glutamate receptors through an apoptotic mechanism. However, the excitotoxic effect of glutamate on all RGCs was eliminated if cells were cultured for an hour with 10 μM ACh, 100 μM nicotine or 100 nM of the α7 nAChR agonist, PNU-282987, before the glutamate insult. Inhibition studies using 10nM methyllycaconitine (MLA) or α-bungarotoxin (α-Bgt) supported the hypothesis that neuroprotection against glutamate-induced excitotoxicity on rat RGCs was mediated through α7 nAChRs. In immunocytochemical studies, double-labeled experiments using antibodies against Thy 1.1 and α7 nAChR subunits demonstrated that both large and small RGCs contained α7 nAChR subunits. The data presented in this study support the hypothesis that ACh and nicotinic acetylcholine receptor (nAChR) agonists provide neuroprotection against glutamate-induced excitotoxicity in adult rat RGCs through activation of α7 nAChR subunits. These studies lay the groundwork required for analyzing the effect of specific α7 nAChR agonists using in vivo models of excitotoxicity. Understanding the type of ACh receptors involved in neuroprotection in the rat retina could ultimately lead to therapeutic treatment for any CNS disease that involves excitotoxicity.

Figure 1. Effects of glutamate on adult cultured RGCs

Glutamate causes significant cell loss in cultured adult rat RGCs, but the neurotoxic effects are blocked by CNQX and MK-801. (A) represents cultured immunostained RGCs under control untreated conditions that were cultured for 3 days. (B) represents cells cultured for 3 days with 500µM glutamate, and (C) depicts labeled RGCs pretreated with both MK-801 and CNQX before addition of 500 µM glutamate.

Figure 2. Evidence of apoptosis in isolated adult rat RGCs

Cultured adult rat RGCs were plated at 1 × 10⁵ cells/ml and cultured for various time points in the presence of 500 µM glutamate. After days 0, 1 and 3, cells were labeled with calcein AM (left column) to label living cells and processed for TUNEL staining (right column) to label cells undergoing apoptosis. Figs. A and B represent images of the same cells obtained at Day 0. Figs. C and D represent images of the same cells after 1 day in culture and figs E and F represent images of the same cells after 3 days in culture with 500 µM glutamate.

Figure 3. ACh and nicotine provide neuroprotection against glutamate-induced excitotoxicity

Each bar graph represents the average percent of RGCs survival compared to untreated conditions after cells were cultured for 3 days under various pharmacological treatments. MLA and α-Bgt were applied 30 minutes before ACh or nicotine. ACh or nicotine was applied to culture for 1 hour before 500 µM glutamate. Averages were obtained from between 9–20 animals. The star represents significance from control. The solid circle represents significance from glutamate’s effect. Experiments were repeated between 3 and 8 times. Error bars represent SEM.

Figure 4. Effects of PNU-282987 on isolated rat RGCs

PNU-282987 provides neuroprotection against glutamate-induced excitotoxicity. Images of labeled RGCs were obtained after 3 days in culture under the following conditions: (A) under control untreated conditions. (B) in 500µM glutamate. (C) pretreated with 100 nM PNU-282987 for 1 hour prior to 500 µM glutamate and (D) when the specific α7 antagonist, MLA (10nM), was applied 30 minutes prior to PNU-282987 and 1 1/2 hours prior to 500 µM glutamate. After 3 days in culture, cells were labeled with 2 µM calcein AM for 1 hour to label viable RGCs.

Figure 5. Dose dependent effects of PNU-282987 on rat RGCs

Each solid circle represents the average percent of PNU-induced neuroprotection against glutamate-induced excitotoxicity compared to control untreated conditions using various concentrations of PNU-282987 that were introduced to cultured RGCs before glutamate insult. PNU-282987 was applied one hour before the glutamate insult. 100nM PNU-282987 showed the highest degree of neuroprotection. The dose response data points were curve fit. Error bars represent SEM. Averages were obtained from N’s of 3 to 20.

Figure 6. PNU-282987 provides neuroprotection in large and small RGCs

PNU-282987 caused an increase in the percent of RGC survival in both large and small RGCs if applied to cultures one hour before 500 µM glutamate. RGCs were cultured for 3 days before quantification processing. Each bar graph represents the average percent of RGC survival compared to control untreated conditions. Bar graphs shown in fig. 6A represent the effect of glutamate and PNU-282987 on large RGCs, fig 6B represent pharmacological effects on small RGCs while fig. 6C represent the effect of glutamate an PNU-282987 on the total RGC population. The star represents significance from control untreated conditions, while the solid circle represents significance from glutamate. Normalized bar graphs were obtained from between 9–20 rats. Error bars represent SEM.

Figure 7. α7nAChRs on large and small rat RGCs

Panels A, B and C represent images obtained from the same isolated RGCs double-labeled with primary antibodies against α7nAChR subunits (left panels) and RGCs (center panels). Panel A illustrates RGCs secondarily labeled with Alexa-Fluor 595, panel B illustrates the same RGCs secondarily labeled with Alexa-Fluor 488, while panel C illustrates the co-localization of the two markers. Panels D, E and F represent images obtained from a dissociated cultured retina were RGCs were not isolated using the panning technique. Panel D illustrates an image obtained after the dissociated tissue was labeled with an antibody against α7nAChR subunits and secondarily labeled with Alexa-Fluor 595, panel E represents the same cultured retinal tissue double-labeled with an antibody against Thy 1.1 and secondarily labeled with Alexa-Fluor 488 and panel F represents the co-localization of the two panels. Note that not all cells double-labeled under these conditions.