Adenosine 2A receptor inhibition protects phrenic motor neurons from cell death induced by protein synthesis inhibition

Abstract

Respiratory motor neuron survival is critical for maintenance of adequate ventilation and airway clearance, preventing dependence to mechanical ventilation and respiratory tract infections. Phrenic motor neurons are highly vulnerable in rodent models of motor neuron disease versus accessory inspiratory motor pools (e.g. intercostals, scalenus). Thus, strategies that promote phrenic motor neuron survival when faced with disease and/or toxic insults are needed to help preserve breathing ability, airway defense and ventilator independence. Adenosine 2A receptors (A2A) are emerging as a potential target to promote neuroprotection, although their activation can have both beneficial and pathogenic effects. Since the role of A2A receptors in the phrenic motor neuron survival/death is not known, we tested the hypothesis that A2A receptor antagonism promotes phrenic motor neuron survival and preserves diaphragm function when faced with toxic, neurodegenerative insults that lead to phrenic motor neuron death. We utilized a novel neurotoxic model of respiratory motor neuron death recently developed in our laboratory: intrapleural injections of cholera toxin B subunit (CtB) conjugated to the ribosomal toxin, saporin (CtB-Saporin). We demonstrate that intrapleural CtB-Saporin causes: 1) profound phrenic motor neuron death (~5% survival); 2) ~7-fold increase in phrenic motor neuron A2A receptor expression prior to cell death; and 3) diaphragm muscle paralysis (inactive in most rats; ~7% residual diaphragm EMG amplitude during room air breathing). The A2A receptor antagonist istradefylline given after CtB-Saporin: 1) reduced phrenic motor neuron death (~20% survival) and 2) preserved diaphragm EMG activity (~46%). Thus, A2A receptors contribute to neurotoxic phrenic motor neuron death, an effect mitigated by A2A receptor antagonism.

Keywords: A2A receptor; ADORA2A; Adenosine; Apoptosis; Neuroprotection; Phrenic motor neuron survival; p38.

Copyright © 2019 Elsevier Inc. All rights reserved.

Figures

Figure 1:

Timeline for experimental procedures: 36 hours after bilateral intrapleural CtB-Saporin injections (Total dose: 50 μg/rat), rats were treated with A2a receptor antagonist (KW6002; also known as Istradefylline) twice daily, attaining a total dose of 1 mg/kg/day. Terminal EMG measurements and phrenic motor neuron counts were performed on day 8 (24 h after the last A2a receptor antagonist injection). Because almost all phrenic motor neurons died by day 8, an earlier time-point when phrenic motor neurons were still viable was selected for immunohistochemical evaluation of protein expression and localization. Thus, for molecular analyses, rats were sacrificed and perfused at Day 5 post-CtB-Saporin injection.

Figure 2:

Adenosine 2A (A2a) receptor expression in phrenic motor neurons 5 days after intrapleural cholera toxin B (CtB)-Saporin injections (i.e. before phrenic motor neuron loss). A-I: Representative images (20x) for CtB (green) and A2a receptors (red) at Day 5. J: A2a receptor fluorescence intensity is increased following intrapleural CtB-Saporin injections versus control (p<0.01; red asterisks). Mean ± 1 SEM. Scale bar = 40 μm.

Figure 3:

Phrenic motor neuron counts at Day 8 post-intrapleural cholera toxin (CtB-Saporin) injections (50 μg/rat total). A-C: Representative images (20x) for CtB-labelled phrenic motor neurons. D: CtB-Saporin causes almost complete loss of phrenic motor neurons (p2a) receptor antagonist (Istradefylline; KW6002) administration following intrapleural CtB-Saporin injections improves phrenic motor neuron survival versus CtB-Saporin injected + vehicle treated rats (p<0.05; #). Mean ± 1 SEM. Scale bar = 40 μm.

Figure 4:

Representative diaphragm EMG recordings during eupnea, maximum chemoreceptor stimulation (10.5% O2 + 7% CO2), and spontaneous deep breaths (sighs) 7 days after CtB (control) and CtB-Saporin injections. Intrapleural CtB-Saporin injections abolished diaphragm EMG activity across all behaviors in 4 of 7 rats. Twice daily treatment of A2a receptor antagonist partially preserved the diaphragm EMG activity.

Figure 5:

Diaphragm EMG activity 8 days after intrapleural cholera toxin B-Saporin (CtB-Saporin) injections (50 μg). Diaphragm EMG amplitude is estimated via root-mean-squared (RMS) calculation over a 50 -ms window. Diaphragm EMG was nearly abolished following CtB-Saporin injections during (A) eupnea, (B) hypoxia (10.5%)-hypercapnia (7%), and (C) augmented breaths (p2a) receptor antagonist (KW6002) following intrapleural CtB-Saporin injections increases preserved diaphragm EMG activity compared to vehicle treatment (p<0.05 during eupnea and augmented breath). (D) EMG activity was normalized to augmented breaths since this normalization reveals the functional reserve of the diaphragm muscle. Normalized EMG value was assumed to be zero in the rats with no EMG activity across all conditions tested, since there was no functional reserve in these rats. Diaphragmatic functional reserve was obliterated following intrapleural CtB-Saporin injections (p<0.001). A2a antagonist treatment restored normalized EMG value (p=0.82 compared to control, p<0.05 compared to CtB-Saporin + Vehicle), suggesting restored functional reserve. Mean ± SEM.

Figure 6:

Phosphorylation of p38 MAPK within the phrenic motor neurons 5 days after intrapleural cholera toxin B-Saporin (CtB-Saporin) injections. A-I. Representative images (20x) for CtB (green) and phospho-p38 MAPK (red) at Day 5. J. Signal-to-background ratio of phospho-p38 fluorescence intensity is increased following intrapleural CtB-Saporin injections compared to control (p2a antagonist treatment reduced phosphor-p38 MAPK levels compared to CtB-Saporin+Vehicle group (p<0.05); however, was still higher than control group (p<0.05). Mean ± SEM. SEM in the control group represents the variability in the raw values normalized to averaged mean value. Scale bar = 40 μm.