Neuromuscular transmission modulation by A2A adenosine receptors in the SOD1G93A mouse model of amyotrophic lateral sclerosis
Year: 2013
Type: dissertation
Abstract: Amyotrophic Lateral Sclerosis (ALS) is an adult-onset progressive neurodegenerative disease characterized by the selective loss of motor neuron function leading to muscle atrophy and weakness. After symptoms onset patients last 4 to 5 years and ultimately die due to bulbar failure. Animal models have been developed to study the neurobiology of ALS, with the SOD1G93A mouse model as the most studied so far. Morphological and functional abnormalities have been reported in both pre-symptomatic and symptomatic stages of ALS progression in this rodent model. Dysfunctions in neuromuscular transmission at phrenic nerve-hemidiaphragm preparations of the SOD1G93A mouse have recently been put in evidence by our group. Since adenosine A2A receptors (A2AR) play a major role in fine-tuning neurotransmitter release in mammalian neuromuscular junctions (NMJs), we decided to evaluate how A2AR modulate acetylcholine (ACh) release in presymptomatic (4-6 weeks old (wo)) and symptomatic (12-14 wo) SOD1G93A mice. Using the selective A2AR agonist CGS 21680, we performed a dose-response study using 3, 5 and 10 nM in pre-symptomatic SOD1G93A mice and age-matched Wild Type (WT) animals. CGS 21680 at 3, 5, 10 and 25 nM was studied in symptomatic SOD1G93A and 12-14 wo WT rodents. 25 nM was used in the previous mentioned group of animals to verify if at concentrations higher than 10nM CGS 21680 effect in NMT would be increased. Intracellular recordings of endplate potentials (EPPs), miniature endplate potentials (MEPPs) and giant miniature endplate potentials (GMEPPs) where performed in high [Mg2+] paralyzed phrenic nerve-hemidiaphragm preparations. Low-frequency stimuli (0.5 Hz) allowed the evaluation of the evoked activity (EPPs) while spontaneous activity (MEPPs and GMEPPs) was measured in gap-free intervals. Quantal content (QC) was measured as the ratio between the mean EPP amplitude and the mean MEPP amplitude recorded in the same period of time. We first validated high [Mg2+] as an useful method to study neuromuscular transmission (NMT) in these mice. The shift in the Ca2+/Mg2+ ratio preserved the previous described features of ACh release in SOD1G93A mice diaphragm fibers. In the pre-symptomatic phase, SOD1G93A mice displayed a significant increase in EPP amplitude and QC in tested concentrations of CGS 21680 when compared to WT mice (p<0.05 Unpaired t-test) except for EPP changes at 3nM (p>0.05 Unpaired t-test). MEPP and GMEPP amplitude were not changed by CGS 21680 (p>0.05 Paired t-test). The A2AR-mediated increase in MEPP frequency was not statistically different between both groups in the presence of 3 and 10nM of the A2AR agonist (p>0.05 Unpaired t-test), but was significantly higher in SOD1G93A mice when perfused at 5nM (p<0.05 Unpaired t-test). We also found that in the pre-symptomatic SOD1G93A fibers GMEPP frequency was statistically higher in the presence of CGS 21680 (5nM) than in age-matched WT NMJs (p<0.05 Unpaired t-test). In the symptomatic phase, CGS 21680 did not elicit any changes in SOD1G93A mice evoked (EPP amplitude and QC) and spontaneous (MEPP and GMEPP frequency and amplitude) activity from SOD1G93A mice (p>0.05 Paired t-test). When 0.5 Hz stimuli were delivered, EPP amplitude, QC and MEPP frequency were increased in 12-14 wo WT mice in the presence of 3, 5, 10 and 25 nM of CGS 21680 (p<0.05 Paired t-test). These parameters together with GMEPP frequency were significantly higher than in symptomatic mice (p<0.05 Unpaired t-test). GMEPP and MEPP amplitude were not changed by CGS 21680 in older WT mice (p>0.05 Paired t-test). The A2AR receptor antagonist SCH 58261 (50nM) was devoid of effect in spontaneous and evoked release (p>0.05 Paired t-test) and effectively blocked CGS 21680 (5nM in 4-6 wo mice and 10 nM in 12-14 wo WT rodents) facilitation (p<0.05 one-way ANOVA followed by Tukey’s pos-hoc). Our results strongly suggest an early functionally enhancement of NMT concerning adenosine modulation before symptoms appear. This dysfunction involves an A2AR functional upregulation at diaphragm NMJs in pre-symptomatic SOD1G93A mice. When symptoms start to develop, A2AR receptor function is lost in symptomatic SOD1G93A mice but remains present in 12-14 wo WT mice. Furthermore, CGS 21680 perfusion triggered a higher A2AR facilitation in 12-14 wo than in 4-6 wo WT mice. This might be attributable to a normal maturation feature of the studied synapse, which contrasted with the observations in transgenic mice. The shift from a functional upregulation of A2AR before symptomatology that arises in SOD1G93A mice towards an apparent loss of A2AR functionality in symptomatic phase, highlights the role of this subtype of P1 receptors in the scope of ALS. A2AR targeted drugs could eventually play an import role in the delay of this disease.
Language: English
Subfield: Clinical Neurology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W2239049017