Deficits in Endogenous Adenosine Formation by Ecto‐5′‐Nucleotidase/CD73 Impair Neuromuscular Transmission and Immune Competence in Experimental Autoimmune Myasthenia Gravis

Abstract

AMP dephosphorylation via ecto-5′-nucleotidase/CD73 is the rate limiting step to generate extracellular adenosine (ADO) from released adenine nucleotides. ADO, via A2A receptors (A2ARs), is a potent modulator of neuromuscular and immunological responses. The pivotal role of ecto-5′-nucleotidase/CD73, in controlling extracellular ADO formation, prompted us to investigate its role in a rat model of experimental autoimmune myasthenia gravis (EAMG). Results show that CD4+CD25+FoxP3+ regulatory T cells express lower amounts of ecto-5′-nucleotidase/CD73 as compared to controls. Reduction of endogenous ADO formation might explain why proliferation of CD4+ T cells failed upon blocking A2A receptors activation with ZM241385 or adenosine deaminase in EAMG animals. Deficits in ADO also contribute to neuromuscular transmission failure in EAMG rats. Rehabilitation of A2AR-mediated immune suppression and facilitation of transmitter release were observed by incubating the cells with the nucleoside precursor, AMP. These findings, together with the characteristic increase in serum adenosine deaminase activity of MG patients, strengthen our hypothesis that the adenosinergic pathway may be dysfunctional in EAMG. Given that endogenous ADO formation is balanced by ecto-5′-nucleotidase/CD73 activity and that A2ARs exert a dual role to restore use-dependent neurocompetence and immune suppression in myasthenics, we hypothesize that stimulation of the two mechanisms may have therapeutic potential in MG.

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A_2A and Muscarinic M₁ Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters

“…Results obtained using this pre-synaptic in vitro model of myasthenia will be compared to previous findings where the muscle component was predominantly affected using rats with toxininduced and experimental autoimmune MG [31,32]. We also hypothesize that by reducing the amount of choline available for neuronal uptake, the acetylcholinesterase inhibitor, neostigmine (NEO), may mimic inhibition of HChT by HC-3 while increasing ACh accumulation at the neuromuscular junction and, hence, the cholinergic tone [34].…”

“…All these properties are mediated by non-genomic pre-synaptic actions of glucocorticoids [56,57] and may contribute to improve neuromuscular transmission deficits in myasthenic patients besides their empirical use as immunosuppressive agents. Facilitation of the neuromuscular transmission by methylprednisolone during high-frequency motor nerve activity coincides with the predominant adenosine A 2A receptor tonus, which coordinates the interplay with other receptors (e.g., muscarinic) on motor nerve endings to sustain ACh release that is required to overcome tetanic neuromuscular depression in myasthenics [31,32]. Recently, we showed that the amplification of neuromuscular transmission by methylprednisolone depends on the tonic activation of presynaptic facilitatory adenosine A 2A receptors secondary to endogenous adenosine generated from ATP released under resting conditions [33].…”

“…However, this mechanism is impaired in rats with toxin-induced and experimental autoimmune Myasthenia gravis (MG). In these studies, no changes were detected in A 2A -receptors expression on motor nerve terminals by immunofluorescence confocal microscopy [31,32]. The molecular paths underlying these deficits in myasthenics are uncertain, but evidence point to an impairment of the adaptive shift from CaV2.1 (P/Q type) to CaV1 (L type) channels leading to tetanic failure associated with deficits in endogenous adenosine accumulation and A 2A receptor tone.…”

The role of T regulatory cells in immunopathogenesis of myasthenia gravis: implications for therapeutics

“…Adenosine/Adenosine A2A receptor Adenosine, via adenosine A2A receptor (A2ARs), is a potent modulator of neuromuscular and immunological responses [120]. Activation of the A2AR has been shown to increase the expression of FOXP3 in cognate antigen-activated T cells, thus promoting the differentiation of iTregs [121].…”

“…On the other hand, Ecto-5¢-nucleotidase (CD73) is the rate limiting enzyme for adenosine production from released adenine nucleotides, which plays a strategic role in calibrating/ controlling extracellular adenosine formation and may assume a pivotal role as a pharmacological target to fine tune A2AR activity. It has been showed that FOXP3 + Tregs from EAMG rats express lower amounts of CD73 as compared to controls [120].…”