Physiological characterization of human muscle acetylcholine receptors from ALS patients

Abstract

Amyotrophic lateral sclerosis (ALS) is characterized by progressive degeneration of motor neurons leading to muscle paralysis. Research in transgenic mice suggests that the muscle actively contributes to the disease onset, but such studies are difficult to pursue in humans and in vitro models would represent a good starting point. In this work we show that tiny amounts of muscle from ALS or from control denervated muscle, obtained by needle biopsy, are amenable to functional characterization by two different technical approaches: "microtransplantation" of muscle membranes into Xenopus oocytes and culture of myogenic satellite cells. Acetylcholine (ACh)-evoked currents and unitary events were characterized in oocytes and multinucleated myotubes. We found that ALS acetylcholine receptors (AChRs) retain their native physiological characteristics, being activated by ACh and nicotine and blocked by α-bungarotoxin (α-BuTX), d-tubocurarine (dTC), and galantamine. The reversal potential of ACh-evoked currents and the unitary channel behavior were also typical of normal muscle AChRs. Interestingly, in oocytes injected with muscle membranes derived from ALS patients, the AChRs showed a significant decrease in ACh affinity, compared with denervated controls. Finally, riluzole, the only drug currently used against ALS, reduced, in a dose-dependent manner, the ACh-evoked currents, indicating that its action remains to be fully characterized. The two methods described here will be important tools for elucidating the role of muscle in ALS pathogenesis and for developing drugs to counter the effects of this disease.voltage-clamp | patch-clamp

Acetylcholine receptors from human muscle as pharmacological targets for ALS therapy

“…We previously reported a significant decrease in ACh sensitivity in oocytes injected with ALS muscle membranes compared with those injected with non-ALS membranes (5). When we mimicked this increase by expressing AChR subunits in Xenopus oocytes, we found reduced ACh sensitivity comparable to that found on microtransplanted ALS membranes (5). This evidence supports the hypothesis that ALS denervation induces changes at the NMJ that are not present after denervation resulting from other neuropathies or trauma.…”

“…Therefore, in muscle from ALS patients, variable proportions of γ-and e-AChRs are present, reflecting cycles of denervation/reinnervation (5,22). Interestingly, we found a selective PEA effect on oocytes expressing e-AChRs, suggesting that it is not mediated by "microtransplanted" accessory proteins and/or signaling molecules.…”

“…The ACh concentrations producing half-maximal effect (EC 50 ) were estimated by fitting the data to Hill equations, using least-square routines as previously described (5).…”

“…Studies have uncovered defects in the skeletal muscle that occur even in the absence of motor neuron anomalies, supporting the "dying-back" hypothesis in which distal motor endplate degeneration plays a key role in the progression of the disease (2-4). NMJs of ALS patients consistently show electrophysiological properties distinctly different from those of patients with pure denervation (5,6). Using muscle biopsies from sporadic ALS patients, we previously characterized the nicotinic acetylcholine currents resulting from the activation of functional acetylcholine receptors (AChRs) (5).…”

The Role of Skeletal Muscle in Amyotrophic Lateral Sclerosis

“…Based on magnetic resonance imaging, other investigations revealed that muscle volume of mutant SOD1 mice was reduced from as early as 8 weeks of age, long before disease (75). By injecting oocytes with muscle membranes derived from ALS patients, it was shown that the affinity of acetycholine receptors for their ligand was lower than that of receptors coming from surgically denervated muscle (88). Additional studies reported electrophysiological postsynaptic alterations in diaphragm at 4-6 weeks of age (99), indicating that neuromuscular transmission could be intrinsically affected.…”