11 December 2011. You can hear the devastation to muscle in the name “amyotrophic lateral sclerosis.” Actually, motor neurons are the main cell type that degenerate in the disease and get the lion’s share of attention. However, scientists are now starting to focus more on muscle pathology in ALS. For example, a paper in the November 29 Proceedings of the National Academy of Sciences online describes two new lab models to move muscle studies along. The study team, led by first author Eleonora Palma of the Sapienza University of Rome and senior author Ricardo Miledi of the University of California, Irvine, used muscle biopsies from people with ALS to culture muscle tissue in vitro. In addition, they transferred human muscle acetylcholine receptors, crucial for controlling contraction, to oocytes of the frog Xenopus laevis so they could study the receptors in a simple system. These models offer a new approach to study human muscle pathology and the effects of potential treatments, Palma said.
ALS is currently undergoing a redefinition, from a motor neuron disease to a multi-systemic disease, wrote Antonio Musarò, who is also at Sapienza University but was not involved in the current study, in an e-mail to ARF (Musarò, 2010). Glia are now recognized as important participants (see ARF related news story on Nagai et al., 2007 and Di Giorgio et al., 2007; ARF related news story on Papadeas et al., 2011), and recent studies point to pathology in muscle, too (see ARF related news story on Dupuis et al., 2009). The earliest discernable events in ALS occur at the neuromuscular junction and proceed with dying back of axons before motor neuron cell bodies are affected (Dadon-Nachum et al., 2010; Fischer et al., 2004). Whether the initial insult begins in the muscle or the nerve is unclear, but mice that express ALS-linked mutant proteins in muscle alone exhibit muscle atrophy (Dobrowolny et al., 2008) and early death (see ARF related news story). “It is very important to develop appropriate tools for elucidating the role of different tissues, including muscle, in ALS pathogenesis, and for developing drugs to counter the effects of this disease,” Musarò wrote.
Mouse models are based on genetic mutations that cause rare familial forms of ALS, and as such, represent only a fraction of the number of people with the disease, Palma noted. To more generally model the disease in vitro, she obtained muscle samples from the deltoid, quadriceps, or anterior tibialis of people with sporadic ALS, and compared those to muscle cells taken from control volunteers.
Palma considered her controls carefully. In ALS, the muscle cells have pulled away from the nerve. This denervation causes a host of complications, culminating in regression of the muscle to a fetal form. Protein subunits making up the acetylcholine receptors change, and the receptors distribute themselves across the entire cell surface instead of clustering in the neuromuscular junction the way they do in innervated muscle. To control for these changes, Palma biopsied people who suffered muscle denervation due to injury.
For the cell model, the scientists isolated immature progenitor cells from the biopsies and then differentiated them into mature, multinucleate muscle cells. They based the acetylcholine receptor system on a Xenopus model previously developed by Miledi to study membrane-based proteins (Miledi et al., 2002; Eusebi et al., 2009). The researchers homogenized the biopsies, isolated the total membrane fraction, and injected it into the frog eggs. Membrane proteins such as acetylcholine receptors incorporated into the oocytes’ plasma membranes. In this way, the oocytes served as crude surrogate muscle cells, presenting the receptors so the scientists could test their electrophysiological properties.
It is important to develop new approaches like these in order to study ALS pathology, and perhaps test therapeutics, in human tissues, Palma said. She focused on the acetylcholine receptors that are responsible for muscle movement. In a healthy neuromuscular junction, motor neurons release acetylcholine, which binds receptors on the muscle side. The acetylcholine receptors open up, allowing sodium and potassium ions to flow into the cell, creating a current that causes muscle contraction. In both model systems derived from ALS biopsies, the researchers found that the receptors had less affinity for acetylcholine than receptors from injury-denervated tissue. “This paper shows that muscles of ALS patients are not ‘simply’ denervated muscles,” wrote Luc Dupuis of the University of Strasbourg, France, who was not involved in the study. “In all, this is another piece of evidence strengthening the idea that ALS is not a disease restricted to motor neurons, but involves also other cell types, including the skeletal muscle.”—Amber Dance.
Palma E, Inghilleri M, Conti L, Deflorio C, Frasca V, Manteca A, Pichiorri F, Roseti C, Torchi G, Limatola C, Grassi F, Miledi R. Physiological characterization of human muscle acetylcholine receptors from ALS patients. Proc Natl Acad Sci U S A. 2011 Nov 29. Abstract