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.

Reference:
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

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  1. This is a highly interesting paper. In my opinion, this paper shows that muscles of ALS patients are not "simply" denervated muscles. Their acetylcholine receptors are different in electrophysiological properties from patients with non-ALS denervation. This suggests intrinsic pathological mechanisms in muscles of ALS patients. This fits with recent studies in the field that show that muscle defects are able, on their own, to trigger neuromuscular changes related to ALS. A major lack of this study is some mechanistic insights: As the authors point out, they do not provide molecular information to explain this difference between ALS and denervated muscles. Future studies should determine whether the transcription of neuromuscular junction genes is somewhat impaired in ALS muscles, or if the defect lies post-transcriptionally. Despite the exciting result, a drawback of this study is that it lacks a healthy control group to compare with ALS patients.

    In all, this is another piece of evidence strengthening the idea that ALS is not a disease restricted to motor neurons, but also involves other cell types, including the skeletal muscle.

    View all comments by Luc Dupuis
  2. In this study, the authors develop an experimental method to further study the muscle pathophysiology of ALS and to better test the so-called “dying-back model.”

    In fact, although most efforts have aimed to define the potential genes and pathways associated with motor neuron degeneration and to understand ALS pathogenesis, no consensus has emerged as to the primary toxicity of gene mutations. The primary causes of ALS are therefore still unknown and no effective or decisive treatments are available.

    Although the steps that lead to the pathological state are well defined, several fundamental issues are still controversial: Are the motor neurons the first and solely direct targets of ALS? And what is the contribution of non-neuronal cells, if any, to the pathogenesis of ALS?

    Several lines of evidence suggested, for example, that the neurodegenerative action of mutant SOD1 genes operate through a dominant paracrine activity that emanates from non-neuronal tissues. The obvious loss of motor neurons in the spinal cord initially focused attention on how mutant SOD1 might act within motor neurons to provoke neuronal degeneration and death. However, the mutant gene products are expressed widely, which raises the possibility that the toxicity might result from the action of mutant SOD1 protein in non-neuronal cells.

    Thus, several recent lines of evidence (including our recent studies) support the redefinition of ALS as a multi-systemic disease in which alterations in structural, physiological, and metabolic parameters in different cell types (motor neurons, glia, and muscle) may act synergistically to exacerbate the pathology.

    Therefore, 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.

    View all comments by Antonio Musaro
  3. This is a very sound paper, and the electrophysiology is well done, as expected from this group. The information is more technical rather than adding new data on ALS specifically, but it is a real breakthrough. They compare well-known differentiated myoblasts to their oocyte “microtransplantation" approach that they present here. They indeed show some (minor) differences in the acetylcholine receptor properties (showing that differentiated myoblasts don't retain original—here ALS—features or cell traits). With this new technique (microtransplantation into Xenopus oocytes), we now have access to (more) original disease traits that are retained. Despite the lack of biological data, this work clearly opens new possibilities to analyze (electrophysiologically or with other approaches) biological material with fewer artifacts, for example, those due to differentiation or a long in-vitro process.

    View all comments by Jean-Phillipe Loeffler

References

News Citations

  1. Glia—Absolving Neurons of Motor Neuron Disease
  2. Believe It: Astrocytes Kill Neurons in New ALS Model
  3. Motors and Muscles—Pacing ALS Progression
  4. DC: Myriad Mice Mimic ALS, FTLD, Loss of TDP-43 Function

Paper Citations

  1. . State of the art and the dark side of amyotrophic lateral sclerosis. World J Biol Chem. 2010 May 26;1(5):62-8. PubMed.
  2. . Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons. Nat Neurosci. 2007 May;10(5):615-22. PubMed.
  3. . Non-cell autonomous effect of glia on motor neurons in an embryonic stem cell-based ALS model. Nat Neurosci. 2007 May;10(5):608-14. PubMed.
  4. . Astrocytes carrying the superoxide dismutase 1 (SOD1G93A) mutation induce wild-type motor neuron degeneration in vivo. Proc Natl Acad Sci U S A. 2011 Oct 25;108(43):17803-8. PubMed.
  5. . Muscle mitochondrial uncoupling dismantles neuromuscular junction and triggers distal degeneration of motor neurons. PLoS One. 2009;4(4):e5390. PubMed.
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  7. . Amyotrophic lateral sclerosis is a distal axonopathy: evidence in mice and man. Exp Neurol. 2004 Feb;185(2):232-40. PubMed.
  8. . Skeletal muscle is a primary target of SOD1G93A-mediated toxicity. Cell Metab. 2008 Nov;8(5):425-36. PubMed.
  9. . Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes. Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):13238-42. PubMed.
  10. . Microtransplantation of ligand-gated receptor-channels from fresh or frozen nervous tissue into Xenopus oocytes: a potent tool for expanding functional information. Prog Neurobiol. 2009 May;88(1):32-40. PubMed.
  11. . Physiological characterization of human muscle acetylcholine receptors from ALS patients. Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):20184-8. PubMed.

Further Reading

Papers

  1. . Physiological characterization of human muscle acetylcholine receptors from ALS patients. Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):20184-8. PubMed.
  2. . Muscle atrophy induced by SOD1G93A expression does not involve the activation of caspase in the absence of denervation. Skelet Muscle. 2011;1(1):3. PubMed.
  3. . Skeletal Muscle in Motor Neuron Diseases: Therapeutic Target and Delivery Route for Potential Treatments. Curr Drug Targets. 2010 Jul 1; PubMed.
  4. . Interaction of high concentrations of riluzole with recombinant skeletal muscle sodium channels and adult-type nicotinic receptor channels. Muscle Nerve. 2002 Oct;26(4):539-45. PubMed.
  5. . Muscle expression of a local Igf-1 isoform protects motor neurons in an ALS mouse model. J Cell Biol. 2005 Jan 17;168(2):193-9. PubMed.
  6. . Microtransplantation of functional receptors and channels from the Alzheimer's brain to frog oocytes. Proc Natl Acad Sci U S A. 2004 Feb 10;101(6):1760-3. PubMed.

News

  1. Muscle MicroRNA Repairs Nerve-Muscle Connection in ALS Model
  2. ALS in a Dish? Studying Motor Neurons from Human Stem Cells
  3. Microglia in ALS: Helpful, Harmful, or Neutral?
  4. ALS: Predicting Prognosis, Banking on Pluripotent Stem Cells
  5. ALS—Is It the Neurons or the Glia?
  6. Glia—Absolving Neurons of Motor Neuron Disease
  7. Believe It: Astrocytes Kill Neurons in New ALS Model
  8. Motors and Muscles—Pacing ALS Progression
  9. DC: Myriad Mice Mimic ALS, FTLD, Loss of TDP-43 Function

Primary Papers

  1. . Physiological characterization of human muscle acetylcholine receptors from ALS patients. Proc Natl Acad Sci U S A. 2011 Dec 13;108(50):20184-8. PubMed.