Keeping muscles strong and active in a mouse model of amyotrophic lateral sclerosis allows the animals to perform better in the face of dwindling motor neuron input, but ultimately cannot slow their demise, according to new research reported in the May 2 Cell Metabolism. Researchers in the laboratory of Don Cleveland at the University of California, San Diego, overexpressed the mitochondria-boosting gene PGC-1α in the skeletal muscles of mice that systemically produced mutant superoxide dismutase 1 (SOD1). The double transgenic mice could run farther and faster than single-mutant mSOD1 mice, but their disease progressed at the same rate. The work confirms that while mSOD1 in muscles causes atrophy, the muscle woes do not contribute to the neurodegeneration that is the primary ALS pathology, said co-first authors Sandrine Da Cruz and Philippe Parone in an interview with ARF.

The study addresses a question ALS researchers have debated—whether muscle pathology could cause the degeneration of neuromuscular junctions (NMJs), axons, and ultimately motor neurons themselves (Wong and Martin, 2010), wrote Ashu Johri and Flint Beal of the Weill Medical College of Cornell University, New York, in a commentary accompanying the Cell Metabolism paper. However, the fact that improving muscle function did not prevent denervation of motor neurons shows “that the disease is not a consequence of a dying back of axons following damage to NMJs in muscle,” Johri and Beal concluded.

PGC-1α (peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α) is “the master regulator of mitochondrial biogenesis and oxidative metabolism,” Parone said. Researchers have already shown that PGC-1α not only increases motor ability, but also extends survival of ALS model mice when constitutively expressed (Zhao et al., 2011; Liang et al., 2011). Parone and Da Cruz, in order to understand the cell-specific contributions of mitochondrial pathology to disease, are developing mSOD1 mouse models that selectively express PGC-1α in motor neurons, astrocytes, or skeletal muscle. The muscle model is the first to be published.

Muscular PGC-1α noticeably benefited the double transgenic mice, even after symptoms developed. Their muscles possessed more mitochondria than did plain mSOD1 mice, and the organelles were bigger. The mice ran farther on a treadmill and faster on a running wheel, and spent more time exploring an open area, than the single mutants. When the researchers electrically stimulated the hind limb muscles, bypassing the motor neurons, the PGC-1α-expressing tissues took twice as long to fatigue as the muscle with only mSOD1. “Even in a mouse that is 100 percent paralyzed [in the hind limbs], the muscle is still very much active,” Parone said.

The scientists reasoned that if dysfunctional muscle sends some toxic signal to motor neurons, then the PGC-1α treatment should dampen that signal and improve the neurological side of the disease. This was not the case. In terms of disease onset, weight loss (of fat), and time of death, “there is absolutely no difference between the single transgenics and the double transgenics,” Parone said. “They still progress through disease at exactly the same speed as the mutant SOD1 animals.” There was also no difference in denervation of NMJs or motor neuron degeneration.

Parone and Da Cruz concluded that muscle cells could not be the most crucial source of ALS pathology. In support of this standpoint, Da Cruz noted that mice with mSOD1 only in muscle do not exhibit motor neuron degeneration (Dobrowolny et al., 2008). SOD1 toxicity in the central nervous system, and the subsequent retraction of neurons from NMJs, is what causes disease, Parone said. Other researchers agree. “The results clearly demonstrate that there is a dissociation between the health of muscle fibers and the denervation process,” wrote Giovanni Manfredi in an e-mail to Alzforum (see full comment below). Manfredi, at Weill Medical College of Cornell University, New York, was not involved in the study.

However, the paper supports the concept that muscle cells suffer direct toxicity from mSOD1, wrote Lee Martin, of the Johns Hopkins Medical School in Baltimore, Maryland, in an e-mail to Alzforum (see full comment below). Martin’s work has supported the contribution of muscles to ALS-like pathology (Wong and Martin, 2010). While that toxicity could be due to mitochondrial dysfunction, as explored by the Cleveland team, it could also be the result of other mechanisms such as misregulated RNA processing, Martin added. Antonio Musarò of the University of Rome, Italy, whose studies have also shown that muscle suffers in the presence of mSOD1 (Dobrowolny et al., 2008), argued in an e-mail to Alzforum that this makes muscles a primary target of the mutant protein, but agreed with the Cleveland team that mSOD1 muscle toxicity is not the main cause of neurodegeneration (see full comment below).

The results, the authors wrote, suggest that amping up muscle PGC-1α expression could improve quality of life for people with ALS, allowing them greater movement for longer. “Anything that could make the muscles stronger…that would translate into a very meaningful treatment,” said Robert Miller of the Forbes Norris MDA/ALS Research Center at the California Pacific Medical Center in San Francisco. Miller was not involved in the Cleveland paper. At this point, there are no drugs specific for PGC-1α that would be appropriate, Parone and Da Cruz said.

There are other options. For example, blocking myostatin activity improved strength and muscle size, but not survival, in ALS mice (Morrison et al., 2009; Holzbaur et al., 2006). Scientists are already testing another muscle-maximizing therapy, CK-2017357, in people (see ARF related news story on Russell et al., 2012). Although model mice with very severe motor neuron disease derive no survival benefit from these treatments, it remains possible that in people, the treatment would extend life by keeping the diaphragm working for longer, speculated Jeremy Shefner of the State University of New York Upstate Medical University in Syracuse. Shefner is lead investigator on a clinical trial in ALS—Amber Dance.

References:
Da Cruz S, Parone PA, Lopes VS, Lillo C, McAlonis-Downes M, Lee SK, Vetto AP, Petrosyan S, Marsala M, Murphy AN, Williams DS, Spiegelman BM, Cleveland DW. Elevated PGC-1β activity sustains mitochondrial biogenesis and muscle function without extending survival in a mouse model of inherited ALS. Cell Metabolism. 2012 May 2;15:778-86. Abstract

Johri A, Beal MF. Muscling in on PGC-1β for improved quality of life in ALS. Cell Metabolism. 2012 May 2;15:567-9. Abstract

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  1. The question that nobody has answered yet is this: Are the motor neurons the first and sole direct targets of ALS?

    In my opinion, the answer is no, based on the following considerations:

    1. Lino and coworkers (Lino et al., 2002) reported that accumulation of SOD1 mutants in postnatal motor neurons does not cause motor neuron pathology or motor neuron disease.

    2. In another work, Pramatarova et al. reported a similar result: "Neuron-specific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment" (Pramatarova et al., 2001). The authors concluded that the accumulation of mutant SOD1 in postnatal motor neurons is thus not sufficient and probably also not critical to induce or accelerate motor neuron disease in FALS mice.

    3. These studies suggest/propose that mutant SOD1 causes the degeneration of motor neurons by a combination of cell-autonomous and non-cell-autonomous processes, requiring the presence of mutant SOD1 in both neurons and glia, and raising the question of whether mutant SOD1 expression in neurons is sufficient to induce disease.

    4. More recently, another group performed a similar experiment: Jaarsma et al. (Jaarsma et al., 2008) have demonstrated that transgenic mice in which mutant "SOD1 was largely restricted to neurons," under the transcriptional control of Thy1.2 promoter, "developed disease but only at an old age." However, "the disease progressed slowly without reaching the same degree of paralysis compared to the classical animal model of ALS in which the same mutant SOD1 gene is ubiquitously expressed."

    So, what is the truth? These studies do not indicate a clear answer, but suggest that the situation is more complicated and the toxicity is not just limited to a single cell type, since in ALS patients and ALS animal models, the mutated toxic form of SOD1 is expressed in several tissues and not just limited to motor neurons.

    What About Skeletal Muscle?
    This issue has been recently investigated by our laboratory, which has demonstrated that muscle-selective expression of SOD1 mutation causes pathological alterations and induces presymptomatic sign of ALS (Dobrowolny et al., 2008). Moreover, other studies support the evidence that skeletal muscle is a primary target of mutant SOD1 toxicity in mice. Wong and Martin have reported that skeletal-muscle-restricted expression of the human mutant SOD1 gene causes motor neuron degeneration in old transgenic mice (Wong and Martin, 2010). Dupuis et al. (Dupuis et al., 2009) have reported that muscle-selective alterations in mitochondrial function might initiate NMJ destruction, which is followed by distal axonopathy, astrocytosis in the spinal cord, and mild motor neuron loss. Moreover, Zhou et al. (Zhou et al., 2010) have reported that alterations in the potential of the mitochondrial inner membrane of fiber segments near NMJs occur in young SOD1G93A mice prior to disease onset. All of the above suggests that skeletal muscle is an important candidate to consider as a primary target of the toxicity that results from mutations in the SOD1 gene.

    In the paper by Cleveland and coworkers, the authors reported that sustained mitochondrial biogenesis and muscle function do not extend survival in a mouse model of inherited ALS. The authors concluded: "Our evidence refutes such a conclusion, demonstrating to the contrary that sustained improvement in muscle activity, including a doubling in endurance, increased energy supply from the mitochondria in muscles, and reducing muscle atrophy throughout ALS-like disease does not prevent or delay retraction of the axons from neuromuscular junctions, loss of motor axons, or death of motor neurons."

    This is, to me, a bias of the authors.

    To my knowledge I do not think that anybody succeeded to cure an ALS mouse model simply by acting on motor neurons, but this does not mean that motor neurons are not primary targets of mutant SOD1 toxicity.

    Moreover, several studies demonstrated that the toxicity of mutant SOD1 is associated with severe alterations (at morphological, functional, and molecular levels) of the tissue in which it is expressed, and since the selective expression of mutant SOD1 induces muscle atrophy, muscle mitochondrial dysfunction, reduced muscle strength, muscle damage, and presymptomatic signs of ALS in the spinal cord of MLC/SOD1G93A mice (Dobrowolny et al., 2008), this is sufficient to me to conclude that skeletal muscle is a primary target (not the sole) of SOD1-mediated toxicity.

    In addition, the fact that increasing PGC-1α activity in the muscles of SOD1 mutant expressing mice produces significantly increased muscle endurance, reduced atrophy, and improved locomotor activity, even at late stages of disease, suggests that the toxic properties of mutant SOD1 in the muscle can be significantly reduced. Of course, this is not sufficient to extend the survival of the ALS mouse model, because the mutant SOD1 protein is also expressed in the spinal cord of transgenic animals, and this is, in my opinion, sufficient to exert its toxic properties.

    Nevertheless, in the paper by Cleveland and coworkers, the authors concluded that “improving muscle activity and reducing atrophy may be effective to improve or preserve daily functioning and quality of life for ALS patients.” This is a good starting point to design more appropriate therapeutic strategies to treat ALS patients.

    Putting together all of this information, I think:

    1. the improvement in muscle mass and function is not sufficient to cure/attenuate significantly the progression of the disease (unless the muscle produces neurotrophic factors, such as IGF-1, that might activate survival pathways at the level of spinal cord; see Dobrowolny et al., 2005; Kaspar et al., 2003);

    2. to date any effort performed to cure ALS mouse models acting specifically on motor neurons failed miserably;

    3. ALS is a multisystemic disease which involves different cell types and tissues, and, in my opinion, we can really attenuate the progression of the disease if we can start to consider using a combinatorial therapeutic approach, acting on motor neurons, glia, and muscle.

    References:

    . Accumulation of SOD1 mutants in postnatal motoneurons does not cause motoneuron pathology or motoneuron disease. J Neurosci. 2002 Jun 15;22(12):4825-32. PubMed.

    . Neuron-specific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment. J Neurosci. 2001 May 15;21(10):3369-74. PubMed.

    . Neuron-specific expression of mutant superoxide dismutase is sufficient to induce amyotrophic lateral sclerosis in transgenic mice. J Neurosci. 2008 Feb 27;28(9):2075-88. PubMed.

    . Skeletal muscle is a primary target of SOD1G93A-mediated toxicity. Cell Metab. 2008 Nov;8(5):425-36. PubMed.

    . Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum Mol Genet. 2010 Jun 1;19(11):2284-302. PubMed.

    . Muscle mitochondrial uncoupling dismantles neuromuscular junction and triggers distal degeneration of motor neurons. PLoS One. 2009;4(4):e5390. PubMed.

    . Hyperactive intracellular calcium signaling associated with localized mitochondrial defects in skeletal muscle of an animal model of amyotrophic lateral sclerosis. J Biol Chem. 2010 Jan 1;285(1):705-12. PubMed.

    . 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.

    . Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science. 2003 Aug 8;301(5634):839-42. PubMed.

  2. I think that these results clearly demonstrate that there is a dissociation between the health of muscle fibers and the denervation process. The former can be prevented by activating mitochondrial biogenesis, but the latter cannot. The conclusion is that enhancing mitochondrial biogenesis exclusively in muscle does not help the motor neurons to maintain their contact with the muscle fibers. There is no benefit on survival, but there is likely an improvement in the quality of life (protection of muscle strength).

    There are some intriguing questions that are not discussed in the manuscript.

    1. Is SOD1 in muscle the cause of muscle atrophy, or is that due to loss of innervation? The results appear to suggest that the former is the case, and that mitochondrial biogenesis in muscle can prevent it. However, it is unclear how this relates to the previous study in which the authors knocked down SOD1 in muscle only.

    2. If muscle mass is preserved, what is the nature of the weight loss that is used as a marker of disease onset in this study. Has lipid metabolism changed, and have the mice lost body fat? Could something else be going on?

    3. The paper does not explore the effects of PGC-1α expression in the CNS. Would that be protective? If a pharmacological approach to enhance mitochondrial biogenesis was to be attempted in patients, it would likely have systemic effects, not limited to muscle.

  3. The recent article by Da Cruz et al. is very exciting in that it supports the concept that skeletal muscle is a primary site of toxicity of ALS-linked mutant SOD1 (Dobrowolny et al., 2008; Wong and Martin, 2010). Moreover, the paper by Da Cruz et al. concludes, as did earlier papers (Dobrowolny et al., 2008; Wong and Martin, 2010), that skeletal muscle could be a tissue target for disease-modifying or palliative therapy in human ALS. Thus, this more recent work vindicates these earlier experiments. The enforcement of PGC-1α, a key regulator of mitochondrial biogenesis and function, in skeletal muscle, however, did not extend survival of the mutant SOD1 mice. This is encouraging news, and not contrary to the skeletal muscle hypothesis for ALS mechanisms, because it suggests that the mechanisms of disease in mutant SOD1 in skeletal muscle are not likely to be driven by mitochondrial pathobiology. The paper by Da Cruz et al. fails to rule out many other possible mechanisms of disease in skeletal muscle, including myofiber nucleus-based pathobiology, which has been recently identified in the CNS of mouse models of ALS (Gertz et al., 2012), as well as faulty RNA processing in skeletal muscle.

    References:

    . Skeletal muscle is a primary target of SOD1G93A-mediated toxicity. Cell Metab. 2008 Nov;8(5):425-36. PubMed.

    . Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum Mol Genet. 2010 Jun 1;19(11):2284-302. PubMed.

    . Nuclear localization of human SOD1 and mutant SOD1-specific disruption of survival motor neuron protein complex in transgenic amyotrophic lateral sclerosis mice. J Neuropathol Exp Neurol. 2012 Feb;71(2):162-77. PubMed.

References

News Citations

  1. More Power! Mechanisms of a Muscle-Maxing Medicine

Paper Citations

  1. . Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum Mol Genet. 2010 Jun 1;19(11):2284-302. PubMed.
  2. . Peroxisome proliferator activator receptor gamma coactivator-1alpha (PGC-1α) improves motor performance and survival in a mouse model of amyotrophic lateral sclerosis. Mol Neurodegener. 2011;6(1):51. PubMed.
  3. . PGC-1α protects neurons and alters disease progression in an amyotrophic lateral sclerosis mouse model. Muscle Nerve. 2011 Dec;44(6):947-56. PubMed.
  4. . Skeletal muscle is a primary target of SOD1G93A-mediated toxicity. Cell Metab. 2008 Nov;8(5):425-36. PubMed.
  5. . A soluble activin type IIB receptor improves function in a mouse model of amyotrophic lateral sclerosis. Exp Neurol. 2009 Jun;217(2):258-68. PubMed.
  6. . Myostatin inhibition slows muscle atrophy in rodent models of amyotrophic lateral sclerosis. Neurobiol Dis. 2006 Sep;23(3):697-707. PubMed.
  7. . Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases. Nat Med. 2012 Mar;18(3):452-5. PubMed.
  8. . Elevated PGC-1α activity sustains mitochondrial biogenesis and muscle function without extending survival in a mouse model of inherited ALS. Cell Metab. 2012 May 2;15(5):778-86. PubMed.
  9. . Muscling in on PGC-1α for improved quality of life in ALS. Cell Metab. 2012 May 2;15(5):567-9. PubMed.

External Citations

  1. clinical trial

Further Reading

Papers

  1. . Muscling in on PGC-1α for improved quality of life in ALS. Cell Metab. 2012 May 2;15(5):567-9. PubMed.
  2. . Elevated PGC-1α activity sustains mitochondrial biogenesis and muscle function without extending survival in a mouse model of inherited ALS. Cell Metab. 2012 May 2;15(5):778-86. PubMed.
  3. . Mitochondrial dysfunction and amyotrophic lateral sclerosis. Muscle Nerve. 2006 May;33(5):598-608. PubMed.
  4. . Metabolic control through the PGC-1 family of transcription coactivators. Cell Metab. 2005 Jun;1(6):361-70. PubMed.
  5. . Gene transfer demonstrates that muscle is not a primary target for non-cell-autonomous toxicity in familial amyotrophic lateral sclerosis. Proc Natl Acad Sci U S A. 2006 Dec 19;103(51):19546-51. PubMed.
  6. . Mitochondria in amyotrophic lateral sclerosis: a trigger and a target. Neurodegener Dis. 2004;1(6):245-54. PubMed.
  7. . 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.
  8. . Hyperactive intracellular calcium signaling associated with localized mitochondrial defects in skeletal muscle of an animal model of amyotrophic lateral sclerosis. J Biol Chem. 2010 Jan 1;285(1):705-12. PubMed.

Primary Papers

  1. . Muscling in on PGC-1α for improved quality of life in ALS. Cell Metab. 2012 May 2;15(5):567-9. PubMed.
  2. . Elevated PGC-1α activity sustains mitochondrial biogenesis and muscle function without extending survival in a mouse model of inherited ALS. Cell Metab. 2012 May 2;15(5):778-86. PubMed.