Muscle MicroRNA Repairs Nerve-Muscle Connection in ALS Model

It may be time to add “slowing amyotrophic lateral sclerosis” to the growing list of tasks accomplished by microRNAs, the tiny snippets of nucleic acid that can silence genes. MicroRNA activity has been linked to many neurodegenerative conditions (Nelson et al., 2008), but a study published today in Science adds a bit of a twist: the miRNA in question, miR-206, is actually expressed in muscle, not the nervous system. Led by principal investigator Eric Olson at the University of Texas Southwestern Medical Center in Dallas, the authors showed that ALS model mice lacking miR-206 decline more quickly than those that have the miRNA. Robert Brown of the University of Massachusetts Medical School in Worcester penned a perspective accompanying the Science article.

“Our paper suggests that factors expressed and secreted from skeletal muscle have a positive role in maintaining the signaling between motor neuron and skeletal muscle during injury and disease,” wrote Andrew Williams of UT Southwestern, joint first author on the paper with Gregorio Valdez of Harvard University, in an e-mail to ARF.

“This appears to introduce a new paradigm: a disease-relevant mechanism in which the post-synaptic segment can influence the presynaptic segment with a non-coding RNA serving as an intermediary,” said Peter T. Nelson of the University of Kentucky in Lexington, who was not involved with the research.

The authors searched for miRNAs involved in amyotrophic lateral sclerosis (ALS) by comparing expression of 320 miRNAs in the lower limb muscles of normal mice and a common model for the disease, animals expressing human superoxide dismutase 1 (SOD1) with a G93A mutation. They found that miR-206 was upregulated to more than three times its normal expression levels with the onset of disease. Expression of miR-206 shot up more than 10-fold when the researchers severed the sciatic nerve of healthy mice, suggesting it may be part of a common response to nerve injury.

The researchers then made miR-206 knockout mice to assess its function. The developed normal neuromuscular junctions (NMJs) and were healthy, suggesting the miRNA is not required for normal NMJ function. However, these knockout mice were slower to reinnervate neuromuscular junctions following injury. In the injured knockout animals, normal numbers of axons grew toward their muscle but sometimes overshot the junction, suggesting they were missing some signal from the muscle end to target their growth.

To further investigate the role of miR-206 in ALS, the scientists crossed the SOD1-G93A animals with the miR-206 knockouts. Comparing these double mutants to single mutants, they found that disease began at the same age of around six months. However, the double mutants declined faster, surviving for a mean of 244 days, compared to 266 days for single mutants. The double mutants also showed increased atrophy of skeletal muscle. The researchers concluded that miR-206 is likely upregulated in ALS to promote NMJ recovery, slowing disease.

Next, the study authors delved into the molecular pathway between miR-206 and muscle reinnervation. Computer models predict that histone deacetylase 4 (HDAC4) is a likely target for miR-206 (Lewis et al., 2005). HDAC4 is concentrated in NMJs, and its expression and gene-activating activity is induced by denervation (Cohen et al., 2007). Williams and colleagues first examined the relationship between miR-206 and HDAC4 in cultured kidney cells. They linked the HDAC4 3’-untranslated region to a luciferase reporter and showed that miR-206 expression decreased HDAC activity, confirming that miR-206 downregulates this epigenetic enzyme.

Animal studies provided further evidence for the miR-206/HDAC4 interaction. In the miR-206 knockout mice, HDAC protein levels were up compared with wild-type mice. HDAC4 knockouts showed the opposite phenotype of miR-206 knockouts: They recovered more quickly from nerve injury. The data suggest that HDAC4 and miR-206 have opposing functions, where the former has a negative effect on NMJ recovery and the latter counteracts this, allowing recovery to proceed.

To look for downstream effectors, the researchers searched for genes that were differentially regulated in the miR-206 knockouts versus the HDAC4 knockouts. They found that expression of fibroblast growth factor binding protein 1 (FGFBP1) was down in the miR-206 knockouts, and up in HDAC4 knockouts. FGFBP1 supports the activity of muscle growth factors that promote formation of the neuromuscular junction.

The work suggests a pathway whereby, in response to injury or degeneration, muscle tissue upregulates miR-206, which indirectly supports recovery by blocking HDAC4. With the deacetylase out of the way, FGFBP1 and the fibroblast growth factors are free to promote reinnervation.

“This work contributes significantly to our knowledge of microRNA function in health and disease,” wrote Sébastien Hébert of Laval University in Québec, Canada, who was not involved in the study, in an e-mail to ARF (see full comment below). One challenge in miRNA research is that one miRNA may regulate dozens or hundreds of genes. “It is important to keep in mind that the observed pathological effects in vivo often result from the abnormal fine-tuning of a limited number of ‘key’ genes.” In the current work, HDAC4 seems to account for the bulk of miR-206’s effects, greatly simplifying study of pathology.

Hébert also noted that miR-206 is but a sideshow in the sense that the work does not provide clues as to the primary cause of neurodegeneration in ALS. However, Williams pointed out that because NMJ degeneration is common to all people with ALS, miR-206 is likely to be important for both sporadic and familial cases.

That assumes, of course, that humans parallel mice on that score. If the miR-206 pathway holds true in people, upregulation of miR-206 might make a useful biomarker for diagnosis and monitoring of ALS, Williams suggested. Increasing miR-206 might also eventually provide a treatment. The predominant muscle expression of the miRNA provides two potential advantages for such an approach. One, therapies targeting miR-206 might avoid serious side effects elsewhere in the body. Two, treatments aimed at miR-206 would not have to cross the blood-spinal cord barrier, neatly sidestepping one of the greatest hurdles in treating neurodegenerative disease.

However, there is a caveat. Symptoms of ALS present only after many nerves have already died, and even then, diagnosing the disease can take months. By the time people receive treatment, would there be enough NMJs left for miR-206 to do any good? “We are hopeful, but unsure,” Williams wrote. In addition, miR-206 only delays disease; so amping its expression would not provide a cure. The researchers are currently testing the effect of miR-206 upregulation and HDAC4 inhibition in ALS mice.—Amber Dance

Comments

  1. The Olson group is a pioneer in the field of microRNA function in muscle cells. Here, the authors provide compelling evidence that miR-206, a skeletal muscle-specific “myomiR,” functions in a complex regulatory pathway to regulate ALS pathology in mice. The strength of this paper relies on the use of different mouse models, including miR-206 knockouts, to characterize the signaling pathway in vivo.

    To obtain insights into the mechanism(s) involved in muscle degeneration in ALS, the authors performed a microRNA array from ALS mice, which harbor the familial SOD1 G93A mutation. MiR-206 was the most significantly changed (upregulated) miRNA in this screen. It is interesting to note that other myomiRs, including miR-1, miR-133b, and miR-133a were, albeit at weaker levels, downregulated in the array; however, the authors could confirm by quantitative PCR the upregulation of miR-206 and miR-133b (which are co-expressed from the same transcript) in the diseased mice. MiR-206 upregulation coincided with denervation and ALS pathology in the mutant mice. To make a long story short, the group identified both upstream (MyoD) and downstream (HDAC4, FGFBP1) effectors of the miR-206 network in vivo. Overall, an elegant study!

    This work contributes significantly to our knowledge with regard to microRNA function in health and disease. Although individual microRNAs can target up to several hundred genes, it’s important to keep in mind that the observed pathological effects in vivo often result from the abnormal fine-tuning of a limited number of "key" genes. With regard to the current study, the effects of miR-206 in reinnervation could be explained, in most part, by the misregulation of HDAC4 (a miR-206 target). One could hypothesize that this mode of “targeted” pathological regulation could function in Alzheimer disease brain, where, for instance, miR-29 could contribute to plaque load by modulating BACE1 regulation.

    Although well structured, this study does not, in my opinion, address the primary cause of motor neuron degeneration in ALS (mice or humans). An interesting follow-up study would be to look for changes in microRNA expression in the CNS. In this sense, this study lacks validation of microRNA (and target gene) expression in humans. Notably, the miR-206/miR-133b locus was originally described as a synapse-associated non-coding RNA (7H4)(see current study). Interestingly, it has been documented that miR-206 can become overexpressed in the brain after cellular insult (Jeyaseelan et al., 2008; Zhang and Pan, 2009). Also, one study associates miR-206 with schizophrenia (Hansen et al., 2007). In the current study, axonal regeneration seemed unaffected in the miR-206 knockout mice. Clearly, future studies are needed to elucidate the role of microRNAs, particularly miR-206, in both neuronal and muscle loss.

    References:

    Hansen T, Olsen L, Lindow M, Jakobsen KD, Ullum H, Jonsson E, Andreassen OA, Djurovic S, Melle I, Agartz I, Hall H, Timm S, Wang AG, Werge T. Brain expressed microRNAs implicated in schizophrenia etiology. PLoS One. 2007;2(9):e873. PubMed.

    Jeyaseelan K, Lim KY, Armugam A. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke. 2008 Mar;39(3):959-66. PubMed.

    Zhang B, Pan X. RDX induces aberrant expression of microRNAs in mouse brain and liver. Environ Health Perspect. 2009 Feb;117(2):231-40. PubMed.

    View all comments by Sébastien S. Hébert

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References

Paper Citations

  2. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell. 2005 Jan 14;120(1):15-20. PubMed.
  3. Cohen TJ, Waddell DS, Barrientos T, Lu Z, Feng G, Cox GA, Bodine SC, Yao TP. The histone deacetylase HDAC4 connects neural activity to muscle transcriptional reprogramming. J Biol Chem. 2007 Nov 16;282(46):33752-9. PubMed.

Further Reading

Papers

  1. Simon DJ, Madison JM, Conery AL, Thompson-Peer KL, Soskis M, Ruvkun GB, Kaplan JM, Kim JK. The microRNA miR-1 regulates a MEF-2-dependent retrograde signal at neuromuscular junctions. Cell. 2008 May 30;133(5):903-15. PubMed.
  2. Hébert SS, Horré K, Nicolaï L, Papadopoulou AS, Mandemakers W, Silahtaroglu AN, Kauppinen S, Delacourte A, De Strooper B. Loss of microRNA cluster miR-29a/b-1 in sporadic Alzheimer's disease correlates with increased BACE1/beta-secretase expression. Proc Natl Acad Sci U S A. 2008 Apr 29;105(17):6415-20. Epub 2008 Apr 23 PubMed.
  3. Wang WX, Rajeev BW, Stromberg AJ, Ren N, Tang G, Huang Q, Rigoutsos I, Nelson PT. The expression of microRNA miR-107 decreases early in Alzheimer's disease and may accelerate disease progression through regulation of beta-site amyloid precursor protein-cleaving enzyme 1. J Neurosci. 2008 Jan 30;28(5):1213-23. PubMed.
  4. Kim J, Inoue K, Ishii J, Vanti WB, Voronov SV, Murchison E, Hannon G, Abeliovich A. A MicroRNA feedback circuit in midbrain dopamine neurons. Science. 2007 Aug 31;317(5842):1220-4. PubMed.
  5. Schaefer A, O'Carroll D, Tan CL, Hillman D, Sugimori M, Llinas R, Greengard P. Cerebellar neurodegeneration in the absence of microRNAs. J Exp Med. 2007 Jul 9;204(7):1553-8. PubMed.

Primary Papers

  1. Williams AH, Valdez G, Moresi V, Qi X, McAnally J, Elliott JL, Bassel-Duby R, Sanes JR, Olson EN. MicroRNA-206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science. 2009 Dec 11;326(5959):1549-54. PubMed.
  2. Brown RH. Medicine. A reinnervating microRNA. Science. 2009 Dec 11;326(5959):1494-5. PubMed.

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