Regeneration of Neuromuscular Synapses

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the loss of both upper and lower motor neurons, denervation of target muscles, paralysis, and respiratory failure.1,2 Decades of research have seen significant advances in ALS genetics, as mutations in SOD1 gene (encoding copper–zinc superoxide dismutase) have been identified to associate with an autosomal dominant form of ALS. Nevertheless, the pathogenesis and pathophysiology of the disease is largely unknown and the prognosis of ALS patients remains very poor. Recently, investigators from University of Texas Southwestern Medical Center discovered a novel pathway for neuromuscular synapse reparation regulated by microRNA-206 in an ALS mouse model. This study was published in Science3 and may represent an important new frontier in neuro-regeneration and neuro-rehabilitation. First described in 1993, microRNA's (miRNA) are small RNA sequences that are approximately 22 nucleotides in length.4,5 They were shown to operate as post-transcriptive modulators and could repress translation by interacting with the 3′ un-translated regions of specific mRNA targets.4,6 It has been reported that miRNA's play an important role in the pathophysiology of cancer7 and heart disease.6 In the current study, Williams et al focused on microRNA-206, which was normally expressed in skeletal muscles exclusively, and used multiple transgenic mouse models to study its function in promoting regeneration of neuromuscular synapses. The authors first observed that, in an ALS mouse model G93A-SOD1, miRNA-206 expression was dramatically up-regulated compared with wide-type (WT) mice, and that the up-regulation coincided with the onset of neurological symptoms. Next, they surgically severed the sciatic nerves in WT, healthy mice and found a robust increase in miRNA-206 levels in fast-twitch muscle fibers 10 days after the initial injury. In ordered to study the developmental importance of miRNA-206, the investigators created transgenic mice that were homozygous for targeted deletion of miRNA-206 (miR-206−/−) and those that were miR-206 knock-out and expressed the G93A-SOD1 transgene (miR-206−/−; G93A–SOD1). The loss of miR-206 gene in a otherwise regular mouse did not affect its weight, behavior, or muscle development, but an miR-206−/−; G93A–SOD1 mouse demonstrated much accelerated ALS disease progression and significantly shortened survival. Moreover, these mice showed overwhelming atrophy of skeletal muscles, resulting in remarkable kyphosis and death (Fig. 1A). Therefore, the presence of miRNA-206 seemed to counteract the pathogenesis of ALS.FIGURE: (A) G93A-SOD1 and miR-206−/−; G93A–SOD1 mice at 7.5 months of age. X-ray demonstrates severe kyphosis in miR-206−/−; G93A–SOD1 mice, and Wheat-germ agglutinin (WGA) staining reveals accelerated muscle atrophy in miR-206−/−; G93A–SOD1 mice. Adapted from Figure 2 of the original article. (B) Immunohistochemistry co-localization with synaptotagmin 2 (Syt 2) staining (green) with BTX (red) staining. There is increased NMJ dysfunction and denervation in miR-206−/−; G93A–SOD1 mice compared with G93A-SOD1 ones. Adapted from Figure 3 of the original article. (C) Proposed mechanism of miRNA-206–dependent reinnervation. Adapted from Figure 4 of the original article.How should one explain the apparent beneficial effects of miRNA-206 on ALS? The authors turned attention to the structure whose functional integrity was critical to motor control of skeletal muscles: the neuromuscular junction (NMJ). Interestingly, the architecture of NMJ's in a miR-206−/− mouse was essentially normal, but depletion of miRNA-206 resulted in significantly slower re-innervation to leg muscles after sciatic nerve severance. Using immunohistochemistry co-localization techniques, the authors demonstrated that miRNA-206 profoundly influenced formation of new NMJ's after nerve injury, and that miR-206−/− mice had delayed presynaptic differentiation and partial reoccupancy of postsynaptic sites. This phenomenon was exacerbated in miR-206−/−; G93A–SOD1 mice, which exhibited increased NMJ dysfunction and irreversible denervation in skeletal muscles (Fig. 1B). Williams et al went further to decipher the downstream targets of miR-206. By producing a knockout mouse lacking histone deacetylase 4 (HDAC4) and using RNA interference technology, they found that miRNA-206 reduced HDAC4 translation and consequently increased the amount of fibroblast growth factor binding protein 1 (FGFBP1) secreted from the muscle fiber. FGFBP1 could potentiate the effects of FGFs to promote presynaptic differentiation at the NMJ.8 The authors postulated that the miRNA-HDAC4 pathway regulated the susceptibility to reinnervation of a muscle fiber and could be a key modulator to achieve the balance between sufficient tissue repair and hyperinnervation after nerve injuries (Fig. 1C). This elegant study not only is a vital breakthrough in understanding the pathophysiology of ALS, it also bears crucial implications in other pathological processes involving motor neuron damage. It is hopeful that continued advancements in microRNA technology may create new pharmatheutical targets and eventually conquer this deadly disease. NING LIN ROBERT M. FRIEDLANDER