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    Topic review

    Age-Related Alterations at Neuromuscular Junction

    Subjects: Cell Biology
    View times: 15

    Definition

    With advancing aging, a decline in physical abilities occurs, leading to reduced mobility and loss of independence. Although many factors contribute to the physio-pathological effects of aging, an important event seems to be related to the compromised integrity of the neuromuscular system, which connects the brain and skeletal muscles via motoneurons and the neuromuscular junctions (NMJs). NMJs undergo severe functional, morphological, and molecular alterations during aging and ultimately degenerate. The effect of this decline is an inexorable decrease in skeletal muscle mass and strength, a condition generally known as sarcopenia. Moreover, several studies have highlighted how the age-related alteration of reactive oxygen species (ROS) homeostasis can contribute to changes in the neuromuscular junction morphology and stability, leading to the reduction in fiber number and innervation. Increasing evidence supports the involvement of epigenetic modifications in age-dependent alterations of the NMJ. In particular, DNA methylation, histone modifications, and miRNA-dependent gene expression represent the major epigenetic mechanisms that play a crucial role in NMJ remodeling. It is established that environmental and lifestyle factors, such as physical exercise and nutrition that are susceptible to change during aging, can modulate epigenetic phenomena and attenuate the age-related NMJs changes.

    1. Introduction

    The decline in physical ability and reduced mobility represent hallmarks of aging. While several events are known to contribute to muscle loss during aging, including chronic low-grade inflammatory status [1], reduced protein synthesis [2], mitochondrial dysfunction [3], and impaired satellite cell function [4], it is generally accepted that the decline in neural function is one of the principal factors leading to the pathological status known as sarcopenia [5][6][7]. Indeed, the nervous system not only controls muscle contraction and voluntary movements [8] but also affects myoblast orientation [9], skeletal muscle fiber specification, and myosin heavy chain (MHC) isoform expression [10]. Skeletal muscle fibers are classified as fast or slow according to the motor neuron activity, their distinct oxidative capacities, and ATPase activities [11]. These properties may be altered by a decrease in motor units, denervation of muscle fibers, and changes in NMJs [12]. Indeed, slow muscle phenotype is promoted by tonic motor neuron activity, while infrequent motor neuron firing results in fast fiber generation [12]. Moreover, cross reinnervation experiments demonstrated that fast muscles switch into slow ones when re-innervated by a slow nerve, whereas slow muscles switch into fast ones once re-innervated by fast nerve [13][14]. It has been reported by Klitgaard et al. that co-expression of different isoforms of MHC in a single muscle fiber increases with age, suggesting a continuous shift in the type of muscle fiber [15], and it is demonstrated that muscles in elderly individuals contain more slow muscle fibers that reduce contraction ability [16]. This also supports endplate fragmentation and functional denervation [17].
    The inability of aged motor axons to efficiently re-innervate muscle fibers following bouts of degeneration [18] contributes to the atrophy of muscle fibers and loss of motor neurons. Motor neuron loss accompanied by muscle fiber loss may result in a consequent age-related increase in intermuscular fat and fibrous tissue [19][20], a minor degree of muscle fiber grouping [21], and a decrease in muscle size [6][22].
    One possible mechanism of this progressive impairment of the re-innervation process seems related to age-associated degeneration of NMJ. NMJ is a synaptic connection where the peripheral nervous system contacts skeletal muscle fibers, thus controlling essential processes such as breathing and body voluntary movements [8]. Significant deterioration, including postsynaptic apparatus fragmentation, denervation, multiple innervations, and axon sprouting, has been observed in this critical region with advancing age [23][24][25]. Interestingly, similar defects characterize several severe motor neuron diseases such as amyotrophic lateral sclerosis (ALS), suggesting that strategies aimed to preserve NMJ integrity may be crucial in understanding the critical components involved in aging and ALS [26][27].
    Although skeletal muscle fibers are characterized by having many nuclei, only those placed in the proximity of the NMJ are important for the transcription of the genes involved in NMJ structure, function, and maintenance [28][29][30]. The correct localization of synaptic nuclei at the NMJ requires the interplay between the cytoskeleton and several nuclear envelope proteins to effectively transduce cytoskeletal forces to the nucleus to regulate signaling pathways, chromatin organization, and gene expression [31][32][33][34]. It has been demonstrated that lamin A/C, an intermediate filament involved in establishing a continuous physical link between the nuclear lamina and the cytoskeleton [35][36][37][38], is reduced in aged muscles, and its mutations are implicated in premature aging disorders [39][40][41]. In particular, a recent paper published by Gao et al. demonstrated that muscle-specific lamin A/C mutation results in progressive denervation, AChR cluster fragmentation, and neuromuscular dysfunction [42]. These results demonstrate that lamin A/C is critical in the maintenance of NMJ and suggest that alterations in the cytoskeleton-nuclear network may contribute to NMJ degeneration in aged muscle.
    Increasing evidence supports the involvement of epigenetic modifications in age-dependent morphological and functional alterations of the NMJ [25][43][44]. In particular, DNA methylation, histone modifications, and miRNA-dependent gene expression represent the major epigenetic mechanisms that play a crucial role in NMJ remodeling [45][46][47].

    2. Age-Related Changes in NMJ

    Three principal elements compose NMJ: the presynaptic motor nerve terminals packed with synaptic vesicles containing the neurotransmitter acetylcholine (ACh); the synaptic cleft; and the deeply folded postsynaptic membrane of the muscle fiber, also referred to as the endplate, where acetylcholine receptors (AChRs) are located. When an action potential reaches the pre-synaptic element, voltage-dependent calcium channels open, allowing calcium to trigger the delivery of ACh in the synaptic cleft. Acetylcholine triggers AChR located in the post-synaptic membrane to produce an action potential, which in turn activates voltage-gated dihydropyridine receptors (DHPRs) located in the sarcolemma, and subsequently ryanodine receptors (RyRs) located in the sarcoplasmic reticulum membrane. The nerve terminal is covered by specialized glial cells, called terminal Schwann cells (tSCs), that produce a basal lamina that fuses with that of the muscle fiber at the boundary of the NMJ [48][49][50]. Moreover, fibroblast-like cells, known as kranocytes or perisynaptic fibroblasts, form a loose cover over the NMJ and participate in nerve repair and regeneration, thus representing other crucial players in neuromuscular functionality [50][51] (Figure 1).
    Figure 1. Schematic representation of NMJ architecture. NMJ is composed of three major elements: the presynaptic motor nerve terminals packed with synaptic vesicles containing the neurotransmitter ACh; the synaptic cleft; and the deeply folded postsynaptic membrane of the muscle fiber, where AChRs are placed. The NMJ-associated cells, tSCs, and kranocytes are also shown.
    During aging, NMJs undergo dramatic morphological, functional, and molecular changes that lead them to ultimately degenerate [52][53][54]. At the level of the presynaptic region, increased axon diameter and larger nerve terminal area have been observed [23][55]. However, this effect is not accompanied by increased Ach stores, since it has been demonstrated that as presynaptic branching increases with age, the quantity of available Ach decreases [56][57]. At the postsynaptic level, the endplates decrease in size and are severely fragmented, and the number and length of postsynaptic folds are reduced, leading to a functional impairment of NMJ response [58]. Along with these modifications, a gradual decrease in the number of AChRs per junction has been observed [59]. Moreover, tSCs migrate away from the motor nerve terminal and protrude branches into the synaptic cleft, contributing to a functional decline of the neuromuscular system in aging [21][60].
    A key role in the communication between nerve and muscle is played by the agrin-muscle-specific kinase (MuSk)–low-density lipoprotein receptor-related protein 4 (Lrp4) signaling pathway, which ensures the correct transmission of the action potential from the nerve terminal to the endplate, thus facilitating myofibrillar excitation [61][62]. It has been demonstrated that the agrin–MuSk–Lrp4 signaling pathway may be dysregulated with advancing aging. Agrin is expressed at NMJs by nerve terminals, muscle fibers, and tSC, where it is necessary for clustering of AchRs in muscle fibers [63][64][65][66][67][68]. In a mouse experimental model, Agrin impaired expression resulted in NMJ fragmentation, similar to what was observed in aged NMJs and premature sarcopenia [69]. Moreover, it has been demonstrated that the expression levels of NMJ-associated genes MuSk and Lrp4 appear to increase in aged muscle [70]. Interestingly, these genes are also upregulated in the denervated skeletal muscle of young mice, suggesting that aging may cause denervation of NMJs [71].
    It has also been demonstrated that mitochondria dysfunction and oxidative stress, along with increased levels of intracellular calcium boost, determine the decline in NMJ [72][73], together with a reduced number of synaptic vesicles, similarly to what was observed in ALS [27][74][75][76].

    3. ROS and NMJ Degeneration

    Reactive oxygen species (ROS) are highly reactive molecules that are generated due to the electron receptivity of O2. In eukaryotic cells, endogenous production of ROS is associated with mitochondrial oxidative phosphorylation, by which a chain of redox reactions transports protons across the inner mitochondrial membrane, generating potential energy that accomplishes cellular ATP production [77].
    One of the major molecules that play an important role in mitochondrial function is calcium (Ca2+). During muscle contraction, a greater influx of Ca2+ into the mitochondria occurs, which results in the activation of specific enzymes that lead to an increased synthesis of ATP to accomplish the energy demand. Moreover, increased mitochondrial Ca2+ levels have also been observed in several pathological conditions such as skeletal muscle denervation or unloading. The result of this steady-state elevation of mitochondrial Ca2+ level leads to increased ROS generation, induction of programmed cell death, and ultimately muscle atrophy [78].
    In physiological conditions, a small number of electrons, passing through the chain, prematurely and incompletely reduces oxygen, producing the superoxide radicals; these are promptly scavenged by the cellular antioxidant enzymes, such as superoxide dismutase 1 (SOD1), to avoid oxidative damage of cellular macromolecules. Therefore, mitochondrial dysfunction and oxidative phosphorylation impairment determine the increase of ROS production and oxidative damage; these events, in turn, induce muscle cells to respond to the unbalanced redox homeostasis, upregulating the antioxidant enzymes [79]. Experimental evidence indicates that ROS may also act as chemical messengers, involved in receptor-mediated signaling pathways and transcriptional activation [80], suggesting that the cellular balance between ROS production and ROS scavenging is crucial to preserve cellular functionality [81].
    Abnormal ROS levels contribute to the etiology of a wide variety of disorders, including aging and neurodegenerative diseases, such as ALS, and most of them are characterized by incoming defects in muscle–nerve communication [82].
    Several animal models have been generated to elucidate the relation between oxidative damage and the alteration of the muscle–nerve endplate; among these are the transgenic mice overexpressing in skeletal muscle tissue the uncoupling protein 1 (UCP1), a mitochondrial protein that uncouples mitochondrial electron transport from ATP synthesis. These mice display deterioration of the NMJ, which correlates with progressive signs of muscle denervation and late-onset motor neuron pathology [83], demonstrating that a relatively mild mitochondrial dysfunction can recapitulate early signs of ALS disease, and it is sufficient to determine NMJ alteration and motor neuron degeneration.
    Several other studies have investigated the relationship between alteration of antioxidant defense and NMJ defects. Data from SOD1 knockout mice (SOD1−/−) [84] and mutant SOD1 gain-of-function models (SOD1G93A mice) [85][86] demonstrate the importance of SOD1 enzyme activity and ROS homeostasis in muscle–nerve communication. Indeed, it has been shown that the ubiquitous expression of the mutant human SOD1G93A gene induces a widespread spinal motor neuron death in mice. Conversely, the genetic ablation of the SOD1 gene (SOD1−/−) in mouse determines, at advanced ages, a motor neuropathy associated with distal motor axon degeneration, without loss of ventral motor neurons, confirming the crucial role of the SOD1 antioxidant activity in the maintenance of NMJ stability. Interestingly, muscle denervation in SOD1−/− strongly correlates with loss of mitochondria at the motor nerve terminal during postnatal life. The defect is rescued by the replacement of SOD1 enzyme in the mitochondrial inner membrane [87], indicating the close relationship between the alteration of mitochondria functionality, induced by oxidative stress, and defects in muscle–nerve communication.
    Although it is widely accepted that NMJ alteration during aging or ALS disease is closely related to axon and synapse damage, controversy exists on whether pathological events could begin at the skeletal muscle, potentially influencing the loss of NMJs and the degeneration of motor neurons. Indeed, the temporal analysis of axon and neuromuscular junction degeneration in an ALS mouse model indicated that motor neuron degeneration starts distally at the NMJ area and occurs earlier than clinical symptoms, proceeding towards neuron soma in a retrograde dying back manner [88].
    The transgenic mice MLC/SOD1G93A, overexpressing the SOD1G93A gene under the control of a muscle-specific promoter, provide further evidence to support the dying-back theory and the concept that skeletal muscle is a primary target of the toxic effect of the SOD1 mutant gene. Indeed, the transgenic MLC/SOD1G93A mice exhibit defects of the mitochondrial membrane potential and reduced integrity of the mitochondrial network, which are associated with higher turnover and fragmentation of the NMJ [75]. This evidence is corroborated by a recent work of Wong and colleagues who demonstrated that skeletal muscle-restricted expression of wild-type or mutant hSOD1 genes causes age-related ALS-like phenotype [89], that is, motor defects, distal axonopathy, NMJ pathology, and motor neuron loss, according to a non-autonomous mechanism for motor-neuron degeneration [90].
    The important role of the antioxidant enzymes in muscle and nerve communication has been investigated in several studies focused on ROS production during muscle contraction [91]. Contractile activity in muscles of young rodents leads to the extracellular release of ATP and the activation of an NADPH oxidase (NOX2) complex in the muscle post-synaptic terminal. NOX2 is responsible for superoxide and hydrogen peroxide (H2O2) production and regulates, together with ATP, the release of ACh from the motor nerve terminal [92]. NOX2 activates redox-sensitive transcription factors that in turn induce higher expression of antioxidant enzymes, such as SOD and catalase, to counteract oxidative damage and facilitate exercise-induced muscle tissue remodeling [93]. It has been reported that exercise, through the activation of mitogen-activated protein kinases (MAPKs) and through nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathways, can cause the expression of antioxidant enzymes that play a crucial role in the protection against ROS, as well as adaptation to exercise [94][95]. However, the beneficial effects of physical activity are lost with exhaustive endurance and resistance exercise since the increased levels of ROS observed in these conditions overwhelm cellular antioxidant defenses, leading to tissue damage [96][97][98][99]. Excessive ROS production during aging or neurodegenerative disorder is responsible not only for mitochondria damage but also for cellular protein oxidation (Figure 2). Elderly muscles accumulate damaged proteins and organelles, since the activity of the autophagy system, a regulated mechanism for the removal of unnecessary or dysfunctional components, declines during aging. Indeed, in a recent paper [100], the authors described how the inhibition of the autophagic flux enhances oxidative stress in young muscles and alters muscle force generation, inducing mitochondrial dysfunction and NMJ instability; in this context, autophagy emerges as a required mechanism for mitochondrial quality control and the correct interplay between muscle and nerve [101].
    Figure 2. Oxidative stress and NMJ instability. Excessive ROS production during aging or neurodegenerative disorder is responsible for the increased denervation and skeletal muscle atrophy, cellular protein oxidation, mitochondrial dysfunction, and impairment of autophagic flux, leading to NMJ instability.
    In conclusion, this evidence demonstrates that the age-related alteration of ROS homeostasis affects muscle–nerve communication; therefore, the maintenance of redox balance can be considered as a good therapeutic tool to counteract the decline of NMJ functionality of elderly individuals.

    The entry is from 10.3390/cells10061307

    References

    1. Chhetri, J.K.; de Souto Barreto, P.; Fougère, B.; Rolland, Y.; Vellas, B.; Cesari, M. Chronic inflammation and sarcopenia: A regenerative cell therapy perspective. Exp. Gerontol. 2018, 103, 115–123.
    2. Dickinson, J.M.; Volpi, E.; Rasmussen, B.B. Exercise and Nutrition to Target Protein Synthesis Impairments in Aging Skeletal Muscle. Exerc. Sport Sci. Rev. 2013, 41, 216–223.
    3. Marzetti, E.; Calvani, R.; Cesari, M.; Buford, T.W.; Lorenzi, M.; Behnke, B.J.; Leeuwenburgh, C. Mitochondrial dysfunction and sarcopenia of aging: From signaling pathways to clinical trials. Int. J. Biochem. Cell Biol. 2013, 45, 2288–2301.
    4. Sousa-Victor, P.; Muñoz-Cánoves, P. Regenerative decline of stem cells in sarcopenia. Mol. Aspects Med. 2016, 50, 109–117.
    5. Larsson, L.; Degens, H.; Li, M.; Salviati, L.; Lee, Y.I.; Thompson, W.; Kirkland, J.L.; Sandri, M. Sarcopenia: Aging-related loss of muscle mass and function. Physiol. Rev. 2019, 99, 427–511.
    6. Hepple, R.T.; Rice, C.L. Innervation and neuromuscular control in ageing skeletal muscle. J. Physiol. 2016, 594, 1965–1978.
    7. Wu, R.; De Vito, G.; Delahunt, E.; Ditroilo, M. Age-related Changes in Motor Function (I)Mechanical and Neuromuscular Factors. Int. J. Sports Med. 2020, 41, 709–719.
    8. Rudolf, R.; Khan, M.M.; Witzemann, V. Motor Endplate—Anatomical, Functional, and Molecular Concepts in the Historical Perspective. Cells 2019, 8, 387.
    9. Mège, R.M.; Goudou, D.; Giaume, C.; Nicolet, M.; Rieger, F. Is intercellular communication via Gap junctions required for myoblast fusion?? Cell Commun. Adhes. 1994, 2, 329–343.
    10. Schiaffino, S.; Reggiani, C. Molecular diversity of myofibrillar proteins: Gene regulation and functional significance. Physiol. Rev. 1996, 76, 371–423.
    11. Hughes, S.M. Muscle development: Electrical control of gene expression. Curr. Biol. 1998, 8, R892–R894.
    12. Olson, E.N.; Williams, R.S. Calcineurin signaling and muscle remodeling. Cell 2000, 101, 689–692.
    13. Buller, A.J.; Eccles, J.C.; Eccles, R.M. Differentiation of fast and slow muscles in the cat hind limb. J. Physiol. 1960, 150, 399–416.
    14. Pette, D.; Vrbová, G. Invited review: Neural control of phenotypic expression in mammalian muscle fibers. Muscle Nerve 1985, 8, 676–689.
    15. Klitgaard, H.; Zhou, M.; Schiaffino, S.; Betto, R.; Salviati, G.; Saltin, B. Ageing alters the myosin heavy chain composition of single fibres from human skeletal muscle. Acta Physiol. Scand. 1990, 140, 55–62.
    16. Choi, S.-J. Age-related functional changes and susceptibility to eccentric contraction-induced damage in skeletal muscle cell. Integr. Med. Res. 2016, 5, 171–175.
    17. Bao, Z.; Cui, C.; Chow, S.K.H.; Qin, L.; Wong, R.M.Y.; Cheung, W.H. AChRs Degeneration at NMJ in Aging-Associated Sarcopenia–A Systematic Review. Front. Aging Neurosci. 2020, 12, 597811.
    18. Vaughan, D.W. Effects of advancing age on peripheral nerve regeneration. J. Comp. Neurol. 1992, 323, 219–237.
    19. Delmonico, M.J.; Harris, T.B.; Visser, M.; Park, S.W.; Conroy, M.B.; Velasquez-Mieyer, P.; Boudreau, R.; Manini, T.M.; Nevitt, M.; Newman, A.B.; et al. Longitudinal study of muscle strength, quality, and adipose tissue infiltration. Am. J. Clin. Nutr. 2009, 90, 1579–1585.
    20. Lexell, J.; Taylor, C.C.; Sjöström, M. What is the cause of the ageing atrophy?. Total number, size and proportion of different fiber types studied in whole vastus lateralis muscle from 15- to 83-year-old men. J. Neurol. Sci. 1988, 84, 275–294.
    21. Wokke, J.H.J.; Jennekens, F.G.I.; van den Oord, C.J.M.; Veldman, H.; Smit, L.M.E.; Leppink, G.J. Morphological changes in the human end plate with age. J. Neurol. Sci. 1990, 95, 291–310.
    22. Rowan, S.L.; Rygiel, K.; Purves-Smith, F.M.; Solbak, N.M.; Turnbull, D.M.; Hepple, R.T. Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle. PLoS ONE 2012, 7, 29082.
    23. Valdez, G.; Tapia, J.C.; Kang, H.; Clemenson, G.D.; Gage, F.H.; Lichtman, J.W.; Sanes, J.R. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc. Natl. Acad. Sci. USA 2010, 107, 14863–14868.
    24. Cerrato, F.; Sparago, A.; Ariani, F.; Brugnoletti, F.; Calzari, L.; Coppedè, F.; De Luca, A.; Gervasini, C.; Giardina, E.; Gurrieri, F.; et al. DNA methylation in the diagnosis of monogenic diseases. Genes 2020, 11, 355.
    25. Bennett, S.A.; Tanaz, R.; Cobos, S.N.; Torrente, M.P. Epigenetics in amyotrophic lateral sclerosis: A role for histone post-translational modifications in neurodegenerative disease. Transl. Res. 2019, 204, 19–30.
    26. Pratt, J.; De Vito, G.; Narici, M.; Boreham, C. Neuromuscular Junction Aging: A Role for Biomarkers and Exercise. J. Gerontol. A Biol. Sci. Med. Sci. 2021, 76, 576–585.
    27. Lepore, E.; Casola, I.; Dobrowolny, G.; Musarò, A. Neuromuscular Junction as an Entity of Nerve-Muscle Communication. Cells 2019, 8, 906.
    28. Ruegg, M.A. Organization of synaptic myonuclei by Syne proteins and their role during the formation of the nerve-muscle synapse. Proc. Natl. Acad. Sci. USA 2005, 102, 5643–5644.
    29. Li, L.; Xiong, W.C.; Mei, L. Neuromuscular Junction Formation, Aging, and Disorders. Annu. Rev. Physiol. 2018, 80, 159–188.
    30. Schaeffer, L.; De Kerchove D’Exaerde, A.; Changeux, J.P. Targeting transcription to the neuromuscular synapse. Neuron 2001, 31, 15–22.
    31. Saha, R.N.; Pahan, K. HATs and HDACs in neurodegeneration: A tale of disconcerted acetylation homeostasis. Cell Death Differ. 2006, 13, 539–550.
    32. Battey, E.; Stroud, M.J.; Ochala, J. Using nuclear envelope mutations to explore age-related skeletal muscle weakness. Clin. Sci. 2020, 134, 2177–2187.
    33. Zhang, X.; Xu, R.; Zhu, B.; Yang, X.; Ding, X.; Duan, S.; Xu, T.; Zhuang, Y.; Han, M. Syne-1 and Syne-2 play crucial roles in myonuclear anchorage and motor neuron innervation. Development 2007, 134, 901–908.
    34. Grady, R.M.; Starr, D.A.; Ackerman, G.L.; Sanes, J.R.; Han, M. Syne proteins anchor muscle nuclei at the neuromuscular junction. Proc. Natl. Acad. Sci. USA 2005, 102, 4359–4364.
    35. Burke, B.; Stewart, C.L. The laminopathies: The functional architecture of the nucleus and its contribution to disease. Annu. Rev. Genomics Hum. Genet. 2006, 7, 369–405.
    36. Burke, B.; Stewart, C.L. The nuclear lamins: Flexibility in function. Nat. Rev. Mol. Cell Biol. 2013, 14, 13–24.
    37. Hutchison, C.J. Lamins: Building blocks or regulators of gene expression? Nat. Rev. Mol. Cell Biol. 2002, 3, 848–858.
    38. Mounkes, L.; Kozlov, S.; Burke, B.; Stewart, C.L. The laminopathies: Nuclear structure meets disease. Curr. Opin. Genet. Dev. 2003, 13, 223–230.
    39. Broers, J.L.V.; Ramaekers, F.C.S.; Bonne, G.; Ben Yaou, R.; Hutchison, C.J. Nuclear lamins: Laminopathies and their role in premature ageing. Physiol. Rev. 2006, 86, 967–1008.
    40. De Sandre-Giovannoli, A.; Chaouch, M.; Kozlov, S.; Vallat, J.M.; Tazir, M.; Kassouri, N.; Szepetowski, P.; Hammadouche, T.; Vandenberghe, A.; Stewart, C.L.; et al. Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse. Am. J. Hum. Genet. 2002, 70, 726–736.
    41. Scaffidi, P.; Misteli, T. Lamin A-dependent nuclear defects in human aging. Science 2006, 312, 1059–1063.
    42. Gao, N.; Zhao, K.; Cao, Y.; Ren, X.; Jing, H.; Xing, G.; Xiong, W.C.; Mei, L. A role of lamin A/C in preventing neuromuscular junction decline in mice. J. Neurosci. 2020, 40, 7203–7215.
    43. Coppedè, F. Epigenetics of neuromuscular disorders. Epigenomics 2020, 12, 2125–2139.
    44. Azpurua, J.; Eaton, B.A. Neuronal epigenetics and the aging synapse. Front. Cell. Neurosci. 2015, 9.
    45. Latcheva, N.K.; Viveiros, J.M.; Waddell, E.A.; Nguyen, P.T.T.; Liebl, F.L.W.; Marenda, D.R. Epigenetic crosstalk: Pharmacological inhibition of HDACs can rescue defective synaptic morphology and neurotransmission phenotypes associated with loss of the chromatin reader Kismet. Mol. Cell. Neurosci. 2018, 87, 77–85.
    46. Osseni, A.; Ravel-Chapuis, A.; Thomas, J.-L.; Gache, V.; Schaeffer, L.; Jasmin, B.J. HDAC6 regulates microtubule stability and clustering of AChRs at neuromuscular junctions. J. Cell Biol. 2020, 219.
    47. Lu, L.; Liu, Y.; Liu, Y.; Zhang, F.; Wang, H.; Zhang, Q.; Pan, F. Secreted miRNAs in the tripartite neuromuscular junction. ExRNA 2019, 1, 1–4.
    48. Castro, R.; Taetzsch, T.; Vaughan, S.K.; Godbe, K.; Chappell, J.; Settlage, R.E.; Valdez, G. Specific labeling of synaptic schwann cells reveals unique cellular and molecular features. Elife 2020, 9, 1–19.
    49. Feng, Z.; Ko, C.P. Schwann cells promote synaptogenesis at the neuromuscular junction via transforming growth factor-β1. J. Neurosci. 2008, 28, 9599–9609.
    50. Sugiura, Y.; Lin, W. Neuron-glia interactions: The roles of Schwann cells in neuromuscular synapse formation and function. Biosci. Rep. 2011, 31, 295–302.
    51. Court, F.A.; Gillingwater, T.H.; Melrose, S.; Sherman, D.L.; Greenshields, K.N.; Morton, A.J.; Harris, J.B.; Willison, H.J.; Ribchester, R.R. Identity, developmental restriction and reactivity of extralaminar cells capping mammalian neuromuscular junctions. J. Cell Sci. 2008, 121, 3901–3911.
    52. Gonzalez-Freire, M.; de Cabo, R.; Studenski, S.A.; Ferrucci, L. The neuromuscular junction: Aging at the crossroad between nerves and muscle. Front. Aging Neurosci. 2014, 6, 208.
    53. Rudolf, R.; Khan, M.M.; Labeit, S.; Deschenes, M.R. Degeneration of neuromuscular junction in age and dystrophy. Front. Aging Neurosci. 2014, 6, 99.
    54. Willadt, S.; Nash, M.; Slater, C. Age-related changes in the structure and function of mammalian neuromuscular junctions. Ann. N. Y. Acad. Sci. 2018, 1412, 41–53.
    55. Deschenes, M.R.; Hurst, T.E.; Ramser, A.E.; Sherman, E.G. Presynaptic to postsynaptic relationships of the neuromuscular junction are held constant across age and muscle fiber type. Dev. Neurobiol. 2013, 73, 744–753.
    56. Bowen, D.C.; Park, J.S.; Bodine, S.; Stark, J.L.; Valenzuela, D.M.; Stitt, T.N.; Yancopoulos, G.D.; Lindsay, R.M.; Glass, D.J.; Distefano, P.S. Localization and regulation of MuSK at the neuromuscular junction. Dev. Biol. 1998, 199, 309–319.
    57. Itou, Y.; Nochi, R.; Kuribayashi, H.; Saito, Y.; Hisatsune, T. Cholinergic activation of hippocampal neural stem cells in aged dentate gyrus. Hippocampus 2011, 21, 446–459.
    58. Taetzsch, T.; Valdez, G. NMJ maintenance and repair in aging. Curr. Opin. Physiol. 2018, 4, 57–64.
    59. Jang, Y.C.; Van Remmen, H. Age-associated alterations of the neuromuscular junction. Exp. Gerontol. 2011, 46, 193–198.
    60. Kang, H.; Tian, L.; Mikesh, M.; Lichtman, J.W.; Thompson, W.J. Terminal schwann cells participate in neuromuscular synapse remodeling during reinnervation following nerve injury. J. Neurosci. 2014, 34, 6323–6333.
    61. Barik, A.; Lu, Y.; Sathyamurthy, A.; Bowman, A.; Shen, C.; Li, L.; Xiong, W.C.; Mei, L. LRP4 is critical for neuromuscular junction maintenance. J. Neurosci. 2014, 34, 13892–13905.
    62. Ohno, K.; Ohkawara, B.; Ito, M. Agrin-LRP4-MuSK signaling as a therapeutic target for myasthenia gravis and other neuromuscular disorders. Expert Opin. Ther. Targets 2017, 21, 949–958.
    63. DeChiara, T.M.; Bowen, D.C.; Valenzuela, D.M.; Simmons, M.V.; Poueymirou, W.T.; Thomas, S.; Kinetz, E.; Compton, D.L.; Rojas, E.; Park, J.S.; et al. The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell 1996, 85, 501–512.
    64. Bowen, D.C.; Sugiyama, J.; Ferns, M.; Hall, Z.W. Neural agrin activates a high-affinity receptor in C2 muscle cells that is unresponsive to muscle agrin. J. Neurosci. 1996, 16, 3791–3797.
    65. Ferns, M.J.; Campanelli, J.T.; Hoch, W.; Scheller, R.H.; Hall, Z. The ability of agrin to cluster AChRs depends on alternative splicing and on cell surface proteoglycans. Neuron 1993, 11, 491–502.
    66. Gesemann, M.; Denzer, A.J.; Ruegg, M.A. Acetylcholine receptor-aggregating activity of agrin isoforms and mapping of the active site. J. Cell Biol. 1995, 128, 625–636.
    67. Lin, W.; Burgess, R.W.; Dominguez, B.; Pfaff, S.L.; Sanes, J.R.; Lee, K.F. Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 2001, 410, 1057–1064.
    68. Yang, J.F.; Cao, G.; Koirala, S.; Reddy, L.V.; Ko, C.P. Schwann cells express active agrin and enhance aggregation of acetylcholine receptors on muscle fibers. J. Neurosci. 2001, 21, 9572–9584.
    69. Bütikofer, L.; Zurlinden, A.; Bolliger, M.F.; Kunz, B.; Sonderegger, P. Destabilization of the neuromuscular junction by proteolytic cleavage of agrin results in precocious sarcopenia. FASEB J. 2011, 25, 4378–4393.
    70. Ibebunjo, C.; Chick, J.M.; Kendall, T.; Eash, J.K.; Li, C.; Zhang, Y.; Vickers, C.; Wu, Z.; Clarke, B.A.; Shi, J.; et al. Genomic and Proteomic Profiling Reveals Reduced Mitochondrial Function and Disruption of the Neuromuscular Junction Driving Rat Sarcopenia. Mol. Cell. Biol. 2013, 33, 194–212.
    71. Dalkin, W.; Taetzsch, T.; Valdez, G. The fibular nerve injury method: A reliable assay to identify and test factors that repair neuromuscular junctions. J. Vis. Exp. 2016, 2016, e54186.
    72. Zhou, J.; Yi, J.; Fu, R.; Liu, E.; Siddique, T.; Rios, E.; Deng, H.X. Hyperactive intracellular calcium signaling associated with localized mitochondrial defects in skeletal muscle of an animal model of amyotrophic lateral sclerosis. J. Biol. Chem. 2010, 285, 705–712.
    73. Muller, F.L.; Song, W.; Jang, Y.C.; Liu, Y.; Sabia, M.; Richardson, A.; Van Remmen, H. Denervation-induced skeletal muscle atrophy is associated with increased mitochondrial ROS production. Am. J. Physiol. Integr. Comp. Physiol. 2007, 293, 1159–1168.
    74. Banker, B.Q.; Kelly, S.S.; Robbins, N. Neuromuscular transmission and correlative morphology in young and old mice. J. Physiol. 1983, 339, 355–377.
    75. Dobrowolny, G.; Martini, M.; Scicchitano, B.M.; Romanello, V.; Boncompagni, S.; Nicoletti, C.; Pietrangelo, L.; De Panfilis, S.; Catizone, A.; Bouchè, M.; et al. Muscle Expression of SOD1 G93A Triggers the Dismantlement of Neuromuscular Junction via PKC-Theta. Antioxid. Redox Signal. 2018, 28, 1105–1119.
    76. Rocha, M.C.; Pousinha, P.A.; Correia, A.M.; Sebastião, A.M.; Ribeiro, J.A. Early Changes of Neuromuscular Transmission in the SOD1(G93A) Mice Model of ALS Start Long before Motor Symptoms Onset. PLoS ONE 2013, 8.
    77. Scialò, F.; Fernández-Ayala, D.J.; Sanz, A. Role of Mitochondrial Reverse Electron Transport in ROS Signaling: Potential Roles in Health and Disease. Front. Physiol. 2017, 8, 428.
    78. Li, A.; Yi, J.; Li, X.; Zhou, J. Physiological Ca2+ Transients Versus Pathological Steady-State Ca2+ Elevation, Who Flips the ROS Coin in Skeletal Muscle Mitochondria. Front. Physiol. 2020, 11.
    79. Hyatt, H.W.; Powers, S.K. Mitochondrial dysfunction is a common denominator linking skeletal muscle wasting due to disease, aging, and prolonged inactivity. Antioxidants 2021, 10, 588.
    80. Simon, H.U.; Haj-Yehia, A.; Levi-Schaffer, F. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 2000, 5, 415–418.
    81. Zhang, J.; Wang, X.; Vikash, V.; Ye, Q.; Wu, D.; Liu, Y.; Dong, W. ROS and ROS-Mediated Cellular Signaling. Oxid. Med. Cell. Longev. 2016, 2016, 1–18.
    82. Scicchitano, B.M.; Pelosi, L.; Sica, G.; Musarò, A. The physiopathologic role of oxidative stress in skeletal muscle. Mech. Ageing Dev. 2018, 170, 37–44.
    83. Dupuis, L.; Gonzalez de Aguilar, J.L.; Echaniz-Laguna, A.; Eschbach, J.; Rene, F.; Oudart, H.; Halter, B.; Huze, C.; Schaeffer, L.; Bouillaud, F.; et al. Muscle mitochondrial uncoupling dismantles neuromuscular junction and triggers distal degeneration of motor neurons. PLoS ONE 2009, 4, e5390.
    84. Fischer, L.R.; Li, Y.; Asress, S.A.; Jones, D.P.; Glass, J.D. Absence of SOD1 leads to oxidative stress in peripheral nerve and causes a progressive distal motor axonopathy. Exp. Neurol. 2012, 233, 163–171.
    85. Fischyer, L.R.; Culver, D.G.; Tennant, P.; Davis, A.A.; Wang, M.; Castellano-Sanchez, A.; Khan, J.; Polak, M.A.; Glass, J.D. Amyotrophic lateral sclerosis is a distal axonopathy: Evidence in mice and man. Exp. Neurol. 2004, 185, 232–240.
    86. Gurney, M.E.; Pu, H.; Chiu, A.Y.; Dal Canto, M.C.; Polchow, C.Y.; Alexander, D.D.; Caliendo, J.; Hentati, A.; Kwon, Y.W.; Deng, H.X.; et al. Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science 1994, 264, 1772–1775.
    87. Hayes, L.R.; Asress, S.A.; Li, Y.; Galkin, A.; Stepanova, A.; Kawamata, H.; Manfredi, G.; Glass, J.D. Distal denervation in the SOD1 knockout mouse correlates with loss of mitochondria at the motor nerve terminal. Exp. Neurol. 2019, 318, 251–257.
    88. Pollari, E.; Goldsteins, G.; Bart, G.; Koistinaho, J.; Giniatullin, R. The role of oxidative stress in degeneration of the neuromuscular junction in amyotrophic lateral sclerosis. Front. Cell. Neurosci. 2014, 8, 131.
    89. Martin, L.J.; Wong, M. Skeletal Muscle-Restricted Expression of Human SOD1 in Transgenic Mice Causes a Fatal ALS-Like Syndrome. Front. Neurol. 2020, 11.
    90. Wong, M.; Martin, L.J. Skeletal muscle-restricted expression of human SOD1 causes motor neuron degeneration in transgenic mice. Hum. Mol. Genet. 2010, 19, 2284–2302.
    91. Ji, L.L.; Yeo, D.; Kang, C.; Zhang, T. The role of mitochondria in redox signaling of muscle homeostasis. J. Sport Heal. Sci. 2020, 9, 386–393.
    92. Giniatullin, A.; Petrov, A.; Giniatullin, R. The involvement of P2Y12 receptors, NADPH oxidase, and lipid rafts in the action of extracellular ATP on synaptic transmission at the frog neuromuscular junction. Neuroscience 2015, 285, 324–332.
    93. Jackson, M.J.; Vasilaki, A.; McArdle, A. Cellular mechanisms underlying oxidative stress in human exercise. Free Radic. Biol. Med. 2016, 98, 13–17.
    94. Gomez-Cabrera, M.C.; Domenech, E.; Viña, J. Moderate exercise is an antioxidant: Upregulation of antioxidant genes by training. Free Radic. Biol. Med. 2008, 44, 126–131.
    95. Wright, V.P.; Reiser, P.J.; Clanton, T.L. Redox modulation of global phosphatase activity and protein phosphorylation in intact skeletal muscle. J. Physiol. 2009, 587, 5767–5781.
    96. Bejma, J.; Ji, L.L. Rapid communication aging and acute exercise enhance free radical generation in rat skeletal muscle. J. Appl. Physiol. 1999, 87, 465–470.
    97. Damiano, S.; Muscariello, E.; La Rosa, G.; Di Maro, M.; Mondola, P.; Santillo, M. Dual role of reactive oxygen species in muscle function: Can antioxidant dietary supplements counteract age-related sarcopenia? Int. J. Mol. Sci. 2019, 20, 3815.
    98. Sinha, S.; Ray, U.S.; Saha, M.; Singh, S.N.; Tomar, O.S. Antioxidant and redox status after maximal aerobic exercise at high altitude in acclimatized lowlanders and native highlanders. Eur. J. Appl. Physiol. 2009, 106, 807–814.
    99. Sinha, S.; Singh, S.N.; Saha, M.; Kain, T.C.; Tyagi, A.K.; Ray, U.S. Antioxidant and oxidative stress responses of sojourners at high altitude in different climatic temperatures. Int. J. Biometeorol. 2010, 54, 85–92.
    100. Carnio, S.; LoVerso, F.; Baraibar, M.A.; Longa, E.; Khan, M.M.; Maffei, M.; Reischl, M.; Canepari, M.; Loefler, S.; Kern, H.; et al. Autophagy Impairment in Muscle Induces Neuromuscular Junction Degeneration and Precocious Aging. Cell Rep. 2014, 8, 1509–1521.
    101. You, J.S.; Singh, N.; Reyes-Ordonez, A.; Khanna, N.; Bao, Z.; Zhao, H.; Chen, J. ARHGEF3 Regulates Skeletal Muscle Regeneration and Strength through Autophagy. Cell Rep. 2021, 34.
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