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Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission Takahiro Eguchi a, Tohru Tezuka a, 1, Takayasu Fukudome b, Yuji Watanabe c, Hiroshi Sagara c, Yuji Yamanashi a, * a
Division of Genetics, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan Department of Clinical Research and Neurology, NHO Nagasaki Kawatana Medical Center, 2005-1 Shimogumigo, Kawatanamachi Higashisonogi-gun, Nagasaki, 859-3615, Japan c Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 November 2019 Accepted 2 December 2019 Available online xxx
Neuromuscular junctions (NMJs) are cholinergic synapses characterized by ultrastructural specializations, including the presynaptic active zones, the acetylcholine (ACh) release sites of the motor nerve terminal, and the postsynaptic junctional folds of muscle membrane, where ACh receptors (AChRs) cluster for efficient neuromuscular transmission. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We had previously demonstrated that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK, and its activation and NMJ formation are enhanced in the Dok-7 transgenic (Tg) mice, in which Dok-7 is specifically overexpressed in skeletal muscle. Although Dok-7 Tg mice develop abnormally large NMJs but show normal motor function, the forced expression of Dok-7 in the muscle improves impaired motor activity in mouse models of neuromuscular disorders with NMJ defects. However, the effect of Dok-7 overexpression in skeletal muscle on ultrastructure and neuromuscular transmission of NMJs is yet to be studied. Here, we investigated the structural and electrophysiological properties of NMJs in the diaphragm muscle of 8week-old Dok-7 Tg mice. The areas of the presynaptic motor nerve terminals and postsynaptic muscle membrane of NMJs were 2.7 and 4.3 times greater in Dok-7 Tg mice than in WT mice, respectively. Electrophysiological analyses revealed that neuromuscular transmission via NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. Consistent with this, the densities of active zones and synaptic vesicles (ACh carriers) in the presynaptic motor nerve terminals were reduced. In addition, the density and size of postsynaptic junctional folds in the muscle membrane were also reduced. Moreover, terminal Schwann cells exhibited significantly greater penetration of their processes into the synaptic clefts, which connect the pre- and post-synaptic specializations. Together, our findings demonstrate that transgenic overexpression of Dok-7 in the skeletal muscle enhances neuromuscular transmission with significant enlargement and ultrastructural alterations of NMJs, the latter of which might prevent toxic overactivation of AChRs at the abnormally enlarged NMJs. © 2019 Published by Elsevier Inc.
Keywords: Neuromuscular junction MuSK Dok-7 Ultrastructure
1. Introduction
* Corresponding author. E-mail address: [email protected] (Y. Yamanashi). 1 Present address: Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606e8501, Japan.
The defining features of a chemical synapse include a presynaptic nerve terminal specialized for the release of neurotransmitter, a postsynaptic apparatus specialized to sense the neurotransmitter, and precise alignment of pre- and post-synaptic specializations [1]. The mammalian neuromuscular junction (NMJ) is a cholinergic synapse essential for skeletal muscle contraction and is marked by
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Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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its ultrastructural features, such as numerous invaginations of the postsynaptic muscle membrane termed junctional folds. The presynaptic active zone is the ultrastructural membrane-associated release site of the neurotransmitter acetylcholine (ACh) and is aligned with the opening of the junctional fold. The ACh is released into the synaptic cleft between pre- and post-synaptic specializations to bind and activate its ligand-gated ion channel receptors (AChRs) clustered at the edge of crests (tops of the folds closest to the presynaptic motor nerve terminal) surrounding the opening of junctional folds [1]. After release, ACh is rapidly hydrolyzed in the synaptic cleft, to prevent excessive activation of AChRs (see below), by acetylcholinesterase (AChE), which is anchored to the synaptic cleft basal lamina by collagen Q (ColQ). Impairments of neuromuscular transmission at NMJs lead to pathologies characterized by fatigable muscle weakness, such as congenital myasthenic syndromes (CMSs) and myasthenia gravis, which are inherited and autoimmune neuromuscular diseases, respectively [2,3]. For example, mutations in AChR subunits that reduce AChR number at NMJs (AChR deficiency syndrome) and/or decrease the synaptic response to ACh (fast-channel syndrome) lead to impaired neuromuscular transmission and consequent fatigable muscle weakness [2]. Interestingly, acute inhibition of AChE induces prolonged endplate potentials and enhanced neuromuscular transmission [4], but long-term inhibition of AChE causes ultrastructural alterations in the NMJs, such as widened synaptic clefts and abnormal junctional folds [5,6]. In addition, mutations in AChR subunits that increase the response to ACh (slow-channel syndrome), or those in ColQ that cause AChE deficiency at NMJs, result in prolonged endplate potentials, degeneration of the junctional folds, and compromised neuromuscular transmission [2]. Thus, excessive activation of AChRs at NMJs leads to the disruption of NMJ structure and impaired neuromuscular transmission. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase MuSK [1]. We previously demonstrated the presence of an essential muscle-intrinsic activator of MuSK, downstream of tyrosine kinases-7 (Dok-7), and found that biallelic DOK7 mutations cause a limb-girdle type of CMS, termed DOK7 myasthenia [7e10]. Just as in MuSK-deficient mice, the Dok-7-deficient mice fail to form NMJs and die of respiratory failure shortly after birth [7,11]. By contrast, skeletal musclespecific Dok-7 overexpression in Dok-7 transgenic (Tg) mice causes strong activation of MuSK, and these mice display abnormally large NMJs but apparently normal motor function [12,13]. Furthermore, we had previously shown that forced expression of Dok-7, specifically in the muscle, ameliorates the shortened lifespan and improves the impaired motor activity in mouse models of neuromuscular disorders [12], such as DOK7 myasthenia and autosomal dominant Emery-Dreifuss muscular dystrophy, a disease associated with defective NMJs due to genetic mutations in the lamin A/C gene [14]. Given that excessive activation of AChRs at NMJs leads to the impaired neuromuscular transmission and consequent muscle weakness mentioned above, the abnormal enlargement of NMJs observed in Dok-7 Tg mice would seem not to cause excess and thus toxic activation of AChRs at the NMJ in as much as motor function is normal. However, it remains to be clarified how Dok-7 overexpression in skeletal muscle affects ultrastructure and neuromuscular transmission of NMJs. Thus, we investigate the structural and electrophysiological properties of NMJs in Dok-7 Tg mice.
Ethics Committee of the Institute of Medical Science, the University of Tokyo. The transgenic mice expressing Dok-7 tagged with the enhanced green fluorescent protein (Dok-7-EGFP) under the control of the skeletal actin promoter were previously described [9]. The C57BL/6J mice were purchased from Japan SLC. 2.2. Whole-mount tissue staining For whole-mount tissue staining, diaphragm muscles of 8week-old WT and Dok-7 Tg mice were dissected and fixed in 1% paraformaldehyde (PFA) in 10 mM phosphate-buffered saline (PBS, pH 7.4). The muscles were permeabilized with 1% Triton X-100 in PBS and then incubated with anti-neurofilament-L and antisynapsin-1 rabbit antibodies (Cell Signaling Technology) to label motor axons and presynaptic nerve terminals, respectively [15]. This was followed by incubation with Alexa 647- or Alexa 594conjugated anti-rabbit IgG and CF 594- or CF 405-conjugated abungarotoxin (Biotium) to label AChR-enriched endplates (the region of synaptic specialization on the myotube). Anti-Bassoon mouse antibodies (Enzo) and Alexa 647-conjugated anti-mouse IgG2a were used to label the active zone. Confocal Z serial images were collected with an FV1000 or FV1200 Confocal Laser Scanning Microscope (Olympus) and collapsed into a single image, and analyzed with cellSens Digital Imaging Software (Olympus) [16]. 2.3. Electrophysiologic examination Diaphragms with phrenic nerve distal endings were dissected from 8-week-old WT and Dok-7 Tg mice and used for the conventional intracellular microelectrode study [17]. Miniature endplate potentials (MEPPs), endplate potentials (EPPs), and resting membrane potentials (RMPs) were recorded. For EPP recording, dTubocurarine chloride (Sigma) was used at a concentration sufficient to inhibit muscle contraction. The potentials were corrected for non-linear summation, and the last 64 responses in a train of 114 were saved for later analysis. The quantal content of EPPs was calculated by the variance method [18,19]. The MEPP and EPP amplitudes were corrected to a standard RMP of 80 mV. 2.4. Electron microscopy For electron microscopy, diaphragm muscles of 8-week-old WT and Dok-7 Tg mice were cut and fixed in 0.1% glutaraldehyde (GA) and 4% PFA in 100 mM phosphate buffer (PB) and then incubated with CF 594-conjugated a-bungarotoxin in 100 mM PB to label the endplates. The endplate-rich region of the muscle was dissected and refixed in 2.5% GA in 100 mM PB. The tissue was postfixed in 2% OsO4 in 100 mM PB, dehydrated, and embedded in Epon812 (Nisshin EM). The sections were cut longitudinally with 50 nm thickness, stained with uranyl acetate and Reynolds’ lead citrate, and photographed in an H7500 transmission electron microscope (Hitachi). The images were analyzed using the National Institutes of Health (NIH) ImageJ software (version 1.48v). Brightness and contrast of representative images were adjusted for better visibility, but all quantitative analyses were performed on original images without adjustments. 2.5. Data analysis
2. Materials and methods 2.1. Mice All animal studies described here were approved by the Animal
Statistical significance was determined by unpaired Student’s ttest or Wilcoxon-Mann-Whitney test. Data were expressed as mean ± SEM, and P < 0.05 was considered to be statistically significant.
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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3. Results 3.1. Dok-7 Tg mice show not only NMJ enlargement but also enhanced neuromuscular transmission We first visualized NMJs in diaphragm muscles of 8-week-old Dok-7 Tg mice and WT mice by whole-mount staining with fluorescently labeled a-bungarotoxin to identify postsynaptic AChR clusters, using antibodies against neurofilament and synapsin-1 to label the motor axons and presynaptic nerve terminals, respectively (Fig. 1A). Consistent with our previous reports [9,12,13,20], the areas of presynaptic motor nerve terminals and AChR clusters were increased by 178% and 332% in Dok-7 Tg mice, respectively, compared with WT mice (Fig. 1B and C). Furthermore, the cover ratio of presynaptic motor nerve terminals to postsynaptic AChR clusters at NMJs decreased from 83% in WT mice to 56% in Dok-7 Tg mice (Fig. 1D). These data indicate that the overexpression of Dok-7, specifically in the skeletal muscle, does not proportionally increase the size of presynaptic motor nerve terminals compared to that of the postsynaptic AChR clusters in NMJs at 8 weeks of age. To examine neuromuscular transmission in Dok-7 Tg mice, we performed electrophysiological recordings on the phrenic nerve/ diaphragm preparation at 8 weeks of age [17]. Recordings of spontaneous miniature endplate potentials (MEPPs) revealed that the amplitudes and frequencies of MEPPs were increased by 32% and 68% in Dok-7 Tg mice, respectively, compared with WT mice (Fig. 2A and B). Note that each MEPP represents the endplate potential evoked by spontaneous release of a single quantum of ACh without nerve impulse [19]. We further examined the endplate potentials (EPPs) by stimulating the phrenic nerve and found that quantal content of EPPs (the number of ACh quanta released per nerve impulse; please refer to Ref. [19] for detailed information) was increased by 73% in Dok-7 Tg mice (Fig. 2C). These results indicate that muscle-specific overexpression of Dok-7 not only enlarges NMJs but also enhances their neuromuscular transmission via the activation of AChRs.
3.2. Dok-7 Tg mice show ultrastructural alterations in pre- and post-synaptic specializations of NMJs The size of the synaptic contact at the NMJs is an important determinant of the amount of ACh release [19]. At 8 weeks of age, the size of the synaptic contact was increased by 178% in Dok-7 Tg mice, based on the size of the presynaptic motor nerve terminals, which were within the postsynaptic AChR clustered area (Fig. 1A
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and B). However, the quantal content of EPPs was increased by only 73% in Dok-7 Tg mice (Fig. 2C), indicating that the ACh release at NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. In addition, given that Dok-7 Tg mice show apparently normal motor function [12,13], the enhanced ACh release appears not to cause excessive and thus toxic AChR activation at the abnormally enlarged NMJs. Therefore, we hypothesized that the ultrastructural specializations of NMJs, such as presynaptic ACh release sites, active zones, and postsynaptic junctional folds, where AChRs cluster, might be altered in Dok-7 Tg mice so as to prevent toxic overactivation of AChRs. York et al. demonstrated that AChRs are concentrated at the edge of the junctional fold crests surrounding the fold openings at NMJs [21]. When viewed en-face by light microscopy, this arrangement shows a regular, striped appearance of AChR clusters at NMJs [22]. To determine whether muscle-specific overexpression of Dok-7 affects this aspect of postsynaptic development, we collected confocal images of the synapses in diaphragm muscles of 8-week-old Dok-7 Tg mice and WT mice (Fig. 3A and B). In WT mice, the postsynaptic AChR clusters are correctly organized into well-defined AChR stripes (Fig. 3B). By contrast, the postsynaptic AChR clusters in Dok-7 Tg mice do not form the regular AChR stripes. We next visualized active zones using an antibody against the active zone marker protein Bassoon (Fig. 3C and D). Consistent with previous studies [22], immunofluorescence of active zones at NMJs appears as discrete puncta, which are largely localized on top of the AChR stripes in WT mice. In Dok-7 Tg mice, although the majority of Bassoon puncta are also localized on the AChR-rich regions of NMJs, the density of active zones in the presynaptic motor nerve terminals was reduced (Fig. 3CeE). These results suggest that overexpression of Dok-7 in the muscle alters the pre- and post-synaptic ultrastructure of NMJs. Thus, we further examined ultrastructural alterations in NMJs using electron microscopy and found that the density of synaptic vesicles in the presynaptic motor nerve terminals was reduced in Dok-7 Tg mice, compared with WT mice (Fig. 4AeC). Furthermore, to estimate the organization and complexity of the postsynaptic membrane, we determined the ratio of postsynaptic versus presynaptic membrane length. Compared with WT mice, the ratio for Dok-7 Tg mice was significantly reduced (Fig. 4A and D). Consistent with this, the density and size of junctional folds in the postsynaptic membrane were reduced in Dok-7 Tg mice (Fig. 4A, E and F). These results indicate that muscle-specific overexpression of Dok-7 induces ultrastructural alterations in the pre- and post-synaptic
Fig. 1. Eight-week-old Dok-7 Tg mice show NMJ enlargement. (A) Whole-mount staining of NMJs in the diaphragm muscles. The AChRs were labeled with a-bungarotoxin (red), and motor axons and presynaptic nerve terminals were stained with antibodies against neurofilament-L and synapsin-1 (green). Scale bars, 20 mm. (BeD) Quantified results for the area of each presynaptic nerve terminal (B) and each AChR cluster (C), and for the cover ratio (presynaptic area divided by AChR cluster area) in each NMJ (D). Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined NMJs (3 mice per group). Asterisks denote a significant statistical difference: ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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Fig. 2. Eight-week-old Dok-7 Tg mice show enhanced neuromuscular transmission. (AeC) Quantified results for the amplitude (A) and frequency (B) of miniature endplate potentials (MEPPs), and for the quantal content of endplate potentials (EPPs) (C) in the diaphragm muscles. Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined NMJs (4e7 mice per group). Asterisks denote a significant statistical difference: *P < 0.05 and ***P < 0.001.
specializations of NMJs, which appears to attenuate the neuromuscular transmission and thus to prevent toxic overactivation of AChRs at the abnormally enlarged NMJs.
3.3. Terminal Schwann cells extend their processes into the synaptic clefts in Dok-7 Tg mice Although the motor axon is wrapped by the myelin sheath formed by Schwann cells (SCs), its terminal region is only capped by terminal SCs (tSCs) without myelin formation [1]. The tSCs are
believed to be required for the maintenance of NMJs. For instance, the ablation of SCs in mice, including tSCs, after NMJ maturation leads to the fragmentation and shrinkage of NMJs, defective neuromuscular transmission, and premature death [23]. In addition, the ablation of tSCs in frogs after NMJ maturation induces retraction of presynaptic motor nerve terminals from the NMJs [24]. However, electron microscopic analysis revealed that tSCs exhibited significantly greater penetration of their processes into the synaptic clefts in Dok-7 Tg mice than in WT mice (Fig. 4B and G), suggesting that muscle-specific overexpression of Dok-7 renders
Fig. 3. Eight-week-old Dok-7 Tg mice show disorganized AChR clustering and reduced density of active zones at NMJs. (A) Whole-mount staining of NMJs in the diaphragm muscles. AChRs, motor axons, and presynaptic nerve terminals were stained as in Fig. 1A. Scale bars, 20 mm. (B) Higher magnification of the white boxed area in (A). Scale bars, 2 mm. (C) Whole-mount staining of active zones and NMJs in the diaphragm muscles. Active zones were visualized using an anti-Bassoon antibody (blue), and AChRs, motor axons, and presynaptic nerve terminals were stained as in Fig. 1A. Scale bars, 5 mm. (D) Higher magnification of the white boxed area in (C). Scale bars, 2.5 mm. (E) Quantified results for the density of active zones in the presynaptic motor nerve terminals (Bassoon-positive area divided by presynaptic area). Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined NMJs (5 mice per group). Asterisks denote a significant statistical difference: ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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Fig. 4. Eight-week-old Dok-7 Tg mice show ultrastructural alterations of pre- and post-synaptic specializations and of tSCs. (A) Representative transmission electron micrographs of NMJs in the diaphragm muscles. NT, nerve terminal; M, myotube (myofiber). Scale bars, 500 nm. (B) Higher magnification of the black boxed area in (A). SV, synaptic vesicle; JF, junctional fold; tSC, terminal Schwann cell. Scale bars, 500 nm. (CeG) Quantified results for the density of synaptic vesicles in the presynaptic area (C), ratio of post-versus presynaptic membrane length (D), density of junctional folds in the postsynaptic membrane that faces the presynaptic membrane (Note that the number of junctional folds was divided by the length of the presynaptic membrane to evaluate the density of junctional folds) (E), size of junctional folds (F), and total length of tSC processes in the synaptic clefts per mm presynaptic membrane length (G). Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined fields (CeE, G) or of examined junctional folds (F) (4 mice per group). Asterisks denote a significant statistical difference: ***P < 0.001.
the synaptic clefts accessible to the processes, which again may serve to attenuate the neuromuscular transmission at the abnormally enlarged NMJs. 4. Discussion In general, NMJs form selectively in the central region of myotubes (also known as myofibers), where MuSK is localized and controls the accumulation of AChR transcripts and proteins [25]. In our previous studies [9,12,13,20], we demonstrated that overexpression of Dok-7 in skeletal muscle in mice enlarges postsynaptic AChR clusters and presynaptic motor nerve terminals in the correct, central region of myotubes, suggesting that Dok-7mediated activation of MuSK induces not only intramuscular signaling but also retrograde signaling that enlarges presynaptic motor nerve terminals. However, in the present study, we demonstrated that the coverage ratio of presynaptic motor nerve terminals over postsynaptic AChR clusters per se decreased in Dok7 Tg mice (Fig. 1D), likely due to lagging enlargement of presynaptic motor nerve terminals in response to the enlarged AChR clusters in the muscle. In addition, muscle-specific overexpression of Dok-7 altered the ultrastructure of pre- and post-synaptic specializations and tSCs (Figs. 3 and 4). One possible explanation for these ultrastructural alterations in Dok-7 Tg mice could be insufficient expression of NMJ components, such as laminin b2 and collagen Q (ColQ); the former is a major component of the synaptic cleft basal lamina and the latter anchors AChE into the synaptic cleft basal lamina. Mice lacking laminin b2 show reduced densities of active
zones in the presynaptic motor nerve terminals and of junctional folds in the postsynaptic membrane, together with enhanced penetration of tSC processes into the synaptic clefts [26]. Moreover, mice lacking ColQ also show reduced density of synaptic vesicles, simplified junctional folds, and enhanced penetration of tSC processes into the synaptic clefts [27]. Since ultrastructural alterations of pre- and post-synaptic specializations and tSCs in Dok-7 Tg mice are similar to those found in laminin b2- or ColQ-deficient mice, it is important to investigate the effects of Dok-7 overexpression in the muscle on the expression of these NMJ components. Patients with neuromuscular diseases often show alterations in the ultrastructure of NMJs. Patients with Lambert-Eaton myasthenic syndrome exhibit impaired neuromuscular transmission and muscle weakness, together with a reduced number of active zones, which are caused by autoantibodies against the presynaptic voltage-gated calcium channel Cav2.1 [28]. Moreover, patients with Pierson syndrome exhibit impaired neuromuscular transmission, reduced density of synaptic vesicles, simplified junctional folds, and enhanced penetration of tSC processes into the synaptic clefts, which are caused by mutations in the laminin b2 genes [29]. In addition, mathematical modeling of neuromuscular transmission in the presence or absence of junctional folds suggests that they may act to reduce the threshold necessary for action potential firing, thus making neuromuscular transmission more efficient [30]. These studies support crucial roles for active zones and junctional folds in neuromuscular transmission. In this study, we demonstrated that muscle-specific overexpression of Dok-7 not only enhanced NMJ formation (Fig. 1B and C) and neuromuscular
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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transmission (Fig. 2) but also reduced the densities of active zones and synaptic vesicles in the presynaptic motor nerve terminals (Figs. 3E and 4C). Furthermore, the density and size of junctional folds in the postsynaptic membrane were reduced in Dok-7 Tg mice (Fig. 4E and F), and tSCs exhibited enhanced penetration of their processes into the synaptic clefts in the mutant mice (Fig. 4G). Together, these findings indicate that muscle-specific overexpression of Dok-7 induces ultrastructural alterations to pre- and post-synaptic specializations and tSCs, which may prevent toxic overactivation of AChRs at the abnormally enlarged NMJs. Thus, further studies to uncover the role of Dok-7-mediated signaling in regulating the size, structure, and function of NMJs will be needed for better understanding NMJs and developing new therapeutic approach against NMJ pathologies. Acknowledgments The authors thank R. F. Whittier, R. Ueta, and A. Inoue-Yamauchi for critically reading the manuscript and for helpful suggestions. We also thank T. Yagami for help in animal care. This work was supported by the Grant for Joint Research Project of the Institute of Medical Science, the University of Tokyo and by JSPS KAKENHI Grant Number 17H01532. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.011 References [1] L. Li, W.C. Xiong, L. Mei, Neuromuscular junction formation, aging and disorders, Annu. Rev. Physiol. 80 (2018) 159e188. [2] A.G. Engel, X.M. Shen, D. Selcen, S.M. Sine, Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment, Lancet Neurol. 14 (2015) 420e434. [3] N.E. Gilhus, G.O. Skeie, F. Romi, K. Lazaridis, P. Zisimopoulou, S. Tzartos, Myasthenia gravis - autoantibody characteristics and their implications for therapy, Nat. Rev. Neurol. 12 (2016) 259e268. [4] R.T. Bois, R.G. Hummel, W.D. Dettbarn, M.B. Laskowski, Presynaptic and postsynaptic neuromuscular effects of a specific inhibitor of acetylcholinesterase, J. Pharmacol. Exp. Ther. 215 (1980) 53e59. [5] A.G. Engel, E.H. Lambert, T. Santa, Study of long-term anticholinesterase therapy. Effects on neuromuscular transmission and on motor end-plate fine structure, Neurology 23 (1973) 1273e1281. [6] M.B. Laskowski, W.H. Olson, W.D. Dettbarn, Ultrastructural changes at the motor end-plate produced by an irreversible cholinesterase inhibitor, Exp. Neurol. 47 (1975) 290e306. [7] K. Okada, A. Inoue, M. Okada, Y. Murata, S. Kakuta, T. Jigami, S. Kubo, H. Shiraishi, K. Eguchi, M. Motomura, T. Akiyama, Y. Iwakura, O. Higuchi, Y. Yamanashi, The muscle protein Dok-7 is essential for neuromuscular synaptogenesis, Science 312 (2006) 1802e1805. [8] D. Beeson, O. Higuchi, J. Palace, J. Cossins, H. Spearman, S. Maxwell, J. Newsom-Davis, G. Burke, P. Fawcett, M. Motomura, J.S. Müller, H. Lochmüller, C. Slater, A. Vincent, Y. Yamanashi, Dok-7 mutations underlie a neuromuscular junction synaptopathy, Science 313 (2006) 1975e1978. [9] A. Inoue, K. Setoguchi, Y. Matsubara, K. Okada, N. Sato, Y. Iwakura, O. Higuchi, Y. Yamanashi, Dok-7 activates the muscle receptor kinase MuSK and shapes
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Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission Takahiro Eguchi a, Tohru Tezuka a, 1, Takayasu Fukudome b, Yuji Watanabe c, Hiroshi Sagara c, Yuji Yamanashi a, * a
Division of Genetics, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan Department of Clinical Research and Neurology, NHO Nagasaki Kawatana Medical Center, 2005-1 Shimogumigo, Kawatanamachi Higashisonogi-gun, Nagasaki, 859-3615, Japan c Medical Proteomics Laboratory, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 November 2019 Accepted 2 December 2019 Available online xxx
Neuromuscular junctions (NMJs) are cholinergic synapses characterized by ultrastructural specializations, including the presynaptic active zones, the acetylcholine (ACh) release sites of the motor nerve terminal, and the postsynaptic junctional folds of muscle membrane, where ACh receptors (AChRs) cluster for efficient neuromuscular transmission. The formation and maintenance of NMJs are governed by the muscle-specific receptor tyrosine kinase MuSK. We had previously demonstrated that the muscle cytoplasmic protein Dok-7 is an essential activator of MuSK, and its activation and NMJ formation are enhanced in the Dok-7 transgenic (Tg) mice, in which Dok-7 is specifically overexpressed in skeletal muscle. Although Dok-7 Tg mice develop abnormally large NMJs but show normal motor function, the forced expression of Dok-7 in the muscle improves impaired motor activity in mouse models of neuromuscular disorders with NMJ defects. However, the effect of Dok-7 overexpression in skeletal muscle on ultrastructure and neuromuscular transmission of NMJs is yet to be studied. Here, we investigated the structural and electrophysiological properties of NMJs in the diaphragm muscle of 8week-old Dok-7 Tg mice. The areas of the presynaptic motor nerve terminals and postsynaptic muscle membrane of NMJs were 2.7 and 4.3 times greater in Dok-7 Tg mice than in WT mice, respectively. Electrophysiological analyses revealed that neuromuscular transmission via NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. Consistent with this, the densities of active zones and synaptic vesicles (ACh carriers) in the presynaptic motor nerve terminals were reduced. In addition, the density and size of postsynaptic junctional folds in the muscle membrane were also reduced. Moreover, terminal Schwann cells exhibited significantly greater penetration of their processes into the synaptic clefts, which connect the pre- and post-synaptic specializations. Together, our findings demonstrate that transgenic overexpression of Dok-7 in the skeletal muscle enhances neuromuscular transmission with significant enlargement and ultrastructural alterations of NMJs, the latter of which might prevent toxic overactivation of AChRs at the abnormally enlarged NMJs. © 2019 Published by Elsevier Inc.
Keywords: Neuromuscular junction MuSK Dok-7 Ultrastructure
1. Introduction
* Corresponding author. E-mail address: [email protected] (Y. Yamanashi). 1 Present address: Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Yoshida Honmachi, Sakyo-ku, Kyoto 606e8501, Japan.
The defining features of a chemical synapse include a presynaptic nerve terminal specialized for the release of neurotransmitter, a postsynaptic apparatus specialized to sense the neurotransmitter, and precise alignment of pre- and post-synaptic specializations [1]. The mammalian neuromuscular junction (NMJ) is a cholinergic synapse essential for skeletal muscle contraction and is marked by
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Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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its ultrastructural features, such as numerous invaginations of the postsynaptic muscle membrane termed junctional folds. The presynaptic active zone is the ultrastructural membrane-associated release site of the neurotransmitter acetylcholine (ACh) and is aligned with the opening of the junctional fold. The ACh is released into the synaptic cleft between pre- and post-synaptic specializations to bind and activate its ligand-gated ion channel receptors (AChRs) clustered at the edge of crests (tops of the folds closest to the presynaptic motor nerve terminal) surrounding the opening of junctional folds [1]. After release, ACh is rapidly hydrolyzed in the synaptic cleft, to prevent excessive activation of AChRs (see below), by acetylcholinesterase (AChE), which is anchored to the synaptic cleft basal lamina by collagen Q (ColQ). Impairments of neuromuscular transmission at NMJs lead to pathologies characterized by fatigable muscle weakness, such as congenital myasthenic syndromes (CMSs) and myasthenia gravis, which are inherited and autoimmune neuromuscular diseases, respectively [2,3]. For example, mutations in AChR subunits that reduce AChR number at NMJs (AChR deficiency syndrome) and/or decrease the synaptic response to ACh (fast-channel syndrome) lead to impaired neuromuscular transmission and consequent fatigable muscle weakness [2]. Interestingly, acute inhibition of AChE induces prolonged endplate potentials and enhanced neuromuscular transmission [4], but long-term inhibition of AChE causes ultrastructural alterations in the NMJs, such as widened synaptic clefts and abnormal junctional folds [5,6]. In addition, mutations in AChR subunits that increase the response to ACh (slow-channel syndrome), or those in ColQ that cause AChE deficiency at NMJs, result in prolonged endplate potentials, degeneration of the junctional folds, and compromised neuromuscular transmission [2]. Thus, excessive activation of AChRs at NMJs leads to the disruption of NMJ structure and impaired neuromuscular transmission. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase MuSK [1]. We previously demonstrated the presence of an essential muscle-intrinsic activator of MuSK, downstream of tyrosine kinases-7 (Dok-7), and found that biallelic DOK7 mutations cause a limb-girdle type of CMS, termed DOK7 myasthenia [7e10]. Just as in MuSK-deficient mice, the Dok-7-deficient mice fail to form NMJs and die of respiratory failure shortly after birth [7,11]. By contrast, skeletal musclespecific Dok-7 overexpression in Dok-7 transgenic (Tg) mice causes strong activation of MuSK, and these mice display abnormally large NMJs but apparently normal motor function [12,13]. Furthermore, we had previously shown that forced expression of Dok-7, specifically in the muscle, ameliorates the shortened lifespan and improves the impaired motor activity in mouse models of neuromuscular disorders [12], such as DOK7 myasthenia and autosomal dominant Emery-Dreifuss muscular dystrophy, a disease associated with defective NMJs due to genetic mutations in the lamin A/C gene [14]. Given that excessive activation of AChRs at NMJs leads to the impaired neuromuscular transmission and consequent muscle weakness mentioned above, the abnormal enlargement of NMJs observed in Dok-7 Tg mice would seem not to cause excess and thus toxic activation of AChRs at the NMJ in as much as motor function is normal. However, it remains to be clarified how Dok-7 overexpression in skeletal muscle affects ultrastructure and neuromuscular transmission of NMJs. Thus, we investigate the structural and electrophysiological properties of NMJs in Dok-7 Tg mice.
Ethics Committee of the Institute of Medical Science, the University of Tokyo. The transgenic mice expressing Dok-7 tagged with the enhanced green fluorescent protein (Dok-7-EGFP) under the control of the skeletal actin promoter were previously described [9]. The C57BL/6J mice were purchased from Japan SLC. 2.2. Whole-mount tissue staining For whole-mount tissue staining, diaphragm muscles of 8week-old WT and Dok-7 Tg mice were dissected and fixed in 1% paraformaldehyde (PFA) in 10 mM phosphate-buffered saline (PBS, pH 7.4). The muscles were permeabilized with 1% Triton X-100 in PBS and then incubated with anti-neurofilament-L and antisynapsin-1 rabbit antibodies (Cell Signaling Technology) to label motor axons and presynaptic nerve terminals, respectively [15]. This was followed by incubation with Alexa 647- or Alexa 594conjugated anti-rabbit IgG and CF 594- or CF 405-conjugated abungarotoxin (Biotium) to label AChR-enriched endplates (the region of synaptic specialization on the myotube). Anti-Bassoon mouse antibodies (Enzo) and Alexa 647-conjugated anti-mouse IgG2a were used to label the active zone. Confocal Z serial images were collected with an FV1000 or FV1200 Confocal Laser Scanning Microscope (Olympus) and collapsed into a single image, and analyzed with cellSens Digital Imaging Software (Olympus) [16]. 2.3. Electrophysiologic examination Diaphragms with phrenic nerve distal endings were dissected from 8-week-old WT and Dok-7 Tg mice and used for the conventional intracellular microelectrode study [17]. Miniature endplate potentials (MEPPs), endplate potentials (EPPs), and resting membrane potentials (RMPs) were recorded. For EPP recording, dTubocurarine chloride (Sigma) was used at a concentration sufficient to inhibit muscle contraction. The potentials were corrected for non-linear summation, and the last 64 responses in a train of 114 were saved for later analysis. The quantal content of EPPs was calculated by the variance method [18,19]. The MEPP and EPP amplitudes were corrected to a standard RMP of 80 mV. 2.4. Electron microscopy For electron microscopy, diaphragm muscles of 8-week-old WT and Dok-7 Tg mice were cut and fixed in 0.1% glutaraldehyde (GA) and 4% PFA in 100 mM phosphate buffer (PB) and then incubated with CF 594-conjugated a-bungarotoxin in 100 mM PB to label the endplates. The endplate-rich region of the muscle was dissected and refixed in 2.5% GA in 100 mM PB. The tissue was postfixed in 2% OsO4 in 100 mM PB, dehydrated, and embedded in Epon812 (Nisshin EM). The sections were cut longitudinally with 50 nm thickness, stained with uranyl acetate and Reynolds’ lead citrate, and photographed in an H7500 transmission electron microscope (Hitachi). The images were analyzed using the National Institutes of Health (NIH) ImageJ software (version 1.48v). Brightness and contrast of representative images were adjusted for better visibility, but all quantitative analyses were performed on original images without adjustments. 2.5. Data analysis
2. Materials and methods 2.1. Mice All animal studies described here were approved by the Animal
Statistical significance was determined by unpaired Student’s ttest or Wilcoxon-Mann-Whitney test. Data were expressed as mean ± SEM, and P < 0.05 was considered to be statistically significant.
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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3. Results 3.1. Dok-7 Tg mice show not only NMJ enlargement but also enhanced neuromuscular transmission We first visualized NMJs in diaphragm muscles of 8-week-old Dok-7 Tg mice and WT mice by whole-mount staining with fluorescently labeled a-bungarotoxin to identify postsynaptic AChR clusters, using antibodies against neurofilament and synapsin-1 to label the motor axons and presynaptic nerve terminals, respectively (Fig. 1A). Consistent with our previous reports [9,12,13,20], the areas of presynaptic motor nerve terminals and AChR clusters were increased by 178% and 332% in Dok-7 Tg mice, respectively, compared with WT mice (Fig. 1B and C). Furthermore, the cover ratio of presynaptic motor nerve terminals to postsynaptic AChR clusters at NMJs decreased from 83% in WT mice to 56% in Dok-7 Tg mice (Fig. 1D). These data indicate that the overexpression of Dok-7, specifically in the skeletal muscle, does not proportionally increase the size of presynaptic motor nerve terminals compared to that of the postsynaptic AChR clusters in NMJs at 8 weeks of age. To examine neuromuscular transmission in Dok-7 Tg mice, we performed electrophysiological recordings on the phrenic nerve/ diaphragm preparation at 8 weeks of age [17]. Recordings of spontaneous miniature endplate potentials (MEPPs) revealed that the amplitudes and frequencies of MEPPs were increased by 32% and 68% in Dok-7 Tg mice, respectively, compared with WT mice (Fig. 2A and B). Note that each MEPP represents the endplate potential evoked by spontaneous release of a single quantum of ACh without nerve impulse [19]. We further examined the endplate potentials (EPPs) by stimulating the phrenic nerve and found that quantal content of EPPs (the number of ACh quanta released per nerve impulse; please refer to Ref. [19] for detailed information) was increased by 73% in Dok-7 Tg mice (Fig. 2C). These results indicate that muscle-specific overexpression of Dok-7 not only enlarges NMJs but also enhances their neuromuscular transmission via the activation of AChRs.
3.2. Dok-7 Tg mice show ultrastructural alterations in pre- and post-synaptic specializations of NMJs The size of the synaptic contact at the NMJs is an important determinant of the amount of ACh release [19]. At 8 weeks of age, the size of the synaptic contact was increased by 178% in Dok-7 Tg mice, based on the size of the presynaptic motor nerve terminals, which were within the postsynaptic AChR clustered area (Fig. 1A
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and B). However, the quantal content of EPPs was increased by only 73% in Dok-7 Tg mice (Fig. 2C), indicating that the ACh release at NMJs in Dok-7 Tg mice was significantly enhanced but not proportionally with the increased size of the synaptic contact. In addition, given that Dok-7 Tg mice show apparently normal motor function [12,13], the enhanced ACh release appears not to cause excessive and thus toxic AChR activation at the abnormally enlarged NMJs. Therefore, we hypothesized that the ultrastructural specializations of NMJs, such as presynaptic ACh release sites, active zones, and postsynaptic junctional folds, where AChRs cluster, might be altered in Dok-7 Tg mice so as to prevent toxic overactivation of AChRs. York et al. demonstrated that AChRs are concentrated at the edge of the junctional fold crests surrounding the fold openings at NMJs [21]. When viewed en-face by light microscopy, this arrangement shows a regular, striped appearance of AChR clusters at NMJs [22]. To determine whether muscle-specific overexpression of Dok-7 affects this aspect of postsynaptic development, we collected confocal images of the synapses in diaphragm muscles of 8-week-old Dok-7 Tg mice and WT mice (Fig. 3A and B). In WT mice, the postsynaptic AChR clusters are correctly organized into well-defined AChR stripes (Fig. 3B). By contrast, the postsynaptic AChR clusters in Dok-7 Tg mice do not form the regular AChR stripes. We next visualized active zones using an antibody against the active zone marker protein Bassoon (Fig. 3C and D). Consistent with previous studies [22], immunofluorescence of active zones at NMJs appears as discrete puncta, which are largely localized on top of the AChR stripes in WT mice. In Dok-7 Tg mice, although the majority of Bassoon puncta are also localized on the AChR-rich regions of NMJs, the density of active zones in the presynaptic motor nerve terminals was reduced (Fig. 3CeE). These results suggest that overexpression of Dok-7 in the muscle alters the pre- and post-synaptic ultrastructure of NMJs. Thus, we further examined ultrastructural alterations in NMJs using electron microscopy and found that the density of synaptic vesicles in the presynaptic motor nerve terminals was reduced in Dok-7 Tg mice, compared with WT mice (Fig. 4AeC). Furthermore, to estimate the organization and complexity of the postsynaptic membrane, we determined the ratio of postsynaptic versus presynaptic membrane length. Compared with WT mice, the ratio for Dok-7 Tg mice was significantly reduced (Fig. 4A and D). Consistent with this, the density and size of junctional folds in the postsynaptic membrane were reduced in Dok-7 Tg mice (Fig. 4A, E and F). These results indicate that muscle-specific overexpression of Dok-7 induces ultrastructural alterations in the pre- and post-synaptic
Fig. 1. Eight-week-old Dok-7 Tg mice show NMJ enlargement. (A) Whole-mount staining of NMJs in the diaphragm muscles. The AChRs were labeled with a-bungarotoxin (red), and motor axons and presynaptic nerve terminals were stained with antibodies against neurofilament-L and synapsin-1 (green). Scale bars, 20 mm. (BeD) Quantified results for the area of each presynaptic nerve terminal (B) and each AChR cluster (C), and for the cover ratio (presynaptic area divided by AChR cluster area) in each NMJ (D). Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined NMJs (3 mice per group). Asterisks denote a significant statistical difference: ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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Fig. 2. Eight-week-old Dok-7 Tg mice show enhanced neuromuscular transmission. (AeC) Quantified results for the amplitude (A) and frequency (B) of miniature endplate potentials (MEPPs), and for the quantal content of endplate potentials (EPPs) (C) in the diaphragm muscles. Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined NMJs (4e7 mice per group). Asterisks denote a significant statistical difference: *P < 0.05 and ***P < 0.001.
specializations of NMJs, which appears to attenuate the neuromuscular transmission and thus to prevent toxic overactivation of AChRs at the abnormally enlarged NMJs.
3.3. Terminal Schwann cells extend their processes into the synaptic clefts in Dok-7 Tg mice Although the motor axon is wrapped by the myelin sheath formed by Schwann cells (SCs), its terminal region is only capped by terminal SCs (tSCs) without myelin formation [1]. The tSCs are
believed to be required for the maintenance of NMJs. For instance, the ablation of SCs in mice, including tSCs, after NMJ maturation leads to the fragmentation and shrinkage of NMJs, defective neuromuscular transmission, and premature death [23]. In addition, the ablation of tSCs in frogs after NMJ maturation induces retraction of presynaptic motor nerve terminals from the NMJs [24]. However, electron microscopic analysis revealed that tSCs exhibited significantly greater penetration of their processes into the synaptic clefts in Dok-7 Tg mice than in WT mice (Fig. 4B and G), suggesting that muscle-specific overexpression of Dok-7 renders
Fig. 3. Eight-week-old Dok-7 Tg mice show disorganized AChR clustering and reduced density of active zones at NMJs. (A) Whole-mount staining of NMJs in the diaphragm muscles. AChRs, motor axons, and presynaptic nerve terminals were stained as in Fig. 1A. Scale bars, 20 mm. (B) Higher magnification of the white boxed area in (A). Scale bars, 2 mm. (C) Whole-mount staining of active zones and NMJs in the diaphragm muscles. Active zones were visualized using an anti-Bassoon antibody (blue), and AChRs, motor axons, and presynaptic nerve terminals were stained as in Fig. 1A. Scale bars, 5 mm. (D) Higher magnification of the white boxed area in (C). Scale bars, 2.5 mm. (E) Quantified results for the density of active zones in the presynaptic motor nerve terminals (Bassoon-positive area divided by presynaptic area). Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined NMJs (5 mice per group). Asterisks denote a significant statistical difference: ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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Fig. 4. Eight-week-old Dok-7 Tg mice show ultrastructural alterations of pre- and post-synaptic specializations and of tSCs. (A) Representative transmission electron micrographs of NMJs in the diaphragm muscles. NT, nerve terminal; M, myotube (myofiber). Scale bars, 500 nm. (B) Higher magnification of the black boxed area in (A). SV, synaptic vesicle; JF, junctional fold; tSC, terminal Schwann cell. Scale bars, 500 nm. (CeG) Quantified results for the density of synaptic vesicles in the presynaptic area (C), ratio of post-versus presynaptic membrane length (D), density of junctional folds in the postsynaptic membrane that faces the presynaptic membrane (Note that the number of junctional folds was divided by the length of the presynaptic membrane to evaluate the density of junctional folds) (E), size of junctional folds (F), and total length of tSC processes in the synaptic clefts per mm presynaptic membrane length (G). Values are represented as means ± SEM. Numbers in parentheses indicate the number of examined fields (CeE, G) or of examined junctional folds (F) (4 mice per group). Asterisks denote a significant statistical difference: ***P < 0.001.
the synaptic clefts accessible to the processes, which again may serve to attenuate the neuromuscular transmission at the abnormally enlarged NMJs. 4. Discussion In general, NMJs form selectively in the central region of myotubes (also known as myofibers), where MuSK is localized and controls the accumulation of AChR transcripts and proteins [25]. In our previous studies [9,12,13,20], we demonstrated that overexpression of Dok-7 in skeletal muscle in mice enlarges postsynaptic AChR clusters and presynaptic motor nerve terminals in the correct, central region of myotubes, suggesting that Dok-7mediated activation of MuSK induces not only intramuscular signaling but also retrograde signaling that enlarges presynaptic motor nerve terminals. However, in the present study, we demonstrated that the coverage ratio of presynaptic motor nerve terminals over postsynaptic AChR clusters per se decreased in Dok7 Tg mice (Fig. 1D), likely due to lagging enlargement of presynaptic motor nerve terminals in response to the enlarged AChR clusters in the muscle. In addition, muscle-specific overexpression of Dok-7 altered the ultrastructure of pre- and post-synaptic specializations and tSCs (Figs. 3 and 4). One possible explanation for these ultrastructural alterations in Dok-7 Tg mice could be insufficient expression of NMJ components, such as laminin b2 and collagen Q (ColQ); the former is a major component of the synaptic cleft basal lamina and the latter anchors AChE into the synaptic cleft basal lamina. Mice lacking laminin b2 show reduced densities of active
zones in the presynaptic motor nerve terminals and of junctional folds in the postsynaptic membrane, together with enhanced penetration of tSC processes into the synaptic clefts [26]. Moreover, mice lacking ColQ also show reduced density of synaptic vesicles, simplified junctional folds, and enhanced penetration of tSC processes into the synaptic clefts [27]. Since ultrastructural alterations of pre- and post-synaptic specializations and tSCs in Dok-7 Tg mice are similar to those found in laminin b2- or ColQ-deficient mice, it is important to investigate the effects of Dok-7 overexpression in the muscle on the expression of these NMJ components. Patients with neuromuscular diseases often show alterations in the ultrastructure of NMJs. Patients with Lambert-Eaton myasthenic syndrome exhibit impaired neuromuscular transmission and muscle weakness, together with a reduced number of active zones, which are caused by autoantibodies against the presynaptic voltage-gated calcium channel Cav2.1 [28]. Moreover, patients with Pierson syndrome exhibit impaired neuromuscular transmission, reduced density of synaptic vesicles, simplified junctional folds, and enhanced penetration of tSC processes into the synaptic clefts, which are caused by mutations in the laminin b2 genes [29]. In addition, mathematical modeling of neuromuscular transmission in the presence or absence of junctional folds suggests that they may act to reduce the threshold necessary for action potential firing, thus making neuromuscular transmission more efficient [30]. These studies support crucial roles for active zones and junctional folds in neuromuscular transmission. In this study, we demonstrated that muscle-specific overexpression of Dok-7 not only enhanced NMJ formation (Fig. 1B and C) and neuromuscular
Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011
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transmission (Fig. 2) but also reduced the densities of active zones and synaptic vesicles in the presynaptic motor nerve terminals (Figs. 3E and 4C). Furthermore, the density and size of junctional folds in the postsynaptic membrane were reduced in Dok-7 Tg mice (Fig. 4E and F), and tSCs exhibited enhanced penetration of their processes into the synaptic clefts in the mutant mice (Fig. 4G). Together, these findings indicate that muscle-specific overexpression of Dok-7 induces ultrastructural alterations to pre- and post-synaptic specializations and tSCs, which may prevent toxic overactivation of AChRs at the abnormally enlarged NMJs. Thus, further studies to uncover the role of Dok-7-mediated signaling in regulating the size, structure, and function of NMJs will be needed for better understanding NMJs and developing new therapeutic approach against NMJ pathologies. Acknowledgments The authors thank R. F. Whittier, R. Ueta, and A. Inoue-Yamauchi for critically reading the manuscript and for helpful suggestions. We also thank T. Yagami for help in animal care. This work was supported by the Grant for Joint Research Project of the Institute of Medical Science, the University of Tokyo and by JSPS KAKENHI Grant Number 17H01532. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.12.011 References [1] L. Li, W.C. Xiong, L. Mei, Neuromuscular junction formation, aging and disorders, Annu. Rev. Physiol. 80 (2018) 159e188. [2] A.G. Engel, X.M. Shen, D. Selcen, S.M. Sine, Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment, Lancet Neurol. 14 (2015) 420e434. [3] N.E. Gilhus, G.O. Skeie, F. Romi, K. Lazaridis, P. Zisimopoulou, S. Tzartos, Myasthenia gravis - autoantibody characteristics and their implications for therapy, Nat. Rev. Neurol. 12 (2016) 259e268. [4] R.T. Bois, R.G. Hummel, W.D. Dettbarn, M.B. Laskowski, Presynaptic and postsynaptic neuromuscular effects of a specific inhibitor of acetylcholinesterase, J. Pharmacol. Exp. Ther. 215 (1980) 53e59. [5] A.G. Engel, E.H. Lambert, T. Santa, Study of long-term anticholinesterase therapy. Effects on neuromuscular transmission and on motor end-plate fine structure, Neurology 23 (1973) 1273e1281. [6] M.B. Laskowski, W.H. Olson, W.D. Dettbarn, Ultrastructural changes at the motor end-plate produced by an irreversible cholinesterase inhibitor, Exp. Neurol. 47 (1975) 290e306. [7] K. Okada, A. Inoue, M. Okada, Y. Murata, S. Kakuta, T. Jigami, S. Kubo, H. Shiraishi, K. Eguchi, M. Motomura, T. Akiyama, Y. Iwakura, O. Higuchi, Y. Yamanashi, The muscle protein Dok-7 is essential for neuromuscular synaptogenesis, Science 312 (2006) 1802e1805. [8] D. Beeson, O. Higuchi, J. Palace, J. Cossins, H. Spearman, S. Maxwell, J. Newsom-Davis, G. Burke, P. Fawcett, M. Motomura, J.S. Müller, H. Lochmüller, C. Slater, A. Vincent, Y. Yamanashi, Dok-7 mutations underlie a neuromuscular junction synaptopathy, Science 313 (2006) 1975e1978. [9] A. Inoue, K. Setoguchi, Y. Matsubara, K. Okada, N. Sato, Y. Iwakura, O. Higuchi, Y. Yamanashi, Dok-7 activates the muscle receptor kinase MuSK and shapes
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Please cite this article as: T. Eguchi et al., Overexpression of Dok-7 in skeletal muscle enhances neuromuscular transmission with structural alterations of neuromuscular junctions: Implications in robustness of neuromuscular transmission, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.011