VAChT overexpression increases acetylcholine at the synaptic cleft and accelerates aging of neuromuscular junctions
© The Author(s). 2016
Received: 4 May 2016
Accepted: 26 August 2016
Published: 5 October 2016
Cholinergic dysfunction occurs during aging and in a variety of diseases, including amyotrophic lateral sclerosis (ALS). However, it remains unknown whether changes in cholinergic transmission contributes to age- and disease-related degeneration of the motor system. Here we investigated the effect of moderately increasing levels of synaptic acetylcholine (ACh) on the neuromuscular junction (NMJ), muscle fibers, and motor neurons during development and aging and in a mouse model for amyotrophic lateral sclerosis (ALS).
Chat-ChR2-EYFP (VAChTHyp) mice containing multiple copies of the vesicular acetylcholine transporter (VAChT), mutant superoxide dismutase 1 (SOD1G93A), and Chat-IRES-Cre and tdTomato transgenic mice were used in this study. NMJs, muscle fibers, and α-motor neurons’ somata and their axons were examined using a light microscope. Transcripts for select genes in muscles and spinal cords were assessed using real-time quantitative PCR. Motor function tests were carried out using an inverted wire mesh and a rotarod. Electrophysiological recordings were collected to examine miniature endplate potentials (MEPP) in muscles.
We show that VAChT is elevated in the spinal cord and at NMJs of VAChTHyp mice. We also show that the amplitude of MEPPs is significantly higher in VAChTHyp muscles, indicating that more ACh is loaded into synaptic vesicles and released into the synaptic cleft at NMJs of VAChTHyp mice compared to control mice. While the development of NMJs was not affected in VAChTHyp mice, NMJs prematurely acquired age-related structural alterations in adult VAChTHyp mice. These structural changes at NMJs were accompanied by motor deficits in VAChTHyp mice. However, cellular features of muscle fibers and levels of molecules with critical functions at the NMJ and in muscle fibers were largely unchanged in VAChTHyp mice. In the SOD1G93A mouse model for ALS, increasing synaptic ACh accelerated degeneration of NMJs caused motor deficits and resulted in premature death specifically in male mice.
The data presented in this manuscript demonstrate that increasing levels of ACh at the synaptic cleft promote degeneration of adult NMJs, contributing to age- and disease-related motor deficits. We thus propose that maintaining normal cholinergic signaling in muscles will slow degeneration of NMJs and attenuate loss of motor function caused by aging and neuromuscular diseases.
KeywordsNeuromuscular junction Acetylcholine VAChT Aging ALS Synapse Cholinergic transmission Motor neuron
The organization and stability of synapses is dictated by the actions of pre- and postsynaptic organizing molecules, including neurotransmitters [1–3]. The vertebrate neuromuscular junction (NMJ), the synapse formed between α-motor neurons and skeletal muscle fibers, is under the influence of the neurotransmitter acetylcholine (ACh) . In addition to promoting muscle contraction, several lines of evidence indicate that ACh acts as an anti-synaptogenic factor [4, 5]. ACh promotes dispersion of nicotinic acetylcholine receptors (AChR) from postsynaptic sites by inducing posttranslational changes and accelerating the endocytosis of AChRs . In mice lacking ACh, developing muscles contain larger and more complex postsynaptic sites that are innervated by silent motor axons. These postsynaptic sites initially form but fail to mature in the absence of neural-derived agrin (z-agrin), a molecule with critical roles in stabilizing AChRs [7, 8]. These findings have led to the hypothesis that ACh acts in concert with z-agrin in sculpting and stabilizing postsynaptic sites .
The recent generation of transgenic mice with altered expression of the vesicular acetylcholine transporter (VAChT) has made it possible to examine the impact of varying levels of ACh throughout the lifespan of mice [9, 10]. VAChT functions to load ACh into synaptic vesicles, thereby regulating the amount of neurotransmitter released . Young adult mice with reduced expression of VAChT (VAChTKD) have reduced cholinergic neurotransmission  while mice overexpressing VAChT (ChAT–ChR2–EYFP; herein called VAChTHyp) [12, 13] release more ACh at cholinergic synapses and thus have heightened cholinergic transmission . Both transgenic lines exhibit cognitive deficits, indicating that cholinergic circuits in the brain are highly sensitive to both increasing and decreasing synaptic ACh. In the lower motor system, decreasing synaptic ACh by approximately 70 %, as is the case in VAChTKD mice, results in symptoms resembling myasthenia gravis . These mice also exhibit moderate changes in the shape and distribution of synaptic vesicles in α-motor axon nerve endings . Conversely, young adult VAChTHyp mice have enhanced motor endurance consistent with increased cholinergic tone .
During aging and throughout the progression of amyotrophic lateral sclerosis (ALS), neuromuscular function decreases suggesting that increasing the release of ACh may potentially preserve muscle tone [1, 15]. Here we used VAChTHyp mice to determine the contribution of chronically increasing synaptic ACh levels on developing, aging, and ALS-afflicted NMJs. We discovered that NMJs prematurely acquire age-related structural alterations in VAChTHyp mice. These structural changes at NMJs are accompanied by a moderate reduction in the size of muscle fibers and motor deficits in aged VAChTHyp mice. To determine if increased cholinergic transmission affects the progression of ALS-like pathology, we examined SOD1G93A mice overexpressing VAChT. Increasing cholinergic transmission selectively affected male SOD1G93A mice, prematurely causing motor deficits, degeneration of NMJs, and accelerating death of these mice. Together, these findings demonstrate that increasing cholinergic transmission accelerates the degeneration of NMJs during aging and progression of ALS-like pathology in a mouse model of the disease.
Several transgenic lines were used in these experiments. We obtained the following lines from The Jackson Laboratory: VAChTHyp (B6.Cg-Tg(Chat-COP4*H134R/EYFP,Slc18a3)6Gfng/J) , SOD1G93A (B6.Cg-Tg(SOD1*G93A)1Gur/J) , ChAT-IRES-Cre (B6;129S6-Chat < tm1(cre)Lowl>/J) , tdTomato (B6;129S6-Gt(ROSA)26Sor tm14(CAG-tdTomato)Hze /J) . Mice overexpressing VAChT  contained several bacterial artificial chromosomes (BAC) modified to express channelrhodopsin from the ChAT locus. However, the BAC construct used to generate these transgenic mice also contained an intact VAChT locus, thus introducing additional functional copies of the VAChT gene. All transgenic mice overexpressing VAChT (VAChTHyp)  were maintained as heterozygous and allowed free access to food and water. For histological and biochemical analysis of NMJs, muscle fibers, motor neurons, and motor axons, we used at least four mice per genotype per experiment. All experiments were carried out under NIH guidelines and animal protocols approved by the Virginia Tech Institutional Animal Care and Use Committee.
We recorded miniature endplate potentials (MEPPs) using an Axoclamp 2A amplifier. Recordings were band-passed filtered at 0.1 to 5 kHz, further amplified 50 times by a Cyberamp, and then sampled on a computer at a frequency of 100 kHz. Microelectrodes were fabricated from borosilicate glass using a Narishige puller (PN-30) and had resistances of 8–15 Mohms when filled with 3 M KCl. The microelectrodes were inserted into the muscle fiber in the endplate region to record MEPPs. Tetrodotoxin (100 nM) was added to the bath solution to prevent muscle contraction. The signals were digitized by a board from National Instruments (NIDAQ-MX) and acquired by the program WinEDR (John Dempster, University of Strathclyde). MEPP amplitudes were corrected to a standard resting potential of −70 mV. Approximately 100 MEPPs in five different synapses per muscle were analyzed. We used three muscles from three different animals per genotype at 4 months of age.
Immunohistochemistry and histological analysis under confocal microscopy
Mice were anesthetized with isoflurane and perfused transcardially with 4 % paraformaldehyde in phosphate buffered saline (PBS). The extensor digitorum longus (EDL) muscle was then dissected. Whole muscles were blocked for 1 h at room temperature (blocking solution; 1 % Triton X-100, 3 % BSA, 5 % goat serum in PBS) and then incubated with primary antibodies for 24 h in blocking solution to visualize AChRs, axons, and synaptic vesicles. Following staining with primary antibodies, muscles were washed three times with PBS-T (0.1 % Triton X-100) and incubated for 2 h with Alexa 488 or 555 conjugated α-bungarotoxin (fBTX, Life Technologies; 1:1000) and secondary antibodies. After washing with PBS-T, the muscles were whole-mounted onto slides using Vectashield (Vector Labs). The primary antibodies used were synaptotagmin-2 (znp-1, Zebrafish International Resource Center; 1:100) and VAChT (Millipore; 1:250). The secondary antibodies used were Alexa-647 anti-mouse IgG2a (Life Technologies; 1:1000) and Alexa-647 anti-guinea pig IgG (Life Technologies; 1:1000).
To analyze structural features at NMJs, maximum intensity projections of confocal stacks were created using ZEN software (Zeiss). We analyzed structural features following the criteria previously described by Valdez et al. . Briefly, fragmented AChRs are defined as five or more AChR clusters in small islands with round shape and/or a segment of the postsynapse. It also includes NMJs with small and/or irregularly shaped AChR clusters. Full or partial denervation describes postsynaptic sites not appropriately opposed by the nerve terminal. Multiple innervations are the simultaneous innervation of the postsynapse by two or more axons. A nerve sprout occurs when the nerve extends beyond AChR clusters in any direction. Colocalization is the extent of pre- and postsynaptic apposition measured using ZEN software. To quantify the size of NMJs, the region occupied by AChRs was measured using ImageJ software.
Fluorescence intensity analysis
To analyze the fluorescence intensity of VAChT immunostaining at NMJs, maximum intensity projections of confocal stacks were created using ZEN software (Zeiss). Using Zen Black software (Zeiss), individual NMJs were outlined and the mean fluorescence intensity was determined by Zen Black software with background fluorescence subtracted. The mean fluorescence intensity for individual NMJs was averaged to find the overall fluorescence intensity for each animal.
Muscle fiber diameter/central nuclei
The tibialis anterior (TA) muscle was dissected from perfused mice, transferred into a 30 % sucrose solution for 2 days, and cut using a cryostat at 14-μm thickness. To visualize muscle fiber size and location of nuclei, the sections were stained by first blocking for 1 h at room temperature (0.1 % Triton X-100, 3 % BSA, 5 % goat serum in PBS) and then incubated with an antibody against laminin (L9393, Sigma; 1:100) for 24 h in blocking solution. The sections were washed three times in PBS and incubated for 3 h with secondary antibodies (Alexa-568 anti-rabbit IgG, Life Technologies; 1:1000). After washing with PBS, sections were incubated with 4′,6-diamidino-2-phenylindole (DAPI; Sigma; 1:1000), washed with PBS, and mounted using Vectashield. The area outlined by laminin was measured using ImageJ software. At least 300 muscle fibers per mouse were randomly selected and used for this analysis. Myonuclei located in the center of muscle fibers were counted. At least 1000 nuclei per mouse were counted.
To visualize motor axons, Chat-Cre;tdTom mice were used. The peroneal nerve was dissected from perfused mice and embedded in a tissue-freezing medium (Tissue-Tek). The specimens were cut in a cryostat at 10-μm thickness. Sections were blocked for 1 h at room temperature (0.1 % Triton X-100, 3 % BSA, 5 % goat serum in PBS) and subsequently incubated with antibodies for both neurofilament (smi-312, Covance; 1:1000) and S-100 (Z0311, Dako; 1:400) for 24 h in blocking solution. Sections were then washed three times with PBS for 5 min each and incubated with secondary antibodies (Alexa-488 anti-mouse IgG1, Alexa-647 anti-rabbit IgG, Life Technologies; 1:1000). After washing with PBS, sections were mounted in Vectashield. Axons expressing tdTom and neurofilament were counted as motor axons. Axons labeled with only neurofilament are sensory axons.
Expression analysis using quantitative PCR
SOD1G93A copy number analysis
DNA isolation was performed with the DNeasy Blood and tissue kit (Qiagen). Changes in transgene copy numbers were evaluated using TaqMan probe-based quantitative real-time PCR by determining the difference in threshold cycle (ΔCT) between the transgene (hSOD1) and an internal positive control gene. Primers for hSOD1 were as follows: IMR9665 or Forward = 5′-GGGAAGCTGTTGTCCCAAG-3′; and IMR9666 or Reverse = 5′-CAAGGGGAGGTAAAAGAGAGC-3′. The TaqMan probe used for hSOD1 is 13854 = 5′-CTGCATCTGGTTCTTGCAAAACACCA-3′. The primers for the internal positive control gene were as follows: IMR1544 or Forward = 5′-CACGTGGGCTCCAGCATT-3′; and IMR3580 or Reverse = 5′-TCACCAGTCATTTCTGCCTTTG-3′. The TaqMan probe for the internal positive control gene is TmoIMR0105 = 5′-CCAATGGTCGGGCACTGCTCAA-3′. These primers and probes are described in the Jackson Laboratories protocol for genotyping hSOD1 transgenic mice. We made minor modifications to the protocol to compare levels of hSOD1 between mice. We used 5 ng of genomic DNA, the TaqMan Universal PCR Mastermix (Thermo Fisher Scientific), and the Bio-Rad CFX Connect Real-Time System (Bio-Rad) to amplify hSOD1. The following PCR settings were used: 50 °C for 2 min, 95 °C for 10 min, 40 PCR cycles of 95 °C for 15 s, 60 °C for 1 min. To determine relative copy numbers, we compared the ΔCT value between SOD1G93A and SOD1G93A;VAChTHyp for hSOD1 after subtracting the CT value of the internal control gene for each sample. The same ΔCT value indicates that there is no difference in the number of hSOD1 between SOD1G93A and SOD1G93A;VAChTHyp mice.
We examined motor function using an inverted grid hanging test . The mice were placed on the center of a wire grid, which was mounted 25 cm above the table. After gently inverting the wire grid, we recorded the time the mouse remained hanging from the wire mesh. Each mouse was tested three times with at least 5-min breaks between trials. When comparing the ability of mice to stay on the wire mesh, we only used the maximum time they spent hanging for each trial.
Mice were placed on a rotarod (Ugo Basile Instruments). The time that the mice were able to spend on the rotating platform was recorded. The following settings were used: for 400-day-old VAChTHyp and wild-type mice, acceleration was set to 8.0 rpm/min with no reverse. The maximum speed was set to 50 rpm and the minimum speed was 4 rpm. To test motor function in SOD1G93A mice, acceleration was set to 5.0 rpm/min with no reverse. The maximum speed was set to 80 rpm and the minimum speed was 2 rpm. Each mouse was tested three times with at least 5-min breaks between trials. When comparing the ability of mice to stay on the rotarod, we only used the maximum time they spent on the rotating platform for each trial.
At least eight animals were tested on both the rotarod and hanging tests. To compare motor function between young adult mice, 5-month-old male wild-type and VAChTHyp mice were used. To compare motor function between middle-aged mice, 13-month-old female wild-type mice and VAChTHyp mice were examined. We examined male mice expressing SOD1G93A.
Analysis of survival rates
All mice affected with ALS were regularly observed. Mice were euthanized when they were unable to right themselves back up after lying on their sides. A Kaplan–Meier log rank test was used to compare the lifespan between the groups of mice affected with ALS.
Student’s t test and a one-way ANOVA followed by Tukey–Kramer and Kolmogorov-Smirnov tests were used to compare differences between groups. Data were expressed as the mean ± SE (standard error). P < 0.05 was considered statistically significant.
VAChT is increased at NMJs of VAChTHyp mice
Miniature endplate potential amplitudes are increased in VAChTHyp muscles
Normal development of NMJs in VAChTHyp mice
Nerve activity has been well documented to influence the maturation and specification of muscle fiber types . There are four major types of skeletal muscle fibers that can be identified based on their expression of myosin heavy chain (MyHC) isoforms (type 1, 2A, 2X, or 2B) . We thus asked if altering levels of ACh affect muscle biogenesis, independently of changes at NMJs. In the tibialis anterior (TA) and EDL muscles of 9-day-old VAChTHyp mice, mRNA levels for MyHC type 2A, 2B, and 2X were similar to those in control mice (Fig. 3g). These findings show that the increased level of synaptic ACh in VAChTHyp does not alter the normal development of muscle fibers and NMJs in the TA and EDL muscles.
NMJs exhibit age-related changes in young adult VAChTHyp mice
NMJ degeneration precedes motor deficits in VAChTHyp mice
NMJ degeneration precedes muscle atrophy in VAChTHyp mice
Presynaptic degeneration occurs in the absence of obvious changes in motor neurons in VAChTHyp mice
As a response to presynaptic degeneration and increased expression of VAChT, motor neurons may increase expression of agrin isoforms that function to stabilize the NMJ . To explore this possibility, we examined transcripts for neural agrin isoforms (z-agrin) in the spinal cord of 5-month-old VAChThyp and control mice. While we found that z-agrin isoforms were not statistically altered in the spinal cord of VAChTHyp mice compared to control mice, it is worth noting that all three z-agrin isoforms were moderately and consistently higher, approximately 1.8-fold for all isoforms in VAChTHyp mice (Fig. 10d). If motor neurons were to increase these z-agrin isoforms in VAChTHyp mice, it would likely be to stabilize nAChRs on the postsynaptic region, and thus prevent further fragmentation and denervation of NMJs. It is also plausible that overexpression of VAChT results in induction of genes involved in the biosynthesis of ACh. This seems unlikely, however, since the choline acetylcholine transferase (ChAT) is expressed at similar levels in the spinal cord of VAChTHyp and control mice (Fig. 10e). Together with the lack of axonal degeneration, these findings indicate that increasing synaptic ACh directly affects the structural integrity of the NMJ.
Increased cholinergic transmission accelerates ALS pathogenesis
Impact of increasing synaptic ACh on NMJs, muscle fibers, and motor neurons
VAChT plays a critical role in the storage of ACh in synaptic vesicles [10, 40, 41]. In VAChTHyp mice, VAChT and ACh secretion is increased in brain tissue . Our qPCR and immunofluorescence analysis revealed that VAChT is similarly increased in the spinal cord and at the NMJ. Previous experiments have examined the amount of ACh released from hippocampal slices from these mice . However, these studies did not address whether increased expression of VAChT results in more ACh loaded into synaptic vesicles in vivo and in motor neurons. Our finding that MEPP amplitudes are increased in VAChTHyp mice strongly suggests that overexpressing VAChT increases ACh accumulation in matured synaptic vesicles. These results suggest that at least a population of mature synaptic vesicles can accumulate higher amounts of ACh upon increased levels of transporter protein. Vesicular ACh accumulation by VAChT is complex and thought to be tightly regulated . Our data suggest that potential changes in expression or trafficking of VAChT to synaptic vesicles can regulate the amount of ACh secreted by nerve endings by direct regulation of neurotransmitter accumulation in synaptic vesicles. Previous reports have shown that overexpression of VAChT in immature nerve-muscle Xenopus cultures increases both amplitude and frequency of MEPPs. This was interpreted as incorporation of VAChT in immature synaptic vesicles can both increase the amount of ACh stored in vesicles as well as increase the number of filled vesicles available for spontaneous release . We did not observe an increased frequency of MEPPs in adult mice overexpressing VAChT, suggesting that the population of vesicles available for spontaneous release is unchanged in the NMJ.
In addition to its function in neurotransmission, ACh plays a critical role in the development of the NMJ. ACh acts as an anti-synaptogenic molecule by decreasing the stability of AChRs, and through this activity, it is believed to function together with the neural-derived factor, z-agrin, in sculpting the NMJ . In this regard, while deletion of genes that prevent the synthesis [5, 7] and transport  of ACh into synaptic vesicles cause significant changes at NMJs, the impact of increasing synaptic ACh levels on developing and adult NMJs had not been examined. We now show that the rate of synapse elimination, the size of AChR clusters, and their apposition with presynaptic sites are unchanged in developing VAChTHyp mice. In addition, we found no difference in the development of muscle fibers. The normal development of NMJs and muscle fibers in VAChTHyp mice may be attributed to different factors. The additional ACh released by motor neurons in VAChTHyp mice may not change muscle action potentials as ACh is already in excess during development . Another plausible explanation, though not necessarily mutually exclusive, is that synaptic ACh and z-agrin are present at saturating levels compared to nAChRs during development. Thus, developing NMJs and muscle fibers are likely immune to further increasing ACh at the synaptic cleft, as is the case in VAChTHyp mice. Regarding synaptic elimination, all motor neurons in VAChTHyp mice are likely to retain their “competitive vigor” [44, 45] since ACh is likely increased by the same magnitude in all NMJs. Hence, the time motor axons spend competing for the same target would not be expected to change in VAChTHyp compared to those in control mice.
In adulthood, the incidence of NMJs with deleterious structural features increases with aging in control mice, and those features are more pronounced in VAChTHyp mice. These findings indicate that NMJs within the EDL muscle vary in their susceptibility to increased levels of ACh at the synaptic cleft. The EDL muscle is primarily composed of muscle fibers that express myosin heavy chain type 2A and type 2B . Thus, it is plausible that one of these muscle fiber type is more susceptible to increased synaptic ACh levels, a possibility not addressed in this work. Another possibility is that NMJs receiving less resource within a motor unit fail to adequately repair following structural changes driven by increased ACh at their synaptic clefts. In this regard, it has been shown that NMJs within the same motor unit respond differently to aging . It is also possible that some motor neurons are more active, resulting in earlier degeneration of NMJs that make up their motor unit. Thus, an increase in the amount of synaptic ACh together with less functional z-agrin in adult NMJs would result in structural changes due to increased destabilization of AChRs. However, this conclusion is not supported by the lack of changes in transcript for the gamma AChR subunit, which is closely associated with increased internalization and degradation of AChRs. While NMJs are clearly affected in adult VAChTHyp mice, it remains possible that cholinergic dysregulation elsewhere in the peripheral and central nervous system, including the spinal cord, may cause or contribute to the precocious degeneration of NMJs observed in young adult VAChTHyp mice. Regardless, the results in this paper do not support previous suggestions [1, 15] that decreased neuromuscular function as a result of aging and ALS should be reversed by increased release of ACh at the NMJ synaptic cleft. Instead, these findings raise the possibility that the age-related increase in cholinergic transmission reported by several published studies contribute to the degeneration of NMJs [46–50].
It is interesting that old VAChTHyp mice present with worse muscle function than age-matched control mice. This is in striking contrast with better performance of young VAChTHyp mice on the treadmill . Interestingly, recent analysis of the cardiac muscle in VAChTHyp mice showed that they have preserved cardiac function when challenged with angiotensin II, a model of heart failure . These data suggest that the better performance of VAChTHyp mice on the treadmill may be a result of better cardiovascular fitness.
Male SOD1G93A mice are selectively susceptible to increased ACh levels
We also showed that increasing cholinergic transmission selectively affects male mice harboring SOD1G93A. Increased synaptic ACh causes NMJs to prematurely degenerate and accelerates death of male SOD1G93A mice. While our findings demonstrate that increasing synaptic ACh does not affect female SOD1G93A mice, it is worth noting that SOD1G93A mice acquire neurological symptoms by 90 days and die within the first 160 days of life. Hence, it remains possible that increasing ACh at NMJs affects the initiation and progression of ALS-related pathology in long-lived female mice expressing mutant genes known to cause ALS, in addition to further exacerbating neurological symptoms in long-lived male mice expressing the same mutant genes. Irrespective, our data corroborates published findings indicating that male animals and humans are more susceptible to ALS [52, 53], and factors that suspect of altering the initiation and progression of the disease [54–58].
Significance to understanding aging- and disease-related changes at NMJs
It is now recognized that cholinergic dysfunction occurs in neurons harboring ALS-causing mutant genes and during normal aging [1, 35, 36, 59]. In addition, perisynaptic Schwann cells were recently shown to be uniquely sensitive to altered cholinergic transmission prior to the initiation of ALS-related symptoms in a mouse model for the disease . The identification of mutations in cholinergic receptors in patients with ALS provides additional evidence that changes in cholinergic transmission directly drives degeneration of motor neurons and their NMJs [38, 39, 60]. In this regard, the only FDA-approved drug to treat ALS, riluzole, was recently shown to bind and to block the activity of muscle nicotinic AChRs obtained from patients with the disease . The results presented in this paper provide evidence that increased cholinergic tone deteriorates NMJs in a mouse model of ALS and during normal aging. Thus, modulation of cholinergic transmission should be considered in approaches aimed at preventing degeneration of the motor system during the progression of ALS and normal aging.
This study sought to determine the impact of increasing synaptic ACh levels on developing, aging, and ALS-afflicted NMJs using mice expressing multiple copies of VAChT. We showed that while NMJs develop normally in VAChTHyp mice, they progressively degenerate as the mice aged. This study also revealed that destruction of NMJs precedes degeneration of muscle fibers and motor neurons during aging and in mice harboring an ALS-causing mutant gene. Together, the data in this paper demonstrate that dysregulated levels of ACh cause pathophysiological changes at NMJs and contribute to loss of motor function during aging and the progression of ALS.
Amyotrophic Lateral Sclerosis
Miniature Endplate Potential
- SOD1G93A :
Superoxide Dismutase 1 mutated from a glycine to alanine at codon 93
Vesicular Acetylcholine Transporter
- VAChTHyp :
Yellow Fluorescence Protein
The authors would like to thank members of the Valdez laboratory, in particular Nicholas Maxwell for genotyping and caring for all the mice used in this study.
This work supported by the following NIH grants from NINDS (K01NS085071) and NIA (R56AG051501).
SS conducted the experiments, analyzed the data, and generated figures 1 and 3–10. LLF and CW generated data for figures 4-5 and 10-11. SKV generated data for figures 1, 3, 6, and 10. MPSMG, WC, LAN, VFP, MAMP, and CG designed and conducted experiments for figure 2. MPSMG and CG generated data for figures 3–4. SS, SKV, MPSMG, CG, VFP, and MAMP contributed to writing and editing the manuscript. GV designed the study, conducted experiments, and prepared the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.
All experiments were carried out under NIH guidelines and animal protocols approved by the Virginia Tech Institutional Animal Care and Use Committee.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Tintignac LA, Brenner H-R, Rüegg MA. Mechanisms regulating neuromuscular junction development and function and causes of muscle wasting. Physiol Rev. 2015;95:809–52.View ArticlePubMedGoogle Scholar
- Harris KP, Littleton JT. Transmission, development, and plasticity of synapses. Genetics. 2015;201:345–75.View ArticlePubMedGoogle Scholar
- Andreae LC, Burrone J. The role of neuronal activity and transmitter release on synapse formation. Curr Opin Neurobiol. 2014;27:47–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin W, Dominguez B, Yang J, Aryal P, Brandon EP, Gage FH, Lee K-F. Neurotransmitter acetylcholine negatively regulates neuromuscular synapse formation by a Cdk5-dependent mechanism. Neuron. 2005;46:569–79.View ArticlePubMedGoogle Scholar
- Misgeld T, Burgess RW, Lewis RM, Cunningham JM, Lichtman JW, Sanes JR. Roles of neurotransmitter in synapse formation: development of neuromuscular junctions lacking choline acetyltransferase. Neuron. 2002;36:635–48.View ArticlePubMedGoogle Scholar
- St John PA, Gordon H. Agonists cause endocytosis of nicotinic acetylcholine receptors on cultured myotubes. J Neurobiol. 2001;49:212–23.View ArticlePubMedGoogle Scholar
- Misgeld T, Kummer TT, Lichtman JW, Sanes JR. Agrin promotes synaptic differentiation by counteracting an inhibitory effect of neurotransmitter. Proc Natl Acad Sci U S A. 2005;102:11088–93.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin W, Burgess RW, Dominguez B, Pfaff SL, Sanes JR, Lee KF. Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature. 2001;410:1057–64.View ArticlePubMedGoogle Scholar
- Kolisnyk B, Guzman MS, Raulic S, Fan J, Magalhães AC, Feng G, Gros R, Prado VF, Prado MAM. ChAT-ChR2-EYFP mice have enhanced motor endurance but show deficits in attention and several additional cognitive domains. J Neurosci. 2013;33:10427–38.View ArticlePubMedGoogle Scholar
- Prado VF, Martins-Silva C, de Castro BM, Lima RF, Barros DM, Amaral E, Ramsey AJ, Sotnikova TD, Ramirez MR, Kim H-G, Rossato JI, Koenen J, Quan H, Cota VR, Moraes MFD, Gomez MV, Guatimosim C, Wetsel WC, Kushmerick C, Pereira GS, Gainetdinov RR, Izquierdo I, Caron MG, Prado MAM. Mice deficient for the vesicular acetylcholine transporter are myasthenic and have deficits in object and social recognition. Neuron. 2006;51:601–12.View ArticlePubMedGoogle Scholar
- Prado VF, Roy A, Kolisnyk B, Gros R, Prado MAM. Regulation of cholinergic activity by the vesicular acetylcholine transporter. Biochem J. 2013;450:265–74.View ArticlePubMedGoogle Scholar
- Ting JT, Feng G. Recombineering strategies for developing next generation BAC transgenic tools for optogenetics and beyond. Front Behav Neurosci. 2014;8:111.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhao S, Ting JT, Atallah HE, Qiu L, Tan J, Gloss B, Augustine GJ, Deisseroth K, Luo M, Graybiel AM, Feng G. Cell type–specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat Methods. 2011;8:745–52.View ArticlePubMedPubMed CentralGoogle Scholar
- Rodrigues HA, Fonseca M de C, Camargo WL, Lima PMA, Martinelli PM, Naves LA, Prado VF, Prado MAM, Guatimosim C. Reduced expression of the vesicular acetylcholine transporter and neurotransmitter content affects synaptic vesicle distribution and shape in mouse neuromuscular junction. PLoS One. 2013;8:e78342.View ArticlePubMedPubMed CentralGoogle Scholar
- Moloney EB, de Winter F, Verhaagen J. ALS as a distal axonopathy: molecular mechanisms affecting neuromuscular junction stability in the presymptomatic stages of the disease. Front Neurosci. 2014;8:252.View ArticlePubMedPubMed CentralGoogle Scholar
- Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, Alexander DD, Caliendo J, Hentati A, Kwon YW, Deng HX. Motor neuron degeneration in mice that express a human Cu, Zn superoxide dismutase mutation. Science. 1994;264:1772–5.View ArticlePubMedGoogle Scholar
- Rossi J, Balthasar N, Olson D, Scott M, Berglund E, Lee CE, Choi MJ, Lauzon D, Lowell BB, Elmquist JK. Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis. Cell Metab. 2011;13:195–204.View ArticlePubMedPubMed CentralGoogle Scholar
- Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci. 2010;13:133–40.View ArticlePubMedGoogle Scholar
- Valdez G, Tapia JC, Kang H, Clemenson Jr GD, Gage FH, Lichtman JW, Sanes JR. Attenuation of age-related changes in mouse neuromuscular synapses by caloric restriction and exercise. Proc Natl Acad Sci U S A. 2010;107:14863–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Pérez-García MJ, Burden SJ. Increasing MuSK activity delays denervation and improves motor function in ALS mice. Cell Rep. 2012;2:497–502.View ArticlePubMedPubMed CentralGoogle Scholar
- Ren J, Qin C, Hu F, Tan J, Qiu L, Zhao S, Feng G, Luo M. Habenula “cholinergic” neurons co-release glutamate and acetylcholine and activate postsynaptic neurons via distinct transmission modes. Neuron. 2011;69:445–52.View ArticlePubMedGoogle Scholar
- Sanes JR, Lichtman JW. Development of the vertebrate neuromuscular junction. Annu Rev Neurosci. 1999;22:389–442.View ArticlePubMedGoogle Scholar
- Missias AC, Chu GC, Klocke BJ, Sanes JR, Merlie JP. Maturation of the acetylcholine receptor in skeletal muscle: regulation of the AChR gamma-to-epsilon switch. Dev Biol. 1996;179:223–38.View ArticlePubMedGoogle Scholar
- Takahashi M, Kubo T, Mizoguchi A, Carlson CG, Endo K, Ohnishi K. Spontaneous muscle action potentials fail to develop without fetal-type acetylcholine receptors. EMBO Rep. 2002;3:674–81.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu Y, Sugiura Y, Padgett D, Lin W. Postsynaptic development of the neuromuscular junction in mice lacking the gamma-subunit of muscle nicotinic acetylcholine receptor. J Mol Neurosci. 2010;40:21–6.View ArticlePubMedGoogle Scholar
- Schiaffino S, Sandri M, Murgia M. Activity-dependent signaling pathways controlling muscle diversity and plasticity. Physiology (Bethesda). 2007;22:269–78.View ArticleGoogle Scholar
- Chakkalakal JV, Kuang S, Buffelli M, Lichtman JW, Sanes JR. Mouse transgenic lines that selectively label Type I, Type IIA, and Types IIX + B skeletal muscle fibers. Genesis. 2012;50:50–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Valdez G, Tapia JC, Lichtman JW, Fox MA, Sanes JR. Shared resistance to aging and ALS in neuromuscular junctions of specific muscles. PLoS One. 2012;7:e34640.View ArticlePubMedPubMed CentralGoogle Scholar
- Frail DE, Musil LS, Buonanno A, Merlie JP. Expression of RAPsyn (43K protein) and nicotinic acetylcholine receptor genes is not coordinately regulated in mouse muscle. Neuron. 1989;2:1077–86.View ArticlePubMedGoogle Scholar
- Bowen DC, Park JS, Bodine S, Stark JL, Valenzuela DM, Stitt TN, Yancopoulos GD, Lindsay RM, Glass DJ, DiStefano PS. Localization and regulation of MuSK at the neuromuscular junction. Dev Biol. 1998;199:309–19.View ArticlePubMedGoogle Scholar
- Apel PJ, Alton T, Northam C, Ma J, Callahan M, Sonntag WE, Li Z. How age impairs the response of the neuromuscular junction to nerve transection and repair: An experimental study in rats. J Orthop Res. 2009;27:385–93.View ArticlePubMedPubMed CentralGoogle Scholar
- Gomes MD, Lecker SH, Jagoe RT, Navon A, Goldberg AL. Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy. Proc Natl Acad Sci U S A. 2001;98:14440–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Sandri M, Sandri C, Gilbert A, Skurk C, Calabria E, Picard A, Walsh K, Schiaffino S, Lecker SH, Goldberg AL. Foxo transcription factors induce the atrophy-related ubiquitin ligase atrogin-1 and cause skeletal muscle atrophy. Cell. 2004;117:399–412.View ArticlePubMedPubMed CentralGoogle Scholar
- Burgess RW, Nguyen QT, Son YJ, Lichtman JW, Sanes JR. Alternatively spliced isoforms of nerve- and muscle-derived agrin: their roles at the neuromuscular junction. Neuron. 1999;23:33–44.View ArticlePubMedGoogle Scholar
- Rocha MC, Pousinha PA, Correia AM, Sebastião AM, Ribeiro JA. Early changes of neuromuscular transmission in the SOD1(G93A) mice model of ALS start long before motor symptoms onset. PLoS One. 2013;8:e73846.View ArticlePubMedPubMed CentralGoogle Scholar
- Wainger BJ, Kiskinis E, Mellin C, Wiskow O, Han SSW, Sandoe J, Perez NP, Williams LA, Lee S, Boulting G, Berry JD, Brown RH, Cudkowicz ME, Bean BP, Eggan K, Woolf CJ. Intrinsic membrane hyperexcitability of amyotrophic lateral sclerosis patient-derived motor neurons. Cell Rep. 2014;7:1–11.View ArticlePubMedPubMed CentralGoogle Scholar
- Arbour D, Tremblay E, Martineau É, Julien J-P, Robitaille R. Early and persistent abnormal decoding by glial cells at the neuromuscular junction in an ALS model. J Neurosci. 2015;35:688–706.View ArticlePubMedGoogle Scholar
- Moriconi C, Di Angelantonio S, Piccioni A, Trettel F, Sabatelli M, Grassi F. Mutant human β4 subunit identified in amyotrophic lateral sclerosis patients impairs nicotinic receptor function. Pflugers Arch. 2011;461:225–33.View ArticlePubMedGoogle Scholar
- Sabatelli M, Eusebi F, Al-Chalabi A, Conte A, Madia F, Luigetti M, Mancuso I, Limatola C, Trettel F, Sobrero F, Di Angelantonio S, Grassi F, Di Castro A, Moriconi C, Fucile S, Lattante S, Marangi G, Murdolo M, Orteschi D, Del Grande A, Tonali P, Neri G, Zollino M. Rare missense variants of neuronal nicotinic acetylcholine receptor altering receptor function are associated with sporadic amyotrophic lateral sclerosis. Hum Mol Genet. 2009;18:3997–4006.View ArticlePubMedGoogle Scholar
- de Castro BM, Pereira GS, Magalhães V, Rossato JI, De Jaeger X, Martins-Silva C, Leles B, Lima P, Gomez MV, Gainetdinov RR, Caron MG, Izquierdo I, Cammarota M, Prado VF, Prado MAM. Reduced expression of the vesicular acetylcholine transporter causes learning deficits in mice. Genes Brain Behav. 2009;8:23–35.View ArticlePubMedGoogle Scholar
- de Castro BM, De Jaeger X, Martins-Silva C, Lima RDF, Amaral E, Menezes C, Lima P, Neves CML, Pires RG, Gould TW, Welch I, Kushmerick C, Guatimosim C, Izquierdo I, Cammarota M, Rylett RJ, Gomez MV, Caron MG, Oppenheim RW, Prado MAM, Prado VF. The vesicular acetylcholine transporter is required for neuromuscular development and function. Mol Cell Biol. 2009;29:5238–50.View ArticlePubMedPubMed CentralGoogle Scholar
- Parsons SM. Transport mechanisms in acetylcholine and monoamine storage. FASEB J. 2000;14:2423–34.View ArticlePubMedGoogle Scholar
- Song H, Ming G, Fon E, Bellocchio E, Edwards RH, Poo M. Expression of a putative vesicular acetylcholine transporter facilitates quantal transmitter packaging. Neuron. 1997;18:815–26.View ArticlePubMedGoogle Scholar
- Buffelli M, Burgess RW, Feng G, Lobe CG, Lichtman JW, Sanes JR. Genetic evidence that relative synaptic efficacy biases the outcome of synaptic competition. Nature. 2003;424:430–4.View ArticlePubMedGoogle Scholar
- Kasthuri N, Lichtman JW. The role of neuronal identity in synaptic competition. Nature. 2003;424:426–30.View ArticlePubMedGoogle Scholar
- Banker BQ, Kelly SS, Robbins N. Neuromuscular transmission and correlative morphology in young and old mice. J Physiol. 1983;339:355–77.View ArticlePubMedPubMed CentralGoogle Scholar
- Bhattacharyya BJ, Tsen K, Sokoll MD. Age-induced alteration of neuromuscular transmission: effect of halothane. Eur J Pharmacol. 1994;254:97–104.View ArticlePubMedGoogle Scholar
- Fahim MA. Endurance exercise modulates neuromuscular junction of C57BL/6NNia aging mice. J Appl Physiol. 1997;83:59–66.PubMedGoogle Scholar
- Smith DO. Acetylcholine storage, release and leakage at the neuromuscular junction of mature adult and aged rats. J Physiol. 1984;347:161–76.View ArticlePubMedPubMed CentralGoogle Scholar
- Willadt S, Nash M, Slater CR. Age-related fragmentation of the motor endplate is not associated with impaired neuromuscular transmission in the mouse diaphragm. Sci Rep. 2016;6:24849.View ArticlePubMedPubMed CentralGoogle Scholar
- Roy A, Dakroub M, Tezini GCSV, Liu Y, Guatimosim S, Feng Q, Salgado HC, Prado VF, Prado MAM, Gros R. Cardiac acetylcholine inhibits ventricular remodeling and dysfunction under pathologic conditions. FASEB J. 2015;30(2):688–701.View ArticlePubMedGoogle Scholar
- Pfohl SR, Halicek MT, Mitchell CS. Characterization of the contribution of genetic background and gender to disease progression in the SOD1 G93A mouse model of amyotrophic lateral sclerosis: a meta-analysis. J Neuromuscul Dis. 2015;2(2):137–50.View ArticlePubMedPubMed CentralGoogle Scholar
- McCombe PA, Henderson RD. Effects of gender in amyotrophic lateral sclerosis. Gend Med. 2010;7:557–70.View ArticlePubMedGoogle Scholar
- McCombe PA, Henderson RD. The Role of immune and inflammatory mechanisms in ALS. Curr Mol Med. 2011;11:246–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Oskarsson B, Horton DK, Mitsumoto H. Potential environmental factors in amyotrophic lateral sclerosis. Neurol Clin. 2015;33:877–88.View ArticlePubMedGoogle Scholar
- Yu Y, Su F-C, Callaghan BC, Goutman SA, Batterman SA, Feldman EL. Environmental risk factors and amyotrophic lateral sclerosis (ALS): a case-control study of ALS in Michigan. PLoS One. 2014;9:e101186.View ArticlePubMedPubMed CentralGoogle Scholar
- de Jong SW, Huisman MHB, Sutedja NA, van der Kooi AJ, de Visser M, Schelhaas HJ, Fischer K, Veldink JH, van den Berg LH. Smoking, alcohol consumption, and the risk of amyotrophic lateral sclerosis: a population-based study. Am J Epidemiol. 2012;176:233–9.View ArticlePubMedGoogle Scholar
- Alonso A, Logroscino G, Hernán MA. Smoking and the risk of amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry. 2010;81:1249–52.View ArticlePubMedGoogle Scholar
- Pousinha PA, Correia AM, Sebastião AM, Ribeiro JA. The giant miniature endplate potentials frequency is increased in aged rats. Neurosci Lett. 2015;584:224–9.View ArticlePubMedGoogle Scholar
- Sabatelli M, Lattante S, Conte A, Marangi G, Luigetti M, Del Grande A, Chiò A, Corbo M, Giannini F, Mandrioli J, Mora G, Calvo A, Restagno G, Lunetta C, Penco S, Battistini S, Zeppilli P, Bizzarro A, Capoluongo E, Neri G, Rossini PM, Zollino M. Replication of association of CHRNA4 rare variants with sporadic amyotrophic lateral sclerosis: the Italian multicentre study. Amyotroph Lateral Scler. 2012;13:580–4.View ArticlePubMedGoogle Scholar
- Deflorio C, Palma E, Conti L, Roseti C, Manteca A, Giacomelli E, Catalano M, Limatola C, Inghilleri M, Grassi F. Riluzole blocks human muscle acetylcholine receptors. J Physiol. 2012;590(Pt 10):2519–28.View ArticlePubMedPubMed CentralGoogle Scholar