G Model
ARTICLE IN PRESS
NSL 31057 1–6
Neuroscience Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
1
3
Q1
Cyclic AMP inhibits neuromuscular junction maturation mediated by intracellular Ca2+
4
Q2
Wei Song a,b,∗ , Xiwan Albert Jin b
2
5 6
a b
Peking University Health Science Center, Beijing 100191, China Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China
7
8 9 10 11 12
h i g h l i g h t s • The first time we report that cyclic AMP inhibits NMJ maturation. • cAMP inhibits NMJ maturation in both physiological and morphological levels. • Intracellular Ca2+ mediates cAMP-dependent inhibition of NMJ maturation.
13
14 26
a r t i c l e
i n f o
a b s t r a c t
15 16 17 18 19 20
Article history: Received 7 December 2014 Received in revised form 9 January 2015 Accepted 12 January 2015 Available online xxx
21
25
Keywords: Synaptic maturation Cyclic AMP Ca2+
27
1. Introduction
22 23 24
28 29 30 31 32 33 34 35 36 37 38 39
The neuromuscular junction (NMJ) is established through initial contact of motor neuron axon with a skeletal muscle cell and the subsequent synaptic maturation. Previous studies have shown that cyclic AMP (cAMP) enhanced spinal neurons’ survival and growth but inhibited synaptogenesis. Here, we found that elevating intracellular cAMP level of presynaptic neurons prevented NMJs from maturation both physiologically and morphologically. Importantly, cytosolic Ca2+ is essential for the inhibitory effects of cAMP on NMJ maturation. We showed that depletion of intracellular Ca2+ store, rather than extracellular Ca2+ , abolished the cAMP-dependent inhibition of synapse maturation. Taken together, we demonstrate that Ca2+ released from intracellular Ca2+ stores regulates neurotrophic actions on NMJ maturation. © 2015 Published by Elsevier Ireland Ltd.
Neuromuscular junction (NMJ) is extensively studied as a simple model of synapse development [20,26]. The formation of NMJ is initiated by the interaction between motor neuron and target muscle cell. The maturation process of NMJ begins when a growth cone of motor neuron reaches the target muscle cell, and is marked by the development of synaptic specializations. On the presynaptic side, acetylcholine (ACh) containing synaptic vesicles (SVs) and mitochondria become enriched at the nerve terminals [7]. On the postsynaptic side, nerve-induced ACh receptor clusters density can reach 10,000/m2 in contrast to 10/m2 at extrajunctional sites [11]. The formation of pre- and postsynaptic specializations makes sure the efficient neurotransmission across synapses.
∗ Corresponding author at: Mailbox #045, Peking University Health Science Center, #38 Xueyuan Road, Haidian District, Beijing 100191, China. Tel.: +86 13520954274. E-mail addresses: [email protected] (W. Song), [email protected] (X.A. Jin).
In the earlier days by using intracellular recordings, people could detect miniature endplate potentials (MEPPs) at postsynaptic endplates because of spontaneous presynaptic quantal secretion of ACh without action potential [10]. Several parameters of MEPPs like amplitude, frequency and amplitude distribution were used to indicate NMJ maturation both in vitro and in vivo [17,18]. Nowadays with the improvement of patch clamp, the spontaneous synaptic currents (SSCs) can be detected from innervated muscles in Xenopus nerve-muscle cocultures [9,36]. Besides, the morphological changes during maturation such as uniform SV clustering within the nerve terminals, pre- and postsynaptic membranes thickening and synaptic cleft narrowing have been reported both in vivo and in vitro [6,18,21]. Cyclic AMP (cAMP) is a second messenger which is derived from ATP by adenylyl cyclase (AC) and is hydrolyzed by phosphodiesterase (PDE). It has neurotrophic effects on neurons and regenerative effects within nervous system [14,19,22]. Traditional neurotrophic factors including neurotrophin-3 (NT-3) and glial cell-derived neurotrophic factor (GDNF), have been reported to promote NMJ maturation and the underlying molecular mechanisms have been studied [15,16,33]. Although acute application of
http://dx.doi.org/10.1016/j.neulet.2015.01.025 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: W. Song, X.A. Jin, Cyclic AMP inhibits neuromuscular junction maturation mediated by intracellular Ca2+ , Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.01.025
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
G Model NSL 31057 1–6
ARTICLE IN PRESS W. Song, X.A. Jin / Neuroscience Letters xxx (2015) xxx–xxx
2
71
cAMP did not potentiate neurotransmission across NMJ, it could significantly enhance the potentiation effects of low concentration of brain-derived neurotrophic factor (BDNF) [4]. However, the long-term effects of cAMP on the developing NMJs remain elusive. cAMP has been shown to inhibit the initiation of NMJ development [24]. Here we used electrophysiological and cell biological approaches to study the effects of cAMP on NMJ maturation process. At both physiologically and morphologically levels, we reported that cAMP as a trophic factor had inhibitory effects on NMJ maturation. In addition, we also showed that the inhibitory effects of cAMP on NMJ maturation were mediated by intracellular Ca2+ .
72
2. Materials and methods
73
2.1. Reagents
61 62 63 64 65 66 67 68 69 70
78
Chlorophenylthio (CPT)-cAMP, forskolin, and thapsigargin (TG) were bought from Sigma (St. Louis, MO). BAPTA-AM was bought from Calbiochem (Merck, Germany). Alexa 488-conjugated ␣bungarotoxin (R-BTX) was bought from Molecular Probes (Eugene, OR).
79
2.2. Xenopus nerve-muscle cocultures preparation
74 75 76 77
80 81 82 83 84 85 86 87 88 89 90 91 92
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94 95 96 97 98 99 100 101 102 103 104 105 106
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108 109 110 111 112 113 114
Xenopus nerve-muscle cocultures were prepared as previously described [23]. In brief, neural tube and myotomal tissue were dissected from Xenopus embryos at stages 20–22 and dissociated in Ca2+ –Mg2+ -free Steinberg’s solution (60 mM NaCl, 0.67 mM KCl, 0.4 mM EDTA, 10 mM HEPES, pH 7.4). After 30 min, cells were plated on ECL (entactin–collagen IV-laminin) coated glass coverslips and kept at 23 ◦ C. The culture medium contained 87% Steinberg’s solution (60 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3 )2 , 0.83 mM MgSO4 , 10 mM HEPES, pH 7.4), 10% Leibovitz L-15 medium, 1% fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml gentamicin and 100 g/ml streptomycin. The cultures were kept for 1 day before experiments. All the reagents were applied at the same time of cell plating on glass coverslips.
2.3. Electrophysiology and data analysis Spontaneous synaptic currents (SSCs) were recorded from innervated muscle cells with patch clamp under whole-cell configuration [12] at room temperature in frog Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2 , 5 mM HEPES, pH 7.4) in the presence of tetrodotoxin. The micropipette solution was consisted of 150 mM KCl, 1 mM NaCl, 1 mM MgCl2 , 10 mM HEPES, pH 7.2) [34]. The membrane potential of the muscle cell was voltage-clamped at −70 mV. The signals were collected by an Axopatch 200B amplifier (Axon Instrument) and sampled at 10 kHz with the filter set at 2 kHz. The amplitudes of SSCs were analyzed using pClamp 10 software (Axon Instrument). A threshold was set at 20 pA for SSC amplitudes because very few currents were
ARTICLE IN PRESS
NSL 31057 1–6
Neuroscience Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
1
3
Q1
Cyclic AMP inhibits neuromuscular junction maturation mediated by intracellular Ca2+
4
Q2
Wei Song a,b,∗ , Xiwan Albert Jin b
2
5 6
a b
Peking University Health Science Center, Beijing 100191, China Division of Life Science, Hong Kong University of Science and Technology, Hong Kong, China
7
8 9 10 11 12
h i g h l i g h t s • The first time we report that cyclic AMP inhibits NMJ maturation. • cAMP inhibits NMJ maturation in both physiological and morphological levels. • Intracellular Ca2+ mediates cAMP-dependent inhibition of NMJ maturation.
13
14 26
a r t i c l e
i n f o
a b s t r a c t
15 16 17 18 19 20
Article history: Received 7 December 2014 Received in revised form 9 January 2015 Accepted 12 January 2015 Available online xxx
21
25
Keywords: Synaptic maturation Cyclic AMP Ca2+
27
1. Introduction
22 23 24
28 29 30 31 32 33 34 35 36 37 38 39
The neuromuscular junction (NMJ) is established through initial contact of motor neuron axon with a skeletal muscle cell and the subsequent synaptic maturation. Previous studies have shown that cyclic AMP (cAMP) enhanced spinal neurons’ survival and growth but inhibited synaptogenesis. Here, we found that elevating intracellular cAMP level of presynaptic neurons prevented NMJs from maturation both physiologically and morphologically. Importantly, cytosolic Ca2+ is essential for the inhibitory effects of cAMP on NMJ maturation. We showed that depletion of intracellular Ca2+ store, rather than extracellular Ca2+ , abolished the cAMP-dependent inhibition of synapse maturation. Taken together, we demonstrate that Ca2+ released from intracellular Ca2+ stores regulates neurotrophic actions on NMJ maturation. © 2015 Published by Elsevier Ireland Ltd.
Neuromuscular junction (NMJ) is extensively studied as a simple model of synapse development [20,26]. The formation of NMJ is initiated by the interaction between motor neuron and target muscle cell. The maturation process of NMJ begins when a growth cone of motor neuron reaches the target muscle cell, and is marked by the development of synaptic specializations. On the presynaptic side, acetylcholine (ACh) containing synaptic vesicles (SVs) and mitochondria become enriched at the nerve terminals [7]. On the postsynaptic side, nerve-induced ACh receptor clusters density can reach 10,000/m2 in contrast to 10/m2 at extrajunctional sites [11]. The formation of pre- and postsynaptic specializations makes sure the efficient neurotransmission across synapses.
∗ Corresponding author at: Mailbox #045, Peking University Health Science Center, #38 Xueyuan Road, Haidian District, Beijing 100191, China. Tel.: +86 13520954274. E-mail addresses: [email protected] (W. Song), [email protected] (X.A. Jin).
In the earlier days by using intracellular recordings, people could detect miniature endplate potentials (MEPPs) at postsynaptic endplates because of spontaneous presynaptic quantal secretion of ACh without action potential [10]. Several parameters of MEPPs like amplitude, frequency and amplitude distribution were used to indicate NMJ maturation both in vitro and in vivo [17,18]. Nowadays with the improvement of patch clamp, the spontaneous synaptic currents (SSCs) can be detected from innervated muscles in Xenopus nerve-muscle cocultures [9,36]. Besides, the morphological changes during maturation such as uniform SV clustering within the nerve terminals, pre- and postsynaptic membranes thickening and synaptic cleft narrowing have been reported both in vivo and in vitro [6,18,21]. Cyclic AMP (cAMP) is a second messenger which is derived from ATP by adenylyl cyclase (AC) and is hydrolyzed by phosphodiesterase (PDE). It has neurotrophic effects on neurons and regenerative effects within nervous system [14,19,22]. Traditional neurotrophic factors including neurotrophin-3 (NT-3) and glial cell-derived neurotrophic factor (GDNF), have been reported to promote NMJ maturation and the underlying molecular mechanisms have been studied [15,16,33]. Although acute application of
http://dx.doi.org/10.1016/j.neulet.2015.01.025 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: W. Song, X.A. Jin, Cyclic AMP inhibits neuromuscular junction maturation mediated by intracellular Ca2+ , Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.01.025
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
G Model NSL 31057 1–6
ARTICLE IN PRESS W. Song, X.A. Jin / Neuroscience Letters xxx (2015) xxx–xxx
2
71
cAMP did not potentiate neurotransmission across NMJ, it could significantly enhance the potentiation effects of low concentration of brain-derived neurotrophic factor (BDNF) [4]. However, the long-term effects of cAMP on the developing NMJs remain elusive. cAMP has been shown to inhibit the initiation of NMJ development [24]. Here we used electrophysiological and cell biological approaches to study the effects of cAMP on NMJ maturation process. At both physiologically and morphologically levels, we reported that cAMP as a trophic factor had inhibitory effects on NMJ maturation. In addition, we also showed that the inhibitory effects of cAMP on NMJ maturation were mediated by intracellular Ca2+ .
72
2. Materials and methods
73
2.1. Reagents
61 62 63 64 65 66 67 68 69 70
78
Chlorophenylthio (CPT)-cAMP, forskolin, and thapsigargin (TG) were bought from Sigma (St. Louis, MO). BAPTA-AM was bought from Calbiochem (Merck, Germany). Alexa 488-conjugated ␣bungarotoxin (R-BTX) was bought from Molecular Probes (Eugene, OR).
79
2.2. Xenopus nerve-muscle cocultures preparation
74 75 76 77
80 81 82 83 84 85 86 87 88 89 90 91 92
93
94 95 96 97 98 99 100 101 102 103 104 105 106
107
108 109 110 111 112 113 114
Xenopus nerve-muscle cocultures were prepared as previously described [23]. In brief, neural tube and myotomal tissue were dissected from Xenopus embryos at stages 20–22 and dissociated in Ca2+ –Mg2+ -free Steinberg’s solution (60 mM NaCl, 0.67 mM KCl, 0.4 mM EDTA, 10 mM HEPES, pH 7.4). After 30 min, cells were plated on ECL (entactin–collagen IV-laminin) coated glass coverslips and kept at 23 ◦ C. The culture medium contained 87% Steinberg’s solution (60 mM NaCl, 0.67 mM KCl, 0.34 mM Ca(NO3 )2 , 0.83 mM MgSO4 , 10 mM HEPES, pH 7.4), 10% Leibovitz L-15 medium, 1% fetal bovine serum (FBS), 100 units/ml penicillin, 100 g/ml gentamicin and 100 g/ml streptomycin. The cultures were kept for 1 day before experiments. All the reagents were applied at the same time of cell plating on glass coverslips.
2.3. Electrophysiology and data analysis Spontaneous synaptic currents (SSCs) were recorded from innervated muscle cells with patch clamp under whole-cell configuration [12] at room temperature in frog Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2 , 5 mM HEPES, pH 7.4) in the presence of tetrodotoxin. The micropipette solution was consisted of 150 mM KCl, 1 mM NaCl, 1 mM MgCl2 , 10 mM HEPES, pH 7.2) [34]. The membrane potential of the muscle cell was voltage-clamped at −70 mV. The signals were collected by an Axopatch 200B amplifier (Axon Instrument) and sampled at 10 kHz with the filter set at 2 kHz. The amplitudes of SSCs were analyzed using pClamp 10 software (Axon Instrument). A threshold was set at 20 pA for SSC amplitudes because very few currents were
