Orai1 plays a crucial role in central sensitization by modulating neuronal excitability

56 Pages·2017·8.48 MB·English

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Research Articles: Cellular/Molecular
Orai1 plays a crucial role in central sensitization by modulating neuronal
excitability
Yannong Dou1,2, Jingsheng Xia1, Ruby Gao1, Xinghua Gao2, Frances M. Munoz1, Dongyu Wei1, Yuzhen
Tian1, James E. Barrett1, Seena Ajit1, Olimpia Meucci1, James W. Putney3, Yue Dai2 and Huijuan Hu1
1Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, PA 19102.
2Department of Pharmacology of Chinese Materia Medica, China Pharmaceutical University, Nanjing, Jiangsu
211198.
3Calcium Regulation Group, National Institute of Environmental Health Sciences, Research Triangle Park, NC
27709.
DOI: 10.1523/JNEUROSCI.3007-17.2017
Received: 18 October 2017
Revised: 19 November 2017
Accepted: 29 November 2017
Published: 11 December 2017
Author contributions: Hu conceived of the project and designed experiments. Dou and Xia designed and
performed experiments, analyzed data. Gao, Wei and Tian performed some experiments. Munoz and Ajit helped
with the genotype protocol. Dou and Hu prepared the manuscript. Meucci provided instruments and input.
Munoz, Barrett, Ajit, Meucci, Dai and Putney edited the manuscript. All authors read and approved the final
manuscript. Dou and Gao were supported by China Scholarship Council. Meucci is supported by DA 15014 and
DA 32444.
Conflict of Interest: The authors declare no competing financial interests.
This work was supported by NIH Grants R21NS077330 and R01NS087033 (to H.H.). We thank Dr. Donald Gill
(Penn State College of Medicine) for providing Orai1-CFP.
Corresponding author: Huijuan Hu, Ph.D. (Associate Professor), 245 N. 15th Street, Philadelphia, PA 19102.
Email: [email protected]; Tel: [phone]; Yue Dai, Ph.D. (Professor), 24 Tong Jia Xiang, Nanjing
210009, China. E-mail: [email protected]; Tel: [phone]
Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3007-17.2017
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Copyright © 2017 the authors
1 Orai1 plays a crucial role in central sensitization by modulating neuronal excitability
2
3 Yannong Dou1,2, Jingsheng Xia1, Ruby Gao1, Xinghua Gao2, Frances M. Munoz1, Dongyu Wei1,
4 Yuzhen Tian1, James E. Barrett1, Seena Ajit1, Olimpia Meucci1, James W. Putney Jr3, Yue Dai2*,
5 and Huijuan Hu1,*
6
7 1Department of Pharmacology and Physiology, Drexel University College of Medicine,
8 Philadelphia, PA 19102. 2Department of Pharmacology of Chinese Materia Medica, China
9 Pharmaceutical University, Nanjing, Jiangsu 211198. 3Calcium Regulation Group, National
10 Institute of Environmental Health Sciences, Research Triangle Park, NC 27709.
11
12 Number of Pages: 44
13 Number of Figures: 11
14 Number of words (significance statement): 104
15 Number of words (abstract): 241
16 Number of words (introduction): 350
17 Number of words (discussion): 981
18
19 *Corresponding author: Huijuan Hu, Ph.D. (Associate Professor), 245 N. 15th Street,
20 Philadelphia, PA 19102. Email: [email protected]; Tel: [phone]
21
22 *Corresponding author: Yue Dai, Ph.D. (Professor), 24 Tong Jia Xiang, Nanjing 210009, China.
23 E-mail: [email protected]; Tel: [phone]
24
25 Conflict of Interest: The authors declare no competing financial interests.
26
27 Acknowledgements: This work was supported by NIH Grants R21NS077330 and
28 R01NS087033 (to H.H.). We thank Dr. Donald Gill (Penn State College of Medicine) for providing
29 Orai1-CFP.
30
31 Author contributions: Hu conceived of the project and designed experiments. Dou and Xia
32 designed and performed experiments, analyzed data. Gao, Wei and Tian performed some
33 experiments. Munoz and Ajit helped with the genotype protocol. Dou and Hu prepared the
34 manuscript. Meucci provided instruments and input. Munoz, Barrett, Ajit, Meucci, Dai and Putney
35 edited the manuscript. All authors read and approved the final manuscript. Dou and Gao were
36 supported by China Scholarship Council. Meucci is supported by DA 15014 and DA 32444.
37
1
38 Abstract
39
40 Pathological pain is a common and debilitating condition that is often poorly managed. Central
41 sensitization is an important mechanism underlying pathological pain. However, candidate
42 molecules involved in central sensitization remain unclear. Store-operated calcium channels
43 (SOCs) mediate important calcium signals in non-excitable and excitable cells. SOCs have been
44 implicated in a wide variety of human pathophysiological conditions including immunodeficiency,
45 occlusive vascular diseases and cancer. However, the role of SOCs in central nervous system
46 (CNS) disorders has been relatively unexplored. Orai1 is a key component of SOCs and
47 expressed in the human and rodent spinal cord dorsal horn, but its functional significance in
48 dorsal horn neurons is poorly understood. Here we sought to explore a potential role of Orai1 in
49 the modulation of neuronal excitability and A-type potassium channels that are involved in pain
50 plasticity. Using both male and female Orai1 knockout mice, we found that activation of Orai1
51 increased neuronal excitability and reduced A-type potassium channels via the PKC-ERK
52 pathway in dorsal horn neurons. Orai1 deficiency significantly decreased acute pain induced by
53 noxious stimuli, nearly eliminated the second phase of formalin-induced nociceptive response,
54 markedly attenuated carrageenan-induced ipsilateral sensory hypersensitivity and completely
55 abolished carrageenan-induced contralateral mechanical allodynia. Consistently,
56 carrageenan-induced increase in neuronal excitability was abolished in the dorsal horn from
57 Orai1 mutant mice. These findings uncover a novel signaling pathway involved in pain process
2
58 and central sensitization. Our study also reveals a novel link among Orai1, ERK, A-type
59 potassium channels and neuronal excitability.
60
61 Keywords: Store-operated calcium channels, Orai1, A-type potassium channels, ERK, spinal
62 cord dorsal horn.
63
64 Significance Statement
65 Orai1 is a key component of store-operated calcium channels (SOCs) in many cell types. It has
66 been implicated in pathological conditions including immunodeficiency, autoimmunity and cancer.
67 However, the role of Orai1 in CNS disorders remains poorly understood. Functional significance
68 of Orai1 in neurons is elusive. Here we demonstrate that activation of Orai1 modulates neuronal
69 excitability and Kv4 containing A-type potassium channels via the PKC-ERK pathway. Genetic
70 knockout of Orai1 nearly eliminates the second phase of formalin-induced pain and markedly
71 attenuates carrageenan-induced sensory hypersensitivity and neuronal excitability. These
72 findings reveal a novel link between Orai1 and neuronal excitability and advance our
73 understanding of central sensitization.
74
75
76 Introduction
77
3
78 Store-operated calcium channels (SOCs) are highly Ca2+-selective cation channels that are
79 activated by depletion of calcium stores from the endoplasmic reticulum (ER) (Smyth et al.,
80 2010). SOCs underlie sustained Ca2+ influx, which is required for Ca2+-dependent cellular
81 functions such as secretion, proliferation, neurotransmitter release and enzymatic activity
82 (Emptage et al., 2001; Chang et al., 2006; Abdullaev et al., 2008; Prakriya, 2009). SOCs are
83 composed of the pore forming Orai proteins (Orai1/2/3, often referred to as calcium release-
84 activated calcium channels, CRAC channels) and are coupled to ER Ca2+ levels by the Ca2+
85 sensing stromal interaction molecules (STIM1 and STIM2) (Liou et al., 2005; Roos et al., 2005;
86 Feske et al., 2006). SOC entry (SOCE) has been extensively studied and its importance is well
87 recognized in non-neuronal cells (Takezawa et al., 2006; Muik et al., 2012). SOCE has been
88 shown in dorsal root ganglion neurons (Gemes et al., 2011), hippocampal neurons (Narayanan
89 et al., 2010), and cortical neurons (Berna-Erro et al., 2009; Gruszczynska-Biegala and Kuznicki,
90 2013). STIM1 and STIM2 have been implicated in brain functions and pathological conditions
91 (Berna-Erro et al., 2009; Hartmann et al., 2014). However, the role of Orai1 in the nervous
92 system remains elusive. A previous study has reported Orai1 expression in the dorsal horn of the
93 human spinal cord (Guzman et al., 2014). Recently, we have demonstrated that SOCs are
94 functional in dorsal horn neurons. Using pharmacological and siRNA knockdown approaches, we
95 have demonstrated that Orai1 is responsible for SOCE in dorsal horn neurons (Xia et al., 2014).
96 However, its functional significance in dorsal horn neurons remains elusive. Here, we show that
97 Orai1-mediated SOCE modulates neuronal excitability and A-type currents in dorsal horn
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98 neurons. These effects are mediated by the PKC-ERK cascade. We also demonstrate that Orai1
99 deficiency reduces acute pain and almost eliminates the second phase of formalin-induced
100 nociceptive behavior through the ERK signaling pathway, while it markedly attenuates
101 carrageenan-induced sensory hypersensitivity and neuronal excitability. These findings reveal a
102 novel link of Orai1 to ERK, A-type channels, neuronal excitability and pain, and provide insights
103 into the molecular mechanisms that underlie central sensitization associated with inflammatory
104 pain.
105
106
107 Materials and Methods
108
109 Animals
110 All experiments were performed in accordance with the guidelines of the National Institutes of
111 Health (NIH) and with the guidelines of the Committee for Research and Ethical Issues of IASP
112 and were approved by the Animal Care and Use Committee of Drexel University College of
113 Medicine. Pregnant mice were purchased from Taconic or Charles River and individually housed
114 in standard cages and maintained in a room with a 12-h light/dark cycle. Both sexes were used
115 for all in vitro and in vivo experiments. Neonatal CD1 mice were used for cell cultures and adult
116 CD1 mice were used for slice preparations (Xia et al., 2014). Orai1 mutant mice were developed
117 in the laboratory of Dr. Kinet by insertion of a β-Geo cassette in the first intron of the Orai1 gene
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118 (Vig et al., 2008), and purchased from Mutant Mouse Regional Resource Centers (MMRRC).
119 The original background of Orai1 mutant mice was a mixed C57BL/6 and 129P2/OlaHsd. These
120 mice had been backcrossed to CD1 for more than 7 generations.
121
122 Genotyping
123 Mice were genotyped by PCR of DNA extracted from tail clips. The following primers were used
124 for β-Geo: 5’-CAAATGGCGATTACCGTTGA (F), 5’-TGCCCAGTCATAGCCGAATA (R); for Tcrd:
125 5’-CAAATGTTGCTTGTCTGGTG (F), 5’-GTCAGTCGAGTGCACAGTTT (R). Genotypes were
126 further determined by the copy number analysis using a TaqMan® Genotyping Master Mix kit
127 (Applied Biosystems, Foster City, CA.) following the manufacturer’s instructions. Briefly, the
128 TaqMan® copy number assay (detecting the β-Geo) was run simultaneously with a TaqMan®
129 copy number reference assay (detecting the telomerase reverse transcriptase (Tert)) in a duplex
130 real-time PCR. Real time quantitative PCR was performed in a 7900HT fast real-time PCR
131 System (Applied Biosystems) using the following amplification conditions: 10 min of initial
132 denaturation at 95°C, then 40 cycles of 95°C for 15 s, 60°C for 1 min. The Applied Biosystems
133 CopyCaller™ software was used for post-PCR data analysis of copy number quantitation.
134
135 Real-time PCR analysis of Orai1 mRNA expression
136 Real-time PCR was performed according to our previous study (Gao et al., 2016). Total RNA was
137 extracted from adult spinal cords and acutely dissociated neurons using TRIzol Reagent
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138 (Molecular Research Center, Cincinnati, OH). RNA concentration was determined by optical
139 density at 260 nm. Total RNA was reverse transcribed into cDNA for each sample using a
140 Fermentas maxima first-strand cDNA synthesis kit (Thermo Scientific, Rockford, IL) following the
141 manufacturer’s instructions. Primers specific for mouse Orai1 (Mm00774349_m1) and GAPDH
142 were purchased from Applied Biosystems. Real-time quantitative PCR was performed in a
143 7900HT fast real-time PCR System (Applied Biosystems) using the following conditions: 5 min of
144 initial denaturation at 96°C, then 35 cycles of 96°C for 30 s, 55°C for 30 s and 72°C for 1.5 min.
145 The threshold cycle for each gene was determined and analyzed using the relative quantitation
146 software (Applied Biosystems). The relative expression of Orai1 was calculated using the
147 2^(-Delta Delta Ct, 2-∆∆Ct) method (Livak and Schmittgen, 2001). The mRNA levels of Orai1 were
148 normalized to the housekeeping gene GAPDH.
149
150 Cell culture and slice preparation
151 Primary cultures of spinal cord superficial dorsal horn neurons were prepared from postnatal day
152 1 or day 2 mice as previously described (Hu et al., 2003). Briefly, neonatal mice were decapitated
153 after inducing hypothermia on ice. A laminectomy was performed and the spinal cord superficial
154 dorsal horn was carefully dissected in the dorsal side of the spinal cord (approximately lamina III)
155 with a surgical blade. The superficial dorsal horn strips were then incubated for 30 min at 37°C in
156 Hank’s balanced saline solution (HBSS; Invitrogen, Carlsbad, CA) (in mM: 137 NaCl, 5.4 KCl,
157 0.4 KH PO , 1 CaCl , 0.5 MgCl , 0.4 MgSO , 4.2 NaHCO , 0.3 Na HPO and 5.6 glucose)
2 4 2 2 4 3 2 4
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158 containing papain (15 U/ml; Worthington Biochemical, Lakewood, NJ), rinsed three times with
159 HBSS, and placed in culture medium containing Neurobasal A (Invitrogen), 2% fetal calf serum
160 (Invitrogen), 2% heat-inactivated horse serum (Invitrogen), 2% B-27 (Invitrogen), 100 U/ml
161 penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen) and 2 mM GlutaMAXTM-1 (Invitrogen).
162 The fragments were mechanically dissociated by gently triturating with a fire-polished Pasteur
163 pipette until the solution turned cloudy. The dispersed cells were plated onto 12-mm
164 poly-D-lysine and laminin (Sigma, St. Louis, MO)-coated coverslips at a density of 3000 cells per
165 well. Neurons were cultured in a humidified atmosphere containing 5% CO at 37°C for 1-2 days
2
166 for A-type currents recording.
167
168 Lumbar spinal cord slices (300-350 μm) were prepared from CD-1 mice as we described
169 previously (Xia et al., 2014). Briefly, after decapitation, the vertebral column and surrounding
170 tissue was isolated and immersed in ice-cold oxygenated NMDG artificial cerebrospinal fluid
171 (ACSF) containing the following (in mM): 93 NMDG, 93 HCl, 30 NaHCO , 20 HEPES, 2.5 KCl,
3
172 1.2 NaH PO , 10 MgCl , 0.5 CaCl , 25 glucose, 3 Myo-inositol, 1 Sodium-L-ascorbate, 5 Ethyl
2 4 2 2
173 pyruvate (osmolality ~305-310 mmol/kg). The lumbar enlargement of the spinal cord was
174 removed and glued onto the cutting platform with the adhesive Loctite 404 (Loctite, Rocky Hill,
175 CT). Transverse spinal cord slices were cut in NMDG ACSF with a Vibratome 3000 (Vibratome,
176 St Louis, MO). Slices were incubated at 32-34°C for 15 min and then maintained at room
177 temperature (20-24°C) for 1 h before electrophysiological recording in the continuously
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178 oxygenated recovery solution containing (in mM): 26 NaHCO , 3 Myo-inositol, 120 NaCl, 5 KCl,
3
179 1.25 NaH PO , 1 MgCl , 1 CaCl , 12.5 glucose (osmolality ~ 305-310 mmol/kg).
2 4 2 2
180
181 Transfection
182 For transfection of Orai1-CFP (inserted in the pIRESneo plasmid, a generous gift from Dr. Gill,
183 Penn State College of Medicine, PA), neurons were electroporated using mouse primary cell
184 Nucleofector kit according to the manufacturer’s instructions (Lonza Group Ltd, Basel,
185 Switzerland). Briefly, neurons were transfected with Orai1-CFP (2.5 μg) per 1 × 106 cells and
186 were seeded on 12-mm glass coverslips. Calcium imaging and patch clamp were performed
187 48-72 h post transfection.
188
189 Calcium imaging
190 Calcium imaging was performed using fura-2-based microfluorimetry and imaging analysis as we
191 previously described (Xia et al., 2014). Neurons were loaded with 4 μM of fura-2AM (Life
192 Technologies, Grand Island, NY) for 30 min at room temperature in HBSS, washed, and further
193 incubated in normal bath solution (Tyrode’s) containing (in mM) 140 NaCl, 5 KCl, 2 CaCl , 1
2
194 MgCl , 10 HEPES, and 5.6 glucose for 20 min. Coverslips were mounted in a small perfusion
2
195 chamber (Model RC-25, Warner Instruments, Hamden, CT) and continuously perfused at 5 to 7
196 ml/min with Tyrode’s solution. Images were acquired at 3-s intervals at room temperature using
197 an Olympus inverted microscope equipped with a CCD camera (Hamamatsu ORCA-03G,
9