Hyperactive Akt-mTOR pathway as a therapeutic target for pain hypersensitivity in Cntnap2-deficient mice

Journal Pre-proof Hyperactive Akt-mTOR pathway as a therapeutic target for pain hypersensitivity in Cntnap2-deficient mice Xiaoliang Xing, Kunyang Wu, Yufan Dong, Yimei Zhou, Jing Zhang, Fang Jiang, Wang-Ping Hu, Jia-Da Li PII:

S0028-3908(19)30382-X

DOI:

https://doi.org/10.1016/j.neuropharm.2019.107816

Reference:

NP 107816

To appear in:

Neuropharmacology

Received Date: 12 July 2019 Revised Date:

9 October 2019

Accepted Date: 16 October 2019

Please cite this article as: Xing, X., Wu, K., Dong, Y., Zhou, Y., Zhang, J., Jiang, F., Hu, W.-P., Li, J.-D., Hyperactive Akt-mTOR pathway as a therapeutic target for pain hypersensitivity in Cntnap2-deficient mice, Neuropharmacology (2020), doi: https://doi.org/10.1016/j.neuropharm.2019.107816. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

Hyperactive

Akt-mTOR

pathway

as

a

therapeutic

target

for

pain

2

hypersensitivity in Cntnap2-deficient mice

3

Xiaoliang Xing1,2,3,4#, Kunyang Wu1,3,4#, Yufan Dong1,3,4, Yimei Zhou5, Jing Zhang1,3,4,

4

Fang Jiang1,3,4, Wang-Ping Hu5*, Jia-Da Li1,3,4*

5

1

6

Changsha 410078, Hunan, P. R. China.

7

2

Hunan University of Medicine, Huaihua 418000, Hunan, P. R. China.

8

3

Hunan Key Laboratory of Animal Models for Human Diseases, Changsha 410078,

9

Hunan, P. R. China.

Center for Medical Genetics, School of Life Sciences, Central South University,

10

4

Hunan Key Laboratory of Medical Genetics, Changsha 410078, Hunan, P. R. China.

11

5

Research Center of Basic Medical Sciences, School of Basic Medical Sciences,

12

Hubei University of Science and Technology, Xianning 437100, Hubei, P. R. China.

13

#

14

*Corresponding author: Correspondence may be addressed to Jia-Da Li. Tel: +86 731

15

84805339; Fax: +86 731 84478152; Email: [email protected]. Correspondence

16

may also be addressed to Wang-Ping Hu. Email: [email protected].

Contributed equally to this work.

17 18

Abstract

19

Contactin-associated protein-like 2 (CNTNAP2 or CASPR2) is a neuronal

20

transmembrane protein of the neurexin superfamily that is involved in many

21

neurological diseases, such as autism and pain hypersensitivity. We recently found

22

that Cntnap2-/- mice showed elevated Akt-mTOR activity in the brain, and

23

suppression of the Akt-mTOR pathway rescued the social deficit in Cntnap2-/- mice.

24

In this study, we found that the dorsal root ganglion (DRG) from Cntnap2-/- mice also

25

showed hyperactive Akt-mTOR signaling. Treatment with the Akt inhibitor LY94002

26

or the mTOR inhibitor rapamycin attenuated pain-related hypersensitivity to noxious

27

mechanical stimuli, heat, and inflammatory substances. Further, suppression of

28

mTOR signaling by rapamycin decreased DRG neuronal hyperexcitability. We further

29

indicated that treatment with the FDA-approved drug metformin normalized the 1

30

hyperactive Akt-mTOR signaling, and attenuated pain-related hypersensitivity in

31

Cntnap2-/- mice. Our results thus identified hyperactive Akt-mTOR signaling pathway

32

as a promising therapeutic target for pain-related hypersensitivity in patients with

33

dysfunction of CNTNAP2.

34

Keywords: Cntnap2; Akt-mTOR signaling pathway; Dorsal root ganglion; Pain

35

1. Introduction

36

Contactin-associated protein-like 2 (CNTNAP2 or CASPR2) is a neuronal

37

transmembrane protein of the neurexin superfamily involved in synapse development

38

and maintenance(Anderson et al., 2012; Varea et al., 2015). Dysfunction in

39

CNTNAP2 has recently been linked to several neurological diseases such as autism

40

spectrum disorders (ASD), epilepsy, and schizophrenia(Alarcon et al., 2008; Arking et

41

al., 2008; Friedman et al., 2008; Li et al., 2010). CNTNAP2 gene is the first widely

42

replicated ASD susceptibility gene(Alarcon et al., 2008; Arking et al., 2008; Li et al.,

43

2010). Indeed, mice deficient in Cntnap2 (Cntnap2-/- mice) show core ASD-like

44

phenotypes, including communication and social behavior abnormalities, repetitive

45

behavior/perseveration(Penagarikano et al., 2011).

46

CNTNAP2 is one of proteins that form the voltage-gated potassium channel

47

complex(Horresh et al., 2008). Autoantibodies against the voltage-gated potassium

48

channel complex have been associated with a number of clinical syndromes, including

49

neuromyotonia, Morvan’s syndrome, and limbic encephalitis(Irani et al., 2012; Klein

50

et al., 2012; Lancaster et al., 2011).Furthermore, human autoantibodies to CNTNAP2

51

are often associated with neuropathic pain, and immunosuppression to reduce the

52

autoantibodies against CNTNAP2 can alleviate pain-related hypersensitivity(Klein et

53

al., 2012). To study the underlying mechanism,Dawes et al found that injection of

54

human CNTNAP2 autoantibodies results in pain-related hypersensitivity in

55

mice(Dawes et al., 2018). Moreover, Cntnap2-/- mice showed enhanced pain-related

56

behaviors to noxious mechanical stimuli, heat, and algogens(Dawes et al., 2018).

57

They further revealed that ablation of Cntnap2 enhanced the excitability of dorsal root

58

ganglion (DRG) neurons by regulating the Kv1 channel expression at the soma

59

membrane(Dawes et al., 2018). 2

60

We recently identified hyperactive Akt-mammalian target of rapamycin (mTOR)

61

signaling in the hippocampus and cortex from Cntnap2-deficient mice(Xing et al.,

62

2019). Suppression of the Akt-mTOR pathway rescued the social deficit in Cntnap2-/-

63

mice(Xing et al., 2019). However, the involvement of Akt-mTOR signaling in

64

pain-related hypersensitivity of Cntnap2-/- mice is unclear.

65

In this study, we found that Akt-mTOR signaling was also elevated in the DRG

66

from Cntnap2-/- mice. Pharmacological inhibition of Akt-mTOR signaling alleviated

67

pain-related hypersensitivity and decreased DRG neuronal hyperexcitability.

68

2. Materials and Methods

69

2.1 Animals

70

Cntnap2+/- mice were obtained from the Jackson Laboratory. Wild-type (WT) and

71

Cntnap2-/- mice were obtained from heterozygous crossing and were born with the

72

expected Mendelian frequencies. The genotyping was performed by PCR as

73

previously described(Xing et al., 2019). Mice were group-housed with 4–6 mice per

74

cage in a room on a 12 h light/12 h dark cycle maintained at 22 ± 2 °C. Male mice at

75

the age of 4-8 weeks were used in all experiments. All procedures were approved by

76

the Ethics Committee of School of Life Sciences, Central South University of China.

77

2.2 Immunoblotting and antibodies

78

DRG tissues were homogenized by a tissue homogenizer in 2×SDS gel-loading

79

buffer (50 mM Tris-HCl at pH 6.8, 2% SDS and 10% glycerol) with 1×NaF,

80

1×NaVO4, and 1×Protein inhibitor cocktail. After centrifugation, the supernatant was

81

collected, and the concentration was measured using the PierceTM BCA protein Assay

82

kit (Thermo Fisher, Waltham mass, USA). Protein extracts were denatured with heat

83

and resolved by SDS-PAGE. Following electrophoresis, proteins were transferred to

84

nitrocellulose membranes for immunoblotting.

85

The following antibodies were used: Cntnap2 (ab33994, Abcam, USA),

86

Phospho-Akt (Ser473) (4060, CST, USA), Akt (4691, CST, USA), Phospho-S6

87

(Ser235/236) (2211, CST, USA), S6 (2217, CST, USA), and β-actin (A2228, Sigma,

88

USA). 3

89

2.3 Drug administration

90

Male mice at the age of 4-8 weeks were used in all experiments. LY294002,

91

rapamycin, and metformin hydrochloride were obtained from Med Chem Express

92

(MCE, New Jersey, USA). LY294002 (25 mg/kg bodyweight)(Lazo et al., 2013; Xing

93

et al., 2019), rapamycin(10mg/kg bodyweight)(Sato et al., 2012; Xing et al., 2019),

94

metformin hydrochloride(200mg/kg bodyweight)(Gantois et al., 2017), or equal

95

volume of saline were intraperitoneally (i.p.) injected into the mice once a day for 2

96

consecutive days. At 60 min after the second injection, the tissues were collected or

97

behavioral tests were performed.

98

2.4 Pain-related tests

99

The mechanical hypersensitivity of mice was measured using Von Frey filaments.

100

Mice were placed in a Perspex box situated on the top of a wire mesh, and they were

101

then calibrated. Von Frey hairs were applied to the plantar surface of the hind paw,

102

and a reflex withdrawal response was used to calculate the 50% withdrawal threshold.

103

The response to a supra-threshold heat stimulus was measured using a hot plate

104

assay. A metallic plate was set with surface temperature of 54.5 °C. Mice were then

105

placed onto the plate and the latency until a response, such as shaking, licking, or

106

biting of the paw, was measured.

107

To assess the capsaicin sensitivity, 1.5 µg of capsaicin (Sigma Aldrich, St Louis,

108

USA) in 10 µl solvent (5% ethanol, 5% Tween-80 and 90% saline) was injected into

109

the dorsal part of the right hind paw by using a 30 G needle. Mice were placed on a

110

Plexiglas column, and the duration of pain-related behaviors, including biting, licking,

111

and paw lifting, was recorded over a period of 5 minutes.

112

For the formalin test, 10 µl of 5% formalin was injected intraplantarly into the mice,

113

and the duration of paw biting, licking, or paw lifting was recorded for 60 minutes.

114

2.5 Electrophysiological recordings

115

Current-clamp recordings were carried out at room temperature (22-25 °C) using a

116

MultiClamp-700B amplifier and Digidata-1440A A/D converter (Axon Instruments,

117

Foster City, CA, USA). The micropipettes were filled with an internal solution

118

containing (mM): KCl 140, MgCl2 2.5, HEPES 10, EGTA 11 and ATP 5; its pH was 4

119

adjusted to 7.2 with KOH and osmolarity was adjusted to 310 mOsm/L with sucrose.

120

Cells were bathed in an external solution containing (mM): NaCl 150, KCl 5, CaCl2

121

2.5, MgCl2 2, HEPES 10, D-glucose 10; the osmolarity of the solution was adjusted to

122

330 mOsm/L with sucrose and its pH was adjusted to 7.4 with NaOH. The resistance

123

of the recording pipette was in the range of 2–5 MΩ. A small patch of membrane

124

underneath the tip of the pipette was aspirated to form a gigaseal, and negative

125

pressure was then applied to rupture it, thus establishing a whole-cell configuration.

126

The series resistance was compensated for by 70-80%. Current-clamp recordings

127

were carried out in the cells with a stable resting membrane potential (more negative

128

than −50 mV). Firing was obtained by a 400 ms depolarizing current injection.

129

Signals were sampled at 10–50 kHz and filtered at 2–10 kHz, and the data were

130

analyzed by the pCLAMP 10 acquisition software (Axon Instruments). The mice

131

treated with vehicle or rapamycin at 60 min after the second injection were sacrificed

132

for DRG collection. The neurons selected for electrophysiological experiment were

133

15–35 µm in diameter, which are thought to be nociceptive neurons(Dawes et al.,

134

2018).

135

2.6 Statistical analysis

136

A repeated-measure ANOVA followed by Bonferroni post hoc tests or unpaired

137

two-tail Student’s t test was used as indicated. All statistical analyses were performed

138

using the Prism 6.01 (GraphPad Software, San Diego, CA).

139

3. Results

140

3.1 Hyperactive Akt-mTOR signaling in the DRG of Cntnap2-/- mice

141

Our previous studies indicated that Akt-mTOR signaling was hyperactive in the

142

hippocampus and cortex of Cntnap2-/- mice(Xing et al., 2019). To assess whether the

143

Akt-mTOR signaling is altered in the DRG from Cntnap2-/- mice, we performed

144

immunoblotting to detect the phosphorylation levels of Akt and its downstream

145

molecule ribosomal protein S6 (S6). As shown in Fig. 1A and B, the phosphorylation

146

levels of Akt and S6 were increased significantly in the DRG from Cntnap2-/- mice as

147

compared with WT controls. 5

148 149

3.2 Inhibition of Akt-mTOR signaling rescued mechanical and thermal hypersensitivity in Cntnap2-/- mice

150

Both immune and genetic-mediated ablation of Cntnap2 in mice led to pain-related

151

hypersensitivity(Dawes et al., 2018), and previous studies have indicated an important

152

role of mTOR signaling in the management of pain in several animal

153

models(Khoutorsky and Price, 2018; Price et al., 2007). To assess whether

154

hyperactive Akt-mTOR signaling was responsible for the pain-related hypersensitivity

155

in Cntnap2-/- mice, we first investigated the effect of the Akt inhibitor LY294002 and

156

the mTOR inhibitor rapamycin on the pain-related sensitivity of Cntnap2-/- mice to

157

mechanical and thermal stimuli. LY294002 (25mg/kg) significantly suppressed the

158

phosphorylation of Akt and S6 in the DRG from Cntnap2-/- mice (Fig. 1C).

159

Rapamycin (10 mg/kg) significantly suppressed the phosphorylation of S6, but had no

160

effect on the phosphorylated Akt in the DRG from Cntnap2-/- mice (Fig. 1D).

161

Consistent with the previous report(Dawes et al., 2018), Cntnap2-/- mice were

162

hypersensitive to Von Frey hair application, with a significantly lower withdrawal

163

threshold than WT mice (Fig. 1E). Both LY294002 and rapamycin treatment

164

significantly increased the withdrawal threshold in Cntnap2-/- mice (Fig. 1E). After

165

drug treatment, there is no significant difference in the mechanical sensitivity between

166

WT and Cntnap2-/- mice (Fig. 1E).

167

We also evaluated the heat hypersensitivity using a hot plate test. As shown in Fig.

168

1F, Cntnap2-/- mice showed a reduced latency to withdraw in the hot plate test as

169

compared to WT mice. However, the withdrawal latency was significantly increased

170

in Cntnap2-/- mice after treatment with LY294002 or rapamycin (Fig. 1F). After drug

171

treatment, there was also no significant difference in the heat sensitivity between WT

172

and Cntnap2-/- mice (Fig. 1F).

173

3.3 Normalization of hypersensitivity to chemical algogens in Cntnap2-/- mice by

174

Akt-mTOR inhibitors

175

The effect of LY294002 and rapamycin on the sensitivity of Cntnap2-/- mice to

176

chemical algogens, such as capsaicin and formalin, was also assessed. Consistent with

177

a previous study(Dawes et al., 2018), the application of capsaicin produced a 6

178

significantly augmented pain response in Cntnap2-/- mice versus WT mice (Fig.

179

2A-B). Cntnap2-/- mice spent significant more time on nocifensive behaviors than WT

180

controls (Fig. 2B). Treatment with LY294002 and rapamycin significantly reduced the

181

duration of nocifensive behaviors in Cntnap2-/- mice, but had no effect on WT mice

182

(Fig. 2A-B).

183

A subcutaneous injection of 5% formalin into the ventral hind paw elicited a

184

biphasic behavioral response, which can be divided into a brief early phase (0–10

185

mins) and a prolonged late phase (10–60 mins). Cntnap2-/- mice exhibited

186

significantly enhanced late-phase responses to subcutaneous administration of

187

formalin, which was significantly normalized by LY294002 and rapamycin (Fig.

188

2D-E).

189

An intraplantar injection of capsaicin or 5% formalin also induces a marked

190

neurogenic inflammation characterized by increased paw diameter. However, a

191

comparable increase in paw diameter was observed in mice regardless of genotypes

192

and treatments (Fig. 2C, 2F). These results indicated that Akt-mTOR inhibition

193

rescued the capsaicin/formalin-evoked pain hypersensitivity in Cntnap2-/- mice

194

without affecting the inflammatory response.

195

3.4 Suppression of the mTOR signaling pathway decreased DRG neuronal

196

hyperexcitability

197

Dawes et al recently reported that ablation of Cntnap2 enhanced the excitability of

198

DRG neurons in a cell-autonomous fashion(Dawes et al., 2018). Consistent with their

199

data, the small/medium-sized DRG neurons from Cntnap2-/- mice showed more action

200

potentials in response to supra-threshold stimulation than those neurons from WT

201

mice (Fig. 3A-B). However, the firing frequency of DRG neurons from

202

rapamycin-treated Cntnap2-/- mice was significantly reduced compared with the

203

vehicle-treated Cntnap2-/- mice (Fig. 3A-B). After rapamycin treatment, there was no

204

significant difference in the firing frequency between DRG neurons from WT and

205

Cntnap2-/- mice (Fig. 3A-B). Nevertheless, the resting membrane potential in DRG

206

neurons was comparable regardless of the genotype and/or drug administration (Fig.

207

3C). 7

208

3.5 Rescue of pain-related hypersensitivity in Cntnap2-/- mice by the FDA-approved

209

drug metformin

210

Metformin is one of the most widely used drugs for the treatment of type 2

211

diabetes(Bennett et al., 2011; Maruthur et al., 2016), but is also used in those with

212

kidney disease, heart failure, or liver problems(Crowley et al., 2017)which may also

213

inhibit the mTOR signaling pathway(Howell et al., 2017; Kalender et al., 2010; Obara

214

et al., 2015; Soares et al., 2013). Indeed, treatment with metformin for two

215

consecutive days normalized the phosphorylated S6 levels in the DRG from

216

Cntnap2-/- mice (Fig. 4A). Metformin significantly suppressed the hypersensitivity of

217

Cntnap2-/- mice to mechanical and thermal stimuli (Fig. 4B-C). Furthermore, the

218

nocifensive behaviors of Cntnap2-/- mice after capsaicin or formalin application were

219

also normalized after metformin treatment (Fig. 4D-G).

220

4. Discussion

221

Cntnap2 is involved in the neuron-glia interaction and clustering of potassium

222

channels in myelinated axons(Poliak et al., 2003). In a seminal study, Dawes and

223

colleagues demonstrated that the ablation of Cntnap2 enhanced the excitability of

224

DRG neurons through the downregulation of Kv1 channel expression at the soma

225

membrane(Dawes et al., 2018). In the present study, we observed hyperactive

226

Akt-mTOR signaling in the DRG neurons of Cntnap2-/- mice. Pharmacological

227

inhibition of Akt-mTOR signaling could attenuate pain-related hypersensitivity in

228

Cntnap2-/- mice. Moreover, suppression of the mTOR signaling pathway decreased

229

DRG neuronal hyperexcitability. Interestingly, Graham et al demonstrated that mTOR

230

signaling could suppress the translation of Kv1 voltage-gated potassium channel,

231

whereas the mTOR inhibitor rapamycin increases the Kv1 protein in hippocampal

232

neurons and promoted Kv1 surface expression(Raab-Graham et al., 2006). Therefore,

233

Cntnap2-deficiency may also affect the translation in addition to clustering of Kv1

234

channels, leading to DRG neuronal hyperactivity and pain-related hypersensitivity. It

235

has also been reported that rapamycin may alleviate the pain hypersensitivity by

236

inhibiting inflammatory related molecules(Duan et al., 2018; Xu et al., 2018).

237

Although LY294002 and rapamycin had no effect on the capsaicin and 8

238

formalin-induced increase in paw diameter in this study, the possible effect of

239

LY294002 and rapamycin on inflammation cannot be totally excluded due to the high

240

doses of capsaicin/formalin used in these assays.

241

mTOR is a serine-threonine kinase involved in regulated key cellular processes,

242

including autophagy, lipogenesis, cell growth and mRNA translation(Costa-Mattioli et

243

al., 2009; Shimobayashi and Hall, 2014). Abnormal activation of mTOR signaling is

244

found in various disorders such as tuberous sclerosis, neurofibromatosis, fragile X

245

syndrome, and epilepsy(Curatolo and Moavero, 2012; Johannessen et al., 2005; Sato

246

et al., 2012; Sha et al., 2012; Sharma et al., 2010). Further, dysregulated mTOR

247

signaling is also involved in pain-related sensitivity(Jimenez-Diaz et al., 2008; Price

248

et al., 2007). Although they were virtually undetectable under normal conditions(Xu

249

et al., 2010), the phosphorylated mTOR and its downstream S6K significantly

250

increased in pain-related animal models(Xu et al., 2011; Zhang et al., 2013)The

251

mTOR inhibitor rapamycin could inhibit nociceptive behaviors induced by formalin

252

and

253

mice(Khoutorsky and Price, 2018; Price et al., 2007). In addition, rats injected with

254

cancer cells showed elevated phosphorylation of mTOR and pS6K and experienced

255

pain hypersensitivity, which is attenuated by an intrathecal injection of

256

rapamycin(Jiang et al., 2016; Shih et al., 2012). An intraplantar injection of complete

257

Freund’s adjuvant (CFA) increased p-mTOR and p-S6K1 levels, and the mechanical

258

and thermal pain hypersensitivity induced by CFA could be alleviated by intrathecally

259

administered rapamycin(Liang et al., 2013). Chronic constriction injury (CCI)

260

induced PI3K, PKB, and mTOR activation. Intrathecal treatment with mTOR

261

inhibitor reversed the CCI-evoked hyperalgesic effect(Zhang et al., 2013).

262

Furthermore, anti-cancer drugs, such as bortezomib, oxaliplatin, could also induce

263

mTOR signaling pathway hyperactive and neuropathic pain. And the hypersensitivity

264

for mechanical and cold stimulate are relieved by treatment with rapamycin(Duan et

265

al., 2018a; Duan et al., 2018b).In this study, we also found pharmacological inhibition

266

of Akt-mTOR signaling attenuated pain-related hypersensitivity in Cntnap2-/- mice.

267

Nevertheless, in contrast to some previous reports(Megat et al., 2019; Price et al.,

DHPG

((RS)-3,5-Dihydroxyphenylglycine,

9

mGluR1/5

agonist)

in

WT

268

2007), the inhibition of Akt-mTOR signaling has no significant e on the pain-related

269

behaviors in wild-type mice in our study. The discrepancy of this finding with other

270

reports may be due to the drug delivery routes (Intrathecal versus Intraperitoneal),

271

drug dosages, or different drugs(Megat et al., 2019).

272

Metformin, one of the most widely used drug for the treatment of type 2

273

diabetes(Kirpichnikov et al., 2002), has been shown to alleviate the pain

274

hypersensitivity in several mouse models(Inyang et al., 2019; La et al., 2017; Ma et

275

al., 2015; Melemedjian et al., 2011; Weng et al., 2019). For instance, metformin

276

reversed spared nerve injury induced mechanical and cold hypersensitivity in male

277

mice(Inyang et al., 2019). Further, metformin selectively inhibited capsaicin-induced

278

secondary

279

allodynia(La et al., 2017). Mechanistically, the pain-relieving effect of metformin may

280

mediated by its function in translation regulation, AMPK activation, autophagy flux

281

stimulation(La et al., 2017; Ma et al., 2015; Melemedjian et al., 2011; Weng et al.,

282

2019). In this study, we showed that treatment with metformin revered the

283

pain-related hypersensitivity in Cntnap2-/- mice.

mechanical

allodynia

and

intrathecal

KO2-induced

mechanical

284

In this study, we showed that treatment with metformin reversed pain-related

285

hypersensitivity in Cntnap2-/- mice. Our data further indicated that the pain-relieving

286

effect of metformin in Cntnap2-/- mice may be mediated by the suppression of mTOR

287

signaling, as treatment with metformin inhibited the phosphorylation of S6 in the

288

DRG neurons. As an activator of AMP-activated protein kinase (AMPK), metformin

289

may suppress mTOR signaling in an AMPK-dependent manner(Xu et al., 2012).

290

However, metformin may also inhibit mTOR signaling in an AMPK-independent

291

manner(Ben Sahra et al., 2011; Chen et al., 2017; Vazquez-Martin et al., 2009).

292

Nevertheless, we cannot exclude the alterative mechanisms underlying the

293

pain-relieving effect of metformin in Cntnap2-/- mice. Indeed, Gantois et al showed

294

that metformin decreased ERK signaling, eIF4E phosphorylation and the expression

295

of MMP-9, but had no effect on the phosphorylated S6 in a mouse model of fragile X

296

syndrome(Gantois et al., 2017).

297

In conclusion, we identified the hyperactive Akt-mTOR pathway as a therapeutic 10

298

target for pain hypersensitivity in Cntnap2-deficient mice. Treatment with the Akt

299

inhibitor LY94002, the mTOR inhibitor rapamycin, or the FDA-approved drug

300

metformin attenuated pain-related hypersensitivity in Cntnap2-/- mice. Our results

301

indicated that the hyperactive Akt-mTOR signaling pathway may be a promising

302

therapeutic target for pain-related hypersensitivity in patients with dysfunction of

303

CNTNAP2.

304

References

305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337

Alarcon, M., Abrahams, B. S., Stone, J. L., Duvall, J. A., Perederiy, J. V., Bomar, J. M., Sebat, J., Wigler, M., Martin, C. L., Ledbetter, D. H., Nelson, S. F., Cantor, R. M., Geschwind, D. H., 2008. Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene. Am J Hum Genet 82, 150-159. Anderson, G. R., Galfin, T., Xu, W., Aoto, J., Malenka, R. C., Sudhof, T. C., 2012. Candidate autism gene screen identifies critical role for cell-adhesion molecule CASPR2 in dendritic arborization and spine development. Proc Natl Acad Sci U S A 109, 18120-18125. Arking, D. E., Cutler, D. J., Brune, C. W., Teslovich, T. M., West, K., Ikeda, M., Rea, A., Guy, M., Lin, S., Cook, E. H., Chakravarti, A., 2008. A common genetic variant in the neurexin superfamily member CNTNAP2 increases familial risk of autism. Am J Hum Genet 82, 160-164. Ben Sahra, I., Regazzetti, C., Robert, G., Laurent, K., Le Marchand-Brustel, Y., Auberger, P., Tanti, J. F., Giorgetti-Peraldi, S., Bost, F., 2011. Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1. Cancer Res 71, 4366-4372. Bennett, W. L., Maruthur, N. M., Singh, S., Segal, J. B., Wilson, L. M., Chatterjee, R., Marinopoulos, S. S., Puhan, M. A., Ranasinghe, P., Block, L., Nicholson, W. K., Hutfless, S., Bass, E. B., Bolen, S., 2011. Comparative effectiveness and safety of medications for type 2 diabetes: an update including new drugs and 2-drug combinations. Ann Intern Med 154, 602-613. Chen, S. C., Brooks, R., Houskeeper, J., Bremner, S. K., Dunlop, J., Viollet, B., Logan, P. J., Salt, I. P., Ahmed, S. F., Yarwood, S. J., 2017. Metformin suppresses adipogenesis through both AMP-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms. Mol Cell Endocrinol 440, 57-68. Costa-Mattioli, M., Sossin, W. S., Klann, E., Sonenberg, N., 2009. Translational control of long-lasting synaptic plasticity and memory. Neuron 61, 10-26. Curatolo, P., Moavero, R., 2012. mTOR Inhibitors in Tuberous Sclerosis Complex. Curr Neuropharmacol 10, 404-415. Dawes, J. M., Weir, G. A., Middleton, S. J., Patel, R., Chisholm, K. I., Pettingill, P., Peck, L. J., Sheridan, J., Shakir, A., Jacobson, L., Gutierrez-Mecinas, M., Galino, J., Walcher, J., Kuhnemund, J., Kuehn, H., Sanna, M. D., Lang, B., Clark, A. J., Themistocleous, A. C., Iwagaki, N., West, S. J., Werynska, K., Carroll, L., Trendafilova, T., Menassa, D. A., Giannoccaro, M. P., Coutinho, E., Cervellini, I., Tewari, D., Buckley, C., Leite, M. I., Wildner, H., Zeilhofer, H. U., Peles, E., Todd, A. J., McMahon, S. B., Dickenson, A. H., Lewin, G. R., Vincent, A., Bennett, D. L., 2018. Immune or Genetic-Mediated Disruption of CASPR2 Causes Pain Hypersensitivity Due to Enhanced Primary Afferent Excitability. Neuron 97, 806-822 e810. Duan, Z., Su, Z., Wang, H., Pang, X., 2018. Involvement of pro-inflammation signal pathway in 11

338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381

inhibitory effects of rapamycin on oxaliplatin-induced neuropathic pain. Mol Pain 14, 1744806918769426. Friedman, J. I., Vrijenhoek, T., Markx, S., Janssen, I. M., van der Vliet, W. A., Faas, B. H., Knoers, N. V., Cahn, W., Kahn, R. S., Edelmann, L., Davis, K. L., Silverman, J. M., Brunner, H. G., van Kessel, A. G., Wijmenga, C., Ophoff, R. A., Veltman, J. A., 2008. CNTNAP2 gene dosage variation is associated with schizophrenia and epilepsy. Mol Psychiatry 13, 261-266. Gantois, I., Khoutorsky, A., Popic, J., Aguilar-Valles, A., Freemantle, E., Cao, R., Sharma, V., Pooters, T., Nagpal, A., Skalecka, A., Truong, V. T., Wiebe, S., Groves, I. A., Jafarnejad, S. M., Chapat, C., McCullagh, E. A., Gamache, K., Nader, K., Lacaille, J. C., Gkogkas, C. G., Sonenberg, N., 2017. Metformin ameliorates core deficits in a mouse model of fragile X syndrome. Nat Med 23, 674-677. Howell, J. J., Hellberg, K., Turner, M., Talbott, G., Kolar, M. J., Ross, D. S., Hoxhaj, G., Saghatelian, A., Shaw, R. J., Manning, B. D., 2017. Metformin Inhibits Hepatic mTORC1 Signaling via Dose-Dependent Mechanisms Involving AMPK and the TSC Complex. Cell Metab 25, 463-471. Inyang, K. E., Szabo-Pardi, T., Wentworth, E., McDougal, T. A., Dussor, G., Burton, M. D., Price, T. J., 2019. The antidiabetic drug metformin prevents and reverses neuropathic pain and spinal cord microglial activation in male but not female mice. Pharmacol Res 139, 1-16. Jiang, Z., Wu, S., Wu, X., Zhong, J., Lv, A., Jiao, J., Chen, Z., 2016. Blocking mammalian target of rapamycin alleviates bone cancer pain and morphine tolerance via micro-opioid receptor. Int J Cancer 138, 2013-2020. Jimenez-Diaz, L., Geranton, S. M., Passmore, G. M., Leith, J. L., Fisher, A. S., Berliocchi, L., Sivasubramaniam, A. K., Sheasby, A., Lumb, B. M., Hunt, S. P., 2008. Local translation in primary afferent fibers regulates nociception. PLoS One 3, e1961. Johannessen, C. M., Reczek, E. E., James, M. F., Brems, H., Legius, E., Cichowski, K., 2005. The NF1 tumor suppressor critically regulates TSC2 and mTOR. Proc Natl Acad Sci U S A 102, 8573-8578. Kalender, A., Selvaraj, A., Kim, S. Y., Gulati, P., Brule, S., Viollet, B., Kemp, B. E., Bardeesy, N., Dennis, P., Schlager, J. J., Marette, A., Kozma, S. C., Thomas, G., 2010. Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner. Cell Metab 11, 390-401. Khoutorsky, A., Price, T. J., 2018. Translational Control Mechanisms in Persistent Pain. Trends Neurosci 41, 100-114. Kirpichnikov, D., McFarlane, S. I., Sowers, J. R., 2002. Metformin: an update. Ann Intern Med 137, 25-33. Klein, C. J., Lennon, V. A., Aston, P. A., McKeon, A., Pittock, S. J., 2012. Chronic pain as a manifestation of potassium channel-complex autoimmunity. Neurology 79, 1136-1144. La, J. H., Wang, J., Bittar, A., Shim, H. S., Bae, C., Chung, J. M., 2017. Differential involvement of reactive oxygen species in a mouse model of capsaicin-induced secondary mechanical hyperalgesia and allodynia. Mol Pain 13, 1744806917713907. Lazo, J. S., Sharlow, E. R., Epperly, M. W., Lira, A., Leimgruber, S., Skoda, E. M., Wipf, P., Greenberger, J. S., 2013. Pharmacologic profiling of phosphoinositide 3-kinase inhibitors as mitigators of ionizing radiation-induced cell death. J Pharmacol Exp Ther 347, 669-680. Li, X., Hu, Z., He, Y., Xiong, Z., Long, Z., Peng, Y., Bu, F., Ling, J., Xun, G., Mo, X., Pan, Q., Zhao, J., Xia, K., 2010. Association analysis of CNTNAP2 polymorphisms with autism in the Chinese Han population. Psychiatr Genet 20, 113-117. Liang, L., Tao, B., Fan, L., Yaster, M., Zhang, Y., Tao, Y. X., 2013. mTOR and its downstream pathway are activated in the dorsal root ganglion and spinal cord after peripheral inflammation, but not after nerve 12

382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425

injury. Brain Res 1513, 17-25. Ma, J., Yu, H., Liu, J., Chen, Y., Wang, Q., Xiang, L., 2015. Metformin attenuates hyperalgesia and allodynia in rats with painful diabetic neuropathy induced by streptozotocin. Eur J Pharmacol 764, 599-606. Maruthur, N. M., Tseng, E., Hutfless, S., Wilson, L. M., Suarez-Cuervo, C., Berger, Z., Chu, Y., Iyoha, E., Segal, J. B., Bolen, S., 2016. Diabetes Medications as Monotherapy or Metformin-Based Combination Therapy for Type 2 Diabetes: A Systematic Review and Meta-analysis. Ann Intern Med 164, 740-751. Megat, S., Ray, P. R., Tavares-Ferreira, D., Moy, J. K., Sankaranarayanan, I., Wanghzou, A., Fang Lou, T., Barragan-Iglesias, P., Campbell, Z. T., Dussor, G., Price, T. J., 2019. Differences between Dorsal Root and Trigeminal Ganglion Nociceptors in Mice Revealed by Translational Profiling. J Neurosci 39, 6829-6847. Melemedjian, O. K., Asiedu, M. N., Tillu, D. V., Sanoja, R., Yan, J., Lark, A., Khoutorsky, A., Johnson, J., Peebles, K. A., Lepow, T., Sonenberg, N., Dussor, G., Price, T. J., 2011. Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain. Mol Pain 7, 70. Obara, I., Medrano, M. C., Signoret-Genest, J., Jimenez-Diaz, L., Geranton, S. M., Hunt, S. P., 2015. Inhibition of the mammalian target of rapamycin complex 1 signaling pathway reduces itch behaviour in mice. Pain 156, 1519-1529. Penagarikano, O., Abrahams, B. S., Herman, E. I., Winden, K. D., Gdalyahu, A., Dong, H., Sonnenblick, L. I., Gruver, R., Almajano, J., Bragin, A., Golshani, P., Trachtenberg, J. T., Peles, E., Geschwind, D. H., 2011. Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities, and core autism-related deficits. Cell 147, 235-246. Poliak, S., Salomon, D., Elhanany, H., Sabanay, H., Kiernan, B., Pevny, L., Stewart, C. L., Xu, X., Chiu, S. Y., Shrager, P., Furley, A. J., Peles, E., 2003. Juxtaparanodal clustering of Shaker-like K+ channels in myelinated axons depends on Caspr2 and TAG-1. J Cell Biol 162, 1149-1160. Price, T. J., Rashid, M. H., Millecamps, M., Sanoja, R., Entrena, J. M., Cervero, F., 2007. Decreased nociceptive sensitization in mice lacking the fragile X mental retardation protein: role of mGluR1/5 and mTOR. J Neurosci 27, 13958-13967. Raab-Graham, K. F., Haddick, P. C., Jan, Y. N., Jan, L. Y., 2006. Activity- and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites. Science 314, 144-148. Sato, A., Kasai, S., Kobayashi, T., Takamatsu, Y., Hino, O., Ikeda, K., Mizuguchi, M., 2012. Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex. Nat Commun 3, 1292. Sha, L. Z., Xing, X. L., Zhang, D., Yao, Y., Dou, W. C., Jin, L. R., Wu, L. W., Xu, Q., 2012. Mapping the spatio-temporal pattern of the mammalian target of rapamycin (mTOR) activation in temporal lobe epilepsy. PLoS One 7, e39152. Sharma, A., Hoeffer, C. A., Takayasu, Y., Miyawaki, T., McBride, S. M., Klann, E., Zukin, R. S., 2010. Dysregulation of mTOR signaling in fragile X syndrome. J Neurosci 30, 694-702. Shih, M. H., Kao, S. C., Wang, W., Yaster, M., Tao, Y. X., 2012. Spinal cord NMDA receptor-mediated activation of mammalian target of rapamycin is required for the development and maintenance of bone cancer-induced pain hypersensitivities in rats. J Pain 13, 338-349. Shimobayashi, M., Hall, M. N., 2014. Making new contacts: the mTOR network in metabolism and signalling crosstalk. Nat Rev Mol Cell Biol 15, 155-162. Soares, H. P., Ni, Y., Kisfalvi, K., Sinnett-Smith, J., Rozengurt, E., 2013. Different patterns of Akt and ERK feedback activation in response to rapamycin, active-site mTOR inhibitors and metformin in pancreatic 13

426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444

cancer cells. PLoS One 8, e57289.

445

Acknowledgement

Varea, O., Martin-de-Saavedra, M. D., Kopeikina, K. J., Schurmann, B., Fleming, H. J., Fawcett-Patel, J. M., Bach, A., Jang, S., Peles, E., Kim, E., Penzes, P., 2015. Synaptic abnormalities and cytoplasmic glutamate receptor aggregates in contactin associated protein-like 2/Caspr2 knockout neurons. Proc Natl Acad Sci U S A 112, 6176-6181. Vazquez-Martin, A., Oliveras-Ferraros, C., Menendez, J. A., 2009. The antidiabetic drug metformin suppresses HER2 (erbB-2) oncoprotein overexpression via inhibition of the mTOR effector p70S6K1 in human breast carcinoma cells. Cell Cycle 8, 88-96. Weng, W., Yao, C., Poonit, K., Zhou, X., Sun, C., Zhang, F., Yan, H., 2019. Metformin relieves neuropathic pain after spinal nerve ligation via autophagy flux stimulation. J Cell Mol Med 23, 1313-1324. Xing, X., Zhang, J., Wu, K., Cao, B., Li, X., Jiang, F., Hu, Z., Xia, K., Li, J. D., 2019. Suppression of Akt-mTOR pathway rescued the social behavior in Cntnap2-deficient mice. Sci Rep 9, 3041. Xu, M., Cheng, Z., Ding, Z., Wang, Y., Guo, Q., Huang, C., 2018. Resveratrol enhances IL-4 receptor-mediated anti-inflammatory effects in spinal cord and attenuates neuropathic pain following sciatic nerve injury. Mol Pain 14, 1744806918767549. Zhang, W., Sun, X. F., Bo, J. H., Zhang, J., Liu, X. J., Wu, L. P., Ma, Z. L., Gu, X. P., 2013. Activation of mTOR in the spinal cord is required for pain hypersensitivity induced by chronic constriction injury in mice. Pharmacol Biochem Behav 111, 64-70.

446

This project is financially supported by the National Natural Science Foundation of

447

China (81728013, 81671101), and Education Department Foundation of Hunan

448

Province (15B165),

449

(2019JJ40204), the Key Research and Development Programs from Hunan Province

450

(2018DK2010 and 2018DK2013), and the Fundamental Research Funds for the

451

Central Universities of Central South University(2018zzts398).

452

Author contribution

the Natural

Science

Foundation

of

Hunan

Province

453

J-D.L., W.H., and F.J., conceived and designed the experiments; X.X., K.W., and

454

Y.Z., performed the experiments; J.Z., and Y.D., helped to analyze the data; X.X.,

455

W.H., and J-D.L. wrote the paper.

456

Conflict of Interest

457 458 459

The authors declare no competing interests. Competing financial interests The authors declare no competing financial interests.

460

14

461

Figure Legend

462

Fig. 1 Inhibition of Akt-mTOR signaling rescued mechanical and thermal

463

hypersensitivity in Cntnap2-/- mice

464

(A) Representative immunoblots of lysates from the DRG of WT and Cntnap2-/- mice.

465

(B) Quantification of phosphorylated and total levels of Akt and S6 in the DRG of

466

WT and Cntnap2-/- mice. (C) Immunoblots of lysates from the DRG of WT and

467

Cntnap2-/- mice treated with LY294002 for two consecutive days. (D) Immunoblots of

468

lysates from the DRG of WT and Cntnap2-/- mice treated with rapamycin for two

469

consecutive days. (E) Treatment with the Akt inhibitor LY294002 and mTOR

470

inhibitor rapamycin rescued the hypersensitivity of Cntnap2-/- mice to mechanical

471

stimuli as assayed with Von Frey hairs test. (F) Treatment with the Akt inhibitor

472

LY294002 and mTOR inhibitor rapamycin rescued the hypersensitivity of Cntnap2-/-

473

mice to heat as assayed with a hot plate set at 54.5°C. The number of mice was

474

indicated in the respective graphs. *p<0.05, **p<0.01, ***p ＜ 0.001. Data are

475

expressed as the mean ± sem (standard error of the mean). A repeated-measure

476

ANOVA followed by Bonferroni post hoc tests or unpaired two-tail Student’s t test

477

was used.

478 479

Fig. 2 Normalization of chemical algogens hypersensitivity in Cntnap2-/- mice by

480

Akt-mTOR inhibitors

481

(A) The nocifensive behavior duration of WT and Cntnap2-/- mice induced by

482

intraplantar injection of capsaicin, after treatment with saline, LY294002 or

483

rapamycin. (B) The total nocifensive behavior duration of WT and Cntnap2-/- mice

484

induced by intraplantar injection of capsaicin after treatment with saline, LY294002

485

and rapamycin. (C) Comparable increase in paw diameter induced by intraplantar

486

injection of capsaicin was observed in mice treatment with saline, LY294002 and

487

rapamycin. (D) The nocifensive behavior duration of WT and Cntnap2-/- mice induced

488

by an intraplantar injection of 5% formalin, after treatment with saline, LY294002, or

489

rapamycin. (E) The total nocifensive behavior duration at the late phase (10-60 min) 15

490

of WT and Cntnap2-/- mice induced by an intraplantar injection of 5% formalin after

491

treatment with saline, LY294002 or rapamycin. (F) Comparable increase in paw

492

diameter induced by an intraplantar injection of 5% formalin was observed in mice

493

treatment with saline, LY294002 and rapamycin. The number of mice was indicated

494

in the respective graphs. *p<0.05, **p<0.01, ***p＜0.001. Data are expressed as

495

mean ± sem. A repeated-measure ANOVA followed by Bonferroni post hoc tests or

496

unpaired two-tail Student’s t test was used.

497 498

Fig. 3 Suppression of the mTOR signaling pathway decreased DRG neuronal

499

hyperexcitability

500

(A) Representative traces showing action potential firing to 300 pA injection in DRG

501

neurons. (B) Quantification of action potentials (APs) firing induced by 100, 200 and

502

300pA current injection in DRG neurons from WT and Cntnap2-/- mice treated with

503

saline and rapamycin. (C) The resting membrane potential (RMP) of DRG neurons

504

were comparable regardless of the genotype and/or drug administration. The number

505

of DRG neuron was indicated in the respective graphs. *p<0.05, **p<0.01, ***p＜

506

0.001. Data are expressed as mean ± sem. A repeated-measure ANOVA followed by

507

Bonferroni post hoc tests or unpaired two-tail Student’s t test was used.

508 509

Fig. 4 Rescue of pain-related hypersensitivity in Cntnap2-/- mice by the FDA drug

510

metformin

511

(A) Immunoblots of lysates from the DRG of WT and Cntnap2-/- mice treated with

512

metformin for two consecutive days. (B) Treatment with metformin rescued the

513

hypersensitivity of Cntnap2-/- mice to mechanical stimuli as assayed with Von Frey

514

hairs test. (C) Treatment with metformin rescued the hypersensitivity of Cntnap2-/-

515

mice to heat as assayed with a hot plate set at 54.5°C. (D) The nocifensive behavior

516

duration of WT and Cntnap2-/- mice induced by intraplantar injection of capsaicin,

517

after treatment with saline or metformin. (E) The total nocifensive behavior duration 16

518

of WT and Cntnap2-/- mice induced by an intraplantar injection of capsaicin, after

519

treatment with saline or metformin. (F) The nocifensive behavior duration of WT and

520

Cntnap2-/- mice induced by an intraplantar injection of 5% formalin, after treatment

521

with saline or metformin. (G) The total nocifensive behavior duration at the late phase

522

(10-60 min) of WT and Cntnap2-/- mice induced by an intraplantar injection of 5%

523

formalin, after treatment with saline or metformin. The number of mice was indicated

524

in the respective graphs. *p<0.05, **p<0.01, ***p＜0.001. Data are expressed as

525

mean ± sem. A repeated-measure ANOVA followed by Bonferroni post hoc tests or

526

unpaired two-tail Student’s t test was used.

17

Highlights Cntnap2 deficiency led to overactivation of the Akt-mTOR pathway in the DRG. Akt-mTOR inhibitors normalized pain-related hypersensitivity of Cntnap2-/- mice. Suppression of mTOR decreased DRG neuronal hyperexcitability of Cntnap2-/mice. FDA drug metformin rescued pain-related hypersensitivity in Cntnap2-/- mice.