bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1

2 The Telomerase Reverse Transcriptase (TERT) and Regulate Mammalian PNS and 3 CNS Regeneration downstream of c- 4 5 Jin-Jin Ma1#, Ren-Jie Xu2,3#, Xin Ju2#, Wei-Hua Wang1, Zong-Ping Luo1,2, Chang-Mei Liu4,5, Lei 6 Yang1,2, Bin Li1,2, Jian-Quan Chen1,2, Bin Meng2, Hui-Lin Yang1,2, Feng-Quan Zhou6, 7* and 7 Saijilafu1,2* 8 9 1Orthopaedic Institute, Medical College, Soochow University, Suzhou, Jiangsu, China 10 2Department of Orthopaedic Surgery, the First Affiliated Hospital, Soochow University, Suzhou, 11 China 12 3Department of Orthopaedics, Suzhou Municipal Hospital/the Affiliated Hospital of Nanjing 13 Medical University, Suzhou, Jiangsu, China 14 4State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese 15 Academy of Science, Beijing, China 16 5Savaid Medical School, University of Chinese Academy of Sciences, Beijing, China 17 6Department of Orthopaedic Surgery, Johns Hopkins University School of Medicine, Baltimore, 18 MD, USA. 19 7The Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of 20 Medicine, Baltimore, MD, USA. 21 22 # These authors contributed equally to this work 23 24 *Correspondence: Saijilafu, MD, Ph.D.；Feng-Quan Zhou, Ph.D. 25 Phone: 0512-6778-0762 26 Email: [email protected]; [email protected] 27

28

29 Running title: The c-Myc / TERT / p53 Pathways regulates axon growth

30

31

32

33

34 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

35 Summary

36

37 Although several genes have been identified to promote axon regeneration in the central nervous

38 system, our understanding of the molecular mechanisms by which mammalian axon regeneration

39 is regulated is still limited and fragmented. Here by using sensory axon and optic

40 regeneration as model systems, we revealed an unexpected role of telomerase reverse

41 transcriptase (TERT) in regulation of axon regeneration. We also provided strong evidence that

42 TERT and p53 acted downstream of c-Myc to control sensory axon regeneration. More

43 importantly, overexpression of p53 in sensory and retinal ganglion cells (RGCs) was

44 sufficient to promote sensory axon and optic never regeneration, respectively. The study revealed

45 a novel c-Myc-TERT-p53 signaling pathway, expanding horizons for novel approaches

46 promoting CNS axon regeneration.

47

48 Keywords: c-Myc, telomerase reverse transcriptase, telomere, p53, axon regeneration, optic

49 nerve regeneration

50

51 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

52 Introduction

53 The central reason of failed axon growth in the adult mammalian

54 (CNS) is the diminished intrinsic axon regeneration ability in neurons during maturation and

55 aging (Sun and He, 2010). For example, RGC neurons lose their intrinsic axon growth ability

56 during development and maturation (Goldberg et al., 2002). During the past decade, studies

57 focusing on the intrinsic axon growth ability have generated by far the most promising results in

58 the CNS axon regeneration field. For instance, genetic deletion of the PTEN could elevate the

59 intrinsic axon regeneration ability of mature CNS neurons and promote regeneration of optic

60 nerve and corticospinal tract (Liu et al., 2010; Park et al., 2008). Similar molecules regulating the

61 intrinsic axon regeneration ability are KLF4 (Moore et al., 2009), SOCS3 (Smith et al., 2009),

62 SOX11 (Wang et al., 2015), Lin28 (Wang et al., 2018), and c-Myc (Belin et al., 2015) etc.

63 Despite these significant progresses during the past decade, our understanding of the molecular

64 mechanisms by which mammalian CNS axon regeneration is regulated is still fragmented.

65 In contrast, neurons from the peripheral nervous system (PNS) can regenerate their

66 by reactivating the intrinsic axon growth abilities in response to peripheral nerve

67 (Chandran et al., 2016; Michaelevski et al., 2010). Such response is mediated by a

68 transcription-dependent process (Saijilafu et al., 2013; Smith and Skene, 1997), in which several

69 transcription factors (TFs) have been identified, such as c-Jun (Raivich et al., 2004; Zhou et al.,

70 2004), Smad1 (Parikh et al., 2011; Saijilafu et al., 2013), ATF3 (Seijffers et al., 2007), and

71 STAT3 (Bareyre et al., 2011; Qiu et al., 2005). Importantly, almost all of these TFs functioned

72 similarly in the CNS to regulate axon regeneration (Bareyre et al., 2011; Fagoe et al., 2015; bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

73 Finelli et al., 2013; Parikh et al., 2011; Qin et al., 2013). Conversely, most of the aforementioned

74 genes regulating the intrinsic axon regeneration ability of CNS neurons also act to control PNS

75 axon regeneration (Jankowski et al., 2009; Jing et al., 2012; Saijilafu et al., 2013; Wang et al.,

76 2018). Thus, currently it is well-recognized that PNS axon regeneration provides a perfect model

77 system to study the molecular mechanisms underlying mammalian axon regeneration.

78 The c-Myc proto-oncogene encodes a that regulates various

79 physiological activities, such as cell growth, cell proliferation, apoptosis, and cellular

80 metabolism (Dang, 1999). Interestingly, a recent study indicated that overexpression of c-Myc in

81 retinal ganglion cells (RGCs), either before or after optic , could promote RGCs

82 survival and axon regeneration (Belin et al., 2015). The study also showed that the expression of

83 c-Myc was significantly increased in sensory neurons following a sciatic nerve lesion. However,

84 the underlying molecular mechanisms by which c-Myc regulates axon regeneration have remain

85 unclear. Previous studies in other non-neuronal systems have identified many downstream target

86 genes regulated by c-Myc. For instance, in some non-neuronal cell lines, c-Myc can transactivate

87 the p53 promoter through its basic-helix-loop-helix (bHLH) recognition motif (Reisman et al.,

88 1993). Moreover, in non-neuronal cells c-Myc has also been shown to be an important

89 transcriptional regulator of telomerase reverse transcriptase (TERT) by directly binding its

90 promoter with other transcription factors (Khattar and Tergaonkar, 2017; Wu et al., 1999). Thus,

91 it is likely that c-Myc might control axon regeneration through these downstream targets. Indeed,

92 during serum induced cell proliferation, c-Myc has been shown to regulate the expression of

93 ATF3 (Tamura et al., 2014), a well-known regulator of axon regeneration. Additionally, several bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

94 studies have shown that p53 signaling is necessary for both PNS and optic nerve regeneration

95 (Di Giovanni and Rathore, 2012; Floriddia et al., 2012; Gaub et al., 2011; Gaub et al., 2010;

96 Joshi et al., 2015; Tedeschi et al., 2009). TERT is the catalytic subunit of the enzyme telomerase,

97 which is well known for its role in regulation of telomere extension, cellular aging, and cancer

98 (Maciejowski and de Lange, 2017). In post-mitotic neurons, TERT has been shown to protect

99 neurons from oxidative damages and degenerative changes during aging (Liu et al., 2018; Miwa

100 and Saretzki, 2017; Spilsbury et al., 2015). Inhibition of TERT during iPSC induced neuronal

101 differentiation can produce aged neurons suitable for studying late onset neurodegenerative

102 diseases (Vera et al., 2016). Whether TERT plays any role in regulation of axon growth or

103 regeneration has not been investigated.

104 In the present study, we demonstrated that c-Myc was both necessary and sufficient for

105 supporting sensory axon growth in vitro and in vivo. Importantly, we provided clear and strong

106 evidence that TERT level was markedly increased in sensory neurons upon peripheral nerve

107 injury. Functionally, inhibition or deletion of TERT in sensory neurons significantly impaired

108 sensory axon regeneration in vitro and in vivo. Moreover, TERT level was regulated by c-Myc in

109 sensory neurons and activation of TERT was able to rescue sensory axon regeneration impaired

110 by down regulation of c-Myc, suggesting that TERT acted downstream of c-Myc to regulate

111 sensory axon regeneration. Lastly, we found that p53 was also up regulated in sensory neurons

112 upon peripheral nerve injury and acted downstream of TERT to regulate sensory axon

113 regeneration. Importantly, overexpression of p53 in sensory neurons and RGCs was sufficient to

114 promote sensory axon regeneration in vivo optic nerve regeneration, respectively. Collectively, bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

115 our data not only revealed an unexpected function of TERT in regulation of axon regeneration,

116 but also suggested that c-Myc, TERT, p53 signaling might act coordinately to regulate both PNS

117 and CNS axon regeneration.

118

119

120 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

121 Results

122

123 C-Myc is necessary and sufficient for supporting sensory axon regeneration in vitro and in

124 vivo

125 A recent study demonstrated that peripheral axotomy could upregulate c-Myc expression in

126 adult dorsal root ganglion (DRG) sensory neurons (Belin et al., 2015). However, the functional

127 roles of c-Myc in peripheral axon regeneration remain unknown. Similarly, our immunostaining

128 results showed that c-Myc expression was significantly upregulated in lumbar L4-L5 DRG

129 neurons after sciatic nerve injury (Supplementary Figure S1A, B). Western blot analysis showed

130 that peripheral nerve injury-induced upregulation of c-Myc expression in sensory neurons

131 reached a peak at approximately 3 days post-injury, and then decreased 7 days post-injury

132 (Supplementary Figure S1C, D). The mRNA levels of c-Myc were also increased after peripheral

133 axotomy, indicating enhanced gene transcription (Supplementary Figure S1E). To investigate the

134 functional role of c-Myc in regulation of sensory axon regeneration, we applied the specific

135 c-Myc inhibitor, 10058-F4 (10 µM) to cultured adult DRG neurons for 3 days. The result showed

136 that 10058-F4 significantly impaired the regenerative axon growth (Supplementary Figure S1F,

137 G). Similarly, downregulation of c-Myc expression via a specific small interfering RNA (siRNA)

138 against c-Myc also blocked axon growth of cultured DRG neurons (Supplementary Figure S1H,

139 I), confirming the pharmacological results. We next examined the role of c-Myc in controlling

140 sensory axon regeneration in vivo using DRG electroporation technique (Saijilafu et al., 2012;

141 Saijilafu et al., 2014). The specific siRNA against c-Myc, together with the plasmid encoding bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

142 enhanced green fluorescent protein (EGFP), were co-electroporated into adult mouse L4-L5

143 DRGs in vivo. Two days later, a sciatic nerve crush was performed using fine forceps and the

144 injured site was labeled with 11–0 nylon epineural sutures as previously described Saijilafu et al.,

145 2014. After three more days, the animals were perfused with paraformaldehyde (PFA) and the

146 entire sciatic nerve was dissected out. The resulting EGFP labeled sensory axons were imaged,

147 manually traced, and measured from the injury site to the distal axon tips to quantify axon

148 regeneration by a different person not directly involved in the experiments. The results showed

149 that the siRNA against c-Myc significantly blocked sensory axon regeneration compared to that

150 of control neurons electroporated with a scrambled siRNA and EGFP plasmid (Figure 1A-C).

151 Overexpression of c-Myc has been shown to increase the intrinsic axon growth ability of

152 RGCs and promote optic nerve regeneration (Belin et al., 2015). We thus tested if c-Myc acted

153 similarly in the PNS to promote axon regeneration. When c-Myc was overexpressed in cultured

154 DRG neurons in vitro, we found that sensory axon growth was significantly enhanced on

155 supportive substrates laminin (Supplementary Figure S2A, B). Moreover, CSPGs and are

156 two well-known major inhibitory substrates of axon regeneration (Lee and Zheng, 2012; Ohtake

157 and Li, 2015). We thus investigated if overexpression of c-Myc was able to overcome the

158 inhibitory effects of these substrates. Dissociated sensory neurons transfected with c-Myc

159 plasmid were cultured on CSPGs or myelin coated cell culture plates for 3 days. The results

160 showed that either CSPGs or myelin substrates significantly inhibited the axon growth of DRG

161 neurons, and overexpression of c-Myc markedly enhanced their axon growth on either CSPGs

162 (Supplementary Figure S2C, D) or myelin (Supplementary Figure S2E, F) substrates. To bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

163 determine the in vivo role of c-Myc in regulation of axon regeneration, we transfected L4-L5

164 DRGs of adult mouse with the c-Myc plasmid via in vivo DRG electroporation, and performed

165 sciatic nerve crush injury two days later. After three days, we found that overexpression of

166 c-Myc significantly promoted sensory axon regeneration in vivo (Figure 1D-F). Taken together,

167 our results provided solid evidence that c-Myc was necessary and sufficient to support sensory

168 axon regeneration in vitro and in vivo.

169

170 TERT functions to regulate sensory axon regeneration in vitro and in vivo

171 To determine the role of TERT in regulation of sensory axon regeneration, we examined the

172 level of TERT in sensory neurons upon peripheral nerve injury. Both immunostaining and

173 western blot analyses showed that TERT expression in DRG neurons was significantly increased

174 after peripheral axotomy (Figure 2A-D). It should be noted that TERT was mostly localized in

175 the cytoplasm of sensory neurons, which is consistent with previous studies of brain neurons

176 (Liu et al., 2018; Spilsbury et al., 2015). One potential explanation is that in post-mitotic neurons

177 TERT is localized in mitochondria to reduce oxidative stress and protect neurons from apoptosis

178 (Liu et al., 2018). Functionally, inhibition of TERT with a specific pharmacological inhibitor

179 BIBR1532 (Lavanya et al., 2018) impaired regenerative axon growth of sensory neurons in a

180 dose dependent manner (Supplementary Figure S3). Importantly, knocking down TERT with a

181 specific siRNA also effectively blocked sensory axon regeneration in vitro (Figure 2E-G),

182 confirming the pharmacological results. To examine if TERT functioned to regulate sensory axon

183 regeneration in vivo, by using in vivo electroporation technique, we transfected L4-L5 DRG bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

184 neurons with the specific TERT siRNA, and performed sciatic nerve crush injury two days later.

185 Three days after the nerve crush, sensory axon regeneration was analyzed. We found that

186 knocking down TERT significantly blocked sensory axon regeneration in vivo (Figure 2H-J),

187 indicating that TERT was required for sensory axon regeneration. These results revealed an

188 unexpected role of telomere regulatory complex in regulating axon regeneration.

189 To determine if TERT acted downstream of c-Myc in sensory neurons to regulate axon

190 regeneration, we examined the expression level of TERT after knocking down c-Myc with

191 siRNA. The results demonstrated that downregulation of c-Myc resulted in significantly reduced

192 level of TERT in cultured sensory neurons (Figure 3A, B). Conversely, overexpression of c-Myc

193 in sensory neurons markedly enhanced TERT expression (Figure 3C, D). Functionally, when we

194 treated sensory neurons with a specific pharmacological TERT activator CAG (Ip et al., 2014),

195 we found that activation of TERT with CAG alone had no promoting effect on sensory axon

196 growth cultured for 3 days, likely due to upregulation of endogenous TERT. However, CAG (25

197 µM) treatment significantly rescued regenerative axon growth inhibited by c-Myc knockdown

198 (Figure 3E, F), suggesting that TERT act downstream of c-Myc to regulate axon regeneration.

199

200 p53 is necessary and sufficient for supporting sensory axon regeneration in vitro and in

201 vivo

202 To investigate if p53 act in the c-Myc-TERT pathway to regulate axon regeneration, we

203 examined the level of p53 in sensory neurons upon peripheral nerve injury. The results showed

204 that p53 was up regulated in sensory neurons following sciatic nerve injury (Figure 4A, B). bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

205 Functionally, the specific membrane permeable p53 inhibitor, PFTα, markedly inhibited sensory

206 axon growth in a dose dependent manner (Figure 4C, D), indicating that p53 is necessary for

207 regenerative sensory axon growth. These results were consistent with previous studies (Di

208 Giovanni and Rathore, 2012; Floriddia et al., 2012; Gaub et al., 2011; Gaub et al., 2010; Joshi et

209 al., 2015; Tedeschi et al., 2009) that p53 was required for mammalian axon regeneration.

210 Conversely, we showed that overexpression of p53 in cultured sensory neurons significantly

211 promoted axon growth (Figure 4E-G), indicating that p53 alone is sufficient to promote sensory

212 axon regeneration. Together, these results demonstrated that p53 was both necessary and

213 sufficient to support sensory axon regeneration in vitro.

214 Previous studies in non-neuronal cells have shown that p53 activity is controlled by

215 c-Myc (Reisman et al., 1993) or TERT (Zhou et al., 2017). Therefore, we investigated if p53

216 acted downstream of c-Myc and TERT in sensory neurons to control axon regeneration. Indeed,

217 the specific TERT inhibitor BIBR1532, which has been shown to reduce the protein level of

218 TERT (Lavanya et al., 2018), also led to reduced p53 level in sensory neurons (Supplementary

219 Figure 4A, B), indicating that p53 is regulated by the cMyc-TERT signaling in sensory neurons.

220 Functionally, the p53 activator Tenovin-6 was able to enhance sensory axon growth inhibited by

221 knocking down c-Myc or TERT via siRNA (Supplementary Figure 4C, D), suggesting that p53

222 act downstream of c-Myc and TERT to regulate axon regeneration. Furthermore, overexpression

223 of p53 significantly enhanced the decreased sensory axon growth caused by c-Myc knockdown

224 in cultured sensory neurons (Figure 4E-G), confirming the pharmacological results.

225 Lastly, we examined the roles of p53 in regulation of sensory axon regeneration in vivo via bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

226 the electroporation approach. The results showed that overexpression of p53 alone was able to

227 enhance sensory axon regeneration in vivo (Figure 5A-C). Moreover, overexpression of p53

228 significantly restored sensory axon regeneration impaired by c-Myc knockdown. Taken together,

229 our results demonstrated clearly that p53 functioned to regulate sensory axon regeneration in

230 vitro and in vivo, likely downstream of c-Myc and TERT signals.

231

232 P53 regulates CNS axon growth and enhances optic nerve regeneration

233 Our results above showed clearly that p53 was able to enhance mature sensory axon

234 regeneration in vitro and in vivo. A previous study has shown that p53 is highly expressed in

235 mammalian hippocampal neurons (Qin et al., 2009). Thus, we thought that p53 overexpression

236 might enhance hippocampal axon growth as well. Therefore, we explored the functional roles of

237 p53 in regulating axon growth of embryonic hippocampal or cortical neurons. Embryonic day 18

238 (E18) hippocampal neurons or E15 cortical neurons were treated with either the p53 inhibitor

239 PFTa or activator Tenovin-6. The results showed that inhibiting p53 with PFTα treatment

240 markedly reduced axonal growth of cortical neurons (Figure 6A, E), whereas activating p53 with

241 Tenovin-6 significantly enhanced cortical axon growth (Figure 6B, F). Similar results were also

242 observed with hippocampal axon growth assay (Figure 6C, D, G, H). These results

243 demonstrated clearly that p53 was also an important regulator of axon growth from embryonic

244 CNS neurons.

245 Next, we investigated if p53 overexpression could enhance optic nerve regeneration in

246 vivo. Adeno-associated virus 2 (AAV2) viral vector encoding p53 was injected into the vitreous bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

247 body between the lens and the retina of the eye in a mouse model. The AAV2-GFP viral vectors

248 were used as the control. After two weeks of AAV2-p53 infection, we found that the expression

249 levels of p53 were significantly enhanced in mouse retina tissue (Supplementary Figure S5A)

250 and RGCs (Supplementary Figure S5B, C). To evaluate the functional role of p53 in optic nerve

251 regeneration, optic nerve crush injury was performed two weeks after AAV2-p53 injection. After

252 twelve more days, the fluorescently Alexa-594 conjugated cholera toxin β-subunit (CTB) was

253 injected into the vitreous body to label regenerating axons. The results showed that the number

254 of axons crossing the lesion site in the AAV2-GFP mice two weeks after the optic nerve crush

255 were minimum (Figure 7A, B). In contrast, p53 overexpression induced strong CTB fluorescent

256 labeled regenerating axons (Figure 7A, B). Because p53 is a well-known regulator of cell

257 apoptosis, we assessed the cell viability of RGCs in whole-mount retina immunostained with the

258 neuron-specific tubulin marker, Tuj1. By quantifying the number of cells positive for Tuj1, we

259 found compared to the AAV2-GFP control group, the survival rates of RGCs were no different in

260 the p53 overexpression experimental group, two weeks after the optic nerve crush (Figure 7C, D).

261 These results demonstrated that overexpression of p53 alone was sufficient to promote optic

262 nerve regeneration without affecting RGCs survival.

263

264

265 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

266 Discussion

267 Identification of novel genes and pathways regulating axon regeneration would not only

268 provide potential new target molecules for treating CNS , but also help us better

269 understand the molecular mechanisms by which mammalian axon regeneration is regulated. Here

270 we demonstrated clearly that c-Myc, previously identified to enhance optic nerve regeneration,

271 was both necessary and sufficient for sensory axon regeneration in vitro and in vivo. Such

272 finding provided a great opportunity and model system to explore novel signaling pathways

273 related to the c-Myc signaling that can be manipulated to regulate mammalian axon regeneration.

274 In this study, we revealed an unexpected role of TERT, a key component of the telomere

275 regulatory complex, in regulating mammalian axon regeneration downstream of c-Myc.

276 Moreover, we provided evidence that p53 likely acted downstream of c-Myc and TERT to

277 regulate sensory axon regeneration. More importantly, we showed that overexpression of p53

278 alone was sufficient to promote sensory axon regeneration in vivo and optic nerve regeneration.

279 Besides its well-known function to maintain telomere length during cell division, TERT

280 has been shown to be involved in many other biological functions independent of its telomere

281 regulatory function, such as the regulation of cell metabolism, growth factor secretion,

282 mitochondria function, energy balance, and apoptosis, among others (Bagheri et al., 2006; Chung

283 et al., 2005; Lin et al., 2007; Liu et al., 2012; Massard et al., 2006; Passos et al., 2007; Smith et

284 al., 2003). In post-mitotic neurons, previous studies (Liu et al., 2018; Miwa and Saretzki, 2017;

285 Spilsbury et al., 2015) have shown that TERT is mainly localized in the cytoplasm and function

286 to protect neurons from oxidative or degenerative stresses in aged neurons. It is likely that TERT bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

287 acts in neurons to regulate mitochondria function by reducing reactive oxidative species (ROS)

288 (Liu et al., 2018; Miwa and Saretzki, 2017; Spilsbury et al., 2015). In this study, we observed

289 that TERT was also mainly localized in the cytoplasm of sensory neurons, similarly to that

290 observed in previous studies, and the expression of TERT was significantly increased in sensory

291 neurons after peripheral injury. Functionally, TERT was required for the sensory axon

292 regeneration in vitro and in vivo. TERT has been shown to be regulated by multiple positive and

293 negative regulators, such as c-Myc, Sp1, p53, Wilms tumor 1 (WT1), E2F, (Cukusic et al., 2008),

294 and the nuclear factor kappa B (NF-kB) (Lingner et al., 1997; Nakamura et al., 1997; Yin et al.,

295 2000). Here we showed that in adult sensory neurons down regulation of c-Myc significantly

296 reduced TERT expression, whereas the upregulation of c-Myc significantly increased TERT

297 expression. Functionally, pharmacological activation of TERT significantly rescued the axon

298 regeneration impaired by c-Myc knockdown in adult sensory neurons. Thus, our data strongly

299 suggest that TERT act as a downstream target of c-Myc during sensory axon regeneration.

300 The p53 protein is well-known for its tumor suppressor role, functioning primarily to

301 promote cell cycle arrest, DNA repair, and apoptosis (Levine and Oren, 2009). In post-mitotic

302 neurons, p53 has been shown to be necessary for proper axon guidance, growth, and regeneration

303 (Arakawa, 2005;Di Giovanni and Rathore, 2012; Floriddia et al., 2012; Gaub et al., 2011; Gaub

304 et al., 2010; Joshi et al., 2015; Tedeschi et al., 2009). However, if p53 is sufficient to promote

305 axon growth and regeneration is unclear. In this study, we showed that overexpression of p53 in

306 adult sensory neurons was sufficient to promote sensory axon regeneration in vitro and in vivo.

307 In addition, the results showed that the protein level p53 was regulated by TERT, suggesting that bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

308 p53 act downstream of c-Myc and TERT to regulate sensory axon regeneration. Indeed,

309 pharmacological activation of p53 was able to significantly restore sensory axon regeneration in

310 vitro impaired by either c-Myc or TERT knockdown. Moreover, overexpression of p53 could

311 significantly restore sensory axon regeneration impaired by c-Myc knockdown both in vitro and

312 in vivo, providing strong evidence that p53 acted downstream of c-Myc and TERT to regulate

313 axon regeneration. Besides peripheral sensory neurons, we also showed that p53 was an

314 important regulator of axon growth of developing cortical or hippocampal neurons, indicating its

315 similar function in controlling CNS axon growth. Most importantly, we showed clearly that

316 overexpression of p53 in RGCs was sufficient to promote optic nerve regeneration.

317 How c-Myc, TERT and p53 regulate axon regeneration remains unclear. One potential

318 mechanism is through regulation of cell metabolism. In support, c-Myc is well known to enhance

319 anabolic metabolism of glycolysis via reprogramming mitochondria (Ward and Thompson, 2012).

320 A latest study (Kim et al., 2019) also showed that wild type p53 can act in the mitochondria to

321 suppress oxidative phosphorylation and thus increase glycolysis, similar to that of c-Myc. As

322 aforementioned, TERT has been shown to be localized in mitochondria of neurons and regulates

323 mitochondria function. It would be interesting in the future to explore if other factors or

324 pathways regulating neuronal metabolism could also promote axon regeneration. bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

325 Materials and methods

326

327 Animals and Surgical Procedures

328 Institute of Cancer Research (ICR) adult female mice, 8 to 12-week-old, (weighing 25 grams [g]

329 to 35 g) were used. All animals were handled according to the guidelines of the Institutional

330 Animal Care and Use Committee of the Soochow University, Suzhou, Jiangsu, China. For

331 surgical procedures, mice were anesthetized with a mixture of ketamine (100 mg/kg) and

332 xylazine (10 mg/kg) via intra-peritoneal (IP) injection. Eye ointment containing atropine sulfate

333 was applied to protect the cornea during surgery, and animals received antibiotics for 24 hours as

334 a post-operative analgesic.

335

336 Reagents and Antibodies

337 10058-F4, BIBR1532, PFTα, and Tenovin-6 were from Selleck Chemicals (Houston, TX, USA),

338 and CAG was from Sigma-Aldrich (Saint Louis, MO, USA). Antibodies against the

339 neuron-specific class III β-tubulin mouse mAb (Tuj1, 1:1000) were from Sigma-Aldrich. The

340 antibody against c-Myc rabbit mAb (1:1000) was from Genetex (Irvine, CA, USA). Antibodies

341 against TERT rabbit mAb (1:1000) and p53 mouse mAb (1:1000) were from Abcam (Cambridge,

342 UK). The siRNA against TERT and c-Myc were from Gene Pharma (Shanghai GenePharma Co.;

343 Shanghai, China). pEX4-c-Myc and pEX3-p53 plasmids were from Gene Pharma. The

344 AAV2-p53 viral vector was purchased from Cyagen Biosciences (Santa Clara, CA, USA). All

345 fluorescence secondary antibodies were purchased from Molecular Probes, Inc. (Eugene, OR, bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

346 USA).

347

348 Cell Cultures and Axon Length Quantification

349 Dissection and culture of adult DRG sensory neurons were performed as described in our

350 previous protocol (Saijilafu et al., 2013). Briefly, DRGs were dissected out from 8-week old

351 adult mice and incubated with collagenase A (1 mg/ml; Roche, Basel, Switzerland) for 90

352 minutes and then with 1X TrypLE (Life Technologies; Carlsbad, CA, USA) for 20 minutes at

353 37°C. Then, DRGs were dissociated in culture medium, which was minimum essential media

354 (MEM) supplemented with 5% fetal bovine serum (FBS), antimitotic agents (20 µM

355 5-fluoro-2-deoxyuridine, 20 µM uridine) and penicillin / streptomycin. The isolated neurons

356 were then centrifuged down and plated onto glass coverslips, which were coated with

357 poly-D-lysine (100 µg/ml, Sigma Aldrich) and laminin (10 µg/ml, Sigma Aldrich). For DRG

358 assays on CSPGs or myelin, culture coverslips were coated with 70 µL CSPGs (5 µg/ml) or

359 purified CNS myelin, which contain a number of axon growth inhibitors. Dissociated DRG

360 neurons were plated onto plastic coverslips in 24-well plates after washing and grown in culture

361 medium (MEM in 5% fetal bovine serum, 20 µM 5-fluoro-2-deoxyuridine, 20 µM uridine, 100

362 units /ml penicillin and 100 ng/ml streptomycin, Sigma Aldrich) for 3 days at 37°C in a

363 CO2-humidified incubator. For DRG neurons obtained from mice used in axotomy procedures,

364 the culture time for axon growth was 20 hours. Cortical and hippocampal neurons were isolated

365 from embryonic day 15 or 18 (E15, E18) ICR mice embryos and treated with TrypLE for 5

366 minutes at 37°C. The supernatants were cultured in neurobasal medium supplemented with bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

367 penicillin / streptomycin, GlutaMAX supplements, and B27 supplements for 3 days.

368 All images were analyzed with the AxioVision 4.7 software (Carl Zeiss MicroImaging, Inc.; Jena,

369 Germany). In each experiment, at least 100 neurons per condition were selected randomly, and

370 three independent experiments were performed to quantify the average axon length. The longest

371 axon of each neuron was traced manually using the “measure / curve” application.

372

373 RNA Interference

374 In order to transfect siRNA and / or DNA plasmids into DRG neurons, the dissociated neurons

375 were centrifuged down to remove the supernatant and resuspended in 80 to 100 µl of Amaxa

376 electroporation buffer for mouse neurons (Lonza Cologne GmbH; Cologne, Germany) with

377 mixtures of siRNA (0.2 nmol per transfection) or plasmids and / or the EGFP plasmid (5 µg per

378 transfection). The suspended cells were then transferred to a 2.0-mm cuvette, followed by

379 electroporation with the Amaxa Nucleofector apparatus. After electroporation, cells were

380 immediately mixed with the desired volume of pre-warmed culture medium and plated onto

381 culture dishes coated with a mixture of poly-D-lysine (100 µg/ml) and laminin (10 µg/ml). After

382 the neurons were fully attached to the coverslips (4 hours), the culture medium was changed to

383 remove the remnant electroporation buffer. After 3 days of culture, the cells were fixed with 4%

384 paraformaldehyde (PFA) and then subjected to immunocytochemistry analysis.

385

386 Myelin Extract

387 Myelin fractions were isolated as described by Norton and colleagues (Norton and Poduslo, bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

388 1973). Briefly, three carefully cleaned adult rat brains were homogenized with a Dounce

389 homogenizer in approximately 20 vol. (w/v) of 0.32 M sucrose. We used three brains per 100 ml.

390 One-third of the homogenate was layered over 25 ml of 0.85 M sucrose in each of three tubes,

391 and the tubes were centrifuged at 75,000 g (25,000 rev/min) for 30 minutes. Layers of crude

392 myelin which formed at the interface of the two sucrose solutions were collected with a Pasteur

393 pipette, everything else was discarded. The combined myelin layers were then suspended in

394 water by homogenization and brought to a final volume of 180 ml. This suspension was

395 centrifuged at 75,000 g (25,000 rev/min) for 15 minutes. The supernatant fluid was subsequently

396 discarded. The three crude myelin pellets were again dispersed in a total volume of 180 ml of

397 water and centrifuged at 12,000 g (10,000 rev/min) for 10 minutes. The cloudy supernatant fluid

398 was discarded. The loosely packed pellets were again dispersed in water and centrifuged again.

399 The myelin pellets were then combined and suspended in 100 ml of 0.32 M sucrose. This

400 suspension was layered over 0.85 M sucrose in three tubes and centrifuged exactly as previously

401 described Norton and Poduslo, 1973. The purified myelin was finally removed from the interface

402 with a Pasteur pipette.

403

404 Immunohistochemistry

405 Mice were deeply anesthetized and perfused with phosphate buffered saline (PBS) and ice-cold 4%

406 PFA. The tissues of lumbar L4-L5 DRG neurons were dissect out and post fixed with 4% PFA at

407 4°C overnight, followed by dehydration in 10%, 20%, and 30% sucrose solution (w/v) overnight,

408 respectively. DRGs were then cryosectioned into slices, 12 µm in thickness. For immunostaining, bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

409 the sections of DRG neurons were then washed three times in PBS containing 0.5% TritonX-100,

410 followed by blocking in PBS containing 5% FBS and 0.3% Triton X-100 for 1 hour at room

411 temperature. Sections were incubated with the indicated primary antibodies overnight at 4°C,

412 washed in PBS containing 0.5% TritonX-100, and then incubated with the corresponding

413 secondary antibodies for 1 hour at room temperature. The sections were mounted with mounting

414 medium (Vector Labs, H-1400) after washing in PBS containing 0.3% Triton X-100.

415

416 RNA Isolation and qRT-PCR

417 The total RNA was extracted from the DRG neurons using the TRIzol reagent (Invitrogen;

418 Carlsbad, CA, USA). The RNA was reverse transcribed into cDNA using Maxima H Minus

419 Reverse Transcriptase according to the manufacturer's instructions (Thermo Scientific, Waltham,

420 MA, USA). Quantitative PCR (qPCR) was carried out using SYBR-Green Real-Time PCR

421 Master Mix (Toyobo Co.; Osaka, Japan). Standard curves (cycle threshold values versus

422 template concentration) were prepared for each target gene and the endogenous reference (18S)

423 in each sample. The primer sequences were as follows; c-Myc: forward,

424 5’-AT CACAG CCCT CA CT CAC -3’ and reverse, 5’-ACAGATTCC ACAAGGTGC -3’; TERT:

425 forward, 5’-TGGTGGAGGTTGCCAA-3’ and reverse, 5’-CCACTGCATACTGGCGGATAC-3’;

426 p53: forward, 5’-CGACGACATTCGGAT AAG-3’ and reverse,

427 5’-TTGCCAGATGAGGGACTA-3’. The amplification step involved an initial denaturation at

428 95°C for 10 seconds (s) followed by 40 cycles of denaturation at 95°C for 15 s and annealing at

429 55°C for 30 s and extension at 72°C for 30 s. Reactions were performed in triplicate and the bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

430 mRNA expression was normalized against the endogenous reference (18S). Data were quantified

431 by using CFX96TM real-time PCR detection system (Bio-Rad; Hercules, CA, USA).

432

433 Western Blot Analysis

434 Tissues or dissociated neurons were collected and lysed using a RIPA buffer for 30 minutes on

435 ice. The protein concentration was measured by using the BCA Kit used according to the

436 manufacturer’s instructions (Beyotime, China). Then, equal amount of extracted proteins were

437 separated with 10% SDS-PAGE, followed by transferring to the polyvinylidene difluoride

438 (PVDF) membrane, Immobilon-P (Millipore; Burlington, MA, USA). Then, PVDF membranes

439 were blocked with 5% non-fat milk in Tris-Buffered Saline (TBS) buffer for 1 hour at room

440 temperature, and reacted with primary antibody overnight at 4°C. Then, the membrane was

441 incubated with horseradish peroxidase-conjugated secondary antibody (dilution of 1:1000) at

442 room temperature for 1 hour. Lastly, the membrane was developed using an ECL Prime Western

443 Blotting Detection Reagent (GE Healthcare; Chicago, IL, USA). The density of protein bands

444 from three independent experiments was quantified using the Image J software (National

445 Institutes of Health [NIH]; Bethesda, MD, USA).

446

447 in vivo Electroporation of Adult Mouse DRGs

448 The in vivo electroporation of adult mouse DRGs was performed as described previously

449 (Saijilafu et al., 2012). Briefly, left L4-L5 DRGs of 10-week old adult ICR mice (weighing from

450 30g to 35g) were surgically exposed under anesthesia. A solution containing siRNAs or c-Myc bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

451 plasmid and/or EGFP plasmid (1µl) was slowly injected into the DRG using a capillary pipette

452 powered by the Picospritzer III (Parker Inc.; Cleveland, OH, USA; pressure: 30 psi; duration: 8

453 ms). The electroporation was performed immediately using a tweezer-like electrode (Ø1.2 mm)

454 and an ECM830 Electro Square Porator BTX (five 15 ms pulses at 35 V with 950 ms interval).

455 The mice were then allowed to recover after surgery after the incision sites were closed. The

456 ipsilateral left sciatic nerve was then crushed with fine forceps two days later, and the crush site

457 was marked with an 11-0 nylon epineural suture. Three days after the nerve crush, the mice were

458 terminally anesthetized and transcardially perfused with ice-cold 4% PFA. The entire sciatic

459 nerve was dissected out and further fixed in 4% PFA overnight at 4°C. The epineural suture was

460 confirmed, and the lengths of all EGFP-labeled regenerating axons were measured from the

461 crush site to the distal axon tips. The number of the independent experiments performed and the

462 number of neurons analyzed in each condition are depicted in the Figure or in the legends.

463

464 Sciatic Nerve Axotomy

465 The sciatic nerve axotomy model was performed as described above. Under anesthesia with

466 ketamine / xylazine, and a 1 cm-long skin incision was made aseptically on the legs. The

467 overlaying muscles were then dissected free from the L4-L5 spinous processes on the two sides.

468 The sciatic nerve was crushed at the sciatic notch with ophthalmic scissors. After a specific

469 period of time, the L4-L5 DRGs were isolated for cell culture, tissue section, qPCR, or western

470 blotting. Mice who received sham surgery were used as “no-injury” controls.

471 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

472 Intravitreal Injection and Optic Nerve Injury

473 After mice were anaesthetized, a glass micropipette was inserted into the vitreous body of

474 4-week old C57 mice avoiding damaging the lens, and a 1 µl of AAV2-GFP or AAV2-p53 (Titer

475 1x1012) viral vector suspension was carefully injected with a Picospritzer III (Parker Inc.

476 pressure: 9 psi; duration: 5 ms). Two weeks after the virus injection, the optic nerve was exposed

477 intraorbitally by blunt dissection, and a crush injury was performed approximately 1 mm behind

478 the optic disc with fine forceps for 2 to 3 seconds.

479

480 RGC Axon Anterograde Labeling and Counting of Regenerating Axons

481 In order to label RGC axons, twelve days after the optic nerve crush injury, 2 µl of fluorescence

482 Alexa-594 conjugated cholera toxin β subunit (CTB) (2 µg/µl, Invitrogen; Carlsbad, CA, USA)

483 was micro-injected into the vitreous body using a Picospritzer III (Parker Inc.; pressure: 9 psi;

484 duration: 5 ms). Two days later, animals were perfused with 4% PFA in 0.01 M phosphate

485 buffered saline via the left ventricle. The entire optic nerve was then harvested. Regenerating

486 RGC axons were calculated as previously described (Kurimoto et al., 2010). 12-µm thick

487 longitudinal sections from the optic were cut using a cryostat. The numbers of

488 CTB-positive axons were counted in each of the five sections (every four sections) at a different

489 distance from the crush site. The cross-sectional width of the nerve was measured at the counting

490 point and used to calculate the number of axons per mm of nerve width. Then, the average of the

491 number of axons per millimeter on all parts was measured. The total number of axons extending

492 from the nerve of radius “r” was estimated by summing all the parts with thickness t (12 µm): bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

493 Σad = πr2 x [average axons/mm]/t.

494

495 Whole Mount Retina Staining

496 After perfusion with PBS and ice cold 4% PFA, whole retinas were dissected out and blocked in

497 PBS containing 10% FBS and 0.3% Triton X-100 for 1 hour. Then, incubated with primary

498 antibody Tuj1 (1:500) overnight at 4°C. After washing the retinas with blocking buffer three

499 times for 30 minutes, the tissues were incubated with Alexa Fluor594-conjugated secondary

500 antibodies for 1 hour at room temperature. Whole-mount retina sections were immunostained

501 with a Tuj1 antibody to label RGC neurons. Using Image J software in eight fields per case

502 distributed in four quadrants of the eye, cell survival was reported as the number of Tuj1 positive

503 cells per mm2 averaged over the eight fields sampled in each retina and then averaged across all

504 cases within each experimental group. The quantitation obtained from the regeneration and cell

505 survival experiments were based on 6 mice per condition.

506

507 Statistics

508 All statistical analyses were expressed as the mean ± standard error mean (SEM). To determine

509 significance between two groups, comparisons between means were made with the Student’s

510 t-test. Multiple group comparisons were performed with one-way ANOVA analysis of variance

511 using the statistical software Prime 5 followed by Bonferroni’s post-test as post hoc test for

512 comparing multiple groups. Statistical significance was considered when the p-value was less

513 than 0.05 for three independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001). bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

514 Author Contributions

515 JJ Ma, RJ Xu, X Ju, WH Wang, ZP Luo, CM Liu, L Yang, B Li, JQ Chen, B Meng, Hl Yang, FQ

516 Zhou, and Saijilafu designed the experiment. JJ Ma, RJ Xu, X Ju and WH Wang performed the

517 experiments and analyzed the data. JJ Ma, FQ Zhou, and Saijilafu co-wrote the paper with all

518 authors’ input.

519

520 Acknowledgments

521 Dr. Saijilafu was supported by the grant from the National Natural Science Foundation of China

522 (Nos. 81772353 and 81571189), the National Key Research and Development Program (Nos.

523 2016YFC 1100203), The Priority Academic Program Development of Jiangsu Higher Education

524 Institutions, and Innovation and Entrepreneurship Program of Jiangsu Province. Dr. Feng-Quan

525 Zhou was supported by NIH (R01NS064288, R01NS085176, R01GM111514, R01EY027347),

526 the Craig H. Neilsen Foundation, and the BrightFocus Foundation.

527

528 Declaration of Interests

529 The authors declare no competing interests.

530

531

532 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

533 Figure legends

534

535 Figure 1. c-Myc is both necessary and sufficient for sensory axon regeneration in vivo

536 (A) Top: time line of the experiments. Bottom: representative images showing that knocking

537 down c-Myc in sensory neurons in vivo significantly impaired sensory axon regeneration. The

538 red line indicates the nerve crush sites, and the red arrows indicate the distal ends of regenerating

539 axons. The images in the white dotted line boxes in the left panel were enlarged and presented in

540 the right panel. Scale bar: 500 µm in the left panel and 250 µm in the right panel.

541 (B) Quantification of (A) showing that knocking down c-Myc significantly impaired sensory

542 axon regeneration in vivo (n=6 mice for each condition, **p < 0.01).

543 (C) Cumulative distribution curves showing that knocking down c-Myc in sensory neurons

544 significantly inhibited sensory axon regeneration in vivo.

545 (D) Top: time line of the experiments. Bottom: representative images showing that c-Myc

546 overexpression promoted sensory axon regeneration in vivo 3 days after sciatic nerve crush

547 injury. The red line indicates the nerve crush sites and the red arrows indicate distal ends of

548 selected regenerating axons. The images in the white dotted line boxes in the left panel were

549 enlarged and presented in the right panel. Scale bar: 500 µm in the left panel and 250 µm in the

550 right panel.

551 (E) Quantification of (D) showing that overexpression of c-Myc significantly promoted sensory

552 axon regeneration in vivo (n=6 mice for each condition, **p < 0.01).

553 (F) Cumulative distribution curves showing that overexpression of c-Myc in sensory neurons bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

554 significantly promoted sensory axon regeneration in vivo.

555 Please note that the control group in A-C is the same as that in D-E.

556

557 Figure 2. TERT is required for sensory axon regeneration in vitro and in vivo

558 (A) Representative immunostaining images of sectioned DRGs showing significantly elevated

559 TERT levels in sensory neurons 3 days after sciatic nerve axotomy. Note that most TERT

560 staining in adult sensory neurons was localized in the cytoplasm. Scale bar: 100 µm.

561 (B) Quantification of fluorescence intensity of TERT staining from three independent

562 experiments; ***p < 0.001.

563 (C) Representative western blot images showing increased protein levels of TERT in DRG

564 tissues after sciatic nerve axotomy.

565 (D) Quantification of (C) from 3 independent experiments; ***p < 0.001.

566 (E) Representative western blot images showing successful knockdown of TERT protein levels

567 in cultured DRG neurons with the siRNA against TERT (siTERT).

568 (F) Quantification of average axon lengths of control neurons or neurons expressing siTERT in

569 cultured sensory neurons from three independent experiments; **p < 0.01.

570 (G) Representative images showing that knocking down of TERT in cultured sensory neurons

571 with a specific siRNA markedly decreased axon growth after 3 days. Scale bar: 50 µm.

572 (H) Top: time line of the experiment. Bottom: representative images of sensory axon

573 regeneration in vivo following TERT knockdown 3 days after sciatic nerve crush injury. The red

574 line indicates the crush sites, and the red arrows indicate the distal ends of selected regenerating bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

575 axons. The images in the white dotted line boxes in the left panel were enlarged and presented in

576 the right panel. Scale bar: 500 µm in the left panel and 250 µm in the right panel.

577 (I) Quantification of (H) showing that knocking down TERT in sensory neurons significantly

578 inhibited axon regeneration in vivo (n=6 mice for each condition, ***p < 0.001).

579 (J) Cumulative distribution curves showing that knocking down TERT in sensory neurons

580 significantly reduced sensory axon regeneration in vivo.

581 Please note the control group in H-J is the same as that shown in Figure 1.

582

583 Figure 3. TERT acts downstream of c-Myc in sensory neuron to control axon regeneration

584 (A) Representative western blot images showing that knocking down c-Myc led to reduced

585 TERT protein levels in sensory neurons.

586 (B) Quantification of (A) showing significantly decreased c-Myc and TERT protein levels in

587 adult sensory neurons after knocking down c-Myc from three independent experiments; ***p <

588 0.001.

589 (C) Representative western blot images showing that overexpression of c-Myc led to increased

590 TERT protein levels in sensory neurons.

591 (D) Quantification of (C) showing significantly increased c-Myc and TERT protein levels of in

592 adult sensory neurons after c-Myc overexpression from three independent experiments; **p <

593 0.01, ***p < 0.001.

594 (E) Representative images showing that treatment of cultured sensory neurons with the specific

595 TERT activator, CAG (25 µM), for 3 days, successfully rescued sensory axon growth inhibited bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

596 by c-Myc knockdown. Scale bar: 50 µm.

597 (F) Quantification of average axon lengths of (E) in different conditions from three independent

598 experiments; **p < 0.01, ***p < 0.001.

599

600 Figure 4. p53 is necessary and sufficient for supporting sensory axon regeneration in vitro

601 (A) Representative western blot images showing markedly increased p53 protein levels in DRG

602 tissues after sciatic nerve axotomy.

603 (B) Quantification of the western blot images in (A) from three independent experiments; ***p <

604 0.001.

605 (C) Quantification of the average axon lengths in (D) from three independent experiments; ***p

606 < 0.001.

607 (D) Representative images showing that inhibition of p53 with PFTα in cultured sensory neurons

608 for 3 days dramatically blocked regenerative axon growth. Scale bar: 200 µm.

609 (E) Representative western blot images showing increased level of p53 after p53 overexpression

610 (OE) in adult sensory neurons.

611 (F) Quantification of the average axon lengths in (G) from three independent experiments; *p <

612 0.05, **p < 0.01.

613 (G) Representative images showing that overexpression of p53 were sufficient to promote

614 sensory axon regeneration by itself in vitro and restored axon regeneration inhibited by c-Myc

615 knockdown. Scale bar: 50 µm.

616 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

617 Figure 5. Overexpression of p53 in sensory neurons is sufficient to promote sensory axon

618 regeneration and restore sensory axon regeneration impaired by c-Myc knockdown in vivo

619 (A) Top: time line of the experiment. Bottom: representative images of sensory axon

620 regeneration in vivo after c-Myc knockdown, overexpression of p53, as well as combined c-Myc

621 knockdown and p53 overexpression. The red line indicates the nerve injury sites and the red

622 arrows indicate the distal ends of selected regenerating axons. The images in the white dotted

623 line boxes in the left panel were enlarged and presented in the right panel. Scale bar: 500 µm in

624 the left panel and 250 µm in the right panel.

625 (B) Quantification of the results in (A) showing that overexpression of p53 alone can promote

626 sensory axon regeneration in vivo and p53 overexpression significantly restores sensory axon

627 regeneration impaired by c-Myc knockdown (n=6 mice for each condition, **p < 0.01, ***p <

628 0.001).

629 (C) Cumulative distribution curves showing the same results as those in (B).

630 Please note the control and sic-Myc groups in this figure are the same as those shown in Figure

631 1.

632

633 Figure 6. p53 regulates axon growth of developing cortical and hippocampal neurons

634 (A) Representative images showing that inhibition of p53 activity with the pharmacological

635 inhibitor, PFTα, blocked cortical neuron axon growth. Scale bar: 200 µm.

636 (B) Representative images showing that activation of p53 activity with the small molecule,

637 Tenovin-6, promoted cortical neuronal axon growth. Scale bar: 200 µm. bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

638 (C) Representative images showing that inhibition of p53 activity with the pharmacological

639 inhibitor, PFTα, blocked hippocampal neuron axon growth. Scale bar: 200 µm.

640 (D) Representative images showing that activation of p53 activity with the small molecule

641 Tenovin-6, promoted hippocampal neuron axon growth. Scale bar, 200 µm.

642 (E) Quantification of the average axon lengths of cortical neurons shown in (A) from three

643 independent experiments; ***p < 0.001.

644 (F) Quantification of the average axon lengths of cortical neurons shown in (B) from three

645 independent experiments; ***p < 0.001.

646 (G) Quantification of the average axon lengths of hippocampal (Hippo.) neurons shown in (C)

647 from three independent experiments; *P<0.05, ***p < 0.001.

648 (H) Quantification of the average axon lengths of hippocampal (Hippo.) neurons shown in (D)

649 from three independent experiments; ***p < 0.001.

650

651 Figure 7. Overexpression of p53 enhances optic nerve regeneration

652 (A) Top: timeline of the experiment. Bottom: representative images showing that p53

653 overexpression in RGCs induced drastic optic nerve regeneration 2 weeks after the optic nerve

654 crush. The right 8 columns show enlarged images of nerves at places marked by dashed white

655 boxes on the left. The red line indicates the crush sites. Scale bar: 50 µm for left, 50 µm for right.

656 (B) Quantification of regenerating optic nerve axons at different distances from the nerve crush

657 site (n = 6 mice for each condition, ***p<0.001).

658 (C) Representative images of whole mount retina stained with the neuron specific bIII tubulin bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

659 antibody Tuj-1. Scale bar: 100 µm.

660 (D) Quantification of Tuj-1 positive cells in (C) showing that overexpression of p53 did not

661 affect RGC survival 2 weeks after optic nerve crush.

662

663 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

664 Supplementary figure legends

665 Supplementary Figure S1. C-Myc is up regulated in adult sensory neurons upon peripheral

666 nerve injury and functionally required for sensory axon regeneration in vitro

667 (A) Representative immunostaining of DRG sections showing nucleus localization of c-Myc in

668 DRG neurons and up regulation of c-Myc levels after sciatic nerve axotomy. Scale bar: 50 µm.

669 (B) Quantification of nucleus fluorescence intensity of c-Myc (n = 3 independent experiments,

670 ***p < 0.001).

671 (C) Representative western blot images showing significantly increased levels of c-Myc in adult

672 DRG neurons in vivo 3 days after sciatic nerve axotomy.

673 (D) Quantification of western blot results from three independent experiments; *p < 0.05.

674 (E) qPCR results showing significantly increased mRNA levels of c-Myc in adult DRG neurons

675 in vivo 3 days after sciatic nerve axotomy, n = 3 independent experiments; *p < 0.05.

676 (F) Representative images showing that inhibition of c-Myc in cultured DRG neurons with the

677 pharmacological agent, 10058-F4, for 3 days dramatically reduced DRG axon growth. Scale bar:

678 200 µm.

679 (G) Quantification of average axon lengths of control neurons and neurons treated with the

680 c-Myc inhibitor from three independent experiments; **p < 0.01.

681 (H) Representative images showing that knocking down c-Myc with a specific siRNA in cultured

682 DRG neurons dramatically reduced axon growth after 3 days in culture. Scale bar: 50 µm.

683 (I) Quantification of average axon lengths of control neurons and neurons expressing sic-Myc

684 from three independent experiments; *p < 0.05. bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

685 Supplementary Figure S2. Overexpression of c-Myc in sensory neurons is sufficient to

686 promote axon growth over both supportive and inhibitory substrates

687 (A) Representative images showing that overexpression of c-Myc significantly enhanced

688 regenerative axon growth of sensory neurons cultured on laminin. Scale bar: 200 µm.

689 (B) Quantification of (A) from three independent experiments; **p < 0.01.

690 (C) Representative images showing that overexpression of c-Myc significantly enhanced

691 regenerative axon growth of sensory neurons cultured on the inhibitory substrates, chondroitin

692 sulfate proteoglycan (CSPGs). Scale bar: 200 µm.

693 (D) Quantification of (C) from three independent experiments; **p < 0.01.

694 (E) Representative images showing that overexpression of c-Myc significantly enhanced

695 regenerative axon growth of sensory neurons cultured on myelin inhibitory substrates. Scale bar:

696 200 µm.

697 (F) Quantification of (E) from three independent experiments; **p < 0.01.

698

699 Supplementary Figure S3. Inhibition of TERT with specific inhibitor BIBR1532 impairs

700 regenerative axon growth of sensory neurons in a dose dependent manner

701 (A) Representative images showing that inhibition of TERT activity with the pharmacological

702 inhibitor, BIBR1532, at different concentrations, blocked regenerative axon growth of sensory

703 neurons. Scale bar: 200 µm.

704 (B) Quantification of the average axon lengths of control neurons and neurons treated with 3

705 different concentration of BIBR1532 from three independent experiments; **p < 0.01, ***p < bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

706 0.001.

707

708 Supplementary Figure S4. p53 expression is increased in adult mouse sensory neurons

709 upon peripheral nerve injury and its activity is required for regenerative sensory axon

710 growth

711 (A) Representative western blot images showing that inhibition of TERT activity with its specific

712 inhibitor BIBR1532 led to reduced levels of TERT and p53 in sensory neurons.

713 (B) Quantification of the Western blot images in (A) from three independent experiments; ***p

714 < 0.001.

715 (C) Representative images showing that activation of p53 activity with Tenovin-6 (0.5 µM)

716 restored sensory axon regeneration defects induced by knocking down c-Myc or TERT. Scale bar:

717 50 µm.

718 (D) Quantification of the average axon lengths in (C) from three independent experiments; ***p

719 < 0.001.

720

721 Supplementary Figure S5. AAV2-p53 mediated overexpression of p53 in retinal ganglion

722 cells (RGCs)

723 (A) Representative western blot images showing increased expression of the p53 protein in

724 mouse retina tissues.

725 (B) Quantification of fluorescence intensity of p53 in RGCs from three independent experiments;

726 ***p < 0.001. bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

727 (C) Representative images of immunostaining of sectioned retina with neuronal marker Tuj-1,

728 p53, and nuclear DNA dye DAPI. Note the markedly increased p53 staining in RGCs 2 weeks

729 after vitreous injection of the adeno-associated virus vector, AAV2-p53. Scale bar: 50 µm.

730

731 bioRxiv preprint doi: https://doi.org/10.1101/547026; this version posted February 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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897 A in vivo electroporation Sciatic nerve crush Axon tracing Day 0 Day 2 Day 5 Ctrl

500μm Myc - sic

100 B ** C

3.0 (%) 80 60 2.0 40 sic-Myc 1.0 20 Ctrl

Axon length (mm) 0 0 0 1 2 3 4 5 6 Ctrl sic-Myc Axon length (mm) Cumulative distribution

D in vivo electroporation Sciatic nerve crush Axon tracing Day 0 Day 2 Day 5 Ctrl Myc - c

** 100 E 5.0 F (%) 80 4.0 60 3.0 40 2.0 Ctrl 20 c-Myc 1.0 Axon length (mm) 0 0 0 1 2 3 4 5 6 7 Ctrl c-Myc Cumulative distribution Axon length (mm)

Figure 1 A TERT Tuj1 merged B

Ctrl 4.0 ***

3.0

2.0

1.0 TERT TERT level

0.0 Ctrl

( fluorescence intensity , a.u.) intensity ( fluorescence Axotomy Axotomy

Axotomy C kDa D F 150 Ctrl 1d 3d 7d *** 130 200 TERT ** 150 100 β-actin 40 100 E siTERT 50

Ctrl 10 25 50 (nM) kDa Ctrl) of (% 50 130 ) Ctrl of (% length TERT

Relative protein level 0 Ctrl 1d 3d 7d 0 Axon β-actin Axotomy Ctrl 40 siTERT G I Ctrl siTERT 3.0 *** ( mm ) ( mm 2.0 length 1.0 Axon

0 in vivo electroporation Sciatic nerve crush Axon tracing Ctrl siTERT H J Day 0 Day 2 Day 5

500μm (%) Ctrl

siTERT Ctrl siTERT Cumulative distribution Axon length (mm)

Figure 2 A B C D ** 150 300 *** Ctrl sic-Myc Ctrl c-Myc kDa *** kDa c-Myc *** c-myc 40 100 40 200 130 130 TERT TERT 50 100

β-actin ) Ctrl of ( % β-actin 40 40 ) Ctrl of ( % Relative protein level 0 Relative protein level 0 Ctrl Ctrl -Myc -Myc c TERT c TERT E F 150 Ctrl sic-Myc sic-Myc+CAG CAG ** *** 100 length 50 (% of Ctrl ) Ctrl of (% Axon

0 sic-Myc - + + - CAG - - + +

Figure 3 A B *** C Axotomy 250 Ctrl 1d 3d 7d kDa 55 200 p53 150 *** 150 *** GAPDH 100 35 100 length

Ctrl 1d 3d 7d ) Ctrl of ( % 50 Axotomy 50 (% of Ctrl ) Ctrl of (% Axon

Relative protein level 0 0 Ctrl 1d 3d 7d 10 20 Ctrl PFTα (μM) D Axotomy

Ctrl 10μM PFTα 20μM PFTα

E G Ctrl sic-Myc Ctrl p53 OE kDa 55 p53

GAPDH 35

F 150 ** sic-Myc+p53 p53 ** 200μm 100 *

50 length (% of Ctrl ) Ctrl of (% length

0 Axon Ctrl Myc p53 -Myc - sic sic +p53

Figure 4 p53 sic-Myc+p53 sic-Myc Ctrl A B in vivo

Axon length (mm) electroporation 1.0 2.0 3.0 4.0 5.0 0 Ctrl Day 0 Day sic ** -Myc sic *** +p53-Myc **

p53 Sciatic nerve crush nerve Sciatic Figure5 Day 2 Day C Cumulative distribution(%) 100 60 80 20 40 0 0 500μm 1 Axon length (mm) length Axon 2 Axon tracing Axon 3 Day 5 Day 4 Ctrl sic sic p53 5 - - Myc Myc+p53 6 7 8 A Ctrl 5μM PFTα 10μM PFTα Cortical neurons

B Ctrl 0.1μM Tev-6 0.25μM Tev-6 Cortical neurons

C Ctrl 5μM PFTα 10μM PFTα Hippocampal neurons

D Ctrl 0.1μM Tev-6 0.25μM Tev-6

Hippocampal neurons *** E F Cortical neurons Cortical neurons G Hippo. neurons H Hippo. neurons 200 200 300 300 *** *** 150 *** *** *** 150 *** 200 200 100 100 *** * 50 50 100 100

0 0 5 0 0 10 5 Axon length (% of Ctrl) Axon length (% of Ctrl) 0.1 Ctrl Ctrl 0.25 Ctrl 10 Axon length (% of Ctrl) 0.1 PFTα μM Axon length (% of Ctrl) Ctrl PFTα μM 0.25 Tev-6 μM Tev-6 μM

Figure 6

p53 -

Day 28

GFP AAV2 - 0

20 40 10 30

Harvest and analyze AAV2 % RGC survival survival RGC % D

*** 3000 p53 - Day 26 CTB injection AAV2 2000 p53 *** - GFP - AAV2 AAV2 Figure 7 Figure 1000 Numbers of regenerating fibers GFP - Day 14

0

Optic nerve crush 2000 500 200 1500 1000 AAV2 Distance from crush site (μm) site crush from Distance B p53 GFP - - Ctrl C Day 0 AAV2 AAV2

Viral injection

p53 - AAV2 GFP - AAV2 A A c-Myc Tuj1 merge B

3.0 *** Ctrl

2.0

1.0 Myc level - c 0.0 Ctrl Axotomy

( fluorescence intensity, a.u.) intensity, ( fluorescence Axotomy

C D * E 8.0 200 * 6.0 Axotomy 150 1d 7d 4.0 Ctrl 3d kDa 100 c-Myc 40 50 2.0 (% of Ctrl) of (% β-actin ) Ctrl of ( fold 40 0 0.0

1d 3d 7d Relative mRNA level Ctrl 1d 3d 7d

Relative protein level Ctrl Axotomy Axotomy

125 ** F Ctrl 10058-F4 G 100 75 length 50 (% of Ctrl) of (%

Axon 25 0 Ctrl -F4 10058

H I 125 * Ctrl sic-Myc 100 75 length 50 (% of Ctrl) of (%

Axon 25 0

Ctrl-Myc sic

Supplementary Figure S1 800 A Ctrl c-Myc B ** 600

400

Laminin 200 Axon length (μm) 0

Ctrl -Myc c C 400 Ctrl c-Myc D ** 300

200

CSPGs 100

Axon length (μm) 0

Ctrl-Myc E c Ctrl c-Myc 600 F **

400

Myelin 200

Axon length (μm) 0 Ctrl -Myc c

Supplementary Figure S2 A Ctrl 25μM BIBR1532

B

150 *** *** ** 100

50μM BIBR1532 100μM BIBR1532 50

0

Axon length (% of Ctrl) 0 25 50 100 BIBR1532 (μM)

Supplementary Figure S3 A B 150 *** *** BIBR 100 Ctrl 1532 TERT 50 p53 ( % of Ctrl ) Ctrl of ( % GAPDH 0 Relative protein level Ctrl p53 C TERT Ctrl sic-Myc sic-Myc+Tev-6

siTERT siTERT+Tev-6 Tev-6

150 *** D *** *** 100 length 50 (% of Ctrl ) Ctrl of (% Axon

0 sic-Myc - + + - - - siTERT - - - + + - Tev-6 - - + - + +

Supplementary Figure S4 B A *** 4.0

kDa 3.0 Ctrl AAV-p53 55 p53 2.0

GAPDH 35 1.0

0.0 P53 staining, a.u.) Ctrl AAV2 p53 ( fluorescence intensity of of intensity ( fluorescence - C Ctrl AAV2-p53

Tuj-1

p53

DAPI

Merged

Supplementary Figure S5