Journal Pre-proof Upregulation of Cdh1 in the trigeminal spinal subnucleus caudalis attenuates trigeminal neuropathic pain via inhibiting GABAergic neuronal apoptosis Jiayan Li, Xuhui Chen, Xuan Li, Rong Hu, Wenlong Yao, Wei Mei, Li Wan, Lingli Gui, Chuanhan Zhang PII:
S0197-0186(19)30484-X
DOI:
https://doi.org/10.1016/j.neuint.2019.104613
Reference:
NCI 104613
To appear in:
Neurochemistry International
Received Date: 26 August 2019 Revised Date:
19 November 2019
Accepted Date: 26 November 2019
Please cite this article as: Li, J., Chen, X., Li, X., Hu, R., Yao, W., Mei, W., Wan, L., Gui, L., Zhang, C., Upregulation of Cdh1 in the trigeminal spinal subnucleus caudalis attenuates trigeminal neuropathic pain via inhibiting GABAergic neuronal apoptosis, Neurochemistry International (2019), doi: https:// doi.org/10.1016/j.neuint.2019.104613. 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.
Upregulation of Cdh1 in the Trigeminal Spinal Subnucleus Caudalis Attenuates Trigeminal Neuropathic Pain via Inhibiting GABAergic Neuronal Apoptosis Jiayan Lia, Xuhui Chena,b , Xuan Lia , Rong Hua, Wenlong Yaoa, Wei Meia, Li Wana, Lingli Gui*a, Chuanhan Zhang*a a
Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, 430030, China b
Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology,
Wuhan, 430030 Hubei Province, China * Lingli Gui and Chuanhan Zhang are co-corresponding authors
ABSTRACT Trigeminal neuropathic pain (TNP) remains a tremendous clinical challenge due to its elusive mechanisms. Previous studies showed that peripheral nerve injury facilitated a selective GABAergic neuronal apoptosis in the superficial dorsal horn and contributed to the development and maintenance of neuropathic pain. It has also demonstrated that downregulation of the anaphase-promoting complex/cyclosome(APC/C) and its coactivator Cdh1 contribute to neuronal apoptosis in diverse neurodegenerative diseases. However, whether APC/C-Cdh1 downregulation could induce GABAergic neuronal apoptosis in trigeminal caudalis nucleus (Vc), and then contribute to the development and maintenance of TNP remains unknown. In this study, we aimed to investigate the role of APC/C-Cdh1 in a TNP rat model and its underlying mechanisms. Our results showed that Cdh1 was primarily distributed in superficial laminae of Vc and significantly downregulated in Vc at day 14 post trigeminal nerve injury. Furthermore, trigerminal nerve injury leads to neuronal apoptosis, espcially GABAergic interneurons in the superficial of Vc. Upregulating Cdh1 in Vc ameliorated mechanical allodynia and inhibited GABAergic neuronal apoptosis induced by chronic constriction injury of trigeminal infraorbital nerve (CCI-ION). Keywords: trigeminal neuropathic pain; Anaphase-promoting complex; Cdh1; Neuronal Apoptosis; GABA Abbreviations:TNP, trigeminal neuropathic pain; APC/C, anaphase-promoting complex/ cyclosome; UPS, ubiquitin-proteasome system; Cdh1, Cdc20-like protein 1; Vc, trigeminal caudalis nucleus; CCI-ION, chronic constriction injury of trigeminal infraorbital nerve.
1. Introduction Trigeminal neuropathic pain (TNP) is one of the most common chronic pain syndromes. Orofacial neuropathic pain following trigeminal nerve injury is intractable and more debilitating than other types of neuropathic pain (Barros et al. 2009; Hitchon et al. 2016). Moreover, TNP lacks effective therapeutic options due to its complicated underlying mechanisms (Woda et al. 2005; Attall et al. 2012; Baron, Förster, and Binder 2012). Although great progress has been made in the pathogenesis of TNP, the detail molecular mechanism remains unknown. Neuropathic pain, defined as pain associated with a lesion or disease of the somatosensory nervous system, was characterized by allodynia and hyperalgesia (Finnerup et al. 2016). Many
previous studies have shown that the mechanism of “central sensitization” or “disinhibition” could account for hyperalgesia and allodynia(Sivilotti and Woolf 1994; Yaksh 1989; Kuner 2010). Recent studies have demonstrated that peripheral nerve injury promotes a selective loss of GABAergic inhibition in the superficial dorsal horn, and the GABAergic interneurons apoptosis in the dorsal horn have been thought to be involved in the maintenance of neuropathic pain(Gwak and Hulsebosch 2011; Meisner and Marsh ADMarsh 2010; Moore et al. 2002). We hypothesize that the apoptosis of GABAergic interneurons after trigeminal nerve injury leads to disinhibition in the trigeminal spinal subnucleus caudalis (Vc, also recognized as the medullary dorsal horn) and contribute to trigeminal neuropathic pain, however, the potential mechanism was still indistinct. Anaphase-promoting complex/cyclosome (APC/C), a multi-subunit RING finger E3 ubiquitin ligase and its co-activator Cdh1, were key components of the ubiquitin-proteasome system (UPS). Previous studies have demonstrated that APC/C-Cdh1 played a significant role in neuronal survival and differentiation, axonal growth and synaptic development in the central nervous system(Pick et al. 2013; Angeles 2012; Fu et al. 2011). The knockdown of Cdh1 in neurons by siRNAs leads to aberrant entry into the S phase and then apoptotic cell death(Almeida, Bolaños, and Moreno 2005). In addition, our previous studies also found that APC/C-Cdh1 was involved in the development of neuropathic pain and the downregulation of Cdh1 signaling in the spinal dorsal horn contributed to the maintenance of mechanical allodynia and hypersensitivity after spinal nerve injury in rats(Hu et al. 2016; Tan et al. 2015). Therefore, we hypothesized that downregulation of APC/C-Cdh1 expressed on GABAergic neurons in Vc following trigeminalnerve injury leads to GABAergic neuronal apoptosis, and then contribute to the development of TNP. To test this hypothesis, we first examined Cdh1 expression in the Vc after trigeminal nerve injuries (CCI-ION) in rats. Simultaneously, we observed the GABAergic neuronal apoptosis. We further microinjected Cdh1-encoding recombinant lentivirus into the Vc of rat, and then evaluated the effect of Cdh1 overexpression on GABAergic neuronal apoptosis and mechanical allodynia induced by CCI-ION.
2. Materials andmethods 2.1. Animals Male Adult Sprague-Dawley rats (200-250 g) used in this study were purchased from Tongji Medical College Experimental Animal Center (Huazhong University of Science and Technology, Wuhan, China (Certificate No. 42009800002519/SCXK(E)2016-0009)). These animals were housed under a 12 h light-dark cycle in a temperature-controlled room (22-25°C) with free access to food and water. All experimental procedures were approved by the Experimental Animal Care and Use Committee of Tongji Hospital, Huazhong University of Science and Technology and were in accordance with the guidelines of the Committee for Research and Ethical Issues of IASP. 2.2. Animal model A model of trigeminal neuropathic pain was established by CCI-ION as described previously (Imamura, Kawamoto, and Nakanishi 1997; Kaushal et al. 2016). Briefly, under anesthesia with sodium pentobarbital (40-50 mg/kg, i.p.), the rat's head was lying on his back and fixed on a table. All incisions were performed intraorally, thus keeping the snout and the vibrissae intact. An incision approximately 8- to 10-mm-long was placed along the gingivobuccal margin of the buccal mucosa
and initiated next to the first molar. The unilateral ION was separated from surrounding connective tissue and was loosely tied with two ligatures (chromic gut 4.0, 2 mm apart) at about 4 mm from the nerve where its branches generate from the infraorbital foramen. To determine the appropriate contractile force, the nerves were observed under a microscope while tightening the knot, slowing the circulation through the superficial vasculature without occlusion. For Sham-operated rats, only a unilateral nerve was exposed without ligatures. The incision was sutured using tissue glue. All surgical procedures were operated aseptically. 2.3. Behavioral testing Pain related behaviors were tested at day 0 (before surgery), 3, 7, 14, 21 and 28 after surgery. In order to minimize the subjective initiative of the observer, the person conducting the behavior test adopts a blind method for the treatment group. All animals were habituated to the experimenter’s hand wearing a regular work glove without restriction and accustomed for one-half an hour daily for at least 3 days before testing as described previously (Ren 1999). A series of calibrated von Frey filaments (Stoelting, Chicago, IL, USA) with incremental stiffness (0.4, 0.6, 1.0, 1.4, 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 26.0g) were applied to the vibrissal whisker pad innervated by the infraorbital nerve(Kaushal et al. 2016). The improved up-down method that was used to assess the mechanical threshold(Dixon 1980; Chaplan et al. 1994; Ma et al. 2012; Kaushal et al. 2016). Initially, the middle von Frey monofilament (2.0 g) applied a slight bending force perpendicular to the vibrating whisker. The rat’s response to facial stimulation consists of one or more elements as follows (Vos, Strassman, and Maciewicz 1994): (1) The rapid head withdrawal reaction; (2) escape or aggressive behavior; (3) asymmetric face grooming, manifested as at least three face-wash strokes directed to the stimulated area. Each von Frey filament was applied five times at intervals of a few seconds for consistency of results. If the rat’s response was observed greater than three times after applying filament, the reaction was considered positive (Ma et al. 2012; Kaushal et al. 2016), and the weaker filament was next applied. In the case of a negative response, a stronger filament was used till positive reaction. Testing proceeded in this manner until two reactions straddling the threshold and afterward four additional fibers applied sequentially up or down, based on the rat's response, allowing the estimation of 50% mechanical withdrawal threshold (Dixon 1980). When using von Frey fiber, care should be taken to avoid direct vibrational stimulation. A mechanical withdrawal threshold decrease indicates mechanical allodynia. 2.4. Construction of Cdh1- encoding lentiviral vector As previously described, for the construction of Cdh1- encoding lentiviral vector (Li et al. 2017; Qi et al. 2014), the encoding sequence of rat Cdh1 gene (NM_001108074.1) was transferred to the pGC-FU vector via geneticrecombination. The package of lentivirus was commercially furnished by Shanghai Gene Chem. The ultimate titer of the lentiviral vector was 2.0 ×109 transduction units(TU/mL) for Lenti-GFP and 2.0× 108 TU/mL for Lenti-Cdh1-GFP, which have been proved effective in our lab(Qi et al. 2014; Tan et al. 2015). 2.5. Stereotaxic injection of the virus in the trigeminal subnucleus caudalis Rats were intraperitoneally anesthetized with sodium pentobarbital (45 mg/kg body weight) and placed in a stereotaxic apparatus (RWD Life Science,Shenzhen, China). A midline opening was performed to expose the skull and a small hole was drilled above the unilateral Vc, -14.52mm
anterior to bregma, 3.2 mm lateral to the midline, and 8.0 mm deep from the dura in the light of stereotaxic coordinate of the rat. Unilateral microinjection was performed using a varimetric micropipettor with a 28-gauge needle (Hamilton, Bonaduz, Switzerland), and the saline, Lenti-GFP or Lenti-Cdh1-GFP was gently injected (0.5µl, over 15 min) at day 7 after CCI-ION when mechanical allodynia has been already developed. After microinjection, the microsyringe was held in place for 15 min to prevent solution-backflow and improve diffusion. The wound was sutured and rats were placed in an incubator before they recoverd from anesthesia. 2.6. Immunofluorescence analysis At the stipulated time points, rats were deeply anesthetized and the brainstem was rapidly removed, sharp-frozen in isopentane and stored at -80 °C. The frozen brain stems were serially sectioned with a freezing microtome (CM1900, Leica, Wiesbaden, Germany) at -20°C. For immunofluorescence, briefly, the tissue sections were fixed in 4% paraformaldehyde/PBS for 10 min, and after strengthening membrane permeability with 0.3% TritonX-100/PBS for 15 min and blocking with 10% goat serum in PBS for 1 h, then sections were incubated overnight at 4 °C with primary antibodies:anti-c-Fos (1:250; Abcam, Cambridge, UK), anti-FZR1/CDH1 (1:100; ABclonalBiotech, College Park, Maryland, USA), anti-NeuN (1:100; MerckMillipore, Darmstadt, Germany), anti-GAD65 (1:100; Abcam, Cambridge, UK), anti-Cleaved Caspase-3 (1:200; CellSignaling Technology, CST,Danvers, MA, USA). After rinsing with PBS, the slides were incubated with secondary antibodies coupled to Dylight-488 or 549(Alexa Fluor, Molecular Probes Inc. USA) for 2 h at room temperature, respectively. Images were collected using a fluorescent microscope (DM2500; Leica Microsystems, Wetzlar, Germany). 2.7. Western blotting After deep anesthetics, sham and treated rats were quickly decapitated, the unilateral Vc tissues were removed and homogenized in ice-cold solubilization buffer consisting of RIPA lysis buffer and phenylmethylsulfonyl fluoride protease inhibitor. The protein concentration from each sample was determined, then equal amounts of protein (40 mg/lane) were separated on 10% SDS-PAGE gels and subsequently blotted to 0.45-mm polyvinylidene difluoride (PVDF) membranes (Roche, USA). After blocked with 5% skim milk in TBST (0.1 % Tween 20, 25 mM Tris, 150 mM NaCl, pH 7.5) for 1.5 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies: anti-β-actin (1:500; Boster Biological Technology, Wuhan,China), anti-FZR1/CDH1 (1:500; ABclonalBiotech, College Park, Maryland, USA), anti-Skp2 (1:500; ABclonalBiotech, College Park, Maryland, USA), anti-GAD65 (1:500; Abcam, Cambridge, UK), anti-cyclinB1(1:1000; ABclonalBiotech, College Park, Maryland, USA), anti-Cleaved Caspase-3 (1:200; Cell Signaling Technology, CST,Danvers, MA, USA), anti-Bax (1:1000; Cell Signaling Technology, CST, Danvers, MA, USA), anti-Bcl-2 (1:1000; Cell Signaling Technology, CST,Danvers, MA, USA). Blots were washed with TBST and incubated for 1.5 h with horseradish peroxidase-linked secondary antibody (1:5000; Boster Biological Technology, Wuhan, China). The immunoreactivity was finally detected using an enhanced chemiluminescence kit (Thermo Scientific, USA) and densitometric quantification of blots was digitally scanned using Scion Image free software (Meyer Instruments). 2.8. Statistical analysis All data are expressed as the mean ± standard deviation(SD). After confirmation that the raw data
for all experiments fitted a normal distribution Shapiro-Wilk's test, two-way ANOVA followed by Bonferroni’s multiple-comparison tests was used for the analysis of the behavioral data. One-way ANOVA followed by Bonferroni’s tests was used for western blot analysis. All comparisons were performed with GraphPad Prism 6.0 for Windows (GraphPad Software, San Diego, CA, USA). A value of P< 0.05 was considered statistically significant.
3. Results 3.1. Chronic Constriction Injury-Evoked Mechanical Allodynia and enhanced C-fos expression in the superficial of Vc after CCI-ION. We first observed the chronic constriction injury of the trigeminal infraorbital nerve (CCI-ION) induce persistent pain in rats. Compared with sham-operated rats, the head-withdrawal threshold was markedly reduced in the ipsilateral ION territory beginning at day 3 and persisting till to day 28 after surgery at least (Fig.1A). Coincide with the behavioral responses, the c-Fos, a neural marker of pain (Takeda et al. 2009), was signifcantly activated in the superficial laminae of Vc on day 14 after CCI-ION as compared with sham-operated rats( Fig.1C(d)). 3.2. The expression and cellular localization of Cdh1 in the Trigeminal subnucleus caudalis after CCI-ION To explore the expression and cellular localization of Cdh1 in the trigeminal subnucleus caudalis, immunofluorescence staining and western blot analysis were performed. Our immunofluorescent staining showed that Cdh1 expression was most prominent in superficial laminae (Fig.2A(a)), and the expression of cdh1 was significantly reduced in the superficial laminae of Vc at day 14 after CCI-ION compared with sham rats (Fig.2A(a-d)). And our double-labelling of Cdh1 with NeuN suggested that Cdh1 was mainly colocalized with NeuN (Fig.2A(c-f)). In addition, our western blot analysis showed that the total level of Cdh1 was significantly downregulated, while downstream substrates of Cdh1 (skp2, cyclinB1) were increased (Fig.2B-C).Our results also showed that Cdh1 was highly expressed in the nuclear of Vc neurons in the sham group, whereas levels increased in the cytoplasm after never injury (Fig.2D-G). Our immunofluorescent staining also showed the Cdh1 transfer out of the nucleus, which confirmed the inactivity of Cdh1 after nerve injury (Fig.2H). 3.3. Neuronal apoptosis in the superficial laminae of Vc after CCI-ION We next found that the CCI-ION could induce neuronal apoptosis in the superficial laminae of Vc. Our immunofluorescent staining showed that the expression of cleaved-caspase-3, a marker of apoptosis-mediated cell death(Meisner and Marsh ADMarsh 2010), was highly activated in the superficial laminae of Vc at day 14 after CCI-ION (Fig.3A(d)). In addition, cleaved-caspase-3 was predominantly colocalized with NeuN (Fig.3A(f)), indicating that CCI could induce neuronal apoptosis in the superficial laminae of Vc. Consistently, quantification of the western blot showed that expression of pro-apoptotic proteins (cleaved-caspase-3 and Bax) in trigeminal subnucleus caudalis of CCI rats were significantly increased, while the level of the anti-apoptotic protein Bcl-2 was decreased compared with the sham rats (Fig.3B-C). 3.4. CCI induced GABAergic neuronal apoptosis and Cdh1 downregulation Accumulated studies demonstrated that reduction of GABAergic interneurons contributed to
neuropathic pain, we further investigated whether CCI-ION induced GABAergic interneurons apoptosis in the superficial laminae of Vc. As shown in Fig .4A, GABAergic interneurons marker GAD65 immunoreactivity in the Vc of CCI rats was significantly reduced. Consistent with the immunofluorescent staining, our western blot analysis also showed the expression of GAD65 in the Vc of CCI rats was decreased compared with the sham rats (Fig4B-C; n=4). To investigate whether CCI-ION could induce GABAergic neuronal apoptosis, we conducted double immunofluorescent staining of cleaved-caspase-3 with GABAergic interneurons marker GAD65. As illustrated in Fig.4D, cleaved-caspase-3 was predominantly colocalized with GAD65, suggesting that CCI induced GABAergic neuronal apoptosis in the superficial laminae of Vc. To further confirm whether Cdh1 was involved in the development of GABAergic neuronal apoptosis after CCI, we performed double immunostaining with Cdh1 and GABAergic interneurons marker GAD65. A reduction of Cdh1 and GAD65 immunostaining in the superficial laminae of Vc was found at day 14 after CCI-ION(Fig.4E). The results also showed that Cdh1 was coexpressed with GAD65 in the superficial laminae of Vc, indicating that the GABAergic neuronal apoptosis may be correlated with the downregulated of Cdh1. 3.5. Upregulation Cdh1 in the superficial laminae of Vc attenuated CCI induced mechanical allodynia in TNP rats. To verify whether upregulation of Cdh1 expression in Vc could attenuate the mechanical allodynia induced by CCI-ION, we stereotactically unilateral microinjected recombinant lentivirus encoding Cdh1 (approximately -14.52mm rostral to the bregma, 3.2 mm lateral to midline, and 8.0 mm deep from the dura) (Fig. 5A), which has been formerly proved to increase the expression of Cdh1 in vitro effectively (Li et al. 2017) , at day 7 post CCI-ION. At 3 days after microinjection of Lenti-GFP and Lenti-Cdh1-GFP, an extensive GFP expression was detected in the superficial laminae of Vc (Fig.5B), suggesting a successful Lentivirus transfection in Vc. In addition, western blotting showed Cdh1 expression in Vc was prominently upregulated in the CCI+Lenti-Cdh1-GFP group at day 3 after microinjection compared with the CCI +saline and CCI + Lenti -GFP group (Fig.5C-D). Simultaneously, the downstream substrate of Cdh1(skp2) was significantly reduced (Fig.5E-F), which in turn confirmed the overexpression of Cdh1. Furthermore, as shown in Fig.5G, microinjection treatment with Lenti-Cdh1-GFP significantly alleviated CCI-induced mechanical allodynia, initiating on 7 days after microinjection and persisting at least to 21 days, as compared with CCI +saline and CCI + Lenti -GFP group. These behavioral data manifested that the upregulation of Cdh1 expression could efficaciously alleviate mechanical allodynia induced by trigeminal nerve injury in rats. 3.6. Cdh1 overexpression reversed CCI-induced GABAergic neuronal apoptosis To further explore the effects of Cdh1 overexpression on CCI-induced GABAergic neuronal apoptosis, western blotting was performed. The results showed that Cdh1 overexpression reversed CCI-induced downregulation of GAD65 in Vc at day14 after Lenti-Cdh1-GFP microinjection, compared with saline and Lenti-GFP injected groups. (Fig6A-B). Additionally, our western blot analysis showed the accumulation of cyclin B1 was significantly
reduced at day 14 after Lenti-Cdh1-GFP microinjection (Fig.6C-D). Furthermore, overexpression of Cdh1 inhibited pro-apoptotic proteins (cleaved-caspase-3 and Bax) in Vc at 14 days, while restored the anti-apoptotic protein Bcl-2 level compared with saline and Lenti -GFP group((Fig.6E-G). Taken together, these results suggested Cdh1 overexpression inhibited GABAergic neuronal apoptosis in Vc during the maintenance of TNP.
4. Discussion In the present study, our results showed that (1) CCI-ION decreased Cdh1 expression in the superficial neurons of Vc, (2) CCI-ION induced GABAergic interneuronal apoptosis in the superficial of Vc, (3) upregulating Cdh1 expression in Vc ameliorated mechanical allodynia via attenuating GABAergic neuronal apoptosis induced by CCI-ION. Taken together, these findings implied that the downregulation of Cdh1 had a significant effect on the development of TNP by promoting GABAergic neuronal apoptosis in Vc. Accumulated studies demonstrated that GABA was an extensively distributed inhibitory neurotransmitter and acts a “counter balance” role in the CNS following neuropathic pain (Gwak and Hulsebosch 2011). Recently, it has been reported that peripheral nerve lesion provoked excitotoxic cell death and GABAergic interneurons apoptosis in the Laminae I–III of the spinal dorsal horn then contributing to persistent pain hypersensitivity (Inquimbert et al. 2018). Our recent studies have also found that peripheral nerve injury causes apoptotic loss of GABAergic neurons via caspase-3 activation (Tan et al. 2018). Our results confirmed that GABAergic neuronal apoptosis in the superficial of Vc, induced by trigeminal nerve injury contributed to TNP. However, the specific mechanism remains unknown. APC/C-Cdh1 was a vital constituent of UPS, which played an important role in the cell cycle by targeting cell cycle proteins for degradation. Differentiated cells, such as neurons, remain resting in the G0 phase due to an active downregulation of cell cycle-related proteins (Angeles 2012). The aberrant re-entry into the cell cycle of neurons contribute to progressive neuronal death under certain pathological circumstances, including both acute injury and chronic neurodegenerative disorders (Timsit and Menn 2010; Shakya et al. 2009; Hernándezortega, Quirozbaez, and Arias 2011; Herrup 2013). Recent studies demonstrated that the dysfunction of APC/C-Cdh1 and the accumulation of its substrates were involved in Alzheimer's disease, stroke and other neurodegenerative diseases (Maestre et al. 2014; Veas-Pérez et al. 2015; Fuchsberger, Lloret, and Viña 2017). It has been reported the Cdh1 inhibiton could trigger cyclinB1-mediated aberrant re-entry into S-phase, leading to apoptotic cell death (Almeida, Bolaños, and Moreno 2005). Afterward, APC/C-Cdh1 was involved in NMDA- or glutamate-induced neural excitotoxicity, and inactivation of Cdh1 could cause abnormal accumulation of cyclin B1 and apoptotic cell death during excitotoxic damage (Maestre et al. 2014; Veas-Pérez et al. 2015) . Our previous studies have found that APC/C–Cdh1 activity was down-regulated in the spinal dorsal horn and the anteriorcingulate cortex (ACC) following spared nerve injury(SNI). Moreover, overexpression of Cdh1 in the spinal dorsal horn and ACC could attenuate SNI-induced mechanical allodynia, indicating that Cdh1 was involved in the development of neuropathic pain (Hu et al. 2016; Tan et al. 2015). Recently, the trigeminal caudalis nucleus is recognized as the anatomical equivalent of the spinal dorsal horn (Kaneko and Hammond 1997). In our studies, we developed a rat model of CCI-ION, which could induce TNP. Our results showed a significant reduction and
nuclear export of Cdh1 in Vc after trigeminal nerve injury, suggesting the activity of Cdh1 in Vc was decreased after CCI-ION. We also found the expression of APC/C-Cdh1 downstream substrates (skp2 and cyclin B1) was significantly increased after CCI-ION, which further confirmed the inactivity of Cdh1. Simultaneously, we observed a corresponding GABAergic neuronal apoptosis. Our double-labeling results also showed that Cdh1 was co-expressed with GABAergic neurons. Additionally, we detected that mechanical allodynia induced by trigeminal nerve injury could be attenuated by overexpression of Cdh1 in Vc , indicating that Cdh1 was involved in the maintenance of TNP. A recent study suggested that inactivation of Cdh1 promoted the Cyclin B1-Cdk1complex to phosphorylate the anti-apoptotic protein Bcl-xL(such as Bcl-2), leading to apoptotic neuronal death during excitotoxic stimuli (Veas-Pérez et al. 2015). Inaddition,knockdown of Cdh1 stabilized modulator of apoptosis protein 1 (MOAP-1), thereby enhancing etoposide-induced Bax (a member of the Bcl-2 family) activation and apoptosis (Huang et al. 2012). Apoptotic pathways initiated by diverse stimuli ultimately aggregate on the activation of the “executioner” protease and cleavedcaspase-3, enhanced cleaved caspase activity induces GABAergic neuronal apoptosis, and then the inhibition is lost in spinal dorsal horn lamina II after peripheral nerve injury (Scholz et al. 2005). Therefore, we observed Bcl-2 proteins , cyclin B1 and cleaved-caspase-3 expression after trigeminal nerve injury in this study. Our results found that Cdh1 overexpression reduced the abnormal accumulation of cyclin B1, Bax activation, and upregulated anti-apoptoticprotein Bcl-2 , thus mitigating neuronal apoptosis. Consistently, cdh1 overexpression inhibited the activation of caspase-3 and rescued the loss of GAD65, which suggested that Cdh1 alleviated GABAergic neuronal apoptosis and disinhibition in Vc induced by trigeminal nerve injury. Besides GABAergic neuronal apoptosis, other mechanisms of APC/C-Cdh1 contributing to the development of trigeminal neuropathic pain. Long-term synaptic plasticity changes in sensory pathways have long been regarded as potential molecular mechanisms of neuropathic pain. And our previous experiments have shown that APC/C-Cdh1 performed a vital role in regulating synaptic plasticity and synaptic development. Inactivity of APC/C-Cdh1 in the ACC and the spinal cord horn contributed to enhanced synaptic activity by upregulating surface expression of AMPA GluR1 subunit through an EphA4-dependent pathway (Hu et al. 2016; Tan et al. 2015). Whether APC/C-Cdh1 alleviated TNP through modulating synaptic plasticity in Vc remained to be determined. Additionally, it is believed that astroglia is involved in the mechanisms of TNP (Akiko et al. 2009). And the application of astroglial inhibitors methionine sulfoximine (MSO) to Vc significantly reduced the elevated nociceptive responses of Vc neurons reflecting orofacial hyperalgesia in rats (Chen-Yu et al. 2007; Xie et al. 2007). The previous study reported that Cdh1 overexpression in flies markedly reduced the number of glial cells (Kaplow, Korayem, and Venkatesh 2008) and Cdh1 inhibited astrocyte proliferation after oxygen–glucose deprivation and reperfusion, suggesting a critical role for APC/C–Cdh1 in astrocyte hyperactivation during nervous system lesion (Qiu et al. 2013). It may be of interest to investigate whether Cdh1 could attenuate TNP through inhibition astroglial toxicity function. Afterward, there exist some limitations in our studies, such as the Cdh1-encording recombinant lentiviral vector was actually non-selective with no particular promoters to target GABAergic inhibitory neurons.
5. Conclusion
In conclusion, our results document the role of APC/C–Cdh1 in CCI-ION induced TNP. We demonstrated here that the downregulation of Cdh1 in Vc promoted GABAergic neuronal apoptosis, and then contributed to mechanical allodynia. Cdh1 overexpression alleviated CCI-induced mechanical allodynia via inhibiting GABAergic neuronal apoptosis. Thus, our present findings indicate that the Cdh1 signaling pathway in Vc may offer a novel therapeutic target for the management of trigeminal neuropathic pain.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements This work was supported by grants from National Natural Science Foundation of PR China (Grant No. 81171158, 81600965), and grants from the Natural Science Foundation of Hubei province (Grant No. 2019CFB444).
References Akiko, Okada Ogawa, Suzuki Ikuko, Barry J Sessle, Chiang Chen-Yu, Michael W Salter, Jonathan O Dostrovsky, Tsuboi Yoshiyuki, Kondo Masahiro, Kitagawa Junichi, and Kobayashi Azusa. 2009. 'Astroglia in medullary dorsal horn (trigeminal spinal subnucleus caudalis) are involved in trigeminal neuropathic pain mechanisms', Journal of Neuroscience, 29: 11161-71. Almeida, Angeles, Juan P. Bolaños, and Sergio Moreno. 2005. 'Cdh1/Hct1-APC Is Essential for the Survival of Postmitotic Neurons', Journal of Neuroscience, 25: 8115-21. Angeles, Almeida. 2012. 'Regulation of APC/C-Cdh1 and Its Function in Neuronal Survival', Molecular Neurobiology, 46: 547-54. Attall, Nadine, Didier Bouhassira, Ralf Baron, Jonathan Dostrovsky, Robert H. Dworkin, Nanna Finnerup, Geoffrey Gourlay, Maija Haanpaa, Srinivasa Raja, and Andrew S. C. Rice. 2012. 'Assessing symptom profiles in neuropathic pain clinical trials: Can it improve outcome?', European Journal of Pain, 15: 441-43. Baron, R, M Förster, and A Binder. 2012. 'Subgrouping of patients with neuropathic pain according to pain-related sensory abnormalities: a first step to a stratified treatment approach', Lancet Neurology, 11: 999-1005. Barros, Vde M, P. I. Seraidarian, M. I. Côrtes, and L. V. de Paula. 2009. 'The impact of orofacial pain on the quality of life of patients with temporomandibular disorder', Journal of Orofacial Pain, 23: 28-37. Chaplan, Sandra R, FW Bach, JW Pogrel, JM Chung, and TL Yaksh. 1994. 'Quantitative assessment of tactile allodynia in the rat paw', Journal of neuroscience methods, 53: 55-63. Chen-Yu, Chiang, Wang Jing, Xie Yu-Feng, Zhang Sun, James W Hu, Jonathan O Dostrovsky, and Barry J Sessle. 2007. 'Astroglial glutamate-glutamine shuttle is involved in central sensitization of nociceptive neurons in rat medullary dorsal horn', Journal of Neuroscience the Official Journal of the Society for Neuroscience, 27: 9068-76. Dixon, Wl J. 1980. 'Efficient analysis of experimental observations', Annual review of pharmacology and toxicology, 20: 441-62. Finnerup, Nanna B., Simon Haroutounian, Peter Kamerman, Ralf Baron, David L. H. Bennett, Didier Bouhassira, Giorgio Cruccu, Roy Freeman, Per Hansson, and Turo Nurmikko. 2016. 'Neuropathic pain: an updated grading system for research and clinical practice', Pain, 157:
1599-606. Fu, Amy K Y, Kwok Wang Hung, Wing Yu Fu, Chong Shen, Yu Chen, Jun Xia, Kwok On Lai, and Nancy Y Ip. 2011. 'APCCdh1 mediates EphA4-dependent downregulation of AMPA receptors in homeostatic plasticity', Nature Neuroscience, 14: 181. Fuchsberger, Tanja, Ana Lloret, and Jose Viña. 2017. 'New Functions of APC/C Ubiquitin Ligase in the Nervous System and Its Role in Alzheimer’s Disease', International Journal of Molecular Sciences, 18. Gwak, Y. S., and C. E. Hulsebosch. 2011. 'GABA and central neuropathic pain following spinal cord injury', Neuropharmacology, 60: 799-808. Hernándezortega, Karina, Ricardo Quirozbaez, and Clorinda Arias. 2011. 'Cell cycle reactivation in mature neurons: a link with brain plasticity, neuronal injury and neurodegenerative diseases?', Neuroscience Bulletin, 27: 185-96. Herrup, Karl. 2013. 'Post-mitotic role of the cell cycle machinery', Current Opinion in Cell Biology, 25: 711-16. Hitchon, Patrick W., Marshall Holland, Jennifer Noeller, Mark C. Smith, Toshio Moritani, Nivedita Jerath, and Wenzhuan He. 2016. 'Options in treating trigeminal neuralgia: Experience with 195 patients', Clinical Neurology & Neurosurgery, 149: 166-70. Hu, R., L. Li, D. Li, W. Tan, L. Wan, C. Zhu, Y. Zhang, C. Zhang, and W. Yao. 2016. 'Downregulation of Cdh1 signalling in spinal dorsal horn contributes to the maintenance of mechanical allodynia after nerve injury in rats', Molecular Pain,12,(2016-5-01), 12. Huang, N. J., L. Zhang, W. Tang, C. Chen, C. S. Yang, and S. Kornbluth. 2012. 'The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1', Journal of Cell Biology, 197: 361. Imamura, Y, Hiroya Kawamoto, and Osamu Nakanishi. 1997. 'Characterization of heat-hyperalgesia in an experimental trigeminal neuropathy in rats', Experimental brain research, 116: 97-103. Inquimbert, P., M. Moll, A. Latremoliere, C. K. Tong, J. Whang, G. F. Sheehan, B. M. Smith, E. Korb, Mcp Athié, and O. Babaniyi. 2018. 'NMDA Receptor Activation Underlies the Loss of Spinal Dorsal Horn Neurons and the Transition to Persistent Pain after Peripheral Nerve Injury', Cell Reports, 23: 2678–89. Kaneko, M, and D. L. Hammond. 1997. 'Role of spinal gamma-aminobutyric acidA receptors in formalin-induced nociception in the rat', Journal of Pharmacology & Experimental Therapeutics, 282: 928-38. Kaplow, Margarita E, Adam H Korayem, and Tadmiri R Venkatesh. 2008. 'Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein', Genetics, 178: 2003-16. Kaushal, R, Bradley K Taylor, AB Jamal, L Zhang, F Ma, R Donahue, and KN Westlund. 2016. 'GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAα1 receptors in the medial prefrontal cortex', Neuroscience, 334: 148-59. Kuner, R. 2010. 'Central mechanisms of pathological pain', Nature Medicine, 16: 1258-66. Li, X., K. Wei, R. Hu, B. Zhang, L. Li, L. Wan, C. Zhang, and W. Yao. 2017. 'Upregulation of Cdh1 Attenuates Isoflurane-Induced Neuronal Apoptosis and Long-Term Cognitive Impairments in Developing Rats', Frontiers in Cellular Neuroscience, 11.
Ma, Fei, Liping Zhang, Danielle Lyons, and Karin N Westlund. 2012. 'Orofacial neuropathic pain mouse model induced by Trigeminal Inflammatory Compression (TIC) of the infraorbital nerve', Molecular brain, 5: 44. Maestre, C, M Delgado-Esteban, J. C. Gomez-Sanchez, J. P. Bolaños, and A Almeida. 2014. 'Cdk5 phosphorylates Cdh1 and modulates cyclin B1 stability in excitotoxicity', Embo Journal, 27: 2736-45. Meisner, J. G., and D. R. Marsh ADMarsh. 2010. 'Loss of GABAergic interneurons in laminae I-III of the spinal cord dorsal horn contributes to reduced GABAergic tone and neuropathic pain after spinal cord injury', J Neurotrauma, 27: 729-37. Moore, Kimberly A., Tatsuro Kohno, Laurie A. Karchewski, Joachim Scholz, Hiroshi Baba, and Clifford J. Woolf. 2002. 'Partial Peripheral Nerve Injury Promotes a Selective Loss of GABAergic Inhibition in the Superficial Dorsal Horn of the Spinal Cord', Journal of Neuroscience, 22: 6724. Pick, Joseph E., Li Wang, Joshua E. Mayfield, and Eric Klann. 2013. 'Neuronal Expression of the Ubiquitin E3 ligase APC/C-Cdh1 During Development is Required for Long-term Potentiation, Behavioral Flexibility, and Extinction', Neurobiology of Learning & Memory, 100: 25-31. Qi, Y. H., W. L. Yao, C. H. Zhang, and Y. Q. Guo. 2014. 'Effect of lentivirus-mediated RNA interference of APC-Cdh1 expression on spinal cord injury in rats', Genetics & Molecular Research Gmr, 13: 1366-72. Qiu, Jin, Chuanhan Zhang, Youyou Lv, Yue Zhang, Chang Zhu, Xueren Wang, and Wenlong Yao. 2013. 'Cdh1 inhibits reactive astrocyte proliferation after oxygen–glucose deprivation and reperfusion', Neurochemistry International, 63: 87-92. Ren, Ke. 1999. 'An Improved Method for Assessing Mechanical Allodynia in the Rat', Physiology & Behavior, 67: 711-16. Scholz, J, D. C. Broom, D. H. Youn, C. D. Mills, T Kohno, M. R. Suter, K. A. Moore, I Decosterd, R. E. Coggeshall, and C. J. Woolf. 2005. 'Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury', Journal of Neuroscience the Official Journal of the Society for Neuroscience, 25: 7317-23. Shakya, Arvind, Robert Cooksey, James E. Cox, Victoria Wang, Donald A. Mcclain, and Dean Tantin. 2009. 'Oct1 loss of function induces a coordinate metabolic shift that opposes tumorigenicity', Nature Cell Biology, 11: 320-27. Sivilotti, L., and C. J. Woolf. 1994. 'The contribution of GABAA and glycine receptors to central sensitization: disinhibition and touch-evoked allodynia in the spinal cord', Journal of Neurophysiology, 72: 169-79. Takeda, Ryuichiro, Yuko Watanabe, Tetsuya Ikeda, Hiroshi Abe, Kosuke Ebihara, Hisae Matsuo, Hiroi Nonaka, Hiroyuki Hashiguchi, Toshikazu Nishimori, and Yasushi Ishida. 2009. 'Analgesic effect of milnacipran is associated with c-Fos expression in the anterior cingulate cortex in the rat neuropathic pain model', Neuroscience Research, 64: 380-84. Tan, Wei, Wen-Long Yao, Bo Zhang, Rong Hu, Li Wan, Chuan-Han Zhang, and Chang Zhu. 2018. 'Neuronal loss in anterior cingulate cortex in spared nerve injury model of neuropathic pain', Neurochemistry International, 118: 127-33. Tan, Wei, Wen Long Yao, Rong Hu, You You Lv, Li Wan, Chuan Han Zhang, and Chang Zhu. 2015.
'Alleviating neuropathic pain mechanical allodynia by increasing Cdh1 in the anterior cingulate cortex', Molecular Pain, 11: 1-13. Timsit, S, and B Menn. 2010. 'Cerebral ischemia, cell cycle elements and Cdk5', Biotechnology Journal, 2: 958-66. Veas-Pérez, de Tudela M, M Delgado-Esteban, C Maestre, V Bobo-Jiménez, D Jiménez-Blasco, R Vecino, J. P. Bolaños, and A Almeida. 2015. 'Regulation of Bcl-xL-ATP Synthase Interaction by Mitochondrial Cyclin B1-Cyclin-Dependent Kinase-1 Determines Neuronal Survival', Journal of Neuroscience the Official Journal of the Society for Neuroscience, 35: 9287-301. Vos, Bart P, Andrew M Strassman, and Raymond J Maciewicz. 1994. 'Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat's infraorbital nerve', Journal of Neuroscience, 14: 2708-23. Woda, A, S Tubert-Jeannin, D Bouhassira, N Attal, B Fleiter, J. P. Goulet, C Gremeau-Richard, M. L. Navez, P Picard, and P Pionchon. 2005. 'Towards a new taxonomy of idiopathic orofacial pain', Pain, 116: 396-406. Xie, Y. F., S. Zhang, C. Y. Chiang, J. W. Hu, J. O. Dostrovsky, and B. J. Sessle. 2007. 'Involvement of glia in central sensitization in trigeminal subnucleus caudalis (medullary dorsal horn)', Brain Behavior & Immunity, 21: 634-41. Yaksh, T. L. 1989. 'Behavioral and autonomic correlates of the tactile evoked allodynia produced by spinal glycine inhibition: effects of modulatory receptor systems and excitatory amino acid antagonists', Pain, 37: 111-23.
Figure Legends Fig.1. Mechanical hyperalgesia/allodynia and c‑ Fos activation in the Trigeminal subnucleus caudalis (Vc) after CCI-ION. A The time course of mechanical allodynia on the ipsilateral vibrissal whisker pad in rats with nerve injury is shown as the Mechanical threshold (g) at baseline through 3, 7, 14, 21, 28 days after surgery. The results are expressed as mean ± SD (BL: baseline, *P< 0.05 compared with the sham group, n = 10 for each group). B The red part marked with the Sp5C represents rat section through the Vc zone, -14.52 mm rostral to the bregma. C C-Fos immunostaining in the Vc of sham (a-c) and CCI (d-f) rats at day 14 after CCI-ION. Sections were labelled with C-fos (red) and nuclei stained with DAPI (blue). The results showed that C-Fos was highly expressed in the superficial laminae of Vc after CCI-ION (d, f).Scale bar : 200 µm. Fig.2. The expression and cellular localization of Cdh1 in the Trigeminal subnucleus caudalis at day 14 after CCI-ION. A (a) Immunostaining showed a higher density of Cdh1 immunoreactivity (red) in the Trigeminal subnucleus caudalis superficial laminae of sham rats .(a-c)Representative photomicrographs of Cdh1(red) labeling with NeuN (green) for neurons, noted that Cdh1 was colocalized with NeuN (yellow). Immunofluorescence showed that Cdh1(a, d) and NeuN (b, e) were both downregulated in the superficial laminae of Vc after CCI-ION, Scale bar :500 µm. B-C Western blot analysis showed the total Cdh1 and downstream substrates (skp2, cyclin B1) expression in the superficial laminae Vc of sham and CCI rats at day 14 after CCI-ION (*P< 0.05, **P < 0.01 ***P < 0.001 compared with the sham group, n = 4 in each group). D-G Representative western blotting of Cdh1 expression in the cytosol and nucleus. (*P< 0.05, compared with the sham group, n = 3 in each group). H Immunostaining showed the subcellular localization of Cdh1 in the Vc after CCI-ION. (a-b) Cdh1(red) double fluorescence labeling with DAPI (blue) for nuclei. (c, f) cdh1 was highly expressed in the nuclei in the sham group, however, poorly expressed in the CCI group and (f) the results showed the translocation of Cdh1 from the nucleus to the cytosol. Scale bar :100 µm. Fig.3. Neuronal apoptosis in the superficial laminae of Vc after CCI-ION. A.(d-f) Immunostaining showed significantly activation of cleaved-caspase3 ,whereas the reduction of NeuN in the superficial laminae of Vc on day 14 after CCI-ION. (d-f) Representative photomicrographs showed cleaved-caspase3 was colocalized with NeuN (yellow) in the superficial laminae of Vc, which indicated that CCI induced Neuronal apoptosis in the superficial laminae of Vc, Scale bar :500 µm. B-C. Western blot analysis showed the expression of pro-apoptotic proteins (cleaved caspase-3 and Bax) and anti-apoptotic protein Bcl-2 in Vc of sham and CCI rats on 14 days after CCI-ION( *P < 0.05, **P < 0.01 compared with the sham group, n = 4 in each group). Fig.4. CCI-ION induced GABAergic neuronal apoptosis A Representative photomicrographs of cleaved-caspase3 (red) double fluorescence labeling with GAD65 (green) for GABAergic neurons in the Vc of Sham and CCI rats, (f) cleaved-caspase3 was colocalized with GAD65 (yellow). B Double immunostaining of Cdh1(red) and GABAergic interneurons marker GAD65(green) in Vc of
Sham and CCI rats on day 14, (c,f) showed that Cdh1 was colocalized with GAD65(yellow). Scale bar: 100 µm.. C Western blot analysis showed that GAD65 expression in Vc of sham and CCI rats at day 14 after CCI-ION (***P< 0.001 compared with the sham group, n = 4 in each group). Fig.5. Microinjection of Cdh1-encoding lentivirus into the the superficial laminae of Vc attenuated CCIffinduced mechanical allodynia in rats. Saline (in the CCI+ Saline group), control lentivirus (in the CCI+ Lenti-GFP group) and Cdh1-expressing lentivirus (in the CCI + Lenti-Cdh1-GFP group ) was injected into the unilateral of superfical laminae of Vc at day 7 after CCI-ION. A Diagram of microinjection location in the Vc . B GFP-positive signals were mainly detected in the superficial laminae of Vc and surrounding area at 3 days after Lenti GFP or Lenti Cdh1-GFP microinjections. Scale bar : 100 µm. C-F Representative bands for the expression of Cdh1 and its downstream substrates (skp2) in the superficial laminae of Vc at day 3 after Lenti-Cdh1-GFP, Lenti-GFP or saline microinjection. ( *P< 0.05, **P< 0.01 compared with the sham group, #P< 0.05 compared with the CCI+Saline, &P< 0.05 compared with CCI+ Lenti-GFP, n = 4 in each group). G Mechanical threshold was measured before and at day7 ,14 and 21 after saline, Lenti-GFP and Lenti-Cdh1-GFP microinjection. Results showed that microinjections of Lenti Cdh1-GFP signifcantly ameliorated CCI induced mechanical allodynia (*P< 0.05 compared with the sham group, #P< 0.05 compared with the CCI+Saline,&P< 0.05 compared with CCI+ Lenti-GFP, n = 4 in each group ). Fig.6. Cdh1 overexpression reversed CCI-induced GABAergic neuronal apoptosis A-B Western blot and data summary showed that at day 14
post microinjection, Cdh1 overexpression restored
the protein expression of GAD65 in Vc (*P< 0.05 compared with the sham group, #P< 0.05 compared with the CCI+Saline , &P< 0.05 compared with CCI+ Lenti-GFP , n = 4 in each group ). C-G Western blot analysis of the effect of Cdh1 overexpression on cyclin B1 and apoptosis-related proteins cleaved-caspase-3, Bax , Bcl-2. (*P< 0.05 compared with the sham group, **P< 0.01 compared with the sham group,
#
P < 0.05 compared with the CCI+Saline, ##P < 0.01 compared
with the CCI+Saline,&P< 0.05 compared with CCI+Lenti-GFP , n = 4 in each group)
Highlights • • • •
CCI-ION decreased Cdh1 expression in the superficial neurons of Vc CCI-ION induced GABAergic interneuronal apoptosis in Vc Upregulation of Cdh1 ameliorated GABAergic interneuronal apoptosis Upregulation of Cdh1 attenuated mechanical allodynia induced by CCI-ION
Author Statement Jiayan Li: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Data Curation, Writing - Original Draft Xuhui Chen: Conceptualization, Investigation, Validation Xuan Li: Methodology, Investigation Rong Hu: Methodology, Investigation Wenlong Yao: Resources, Project administration, Wei Mei: Resources, Project administration, Li Wan: Resources, Project administration, Funding acquisition Lingli Gui: Resources, Project administration, Supervision, Writing, Reviewing and Editing Chuanhan Zhang: Resources, Supervision, Funding acquisition, Writing, Reviewing and Editing
All authors read and approved the final manuscript.