Electroacupuncture at Governor Vessel improves neurobehavioral function via 1 silencing complexin I

31 Governor Vessel electro-acupuncture (GV), as a traditional Chinese medicine, has been 32 proved that it can reduce scar and promote axon regeneration. However, the underlying 33 mechanism remains unclear. Herein, complexin I (CPLX1), as a candidate protein 34 involved in the process of GV treatment on spinal cord contusion (SCC), was found by 35 using protein chip. Therefore, using a CRISPR/Cas9 knockout approach, we silenced 36 CPLX1 to assess its role in the process of GV treatment. Additionally, eIF5A1 promotes 37 translation of CPLX1 with PPG sequence, we attempt to uncover whether eIF5A1 play 38 a role in GV treatment. Indeed, GV can reduce scar and promote axon regeneration after 39 SCC. CPLX1 -/+ SCC rats demonstrated that decreased CPLX1 improved the 40 microenvironment of injured area via reducing the components of fibrotic scar and 41 further enhanced the synaptic plasticity, which benefit the regeneration of axons. And 42 eIF5A1 could regulate the expression of CPLX1 in the process of GV treatment. 43 Therefore, GV contributes to axon regeneration and synapse plasticity via eIF5A1 44 regulating CPLX1 following SCC, providing a convincible mechanism for improving 45 the therapeutic efficacy of GV for SCC. 46 47 48 49 50 51 52 53 acquire mechanistic insight, we performed protein mass spectrometry analysis of spinal cord injured segments at 4wpi after GV treatment. We identified 379 proteins that were differentially expressed including 226 up-regulated and 153 down-regulated


Introduction
CPLX1 with PPG sequence (26, 27), we attempt to uncover whether eIF5A1 play a role 119 in the GV treatment. Herein, we provide evidence that GV promote the recovery of 120 motor function via regulating CPLX1 using immunofluorescence double labeling 121 analysis, western blot (WB), electromyography (EMG), motor evoked potential (MEP), 122 field potential, diffusion tensor imaging (DTI), and Golgi staining. Taken together, 123 these data evidence the efficacy of GV on the treatment for SCC and demonstrate the 124 underlying mechanism in the process of GV treatment on SCC, establishing a reliable 125 molecular theoretical basis for clinical application of GV in the treatment of SCI.  These data suggested that GV treatment increase the axonal plasticity. 145 To acquire mechanistic insight, we performed protein mass spectrometry analysis of assessed by WB in the same sites was made between the SCC and GV-SCC group 179 demonstrating that the CPLX1 expression of rostral and caudal segments significantly 180 decreased after GV treated group comparing to the SCC group ( Figure 2C). Thus, these 181 implied that silencing CPLX1 expression may be the reason for the effectiveness of GV 182 treatment.

183
For further researching, we used a transgenic rat line with homozygous knockout of 184 CPLX1 generated by CRISPR/Cas9 gene editing system. Due to CPLX1 -/rats had a 185 profound ataxia and short lifespan, CPLX1 -/+ rats were used for the following 186 experiment, which have been verified with significant levels of CPLX1 gene silencing 187 (Supplemental Figure 2). Then, CPLX1 -/+ and GV-WT group of SCC models were 188 conducted for further investigating. Reduced CPLX1 expression in SCC models 189 resulted in significant improvements in hindlimb moving ability within the first 3 weeks.

190
However, in GV-treated rats, functional recovery first appeared by 4 weeks and became 191 significant gradually. This trend of those two groups kept extending even at 9, 10, 11 192 week, on those period no GV carry out, indicating the functional sustainability in the 193 GV treating and partial delection of CPLX1 in SCC rats ( Figure 2D). When the nervi  and reduces fibrotic scar tissue after SCC even more than GV application. As 207 previously mentioned, reduction of CPLX1 using genetic method in the SCC models 208 significantly ameliorated the motor dysfunction. Eleven weeks later, the content of 209 CPLX1 at the rostral and caudal spinal cord extraction in the CPLX1 -/+ SCC models 210 was lower than that of WT SCC group, respectively ( Figure 3A). Further, Diffusion 211 tensor imaging (DTI) scans was used noninvasively to longitudinally track neural tissue 212 regeneration progress, which was an ideal measurement for both human and animal SCI 213 studies. Different colors of tracking were used to mark the direction of fiber orientations.

214
Results demonstrated that spinal cord neural regeneration was more obvious in CPLX1 -215 /+ SCC rats. But, in the WT rats, the ascending and descending blue fiber tracks were   which can be rescued by overexpressing CPLX1 but not eIF5A1 ( Figure 6K).

318
To evaluate whether eIF5A1-dependent CPLX1 expression can affect the 319 neurobehavioral recovery after SCC, we used CPLX1 -/+ rats to generate SCC model 320 and HSV-eIF5A1-ORF was injected into the spinal cord lesion area of CPLX1 -/+ rats.

321
WB results confirmed that the content of CPLX1 was decreased as mentioned above in 322 CPLX1 -/+ SCC group compared to WT SCC group (P<0.001), but increased after over- showed that CPLX1 -/+ -eIF5A1-ORF group had a lower BBB scores compared to 331 CPLX1 -/+ SCC models begin 4wpi, revealing that over-expression of eIF5A1 in 332 CPLX1 -/+ SCC models reversed the improved neurobehavioral defects ( Figure 7G). We 333 finally investigated whether the motor function recovery and amelioration after GV 334 treatment was due to down-regulated CPLX1 through eIF5A1. HSV-eIF5A1-ORF was 335 injected into the spinal cord lesion area of WT SCC models whose were given the GV   In order to investigate the underlying mechanism, results of protein chip show that 375 the expression of CPLX1 decreased obviously after GV treatment. CPLX1 is a highly 376 charged protein that is essential for Ca 2+ -mediated neurotransmission that appears to 377 act by interacting with and regulating the SNAREs (38). To date, CPLX1 levels are 378 differentially expressed in many psychiatric and neurodegenerative disorders (39).

494
After obtaining the image, each data was corrected using DTI studio (Jiang et al., 2006).

495
Then the Diffusion Toolkit software was used to perform white matter fiber tracking on 496 the corrected DTI data and display the image using Trackvis (http://www.trackvis.org).   Reporting summary. 686 Further information on experimental design is available in the Nature Research

687
Reporting Summary linked to this article.

688
Data availability 690 The data that support the findings of this study are available from the corresponding 691 author upon reasonable request.