RhoA Inhibitor Treatment At Acute Phase of Spinal Cord Injury May Induce Neurite Outgrowth and Synaptogenesis*

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomic-systems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these results by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement.

The therapeutic use of RhoA inhibitors (RhoAi) has been experimentally tested in spinal cord injury (SCI). In order to decipher the underlying molecular mechanisms involved in such a process, an in vitro neuroproteomicsystems biology platform was developed in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. A fine mapping of the spatio-temporal molecular events of the RhoAi treatment in SCI was performed. The data obtained allow a better understanding of regeneration/degeneration induced above and below the lesion site. Results notably showed a time-dependent alteration of the transcription factors profile along with the synthesis of growth cone-related factors (receptors, ligands, and signaling pathways) in RhoAi treated DRG cells. Furthermore, we assessed in a rat SCI model the in vivo impact of RhoAi treatment administered in situ via alginate scaffold that was combined with FK506 delivery. The improved recovery of locomotion was detected only at the early postinjury time points, whereas after overall survival a dramatic increase of synaptic contacts on outgrowing neurites in affected segments was observed. We validate these re-sults by in vivo proteomic studies along the spinal cord segments from tissue and secreted media analyses, confirming the increase of the synaptogenesis expression factors under RhoAi treatment. Taken together, we demonstrate that RhoAi treatment seems to be useful to stimulate neurite outgrowth in both in vitro as well in vivo environments. However, for in vivo experiments there is a need for sustained delivery regiment to facilitate axon regeneration and promote synaptic reconnections with appropriate target neurons also at chronic phase, which in turn may lead to higher assumption for functional improvement. Among the inhibitory factors that prevent axonal regrowth in spinal cord injury (SCI) 1 , RhoA, an intracellular GTPase, is considered as a key target for the design of proregenerative strategies. Previous experiments have shown that lysophosphatidic acid, via activation of the RhoA pathway, induced neurite retraction and neuronal soma rounding (1). Conversely, the use of C3 transferase to inactivate Rho in primary neuronal culture confirmed the role of Rho in neurite outgrowth inhibition (2)(3)(4). Thus, blockers of the post-receptor components of RhoA are now used to improve long-distance axon regeneration and sprouting (5). Furthermore, there is evidence that RhoA-ROCK signaling mediates the inhibitory effects of chondroitin sulfate proteoglycans (CPSG) in neurons; whereas, the sustained delivery of Rho inhibitor and BDNF promotes axonal growth in CPSG region after SCI. Along this line, novel inhibitors i.e. cholesterol and sphingomyelin as novel myelin-associated inhibitors have also demonstrated to operate via RhoA-dependent mechanism(s) (6 -8). On this basis, the RhoA pathway in neurons is considered to mediate the intracellular signaling of several major extracellular cues that inhibit neuroregeneration in SCI. Accord-ingly, the RhoA inhibitor Cethrin is currently under phase I/IIa clinical trials for the treatment of SCI (9).
One of the mechanism by which RhoA signaling inhibits neurite growth involves the p75 neurotrophin receptor. Indeed, several studies, using for some of them the p75 neurotrophin receptor-(p75 NTR ) -null mutant mice (7) showed that RhoA binds to p75 NTR and forms part of the membrane raft receptor complex responsible for growth inhibition signaling (10 -12). However, a pan-proteomic approach that would identify the whole range of effects exerted by RhoA inhibition on neurons is still missing. In this context, we have recently demonstrated, based on spatial and temporal proteomic studies, that major differences between the rostral and caudal segments adjacent to the lesion could be demonstrated at day 3 post-SCI, in terms of injury mechanisms, inflammatory regulation and regeneration processes (13). In the rostral or lesion segments, multiple proteins belonging to the chemokines/cytokines family or exerting neurotrophic functions were identified. In contrast, multiple proteins identified in caudal segments appeared to relate with injury and necrosis events. Our data suggest that in acute SCI regionalization in terms of inflammatory and neurotrophic responses may occur because of alterations in protein dynamics between rostral and caudal segments (13). In addition, the proteomic profile in caudal segments was characterized by the neuronal expression of IgG2a neuronal and by a signature of axonal regrowth inhibition associating CSPG and proteins of the MEMO1-RHOA-DIAPH1 signaling pathway (14). The MEMO1-RHOA-DIAPH1 signaling pathway plays an important role in ERBB2dependent stabilization of microtubules at the cell cortex and inhibits neurite outgrowth. Interestingly, a comparative proteomic approach performed by Liu et al. (2015) (15) at the caudal segment level has shown that the eukaryotic translation initiation factor 5A1 (eIF5A1) and Rho GDP dissociation inhibitor alpha (RhoGDI␣), a member of Rho GDI family, played a major role in determining the extent of spontaneous functional recovery (15). In vitro, eIF5A1 overexpression in primary neurons increased cell survival and elongated neurite length whereas eIF5A1 knockdown reversed these effects (15). Moreover, eIF5A1 and RhoGDI␣ were involved in the same pathway as, both in vivo and in vitro, RhoGDI␣ upregulation or down-regulation rescued the neuroregeneration impact of eIF5A1 down-or upregulation respectively (15).
In this context, the present study was designed to: (1) optimize SCI neurotherapy with RhoA inhibitors (RhoAi) and (2) gain further molecular insights on the mechanism(s) by which RhoAi may exert its neuroregenerative effects in SCI. For this purpose, we developed an in vitro neuroproteomicsystems biology platform in which the pan-proteomic profile of the dorsal root ganglia (DRG) cell line ND7/23 DRG was assessed in a large array of culture conditions using RhoAi and/or conditioned media obtained from SCI ex vivo derived spinal cord slices. In addition, pan-proteomic analyses and identification of functional biochemical pathways were cou-pled to the assessment of a large array of transcription factors.
This innovative analytical platform allowed a fine mapping of the spatio-temporal molecular events supporting the neuroregenerative impact of RhoAi in SCI. We then validate our finding by in vivo proteomic study at the level of the tissue segments and conditioned media. Our findings highlight the large molecular effects of RhoAi and provide an integrated mapping of such effects on the secretome, regulome and intra-cellular proteome of injured neurons.
Finally, our work points to the possible therapeutic potential of RhoAi administered in alginate scaffolds and delivered in a time-and segment-specific fashion. In particular, we show that RhoAi is able to promote neurite outgrowth and synaptic reconnection, but is not sufficient to induce and maintain a real beneficial outcome evaluated by BBB score. Thus our work open the door for new treatment scenario where a RhoA is a key player.
Experimental Design and Statistical Rational-All the experiments were performed with biological replicates. For protein extraction was performed from SCI tissues from control rats (n ϭ 3) and rats 12 h after SCI treated with RhoAi ϩ FK506 (n ϭ 3) or not treated (n ϭ 3). For the collection of the conditioned media, control rats (no balloon inflation, n ϭ 6), rats 12 h post-injury (n ϭ 6) and rats 3 days post-injury (n ϭ 6) were sacrificed. For the behavioral experiments, 5 rats received saline and 5 rats received RhoAi ϩ FK506. Statistical analysis: For the proteomic statistical analysis of conditioned media, as a criterion of significance, we applied an ANOVA significance threshold of p Ͻ 0.05, and heat maps were generated. Normalization was achieved using a Z-score with a matrix access by rows. Obtained data from tissue analyses and behavioral testing were reported as mean Ϯ S.E. Mean values among different experimental groups were statistically compared by one-way ANOVA and Tukey's post hock tests using Graph pad PRISM software. Values of p Ͻ 0.05 were considered statistically significant (*p value of Ͻ 0.05, **p value of Ͻ 0.01, ***p Ͻ 0.001).
Intraspinal Delivery of RhoAi-Seven days after SCI, animals (n ϭ 10) were anesthetized with 1.5-2% isoflurane and partial laminectomy at Th6 -12 level was performed. Using a 50-l Hamilton syringe (30G needle, Cole Parmer, Anjou, Quebec) connected to Ultra-MicroPump III with Micro4 Controller, 4-Channel (World Precious Instruments, Inc., Sarasota, FL) and stereotactic device, 2 intraspinal injections per animal were applied bilaterally to the lesion site and to the rostral and caudal segments (6 injections total). In most cases the lesion cavity was apparent through the dorsal site of spinal cord. Bilateral delivery of (1) saline (n ϭ 5), (2) RhoAi, 0.1 g/l (n ϭ 5) (2 injections of 2 l of alginate containing RhoAi per injection on left and right sides with delivery rate of 0.5 l/min, was performed at lesion cavity and 1 l of pure RhoAi per injection at rostral and caudal segments. The volume of 1 l was used in the case of intraspinal injections, whereas 2 l injections for administration to the cavity site. Each delivery was positioned 1 mm from the spinal cord midline and injected at the depth of 1.8 -2 mm from the pial surface of the spinal cord. The distance between injections was 1 mm, avoiding vessels. Intraspinal injections were followed by procedure published in our study (16). After injecting the dose of saline or RhoAi, the needle was maintained in the tissue for an additional 30 s. No antibiotic treatment was performed. Rats treated with RhoAi received daily intraperitoneal (i.p) injection of FK506 0.5 mg/kg/animal/3ϫ during the first week, followed by dose of 0.25 mg/kg/animal/3ϫ during the second week, whereas rats with vehicles received i.p saline. A separate group of SCI rats (n ϭ 6) injected with RhoAi with respective saline controls (n ϭ 6) that survived 12 h was performed for proteomic analyses.
Collection of Conditioned Media (CM) from Control and Lesioned Spinal Cord Segments-Experimental SCI rats at 3 days (n ϭ 3) and at 12 h with (n ϭ 3) or without RhoAi treatment (n ϭ 3) and respective controls (n ϭ 3/3D; n ϭ 3/12h) were sacrificed by isoflurane anesthesia followed by decapitation. The spinal cord was pressure expressed by injecting sterile saline (10 ml) throughout the vertebrate canal, along the caudo-rostral axis. Each spinal cord was macroscopically observed to check that lesion was well centered at the Th8-Th9 level on the longitudinal axis. Entire spinal cord was divided into transversally sectioned slides (ϳ1.0 cm thick each) obtained from the lesion site (Th7-Th11) and from the segments rostral (C1-Th6) and caudal (Th12-L6) to the site of injury. Slides were then chopped into 0.5 cm thick sections (2 sections per segment) and deposited into a 12-well culture plate containing 1 ml DMEM without FCS. After 24 h incubation in a humidified atmosphere with 5% CO 2 at 37°C, 1 ml of SCI-derived conditioned media (SCI-CM) were collected (rostral (R1), lesion (L), caudal (C1) segments) and centrifuged 30 min at 15,000 rpm at 4°C. Samples were stored at Ϫ80°C.
Protein Extraction from SCI Tissues-1 mm thick sections from rostral, lesioned and caudal segments from 12 h post-SCI rats (n ϭ 3) were ground in 1.5 ml tubes. Two hundred microliters of extraction buffer (CHAPS 3.5%, Tris 0.1 M, Dithiothreitol (DTT) 50 mM, pH 10.0) were added in each tube. Samples were mixed for 5 min and sonicated for 20 min. Cell debris were removed by centrifugation (15 min, 15000 ϫ g). 30 l of supernatant were used for FASP analysis using Amicon 30 kDa (Millipore) and LysC/trypsin enzymatic mix (30 g/ml in 0.05 M NH 4 HCO 3 ) (17). After overnight incubation at 37°C, the digests were collected by centrifugation. The filters were rinsed with 50 l of NaCl 0.5 M. Digestion was quenched by adding TFA 5% to the digests. The peptides were desalted with a Millipore ZipTip device before LC-MS/MS analysis.
Protein Digestion of SCI-CM-One hundred fifty microliters of SCI-CM and control CM were denatured with 6 M urea in 40 mM HEPES, pH 8.0 by sonication on ice. The proteins were reduced with 50 l DTT 10 mM for 40 min at 56°C and alkylated with iodoacetamide 55 mM for 40 min in the dark. The alkylation reaction was quenched with thiourea 100 mM. The proteins were digested overnight at 37°C with 30 g/ml LysC/Trypsin mixture. The digestion was stopped with 10 l TFA 17.5%. The peptides were desalted with a Millipore ZipTip device before LC MS/MS analysis.
In Vitro Neurite Outgrowth With SCI-CM-ND7/23 cell line (Sigma mouse neuroblastoma X rat neuron hybrid) was used to visualize in vitro the neurite outgrowth in presence of R1 and C1 conditioned media 3 days post injury in combination or not with RhoAi. ND7/23 cells were plated at a density of 18,000 cells/per well in 96-wells plate. The cells were starved overnight with DMEM medium supplemented with 2% Fetal bovine serum (FBS) ϩ 1% antibiotics ϩ 1% L-glutamine. Afterward, cells were stimulated with 1/3 R1 or C1 CM and 2/3 DMEM ϩ 1% L-1% L-Glutamine ϩ 1% penicillin-streptomy-cin (serum free medium) (14). The cells were treated or not with 1 g/ml RhoAi in combination with 1/3 of CM with 2/3 DMEM supplemented medium 24 h after C1 or R1 stimulation in order to reproduce the injured environment. The optimum split ratio 1/3 (CM, RhAi): 2/3 culture DMEM was set to perform sustained culture conditions for cells, according to our previous studies (14). Live images of cells not stimulated with RhoAi were captured 48 h after R1 or C1 CM stimulation and the images of ND7/23 cells stimulated with CM and RhoAi were captured 24 h after RhoAi stimulation (corresponding to 48 h after CM stimulation) with a camera mounted on a phase-contrast microscope (Nikon Eclipse TS100). Measurements were performed by using ImageJ software to determine the neurite length and statistical significance evaluated with One-Way ANOVA followed by Tukey Kramer Test (GraphPadInStat 3.0).
Total Protein Extracts and Conditioned Media (CM) Collection-ND7/23 cells were plated in 6-well plates until confluent. The cells were starved overnight with DMEM supplemented with 2% FBS, 1% L-Glutamine and 1% penicillin-streptomycin. Cells were first stimulated with 1/3 of R1 or lesion or C1 CM 3 days post injury and 2/3 DMEM ϩ 1% L-Glu ϩ 1% antibiotics, or left untreated. After 24 h of CM stimulation, 1 g/ml of RhoAi is added to the media. 24 h after RhoAi stimulation, the cell supernatants were collected, centrifuged (1000 rpm, 5 min) and immediately frozen at Ϫ80°C, and the cells were collected and then lysed with RIPA buffer for total protein extraction (150 mM NaCl, 50 mM Tris, 5 mM EGTA, 2 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1% Nonidet P-40, 1 mM PMSF, 1ϫ protease inhibitors). Cell debris was removed by centrifugation (20000 ϫ g, 10 min, 4°C). For the time course experiment after RhoAi, the cells were starved overnight. Cells were stimulated with RhoAi for 30 min (T30), 1 h (T1) and 4 h (T4) followed by the same protocol for total proteins extraction. The supernatants were collected and the protein concentrations were measured using the Bio-Rad Protein Assay. Experiments were performed in triplicates but not for T0, T30 min, T1 h, T4 h experiment with only one replicate.
Filter-aided Sample Preparation (FASP)-The total protein extract (0.1 mg) was used for FASP analysis as described previously (17). We performed FASP using Microcon devices YM-10 (Millipore) before adding trypsin for protein digestion (40 g/ml in 0.05 M NH4HCO3). The samples were incubated overnight at 37°C. The digests were collected by centrifugation, and the filter device was rinsed with 50 l of NaCl 0.5 M. Next, 5% TFA was added to the digests, and the peptides were desalted with a Millipore ZipTip device before LC-MS/MS analysis.
Protein Digestion of Condition Medium-One hundred microliters of the CM were collected for each condition. Secretome digestion was performed as previously described (18). In brief, the cell supernatants were denatured with 2 M urea in 10 mM HEPES, pH 8.0 by sonication on ice. The proteins were reduced with 10 mM DTT for 40 min followed by alkylation with 55 mM iodoacetamide for 40 min in the dark. The iodoacetamide was quenched with 100 mM thiourea. The proteins were digested with 20 g/ml LysC/Trypsin mixture overnight at 37°C. The digestion was stopped with 0.5% TFA. The peptides were desalted with a Millipore ZipTip device in a final volume of 20 l of 80% ACN elution solution. The solution was then dried using the SpeedVac. Dried samples were solubilized in water/0.1% formic acid before LC MS/MS analysis.
LC MS/MS Analysis-Samples were separated by online reversedphase chromatography using a Thermo Scientific Proxeon Easy-nLC1000 system equipped with a Proxeon trap column (100 m ID ϫ 2 cm, Thermo Scientific) and a C18 packed-tip column (Acclaim PepMap, 75 m ID ϫ 15 cm, Thermo Scientific). Peptides were separated using an increasing amount of acetonitrile (5-35% over 120 min) at a flow rate of 300 nL/min. The LC eluent was electrosprayed directly from the analytical column and a voltage of 1.7 kV was applied via the liquid junction of the nanospray source. The chromatography system was coupled to a Thermo Scientific Q-exactive mass spectrometer programmed to acquire in a data-dependent mode Top 10 most intense ion method. The survey scans were done at a resolving power of 70,000 FWHM (m/z 400), in positive mode and using an AGC target of 3e6. Default charge state was set at 2, unassigned and ϩ1 charge states were rejected and dynamic exclusion was enabled for 25 s. The scan range was set to 300-1600 m/z. For ddMS 2 , the scan range was between 200 -2000 m/z, 1 microscan was acquired at 17,500 FWHM and an isolation window of 4.0 m/z was used.
MS Data Analysis of T0, T30 min, T1 h and T4 h Protein Extract After RhoAi Treatment-Tandem mass spectra were processed with Thermo Scientific Proteome Discoverer software version 1.4. Spectra were searched against UniprotKB/Swiss-Prot (version January 2016) filtered with Rattus norvegicus (31093 sequences) taxonomy using the SEQUEST HT algorithm (version 1.4.1.14). The search was performed choosing trypsin as the enzyme with one missed cleavage allowed. Precursor mass tolerance was 10 ppm, and fragment mass tolerance was 0.1 Da. N-terminal acetylation; and cysteine carbamidomethylation; methionine oxidation were set as variable modifications and cysteine carbamidomethylation as fixed modification. Peptide validation was performed with the Percolator algorithm by filtering based on a q-value below 0.01, which corresponds to a false discovery rate (FDR) of 1%. Proteins were identified with a minimum of 2 peptides with at least one unique peptide per protein. The data sets used for analysis and the annotated MS/MS spectra were deposited at the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD004639 (for review: Username: reviewer60033@ ebi.ac.uk Password: 7O08FxXe).

MS Data Analysis of Protein Extract and Secretome
After SCI-CM With or Without RhoAi Treatment-All the MS data were processed with MaxQuant (version 1.5.6.5) (19) using the Andromeda (20) search engine. Secretome and protein extract from ND7/23 cell line were processed in two different files. Proteins were identified by searching MS and MS/MS data against Decoy version of the complete proteome for Rattusnorvegicus of the UniProt database (21) (Release June 2014, 33,675 entries) combined with 262 commonly detected contaminants. Trypsin specificity was used for the digestion mode with N-terminal acetylation and methionine oxidation selected as the variable. Carbarmidomethylation of cysteines was set as a fixed modification, with up to two missed cleavages. For MS spectra, an initial mass accuracy of 6 ppm was selected, with a minimum of 2 peptides and at least 1 unique peptide per protein, and the MS/MS tolerance was set to 20 ppm for HCD data. For identification, the FDR at the peptide spectrum matches (PSMs) and protein level was set to 0.01. Relative, label-free quantification of proteins was performed using the MaxLFQ algorithm (22) integrated into MaxQuant with the default parameters. The data sets, the Perseus result files used for analysis and the annotated MS/MS spectra were deposited at the ProteomeXchange Consortium (23) (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository (24) with the data set identifier PXD004639 (for review: Username: reviewer 60033@ebi.ac.uk Password: 7O08FxXe) for cellular extracts and secretomes. Analysis of the proteins identified was performed using Perseus software (http://www.perseus-framework.org/) (version 1.5.6.0). The file containing the information from identification was used with hits to the reverse database, and proteins only identified with modified peptides and potential contaminants were removed. Then, the LFQ intensity was logarithmized (log2[x]). Categorical annotation of rows was used to defined different groups after grouping replicates (1) Replicate (DMEM, R1, L, C1, R1 RhoAi, L RhoAi, C1 RhoAi), (2) Rho versus NT (DMEM, DMEM RhoAi, Rho (R1, L and C1 ϩ RhoAi) and NT (R1, L and C1 without RhoAi). Multiple-samples tests were performed using ANOVA test with a FDR of 5% and preserving grouping in randomization. Normalization was achieved using a Z-score with a matrix access by rows.
For the statistical analysis, only proteins presenting as significant by the ANOVA test were used for statistical analysis. Hierarchical clustering depending protein extract or secretome were first performed using the Euclidean parameter for distance calculation and average option for linkage in row and column trees using a maximum of 300 clusters. For visualization of the variation of proteins expression depending to the condition, the profile plot tool was used with a reference profile and an automatic selection of the 10 or 15 correlated profiles. To quantify fold changes of proteins across samples, we used MaxLFQ. To visualize these fold changes in the context of individual protein abundances in the proteome, we projected them onto the summed peptide intensities normalized by the number of theoretically observable peptides. Specifically, to compare relative protein abundances between and within samples, protein lengths normalized to log 2 protein intensities (termed "iBAQ" value in Max-Quant) were added to the MaxLFQ differences. Functional annotation and characterization of identified proteins were obtained using PANTHER software (version 9.0, http://www.pantherdb.org) and STRING (version 9.1, http://string-db.org).

Subnetwork Enrichment Pathway Analyses and Statistical Testing-The
Elsevier's Pathway Studio version 9.0 (Ariadne Genomics/ Elsevier) was used to deduce relationships among differentially expressed proteomics protein candidates using the Ariadne ResNet database (25,26). "Subnetwork Enrichment Analysis" (SNEA) algorithm was selected to extract statistically significant altered biological and functional pathways pertaining to each identified set of protein hits (C1, R1, L after RhoA inhibitor treatment sets). SNEA utilizes Fisher's statistical test used to determine if there are nonrandomized associations between two categorical variables organized by specific relationship. SNEA starts by creating a central "seed" from all relevant entities in the database, and retrieving associated entities based on their relationship with the "seed" (i.e. binding partners, expression targets, protein modification targets, regulation). The algorithm compares the sub-network distribution to the background distribution using one-sided Mann-Whitney U-Test, and calculates a p value indicating the statistical significance of difference between two distributions. In our analysis, "GenBank" ID and gene symbols from each set were imported to the software to form an experimental data set. For the reconstruction of networks of pathways, biological processes and molecular function were evaluated for each single protein hit and its associated targets (networks and pathways) (27,28). Integrated Venn diagram analysis was performed using "the InteractiVenn"; a web-based tool for the analysis of complex data sets.
Behavioral Testing-Animals were evaluated using Basso, Beattie, and Bresnahan (BBB) open-field test to assess motor function after SCI at day 0, 7, 14, 21, 28, 35, 42 and 49 days post injury. Each rat was tested for 5 min by two blinded examiners. BBB test measures locomotor outcome (hind limb activity, body position, trunk stability, tail position and walking paw placement) of rats utilizing the rating scale ranges from 0 (no observable hind limbs movements) to a maximum of 21 (plantar stepping, coordination and trunk stability like control rats).
Quantification Analysis-Immunohistochemically stained sections were analyzed using confocal microscope (Leica DM1500) and quantification was performed by ImageJ software. Five sections per animal were analyzed for each staining in rostral, lesion and caudal segments. For synaptophysin quantification analysis, images were first transformed into monochromatic 8-bit images and then threshold was adjusted to optimal value. Synaptophysin positivity was evaluated as a percentage of black pixels in overall image (value 0 -255, where 0 ϭ white pixels, 255 ϭ black pixels). For axonal regrowth evaluation, GAP-43 positive fibers were measured manually in micrometers. Total length of axon fibers was averaged for each image. RESULTS We previously performed a spatio-temporal study of the SCI from 3 days to 10 days after lesion. Our data suggest that in acute SCI regionalization in terms of inflammatory and neurotrophic responses may occur because of alterations in protein dynamics between rostral and caudal segments (13). In addition, the proteomic profile in caudal segments was characterized by the neuronal expression of IgG2a and by a signature of axonal regrowth inhibition associating CSPG and proteins of the MEMO1-RHOA-DIAPH1 signaling pathway (14). The MEMO1-RHOA-DIAPH1 signaling pathway plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex and inhibits neurite outgrowth. In this context, we established an in vitro proteomic system biology platform in order to better understand the impact of RhoAi on DRG neurons in time course, followed by in vivo SCI experiments to further compare and validate the similarities and differences of treatment approaches.
In Vitro Neuroproteomic-systems Biology Platform Targeting RhoA Signaling-Impact of RhoAi Treatment on Neurite Outgrowth-In vitro, RhoAi was added to ND7/23 DRGs cell line cultivated with spinal cord conditioned media (CM-SCI) collected from lesion (L), rostral (R1) and caudal (C1) segments (Fig. 1). Time course analysis showed that ND7/23 cells in presence of R1 or C1 conditioned media initiated neurite outgrowth at 24 h after cultivation and the results were statistically significant at 48h (Figs. 1A, 1B). Afterward, the ND7/23 cells were treated with 1 g/ml of RhoAi in combination with 1/3 of CM and 2/3 DMEM supplemented medium 24 h after C1 or R1 stimulation to reproduce the injured environment. In this context, neurite outgrowth appeared at 48 h with longer extensions when compared with non-RhoAi treatment (Figs. 1A, 1B). These results established the ability to block the MEMO1-RHOA-DIAPH1 signaling pathway and stimulate neurogenesis using such RhoAi.
Impact of RhoAi Treatment on ND7/23 DRGs Proteome-In order to identify proteins involved with RhoAi the proteomic approach was then performed with ND7/23 DRGs cell lines incubated with CM-SCI collected from L, R1, and C1 segments in presence or absence of RhoA inhibitor, using identical scenario as in experiments for neurite outgrowth evaluation (Fig. 1C). Secretomes collected in each condition have been processed by shotgun analyses. Proteins with an abundance that was significantly different among the samples were determined according to the MaxQuant and Perseus software. As a criterion of significance, we applied an ANOVA significance threshold of p Ͻ 0.05, and heat maps were generated (Fig. 1C, supplemental Data S1). Heat maps were performed and hierarchical clustering indicated two main branches i.e. one for Control branch (CTB)(DMEM conditions with or without RhoAi) and the second one is related to Conditioned medium branch (CMB) (L, R1, or C1 conditions with or without RhoAi). This branch is then sub-divided into two sub-branches: lesion on one side and R1 or C1 on the other side (Fig. 1Ca). From these data, clear clusters could be retrieved between the two branches. By contrast, in the CMB, only one main cluster allowed to differentiate all media in presence or absence of Rhoi (a yellow boxed area). A zoom of this cluster is presented in Fig. 1Cb and the ibaq quantitative values in Table I. Main proteins found in this cluster that could be sorted according to their over-expressed intensities are in the following order: immunoglobulins (IgG chains light and heavy), AKT proteins (AKT1, AKT2, and AKT3), BMP1, syntaxin 12, serpin 3, GMP ganglioside activator, meosin, hemopexin, protein VSP26b, 14-3-3 protein theta, and protein disulfide isomerase. The important finding is that the ibaq value showed that most of these proteins are under-expressed under DMEM conditions whereas, in presence of CMB alone or with RhoAi, they are over expressed, with some exemptions (Table I). Immunoglobulins were overexpressed particularly in the samples associated with lesion, treated with CM from Lesion segment alone and with RhoAi and in CM from C1 alone. For AKT proteins family, real differences could be registered between AKT3, AKT1, and AKT2 in relation to CM from rostral and lesion segments. With RhoAi, the level of AKT3 was diminished when compared with untreated cells. For AKT1 and AKT2 proteins, RhoAi increased their level in R1 and diminished these in lesion and C1. Serpina3c, Snx12, Gm2a, meosin, Timm44, Cthrc1, Stx6, vsp26b, Itih1, Aqp4, aggrecan core protein, BMP1 were over-expressed in C1 in presence of RhoAi compared with R1 or lesion with or without treatment. In R1, with RhoA inhibitor, only AK1 and AKT2 were Proteomic Analyses of RhoAi Effect on ND7/23 DRGs Cell Line-Global analysis was then performed by regrouping all conditioned medium treatment with RhoAi samples compared with nontreated (NT) samples with RhoA and compared with control i.e. DRG cells cultivated with only DMEM with or without RhoAi. We have identified 3133 proteins ( Fig. 2A) that clustered (Fig. 2B). In this context, two branches separated the secreted factors. The first branch separates the factors detected in control (culture medium with DMEM) from the ones cultivated with SCI CM. The second branches separated the ones treated with or without RhoAi (Fig. 2B). Of the 5 differentiated clusters that were identified (See yellow boxes), 2 contained over-expressed proteins (clusters 1,2) and 3 under-expressed proteins (clusters 3,4,5) (Fig. 2B). These clusters have been regrouped (Table II) and functional pathways extracted from Subnetwork Enrichment Analysis (SEA) was generated (Fig. 2C). Although 16 secreted proteins from ND7/23 DRGs cell line were overexpressed after RhoAi treatment, 23 were under-expressed (Table II, supplemental Data  S2). Among the 16 overexpressed proteins, some were already known to be implicated in neurites outgrowth or neurogenesis e.g. Pde6d (29), Ltbp4 (30), Clip2 (31), Enah (32), Vps26b (33), Sema7a (34), BDNF/NT3 (35), UNC5C (36), Ephrin A5 and Ephrin B receptor (34), VEGF (37). Pathway analyses reflected that nucleic proteins involved in cell cycle regulation, transcription activation, and cell survival were under-expressed in cells treated with RhoAi. On the other hand, proteins involved in stem cell proliferation, neuronal migration, axon guidance, neurotransmission, synaptic transmission, and nerve development were over-expressed (Fig. 2C). These results confirm that despite the presence of an inflammatory medium containing neurites outgrowth inhibitors, RhoAi still positively impacts the functional behavior of DRG cells and stimulates the neurite outgrowth process. On this basis, a time course proteomic study was undertaken in order to significance at 48 h (A). Arrows indicate the neurite outgrowth (A). Control was done in DMEM media without serum, to be in the same media than CM after SCI. Enhanced outgrowth referred to dense network of elongated processes interconnecting cells was documented in treatment group. Quantification of neurite outgrowth by ImageJ demonstrates the effect of RhoAi on neurite outgrowth (B) (One Way ANOVA followed by Tukey-Kramer test *p Ͻ 0.05, **p Ͻ 0.01, ***p Ͻ 0.001, ns ϭ nonsignificant).  treatment, allowed to identify 4030 proteins from which 179 modulated proteins were found between nontreated and treated cells (supplemental Data S2, supplemental Data S3A). From these 179 proteins, numerous factors were identified that could regulate the intrinsic growth capacity, including certain transcription factors (TF), such as cAMPresponsive element binding protein (CREB), signal transducer and activator of transcription 3 (STAT3), nuclear factor of activated T cell (NFAT), c-Jun activating transcription factor 3 (AFT3) and Krü ppel-like factors (KLFs), and intracellular signaling proteins, such as PI3 kinase, Akt, phosphatase and tensin homolog (PTEN), suppressor of cytokine signaling 3 (SOCS3), B-RAF, dual leucine zipper kinase (DLK), and insulin/insulin-like growth factor-1 (IGF-1) signaling have been detected (38,39). The Ibaq value confirmed the over-expression of Tp53, Stat2, Stat3, Proteins of the Smad family (smad1, smad2, smad3, smad4, and smad5), Smarcc1 (Baf155), and Smarcc2 (Baf170), Akt3, rpap3, b-raf, and PTEN (Table III). The string protein analysis confirms that all these proteins can be gathered in the same network (supplemental Data S3B). Nevertheless, one most intriguing is the presence of PTEN. Subnetwork global Analysis was generated between RhoAi treated DRG cells incubated with conditioned medium of R1, Lesion or C1 (Fig. 3). 24 h after treatment, complete disparities are observed between the 3 conditioned media after treatment. Only C1 medium clearly showed over-expressed proteins involved in neurite outgrowth, neuronal migration, and neurogenesis (Fig. 3A). Whereas with R1 medium, proteins detected are involved in neurite outgrowth, neuronal cell death, inflammation and cell proliferation and differentiation (Fig. 3B). The proteomic profile of DRG cells stimulated with lesion medium showed a unique enrichment in molecules involved in apoptosis and necrosis, inflammation, T cell response and, as well as neutrophils chemotaxis (Fig. 3C). Subnetwork Enrichment Analysis (supplemental Data S3) confirmed the presence of specific complementary proteins involved in dendrite morphogenesis such as CAMK1, SIPA1L1 and L1CAM (supplemental Data S3Ca). Proteins involved in cell death and proteins degradation such as TSC1, WDFY3 and OGT were found to be specific to lesion treatment (supplemental Data S3Cb). Proteins involved in neurite outgrowth and neuronal migration such as   (Fig. 4), from T0 to T4 h, a majority of the differentially expressed proteins were implicated in ER-associated protein catabolism (Fig. 4), from T0 to T30 min and to T4 h, two clusters of proteins were detected, namely nonselective vesicle building and cell spreading (data not shown). From T1 h to T4 h, chaperones proteins are identified (Fig. 4). Finally, transition from T0, T30 min, and T1 h to T4 h involved several clusters linked to oxidative stress, cell proliferation, differentiation and migration (data not shown) and in particularly some TF are observed at specific time points after treatment (Table  IV). In particular, BAF155 and BAF170 were present from time T0 to T24 h, whereas Smad2 was detected from T0 to T1h, and then at T24 h. AKT3 protein only showed at 1h with no detection before and after this time point. All the other TFs ware detected only after 24 h of treatment (Table IV). Investigate RhoA inhibitor in Vivo-According to the time course experiments results (Fig. 4) and the fact that at 24 h exposure to RhoA inhibitor all transcription processes in DRG cells are impacted, we decided to investigate the treatment 12 h after SCI. Proteomic studies were realized from tissues in the 3 segments at both side of the lesion and also in secretome (Fig. 5).
Tissue extracted proteins collected in each condition have been processed by shotgun analyses. Heat maps of proteins with an ANOVA significance threshold of p Ͻ 0.05, were generated (Fig. 5A, supplemental Data S6). Heat maps were performed and hierarchical clustering indicated two main branches i.e. one for SCI and the second one is related to treated animal with RhoA Inhibitor and control (without lesion). This branch is then subdivided between RhoA inhibitor treatment in function of the considered segment (Rostral, Lesion and Caudal) on one side and control in the other side (Fig. 5A). From these data, clear clusters could be retrieved between the two branches. Clusters 1 and 2 are specifically found in Rostral and Caudal segments after SCI. Cluster 1 integrates many subexpressed proteins in SCI segments (Fig. 5A, supplemental Data S6). By contrast, cluster 2 constitutes the one of overexpressed proteins in SCI. Such proteins are in control and treated animals sub-expressed in caudal and lesion segments. A slight overexpression is registered in rostral segments (control and treated animals). No inflammatory proteins were detected in cluster 2 whereas proteins involved in neurites outgrowth are present (e.g. CD166, advilin, neuritin, Neurocan, L1Cam, Vcan, Limbic system-associated membrane protein, neuronal growth regulator 1 precursor, C1qBp, SLIT-ROBO Rho GTPase-activating protein 2, Roundabout homolog 1, Ciliary neurotrophic factor) ( Table V). In RhoA inhibitor treatment, these neurite outgrowth factors expression is less important in both tissue (Table V) but also in secretome (Table  VI). However, proteins involved in synaptogenesis, their level of expression is increased after RhoA inhibitor treatment. The LFQ of synapsins, synthaxins, GAP43, Synaptojanin-1 is higher in lesions segment in treated animals compared with the only injured ones (Tables V and VI).
Interestingly, it must be noticed that Rock1 and RhoA are overexpressed in SCI and inhibited in RhoA inhibitory treated samples, confirming the efficiency of the treatment. Immune components are only detected in cluster 4. These are overexpressed in lesion after SCI, sub expressed in control and modulated in RhoA inhibitor treated animals (Fig. 5A, supplemental Data S6, Table V). Among the identified inflammatory proteins like complement proteins family (C3, C4a, C9), C-reactive protein, Alpha-1-macroglobulin, Alpha-2-macroglobulin, Plasminogen Plasmin heavy chain A Activation peptide are detected in segments after SCI with a higher ratio in Lesion compared with rostral and caudal segments (Table V, supplemental Data S6) which is in line with the data obtained in collected secretome (Fig. 5B, Table VI, supplemental Data S7). RhoA inhibitor treatment increased the level of IgG2b and IgG2c in lesion and caudal segments. In summary, RhoA inhibitor did not diminished the level of the antibodies in segments but their sub classes. The proportion of IgG2 (a,b,c) compared with IgG1 is higher in treated animals. Moreover, concerning the global impact of the RhoA inhibitor treatment 12 h after SCI on the proteome pattern in both sides of lesion segment (Tables V and VI). It is clearly that the treatment turned the proteome of the lesion and the caudal segments close to the one found in control, except for the rostral which is more divergent (Fig. 5A). Moreover, what was the most surprising is the low level of proteins involved in inflammation in tissue and in secretome (Tables V and VI). More proteins which are involved in neurite outgrowth, neurogenesis and synaptogenesis are identified in SCI compared with ones identified with RhoA inhibitor treatment (Tables V and VI). It seems that factors produced by the cells in tissue promote neurogenesis itself and RhoA will modify such process.
To confirm the proteomic data outlining the role of the RhoAi treatment at early stage of the lesion, we decided to design in vivo experiments consisting in the local intraspinal delivery of RhoAi and by an intraperitoneal injection of an immunosuppressant, calcineurin inhibitor (FK506) to diminish inflammation (14). Here were administered RhoAi in an alginate scaffold (with no growth factors) which biocompatibility and its intrinsic beneficial impact on neurite outgrowth was previously showed (16).
Behavioral assessment by BBB open field scale showed that 7 days after treatment with RhoAi and FK506, the score significantly increased to score 5.0 when compared with SCI group (Fig. 6J). Nevertheless, the locomotor function remained unchanged during the entire survival and reached a plateau. In contrary, SCI group showed slow gradual improvement from beginning reaching score 5.0 with certain time delay at 30 days when compared with treated group, but still slightly improving with score around 6 at final 49 days. These data clearly demonstrated that compared with SCI without treatment, the beneficial effect of RhoAi was seen only at early time points of the treatment but not during later survival, even in combination with alginate and anti-inflammatory compound. On the other hand the immunohistochemical analyses of spinal cord tissue showed that RhoAi ϩ FK506 treated group exhibited significantly higher density of synaptophysin (SYN)ϩvesicles at lesion site (Figs. 6A) in comparison to SCI group (Figs. 6A, 6C) but no apparent differences between rostral and caudal segments were detected (Figs. 6E). Similarly, quantification of GAP-43 immunoreactivity outlining regrowth axons within damaged dorsal and lateral white matter tracts did not show significant differences between SCI and SCI RhoAi ϩ FK506 treated groups (Fig. 6I). Dense network of GAP-43 immunoreactive axons of different thickness oriented in various directions were present in both rostral and caudal segments as well as at the lesion epicenter in both experi-  mental groups (Fig. 6I). Furthermore, the sections taken from control-naive rats revealed no GAP-43 immunoreactivity, nor in the gray or white matter regions (data not shown), confirming that GAP-43 positivity strictly correlates with axonal outgrowth after SCI (Fig. 6Ha, b). These data show that single intraspinal delivery of RhoAi in combination with FK506 promote neurite outgrowth and synaptogenesis in distinct segments, but without the ultimate clinical improvement of locomotion. DISCUSSION We previously demonstrated the benefic impact on neurite outgrowth in vivo after delivery of functionalized alginate scaffold loaded with Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) (16). Significant enhancement of spinal cord tissue sparing and an increased number of choline acetyltransferase motoneurons and sensory fibers were registered. We also document the enhancement of axonal outgrowth in corticospinal tracts and an increased density of blood vessels in central lesion. However, although a switch of microglia functional behavior was observed, this therapeutic strategy did not appear to impact astrocytes functions (16). In our recently published spatio-temporal study of acute SCI (13), we demonstrated that in terms of inflammatory and neurotrophic responses, the rostral segments could be clearly distinguished from caudal ones, which indicated a regionalization effect. Among the factors detected in caudal segments, CSPG, neuronal IgG2a were identified along with the MEMO1-RHOA-DIAPH1 signaling pathway (14) which is known to inhibit neurite outgrowth.
In vitro and in vivo studies confirmed the effect of the RhoA inhibitor on synaptogenesis and modulation of neurogenesis. In fact, DRG cell line incubated with conditioned media obtained from 24 h conditioned medium of rostral, lesion and caudal segments 3 days after SCI, as we previously published (14), showed a slight increase of neurites outgrowth whereas in presence of RhoA inhibitor, this outgrowth is significant. The proteomic analyses of the secreted factors of the DRG cells under RhoA inhibitor treatment in presence of the different collected conditioned medium clearly showed difference between segments. Immunoglobulins are overexpressed particularly in the samples associated with lesion, and from caudal segment. AKT proteins family expressed real differences between rostral and lesion segments. Level of AKT3 diminished whereas the ones of AKT1 and AKT2 proteins, RhoAi increased their level in rostral and diminished in lesion and caudal segments. Serpina3c, Snx12, Gm2a, meosin, Timm44, Cthrc1, Stx6, vsp26b, Itih1, Aqp4, aggrecan core protein, BMP1 are over-expressed in caudal in presence of RhoAi compared with rostral segment or lesion. In rostal segment, with RhoA inhibitor, only AK1 and AKT2 are overexpressed, by contrast Timm44, STX6, Cthrc1, AKT3, STX6 are underexpressed. In lesion, most of proteins present in this cluster were under-expressed or had the same level with RhoAi treatment except of Gm2a, hemopexin, Protein disulfide-isomerase, Stx6 that are over-expressed. Global proteomic analyses, confirmed that among the 16 over-expressed proteins under RhoAi treatments, some were already known to be implicated in neurites outgrowth or neurogenesis e.g. Pde6d (29), Ltbp4 (30), Clip2 (31), Enah (32), Vps26b (33), Sema7a  We also showed by our time course proteomic experiments that several transcription factors are produced. Smad proteins family is one of the key players in the regeneration process. Smad1 is known to integrate signals from BMP receptors. Together with Smad4, phosphorylated Smad1 assembles a multi-subunits complex that regulates transcription (40). In the absence of Smad1, conditioned DRG neurons show impairment in axon elongation in vitro (40). Moreover, blockade of BMP signaling with the BMP antagonist Noggin inhibits axonal growth in both naive and preconditioned DRG neurons (40). The LFQ results reflected that Smad1, Smad5 and Smad9 appeared at 24 h whereas Smad2 is always present except at 4 Hours (Table IV). The second important player appears to be the tumor suppressor p53. Previous studies have shown that following SCI, transcriptionally active p53 undergoes a series of acetylation events on its C-terminal domain (41,42). After injury, active gene transcription is necessary to synthesize new proteins needed for axon growth. Acetylated-p53, together with CBP/p300 and PCAF, selectively occupies regulatory regions upstream to the TSS of proneurite and axon-outgrowth genes such as Coronin1b, Rab13, and GAP-43 during an early regenerative response (43). Acetylated-p53 may have a critical role in modulating different transcriptional responses during axonal regeneration (44 -46). For STATs proteins, absence of STAT3, peripheral nerve regeneration is impaired in DRG neurons (47,48). Interestingly, sustained STAT3 expression promotes terminal and collateral sprouting by controlling initiation of axon growth after dorsal columns injury (47,48). Stat3 is detected in detected only at 1 h. Interestingly is the presence of SWI/SNF complex subunit SMARCC1 (BAF155) and SMARCC2 (BAF170) proteins (49). These two proteins belong to the neural progenitors-specific chromatin remodeling complex  6. Quantification of synaptophysin (SYN) positivity at the lesion site (A) and rostral-caudal segments (E) showed significant decrease of SYN after injury, whereas RhoAi ؉ FK506 treatment increased SYN expression significantly at lesion, but not in rostral or caudal segments (E), *p < 0. 05, ** p < 0.001, *** p < 0.0001, One-way ANOVA. Representative images of synaptophysin immunoreactivity (SYN, green) revealed intensely stained synaptic vesicles -punctate structures within the spinal cord-lesion site in control (B) and treated group (D), note only occasional synaptic vesicles on sections from SCI rats (C). Confocal imaging with double labeling of GAP-43 (red) and SYN (green) antibodies, confirmed enhanced growth of axons with dense synaptic vesicles distribution after RhoAi ϩ FK506 treatment at lesion (G). Note, areas containing GAP-43 positive fibers, but only occasional SYN expression at lesion in SCI group (F). Quantification of GAP-43 positive fibers did not reveal significant differences between SCI and SCI RhoAiϩ FK506 groups (I), outlining growing axons within damaged dorsal and lateral white matter tracts (Ha, Hb). Note, high number of GAP-43 axons penetrating the lesion site, with dense (arrowheads) or sporadic positive synaptic vesicles (asterisk) (Ha). Scale bar ϭ 25 m. BBB open field test in SCI rats (blue line) and SCI rats treated with RhoAi ϩ FK506 (red line) at 0, 7, 14, 21, 28, 35, 42 and 49 days post injury, reveals that BBB score in treated rats reached 5 at 14 days and remained unchanged, whereas, rats without treatment reached score 5 at 30 days and further slightly improved (J).
(npBAF complex) and the neuron-specific chromatin remodeling complex (nBAF complex). The npBAF complex is essential for the self-renewal/proliferative capacity of the multipotent neural stem cells. The nBAF complex along with CREST plays a role regulating the activity of genes essential for dendrite growth (50). These two proteins are overexpressed after RhoAi treatment. Altogether these data pointed out that the chromatin-remodeling BAF complex (formed by the two subunits BAF155 and BAF170) known to play a role in brain development (51) is a key target of RhoAi treatment in acute SCI. BAF (Brg1/Brm Associated Factors) complex is a multisubunit chromatin remodeling complex that alters the position of nucleosomes thereby regulating gene expression. Although, specific BAF subunits selectively interact with transcription factors to regulate gene expression programs, the logic underlying the composition of the BAF complex remains largely unknown. Here we showed that this complex can interact with Smad2/3 and TP53 transcription factors at the early stage of the treatment impacting the cellular traffic and increase vesicles production that are secreted at least 24 h after treatment. The transcription factors Smad2 and Smad3 are known to mediate a large set of gene responses induced by TGF-␤ and recent observations have showed interactions between the two Smads and BAF complex. BAF complex is incorporated into transcriptional complexes that are formed by activated Smads in the nucleus, on target promoters (52). At 24 h, all TF implicated in control of neurites outgrowth factors expression are present. Expression in DRG cell of robo1, nestin, N chimaerin, glomulin, MAGED 1, TRPV2 in presence of R1 conditioned medium or, slit, FARP1, srGAP2, and STK25 in C1 conditioned medium confirms the differential activation of the DRG cells dependently to the medium considered.
In this context, we investigated the impact of a local treatment of RhoAi in conjunction with an intraperitoneal injection of FK506. The main scenario was to combine factors with both anti-inflammatory and neuro-stimulatory potential to scale up the treatment. Because in our previous study, we did not observe beneficial effect of sustained, long term FK506 delivery, we have decided to shorten the delivery regiment up to 14 days (53). Spinal cord sections dissected from different segments revealed numerous synaptophysin labeling at the FIG. 7. Schematic representation of the positive growth cone guidance after RhoA inhibitor treatment. The scheme integrates the specific proteins identified after proteins cell extraction or from the secretome. Signaling pathways linked to identify proteins are also presented. lesion site and adjacent segments. Moreover, dense network of GAP-43 immunoreactive axons of different thickness oriented in various directions were present in both rostral and caudal segments as well as at the lesion epicenter. These data confirmed that the treatment has enhanced neurite outgrowth in both segments with dense synaptic contacts at the epicenter of the lesion. The BBB score showed a significant improvement at 7 days after treatment and continued with plateau characteristics, whereas the SCI group revealed delayed and gradual locomotor improvement during entire survival. These data clearly showed different locomotor outcome between both groups, thus revealing beneficial effect of RhoAi ϩ FK 506 delivery at the initial phase of the treatment, but not at longer survival. Thus, treated group launched recovery much earlier than SCI, which regenerate more slowly but at overall survival both groups revealed similar recovery pattern at long term (49 days). This could be caused by low dose of RhoAi delivered via single application that was probably not sufficient for long term stimulation and inhibition of RhoA pathways. For example, previous study demonstrating beneficial recovery of injured CNS axons treated with RhoAinhibiting NSAID ibuprofen delivery was initiated 1 h after the injury until 5 days post-trauma, via daily subcutaneous injections (54). It is also difficult to determine whether concentration of RhoAi (1 g/10 l) that was set according to published studies, represented an optimal concentration and was biologically attainable to the concentration used in vitro. To address this, it would require a complex of comparative and dose response studies processed under in vitro and in vivo conditions. Second important factor that should be mentioned is the route of RhoAi administration. Oral, intramuscular, subcutaneous, or intravenous drug deliveries which imposes a minimal burden on the animals could be applied on daily basis, but not intraspinal-local delivery which requires surgery. Thus, complex factors have to be taken in account to develop an optimal treatment scenario that could complementary sum the efficacy of RhoAi treatment.
Taken together, we demonstrated here that RhoAi treatment provokes sequential activation events in time course resulting in chromatin remodeling, selective and timely activation of transcription factors leading to the expression of a large array of factors involved in neurite outgrowth. Major factors include receptors (Robo1, Plexin A3, Plexin B2, UNC5C, neuropilin 1), ligands (semaphorin 7A, netrin, Ephrin A5, Slit2, BDNF/NT3) and transcription factor (␤ catenin, WLS, Phox2a, Pho2b) previously shown to regulate axonal regrowth (Fig. 7). We confirm in vivo their presence under RhoAi treatment in tissue and in secreted factors. Regional differences regarding the effects of conditioned medium generated from distinct spinal cord segments indicate that each segment is endowed with a specific ability to secrete axonal regrowth-modifying molecules. Interestingly, in this context, both the R1 and C1 segments harbor a potential to produce such neurites outgrowth factors allowing growth cone formation and activation as we evidence by in vitro and in vivo experiments. These segments are the most impacted by the RhoAi treatment at the early stage of the growth cone formation leading enhanced neurite outgrowth and synaptogenesis. Thus, to improve the efficiency of SCI treatment with RhoAi, it appears essential to specifically target the R1 and C1 segments and to operate in a timely fashion to bypass the regeneration plateau observed 7 days after the treatment.

DATA AVAILABILITY
The raw data and annotated MS/MS spectra were deposited at the ProteomeXchange Consortium (http://proteome central.proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD004639.