Animal surgery
Specific pathogen-free degree Sprague-Dawley (SD) rats used in this study were purchased from the Animal Center of Nantong University (animal licenses No. SCXK [Su] 2014-0001 and SYXK [Su] 2012-0031). All animal experimental procedures were approved by the Ethics Committees of Experimental Animals, Jiangsu Province, China (approval ID: S20231219-041) and performed in accordance with the guidelines of Nantong University Institutional Animal Care.
Male adult SD rats (8-week-old, weighting 180–220 g) were subjected to sciatic nerve axotomy-induced peripheral axon injury or dorsal root axotomy-induced central axon injury, as previously described with modifications(Avraham et al. 2021). Briefly, for sciatic nerve axotomy, after anesthetization, a skin incision on the lateral aspect of the mid-thigh of rat hind limb was made and exposed rat sciatic nerves were subjected to a sharp axotomy. For dorsal root axotomy, a midline incision at the lumbar (L)2-L3 vertebral level was made, the dura mater was removed, and exposed rat L4 and L5 dorsal roots were subjected to axotomy. Rats underwent sciatic nerve or dorsal root exposure without axotomy were designated as sham-operated. L4 and L5 DRGs were collected at 1 day after axotomy or sham surgery and subjected to RNA sequencing.
Sequencing
RNA sequencing of L4 and L5 DRGs following sciatic nerve injury versus sciatic nerve sham surgery and dorsal root injury versus dorsal root sham surgery has been published with sequencing data stored in Genome Sequence Archive database (accession number CRA006070)(Cao et al. 2022). RNA sequencing was performed on an HiSeq™ 4000 by Genedenovo Biotechnology Co., Ltd. (Guangzhou, Guangdong, China). Transcripts abundances were quantified using StringTie and differential expression testing was performed using edgeR(Robinson et al. 2010).
Single-cell sequencing of 1-day-old neonatal and 8-week-old adult rat DRGs was performed as previously described with sequencing data stored in NCBI database (accession number GSE147615)(Zhang et al. 2021). Digested single cell suspensions were loaded on the 10 × Chromium system, libraries were prepared using 10 × Genomics GemCode Single-Cell 3′ Gel Bead and Library Kit, and sequencing was conducted using Illumina NovaSeq platform by NovelBioinformatics Ltd., Co. (Shanghai, China). Raw data was processed by fastp quality control and analyzed with Cell Ranger (v3.0.0) for barcode identification, mapping, and gene counting. Transcripts abundances were quantified after normalization. Sequencing data were categorized to clusters using Seurat 3.1 software package. Clusters were presented in a t-distributed stochastic neighbor embedding (tSNE) plot using a dimensional reduction algorithm.
Quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA of collected DRG tissues or cultured DRG neurons was extracted using RNA-Quick Purification Kit (Yeasen Biotechnology Co., Beijing, China) or Cell RNA Extraction Kit (UU-Bio Technology Co., Suzhou, Jiangsu, China), respectively, and then treated with amplification-grade DNase I (Thermo Fisher Scientific). After RNA quantification using a Nanodrop 1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA), total RNA was reverse transcribed to cDNA using HiScript®ⅡQ RT SuperMix for qPCR (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. Quantitative RT-PCR was then performed using ChamQ™ SYBR® qPCR Master Mix (Vazyme) on an ABI StepOne system (Applied Biosystems, Foster City, CA, USA). Experiments were repeated in triplicate. The Ct values of target gene DBNDD2 were compared with the Ct values of the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative abundance of DBNDD2 was quantitated using the comparative 2−ΔΔCt method. Primers were synthesized by Sangon Biotech (Shanghai, China). The sequences of specific primers for target gene DBNDD2 were DBNDD2 (forward) 5’-CGTCAGACAGGACCACATCC-3’ and DBNDD2 (reverse) 5’-TGTCTCCTCCCCCATCACTT-3’ while the sequences of specific primers for reference gene GAPDH were GAPDH (forward) 5’-ACAGCAACAGGGTGGTGGAC-3’ and GAPDH (reverse) 5’-TTTGAGGTGCAGCGAACTT-3’.
Immunohistochemistry
Rat L4 and L5 DRGs were washed with PBS, fixed with 4% paraformaldehyde, and cryoprotected using 30% sucrose. Rat DRGs were then embedded in O.C.T., frozen, and cut to tissue sections. DRG sections were incubated with anti-Tuj1 (1:1000; Abcam, catalog # ab18207, Cambridge, Massachusetts, USA), anti-DBNDD2 (1:200; Proteintech, catalog # 27623-1-AP, Rosemont, Illinois, USA), anti-enhanced green fluorescent protein (EGFP; 1:100; Abcam, catalog # ab184601), or anti-superior cervical ganglion-10 protein (SCG10; 1:500; Novus Biologicals, catalog # NBP1-49461, Littleton, Colorado, USA) primary antibodies at 4°C overnight and subsequently with Alexa Fluor 488-conjugated donkey anti-mouse IgG (1:500; Proteintech, catalog # SA00013-5), Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500; Proteintech, catalog # SA00013-6), Cy3 goat anti-mouse IgG (H + L) (1:500; Proteintech, catalog #SA00009-1), or Cy3 goat anti-rabbit IgG (H + L) (1:500; Proteintech, catalog #SA00009-2) secondary antibodies. Nuclei were counterstained with DAPI Fluoromount-G stain (SouthernBiotech, catalog # 0100 − 20, Birmingham, Alabama, USA). Immunofluorescence images were captured using a Zeiss Axio Imager M2 microscope (Jena, Germany). Exposure time and gain were maintained constant for each fluorescence channel during image capture.
Primary DRG neuron isolation and culture
DRGs collected from 1-day-old neonatal and 8-week-old adult rats were dissected into small pieces and subjected to tissue digestion. For neonatal rats, dissected DRGs were digested with 3 mg/ml collagenaseⅠfor 30 minutes and 0.25% trypsin for 20 minutes while for adult rats, DRGs were digested with 3 mg/ml collagenaseⅠfor 90 minutes and 0.25% trypsin for 5 minutes. After adding complete culture medium containing 10% fetal bovine serum albumin (Sigma, St. Louis, MO, USA) to terminate digestion, cells were filtered through a 70-µm cell strainer. Cell pellets were re-suspended in 15% BSA Albumine Bovine V (BioFroxx, Einhausen, Germany) and then subjected to centrifugation. Seperated neonatal or adult rat DRG neurons were cultured in Neurobasal medium (Gibco, Grand Island, New York, USA) containing 2% B27 supplement (Gibco) and 2 mM L-glutamine (ThermoFisher Scientific, Waltham, MA, USA) and plated in cell culture dishes pre-coated with poly-L-lysine (PLL).
DRG neuron transfection
Primary cultured neonatal or adult rat DRG neurons were transfected with the small interfering RNA (siRNA) fragments against DBNDD2 to knockdown DBNDD2 expression in neurons. DRG neurons were transfected with siRNAs targeting DBNDD2 (si-DBNDD2) or a control scrambled siRNA with a random sequence using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer’s instructions. The sequences of siRNAs targeting DBNDD2 were as follows: DBNDD2-siRNA-1, 5’-GAAGTTCTTCGAGGACATT-3’; DBNDD2-siRNA-2, 5’-GGTGGAATTTATTGACCTT-3’; and DBNDD2-siRNA-3, 5’-GCAGTCCAAATCCAAGTGA-3’. The sequence of the control siRNA was 5’-GGCUCUAGAAAAGCCUAUGC-3’. The siRNA fragments were synthesized by RibiBio Biotechnology Co., Ltd. (Guangzhou, Guangdong, China).
Western blot
Cultured DRG neurons were lysed with RIPA buffer (Thermo Fisher Scientific). After measuring the concentrations of protein extracts using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific), equal amounts of protein samples were electrophoresed on SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Membranes were incubated with anti-DBNDD2 (1:200; Proteintech, catalog # 27623-1-AP) or anti-β-actin (1:1000) primary antibodies at 4°C overnight and subsequently with horseradish peroxidase-conjugated secondary antibodies. Protein bands were developed using the ECL Western blotting detection kit (Thermo Fisher Scientific).
In vitro neurite growth assay
Cultured neonatal rat DRG neurons were fixed with 4% paraformaldehyde at 36 hours after transfection and subjected to anti-Tuj1 immunostaining. Adult rat DRG neurons were replaced by trypsin treatment and seeded on to glass coverslips pre-coated with PLL. At 24 hours after cell culture, neurons were washed with PBS, fixed with 4% paraformaldehyde, and immunostained with anti-Tuj1 antibody. The longest and total lengths of neurites from each neonatal or adult rat DRG neuron were measured and quantified using the Image J software.
For myelin experiments, myelin fractions were extracted from adult rat brain tissues as previously described with modifications(Ma et al. 2019). Briefly, adult rat brain tissues were homogenized in 0.30 M sucrose, layered over 0.83 M sucrose, and centrifuged to gather the crude myelin layers between the interfaces. The process was repeated to purify collected myelin extracts and the glass coverslips were coated with PLL and myelin extracts.
In vitro neurite regeneration assay
Adult rat DRG neurons were cultured onto the somal compartments of microfluidic chambers pre-coated with PLL (catalog # SND150, Xona 2-compartment SND 150, Xona Microfluidics LLC). Neurites that entered the axonal compartments after cell culture were dissected and removed using a 0.08 mpa vacuum suction 3 times with 20 seconds each time. Dissected neurites were cultured for additional 24 hours, fixed with 4% paraformaldehyde, and immunostained with anti-Tuj1 antibody to observe neurite elongation and regeneration. The lengths of regenerated neurites were measured and quantified using the Image J software.
Intrathecal injection of adeno-associated virus (AAVs)
Adult SD rats were anesthetized, shaved to expose the skin around the lumbar region, and injected with AAV that carry shRNA against DBNDD2 (pAAV-U6-shRNA(DBNDD2)-CMV-EGFP-WPRE) or a control AAV (pAAV-U6-shRNA(NC)-CMV-EGFP-WPRE). The AAVs were packaged by OBiO Biotechnology Co., Ltd. (Shanghai, China). A total of 10 µl of AAV solution was slowly injected into the cerebrospinal fluid between vertebrae L4 and L5 using a 25 µL Hamilton syringe and the needle was left in place for additional 2 minutes. After leaving rats injected with AAVs to recover for 21 days, the left sciatic nerve at 10 mm above the bifurcation into the tibial and common fibular nerves was crushed with forceps as previously described(Yi et al. 2015). At 3 days after sciatic nerve crush injury, sciatic nerve tissues were collected and then subjected to SCG10 immunostaining. The lengths of regenerated nerves were measured and quantified using the Image J software.
Bioinformatic analysis
Molecules that interact with DBNDD2 were discovered and visualized using the STRING data resource(Szklarczyk et al. 2023). Molecules that interact with DBNDD2 or controls the expression of DBNDD2 were determined using the Pathway Commons(Rodchenkov et al. 2020). Upstream transcription factors of DBNDD2 were predicted using JASPAR database(Castro-Mondragon et al. 2022), animalTFDB 3.0 database(Hu et al. 2019), Gene Transcription Regulation database (GTRD)(Kolmykov et al. 2021), and hTFtarget database(Zhang et al. 2020). The intersection of potential upstream transcription factors of DBNDD2 was obtained using Venny (https://bioinfogp.cnb.csic.es/tools/venny/index.html). The binding of transcription factor estrogen receptor 1 (ESR1) to the promoter region of DBNDD2 was generated using motif-based sequence analysis tool FIMO-MEME Suite(Bailey et al. 2009).
Statistical analysis
All quantitative data were presented as the mean ± the standard error of the mean (SEM). The numbers of independent experiments were indicated in the figure legends. Unpaired two-tailed student’s t-test or one-way ANOVA followed by a Dunnett’s or a Tukey’s multiple comparison post hoc test was performed using GraphPad Prism, and significance was set at p-value < 0.05.