Suppressing DBNDD2 promotes neuron growth and axon regeneration in adult mammals

doi:10.21203/rs.3.rs-5265998/v1

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Suppressing DBNDD2 promotes neuron growth and axon regeneration in adult mammals

https://doi.org/10.21203/rs.3.rs-5265998/v1

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Effective axon regeneration is essential for the successful recovery of nerve functions in patients with axon injury-associated neurological diseases. Certain self-regeneration occurs in injured peripheral axonal branches of dorsal root ganglion (DRG) neurons but does not occur in their central axonal branches. By performing rat sciatic nerve or dorsal root axotomy, we determined the expression of the dysbindin domain containing 2 (DBNDD2) in the DRGs after the regenerative peripheral axon injury or the non-regenerative central axon injury, respectively, and found that DBNDD2 is down-regulated in the DRGs after peripheral axon injury but up-regulated after central axon injury. Moreover, we found that DBNDD2 expression differs in neonatal and adult rat DRGs and is gradually increased during development. DBNDD2 knockdown promotes the outgrowth of neurites in both neonatal and adult rat DRG neurons and stimulates robust axon regeneration in adult rats after sciatic nerve crush injury. Bioinformatic analysis data showed that transcription factor estrogen receptor 1 (ESR1) interacts with DBNDD2, exhibits a similar expression trend as DBNDD2 after axon injury, and may targets DBDNN2. These studies indicate that reduced level of DBNDD2 after peripheral axon injury and low abundance of DBNDD2 in neonates contribute to axon regeneration and thus suggest the manipulation of DBNDD2 expression as a promising therapeutic approach for improving recovery after axon damage.

axon damage

peripheral axon injury

central axon injury

RNA sequencing

development

single-cell sequencing

DBNDD2

DRG neuron

neuron growth

axon regeneration

Axon damage interrupts neuronal communications and signal communications between neurons and their target cells, disrupts the physiological functions of neurons, results in partial or complete loss of motor, sensory, and autonomic functions, and normally induces neuropathological disorders(Winter et al. 2022; Williams et al. 2020). Success axon growth and regeneration is fundamental for the re-establishment of neural circuits and the recovery of impaired nerve functions. Unfortunately, in the adult mammalian central nervous system, injured axons are incapable of regenerating, leading to failure of the restoration of normal nerve function(Uyeda and Muramatsu 2020; Li et al. 2020a; Bradke 2022). On the contrast, in immature mammals, injured central nerves are able to regrow following axon injury, indicating that adult mammals process certain inherent regeneration potential (Montero and Huang 2022). More importantly, except for immature mammalian central nerves, mammalian peripheral nerves have a regenerative ability following axon injury, albeit their regeneration is limited and the regeneration outcome is often incomplete, especially after severe axon injury such as nerve transection with long distance(He and Jin 2016) (Mahar and Cavalli 2018; Robinson 2022). Exploring the intrinsic factors underpinning successful axon regrowth following peripheral nerve injury as well as the unsuccessful axon regrowth following central nerve injury is therefore essential for the understanding of the molecular mechanisms that controls the neuronal intrinsic regeneration capacity(Mahar and Cavalli 2018).

Sensory neurons in dorsal root ganglion (DRG) are pseudounipolar neurons with peripheral axonal branches that proceed along peripheral nerves and central axonal branches that proceed along the dorsal roots into the spinal cord(Hutson et al. 2019) (Zhao et al. 2024). Peripheral and central axonal branches behave differently after axon injury. The regeneration rate of injured dorsal roots is only at half the rate of injured peripheral axonal branches and the regrowth of injured dorsal roots stops at the dorsal root entry zone(Avraham et al. 2021). DRG neurons are thus commonly used to discriminate injury responses in the same neuronal soma following damage to the regenerative peripheral axonal branches or the non-regenerative central axonal branches(Avraham et al. 2021; Kong et al. 2020; Palmisano et al. 2019). For instance, the comparison of the levels of reactive oxygen species after sciatic nerve crush or dorsal column crush demonstrates that the production of reactive oxygen species is increased in DRGs after peripheral axon injury but not altered after central axon injury(Hervera et al. 2019). Elevated reactive oxygen species promotes the outgrowth of axons and the recovery of nerve functions, indicating that the assessment of diverse molecular changes after peripheral or central axon injury is critical for the identification of regeneration-associated molecules(Hervera et al. 2019). Likewise, calcium activation of protein phosphatase 4 and protein phosphatase 4–dependent de-phosphorylation of histone deacetylase 3, a process that inhibits the activity of histone deacetylase 3, are only detected after peripheral axon injury but not observed after central axon injury(Hervera et al. 2019). Histone deacetylase 3 inhibition modulates several regenerative pathways, activates the regenerative program, benefits neurite outgrowth, and is even capable of promoting the regeneration of sensory axons following spinal cord injury(Hervera et al. 2019). Deeper understanding of differentially expressed molecules after the regenerative peripheral axon injury and the non-regenerative central axon injury of DRG neurons is hence crucial for revealing novel regeneration associated factors and developing successful therapeutics.

The dysbindin domain containing 2 (DBNDD2), also named casein kinase-1 binding protein, is a dysbindin protein family member involved in the negative regulation of casein kinase-1 activity(Yin et al. 2006). A recent study demonstrates that by binding to casein kinase-1 and inhibiting casein kinase-1 activity, DBNDD2 reduces the amounts of total and insoluble α-synuclein and thus may be conductive to the treatment of Parkinsons's disease(Elsholz et al. 2021). However, the biological effects of DBDNN2 on axon regeneration during nerve injury and regeneration process remains to be explored.

Taking advantage of high throughput sequencing data, in the current study, we found that in adult rats, DBNDD2 expression is reduced after peripheral axon injury but increased after central axon injury and thus predicted that DBNDD2 might be a negative regulator of axon regeneration. Indeed, we showed that DBNDD2 knockdown in neurons facilitates neurite growth and axon elongation. Our results uncover the functional roles of DBNDD2 in axon regeneration and imply that drugs targeting DBNDD2 may be of benefit in treating traumatic nerve injury as well as other axon damage-associated neurological disorders such as stroke and glaucoma.

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.

DBNDD2 is differentially expressed in adult rat DRGs following nerve injury

We analyzed sequencing data of rat DRGs after sciatic nerve or dorsal root axotomy and examined the expression levels of DBNDD2 gene in rat DRGs after peripheral and central axon injuries. At 24 hours after sciatic nerve injury, the expression of DBNDD2 gene in rat DRGs was dropped by more than 65% as compared with its expressions in sciatic nerve sham injured animals. Following dorsal root injury, the expression of DBNDD2 gene in rat DRGs increased to 1.85 folds as compared to the baseline expression of DBNDD2 gene in sciatic dorsal root injured animals (Fig. 1A). In consistent with sequencing data, RT-PCR results showed that DBNDD2 gene expression in the DRGs was remarkably decreased after sciatic nerve injury but significantly increased after dorsal root injury (Fig. 1B). DBNDD2 protein expression in the DRGs was also found to be affected by axonal injury. The fluorescence intensity of DBNDD2 in the DRGs seemed to be much lower following sciatic nerve injury relative to that of DBNDD2 in the DRGs under the uninjured naïve state while the intensity of DBNDD2 seemed to be much higher following dorsal root injury (Fig. 1C). These observations from RNA sequencing, RT-PCR validation, and immunohistochemistry immunostaining fully demonstrate that in the DRGs, DBNDD2 presented an opposite following injury to the peripheral and central axonal branches. In addition, DBNDD2 signal was found to co-localized with those of a neuronal cell marker Tuj1 (Fig. 1C). It demonstrates that injury to peripheral and central axonal branches of the DRGs elicits different changes of DBNDD2 in DRG neuronal somas and hints that differentially expressed DBNDD2 contribute to the different regeneration capacity of injured peripheral and central axons.

DBNDD2 is differentially expressed in the DRGs at different developmental stages

We next compared the expression levels of DBNDD2 gene in the DRG neurons of neonatal and adult rats, rats at two diverse developmental stages with different regeneration capacities. Using single-cell sequencing, a high-throughput analysis tool that identifies cellular heterogeneity and seperates diverse cell populations, neurons were distinguished from other cell types in the DRGs and DRG neurons in neonatal and adult rats were recognized (Fig. 2A). The tSNE plot showed that DBNDD2 gene was expressed in DRG neurons of both neonatal and adult rats and was present in a larger number of DRG neurons in adult rats as compared with in neonatal rats (Fig. 2B). Quantification of DBNDD2 gene expression in the DRG neurons of neonatal and adult rats demonstrated an obviously higher expression level of DBNDD2 gene in adult rats (Fig. 2C). Besides 1-old-old neonatal and 8-week-old adult rats, the temporal expression pattern of DBNDD2 gene in 2-week-old and 4-week-old rats were also determined. Similar as in 8-week-old adult rats, elevated expression of DBNDD2 gene was detected in the DRGs of 2-week-old and 4-week-old rats as compared with the DRGs of neonatal rats (Fig. 2D). The observed elevation of DBNDD2 gene expression in developing animals with reduced regeneration capacity implies that DBNDD2 may be a negative regulator of axon regeneration.

DBNDD2 deficiency promotes the growth of neonatal rat DRG neurons

Primary neonatal rat DRG neurons were isolated, cultured, and gene manipulated to assess the role of DBNDD2 (Fig. 3A). RT-PCR experiments showed that transfection of neonatal rat DRG neurons with three siRNA fragments against DBNDD2 successfully reduced DBNDD2 gene expression to less than 20% (Fig. 3B). DBNDD2-siRNA-1 and DBNDD2-siRNA-2, two siRNA fragments with relative higher knockdown efficiency, were subsequently used for functional investigation. Following the transfection of DBNDD2-siRNA-1, the intrinsic axon growth ability of neonatal rat DRG neurons seemed to be activated and the neurites of neonatal neurons were noticeably much longer (Fig. 3C). Summarized data of more than 400 DRG neurons showed that the transfection of DBNDD2-siRNA-1 increased the lengths of total neurites from 180.09 µm in the control-siRNA treated group to 247.44 µm in the DBNDD2-siRNA-1-siRNA transfected group. The lengths of the longest neurites also increased from 113.68 µm in the control-siRNA treated group to 142.20 µm in the DBNDD2-siRNA-1-siRNA transfected group (Fig. 3D). DBNDD2-siRNA-2 transfection induces comparable cellular responses, with the lengths of total neurites increased from 167.06 µm in the control-siRNA treated group to 255.98 µm in the DBNDD2-siRNA-1-siRNA transfected group and the length of the longest neurites increased from 109.85 µm to 154.40 µm (Figs. 3C and 3D). These data indicate that in neonatal DRG neurons, silencing DBNDD2 is able to enhance neuronal sprouting and neurite growth.

DBNDD2 deficiency promotes the growth of adult rat DRG neurons and the elongation of injured axons

Next, we explored the effects of DBNDD2 knockdown on primary cultured adult DRG neurons. Given that adult neurons have diminished growth ability relative to neonatal rats, adult DRG neurons were subjected to a culture-and-replating protocol to recapitulate nerve injury process and to boost the intrinsic growth capacity of adult neurons (Fig. 4A). Transfection of adult DRG neurons with DBNDD2-siRNA-1 and DBNDD2-siRNA-2 decreased the expression of DBNDD2 gene to less than 50% (Fig. 4B). Western blot result also demonstrates that the protein abundance of DBNDD2 was effectively reduced after siRNA transfection (Figure S1). Similar as neonatal DRG neurons, in adult DRG neurons, decline expression of DBNDD2 contributed to significantly enhanced neurite extension (Figs. 4C and 4D). The growth conditions of adult DRG neurons cultured on myelin inhibitory substrates were further investigated. For adult DRG neurons transfected with the control siRNA, compared with neurons cultured on PLL-coated plates, the total lengths and the longest lengths of neurites of adult DRG neurons cultured on plates coated with PLL and extracted myelin were reduced by approximately 25%. Knockdown of DBNDD2 not only boosted the growth of neurons cultured on LL-coated plates, but also supported the elongation of neurites when adult DRG neurons were cultured on myelin substrates (Figs. 4E and 4F). These results show that myelin substrates considerably suppress neuronal growth while DBNDD2 silencing prominently augments neuronal growth on myelin substrates.

In addition to the culture-and-replating cell model, by using a two-compartment microfluidic chamber, the axons of adult DRG neurons were directly severed in vitro and the subsequent regrowth of injured neurites were visualized. In control siRNA-transfected neurons, regenerated neurites were observed in the axonal compartments. Adult DRG neurons transfected with DBNDD2-siRNA-1 had elongated neurites as compared with neurons transfected with the control siRNA. Likewise, following the transfection of DBNDD2-siRNA-2, the average lengths of regenerated neurites seemed to be longer relative to the control group, although not significant (Figs. 4G and 4H). These straightforward observations demonstrate that knockdown of DBNDD2 in DRG neurons advances the regrowth and elongation of injured neurites, indicating that DBNDD2 may function as an important molecular target for enhancing the intrinsic regeneration capacity of adult neurons.

DBNDD2 deficiency facilitates nerve regeneration after sciatic nerve injury

To determine the in vivo roles of DBNDD2 knockdown, a AAV-EGFP-DBNDD2 shRNA or a control AAV was intrathecally administered to adult rats (Fig. 5A). RT-PCR results showed that at 21 days after the injection of AAV-DBNDD2-shRNA, the expression of DBNDD2 gene in the DRGs was much lower relative to in the DRGs of rats injected with the control AAV (Fig. 5B). Immunostaining images displayed the co-labelling of EGFP and neuronal marker Tuj1 in rat DRGs (Fig. 5C) as well as noticeably diminished immunofluorescence signals of DBNDD2 in Tuj1-labelled neurons (Fig. 5D), indicating that AAV delivery of DBNDD2shRNA effectively silenced DBNDD2 expression in neurons. After the validation of success knockdown of DBNDD2 in DRG neurons, rats were subjected to sciatic nerve crush injury. We collected rat sciatic nerves at 3 days after crush injury, labelled sciatic nerves with regenerating sensory axon marker SCG10, and observed that in rats injected with AAV expressing DBNDD2 shRNA, the length of regenerated axons increased to roughly 1.5-fold relative to rats injected with control AAV (Figs. 5E and 5F). Observations from animal studies indicate that AAV delivery of shRNA against DBNDD2 benefits axon elongation and nerve regeneration.

Identification of DBNDD2-associated molecules

To determine molecules that interact with DBNDD2, top 5 STRING interactants of DBNDD2 calculated based experimental scores, including casein kinase 1 isoform delta (CSNK1D), casein kinase 1 epsilon (CSNK1E), casein kinase 1 isoform gamma 2 (CSNK1G2), casein kinase 1 isoform gamma 3 (CSNK1G3), and secreted frizzled related protein 2 (SFRP2), were displayed in a STRING interaction network (Fig. 6A). DBNDD2 closely interacts with CSNK1D and CSNK1G2 while CSNK1D and CSNK1G2 have combined confidence of the functional interactions with CSNK1E and CSNK1G3, respectively(Yin et al. 2006). A more comprehensive DBNDD2-centered molecular network was generated using the integrated Pathway Commons web resource (Fig. 6B). Besides molecules in the STRING protein-protein network, DBNDD2 is found to be interacted with vasoactive intestinal peptide receptor 2 (VIRP2), angiotensin II receptor type 1 (AGTR1), lamin A/C (LMNA), and calcium binding and coiled-coil domain 2 (CALCOCO2). DBNDD2 is also identified to be controlled by zine finger E-box binding homeobox 1 (ZEB1), forkhead box F2 (FOXF2), transforming growth factor beta 1 (TGFB1), signal transducer and activator of transcription 5B (STAT5B), ESR1, myocyte enhancer factor 2A (MEF2A), MDS1 and EVI1 complex locus (MECOM), paired box 4 (PAX4), forkhead box O4 (FOXO4), transcription factor CP2 (TFCP2), androgen receptor (AR), and forkhead box O1 (FOXO1).

Next, we explored the expression levels of these DBNDD2-associated molecules in the DRGs after peripheral or central axon injury according to sequencing data. The heatmap displayed the relative fold changes of these DBNDD2-associated molecules and labeled genes with an elevated expression relative to the corresponding sham control with red color and genes with a reduced expression relative to the corresponding sham control with green color. It is demonstrated that although many of these DBNDD2-associated molecules did not show apparent expression changes after dorsal root axotomy-induced central axon injury, some molecules were differentially expressed in the DRGs after sciatic nerve axotomy-induced peripheral axon injury. For instance, VIPR2, AGTR1A, AGTR1B, ESR1, MECOM had a similar expression trend as DBNDD2 and were down-regulated in the DRGs after sciatic nerve injury. On the contrast, SFRP2 and PAX4 exhibited an opposite expression pattern as DBNDD2 and were up-regulated in the DRGs after sciatic nerve injury (Fig. 6C).

Given that many DBNDD2-associated molecules that exhibit similar expression trends as DBNDD2 are transcription factor-coding genes, we then predicted the potential upstream transcription factors of DBNDD2. A total of 23, 5330, 1490, and 268 transcription factors of DBNDD2 were screened using JASPAR, animalTFDB 3.0, GTRD, and hTFtarget databases. Three common elements, including CAMP responsive element binding protein 1 (CREB1), transcription factor specificity protein 1 (SP1), and ESR1, a molecule that controls DBNDD2 according to Pathway Commons web resource, located at the intersection of these databases (Fig. 6D). Transcription factor ESR1 may directly bind to the putative binding site located at -1915 ~ -1898 in the promoter region of DBNDD2 and regulate DBNDD2 expression (Fig. 6E).

The molecular functions of DBNDD2, a protein that is highly expressed in the nervous system, are largely unclear besides its casein kinase-1 binding and inhibiting roles(Elsholz et al. 2021). Herein, we examined the expression changes of DBNDD2 in rat DRGs during regeneration and development by sequencing, RT-PCR, and immunostaining, studied the biological effects of DBNDD2 on DRG neurons by siRNA transfection, and found that DBNDD2 knockdown is beneficial for axon growth and nerve regeneration.

Rats subjected to partial or complete nerve injury are widely applied as appropriate models to explore the pathological basis of nerve injury and to assess novel medications and profound treatment strategies(Gordon and Borschel 2017). DRG neurons process peripheral and central axon branches with different regeneration capacities and thus are valuable for the investigation of the intrinsic mechanisms underlying success axon regeneration. The peripheral projecting axons of rat L4 and L5 DRGs, together with nerve fibers of spinal cord motor neurons, make up peripheral nerves with the largest diameters, that are sciatic nerves(Bobkiewicz et al. 2017). Hence, surgeries such as crush, stretch, percussion, and transection to sciatic nerves induce injuries to peripheral axonal branches of L4 and L5 DRG neurons. The central axonal branches projected from L4 and L5 DRG neuronal somas extend along dorsal roots, enter the spinal cord via the dorsal root entry zone, and then bifurcate to ascending central axonal branches towards the brain and descending central axonal branches towards the cauda equine(Smith et al. 2012; Zheng et al. 2019). Compared with spinal cord injury that impairs the ascending central axonal branches only, injury at the dorsal root before the branch point impairs the whole central axonal branches and is considered as a well suited surgical model to study central axon regeneration(Smith et al. 2012).

Consequently, in the current study, we first investigated sequencing data of rat L4 and L5 DRGs after sciatic nerve axotomy-induced peripheral axon injury and dorsal root axotomy-induced central axon injury, aiming to decipher the expression changes of DBNDD2 gene after injury to DRG neuron peripheral and central axonal branches with dissimilar generation capacities. RNA bulk sequencing, together with consistent validation outcomes, showed that DBNDD2 gene expression in rat DRGs is substantially reduced after peripheral axon injury but elevated after central axon injury. It is worth noting that besides DRG neurons, there exist a large number of different cell types in rat DRGs whereas RNA bulk sequencing determines the global gene expression in tissues and organs without distinguishing the transcriptional heterogeneity of cell populations. To verify whether DBNDD2 is indeed altered expressed in DRG neurons, we collected L4 and L5 DRGs from rats underwent peripheral or central axon injury as well as uninjured rats, double immunostained rat DRGs with DBNDD2 and neuronal marker Tuj1 in frozen DRG specimens, and then determined the expression of DBNDD2 protein in Tuj1-labelled neurons. The fact that compared with uninjured rats, obviously weaker signals of DBNDD2 protein that are co-labeled with Tuj1 are observed in peripheral axon-injured rats indicates that reduced DBNDD2 expression in DRG neurons may contribute to enhanced axon regeneration. On the other hand, stronger signals of DBNDD2 protein that are co-labeled with Tuj1 observed in central axon-injured rats indicates that increased DBNDD2 expression in DRG neurons may contribute to compromised axon regeneration.

Tissue and organ regeneration shares common mechanisms with morphogenesis and, to certain degree, recapitulates the development process. Actually, neonatal mammals, different from adult mammals, have remarkable regeneration potentials after both peripheral and central nerve injuries(Li et al. 2020b). Along with the down-regulation of many regeneration promoting molecules and the up-regulation of many regeneration inhibiting molecules during development, the regeneration capacity of the nervous system decline step wisely(Park et al. 2010). Phosphatase and tensin homolog (PTEN) is one of the most well-known neuron-intrinsic inhibitors whose deletion is capable of enhancing the regeneration ability of retinal ganglion cells, corticospinal neurons, and DRG neurons in adult mammals(Park et al. 2008) (Liu et al. 2010; Zhou et al. 2020). The investigation of the expression patterns of PTEN during regeneration and development showed that showed that during regeneration, PTEN expressions is reduced in peripheral nerves while during development, PTEN is expressed at low levels in DRG neuronal cell bodies and axons during the early prenatal stages but expressed at higher levels as the nerves system develops(Chen et al. 2018).

Given that similar as PTEN, DBNDD2 is differentially expressed after nerve injury, we next evaluated that whether the expression levels of DBNDD2 is altered during development. We compared the abundances of DBNDD2 in neonatal and adult rat DRGs using single-cell sequencing data, determined the expression trends of DBNDD2 in the DRGs of rats at different ages (1-day-old neonatal, 2-week-old, and 4-week-old) using RT-PCR, and found development-dependent increase of DBNDD2 gene expression. The application of single-cell sequencing separates neurons from other different types of cells and allows the identification of the transcription programs in neurons under various physiological and pathological conditions(Zeisel et al. 2018; Renthal et al. 2020; Wang et al. 2021a). Here, using single-cell sequencing data, we distinguished DRG neurons from glial cells and immune cells in neonatal and adult rat DRGs and found that DRG neurons occupy a large cell population in the DRGs of rats at different ages. Using t-SNE plot, we visualized the presence of DBNDD2 in both neonatal and adult rat DRGs and found the obviously higher expression of DBNDD2 in adult rats. The increased expression trend of DBNDD2 in DRG neurons during development, together with the elevated amount of DBNDD2 after the non-regenerative central axon injury and the reduced amount of DBNDD2 after the regenerative peripheral axon injury, imply that DBNDD2 may be an inhibitory factor for neuron growth and axon regeneration.

To explore the biological effects of DBNDD2 on neurons, RNA interference, an effective technology that mediates sequence-specific gene knockdown, was applied and rat DRG neurons were transfected with siRNA segments against DBNDD2. Three siRNA segments targeting different portions of the target gene DBNDD2 and a siRNA segment targeting sequences altered from the target were utilized to examine the knockdown efficiency. DBNDD2-siRNA-1 and DBNDD2-siRNA-2, two siRNA segments that robustly suppressed DBNDD2 gene expression, were utilized together for the success manipulation of DBNDD2 gene expression. In vitro monitoring of neurite outgrowth showed that DBNDD2 siRNA transfection leads to enhanced neuron growth in both neonatal rat DRG neurons with certain regeneration capacity and adult rat DRG neurons with limited regeneration capacity. More importantly, in adult rats, via an intrathecal injection of DBNDD2 shRNA-expressing AAV, it is found that silencing DBNDD2 is sufficient to promote the elongation of injured axons of cultured adult DRG neurons as well as the regeneration of injured sciatic nerves. These studies fully indicate that DBNDD2 is a key regulating factor of neuron growth and axon regeneration and imply that knocking down DBNDD2 in neurons is an effective strategy for restoring impaired nerve functions. It is worth raising that in the current study, the in vivo roles of reduced DBNDD2 expression in DRG neurons is examined by immunostained the regenerating sciatic nerves with SCG10 at 3 days after rat sciatic nerve crush injury. Sciatic nerve crush injury, as previous mentioned, is a commonly used peripheral nerve injury model. Crush injury induces a modest damage and elicits axonotmesis without disrupting the epineurium. Nerve regeneration following crush injury is hence more rapid and effective as compared with transection injury that disrupts the entire nerve stump, including the endoneurium, the perineurium, as well as the epineurium(Renthal et al. 2020; Yi et al. 2020). To further evaluate the clinical potential of the manipulation of DBNDD2, the functional roles of DBNDD2 knockdown in axon regeneration after a more severe injury to peripheral axon branches of DRG neurons can be determined using a transection injury model and a long gap peripheral nerve injury model. The effects of DBNDD2 deficiency on axon regeneration following injury to central axon branches of DRG neurons and even injury to central nerves can be further assessed to examine whether DBNDD2 deficiency is capable of triggering central axon-injured DRG neurons and/or central neurons to switch to a pro-regenerative state. Actually, the fact that DBNDD2 knockdown boosts the regenerative axon growth of DRG neurons cultured on myelin, a well demonstrated inhibitor in axonal repair(Lee and Zheng 2012), implies that DBNDD2 deficiency may be able to enabling neurons to overcome the inhibitory microenvironment in the nervous system.

The construction of molecular interaction network is valuable for the discovery of functionally associated molecules and the systemic understanding of biological processes(Cowen et al. 2017). For the investigation of DBNDD2-associated molecules, in the current study, we analyzed molecules that interact with DBNDD2 using the STRING data resource and the Pathway Commons website and screened transcription factors targeting DBNDD2 using JASPAR, animalTFDB 3.0, GTRD, and hTFtarget databases. It is worth mention that databases of human transcriptional regulatory interactions are comprehensive while there are less number of databases that recognize the regulations of rat transcription factors and their downstream target genes. Still, it is demonstrated that the cis-regulatory modules and transcription factor binding locations among species are relatively conserved(Ballester et al. 2014). Hence, except for selecting rat species in JASPAR database, we used all animal species in animalTFDB 3.0 database, human species in GTRD database, and human species in hTFtarget database to predict potential upstream transcription factors targeting DBNDD2 and then discovered ESR1 as a potential upstream regulator of DBNDD2.

Transcription factors are important gene regulating factors in numerous biological phenomena, including nerve injury and regeneration(Lambert et al. 2018; Zhang et al. 2023). For instance, activating transcription factor 3 (ATF3) and AP-1 transcription factor subunit Jun proto-oncogene (JUN), two transcription factors that are up-regulated in the DRGs after sciatic nerve injury, regulates many regeneration-associated molecules and enhances neurite outgrowth(Chandran et al. 2016; Renthal et al. 2020). It has been demonstrated that, in the nervous system, transcription factor ESR1 in glutamatergic and GABAergic neurons is important for normal puberty phenotype(Cheong et al. 2015). The effect of ESR1 on axon growth and regeneration remains largely undetermined. Notably, emerging studies demonstrate that ESR1 is expressed at low abundance in various types of cancers, such as hepatocellular carcinoma(Hishida et al. 2013), endometrioid endometrial cancer(Backes et al. 2016), breast cancer(Król et al. 2018), non-small cell lung cancer(Aresti et al. 2014), and bladder cancer(Ge et al. 2019). Overexpression of ESR1 mediates cellular apoptosis and hinders cellular proliferation and invasion(Tu et al. 2013; Zhou et al. 2013). On the other hand, reduced ESR1 expression stimulates cellular proliferation, migration, and invasion(Wang et al. 2021b). The reduced expression patterns of ESR1 in tumor tissues as compared with in non-tumor tissues as well as the inhibiting roles of ESR1 on cell growth indicate that ESR1 functions as a tumor suppressor gene(Hishida et al. 2013; Li et al. 2021). Tumor suppressor genes may be key regulators of nerve regeneration as the reduced expressions of many tumor suppressor genes, including PTEN, adenomatous polyposis coli (APC), and retinoblastoma (Rb), modulates neurite plasticity, supports axon regeneration, and facilities the recovery of injured nerves(Christie et al. 2014; Duraikannu et al. 2019; Liu et al. 2010; Meyer Zu Reckendorf et al. 2022). Our bioinformatic analysis indicates that it is likely that tumor suppressor gene ESR1 is also a negative regulator of nerve regeneration and ESR1 may inhibit neurite growth and axon regeneration by targeting DBNDD2. Whether knockdown or deletion of ESR1 would lead to reduced DBNDD2 expression and drive axon regeneration may be explored in further studies.

Taken together, our study reveals that in the DRGs, DBNDD2 shows reduced expression after peripheral axotomy but elevated expression after central axotomy to adult rats and exhibits an increased expression trend during development. We find that reduced expression of DBNDD2 contributes to enhanced neurite growth and nerve regeneration and demonstrate that DBNDD2, as well as its potential upstream regulator ESR1, may be novel suppressors of success axon regeneration. Molecular manipulation approaches that decrease DBNDD2 expression represents an attractive therapeutic strategy to modify the regeneration ability of neurons and to improve nerve regeneration.

Conflict of interest: The authors declare no competing interests.

Authors contributions

L.Z., W.D., Y.W., T.C., Y.Q., Y.T. and P.Y. collected data, prepared figure, and analyzed data. S.Y. and L.G. conceived the project and wrote the manuscript. All authors contributed to the article and approved the submitted version.

Funding and additional information

This work was supported by National Key R&D Program of China [2022YFC2409800 and 2022YFC2409802], Collegiate Natural Science Fund of Jiangsu Province [23KJA180006], and Nantong University College Students’ Innovative Entrepreneurial Training Plan Program [2023159].

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No competing interests reported.

DBNDD2.figureS1.tif
Figure S1. SiRNA transfection reduced the protein expression of DBNDD2. Western blot images of the protein expressions of DBNDD2 in adult rat DRG neurons transfected with DBNDD2-siRNA-1 or control siRNA.

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Suppressing DBNDD2 promotes neuron growth and axon regeneration in adult mammals

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Abstract

Figures

Introduction

Materials and methods

Animal surgery

Sequencing

Quantitative real-time polymerase chain reaction (RT-PCR)

Immunohistochemistry

Primary DRG neuron isolation and culture

DRG neuron transfection

Western blot

In vitro neurite growth assay

In vitro neurite regeneration assay

Intrathecal injection of adeno-associated virus (AAVs)

Bioinformatic analysis

Statistical analysis

Results

DBNDD2 is differentially expressed in adult rat DRGs following nerve injury

DBNDD2 is differentially expressed in the DRGs at different developmental stages

DBNDD2 deficiency promotes the growth of neonatal rat DRG neurons

DBNDD2 deficiency facilitates nerve regeneration after sciatic nerve injury

Identification of DBNDD2-associated molecules

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1