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Sexually dimorphic transcriptional programs of early-phase response in regenerating peripheral nerves

The convergence of transcriptional and epigenetic changes in the peripheral nervous system (PNS) reshapes the spatiotemporal gene expression landscape in response to nerve transection. The control of these molecular programs exhibits sexually dimorphic characteristics that remain not sufficiently characterized. In the present study, we recorded genome-wide and sex-dependent early-phase transcriptional changes in regenerating (proximal) sciatic nerve 24 h after axotomy. Male nerves exhibited more extensive transcriptional changes with male-dominant upregulation of cytoskeletal binding and structural protein genes. Regulation of mRNAs encoding ion and ionotropic neurotransmitter channels displayed prominent sexual dimorphism consistent with sex-specific mRNA axonal transport in an early-phase regenerative response. Protein kinases and axonal transport genes showed sexually dimorphic regulation. Genes encoding components of synaptic vesicles were at high baseline expression in females and showed post-injury induction selectively in males. Predictive bioinformatic analyses established patterns of sexually dimorphic regulation of neurotrophic and immune genes, including activation of glial cell line-derived neurotrophic factor Gfra1 receptor and immune checkpoint cyclin D1 (Ccnd1) potentially linked to X-chromosome encoded tissue inhibitor of matrix metallo proteinases 1 (Timp1). Regulatory networks involving Olig1, Pou3f3/Oct6, Myrf, and Myt1l transcription factors were linked to sex-dependent reprogramming in regenerating nerves. Differential expression patterns of non-coding RNAs motivate a model of sexually dimorphic nerve regenerative responses to injury determined by epigenetic factors. Combined with our findings in the corresponding dorsal root ganglia (DRG), unique early-phase sex-specific molecular triggers could enrich the mechanistic understanding of peripheral neuropathies.

The convergence of transcriptional and epigenetic changes in the peripheral nervous system (PNS) reshapes the spatiotemporal gene expression landscape in response to nerve transection. The control of these molecular programs exhibits sexually dimorphic characteristics that remain not sufficiently characterized. In the present study, we recorded genome-wide and sexdependent early-phase transcriptional changes in regenerating (proximal) sciatic nerve 24 h after axotomy. Male nerves exhibited more extensive transcriptional changes with male-dominant upregulation of cytoskeletal binding and structural protein genes. Regulation of mRNAs encoding ion and ionotropic neurotransmitter channels displayed prominent sexual dimorphism consistent with sex-specific mRNA axonal transport in an early-phase regenerative response. Protein kinases and axonal transport genes showed sexually dimorphic regulation. Genes encoding components of synaptic vesicles were at high baseline expression in females and showed post-injury induction selectively in males. Predictive bioinformatic analyses established patterns of sexually dimorphic regulation of neurotrophic and immune genes, including activation of glial cell line-derived neurotrophic factor Gfra1 receptor and immune checkpoint cyclin D1 (Ccnd1) potentially linked to X-chromosome encoded tissue inhibitor of matrix metallo proteinases 1 (Timp1). Regulatory networks involving Olig1, Pou3f3/Oct6, Myrf, and Myt1l transcription factors were linked to sex-dependent reprogramming in regenerating nerves. Differential expression patterns of non-coding RNAs motivate a model of sexually dimorphic nerve regenerative responses to injury determined by epigenetic factors. Combined with our findings in the corresponding dorsal root ganglia (DRG), unique early-phase sexspecific molecular triggers could enrich the mechanistic understanding of peripheral neuropathies.

Introduction
The peripheral nervous system (PNS) displays strong regenerative potential compared to the brain and spinal cord. Substantial knowledge of mechanisms involved in PNS injury has been acquired using rodent models of sciatic nerve axotomy originally described by Augustus Waller in 1850 (Fawcett and Keynes, 1990;Stoll et al., 2002). The success of sensory recovery after PNS injury depends on molecular remodeling ensuing within 24 h post-axotomy in the proximal nerve segment (Gordon et al., 2003;McDonald et al., 2006;Zochodne, 2012) coordinated with an extensive transcriptional response within neuronal somas in the dorsal root ganglia (DRG) (McDonald et al., 2006;Chernov and Shubayev, 2021). Upon an initial die-back toward DRG, the proximal axotomized axons form attributed to metabolic, immune, neuroendocrine, and sex chromosome-related genetic programs that have shown sexspecific regulation in the damaged PNS (North et al., 2019;Ray et al., 2019;Stephens et al., 2019;Chernov et al., 2020;Mecklenburg et al., 2020;Paige et al., 2020;Tavares-Ferreira et al., 2020;Ahlström et al., 2021). Unique to females, the chromosome-wide epigenetic inactivation of one of the two X chromosomes (Xi) regulates the normal and aberrant activity of X-linked immunity-related genes (Bianchi et al., 2012), including that in DRG at 24 h after sciatic nerve axotomy (Chernov and Shubayev, 2021). Whether the corresponding regenerating nerve segments exhibit sexual dimorphism in response to PNS axotomy remains unknown.
Using high-depth RNA-seq, the present study identified sexspecific early-phase transcriptional changes in protein-coding and ncRNAs in regenerating (proximal) nerves at 24 h after sciatic nerve axotomy in male and female mice. Applying predictive bioinformatics, we determined unique signaling events related to regenerative, trophic, metabolic, and sex chromosome-linked systems.

Results
To determine the sex specificity of an early-phase PNS regenerative transcriptional program, sciatic nerve axotomy or sham operation was conducted in female and male mice. At 24 h post-axotomy, regenerating (proximal segment) tissues ( Figure 1A) were subjected to wholegenome transcriptomics analysis by high-depth RNA-seq (Supplementary Figure 1, n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group). At 24 h after the sham operation, nerve tissues were assessed for transcriptomic signatures without injury. Transcriptomics of the respective DRG tissues in the same animal cohorts was reported earlier (Chernov and Shubayev, 2021).
Males exhibited more extensive transcriptional changes in regenerating sciatic nerve A total of 25,788 genes were detected by RNA-seq in the proximal nerve stumps, including 2,553 differentially expressed genes (DEGs, Supplementary Table 1) that conformed to the significance criteria (adjusted p-values (P adj ) < 0.1) and the expression fold change (FC) filter (log 2 FC > 1 or log 2 FC < -1, Figure 1A). In summary, more unique, highly expressed upregulated DEGs (n = 232) were identified in males relative to females as presented on MA and scatter plots (Figures 1B,C). In addition, 65 and 159 DEGs were downregulated in female and male nerves, respectively. Global transcriptional changes in proximal nerve 24 h post-axotomy lead to a larger number of increased transcripts in males. (A) A schematic of the sciatic nerve axotomy followed by ipsilateral proximal stump tissue analysis. (B) MA plots display log 2 FC and mean counts normalized using DESeq2 and adaptive t-prior apeglm method. The left and right panels show up-and downregulated genes in females (red dots) and males (blue dots), respectively. Dots indicate differentially expressed genes (DEGs) with P adj < 0.1. Orange dashed lines show log 2 FC thresholds above 1 or below -1. (C) Scatter plots of significant up-and downregulated DEGs in females (light and dark red dots) and males (light and dark blue dots). Light and dark color intensity indicate DEGs in sham or axotomy samples (n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group), respectively. DEGs (P adj < 0.1) were filtered by log 2 FC either above 1 or below -1 (two-fold change or larger). Dots on the Y-axis correspond to the absolute gene expression measured as normalized mean counts following logarithm transformation of gene counts [log 2 (counts)] by the normTransform method in DESeq2. Specific expression data for DEGs is available in Supplementary Table 1 and the GEO repository (accession numbers GSE182713 and GSE182709). Pairwise comparisons were made using the analysis of variance (ANOVA) with the Tukey post hoc test: ****, p ≤ 0.00005; ns, not significant.

Sexually dimorphic gene regulation in injured nerves
Heatmaps (Figure 2A) and volcano plots ( Figure 2B) present the identity of the most significant protein-coding DEGs in each sex. Principal component analysis (PCA) identified their contribution to variance in axotomized samples from males and females based on normalized count ranking. Sample-tosample comparisons using PCA demonstrated that female and male samples aggregated as distinct clusters in the PC1/PC2 dimensions ( Figure 2C).
Gene ontology analysis identified sexual dimorphic genes relevant to peripheral nervous system (PNS) regeneration Many sexually dimorphic and monomorphic DEGs were recognized as important components of cell signaling pathways in the nervous and immune systems. To gain further mechanistic insight into their role in nerve regeneration, gene ontology (GO) enrichment analysis was conducted. Molecular-level biochemical characteristics of DEGs were used to identify the most relevant GO clusters of molecular function (Figures 3A,B). GO clusters that included sexually dimorphic DEGs relevant to nerve injury response are illustrated in Figure 3C and detailed below.

Cytokine binding
Reduced expression of many cytokine receptors was observed in both sexes (cluster 9), including interleukin receptors (Il31ra, Il5ra, Il12rb2), chemokine receptors (Ccrl2, Ccr5, Ccr2, Ccr9, Ccr1l1, and Ccr3), and other receptors (Tnfrsf14, Csf1r, Ackr1, and Ackr4). Despite the decrease, the absolute expression levels of many cytokine receptor genes remained high. Only cytokine receptor-like factor 1 (Crlf1) Nerve axotomy induced distinct sexually dimorphic regulation of differentially expressed genes (DEGs) in female and male mice. (A) Hierarchical clustering plot of 300 most significant upregulated DEGs (log 2 FC > 1, P adj < 0.1, n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group). Heatmap colors correspond to mean log 2 (counts)-Blue, yellow, and red -low, medium, and high expression, respectively. DEGs were sorted by log 2 FC. (B) Significant DEGs in female (left panel) and male (right panel) mice. Volcano scatter plots show -log 10 P adj and log 2 FC. Red and green colors indicate up-and down-regulated DEGs, respectively. Thresholds (log 2 FC > 1 or log 2 FC < -1) and -log 10 P adj < 0.1 are indicated by yellow dashed lines. Selected DEG symbols are shown. (C) Principal component analysis of female (red) and male (blue) groups of sham (rectangles) and axotomy (circles) samples (n = 3 samples/group). Red and blue arrows indicate genes with the most influence on variance in females and males, respectively. (D) A principal component analysis (PCA) loading plot indicates genes influencing variance in the top five PCA projections (PC1 to PC5) within the top/bottom 5% of the loadings range. The dot color scale corresponds to component loading coefficients defined as the coordinates of the variables divided by the square root of the eigenvalue associated with the component. Axotomy influenced distinct molecular functions in each sex. (A) Clustering of significant gene ontology (GO) molecular functions based on best-match average (BMA) distances. (B) Wang distances of GO molecular function clusters projected on a two-dimensional scaling plot using the ViSEAGO R package. (C) Heatmaps of differentially expressed genes (DEGs) associated with eight GO molecular functions: heterocyclic compound binding (cluster 1), ion binding (cluster 2), carbohydrate derivative binding (cluster 4), cytoskeletal protein binding (cluster 8), cytokine binding (cluster 9), receptor-ligand activity (cluster 10), phosphotransferase activity (cluster 15), and structural molecule activity (cluster 24). Heatmaps display normalized mean log 2 (counts) in sample groups (F S , female sham; M S , male sham; F A , female axotomy; M A , male axotomy) and log 2 FC in female (F) and male (M); (n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group) of respective DEGs. Log 2 FC significance was determined in DESeq2 using Wald test: *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005. DEGs were sorted by log 2 FC. Canonical signaling pathways demonstrate distinct regulation patterns in females and males. Canonical pathways were predicted in Ingenuity Pathway Analysis (IPA) and ranked by z-scores. Positive (red) and negative (green) z-scores indicate pathway up-regulation or down-regulation, respectively, according to the color scale. Fisher's exact test p-values [-log 10 (P)] predicted for each canonical pathway are shown by purple (females) and blue (males) horizontal bars. Asterisks indicate significance *, z > 2.0 or z < −2; **, z > 3.0 or z < −3; ***, z > 4.0 or z < −4. and corticotrophin-like cytokine factor 1 (Clcf1) demonstrated significant upregulation in both sexes.

Axotomy significantly reshaped cell signaling in regenerating nerves
Signaling pathways, upstream regulation, and interactive networks were predicted by Ingenuity Pathway Analysis (IPA) software using a highly stringent subset of DEGs (P adj < 0.001 and log 2 FC > 1 or log 2 FC < -1). Significant canonical pathways were further ranked based on Fisher's exact test (p < 0.05). Positive or negative z-scores determined pathways' activation and deactivation.
Axotomy-induced significant changes in cellular signaling processes in both sexes (Figure 4)

Upstream regulator molecules
Predictive analysis of upstream regulators was conducted in IPA to identify regulatory molecules, including transcription factors (TFs), any gene or small molecule that, with high probability, could affect the expression of their target DEGs. The activation or inhibition efficiency of each upstream regulator on target DEGs could be defined by positive or negative z-scores, respectively, as illustrated in Figure 5 and summarized in Supplementary Table 2. Worth noting that upstream regulator z-scores do not directly reflect the expression of the regulator itself (Krämer et al., 2014), as different organs or tissues can contribute regulatory molecules. IPA identified many sexually dimorphic upstream regulators, including the brain-derived neurotrophic factor (Bdnf), a known stimulator of the nerve axon growth (Zhou and Shine, 2003). Bdnf was predicted to strongly affect gene expression in males but not females. Bdnf level was low in the proximal nerve stumps in both sexes. However, Bdnf mRNA was highly abundant in DRG and demonstrated a male-dominant increase postaxotomy (Chernov and Shubayev, 2021). Other male-specific upstream positive regulators included Sox2, Aire, Egr2, Cdkn1a, Map2k1/2, and other molecules. Ikzf3, Mek, Erk, and Akt1 were predicted as prospective positive regulators in females. Remarkably, IRFs, STATs, and cytokines were identified among significant negative regulators in both sexes.

Sexually dimorphic protein phosphorylation
Protein kinases and their regulatory molecules were identified as significant sexually dimorphic upstream regulators ( Figure 6A and Supplementary Table 2). Male-specific upregulation of Kndc1 (also known as Very-KIND) may modulate Hras, Myc, Neu1, Ras, and Map2 ( Figure 6B) during neuronal growth (Huang et al., 2007). Highly expressed Cdk5 was moderately activated by axotomy in male nerves. Cdk5 activator molecule that also controls cytoplasmic or nuclear localization of the Cdk5 complex demonstrated maledominant upregulation. Co-upregulation of Cdk5-interacting and target molecules in males was significantly stronger than in females ( Figure 6C). While female nerves upregulated fewer protein-coding DEGs than males, two female-specific interactive networks were determined using the STRING database (Szklarczyk et al., 2021). The first proposed network potentially conveys Gdnf signaling via the neuron-specific adaptor Shc3, the Gdnf receptor α1 (Gfra1), and neural cell adhesion proteins Chl1 and Cadm1 to regulate cell-cell adhesion and neuronal plasticity ( Figure 6D). Another network is centered around the regulatory component of the Cyclin D1 (Ccnd1)-Cdk4 complex ( Figure 6E). It is known to respond to Timp1 that activates cyclic AMP-induced regenerative program gene post-axotomy  partly via Cd63/Pi3k/Akt signaling (Rossi et al., 2015).

Cell signaling engages sexually dimorphic molecular programs
Regulatory signaling events reflect distinct molecular programs involved in regenerating nerves. Programs exhibiting sexually dimorphic regulation are as follows: Neuronal survival and regeneration programs GDNF Family Ligand Receptor Interactions pathway is modulated by the Gdnf and related ligands: neurturin (Nrtn), artemin (Artn), and persephin (Pspn). Notably, the Gdnf gene was highly upregulated in both sexes in the proximal stumps ( Figure 7A) but not in DRG (Chernov and Shubayev, 2021). Neurturin was upregulated in males. Artemin was mildly expressed in distal stumps but significantly upregulated in male DRG. The ligand receptors encoded by Gfra1 and Gfra3 genes increased in both sexes.

Regulation of synaptogenesis signaling
Synaptogenesis Signaling Pathway was distinctly regulated in both sexes. Genes encoding components of synaptic vesicles (SV) showed greater activation in males ( Figure 7D). Consequently, the male-dominant increase of the pre-synapticlike activity and vesicular transport can be mediated by synuclein subunits α, β, and γ, Rab3A oncogene, and vesicleassociated membrane protein (Vamp2). It is important to note that SV-related genes show high levels of expression in sham and axotomized female nerves; therefore, male-prevalent gene activation could level up pre-synaptic activity to match the activity in females.

Axonal transport
Axonal transport is essential for neuronal survival and function restoration after damage. Many kinesins involved in anterograde axonal transport decreased in males (Figure 7E)

Immune regulation
Cytokines Il11 and Il17b were upregulated in both sexes, while Il6 and Il19 were predominantly activated in males ( Figure 8B). Genes related to adaptive (GO:0002250) and innate (GO:0045087) immunity showed a system-wide malespecific decrease. Many immunity-related canonical pathways were significantly downregulated after axotomy in both sexes' proximal nerve segments, including Neuroinflammation Signaling Pathway, Activation of IRF, Leukocyte Extravasation, Signaling, Interferon Signaling, Pattern Recognition Receptors, and Phagosome Formation pathways (Figure 4). The following immunity-related pathways demonstrated male-specific negative regulation: Inflammasome Pathway, Toll-like Receptor Signaling, IL-15 Production, PI3K Signaling in B Lymphocytes, Phagocytosis in Macrophages and Monocytes, and IL-8 Signaling.

Cation transporting ATPases
Males but not females upregulated plasma membrane Ca 2+transporting ATPase subunits Atp2b2 and Atp2b3 (Figure 8D). mRNAs of Na + /K + -transporting ATPase subunits Atp1b1 and Atp1b2 were abundant in both sexes and exhibited further increase in males.

Non-coding RNAs
Levels of Mir124-1hg and Mir124-hg2 RNAs, the precursors of the ubiquitous neuronal microRNA Mir-124, were higher in males than in females ( Figure 9A). In addition, small nucleolar RNAs (snoRNAs) encoded by Snhg9, Snhg6, and Snora57 host genes showed male-dominant expression. The erythroid differentiation regulator 1 ncRNA encoded by the X-linked Erdr1 gene was upregulated in males but downregulated in females. U1 spliceosome RNA (U1) and ribonuclease P RNA component H1 (Rpph1) were upregulated in females.

Discussion
We obtained novel evidence of sexually monomorphic and dimorphic regulation of the early-phase transcriptional programs in regenerating segments of injured peripheral nerves (Figure 10). The concerted accumulation of mRNAs that encode actin, microtubules, neurofilaments, cytoskeletal regulators, Males exhibited stronger activation of ion channels, transcriptional regulators, cation transporting ATPases, and select cytokines. Innate and adaptive immunity exhibited a male-specific decrease. (A) Ion channel genes. (B) Cytokines, adaptive (Go term GO:0002250), and innate (GO term GO:0045087) immunity-related DEGs. (C) DNA-binding transcriptional regulators (GO term GO:0003700). (D) Cation transporting ATPases. Heatmaps display normalized mean log 2 (counts) in sample groups (F S , female sham; M S , male sham; F A , female axotomy; M A , male axotomy) and log 2 FC in female (F) and male (M); (n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group) of respective DEGs. Heatmap colors correspond to the respective scales. Log 2 FC significance was determined in DESeq2 using Wald test: *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005. DEGs were sorted by log 2 FC. Regulated ncRNA and sex chromosome-linked differentially expressed genes (DEGs) exhibited profound sexual dimorphism. (A) Significant ncRNA DEGs in female (left panel) and male (right panel) mice. Volcano scatter plots show -log 10 P adj and log 2 FC. Red and green colors indicate up-and downregulated ncRNAs, respectively. Thresholds (log 2 FC > 1 or log 2 FC < -1) and -log 10 P adj < 0.1 are shown by yellow dotted lines. Selected DEGs are labeled by gene symbols. (B) Regulated antisense lncRNA DEGs. (C) Regulated sex chromosome-linked DEGs. Heatmaps display normalized mean log 2 (counts) in sample groups (F S , female sham; M S , male sham; F A , female axotomy; M A , male axotomy) and log 2 FC in female (F) and male (M); (n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group) of respective DEGs. Heatmap colors correspond to the respective scales. Log 2 FC significance was determined in DESeq2 using Wald test: *, p ≤ 0.05; **, p ≤ 0.005; ***, p ≤ 0.0005. DEGs were sorted by log 2 FC. and cytoskeleton-binding proteins was observed in proximal nerve stumps of both sexes, confirming extensive cytoskeletal remodeling aimed at rebuilding the growth cone (Lundborg, 1987;Burnett and Zager, 2004;Radtke and Vogt, 2009).

Schwann cell reprogramming
After nerve injury, Schwann cells assume a proregenerative function Mirsky, 2016, 2021;FIGURE 10 A summary of activated and inhibited transcriptional programs detected in the proximal nerve stumps 24 h post-axotomy in female and male mice. Merrell and Stanger, 2016;Milichko and Dyachuk, 2020), exhibiting an exceptional phenotypic plasticity (Jessen and Arthur-Farraj, 2019;Stierli et al., 2019;Nocera and Jacob, 2020) that supports their de-differentiation, proliferation, and re-differentiation into myelinating and non-myelinating phenotype to facilitate repair of the respective axons. Schwann cell reprogramming is controlled by a set of TFs (Balakrishnan et al., 2021), including Myt1l, Pou3f1/Oct6, Myrf, Olig1/2, Jun, Sox-, Hox-, and Fox-family members TFs upregulated at 24 h post-axotomy predominantly in males. Transcripts of TF genes, such as Fos, Smad3, Creb1, Rac2, Klf6, and Atf3, that function as RAGs (reviewed in [Van Kesteren et al., 2011;Ma and Willis, 2015)] in the corresponding DRGs after PNS injury (Chernov and Shubayev, 2021) were detected in proximal nerve segments. In addition to transcriptional activity in cells of the PNS, this accumulation may reflect an anterograde transport from DRG in coordinated axonal regeneration processes post-axotomy. Single-cell RNA-seq and spatial transcriptomics analysis could provide crucial information on the identity of cell lineages activating these sexually dimorphic TFs.
The GDNF family of ligands (GDNF, neurturin, artemin, and persephin), known survival factors for neurons, bind to GFRA receptors, trigger the phosphorylation of RET tyrosine kinase receptor (Durbec et al., 1996) that is known to regulate Pi3k/Akt and Plcγ/Ip3r dependent Ca 2+ signaling during neurogenesis (Fukuda et al., 2002;Lundgren et al., 2012) and, sometimes, hyperalgesia (Bogen et al., 2008). Furthermore, GDNF/Gfra1 could interact with neural cell adhesion molecules such as NCAM to induce an axonal expansion (Nielsen et al., 2009). GDNF family of ligands can act in a synergetic manner with other growth factors, including transforming growth factor-β (Tgf-β) and sonic hedgehog (Shh) [reviewed in Jessen and Mirsky (2021)]. GDNF signaling can stimulate the migration of neuronal precursors and Schwann cells (Iwase et al., 2005) to the injury site in both sexes.

Extracellular proteolysis
The outcome of nerve regeneration after sciatic nerve injury is partly dependent on the integrity of ECM-rich Schwann cell basal lamina and endoneurial tubes (Fawcett and Keynes, 1990). Proteolytic cleavage mediated by ECM proteinases and their endogenous inhibitors is indispensable for morphological remodeling of regenerating axons (Rivera et al., 2010;Fujioka et al., 2012;Bajor and Kaczmarek, 2013), after sciatic nerve injury (Liu et al., 2010). In addition, MMPs control the activity of critical growth factors, their ligands, and receptors in peripheral nerve, including IGF and neuregulin/ErbB (Chattopadhyay and Shubayev, 2009). Mmp17 was upregulated in both sexes and can convey inflammatory responses due to the TNFα-converting activity (English et al., 2000).
Intriguingly, a group of MMPs co-localized on the mouse chromosome 9 (Mmp3, Mmp8, Mmp12, Mmp13, and Mmp27) have decreased in both sexes. Because MMPs in the orthologous human chromosome 11q22.3 region could carry different histone modification marks (Chernov et al., 2010), epigenetic control of this set of metzincins may be involved. Consistent with our previous report, MMPs, and many disintegrin ADAM/ADAM-TS families of the ECM-remodeling metzincins demonstrate regulated expression in injured nerves (Chernov et al., 2015).
Notably, protease expression levels were high in intact and axotomized nerves. Processes of post-translational protease activation and binding to intrinsic proteinase inhibitors control potentially cytotoxic proteolytic activities. Aberrant cleavage of mediators of nociceptive signaling during neurogenesis potentially causes neuropathic pain . Previously we identified Timps as key genes in the normal and damaged nerves (Kim et al., 2012;Nishihara et al., 2015) and post-axotomy DRG Chernov and Shubayev, 2021). Timp1 binds and inhibits all MMPs, except Mmp14; Timp2 inhibits Mmp14 (Brew and Nagase, 2010). In the proximal nerve, Timp1 demonstrated strong upregulation in both sexes. Other upregulated endogenous inhibitors of proteinases Wfdc5, Fetuin B, Spink1, Itih3, and Serpineb1b could serve a not well-understood role in nerve damage response.

Sex-specific control of axonal trafficking
An intact connection to DRG allows for dynamic axonal transport of protein and mRNA content between neuronal soma and the proximal segment of the axotomized nerve (Cavalli et al., 2005). The anterogradely transported mRNAs from DRG are directly used for the local protein synthesis at the site of the injury (Sahoo et al., 2018) and complemented by the transcriptional repertoire of the resident non-neuronal cells in the damaged nerve (e.g., Schwann cells, macrophages) that establish a microenvironment generally permissive for axon regeneration (Avraham et al., 2021).
Kinesins facilitate anterograde axonal transport of neurotransmitter receptors, mitochondria [reviewed in (Fan and Lai, 2021)], and also mRNAs [reviewed in (Kanai et al., 2004;Turner-Bridger et al., 2020)]. Remarkably, most kinesin mRNAs were depleted in proximal nerve segments at 24 h post-axotomy, except for members of the conventional kinesin-1 family Kif5a/b/c increased in males.
Retrograde transport of transcription factors from the site of nerve injury to DRG has been shown to facilitate the neuronal survival (Cox et al., 2008) and axon regeneration (Ito and Enomoto, 2016). In the corresponding DRGs of the same animal cohort, at 24 h post-axotomy, we observed male-specific reduction of mRNAs encoding calcium, sodium, and potassium ion channels as well as ionotropic AMPA, NMDA, and GABA receptors (Chernov and Shubayev, 2021) that correlated to their increase in proximal segments in the present study and presumably, anterograde axonal transport. We conclude that axonal transport of mRNAs contributes to sex-specific protein synthesis and downstream signaling programs both in DRG neurons and regenerating axons at the early stages of response to sensory nerve damage.

Sex chromosome encoded genes
Sex chromosome-encoded genes were activated in regenerating sciatic nerves. Several X-linked genes were differentially regulated in male and female nerves. Among X-linked genes induced in nerves of both sexes were Timp1, known to promote regeneration (Kim et al., 2012;Liu et al., 2015;Nishihara et al., 2015), and Map7d2, involved in kinesinmediated cargo transport into the axon (Pan et al., 2019). It is worth noting that genes associated with the female-specific epigenetic X-chromosome inactivation (Xi) process were differentially expressed in DRGs within 24 h after post-axotomy in the same animal cohort (Chernov and Shubayev, 2021).

Conclusion
The noted remodeling represents baseline sexual dimorphism in uninjured peripheral nerves and an immediate regenerative response to nerve injury. The functional significance of the reported sexual dimorphism remains to be determined. It is conceivable that, at least partially, sex differences were equalized over the time-course of nerve injury. Single-cell RNA-seq and spatial transcriptomics analysis could provide crucial information on the identity of cell lineages activating these sexually dimorphic coding and non-coding RNAs.

Reagents
Detailed descriptions of reagents and resources are included in Supplementary Table 3.

Animals
Female and male C57BL6/J mice (6-8 weeks old, Jackson Labs, n = 24) were housed in a temperature-controlled room (22 • C) on a 12 h light/dark cycle with ad libitum access to food and water. The mice were randomly assigned to axotomy and sham groups by sex (n = 6/group). Under isoflurane anesthesia, the left sciatic nerve was exposed at the mid-thigh level, followed by a complete transection using sterile microsurgery scissors. In the sham group, anatomically equivalent nerves were subjected to exposure without transection. The muscle was sutured, and the skin stapled. At 24 h after surgery, proximal segments of the sciatic nerve, corresponding to previously investigated lumbar (L)4 and L5 DRG tissues (Chernov and Shubayev, 2021), were collected for RNA isolation. All animal procedures were performed according to the Policy on Humane Care and Use of Laboratory Animals and the protocol approved by the Institutional Animal Care and Use Committee at the VA San Diego Healthcare System.

Samples
All surgical and tissue harvesting instruments were sterilized and repeatedly treated with RNase Away reagent followed by RNase-free water rinse. Tissues were immediately submerged in 500 µl RNAlater Stabilization Solution to preserve RNA integrity, placed at 4 • C overnight, then transferred for storage at -20 • C. All sample groups were processed in parallel to minimize batch effects. Proximal stumps of sciatic nerve tissues were pooled from 2 mice per group for RNA purification.

RNA purification
Nerve tissues were transferred in Trizol solution (Invitrogen) and disrupted by mechanical homogenization. Total RNAs were purified using RNeasy RNA purification reagents. RNA concentrations and quality were determined using Nanodrop absorbance ratios at 260/280 nm and 260/230 nm. RNA integrity indices were determined using the Agilent Bioanalyzer Nano RNA chip. 500 ng of total RNA samples (n = 6 mice/group, 2 mice/sample (pooled), 3 sample/group) with RIN ≥ 7.0 were used for RNA-seq.
RNA sequencing (RNA-seq) mRNA libraries were generated following the TruSeq Stranded mRNA library preparation protocol (Illumina). In brief, the Poly-A enriched mRNAs were purified using poly-T oligo coupled magnetic beads, followed by mRNA fragmentation, first and second strands synthesis, cleaning on AMPure XP magnetic beads, and 3'-adenylation. Ligation of TruSeq dual-index adapters was used for barcoding. The quality of RNA-seq libraries was validated using qPCR. Libraries were sized on Agilent Bioanalyzer DNA high sensitivity chip and normalized. RNA-seq was performed using the pairedend 100 cycle program on the NovaSeq 6000 system at the Genomics High Throughput Facility (University of California Irvine). Base calls were recorded and converted to FASTQ files containing sequencing reads and the corresponding quality scores using Illumina software. Sequencing was conducted until we acquired at least 50 million paired-end reads per sample.
RNA sequencing (RNA-seq) data analysis FASTQ files were uploaded to the Amazon S3 server and processed using Elastic Compute Cloud (EC2) [Amazon Web Services (AWS)] running Ubuntu Server 20.04 LTS (64-bit ARM). Data analysis steps are summarized in Supplementary Figure 1 and Supplementary Table 3. FASTQ files were filtered to remove low-quality bases, TruSeq dual-index adapter sequences, and unpaired reads using Trimmomatic (Bolger et al., 2014). Transcript-level quantification and mapping were performed using Salmon (Patro et al., 2017) and the Gencode M29 mouse genome.
Mapping coverage was estimated in MultiQC (Ewels et al., 2016). Transcript-to gene-level quantifications were done using Tximeta (Love et al., 2020). Gene count matrices were processed in the DESeq2 (Love et al., 2014). Log 2 FC values were calculated using the Wald test and adjusted using the adaptive t-prior apeglm method (Love et al., 2014). Significant DEGs were identified by P adj values below a false discovery rate (FDR) cutoff (P adj < 0.1) (Supplementary Table 1). P adj < 0.1 were used in downstream analyses unless otherwise noted. Batch effects were controlled using the removeBatchEffect tool (Ritchie et al., 2015) and RUVseq (Risso et al., 2014) functions. DEGs were visualized using ggVennDiagramm, PCAtools, ComplexHeatmap, and EnhancedVolcano R packages.

Signaling pathway analysis
Prediction and biological interpretation of the regulated signaling pathways and upstream regulators in female and male animals was made in IPA using the default parameters (Krämer et al., 2014). Significance criteria of P adj < 0.1, log 2 FC > 1 or log 2 FC < -1 were applied. Regulation directionality was estimated in IPA based on z-scores. Gene ontology terms were determined using Geneontology 1 and the STRING database using the default parameters.

Data availability statement
The datasets presented in this study can be found in the Gene Expression Omnibus (GEO) public repository (accession numbers: GSE182713 and GSE182709).

Ethics statement
The animal study was reviewed and approved by Institutional Animal Care and Use Committee at the VA San Diego Healthcare System.

Author contributions
AC: conceptualization, methodology, software, formal analysis, investigation, data curation, writing -original draft, review and editing, and visualization. VS: conceptualization, resources, project administration, funding acquisition, and writing -original draft, review and editing. All authors contributed to the article and approved the submitted version.

Funding
The research was supported by the National Institutes of Health (NIH) RO1 DE022757 (to VS), and the Department of Veterans Affairs Merit Review Award 5I01BX000638 (to VS). The content is solely the authors' responsibility and does not necessarily represent the official views of the funding agencies.