Modulation of microtubule dynamics as therapeutic targets for neural restoration

Abstract

The present disclosure relates to modulation of microtubule dynamics as therapeutic targets for neural restoration. There is provided a method of treating nerve injury in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a therapeutic agent wherein the therapeutic agent is selected from the group consisting of metaxalone and a formamidin-2 inhibitor, such as an antibody, an antibody fragment, an inhibitory nucleic acid molecule or a small molecule, wherein the inhibitory nucleic acid molecule substantially silences Fmn2.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 590,594, filed on October 16, 2023, which is incorporated herein by reference in its entirety.

[0003] Reference to a sequence listing

[0004] The Sequence Listing is identified as Sequence_Listing_P25599US01.xml; size: 101 kilobytes; and creation date: January 9, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0005] The present disclosure generally relates to the use of metaxalone and Formin-2 inhibitors in treating nerve damage. Background Art

[0006] Peripheral nervous system (PNS) injuries induce either adaptive (growth and survival) or maladaptive responses (cell death, neuropathic pain, and regeneration failure) that are precisely determined by the expression levels of a subset of genes (intrinsic growth programs). The first step in successful peripheral nerve regeneration is to induce axonal regeneration from damaged neurons. At the cytoskeletal level, this requires a precise balance between stable and dynamic microtubules, as observed during neuronal development. The rate of axonal regrowth after peripheral nerve injury is slow and is associated with suboptimal functional recovery. For example, the most common types of proximal nerve injuries in humans, such as brachial plexus injuries and injuries involving complete transection of peripheral nerves, require long-distance axonal regeneration to reinnervate their target muscles. Even after surgical repair, function is virtually unrecoverable, mainly due to the slow rate of axonal regrowth and limited reinnervation of target muscles. Therefore, one of the major challenges is to identify the gene or genes involved in this adaptive response.

[0007] DNA methylation is the most studied and common epigenetic modification in gene expression. Recent reports suggest that the coordination of global DNA methylation and demethylation is a fundamental mechanism to promote axon regeneration. Epigenetic regulators such as 10-11 translocation methylcytosine dioxygenase (Tet) and ubiquitin-like PHD ring finger containing 1 (UHRF1) have been shown to be important for peripheral nerve regeneration. Tet is an enzyme that regulates DNA demethylation, and their expression is increased in dorsal root ganglia (DRG) after sciatic nerve injury. TET3 is highly upregulated in DRG neurons 1-3 days after sciatic nerve injury and promotes the expression of multiple regeneration-related genes (RAGs). UHRF1 enhances the regenerative capacity of DRG neurons through promoter methylation (gene silencing) of PTEN and REST. However, the correlation between global DNA methylation and RAG expression after peripheral nerve injury (PNI) is weak and more precisely descriptive. There is still much uncertainty regarding the importance of DNA methylation after peripheral axon injury. Pharmacological perturbations of PNI-induced changes in DNA methylation status (activation or inhibition of DNA methylation) resulted in significant attenuation of axonal regeneration in DRG neurons. Administration of folic acid, which increases global DNA methylation in the spinal cord, enhances axonal regeneration in spinal neurons after spinal cord injury. However, how changes in DNA methylation functionally affect individual gene expression and coordinate downstream effectors to contribute to successful axon regeneration remains elusive.

[0008] Histone modifications, together with DNA methylation, represent a classic and important epigenetic mechanism. Histone deacetylase (HDAC) is not only an enzyme that catalyzes histone deacetylation in the nucleus, but also catalyzes the deacetylation of many cytoplasmic proteins, such as α-tubulin. The effect of post-translational modification of tubulin on microtubule dynamics, such as acetylation, is one of the key events regulating axon regeneration. Previous studies have reported that inhibition of microtubule dynamics without destroying the integrity of axonal microtubules leads to reduced axonal growth. Tubulin is the main building block of microtubules. Stable and dynamic microtubules can be distinguished by the degree of tubulin acetylation in the axon shaft. For example, acetylated tubulin (stable) is mainly present in stable microtubules in the proximal part of the axon, where active growth is restricted. Highly deacetylated tubulin (dynamic) is located at the distal end of the axon, where active tubulin polymerization occurs at the growth cone. PNI induces microtubule remodeling, promotes the formation of growth cones and promotes axon regeneration. PNI induces tubulin deacetylation in the distal end of injured peripheral nerves, where axonal regrowth is occurring. ScriptAid, a pan-HDAC inhibitor that blocks tubulin deacetylation in injured axons, significantly inhibits axonal regrowth after PNI.

[0009] The beneficial effects of low-dose ionizing radiation (LDIR) have been documented for decades. LDIR induces adaptive responses that enhance the overall functional capacity of an organism and its intrinsic growth capacity. LDIR (e.g., X-rays) have been reported to have beneficial effects on cell growth stimulation, increased mean lifespan in rodents, and neuroprotection in animal disease models as well as in human leukemia and Alzheimer's disease. Localized low-dose X-ray irradiation of the sciatic nerve improved neural repair and functional recovery in rats, but the mechanisms are unclear. LDIR is known to induce DNA methylation and transcriptional changes in different in vitro and in vivo models. These studies suggest that alterations in gene expression profiles following LDIR contribute to the beneficial adaptive responses of the organism. There is still much to be learned before LDIR can become a routine, effective, and safe medical approach. By identifying key genes and small molecules associated with the protective effects induced by LDIR, it may become an attractive and safe pharmacological approach for PNI.

[0010] Therefore, there is a need for improved methods of treating nerve damage. Summary of the invention

[0011] Here we show that two commonly used sources of ionizing radiation, alpha particles and X-rays, promote neurite outgrowth in purified DRG neurons in culture. Whole-body X-ray irradiation with LDIR enhanced the intrinsic growth capacity of injured neurons and induced robust axonal regeneration of axotomized DRG neurons in ex vivo explant cultures and accelerated axonal regeneration and functional recovery in vivo after sciatic nerve crush injury. In-depth genome-wide CpG methylation profiling and bioinformatics analysis were performed to identify hypermethylation of the morphogen-2 (Fmn2) promoter as the major contributor associated with the promoting effect of LDIR. To determine the role of Fmn2 in peripheral nerve regeneration, the temporal expression profile of Fmn2 was investigated after sciatic nerve crush injury, in which PNI induced downregulation of Fmn2 expression in DRG. Complete Fmn2 knockout (Fmn2 - / -) accelerated axonal regeneration and functional recovery after PNI. To further determine the specific role of Fmn2 in neurons, Fmn2 expression in DRG and motor neurons was knocked down using highly neuron-tropic AAV serotype 2 / 9, and the specificity of neuronal knockdown was verified by eGFP-directed fluorescence-activated cell sorting (FACS). Similar growth-promoting effects were observed in AAV2 / 9-Fmn2-shRNA mice. Mechanistically, Fmn2 knockdown increased microtubule dynamics in the growth cones of DRG neurons by increasing the displacement rate of microtubule end-binding protein 3 (EB3) comets. In AAV2 / 9-Fmn2-shRNA mice, tubulin deacetylation was significantly increased in distal regenerating axons, while the phosphorylated form of HDAC5 was upregulated. The promotion of axonal regeneration by neuronal Fmn2 knockdown was completely abolished by inhibiting HDAC5 with the selective HDAC4 / 5 inhibitor LMK-235 or neuronal Hdac5 knockout (AAV2 / 9-Hdac5-shRNA). Finally, for Fmn 2- / - Small molecule bioinformatics analysis was performed to identify the FDA-approved small molecule metaxalone, which promoted axonal regeneration and functional recovery in vitro / in vivo. Metaxalone treatment delayed 24 hours after injury still maintained a full therapeutic effect in terms of functional recovery.

[0012] In a first aspect, provided herein is a method of treating nerve damage in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent is selected from the group consisting of metaxalone and a morphogen-2 (Fmn2) inhibitor.

[0013] In certain embodiments, the Fmn2 inhibitor is an antibody, an antibody fragment, an inhibitory nucleic acid molecule, or a small molecule, wherein the inhibitory nucleic acid molecule substantially silences Fmn2.

[0014] In certain embodiments, the inhibitory nucleic acid molecule is selected from the group consisting of short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotides, short interfering nucleic acid and post-transcriptional gene silencing RNA (ptgsRNA).

[0015] In certain embodiments, the medicament is formulated for intraperitoneal injection.

[0016] In certain embodiments, the inhibitory nucleic acid molecule is selected from the group consisting of an antisense oligonucleotide (ASO) and a single guide RNA molecule (sgRNA).

[0017] In certain embodiments, the inhibitory nucleic acid molecule comprises contiguous nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0018] In certain embodiments, the inhibitory nucleic acid molecule comprises SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:12.

[0019] In certain embodiments, administration of an inhibitory nucleic acid molecule reduces Fmn2 protein expression by 40%-100%.

[0020] In certain embodiments, the therapeutic agent is administered within 24 hours of onset of neural injury.

[0021] In certain embodiments, the therapeutic agent is administered parenterally, peritumorally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, perineurally, intraneurally, or directly at the site of a central nervous system lesion, including a nerve injury, or a peripheral nervous system lesion.

[0022] In certain embodiments, the inhibitory nucleic acid molecule is administered directly to a central nervous system lesion or a peripheral nervous system lesion containing a neural injury.

[0023] In certain embodiments, the therapeutic agent is metaxalone.

[0024] In certain embodiments, the nerve injury comprises at least one of central nervous system injury or peripheral nervous system injury.

[0025] In certain embodiments, the nerve damage is the result of physical trauma.

[0026] In certain embodiments, the nerve injury comprises damaged dorsal root ganglia.

[0027] In certain embodiments, the subject is a human, non-human primate, equine, bovine, canine, feline, or rodent.

[0028] In certain embodiments, the subject does not suffer from a muscle-related disorder.

[0029] In certain embodiments, the muscle-related disorder is selected from muscle spasm, muscle spasticity, and musculoskeletal pain.

[0030] In certain embodiments, the nerve damage is the result of physical trauma.

[0031] In certain embodiments, administration of metaxalone is initiated within 24 hours of onset of nerve damage.

[0032] In certain embodiments, metaxalone is administered orally.

[0033] In certain embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The foregoing aspects and many of the attendant advantages of the present invention will become more readily appreciated as they become better understood with reference to the following detailed description when taken in conjunction with the accompanying drawings, wherein:

[0035] Figure 1Low-dose ionizing radiation (LDIR) promotes robust axonal regeneration and functional recovery after peripheral nerve injury. (A) Schematic illustrating the LDIR treatment paradigm for the sciatic nerve pinch test. Immediately after the crush injury, mice were exposed to a single dose of 2 Gy of X-ray irradiation. The sciatic nerve pinch test was then performed 3 days after injury and X-ray irradiation. (B) LD-X-ray irradiation accelerated sensory fiber regeneration (as measured by the sciatic nerve pinch test) compared with sham-irradiated controls (n = 8 per group). Each dot represents the length of axonal regrowth from one mouse. (C) Representative longitudinal sections of sciatic nerves immunostained with anti-SCG10 antibodies from control or X-ray irradiated mice 3 days after the crush injury, demonstrating accelerated axonal regeneration in X-ray irradiated mice. The red dashed line indicates the injury site, and the red arrows indicate SCG10-positive regenerating axons in the distal nerve. Scale bar: 500 μm. (D) Regeneration index was measured as SCG10 immunoreactivity normalized to the crush site (n=4 per group). Each point represents the normalized SCG10 immunoreactivity of one mouse. (E) Schematic diagram illustrating the sciatic nerve injury treatment paradigm. Adult mice were exposed to a single dose of 2 Gy of X-ray irradiation immediately after crush injury. Sensory and motor function recovery was monitored for 28 days by animal behavioral tests. (FI) X-ray irradiated mice showed significant and lasting promotion in sensory (F) and motor (GI) function recovery (n=8 per group). (JK) Accelerated motor function recovery was verified by electromyographic recordings, showing that in X-ray irradiated mice, the compound muscle action potential amplitude in the proximal gastrocnemius (J) and distal interosseous muscles (plantar) (K) was significantly increased from 7 to 21 days after injury (n=8 per group). Each point represents the average CMAP amplitude of one mouse. Mean ± SEM. *P<0.05, ***P<0.001; Student's t-test in (B), two-way ANOVA followed by post hoc Bonferroni test in (D), or two-way ANOVA with repeated measures followed by post hoc Bonferroni test in (FK).

[0036] Figure 2.Fmn2 is a negative regulator of axon regeneration in mice. (A) The left sciatic nerve of adult C57BL / 6 mice was subjected to sciatic nerve transection, and the ipsilateral lumbar 4 and 5 DRGs that directly supply the sciatic nerve were harvested on days 1, 2, 3, and 5 for Fmn2 mRNA and protein expression analysis. (BC) In response to sciatic nerve injury, Fmn2 expression was significantly downregulated at the mRNA (B) and protein (C) levels (n=3 per time point). Each point represents a pooled mRNA or protein sample from L4 / 5 DRGs of 3 individual mice. FMN2 protein expression levels were normalized to GAPDH. (D) Schematic diagram illustrating the expression of Fmn2 - / - Example of the sciatic nerve crush test in mice. (E) Fmn2 mice were expressed in 96 mice compared to WT littermates (n = 6 per group). - / - The distal extent of sensory axon regrowth (measured by the sciatic nerve crush test) was significantly increased in mice. Each dot represents the length of axon regrowth from one mouse. (F) Representative photomicrographs showing that Fmn2 - / - Mice have an increase in the number of SCG10-positive regenerating axons distal to the crush injury site. The red dashed line indicates the injury site, and the red arrow indicates the SCG10-positive regenerating axon at the distal nerve. Scale bar: 500 μm. (G) Regeneration index was measured as SCG10 immunoreactivity normalized to the crush site (n = 4-5 per group). Each point represents the normalized SCG10 immunoreactivity of one mouse. Mean ± SEM. *P < 0.05, ***P < 0.001; one-way ANOVA followed by post hoc Bonferroni test in (B and C), Student's t test in (E), and two-way ANOVA followed by post hoc Bonferroni test in (G). SNT, sciatic nerve transection; N, naive control mouse; WT, wild type.

[0037] Figure 3Silencing the neuronal Fmn2 gene increases microtubule dynamics in cultured DRG neurons. (A) Representative micrographs show growth cones of DRG neurons expressing the microtubule plus-end marker EB3-mCherry (red) and the neuronal marker βIII-tubulin (green). Scale bar: 5 μm. (B) Series of time-lapse images showing the movement of EB3 comets at the plus-ends of growing microtubules in AAV2 / 9-scr-shRNA- or AAV2 / 9-Fmn2-shRNA-treated DRG neurons. Arrows of the same color indicate the movement of individual EB3 comets over time. Scale bar: 2 μm. (C) Representative kymographs of EB3 comet movement at the plus-ends of growing microtubules in DRG neurons treated with AAV2 / 9-scr-shRNA (as control) or AAV2 / 9-Fmn2-shRNA. The movement of individual EB3 comets (black lines) was recorded over 5 minutes at a rate of one frame per 2 seconds. Vertical scale bar: 1 min; horizontal scale bar: 1 μm. (D) The movement of each EB3 comet was automatically tracked, and microtubule dynamics were assessed using plusTipTracker software. Fmn2 knockdown resulted in a significant increase in the average microtubule growth velocity. Each dot represents the average microtubule growth velocity of EB3 comets from one growth cone. (E) Fmn2 deficiency significantly increased the growth length of EB3 comets at the distal growth tip of DRG neurons. Each dot represents the average growth length of EB3 comets from one growth cone. (F) Microtubule growth lifespan remained unchanged after genetic silencing of Fmn2. Each dot represents the average growth lifespan of EB3 comets from one growth cone. (G) Genetic silencing of Fmn2 did not change the EB3 comet density at the plus end of growing microtubules in DRG neurons. Each dot represents the average density of EB3 comets from one growth cone. A total of 31 (AAV2 / 9-scr-shRNA-treated neurons) and 39 (AAV2 / 9-Fmn2-shRNA-treated neurons) growth cones were analyzed from 3 independent experiments. Mean ± SEM. **P < 0.01, ***P < 0.001; Mann–Whitney U test was performed in (DG). ns, not significant.

[0038] Figure 4Fmn2 expression is a key determinant of α-tubulin deacetylation during axon regeneration in an HDAC5-dependent manner. (A) To knock down the genetic expression of Fmn2 and Hdac5 in DRG, AAV2 / 9-Fmn2-shRNA, AAV2 / 9-Hdac5-shRNA, or AAV2 / 9-scrambled (scr)-shRNA was directly injected into the sciatic nerve two weeks before sciatic nerve crush injury. LMK-235 (20 mg / kg) was injected intraperitoneally immediately after crush for 3 consecutive days to achieve selective blockade of HDAC5. Sciatic nerve crush test and axon quantification were performed three days after sciatic nerve crush injury to assess the distal extent of axon regeneration. (B) The distal extent of axon regeneration was significantly increased in AAV2 / 9-Fmn2-shRNA mice compared with AAV2 / 9-scr-shRNA mice. Drug blockade of HDAC5 activity using LMK-235 or genetic silencing of HDAC5 using AAV2 / 9-Hdac5-shRNA significantly reduced the growth-promoting effect induced by Fmn2 deletion (n = 6 per group). Each dot represents the length of axonal regrowth from one mouse. (C) Representative SCG10 immunostaining of sciatic nerves treated with different AAV2 / 9-shRNA and LMK-235 3 days after crush injury. The red dashed line indicates the injury site, and the red arrows indicate SCG10-positive regenerating axons at the distal nerve. Scale bar: 500 μm. (D) SCG10 immunoreactivity was measured at different distal distances and normalized to the level at the crush site as the regeneration index (n = 4 per group). Each dot represents the normalized SCG10 immunoreactivity from one mouse. (E) Representative immunoblots showing activation of HDAC5 phosphorylation correlated with a decrease in acetylated tubulin in regenerating axons of AAV2 / 9-Fmn2-shRNA mice 3 days after injury. Both drug blockade and knockdown of HDAC5 abolished tubulin deacetylation induced by Fmn2 deletion at growing axons. (F) Bar graph showing normalized fold change of pHDAC5 protein expression. pHDAC5 protein expression levels were normalized to GAPDH. Each point represents a pooled protein sample from segmented sciatic nerves (located 0-6 mm distal to the crush site) from 3 individual mice. (G) Bar graph showing normalized fold change of acetylated tubulin / total tubulin protein expression. Acetylated tubulin expression levels were first normalized to total tubulin levels and then to GAPDH expression. Each point represents a pooled protein sample from segmented sciatic nerves (located 0-6 mm distal to the crush site) from 3 individual mice. Mean ± SEM.*P<0.05 compared with AAV2 / 9-scr-shRNA-treated control mice; #P<0.05 compared with AAV2 / 9-Fmn2-shRNA-treated mice; one-way ANOVA followed by post hoc Bonferroni test was performed in (B, EF), and two-way ANOVA followed by post hoc Bonferroni test was performed in (D).

[0039] Figure 5 Fmn2 deficiency induces unique transcriptional reprogramming, serving as a unique gene signature for in silico screening of FDA-approved small molecules. Whole transcriptome analysis using RNA-seq was performed using L4 / 5 DRGs 5 days after sciatic nerve injury. (AB) Hierarchical clustering and principal component analysis revealed clear distinctions between naive (uninjured) and injured DRG neurons, regardless of genotype differences. (C) In injured wild-type (WT) and Fmn2 - / - A total of 397 genes were upregulated in DRG, representing common core transcriptional changes in response to injury in both genotype groups. - / - There are 87 upregulated genes and 58 downregulated genes differentially expressed in the injured DRG of mice, representing genotype-specific transcriptional regulation caused by genetic ablation of Fmn2. (D) Heat map of differentially expressed genes, including a total of 145 genes that were only expressed in Fmn2 - / - Differential expression in injured DRG of mice. Color intensity represents the change in log2FC from negative (light blue) to positive (dark brown).

[0040] Figure 6.Clinical relevance of FDA-approved metaxalone in promoting axonal regeneration in vivo after PNI. Whole transcriptome analysis and small molecule bioinformatics analysis identified four top-ranked bioactive small molecules for testing. (A) Schematic diagram showing the direct application of small molecules to the injury site immediately after sciatic nerve crush for 3 consecutive days (twice a day, 8 hours apart). Sciatic nerve crush testing was performed 72 hours after injury. (B) FDA-approved small molecule metaxalone induced robust axon regeneration in vivo (n=4 per group). Each point represents the length of axonal regrowth from one mouse. (C) Micrographs of longitudinal sections of sciatic nerves show a greater number of SCG10-positive regenerating axons distal to the crush injury site in metaxalone-treated mice compared to vehicle controls. (n=4 per group). The red dashed line indicates the injury site and the red arrows indicate SCG10-positive regenerating axons in the distal nerve. Scale bar: 500 μm. (D) Regeneration index was measured as SCG10 immunoreactivity normalized to the crush site. Each dot represents the normalized SCG10 immunoreactivity of one mouse. (E) Representative immunoblots show that metaxalone treatment induced robust activation of HDAC5 phosphorylation and reduced the expression of acetylated tubulin in the sciatic nerve 3 days after injury. (F) Bar graph shows the normalized fold change of pHDAC5 protein expression. pHDAC5 protein expression levels were normalized to GAPDH. Each dot represents a pooled protein sample of segmented sciatic nerves (located 0-6 mm distal to the crush site) from 3 individual mice (n=3 per group). (G) Bar graph shows the normalized fold change of acetylated tubulin / total tubulin protein expression. Acetylated tubulin expression levels were first normalized to total tubulin levels and then to GAPDH expression. Each dot represents a pooled protein sample of segmented sciatic nerves (located 0-6 mm distal to the crush site) from 3 individual mice (n=3 per group). Mean ± SEM. *P<0.05, **P<0.01, ***P<0.001; one-way ANOVA followed by post hoc Bonferroni test was performed in (B); two-way ANOVA followed by post hoc Bonferroni test was performed in (D); Student's t test was performed in (F and G).

[0041] Figure 7.Delayed metaxalone treatment still promotes robust axon regeneration and functional recovery in vivo after PNI. (A) Schematic diagram illustrates the experimental paradigm of delayed metaxalone treatment. One day after sciatic nerve crush, metaxalone (0.25 μg / μl) was applied to the injury site for 2 consecutive days (twice a day, 8 hours apart). Sciatic nerve crush test was performed on the 3rd day after injury to assess the extent of distal sensory axon regrowth. (B) Delayed metaxalone treatment for 24 hours was as effective as conventional metaxalone treatment in promoting axon regeneration in vivo (n=5 per group). Each point represents the length of axon regrowth in one mouse. (C) Representative confocal micrographs show that delayed metaxalone treatment regenerates more and longer SCG10-positive regenerated axons compared to vehicle controls. The red dotted line indicates the injury site, and the red arrow indicates the SCG10-positive regenerated axons of the distal nerve. Scale bar: 500 μm. (D) Regeneration index was measured as SCG10 immunoreactivity normalized to the crush site (n=4 per group). Each point represents the normalized SCG10 immunoreactivity of one mouse. (E) Adult mice were injected intraperitoneally with metaxalone (10 mg / kg) immediately after sciatic nerve crush injury for 21 consecutive days, or with a daily intraperitoneal injection of metaxalone (10 mg / kg) for 20 consecutive days 24 hours after injury. Animal behavioral assessments and weekly EMG recordings were performed. (FI) Both metaxalone-treated and delayed metaxalone-treated mice showed similar degrees of accelerated recovery of sensory (F) and motor (GI) function compared to vehicle controls (n=8-11 per group). (JK) Motor function recovery was improved in metaxalone-treated and delayed metaxalone-treated mice, as confirmed by a significant increase in CMAP amplitudes in proximal (gastrocnemius; J) and distal plantar (interosseous; K) muscles 7 to 21 days after injury compared to vehicle controls. (n=7-8 per group). Each point represents the average CMAP amplitude of one mouse. Mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Student's t-test in (B), two-way ANOVA followed by post hoc Bonferroni test in (D), and repeated measures two-way ANOVA followed by post hoc Bonferroni test in (FK).

[0042] Figure 8.Low-dose ionizing radiation enhances axonal regrowth of axotomized DRG neurons in culture. (A) Schematic illustrating the alpha particle treatment paradigm of DRG culture. A 3.5 μm thick Mylar membrane was cut into 18 × 18 mm dimensions and taped to the bottom of a 9 mm well located in the center of a 35 mm culture dish. A total of 2,000 DRG neurons were plated on the Mylar membrane (yellow highlighted area) pre-coated with poly-D-lysine (100 μg / ml) and laminin (10 μg / ml), as indicated by the black arrow. A planar 241Am alpha particle source was placed below the 9 mm well covered by the Mylar membrane. DRG neurons were exposed to different doses of single alpha particle irradiation (green arrows) and allowed to grow for 17 h for neurite outgrowth assay. (B) DRG neurons immunostained with anti-βIII-tubulin antibody exhibited significantly longer neurites after a single exposure to 20 mGy alpha particle irradiation compared to other treatment groups. Each dot represents the total neurite length or the average longest neurite from an independent cell culture experiment. Scale bar: 500 μm. (C) DRG neurons were placed in the center of the X-ray irradiator, irradiated with a single dose of X-rays using a cabinet irradiator X-Rad 320 (Precision X-Ray), and allowed to grow for 17 hours for neurite growth assays. (D) DRG neurons subjected to a single X-ray irradiation of 150 mGy exhibited significantly longer neurites compared to sham-irradiated controls. Each dot represents the total neurite length or the average longest neurite from an independent cell culture experiment. Scale bar: 500 μm. (E) Neither alpha particles nor X-ray irradiation affected cell survival of cultured DRG neurons. Each dot represents the average of an independent cell culture experiment. (F) Schematic diagram illustrating an example of LDIR treatment for ex vivo DRG explant cultures. Scale bar: 500 μm. (G) Whole-body exposure to 2 Gy of X-ray irradiation has the greatest promoting effect on neurite outgrowth of axotomized DRG explants (n = 5 DRG explants per group, from 3 independent experiments). Scale bar: 500 μm. Each point represents the total neurite outgrowth or the average longest neurite of one DRG. Mean ± SEM of triplicates. *P < 0.05, **P < 0.01; one-way ANOVA followed by post hoc Bonferroni test. ns, not significantly different; CTL, control.

[0043] Fig. 9.Alpha particle and X-ray irradiation induces gene-specific DNA methylation at CpG sites in promoter regions without affecting global DNA methylation and expression of key regeneration-related genes (RAGs) in DRG neurons. DRG neurons were irradiated with alpha particles (20 mGy) and X-rays (150 mGy), grown for 17 h, and genomic DNA was extracted. Whole-genome reduced representation bisulfite sequencing (RRBS) was performed. (A) Global DNA methylation in DRG neurons was not affected by alpha particle or X-ray irradiation. (B) Alpha particle and X-ray irradiation did not alter gene expression of DNA methylation machinery. Each dot represents mRNA expression from an independent cell culture experiment. (C) No significant differences in gene expression levels of key RAGs in DRG neurons were observed after treatment with alpha particles or X-ray irradiation. Each dot represents mRNA expression from an independent cell culture experiment. (D) Number of genes with significant hypermethylation and hypomethylation at promoter CpG sites in axotomized DRG neurons after exposure to 20 mGy alpha particle irradiation. (E) A total of 124 genes (44 hypomethylated + 80 hypermethylated) showed differential methylation at CpG sites in promoter regions in X-ray irradiated DRG neurons compared with sham irradiated controls. Mean ± SEM of triplicates. P > 0.05, two-way ANONA followed by post hoc Bonferroni test.

[0044] Fig.10 .Hypermethylation of the Fmn2 promoter was associated with significant downregulation of Fmn2 transcripts. Primary dissociated cultures of DRG neurons were irradiated with 20 mGy of alpha particles or 150 mGy of X-rays. Irradiated neurons were cultured for 17 h, and total RNA was extracted and subjected to quantitative polymerase chain reaction analysis to examine transcriptional changes. Among the seven candidate genes, only Fmn2 showed significant downregulation in both LDIR-treated cultures, which was closely associated with hypermethylation of CpG dinucleotides in its promoter region (n=4 per group). Each point represents mRNA expression from an independent cell culture experiment. Mean ± SEM. *P<0.05, Kruskal-Wallis test, followed by Dunn's post hoc test.

[0045] Fig.11 Genetic ablation of Fmn2 enhances the intrinsic growth capacity of preconditioned DRG neurons. (A) FMN2 protein is abundantly expressed in axotomized DRG neurons prepared from WT mice. However, in Fmn2 - / - FMN2 immunoreactivity was not detected in DRG neurons of mice. - / -DRG neurons prepared from mice exhibit robust neurite outgrowth (n=3 per group). Scale bar: 500 μm. Each point represents the total neurite outgrowth or the average longest neurite from an independent cell culture. (B) Fmn2 was observed - / - Neurite outgrowth was significantly increased in the contralateral DRG of mice. Removal of Fmn2 further enhanced the pretreatment effect on neurite outgrowth (n=8-11 DRG explants per group from 3 independent experiments). Each point represents the total neurite outgrowth or the average longest neurite of one DRG. Scale bar: 500 μm. Mean ± SEM. *P<0.05 compared with WT control; #P<0.05 compared with pretreated (ipsilateral) WT DRG explants; Student's t-test in (A) and two-way ANOVA followed by post hoc Bonferroni test in (B). WT, wild type.

[0046] Fig.12 Genetic ablation of Fmn2 promotes recovery of sensory and motor function after sciatic nerve injury. (A) As assessed by response to acupuncture stimulation, Fmn2 was expressed on days 7-15 after injury. - / - The recovery of sensory function in mice was significantly accelerated. (B) Fmn2 - / - Mice showed better recovery of toe extension reflex motor function on days 11-21 after injury. (C) On days 7-23 after injury, Fmn2 - / - The hind limb grip strength of mice was significantly higher than that of the control group. (D) Fmn2 - / - Mice showed an improvement in the sciatic nerve function index (SFI) during recovery. (EF) At days 7-21 after injury, Fmn2 - / - The CMAP amplitudes of the gastrocnemius and interosseous muscles of mice were significantly higher than those of their age-matched WT littermates. Each point represents the mean CMAP amplitude of one mouse. Mean ± SEM (n = 8-11 per group). *P < 0.05; two-way ANOVA with repeated measures followed by Bonferroni post hoc test. WT, wild type.

[0047] Fig.13.Adeno-associated virus 2 / 9 (AAV2 / 9)-based gene delivery system shows preferential transduction of DRG and motor neurons after sciatic nerve injection. (A) Representative fluorescent micrographs show that AAV2 / 9-scr-shRNA (labeled with eGFP) is preferentially transduced into DRG neurons 2 weeks after sciatic nerve injection. Approximately 72% of all βIII-tubulin-positive DRG neurons are GFP-positive (n=5 per group). Each dot represents the percentage of GFP and βIII-tubulin-positive DRG neurons from one mouse. Scale bar: 100 μm. (B) Representative confocal micrographs show that AAV2 / 9scr-shRNA (labeled with eGFP) successfully transduces ChAT-positive motor neurons in the anterior horn of the spinal cord. Scale bar: 100 μm. Approximately 75% of all ChAT-positive motor neurons are GFP-positive (n=5 per group). Each dot represents the percentage of GFP and ChAT-positive motor neurons from one mouse. (C) GFP immunoreactivity showed minimal overlap with SOX10-positive Schwann cells. Scale bar: 100 μm. (D) Representative confocal micrographs showing that AAV2 / 9-scr shRNA (labeled with eGFP) does not transduce SOX10-positive Schwann cells near the sciatic nerve injection site. Scale bar: 100 μm. (E) GFP immunoreactivity showed virtually no overlap with CD68-positive macrophages. Scale bar: 100 μm.

[0048] Fig.14.Adeno-associated virus 2 / 9 (AAV2 / 9)-based gene delivery system effectively transduces DRG and motor neurons after sciatic nerve injection. (AB) Representative FACS plots showing the gating strategy for isolating GFP-positive cells from DRG for western blot analysis. After enzymatic and mechanical dissociation of DRG, the cell suspension was first gated based on cell size (forward scatter; FSC-A, x-axis) and surface characteristics (side scatter; SSC-A, y-axis) (left panel of A). Next, single cells were selected and aggregated cells were discarded based on the ratio of FSC-A and FSC-H (right panel in A). Dissociated cells from non-transduced L4 / 5 DRG neurons (i.e., no GFP expression) were used as negative controls (left panel in B) to set the threshold of FITC-A (x-axis represents the fluorescence intensity of GFP) for selecting GFP-positive cells. GFP-positive cells within the gate (yellow inset in the right panel in B) were collected for subsequent western blot analysis. (C) Sorted GFP-positive cells expressed high levels of NeuN (neuronal marker) and GFP (fluorescent tag), and barely expressed major glial markers such as SOX10 (marker of Schwann cells) and IBA-1 (marker of microglia), indicating that AAV2 / 9 preferentially transduced DRG neurons rather than non-neuronal cells after injection into the sciatic nerve. Sorted GFP-positive cells from AAV2 / 9-Fmn2-shRNA mice expressed significantly lower levels of FMN2 protein compared to AAV2 / 9-scr-shRNA controls (n=3 per group). Each point represents one protein sample of sorted GFP-positive cells pooled from 3 individual mice. (D) Western blot analysis showed that FMN2 protein expression was significantly downregulated in the anterior horn of the lumbar spinal cord of the spinal cord of AAV2 / 9-Fmn2-shRNA-treated mice. Each point represents one protein sample pooled from the anterior horn of the spinal cord of 3 individual mice. Mean ± SEM. *P<0.05; Student's t test was performed in (C and D). (E) FMN2 protein expression in transduced GFP-positive and ChAT-positive motor neurons was significantly reduced in AAV2 / 9-Fmn2-shRNA-treated mice (white arrows). Untransduced ChAT-positive motor neurons without GFP (yellow arrows) showed high immunoreactivity for FMN2 protein expression. Scale bar: 100 μm.

[0049] Fig.15AAV2 / 9-mediated Fmn2 gene silencing in DRG accelerates axonal regeneration in vivo and promotes recovery of sensory and motor functions after sciatic nerve crush injury in adult mice. (A) To knock down gene expression of Fmn2 in DRG, AAV2 / 9-Fmn2-shRNA or AAV2 / 9-scrambled (scr)-shRNA was injected directly into the sciatic nerve two weeks before sciatic nerve crush injury. Axon quantification was performed three days after sciatic nerve crush injury to assess the distal extent of axonal regeneration. (B) Injection of AAV2 / 9-Fmn2-shRNA into the sciatic nerve significantly reduced FMN2 protein expression in L4 / 5 DRG, which directly supplies the sciatic nerve, two weeks after injection (n=3 per group). Each point represents a pooled protein sample of L4 / 5 DRG from 3 separate mice. (CD) Representative confocal micrographs showing that significantly higher levels of SCG10 immunoreactivity were detected in the distal axonal segments of injured sciatic nerves in AAV2 / 9-Fmn2-shRNA mice. SCG10 immunoreactivity was measured at different distal distances and normalized to the level at the crush site as the regeneration index (n = 4 per group). Quantitative data of SCG10 immunoreactivity in AAV2 / 9-scr-shRNA and AAV2 / 9-Fmn2-shRNA mice were obtained from Figure 4Extracted from C. Each dot represents the normalized SCG10 immunoreactivity of one mouse. The red dashed line indicates the injury site and the red arrows indicate the SCG10-positive regenerating axons of the distal nerve. Scale bar: 500 μm. (E) To knock down Fmn2 gene expression in DRG in vivo, AAV2 / 9-Fmn2-shRNA was directly injected into the left sciatic nerve of adult male C57BL / 6 mice two weeks before sciatic nerve crush injury. AAV2 / 9-scrambled (scr)-shRNA was used as a control. Sensory and motor function recovery was monitored for 29 days by an exhaustive list of animal behavioral tests. (FI) AAV2 / 9-Fmn2-shRNA-treated mice showed improved sensory (F) and motor (GI) function recovery compared to AAV2 / 9-scr shRNA-treated controls. (JK) AAV2 / 9-Fmn2-shRNA treated mice had accelerated recovery of motor function, as evidenced by significant increases in CMAP amplitudes in proximal (gastrocnemius; F) and distal plantar (interosseous; G) muscles from 7 to 21 days after injury compared with AAV2 / 9-scr-shRNA treated controls. Each point represents the mean CMAP amplitude of one mouse (n = 7-8 per group in FK). Mean ± SEM. *P < 0.05; Student's t test in (B), two-way ANOVA followed by post hoc Bonferroni test in (D), or two-way ANOVA with repeated measures followed by post hoc Bonferroni test in (FK).

[0050] Fig.16 .Efficient gene silencing of HDAC5 protein in lumbar 4 and 5 (L4 / 5) DRGs by sciatic nerve injection of AAV2 / 9-Hdac5-shRNA. Sciatic nerve injection of AAV-Hdac5-shRNA significantly reduced protein expression of HDAC5 in L4 / 5 DRGs two weeks after injection. Each point represents a pooled protein sample of L4 / 5 DRGs from 3 individual mice. Mean ± SEM of triplicates. *P < 0.05; Student's t-test.

[0051] Fig.17 Fmn2 deficiency induced upregulation of regeneration-related genes at similar levels to those in wild-type mice and activated inflammatory responses in injured DRG. (A) Upregulation of core regeneration-related genes in injured WT and Fmn2 mice - / -DRGs were induced in both, indicating that the two genotypes have a common core transcriptional reprogramming in response to injury (n=4 per group). Each point represents a pooled mRNA sample from L4 / 5 DRGs of 3 separate mice. Mean ± SEM. *P < 0.05 compared to WT naive (i.e., uninjured) DRGs; Kruskal Wallis test followed by Dunn post hoc test. (B) Gene set enrichment analysis was performed for a total of 145 genes that were only expressed in Fmn2 - / - Differential expression of these genes in injured DRG of mice was detected, and core gene regulatory networks, such as Jak-Stat and chemokine / cytokine signaling pathways, were identified.

[0052] Fig.18 Metaxalone enhances the intrinsic growth capacity of axotomized DRG neurons. (A) DRG neurons immunostained with anti-βIII-tubulin antibody exhibited extensive neurite outgrowth after treatment with increasing doses of metaxalone, and neurite outgrowth increased almost linearly until reaching a plateau at 20 μM. Scale bar: 500 μm. (BC) Total neurite outgrowth (B) and longest neurite length (C) of DRG neurons are the averages of at least 250 neurons per treatment group from an independent cell culture experiment (n=4 per group). Each point represents the total neurite length or the average longest neurite from an independent cell culture experiment. Mean ± SEM. *P < 0.05; one-way ANOVA followed by post hoc Bonferroni test was performed in (B and C).

[0053] Fig.19 .Percentage changes in DNA methylation levels in the promoter regions of seven candidate genes in DRG neurons after α-particle (20 mGy) and X-ray (150 mGy) LDIR. Positive and negative values ​​indicate hypermethylation and hypomethylation, respectively.

[0054] Fig. 20 The top-ranked bioactive small molecules with connectivity scores greater than 95 were shortlisted for functional assessment using the sciatic nerve crush test. Metaxalone and acamprosate are FDA-approved drugs.

[0055] Fig.21 . qPCR primer sequences used in this article.

[0056] Fig. 22 . shRNA sequences targeting mouse Fmn2 and Hdac5 mRNA transcripts and non-targeting scrambled sequences for AAV vector construction. DETAILED DESCRIPTION

[0057] definition

[0058] Throughout this disclosure, unless the context requires otherwise, the words "comprise" or variations thereof will be understood to imply the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers. It should also be noted that in this disclosure, particularly in the claims and / or paragraphs, terms such as "comprise", "include", "contain", etc. may have the meanings assigned to them by U.S. patent law; for example, they may represent open-ended "include", "include", "contain", etc.; and terms such as "consisting essentially of / consists essentially of" have the meanings assigned to them by U.S. patent law, such as allowing the presence of elements not explicitly stated, but excluding elements existing in the prior art or elements that affect the basic or novel characteristics of the invention.

[0059] Furthermore, throughout the present disclosure and claims, unless the context requires otherwise, the words "comprises / comprising" or variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0060] Unless otherwise expressly stated, nouns modified by quantifiers used herein include the singular and plural nouns. In addition, if the term "about" is used before a quantitative value, the present teaching also includes the specific quantitative value itself, unless otherwise specifically stated. As used herein, unless otherwise stated or inferred, the term "about" refers to a variation of ±10%, ±7%, ±5%, ±3%, ±1%, or ±0% of the nominal value.

[0061] As used herein, the term "treatment" and the like refers to the alleviation or improvement of a condition / disease and / or symptoms associated therewith. It should be understood that, although it cannot be excluded, the treatment of a disorder or condition does not require the complete elimination of the disorder, condition or symptoms associated therewith. In certain embodiments, treatment includes the prevention of a disorder or condition and / or symptoms associated therewith. The term "prevention" or "prevent" used herein refers to any effect that inhibits or at least delays the development of a disorder, symptom or condition associated therewith. Prevention may include primary, secondary and tertiary prevention, wherein: a) primary prevention avoids the development of the disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing the chances of intervention to prevent disease progression and symptom appearance; and c) tertiary prevention reduces the negative effects of existing diseases by restoring function and reducing complications associated with the disease.

[0062] As used herein, the term "subject" refers to any animal (eg, mammal), including, but not limited to, humans, non-human primates, canines, felines, and rodents.

[0063] As used herein, the term "substantially silencing Fmn2" means that the expression or protein activity or level of the target gene or the protein encoded by the target gene is reduced by about 10% to 100%, 10% to 90%, 20% to 90%, 30% to 90%, 40% to 90%, 50% to 90%, 50% to 80%, 50% to 70%, 55% to 70%, 55% to 69.2%, 70% to 90%, or 80% to 90% in the presence of an inhibitory nucleic acid molecule targeting an Fmn2 nucleic acid sequence introduced compared to the expression or protein activity or level of the target gene or the protein encoded by the target gene observed in the absence of the inhibitory nucleic acid molecule targeting the Fmn2 nucleic acid sequence. Generally speaking, when a gene is substantially silenced, the expression or protein activity or level of the target gene or protein encoded by the target gene will be reduced by at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or even 100% compared to when the inhibitory nucleic acid molecule is not present.

[0064] As used herein, the term "therapeutically effective amount" refers to that amount of a compound or therapeutic agent that elicits the biological and / or pharmaceutical response sought by the researcher, veterinarian, clinician or physician, including the alleviation of symptoms of the disease, condition or disorder being treated, in a cell culture, tissue system, subject, animal or human.

[0065] The present disclosure provides a method of treating nerve damage in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a therapeutic agent, wherein the therapeutic agent is selected from the group consisting of metaxalone and an Fmn2 inhibitor.

[0066] In certain embodiments where the therapeutic agent is metaxalone, the subject does not suffer from muscle-related disorders. The term "musculoskeletal disorders" may include any disorder of muscles, tendons, ligaments, bones, joints and related tissues that affect body movement and maintain its form. Such disorders include disorders that may originate from muscles, tendons, ligaments or bones and related tissues or disorders that originate from other parts of the body (such as the central or peripheral nervous system) and are manifested in muscles, tendons, ligaments, bones, joints or related tissues. In certain embodiments, muscle-related disorders are selected from muscle twitches, muscle spasms and muscle and / or bone pain.

[0067] The Fmn2 inhibitor can be an antibody, an antibody fragment, an inhibitory nucleic acid molecule, a small molecule (synthetic or naturally isolated or derived), a protein, a peptide, a carbohydrate or an antibody, wherein the inhibitory nucleic acid molecule substantially silences Fmn2.

[0068] The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be a single-chain antibody. The antibody may be a chimeric antibody. The antibody may be a human antibody or a humanized antibody.

[0069] The inhibitory nucleic acid molecule can be a short interfering nucleic acid (siNA), a short interfering nucleic acid (siRNA), a double-stranded RNA (dsRNA), a micro RNA (miRNA), a short hairpin RNA (shRNA), a short interfering oligonucleotide, a short interfering nucleic acid, or a post-transcriptional gene silencing RNA (ptgsRNA).

[0070] In general, the inhibitory nucleic acid molecule can be unmodified or modified. In certain embodiments, the inhibitory nucleic acid molecule comprises one or more modified oligonucleotides, such as oligonucleotides modified with phosphorothioate-, 2'-O-methyl-, etc., which are methods known in the art to improve the in vivo stability of oligonucleotides.

[0071] In certain embodiments, the inhibitory nucleic acid molecule comprises a sequence that is complementary to 5 to 50 consecutive nucleotides (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, about 30, about 35, about 40, or about 50 consecutive nucleotides) of a nucleic acid sequence encoding Fmn2 (e.g., an RNA sequence such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9). In certain embodiments, the inhibitory nucleic acid molecule comprises 15-50 nucleotides, 15-45 nucleotides, 15-40 nucleotides, 15-35 nucleotides, 15-30 nucleotides, 15-25, or 20-29 nucleotides and may include 3' and / or 5' overhangs of about 0, 1, 2, 3, 4, or 5 nucleotides in length on each strand. Inhibitory nucleic acid molecules useful in the methods described herein also include sequences that bind to nucleic acids under stringent conditions, wherein the nucleic acids have 80%, 85%, 90% or 95% homology to 5 to 50 consecutive nucleotides of the following: NM_001305424.2 (SEQ ID NO: 1), NM_020066.5 (SEQ ID NO: 2), NM_001348094.2 (SEQ ID NO: 3), XM_017001837.2 (SEQ ID NO: 4), XM_017001838.2 (SEQ ID NO: 5), XM_011544237.4 (SEQ ID NO: 6), XM_047425620.1 (SEQ ID NO: 7), XM_017001840.3 (SEQ ID NO: 8) and XM_017001841.3 (SEQ ID NO: 9).

[0072] In certain embodiments, the inhibitory nucleic acid molecule comprises SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In certain embodiments, the inhibitory nucleic acid molecule is a shRNA comprising SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:12.

[0073] Nerve damage can include damage to the central nervous system or the peripheral nervous system.

[0074] Nerve injury can be acute or chronic. Nerve injury can include complete or partial cutting of neurons or compression or compression injury of neurons. In certain embodiments, nerve injury directly damages the normal function of one or more neurons. In certain embodiments, nerve injury indirectly damages the normal function of one or more neurons. Nerve injury can be caused by acute or traumatic events, chronic events, pressure accumulation or chronic neurodegeneration. Subject injury may cause neuronal injury. Common causes of nerve injury include but are not limited to disease and / or infection, ischemia, hypoxia, hypoglycemia, contusion, tearing, brain or spinal cord trauma (e.g., caused by acute spinal cord injury or stroke), damage caused by exogenous chemicals and combinations thereof.

[0075] The neural injury can be the result of a disease, disorder or condition of the subject, such as injury to the dorsal root ganglia; traumatic brain injury; stroke-related injury; cerebral aneurysm-related injury; spinal cord injury, including monoplegia, diplegia, paraplegia, hemiplegia and quadriplegia; neuroproliferative disease or a neuropathic pain syndrome.

[0076] In certain embodiments, the subject suffers from nerve damage caused by trauma. The nerve damage may include optic nerve damage, spinal cord damage, or peripheral nerve damage. In certain embodiments, the spinal cord damage or peripheral nerve damage includes damaged dorsal root ganglion neurons.

[0077] In certain embodiments, the subject suffers from a disease or condition that causes neural damage. In certain embodiments, the disease or condition is selected from the group consisting of: stroke, spinal cord injury, Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple system atrophy, spinocerebellar atrophy, motor neuropathy, epilepsy or seizures, peripheral neuropathy, cerebral palsy, glaucoma, age-related loss of neurons or neuronal connectivity, and associated sensory, motor, reflex, and cognitive impairment.

[0078] In certain embodiments, the subject suffers from an injury caused by or associated with a peripheral neuropathy, e.g., diabetic neuropathy, viral-related neuropathy, botulism-related neuropathy; toxic polyneuropathy, nutritional neuropathy, vasculopathic neuropathy, sarcoidosis-related neuropathy; cancerous neuropathy; compressive neuropathy and / or hereditary neuropathy; and / or peripheral nerve injury associated with a spinal cord injury.

[0079] The specific route of administration and dosage regimen of the therapeutic agent can be determined by the skilled clinician based on factors such as the exact nature of the condition being treated, the severity of the condition, and the age and general physical condition of the patient.

[0080] In certain embodiments, the therapeutic agent is administered parenterally, peritumorally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, perineurally, intraneurally, or directly at a central nervous system lesion site including a nerve injury or a peripheral nervous system lesion site. In the case where the therapeutic agent is metaxalone or a small molecule, the therapeutic agent can be administered orally.

[0081] The exact dosage of the therapeutic agent can vary according to factors known in the art, including but not limited to the physical / chemical properties of the compound, the properties of the pharmaceutically acceptable carrier, the intended dosing regimen, the state of the subject's nervous system damage, and the method of administration of the compound. However, one of ordinary skill in the art can easily determine the appropriate amount taking these factors into account.

[0082] In certain embodiments, the therapeutic agent is administered to the subject within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, or 72 hours of onset of neural injury.

[0083] Example

[0084] result

[0085] LDIR enhances the intrinsic growth capacity and functional recovery of injured neurons

[0086] Ionizing radiations such as alpha particles and X-rays are commonly used in biological research and medical applications. LDIR exerts neuroprotective effects in mouse models of Alzheimer's disease, Parkinson's disease, glaucoma, diabetic neuropathy, and retinitis pigmentosa. In a recent study, the beneficial effects of LDIR in rats were observed by direct local irradiation of the sciatic nerve, but the underlying mechanism remains unclear. Therefore, it was hypothesized that LDIR may not only promote the overall physiological function of the organism but also enhance the intrinsic growth capacity of injured DRG neurons because DRG directly supplies the sciatic nerve. For the first time, it was demonstrated that 20 mGy alpha particles induced an increase in the total neurite outgrowth and the average longest neurite length of DRG neurons by 39.9% and 35.5%, respectively ( Figure 8 A-8B). In addition, robust neurite outgrowth was observed after irradiation with LD X-rays, which have higher penetrating power than α particles, with 150 mGy having the greatest promotion effect on total neurite outgrowth (89.0%) and longest neurite length (66.7%) ( Figure 8 C-8D). Irradiation with alpha-particles (α-particles) and X-rays had no adverse effects on cell survival as measured by the WST-1 cytotoxicity assay ( Figure 8 E).

[0087] These in vitro findings were then extended to whole-body X-ray irradiation ( Figure 8 F), and investigated whether LD X-ray irradiation could increase the intrinsic growth capacity of axotomized neurons in DRG explant culture. Notably, 2 Gy of whole-body X-ray irradiation showed a significant increase in total neurite outgrowth and longest neurite length by 54.8% and 68.4%, respectively ( Figure 8 G). The ex vivo DRG explant results were validated by the sciatic nerve crush test to measure the rate of axonal regeneration in vivo ( Figure 1 A). Mice exposed to a single X-ray dose of 2 Gy immediately after crush had a 41.2% increase in axonal regeneration compared to sham-irradiated mice ( Figure 1 B). The results of the nerve crush test were validated by immunostaining for SCG10, which is preferentially expressed in regenerating sensory axons. The regeneration index was calculated by normalizing the mean SCG10 immunoreactivity of longitudinal sections of the sciatic nerve measured at different distal distances relative to the SCG10 immunoreactivity at the crush site, indicating that a certain length and number of axons have been regenerated across the crush site ( Figure 1 C). Three days after sciatic nerve crush, the regeneration index was significantly higher in the region beyond 4 mm from the injury site in the 2 Gy X-ray-treated nerves compared with the sham-irradiated controls, indicating that X-ray irradiation accelerates axonal regeneration in injured sciatic nerves in mice ( Figure 1 D).

[0088] To examine whether this increase in intrinsic growth capacity could promote functional recovery in vivo over an extended period of time (28 days), adult mice were exposed to a single dose of 2 Gy of X-ray irradiation immediately after crushing. Sensory and motor function recovery was monitored for 28 days using a battery of behavioral tests ( Figure 1 E). X-ray-irradiated mice showed an early initial pinprick sensory response, 4 days earlier than sham-irradiated mice ( Figure 1 F). Regarding motor function recovery, X-ray-irradiated mice recovered full toe extension reflex 6 days earlier than sham-irradiated mice ( Figure 1 G ) and showed faster recovery of hindlimb grip strength than sham-irradiated mice ( Figure 1 H). Gait analysis of walking trajectories by sciatic function index (SFI) showed that motor recovery in X-ray-irradiated mice was significantly greater than that in sham-irradiated mice on days 11-19 after injury ( Figure 1 I). Electromyographic (EMG) recordings confirmed the accelerated motor recovery, showing significant increases in compound muscle action potential (CMAP) amplitudes in the proximal (gastrocnemius) and distal plantar (interosseous) hindlimb muscles of X-ray-irradiated mice at 7, 14, and 21 days after injury compared with sham-irradiated controls ( Figure 1 J-1K).

[0089] LDIR induces Fmn2 promoter hypermethylation, and Fmn2 expression is downregulated in DRG in response to PNI

[0090] To delineate the intrinsic growth program induced by LDIR, genome-wide CpG methylation profiling was performed by reduced representation bisulfite sequencing (RRBS) of purified DRG neurons after treatment with an optimal dose of α-particles (20 mGy) or X-ray irradiation (150 mGy). Global DNA methylation was found to be unaffected by LDIR. The methylation levels of CpG sites in α-particle or X-ray irradiated DRG neurons were comparable to their sham-irradiated controls: approximately 60% of CpG sites were weakly methylated (0-10% methylation), while approximately 28% of CpG sites were highly methylated (>70% methylation) ( Fig. 9 A). Loss of global DNA methylation in LDIR-treated DRG neurons indicated no changes in the expression of genes of the methylation machinery, such as DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b, and Gadd45b), as confirmed by qPCR analysis ( Fig. 9 B). No significant differences were detected in the gene expression levels of key RAGs in DRG neurons after α-particle or X-ray irradiation treatment ( Fig. 9 C).

[0091] We then explored the possibility that DNA methylation at the promoter region might be associated with transcriptional activation / repression of the corresponding gene after LDIR. We found that CpG sites in the promoter region of 206 genes were differentially methylated between α-particle-irradiated and sham-irradiated neurons (difference in differential methylation > 20%). Among these differentially methylated genes, 117 genes were hypermethylated and 89 genes were hypomethylated ( Fig. 9 D) In ​​X-ray-irradiated neurons, the CpG island promoters of 80 genes were hypermethylated and 44 genes were hypomethylated ( Fig. 9 E). Among these differentially methylated genes, 7 genes were uniquely expressed in α-particle and X-ray irradiated DRGs with similar overall CpG island promoter hypermethylation or hypomethylation percentages ( Fig.19 ). Correlation analysis was performed between the mRNA expression levels and DNA methylation levels of these seven genes. Notably, Fmn2 mRNA expression was observed to be significantly downregulated (hypermethylated) in cultured DRG neurons irradiated with α particles (32%) and X-rays (22%) compared with sham irradiated controls. Gene expression of the remaining six genes was largely unaffected, regardless of their hypermethylation / hypomethylation status ( Fig.10). In fact, the actual gene expression of the hypermethylated / hypomethylated genes may not be affected or even up-regulated / down-regulated in some cases.

[0092] After PNI, thousands of genes are differentially expressed, coordinating the intrinsic growth program that allows injured neurons to enter an active growth state, leading to successful axon regeneration. To verify the involvement of Fmn2 in the intrinsic growth program, the mRNA and protein expression of Fmn2 were examined in injured lumbar 4 and 5 (L4 / 5) DRGs, which directly supply the sciatic nerve. Figure 2 A). 2-3 days after sciatic nerve injury, Fmn2 mRNA (38.6%) was detected in DRG ( Figure 2 B) and protein expression (68.2-81.6%) ( Figure 2 C) Significant downregulation. These results confirm injury-induced downregulation of Fmn2 in injured DRG and suggest that Fmn2 is a potential target gene for axon regeneration.

[0093] Fmn2 gene ablation enhances the intrinsic growth capacity of injured neurons and accelerates functional recovery after PNI

[0094] These findings were then extended to examine the effects of Fmn2 knockout mice (Fmn2 - / - The intrinsic growth capacity of DRG neurons in axotomized and preconditioned (to induce maximal growth capacity) mice has been extensively used to study the role of FMN2 in oocyte development. - / - The mice were viable, had no major defects other than poor female fertility, and showed no gross or histological brain differences compared to wild-type mice. - / - Primary DRG neurons from Fmn2 mice exhibited significantly more neurite outgrowth and longer neurite length - / - Total neurite outgrowth and mean longest neurite length in DRG neurons increased by 71.5% and 82.1%, respectively ( Fig.11 A). The results showed that preconditioning lesions of the sciatic nerve increased neurite outgrowth in DRG explants of WT mice, whereas Fmn2 ablation significantly enhanced the preconditioning effect of neurite outgrowth, increasing total neurite outgrowth by 81.8% and mean longest neurite by 78.4%, respectively ( Fig.11 B) Next, for Fmn2 - / - Mice were subjected to sciatic nerve crush injury, and the extent of axonal regrowth was assessed by sciatic nerve crush test 3 days after injury ( Figure 2 D). Consistent with the in vitro results, Fmn2 depletion significantly enhanced the distal extent of axonal regrowth by 83.5% (Fmn2 - / -5.4±0.3 mm in WT mice compared to 2.9±0.2 mm in WT mice) Figure 2 E) The results showed that compared with WT control neurons, Fmn2 - / - Mice exhibited longer SCG10-positive regenerating axons in injured sciatic nerves ( Figure 2 F). Significant SCG10 immunoreactivity was observed even in the distal region of the injured nerve 5 mm from the injury site ( Figure 2 G).

[0095] To detect Fmn2 - / - Whether the increased intrinsic growth capacity and axonal regeneration in vivo in mice contribute to accelerated functional recovery after PNI was evaluated by exhaustive animal behavioral and electrophysiological tests to assess sensory and motor function recovery. - / - In Fmn2 mice, initial recovery of sensory function measured by the pinprick test was significantly earlier than in WT mice by 4 days. - / - The mice fully recovered sensory function on day 17, 2 days earlier than WT mice ( Fig.19 A) For motor function recovery, Fmn2 - / - Mice fully recovered their toe extension reflex on day 19 after injury, 4 days earlier than WT mice ( Fig.12 B) Through Fmn2 - / - Grip strength testing of mice confirmed accelerated motor recovery, with mice showing significantly higher grip strength than controls from day 7 to day 23 ( Fig.12 C) Gait movements assessed by SFI showed that Fmn2 - / - The mice showed significant recovery of motor function from day 9 to day 17 ( Fig.12 D) The facilitatory effect observed in behavioral tests was confirmed by muscle EMG recordings, showing that Fmn2 - / - Proximal (gastrocnemius: 86.6% at 1 week; 56.4% at 2 weeks; 42.0% at 3 weeks) of mice ( Fig.12 E) and distal plantar muscles (interosseous muscles: 51.9% at 1 week; 61.6% at 2 weeks; 28.0% at 3 weeks) ( Fig.12 F) CMAP amplitude increased.

[0096] Ablation of FMN2 expression in DRGs using a highly neuron-tropic AAV serotype promotes axonal regeneration and functional recovery in vivo after PNI

[0097] During mouse development, Fmn2 is expressed almost exclusively in the nervous system, including the brain, spinal cord, and DRG; however, little is known about its mechanistic role in axon regeneration after PNI. To overcome the limitations of global Fmn2 knockout mice, neuronal Fmn2 expression was knocked down by expressing short hairpin RNA (shRNA) targeting Fmn2 mRNA via recombinant adeno-associated virus (AAV2 / 9-Fmn2-shRNA) injected into the sciatic nerve. Among the commonly used AAV serotypes, AAV2 / 9 showed a superior ability to transduce neurons in DRGs following a peripheral delivery route. Mice injected with AAV2 / 9-scrambled (scr)shRNA-eGFP showed high levels of lumbar DRG and motor neuron transduction 2 weeks after sciatic nerve injection. In fact, in 72% of total βIII-tubulin-positive neurons ( Fig.13 A) and 75% of ChAT-positive neurons ( Fig.13 In contrast, no eGFP signal was found in SOX10-positive Schwann cells ( Fig.13 C). To examine whether AAV2 / 9 targeted gene delivery to non-neuronal cell populations in the sciatic nerve near the AAV2 / 9 injection site, colocalization of eGFP and SOX10 / CD68 immunofluorescence signals was examined in the sciatic nerve, and it was found that virtually no eGFP immunofluorescence was associated with SOX10-positive Schwann cells ( Fig.13 D) or CD68 positive macrophages ( Fig.13 E), indicating that AAV2 / 9 preferentially transduces neurons rather than non-neuronal cells. Two weeks after AAV2 / 9 injection into the sciatic nerve, the transduced cell population was further analyzed by flow cytometry of L4 / 5 DRG ( Fig.14 A-14B). Western blot analysis showed that NeuN (a pan-neuronal marker) was strongly expressed in sorted eGFP-positive cells from AAV2 / 9-scr-shRNA-treated mice or AAV2 / 9-Fmn2-shRNA-treated mice, but glial markers such as SOX10 (Schwann cells) and IBA-1 (microglia) were not. A high knockdown efficiency (69.2%) of Fmn2 protein expression was detected in those sorted eGFP-positive neurons collected from DRGs of AAV2 / 9-Fmn2-shRNA-treated mice ( Fig.14 C). In addition, Fmn2 expression in the anterior horn of the spinal cord of AAV2 / 9-Fmn2-shRNA-treated mice showed a higher knockdown efficiency (55%) compared with that of AAV2 / 9-scr-shRNA-treated mice. Fig.14D). To confirm the western blot results, immunohistological studies were performed on the spinal cord to observe the knockdown efficiency of Fmn2 in motor neurons. In AAV2 / 9-Fmn2-shRNA treated mice, GFP expression was highly abundant in ChAT-positive motor neurons (white arrows), while Fmn2 expression was greatly reduced to undetectable expression levels. In contrast, Fmn2 expression in non-transduced ChAT-positive motor neurons remained at a high level (yellow arrows) ( Fig.14 E). These results clearly demonstrate the high neuronal tropism of AAV serotype 2 / 9 and the specificity of Fmn2 knockdown in neurons in our study.

[0098] To determine the effects of neuronal Fmn2 knockdown on axonal regeneration and functional recovery in vivo, AAV2 / 9-Fmn2-shRNA was injected into the sciatic nerve ( Fig.15 A). Similarly, a significantly high knockdown efficiency of Fmn2 (69.2%) was shown in L4 / 5 DRG 2 weeks after injection ( Fig.15 B). A significant increase in axonal regeneration (5.3±0.3 mm) was observed in AAV2 / 9-Fmn2-shRNA mice ( Fig.15 C). The number of SCG10-positive regenerating axons located 5 mm distal to the crush site in AAV2 / 9-Fmn2-shRNA mice was significantly higher than that in AAV2 / 9-scr-shRNA control mice ( Fig.15 D), indicating that the axonal regrowth-promoting effect of Fmn2 knockout is mainly mediated by neurons. As expected, our behavioral and electrophysiological studies ( Fig.15 E) shows that accelerated axon regeneration in AAV2 / 9-Fmn2-shRNA mice promotes sensory ( Fig.15 F) and recovery of motor function ( Fig.15 G-15I), and promotes the formation of functional neuromuscular junctions after sciatic nerve compression ( Fig.15 J-15K).

[0099] Mechanistic role of Fmn2 in microtubule dynamics and post-translational modification of tubulin

[0100] Microtubules are highly dynamic structures that undergo continuous assembly (polymerization) and disassembly (depolymerization). The plus ends of dynamically growing microtubules act as "sensors" of the cellular microenvironment, allowing rapid reorganization of the cytoskeleton for successful axon regeneration. Microtubule dynamics may also be affected by post-translational modifications, such as tubulin acetylation and deacetylation. Therefore, microtubule dynamics were first quantitatively analyzed in peripheral axons using high-resolution time-lapse confocal microscopy and fluorescently labeled microtubule end-binding protein 3 (EB3). To this end, DRG neurons from adult mice were purified and transduced with AAV2 / 9-EB3-mCherry. Four days after transduction, growth cones were sorted as GFP-positive and EB3-positive during live cell imaging, and immunoreactivity for βIII-tubulin was confirmed after fixation ( Figure 3 A), and a series of 5-minute time-lapse images were captured (with a 2-second time interval between each frame). Microtubule dynamics were quantified by tracking the displacement of EB3 comets (and thus growing microtubules) at the plus ends of microtubules in successive frames of the time-lapse images. ( Figure 3 B) (Dynamic Image S1). Curve recording analysis of EB3-mCherry signals revealed EB3-mCherry comet tracks, with comet velocity increased in the distal end of Fmn2-deficient DRG neurons ( Figure 3 C). Using the MATLAB-based automated software plusTipTracker, the movement of these EB3 comets can be used to quantitatively determine microtubule dynamics. The microtubule polymerization rate (growth velocity) in Fmn2-deficient DRG neurons was significantly increased by 16.1% compared to AAV2 / 9-scr-shRNA control DRG neurons, indicating that Fmn2-deficient microtubules grew significantly faster than control microtubules (8.20±0.21μm / min compared to 9.52±0.17μm / min). Fmn2-deficient DRG neurons also exhibited a significant increase in the length of microtubule growth, which represents the total displacement of EB3 comets before pausing or collapsing (1.71±0.03μm in AAV2 / 9-scr-shRNA neurons and 1.81±0.03μm in AAV2 / 9-Fmn2-shRNA neurons) ( Figure 3 E). Knockdown of Fmn2 does not affect the duration of microtubule polymerization (growth lifetime) ( Figure 3 F) and the density of EB3 comets in the growth cone ( Figure 3 G). Data indicate that Fmn2 knockdown increases microtubule dynamics in growth cones, consistent with previous studies on the role of Fmn2 as a regulator of growth cone dynamics in cultured chick spinal cord commissural neurons and zebrafish.

[0101] Morphogenetic proteins, including Fmn2, contain highly conserved morphogen homolog 1 (FH1) and FH2 domains at the C-terminus. An increasing number of studies have shown that the FH1 and FH2 domains regulate microtubule stability and dynamics by inducing α-tubulin acetylation. Histone deacetylase 5 phosphorylation (HDAC5)-mediated α-tubulin deacetylation is essential for axon regeneration. For example, sciatic nerve injury induces HDAC5 phosphorylation in injured axons in vivo, and pHDAC5 has been shown to catalyze the deacetylation of α-tubulin in axons to promote axon regeneration. To examine the mechanistic role of neuronal Fmn2 expression in axon regeneration, more specifically, whether HDAC5 is responsible for the growth-promoting effect induced by Fmn2 loss, LMK-235, a selective inhibitor of HDAC4 and HDAC5 that has been reported to preferentially inhibit HDAC5 activity at low-dose exposures immediately after sciatic nerve crush, was injected intraperitoneally, and axon regeneration in vivo was assessed by the nerve crush test ( Figure 4 A). Three days after sciatic nerve injury, LMK-235 completely abolished the axonal regrowth promotion effect in AAV2 / 9-Fmn2-shRNA mice, reaching a level comparable to that of AAV2 / 9-scr-shRNA control mice. To exclude the possibility of any nonspecific inhibition of HDAC4 even at low concentrations of LMK-235, knockdown of HDAC5 expression by expressing shRNA against Hdac5 mRNA using AAV2 / 9 (AAV2 / 9-Hdac5-shRNA) injected into the sciatic nerve resulted in a similar reduction in the rate of distal axon regeneration and the number of SCG10-positive regenerating axons three days after injury ( Figure 4 B-4D). Consistent with the observations in the AAV2 / 9-Fmn2-shRNA knockdown experiment, AAV2 / 9-Hdac5-shRNA also significantly reduced HDAC5 protein expression in L4 / 5DRG by 57.8% ( Fig.16 Next, pHDAC5 and acetylated tubulin expression in the sciatic nerve segments 0-6 mm distal to the crush injury site of AAV2 / 9-scr-shRNA and AAV2 / 9-Fmn2-shRNA mice were examined by Western blot analysis 3 days after injury ( Figure 4 E) According to Figure 4 Results of the nerve crush test in B show that in AAV2 / 9-Fmn2-shRNA mice, active axon regeneration occurred in the sciatic nerve segment 0-6 mm distal to the crush site, and optical density analysis of the resulting strips showed that pHDAC5 protein increased 5.13-fold on day 3 after injury ( Figure 4 F), which appeared to be associated with a decrease in the expression of acetylated tubulin (70.3%) in AAV2 / 9-Fmn2-shRNA mice ( Figure 4G). Similarly, pharmacological blockade of HDAC5 with LMK-235 (70%) or genetic silencing of HDAC5 using AAV2 / 9-Hdac5-shRNA (77%) significantly reduced pHDAC5 protein expression, leading to increased expression of acetylated tubulin in the sciatic nerves of AAV2 / 9-Fmn2-shRNA mice 3 days after injury ( Figure 4 E-4G).

[0102] Fmn2 - / - Small molecule bioinformatics analysis of gene expression signatures and identification of the therapeutic small molecule metaxalone

[0103] Axonal injury in the PNS induces the intrinsic growth capacity of neurons and transcriptional changes in gene expression that promote axon regeneration. Therefore, it is hypothesized that bioactive small molecules associated with the gene regulatory network of Fmn2 deficiency could be identified that could activate the intrinsic growth capacity of injured neurons, leading to accelerated axonal growth in vivo, which could be exploited for regeneration and functional recovery. To achieve this goal, we injected Fmn2-derived mitochondria into the PNS 5 days after PNI. - / - Whole transcriptome analysis and small molecule bioinformatics analysis were performed on L4 / 5 DRGs of WT mice and their age-matched WT mice. Hierarchical clustering and principal component analysis showed that there were clear distinctions between contralateral and ipsilateral DRGs regardless of genotype differences ( Figure 5 A-5B). After injury, Fmn2 - / - There were 563 and 636 differentially expressed (DE) genes in DRG and WT DRG, respectively. Among these DE genes, Fmn2 - / - In DRGs of WT and WT injured groups, 397 genes were upregulated and 21 genes were downregulated ( Figure 5 C). Specifically, key RAGs such as Atf3, Sprr1a, Jun, Stmn4, Fabp5, Hspb1, Sox11, and Gap43 are involved in the regulation of Fmn2 - / - Fmn2 deficiency did not affect RAG expression after PNI ( Fig.17 A) A total of 145 genes (87 up-regulated and 58 down-regulated) were expressed only in Fmn2 - / - mice, but not in WT mice after injury ( Figure 5 D). Gene set enrichment analysis (GSEA) was performed to identify regulatory Fmn2 - / - Core gene regulatory network for enhanced regenerative capacity in mice. Significant enrichment of signaling pathways associated with inflammatory response, such as Jak / Stat and chemokine / cytokine-related signaling pathways, was observed ( Fig.17 B).

[0104] Next, these 145 DE genes were used as gene expression signatures to query the public database of integrated network-based cell signaling (LINCS) and matched with gene expression profiles derived from cell lines treated with small molecules based on connectivity and specificity scores (see Methods for details). Four small molecules with connectivity scores exceeding 95 (100 being the highest score) were then selected and tested using the sciatic nerve crush test ( Figure 6 A) Experimental verification ( Fig. 20 The FDA-approved small molecule metaxalone showed a significant increase in distal sensory axon regrowth 3 days after sciatic nerve crush injury (4.82 ± 0.32 mm in metaxalone-treated mice versus 3.05 ± 0.16 mm in vehicle controls) ( Figure 6 B). Consistent with the results of the crush test, a significant increase in the number of SCG10-positive regenerating axons was observed 4.5 mm distal to the crush injury site in metaxalone-treated mice compared with vehicle controls ( Figure 6 C-6D). As expected, metaxalone induced axonal regeneration in sciatic nerves 3 days after injury, which was associated with a decrease in tubulin acetylation ( Figure 6 G) Related pHDAC5 protein expression ( Figure 6 E-6F), demonstrating the recapitulation of the growth-promoting mechanism induced by Fmn2-deficiency. To assess the direct effects of metaxalone on purified DRG neurons, axotomized DRG neurons were treated with increasing doses of metaxalone, which resulted in an almost linear increase in neurite outgrowth until a plateau was reached at 20 μM ( Fig.18 ). The results suggest that metaxalone can directly enhance the intrinsic growth capacity of damaged DRG neurons.

[0105] Delayed metaxalone treatment promotes axonal regeneration and functional recovery

[0106] To simulate a clinically relevant situation, where it may take hours before any treatment can be administered, we first evaluated the efficacy of a 24-hour delayed post-injury treatment with metaxalone in promoting axonal regeneration in vivo. Metaxalone was administered one day after the sciatic nerve crush injury. Three days after injury, a nerve crush test was performed to assess the extent of axonal regrowth ( Figure 7 A). This is similar to that of mice treated with metaxalone immediately after injury (4.82 ± 0.32 mm) (see Figure 6 B), Accelerated axonal regeneration was observed in mice with a 24-hour delay in metaxalone treatment (4.50 ± 0.25 mm) ( Figure 7 B). By examining the expression of SCG10, it was found that metaxalone-treated mice (0.34) (see Figure 6 D) and delayed metaxalone-treated mice (0.40) ( Figure 7C-7D) The regeneration index was similar in areas beyond 4.5 mm, indicating that metaxalone treatment, even delayed for 24 h, did not affect the length and number of regenerating axons beyond the crush site.

[0107] Finally, we further validated the therapeutic applicability of metaxalone in terms of functional recovery when treatment was delayed until 24 hours after PNI. Adult mice received 10 mg / kg of metaxalone intraperitoneally once daily for 21 days immediately after PNI or delayed daily metaxalone treatment until 24 hours after injury for 20 days ( Figure 7 E). Mice treated with metaxalone and delayed metaxalone showed initial responses to acupuncture stimulation as early as 9 days and 10 days after injury, respectively (scoring a score of 1). Mice recovered full sensory acupuncture responses 4 days (metaxalone treatment) and 2 days (delayed metaxalone treatment) earlier than vehicle controls ( Figure 7 F). For motor function recovery, metaxalone-treated mice required 14 days to recover 50% of the toe extension reflex (scoring 1 point) and 19 days to fully recover the toe extension reflex. Delayed metaxalone-treated mice took 1 day longer to recover 50% of the toe extension reflex and fully recover the toe extension reflex than metaxalone-treated mice. Vehicle-treated mice recovered only 50% of the toe extension reflex in 18 days and required another 7 days to fully recover ( Figure 7 G). From day 11 to day 23, metaxalone-treated mice and delayed metaxalone-treated mice showed significantly higher recovery of hindlimb grip strength than controls ( Figure 7 H). Metaxalone and delayed metaxalone treatment showed the most rapid improvement in SFI during recovery from injury compared with vehicle alone. At 15 days post-injury (halfway through the recovery period), mean SFI increased significantly in mice treated with immediate metaxalone (73.0%) and mice treated with 24-hour delayed metaxalone (71.4%) compared with SFI values ​​on day 3 post-injury, whereas mean SFI increased by 49.3% in vehicle-treated mice ( Figure 7 I).

[0108] Consistent with the animal behavioral data, EMG recordings from the proximal gastrocnemius muscles of metaxalone-treated and delayed metaxalone-treated mice showed significant improvements in muscle activity from 1 to 3 weeks after injury compared with vehicle controls. Compared with vehicle controls, increased muscle activity in the most distal interosseous muscles of metaxalone-treated mice reached significant levels during the first 3 weeks after injury, while significant increases in muscle activity were detected in delayed metaxalone-treated mice at 2-3 weeks after injury ( Figure 7In conclusion, bioinformatics analysis successfully identified that the FDA-approved small molecule metaxalone not only recapitulates the growth-promoting effects of Fmn2 ablation in peripheral nerve repair but is also able to delay intervention for such a long therapeutic window, which is beneficial for translating the findings into clinical applications.

[0109] This work reveals that neuronal Fmn2 deficiency promotes axon regeneration and functional recovery by increasing microtubule dynamics and post-translational modifications, such as tubulin deacetylation at the distal ends of growing axons. Fmn2 has been shown to bind and interact with microtubules and induce α-tubulin acetylation. HDAC-induced tubulin deacetylation generates highly dynamic microtubules and plays a key role in axon regeneration. However, little is known about the mechanisms underlying Fmn2 function in peripheral nerve regeneration. In this study, we first examined the potential beneficial effects of LDIR on axonal regrowth in vitro and in vivo in axotomized DRG neurons. It was hypothesized that changes in gene expression profiles after LDIR contribute to the beneficial adaptive responses after PNI. However, RRBS and transcriptional analysis showed that LDIR did not affect global DNA methylation and key RAG expression in axotomized DRG neurons. There was a lack of consistent correlation between DNA methylation and RAG expression. For example, drug-induced or inhibition of global DNA methylation reduced axonal growth in axotomized DRG neurons by repressing gene expression of many key RAGs, including Arg1, Sprr1a, Gadd45a, and Gap43. In-depth bioinformatics analysis revealed a previously unrecognized functional role for Fmn2, in which more than 96% of DNA hypermethylation in axon regeneration occurred in its promoter region (Fmn2 expression was downregulated). RNA-seq analysis showed that removal of Fmn2 did not alter RAG expression in damaged DRGs, but interestingly, loss of Fmn2 induced activation of transcriptional networks regulating immune responses, such as the Jak-Stat signaling pathway, and pathways involved in chemokine and cytokine production were upregulated only in the presence of impaired Fmn2. - / - pSTAT3 is uniquely enriched in DRG. It is well documented that pSTAT3, as a transcription factor mediating nearly all cytokine-driven signaling, is a key switch to a pro-regenerative state in neurons. Our GSEA analysis suggests that Fmn2 deficiency may create a favorable anti-inflammatory microenvironment for peripheral nerve repair. We subsequently demonstrated that injury-induced Fmn2 downregulation occurs in DRG at early time points after injury, concurrent with robust axonal regeneration. Using a combination of approaches, we elucidated the relationship between Fmn2 deficiency and acceleration of microtubule dynamics and axonal regeneration and functional recovery.

[0110] Microtubule plus-end polymerization is required for regenerating axons through cycles of polymerization and depolymerization (dynamic instability), which enables the microtubules at the growing end to probe the intracellular environment and promote the projection of the regenerating axon back to its original target area (i.e., skin, muscle). Therefore, microtubule dynamic instability is essential for growth cone guidance, directing the regenerating axon along the correct path. In the current study, it was shown that Fmn2-deficiency prolongs the phase of high microtubule dynamics at the microtubule plus ends of growing axons by increasing the rate of microtubule polymerization (growth rate) and increasing the duration of microtubule polymerization (growth length). It was also hypothesized that after microtubule polymerization; post-translational modifications will have a direct impact on microtubule dynamics through tubulin acetylation. The data herein show that pHDAC5 is highly upregulated only at regenerating axons of AAV2 / 9-Fmn2-shRNA mice 3 days after sciatic nerve crush. In the case of HDAC5 activity inhibition by LMK-235 or genetic silencing of Hdac5 using AAV2 / 9-Hdac5-shRNA, both treatments completely abolished the axon growth-promoting effect of Fmn2 deletion in AAV2 / 9-Fmn2-shRNA mice ( Figure 4B-4C). These results are in good agreement with the observation that peripheral axonal injury induces nuclear export of HDAC5 from the nucleus to the cytoplasm and subsequent anterograde transport of HDAC5 into axons. In addition, HDAC5, but not HDAC1-4 and 6, was identified as a novel injury-dependent tubulin deacetylase that catalyzes the deacetylation of α-tubulin in axons. Inhibition of HDAC6 expression and pharmacological blockade of HDAC6 activity improved neurite outgrowth of cortical neurons on myelin inhibitory substrates such as myelin-associated glycoprotein and chondroitin sulfate proteoglycans. However, the promoting effect of HDAC6 in the permissive PNS microenvironment, in which HDAC6 knockdown had no significant effect on axon regeneration, was negligible. Unlike HDAC6, HDAC5 knockdown in DRG neurons did not affect the basal levels of acetylated tubulin, indicating that HDAC5 is not involved in the regulation of tubulin acetylation under basal conditions but only in response to injury. Although sciatic nerve injury induces phosphorylation (activation) of HDAC5 in vivo in injured axons, relatively low pHDAC5 expression was detected in uninjured axons. Recent reports have also highlighted the fact that increased tubulin polymerization and microtubule dynamics show promising results in axonal regeneration and functional recovery after injury. For example, genetic ablation of TUBB3 significantly increased the level of acetylated tubulin (stable form) and impaired microtubule polymerization at the growth cones of cultured DRG neurons. Reduced microtubule dynamics impaired neurite outgrowth in TUBB3-deficient neurons, resulting in delayed recovery of sensory function after PNI. Epothilone B induced rapid tubulin polymerization at the growth tips of cultured rat cortical neurons and promoted axonal regeneration in vivo after spinal cord injury. Similarly, paclitaxel induced active tubulin polymerization at growth cones and promoted axonal regeneration and partial functional recovery in vivo in rats with spinal cord injury. However, when used in animals at high doses, long-term exposure to epothilone B or paclitaxel is known to cause chemotherapy-induced peripheral neuropathy. It is also worth noting that morphogen proteins are known as actin nucleators and play a key role in morphogen-dependent actin nucleation, the first step in actin polymerization. Understanding how the actin cytoskeleton is altered in the growth cones of Fmn2-deficient neurons and how Fmn2 participates in actin-microtubule crosstalk will be important areas for further investigation in axon regeneration.

[0111] In a final proof-of-concept experiment, Fmn2 - / -The gene expression profile features of the 1,234 FDA-approved small molecules were queried against the public database LINCS for small molecule bioinformatics analysis. Metaxalone was identified, which is an FDA-approved drug that has been used as a skeletal muscle relaxant for more than two decades to treat a wide range of skeletal muscle diseases associated with acute and painful musculoskeletal diseases (i.e., diabetic neuropathy). In addition, the study showed that metaxalone treatment delayed by 8 hours after injury was still able to accelerate substantial axonal regeneration and functional recovery to a level comparable to metaxalone treatment immediately after injury, providing a highly relevant treatment strategy for the clinical setting.

[0112] In summary, the current study provides new insights into the development of FDA-approved small molecule-based therapies for the treatment of PNI. It typically takes more than 15 years from the identification of a bioactive compound to FDA approval for clinical use. Repurposing existing FDA-approved drugs, such as metaxalone, will save time and can often skip Phase I clinical trials. This is an important proof-of-concept study designed to demonstrate that FDA-approved small molecules identified through bioinformatics analysis can be used not only as potential therapies for PNI, but can also be used for transformational drug development for a range of different neurological diseases.

[0113] STAR Method

[0114] Resource availability

[0115] Material availability

[0116] All unique reagents generated in this study are available from the primary contact upon reasonable request.

[0117] animal

[0118] Animal experiments were performed in accordance with experimental procedures approved by the Animal Research Ethics Subcommittee of City University of Hong Kong. Animals were euthanized by carbon dioxide asphyxiation according to the American Veterinary Medical Association (AVMA) euthanasia guidelines. Adult male C57BL / 6 or Fmn2 knockout (Fmn2) mice were used in in vitro and in vivo experiments. - / - ) mice (8-12 weeks old). The mice were provided with food and water ad libitum and a light-dark cycle of 12:12 h.

[0119] Fmn2 with 129S6 / SvEvTac background - / - Mice were obtained from Jackson Laboratory. To delete Fmn2, a 1,300 base pair (bp) sequence of the proline-rich FH1 domain in the Fmn2 gene was replaced with a 1,257 bp neomycin resistance gene followed by a stop codon to terminate Fmn2 transcription.- / - The mice were viable, and no morphological or functional defects were observed in the nervous system. Male Fmn2 - / - Mice are fertile, and female Fmn2 - / - The fertility of mice is reduced. Therefore, only male Fmn2 - / - Mouse. PCR genotyping was performed using genomic DNA isolated from the tail. Forward 5'-CTAATTGTGGCTGCCCTTGT-3' (SEQ ID NO: 13) and reverse 5'-AGGTGGCAATGTCAGGATTC-3' (SEQ ID NO: 14) primers were used for PCR amplification of wild-type Fmn2, with a product size of 318 bp. Forward 5'-CTTGGGTGGAGAGGCTATTC-3' (SEQ ID NO: 15) and reverse 5'-AGGTGAGATGACAGGAGATC-3' (SEQ ID NO: 16) primers were used for PCR amplification of mutant Fmn2, with a product size of 280 bp.

[0120] Operation

[0121] As previously described, 24 h of anesthetized adult male C57BL / 6 or Fmn2 - / - Sciatic nerve crush or transection injury was performed in mice (8-12 weeks old). In short, the left sciatic nerve was exposed in the middle of the thigh. For crush injury, the exposed nerve was crushed at the sciatic notch for 15 seconds with smooth forceps (Dumont #5 / 45 forceps, Fine Science Tools). For sciatic nerve transection, the epineurium was first sutured at the level of the external rotator muscle at the distal end of the sciatic notch. The exposed nerve was then transected using ophthalmic microscissors at the distal end of the suture. After injury, the overlying muscles and skin were sutured using 5-0 sutures (Ethilon), and the mice were allowed to recover on a heating pad and returned to their home cages after recovery. The surgeon was unaware of the genotype and treatment.

[0122] Whole body X-ray

[0123] Adult male C57BL / 6 mice (8-12 weeks old) were irradiated with a cabinet irradiator X-Rad 320 (Precision X-Ray) equipped with beam conditioning filters (1.5 mm aluminum, 0.25 mm copper, and 0.75 mm tin) at 200 kV and 10 mA, with a focus-to-surface distance of 70 cm. To ensure that each mouse in the pie-shaped cage received the same amount of radiation, the dose distribution in the pie-shaped cage was measured with a dosimeter before the experiment. During irradiation, the pie-shaped cage was placed on a slowly rotating turntable to evenly distribute the X-ray irradiation. Mice received a single X-ray irradiation of different doses (1-5 Gy). Sham-irradiated control mice were placed in the irradiator without turning on the X-ray. In vitro DRG explant culture was performed 5 days after whole-body X-ray irradiation (see for detailed experimental examples). Figure 8 F). For the sciatic nerve crush test and assessment of functional recovery after PNI, injured mice were subjected to whole-body X-rays immediately after the sciatic nerve crush injury (see for detailed experimental paradigm). Figure 1 A and 1D).

[0124] In vitro DRG explant culture

[0125] In vitro DRG explant cultures were prepared from adult (8-12 weeks old) male C57BL / 6, Fmn2 - / - Mice and their age-matched WT littermates were prepared as described. In brief, DRGs were dissected out, the spinal cord and peripheral roots were removed, and they were plated on 8-well chambers (Millipore) coated with poly-D-lysine (100 μg / ml) (Sigma-Aldrich) and a thin layer of matrigel (BD Biosciences). DRG explants were then incubated in complete Neurobasal (NB) culture medium supplemented with B27, 200 mM L-glutamine, penicillin / streptomycin, 50 ng / ml NGF (Gibco), 2 ng / ml GDNF, and 10 μM Ara-C (Sigma-Aldrich). After 48 hours, DRG explant cultures were fixed with 4% paraformaldehyde (PFA) and immunostained with anti-βIII-tubulin antibodies for neurite outgrowth assays.

[0126] Primary dissociated DRG cultures

[0127] Primary cultures of dissociated DRG neurons were generated from adult male C57BL / 6 or Fmn2 - / -Mice (8-12 weeks old) and their age-matched WT littermates were prepared. Briefly, DRGs were excised from mice, moderately digested in collagenase / dispase II (Roche Diagnostics) solution, trypsinized, and mechanically dissociated using three different diameters of flame-polished Pasteur pipettes. DRG neurons were incubated in complete NB medium.

[0128] To extract genomic DNA or total RNA from cultured DRG neurons, DRG neurons were plated on thin layers of Mylar membrane (for α-particle irradiation) or glass-bottom culture dishes (for X-ray irradiation) pre-coated with poly-D-lysine (100 μg / ml) and laminin (10 μg / ml) (Sigma-Aldrich) at a fixed cell density of 3,000 cells / cm 2 DRG neurons were cultured in Neurobasal (NB) medium supplemented with B27, 200 mM L-glutamine, penicillin / streptomycin, 50 ng / ml NGF (Gibco), 2 ng / ml GDNF and 10 μM Ara-C (Sigma-Aldrich) for 17 hours.

[0129] Alpha particle and X-ray irradiation of cultured DRG neurons

[0130] For alpha particle irradiation, a plane with an alpha particle energy of 5.49 MeV and an activity of 4.26 kBq in vacuum is placed 241 Am was placed under a 9 mm hole covered by Mylar film. DRG neurons were exposed to alpha particle irradiation for 1 min, equivalent to an absorbed dose of 20 mGy. Sham irradiation was performed by exposing neurons to a non-radioactive source (i.e. plastic film) for 1 min.

[0131] For X-ray irradiation, a glass-bottomed culture dish containing DRG neurons was placed in the center of the X-ray irradiator and exposed to a single dose of 150 mGy using an X-Rad 320 (Precision X-Ray) cabinet irradiator equipped with a beam adjustment filter (1.5 mm aluminum, 0.25 mm copper, and 0.75 mm tin) with a focus-to-surface distance (FSD) of 70 cm. The cultured neurons were placed in the center of the X-ray irradiator for 2.5 minutes (i.e., the duration of 150 mGy X-ray irradiation) without turning on the machine for sham irradiation. Alpha particle and X-ray irradiation was performed one hour after cell plating.

[0132] Neurite outgrowth and cell survival assays

[0133] For ex vivo DRG explant cultures, non-overlapping quadrant images were obtained from each DRG explant, and total neurite length was measured using automated WIS-NeuroMath software (Weizmann Institute of Science). Data are from at least 3 separate experiments, n = 8 explants per dose. For dissociated DRG cultures, DRG cultures were fixed with 4% paraformaldehyde (PFA) after 17 hours of incubation, blocked with 0.5% bovine serum albumin / 0.1% Triton X-100 (Sigma-Aldrich), incubated with anti-βIII-tubulin (1:800; Sigma-Aldrich), and then incubated with the corresponding secondary antibody conjugated with AlexaFluor 488 (1:300; Molecular Probes). Neurite outgrowth assays were performed as described. In brief, 30 non-overlapping images were taken at 10 times magnification using an epifluorescence microscope (Nikon Eclipse Ni-E) equipped with a motorized stage. Total neurite length and average longest neurite length were quantified using automated WIS-NeuroMath software (Weizmann Institute of Science). Data were from 3 independent experiments repeated in duplicate. DRG neurons with βIII-tubulin-positive intact neurites adjacent to the neuronal cell body were counted as healthy neurons for neurite outgrowth assays.

[0134] Cell survival assays were performed using WST-1 reagent according to the manufacturer's instructions (ClonTech). Briefly, DRG neurons were plated at a cell density of 500 cells per well on 96-well plates coated with poly-D-lysine (100 μg / ml) and laminin (10 μg / ml) (Sigma-Aldrich) and cultured in complete NB medium. After LDIR or siRNA treatment, 10 μl of WST-1 reagent was added to each well and incubated for another 3.5 hours. The absorbance at 460 nm was measured using a microplate reader (BioTek Powerwave XS MQX200R). Data are from three independent experiments repeated in triplicate.

[0135] Genomic DNA extraction and reduced genome methylation sequencing (RRBS)

[0136] After 17 h of incubation, DRG neurons treated with alpha particles or X-rays were trypsinized and resuspended in cell lysis buffer. Genomic DNA was isolated using the PureLink Genomic DNA Mini Kit (Invitrogen). Genomic DNA was digested with MspI and then library preparation was performed. After bisulfite conversion, which converts unmethylated cytosine nucleotides to uracil while methylated cytosine nucleotides remain unmodified, the bisulfite-converted DNA was amplified by PCR and the amplified PCR fragments were sequenced using an Illumia HiSeq 1500.

[0137] Bioinformatics analysis of RRBS was performed as previously described. In brief, the raw reads of RRBS were first aligned to the mouse reference genome (UCSC GRCm38 / mm10) using the Bismark (version 0.13) and bowtie2 (version 2.3.5) programs with default parameters. The generated BAM files were then imported into the R package methylKit (version 1.2.10) to calculate the DNA methylation level of the promoter region (5,000 bp upstream of the transcription start site) of each gene. Several studies have shown that differential methylation is highly enriched in the promoter region (5,000 bp upstream of the transcription start site), so it is likely that the putative promoter sequence of the gene was found and its expression was affected by DNA methylation at the promoter region.

[0138] Total RNA extraction and qPCR analysis

[0139] After 17 h of incubation, total RNA was isolated from DRG cultures treated with 20 mGy of alpha particles or 150 mGy of X-rays and their respective sham-irradiated controls using Trizol reagent (Invitrogen). DRG neurons from lumbar vertebrae 4 and 5 (L4 / 5), which directly supply the sciatic nerve injury, were harvested at multiple different time points after sciatic nerve transection, and total RNA was extracted using Trizol reagent (Invitrogen). Reverse transcription was performed using Superscript III First-Strand Synthesis SuperMix (Invitrogen). Quantitative PCR (qPCR) was performed in triplicate using the KAPA SYBR Fast qPCR Kit (KAPA) on the QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems). Gapdh was used as an endogenous control and 2 -ΔΔct The relative fold change of each candidate gene was calculated using the formula. The qPCR primer sequences are listed in Fig.21 middle.

[0140] AAV2 / 9-mediated knockdown of Fmn2 and Hdac5 expression in vivo

[0141] For in vivo gene silencing of Fmn2 and / or Hdac5 in DRG, AAV2 / 9-Fmn2-shRNA and AAV2 / 9-Hdac5-shRNA were injected into DRG at a concentration of 5 × 10 12 vg / mL (OBiO Technology) was injected directly into the sciatic nerve using a glass micropipette. The mice were then subjected to sciatic nerve crush injury and sciatic nerve clamp test ( Figure 4 ) and animal behavior assessment ( Fig.15 ). The expression of shRNA was under the control of U6 promoter, and the expression of reporter gene eGFP was under the control of CAG promoter. AAV2 / 9-scr-shRNA (Packgene) was used as negative control (shRNA sequence see Fig. 22 In another batch of animals, two weeks after AAV injection, the knockdown efficiency in DRGs of AAV2-Fmn2-shRNA and AAV2-Hdac5-shRNA mice was assessed by Western blot analysis ( Fig.15 and Fig.16 To confirm successful knockdown of Fmn2 in motor neurons, the anterior horns of the lumbar spinal cord were harvested from AAV2-Fmn2-shRNA mice two weeks after AAV injection for Western blot analysis ( Fig.14 D) and immunohistochemical studies ( Fig.14 E).

[0142] Sciatic nerve compression test

[0143] Three days after sciatic nerve crush injury, sciatic nerve clamp test was performed on the ipsilateral side. Mice were anesthetized under 2.5% isoflurane and the left sciatic nerve was exposed in the middle of the thigh. The mice were then slowly awakened under light anesthesia (1% isoflurane). A series of clamps were applied using smooth forceps (Dumont #5 / 45 forceps, Fine Science Tools) from the most distal part of the sciatic nerve near the trifurcation to the proximal end to the compression site. The distance representing the length of axonal regrowth (in mm) was recorded from the compression site to the farthest point of the sciatic nerve, and the initial flexor reflex was observed after the clamp. The observer was unaware of the treatment and genotype.

[0144] Quantification of axon regeneration in vivo

[0145] Mice were perfused transcardially with 4% PFA, and L4 / 5DRG and sciatic nerves were harvested, post-fixed, cryoprotected, and frozen in OCT compound (Tissue-Tek). For quantification of regenerated axons, 12 μm thick longitudinal serial sections of sciatic nerves were blocked with 0.5% BSA / 0.1% Triton X-100 (Sigma-Aldrich) and incubated with primary antibodies against SCG10 (a marker of regenerated sensory axons; 1:5,000; Novus Biologicals). The sections were then incubated with secondary antibodies conjugated with Alexa Fluor 555 (1:300; Molecular Probes). A series of z-stack images were taken at 20x magnification using a Nikon AXR confocal microscope equipped with a motorized stage and a resonant scanner, and stitching and maximum projection were performed using NIS-Elements software.

[0146] To quantify regenerating axons, SCG10 fluorescence intensity was measured at different distal distances from the crush site using a line scan macro in ImageJ (NIH). The fluorescence intensity profile of SCG10 immunoreactivity was plotted by the average SCG10 intensity from 100 adjacent pixels and normalized to the value measured at the crush site where SCG10 immunoreactivity was maximal. For each mouse, at least 3 non-overlapping sections (at least 36 μm apart) were used, and 12-15 sections were averaged for each genotype or treatment group (n=4-5 per genotype or treatment group).

[0147] Sensory and motor function recovery tests

[0148] Fmn2 in adult males before sciatic nerve crush injury - / - Mice (8-12 weeks old) and their age-matched WT littermates, or adult male C57BL / 6 mice (for whole-body X-ray, AAV2 / 9-Fmn2-shRNA, and metaxalone experiments) were trained and habituated for one week. After three independent training sessions (30 min each, two days of rest), baseline values ​​were obtained, and sciatic nerve crush injury was performed one day after the baseline recording. Functional recovery was monitored for 30 days. On the third day after sciatic nerve crush, acupuncture measurements were first performed, and then motor function measurements (toe extension, grip strength, and sciatic nerve function index) were performed sequentially every other day, with a 30-min interval between each test. Animal behavioral testing was performed without knowledge of surgery and genotype.

[0149] Acupuncture test

[0150] Mice were placed in small wire mesh cages for 30 minutes of habituation, and insect pins (FineScience Tools) were applied to the lateral plantar surface of the ipsilateral hind limb, from the toes to the heel. The lateral plantar surface was divided into 5 different areas, from the farthest toes (score = 5) to the heels (score = 1). If a rapid retraction response of the hind paw was observed, the area of ​​the mouse was scored. If no retraction response was observed after the insect pin was applied to all 5 areas, the mouse was scored as 0. If there was no positive response in all five areas, the saphenous nerve innervation area of ​​the same hind limb was tested as a positive control. Each mouse was tested for two rounds of acupuncture (time interval of 30 minutes) to confirm the score.

[0151] Toe extension reflex

[0152] To evaluate the toe extension reflex after PNI, mice were covered with a piece of cotton, lifted by the tail, and the hind limbs were exposed for evaluation. The toe extension reflex score is as follows: 0 means no extension, 1 means moderate extension, all toes are separated for less than 2 seconds, 2 means full extension, all toes are fully and widely extended, and last for at least two seconds. Mice were scored only when a complete response was observed on the contralateral side (uninjured hind limb). Mice were evaluated twice, with a time interval of 30 minutes between each evaluation.

[0153] Grip strength measurement

[0154] Hindlimb muscle strength was measured using a grip dynamometer (BIO-GS3, Bioseb). To measure the grip strength of both hindlimbs, their hindlimbs were positioned to grasp a metal T-bar attached to the grip dynamometer, while their forelimbs were placed on a plastic bar. The mice were then gently pulled off the T-bar. The value when the mouse left the T-bar reflected the grip strength (in grams). The average grip strength was taken from at least 5 consecutive grip strength measurements for each animal.

[0155] Sciatic Function Index (SFI)

[0156] Walking track analysis was performed by training mice to walk along a narrow corridor lined with white paper strips (10 x 60 cm), and the hind limbs of the mice were painted with red watercolor. SFI baseline values ​​were obtained after 3 independent training sessions one week before injury. SFI values ​​were calculated based on the footprints as follows:

[0157] -38.3×(experimental footprint length-original footprint length) / original footprint length+109.5×(experimental toe width-original toe width) / original toe width+13.3×(experimental middle toe width-original middle toe width) / original middle toe width-8.8. The footprint length is considered to be the distance between the third toe to the heel. The toe width is considered to be the distance between the first and fifth toes. The middle toe width is considered to be the distance between the second and fourth toes. Four clear and different footprints were collected from the ipsilateral experimental paw of the left injury and the contralateral original paw of the right uninjured side for SFI calculation. Mice with SFI values ​​close to 0 indicate normal gait movement, while if the SFI value is close to -100, the motor function is severely impaired.

[0158] Electromyography (EMG) recording

[0159] Electromyography (EMG) recordings were performed on anesthetized mice as described. In brief, mice were anesthetized with ketamine (100 mg / kg) / xylazine (10 mg / kg) and placed on a heat pad set to 37°C to avoid hypothermia during EMG recording. Baseline EMG was recorded before sciatic nerve crush injury using custom-made unipolar Teflon-coated electrodes. After sciatic nerve crush injury, EMG was recorded once a week for one month (days 7, 14, 21, and 28). For EMG recordings of the proximal gastrocnemius muscle, the recording electrode was inserted into the gastrocnemius muscle, and the Achilles tendon electrode was used as a reference. For EMG recordings of the distal interosseous muscles, the recording electrode and the reference electrode were inserted into the first and fourth muscles of the same paw, respectively. The sciatic nerve was then stimulated proximally or distally, and both proximal and distal stimulations were used for the CMAP of the interosseous muscles. Only proximal stimulation was used for the CMAP of the gastrocnemius muscle. Record (Blackrock microsystem, USA) and calculate (Spike 2, UK) the average CMAP amplitude.

[0160] Immunohistochemical analysis of neuron-specific AAV serotype tropism in DRG, sciatic nerve, and ventral horn motor neurons of the lumbar spinal cord

[0161] Two weeks after injection of AAV2 / 9-scr-shRNA (labeled with eGFP) into the sciatic nerve, mice were transcardially perfused with 4% PFA. Sciatic nerves, L4 / 5 DRGs, and lumbar anterior horns of the spinal cord were harvested, post-fixed, cryoprotected, and frozen in OCT compound (Tissue-Tek). 5 μm thick DRG cryosections, 12 μm thick longitudinal sciatic nerve cryosections, and 30 μm thick transverse spinal cord cryosections were blocked with 0.5% BSA / 0.5% Triton X-100 (Sigma-Aldrich) and incubated with anti-βIII-tubulin (neuronal marker; 1:800; Sigma-Aldrich), anti-ChAT (motor neuron marker; 1:100; Millipore), anti-FMN2 (ProteinTech; 1:200), anti-SOX10 (Schwann cell marker; 1:250; Abcam), or CD68 (macrophage marker; 1:250; Abcam) primary antibodies as indicated. Sections were then incubated with secondary antibodies conjugated with Alexa Fluor 555 (1:300; Molecular Probes) or Alexa Fluor 647 (1:300; Molecular Probes). For DRG and sciatic nerve cryosections, images were taken at 20× magnification using a Nikon Ni-E epifluorescence microscope (for DRG and sciatic nerve cryosections) equipped with a motorized stage. For spinal cord cryosections, images were taken at 20× magnification using a Nikon AXR confocal microscope equipped with a motorized stage and a resonant scanner.

[0162] Fluorescence activated cell sorting (FACS)

[0163] Two weeks after injection of AAV2 / 9-scr-shRNA or AAV2 / 9-Fmn2-shRNA into the sciatic nerve, L4 / 5 DRGs were harvested, digested with collagenase / dispase, mechanically dissociated using three different diameter fire-polished Pasteur pipettes in dissociation buffer (1× PBS with 10% FBS), and then filtered through a 70 μm cell strainer (BD Falcon) to generate single-cell suspensions before FACS. All AAV2 / 9 used in the current study were tagged with eGFP.

[0164] FACS was performed using a SONY SH800Z cell sorter. To select DRG neurons, dissociated cells were first separated based on cell size (forward scatter) and cell surface characteristics (side scatter), and aggregated cells were eliminated based on the FSC-H:FSC-A ratio ( Fig.14A). In each experiment, dissociated DRG neurons not transduced with AAV were used as negative controls to set the detection gate for eGFP-positive cells ( Fig.14 B) Sorted eGFP-positive cells were immediately lysed and subjected to Western blot analysis.

[0165] Western blot analysis

[0166] As indicated, ipsilateral L4 / 5DRG neurons were harvested at different time points after sciatic nerve injury. The sciatic nerve was harvested 3 days after crush injury and segmented into 0-6mm at the distal end of the crush site. Each protein sample was merged from 3 mice. The DRG neurons and segmented sciatic nerves were mechanically dissociated with protein lysis buffer, and protein BCA assay (Pierce) was used to measure protein concentration. Protein lysates (30 μg) were separated on 4-12% NuPAGE Tris-glycine precast gels (Invitrogen). The protein was then transferred to a PVDF membrane (Bio-Rad) and sealed with 5% skim milk (Bio-Rad) in TBS-T. The membrane was incubated with anti-FMN2 (1:500; ProteinTech), anti-phospho-HDAC5 (1:500, ThermoFisher), anti-HDAC5 (1:500, Novus Biologicals), anti-acetylated tubulin (1:10,000; Sigma-Aldrich), anti-NeuN (1:500; Millipore), anti-SOX10 (1:500; Abcam), anti-IBA-1 (1:1,000; Wako), anti-GFP (1:1,000; Cell Signaling), and anti-GAPDH antibody (1:2,000; ProteinTech) as a loading control. The membrane was incubated with a secondary antibody conjugated to horseradish peroxidase (1:2,000; Thermo-Fisher). Signals were detected using the WestFemto Maximum Sensitivity Substrate Kit (Thermo Scientific). The membrane was then stripped and reblotted with an anti-total tubulin antibody (1:2,000; Sigma-Aldrich). Band intensities were measured using Image Lab software (Bio-Rad). FMN2 and pHDAC5 protein expression levels were normalized to GAPDH. Acetylated tubulin expression levels were first normalized to total tubulin levels and then to GAPDH.

[0167] Live cell imaging and analysis of microtubule dynamics at growth cones of DRG neurons

[0168] 2000 DRG neurons were plated onto poly-D-lysine and laminin-coated glass-bottom culture dishes and transduced with AAV2 / 9-EB3-mCherry (Packgene) one hour after plating. On day 4, EB3-mCherry tracking was performed in vitro and time-lapse images were taken at 100× magnification using a Nikon AXR confocal microscope equipped with a motorized stage and a resonant scanner. Fluctuation curve records of EB3 comet motion at growth cones were generated using NIS-Elements software. For microtubule dynamics analysis, EB3 comets at growth cones were automatically tracked, and microtubule dynamics parameters including microtubule growth velocity, growth lifetime, growth length, and EB3 comet density were determined using MATLAB-based software plusTipTracker. For each experimental group, at least 30 growth cones were analyzed based on three independent experiments.

[0169] Treatment with HDAC5 inhibitor LMK-235

[0170] To inhibit HDAC5-mediated tubulin deacetylation in mice, LMK-235 (20 mg / kg), a selective inhibitor of HDAC4 and HDAC5, was intraperitoneally injected into adult male C57BL / 6 mice (8-12 weeks old) pretreated with AAV2 / 9-Fmn2-shRNA for 3 consecutive days. The mice were then subjected to sciatic nerve crush test and immunohistochemical analysis.

[0171] RNA-seq whole transcriptome analysis

[0172] In adult males, Fmn2 - / - Sciatic nerve transection was performed in mice and WT littermates (8-12 weeks old), and L4 / 5 DRGs were collected from the contralateral (uninjured) and ipsilateral (injured) sides 5 days after injury. Total RNA was extracted using Trizol reagent, and 100 ng of total RNA from each sample (n=3 per group) was used for library preparation after ribosomal RNA removal. The library of each sample was then sequenced using Illumina Novasek6000 with a depth of 75 million reads per sample. After quality control using FastQC, the sequencing data were plotted and annotated using the reference genome GRCm38 and quantified using DESeq. Genes with log2-fold >|1| and Benjamini-Hochberg adjusted P<0.05 were considered differentially expressed. DESeq2 normalized counts with log2 transformation were used for principal component analysis and hierarchical cluster analysis.

[0173] Functional enrichment analysis

[0174] To check Fmn2 - / -The biological functions associated with differentially expressed genes in mice were analyzed by GSEA and gene set overrepresentation analysis using the “HTSanalyzeR” R package for Gene Ontology (GO) gene sets (biological process, cellular component, and molecular function categories) and KEGG pathways. Significantly enriched GO terms and pathways (Benjamini-Hochberg adjusted P-value < 0.05) were clustered using the Markov clustering method by AutoAnnotate (version 1.3.2) and visualized using Cytoscape (v.3.7.2).

[0175] In silico small molecule screening

[0176] The Library of Integrated Network-Based Cellular Characterizations (LINCS) database was used for in silico small molecule screening. LINCS is a public web-based database containing more than 1 million gene expression profiles from 50 human cell lines (including neuronal cell lines such as fibroblast-derived neuronal progenitor cells and terminally differentiated neurons) treated with 44,328 bioactive small molecules (including 1,234 FDA-approved small molecules). A total of 145 genes were expressed only in the impaired Fmn2 compared to the gene expression profiles of the neuronal cell lines in the public LINCS database. - / - A metric was used to estimate impaired Fmn2 expression based on the nonparametric, rank-based pattern matching Kolmogorov-Smirnov statistic. - / - Each query feature in the DRG neuron gene expression profile was evaluated for similarity with the gene expression profile from the cell line treated with the small molecule (reference feature). This generated a connectivity score ranging from +100 (strong positive correlation) to -100 (strong negative correlation). The average of the connectivity scores was used to rank the small molecules. Small molecules with connectivity scores greater than +95 (strong overlap between the query feature and the reference feature) were shortlisted for experimental validation using the mouse sciatic nerve crush test.

[0177] Evaluation of small molecules for in vivo axonal regeneration and functional recovery after PNI

[0178] To evaluate the effects of the top ranked small molecules on promoting axonal regeneration in vivo, 40 μl of alverine (0.5 μg / μl), cilnidipine (0.5 μg / μl), metaxalone (0.25 μg / μl), or acamprosate (0.25 μg / μl) was directly administered to the injury site of adult male C57BL / 6 mice (8-12 weeks old) immediately after sciatic nerve crush for 3 consecutive days (daily morning and evening dosing schedule, with an interval of more than 8 hours between dosings), because the half-life of small molecules ranged from 5.7 to 33 hours (i.e., 9 hours for metaxalone). The mice were then subjected to sciatic nerve crush test and immunohistochemical analysis.

[0179] To examine the therapeutic potential of metaxalone, adult mice were injected intraperitoneally with 10 mg / kg of metaxalone daily for 21 consecutive days immediately after injury (i.e., metaxalone treatment) or with 10 mg / kg of metaxalone daily for 20 consecutive days 24 hours after injury (i.e., delayed metaxalone treatment), and all metaxalone treatments ended on day 21 after injury. The mice were then tested for sensory and motor function recovery, and EMG recordings were performed weekly for 28 days after injury.

[0180] Statistical analysis

[0181] Data are expressed as mean ± SEM. Where appropriate, data analysis was performed using Student's t-test (2 groups) or one-way ANOVA (more than 2 groups), followed by a post hoc Bonferroni test. SCG10 immunoreactivity was analyzed using a two-way ANOVA, followed by a post hoc Bonferroni test. qPCR experiments were analyzed using a nonparametric Mann-Whitney U test (2 groups) or a Kruskal-Wallis test, followed by a Dunn post hoc test (more than 2 groups). Animal neurobehavior and electrophysiology experiments were analyzed using a two-way ANOVA with repeated measures and a post hoc Bonferroni test. Microtubule growth velocity, microtubule growth length, microtubule growth lifespan, and EB3 comet density were analyzed using the Mann-Whitney U test. P<0.05 was considered statistically significant. GraphPad Prism 9.0 was used for all statistical analyses and plots.

[0182] Data and code availability

[0183] The sequencing data were deposited in the Gene Expression Omnibus (GEO) under accession number GSE243896 and are publicly available from the date of publication.

Claims

1. Use of a therapeutic agent in the preparation of a medicament for treating nerve damage in a subject in need thereof, wherein the therapeutic agent is selected from the group consisting of metaxalone and a morphogen-2 (Fmn2) inhibitor.

2. The use of claim 1, wherein the Fmn2 inhibitor is an antibody, an antibody fragment, an inhibitory nucleic acid molecule or a small molecule, wherein the inhibitory nucleic acid molecule substantially silences Fmn2.

3. The use according to claim 2, wherein the inhibitory nucleic acid molecule is selected from the group consisting of short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid and post-transcriptional gene silencing RNA (ptgsRNA).

4. The use of claim 2, wherein the inhibitory nucleic acid molecule comprises consecutive nucleotides complementary to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:

9.

5. The use of claim 2, wherein the inhibitory nucleic acid molecule comprises SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO:

12.

6. The use according to claim 2, wherein the administration of the inhibitory nucleic acid molecule in the medicament reduces the expression of Fmn2 protein by 40%-100%.

7. The use of claim 1, wherein the drug is administered within 24 hours of the onset of the nerve damage.

8. purposes as claimed in claim 1, wherein the medicament is formulated for parenteral, paracancer, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, perineural, intraneural administration or directly at the central nervous system lesion site or peripheral nervous system lesion site including the nerve damage.

9. The use according to claim 3, wherein the medicament is formulated for direct administration of the inhibitory nucleic acid molecule to a central nervous system lesion site or a peripheral nervous system lesion site containing the nerve damage.

10. The use of claim 1, wherein the therapeutic agent is metaxalone.

11. The use according to claim 1, wherein the nerve damage comprises at least one of central nervous system damage or peripheral nervous system damage.

12. The use of claim 1, wherein the nerve damage is the result of physical trauma.

13. The use of claim 1, wherein the nerve damage comprises damaged dorsal root ganglia.

14. The use of claim 1, wherein the subject is a human, a non-human primate, an equine, a bovine, a canine, a feline, or a rodent.

15. The use of claim 10, wherein the subject does not suffer from a muscle-related disorder.

16. The use according to claim 15, wherein the muscle-related disorder is selected from the group consisting of muscle twitches, muscle spasms and musculoskeletal pain.

17. The use of claim 10, wherein the nerve damage is the result of physical trauma.

18. The use of claim 10, wherein administration of the drug is initiated within 24 hours of the onset of the nerve damage.

19. The use according to claim 10, wherein the medicament is formulated for oral administration.

20. The use of claim 10, wherein the subject is a human.

21. purposes as claimed in claim 10, wherein the medicament is formulated for parenteral, paracancer, transmucosal, transdermal, intramuscular, intravenous, intradermal, subcutaneous, intraperitoneal, intraventricular, intracranial, perineural, intraneural administration or directly at the central nervous system lesion site or peripheral nervous system lesion site including the nerve damage.

22. The use of claim 10, wherein the medicament is formulated for intraperitoneal injection.

23. The use of claim 2, wherein the inhibitory nucleic acid molecule is selected from the group consisting of: antisense oligonucleotides (ASOs) and single guide RNA molecules (sgRNAs).

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