Posttranscriptional Regulation of Gene Expression Participates in the Myelin Restoration in Mouse Models of Multiple Sclerosis: Antisense Modulation of HuR and HuD ELAV RNA Binding Protein

**Background:** Multiple sclerosis (MS) is a chronic inflammatory CNS condition characterized by demyelination and neurodegeneration. Neuropathic pain affects about 70% of MS patients and is difficult to treat. Recent evidence links neuropathic pain to demyelination driven by unresolved chronic inflammation. The ELAV/Hu family of RNA-binding proteins (RBPs)—particularly HuR (ubiquitous, pro-inflammatory) and HuD (neuron-specific, neuroprotective)—regulate gene expression posttranscriptionally. This study hypothesized that aberrant HuR expression disrupts the HuD/HuR ratio, promoting demyelination and neurodegeneration, and that antisense-mediated silencing of HuR could attenuate these processes.

**Methods:** Three mouse models were used: (1) MOG₃₅–₅₅-induced chronic progressive EAE in CB57BL/6 female mice, (2) PLP₁₃₉–₁₅₁-induced relapsing–remitting EAE in SJL female mice, and (3) spared nerve injury (SNI) in male CD1 mice. Phosphorothioate antisense oligonucleotides (ASOs) against HuR or HuD (1 nmol/mouse) were administered intranasally every 2 days starting from day 14 post-immunization (EAE) or day 3 post-surgery (SNI). Behavioral testing included von Frey (mechanical allodynia), Hargreaves' plantar test (thermal hyperalgesia), clinical disease scoring (0–3.5 scale), and rotarod performance. Biochemical analyses included Western blotting, immunofluorescence, Luxol Fast Blue (LFB) myelin staining, Evans blue BBB permeability assay, and plasma cytokine ELISA. Statistical analysis used two-way ANOVA with Bonferroni post-hoc, Kruskal–Wallis with Dunn's test, or one-way ANOVA with Tukey post-hoc as appropriate (P < 0.05).

**Key Results:** In MOG-EAE mice, anti-HuR ASO reduced mechanical allodynia from day 28 (P < 0.05 vs MOG dODN), attenuated rotarod impairment, and progressively reduced body weight loss (significant from day 28). Anti-HuD ASO improved motor dysfunction but did not modify pain hypersensitivity. In PLP-EAE mice, anti-HuR ASO attenuated both mechanical and thermal allodynia and improved clinical score and locomotor disability at day 28. LFB staining showed robust myelin loss in MOG-EAE mice that was significantly increased by anti-HuR ASO (P < 0.05). MBP expression was markedly upregulated by anti-HuR ASO in MOG-EAE mice, while anti-HuD ASO had no effect. GAP43 and neurofilament H levels, drastically reduced in MOG-EAE, were restored to control values by anti-HuR ASO. Anti-HuR ASO reduced Evans blue dye extravasation in both brain and spinal cord of MOG-EAE mice (P < 0.05 vs MOG dODN). Plasma IL-17, TNF-α, and IL-6 were elevated in both EAE models; anti-HuR ASO reduced IL-17, IL-1β, and IL-6 in PLP mice but had no effect on circulating cytokines in MOG mice. IBA1 and CD11b expression were reduced by anti-HuR ASO in PLP mice but not in MOG mice. HuR was overexpressed in both EAE models and SNI mice. HuD was drastically downregulated in MOG-EAE but unaltered in PLP-EAE and upregulated in SNI. Anti-HuR ASO increased HuD expression in both EAE models. In SNI mice, anti-HuR ASO reduced thermal and mechanical allodynia from day 7 and increased spinal MBP and LFB staining, while anti-HuD ASO reduced pain hypersensitivity from day 10. Neither ASO altered S100 expression in the sciatic nerve.

**Clinical Implications:** This study demonstrates that intranasal anti-HuR ASO attenuates neuropathic pain and promotes myelin restoration in MS models through dual mechanisms: dampening microglia-mediated neuroinflammation (prominent in relapsing–remitting PLP-EAE) and restoring HuD-mediated neuroprotection (prominent in chronic MOG-EAE with neurodegeneration). The intranasal route offers a noninvasive alternative to intrathecal delivery for ASO-based therapies. These findings position HuR as a promising disease-modifying target for neuropathic pain in MS, addressing both early inflammatory and late neurodegenerative disease stages.