Journal: bioRxiv

Article Title: Disruption of the hypothalamic orexin system links SARS-CoV-2 infection to persistent cortical neuronal pathology

doi: 10.64898/2026.01.22.701182

Figure Lengend Snippet: a , Schematic illustration of the in vitro experimental design used to assess the specificity of orexin signalling. Human iPSC-derived glutamatergic neurones were pre-treated with the dual orexin receptor antagonist suvorexant (10 µM) or vehicle for 2 h, followed by treatment with recombinant orexin-A/B peptides (γORX-A/B; 5 μM each) at the indicated concentrations; the cells were then harvested at 72 h post-treatment (hpt) for immunoblotting. b, Western blot analysis showing the effect of suvorexant pretreatment on γORX-A/B-mediated NeuN induction. Representative blots (top) and quantification of NeuN protein levels normalized to β-actin (bottom) are shown. The fold change relative to the mock control is indicated below the blots. c, Dose-dependent effects of orexin on NeuN expression. Neurones were treated with increasing concentrations of γORX-A/B (0, 10, and 100 nM) and analysed by western blotting at 72 h post-treatment (hpt). d, Schematic representation of the in vivo rescue experiments. BALB/c mice were intranasally infected with MA10 SARS-CoV-2 (4 × 10 4 PFU) and administered γORX-A/B (2 mg kg ¹) or vehicle daily from 0 to 5 days post-infection (dpi). Brain tissues were collected at 6 dpi. e,f, Quantification of viral burden ( e ) and Hcrt mRNA levels ( f ) in whole-brain lysates at 6 dpi using RT–qPCR. g, ELISA quantification of the orexin-A/B peptide concentrations in brain homogenates. h, Representative Western blots of NeuN expression in whole-brain lysates from mock, vehicle-treated and γORX-A/B-treated mice at 6 dpi. i, Densitometric quantification of NeuN protein levels normalised to β-actin. Data in e – g and i represent mean ± s.e.m. (n = 4 mice per group). Statistical significance was determined using one-way ANOVA with Dunnett’s multiple comparison test ( f , g , i ) or a two-tailed unpaired Student’s t-test ( e ). The exact P values are indicated. N.D., not detected.

Article Snippet: For orexin rescue experiments, recombinant orexin-A and orexin-B (MCE, HY-106224B, HY-P1339B) were added at the indicated concentrations (10 nM, 100 nM, or 5 μM) immediately after infection.

Techniques: In Vitro, Derivative Assay, Recombinant, Western Blot, Control, Expressing, In Vivo, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test