Fig 1: Proteomic analysis of aortic samples from DOX-treated mice at Weeks 2 and 6 and validation of two candidate biomarkers with western blotting. Plots of differentially expressed proteins at Weeks 2 and 6 (A). For A, horizontal grey line indicates significance threshold (P < 0.05); up- and down-regulated proteins are indicated in blue and red, respectively; differentially expressed proteins are abbreviated (full names are listed in abbreviations list). Western blotting shows and corroborates higher levels of SERPINA3 in DOX-treated mice at Weeks 2 and 6, which persist at Week 9 (B). Western blotting also confirms higher levels of THBS1 in the DOX-treated group at Weeks 2 and 6 and shows a trend (P = 0.052) towards increased THBS1 levels at Week 9 (B). Representative western blot images for SERPINA3 and THBS1 at Weeks 2, 6, and 9 (C). For C, ‘V’ and ‘D’ indicate lanes with vehicle and DOX aortic samples, respectively. D* on the blot at Week 9 contains a DOX sample for which no protein was detected and was excluded from analysis as such. For A, n = 8 in vehicle and DOX group for both Week 2 and Week 6. For B, vehicle group: n = 5, n = 6, and n = 6 at Weeks 2, 6, and 9, respectively; DOX group: n = 5, n = 6, and n = 5 at Weeks 2, 6, and 9, respectively. For B, Mann–Whitney U test for each time point. * and ** P < 0.05 and 0.01. Abbreviations for differentially expressed proteins in proteomics: SERPINA1E, α-1-antitrypsin; HMGN2, Non-histone chromosomal protein HMG-17; SERPINA3N, α-1-antichymotrypsin orthologue n; MT2, Metallothionein-2; MBL2, Mannose-binding protein C; MT1, Metallothionein-1; THBS1, Thrombospondin-1; FTL1, Ferritin light chain 1; COL14A1, Collagen α-1 (XIV) chain; LGALS3BP, Galectin-3-binding protein; RPL26, 60S ribosomal protein L26; IGHM, Immunoglobulin heavy constant µ; HAMP, Hepcidin; APOA2, Apolipoprotein A-II; HBA, Hemoglobin subunit α; HBB-B1, Hemoglobin subunit β-1; CA1, Carbonic anhydrase 1; KRT15, Keratin (type I cytoskeletal 15); SPTA1, Spectrin α; CA2; Carbonic anhydrase 2; RPL6, 60S ribosomal protein L6; VWA1, von Willebrand factor A domain-containing protein 1; RPL37A, 60S ribosomal protein L37a; CAPG, Macrophage-capping protein; RAD23A, UV excision repair protein RAD23 homolog A; IGHG1, Ig γ-1 chain C region (membrane-bound form); SLC4A1, Band 3 anion transport protein; ACLY, ATP-citrate synthase; ATPA1, Sodium/potassium-transporting ATPase subunit alpha-1.
Fig 2: SerpinA3N is neuroprotective in models of flavivirus encephalitis(A) SerpinA3N concentrations (measured via ELISA) in whole brain homogenates derived from mice of indicated genotypes four days following intracranial (i.c.) ZIKV-MR766 infection. (B) Schematic illustrating timeline of i.c. ZIKV-MR766 infection and i.c.v. treatment with recombinant SerpinA3N, prior to various endpoint readouts. (C) Collagenase/gelatinase activity assay in whole brain homogenates derived from mice of indicated genotypes following ZIKV-MR766 infection and SerpinA3N treatment, as described in (B). (D) Immunohistochemical (IHC) staining of Claudin5 (magenta), Zo-1 (yellow), and DAPI (cyan) in cortical brain tissue of mice of indicated genotypes following ZIKV-MR766 infection and SerpinA3N treatment, as described in (B). Scale bar = 10μm. Images are at 63x magnification. (E) Colocalization index of Claudin5 and Zo-1 staining in cortical brain sections described in (D). (F) Schematic illustrating survival studies in which mice received i.c. ZIKV-MR766 infection or subcutaneous (s.c.) WNVWN02-Bird114 infection, followed by i.c.v. administration of recombinant SerpinA3N at indicated intervals following infection. (G-H) Survival and weight measurements in mice of indicated genotypes following ZIKV-MR766 infection (G) or WNV-WN02-Bird114 infection (H) with or without i.c.v. SerpinA3N treatment, as described in (F). n=6–10 mice/group. *p<0.05, **p < 0.01, ***p < 0.001. Error bars represent SEM.
Fig 3: SerpinA3N suppresses deleterious neuroinflammation during flavivirus encephalitis(A) Representative flow cytometry plots depicting resident (CD45.2int) or infiltrating (CD45.2hi) CNS leukocytes derived from mice of indicated genotypes infected with ZIKV-MR766 with or without i.c.v. treatment with SerpinA3N two days following infection. Flow cytometry was performed four days following infection. (B) Total numbers of indicated leukocyte populations in brain tissue derived from mice described in (A). (C) Schematic illustrating survival study and immunohistochemical (IHC) analysis in which mice received intraperitoneal administration of an anti-CD8 neutralizing antibody (αCD8) two days following intracranial (i.c.) ZIKV-MR766 infection. (D) Survival and weight measurements in mice of indicated genotypes following ZIKV-MR766 infection with or without depletion of CD8+ T cells, as described in (C). (E) IHC analysis of GFAP (green) and DAPI (blue) in cortical brain tissue derived from mice of indicated genotypes treated as described in (C). Images are 2×2 tiled composites at 20x magnification. Scale bar = 50μm. (F) Mean fluorescence intensity of GFAP staining in cortical brain tissue derived from mice of indicated genotypes and treated as described in (C). *p<0.05, **p < 0.01. Error bars represent SEM.
Fig 4: Genetic evidence for SERPINA3N expression in oligodendrocytes.a Schematic of Serpina3n-tdTom reporter design. The P2A self-cleaving site separates dTom from SERPINA3N-tdTom, allowing tdTom to localize in cell bodies while SERPINA3N remains intracellular or is secreted.b Double fluorescent IHC for SERPINA3N and tdTom in D30 MOG/EAE ventral white matter of spinal cord. Arrowheads, tdTom+ SERPINA3N+ cell bodies; arrows, extracellular SERPINA3N.c-d IHC of tdTom and SOX10 (c) or GFAP (d) in D30 MOG/EAE spinal cord.e Quantification of tdTom+ cells co-expressing indicated marker in D30 MOG/EAE spinal cord. n = 3 mice each group.f-g IHC of tdTom and SOX10 (f) or GFAP (g) in 3-week CPZ corpus callosum.h Quantification of tdTom+ cells co-expressing indicated markers in 3-week CPZ corpus callosum. n = 3 mice each group.i IHC for SERPINA3N and SOX10 in spinal cord of Serpina3n control (Ctrl) and conditional knockout (cKO, Olig2-Cre:Serpina3nfl/fl) mice at D30 post-MOG/EAE.j IHC of SERPINA3N and SOX10 in corpus callosum of Serpina3n Ctrl and cKO mice after 4-week CPZ (or Norm) diet. Scale bars: b, c, d, f, g, i 10 μm, j 20 μm.Data were presented as mean ± s.e.m. (e, h).
Fig 5: SerpinOLs regulate CNS inflammation and microglial activation in response to demyelination.a UMAP clusters of single cells from spinal cord of three groups of mice at D30 post-MOG/EAE (or CFA).b Percentage of cell types.c Numbers of dendritic cells (DCs) and microglia/macrophages (MG/MØ) in each group.d Upregulated DEGs in Ctrl_MOG vs. Ctrl_CFA revealed by psuedobulk analysis of MG/MΦ (C11, C13, C38) (Suppl Table 8).e Biological processes of upregulated DEGs in Ctrl_MOG mice (Suppl Table 8).f Downregulated DEGs in cKO_MOG versus Ctrl_MOG mice revealed by psuedobulk analysis of MG/MΦ (Suppl Table 9).g Biological processes of downregulated DEGs in Ctrl_MOG mice (Suppl Table 9).h purity check of microglia (MG) by RT-qPCR assay of lineage-specific marker genes. MG were purified from Serpina3n Ctrl brain of normal diet (Norm_Ctrl, n = 3) and 4 weeks CPZ (CPZ_Ctrl, n = 3) and Serpina3n cKO brain of 4 weeks CPZ (CPZ_cKO, n = 4)). Microglial RNA was used for RNA-seq and bioinformatics in Panels k-q. **** P < 0.0001, * P = 0.0104.i Principal component analysis of MG samples. N = 3 mice Norm_Ctrl and CPZ_Ctrl; n = 4 mice CPZ_cKOj Upregulation of core neurodegeneration-related genes62 in MG of CPZ_Ctrl versus Norm_Ctrl mice (Suppl Table 10).k Downregulation of core neurodegeneration-related genes62 in MG of CPZ_cKO versus CPZ_Ctrl mice (Suppl Table 10).l, heatmap and cumulative scores of core neurodegeneration-related gene set (Suppl Table 11). One way ANOVA and Tukey’s post-hoc test, P < 0.0001 CPZ_Ctrl vs Norm_Ctrl, P < 0.0001 CPZ_cKO vs CPZ_Ctrl. N = 3 mice Norm_Ctrl and CPZ_Ctrl; n = 4 mice CPZ_cKOm-n, cumulative scores of disease-associated (m)77 and LPS-related (n)62 microglial gene sets (Suppl Table 11) showing an attenuated transcriptomic response of microglia to demyelination in CPZ_cKO mice. One way ANOVA and Tukey’s post-hoc test, **** P < 0.0001, *** P = 0.0006, * P = 0.0493. N = 3 mice Norm_Ctrl and CPZ_Ctrl; n = 4 mice CPZ_cKOo, heatmap and cumulative scores of homeostatic microglial gene set (Suppl Table 11). One way ANOVA and Tukey’s post-hoc test, P < 0.0001 CPZ_Ctrl vs Norm_Ctrl, P = 0.0034 CPZ_cKO vs CPZ_Ctrl. N = 3 mice Norm_Ctrl and CPZ_Ctrl; n = 4 mice CPZ_cKOp, graphic conclusion of microglial RNA-seq. Oligodendrocyte-derived SERPINA3N promotes the activation of homeostatic microglia towards disease-associated and neurodegeneration-related states.Data were presented as mean ± s.e.m. (h, i, m, n, o).
Supplier Page from R&D Systems, a Bio-Techne Brand for Serpin A3N Protein
Available conjugates: Sizes Available: 10 ug