Fig 1: Down-regulation of Nr4a factors and their effector genes is linked to ADRD pathology.(A and B) Expression profiles of NR4A1, NR4A2, and NR4A3 in the hippocampus, from the Allen Brain Institute Study of Aging, Dementia, and Traumatic Brain Injury, correlated with (A) Cerad scores, which reflect the density of neuritic plaques, and (B) Braak stages, which reflect the severity of neurofibrillary tangles. (C) Schematic depiction of control (CaMKIIα-tTA) or rTg4510 (CaMKIIα-tTA and TetO-hMAPT P301L) mice and Western blots of tau phosphorylation (AT8, phosphorylation at both Ser202 and Thr205) in the dorsal hippocampus in the presence or absence of doxycycline (Dox). (D) Long-term memory in rTg4510 and control mice at 4 months of age, following training in SOR. Two-way ANOVA: significant main effect of genotype/treatment (F2,29 = 4.792, P = 0.0159) and significant main effect of sessions (F1,29 = 9.221, P = 0.0050). Sidak’s multiple comparisons test: **P = 0.0096 (control mice, train versus test), *P = 0.0439 (rTg4510-Dox mice, train versus test), **P = 0.0016 (rTg4510 mice, 24-hour test versus control mice, 24-hour test), and *P = 0.0140 (rTg4510 mice, 24-hour test versus rTg4510 Dox, 24-hour test). Control, n = 11 (3F); rTg4510, n = 8 (4F); and rTg4510 + Dox, n = 13 (7F). (E and F) rTg4510 and control mice (n = 9 per group) were trained in SOR, and 2 hours later, the dorsal hippocampus was collected and processed for RNA extraction and the analysis of gene expression. (E) Nr4a subfamily gene expression: unpaired t test: Nr4a1: t16 = 4.878, ***P = 0.0002; Nr4a2: t16 = 2.621, *P = 0.0185. (F) Hspa5 and Pdia6 gene expression: unpaired t test: Hspa5: t16 = 3.692, **P = 0.0020; Pdia6: t16 = 4.177, ***P = 0.0007.
Fig 2: IC87114 and 4‐PBA alleviate A. fumigatus‐induced NLRP3 inflammasome activation of IRE1α. Lung tissues were obtained from A. fumigatus‐challenged, saline‐treated (Af) mice, and A. fumigatus‐challenged mice treated with 1 mg kg−1 IC87114 (Af + IC) or 80 mg kg−1 4‐PBA (Af + 4‐PBA). (a) Lung tissue were immunoprecipitated and immunoblotted with anti‐IRE1α antibody and immunoblotted with anti‐PDIA6 antibody. (b) Subcellular localisation of TXNIP in membrane fractions of lung tissue was determined by immunoblotting and quantified. (c) Lung tissue lysates were immunoprecipitated with anti‐TXNIP antibody and immunoblotted with antibodies against TXNIP and NLRP3. (d) Lung tissue lysates were immunoprecipitated with anti‐NLRP3 antibodies and immunoblotted with antibodies against NLRP3, ASC, or caspase‐1. (e) Quantification of caspase‐1. ELISA analysis of released cytokines IL‐1β (f) and IL‐18 (g) in BALF. (h) Immunoblot analysis of nuclear NF‐κB p65/nuclear histone, cytosol p65/cytosolic tubulin, and IκB‐α/β‐actin; bands were quantified by densitometry and normalised to histone (nuclear), tubulin (cytosol), and β‐actin (total) as the control. Data are expressed as the mean ± SD (n = 10). # P < .05, significantly different from saline; *P < .05, significantly different from A. fumigatus alone; ANOVA. ER, endoplasmic reticulum; IC, IC87114; TXNIP, thioredoxin interacting protein; NLRP3, NOD‐like receptor protein
Fig 3: A subset of the genes downstream of Nr4a is induced by learning.(A) Quadrant plot based on total RNA-seq data. Genes induced by learning were identified on the basis of comparison of genes regulated in dorsal hippocampus of control male mice 2 hours after SOR (tTA+ Nr4ADN−, n = 2; tTA− Nr4ADN−, n = 2) and homecage control mice (tTA+ Nr4ADN−, n = 2; tTA− Nr4ADN−, n = 2). Quadrant plot comparing genes regulated by learning in control mice to genes regulated in Nr4ADN mice by learning. Genes up-regulated by SOR are down-regulated in Nr4ADN mice (labeled points). Size, opacity, and color intensity of each point reflect the minimum false discovery rate value for a gene between each experiment. (B and C) Expression of (B) Hspa5 and (C) Pdia6 mRNAs in C57BL/6J male mice trained in SOR and euthanized at the indicated times after training (1 hour: n = 9; 2 hours: n = 9; 3 hours: n = 9; 4 hours: n = 9), expressed as fold difference from that of mice handled only in the homecage (HC) (baseline controls, n = 10). One-way ANOVA: Hspa5: F4,41 = 13.00, P < 0.0001. Sidak’s multiple comparisons tests: ***P < 0.0001 (HC versus 1 hour), ***P < 0.0001 (HC versus 2 hours), ***P < 0.0001 (HC versus 3 hours), and *P = 0.0158 (HC versus 4 hours); Pdia6: F4,41 = 8.442, P < 0.0001. Sidak’s multiple comparisons tests: ***P = 0.0008 (HC versus 1 hour), ***P = 0.0001 (HC versus 2 hours), ***P < 0.0001 (HC versus 3 hours), and **P = 0.0054 (HC versus 4 hours). (D and E) Down-regulation of gene expression at 2 hours after SOR training in male Nr4ADN (n = 9) and control (n = 10) mice, as validated by qPCR. Unpaired t test: t17 = 3.305, **P = 0.0042 (Hspa5); t17 = 3.630, **P = 0.0021 (Pdia6). (F and G) Quantification of Western blot of lysates of synaptosomes isolated from the dorsal hippocampus of male Nr4ADN (n = 4) and control mice 2 hours after SOR training (n = 4). Unpaired t test: t6 = 4.011, **P = 0.0070 (Hspa5); t6 = 2.982, *P = 0.0246 (Pdia6).
Fig 4: Meprin β WT and meprin β R238Q do not differ in cellular localization and shedding.(A) For cell surface biotinylation assays, membrane proteins on intact cells were labeled with thiol-cleavable amine–reactive biotin and pulled down from lysates with magnetic streptavidin beads. (B) Cell surface biotinylation assay of HEK 293T cells overexpressing meprin β variants, analyzed by Western blot. Pan-cadherin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as controls for cell surface and intracellular proteins, respectively. (C and D) Immunofluorescence microscopy images of HEK 293T transfected with the indicated meprin β variants, stained with anti–meprin β antibody (hEcto1, green) (C) or anti–flag tag antibody (green) (D). Antibody against PDIA6 (red) served as ER marker (D) and nuclei were stained with DAPI (blue). (E) Inactive pro–meprin β can be shed from the cell surface by ADAM10/17 and MT1-MMP. Soluble pro–meprin β can be activated by trypsin. (F) HEK 293T WT/ADAM10/17−/− cells were transfected with meprin β variants and ADAM10. Lysates and ultracentrifuged supernatants were analyzed by Western blot. GAPDH served as loading control. (G) Activity of shed meprin β variants in ultracentrifuged cell supernatants [untreated or activated with trypsin, measured using (mca)-EDAEDA-(dnp)] (n = 3) [means ± SD; two-way ANOVA, followed by a Tukey posttest (* ≙ P < 0.05; ** ≙ P < 0.01; *** ≙ P < 0.001)].
Supplier Page from Abcam for Anti-PDIA6 antibody