The NORAD -Pumilio regulatory axis in the evolution of inclusion body myositis

Figure 1. Disease activity related increase in (A) NORAD expression in muscle biopsies from sporadic IBM patients showing mild (mIBM) and severe (IBM) pathology compared to control muscle with no pathologic diagnosis. Increased (B) PUM 1 and (C) PUM 2 levels from the same samples compared to control muscle. Increased (D) NORAD expression in biopsies from vacuolar myopathy (VM) and dermatomyositis (DM) cases and increased (E) PUM 1 and (F) PUM 2 levels from the same diseased samples compared to control muscle are shown. Representative confocal images showing NORAD probe-positive staining in (G) IBM and (H) control muscle samples. (I) shows NORAD probe-positive staining around nucleus at high magnification and (J) colocalization of NORAD probe with PUM2 as NP bodies in muscle from an IBM patient. Scale bars: G, H = 50 µm; I, J = 5 µm. Data are represented as mean ± SEM, 1-way ANOVA, *P < .05, **P < .01, ***P < .001. n numbers: Control: 6 to 7, mIBM: 6, IBM: 8-9, DM: 9-10, VM: 5.

Figure 2. Representative H&E-stained images from gastrocnemius muscle of young (A) and old (B) VCP-A232E (Vv) mice showing increased internal nuclei and fiber size variability with aging. (C) shows percent distribution of fibers with internal nuclei (n = 5 for 2-3 months old, n = 7 for 1-year-old Vv mice). SDH enzyme histochemical stain on muscle sections from young (D) and old (E) Vv mice. (F) shows marked increase in number of fibers with abnormal mitochondria content and distribution (arrows in E) with aging (n = 3 for each cohort). (H-I) show relative mRNA expression levels of Norad, Pum1, and Pum2 in young and old Vv mice and age-matched controls. n numbers: C57BL/6 (2-3 mos): 6, C57BL/6 (1 yr): C57BL/6 (2 yrs): 8, Vv (2-3 mos): 9, Vv (1 yr): 11-12 Data are represented as mean ± SEM, unpaired t-test, 1-way ANOVA, 2-way ANOVA, *P < .05, **P < .01, ***P < .001, ****P < .0001. Scale bars: A, B = 50 µm; D, E = 30 µm.

Figure 3. Representative H&E-stained images from gastrocnemius muscle of (A) VCP-A232E (Vv), (B) VCP-A232E; Norad+/− (VvNn), and (C) VCP-A232E; Norad−/− (Vvnn) mice at 6 weeks of age showing (D) significant increase in muscle fibers with diameter < 25 µm and increased fiber density per unit area in the Vvnn mice. Images with SDH enzyme histochemical stain on muscle from (E) Vv, (F) VvNn, and Vvnn (G) mice showing muscle fibers with increased subsarcolemmal mitochondria (arrows) in F and G. (H) Marked increase in number of fibers with abnormal mitochondria content and distribution are seen in VvNn and Vvnn compared to Vv. Representative gastrocnemius muscle sections with H&E stain from Vvnn mice at 6 weeks of age show (I) muscle fiber necrosis, phagocytosis, (J) regenerating basophilic fibers (arrow), and (K) muscle fiber loss with fat replacement, which was prominent in (L) some samples. Images of gastrocnemius muscle from VvNn mice at 6 weeks of age show scattered, rare (M) necrotic or (N) basophilic regenerating fibers and (O) subtle fat replacement. Data are represented as mean ± SEM, n = 3 for all experimental cohorts, 1-way ANOVA, 2-way ANOVA, *P < .05, **P < .01, ***P < .001, ****P <…

Figure 4. Relative mRNA expression levels of (A) Norad, (B) Pum1, and (C) Pum2 in young (solid bars) and old (crossed bars) Vv, VvNn, and Vvnn mice. Data are represented as mean ± SEM, n numbers: Vv (2-3 mos): 9, Vv (1 yr): 11 to 12, VvNn (2-3 mos): 5-6, VvNn (1 yr): 3, Vvnn (2-3 mos): 6, unpaired t-test, 1-way ANOVA, *P < .05, **P < .01, ***P < .001, ****P < .0001.

Figure 5. Elevated Norad (A) and (B) Pum2 mRNA levels in the gastrocnemius muscle of Pum2; rtTA3 double transgenic mouse model at 15 weeks of age 6 weeks following enforced Pum2 expression (Pum2-OE). Representative confocal images showing Norad probe-positive staining in (C) Pum2-OE and (D) control gastrocnemius muscle samples. High magnification mages show (E) Norad probe-positive staining, (F) PUM2 antibody staining, and (G) their colocalization as NP bodies coalescing around nucleus in the Pum2-OE muscle. Scale bars: C, D = 20 µm; I, J. Data are represented as mean ± SEM, 2-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.