Nuclear pore complex remodeling by p75(NTR) cleavage controls TGF-β signaling and astrocyte functions

Abstract

Astrocytes modulate neuronal activity and inhibit regeneration. We show that cleaved p75 neurotrophin receptor (p75(NTR)) is a component of the nuclear pore complex (NPC) required for glial scar formation and reduced gamma oscillations in mice via regulation of transforming growth factor (TGF)-β signaling. Cleaved p75(NTR) interacts with nucleoporins to promote Smad2 nucleocytoplasmic shuttling. Thus, NPC remodeling by regulated intramembrane cleavage of p75(NTR) controls astrocyte-neuronal communication in response to profibrotic factors.

Figure 1

p75^NTR deficiency rescues TGF-β-induced hydrocephalus, astrocyte activation and neuronal dysfunction. (a) Hematoxylin and eosin stain and GFAP immunostaining (red) of representative brain sections of 4-week old WT, p75^NTR−/−, GFAP-TGF-β, and GFAP-TGF-β: p75^NTR−/− mice. Nuclei are stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). Representative images are shown from n = 4 mice. (b) Quantification of ventricle size and GFAP intensity (n = 4 mice per group). Values are mean ± SEM. Ventricle size (top), *P = 4.995 × 10⁻⁶; GFAP intensity (bottom), *P = 2.452 × 10⁻⁶ and ns, not significant, by one-way ANOVA. Scale bar, 750 μm (upper) and 15 μm (lower). (c) Locomotor activity in the open field during the 50 min of EEG recordings. p75^NTR deficiency normalized locomotor activity in GFAP-TGF-β mice (n = 6–8 mice per group). ** P = 0.003 by repeated-measures ANOVA and Bonferroni post-hoc multiple comparisons test. (d) Gamma oscillatory power (30–80Hz) during different locomotor activity intervals in an open field from (c). GFAP-TGF-β, but not GFAP-TGF-β:p75^NTR−/−, mice had impaired activity-dependent inductions of gamma oscillatory activity (n = 6–8 mice per group). *P < 0.05, ** P < 0.01, *** P < 0.001 by one-way ANOVA and Bonferroni post-hoc multiple comparisons test; 1–5 movements/min, P = 0.009; 6–25 movements/min, P = 9.568 × 10⁻⁵; 26-100 movements/min, P = 4.793 × 10⁻¹⁷; >100 movements/min, P = 0.0002.

Figure 2

p75^NTR is a component of the NPC regulating TGF-β-induced P-Smad2 nuclear translocation. (a) P-Smad2 in the cytosolic and nuclear fractions of WT and p75^NTR−/− astrocytes. Representative immunoblots from three independent experiments are shown. (b) P-Smad2 in cytosol and nuclear fractions of TGF-β-treated astrocytes in the presence of the γ-secretase inhibitor compound E. Representative immunoblots from three independent experiments are shown. (c) 3D-SIM of WT astrocytes stained for p75^NTR (green) and FG-Nup (red) shows abundant p75^NTR staining at the nuclear surface (top) and sparse p75^NTR staining in the nuclear center (bottom). Enlargement of regions indicated by a rectangle show p75^NTR adjacent to FG-Nup proteins at the nuclear surface (top inset) and the nuclear center (bottom inset). Scale bars: 250 nm, top and bottom panel and 100 nm, insets. Nuclei are stained with DAPI (blue). Representative images from three independent experiments are shown. (d) Endogenous co-immunoprecipitation of p75^NTR with individual FG-Nup proteins in whole cell lysates. Representative immunoblots are shown from two independent experiments. (e) Nuclear fraction of WT astrocytes treated with TGF-β for 1 h, or after 4 h pre-treatment with γ-secretase inhibitor compound E. Blots were developed with anti-p75^NTR and H3 antibodies. Representative immunoblots from three independent experiments are shown. (f) 3D-STED of WT astrocytes stained for p75^NTR (green) and FG-Nup358 (red). TGF-β-induced relocalization of p75^NTR signal within the NPC is blocked by Compound E. Scale bar: 100 nm. Nuclei are stained with DRAQ5 (blue). Representative images are shown from two independent experiments. Full-length blots are shown in Supplemental Figure 15.

Figure 3

TGF-β-induced p75^NTR intramembrane cleavage regulates the NPC structure and function. (a) Cherry-p75^NTR-EGFP fusion protein. p75^NTR N-terminal, Cherry (red); p75^NTR C-terminal, EGFP (green); uncleaved p75FL (yellow). EGFP signal reduction after γ-secretase inhibition indicates reduced p75ICD formation. (b) Real-time imaging of TGF-β-treated astrocytes transfected with cherry-p75^NTR-EGFP in the presence of the γ-secretase inhibitor compound E or the α-secretase inhibitor TAPI-2. Representative merged cherry/EGFP images from three independent experiments are shown. Scale bar, 5 μm. (c) Quantification of nuclear EGFP signal. Values from the mean of 20 nuclei obtained from three independent experiments. (d) TGF-β-treated WT astrocytes show increased nuclear p75ICD (right) compared to control (left). Scale bar: 200 nm. Representative images from five independent experiments are shown. (e) Averaged signal for p75^NTR (white) revealed relocalization of p75^NTR into the NPC in TGF-β-treated astrocytes. p75^NTR signal is absent (black) in the NPC in untreated astrocytes. Scale bar, 100 nm. (f) Quantification of p75^NTR (green), FG-Nup (red) and DAPI signals (blue) in ~800 NPCs obtained from three independent experiments. (g) Nuclear pore diameter measurements by 3D electron tomography in ~16 nuclear pores per condition. Values are mean ± SEM. *P < 0.01, ** P < 0.0001, and ns, not significant, by one-way ANOVA and Bonferroni post-hoc multiple comparisons; WT untreated vs WT + TGF-β, P = 0.0003; WT untreated vs p75^NTR−/− untreated, P = 3.794 × 10⁻⁷; WT + TGF-β vs p75^NTR−/− + TGF-β, P = 8.774 ×10⁻¹⁰; p75^NTR−/− untreated vs p75^NTR−/− + TGF-β, P = 0.054.