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. 2019 Aug 20:10:569.
doi: 10.3389/fendo.2019.00569. eCollection 2019.

Autophagic and Proteasomal Mediated Removal of Mutant Androgen Receptor in Muscle Models of Spinal and Bulbar Muscular Atrophy

Maria Elena Cicardi  1 , Riccardo Cristofani  1 , Valeria Crippa  1 , Veronica Ferrari  1 , Barbara Tedesco  1 , Elena Casarotto  1 , Marta Chierichetti  1 , Mariarita Galbiati  1 , Margherita Piccolella  1 , Elio Messi  1 , Serena Carra  2 , Maria Pennuto  3   4   5 , Paola Rusmini  1 , Angelo Poletti  1   6
Affiliations

Autophagic and Proteasomal Mediated Removal of Mutant Androgen Receptor in Muscle Models of Spinal and Bulbar Muscular Atrophy

Maria Elena Cicardi et al. Front Endocrinol (Lausanne). .

Abstract

Spinal and bulbar muscular atrophy (SBMA) is an X-linked motoneuron disease (MND) caused by a mutant androgen receptor (AR) containing an elongated polyglutamine (polyQ) tract. ARpolyQ toxicity is triggered by androgenic AR ligands, which induce aberrant conformations (misfolding) of the ARpolyQ protein that aggregates. Misfolded proteins perturb the protein quality control (PQC) system leading to cell dysfunction and death. Spinal cord motoneurons, dorsal root ganglia neurons and skeletal muscle cells are affected by ARpolyQ toxicity. Here, we found that, in stabilized skeletal myoblasts (s-myoblasts), ARpolyQ formed testosterone-inducible aggregates resistant to NP-40 solubilization; these aggregates did not affect s-myoblasts survival or viability. Both wild type AR and ARpolyQ were processed via proteasome, but ARpolyQ triggered (and it was also cleared via) autophagy. ARpolyQ reduced two pro-autophagic proteins expression (BAG3 and VCP), leading to decreased autophagic response in ARpolyQ s-myoblasts. Overexpression of two components of the chaperone assisted selective autophagy (CASA) complex (BAG3 and HSPB8), enhanced ARpolyQ clearance, while the treatment with the mTOR independent autophagy activator trehalose induced complete ARpolyQ degradation. Thus, trehalose has beneficial effects in SBMA skeletal muscle models even when autophagy is impaired, possibly by stimulating CASA to assist the removal of ARpolyQ misfolded species/aggregates.

Keywords: androgen receptor; autophagy; chaperones; motoneuron disease; protein aggregation.

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Figures

Figure 1
Figure 1
Characterization of the cellular model. (A) Immunostaining for AR. Nuclei staining: DAPI. 60X magnification. Confocal microscope: Eclipse Ti2 (Nikon). Scale bar = 10 μm (B) WB (upper inset) and FRA (middle inset) of PBS extracts. Optical densitometry quantification of FRA (lower inset). ***p < 0.001 vs. ARQ24+T; **p > 0.01 vs. ARQ100-T. (C) WB (upper inset) and FRA (middle inset) of NP-40 soluble extracts. Optical densitometry quantification of FRA (lower inset). (**p < 0.01 vs. ARQ24+T or vs. ARQ100-T). (D) WB (upper inset) and FRA (middle inset) of NP-40 insoluble extracts. Optical densitometry quantification of FRA (lower inset). (**p < 0.01 vs. ARQ24+T; *p < 0.05 vs. ARQ100-T). Two-way ANOVA followed by Bonferroni post-hoc test was used. Each experiment was independently replicated three times. Graphs show quantification of three independent biological samples (n = 3).
Figure 2
Figure 2
Degradative systems involvement. (A) WB (upper inset) and FRA (middle inset) of PBS extract of cells treated with testosterone, MG132 and bafilomycin A1. Optical densitometry quantification of FRA (lower inset). (**p < 0.01; ***p < 0.001 vs. relative control conditions –T/+T). (B) FRA (upper inset) of NP-40 soluble extracts of cells treated with testosterone, MG132 and bafilomycin A1. Optical densitometry quantification of FRA (lower inset). (***p < 0.001 vs. relative control conditions –T/+T; *p < 0.05 vs. ARQ24 -T). (C) FRA (upper inset) of NP-40 insoluble extracts of cells treated with testosterone, MG132 and bafilomycin A1. Optical densitometry quantification of FRA (lower inset) (***p < 0.001 vs. relative control conditions +T). For each panel, FRA images derive from the same membranes with identical exposure time to permit direct comparison of wtAR and ARpolyQ levels. Two-way ANOVA followed by Bonferroni post-hoc test was used. Each experiment was independently replicated three times. Graphs show quantification of three independent biological samples (n = 3).
Figure 3
Figure 3
PQC activation and role against AR accumulation. (A) RT-qPCR of PQC system related genes performed on C2C12_ARQ24 and C2C12_ARQ100. *p < 0.05; **p < 0.01; ***p < 0.001 vs. ARQ24 in the same conditions. Graphs show quantification of four independent biological samples (n = 4). (B) WB (upper inset) and FRA (middle inset) of C2C12 transiently transfected with p5HBhARQ112 and co-transfected with plasmids coding for HSPB8, BAG3 and BAG1. Optical densitometry quantification of FRA (lower inset). (***p < 0.001 vs. relative control conditions +T). (C) WB (upper inset) and FRA (middle inset) of C2C12 transiently transfected with pARQ112ΔHA and co-transfected with plasmids coding for HSPB8, BAG3 and BAG1. Optical densitometry quantification of FRA (lower inset). (**p < 0.01; ***p < 0.001 vs. relative control conditions pcDNA3). Each experiment was independently replicated three times. Graphs show quantification of three independent biological samples (n = 3).
Figure 4
Figure 4
Trehalose activates autophagy and reduces ARpolyQ accumulation (A) Immunostaining for LC3 (left inset) and p62 (right inset) of C2C12_ARQ100 in presence of trehalose. Nuclei staining: DAPI. Magnification: 63X. Microscope: Axiovert 2000 (B) C2C12_ARQ100 treated with trehalose. RTqPCR of PQC system related genes. (**p < 0.01; ***p < 0.001 vs. relative untreated control). (C) C2C12_ARQ100 treated with trehalose and bafilomycin A1. WB (upper inset) and FRA (middle inset) of PBS extracts of Optical densitometry quantification of FRA (lower panel) (**p < 0.01 vs. relative untreated control; ***p < 0.001 vs. trehalose +T). (D,E) C2C12 transiently transfected with p5HBhARQ112 and treated with testosterone and trehalose. (D) WB (upper inset) and FRA (middle inset). Optical densitometry quantification of FRA (lower inset). *p < 0.05 vs. relative control conditions +T. (E) Immunostaining for AR. Nuclei staining: DAPI. Magnification: 63X. Microscope: confocal LSM510 Zeiss (F,G) C2C12 transiently transfected with pARQ16ΔHA or pARQ112ΔHA and treated with trehalose. (F) WB (upper inset) and FRA (middle inset). Optical densitometry quantification of FRA (lower inset). **p < 0.01 vs. relative control conditions +T. (G) Immunostaining for AR. Nuclei staining: DAPI. Magnification: 63X. Microscope: confocal LSM510 Zeiss. Each experiment was independently replicated three times (n = 3). Graphs show quantification of three independent biological samples (n = 3).
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