Background: Spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are characterized by the presence of aggregates of aberrant proteins (androgen receptors (AR), superoxide dismutase 1 (SOD1), TAR DNA binding protein 43 (TDP-43), etc.) with abnormal conformations (misfolding). These accumulate into aggregates because the protein quality control system is not suffi ciently active for their correct removal. Dynein motor complex seems to play a crucial role to maintain an effi cient clearance of misfolded proteins by autophagy. Dynein mediated the transport of misfolded proteins/aggregates near microtubule organization center (MTOC). In addition dynein allows autophagosome nucleation by binding Vps34-Beclin1-Ambra1 complex, and assists the fusion of autophagosome to lysosome. Objectives: In this study we aimed to identify the contribution of dynein motor complex in the removal of misfolded proteins which accumulate in motoneuron diseases. Methods: We used NSC34 cells transfected with mutant SBMA-AR, G93A-SOD1 and Δ C-TDP-43. Dynein inhibition was obtained using the selective dynein ATPase inhibitor EHNA at 0,1mM for 48h. Protein accumulation was quantified by retained protein on cellulose acetate membrane in fi lter retardation assay (FRA). Autophagy activity was analyzed using the LC3 marker by western blotting. Misfolded proteins rerouting to proteasome was analyzed measuring the BAG1/BAG3 ratio in RT-qPCR.Results: Inhibition of dynein, drastically reduce the LC3II/LC3I ratio when autophagy is induced by trehalose. Immunofl uorescence on NSC34 transfected with SBMA-AR showed that dynein is sequestered into mutant SBMA-AR aggregates. Unexpectedly, EHNA reduces aggregates of mutant proteins in FTA also in presence of autophagy inhibitor (3-MA), but not in presence of proteasome inhibitor (MG132). In order to confi rm these data we produced an inducible stable transfected GFP-SBMA-AR cell line. Also in this case EHNA treatment reduces aggregate retained in FRA. Moreover, EHNA treatment increases the levels of mutated AR in PBS and Triton-X100 fraction in progressive extraction. When we analyzed the mRNA levels of the co-chaperones BAG1 and BAG3, which route misfolding proteins to proteasome degradation or chaperone-mediated-autophagy (BAG1) and autophagy (BAG3), we found that BAG1/BAG3 ratio is increased after EHNA treatment. Conclusion: These data suggest that dynein impairment, which results in autophagy blockage, also reduces aggregation of misfolding proteins involved in MNDs, by increasing their solubility and possibly through induction of alternative degradative pathways. Acknowledgements: Telethon; Fondazione AriSLA; AFM, France; Regione Lombardia; Universit à degli Studi di Milano; Ministero della Salute.

Inhibition of dynein atpase activity reduces aggregation of misfolded protein responsible for SBMA and ALS / R. Cristofani, E. Giorgetti, V. Crippa, A. Boncoraglio, M.E. Cicardi, P. Rusmini, S. Carra, A. Poletti. - In: AMYOTROPHIC LATERAL SCLEROSIS AND FRONTOTEMPORAL DEGENERATION. - ISSN 2167-8421. - 15:suppl. 1(2014), pp. 173-174. ((Intervento presentato al 25. convegno International Symposium on ALS/MND tenutosi a Brussels nel 2014 [10.3109/21678421.2014.960172A].

Inhibition of dynein atpase activity reduces aggregation of misfolded protein responsible for SBMA and ALS

R. Cristofani;E. Giorgetti;V. Crippa;A. Boncoraglio;M.E. Cicardi;P. Rusmini;A. Poletti
2014

Abstract

Background: Spinobulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS) are characterized by the presence of aggregates of aberrant proteins (androgen receptors (AR), superoxide dismutase 1 (SOD1), TAR DNA binding protein 43 (TDP-43), etc.) with abnormal conformations (misfolding). These accumulate into aggregates because the protein quality control system is not suffi ciently active for their correct removal. Dynein motor complex seems to play a crucial role to maintain an effi cient clearance of misfolded proteins by autophagy. Dynein mediated the transport of misfolded proteins/aggregates near microtubule organization center (MTOC). In addition dynein allows autophagosome nucleation by binding Vps34-Beclin1-Ambra1 complex, and assists the fusion of autophagosome to lysosome. Objectives: In this study we aimed to identify the contribution of dynein motor complex in the removal of misfolded proteins which accumulate in motoneuron diseases. Methods: We used NSC34 cells transfected with mutant SBMA-AR, G93A-SOD1 and Δ C-TDP-43. Dynein inhibition was obtained using the selective dynein ATPase inhibitor EHNA at 0,1mM for 48h. Protein accumulation was quantified by retained protein on cellulose acetate membrane in fi lter retardation assay (FRA). Autophagy activity was analyzed using the LC3 marker by western blotting. Misfolded proteins rerouting to proteasome was analyzed measuring the BAG1/BAG3 ratio in RT-qPCR.Results: Inhibition of dynein, drastically reduce the LC3II/LC3I ratio when autophagy is induced by trehalose. Immunofl uorescence on NSC34 transfected with SBMA-AR showed that dynein is sequestered into mutant SBMA-AR aggregates. Unexpectedly, EHNA reduces aggregates of mutant proteins in FTA also in presence of autophagy inhibitor (3-MA), but not in presence of proteasome inhibitor (MG132). In order to confi rm these data we produced an inducible stable transfected GFP-SBMA-AR cell line. Also in this case EHNA treatment reduces aggregate retained in FRA. Moreover, EHNA treatment increases the levels of mutated AR in PBS and Triton-X100 fraction in progressive extraction. When we analyzed the mRNA levels of the co-chaperones BAG1 and BAG3, which route misfolding proteins to proteasome degradation or chaperone-mediated-autophagy (BAG1) and autophagy (BAG3), we found that BAG1/BAG3 ratio is increased after EHNA treatment. Conclusion: These data suggest that dynein impairment, which results in autophagy blockage, also reduces aggregation of misfolding proteins involved in MNDs, by increasing their solubility and possibly through induction of alternative degradative pathways. Acknowledgements: Telethon; Fondazione AriSLA; AFM, France; Regione Lombardia; Universit à degli Studi di Milano; Ministero della Salute.
retrograde transport; autophagy; proteasome
Settore BIO/13 - Biologia Applicata
Motor Neuron Diseases Association
Centro Interdipartimentale di Eccellenza per le Malattie Neurodegenerative CEND
Centro Interuniversitario di Ricerca sulle Basi Molecolari delle Malattie Neurodegenerative
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