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Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48‑linked ubiquitylation and defective autophagy

Journal Article


Abstract


  • Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

Authors


  •   Lee, Albert (external author)
  •   Rayner, Stephanie L. (external author)
  •   Gwee, Serene S. L. (external author)
  •   De Luca, Alana (external author)
  •   Shahheydari, Hamideh (external author)
  •   Sundaramoorthy, Vinod (external author)
  •   Ragagnin, Audrey (external author)
  •   Morsch, Marco (external author)
  •   Radford, Rowan (external author)
  •   Galper, Jasmin (external author)
  •   Yerbury, Justin J.

Publication Date


  • 2018

Citation


  • Lee, A., Rayner, S. L., Gwee, S. S. L., De Luca, A., Shahheydari, H., Sundaramoorthy, V., Ragagnin, A., Morsch, M., Radford, R., Galper, J., Yerbury, J. J. et al (2018). Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48‑linked ubiquitylation and defective autophagy. Cellular and Molecular Life Sciences, 75 (2), 335-354.

Scopus Eid


  • 2-s2.0-85028760989

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/1123

Number Of Pages


  • 19

Start Page


  • 335

End Page


  • 354

Volume


  • 75

Issue


  • 2

Place Of Publication


  • Switzerland

Abstract


  • Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

Authors


  •   Lee, Albert (external author)
  •   Rayner, Stephanie L. (external author)
  •   Gwee, Serene S. L. (external author)
  •   De Luca, Alana (external author)
  •   Shahheydari, Hamideh (external author)
  •   Sundaramoorthy, Vinod (external author)
  •   Ragagnin, Audrey (external author)
  •   Morsch, Marco (external author)
  •   Radford, Rowan (external author)
  •   Galper, Jasmin (external author)
  •   Yerbury, Justin J.

Publication Date


  • 2018

Citation


  • Lee, A., Rayner, S. L., Gwee, S. S. L., De Luca, A., Shahheydari, H., Sundaramoorthy, V., Ragagnin, A., Morsch, M., Radford, R., Galper, J., Yerbury, J. J. et al (2018). Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48‑linked ubiquitylation and defective autophagy. Cellular and Molecular Life Sciences, 75 (2), 335-354.

Scopus Eid


  • 2-s2.0-85028760989

Ro Metadata Url


  • http://ro.uow.edu.au/ihmri/1123

Number Of Pages


  • 19

Start Page


  • 335

End Page


  • 354

Volume


  • 75

Issue


  • 2

Place Of Publication


  • Switzerland