Alterations in ROS activity and lysosomal pH account for distinct patterns of macroautophagy in LINCL and JNCL fibroblasts

PLoS One. 2013;8(2):e55526. doi: 10.1371/journal.pone.0055526. Epub 2013 Feb 7.


Neuronal ceroid lipofuscinoses (NCL) are lysosomal storage disorders characterized by the accumulation of lipofuscin within lysosomes. Late infantile (LINCL) and juvenile (JNCL) are their most common forms and are caused by loss-of-function mutations in tripeptidyl peptidase 1 (TPP1), a lysosomal endopeptidase, and CLN3 protein (CLN3p), whose location and function is still controversial. LINCL patients suffer more severely from NCL consequences than JNCL patients, in spite of having in common an abnormal accumulation of material with a similar composition in the lysosomes. To identify distinctive characteristics that could explain the differences in the severity of LINCL and JNCL pathologies, we compared the protein degradation mechanisms in patientś fibroblasts. Pulse-chase experiments show a significant decrease in protein degradation by macroautophagy in fibroblasts bearing TPP1 (CLN2) and CLN3p (CLN3) mutations. In CLN2 fibroblasts, LC3-II levels and other procedures indicate an impaired formation of autophagosomes, which confirms the pulse-chase experiments. This defect is linked to an accumulation of reactive oxygen species (ROS), an upregulation of the Akt-mTOR signalling pathway and increased activities of the p38α and ERK1/2 MAPKs. In CLN3 fibroblasts, LC3-II analysis indicates impairment in autophagosome maturation and there is also a defect in fluid phase endocytosis, two alterations that can be related to an observed increase of 0.5 units in lysosomal pH. CLN3 fibroblasts also accumulate ROS but to a lower extent than CLN2. TPP1 activity is completely abrogated in CLN2 and partially diminished in CLN3 fibroblasts. TPP1 cleaves small hydrophobic proteins like subunit c of mitochondrial ATP synthase and the lack or a lower activity of this enzyme can contribute to lipofuscin accumulation. These alterations in TPP1 activity lead to an increased ROS production, especially in CLN2 in which it is aggravated by a decrease in catalase activity. This could explain the earlier appearance of the symptoms in the LINCL form.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy*
  • Catalase / metabolism
  • Cells, Cultured
  • Fibroblasts / enzymology
  • Fibroblasts / immunology*
  • Fibroblasts / metabolism
  • Humans
  • Hydrogen-Ion Concentration*
  • Lysosomes / metabolism*
  • Neuronal Ceroid-Lipofuscinoses / enzymology
  • Neuronal Ceroid-Lipofuscinoses / immunology*
  • Neuronal Ceroid-Lipofuscinoses / metabolism
  • Oxidative Stress
  • Reactive Oxygen Species / metabolism*


  • Reactive Oxygen Species
  • Catalase

Grant support

This work was supported by the Spanish Ministry of Science and Innovation (BFU2011-22630), Generalitat Valenciana (PROMETEO/2012/061), Fundació Marató TV3 (Grant number 100130) and the Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). JMVD holds a fellowship from the Spanish Ministry of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.