Alterations in ROS Activity and Lysosomal pH Account for Distinct Patterns of Macroautophagy in LINCL and JNCL Fibroblasts

Abstract

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.

Lysosomal storage diseases and the heat shock response: convergences and therapeutic opportunities

“…This was consistent with SCMAS being released from lysosomes through LMP, suggesting that LMP is a previously unrecognized pathogenic event in CLN2 disease linking LMP and the autophagic machinery ( 67 ). This is supported by studies in human fi broblasts from patients with CLN2 disease showing increased levels of ROS, a well-described inducer of LMP ( 135 ).…”

Modulating membrane fluidity corrects Batten disease phenotypes in vitro and in vivo

“…Impaired fluid phase endocytosis has been reported for Cln3 mutant yeast, Cln3 −/− mouse brain endothelial cells, Cln3 −/− neuronal cells, and in fibroblasts harvested from patients with CLN3 deficiency (Codlin et al, 2008; Fossale et al, 2004; Luiro et al, 2004; Schultz et al, 2014; Vidal-Donet et al, 2013). We therefore measured fluid-phase endocytosis by uptake of fluorescently labeled dextran in cells treated with CBX or vehicle.…”

“…Work in model systems over the last two decades suggest that CLN3 impacts multiple cellular functions including lysosomal pH (Golabek et al, 2000; Holopainen et al, 2001; Pearce et al, 1999; Pearce and Sherman, 1998), vesicular trafficking (Cao et al, 2006; Codlin and Mole, 2009; Fossale et al, 2004; Kama et al, 2011; Metcalf et al, 2008), palmitoyl desaturase activity (Narayan et al, 2006), endocytosis (Codlin et al, 2008; Fossale et al, 2004; Luiro et al, 2001; Luiro et al, 2004; Schultz et al, 2014; Tecedor et al, 2013; Vidal-Donet et al, 2013), and membrane microdomain formation or stability (Tecedor et al, 2013). …”

Unbiased Cell-based Screening in a Neuronal Cell Model of Batten Disease Highlights an Interaction between Ca2+ Homeostasis, Autophagy, and CLN3 Protein Function

“…Although the established autophagy-related phenotypes in CLN3 models and the relationship of these to neurodegeneration in JNCL are incompletely understood, we further reasoned that developing a screening assay around autophagy in an accurate genetic model would serve as a useful starting point because abnormalities in autophagy are detected early in the disease process (17). Furthermore, the CLN3 protein is documented to be present in autophagosomes and autolysosomes/lysosomes (17), and defects in this pathway are hypothesized to be important in the development of mitochondrial ATPase subunit c-containing lysosomal storage material, a pathological hallmark in JNCL and other forms of NCL (17,42,50). Here, we have successfully developed a GFP-LC3-based autophagy assay in our murine cerebellar neuronal progenitor cell model of JNCL, and importantly, we have demonstrated that chemical biology studies using this model can successfully identify compounds with conserved bioactivity in human JNCL patient-derived disease relevant cell types.…”