Role of the Lysosomal Membrane Protein, CLN3, in the Regulation of Cathepsin D Activity

Abstract

Among Neuronal Ceroid Lipofuscinoses (NCLs), which are childhood fatal neurodegenerative disorders, the juvenile onset form (JNCL) is the most common. JNCL is caused by recessive mutations in the CLN3 gene. CLN3 encodes a lysosomal/endosomal transmembrane protein but its precise function is not completely known. We have previously reported that in baby hamster kidney (BHK) cells stably expressing myc-tagged human CLN3 (myc-CLN3), hyperosmotic conditions drastically increased myc-CLN3 mRNA and protein expression. In the present study, we analyzed the consequences of hyperosmolarity and increased CLN3 expression on cathepsin D activity and prosaposin processing using BHK cells transiently or stably expressing myc-CLN3. We found that hyperosmolarity increased lysotracker staining of lysosomes and elevated the levels of myc-CLN3 and lysosome-associated membrane protein-1 (LAMP1). Hyperosmolarity, independently of the expression level of myc-CLN3, decreased the levels of prosaposin and saposin D, which are protein cofactors in sphingolipid metabolism. The lysosomal enzyme cathepsin D (CTSD) mediates the proteolytic cleavage of prosaposin precursor into saposins A–D. Myc-CLN3 colocalized with CTSD and activity of CTSD decreased as myc-CLN3 expression increased and clearly decreased under hyperosmotic conditions. Nevertheless, levels of CTSD measured by Western blotting were not altered under any studied condition. Our results suggest a direct involvement of CLN3 in the regulation of CTSD activity.

Molecular networking in the neuronal ceroid lipofuscinoses: insights from mammalian models and the social amoeba Dictyostelium discoideum

“…In baby hamster kidney cells, hypertonic stress increases the expression of human myc-tagged CLN3 mRNA and protein [55]. Under these conditions, human myc-tagged CLN3 colocalizes with CTSD/CLN10 and decreases the activity of CTSD/CLN10 but has no effect on the amount of CTSD/CLN10 protein [82] (Table 2). In Dictyostelium, loss of cln3 alters the expression of ctsD, the intracellular and extracellular activity of CtsD, and the secretion of Cln5 [48,94] (Fig.…”

Differential accumulation of storage bodies with aging defines discrete subsets of microglia in the healthy brain

“…In contrast, we established that lysosomal and autophagosomal degradation pathways are primary mechanisms of accumulation of AF material, as evidenced by the accelerated and decreased AF accumulation respectively observed in AF + microglia from Cln3 Δex7-8 mice and Atg5 -deficient microglia. Of all cell types in the brain, microglia express the highest transcript levels of Cln3 , which is involved in the regulation of lysosomal pH, cathepsin activity, and endocytic trafficking ( Cárcel-Trullols et al, 2017 ; Cotman and Staropoli, 2012 ; Golabek et al, 2000 ; Schmidtke et al, 2019 ). Although the disruption of Cln3 expression was present in both AF + and AF − cells in Cln3 Δex7-8 mice, the latter subset was unaffected by the perturbation of the lysosomal pathway, suggesting that CLN3-dependent lysosomal degradation is dispensable in AF − microglia, which further highlights the molecular differences between AF + and AF − microglia.…”

Immunomodulation with minocycline rescues retinal degeneration in juvenile neuronal ceroid lipofuscinosis mice highly susceptible to light damage

“…Our data support this hypothesis in several ways. First, besides the accumulation of SubC as discussed above, a dysregulation of the lysosomal enzyme CatD has been connected with the absence of CLN3 ( Cárcel-Trullols et al, 2017 ; Fossale et al, 2004 ; Golabek et al, 2000 ; Kyttälä et al, 2006 ). Indeed, here we showed enrichment of CatD in ‘low light’-triggered retinal degeneration in young Cln3 Δex7/8 mice that was normally only present in aged Cln3 Δex7/8 mice.…”