Converging roles of PSENEN/PEN2 and CLN3 in the autophagy-lysosome system

Abstract

PSENEN/PEN2 is the smallest subunit of the γ-secretase complex, an intramembrane protease that cleaves proteins within their transmembrane domains. Mutations in components of the γ-secretase underlie familial Alzheimer disease. In addition to its proteolytic activity, supplementary, γ-secretase independent, functions in the macroautophagy/autophagy-lysosome system have been proposed. Here, we screened for PSENEN-interacting proteins and identified CLN3. Mutations in CLN3 are causative for juvenile neuronal ceroid lipofuscinosis, a rare lysosomal storage disorder considered the most common neurodegenerative disease in children. As mutations in the PSENEN and CLN3 genes cause different neurodegenerative diseases, understanding shared cellular functions of both proteins might be pertinent for understanding general cellular mechanisms underlying neurodegeneration. We hypothesized that CLN3 modulates γ-secretase activity and that PSENEN and CLN3 play associated roles in the autophagy-lysosome system. We applied CRISPR gene-editing and obtained independent isogenic HeLa knockout cell lines for PSENEN and CLN3 . Following previous studies, we demonstrate that PSENEN is essential for forming a functional γ-secretase complex and is indispensable for γ-secretase activity. In contrast, CLN3 does not modulate γ-secretase activity to a significant degree. We observed in PSENEN - and CLN3 -knockout cells corresponding alterations in the autophagy-lysosome system. These include reduced activity of lysosomal enzymes and lysosome number, an increased number of autophagosomes, increased lysosome-autophagosome fusion, and elevated levels of TFEB (transcription factor EB). Our study strongly suggests converging roles of PSENEN and CLN3 in the autophagy-lysosome system in a γ-secretase activity-independent manner, supporting the idea of common cytopathological processes underlying different neurodegenerative diseases. Abbreviations: Aβ, amyloid-beta; AD, Alzheimer disease; APP, amyloid precursor protein; ATP5MC, ATP synthase membrane subunit c; DQ-BSA, dye-quenched bovine serum albumin; ER, endoplasmic reticulum; GFP, green fluorescent protein; ICC, immunocytochemistry; ICD, intracellular domain; JNCL, juvenile neuronal ceroid lipofuscinosis; KO, knockout; LC3, microtubule associated protein 1 light chain 3; NCL, neuronal ceroid lipofuscinoses; PSEN, presenilin; PSENEN/PEN2: presenilin enhancer, gamma-secretase subunit; TAP, tandem affinity purification; TEV, tobacco etch virus; TF, transferrin; WB, Western blot; WT, wild type.

Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex

“…We provide here reassembled figures that should restore trust in the conclusions of our previous work. The Notch deficient phenotype of the Psenen −/− mice ( 17 , 51 , 52 , 53 , 54 , 55 ), the Psenen −/− fibroblasts to study structure–function ( 56 , 57 , 58 , 59 ), the role of PSENEN in complex formation ( 52 , 54 , 60 , 61 , 62 ), and the data of the cysteine scan ( 19 , 28 , 59 ) were used and cited in other publications, and based on the reported data here, these citations remain fully valid. One major change in the current article is the updated interpretation of the cysteine scan data presented in Figure 5 , Figure 6 , Figure 7 and S1 .…”

The Batten disease protein CLN3 is important for stress granules dynamics and translational activity

“…at 37 °C in a humidified chamber at 95% air and 5% CO2. CLN3 KO , CLN5 KO , and Rab7A KO HeLa cells have been described ( 17 , 43 , 77 ); the same parental line was used to generate these KO lines ( 17 , 43 , 77 ). Fibroblast cells were derived from skin biopsies taken from two unrelated healthy individuals (523N and 526N) and from two unrelated patients (478Pa and 481Pa) homozygous for the 1-kb deletion that causes classic juvenile CLN3 disease.…”

The Batten disease protein CLN3 is important for stress granules dynamics and translational activity

“…at 37°C in a humidified chamber at 95% air and 5% CO2. CLN3 KO , CLN3 KO and Rab7A KO HeLa cells have been described previously (13,39,70). Cells were seeded at a density of 5×10 6 / well in 24 well plates, 2×10 6 / well in 6 well plates and 10×10 6 /well in 15cm dishes.…”