Loss of P-type ATPase ATP13A2/PARK9 function induces general lysosomal deficiency and leads to Parkinson disease neurodegeneration

Abstract

Parkinson disease (PD) is a progressive neurodegenerative disorder pathologically characterized by the loss of dopaminergic neurons from the substantia nigra pars compacta and the presence, in affected brain regions, of protein inclusions named Lewy bodies (LBs). The ATP13A2 gene (locus PARK9) encodes the protein ATP13A2, a lysosomal type 5 P-type ATPase that is linked to autosomal recessive familial parkinsonism. The physiological function of ATP13A2, and hence its role in PD, remains to be elucidated. Here, we show that PD-linked mutations in ATP13A2 lead to several lysosomal alterations in ATP13A2 PD patient-derived fibroblasts, including impaired lysosomal acidification, decreased proteolytic processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished lysosomal-mediated clearance of autophagosomes. Similar alterations are observed in stable ATP13A2-knockdown dopaminergic cell lines, which are associated with cell death. Restoration of ATP13A2 levels in ATP13A2-mutant/depleted cells restores lysosomal function and attenuates cell death. Relevant to PD, ATP13A2 levels are decreased in dopaminergic nigral neurons from patients with PD, in which ATP13A2 mostly accumulates within Lewy bodies. Our results unravel an instrumental role of ATP13A2 deficiency on lysosomal function and cell viability and demonstrate the feasibility and therapeutic potential of modulating ATP13A2 levels in the context of PD.

Fig. 1.

Accumulation of lysosomal-related structures in ATP13A2 mutant fibroblasts. (A) Accumulation of lysosomes and/or ALs in mutant ATP13A2 fibroblasts (L3292 and L6025) under basal conditions, as evidenced by lysosomal marker LysoTracker staining (red; nucleus in blue) and immunostaining for cath-B, cath-D, and LAMP-2 (green; nucleus in blue). *P < 0.05 compared with control fibroblasts. (Scale bars, 10 μm.) (B) Ultrastructural examination of mutant ATP13A2 fibroblasts by EM. Note the accumulation of AVs/ALs in mutant ATP13A2 fibroblasts (B2, B3) compared with control fibroblasts (B1). B1′–B3′ correspond to high-magnification images. (C) AVs were subcategorized based on their morphology. AP, autophagosome-double membrane; EAL, early AL-single membrane with relatively undigested material; LAL, late AL-single membrane with amorphous electron-dense material. Double arrows represent double membranes, and single arrows represent single membranes. (Scale bars, 500 nm.) *P < 0.05 compared with control fibroblasts. (D) (Upper) Immunofluorescent (IF) labeling for LAMP-1 in control and mutant ATP13A2 fibroblasts. (Scale bars, 1 μm.) (Lower) EM-immunogold labeling for LAMP-1 in control and mutant ATP13A2 fibroblasts. (Insets) Higher magnification images of single vesicles. (Scale bars, 500 nm.) (E) Representative immunoblot levels of LAMP-2 and LAMP-1 in mutant ATP13A2 and control (Ctrl) fibroblast under basal conditions.

Fig. 2.

Impaired lysosomal degradation in ATP13A2 mutant fibroblasts. (A and B) Cath-D (CTSD) and Cath-B (CTSB) immunoblot levels in mutant ATP13A2 and control (Ctrl) fibroblasts. (C) In vitro assay of cath-D enzyme activity in lysosomal fractions of control and mutant ATP13A2 fibroblasts. (D) Lysosomal pH values as measured ratiometrically using Lysosensor Yellow/Blue DND-160 in control and mutant ATP13A2 fibroblasts. (E and F) Degradation of long-lived proteins in control, mutant L6025, and WT ATP13A2-transfected L6025 fibroblasts. After incorporation of [³H]-valine, cells were incubated in serum-supplemented or -deprived medium with or without lysosomal inhibitors (20 mM NH₄Cl, 100 μM leupeptin) during the chase period (up to 24 h). (F) Lysosomal degradation calculated as degradation sensitive to NH₄Cl/leupeptin. LC3 immunoblot levels in mutant ATP13A2 L6025 (G) and L3292 (H) fibroblasts were treated or not treated with 5 nM bafilomycin A₁ for 1 h. (I) MEF2D and GAPDH immunoblot levels in mutant ATP13A2 and control fibroblasts. In all panels, n = 3–5 per experimental group. *P < 0.05 compared with control untreated cells.

Fig. 3.

Characterization of ATP13A2-knockdown cell lines. (A) Accumulation of lysosomes and/or ALs in M17 cells with stable knockdown of ATP13A2 (sh403-1) under basal conditions, as evidenced by LysoTracker staining and immunohistochemistry for cath-D and LAMP-2. (Scale bars, 10 μm.) (B and C) Representative immunoblot levels of LAMP-2, LAMP-1, and cath-D in sh403-1 and control (Ctrl) cells (shScr-1). (D) In vitro assay of cath-D enzyme activity in lysosomal fractions of sh403-1 and shScr-1 cells. (E) Cath-B (CTSB) immunoblot levels in sh403-1 and shScr-1 cells. (F) Lysosomal pH values in sh403-1 and shScr-1 cell lines. (G and H) Degradation of long-lived proteins in shScr-1 and sh403-1 cells and in lentiviral (LV)-transduced sh403-1 cells. (H) Lysosomal degradation (i.e., sensitive to NH₄Cl/leupeptin) in shScr-1 and sh403-1 cells and LV-transduced sh403-1 cells. (I) LC3 immunoblot levels in M17 cells treated or not treated with 5 nM bafilomycin A₁ for 1 h. (J) Immunoblot levels of p62, MEF2D, GAPDH, and α-synuclein in control and ATP13A2-knockdown cells. In all panels, n = 3–5 per experimental group. *P < 0.05 compared with shScr-1.

Fig. 4.

Overexpression of WT ATP13A2 in ATP13A2-deficient cells rescues lysosomal deficiency. (A) Overexpression of ATP13A2 assessed by Western blot in stable cell lines. (B) Colocalization of LysoTracker staining and GFP immunofluorescence in sh403-1 cells transfected with ATP13A2-GFP. (Scale bars, 10 μm.) (C and D) Cath-D activity in lysosomal fractions from sh403-1 cells and mutant ATP13A2 fibroblasts overexpressing or not overexpressing WT ATP13A2. Effects of ATP13A2 overexpression on lysosomal stress susceptibility to lysosomal inhibitor NH₄Cl (40 mM for 24 h) in sh403-1 cells and mutant ATP13A2 fibroblasts (L3292), as evidenced by the presence of acid phosphatase (E and G) and β-hexosaminidase (F and H) activities in the cytosol of NH₄Cl-treated cells after removing lysosomes by differential centrifugation. (I) Cell death in sh403-1 cells overexpressing or not overexpressing ATP13A2. In all panels, n = 3–5 per experimental group. *P < 0.05 compared with control (Ctrl) untreated (UT) cells; ^#P < 0.05 compared with control treated cells.