LIMP-2 expression is critical for β-glucocerebrosidase activity and α-synuclein clearance

Abstract

Mutations within the lysosomal enzyme β-glucocerebrosidase (GC) result in Gaucher disease and represent a major risk factor for developing Parkinson disease (PD). Loss of GC activity leads to accumulation of its substrate glucosylceramide and α-synuclein. Since lysosomal activity of GC is tightly linked to expression of its trafficking receptor, the lysosomal integral membrane protein type-2 (LIMP-2), we studied α-synuclein metabolism in LIMP-2-deficient mice. These mice showed an α-synuclein dosage-dependent phenotype, including severe neurological impairments and premature death. In LIMP-2-deficient brains a significant reduction in GC activity led to lipid storage, disturbed autophagic/lysosomal function, and α-synuclein accumulation mediating neurotoxicity of dopaminergic (DA) neurons, apoptotic cell death, and inflammation. Heterologous expression of LIMP-2 accelerated clearance of overexpressed α-synuclein, possibly through increasing lysosomal GC activity. In surviving DA neurons of human PD midbrain, LIMP-2 levels were increased, probably to compensate for lysosomal GC deficiency. Therefore, we suggest that manipulating LIMP-2 expression to increase lysosomal GC activity is a promising strategy for the treatment of synucleinopathies.

Keywords: AMRF; C57/BL6-J; GD; PME; SCARB2.

Fig. 1.

Accumulation of α-syn, apoptosis, and gliosis in LIMP-2^−/− brain. Immunoblot of α-syn (A) and quantitative RT-PCR of α-syn and LIMP-2 (B) from brain samples of mice with different genetic backgrounds. (C) Immunoblots of α-syn in wild-type (WT) and LIMP-2^−/− soluble (T-Sol) and insoluble (SDS) fractions of midbrain (arrowheads indicate α-syn species, and the asterisk indicates a nonspecific band; GAPDH was used to control loading). (D) Native SEC/immunoblot analysis of T-Sol lysates (radius in angstroms). GAPDH was used as a loading control. (E) Immunofluorescent staining of α-syn (green) colabeled with NeuN (red) and TH (red) in the pons and midbrain of 10-month-old littermates (arrowheads highlight accumulated α-syn). S. Nigra, substantia nigra. (F) Quantification of the soma area of DAB-stained TH-positive neurons within the S. Nigra (WT, n = 3; LIMP-2^−/−, n = 4). Quantification and representative DAB staining from 10-month-old mice show TUNEL-positive apoptotic cells (G; arrows), astrogliosis (H; GFAP), and microgliosis (I; CD68) in LIMP-2^−/− mice but not in WT mice (also Fig. S1). m, months of age. ^#P > 0.05; *P < 0.05; ***P < 0.001.

Fig. 2.

Reduced GC activity accompanied by lipid accumulation and lysosomal dysfunction. Reduced GC protein levels and enzyme activity in LIMP-2^−/− midbrain, pons, and whole-brain lysates assessed by immunoblot analysis (A), activity assay (B), and Inhibody Red staining (C) [neuron-specific enolase (NSE) or Coomassie Brilliant Blue (CBB) was used to control loading]. (D) Ultrastructural analysis of the pons. Toluidine blue-stained sections of neurons (arrows highlight storage material) are shown. Electron micrographs show electron-dense rich deposits (arrows) and lipid droplets (red asterisks) within the soma of a LIMP-2^−/− neuron. Accumulation of Bodipy 493/503-stained lipid droplets (green, arrows) within the soma of MAP-2–labeled neurons (red) (E) that show increased levels of α-syn (green) in the absence of LIMP-2 (red) (F) is illustrated. (G) Immunological staining of LIMP-2 (green) and respective densitometric analysis in PD and control midbrain sections identified via their high neuromelanin content [black structures in differential interference contrast (DIC) image]. The circumference of a randomly chosen lysosome is highlighted in each sample, and the total intensity of the LIMP-2 signal per cell was quantified and divided by the area of the cell to compensate for variation in cell size (also Fig. S2). *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Heterologous overexpression of LIMP-2 is beneficial for α-syn clearance. (A) Immunoblotting and densitometry of HA-tagged α-syn (α-syn-HA) in N2a cells after overexpression of LIMP-2 or LIMP-2 ERret. (B) GC activity assay of N2a cells with GFP control (GFP cont.) or overexpressed LIMP-2. (C) Immunoblot and densitometry of α-syn and post-ER/ER GC levels in H4 cells after 48 h of LIMP-2 overexpression. (D) Immunoblot and densitometry of α-syn turnover in H4 cells after treatment with doxycycline to suppress further α-syn transcription. Enhanced clearance of α-syn is highlighted with the larger absolute value of the slope compared with a mock-transfected control (also Fig. S3). ^#P > 0.05; *P < 0.05.