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. 2020 May;35(5):760-773.
doi: 10.1002/mds.27994. Epub 2020 Feb 8.

Lysosome and Inflammatory Defects in GBA1-Mutant Astrocytes Are Normalized by LRRK2 Inhibition

Anwesha Sanyal  1 , Mark P DeAndrade  1 , Hailey S Novis  1 , Steven Lin  1 , Jianjun Chang  2 , Nathalie Lengacher  3 , Julianna J Tomlinson  3 , Malú G Tansey  4 , Matthew J LaVoie  1
Affiliations

Lysosome and Inflammatory Defects in GBA1-Mutant Astrocytes Are Normalized by LRRK2 Inhibition

Anwesha Sanyal et al. Mov Disord. 2020 May.

Abstract

Background: Autosomal recessive mutations in the glucocerebrosidase gene, Beta-glucocerebrosidase 1 (GBA1), cause the lysosomal storage disorder Gaucher's disease. Heterozygous carriers of most GBA1 mutations have dramatically increased Parkinson's disease (PD) risk, but the mechanisms and cells affected remain unknown. Glucocerebrosidase expression is relatively enriched in astrocytes, yet the impact of its mutation in these cells has not yet been addressed.

Objectives: Emerging data supporting non-cell-autonomous mechanisms driving PD pathogenesis inspired the first characterization of GBA1-mutant astrocytes. In addition, we asked whether LRRK2, likewise linked to PD and enriched in astrocytes, intersected with GBA1 phenotypes.

Methods: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels.

Results: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes.

Conclusions: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention. © 2020 International Parkinson and Movement Disorder Society.

Keywords: GBA1; LRRK2; Parkinson's disease; astrocyte dysfunction; neuroinflammation.

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Conflict of interest statement

Relevant conflicts of interests/financial disclosures: Nothing to report.

Figures

FIG. 1.
FIG. 1.
GBA1 D409V knockin mutation causes morphologic defects in the lysosomes of primary murine astrocytes. (A) Flow cytometry analysis (left panel) of primary mouse astrocyte cultures stained with GFAP detected in Q3 and ~2% microglial contamination detected in Q4. These cultures, analyzed by immunofluorescence assay (right panel) detecting astrocytes stained with GFAP (red), microglia stained with Cd11B (green) and neurons stained with NeuN (yellow) reveal similar levels of microglial contamination. Nucleus is stained with DAPI (blue). (B) Immunologic responsiveness of WT astrocytes tested by LPS induction (100 ng/mL or 50 EU/mL; 6 and 24 hours) display a classic TLR4-dependent upregulation of IL6, IL1β, TNF, and iNOS mRNA (N = 3). (C) GCase activity assay using GCase-specific fluorogenic substrate show 47% activity in heterozygous (GBA HET) and 8% activity in homozygous (GBA KI) knockin astrocytes. CBE treatment almost completely inhibits GCase activity, confirming specificity of the assay (N = 3). High-content image analysis, as detected by Lysotracker staining normalized to number of cells, show (D) a significant decrease in the lysosomal numbers in heterozygous (GBA HET) and homozygous (GBA KI) knockin astrocytes and (E) unchanged average lysosomal area in these cells. Yellow fluorescence from Lysotracker staining is observed in representative microscopy images. All lysosomal analyses were collated from 3 independent experiments with 20 wells per genotype per experiment (N = 3, F = 49, P < 0.0001). (F) Protein expression of lysosomal genes, LAMP1, GCase, and nTFEB was not affected by GBA1 mutation, as detected by Western blot. Actin was used as a loading control (G). The immunofluorescence of LAMP1 (red) and LAMP2 (green) shows reduced colocalization of LAMP1 at lysosomes detected by LAMP2 in heterozygous (Spearman’s rank correlation of 0.49; GBA HET) and homozygous (Spearman’s rank correlation of 0.45; GBA KI) knockin astrocytes when compared with WT (Spearman’s rank correlation of 0.56). Nuclei are detected by DAPI (blue) stain. *P < 0.05, analysis of variance followed by Tukey’s post hoc test. Cd11B, integrin alpha M subunit.
FIG. 2.
FIG. 2.
GBA1 D409V knockin mutation reduces lysosomal protease activity. (A) Determination of lysosomal pH using LysoSensor displays increased alkalinization to pH 6 in heterozygous (GBA HET) and homozygous (GBA KI) knockin astrocytes. Data are collated from 3 independent experiments with 20 wells per genotype (N = 3, F = 5.017, P = 0.0089). (B) General lysosomal protease activity, as detected by DQ-BSA cleavage (green), shows a significant reduction in homozygous (GBA HET), but not heterozygous (GBA KI), knockin astrocytes. Green fluorescence as a result of cleavage of DQ-BSA is observed in representative microscopy images (N = 3, F = 55.41, P < 0.0001). (C) Cathepsin D activity, as detected by cathepsin D activity assay and Western blot of cathepsin D protein band and (D) cathepsin L activity, as detected by cleavage of Magic-Red substrate (red), exhibit no change as a result of a GBA1 mutation (N = 3, F = 3,783, P = 0.0309). (E) Cathepsin B activity, as detected by cleavage of Magic-Red substrate (red), show a significant dose-dependent reduction in heterozygous (GBA HET) and homozygous (GBA KI) knockin astrocytes. Red fluorescence from cathepsin B/L-specific substrate cleavage is observed in representative microscopy images (N = 3, F = 161.3, P < 0.0001). In all fluorogenic plate-based activity assays, the nuclei were detected by Hoechst 33258 (blue) for normalization of the fluorescent signal. Data were collated from 3 independent experiments with 5 to 10 wells per genotype per experiment (N = 3). *P < 0.05, analysis of variance followed by Tukey’s post hoc test.
FIG. 3.
FIG. 3.
LRRK2 kinase inhibition rescues lysosomal defects caused by the GBA1 D409V mutation. (A) 3 days treatment with the LRRK2 kinase inhibitor, MLi-2 (15 nM), normalizes GBA1-mutant lysosomes to pH ~5.5 while not affecting WT lysosomal pH (N = 3, F = 8.731, P < 0.0001). (B) Lysosomal number (N = 3, F = 220.6, P < 0.0001) and (C) average lysosomal area in WT and GBA1-mutant astrocytes remain unaffected by MLi-2 treatment. (D) 7 days treatment with MLi-2 (15 nM) rescues cathepsin B activity, as detected by enzyme-specific Magic-Red dye cleavage in heterozygous (GBA HET), but not homozygous (GBA KI), knockin astrocytes (N = 3, F = 31.53, P < 0.0001). All lysosomal analyses were collated from 3 independent experiments with 10 wells per genotype per experiment (N = 3). Two-way analysis of variance followed by Tukey’s post hoc test.
FIG. 4.
FIG. 4.
GBA1 D490V knockin astrocytes show dysregulated inflammatory responses. Quantitative polymerase chain reaction of (A) basal and (B) LPS-induced (100 ng/mL or 50 EU/mL, 6 and 24 hours) proinflammatory cytokines IL6, IL1β, TNF, and Interleukin 12 and chemokines iNOS, CXCL1, LCN2 show significant reductions of mRNA levels in GBA1-mutant astrocytes when compared with WT astrocytes. Data are normalized to actin expression and collated from 3 independent experiments including 3 biological replicates per experiment (N = 3). *P < 0.05, analysis of variance followed by Tukey’s post hoc test.
FIG. 5.
FIG. 5.
MLi-2 normalizes the deficits in inflammatory response caused by GBA1 D409V knockin mutation. (A) Quantitative polymerase chain reaction of MLi-2 treated (15 nM, 3 days) and LPS-induced (100 ng/mL or 50 EU/mL, 6 hours) proinflammatory cytokines IL6, IL1β, TNF, and IL12p70 and chemokines iNOS, CXCL1, and LCN2 in GBA1-mutant astrocytes display normalization of mRNA expression to WT levels when compared with the DMSO-treated control. Data are normalized to actin expression and collated from 3 independent experiments including 3 biological replicates per experiment (N = 3). *P < 0.05, analysis of variance followed by Tukey’s post hoc test. (B) Meso Scale Discovery (Rockville, MD) immunoassay of MLi-2 treated (15 nM, 3 days) basal levels of proinflammatory cytokines IL6, IL10, and TNF and chemokine CXCL1 secreted in the conditioned media show robust normalization beyond WT levels (n = 3). *P < 0.05, analysis of variance followed by Tukey’s post hoc test.
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