Direct and indirect regulation of β-glucocerebrosidase by the transcription factors USF2 and ONECUT2

Abstract

Mutations in GBA1 encoding the lysosomal enzyme β-glucocerebrosidase (GCase) are among the most prevalent genetic susceptibility factors for Parkinson's disease (PD), with 10-30% of carriers developing the disease. To identify genetic modifiers contributing to the incomplete penetrance, we examined the effect of 1634 human transcription factors (TFs) on GCase activity in lysates of an engineered human glioblastoma line homozygous for the pathogenic GBA1 L444P variant. Using an arrayed CRISPR activation library, we uncovered 11 TFs as regulators of GCase activity. Among these, activation of MITF and TFEC increased lysosomal GCase activity in live cells, while activation of ONECUT2 and USF2 decreased it. While MITF, TFEC, and USF2 affected GBA1 transcription, ONECUT2 might control GCase trafficking. The effects of MITF, TFEC, and USF2 on lysosomal GCase activity were reproducible in iPSC-derived neurons from PD patients. Our study provides a systematic approach to identifying modulators of GCase activity and deepens our understanding of the mechanisms regulating GCase.

Fig. 1. A forward-genetics screen for GCase activity modulators.

a Study workflow. b Read frequencies (next-generation sequencing) of the desired T > C point mutation in the base-edited LN-229^L444P (orange) and its parental LN-229^WT (blue) line. c GCase enzymatic activity in cell lysates of LN-229^WT (WT), LN-229^L444P (L444P), and GBA1-ablated (KO) LN-229 cells. 30 wells/genotype, two independent experiments. AT3375: GCase-selective inhibitor. d. Immunoblot of cellular GCase protein in LN-229^ΔGBA1 (KO), LN-229^L444P (L444P), and LN-229^WT (WT) after treatment with the proteasome inhibitor MG-132 (+) or with DMSO vehicle (−). N = 4 experiments, unpaired two-samples t-test. e. Lysate-based GCase assay in LN-229^WT (WT) and LN-229^L444P (L444P) cells after treatment with the proteasome inhibitor MG-132 or with DMSO vehicle. N = 18 wells/condition, unpaired two-samples t-test fluorescence intensity was normalized to average cell number/well. DMSO-treated WT cells were taken as reference (100%).

Fig. 2. Arrayed CRISPR activation screen of human TFs in LN-229^L444P cells.

a Overview of the screening workflow. b Z’ factor of each assay plate reporting on the separation between cells exposed to qgRNA lentiviruses targeting GBA1 (positive controls) vs. nontargeting (NT) qgRNA (negative controls). Plates are ordered in ascending order of Z’ factor of the replicate plates, with plate numbers (order of pipetting) provided on the x-axis. Histograms of enzymatic activity (c) and viability (d) of LN-229^L444P cells treated with TF-activating (grey), GBA1-specific (red), and nontargeting qgRNAs (blue). GCase activity was normalized to wells infected with NT controls. e Changes in GCase activity (abscissa) and ps (ordinate) after activation of designated transcription factors. f Re-testing of 29 candidates with a higher number of replicates. Grey: NT controls and TFs previously found to regulate GCase expression (TFE3, TFEB, and MYC). Purple and blue: TFs increasing and decreasing GCase activity in the primary screen, respectively. Red: positive controls (GBA1-activated cells). N ≥ 10 transductions in ≥2 independent experiments; unpaired t-test. Asterisks: ps (here and henceforth). ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Fig. 3. GBA1 expression, GCase protein levels, and lysosomal hydrolase activities in LN-229^L444P cells following activation of TFs.

a GBA1 and GBAP1 genes approximately to scale. Boxes: exons; lines: introns. Purple: 55-bp sequence unique to GBA1 exon 9. Pink: GBA1 RT-PCR primers. b Fold changes GBA1 mRNA (RT-qPCR) after CRISPRa of TFs in LN-229^L444P cells. -actin (ActB) was used for normalization (N = 3–8 repeats). c GCase protein levels after CRISPRa of TFs in LN-229^L444P cells. Cells were harvested 5 days post-transduction. d GCase protein quantification after CRIPSRa of TFs in LN-229^L444P cells (N = 5–11 experiments). e Microscopy-based assessment of lysosomal hydrolase activities in live LN-229^L444P cells. f GCase activity assessed by LysoFQ-GBA1 in live LN-229^L444P cells after CRISPRa (N = 3–9 experiments). g α-N-acetylgalactosaminidase (NAGAL) activity in live LN-229^L444P cells after CRISPRa of TFs. DGJNAc: NAGAL inhibitor (3 experiments). h Cathepsin B activity assessed in live LN-229^L444P cells after CRISPRa of TFs. Bafilomycin A1: v-ATPase inhibitor (N = 3 experiments). i Hit selection process. Solid lines in C and D: medians, 25%, and 75% quartiles. Medians of NT controls: 100%. Unpaired t-test.

Fig. 4. Relative lysosomal GCase activity in iPSC-derived forebrain neurons.

Relative lysosomal GCase activity assessed with LysoFQ-GBA1 in live neurons derived from a healthy individual (WT neurons), from a Parkinson patient carrying a heterozygous GBA1 mutation (PD^N370S/WT) or a Gaucher patient with compound heterozygous GBA1 mutations (GD^L444P/P415R) after overexpression of candidate TFs at 7 (top row) or 14 days (bottom row) post-transduction. LN-229^WT (WT), LN-229^L444P (L444P), and GBA1-ablated (KO) LN-229 cells. 30 wells/genotype, two independent experiments. AT3375: GCase-selective inhibitor. Six replicate wells/candidate and 26 replicates for the empty vector; N = 1 experiment. Lines: medians and 25-75% quartiles. 100% Lysosomal GCase activity represents the relative activity seen in each of the three cell lines transduced with empty vector. Unpaired t-test.

Fig. 5. USF2 ablation increases GBA1 transcription and GCase activity/abundance.

a GCase activity in cell lysates of LN-229^L444P expressing Cas9 following ablations of TFs (N = 3 experiments). b GCase activity in live LN-229^L444P cells expressing Cas9 assessed with LysoFQ-GBA1 after USF2 or GBA1 ablation (N = 3). c GCase immunoblot after USF2 ablation in LN-229^L444P cells expressing Cas9. d GCase protein levels after ablation of USF2 in LN-229^L444P expressing Cas9 (N = 4). e Immunoblot demonstrating 75% decrease of USF2 in LN-229^L444P cells expressing Cas9 after ablation. f GCase activity in LN-229^WT cell lysates after ablating candidate TFs (N = 3). g Immunoblot for GCase after ablating USF2 in LN-229^WT expressing Cas9. h Selected top DEGs following transcriptomic profiling after ablation of USF1 and USF2 in LN-229^L444P cells. i Transcriptional profiling of select genes following ablation of USF1, USF2, or USF1/2 in LN-229^L444P cells. Horizontal axes: log₂ fold-change (log2FC) in gene expression. j Overrepresented gene-ontology terms. Bar length: −log₁₀ adjusted p. ((h–j): N = 3 replicates). Median values of NT controls: 100%. Solid lines: medians, 25%, and 75% quartiles.

Fig. 6. Transcriptional versus post-transcriptional mechanisms involved in GBA1 regulation.

a Transcriptional profiling of selected genes of interest following CRISPRa of candidate TFs in LN-229^L444P cells. Horizontal axes: log2 fold-change (log2FC) in gene expression. b Top gene ontology terms of overrepresentation analysis (biological processes). Bar length: -log₁₀ adjusted p. c Differentially expressed genes of GO term 0090383 (phagosome acidification) (N = 3 replicates per candidate TF).

Fig. 7. Identification of downstream mediators of ONECUT2’s effect on GCase activity.

a Transcriptomic profiling of selected DEGs following after CRISPRa of ONECUT2 in LN-229^L444P cells (N = 3 replicates). Enzymatic activity in cell lysates following activation of top 30 DEGs in LN-229^WT (b) or LN-229^L444P (c) (N = 7 to 10). d Lysosomal enzymatic activity assessed with LysoFQ-GBA1 following CRISPRa of PLEKHG4 or PLEKHG4B in LN-229^WT and LN-229^L444P cells (N = 3). Median values of the NT controls set as 100%. Solid lines: medians and quartiles. Unpaired t-test.