Post-translational proteomics platform identifies neurite outgrowth impairments in Parkinson's disease GBA-N370S dopamine neurons

Abstract

Variants at the GBA locus, encoding glucocerebrosidase, are the strongest common genetic risk factor for Parkinson's disease (PD). To understand GBA-related disease mechanisms, we use a multi-part-enrichment proteomics and post-translational modification (PTM) workflow, identifying large numbers of dysregulated proteins and PTMs in heterozygous GBA-N370S PD patient induced pluripotent stem cell (iPSC) dopamine neurons. Alterations in glycosylation status show disturbances in the autophagy-lysosomal pathway, which concur with upstream perturbations in mammalian target of rapamycin (mTOR) activation in GBA-PD neurons. Several native and modified proteins encoded by PD-associated genes are dysregulated in GBA-PD neurons. Integrated pathway analysis reveals impaired neuritogenesis in GBA-PD neurons and identify tau as a key pathway mediator. Functional assays confirm neurite outgrowth deficits and identify impaired mitochondrial movement in GBA-PD neurons. Furthermore, pharmacological rescue of glucocerebrosidase activity in GBA-PD neurons improves the neurite outgrowth deficit. Overall, this study demonstrates the potential of PTMomics to elucidate neurodegeneration-associated pathways and potential drug targets in complex disease models.

Keywords: CP: Neuroscience; Parkinson’s; glucocerebrosidase; glycosylation; iPSC; lysosome; neuritogenesis; phosphoproteomics; post-translational modifications; proteomics; stem cells.

Graphical abstract

Figure 1. Proteomic analysis on GBA and control iPSC-dopamine neurons achieves clear patient stratification

(A) Immunofluorescence staining for tyrosine hydroxylase (TH; green), β-III-tubulin (TUJ1, yellow), and FOXA2 (red) on differentiation day 35 GBA patient and control neurons. Nuclei stained with DAPI (blue). Scale bar: 50 μm. (B) Glucocerebrosidase (GCase) enzyme activity, relative to average of controls, of the GBA patient and control iPSC-dopamine neurons included in the proteomic analysis (n = 4 patients with GBA and 4 controls; mean ± SEM). *p % 0.05 (Student’s t test). (C) Schematic representation of the preparation and enrichment workflow for the proteomic analysis of GBA patient and control iPSC-dopamine neuron cell lysates. (D) Overview of the identified non-modified proteins and phosphorylated/glycosylated/cysteine-modified peptides. Normalized peptides represent number of individual phosphorylated/glycosylated/cysteine-modified peptides normalized to levels of the corresponding non-modified proteins. (E) Venn diagram showing the overlap between the resulting differentially abundant non-modified proteins (GBA-PD/control abundance ratio > 1.2, p ≤ 0.05 [t test with Benjamini-Hochberg correction (false discovery rate [FDR] 0.1)]) and modified peptides (GBA-PD/control abundance ratio >1.3, coefficient of variation [CV]% ≤ 30) in the four groups. (F and G) Principal-component analysis (PCA) plot based on the protein expression data from iPSC-dopamine neurons derived from the four patients with GBA (GBA 2; GBA-PSP) and healthy controls (F) with GBA 2 and (G) without GBA 2 included in the analysis.

Figure 2. Assessment of glycosylated peptides reveals widespread changes in the lysosomal proteome in GBA-PD iPSC-dopamine neurons

(A and B) PCA plot based on glycosylated protein levels in iPSC-dopamine neurons derived from the four patients with GBA (GBA 2; GBA-PSP) and healthy controls (A) with GBA 2 and (B) without GBA 2 included in the analysis. (C) Heatmap of the abundance ratios (GBA-PD/control) of all glycosylated proteins identified by the proteomic analysis showing the three main functional clusters that segregate patients and controls. (D) Heatmap of the abundance ratios (GBA-PD/control) of non-modified protein and PTMs for all established lysosomal proteins identified by the proteomic analysis. Unidentified proteins/PTMs are marked with gray. An asterisk (*) indicates PTM abundance ratio >1.3 and CV% <30. None of the non-modified proteins were significantly regulated. (E) Visualization of enriched functional Gene Ontology (GO) term networks based on annotations of significantly regulated glycosylated proteins. The node size indicates the number of proteins connected to the GO term, and the color reflects functionally connected groups of terms. Only terms with an adjusted p value ≤ 0.05 shown (two-sided hypergeometric test with Bonferroni stepdown). (F) Proteomic data from proteins related to ceramide catabolism with graph showing the levels of glycosylated peptides normalized to control and table showing the abundance ratio (GBA-PD/control) for the non-modified and glycosylated form including the glycosylation site (n = 3 GBA patients and 4 controls; mean ± SEM).

Figure 3. Tau and mammalian target of rapamycin (mTOR) are predicted as key regulators of the proteomic changes detected in GBA-PD iPSC-dopamine neurons

(A) Volcano plot showing the fold change and significance level (−log10 [p value]) of all non-modified proteins with the dashed line corresponding to a p = 0.05. The labeled points represent the ten most highly up-/downregulated proteins in patients with GBA-PD relative to control. (B) STRING network of connected, significantly regulated non-modified proteins with the circle color displaying protein abundance ratios (GBA-PD/control) and significantly regulated phospho-sites marked by surrounding halo, displaying the phosphorylation abundance ratio (GBA-PD/control). (C) Heatmap of PD-GWAS proteins with altered protein/PTM levels displaying the abundance ratio (GBA-PD/control). Unidentified proteins/PTMs are marked with gray. An asterisk (*) indicates non-modified protein abundance ratio >1.2 (p < 0.05) or PTM abundance ratio >1.3 (CV% < 30). (D) Pathway analysis identifying the predicted upstream regulators most highly enriched across all datasets. Data are presented as a heatmap of –log(p value), where increasing color intensity corresponds to increasing significance in enrichment, as indicated. (E–J) Western blotting (E) and quantification of (F) microtubule-associated protein tau (Tau), (G) mTOR, (H) phospho-mTOR, (I) phospho-mTOR/mTOR ratio, and (J) the lipidated form of microtubule-associated protein light chain 3 (LC3II) levels in GBA-PD patient and control neurons from a representative independent differentiation (n = 3–5 iPSC lines). GBA 2; GBA-PSP shown on blots and excluded from quantification. Protein expression levels were normalized to β-actin and are shown relative to control. Mean ± SEM, *p % 0.05 (Student’s t test).

Figure 4. Proteomics and PTMomics indicate cytoskeletal organization and axon extension defects in GBA-PD iPSC-dopamine neurons

Visualization of functional GO term networks based on annotations of significantly regulated (A and B) non-modified, (C and E) phosphorylated, and (D and F) cysteine-modified proteins. (A, C, and D) The node size indicates the number of proteins connected to the GO term, and the color reflects functionally connected groups of terms. (B, E, and F) Subnetworks related to axogenesis and cytoskeletal organization with the node size reflecting the enrichment significance of the terms and the leading group term being that of the highest significance. Nodes with similarity in the associated proteins are connected by lines with arrows indicating positive regulation, cropped lines negative regulation, and diamonds undefined regulation. Only terms with adjusted p values ≤0.05 are shown (two-sided hypergeometric test with Bonferroni stepdown).

Figure 5. Functional assay confirms defects in neurite outgrowth predicted by pathway analysis

(A) Pathway analysis identified the diseases and functions most highly enriched across all datasets. Comparison analysis was performed using Ingenuity Pathway Analysis on the four datasets (non-modified, phosphorylated, glycosylated, and cysteine modified) from the proteomic characterization of GBA-PD iPSC-dopamine neurons. Data are presented as a heatmap of –log(p value), where increasing color intensity corresponds to increasing significance in enrichment. (B) Representative bright-field images of GBA-PD and control iPSC-dopamine neurons from neurite outgrowth assay day 0, 2, 4, and 6 post-scratch. Scratch areas are marked with blue dotted lines. Scale bar: 100 μm. Representative closeup of processed binary image used for quantification of neuronal processes in the scratch areas from 6 days post-scratch. (C) Quantification of neurite outgrowth as measured by the percentage of the scratch area covered by processes. Values from day 0 subtracted as background. Data are from 3 independent differentiations (n = 4–5 iPSC lines per group). Mean ± SEM, **p ≤ 0.01 (paired Student’s t test). (D–F) Analysis of mitochondrial motility and morphology showing (D) representative images of mitochondrial movement visualized with MitoTracker deep red (Red) at 12 time points from T = 0–484. Nuclei stained with NucBlue LiveReady stain (blue). Arrows indicate individual tracked mitochondria. Scale bar: 25 μm. Quantification of (E) mitochondrial movement speed (nm/s) and (F) mitochondrial width-to-length ratio (n = 3 iPSC lines per group). Mean ± SEM, **p ≤ 0.01 (Student’s t test). (G) Western blotting confirmed lentiviral-mediated shRNA knockdown (KD) of tau in GBA-PD iPSC-dopamine neurons (GBA 3) and no effect of scrambled shRNA. Expression levels were normalized to β-actin and are shown relative to untreated GBA-PD iPSC-dopamine neurons (n = 3 technical replicates). Mean ± SEM, *p ≤ 0.05 (Student’s t test). (H) Representative bright-field images of one line of GBA-PD iPSC-dopamine neurons (GBA 3) with lentiviral-mediated shRNA tau KD and untreated from neurite outgrowth assay day 0 and 6. Scale bar: 100 μm. (I–J) Quantification of neurite outgrowth as measured by the percentage of the scratch area covered by processes over time. Data from (I) GBA-PD (GBA 3) and (J) control (Con 3) iPSC-dopamine neurons with shRNA tau KD or a scrambled shRNA control. Values from day 0 subtracted as background (n = 8–12 wells per group). Mean ± SEM, *p ≤ 0.05, **p ≤ 0.01 (one-way ANOVA, Dunnett’s multiple comparisons).

Figure 6. GCase chaperoning rescues neurite outgrowth defects in GBA-PD iPSC-dopamine neurons

(A) Representative graph of GCase activity in GBA-PD iPSC-dopamine neurons treated for 48 h, in response to increasing concentrations of the GCase activator NCGC758 (758), relative to healthy control neurons (n = 3–26 wells per condition). Mean ± SEM (non-linear regression). (B) Quantification of scratch repair as measured by the percentage of the scratch area covered by processes on day 6. Values from day 0 subtracted as background (n = 2–3 iPSC lines per group). Mean ± SEM, *p ≤ 0.05 (one-way ANOVA, Tukey’s multiple comparisons). (C) Representative bright-field images of control and GBA-PD iPSC-dopamine neurons treated with 5 or 15 μM 758 for 7 days. Scale bar: 100 μm. (D) Quantification of scratch repair with increasing doses of 758. Data are from one control and one line of GBA-PD iPSC-dopamine neurons (n = 3 independent differentiations). Mean ± SEM, *p ≤ 0.05, **p ≤ 0.01 (one-way ANOVA, Tukey’s multiple comparisons).