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The Ubiquitin Proteasome System Is a Key Regulator of Pluripotent Stem Cell Survival and Motor Neuron Differentiation - PubMed

  • ️Tue Jan 01 2019

The Ubiquitin Proteasome System Is a Key Regulator of Pluripotent Stem Cell Survival and Motor Neuron Differentiation

Monique Bax et al. Cells. 2019.

Abstract

The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons. The UPS regulator, ubiquitin-like modifier activating enzyme 1 (UBA1), was increased 36-fold in the ubiquitome of motor neurons compared to pluripotent stem cells. Thus, we further investigated the functional consequences of inhibiting the UPS and UBA1 on motor neurons. The proteasome inhibitor MG132, or the UBA1-specific inhibitor PYR41, significantly decreased the viability of motor neurons. Consistent with a role of the UPS in maintaining the cytoskeleton and regulating motor neuron differentiation, UBA1 inhibition also reduced neurite length. Pluripotent stem cells were extremely sensitive to MG132, showing toxicity at nanomolar concentrations. The motor neurons were more resilient to MG132 than pluripotent stem cells but demonstrated higher sensitivity than fibroblasts. Together, this data highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation.

Keywords: UBA1; amyotrophic lateral sclerosis; induced pluripotent stem cell; motor neuron; motor neurone disease; ubiquitin; ubiquitinomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Generation of patient induced pluripotent stem cell (iPSC)-derived motor neurons from fibroblasts. (A) Schematic timeline of iPSC reprogramming and motor neuron differentiation. Cells were characterised by immunocytochemistry and ubiquitomics at the pluripotent stem cell stage and motor neuron stage. (B) Representative brightfield images of fibroblasts at day 0 of reprogramming, iPSCs on day 2 after passaging, and motor neurons on day 76 of differentiation. Scale bar represents 100 µm. (C) Representative epifluorescent images of OCT4 expression (immunofluorescence) in an iPSC colony grown on Matrigel-coated tissue culture plates in TeSR E8, 2 days following passage, with DAPI staining (nucleus). Scale bar represents 100 µm. (D) Representative iPSC karyogram confirms the absence of introduced chromosomal abnormalities. (E) Representative epifluorescent images of TRA-1-60 expression (immunofluorescence) in an iPSC colony grown on Matrigel-coated tissue culture plates in TeSR E8, 2 days following passage. Scale bar represents 50 µm. (F) PluriTest results following comparison of the reprogrammed stem cells to previous characterised cells. (G) Representative scanning confocal micrographs of iPSC-derived motor neurons stained showing expression of neuronal marker neurofilament heavy (SMI32) by immunofluorescence, with RedDot2 staining (nucleus). Motor neurons were cultured for 76 days on laminin, collagen I and fibronectin-coated plates. Scale bar represents 100 µm. (H) Representative scanning confocal micrographs of iPSC-derived motor neurons stained showing expression of neuronal marker Islet 1 by immunofluorescence, with RedDot2 staining (nucleus), and image quantification via Image J analysis in six cell lines. NS = no significant difference by one-way ANOVA. Scale bar represents 50 µm. (I) Representative scanning confocal micrographs of iPSC-derived motor neurons stained showing expression of neuronal marker HB9 by immunofluorescence, with RedDot2 staining (nucleus), and image quantification via Image J analysis in six cell lines. Scale bar represents 25 µm.

Figure 2
Figure 2

Defining the ubiquitome in iPSCs and iPSC-derived motor neurons. (A) Schematic of sample collection from iPSC and iPSC-derived motor neurons (n = 4), and processing for analysis. (B) Histograms showing distribution of peptide intensities in individual replicates and Venn diagrams showing number of proteins identified in the ubiquitome in individual replicates of iPSC. (C) Histograms showing distribution of peptide intensities in individual replicates and Venn diagrams showing number of proteins identified in the ubiquitome in individual replicates of motor neurons. (D) Venn diagram showing number of individual proteins identified in the ubiquitome of iPSC and iPSC-derived motor neurons. (E) Hierarchical clustering showing relationship of individual replicates of iPSC and motor neuron ubiquitomes. (F) Abundance of individual Ub pathway components in the ubiquitome of iPSC and iPSC-derived motor neurons. Data shown are mean ± SEM; n = 4. (G) Multiple regression analysis of protein abundances (LFQ) in individual replicates, showing Pearson’s correlation values. (H) Abundance (LFQ) of histone variants in the ubiquitome of iPSC and motor neurons. (I) Heat map showing functional enrichment (using KEGG pathways) in the ubiquitome of iPSC and motor neurons (shading relative to number of individual proteins represented in each pathway). (J) Abundance (LFQ) of proteasome subunits in the ubiquitomes of iPSCs and motor neurons.

Figure 3
Figure 3

Ubiquitome changes following iPSC differentiation to motor neurons. (A) Volcano plot (Log10 abundance vs. −log10 p-value) to determine significantly enriched proteins in the ubiquitome of iPSC and iPSC-derived motor neurons. Proteins with adjusted p < 0.01 and fold change >2 are shown in red (B) Protein–protein interaction map of differentially enriched proteins in the ubiquitome of iPSCs (blue) and motor neurons (red) as determined by STRING analysis (confidence score > 0.700). (C) Functional enrichment of differentially expressed proteins within the ubiquitome of iPSCs and motor neurons, showing the number of individual proteins present in each KEGG pathway in each cell state (data from n = 4).

Figure 4
Figure 4

Differential sensitivity of various cell states to UPS inhibition. (A) Viability of iPSCs, ESCs, motor neurons and fibroblasts following 16 h treatment with increasing doses of the proteasome inhibitor MG132. (B) Neurite outgrowth of motor neuron precursors following 24 h treatment with 1 µM–10 µM UBA1 inhibitor (PYR41). (C) Viability of motor neurons subjected to long-term (4 week) treatment with 1 µM PYR41 UBA1 inhibitor (red) or vehicle control (black). Data shown are mean ± SEM, n = 3–4, * p < 0.05 (one-way ANOVA Brown-Forsythe test (B), or two-tailed paired t-test (C)).

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