Transcriptome and Proteome Analysis in LUHMES Cells Overexpressing Alpha-Synuclein

Abstract

LUHMES cells share many characteristics with human dopaminergic neurons in the substantia nigra, the cells, the demise of which is responsible for the motor symptoms in Parkinson's disease (PD). LUHMES cells can, therefore, be used bona fide as a model to study pathophysiological processes involved in PD. Previously, we showed that LUHMES cells degenerate after 6 days upon overexpression of wild-type alpha-synuclein. In the present study, we performed a transcriptome and proteome expression analysis in alpha-synuclein-overexpressing cells and GFP-expressing control cells in order to identify genes and proteins that are differentially regulated upon overexpression of alpha-synuclein. The analysis was performed 4 days after the initiation of alpha-synuclein or GFP overexpression, before the cells died, in order to identify processes that preceded cell death. After adjustments for multiple testing, we found 765 genes being differentially regulated (439 upregulated, 326 downregulated) and 122 proteins being differentially expressed (75 upregulated, 47 downregulated). In total, 21 genes and corresponding proteins were significantly differentially regulated in the same direction in both datasets, of these 13 were upregulated and 8 were downregulated. In total, 13 genes and 9 proteins were differentially regulated in our cell model, which had been previously associated with PD in recent genome-wide association studies (GWAS). In the gene ontology (GO) analysis of all upregulated genes, the top terms were "regulation of cell death," "positive regulation of programmed cell death," and "regulation of apoptotic signaling pathway," showing a regulation of cell death-associated genes and proteins already 2 days before the cells started to die. In the GO analysis of the regulated proteins, among the strongest enriched GO terms were "vesicle," "synapse," and "lysosome." In total, 33 differentially regulated proteins were associated with synapses, and 12 differentially regulated proteins were associated with the "lysosome", suggesting that these intracellular mechanisms, which had been previously associated with PD, also play an important role in our cell model.

Keywords: Parkinson's disease; alpha-synuclein; lysosome; proteome; synapse; transcriptome; vesicular transport.

Figure 1

Description of study design and samples. (A) Schematic of the experimental design. Cells were transduced 2 days into the differentiation process and collected 4 days after transduction. AV, adenoviral vectors. The upper timeline indicates days post transduction; the lower timeline indicates days into the differentiation process. (B) In total, three samples of αSyn-overexpressing cells and GFP-expressing cells were collected for the transcriptome analysis, and nine samples of αSyn-overexpressing cells and GFP-expressing cells were collected for the proteome analysis. (C) Overlap between detected genes in the transcriptome analysis and corresponding proteins in the proteome analysis. N = 15,516 transcripts were detected and 3,537 proteins. The yellow circle shows items exclusively detected in the transcriptome (N = 12,157). The green circle indicates items detected in both, the transcriptome and the proteome (N = 3,358). The blue circle indicates items exclusively detected in the proteome (N = 179). (D) Depiction of differentially regulated transcripts (an upper panel) and proteins (a lower panel) with an adjusted p-value < 0.05 (Benjamini-Hochberg false-discovery rate). Red indicates downregulation. Green indicates upregulation.

Figure 2

Volcano plots of differentially regulated genes and proteins. (A) The volcano plot of transcripts of genes that were differentially regulated between αSyn-overexpressing and GFP-expressing LUHMES cells. Blue dots indicate all transcripts that were above the threshold for statistical significance after correction for multiple testings (adjusted p-value < 0.05). Orange dots indicate those of the statistically significant regulated transcripts were regulated more than 2-fold. Of these, 27 were downregulated (orange dots on the left), and 130 were upregulated (orange dots on the right). (B) The volcano plot of proteins that were differentially regulated between αSyn-overexpressing and GFP-expressing LUHMES cells. Blue dots indicate all proteins that were above the threshold for statistical significance after correction for multiple testings (adjusted p-value < 0.05). Orange dots indicate those of the statistically significant regulated proteins that were regulated more than 2-fold. Of these, three were downregulated (orange dots on the left), and eight were upregulated (orange dots on the right).

Figure 3

Western blots confirmation. Western blots with antibodies against the 21 proteins that were differentially regulated in the proteomics, of which the corresponding genes were also differentially regulated. For the Western blot investigation, samples from untransduced cells (untr), αSyn-overexpressing cells (αSyn), and GFP-expressing cells (GFP) cells were used. The proteins are presented in the same order as in Table 3. (A) SNCA, synuclein alpha; (B) SCG2, Secretogranin II; (C) MTHFD2, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate; (D) SHMT2, serine hydroxymethyltransferase 2; (E) WARS1, tryptophanyl-tRNA synthetase 1; (F) PSAT1, phosphoserine aminotransferase 1; (G) TFRC, transferrin receptor; (H) SERPINB6, serpin Family B, Member 6; (I) ASNS, asparagine synthetase (glutamine-hydrolyzing); (J) ARHGEF2, Rho/Rac guanine nucleotide exchange Factor 2; (K) SLC7A1, solute carrier Family 7, Member 1; (L) EDF1, endothelial differentiation-related Factor 1; (M) SCARB2, scavenger receptor Class B, Member 2; (N) CNTN2, contactin 2; (O) MDGA1, MAM domain containing glycosylphosphatidylinositol Anchor 1; (P) NCALD, neurocalcin delta; (Q) ADD1, Adducin 1; (R) STXBP1, syntaxin-binding Protein 1; (S) ALDH6A1, aldehyde dehydrogenase 6 Family, Member A1; (T): LHX9, LIM Homeobox 9; (U) DLG4, discs large MAGUK scaffold Protein 4. Twenty out of 21 proteins (A–S,U) were differentially regulated between αSyn-overexpressing cells and GFP-expressing cells in the same direction as in the LC-MS investigation and thereby confirming these findings. With the exception of TFRC (G), ADD1 (Q), ALDH6A1 (S), and DLG4 (U), there was no difference in protein levels between untransduced cells and GFP-expressing cells, suggesting an αSyn-specific effect of the protein regulation. One protein (LHX9, T) could not be confirmed. *p < 0.05, **p < 0.01, ***p < 0.001 vs. untransduced cells. ^§p < 0.05, ^§§p < 0.01, ^§§§p < 0.001 vs. GFP transduced cells.

Figure 4

Overlap to existing datasets. (A) Overlaps between N = 357 genes that were associated with risk loci in two recent Parkinson's disease genome-wide associated studies (GWAS) (an orange circle) and transcripts (a yellow circle) and protein (a blue circle) that were differentially regulated in our cell model. Of the 357 genes associated with PD, N = 11 were differentially regulated in our cell model (a brown area). Furthermore, N = 7 proteins encoded by these genes were differentially regulated in our cell model (a pink area). In addition to SNCA (alpha-synuclein) that was overexpressed in our cells model, also SCARB2 (scavenger receptor Class B, Member 2) was regulated on the transcriptomic and proteomic level (a red area). (B) Overlap between N = 434 genes present in the human lysosome gene database (an orange circle) and differentially regulated genes (a yellow circle) or proteins (a blue circle) in our cell model. N = 15 of the 434 lysosomal genes were differentially regulated in our cell model (a brown area). N = 11 proteins that were present in the human lysosome gene database were differentially regulated in our cell model (a pink area). Red characters indicate genes/proteins (SCARB2, scavenger receptor class B, member 2; CTSB, cathepsin B) that were found in the recent GWAS meta-analyses. (C) Overlap between N = 1,233 synaptic genes (an orange circle) and genes (a yellow circle) or proteins (a blue circle) that were regulated in our cell model. N = 61 synaptic genes were differentially regulated in our cell model (a brown area). Furthermore, N = 25 of the proteins that were differentially regulated in our cell model were encoded by synaptic genes (a pink area). Red characters mark genes/proteins that have been found in the recent GWAS meta-analyses (FYN, FYN proto-oncogene, Src family tyrosine kinase; STX1B, syntaxin 1B). In addition to SNCA, 7 more genes/proteins were regulated on the transcriptomic and proteomic level in our cell model (a red area). Diff. reg., differentially regulated (adjusted p-value < 0.05, Benjanimi-Hochberg false discovery rate).

Figure 5

STRING network analysis. (A) Interaction network of all differentially regulated proteins with a high confidence level (0.7). (B) Protein associated with PD without interactors. (C) Proteins associated with the lysosome. (D) Proteins associated with synapses. (E) Proteins with one interactor. (F) Proteins without interactors. Diamonds show proteins only differentially regulated in the proteome. Hexagons indicate proteins with corresponding differentially regulated transcripts. Upregulated proteins are shown in green; downregulated proteins are shown in purple. Red borders indicate proteins with association with the lysosome. Blue borders indicate proteins with association with synapses. A turquois border indicates association with synapses and the lysosome. A yellow label indicates genes that were previously associated with PD in the recent GWAS meta-analyses.