A Comparative Analysis of Erythropoietin and Carbamoylated Erythropoietin Proteome Profiles

Abstract

In recent years, erythropoietin (EPO) has emerged as a useful neuroprotective and neurotrophic molecule that produces antidepressant and cognitive-enhancing effects in psychiatric disorders. However, EPO robustly induces erythropoiesis and elevates red blood cell counts. Chronic administration is therefore likely to increase blood viscosity and produce adverse effects in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered modification of EPO, is non-erythropoietic but retains the neurotrophic and neurotrophic activity of EPO. Blood profile analysis after EPO and CEPO administration showed that CEPO has no effect on red blood cell or platelet counts. We conducted an unbiased, quantitative, mass spectrometry-based proteomics study to comparatively investigate EPO and CEPO-induced protein profiles in neuronal phenotype PC12 cells. Bioinformatics enrichment analysis of the protein expression profiles revealed the upregulation of protein functions related to memory formation such as synaptic plasticity, long term potentiation (LTP), neurotransmitter transport, synaptic vesicle priming, and dendritic spine development. The regulated proteins, with roles in LTP and synaptic plasticity, include calcium/calmodulin-dependent protein kinase type 1 (Camk1), Synaptosomal-Associated Protein, 25 kDa (SNAP-25), Sectretogranin-1 (Chgb), Cortactin (Cttn), Elongation initiation factor 3a (Eif3a) and 60S acidic ribosomal protein P2 (Rplp2). We examined the expression of a subset of regulated proteins, Cortactin, Grb2 and Pleiotrophin, by immunofluorescence analysis in the rat brain. Grb2 was increased in the dentate gyrus by EPO and CEPO. Cortactin was induced by CEPO in the molecular layer, and pleiotrophin was increased in the vasculature by EPO. The results of our study shed light on potential mechanisms whereby EPO and CEPO produce cognitive-enhancing effects in clinical and preclinical studies.

Keywords: cognition; hippocampus; neurotrophic factors; protein regulation.

Figure 1

Exploratory analysis of the LFQ data for EPO- and CEPO-treated neuronal cell cultures. (A) Hierarchical clustering of all samples is based on Pearson correlation coefficients. Correlation values were color-coded from blue to red, corresponding to lower or higher values. (B) Principal component analysis (PCA) of the LFQ intensities obtained from the control, EPO-, and CEPO-treated samples.

Figure 2

Enrichment analysis of the LFQ data for EPO- and CEPO-treated neuronal cell cultures. (A) Heatmap of the significant proteins expressed in CEPO, EPO and Control samples. Hierarchical clustering was performed for tables, where rows represent one protein, and columns represent biological replicates. Significant proteins were calculated with multi-sample ANOVA tests with a permutation-based cutoff of 0.05 applied on the logarithmic intensities. Intensity values were color-coded from green to red, corresponding to downregulation or upregulation, respectively. (B) Profile plot for three selected clusters showing distinct behavior with respect to different treatments includes Cluster 1, strongly expressed with both EPO and CEPO treatment; Cluster 3, strongly expressed with CEPO treatment; and Cluster 7, strongly expressed with EPO treatment. (C) Enrichment analysis of protein annotations shows functional categories enriched in the three selected clusters, 1, 3, and 7. The enriched terms, the corresponding enrichment factor, and p-value are shown.

Figure 3

The signaling pathways upregulated in EPO- and CEPO-treated neuronal cell cultures. (A) EPO vs. Control. (B) CEPO vs. Control. The canonical pathways are represented on the x-axis. The y-axis represents the significance scores as −log p-value. The threshold line indicates the significance (p < 0.05) cutoff. The height of the bar shows the level of significance.

Figure 4

EPO and CEPO treatment upregulates pAKT and pERK signaling in rat hippocampal samples. (A) Western blot. (B) Graphical representation of Western blot results showing an increase in phosphorylated-AKT in the hippocampus after 4 days of EPO and CEPO (30 µg/kg) treatment in Sprague Dawley rats (n = 6). (C) Western blot. (D) Graphical representation of Western blot results showing an increase in phosphorylated-ERK1/2 in hippocampus after 4 days of EPO and CEPO (30 µg/kg) treatment in Sprague Dawley rats (n = 6). The data are reported as mean (±SEM) and p-values < 0.05 (*) were considered as significant.

Figure 5

Differentially expressed proteins in EPO- and CEPO-treated neuronal cell cultures. Volcano plots of differentially expressed proteins between the experimental groups are shown. (A) EPO vs. Control: 104 were upregulated and 18 were downregulated in EPO as compared to the control. (B) CEPO vs. Control: 85 proteins were upregulated and 148 were downregulated in CEPO as compared to the control. (C) EPO vs. CEPO: 443 proteins were upregulated and 101 proteins were downregulated in EPO as compared to CEPO. For the graph, −log (p-value) is plotted against the t-test difference. The downregulated proteins are on the left and significant ones are in blue; the upregulated proteins are on the right and significant ones are in red. The cutoff value for differentially expressed proteins was set at ±1.3-fold (0.379 in log2-transformed values).

Figure 6

Immuno-fluorescence analysis of protein expression in the rat brain. Rats were administered EPO or CEPO for 4 days (30 µg/kg/day). Cryocut hippocampal brain sections were processed for immunofluorescence detection of 3 proteins in the 3 experimental groups, EPO, CEPO and Control (PBS). Representative images are shown from n = 4 analyses. (A) Growth factor-bound 2 (Grb2); (B) Cortactin; and (C) Pleiotrophin. Dotted ovals indicate vasculature in the cortex. DG, dentate gyrus; DGml, dentate gyrus molecular layer; Ctx, cortex.

Figure 7

Model of EPO and CEPO actions. Molecules and signaling pathways regulated by EPO and CEPO were integrated to develop a mechanistic model involving synapse activity, LTP, and spine generation. NMDAR, glutamate receptor; CD131, beta common receptor; mTOR, mammalian target of rapamycin; Eif3a, elongation initiation factor 3a; Rplp2, 60S acidic ribosomal protein P2; CamKI, calcium/calmodulin-dependent protein kinase I; Cttn, cortactin; LTP, long-term potentiation. Model was adapted from [41].