Neuroprotective effects of EpoL against oxidative stress induced by soluble oligomers of Aβ peptide

Abstract

Erythropoietin is a glycoproteic hormone that regulates hematopoiesis by acting on its specific receptor (EpoR). The expression of EpoR in the central nervous system (CNS) suggests a role for this hormone in the brain. Recently, we developed a new Epo variant without hematopoietic activity called EpoL, which showed marked neuroprotective effects against oxidative stress in brain ischemia related models. In this study, we have evaluated the neuroprotective effects of EpoL against oxidative stress induced by chronic treatment with Aβ. Our results show that EpoL was neuroprotective against Aβ-induced toxicity by a mechanism that implicates EpoR, reduction in reactive oxygen species, and reduction in astrogliosis. Furthermore, EpoL treatment improved calcium handling and SV2 levels. Interestingly, the neuroprotective effect of EpoL against oxidative stress induced by chronic Aβ treatment was achieved at a concentration 10 times lower than that of Epo. In conclusion, EpoL, a new variant of Epo without hematopoietic activity, is of potential interest for the treatment of diseases related to oxidative stress in the CNS such as Alzheimer disease.

Keywords: Alzheimer disease; Amyloid beta; Erythropoietin; Glycosylation; Neuroprotection; Oxidative stress.

Graphical abstract

Fig. 1

EpoL induced a protective effect against stress induced by chronic Aβ treatment. (A) Viability assay in PC12 cells treated with oligomers of Aβ₄₀ (1uM) for 24 h and Epo or EpoL at different concentrations. The percentage of viability response was evaluated by MTT. (B) Viability assay using a specific antibody to block interaction between EpoR and EpoL or EpoL. (C) Evaluation of the relative expression of the Bcl-2 gene using β-actin as a housekeeping gene after 24 h of treatment with Aβ and co-treatment with Epo or EpoL, at the neuroprotective concentrations observed. Values are mean ± SEM, n = 3 using one-way ANOVA and Dunns test. *:p < 0.05; **:p < 0.01 versus PC12 cells treated with Ab; ++++:p < 0.0001 versus control cells.

Fig. 2

EpoL treatment restored calcium homeostasis in neuronal cells exposed to Aβ. (A) Hippocampal neurons were treated for 24 h with oligomers of Aβ₄₀ and co-incubated with Epo or EpoL at neuroprotective concentrations, as represented in the scheme on the top part of figure. (B) Quantification of percentage frequency of calcium transients using Fluo4AM loaded cells treated for 24 h with the different treatments. (C) Representative images of calcium transients over a 200 s period in cells exposed to the different indicated treatments. Values are mean ± SEM, n = 3 using one-way ANOVA and Dunns test. ***:p < 0.001; ****:p < 0.0001 versus neuronal cells treated with Aβ; ++++:p < 0.0001 versus control cells; ###:p < 0.001; ####:p < 0.0001 versus EpoL or Epo treatment.

Fig. 3

The intracellular calcium modulating effects of EpoL in Aβ treated neurons is EpoR dependent. (A) Hippocampal neurons were treated for 24 h with oligomers of Aβ₄₀ and co-incubated with EpoL and a specific antibody at neuroprotective concentrations, as represented in the schematic. (B) Quantification of percentage frequency of calcium transients in Fluo4AM loaded cells treated for 24 h with Aβ or Aβ+EpoL or Aβ+ EpoL + Aβ, respectively. (C) Representative images of calcium transients during 200 s, under the indicated treatments. Values are mean ± SEM, n = 3 using one-way ANOVA and Dunns test. ****:p < 0.0001 versus neuronal culture treated with Aβ; ++++:p < 0.0001 versus control cells; ####:p < 0.0001 versus EpoL or Epo treatment.

Fig. 4

Neuroprotective effect of EpoL on synaptic markers induced by chronic treatments of Aβ. (A) Confocal images of neuronal cultures treated for 24 h with oligomers of Aβ₄₀ and co-incubated with EpoL at neuroprotective concentrations to analyze Map2 and SV2. (B) The number of SV2 punctas observed in the first 20 μm of each neuron after 24 h of co-incubation is represented. Values are mean ± SEM, n = 3 using one-way ANOVA and Dunns test. ***:p < 0.001 versus neuronal culture treated with Aβ; +++:p < 0.001 versus control cells.

Fig. 5

EpoL treatment decreased oxidative stress in hippocampal organotypic cultures. (A) Epifluorescence images of hippocampal organotypic cultures treated for 4 days with Aβ_25-35 (0.5 μM) and co-incubated with EpoL or Epo at neuroprotective concentrations. (B) Images showing the fluorescence intensity of DCF-DA/Hoechst signal in CA1 region. Values are mean ± SEM, n = 5 using one-way ANOVA and Tukey test. ****:p < 0.0001 versus neuronal culture treated with Aβ; ++++:p < 0.001 versus control cells; ###:p < 0.001 versus Epo treatment.

Fig. 6

Neuroprotective effect of EpoL in hippocampal organotypic cultures against stress induced by chronic treatments of Aβ. (A) Epifluorescence images of hippocampal organotypic cultures treated for 4 days with Aβ_25-35 (0.5 μM) and co-incubated with EpoL or Epo at neuroprotective concentrations. (B) Representative fluorescence images of PI/Hoechst signal of the CA1 region. Values are mean ± SEM, n = 5 using one-way ANOVA and Tukey test. ****:p < 0.0001 versus neuronal culture treated with Aβ; ++++:p < 0.001 versus control cells; ###:p < 0.001 versus Epo treatment.

Fig. 7

The neuroprotective effect of EpoL in hippocampal organotypic cultures against Aβ stress depended on EpoR interaction: (A) Quantification of fluorescence signal intensity of DCF-DA/Hoechst on the CA1 region of hippocampus obtained by fluorescence images of hippocampi organotypic cultures treated for 4 days with Aβ_25-35 (0.5 μM) and co-incubated with EpoL or EpoL + Anti-EpoR (Ab). (B) Quantification of PI/Hoechst signal of the CA1 region, as the same conditions used in A. Values are mean ± SEM, n = 7 using One-way ANOVA and Tukey test. ***:p < 0.001, “ns” versus Aβ; ###:p < 0.001 versus ctrl.

Fig. 8

EpoL and Epo prevented astrogliosis induced by chronic treatment with Aβ. (A) Immunofluorescence confocal images of organotypic hippocampal cultures treated for 4 days with Aβ_25-35 (0.5 μM) and co-incubated with EpoL or Epo at a neuroprotective concentration to analyze GFAP intensity signal. (B) Scheme of protocol used. (C) Mean intensity of GFPA fluorescence in the CA1 observed under each experimental condition using ImageJ software. Values are mean ± SEM, n = 3 using one-way ANOVA and Dunns test. ***:p < 0.001 versus culture treated with Aβ_25-35; ++++:p < 0.001 versus control cells.