Harmine stimulates proliferation of human neural progenitors

Abstract

Harmine is the β-carboline alkaloid with the highest concentration in the psychotropic plant decoction Ayahuasca. In rodents, classical antidepressants reverse the symptoms of depression by stimulating neuronal proliferation. It has been shown that Ayahuasca presents antidepressant effects in patients with depressive disorder. In the present study, we investigated the effects of harmine in cell cultures containing human neural progenitor cells (hNPCs, 97% nestin-positive) derived from pluripotent stem cells. After 4 days of treatment, the pool of proliferating hNPCs increased by 71.5%. Harmine has been reported as a potent inhibitor of the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK1A), which regulates cell proliferation and brain development. We tested the effect of analogs of harmine, an inhibitor of DYRK1A (INDY), and an irreversible selective inhibitor of monoamine oxidase (MAO) but not DYRK1A (pargyline). INDY but not pargyline induced proliferation of hNPCs similarly to harmine, suggesting that inhibition of DYRK1A is a possible mechanism to explain harmine effects upon the proliferation of hNPCs. Our findings show that harmine enhances proliferation of hNPCs and suggest that inhibition of DYRK1A may explain its effects upon proliferation in vitro and antidepressant effects in vivo.

Keywords: Antidepressant; Ayahuasca; DYRK1A; Proliferation; hNPC.

Figure 1. Characterization of neural progenitor cells derived from human embryonic stem cells.

Representative images showing hNPCs stained for (A) TBR2 and Nestin, (B) PAX6 and β-Tubulin III, (C) GFAP and MAP2, (D) SOX2, (E) FOXG1, (F) DYRK1A. (G) Quantification of cell markers. (H) Venn diagram showing percentage of double stained cells. A minimum of 10,000 hNPCs was counted per marker. Scale bar: 25 µm.

Figure 2. Quantification of the proliferation and differentiation of hNPCs after treatment with harmine.

(A) Representative images of EdU staining. (B) Cell proliferation (S phase) relative to DMSO control, measured by EdU incorporation. (C) Representative images of GFAP and Nestin staining. (D) Expression of Nestin and GFAP proteins relative to DMSO control. A minimum of 10,000 hNPCs was counted per condition/per experiment. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test, ^∗∗p < 0.001, ^∗∗∗ < 0.0001. Values represent mean ± sem. The number inside the bar represents the number of experiments in each group. Scale bar: 100 µm.

Figure 3. Percentage of cell death and DNA damage in neural cells treated with harmine.

(A) Representative immunostaining images of BOBO-3 (red) positive cells. Nuclei are stained with Hoechst (blue). (B) Cell death relative to DMSO control, measured as the percentage of cells stained with BOBO-3. (C) Representative images of cells positive for H2AX (green). Nuclei are stained with DAPI (blue). (D) Quantification of H2AX positive cells relative to DMSO control, as percentage of cells stained with H2AX. H₂O₂ was used as positive control for both cell death and DNA damage. A minimum of 10,000 hNPCs was counted per condition/per experiment. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test, ^∗p < 0.05, ^∗∗∗ < 0.0001. Values represent mean ± sem. The number inside the bar represents the number of experiments in each group. Scale bar: 100 µm.

Figure 4. Quantification of EdU and Ki-67 labelling in human neural progenitor cells in response to harmine and its functional analogs.

(A) Cell proliferation relative to DMSO control, measured by EdU incorporation. (B) Cell proliferation relative to DMSO control, measured by Ki-67. Values represent mean ± sem. A minimum of 10,000 hNPCs was counted per condition/per experiment. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test, ^∗p < 0.05, ^∗∗p < 0.001, ^∗∗∗ < 0.0001. Values represent mean ± sem. The number inside the bar represents the number of experiments in each group.