Harmine stimulates proliferation of human neural progenitors

This cell-based study found that harmine (found in ayahuasca) leads to the rapid growth of human neural progenitor cells (hNPCs). The inhibition of a gene (DYRK1A) could possibly explain it’s cell-growth and anti-depressant effects.

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.“

Authors: Vanja Dakic, Renata de Moraes Maciel, Hannah Drummond, Juliana M. Nascimento, Pablo Trindade & Stevens K. Rehen

Summary

ABSTRACT

Harmine, an alkaloid in the psychotropic plant decoction Ayahuasca, stimulates proliferation of human neural progenitor cells derived from pluripotent stem cells. Its effects on proliferation may be explained by inhibition of the dual specificity tyrosine-phosphorylation-regulated kinase (DYRK1A), which regulates cell proliferation and brain development.

INTRODUCTION

Throughout life, specific regions of the human adult brain continuously generate neural cells from a pool of neural progenitor cells. Chronic stress, depression, aging, and neurodegenerative diseases can disrupt this process.

Classical antidepressants can reverse stress-induced hippocampal atrophy in rodents, but they cause side effects and the time required for achieving therapeutic response is usually measured in weeks.

Ayahuasca, a psychotropic beverage traditionally used in the Amazonian region of South America, contains two members of the beta-carboline family, harmine and harmaline. These alkaloids have fast-acting anxiolytic and antidepressant effects.

We examined the effects of harmine on human neural progenitor cells and found that it increased the pool of neural progenitor cells.

Chemicals

Harmine, INDY and pargyline hydrochloride were diluted in DMSO and then made into aqueous solutions. The controls received DMSO.

Human pluripotent stem cells

Human embryonic stem cells were cultured under feeder-free conditions on Matrigel coated dishes in Essential 8TM Medium and passedaging was performed enzymatically using Accutase.

Human neural progenitor cells

We performed an adaptation of Baharvand et al. (2007) protocol to induce embryonic stem cells to direct neural differentiation. The cells were expanded in N2B27 medium supplemented with 25 ng/mL bFGF and 20 ng/mL EGF and incubated at 37 °C and 5% CO2.

High content screening

Cell proliferation, cell death and DNA damage experiments were performed in a High Content Screening (HCS) format using hNPCs (1,500 cells/per well) on a multiwell 384 Clear plate.

High content analysis

All images were acquired on Operetta high-content imaging system (Perkin Elmer). Cell proliferation was measured by incorporating EdU with Alexa Fluor 488 and Ki-67 with a 10 objective with high numerical aperture.

For cell death analysis, cells were labelled with Hoechst and BOBOTM -3, and live cell imaging was performed using temperature and CO2 control option (TCO) of Operetta at 10 magnification. DNA damage analysis was performed using H2AX antibody, and images were acquired at 10 magnification.

hNPCs were fixed in formaldehyde 3.7% for 15 min at RT, permeabilized in 0.2% Triton X-100 for 15 min, and stained with primary antibodies, followed by blocking in 2% BSA for 40 min. Images were acquired using confocal microscopy and Operetta.

RESULTS

We generated hNPCs from human embryonic stem cells using a protocol that recapitulates the early steps of neural development. Ninety percent of the cells expressed markers of neural progenitors, and 90% expressed the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A).

Harmine increases proliferation of human neural progenitors: a possible role of DYRK1A

Pharmacokinetic studies have shown that the bioavailability of ayahuasca alkaloids in the human plasma varies from 0.5 ng/mL to 222.3 ng/mL, and in vitro studies have used a wide spectrum of harmine concentrations ranging from 333 nM to 22.5 mM.

Harmine, an inhibitor of DYRK1A, MAO, and other DYRK family members, increased proliferation of human neural cells in comparison with control. Concomitant inhibition of both DYRK1A and MAOs produced similar effect as harmine or INDY alone.

DISCUSSION

In vitro models can potentially clarify mechanisms related to proliferation and differentiation of newborn neurons. Harmine, a compound with potential antidepressant properties, increases proliferation of human neural progenitor cells derived from pluripotent stem cells without DNA damage or cell death.

GFAP+ cells can shift to a phenotype PSA-NCAM+, which gives rise to neurons. Harmine can potentiate proliferation of radial glia-like cells derived from hNPCs, which can generate both neurons and astrocytes.

Harmine, an inhibitor of MAO and DYRK1A, increases serotonergic neurotransmission in the adult brain and is a key component of the classical antidepressant action. It has been reported that mice lacking MAO present reduced proliferation of neural progenitor cells.

Harmine increased the proliferation of pancreatic beta cells, and INDY, an inhibitor of DYRK1, induced proliferation similarly to harmine. INDY and harmine have multiple targets on DYRK family, including regulators of cell cycle, and DYRK1A is the major candidate for mediating the increase in proliferation seen in this study.

Harmine stimulates proliferation of human neural progenitor cells (hNPCs) by inhibiting DYRK1A. This finding sheds light on the possible mechanisms behind the antidepressant effects of Ayahuasca described in patients.

Funding

This study was funded by several Brazilian agencies, but the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures

The following grants were disclosed by the authors: CNPq, FAPERJ, CAPES. Übrigens: Renata de Moraes Maciel designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, reviewed drafts of the paper.