The Endogenous Hallucinogen and Trace Amine N,N-Dimethyltryptamine (DMT) Displays Potent Protective Effects against Hypoxia via Sigma-1 Receptor Activation in Human Primary iPSC-Derived Cortical Neurons and Microglia-Like Immune Cells

This in vitro study investigated whether DMT acts neuroprotective against oxidative stress within cultured neurons and immune cells derived from human precursor cells. Results indicate that DMT robustly increases the survival of these cells in response to severe oxygen deprivation, through activation of the Sig-1 receptor, a key modulator of cellular oxidative stress. The authors postulate that DMT may be endogenously generated to mitigate oxidative stress occasioned by adverse brain injuries such as ischemic infarcts.

Abstract

“Introduction: N,N-dimethyltryptamine (DMT) is a potent endogenous hallucinogen present in the brain of humans and other mammals. Despite extensive research, its physiological role remains largely unknown. Recently, DMT has been found to activate the sigma-1 receptor (Sig-1R), an intracellular chaperone fulfilling an interface role between the endoplasmic reticulum (ER) and mitochondria. It ensures the correct transmission of ER stress into the nucleus resulting in the enhanced production of antistress and antioxidant proteins. Due to this function, the activation of Sig-1R can mitigate the outcome of hypoxia or oxidative stress.

Methods: In this paper, we aimed to test the hypothesis that DMT plays a neuroprotective role in the brain by activating the Sig-1R. We tested whether DMT can mitigate hypoxic stress in in vitro cultured human cortical neurons (derived from induced pluripotent stem cells, iPSCs), monocyte-derived macrophages (moMACs), and dendritic cells (moDCs).

Results: DMT robustly increases the survival of these cell types in severe hypoxia (0.5% O₂) through the Sig-1R. Furthermore, this phenomenon is associated with the decreased expression and function of the alpha subunit of the hypoxia-inducible factor 1 (HIF-1) suggesting that DMT-mediated Sig-1R activation may alleviate hypoxia-induced cellular stress and increase survival in a HIF-1-independent manner.

Discussion: Our results reveal a novel and important role of DMT in human cellular physiology. We postulate that this compound may be endogenously generated in situations of stress, ameliorating the adverse effects of hypoxic/ischemic insult to the brain.”

Authors: Attila Szabo, Attila Kovacs, Jordi Riba, Srdjan Djurovic, Eva Rajnavolgyi & Ede Frecska

Summary

INTRODUCTION

The sigma-1 receptor (Sig-1R) is a non-opioid receptor located on the mitochondria-associated endoplasmic reticulum membrane (MAM). It regulates ATP synthesis and stress signaling through the regulation of Ca2+ signaling and chaperoning the inositol requiring enzyme 1 (IRE1) and thereby increasing the intracellular levels of antistress and antioxidant proteins.

Sig-1R has been found to regulate a variety of different physiological processes, including cell differentiation, survival, and immunity. It has been shown to be involved in many human diseases, including cancer, pain, addiction, stroke, ischemic heart disease, and many neuropsychiatric disorders.

The Sig-1R is an endogenous trace amine neurotransmitter that regulates several physiological functions including neural signaling and brain/peripheral immunological processes. It is found in the mammalian lung, brain, and blood, and is increased by environmental stress.

Hypoxia induces alterations in the phenotype and function of cells by provoking increased expression of numerous genes, including the hypoxia-inducible factor (HIF)-1, which is widely considered as a cellular indicator of hypoxic stress or state.

In vitro induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) are emerging as promising models for neurobiological research.

Monocyte-derived macrophages and dendritic cells are critical players of immune defense in higher vertebrates. They are frequently used in different clinical and experimental settings and may represent microglia-like cell types that could significantly contribute to the physiological regulation of the neural tissue.

We tested the hypothesis that DMT-mediated activation of Sig-1R alleviates the effects of hypoxic stress on human primary cells using iPSC, moMAC, and moDC models.

Cell Types, Isolation, Culturing, and Phenotyping

Human iPSC-derived neural progenitor stem cells were differentiated to cerebral cortical neurons in 35 – 40 days and phenotyped using anti-CUTL1 and anti-Ctip2 antibodies. Data were analyzed using the FlowJo software.

Healthy blood donors gave blood to the Regional Blood Center of the Hungarian National Blood Transfusion Service. The blood was processed and stored according to European Union directives. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation, and monocytes were purified using anti-CD14 microbeads. Monocytes were cultured in AIMV medium with 80 ng/ml GM-CSF and 100 ng/ml IL-4, and moDCs and moMACs were harvested on day 5.

Cultures

After pre-incubation in a low-oxygen atmosphere for 4 h, cells were placed in a hypoxia chamber and incubated for 6 h under similar hypoxia conditions. Cells were either immediately lysed or placed on ice for analysis.

DMT Treatment and Sampling of Cells

N,N-dimethyltryptamine and BD1063 dihydrochloride were used to treat mice with a combination of DMT and BD1063 to determine the effects of DMT.

RNA Isolation, cDNA Synthesis, and QPCR

Real-time quantitative polymerase chain reaction (QPCR) was performed as described previously (Szabo et al., 2016). Total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA), reverse transcribed, and TaqMan assays were used for QPCR.

Western Blotting

Cells were lysed in Laemmli buffer and probed with Ab specific for Sig-1R/OPRS1, HIF-1, ATF6, p65, phospho-p65 (S536) and -actin. The SuperSignal enhanced chemiluminescence system was used for probing target proteins.

Cellular Viability Assays

The percentage of apoptotic cells was assessed by using an Annexin V apoptosis kit, and the rate of necrotic cell death was monitored simultaneously by measuring membrane integrity.

RNA Interference

Gene-specific siRNA knockdown was performed by Gene Pulser Xcell instrument using Silencer Select siRNA and LyoVec transfection system. The efficacy of siRNA treatments was tested 2 days post-transfection by Western blotting.

Differentiation-Dependent Expression of Sig-1R in Human iPSC-Derived Cortical Neurons

In this work, we investigated the effects of DMT-mediated activation of the Sig-1R in hypoxia using moMACs, moDCs, and iPSC-derived neurons. We found that the expression of Sig-1R increases during the differentiation process of iPSC-derived cortical neurons.

In vitro DMT-Treatment of Human Primary Cells Results in Increased Survival in Severe Hypoxic Environment

Based on our previous findings, we treated human iPSC-derived neurons, moMACs, and moDCs with DMT, a natural endogenous ligand of Sig-1R, to investigate whether this ligand influences their survival in hypoxia. The results indicate that DMT increases the survival of cells in hypoxia.

Incubation in hypoxic environment rapidly induced cell death in all cultures, as measured by Annexin V-FITC staining and subsequent flow cytometry analysis. We found that administration of DMT to iPSC-derived cortical neurons, moMACs, and moDCs increased their survival in hypoxia. Non-DMT treated cultures of iPSC-derived cortical neurons showed significant increase in apoptotic cells even after 1 h of hypoxia treatment, while DMT treated cultures of moMACs and moDCs showed no change in cellular viability after 6 h of hypoxia treatment.

HIF-1, a transcription factor involved in hypoxia-induced gene expression, is subject to ubiquitination and rapid degradation under normoxia. Therefore, we aimed to investigate whether HIF-1 was involved or affected in this process.

We found that 6 h of hypoxia treatment greatly induced the protein-level expression of HIF-1 in human iPSC-derived neurons, moMACs, and moDCs, and that 50 m DMT prevented this increase in all cell types. Furthermore, the level of the ER-stress sensor ATF6 showed some degree of decrease when treated with DMT under hypoxia.

Sig-1R Is Indispensable for the DMT-Mediated Modulation of Cellular Survival and HIF-1α Expression in Human iPSC-Derived Neurons, moMACs, and moDCs under Hypoxia

Sig-1R is expressed in human iPSC-derived neurons, moMACs, and moDCs, and DMT treatment increases their survival under severe hypoxia. DMT has been shown to be an endogenous agonist of Sig-1R in human primary cells. Using the same treatment protocols as in Figures 2, 3, we found that Sig-1R was required for the modulatory effects of DMT on cellular survival and HIF-1 protein expression.

DISCUSSION

Sig-1R was found to be expressed in human in vitro differentiated iPSC-derived cortical neurons during the process of differentiation. The expression of Sig-1R was statistically significant from the 21st day of differentiation as compared to baseline (NSC) values.

After demonstrating that Sig-1R is expressed in iPSC-derived cortical neurons, we next sought to investigate the effects of DMT treatment in hypoxic stress. DMT treatment significantly increased the survival of cells as measured by flow cytometry.

We found that the endogenous indicator of hypoxic stress, HIF-1, was rapidly upregulated in all cell types following 6 h of hypoxia exposure, and that DMT significantly prevented this phenomenon.

We used gene-specific silencing to test the contribution of Sig-1R to the observed phenomenon. The results suggested that Sig-1R is a critical, indispensable role player in the protective and antistress effects of DMT in human iPSC-derived cortical neurons, moMACs and moDCs in hypoxic environment.

The observed, Sig-1R-mediated protective and antistress effects of DMT in hypoxia may be based on multiple mechanisms, including the fine-tuning of mitochondrial functions and the regulation of cellular oxygen metabolism, as well as the modulation of Ca2+ signaling altering the function of intracellular kinases involved in cellular survival.

This study shows that DMT, through the Sig-1R of human primary cells, can increase survival and alleviate cellular stress in hypoxic environments. DMT may also contribute to neuroregenerative and neurorestorative processes by modulating the survival of microglia-like cells.