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.
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.
Papers cited by this study that are also in Blossom
Frecska, E., Bokor, P., Winkelman, M. J. · Frontiers in Pharmacology (2016)
Frecska, E., Szabo, A., Winkelman, M. J. et al. · Translational Neurosciences (2013)
McKenna, D., Riba, J. · Current Topics in Behavioral Neurosciences (2016)
Shen, H. W., Jiang, X. L., Winter, J. C. et al. · Current Drug Metabolism (2010)
Ahmad and colleagues situate their study within research on the sigma-1 receptor (Sig-1R), an endoplasmic reticulum (ER) chaperone located at mitochondria-associated ER membranes (MAM). Sig-1R has been implicated in regulating Ca2+ signalling, ATP synthesis, ER-to-nucleus stress signalling (via chaperoning proteins such as IRE1), and in modulating cell survival, differentiation and immune responses. Previous in vitro and in vivo work indicates that Sig-1R agonists can protect against hypoxia and ischemic injury, and Sig-1R is expressed across neural and immune tissues. N,N-dimethyltryptamine (DMT), an endogenous tryptamine and known Sig-1R agonist, has been detected in mammalian tissues and is proposed to be sequestered in vesicles and increased by stress, but its physiological role remains unclear. The study therefore tests the hypothesis that DMT confers cellular protection against hypoxic stress via Sig-1R activation. Using human induced pluripotent stem cell (iPSC)-derived cortical neurons and monocyte-derived macrophages and dendritic cells (moMACs and moDCs, considered microglia-like), the investigators examine whether DMT affects survival in severe hypoxia and whether these effects involve modulation of hypoxia-inducible factor 1 alpha (HIF-1α) and dependence on Sig-1R signalling. The work aims to clarify a potential endogenous neuroprotective role for DMT and the mechanistic involvement of Sig-1R in human primary cell models.
The experimental models comprised three human primary cell types: iPSC-derived cortical neurons (differentiated over ~35–40 days from neural progenitors obtained commercially), monocyte-derived macrophages (moMACs) and monocyte-derived dendritic cells (moDCs) generated from healthy donor peripheral blood mononuclear cells. Monocytes were isolated by CD14-positive selection; moDCs were differentiated with GM-CSF and IL-4 and harvested on day 5, while moMACs were generated with M-CSF and collected on day 4. Phenotyping of neurons used antibodies to CUTL1 and Ctip2; moMAC/moDC phenotyping used markers including CD14, CD68, CD209 and HLA-DR. Hypoxia was induced in vitro using a hypoxia chamber set to 0.5% O2 (94.5% N2, 5% CO2). Culture plates with serum-free medium were pre-incubated in low oxygen for 4 h to de-gas, then cells were seeded and incubated at 37 °C for up to 6 h under the same hypoxic conditions; an automated regulator maintained gas composition. DMT (1–200 µM) and the selective Sig-1R antagonist BD1063 (1–100 µM) were applied in vitro, with BD1063 added 30 min prior to DMT when used. Cells were generally sampled after 6 h of treatment for downstream assays. Outcomes and assays included cellular viability by Annexin V-FITC apoptosis staining and propidium iodide (PI) uptake for necrosis measured by flow cytometry; protein expression by Western blot (targets included Sig-1R, HIF-1α, ATF6, p65 and phospho-p65); and mRNA quantification by real-time QPCR (TaqMan assays, 36B4 as normaliser). Sig-1R was silenced using pooled Silencer Select siRNAs delivered by electroporation (monocytes/macrophages/dendritic cells) or LyoVec (neurons), with knockdown efficacy checked by Western blot 48 h post-transfection. Statistical analysis used t-tests with Bonferroni correction for pairwise comparisons and two-way ANOVA for multiple comparisons, with p < 0.05 considered significant.
Sig-1R expression increased during neuronal differentiation. Neural stem cells displayed low baseline Sig-1R mRNA and protein, with mRNA rising significantly by day 14 and protein by day 21; the highest levels were observed at the end of the ~35–40 day differentiation protocol. Previously reported high Sig-1R expression in moMACs and moDCs was confirmed. Severe hypoxia (0.5% O2) induced predominantly apoptotic cell death across all cultures within 6 h; necrosis remained low (median PI-positive values never exceeded 6%). DMT treatment increased survival in all three cell types in hypoxia in a concentration-dependent manner. For iPSC-derived cortical neurons, DMT concentrations of 10–200 µM produced significant survival benefits, with 10 µM and 50 µM increasing median survival after 6 h from 19% (±2%, n = 3) in untreated hypoxia controls to 31% (±6%, p = 0.037) and 64% (±5%, p = 0.006), respectively. For moMACs and moDCs, significant protective effects were observed at 50–200 µM; no additional benefit was seen at 200 µM versus 50 µM. Normoxic cultures showed no change in viability with DMT treatment. The investigators selected 50 µM DMT for subsequent experiments, noting this is an achievable serum concentration in prior in vivo reports. At the molecular level, 6 h of hypoxia strongly induced HIF-1α protein in neurons, moMACs and moDCs; DMT (50 µM) prevented this hypoxia-induced increase in HIF-1α protein in all cell types. Concordantly, VEGF mRNA, a HIF-1α target, was reduced in DMT-treated cultures under hypoxia. Analyses of other stress pathways showed no detectable change in native or phosphorylated NF-κB p65 within the observation window; the 50 kDa active form of ATF6 trended downward with DMT under hypoxia but this did not reach statistical significance. To test dependence on Sig-1R, the researchers performed gene-specific knockdown and pharmacological antagonism. siRNA-mediated Sig-1R silencing reduced Sig-1R protein by >93% (±5%, n = 3) in neurons, >96% (±3%) in moMACs and >91% (±5%, n = 4) in moDCs. Sig-1R knockdown abolished the DMT-induced survival benefit and the DMT-mediated reduction in HIF-1α expression across all cell types. Similarly, pre-treatment with the selective Sig-1R antagonist BD1063 prevented the protective effects of DMT. These findings indicate that the observed DMT effects on survival and HIF-1α expression are Sig-1R-dependent.
Ahmad and colleagues interpret their data as evidence that DMT, acting via Sig-1R, confers protection to human iPSC-derived cortical neurons and monocyte-derived microglia-like cells under severe hypoxia. The key findings are increased cell viability in 0.5% O2 with DMT treatment, prevention of hypoxia-induced HIF-1α protein accumulation and reduced VEGF mRNA, and loss of these effects when Sig-1R is silenced or pharmacologically blocked. The investigators note this is the first report demonstrating DMT’s Sig-1R-mediated protective and antistress effects in human primary cell cultures subjected to hypoxia. They position these results relative to prior studies showing Sig-1R agonists protect against ischemia and oxidative stress, and suggest possible mechanisms: modulation of mitochondrial function and cellular oxygen metabolism, and altered Ca2+ signalling that affects intracellular kinases involved in survival. The authors highlight that Sig-1R’s localisation to the MAM means its activation could channel stress responses via HIF-1α-independent routes; their observations of increased survival despite decreased HIF-1α support this possibility. The paper reports that NF-κB and ATF6 were not evidently altered within the time-frame examined, but acknowledges that biochemical pathways mediating the protective effect remain to be elucidated. Finally, the investigators discuss potential physiological and translational relevance: because microglia and microglia-like cells are important in CNS injury and recovery, DMT-mediated modulation of their survival could influence neuroregenerative processes. They propose that endogenous DMT generation during stress might ameliorate hypoxic or ischaemic insults and suggest that Sig-1R modulation by DMT may be relevant for future therapies targeting hypoxia/ischemia-related pathologies, while noting further mechanistic and translational investigations are required.
Originally thought to be an opioid receptor, the sigma-1 receptor (Sig-1R) is now classified as member of an orphan family after Su and colleagues had characterized the structural and biochemical features of the ligand binding site and identified the class as a non-opioid one. Later, based on their pharmacological characteristics and tissue expression, the distinction of Sig-1R and Sig-2R subtypes was proposed. Previous studies showed that Sig-1R is expressed not only in different regions of the brain but also in immune cells, and organs like liver, kidney, and gut. Sig-1R is chiefly an endoplasmic reticulum (ER) protein located on the mitochondria-associated endoplasmic reticulum membrane (MAM) where its main role is to regulate ATP synthesis through the regulation of Ca 2+ signaling by primarily acting as a molecular chaperone. Another MAM-related role of Sig-1R is to facilitate stress signaling from the ER to the nucleus through chaperoning the inositol requiring enzyme 1 (IRE1) and thereby increasing the intracellular levels of antistress and antioxidant proteins. Upon cellular stress, including hypoxia or oxidative stress, Sig-1R interacts with numerous receptors, ion channels, kinases, and various master regulator proteins residing on the ER, MAM, nucleus, or even in the cytosol to mobilize and fine-tune antistress responses. Based on these complex intracellular actions the Sig-1R has been conceptualized as a "pluripotent modulator" in living systems, as a controller of cell survival and differentiationwhich may be involved in many human diseases. Indeed, in the last two decades a considerable amount of clinical data demonstrated the involvement of Sig-1R in various pathologies including cancer, pain, addiction, stroke, ischemic heart disease, and many neuropsychiatric disorders. It has been reported that Sig-1R regulates a plethora of different physiological processes predominantly associated with cellular differentiation, survival, and immunity. Recent in vitro and in vivo reports suggest that Sig-1R agonists possess potent protective effects in hypoxia and neurotoxicity models. Sig-1R and Sig-2R both have been found to modulate neuronal and microglial responses to ischemia, and specific Sig-1R stimulation was shown to protect against the formation of ischemic lesions subsequent to stroke. In mammals, the endogenous ligands of Sig-1R include neurosteroids (e.g., pregnenolone, dehydroepiandrosterone, progesterone, etc.,, and naturally occurring tryptamines such as N,N-dimethyltryptamine (DMT;. In early studies, DMT was shown to be present in various animal tissues and now is considered to be an endogenous trace amine neurotransmitter that regulates several physiological functions including neural signaling and brain/peripheral immunological processes through the Sig-1R. In addition to its centuries-long use as a sacramental medicine within the circles of South American natives (e.g., yopo, ayahuasca, yagé), DMT was shown to be synthesized in the mammalian lungand brainand was found in human blood, urine, and cerebrospinal fluid. Furthermore, evidence suggests that DMT can be sequestered into and stored in the vesicle system of the brain and environmental stress increases its CNS levels in mammals. However, the exact role of DMT in mammalian physiology is yet to be understood. Hypoxia induces immense alterations in the phenotype and function of cells by provoking increased expression of numerous genes. One of these major changes include the hypoxic upregulation of the hypoxia-inducible factor (HIF)-1 which consists of an α subunit (HIF-1α) and a constitutively expressed β subunit. The presence of oxygen causes the immediate cytoplasmic degradation of HIF-1α, while in hypoxia the rapid accumulation of HIF-1α and its subsequent association with the β subunit leads to the formation of an active transcription factor that translocates to the nucleus and binds to the promoters of oxygen-sensitive genes, such as the vascular endothelial growth factor (VEGF). Thus, HIF-1α is widely considered as a cellular indicator of hypoxic stress or state. The application of human induced pluripotent stem cells (iPSCs)/neural stem cells (NSCs) in order to elucidate the cellular and molecular details of neurological and psychiatric disorders has become an increasing trend in modern science. The lack of appropriate animal models and the unavailability of human brain tissue pose a significant drawback in biomedical investigations. Thus, in vitro iPSC-derived neurons are emerging as promising models both in single-cell and in simple network-based neurobiological research. Monocyte-derived macrophages (moMACs) and dendritic cells (moDCs) are critical players of immune defense in higher vertebrates. They are present in virtually all tissues of the body and, by continuously sampling their environment for selfand non-self-ligands, maintain immunosurveillance and control tissue protection and regeneration. In vitro differentiated moMACs and moDCs are frequently used in different clinical and experimental settings. Furthermore, microglial and moMAC populations of the CNS are comparable concerning their phenotypic and functional propertiesand are considered as gold standards in immunology and regularly used in various clinical and experimental settings. They have been suggested as comparable with the microglial populations of the brain and thus may be considered as microglia-like cells. Very recently, human monocytes have been reported to migrate to the brain and are able to modulate the neuroimmune profile of the CNS. Thus, within the specific tissue setting of the brain, moMACs and moDCs may represent microglia-like cell types which-besides, in concert with, or similar to microglia-could significantly contribute to the physiological regulation of the neural tissue. As Sig-1R activation has already been reported to be massively protective in various in vitro and in vivo ischemia and hypoxicshock settings, our goal was to test the hypothesis whether the DMT-mediated activation of Sig-1R alleviates the effects of hypoxic stress on human primary cells using iPSC, moMAC, and moDC models.
Human iPSC-derived neural progenitor stem cells were obtained from Axol Bioscience (Little Chesterford, UK) and were differentiated to cerebral cortical neurons in approximately 35-40 days following the recommended protocol. Phenotyping of fully differentiated cortical neurons was performed by flow cytometry using anti-CUTL1 and anti-Ctip2 (both from Abcam, Cambridge, UK) and isotype-matched control antibodies (BD Biosciences, Franklin Lakes, NJ) in accordance with the recent literature. Fluorescence intensities were measured by FACS Calibur (BD Biosciences) and data were analyzed by the FlowJo software. Further information about the characterization of cerebral cortical neurons (phenotyping and transcriptome analysis) is available at the company website (). Leukocyte-enriched buffy coats were obtained from healthy blood donors drawn at the Regional Blood Center of the Hungarian National Blood Transfusion Service (Debrecen, Hungary) in accordance with the written approval of the Director of the National Blood Transfusion Service and the Regional and Institutional Ethics Committee of the University of Debrecen, Faculty of Medicine (Debrecen, Hungary). Written informed consent was obtained from the donors prior blood donation and their data were processed and stored according to the directives of the European Union. Peripheral blood mononuclear cells (PBMCs) were separated by a standard density gradient centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Uppsala, Sweden). Monocytes were purified from PBMCs by positive selection using immunomagnetic cell separation with anti-CD14 microbeads according to the manufacturer's instruction. After separation on a VarioMACS magnet, 96-99% of the cells were CD14 + monocytes as measured by flow cytometry. For moDC generation, monocytes were cultured in 12-well tissue culture plates at a density of 2 × 10 6 cells/ml in AIMV medium (Invitrogen, Carlsbad, CA) supplemented with 80 ng/ml GM-CSF (Gentaur Molecular Products, Brussels, Belgium) and 100 ng/ml IL-4 (Peprotech EC, London, UK). On day 2, the same amounts of GM-CSF and IL-4 were added to the cell cultures, and moDCs were harvested on day 5. For moMAC differentiation, 50 ng/ml M-CSF (Gentaur) was added to the monocyte cultures on days 0 and 2, and fully differentiated macrophages were collected on day 4. Phenotyping of moDCs and moMACs was carried out by flow cytometry using anti-CD209-PE, anti-CD14-PE (Beckman Coulter, Hialeah, FL), anti-CD68-PE, anti-HLA-DR-FITC, and isotype-matched control antibodies (BD Biosciences).
Induction of hypoxia was performed following the modified version of a previously described protocol. To allow culture media to de-gas, 12-well plates containing serum-free AIMV were pre-incubated in low-oxygen atmosphere (94.5% N 2 , 5% CO 2 , 0.5% O 2 ) for 4 h using a hypoxia chamber (Billups-Rothenberg, San Diego, CA). Cells were then seeded on the plates, placed in the chamber and were incubated at 37 • C for up to 6 h under similar hypoxia conditions. An automated regulator (with built-in flow meter and oxygen-sensor) was used to ensure and maintain the proper composition of gas mixture within the chamber. After hypoxia treatment cells were removed from the chamber and were either immediately lysed (for Western blot or QPCR), or placed on ice (for Annexin V-FITC staining and flow cytometry analysis).
N,N-dimethyltryptamine (R&D Systems, Abingdon, UK) and BD1063 dihydrochloride (Tocris, Bristol, UK) were used at working concentrations of 1-200 and 1-100 µM, respectively. BD1063 treatments always preceded the addition of DMT by 30 min to allow successful antagonism. DMT, as a controlled substance (Schedule I drug), was used with the approval and monitoring of the Hungarian Institute for Forensic Sciences and the Hungarian National Police Department. To prepare cell lysates for Western blotting and QPCR measurements, in vitro cell cultures were sampled after 6 h of treatment (if not stated otherwise).
Real-time quantitative polymerase chain reaction (QPCR) was performed as described previously. Briefly, total RNA was isolated by TRIzol reagent (Invitrogen, Carlsbad, CA). 1.5-2 µg of total RNA were reverse transcribed using SuperScript II RNase H reverse transcriptase (Invitrogen) and Oligo(dT)15 primers (Promega, Madison, WI). Gene-specific TaqMan assays (Applied Biosystems, Foster City, CA) were used to perform QPCR in a final volume of 12 µl in triplicates using AmpliTaq Gold DNA polymerase and ABI StepOnePlus real-time PCR instrument (Applied Biosystems). Amplification of 36B4 was used as a normalizing control. Cycle threshold values (Ct) were determined by using the StepOne 2.1 software. Constant threshold values were set for each gene throughout the study. The sequence of the primers and probes are available upon request.
Cells were lysed in Laemmli buffer and the protein extracts were tested by Ab specific for Sig-1R/OPRS1, HIF-1α, ATF6, p65, phospho-p65 (S536) (all from Abcam), and β-actin (Sigma, Schnelldorf, Germany) diluted at 1:500 and 1:000, respectively. Anti-rabbit Ab conjugated to horseradish peroxidase (GE Healthcare, Little Chalfont Buckinghamshire, UK) was used as the secondary Ab at a dilution of 1:5000. The SuperSignal enhanced chemiluminescence system was used for probing target proteins (Thermo Scientific, Rockford, IL). After the membranes had been probed for Sig-1R/OPRS1 or HIF-1α, they were stripped and re-probed for β-actin.
The percentage of apoptotic cells was assessed by using an Annexin V apoptosis kit (BioVision, CA, USA) following the manufacturer's recommendations. The rate of necrotic cell death was also monitored simultaneously by measuring membrane integrity. Necrotic cell death was quantified based on the loss of membrane integrity and the uptake of propidium iodide (PI). Upon stimulation cells were harvested and stained with PI (10 µg/ml) and analyzed immediately by flow cytometry.
Gene-specific siRNA knockdown was performed by Silencer Select siRNA (Applied Biosystems) transfection using Gene Pulser Xcell instrument (Bio-Rad, Hercules, CA). Pulse conditions were square-wave pulse, 500 V, 0.5 ms. Immediately after electroporation, moMACs/moDCs were transferred to pre-warmed, fresh medium supplemented with penicillin, streptomycin, and L-glutamine. Gene knockdown in neurons was performed with the LyoVec transfection system (InvivoGen, San Diego, CA) according to the manufacturer's recommendations. Silencing of Sig-1R gene expression was performed by using a mix of three of the available Sig-1R siRNAs. Silencer negative control non-targeting siRNA (Applied Biosystems) was used as a negative control. The efficacy of siRNA treatments was tested 2 days post-transfection by Western blotting.
Data are presented as mean ± SEM. A t-test was used for comparison of two groups followed by Bonferroni correction. Two-way ANOVA was used for multiple comparisons. Differences were considered to be statistically significant at p < 0.05 ( * ).
In this work, we used three experimental models-moMACs, moDCs, and iPSC-derived neurons-to investigate the effects of DMT-mediated activation of the Sig-1R in hypoxia. In a previous study, we have already showed that human moMACs and moDCs express high levels of the Sig-1R gene and protein. Though the expression of Sig-1R has also been demonstrated in human neural cell types (reviewed in, it has not been investigated in NSCs and in iPSC-derived cortical neurons. Therefore, we first sought for the Sig-1R expression profile of iPSC-derived neurons during the differentiation process. We found that NSCs have a low baseline expression of Sig-1R at both the mRNA and protein levels and its expression increases during the differentiation of cells into cortical neurons (Figure). The expression of Sig-1R becomes prominent after the 14th (mRNA) and 21st (protein) days of differentiation where significant changes were detected as compared to the baseline Sig-1R expression of NSCs (Figures). The highest level of Sig-1R was detected at the end of the differentiation process (Figure).
Based on our previous findings about the detectable levels of Sig-1R in human iPSC-derived neurons (Figure), moMACs, and moDCs, we next investigated whether the in vitro treatment of these cell types with DMT, as a natural endogenous ligand of Sig-1R, influences their survival in hypoxia. Human tissues experience a wide range of diverse oxygen tensions that profoundly differ from that of the inhaled ambient oxygen (21%, 160 mm Hg). By the time inhaled oxygen reaches tissues and organs its tension drops to 2-9% (14-65 mm Hg;. Thus, in various experimental human tissue cultures, 2-9% in vitro oxygen level is considered as physiologic normoxia. Certain low vascular density tissues may experience even lower oxygen tensions, however, 1% or lower level of oxygen is often regarded as a hypoxic environment in the literature. In our experiments we used severe hypoxic treatment of cells where, in accordance with other studies, the level of oxygen was set to 0.5% in a hypoxia chamber. Incubation in hypoxic environment rapidly induced cell death in all cultures as assessed by Annexin V-FITC staining and subsequent flow cytometry analysis (Figure). The ratio of necrotic cells, as measured with PI-staining, never exceeded 6% median values (±1%, n = 3 in neuron, and ±3%, n = 6 in moMAC/DC cultures; data not included in Figure). By using different concentrations (1-200 µM) we found that in vitro administration of 10-200 µM DMT increases the survival of iPSC-derived cortical neurons (Figure), moMACs (Figure), and moDCs (Figure) in hypoxia. This survivalboosting effect was statistically significant at DMT concentrations of as low as 10 µM (neurons) or 50 µM (moMAC/DCs) as compared to the non-DMT-treated hypoxia controls (Figure). Non-DMT-treated control cultures (hypoxia controls) of iPSCderived neurons showed significant increase in the ratio of apoptotic cells even after 1 h of hypoxia treatment as compared to normoxic controls (Figure). Six hours of hypoxia resulted in a median of 19% (±2%, n = 3) survival rate in case of nontreated iPSC-derived cortical neuron control cultures, while the administration of 10 and 50 µMs DMT elevated this value to a median of 31% (±6%, p = 0.037) and 64% (±5%, p = 0.006), respectively (Figure). Interestingly, in cases of moMACs and moDCs significant amounts of apoptotic cells could be detected only after 6 h of hypoxia (Figures). Furthermore, no difference was seen between the modulating effects of 50 and 200 µM concentrations of DMT (Figure). Normoxic cultures exhibited no sign of change in cellular viability neither in controls (Figures) nor in DMT-treated (normoxia+1-200 µM DMT) cases (data not shown). Since 50 µM DMT, an achievable serum concentration reported by previous in vivo humanand animal studies, was found to be optimal for modulating the survival capacity of all cell types this concentration was used in further experiments.
Our results demonstrated that severe hypoxia (0.5% O 2 ) induced apoptotic death of human primary cells and DMT treatment could prevent this phenomenon (Figure). The expression of the transcription factor HIF-1α is strongly induced by hypoxia in many cell types, however, it is subject to ubiquitination and rapid degradation under normoxia. The molecular basis of this process is an O 2 -dependent hydroxylation of proline residues. Under hypoxia, HIF-1α is promptly assembled and carries out the downstream control of the expression of many genes related to hypoxic stress including VEGF. Thus, increased expressions of both HIF-1α and its target gene VEGF often signify hypoxic stress or state. We next aimed to investigate whether HIF-1α was involved or affected in this process. We found that 6 h of hypoxia treatment greatly induced the protein-level expression of HIF-1α in human iPSC-derived neurons, moMACs, and moDCs, and the administration of 50 µM DMT prevented this increase in all cell types (Figures). In normoxia control experiments, DMT alone did not influence HIF-1α expressions (Figure). The DMTmediated inhibition of HIF-1α protein expression under hypoxia was found to be statistically significant as compared to hypoxia controls (Figure). These results were consistent with our subsequent findings showing significantly decreased mRNA expressions of VEGF upon DMT treatment in all cell types under hypoxia (Figure). Furthermore, we also monitored other stress-related pathways including NF-κB and the ER-stress sensor Activating transcription factor 6 (ATF6;. We found no alterations in the protein level expression of native p65 NF-κB subunit neither could detect its phosphorylated form in our cell types in hypoxic vs. normoxic conditions within the time-range of observation (data not shown). Interestingly, the level of the 50 kDa transcriptionally active form of ATF6 showed some degree of decrease when cells were treated with DMT under hypoxia as compared to non-treated controls. However, this change did not appear to be statistically significant (Figure).
In previous experiments we showed that Sig-1R is expressed in human iPSC-derived neurons (Figure), moMACs, and moDCs. We also demonstrated that DMT treatment of these cell types can significantly increase their survival under severe hypoxia (Figure) and decrease their expression of HIF-1α and VEGF (Figure). As DMT has previously been described as a natural endogenous agonist of Sig-1Rwe next tested whether Sig-1R was involved in the observed cell biological effects of DMT. To clarify the DMT-dependent modulatory role of Sig-1R in human primary cells and to check its contribution to the observed phenomena we performed Sig-1R gene knockdown experiments. Specific silencing of the Sig-1R gene resulted in a >93% (±5%, n = 3) of downregulation of the Sig-1R protein in human iPSC-derived neurons as compared to nontransfected and scrambled siRNA controls, and >96% (±3%, FIGURE 2 | The effect of DMT treatment on the survival of human iPSC-derived cortical neurons, monocyte-derived macrophages and dendritic cells in hypoxic environment. Hypoxia treatment (0.5% O 2 ) of in vitro cell cultures and cellular viability assays were carried out as described in the Section Materials and Methods. Prior to hypoxia treatment, human primary iPSC-derived cortical neurons (A), moMACs (B), and moDCs (C) were either treated with 1-200 µM DMT or left untreated (hypoxia ctrl). Normoxic cultures were used as positive controls (normoxia). Induction of cellular death was assessed by Annexin V-FITC staining. Flow cytometry data of three (neurons) or six (macrophages and dendritic cells) independent experiments are shown as Mean ± SEM. In each case, asterisk indicates significance compared to hypoxia controls (p < 0.05). n = 4) and >91% (±5%, n = 4) in human moMACs and moDCs, respectively (Figure). Using the same treatment protocols as in Figures, we found that specific silencing of Sig-1R abrogated the modulatory potential of DMT on the survival (Figure) and HIF-1α protein expression of human iPSC-derived neurons, moMACs, and moDCs (Figure). In DMT-treated Sig-1R knockdown cultures, the survival of all cell types was found to be significantly lower as compared to controls (Figure). Similarly, Sig-1R gene silencing resulted in the ablation of the modulatory capacity of DMT on HIF-1α protein expression (Figure). These findings were further validated by an additional set of experiments in which we used the selective Sig-1R antagonist BD1063 to block Sig-1R-mediated signals (Figure). The effects of DMT on cellular survival and HIF-1α expression is thus dependent on the Sig-1R in human primary iPSC-derived neurons and monocyte-derived microglialike cells.
In a previous study we reported the expression of Sig-1R in human moMACs and moDCs. Although the expression of Sig-1R has been described in several human tissues its expression has not been investigated in iPSC-derived neurons. We therefore first examined the mRNA and protein level expression of Sig-1R in human in vitro differentiated iPSCderived cortical neurons during the process of differentiation. We found, that NSCs have low baseline levels of Sig-1R mRNA and protein which are increasing during the differentiation process and both peaking at the end of differentiation (Figure). The increase in Sig-1R protein levels exhibited statistical significance from the 21st day of differentiation as compared to baseline (NSC) values (Figure). It is in agreement with recent findings showing alterations in phenotypic similarities between primary tissue and iPSC-derived cortical neurons by the analysis of transcriptomic changes in several typical neural marker genes. After demonstrating the expression of Sig-1R in our in vitro cultures we next sought to investigate the effects of DMT treatment in hypoxic stress. We applied a severe hypoxia model setting oxygen levels to 0.5% (94.5% N 2 , 5% CO 2 ) in accordance with recent studies. Hypoxia caused-dominantly apoptotic-cell death in all types of cultures within 6 h. Interestingly, DMT-treated cultures exhibited significantly higher survival rates as measured by flow cytometry. Relatively low in vitro working concentrations of DMT were found to significantly increase the survival of cells (10-50 µM for iPSC-derived cortical neurons, and 50 µM for moMACs/moDCs) as compared to non-treated controls (Figure). Cellular viability was less affected in hypoxia-exposed moMAC and moDC cultures (Figures) in concert with recent findings reporting relative resistance to hypoxia of monocytes and monocyte-derived myeloid cells. To assess the influence of DMT on cellular physiology under hypoxia we monitored the expression and function of HIF-1α, an endogenous indicator of hypoxic stress. We found that HIF-1α was rapidly upregulated in all cell types following 6 h of hypoxia exposure, a phenomenon that was significantly prevented by the administration of DMT (Figure). Consistent with these results, under severe hypoxia, mRNA expression of the HIF-1α target gene VEGF was also significantly lower in DMT-treated human primary cells as compared to control cultures (Figure). Taken together, our results suggested that DMT may prevent or mitigate cellular stress in hypoxic environments. This protective effect may be brought about through various mechanisms. Since one of the proposed mechanism is the DMT-mediated activation of Sig-1R, we used the approach of gene-specific silencing in order to test the contribution of Sig-1R to the observed phenomenon. Our findings demonstrated that the downregulation of Sig-1R leads to the abrogation of DMT-mediated effects, as far as cellular survival and HIF-1α expressions are concerned, in hypoxic in vitro cultures of human primary cells (Figure). Furthermore, by using a Sig-1R-specific inhibitor we could further verify our results in all cell types in terms of cellular survival (Figure). These data suggest a critical, indispensable role of Sig-1R in the protective and antistress effects of DMT in human iPSCderived 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. Since the Sig-1R has been shown to promote neuronal survival against oxidative stressas well as to modulate immune processes, it is tempting to speculate that its activation by DMT, a natural, endogenous ligand, also results in similar physiological phenomena. One of the possible mechanisms is the fine-tuning of mitochondrial functions and consequently the regulation of cellular oxygen metabolism, a process that is indirectly related to HIF-1α expression and function. Secondly, Sig-1R activation by DMT may also result in modulated Ca 2+ signaling altering the function of intracellular kinases involved in cellular survival. Although altered mitochondrial functions may interfere with HIF-1α expression and activation, it has been shown that hypoxic stress can also be canalized downstream toward the nucleus through mitochondrion-associated intracellular stress pathways irrespective of HIF-1α (reviewed in. Since Sig-1R is primarily residing in the MAM membrane system, its activation likely affects an HIF-1α-independent pathway. Our results support this hypothesis as DMT-treated human primary cells exhibited higher survival rates despite their decreased HIF-1α expression, a phenomenon that suggest either moderate hypoxic stress response or the activation of a HIF-1α-independent pathway (Figure). Although we could exclude some of these additional stress-related pathways, such as NF-κB and ATF6 (Figure), the biochemical elucidation of these background mechanisms needs further investigations. This is the first study reporting that DMT, through the Sig-1R of human primary cells, can increase survival and alleviate cellular stress in hypoxic environments. This phenomenon is associated with increased cell viability and decreased expression and function of the stress factor HIF-1α in severe hypoxia-exposed, DMT-treated iPSC-derived cortical neurons, and monocyte-derived immune cells. The importance of microglia and microglia-like cells, such as monocytes, macrophages, and dendritic cells in hypoxia and post-injury recovery of the CNS has been recently reported. Thus, DMT may also notably contribute to neuroregenerative and neurorestorative processes by modulating the survival of microglia-like cells, such as moMACs and moDCs. In conclusion, our results suggest a novel and important role of DMT in human cellular physiology and point out to the relevance of DMT-mediated Sig-1R modulation in future therapies concerning hypoxia/ischemia-related pathologies.
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