Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT

Earlier research has identified dimethyltryptamines — including N,N-DMT, 5-HO-DMT and 5-MeO-DMT — as serotonin-like entheogens with reported antidepressant and cognitive effects and with activity at serotonin (5-HT) receptors and sigma-1 receptors (σ-1R). However, legal constraints and a lack of suitable human experimental models have limited mechanistic understanding of how these compounds alter human brain biology. Advances in differentiation of human pluripotent stem cells into three-dimensional cerebral organoids provide a system that recapitulates multiple brain cell types and aspects of cortical architecture, offering a platform to study drug effects on human neural tissue and plasticity that may not be captured in simple monolayer cultures or animal models. Dakic and colleagues set out to characterise short-term molecular effects of 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human neural preparations, comparing simple human neural progenitor cell (hNPC) monolayers with 3D cerebral organoids. Using unbiased, mass spectrometry-based shotgun proteomics, the study aimed to identify proteome-wide changes after a 24-hour exposure and to infer affected pathways related to inflammation, synaptic plasticity (including long-term potentiation, LTP), cytoskeletal dynamics and cell survival.

The investigators used human embryonic stem cells (BR1 line) to generate two preparations: adherent human neural progenitor cells (hNPCs) and 45-day-old cerebral organoids. hNPCs were derived by directed differentiation and expanded for up to five passages; assays on these cells included high-content screening (HCS) for proliferation, cell death and early neuronal arborisation. Cerebral organoids were grown in spinner flasks following an established protocol, embedded in Matrigel and matured for 45 days before experimentation. Treatment conditions for organoids comprised 24-hour exposures to 13 µM 5-MeO-DMT, a vehicle control (0.3% ethanol) or medium alone; four to five organoids were allocated per group and the entire experiment was repeated three times using three different organoid derivations. For hNPCs, a concentration range of 23 nM to 7.11 µM 5-MeO-DMT was tested in quintuplicate wells for four days in HCS assays. Expression of receptors (5-HT2A, 5-HT2C, σ-1R) and synaptic markers (AMPAR, NMDAR, MAP2, GFAP) was assessed by PCR and immunostaining. Proteomic analysis of whole-organoid homogenates used a label-free, high-definition data-independent acquisition (HDMSE) workflow on a 2D-LC–MS/MS platform (Synapt G2-Si). Samples (50 µg protein per sample) were run in technical and biological triplicates (nine runs per sample). Peptide and protein identification required at least two unique peptides and an overall false discovery rate (FDR) below 1%. Differential expression was determined using a log2 fold-change threshold (reported as >2 or <−2 in the extracted text). In silico pathway and network analyses of differentially expressed proteins were performed with Ingenuity Pathway Analysis (IPA), applying Fisher’s exact test and Benjamini–Hochberg correction at a 1% FDR for multiple comparisons.

In monolayer hNPC cultures, Dakic et al. found no significant effects of 5-MeO-DMT on cell death, proliferation or early neuronal arborisation across the tested concentration range (23 nM to 7.11 µM); hNPCs expressed σ-1R but lacked detectable 5-HT2A/2C receptor expression. By contrast, 45-day-old cerebral organoids showed expression of neuronal markers (MAP2), glutamate receptors (AMPAR, NMDAR) and glial marker GFAP, and PCR/immunostaining detected 5-HT2A, 5-HT2C and σ-1R, supporting organoids as a relevant model for 5-MeO-DMT actions. Proteomic profiling after 24-hour exposure revealed deep coverage: 144,700 peptides (FDR < 1%) mapping to 6,728 unique proteins (identified by at least two unique peptides and present in at least two of three biological replicates). Of the proteins commonly identified across treatment groups, 934 were reported as differentially expressed using the stated log2 ratio cut-off, with 574 upregulated and 360 downregulated in 5-MeO-DMT-treated organoids versus vehicle. Functional enrichment of these combined changes predicted activation (IPA z-score) of dendritic spine and cellular protrusion formation, microtubule and cytoskeletal organisation, and a mild activation of T lymphocyte differentiation; conversely, processes related to neurodegeneration, cell death and brain lesion were predicted to be inhibited. Pathway-level analysis indicated inhibition of nuclear factor of activated T-cells (NFAT) and nuclear factor kappa B (NF-κB) signalling, via toll-like receptors (TLRs) and Gq-coupled receptors — the latter relevant because 5-HT2A/2C are Gq-coupled. Regarding molecules implicated in long-term potentiation (LTP), several components were differentially regulated: upregulated proteins included NMDAR, CaMK2 and CREB, while a group of signalling proteins were downregulated, notably mGluR5, Gαq, PKC, PLC, calmodulin, adenylyl cyclases AC1/8, IP3R, EPAC1 and PKA. AMPAR and the downstream cascade to c-Raf, MEK1/2 and ERK1/2 were upregulated, consistent with a signalling route that can activate CREB-dependent gene expression. Proteins linked to cytoskeletal reorganisation and dendritic spine morphogenesis were also altered. Ephrin-B2 (EFNB2) and EphB receptors (EPHB) were upregulated, together with intersectin, CDC42, N-WASP and ARP2/3, and ELMO1-mediated activation of RAC1, indicating activation of actin-regulatory pathways that promote spine morphogenesis. Reverse ephrin signalling components (plexins, NCK/GRB4, FAK, paxillin) and changes in plexins, integrins, srGAP, the netrin receptor DCC and metalloproteinases were also reported, supporting broader modulation of synaptic architecture. Finally, the authors noted that cell-death–associated pathways were predicted to be inhibited, a pattern consistent with known σ-1R–mediated neuroprotective mechanisms.

Dakic and colleagues interpret their data as evidence that a single 24-hour exposure to 5-MeO-DMT produces proteomic changes in human cerebral organoids consistent with anti-inflammatory effects, enhanced dendritic spine formation and modulation of signalling pathways linked to synaptic plasticity and cell survival. The observed inhibition of NFAT and NF-κB pathways via TLR and Gq-coupled receptor signalling is taken as molecular support for an anti-inflammatory profile previously reported in immune cells for 5-MeO-DMT and other serotonergic psychedelics. The requirement for a multicellular, 3D organoid context — since monolayer hNPCs showed no changes — led the authors to emphasise that network complexity and cellular diversity are important for the drug’s actions. With respect to synaptic plasticity, the authors describe a complex pattern of LTP-related protein modulation that could produce cell-type–specific augmentation or inhibition of LTP; they note that functional confirmation (for example electrophysiology) was not performed. Downregulation of mGluR5 is highlighted as potentially relevant to addiction biology, as reduced mGluR5 activity has been associated with decreased drug-seeking behaviours in preclinical models and may help to explain reports of Ayahuasca-related reductions in substance dependence. Changes in ephrin/EphB pathway components, σ-1R–related effectors and multiple actin-regulatory proteins are discussed as mechanisms capable of promoting dendritic spine morphogenesis and remodelling of synaptic architecture. The authors acknowledge important limitations: the findings derive from an in vitro organoid model and reflect proteomic inferences rather than direct functional or behavioural outcomes; electrophysiological and in vivo studies are necessary to establish how the molecular changes translate to synaptic function and behaviour. They also note that the study examined a single acute (24-hour) exposure and that effects of other dimethyltryptamines or non-psychoactive tryptamines cannot be assumed to be the same. Finally, Dakic et al. position organoids as a promising platform to investigate human-specific neural responses to psychoactive compounds but call for further investigations to link acute molecular effects with longer-term and organism-level consequences.