Psilocin fosters neuroplasticity in iPSC-derived human cortical neurons

Reviewer #1 (Public review):

Summary:

This study reports the effects of psilocin on iPSC-derived human cortical neurons.

Strengths:

The characterization was comprehensive, involving immunohistochemistry of various markers, 5-HT2A receptors, BDNF, and TrkB, transcriptomics analyses, morphological determination, electrophysiology, and finally synaptic protein measurements. The results are in close agreement with prior work (PMID 29898390) on rat-cultured cortical neurons. Nevertheless, there is value in confirming those earlier findings and furthermore demonstrating the effects in human neurons, which are important for translation. The genetic, proteomics, and cell structure analyses used in this paper are its major strengths. The study supports the value of using iPSC-derived human cortical neurons for drug development involving psychedelics-related compounds.

Weaknesses:

(1) Line 140: 5-HT2A receptor expression was found via immunocytochemistry to reside in the somatodendritic and axonal compartments. However, prior work from ex vivo tissue using electron microscopy has found predominantly 5-HT2A receptor expression in the somatodendritic compartment (PMID: 12535944). Was this antibody validated to be 5-HT2A receptor-specific? Can the authors reason why the discrepancy may arise, and if the axonal expression is specific to the cultured neurons?

(2) Line 143: It would be helpful to specify the dose of psilocin tested, and describe how this dose was chosen.

(3) Figure 1: The interpretation is that the differential internalization in the axonal and somatodendritic compartments is time-dependent. However, given that only one dose is tested, it is also possible that this reflects dose dependence, with the longer time exposure leading to higher dose exposure, so these variables are related. That is, if a higher dose is given, internalization may also be observed after 10 minutes in the dendritic compartment.

(4) Figure 3 & 4: What is the 'control' here? A more appropriate control for the 24 hours after psilocin application would be 24 hours after vehicle application. Here the authors are looking at before and after, but the factor of time elapsed and perturbation via application is not controlled for.

(5) The sample size was not clearly described. In the figure legend, N = the number of neurites is provided, but it is unclear how many cells have been analyzed, and then how many of those cells belong to the same culture. These are important sample size information that should be provided. Relatedly, statistical analyses should consider that the neurites from the same cells are not independent. If the neurites indeed come from the same cells, then the sample size is much smaller and a statistical analysis considering the nested nature of the data should be used.

Reviewer #2 (Public review):

In this article, Schmidt et al use iPSC-derived human cortical neurons to test the effects the psychedelic psilocin in different models of neuroplasticity.

Using human iPSC-derived cortical neurons, the authors test the expression of 5-HT2A and subcellular distribution, as well as the effect of different times of exposure to psilocin on 5-HT2A expression. The authors evaluated the effect of the 5-HT2 antagonist ketanserin, as well as the inhibition of dynamin-dependent endocytic pathways with dynasore. Gene expression and plasticity (structural and functional) was also evaluated after different times of exposure to psilocin.

In general, results are interesting since they use the iPSC to evaluate the potentially translationally relevant effects of psilocin (the active metabolite of the psychedelic psilocybin). However, there are a few concerns that need to be addressed:

(1) My main critique is the lack of experimental validation of selectivity and/or specificity of the anti-5-HT2A antibody targeting the extracellular loop of the 5-HT2A receptor (Alomone labs, cat # ASR-033). Most of the primary antibodies targeting class A GPCRs (including the 5-HT2A receptor) have very limited selectivity. Without validation (using for example knockdown techniques to decrease expression of 5-HT2A in their iPSC-derived human cortical neurons), the experiments using this antibody should be excluded from the manuscript.

(2) Did the author evaluate whether 5-HT is present in the cell media? If it is, this may affect the functional outcomes evaluated throughout, since as the endogenous ligand it would in principle activate the 5-HT2A receptor.

(3) Some of the datasets are not statistically analyzed (or quantified), such as Figure S1F.

(4) Another important concern is the experimental design used to evaluate the effect of psilocin at different time points (24h, 4 days and 10 days). One of the unique and translationally interesting effects of psychedelics including psilocybin is that the in vivo plasticity-related effects (increased structural or synaptic plasticity for example) are observed post-acutely, or once the active compound psilocin is fully metabolized, or not present in the CNS directly targeting the 5-HT2A. Using the iPSC, it seems that the authors continuously exposed cells to psilocin (for hours or even days) at least for some of the experimental techniques. Since this is not the model of what occurs using an in vivo model (such as a single dose of psilocybin to mice, collecting frontal cortex samples 24-h after drug administration, once the active compound is fully metabolized), the authors' findings lack translational validity. Can the authors comment on this?

(5) In Figure 2E, it seems that ketamine by itself is reducing BDNF density. How then the authors conclude that ketamine blocks psi-induced effects? Using a more selective 5-HT2A antagonist such as M100907 could also improve the outcome (in terms of selectivity) of this experiment.

(6) To evaluate neurite complexity, the authors used the AAV-CamKII-mCherry viral vector, but mCherry (Fig 4A) seems to be retained in the nucleus.

(7) Minor: Reference 36- this is a review article that does not mention the psychedelic psilocin

Author response:

We sincerely thank the reviewers for their thorough and constructive evaluation of our manuscript. We particularly appreciate their recognition of our comprehensive characterization approach, which integrates immunohistochemistry, transcriptomics, morphological assessments, and electrophysiology to understand psilocin's effects on human neurons. The reviewers highlighted that our findings closely align with and validate prior work on rat cortical neurons, while importantly extending these insights to human cells. We are encouraged by their acknowledgment that our study demonstrates the value of using iPSC-derived human cortical neurons for testing potentially translatable effects of psychedelic compounds. Their positive assessment of our work's implications for psychedelic drug development is particularly valuable, as it supports our goal of advancing the understanding of these compounds' therapeutic potential and their possible application in treating neuropsychiatric disorders.

We are also very grateful for the reviewers' constructive criticism which will help strengthen our manuscript significantly. Based on their detailed feedback, we plan to perform several additional experiments for inclusion in the revised manuscript.

The most important concern raised by both reviewers is about the specificity of the antibody used to detect the expression pattern and abundance of 5-HT2A receptors at the cells' surface. We acknowledge that GPCR antibodies, including those targeting 5-HT2A receptors, can be challenging in terms of specificity and reliability, particularly given the structural similarities within this receptor family. To address these concerns comprehensively, we propose the following systematic validation strategy:

(1) Cell-Type Specific Expression Analysis: We will systematically evaluate the antibody across different developmental stages and cell lines. The results from the stainings will be correlated with RNA sequencing data to provide quantitative validation of expression patterns. Cell types to be included will be:

· iPSCs (expected negative)

· Neural progenitors (expected positive)

· Mature neurons (expected positive)

· HEK cells (expected negative) This multi-stage analysis will allow us to track receptor expression through development and verify antibody specificity across distinct cellular contexts.

(2) Peptide Competition Study: We will perform blocking experiments using the specific peptide sequence against which the antibody was raised. By pre-incubating the antibody with its cognate peptide at established working concentration, followed by detailed documentation of signal reduction in peptide-blocked condition versus standard staining, we can demonstrate binding specificity. This approach will provide direct evidence of antibody selectivity for its intended target.

(3) Sequence Analysis and Specificity: We will perform a comprehensive protein BLAST analysis of the antigenic peptide sequence, assess potential cross-reactivity with related receptors, and evaluate species conservation and specificity. This in silico approach will complement our experimental validation and help identify any potential off-target binding sites.

(4) Additional Validation: While technically challenging, we will attempt knockdown studies using siRNA/shRNA approaches to provide additional validation of antibody specificity. This molecular intervention will offer another layer of validation through targeted reduction of the receptor.

We plan to present these results in a new supplementary figure that will provide a comprehensive overview of our validation efforts. Should we not be able to convincingly demonstrate the specificity of the antibody, we will discuss with the editors and reviewers to modify Figure 1 and exclude critical parts from the manuscript. While we find the results interesting and important to communicate, an omission would not critically impact the key message of the manuscript, which is the structural and molecular changes elicited by psilocin on human neurons. The strength of our multi-modal approach means that our core findings are supported by several independent lines of evidence beyond antibody-based detection.