Impact of Psilocybin on Vitreous Induced Inflammation in Lens Epithelial Explants

Research Group

Robinson Lab, Miami University

Jeffery T Fletcher*, Jacob M Weaver, Brad D Wagner, Michael L Robinson

*undergraduate presenter

Introduction

•Psilocybin is a psychoactive tryptamine found in psychedelic mushrooms

•Psilocybin has been shown as a promising treatment for diseases1 like Major Depressive Disorder, OCD, and addiction

•Believed to act on serotonin receptor 5HT-2A

•A link has been established between inflammation and depression (as well as other neurodegenerative diseases)

•The ocular lens is an ideal model for monitoring cell development due to its simple structure and presence of only two cell types

•Vitreous humor induces inflammation in lens epithelial explants2

•RNA-Seq has revealed that the 5HT-2A receptor is expressed in the lens epithelium

•Other treatments for inflammation, like NSAIDs and immunosuppressants, can cause GI issues and immunodeficiency, respectively – psilocybin has mild effects in comparison

Hypothesis: Treatment of lens epithelial explants with psilocybin will inhibit vitreous-induced inflammation through changes in gene expression

Methodology

Image showing the lens explantation process. 4 step figure shows the lens capsule being opened with forceps, and removal of fiber cells in blue, leaving a sheet of lens epithelial cells in red.

1. Lens Epithelium Explant System

The lens of a P8 mouse will be positioned so that the posterior side is upward, the lens capsule will then be opened, and the fiber cells (blue) will be removed. The capsule will then be pinned to the culture dish, resulting in the epithelial cells being exposed (red).

Image of experiemental design. Timeline shows explant process occurs on day 0, treatment occurs on day 1, and rna extraction occurs on days 2 or 6 depending on the experimental group.

2. Experimental Design

The epithelial cells of P8 (8 days after birth) mice will be explanted and incubated with culture media for 24 hours. They will then be treated with either differentiation media alone or differentiation media and psilocybin. The RNA from these cells will be extracted at either 1 day following treatment or 5 days. This RNA will then be sequenced by an outside lab.

3. Statistical Analysis

From the raw data we receive from the sequencing lab, we will process it using bioinformatics software to understand the trends in the data, which are used to make the resulting figures.

Results

Bar graph with 4 groups: DM1, DM5, PSI1, and PSI5. The bars show that the gene Htr2a has 38, 7, 51, and 14 transcripts, respectively for the 4 groups.

Supplemental Figure S1

Figure S1: Bar graph from RNA-seq data showing that the 5HT-2A receptor is present in our lens epithelial explant samples at transcript level.

PCA plot showing similarity of samples based on their clustering. DM5 samples A, B, and C all cluster close to one another and distinct from other groups. This is observed for PSI5 samples A, B, and C as well. The samples of PSI1 and DM1 cluster close to one another.

Figure 1

Figure 1: PCA plot of lens epithelial explant samples treated with vitreous humor or both vitreous and psilocybin for 1 or 5 days prior to RNA extraction and subsequent RNA sequencing.

Heat map showing relative gene expression for genes associated with the lens. The 6 genes shown: Crygf, Crygc, Crygb, Crybb1, Cryab, and Emilin3 all have significantly higher expression following psilocybin treatment.

Figure 2A

Heat map showing relative gene expression for genes associated with the lens. The 8 genes shown: Bnip3, Pitx2, Pdgfra, Fgfr5, Eln, Cdh5, Sox5, and Fbn2 all have significantly lower expression following psilocybin treatment.

Figure 2B

Figure 2: Heat maps of differentially expressed lens genes that are either A) upregulated or B) downregulated upon psilocybin treatment at D5 (RNA extracted 5 days following treatment).

Differentially expressed: Log2FC > +/- 1, p-adj < 0.05

Heat map showing relative gene expression for genes associated with increased inflammation. The 15 genes shown all have significantly lower expression following psilocybin treatment.

Figure 3A

Heat map showing relative gene expression for genes associated with BMP signaling. The 12 genes shown all have significantly lower expression following psilocybin treatment.

Figure 3B

Figure 3: Heat maps of differentially expressed genes related to A) inflammation and B) BMP signaling at D5 (RNA extracted 5 days following treatment).

Differentially expressed: Log2FC > +/- 1, p-adj < 0.05

Table listing categories for genes upregulated by the addition of psilocybin. Key categories include: Detoxification, cellular detoxification, cellular response to toxic substance, and cellular oxidant detoxification.

Table listing categories for genes downregulated by the addition of psilocybin. Key categories include: anatomical structure morphogenesis, system development, developmental process, and epithelium development.

Figure 4A

Figure 4B

Figure 4: Gene Ontology (GO) Terms for differentially expressed genes between vitreous treated samples and samples treated with both vitreous and psilocybin at D5. A) GO terms for genes upregulated by the addition of psilocybin. B) GO terms for genes downregulated by the addition of psilocybin.

Venn diagram comparing genes whose expression was significantly changed at Day 1 and Day 5. 23 genes were dysregulated only at Day 1, 1259 genes were dysregulated only at Day 5, and 10 were dysregulated at both Day 1 and Day 5. The list of those 10 genes is given to the right of the Venn diagram, with key transcription factors Zic1 and Otx1 listed in bold.

Figure 5

Figure 5: Venn Diagram of genes differentially expressed at Day 1 and/or Day 5 following psilocybin treatment. The 10 genes expressed at both time points are listed to the right, with key transcription factors Zic1 and Otx1 in bold.

Heat map showing relative gene expression for genes encoding key transcription factors. Of the 5 genes shown, 4 have significantly lower expression 1 day following psilocybin treatment: Zic1, Zic4, Six6, and Otx1.

Figure 6

Figure 6: Heat maps displaying the z scores of key transcription factors Egr2, Zic1, Zic4, Six6, and Otx1 at D1 (RNA extracted 1 day following treatment with or without 100 uM psilocybin). Differentially expressed: Log2FC > +/- 1, p-adj < 0.05

Conclusions

Serotonin receptor signaling causes deviation from traditional lens differentiation trajectory following vitreous treatment
- May be due to altered Fgf signaling, which is responsible for fiber cell differentiation
Psilocybin treatment is associated with decreased BMP signaling genes, which is known to be important for lens epithelial cell polarity and has implications in inflammation
Zic1 and Otx1 are downregulated at D1 and D5 (key transcription factors)
- This downregulation may permanently alter fate of cell differentiation
- Serotonin binding/psilocybin may be interfering with Fgf signaling induced by vitreous humor
Upregulation of detoxifying genes at D5 may be delayed/long term response to psilocybin

Future Directions

Compare this data to culture media for both D1 and D5
- Provides clarity on the impact of vitreous and the comparison between psilocybin treated cells and those without any vitreous treatment
Test psilocybin’s ability to reduce already present inflammation, rather than resisting it when both are added simultaneously
- This has more therapeutic potential than psilocybin treatment in anticipation of inflammation
Use norbaeocystin, a reportedly non-hallucinogenic derivative of psilocybin to see its effectiveness
- Will also show whether the effects are due to psilocybin specifically or the binding of the 5HT-2A receptor regardless of the molecule

References

1. Davis AK, Barrett FS, May DG, Cosimano MP, Sepeda ND, Johnson MW, et al. Effects of Psilocybin-Assisted Therapy on Major Depressive Disorder: A Randomized Clinical Trial. JAMA Psychiatry 2021;78:481–9.

2. Upreti A, Padula SL, Tangeman JA, Wagner BD, O’Connell MJ, Jaquish TJ, et al. Lens Epithelial Explants Treated with Vitreous Humor Undergo Alterations in Chromatin Landscape with Concurrent Activation of Genes Associated with Fiber Cell Differentiation and Innate Immune Response. Cells 2023;12.: https://doi.org/10.3390/cells12030501.

3. Adams, Teri & Adler, Ruben & Beebe, David. (2002). Bone morphogenic protein signaling and the initiation of lens fiber cell differentiation. Development (Cambridge, England). 129. 3795-802. 10.1242/dev.129.16.3795.

Acknowledgments

Center for Bioinformatics and Functional Genomics (CBFG)
Brad D. Wagner
Abhishek Kumar Sen

NACE Competencies

From this research experience, the three most significant NACE competencies which I have gained are Leadership, Communication, and Technology.

Leadership - I was the first undergrad to join this project, which was significantly different than the traditional research of this group. Research on psilocybin has not been going on for very long as it had been a federally banned substance, so taking up this project and leading the charge is something that I am proud of.
Communication - There have been several bumps in the road regarding this project, and it has been important for me to constantly be in communication with the other people working on the project to ensure that we are accomplishing our goals.
Technology - A large part of this research is data analysis. Before working in this research group, and specifically on this project, I had little to no bioinformatics experience. However, as a result of this project, I have learned and refined my skills using bioinformatics software to process, analyze, and present complex data sets.

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