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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C & D

W8 - Abstract for Poster Presentation for Project - Engineering 2D Neurons: Modeling Alzheimer’s Disease

Friday, October 10, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Alzheimer’s Disease (AD) affects 24 million worldwide, and early-onset familial AD (EOFAD)–caused by disease-penetrant mutations--offers a valuable model for studying AD. The repressor element-1 silencing transcription factor (REST) is a master transcriptional repressor of neuronal genes in non-neuronal cells, and has been previously shown to be aberrantly activated in neurons as a component of AD pathology. This results in dedifferentiation and a loss of neuronal identity as best indicated by alterations in chromatin topography and cell cycle re-entry. As a result, AD-afflicted neurons exist in a less neuronal state, leading to compromised function and eventual cell death. The inhibition of REST has unknown effects on the human brain, however, we hypothesize that REST inhibition can restore the neural cell fate of afflicted neurons. Currently, there remains a lack of mechanistic insight for the cause of AD and therefore inadequate treatment solutions available. Here, we investigate REST activation in AD iPSC-derived neurons and how dynamic inhibition of REST impacts neuron identity.

Materials and

Methods: : In our experiment, we grew iPSC-derived neurons in 6-cm dishes over 6 weeks using control and isogenically modified EOFAD mutant iPSCs in a two-fold experiment. In the first phase, we ran a pilot neuron generation to determine the optimal dose of X5050. To do this, we dosed the neurons with 0 µM, 1 µM, 10µM, and 100µM of X5050 REST inhibitor before conducting quantitative Polymerase Chain Reaction (qPCR) to extract data for RNA-Sequencing (RNA-Seq) to analyze transcriptomic changes and immunohistochemistry (IHC) to find common neuronal markers to characterize the health of the 2D neurons. In the second phase, we expanded biological replicates with 0µM and 1µM across AD and NDC conditions.

Results, Conclusions, and Discussions:: The qPCR analysis presented here assesses the expression of neuron function genes: SNAP25, SYP, BDNF, and SLC17A7. Since these genes are also REST targets, we then assessed the effect of a REST inhibitor, X5050, on neural gene expression. From the qPCR, we saw an increase in neuronal gene expression. The IHC analysis showed postmitotic neurons with complex neuron branching. Additionally, we observed clear nuclear degradation in the 100µM and 10µM X5050 dosage for both non-demented control (NDC) and AD neurons, leading us to move forward with only 1µM dose. In general, we found that certain conditions of REST inhibition did upregulate some neuronal genes, as hypothesized. However, REST inhibition did not completely restore their neural cell fate (cure the disease state), showing that REST inhibition likely targets a specific aspect of AD and suggesting that it is effective in addressing the onset of AD. Further analyses are underway to investigate potential mechanisms underlying this finding.
In this project, we were successful in completing a two-phase, 6-week differentiation of isogenic iPSC-derived neurons across multiple experimental conditions. First, we found an effective X5050 dosage of 1µM. Next, we expanded biological replicates to test our hypothesis that inhibiting REST would restore neuron function and neuron identity in disease samples. Analysis from qPCR and IHC showed inhibition of REST downstream targets in AD conditions. These findings provide newly discovered insights that REST is involved in neuron differentiation potentially playing a role in the progression of AD, which could inform future diagnostics to identify AD in its earlier stages. From there, we could also develop more effective interventions that target the underlying causes of AD by identifying the role of cell state changes in AD. Future directions include implementing additional downstream analyses, such as gene enrichment analysis via Gene Ontology databases and targeted exploration of REST regulatory networks that provide deeper insights into potential mechanisms in EOFAD. Alleviating cell state phenotypes using REST inhibition presents a new approach to AD treatment. Combining more advanced models in the future with relevant assays can offer insights into the underlying mechanism, and potential cause, of Alzheimer’s Disease.

Acknowledgements and/or References (Optional): : We would like to thank Tiffany Chu, Michael Tenreiro for their expertise and guidance and Jen Nguyen and Paul Savarino for their work throughout the project. We would also like to thank Dr. Alysson Muotri for allowing us to use his lab space for our project.