A High-Throughput Single-Cell Sequencing Screen Identifies Small Molecules that Enhance Neurogenic Reprogramming of Murine Müller Glia

Core Concepts

High-throughput single-cell sequencing can identify small molecules that significantly enhance the neurogenic reprogramming of mouse Müller glia, a promising approach for restoring vision in retinal degeneration.

Abstract

The content describes the use of a high-throughput single-cell sequencing approach called sci-Plex to screen a library of 92 small molecules and identify compounds that can improve the neurogenic reprogramming of mouse Müller glia (MG).
The key highlights are:

The sci-Plex method was first used to study the temporal dynamics of MG reprogramming induced by the transcription factor Ascl1. This revealed insights into the gene expression changes and potential regulators like Myt1 that drive the transition from MG to neuronal fates.

The sci-Plex screen was then applied to test the effects of 92 small molecules on Ascl1-induced MG reprogramming. This identified several compounds, including the Notch inhibitor DBZ and the metabolic regulator metformin, that significantly enhanced neurogenesis.

The top hits from the in vitro screen were further validated in vivo using a mouse model of Ascl1-induced MG reprogramming. Both DBZ and metformin were found to boost the generation of MG-derived neurons compared to Ascl1 alone.

The study demonstrates the power of high-throughput single-cell sequencing approaches like sci-Plex to efficiently identify small molecule modulators of cell fate reprogramming, which could have important implications for regenerative therapies targeting the retina and other parts of the central nervous system.

Stats

Metformin treatment increased the percentage of GFP+ Müller glia-derived cells expressing the neuronal marker Otx2 from 50% to 75% compared to the Ascl1/NMDA/TSA control condition (p=0.017).
DBZ treatment increased the percentage of GFP+/Otx2+ Müller glia-derived neurons from 50% to 75% compared to the Ascl1/NMDA/TSA control (p=0.0009).

Quotes

"Repairing the nervous system has been an elusive goal, and while progress is being made with transplantation of pluripotent stem cell derived neurons, there are many complexities in neural transplantation. Reprogramming glia to serve as a source of neural repair is a strategy utilized by non-mammalian vertebrates, and inspired by nature, we and others have shown that harnessing this latent neural regenerative capacity by proneural TF over-expression can revive the neuronal reprogramming program in mammals."

Key Insights Distilled From

A multiplexed, single-cell sequencing screen identifies compounds that increase neurogenic reprogramming of murine Muller glia

by Tresenrider,... at www.biorxiv.org 09-27-2023

https://www.biorxiv.org/content/10.1101/2023.09.26.559569v2

Deeper Inquiries

What other signaling pathways or transcriptional regulators could be targeted to further enhance the neurogenic reprogramming of Müller glia?

To further enhance the neurogenic reprogramming of Müller glia, targeting additional signaling pathways and transcriptional regulators could be beneficial. One potential target could be the Wnt signaling pathway, which plays a crucial role in regulating cell fate determination and differentiation in various tissues, including the retina. Activation of the Wnt pathway has been shown to promote neurogenesis in different contexts, and modulating this pathway in Müller glia could potentially enhance their reprogramming into neurons.
Another promising target is the Sonic Hedgehog (Shh) signaling pathway, which is involved in regulating cell proliferation and differentiation during development. Activation of the Shh pathway has been linked to neurogenesis in the retina, and targeting this pathway in Müller glia could stimulate their conversion into neuronal cells.
In terms of transcriptional regulators, factors like Neurog2, Sox4, Myt1, and Rorb, which were identified in the study as being associated with the progression towards a neuronal fate, could be further explored. Modulating the expression or activity of these transcription factors may help in driving Müller glia towards a more efficient and complete neurogenic reprogramming process.

How might the effects of the identified small molecule hits, like metformin and DBZ, be combined with other approaches like gene therapy to maximize the regenerative potential of Müller glia?

Combining the effects of small molecule hits like metformin and DBZ with gene therapy approaches could potentially maximize the regenerative potential of Müller glia in the context of neural regeneration. One strategy could involve using gene therapy to overexpress key transcription factors like Ascl1 in Müller glia to initiate the reprogramming process towards a neuronal fate. Subsequently, the administration of small molecules like metformin and DBZ could enhance this reprogramming process by modulating specific signaling pathways or transcriptional regulators.
Metformin, known for its neuroprotective and anti-neuroinflammatory effects, could be used in conjunction with gene therapy to promote the survival and differentiation of reprogrammed Müller glia into functional neurons. By combining the neurogenic effects of metformin with the pro-neurogenic properties of Ascl1 overexpression, the overall efficiency and success of the reprogramming process could be significantly enhanced.
Similarly, DBZ, a γ-secretase inhibitor targeting the Notch pathway, could be used in combination with gene therapy to suppress inhibitory signaling pathways that hinder neurogenesis. By blocking Notch signaling with DBZ while simultaneously inducing the expression of proneural transcription factors through gene therapy, Müller glia could be pushed towards a more efficient and complete neuronal reprogramming process.

Given the broader implications for neural regeneration, could similar high-throughput single-cell screening strategies be applied to study reprogramming of other glial cell types in the central nervous system?

Yes, similar high-throughput single-cell screening strategies could be applied to study the reprogramming of other glial cell types in the central nervous system. The success of the sci-Plex screening approach in identifying small molecules that enhance neurogenic reprogramming of Müller glia demonstrates the potential of this method for studying glial cell reprogramming in other contexts.
For example, oligodendrocyte precursor cells (OPCs) in the central nervous system have the potential to be reprogrammed into neurons or other cell types. By applying high-throughput single-cell screening strategies like sci-Plex to OPC cultures, researchers could identify novel compounds or factors that promote the conversion of OPCs into functional neurons, offering new avenues for neural regeneration and repair in conditions like spinal cord injury or demyelinating diseases.
Similarly, astrocytes, another type of glial cell in the central nervous system, could be targeted for reprogramming towards a neuronal fate using similar screening approaches. By screening libraries of small molecules or biologics in astrocyte cultures and combining them with gene therapy techniques, researchers could uncover new strategies for enhancing neurogenesis and neural repair in various neurological disorders.
Overall, the application of high-throughput single-cell screening strategies to study glial cell reprogramming holds great promise for advancing our understanding of neural regeneration and developing novel therapeutic interventions for neurological conditions.