Deconstructing in vivo glia-to-neuron conversion

Project Information

Neuro-ReprOmics

Grant agreement ID: 101030217

DOI

10.3030/101030217

Project closed

EC signature date 2 August 2021

Start date 1 September 2022

End date 31 August 2024

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 212 933,76

EU contribution

€ 212 933,76

Coordinated by

KING'S COLLEGE LONDON
United Kingdom

Periodic Reporting for period 1 - Neuro-ReprOmics (Deconstructing in vivo glia-to-neuron conversion)

Reporting period: 2022-09-01 to 2024-08-31

Summary of the context and overall objectives of the project

The project addresses the challenge of understanding and influencing neuronal reprogramming in the brain, specifically focusing on how certain transcription factors can drive cellular transitions to neuronal states. This research involves examining gene expression, chromatin accessibility, and cellular phenotypes post-reprogramming, aiming to decode key molecular mechanisms. By leveraging advanced multiomic techniques such as single-cell RNA sequencing, ATAC sequencing, and spatial transcriptomics, the project seeks to uncover the intricate regulatory networks involved in neuronal reprogramming.

This work is of immense importance for society, particularly in the context of neurodegenerative diseases. The ability to reprogram cells into functional neurons offers transformative therapeutic potential, paving the way for innovative treatments aimed at replacing damaged or lost neurons. Additionally, understanding the molecular processes behind neuronal reprogramming could lead to breakthroughs in neuroplasticity and brain regeneration, providing insights into strategies for cognitive health preservation and recovery.

The overall objectives of the project include profiling gene expression and chromatin states across various reprogramming stages, establishing robust methodologies for combined single-cell RNA sequencing, ATAC sequencing, and spatial transcriptomics, and developing experimental techniques for in vivo tracking and analysis of reprogrammed cells at different stages. These objectives aim to contribute to a detailed understanding of cell fate transitions in the central nervous system and the role of transcription factors in neuronal development and plasticity.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project began with the profiling of gene expression in reprogrammed cells at various time points, focusing on the early stages of reprogramming (7, 14, and 28 days post-injection). Single-cell RNA sequencing was employed to capture the transcriptional landscape of reprogrammed cells and to identify significant gene markers that could indicate the progression of reprogramming. Early analyses revealed important changes in gene expression associated with neuronal differentiation, providing insight into the regulatory networks driving cell fate transitions.

One of the major advancements during this period was the optimization of single-nucleus RNA sequencing protocols. This was necessary to analyze more mature neuronal populations, particularly in samples collected at later time points. These optimized protocols provided high-quality nuclei profiles, enabling more reliable analysis of gene expression changes in later stages of neuronal reprogramming. This step was critical for improving the resolution of temporal changes in gene expression and for understanding how reprogrammed cells evolve over time.

Additionally, viral vector designs were improved to enable more efficient tracking of reprogrammed cells over extended periods. The incorporation of enhanced fluorophores in these vectors facilitated the long-term tracking of cell morphology, which was essential for understanding the sustained neuronal features and ensuring that reprogrammed cells maintained their identity over time. This improvement overcomes previous limitations in fluorescence stability, allowing for better visualization of cellular dynamics during the reprogramming process.

The results so far have contributed to a deeper understanding of the molecular changes that occur during neuronal reprogramming. Gene expression profiles have been integrated with chromatin accessibility data to provide a more complete picture of the transcriptional and epigenetic changes involved in neuronal differentiation. These results have provided valuable insights into the roles of specific transcription factors in driving reprogramming and have identified potential biomarkers for further study.

In terms of dissemination, findings have been shared at several workshops and scientific conferences, where the focus has been on methodological advancements and initial results related to gene expression changes during neuronal reprogramming. The development of the bioinformatics pipeline, which integrates data from various high-throughput sequencing techniques, has been a major highlight of the project and will be featured in upcoming publications. Manuscripts are currently being prepared for submission, detailing the techniques and results obtained during the project.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project has made significant progress beyond the state of the art in the field of neuronal reprogramming. One of the key innovations is the development of a pipeline that integrates single-cell RNA sequencing transcriptomics. This cutting-edge approach allows for highly detailed views of the reprogramming process at the transcriptional level.The project provides a more comprehensive understanding of how transcriptional networks influence neuronal reprogramming in situ.

Another major advancement is the design of novel viral constructs containing enhanced fluorophores. These vectors allow for sustained in vivo tracking of reprogrammed cells, surpassing previous limitations in fluorescence stability. This improvement enables better visualization of cell morphology over time, which is essential for studying long-term cellular behavior in reprogramming experiments.

The integration of gene expression data has led to a more nuanced understanding of the molecular processes that drive neuronal differentiation. By identifying key transcription factors and their binding sites, the project has uncovered important regulatory networks involved in reprogramming. This represents a significant step forward in understanding the molecular underpinnings of neuronal plasticity and could lead to the identification of novel targets for therapeutic intervention in neurodegenerative diseases.

Looking forward, the expected results until the end of the project include a comprehensive mapping of the transcription factor networks involved in neuronal reprogramming. This will include a deeper investigation into proneural factor binding and how it influences gene expression during reprogramming. The results will provide valuable insights into the dynamic changes that occur in reprogrammed neurons and how these changes can be manipulated to improve reprogramming efficiency and stability.

The potential impacts of this project are significant. Socioeconomically, the ability to reprogram neurons could revolutionize the treatment of neurodegenerative diseases, such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis (ALS). By providing a way to generate new neurons from existing cells, this approach could offer a sustainable solution to replacing damaged neurons, reducing the need for costly long-term care, and improving the quality of life for patients.

From a broader societal perspective, this research contributes to our understanding of brain health, plasticity, and regeneration. The knowledge gained from this project could have wide-ranging implications not only in neurodegenerative diseases but also in mental health and cognitive resilience. As the population ages and the prevalence of neurological disorders increases, the potential for neuronal reprogramming to provide therapeutic solutions becomes ever more important, making this research a valuable contribution to society.

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