Brain development and response to injury : focus on microglia and cranial irradiation

Postnatal brain development is crucial for proper brain formation and function in adulthood. Impairment of this process due to external insults such as cranial radiotherapy, used to treat brain tumors and metastases, can lead to severe cognitive sequelae in cancer survivors, particularly children. However, currently, neither a treatment nor established biomarkers exist to alleviate or predict the extent of brain injury and subsequent cognitive impairments following radiotherapy.

Microglia, the resident immune cells and macrophages of the brain, play a vital role in the refinement, maturation, and maintenance of neuronal circuits during development. Therefore, understanding microglial dysregulation caused by ionizing radiation (IR) and its impact on neurocognitive outcomes is of great importance.

Consequently, the primary objective of this thesis was to analyze the role of microglia in brain development and their response to cranial irradiation. This includes a specific focus on temporary microglial subtypes and their contribution to irradiation-associated neuroinflammation. Additionally, we aimed to identify potential biomarkers for irradiation-induced brain damage that can be measured in easily accessible clinical samples.

First, we identified a spatiotemporally distinct microglial subtype, arginase 1- expressing (ARG1+) microglia. This subtype, while morphologically similar to other microglial cells, exhibits a unique molecular profile and is crucial for the maturation of the basal forebrain cholinergic system, synaptic plasticity, and cognitive functions in female mice.

Next, we developed a simple, rapid, and reproducible cell isolation method to generate single-cell suspensions from brain subregions, enriched with microglial and vascular cells. This method is suitable for integrative longitudinal molecular studies using single-cell RNA sequencing.

Using this method, we performed a single-cell longitudinal analysis of the microglial responses following cranial IR and identified several hippocampal radiation-associated microglia (RAM) subtypes that emerge from hours to one year after IR. We found that IR causes progressive decrease in microglial cells, that fail to repopulate their loss by self-renewal, and this leads to the infiltration of monocytes-derived macrophages that differentiate into microglial-like cells to compensate for the microglial loss. Moreover, all these events were correlated with episodes of neuronal asynchrony.

Finally, we propose EDA2R as a promising biomarker candidate for cranial IR- induced brain injury, as its levels rapidly increase in liquid biopsies (cerebrospinal fluid and plasma) following cranial IR.

The results presented in this thesis provide new insights into the role of microglia during postnatal brain development and their response to cranial irradiation. They also highlight a potential biomarker for radiotherapy-induced brain damage.

In summary, this research is important for the field of neuroscience. It is particularly significant for advancing our understanding of radiotherapy-related neurocognitive complications, with the aim of improving the quality of life of pediatric brain cancer survivors.

List of scientific papers

I. Vassilis Stratoulias, Rocío Ruiz, Shigeaki Kanatani, Ahmed M. Osman, Lily Keane, Jose A. Armengol, Antonio Rodríguez-Moreno, Adriana- Natalia Murgoci, Irene García-Domínguez, Isabel Alonso-Bellido, Fernando González Ibáñez, Katherine Picard, Guillermo Vázquez- Cabrera, Mercedes Posada-Pérez, Nathalie Vernoux, Dario Tejera, Kathleen Grabert, Mathilde Cheray, Patricia González-Rodríguez, Eva M. Pérez-Villegas, Irene Martínez-Gallego, Alejandro Lastra- Romero, David Brodin, Javier Avila-Cariño, Yang Cao, Mikko Airavaara, Per Uhlen, Michael T. Heneka, Marie-Ève Tremblay, Klas Blomgren, Jose L. Venero & Bertrand Joseph. ARG1-expressing microglia show a distinct molecular signature and modulate postnatal development and function of the mouse brain. Nature Neuroscience, volume 26, pages 1008-1020 (2023).
https://doi.org/10.1038/s41593-023-01326-3

II. Efthalia Preka*, Alejandro Lastra Romero*, Ying Sun, Yara Onetti Vilalta, Thea Seitz, Adamantia Fragkopoulou, Christer Betsholtz, Ahmed M Osman, Klas Blomgren. Rapid and robust isolation of microglia and vascular cells from brain subregions for integrative single-cell analyses. Heliyon. volume 10, issue 16 (2024); e35838. * Denotes equal first-author contribution
https://doi.org/10.1016/j.heliyon.2024.e35838

III. Alejandro Lastra Romero*, Efthalia Preka*, Giusy Pizzirusso, Luis Enrique Arroyo- Garcia, Georgios Alkis Zisiadis, Nuria Oliva-Vilarnau, Thea Seitz, Kai Zhou, Arturo Gonzalez Isla, Lara Friess, Ying Sun, Alia Shamik, Changlian Zhu, Carlos F. D. Rodrigues, André Fisahn, Bertrand Joseph, Lena-Maria Carlson, Adamantia Fragkopoulou, Christer Betsholtz, Volker M Lauschke, Ahmed M Osman, Klas Blomgren. Microglia Adopt Temporally Specific Subtypes after Irradiation, Correlating with Neuronal Asynchrony. * Denotes equal first-author contribution. [Manuscript]

IV. Alejandro Lastra Romero, Thea Seitz, Georgios Alkis Zisiadis, Holli Jeffery, Ahmed M Osman. EDA2R reflects the acute brain response to cranial irradiation in liquid biopsies. Neuro-Oncology (2024); noae077.
https://doi.org/10.1093/neuonc/noae077