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

Abstract

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 IRinduced 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.