Prevention and treatment of cognitive decline in a mouse model of cranial radiotherapy

Pediatric brain cancers remain the leading cause of cancer-related mortality in children. With advancements in medical technology, survival rates have significantly improved, with more children reaching the five-year survival milestone. However, for many, survival comes at a high cost. Cranial radiotherapy, a cornerstone in tumor management and metastasis control, leaves lasting imprints on the developing brain. Among its most devastating consequences are late-emerging cognitive complications that persist throughout life, profoundly affecting the quality of life for survivors. Despite growing recognition of these impairments, the underlying mechanisms remain elusive, and no effective treatments exist.

Furthermore, sex has emerged as a potential risk factor influencing the severity of these deficits, adding another layer of complexity to an already pressing clinical challenge.

One of the leading hypotheses attributes these cognitive deficits to the depletion of neurogenesis, the birth of new neurons, in the hippocampus, a central hub for learning and memory. The failure of hippocampal neurogenesis to recover after radiation is thought to stem from chronic microglial activation. As the primary immune cells of the central nervous system (CNS), microglia are essential for normal brain maturation, synaptic refinement, and immune surveillance. Hence, understanding how microglia respond to irradiation over time, could provide critical insights into the mechanisms driving radiation-induced cognitive impairments.

This thesis investigates the long-term alterations in microglia across the mouse lifespan, both in normal aging and following cranial irradiation. A particular focus is also placed on the sex-dependent neuroinflammatory response. Beyond identifying key molecular events after irradiation, we sought to identify key components of the inflammatory cascade that could be therapeutically targeted to mitigate radiation-induced brain injury.

To enable this investigation, we developed a rapid and robust single-cell isolation protocol for brain subregions, facilitating the enrichment of traditionally hard-to- isolate microglia and vascular cells. Importantly, our method eliminates the need for additional enrichment steps, making it highly reproducible and well-suited for unbiased longitudinal single-cell RNA sequencing studies across different brain regions, time points, and cell types.

Leveraging this novel approach, we conducted a comprehensive longitudinal analysis of the hippocampal response to cranial irradiation, spanning up to one year post-exposure. Microglia emerged as the most distinct and dynamically affected population, prompting an in-depth examination of their radiation- induced states. Our findings revealed the presence of temporally specific radiation-associated microglia (RAM) subtypes, characterized by a delayed yet sustained inflammatory response in the hippocampus. This persistent activation, spearheaded by microglia, appeared to play a crucial role in shaping long-term microglial dynamics following irradiation. We observed a progressive loss of microglia, which failed to compensate for their depletion through self-renewal. This deficit ultimately led to the infiltration of monocyte-derived macrophages, which gradually adopted a microglial-like phenotype. Functional analysis revealed a correlation between these molecular events and episodes of neuronal asynchrony, suggesting a potential link between radiation-induced microglial dysfunction and impaired network activity.

Building upon these findings, we explored sex-dependent differences in the inflammatory response to cranial irradiation. Transcriptomic profiling revealed a more pronounced inflammatory signature in female hippocampi compared to males, driven primarily by microglia and likely mediated by distinct molecular mechanisms. Expanding our analysis beyond the hippocampus to cognition- related areas such as the cortex and cerebellum, we uncovered differential susceptibilities across brain regions. While females exhibited the strongest inflammatory response in the hippocampus, they also showed the greatest microglial loss in the cortex-a pattern mirrored in males, albeit to a lesser extent.

To translate these insights into potential therapeutic strategies, we employed a combination of computational and in vivo approaches to identify key regulators of the inflammatory pathway. By modulating the expression of these targets at critical time points using clinically approved antisense oligonucleotides, we demonstrated that dampening their activity could effectively reduce irradiation- induced neuroinflammation in the hippocampus. These findings highlight a promising avenue for mitigating cognitive decline in pediatric brain cancer survivors.

Finally, to contextualize our findings within the broader landscape of neurodegenerative processes, we examined microglial dynamics in an AppNL-G-F knock-in mouse model, resembling amyloid pathology and Alzheimer's disease. Our analysis revealed dynamic transcriptional changes in microglia before and after plaque accumulation, coinciding with disruptions in hippocampal network activity, impaired synchronization of fast-spiking interneurons and plaque formation.

Overall, this thesis provides novel and critical insights into microglial responses during normal brain aging and in the aftermath of cranial irradiation. By elucidating sex-specific vulnerabilities and identifying key inflammatory mediators, our work lays the foundation for potential therapeutic interventions aimed at preserving cognitive function. Ultimately, these findings bring us one step closer to improving the quality of life for pediatric brain tumor survivors and their families.

List of scientific papers

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

II. Microglia Adopt Temporally Specific States after Irradiation, Correlating with Neuronal Asynchrony

Alejandro Lastra Romero#, Efthalia Preka#, Giusy Pizzirusso, Luis Enrique Arroyo-García, Georgios Alkis Zisiadis, Nuria Oliva-Vilarnau, Yana Ruchiy, Thea Seitz, Kai Zhou, Arturo Gonzalez Isla, Lara Friess, Ying Sun, Maria Querol Canut, Alia Shamik, Yiran Xu, Changlian Zhu, Carlos F. D. Rodrigues, André Fisahn, Bertrand Joseph, Lena-Maria Carlson, Adamantia Fragkopoulou, Volker M Lauschke, Christer Betsholtz, Ahmed M Osman*, Klas Blomgren* #Shared first authorship, *Shared senior authorship [Manuscript]

III. Sex- and brain region-dependent inflammatory responses to cranial irradiation

Efthalia Preka, Maria Querol Canut, Sophie Sandner, Allanah Greer, Kalliopi-Maria Tamvaki, Xeni Androni, Yara Onetti Vilalta, Ahmed M. Osman, Klas Blomgren, Adamantia Fragkopoulou [Manuscript]

IV. Antisense Therapy Targeting the cGAS-STING Pathway to Alleviate Neuroinflammation after Cranial Radiotherapy

Efthalia Preka, Maria Querol Canut, Sophie Sandner, Allanah Greer, Apostolia-Ioli Diamanti, Niki Throumpari, Renato Esposito, Lei Huang, Zunpeng Liu, Yara Onetti Vilalta, Manolis Kellis, Ahmed M. Osman, Fredrik Kamme, Klas Blomgren, Adamantia Fragkopoulou [Manuscript]

V. Dynamic microglia alterations associate with hippocampal network impairments: A turning point in amyloid pathology progression Giusy Pizzirusso, Efthalia Preka#, Julen Goikolea#, Celia Aguilar-Ruiz, Patricia Rodriguez-Rodriguez, Guillermo Vazquez-Cabrera, Simona Laterza, Maria Latorre-Leal, Francesca Eroli, Klas Blomgren, Silvia Maioli, Per Nilsson, Adamantia Fragkopoulou, André Fisahn, Luis Enrique Arroyo-García #Equal authorship

Brain, Behavior and Immunity, July 2024, Volume 119, Pages 286-300
https://doi.org/10.1016/j.bbi.2024.04.009