Prevention of cognitive decline after radiation therapy

Central nervous system tumours are the leading cause of cancer-related deaths in children and the second-most-common malignancy diagnosed after leukaemia. Advances in treatment regimens and multimodal strategies, from early surgical resection, to radiotherapy, complete craniospinal radiation, and chemotherapeutic treatments have greatly improved the chances of survival. However, they are usually accompanied by long-term, late neurocognitive deficits, which, in totality, contribute to reduced quality of life. The aim of this thesis was to illuminate the mechanisms behind the cognitive decline that arises after cranial radiotherapy in childhood cancer survivors and to explore potential treatment strategies to prevent it.

Despite the effectiveness of radiotherapy in treating brain cancer, a biomarker that can estimate the extent of IR-induced brain injury and can be correlated with the clinical outcomes has yet to be established. Discovering a measurable molecule, that can be easily quantified in clinical samples that can be obtained through minimally invasive procedures would prove instrumental in predicting the severity of the neurocognitive complications and stratifying patients to the best treatment scheme to prevent or reduce the debilitating decline. In the first study of this thesis, we identified a very promising molecule, EDA2R (ectodysplasin A2 receptor), and we propose it as a potential biomarker. Ectodysplasin A2 receptor is a protein that belongs to the tumour necrosis factor family and is involved a variety of biological processes. After irradiation, we show that EDA2R is highly elevated in the cerebrospinal fluid as well as the blood of mice and is the only marker that remains at high concentrations even at two weeks after the treatment completion. Fulfilling the criteria of a biomarker, it remains to be correlated with cognitive performance in a clinical setting.

Among the most dominant hypotheses on the explanation of radiotherapy-induced cognitive decline is the depletion of hippocampal neurogenesis, a physiological process through which new neurons are constantly being born in the from neural stem and progenitor cells (NSPCs), which reside deep in the hippocampus, the centre of memory and learning. The depletion of neurogenesis has also been theorized to derive from the neuroinflammatory microenvironment that radiotherapy induces, which hinders the abilities of NSPCs to differentiate into neurons, and is governed by aberrantly activated microglia. As the resident immune cells of the brain, microglia react immediately to irradiation and undergo a series of changes to adapt to the needs of their niche, and their irradiation-induced diminishing numbers. Through unbiased, longitudinal in vivo studies, we showed that microglia reactivate long after irradiation and adopt unique morphologies and inflammatory profiles in a temporal manner. We observed a biphasic response in the hippocampal tissue, characterized by interferon signalling and proliferation of microglia that leads to neuronal asynchrony, which improves over time, due to the arrival of macrophage-derived microglia. In addition, our investigation revealed subsets of microglia that try to divide even in the presence of DNA damage, leading to senescence.

Besides raising and maintaining a neuroinflammatory alarm, microglia are also responsible for clearing dead neural progenitors and cell debris. Irradiation has also been shown to increase the levels of expression of genes that are related to phagocytosis. Several genetic and pharmacological tools have been developed to deplete microglial populations over the years. The usage of these approaches could prove instrumental in understanding the role of microglia in the depletion of neurogenesis. In addition, a study by Willis et al. in 2020 showed that depleting microglia and their subsequent repopulation attenuated cognitive decline in a traumatic brain injury model. With these in mind, we utilized the Cx3cr1CreERt2- YFP/+Rosa26DTA/+ mouse model to deplete microglia before subjecting the animals to cranial radiotherapy. Contrary to what we expected, the absence of microglia increased the production of pro-inflammatory factors and neither the depletion nor the subsequent repopulation had any impact on the loss of immature or proliferating neurons. These findings challenge the proposed role for a pro-inflammatory microenvironment in the dysregulation of hippocampal neurogenesis and suggest that the observed reduction of neurogenesis was solely due to IR..

Cranial radiotherapy has been documented to lead to decreases in dendritic density and changes in the spine morphology of the hippocampal neurons. As the main sites of the excitatory synapses, changes in dendrites and spines will often lead to abnormal signaling and dysregulation of the circuit, functions that are essential for cognition, and might also lead to glutamatergic excitotoxicity and neuronal death. Since newborn neurons rely on external inputs from the hippocampal circuit to pass survival checkpoints, strengthening synaptic connectivity might increase the chances of survival of the damaged immature neurons and improve cognitive functions in patients.

Memantine is an uncompetitive antagonist of the N-methyl-D-aspartate receptor (NMDAR), which has been shown to exert neuroprotective properties and increases synaptic plasticity and is currently used in the treatment of AlzheimerŐs disease patients. Due to these factors, memantine was considered as a great candidate for ameliorating radiation-induced cognitive decline, and several clinical trials have been completed and are underway since 2013, with very promising results. However, the exact mechanisms of how memantine exerts its neuroprotective effects are still unknown. For the final study of this thesis, we investigated the potential of memantine in preventing loss of neurogenesis. Memantine was administered in mice intraperitonially 30 minutes after cranial irradiation, and then it was supplied in their drinking water to achieve a steady state plasma concentration that recapitulates the clinical practice. Even though our results suggest no change in the numbers of surviving neurons in the hippocampal neurogenic niche, we showed that memantine led to an increase in the arborization of the dendritic processes of the young neurons. These data highlight memantineŐs potential in improving synaptic plasticity, the incorporation of immature neurons in the hippocampal circuitry and, by extension, in attenuating cognitive decline.

List of scientific papers

I. EDA2R reflects the acute brain response to cranial irradiation in liquid biopsies. Alejandro Lastra Romero, Thea Seitz, Georgios Alkis Zisiadis, Holli Jeffery, Ahmed M Osman*. Neuro Oncol. 2024 Apr 29:noae077. *: Corresponding author(s).
https://doi.org/10.1093/neuonc/noae077

II. Microglia Adopt Temporally Specific Subtypes after Irradiation, Correlating with Neuronal Asynchrony. 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, AndrŽ Fisahn, Bertrand Joseph, Lena-Maria Carlson, Adamantia Fragkopoulou, Volker M Lauschke, Christer Betsholtz, Ahmed M Osman*, Klas Blomgren*. #These authors contributed equally to this study. *Corresponding author(s). [Manuscript]

III. Microglia depletion and repopulation do not alter the effects of cranial irradiation on hippocampal neurogenesis. Kai Zhou#, Georgios Alkis Zisiadis#, Monique Havermans, Adamantia Fragkopoulou, Cecilia Dominguez, Makiko Ohshima, Ahmed M Osman, Carlos F. D. Rodrigues, Klas Blomgren*. #These authors contributed equally to this study. *Corresponding author(s). [Manuscript]

IV. Memantine increases the dendritic complexity of hippocampal young neurons in the juvenile brain after cranial irradiation. Georgios Alkis Zisiadis, Androniki Alevyzaki, Elene Nicola, Carlos F. D. Rodrigues, Klas Blomgren, Ahmed M Osman*. Front Oncol. 2023 Oct 4;13:1202200. *Corresponding author(s).
https://doi.org/10.3389/fonc.2023.1202200