Long-term Replacement of Endogenous Microglia in Mice

by Konstantina Kolotourou
Doctoral thesis
Date of Examination:2024-06-18
Date of issue:2024-12-19
Advisor:Prof. Dr. Christine Stadelmann-Nessler
Referee:Prof. Dr. Alexander Flügel
Referee:Prof. Dr. Oliver Wirths
Referee:Prof. Dr. Frauke Alves
Referee:Prof. Dr. Susann Boretius
Referee:Prof. Dr. Tiago Fleming Outeiro
Persistent Address: http://dx.doi.org/10.53846/goediss-10953

 

 

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Abstract

English

Microglia are the resident immune cells of the central nervous system (CNS). They play pivotal roles in the development and homeostasis of the CNS and have been implicated in neurodegenerative disorders, such as Alzheimer´s Disease (AD). Microglia are highly responsive to environmental signals, making it invaluable to study them in vivo. Here we generated and characterized CX3CR1creERT2 CSF1Rflox/flox mice, in which microglia can be conditionally depleted by tamoxifen injection. We devised a transplantation protocol in these mice with the goal of replacing endogenous microglia with red fluorescent protein (RFP)-tagged bone marrow (BM) cells injected intravenously (i.v.). These cells migrate to the microglia-depleted central nervous system (CNS) where they adopt a microglia-like cell (MLC) phenotype and remain stable for months. We purified hematopoietic stem cells (HSCs), granulocyte-monocyte progenitors (GMPs) and inflammatory monocytes from BM and performed competition experiments to define the most effective donor cell type for microglia replacement. Both HSCs and GMPs performed equally well in repopulating the microglia-depleted CNS as MLCs, whereas inflammatory monocytes did not. Competition experiments with chemokine receptor-deficient or -competent BM donor cells transplanted either i.v. or intracortically (i.c.) into microglia-depleted recipients revealed that CCR2 was relevant for BM cells to migrate to the CNS and that CX3CR1 was required for BM cells to differentiate into MLCs. We evaluated the potential differences between endogenous and BM-derived MLCs by bulk RNA sequencing, flow cytometry and immunohistochemistry. MLCs expressed homeostatic microglia genes such as P2RY12 or TMEM119, although expression levels were significantly lower than in endogenous microglia. Conversely, disease-associated microglia (DAM) marker genes, as well as genes associated with antigen presentation and phagocytosis were upregulated, all in all suggesting that MLCs are in a higher activation state than microglia. We tested whether MLCs could ameliorate the disease phenotype of 5xFAD mice, but achieved only a patchy GMP-derived MLC replacement, because conditional microglia depletion was less efficient in 5xFAD CX3CR1creERT2 CSF1Rflox/flox mice compared to CX3CR1creERT2 CSF1Rflox/flox mice without the 5xFAD transgene. The ß-amyloid plaque load was however reduced in patches with GMP-derived MLCs compared to areas with endogenous microglia, suggesting that microglia replacement by MLCs could help in diminishing plaque load in AD. Finally, we crossbred CX3CR1creERT2 CSF1Rflox/flox mice to immunodeficient mice to accommodate human induced pluripotent stem cell (hiPSC)-derived MLCs. These hMLCs demonstrated expression of key human microglial markers, including P2RY12, TMEM119, and the transcription factor SALL1 and remained exceptionally stable within the murine brain. Transplanted mice with hiPSC-derived MLCs exhibited proficient performances in motor and cognitive tasks and nuanced functional attributes will be explored in future experiments. In summary, we have developed and characterized a model system poised to elucidate both mouse and human microglia-like cell features in vivo across prolonged periods of time.
Keywords: microglia; microglia-like cells; Alzheimer´s Disease; depletion