How gut metabolites dictate CNS macrophages functi on in steady state, aging and neurodegenerati on

Prof. Dr. Daniel Erny

• A07

PD Dr. Thomas Blank

• A07

Project Summary

As innate immune cells, central nervous system (CNS)-associated macrophages (CAMs) and microglia support tissue homeostasis and immune surveillance in the CNS. The communication between the gut and the brain becomes increasingly important to understand how microglia and CAMs are regulated by specific gut microbiota-derived compounds during normal and pathological aging. As an example, the dysregulation of toll-like receptor (TLR) 5, which is activated by flagellin, a TLR5 agonist originating from the gut, is suggested to contribute to Alzheimer`s disease (AD). N(6)-Carboxymethyllysine (CML) is an additional gut-derived metabolite, which accumulates in microglia and CAMs during aging and inflicts mitochondrial damage. Although TLR5 and the Receptor for Advanced Glycati on Endproducts (RAGE), which binds CML, are both expressed by brain macrophages, it is sti ll unclear how both signaling pathways in microglia and CAMS affect healthy brain aging and how the functional regulation of both receptor complexes drives neurodegenerative processes in AD pathology in both sexes. With this present research proposal, we will address these questions in male and female mice by clarifying the TLR5-dependent effects on microglia and CAMs using histology, single-cell (sc) and bulk RNA-sequencing (seq) in Cx3cr1^CreERT2/+:Tlr5^{fl /fl} and Mrc1^CreERT2/+:Tlr5^{fl /fl} mice during aging (Aim1). We expect that the detrimental eff ect of CML on microglia increases with age and will manifest itself in CAMs as well. Thus, we will map microglia and CAMs with the help of scRNA-seq for their transcriptional and functional signatures and validate the transcriptional changes on protein level by imaging mass cytometry (IMC) (Aim 2). TLR5 and CML are both implicated in the severity and progression of AD, however without any present mechanistic insights. We will address this issue and will determine the outcomes of CML applied to Cx3cr1^CreERT2/+:Tlr5^{fl /fl} and Mrc1^CreERT2/+:Tlr5^{fl /fl} mice crossed with 5xFAD mice. We will perform behavioral testing, determine amyloid beta (Aβ) deposits and clearance, the impact on the mitochondria ultrastructure of microglia and perivascular macrophages (pvMΦ), cellular morphology and gene expression (Aim 3). In order to translate our findings to the human situation, human microglia will be exposed to flagellin alone or in combination with Aβ. Changes in gene expression (bulkRNA-seq) and Aβ-uptake will be analyzed (Aim 4). Using a further translational approach, we will analyze the impact of CML on hiPSC-derived microglia engrafted in Rag2^−/−:hCSF1 and Rag2^−/−:hCSF1 mice crossed with 5xFAD mice. The analysis techniques include electron microscopy, scRNA-seq, histology, Aβ-phagocytosis assay and IMC (Aim 5). With the proposed aims we will identify novel gut metabolite-dependent molecular mechanisms that regulate the genetic and functional characteristics of CAMs and microglia in both, the healthy and pathologically aging CNS. The expected data may lead to the development of innovative therapeutic intervention techniques in the field of cognitive aging and dementia.