Imaging the metabolic and phagocytic landscape of microglia in Alzheimer’s disease

Imaging the Metabolic and Phagocytic Landscape of Microglia in Alzheimer’s Disease Genome-wide association studies show that some of the strongest genetic risk variants for Alzheimer’s disease (AD) involve genes exclusively expressed in microglia, indicating its central role in AD pathology. Microglia are

the resident immune cells of the brain, essential for maintaining the health and function of the brain, as well as providing a first line of defense by phagocytizing debris and secreting cytokines. In AD, characterized by a CNS environment with chronic exposure to cellular debris and protein aggregation, recent single-cell RNA sequencing

has uncovered a variety of microglial transcriptional states specific to AD, indicating both protective and detrimental functions. While their transcriptional profiles are well-characterized, we lack an understanding of the molecular mechanisms that drive the formation of protective/detrimental microglial phenotypes, their functional

characteristics and how they inform AD disease pathology. Only by connecting transcriptional profiles to functional cell-states can we identify promising, new therapeutic targets. Subpopulations with detrimental functional signatures may represent novel therapeutic targets for AD. This requires the integration of new

technologies into the field, where the transcriptional profile of individual cells can be complemented by their functional signatures and correlated with microglia-activating agents and pathological hallmarks. Here, we propose to complement available transcriptional data with microscopy of the metabolic and

phagocytic landscape of microglia in the human AD brain. The heterogeneity of microglial transcription makes it difficult to unambiguously distinguish phenotypes based on immunostaining in conventional fluorescence microscopy. Instead, we have developed a front-line nonlinear microscopy platform, where

microglial phenotypes can be distinguished based on their metabolic and phagocytic profiles using spectral coherent anti-Stokes Raman (CARS) and simultaneous two-photon excited fluorescence (TPEF) microscopy. The profiles will be compiled from quantitative data extracted from the microscopy images;

amounts of (i) intracellular lipid stores, (ii) mitochondria, and (iii) the cellular redox ratio will be integrated into the metabolic profile, while (iv) lysosomal myelin/amyloid debris, (v) cytosolic myelin debris, and (vi) accumulating undegradable waste as lipofuscin will form the phagocytic profile. By further integrating a capability to map the

distribution of specific RNA transcripts using RNA probes (RNAScope), we will be able to link transcriptional expression to the metabolic and phagocytic profiles at the single cell level. Specifically, we will investigate the metabolic and phagocytic signatures of microglia in human AD brain tissues that express

a set of genes, which we have discovered to modulate lipid accumulation in human immune cells through our functional genome-wide CRISPR knock-out screens. This will reveal genetic regulators of dysfunctional lipid accumulation, characteristic for detrimental microglia, which may represent novel therapeutic targets. We

envision that metabolic reprogramming of microglia will become a new therapeutic route for AD.