Advances in proteomic phenotyping of microglia in neurodegeneration

Abstract

Microglia are dynamic resident immune cells of the central nervous system (CNS) that sense, survey, and respond to changes in their environment. In disease states, microglia transform from homeostatic to diverse molecular phenotypic states that play complex and causal roles in neurologic disease pathogenesis, as evidenced by the identification of microglial genes as genetic risk factors for neurodegenerative disease. While advances in transcriptomic profiling of microglia from the CNS of humans and animal models have provided transformative insights, the transcriptome is only modestly reflective of the proteome. Proteomic profiling of microglia is therefore more likely to provide functionally and therapeutically relevant targets. In this review, we discuss molecular insights gained from transcriptomic studies of microglia in the context of Alzheimer's disease as a prototypic neurodegenerative disease, and highlight existing and emerging approaches for proteomic profiling of microglia derived from in vivo model systems and human brain.

Keywords: inflammation; microglia; neurodegeneration; proteomics; signaling.

Figure 1.. Microglial responses in Alzheimer’s Disease.

Several genetic risk loci for AD (TREM2, CD33, CSF1R, CR1, APOE) encode proteins involved in signaling cascades that support broad phenotypic shifts in motility, metabolism, phagocytosis, proliferation, cytokine production, exosome production and release, and apoptosis. Microglia use complement signaling to identify both healthy and dead neurons for degradation. Microglia can use signaling cascades to detect and recruit to pathological proteins including TAU and Aβ. For example, fractalkine signaling (via fractalkine receptor CX3CR1) allows microglia to detect and phagocytose Tau ^[54,216,217]. Microglial interactions with Aβ plaques are distinct, where microglia actively surround Aβ plaques and interact with them via mechanisms involving APOE, TREM2 and its receptor. This receptor is responsible for the TREM2-dependent signaling pathway which results in activation of SYK and ERK ^[218,219]. ERK then crosses the nucleus to allow for transcription of key inflammatory signaling molecules such as STAT1. Whereas activation of the mTOR pathway leads to further phagocytosis and cell proliferation. TAU and Aβ can also directly interact with each other, where tau tangles can act as a seed for Aβ plaque accumulation and Aβ can promote the phosphorylation of tau necessary for tau fibrillization. Cross-talk between microglia and astrocytes is largely responsible for activation of the TLR4 and IL1R on both cell types which can shift astrocytes and microglia toward more proinflammatory cytokine release ^[77]. Proteomic studies evidence an increase in CASP1 as well as IL-1β indicating formation and activation of the NLRP3 inflammasome which cleaves proIL-1β and proIL-18 prior to release from microglia. These signaling events also result in increased production and release of exosomes that contain key signaling proteins like TNF, and other cargo. This corresponds with metabolic reprograming in microglia, where there is an increased movement of glucose to glycolysis but a reduction in mitochondrial activity, which is in turn sustains ATP production and calcium-dependent mechanisms necessary for DAM function. Aβ and other neuropathologies also transform microglia from homeostatic and disease-associated microglia (DAM) states, and key markers of these states also shown below. ^[16,220]

Figure 2.. Isolation-dependent approaches for microglial proteomics.

Acute isolation of microglia and other brain cell types requires fresh brain tissue that undergoes mechanical or enzymatic dissociation to generate a heterogenous single-cell suspension that can be prepared for MACS, FACS, or immunopanning methods. For MACS, the single-cell suspension is incubated with a magnetic microbead conjugated to an antibody that binds a cell surface receptor. Then, a magnet is used to select for the desired cell type and the unbound cells are washed away. The magnetically bound cells are released and collected for downstream analysis. For FACS, the single-cell suspension is incubated with a fluorophore-conjugated antibody specific to a cell surface receptor. Following, the desired cell type is sorted based on size and fluorescent signal. The sorted cells are collected for downstream analysis. For immunopanning, single-cell suspensions are plated on a cell culture dish coated with an antibody specific to a cell surface receptor. The unbound cells are washed away, and the bound cells are collected for downstream analysis.

Figure 3.. Isolation-independent methods for in vivo proteomic labeling of microglia.

In vivo biorthogonal amino acid tagging (BONCAT) of proteins (red panel) and cell type-specific in vivo biotinylation of proteins (CIBOP) (blue panel) is achieved by inserting mutant MetRS (L274G) or MetRS*, or TurboID, into the Rosa26 locus to generate in vivo proteomic labeling in mouse models. Following, microglia specificity can be achieved by either breeding the MetRS* or TurboID models with a microglia-specific Cre mouse, with or without inducibility, or by injecting a microglial-specific adeno-associated virus (AAV) to deliver Cre. MetRS* contains a mutation (L247G) in the amino acid binding site which tags nascent proteins with an azide tagged methionine analog, azidonorleucine (ANL). The azide residue of ANL can undergo “click” chemistry in which ANL-tagged proteins residues are “clicked” with a PEG-biotin-alkyne. Proximity labeling using CIBOP is achieved by the biotin ligase, TurboID, that biotinylates endogenous proteins in close proximity. After, MetRS* or TurboID tagged proteins can undergo biotin affinity capture using streptavidin-coated beads and processed for downstream mass MS-based proteomics.