Identification and therapeutic modulation of a pro-inflammatory subset of disease-associated-microglia in Alzheimer’s disease

Abstract

BackgroundDisease-associated-microglia (DAM) represent transcriptionally-distinct and neurodegeneration-specific microglial profiles with unclear significance in Alzheimer’s disease (AD). An understanding of heterogeneity within DAM and their key regulators may guide pre-clinical experimentation and drug discovery.MethodsWeighted co-expression network analysis (WGCNA) was applied to existing microglial transcriptomic datasets from neuroinflammatory and neurodegenerative disease mouse models to identify modules of highly co-expressed genes. These modules were contrasted with known signatures of homeostatic microglia and DAM to reveal novel molecular heterogeneity within DAM. Flow cytometric validation studies were performed to confirm existence of distinct DAM sub-populations in AD mouse models predicted by WGCNA. Gene ontology analyses coupled with bioinformatics approaches revealed drug targets and transcriptional regulators of microglial modules predicted to favorably modulate neuroinflammation in AD. These guided in-vivo and in-vitro studies in mouse models of neuroinflammation and neurodegeneration (5xFAD) to determine whether inhibition of pro-inflammatory gene expression and promotion of amyloid clearance was feasible. We determined the human relevance of these findings by integrating our results with AD genome-wide association studies and human AD and non-disease post-mortem brain proteomes.ResultsWGCNA applied to microglial gene expression data revealed a transcriptomic framework of microglial activation that predicted distinct pro-inflammatory and anti-inflammatory phenotypes within DAM, which we confirmed in AD and aging models by flow cytometry. Pro-inflammatory DAM emerged earlier in mouse models of AD and were characterized by pro-inflammatory genes (Tlr2, Ptgs2, Il12b, Il1b), surface marker CD44, potassium channel Kv1.3 and regulators (NFkb, Stat1, RelA) while anti-inflammatory DAM expressed phagocytic genes (Igf1, Apoe, Myo1e), surface marker CXCR4 with distinct regulators (LXRα/β, Atf1). As neuro-immunomodulatory strategies, we validated LXRα/β agonism and Kv1.3 blockade by ShK-223 peptide that promoted anti-inflammatory DAM, inhibited pro-inflammatory DAM and augmented Aβ clearance in AD models. Human AD-risk genes were highly represented within homeostatic microglia suggesting causal roles for early microglial dysregulation in AD. Pro-inflammatory DAM proteins were positively associated with neuropathology and preceded cognitive decline confirming the therapeutic relevance of inhibiting pro-inflammatory DAM in AD.ConclusionsWe provide a predictive transcriptomic framework of microglial activation in neurodegeneration that can guide pre-clinical studies to characterize and therapeutically modulate neuroinflammation in AD.Electronic supplementary materialThe online version of this article (10.1186/s13024-018-0254-8) contains supplementary material, which is available to authorized users.

Enforced microglial depletion and repopulation as a promising strategy for the treatment of neurological disorders

“…Compared with normal microglia the disease‐associated microglia progressively increase lipid metabolism and expression of phagocytic genes, and are specifically localized around AD plaques, thereby engulfing neurotoxic proteins and protecting against neurodegenerative diseases (Keren‐Shaul et al, ). However, recent discoveries using unbiased weighted co‐expression network analysis have shed light on another distinct pro‐inflammatory subpopulation of disease‐associated microglia in the context of AD (Rangaraju et al, ).…”

“…Currently, it is still unknown whether other disease‐associated microglia subsets in different neurological disorders exist or not, or indeed whether specific subtypes are common to several neurological disease states. However, we are optimistic that further studies employing comprehensive combined transcriptome, proteomic and metabolomic approaches will define a wider spectrum of microglia subsets in different neurological disorders and facilitate targeting microglia with unprecedented precision (Haimon et al, ; Rangaraju et al, ). Thus further studies are warranted to characterize the scope of intrinsic mechanisms of disease‐associated microglia during diverse disease conditions.…”

Transcriptional regulation of homeostatic and disease‐associated‐microglial genes by IRF1, LXRβ, and CEBPα

“…Based on single cell RNAseq data, homeostatic microglia adopt distinct disease‐associated‐microglia (DAM) profiles in aging and chronic neurodegenerative diseases (Keren‐Shaul et al, ; Orre et al, ; Wang et al, ). DAM also appears to be a heterogeneous group of cells and we recently identified distinct pro‐inflammatory DAM and anti‐inflammatory DAM subsets within DAM (Rangaraju, Dammer, et al, ; Sarlus & Heneka, ). Flow cytometry studies confirmed that among CD11c + DAM, distinct CD44 + , and CXCR4 + subsets exist, and pharmacologic studies demonstrated that these sub‐profiles can be selectively modulated in‐vivo (Rangaraju, Dammer, et al, ).…”

“…DAM also appears to be a heterogeneous group of cells and we recently identified distinct pro‐inflammatory DAM and anti‐inflammatory DAM subsets within DAM (Rangaraju, Dammer, et al, ; Sarlus & Heneka, ). Flow cytometry studies confirmed that among CD11c + DAM, distinct CD44 + , and CXCR4 + subsets exist, and pharmacologic studies demonstrated that these sub‐profiles can be selectively modulated in‐vivo (Rangaraju, Dammer, et al, ). Although signature genes highly expressed by these microglial states and transcriptional regulators of microglial development and survival (PU.1, MAFB, SALL1, IRF8) are now known (Buttgereit et al, ; Kierdorf et al, ; Koshida, Oishi, Hamada, & Takahashi, ; Koso et al, ), the key upstream regulators of homeostatic, pro‐inflammatory DAM and anti‐inflammatory DAM transcriptional programs have not been identified.…”

“…In our recent network analysis of microglial transcriptomics data, we used pathway analyses to identify several TFs that may regulate homeostatic and distinct DAM phenotypes in the context of neurodegeneration (Rangaraju, Dammer, et al, ). However, confirmatory studies of regulatory roles for these putative TFs have not been performed.…”