Unraveling metabolic features of microglia and macrophages highlighting the role of peroxisomal β-oxidation

Background

All neurodegenerative diseases including Alzheimer’s and Parkinson’s disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis but also rare inherited metabolic diseases have an inflammatory component (1). Microglia, the immune cells of the brain, are pivotal cells in the generation of this inflammatory response to acute and chronic alterations in brain homeostasis (2). These cells convert from a surveying phenotype in which they constantly scan their environment into a multitude of activated states. Only recently, the molecular signature of microglia has begun to be defined by using transcriptomic and proteomic approaches (3-6). However, with regard to the metabolic program of microglia in their surveying or diverse activation states virtually nothing is known. Still, it can be expected that metabolism has a major impact on the function of microglia. It has indeed become clear that metabolism is reprogrammed in diverse immune cells such as T cells, dendritic cells and macrophages, depending on their activation state (7). For example, classically activated macrophages acquire a glycolytic state together with their pro-inflammatory phenotype (8). In contrast, alternatively activated macrophages require oxidative phosphorylation and mitochondrial fatty acid oxidation for their anti-inflammatory function (9). Although microglia are related to macrophages, they profoundly differ in genetic profile (3) and it is an open question whether metabolic pathways impact in a similar way on their activation state. 

Objectives

The goal of this PhD project is to investigate the metabolic characteristics of microglia in diverse activation states. In addition it will be explored whether inactivation of specific metabolic pathways in microglia affects their phenotype. In view of the extensive microglia activation in a mouse model with peroxisome deficiency (MFP2 knockout) special attention will be drawn to the importance of these poorly understood organelles in microglia as well as in macrophages (10).

Experimental approaches

In order to define which metabolic pathways are active in microglia with diverse activation states, primary microglia cultures derived from newborn mice will be treated with vehicle, IL-1beta/IFNgamma or IL-4 in order to skew them into sensing and surveying (M0), pro-inflammatory (M1) and anti-inflammatory (M2) states, respectively. To initially characterize some key metabolic pathways (glycolysis, fatty acid oxidation, glutamine oxidation), classic radioactive tracer studies will be performed. Subsequently, metabolic flux analysis (MFA) will be performed to trace the metabolic fate of 13C-labeled nutrients (glucose, palmitate, glutamine) into metabolites of multiple metabolic pathways. These data will be corroborated by gene expression analysis.

To further investigate the importance of key metabolic pathways in microglia activation, crucial enzymes of these pathways will be blocked in vitro using genetic or pharmacological tools and the consequences on diverse aspects of microglia functioning including M1-M2 gene expression and phagocytosis will be investigated.

In addition, in order to clarify the importance of peroxisomes in immune cells, MFP2-deficient macrophages and microglia in different activation states will be characterized.

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2          Kettenmann, H., Hanisch, U. K., Noda, M. & Verkhratsky, A. Physiology of microglia. Physiol Rev. 91, 461-553 (2011).

3          Gautier, E. L. et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nature immunology 13, 1118-1128, doi:10.1038/ni.2419 (2012).

4          Chiu, I. M. et al. A neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model. Cell reports 4, 385-401, doi:10.1016/j.celrep.2013.06.018 (2013).

5          Hickman, S. E. et al. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci 16, 1896-1905, doi:10.1038/nn.3554 (2013).

6          Butovsky, O. et al. Identification of a unique TGF-beta-dependent molecular and functional signature in microglia. Nat Neurosci 17, 131-143, doi:10.1038/nn.3599 (2014).

7          Pearce, E. L. & Pearce, E. J. Metabolic pathways in immune cell activation and quiescence. Immunity. 38, 633-643 (2013).

8          Krawczyk CM, Holowka T, Sun J, Blagih J, Amiel E, DeBerardinis RJ, et al. Toll-like receptor-induced changes in glycolytic metabolism regulate dendritic cell activation. Blood. 2010;115(23):4742-9. Epub 2010/03/31.

9          Vats D, Mukundan L, Odegaard JI, Zhang L, Smith KL, Morel CR, et al. Oxidative metabolism and PGC-1beta attenuate macrophage-mediated inflammation. Cell Metab. 2006;4(1):13-24. Epub 2006/07/04.

10        Verheijden, S. et al. Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation. Neurobiology of disease 58, 258-269 (2013).