Author:

Abstract:

The interest for the highly dynamic microglial cells, the unique immune cells of the central nervous system (CNS), has been growing exponentially in the last decade and expanded the field of neurodegenerative diseases. Microglia play an essential role in brain development, in protecting the CNS from injury and pathological intruders, and in several adult physiological functions such as normal neuronal functioning and connectivity, learning processes, and social behavior. During my PhD project, microglia were also shown to be key players in a mouse model of multifunctional protein-2 (MFP2) deficiency, a neurometabolic disorder. MFP2 is an essential enzyme that plays a key role in peroxisomal β-oxidation, a pathway responsible for the synthesis and breakdown of fatty acids. Both constitutive and conditional cell type-specific Mfp2-/- mouse models were used as unique tools to study the cellular and molecular aspects of microgliosis (= activation, proliferation and transformation of microglia) in peroxisomal β-oxidation deficiency. We aimed to molecularly define the microglial phenotype, to uncover the cellular mechanisms driving the neuroinflammatory response, and to reveal the impact of microgliosis on neuronal integrity and cognitive and behavioral aspects. We determined the unique microglial signature in the constitutive murine model of MFP2 deficiency in which extensive neuroinflammation occurs in the absence of neuronal loss. We defined that the resident microglia in constitutive Mfp2-/- mice are chronically activated, adopt a primed and mixed pro- and anti-inflammatory phenotype, and lose their typical homeostatic markers. Surprisingly, Mfp2-/- microglia remain in an activated and proliferative state without acquiring overt neurotoxic and phagocytic properties. This provides novel insight into the functional diversity of microglia as it has been assumed that chronic microgliosis is detrimental to the brain and neurons in particular. However, Mfp2-/- microglia might exert an adverse effect on neuronal functioning without inducing neuronal loss. In order to reveal the cause and consequences of microglial pathology in MFP2 deficiency, we investigated the impact of selective loss of MFP2 by generating either neural-specific (i.e. neurons, astrocytes and oligodendrocytes) Nestin-Mfp2-/- or microglia/monocyte-specific Cx3cr1-Mfp2-/- mice, and comparing them with the constitutive Mfp2-/- mice. A comparative study demonstrated that microgliosis in Nestin-Mfp2-/- mice is delayed and is never as extensive as compared to Mfp2-/- mice. The excessive activation state of microglia in Mfp2-/- mice refers to more aberrant microglial morphology, priming, loss of homeostatic signature, and induction of activation markers. This was paralleled by a more prominently disturbed neuronal transmission in Mfp2-/- compared to Nestin-Mfp2-/- mice. Our results suggest that mild microgliosis induced by absence of MFP2 in neural cells, is amplified by additional loss of MFP2 from microglia, jointly leading to a severe neurological pathology as in constitutive Mfp2-/- mice. The disturbed interplay between neurons and microglia in Mfp2-/- mice was proven by decreased expression of neuronal restraint markers that normally maintain microglial quiescence in the healthy brain. Of note, it cannot be excluded that preserved MFP2 function in non-neural cells contributes to the milder phenotype of Nestin-Mfp2-/- mice. Examination of the microglia/monocyte-specific Cx3cr1-Mfp2-/- mouse model revealed that Mfp2-/- microglia in an intact brain environment modify their phenotype in a cell-autonomous manner. Numbers of Mfp2-/- microglia progressively increase and their morphology changes enormously throughout the brain whereas neuronal functional and clinical parameters are unaffected, at least until the age of 8 months. In addition, Mfp2-/- microglia seem to adopt a pro-inflammatory state. Interestingly, we proved that Mfp2-/- microglia in vivo respond normally to disturbances in their CNS environment such as to an acute inflammatory and a neurodegenerative trigger. In a complementary approach, we attempted to eliminate microglial cells from the CNS in order to verify whether Mfp2-/- microglia are detrimental to neurons and brain integrity. However, treatment of mice with the specific CSF1R inhibitor PLX5622 only depleted 70% of microglial cells in Mfp2-/- mice in contrast to 95-98% of microglia in control mice. The remaining strongly activated Mfp2-/- microglia were sufficient to induce a strong inflammatory response, shown by the unchanged levels of inflammatory mediators in PLX-treated Mfp2-/- mice. Consequently, the PLX-treatment did not improve the clinical score, cognition or neuronal and behavioral impairments of Mfp2-/- mice. In conclusion, we propose that crosstalk between microglial cell-autonomous pathology and dysfunctional signaling from neural cells, neurons in particular, causes a synergistic pathological effect leading to microglial priming as well as initiation and progression of neurological disease in mice lacking MFP2.