Lien Beckers

Peroxisomal multifunctional protein-2 deficiency causes neuroinflammation and degeneration of Purkinje cells independent of very long chain fatty acid accumulation

Although peroxisome biogenesis and β-oxidation disorders are well known for their neurodevelopmental defects, patients with these disorders are increasingly diagnosed with neurodegenerative pathologies. In order to investigate the cellular mechanisms of neurodegeneration in these patients, we developed a mouse model lacking multifunctional protein 2 (MFP2, also called D-bifunctional protein), a central enzyme of peroxisomal β-oxidation, in all neural cells (Nestin-Mfp2(-/-)) or in oligodendrocytes (Cnp-Mfp2(-/-)) and compared these models with an already established general Mfp2 knockout. Nestin-Mfp2 but not Cnp-Mfp2 knockout mice develop motor disabilities and ataxia, similar to the general mutant. Deterioration of motor performance correlates with the demise of Purkinje cell axons in the cerebellum, which precedes loss of Purkinje cells and cerebellar atrophy. This closely mimics spinocerebellar ataxias of patients affected with mild peroxisome β-oxidation disorders. However, general knockouts have a much shorter life span than Nestin-Mfp2 knockouts which is paralleled by a disparity in activation of the innate immune system. Whereas in general mutants a strong and chronic proinflammatory reaction proceeds throughout the brain, elimination of MFP2 from neural cells results in minor neuroinflammation. Neither the extent of the inflammatory reaction nor the cerebellar degeneration could be correlated with levels of very long chain fatty acids, substrates of peroxisomal β-oxidation. In conclusion, MFP2 has multiple tasks in the adult brain, including the maintenance of Purkinje cells and the prevention of neuroinflammation but this is not mediated by its activity in oligodendrocytes nor by its role in very long chain fatty acid degradation.

Identification of a chronic non-neurodegenerative microglia activation state in a mouse model of peroxisomal β-oxidation deficiency.

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β‐oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2−/− microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2−/− microglia were compared with those from SOD1G93A mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1G93A spinal cord, neurons were intact in Mfp2−/− brain and Mfp2−/− microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2−/− microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2−/− microglia were immunologically activated but not polarized to a pro‐ or anti‐inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2−/− brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity. GLIA 2015;63:1606–1620

The oxysterol and cholestenoic acid profile of mouse cerebrospinal fluid

Graphical abstract

Increased Expression of Translocator Protein (TSPO) Marks Pro-inflammatory Microglia but Does Not Predict Neurodegeneration.

PurposeActivation of the innate immune system plays a significant role in pathologies of the central nervous system (CNS). In order to follow disease progression and evaluate effectiveness of potential treatments involved in neuroinflammation, it is important to track neuroinflammatory markers in vivo longitudinally. The translocator protein (TSPO) is used as a target to image neuroinflammation as its expression is upregulated in reactive glial cells during CNS pathologies. However, it remains unclear in which microglial phenotypes TSPO levels are upregulated, as microglia can display a plethora of activation states that can be protective or detrimental to the CNS.ProceduresWe assessed the levels of TSPO transcripts in cultured microglia that were polarized into pro- and anti-inflammatory states in vitro and in the brain of mice in which an anti-inflammatory environment was induced in vivo. In addition, we used a mouse model of peroxisomal multifunctional protein-2 (MFP2) deficiency that exhibits widespread neuroinflammation despite no neuronal loss and monitored TSPO expression by immunohistochemistry and by imaging using the TSPO radiotracer [18F]DPA-714.ResultsTSPO expression was selectively increased in so-called classically activated or M1 microglia but not in alternatively activated or M2 microglia in vitro. In agreement, TSPO transcript levels were not induced in an anti-inflammatory brain environment. We found that both transcript and protein levels of TSPO are significantly increased in the brain of Mfp2−/− compared to those of the control mice and TSPO immunoreactivity colocalized predominantly with microglia in Mfp2−/− brain. In vitro and ex vivo autoradiography in Mfp2−/− mice using the TSPO radiotracer [18F]DPA-714 confirmed increased expression of TSPO. These data demonstrate that TSPO imaging reveals microgliosis in non-neurodegenerative brain pathologies.ConclusionsWe show that induced TSPO expression marks a pro-inflammatory brain environment that is not necessarily accompanied by neuronal loss.

Central nervous system pathology in MFP2 deficiency: Insights from general and conditional knockout mouse models

Multifunctional protein-2 (MFP2), also known as D-bifunctional protein, is a central enzyme of the peroxisomal β-oxidation pathway. Defects in this enzyme are associated with a spectrum of neurological disorders encompassing developmental and degenerative pathologies. In order to investigate the cellular and molecular mechanisms of these neuropathologies, mouse models with general and cell type selective loss of MFP2 were generated. In this review the distinct anomalies in the CNS of adult Mfp2 knockout mice are discussed, in particular the cerebellar degeneration and neuroinflammation. The potential underlying mechanisms are considered with regard to the cellular origin and biochemical causes. Finally, the similarities and differences between the CNS phenotypes of mice lacking MFP2 and mice with peroxisome biogenesis disorders are assessed.

Specific suppression of microgliosis cannot circumvent the severe neuropathology in peroxisomal β-oxidation-deficient mice

Abstract An important hallmark of various neurodegenerative disorders is the proliferation and activation of microglial cells, the resident immune cells of the central nervous system (CNS). Mice that lack multifunctional protein‐2 (MFP2), the key enzyme in peroxisomal &bgr;‐oxidation, develop excessive microgliosis that positively correlates with behavioral deficits whereas no neuronal loss occurs. However, the precise contribution of neuroinflammation to the fatal neuropathology of MFP2 deficiency remains largely unknown. Here, we first attempted to suppress the inflammatory response by administering various anti‐inflammatory drugs but they failed to reduce microgliosis. Subsequently, Mfp2−/− mice were treated with the selective colony‐stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 as microglial proliferation and survival is dependent on CSF1R signaling. This resulted in the elimination of > 95% of microglia from control mice but only 70% of the expanded microglial population from Mfp2−/− mice. Despite microglial diminution in Mfp2−/− brain, inflammatory markers remained unaltered and residual microglia persisted in a reactive state. CSF1R inhibition did not prevent neuronal dysfunction, cognitive decline and clinical deterioration of Mfp2−/− mice. Collectively, the unaltered inflammatory profile despite suppressed microgliosis concurrent with persevering clinical decline strengthens our hypothesis that neuroinflammation importantly contributes to the Mfp2−/− phenotype. Graphical abstract Figure. No caption available. HighlightsCSF1R blocker PLX5622 suppresses microgliosis in Mfp2−/− mice.Residual microglia remain morphologically reactive.Inflammatory parameters are not impaired.CSF1R inhibition did not counteract behavioral deficits and clinical deterioration.

Response to Notter and Meyer’s Letter to the Editor Regarding Increased Expression of Translocator Protein (TSPO) Marks Pro-inflammatory Microglia but Does Not Predict Neurodegeneration

Purpose: Activation of the innate immune system plays a significant role in pathologies of the central nervous system (CNS). In order to follow disease progression and evaluate effectiveness of potential treatments involved in neuroinflammation, it is important to track neuroinflammatory markers in vivo longitudinally. The translocator protein (TSPO) is used as a target to image neuroinflammation as its expression is upregulated in reactive glial cells during CNS pathologies. However, it remains unclear in which microglial phenotypes TSPO levels are upregulated, as microglia can display a plethora of activation states that can be protective or detrimental to the CNS. Procedures: We assessed the levels of TSPO transcripts in cultured microglia that were polarized into proand anti-inflammatory states in vitro and in the brain of mice in which an antiinflammatory environment was induced in vivo. In addition, we used a mouse model of peroxisomal multifunctional protein-2 (MFP2) deficiency that exhibits widespread neuroinflammation despite no neuronal loss and monitored TSPO expression by immunohistochemistry and by imaging using the TSPO radiotracer [F]DPA-714. Results: TSPO expression was selectively increased in so-called classically activated or M1 microglia but not in alternatively activated or M2 microglia in vitro. In agreement, TSPO transcript levels were not induced in an anti-inflammatory brain environment. We found that both transcript and protein levels of TSPO are significantly increased in the brain of Mfp2 compared to those of the control mice and TSPO immunoreactivity colocalized predominantly with microglia in Mfp2 brain. In vitro and ex vivo autoradiography in Mfp2 mice using the TSPO radiotracer [F]DPA-714 confirmed increased expression of TSPO. These data demonstrate that TSPO imaging reveals microgliosis in non-neurodegenerative brain pathologies. Lien Beckers and Dieter Ory are equal first authors Guy Bormans and Myriam Baes are equal last authors Electronic supplementary material The online version of this article (doi:10.1007/s11307-017-1099-1) contains supplementary material, which is available to authorized users. Correspondence to: Myriam Baes; e-mail: Myriam.Baes@kuleuven.be Conclusions: We show that induced TSPO expression marks a pro-inflammatory brain environment that is not necessarily accompanied by neuronal loss.