Cerebellaire degeneratie en microglia activatie in peroxisomale aandoeningen: nieuwe inzichten met behulp van multifunctioneel proteïne-2 knockout muizen

It is well established that peroxisomes are vital for central nervous system development and maintenance, as patients with peroxisomal defects face pronounced neurological regression. The complexity of peroxisomal function in the CNS is illustrated by the heterogeneous disease spectrum associated with complete peroxisomal dysfunction. The majority of these patients show severe neurodevelopmental abnormalities in the neonatal period and generally dies before the age of one year. However, a substantial fraction of patients develops a milder clinical disease course that can present as a childhood leukodystrophy or as isolated cerebellar ataxia. Until now, the precise metabolic defects underlying this heterogeneous neurological spectrum are poorly understood. Notably, patients with defects in MFP2, a central enzyme of the peroxisomal ß-oxidation pathway, are generally indistinguishable from patients with total peroxisomal dysfunction, indicating that MFP2 is crucial for CNS homeostasis. Hence, knowledge on the role of MFP2 in the CNS might provide us with significant mechanistic insights on the pathogenesis of peroxisomal disorders. A mouse model lacking MFP2 (Mfp2-/-) does not show pronounced neurodevelopmental abnormalities, but a regressive neurological disease course develops involving motor defects, lethargic behavior and death before the age of 6 months. In the preterminal stage, strong astro- and microgliosis is present throughout the brain, but there are no obvious signs of neurodegeneration. In this study, we uncovered two distinct components involved in the neuropathology in MFP2 deficiency by generating mouse models with cell type selective elimination of MFP2. Firstly, MFP2 deficiency induces a dying back neuropathy of Purkinje cells leading to cerebellar atrophy and severe ataxia. Although Purkinje cell axonal integrity is lost before cell bodies are affected, we were able to exclude that the axonopathy is mediated by primary demyelination or oligodendrocyte dysfunction. Furthermore, by comparing metabolic abnormalities in general and cell type selective knockouts of MFP2, we show that loss of cerebellar integrity does not correlate with the accumulation of known peroxisomal ß-oxidation substrates such as very long chain fatty acids and branched-chain fatty acids. Secondly, Mfp2-/- mice show a strong neuroinflammatory response characterized by increased pro-inflammatory gene expression and reactive microgliosis. Remarkably, selective elimination of MFP2 from neural cells (neurons, astrocytes and oligodendrocytes) only induces minor neuroinflammation and limited microgliosis, suggesting a role of non-neural cells in the inflammatory response. We excluded a possible contribution of peripheral immune cells to the inflammatory response in brain of Mfp2-/- mice, suggesting that intrinsic misbehavior of MFP2 deficient microglia underlies the excessive neuroinflammation. In order to characterize the microglia mediated inflammatory response, transcriptomics analysis on isolated microglia from Mfp2-/- mice was performed. This revealed a significant downregulation of markers characteristic of resting/surveillant microglia whereas markers of activated macrophages were induced. The latter did not coincide with a typical M1 or M2 macrophage signature. Pathway analysis indicated activation of the mTOR pathway which was predicted to be regulated upstream by growth factor-AKT-MAPK signaling. Comparative analysis with a mouse model of amyotrophic lateral sclerosis showed that microglia in the MFP2 deficient brain display a unique activated phenotype, lacking signs of phagocytic and neurotoxic activity but with a pronounced induction of pathways associated with growth and proliferation. In conclusion, using knockout mice as a modeling system, we show that MFP2 deficiency induces multiple pathologies in the CNS. The finding that MFP2 deficiency leads to cerebellar degeneration introduces a novel concept in the research on peroxisomal disorders that might be of particular relevance for patients with a mild form of peroxisomal disease presenting with isolated cerebellar ataxia. Furthermore, the distinct microglia mediated inflammation in Mfp2-/- and Nestin-Mfp2-/- mice is the first in vivo experimental evidence that peroxisomal dysfunction in microglia impacts on responses of these cells. Although we were able to annotate a peculiar activation state to MFP2 deficient microglia, its contribution to disease progression in MFP2 deficient mice needs further investigation.status: publishe