The role of peroxisomes in the formation and maintenance of myelinated axons.

Abstract

Patients with Zellweger syndrome or other peroxisomal disorders underscore that peroxisomes are indispensible organelles for the development of the CNS. However, milder affected patients do not only display developmental problems, like a neuronal migration defect, hypotonia and seizures, but also degenerative changes, including inflammatory demyelination. Mouse models, mimicking human pathology, are valuable tools to investigate the pathomechanisms triggered by peroxisomal dysfunction. The Nestin-Pex5 mouse model, lacking functional peroxisomes in neural cells, was developed to circumvent the very early death of the general Pex5 knockout model, which hampered postnatal research. As described earlier, Nestin-Pex5 knockout mice do not display pronounced developmental problems, but show remarkable regressive pathology after birth. Three major findings have been described at the microscopic level, including demyelination, axonal degeneration and neuroinflammation, comprising astro- and microgliosis. Microscopic brain pathology is associated with an abnormal phenotype characterized by disturbed motor performance, progressively worsening with increasing age and evolving in lethargy and early death. As expected, metabolic alterations were also observed in these mice, including an accumulation of VLCFAs and a depletion of plasmalogens. Until now, the link between all these changes is still not elucidated. The Cnp-Pex5 model, with dysfunctional peroxisomes specifically in oligodendrocytes, proved that functional peroxisomes in these myelinating cells are of major importance for the preservation of normal myelinated axons. This mouse model developed similar pathology as the Nestin-Pex5 knockout, although with a later onset and a slower progression. The main goal of this thesis was to clarify the role of peroxisomes in the maintenance of healthy myelinated nerve fibers, which are crucial for the normal functioning of the brain. In view of the less severe phenotype of the oligodendrocyte specific Cnp-Pex5-/- compared with the neural Nestin-Pex5-/- mice, the role of peroxisomes in neurons and astrocytes was investigated, by the use of the NEX- and GFAP-Pex5 mouse models. Based on the normal phenotype of the NEX-Pex5-/-, peroxisomes are not important in neurons, provided that there are still peroxisomes in other brain cell types. Several abnormalities were observed in the astrocyte selective GFAP-Pex5 knockout, including an accumulation of VLCFAs, a small reduction of plasmalogens, an accumulation of lipid droplets and elevated levels of catalase. However, this had no major repercussion on the health and survival of these mice. Taking into account the phenotype of the three cell type selective Pex5 knockouts, we can conclude that peroxisomes in oligodendrocytes are the most important for normal brain functioning and that peroxisomes in astrocytes may have a supportive function. The second aim of this manuscript was to decipher the onset and relationship between neuroinflammation, demyelination and axonal degeneration. Immunohistochemical investigations revealed that demyelination and microgliosis are observed in young Nestin-Pex5 knockout mice and in close association with each other. Axonal degeneration was mostly observed after demyelination, indicating that this is presumably a consequence of demyelination. In order to rule out that these defects are a consequence of developmental abnormalities, a mouse model was generated and analyzed with deletion of functional peroxisomes after the myelination period. This CMV-Tx-Pex5 knockout model developed similar pathologies of inflammation, demyelination and axon degeneration. The analysis of mRNA levels of inflammatory markers like TNFα, interleukins and chemokines showed that pro-inflammatory markers were highly up regulated and clearly before demyelination was observed by fluorescence microscopy. Interestingly, also anti-inflammatory markers were up regulated in Nestin-Pex5 knockout but this could not prevent the evolvement in a chronic inflammatory reaction. As activated microglial cells may have different shapes and functions, we demonstrated their phagocytotic activity by their swollen morphology, the presence of myelin debris within several microglial cells, and the up regulation of mRNA levels of macrophage markers. This proves that inflammatory demyelination is ongoing in the Nestin-Pex5 knockout. C1q, the first component of the complement system, was detected on neurons and oligodendrocytes before significant microgliosis was observed. This component is considered to be present on damaged cells as an alert signal towards inflammatory cells. We hypothesize that dysfunction of peroxisomes in oligodendrocytes triggers the activation of the innate immune system. If peroxisomes are also absent from other neural cell types, this reaction occurs much earlier and to a larger extent. Direct comparison of the Nestin-Pex5 model with the Gnpat knockout model, characterized by plasmalogen depletion, demonstrated that the absence of plasmalogens per se does not trigger an inflammatory reaction. Markers of oxidative stress were detected in both models in Purkinje cells and only in a late phase in the corpus callosum of Nestin-Pex5-/- mice. Thus, oxidative stress is not considered to be a trigger for neuroinflammation. The latter is confirmed by the observation that treatment of Nestin-Pex5 knockout mice with anti-oxidant drugs did not improve pathology.status: publishe