Neuron-Glia Crosstalk: Investigating Immune-Cell Modulation in the CNP1 Null Mutant Mouse
by Marie Louise Aicher
Date of Examination:2023-03-01
Date of issue:2023-03-01
Advisor:Prof. Dr. Klaus-Armin Nave
Referee:Prof. Dr. Klaus-Armin Nave
Referee:Prof. Dr. Christine Stadelmann-Nessler
Persistent Address:
http://dx.doi.org/10.53846/goediss-9749
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Abstract
English
Myelin sheaths built by oligodendrocytes in the central nervous system (CNS) are not only important insulators that enable the fast saltatory propagation of action potentials but also fulfill metabolic support functions to their underlying axons. 2’,3’-cyclic nucleotide 3’ phos- phodiesterase (CNP1) constitutes 4% of total CNS myelin proteins and is disease-relevant in humans and mice. First, a homozygous missense mutation in CNP1 was recently associated with a hypomyelinating dystrophy in humans and second, a reduced CNP1 gene expression causes an age-dependent catatonia-depression syndrome in mouse and man. By immunohistochemistry and RNA-Seq. on purified myelin-enriched fractions, we aimed at investigating which changes occur at early ages in CNP1 null mutants and may thus be initial mediators of disease pathology. We found that signs of neuroinflammation and neu- rodegeneration were largely absent at the age of P18 but obvious at P75, suggesting that the earlier age represents a prodromal phase of pathology. Nevertheless, by sensitive qRT-PCR, we identified the anti-inflammatory Tnfaip6 and the alarmin Il-33 upregulated in mutant myelin as early as P20 consistent with the idea that these molecules are secreted by CNP1 deprived oligodendrocytes. Although these candidates are thus possible initial disease modi- fiers, it remains to be analyzed in future experiments, whether they are indeed oligodendro- cyte-derived, since subsequent analyses by qRT-PCR questioned, that P20 can be fully re- garded as prodromal stage of CNP1-related pathology. When analyzing astroglial and microglial RNAs in purified myelin at P75, we noted that as- trocytes exhibit features of A1 (C3, Ggta1, Gbp2) and A2 (Cd109, Cd14, Emp1) cells, and that microglia express pro-inflammatory M1 markers (Ccl6, Fcgr3, Cd86) as well as M2 mark- ers (Clec7a, Trem2) typical for phagocytosing cells. Additionally, Toll-like-receptors (Tlr) 1 and 2 were found upregulated. A subsequent immunohistochemical analysis of CNP1- MyD88-double mutants identified the TLR-associated MyD88-pathway in microglia as in- strumental in inducing axonal pathology in CNP1-deficient mice. Finally, we hypothesized that the previously reported pathology of cytosolic channels in CNP1 null mutant myelin results in a quantitative alteration of transported mRNAs and pro- teins. By qRT-PCR we found Mbp-mRNA higher in abundance in myelin of mutants at P20, compatible with the idea that an increased local translation of this mRNA is causatively linked to the reported earlier closure of cytosolic myelin channels due to an increased presence of MPB-protein, the only myelin protein absolutely necessary for myelin compaction. At P75 we found Fth1-mRNA, which is reported to be extremely abundant in myelin, reduced by 70% in CNP1 null mutants. This may be of relevance for the pathology since the secretion of Fth1 by oligodendrocytes is suggested to serve as a protective mechanism against iron- induced axonopathy. At P75 we also found the abundance of MCT1 protein, an oligoden- droglial axon supporting monocarboxylate transporter, reduced. Hence, the pathology of cytosolic myelin channels may indeed disrupt the transport of proteins essential for axonal support.
Keywords: Myelin; Oligodendrocytes; CNP1; Glial Crosstalk