Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous
system (CNS) characterized by inflammation and the formation of demyelinating lesions.
Remyelination ensures the survival of the denuded axons. The clearance of myelin debris,
oligodendrocyte (OL) precursor cell (OPC) proliferation, migration, and differentiation,
known as oligodendrogenesis, are crucial for remyelination. Earlier studies have shown that
myelin-specific T cells can stimulate oligodendrogenesis in mice, and that the ferroxidase
ceruloplasmin (Cp) is a potential OPC-expressed gene involved in the T-cell enhanced
oligodendrogenesis. The aim of this thesis was to test the hypothesis that Cp is involved in
T cell-enhanced oligodendrogenesis.
Central to this PhD is the use of a Cp deficient mouse (Cptm1b(KOMP)Wtsi), which is not widely
used in scientific publications. Therefore, the first study in the PhD was dedicated to examine
the proteome of brains from asymptomatic 4-6-month-old Cp deficient (knockouts and
heterozygotes) mice in comparison to wildtype mice. More than 5600 proteins were
identified, out of which 23 were regulated in our mice, together with more than 600 proteins
having post-translational modifications (PTMs). The genes coding for these proteins were
mostly expressed in neurons and OPCs. In addition, the relation to neurons was clear from
the GO-term analysis, where some of the most affected terms were synaptic vesicle
budding, neuronal projection development, and dendrite development. Because of the
ferroxidase activity of Cp, the observed changes might also be due to altered iron
metabolism. Haematological examination of 3-5-month-old Cp deficient mice showed a
reduction in serum iron levels, transferrin saturation and mean corpuscular volume, showing
that iron metabolism is affected even in asymptomatic Cp deficient mice.
The characterisation of the Cp deficient mice was followed by a developmental study
examining the effect of Cp on the primary myelination and the mRNA expression of ironrelated genes from postnatal day 9, through postnatal day 22, and until 4-5 months old.
Luxol fast blue (LFB), Olig2 stainings and Q-PCR data, for myelin and oligodendrocyte
lineage cells, suggested that the primary myelination in the brain of Cp deficient mice is
comparable to that of wildtype mice. Additionally, the mRNA expression of iron-related
genes did not show any compensatory changes in absence of Cp.
The finding of a normal primary myelination in the Cp deficient mice made it possible to
examine the effect of Cp on de- and remyelination processes by using the cuprizone (CPZ)-
model. The CPZ-induced de- and remyelination in 3-6-month-old mice was examined by
LFB stainings and showed no effect of Cp on the completeness of the demyelination of the
corpus callosum (CC) after 6 weeks of treatment. However, Cp deficient mice showed a
lower cellularity and CD11b immunopositivity in the CC after 6 weeks of CPZ treatment,
which suggests an effect of Cp on the demyelination-induced microglial activation state. QPCR analysis further suggested a connection between Cp, the OPC expressed gene Pdgfra
and microglial CD11b and CD11c. The mRNA expression of Cp and the homologue Heph
were both upregulated during CPZ treatment, but Heph was not affected by Cp deficiency.
This suggests that Heph did not compensate for the Cp deficiency even during
demyelination, where Cp is upregulated in wildtype mice.
Lastly, a model combining CPZ treatment and active immunisation was used to examine the
hypothesis, that Cp is involved in T cell-enhanced oligodendrogenesis. The mice were
treated with CPZ for three weeks with a MOG(35-55) injection at day 17, which should induce
myelin-reactive T cells to infiltrate the brain and enhance the generation of new OLs in the
CC. The treated mice were sacrificed one week after CPZ withdrawal and showed no
symptoms of the immunisation. The model, including the T cell infiltration, needs to be
additionally validated, but preliminary results show some degree of demyelination in the
mice, an increase in cellularity, and an activation of CD11b microglia in the CC. Of potential
interest, this activation of microglia and demyelination were not observed in the Cp deficient
mice, all of which needs to be investigated in future studies.
In conclusion, this thesis suggests interesting new functions of Cp in the brain during
development and CPZ-induced demyelination. The characterisation of the Cp deficient mice
in relation to the brain proteome, haematology and primary myelination was performed as
an introductory study before examining the hypothesis. The results generated up till now
neither confirm nor reject the hypothesis that Cp is involved in T cell-enhanced
oligodendrogenesis, however, the results suggest that Cp is important for the activation of
CD11b microglia in response to CPZ-treatment. The latter might impact both myelin
clearance and remyelination.
Print copy of the thesis is restricted to reference use in the Library.