Multiple sclerosis (MS) is the most common neurological disease affecting
young adults. The incidence rates in Scotland are among the highest in the
world. Virus infection may have a precipitating role, or exacerbate
symptoms. That viruses can produce inflammatory central nervous system
(CNS) demyelinating disease has been well established from study of several
natural and experimental infections; one model system is Semliki Forest
virus (SFV) infection of mice.
SFV infection of BALB/c mice infected at less than 12 days of age (P12) causes
fulminant encephalitis, characterised by apoptotic death of neuronal cell
populations. In contrast, virus-infected neurons in mice older than P14
survive. In immunocompetent animals lesions of inflammatory
demyelination develop. In immunocompromised animals (for example
nu/nu mice) virus persists but no lesions of demyelination develop. SFV had
previously been observed to trigger apoptosis in numerous proliferating cell
lines and in immature differentiating neuronal cell cultures. One explanation
for the dichotomy in the outcome of neuronal infection is that levels of key
pro- or anti-apoptotic proteins differ as a function of maturational state.
Oligodendrocytes are infected by SFV but little is known about the outcome
of infection in these cells. The absence of demyelination in
immunocompromised animals suggests these cells can survive infection.
The aim of this thesis is to determine the fate of oligodendrocytes upon SFV
infection, to ascertain the nature of any cell death and, if observed, to
determine whether this is dependent on cell maturation state. Three
experimental systems are used; glial cell lines, mixed glial cell cultures, and
tissue sections from infected adult BALB/c and nu/nu mice.
Two differentiation-inducible oligodendrocyte-type-2 astrocyte (0-2A)
progenitor cell lines, SS5 and BC30, were utilised. 0-2A progenitor cells died
rapidly of apoptosis as determined by DAPI labelling, DNA fragmentation
and morphological features such as cell blistering and cell blebbing.
Differentiation delayed virus induced cell death between 12 and 24 hours but
did not prevent it: again cell death occurred by apoptosis.
The outcome of SFV infection of oligodendroglia derived from mixed
primary cultures was dependent on the degree of cellular homogeneity.
Immunocytochemical studies revealed that immature 0-2A progenitors died
rapidly, as revealed by double staining for caspase-3 and early 0-2A cell
markers. The susceptibility of mature oligodendrocytes was found to be
dependent on cellular environment. Mature oligodendrocytes were equally
susceptible to virus-induced apoptosis as immature progenitors when
cultured in isolation i.e. as enriched purified populations. On the contrary,
cultures of mature oligodendrocytes maintained in the presence of other glial
cells, or in contact with extracellular matrix molecules present in vivo,
survived infection for the period under study: two weeks.
The fate of the mature oligodendrocyte was further investigated in the
corpus callosum in vivo using sections from SFV-infected adult BALB/c and
nu/nu mice. Dual immunolabelling for virus and apoptosis (TUNEL
staining) revealed an active period of cell death between PID 4 and 6. Dual
immunofluorescence for 2'/-3'-cyclic nucleotide 3'-phosphodiesterase
(CNPase) and SFV or caspase-3 visualised by confocal microscopy indicated
that although mature oligodendrocytes were infected with SFV they did not
die by apoptosis.
In conclusion, this study presents evidence that isolated enriched cultures of
mature oligodendrocytes are susceptible to apoptosis as are their immature
counterparts. Paradoxically, mature oligodendrocytes studied in vivo or
maintained in the presence of other glial cells factors in vitro, did not undergo
virus-induced apoptosis, unlike their immature relatives.