| dc.description.abstract | Axonal degeneration is the major cause of disability in progressive
multiple sclerosis (MS). It has been shown that in MS and relevant disease
models, demyelinated axons harbor an increased number of mitochondria,
which is reflected in bigger stationary sites of mitochondria, increased
mitochondrial activity and increased transport speed of mitochondria. This
axonal response of mitochondria to demyelination (ARMD) is protective, as
there is an increase in energy demand due to the redistribution of sodium
channels along the axon following demyelination. However, it remains to be
determined how this ARMD is mounted and how mitochondrial dynamics
are involved. By using in vivo and in vitro systems we are determined to
elucidate the transport and fusion dynamics of the ARMD and where these
additional mitochondria come from. Using a cerebellar slice culture system
with lysolecithin induced demyelination, we show that the increase in
mitochondrial occupancy of the axon already occurs at 24 hours after
demyelination and plateaus around 3 to 4 days after demyelination. At 24
hours, there was a steep increase in the mitochondrial numbers inside the
axon, which is then followed by an increase in mitochondrial size over the
following days. All parameters decrease again over the following days, but
remain elevated compared to baseline even 12 days after demyelination. To
determine the source of these additional mitochondria and to assess the
fusion dynamics within the axon, we used a lentivirus expressing a
mitochondrial targeted photoconvertible dye (mEOS2) to label
mitochondria in Purkinje cells. The mitochondria that are labelled green in
the Purkinje cell axons are then photoconverted to red by illuminating the
initial part of the axon with a 405-nm laser and imaged over the following
20 minutes to determine the transport and fusion dynamics. This showed an
increased number of mitochondria moving from the cell body into the axon,
as well as an increase in retrograde transport of mitochondria in the
demyelinated compared to the myelinated axons. Furthermore the size of
newly transported mitochondria and their speed was increased in the
anterograde direction. Furthermore, the fusion rate of newly transported
mitochondria with stationary converted mitochondria was increased in the
demyelinated axons compared to myelinated control. These changes can
also be observed in unmyelinated axons, as well as axons of cerebellar slices
of the dysmyelinating shiverer mutant with or without lysolecithin
treatment. The manipulation of mitochondrial dynamics after
demyelination with the fission inhibitor mdivi-1 and the ATPase inhibitor
oligomycin both showed an increasing or decreasing effect on the
mitochondrial parameters after demyelination respectively. The effect on
the axonal health after demyelination was detrimental with both of these
treatments. Increasing mitochondrial biogenesis with pioglitazone
increased axonal mitochondrial parameters, as well as ameliorated axonal
damage after demyelination with lysolecithin. As the neuronal cell bodies in
MS harbour mitochondrial DNA deletions, which affects their physiology,
including energy production efficiency, another aim of this thesis was to
model this deficiency in vitro. As it was not possible to model these
mitochondrial defects in vitro within the experiments of this thesis, the
characterization of a mitochondrial mutant in vivo model was done as a
contribution to a greater set of experiments performed by other members of
the Mahad lab. | en |
