Age-associated ROS production during remyelination in Multiple Sclerosis (MS)
Creators: Panda, Sharmistha
Publisher: name: University of Alberta Library
Year: 2024

<p>Multiple sclerosis (MS) is a chronic inflammatory disease characterized by central<br>
nervous system (CNS) lesions, resulting in axonal loss and physical and cognitive disability.<br>
Loss of myelin sheath known as demyelination, is followed by its regeneration in a process,<br>
known as remyelination, that protects axons from degeneration, thereby slowing the permanent<br>
disability related to axonal loss. Acute demyelination is followed by myelin debris removal<br>
which is crucial for remyelination. The process of remyelination is then characterized by the<br>
recruitment and proliferation of oligodendrocyte progenitor cells (OPCs) and their subsequent<br>
differentiation into myelinating oligodendrocytes. Microglia and monocyte-derived macrophages<br>
(MDMs) play multiple roles during remyelination. Microglia can stimulate OPC proliferation in<br>
vitro, and microglia/MDMs can secrete factors, such as activin, that promote oligodendrocyte<br>
differentiation. Both microglia and MDMs also phagocytose debris from injured myelin, which<br>
is a known remyelination inhibitor.<br>
Remyelination declines during aging. However, it is still unknown what causes this agedependent<br>
decline. Previous studies have shown that microglia and MDMs produce reactive<br>
oxygen species(ROS) up to toxic levels in middle-aged mice. Given that aging is associated with<br>
increased ROS in the CNS, we hypothesized that a population(s) of aged microglia/macrophages<br>
in the CNS produce excessive ROS contributing to age-associated remyelination decline. I used<br>
the LPC (lysophosphatidylcholine) model of demyelination and examined the presence of ageassociated<br>
ROS production during remyelination in young (2-3months) and middle-aged (8-10<br>
months) mice receiving intraspinal LPC injections. I first characterized the accumulation of<br>
microglia and MDMs using lineage tracing with young and middle-aged male and female<br>
CX3CR1CreEr; ROSA26tdT mice. This mouse line expresses tdTomato (tdTom) in microglia under<br>
the ROSA26 promotor. The benefit of this method is that microglia and their progeny are<br>
permanently labelled using a reporter that does not change during inflammation. I found a<br>
delayed accumulation of microglia but not monocyte-derived macrophages in middle-aged<br>
CX3CR1CreEr; ROSA26tdT mice. To evaluate ROS production, I quantified oxidative damage to<br>
lipids by immunostaining against different lipid peroxidation markers within remyelinating<br>
lesions. Based on MDA and E06 immunoreactivity, I found that lipid peroxidation peaks in<br>
middle-aged mice at early to later stages of remyelination, i.e. 7 and 21DPL. In young mice,<br>
based on MDA immunostaining, lipid peroxidation increases from 3 to 21 DPL.<br>
I next inhibited elevated ROS production by orally administering Setanaxib<br>
(GKT137831), an inhibitor of ROS-producing enzyme, NADPH oxidase (NOX1/4 isoform), to<br>
middle-aged female C57BL/6 mice after LPC injection, to see if reduced ROS production can<br>
boost remyelination. I found that Setanaxib reduced excessive ROS production marked by<br>
decreased E06 immunoreactivity in the lesion at 7DPL. I then immunostained against OPC<br>
marker PDGFRa and proliferation marker Ki67 to quantify proliferating OPCs and found no<br>
difference in proliferating OPC densities in Setanaxib and Vehicle groups. Similarly, upon<br>
immunostaining against transcription factor Myrf+, there were no differences in Myrf+<br>
oligodendrocytes in Setanaxib and Vehicle groups. Also, immunostaining against pan-leukocyte<br>
marker CD45, microglia/MDM marker IBA1 and myelin basic protein (MBP), revealed that<br>
Setanaxib did not boost MBP+myelin debris clearance and accumulation of microglia/MDMs in<br>
the lesion. Taken together, Setanaxib did not boost remyelination in middle-aged female<br>
C57BL/6 mice and therefore, reducing excessive ROS production alone, cannot rescue age-associated<br>
delayed remyelination.</p>