Age-associated ROS production during remyelination in Multiple Sclerosis (MS)

• Author / Creator

  Panda, Sharmistha

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

• Subjects / Keywords

  • Multiple Sclerosis
  • Aging
  • Remyelination

• Graduation date

  Fall 2024

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-yah3-6x61

• License

  This thesis is made available by the University of Alberta Library with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.