(W1149) THE DELETERIOUS IMPACTS OF INTERFERON-GAMMA AND NEUROTOXIN QUINOLINIC ACID ON CELLULAR HEALTH OF SVZ NEURAL STEM CELLS AND CONSEQUENCES FOR REGENERATION
Senior Postdoctoral Scientist St. Vincent's Centre for Applied Medical Research, New South Wales, Australia
Abstract: Current treatments for Multiple Sclerosis (MS) reduce the autoimmune-driven relapses, but are ineffective at preventing neurological disability arising in the progressive phase, where brain cells die. The Kynurenine Pathway (KP) contributes to MS pathogenesis by it’s dysregulation in cells like proinflammatory monocytes, producing neurotoxins including Quinolinic acid (QUIN) that our group showed potently kills brain cells, particularly oligodendrocytes. Circulating blood/CSF QUIN correlates with increased MS severity. The KP is activated by the proinflammatory cytokine interferon-gamma (IFN-g), upregulating rate-limiting enzymes including indoleamine-2,3-dioxygenase (IDO-1), however whether this pathway is active in adult subventricular zone (SVZ) mouse neural stem cells (mNSCs) is unknown. IFN-g reduces proliferation of other stem cells, and could compromise repair mechanisms. We further hypothesised QUIN might damage the cellular health of mNSCs, and have similar sequelae. QUIN can be taken up by amino acid transporters and localise to lysosomes, where it can produce reactive oxygen species (ROS) via the Fenton reaction.
A Muse cytometer was used to measure cell-health in neurospheres grown from the subventricular zone, passaged and plated as monolayers, over 24-72 hour timepoints; analysis used an ANOVA. IFN-g significantly increased oxidative stress in mNSCs (72 hour control 14.26±3.28% versus IFN-γ 26.03±4.17%, *p0.0183). IFN-g significantly increased G0/G1 NSCs, signifying reduced proliferation (72hr control 56.54±2.44% versus IFN-g 67.09±1.60%, *p0.037) – this was also observed in significantly reduced neurosphere size. QUIN dose-dependently significantly increased activated caspases, even at the 24 hour timepoint - control (36.45%±3.09%) vs 3uM QUIN (70.92%±6.82%), demonstrating QUIN activates the apoptosis cell death pathway. Live cell imaging was then used to assess lysosomes and their Fe2+-iron ion content, linked with ROS production, demonstrating substantial pools of iron in NSCs and reduced confluence in 50nM/250nM-treated cells (14 hour imaging). In conclusion, reducing IFN-g and QUIN production by other cells could reduce cell death, potentially improve the regenerative capacity of NSCs and in turn be effective in treating the neurodegeneration in MS.
Funding Source: We acknowledge funding of the Peter Duncan Neurosciences Unit.
