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Abdul-Rahman, Raneen

University of British Columbia

Multiple sclerosis (MS) is characterized by inflammatory demyelination and axonal loss, which contribute to irreversible clinical disability. Both hallmarks affect calcium homeostasis, partly due to mediators released by inflammatory cells, like glutamate and hydrogen peroxide (H₂O₂). Nuclear calcium transients can activate neuroprotective factors, including neuronal PAS-domain containing protein 4 (NPAS4), a neuronal activity-dependent gene. NPAS4 and aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) work independently or in combination to regulate genes responsible for neuronal survival. We sought to determine the effects of soluble inflammatory mediators and inflammatory cells relevant to MS pathogenesis on NPAS4 and ARNT2 expression. Murine neuron-enriched cortical cultures were treated with increasing concentrations of glutamate (1-30µM) and H₂O₂ (12.5-100µM) or co-cultured in pilot experiments with resting or activated CD4⁺ T-cells. NPAS4 and ARNT2 protein expression were characterized via immunocytochemistry by examining the neuronal nuclear mean fluorescent intensity (MFI), the percentage of positive cells, and staining intensity changes using fluorescence microscopy across time and concentrations. In untreated conditions, NPAS4 was detected in less than 10% of neurons, while greater than 70% of neurons expressed low-medium ARNT2 levels. Glutamate increased the NPAS4 MFI and percentage of NPAS4-positive neurons, with increased staining intensity observed early at all doses. ARNT2 staining levels were similar across the glutamate doses and times. Prolonged glutamate exposure (10-30µM) was associated with morphological changes and cell loss. H₂O₂ (50-100µM) increased the NPAS4 MFI and percentage of NPAS4-expressing cells at later time points (5hrs and greater), with a simultaneous decrease and loss of ARNT2 expression. Exposure to H₂O₂ at 50-100µM tended to impact neuronal cell viability with significant cell loss by 9 hours compared to controls. Lastly, the addition of resting or activated CD4⁺ T-cells, at a 10- to 20-fold higher ratio to neurons, tended to increase the NPAS4 MFI and rapidly increased the percentage of high-intensely expressing cells without affecting ARNT2 expression. This study shows that MS-related inflammatory mediators and CD4⁺ lymphocytes modulate in vitro neuronal NPAS4 and ARNT2 protein expression. Further research is required to elucidate the relationship between these proteins and how changes in their expression might impact neuronal health in inflammatory degenerative settings.

Text

2024-11-20

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89692

Pathology and Laboratory Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/