Electronic Thesis and Dissertation Repository

Degree

Master of Science

Program

Anatomy and Cell Biology

Supervisor

Whitehead, Shawn N

Abstract

Gangliosides are membrane glycosphingolipids which are widely distributed within cells of the central nervous system and play a pivotal role in cell repair and injury. Gangliosides exist in equilibrium within the cell; however, this equilibrium shifts in response to stress such that the complex ganglioside GM1 is degraded to simpler species GM2 and GM3. Complex gangliosides are linked to an upregulation of neuroprotective signaling while simple species have been associated with neurodegeneration and cell death. In this study, we therefore sought to perturb the gangliosides homeostasis using Ambroxol; a ganglioside modifying agent that has been shown to perturb enzymes in the ganglioside degradation pathway, in order to determine if Ambroxol has any effects on neuronal ganglioside expression profiles, and to compare the neuroprotective potential of Ambroxol to exogenous GM1 treatment in neurons undergoing oxygen glucose deprivation (OGD). Primary embryonic (E18) rat cortical neurons were cultured, and at DIV (days in vitro) 10 treated with different concentrations of Ambroxol for up to 24 h. GM1 and GM3 gangliosides were visualized using immunocytochemistry. GM1, GM2 and GM3 species were quantified using electrospray ionization mass spectrometry (ESI-MS). Immunofluorescence demonstrated that GM1 significantly increased 6 h following treatment with Ambroxol. ESI-MS data revealed that GM1 increased at 6 h and 24 h post treatment. To test the neuroprotective potential of Ambroxol, neurons were pre-treated with either Ambroxol for 6 h or exogenous GM1 for 24 h prior to 1 h oxygen glucose deprivation (OGD), and re-perfused for 6 h and 24 h. Cell viability was assessed using propidium iodide (PI) staining. Compared to untreated neurons and to neurons only exposed to OGD, neurons treated with Ambroxol and exposed to OGD showed no increase in cell death after OGD/R injury. Exogenous GM1 pretreatment conferred neuroprotection and enhanced endogenous GM1 levels. These preliminary results suggest that Ambroxol increases GM1levels in neurons and may confer a neuroprotective effect. All in all, modulating ganglioside levels by ganglioside enzyme modifiers may uncover a new lipid based therapeutic approach to protect neurons and promote their survival following stress or injury.

Download

Share

COinS