Dynamic regulation of the cellular prion protein in microglia following histamine-induced stimulation
Creators: Pehar, Marcus F
Publisher: name: University of Alberta Library
Year: 2024

<p>Although the cellular prion protein (PrPC) has been evolutionarily conserved throughout evolution, its precise role remains elusive. Recent findings suggest that PrPC may be involved in neuroinflammation and immune responses in the brain, and its expression can be modulated through various mechanisms. Additionally, the role of mast cells in the central nervous system is a growing topic of interest, with evidence that histamine released by mast cells in mice can stimulate various cells, including microglia, an innate immune cell of the central nervous system. This thesis investigates the expression and regulation of PrPC in immune and neurological cell models, with a focus on human microglial cells and their response to histamine. </p>

<p>We first established a reliable method to measure total and surface PrPC in various immune, neurological, and microglial cell models using flow cytometry and western blot. This included confirmation that PrPC was present on the human microglial cell line, HMC3, which was then used for the remaining studies to examine the effects of histamine on microglia.</p>

<p>Western blotting and quantitative reverse transcription-polymerase chain reaction were used to measure protein and gene expression levels of histamine receptors HRH1-4. HMC3 cells expressed all but HRH4. Histamine-induced responses, including metabolic activity, cytokine release, and intracellular calcium levels were assessed. Metabolic activity was measured via XTT assay and calcium flux was assessed following histamine stimulation. Cytokine production was monitored by enzyme-linked immunosorbent assay (ELISA), with IL-6 and IL-8 levels increasing following histamine treatment.</p>

<p>We then examined how HMC3 PrPC levels were affected by different doses and durations of histamine treatment. Histamine-induced stimulation for 6 and 24 hours increased HMC3 surface PrPC expression, with a subsequent decrease after 48 and 72 hours. Antagonist studies confirmed that these changes in surface PrPC were mediated via stimulation of the HRH2 receptor. Total PrPC levels were unchanged, suggesting the surface PrPC changes were secondary to a change in localization. Fluorescence microscopy of the cells further supported this hypothesis, as more surface PrPC was seen on the cells after 24 hours of histamine stimulation. Notably, while the HRH2 antagonist blocked the histamine-induced increase in surface PrPC, it did not inhibit IL-8 release and only partially reduced IL-6 release.</p>

<p>In conclusion, we demonstrated that the HMC3 human microglial cell line expresses PrPC and histamine receptors 1-3. HMC3 cells can be stimulated by histamine to release IL-6 and IL-8 and undergo intracellular calcium influx. More notably, the surface levels of PrPC on HMC3 cells are altered by histamine exposure, primarily mediated by HRH2 in a time- and dose-dependent manner. While histamine exposure also leads to the release of IL-6 and IL-8 in these cells, this cytokine release is not fully dependent on PrPC levels, as IL-6 release is only partially reduced and IL-8 release is unchanged under the conditions of HRH2 blockade that prevent PrPC changes. Overall, this suggests that PrPC may play a role in modulating microglial responses to histamine but does so through pathways other than cytokine release.</p>