Effect of PAR‐1 Receptor Inhibition in an In Vitro 6‐hydroxydopamine Model of Parkinson’s Disease Using Differentiated PC12 Cell Line and Undifferentiated SH‐SY5Y Cell Line.

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative condition affecting dopaminergic neurons of the A9 nigrostriatal pathway. Besides the hallmark features of bradykinesia, cogwheel rigidity, and resting tremor, PD patients are at an increased risk of strokes. Protease‐activated receptors (PARs) and thrombin have been implicated in the neuronal damage following both ischemic and hemorrhagic strokes, and may play a role in the neurodegeneration observed in PD. Furthermore, brain PAR receptor expression appears to be upregulated in PD patients. The purpose of this study is to determine if PAR‐1 receptor inhibition can provide neuroprotection in two commonly used in vitro 6‐hydroxydopamine (6‐OHDA) models of PD: differentiated PC12 cells vs. undifferentiated SH‐SY5Y cells. In a previous study, we demonstrated an antiproliferative effect on the A549 cancer cell line via inhibition of PAR‐1 receptors with enoxaparin. We hypothesize that PAR‐1 receptor inhibition will result in neuroprotection from 6‐OHDA toxicity in differentiated amitotic PC12 cells and will promote neurotoxicity in undifferentiated SH‐SY5Y cells. This study hopes to elucidate the mechanisms behind this differential effect of PAR‐1 receptor inhibition in the two cell lines. To this end, Western Blot analysis was conducted to confirm that PAR‐1 receptors were expressed in differentiated PC12 cells and undifferentiated SH‐SY5Y cells. Then, both cell lines were treated with a potent PAR‐1 antagonist, SCH79797 dihydrochloride (SCH), and cell viability was assessed by the MTS Assay. PAR‐1 blockade did in fact have a differential effect in the two cell lines by reducing the cell viability of undifferentiated SH‐SY5Y cells, while having no effect on the cell viability of differentiated PC12 cells. Consequently, both the cell lines were exposed to 6‐OHDA and differentiated PC12 cells were treated with SCH after inducing toxicity with 6‐OHDA. Preliminary results suggest that the selective PAR‐1 antagonist, SCH79797 dihydrochloride, may provide a protective effect against 6‐OHDA toxicity in differentiated PC12 cells. Additional studies will be conducted to determine the effect of SCH on 6‐OHDA treated undifferentiated SH‐SY5Y cells, and the mechanisms involved in this differential effect of PAR‐1 receptor inhibition.Support or Funding InformationDepartment of Pharmaceutical Sciences, MCPHS University, Boston, MA.PAR‐1 Receptor Inhibition Increases Cell Viability in a PC12 model of Parkinson’s Disease:Effect of treatment with SCH79797 dihydrochloride (SCH; 1 μM) on differentiated PC12 cells exposed to 6‐hydroxydopamine (6‐OHDA; 250 μM) toxicity as assessed by the MTS Assay. Data is represented as a mean ± S.D. of three independent experiments (n=3). * p<0.0001 as compared to the untreated control, # p<0.005 as compared to the model group.Figure 1