Investigating microRNA miR-34b and miR-34c Expression in a Parkinson’s Disease Cell Line Model
Description
PURPOSE: The motor symptoms of Parkinson’s disease (PD) are caused by the degeneration of dopaminergic neurons in the midbrain. These dying neurons are characterized primarily by large aggregates of alpha-synuclein protein. miR-34b and miR-34c are two microRNAs that repress alpha-synuclein expression. We hypothesize that treating degenerating neurons with these microRNAs will decrease alpha-synuclein aggregation and increase cell survival. SUBJECTS: SH-SY5Y, a neuroblastoma cell line, was cultured and treated with retinoic acid, brain-derived neurotrophic factor (BDNF), and rotenone to induce a PD-like phenotype. METHODS AND MATERIALS: Live cells were counted on a hemocytometer after staining with trypan blue. Brightfield images were taken using an inverted compound microscope. Cells will be stained using specific antibodies for tyrosine hydroxylase (TH) and alpha-synuclein. ANALYSES: Quantification of proteins in fluorescence microscopy images will be done using ImageJ software. All experiments will be run in triplicate and statistical significance will be evaluated with t-test. RESULTS: Cell counts in brightfield microscopy confirmed that SH-SY5Y cells can be differentiated to a neuronal phenotype after 10 days of retinoic acid and BDNF treatment. CONCLUSIONS: Our next step is to use immunofluorescent staining and fluorescence microscopy to visualize TH and alpha-synuclein in differentiated and rotenone-treated cells. We will verify that differentiated cells express TH, a dopaminergic marker, at higher levels, and that rotenone treatment induces the formation of alpha-synuclein aggregates. We will then transfect these PD model cells with miR-34b and miR-34c and use additional immunostaining to measure their effects on alpha-synuclein aggregation.
Investigating microRNA miR-34b and miR-34c Expression in a Parkinson’s Disease Cell Line Model
PURPOSE: The motor symptoms of Parkinson’s disease (PD) are caused by the degeneration of dopaminergic neurons in the midbrain. These dying neurons are characterized primarily by large aggregates of alpha-synuclein protein. miR-34b and miR-34c are two microRNAs that repress alpha-synuclein expression. We hypothesize that treating degenerating neurons with these microRNAs will decrease alpha-synuclein aggregation and increase cell survival. SUBJECTS: SH-SY5Y, a neuroblastoma cell line, was cultured and treated with retinoic acid, brain-derived neurotrophic factor (BDNF), and rotenone to induce a PD-like phenotype. METHODS AND MATERIALS: Live cells were counted on a hemocytometer after staining with trypan blue. Brightfield images were taken using an inverted compound microscope. Cells will be stained using specific antibodies for tyrosine hydroxylase (TH) and alpha-synuclein. ANALYSES: Quantification of proteins in fluorescence microscopy images will be done using ImageJ software. All experiments will be run in triplicate and statistical significance will be evaluated with t-test. RESULTS: Cell counts in brightfield microscopy confirmed that SH-SY5Y cells can be differentiated to a neuronal phenotype after 10 days of retinoic acid and BDNF treatment. CONCLUSIONS: Our next step is to use immunofluorescent staining and fluorescence microscopy to visualize TH and alpha-synuclein in differentiated and rotenone-treated cells. We will verify that differentiated cells express TH, a dopaminergic marker, at higher levels, and that rotenone treatment induces the formation of alpha-synuclein aggregates. We will then transfect these PD model cells with miR-34b and miR-34c and use additional immunostaining to measure their effects on alpha-synuclein aggregation.