Quantification of the Metabolic State in Cell-Model of Parkinson's Disease by Fluorescence Lifetime Imaging Microscopy

Sandeep Chakraborty, Fang-Shin Nian, Jin-Wu Tsai, Artashes Karmenyan, Arthur Chiou, Sandeep Chakraborty, Fang-Shin Nian, Jin-Wu Tsai, Artashes Karmenyan, Arthur Chiou

Abstract

Intracellular endogenous fluorescent co-enzymes, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), play a pivotal role in cellular metabolism; quantitative assessment of their presence in living cells can be exploited to monitor cellular energetics in Parkinson's disease (PD), a neurodegenerative disorder. Here, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to noninvasively measure the fluorescence lifetime components of NADH and FAD, and their relative contributions in MPP(+) (1-methyl-4-phenylpyridinium) treated neuronal cells, derived from PC12 cells treated with nerve growth factor (NGF), to mimic PD conditions. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP(+) treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increase significantly (p < 0.001) in MPP(+) treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP(+) treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level.

Figures

Figure 1. PC12 cells’ response to NGF…
Figure 1. PC12 cells’ response to NGF (nerve growth factor) treatment.
Differential interference contrast (DIC) microscopy images of (a) undifferentiated, and (b) differentiated PC12 cells after 200 ng/ml of NGF treatment for three days. The arrows in micrograph (b) point to the neuronal-like branching extended from PC12 cells after NGF treatment. Objective: 40x, NA 0.6, air. Scale bar: 20 μm.
Figure 2. Micrographs of average NADH fluorescence…
Figure 2. Micrographs of average NADH fluorescence lifetime.
Pseudocolor mapping of average fluorescence lifetime (τavg) of NADH of (a) the untreated differentiated PC12 cells, and of the cells treated with (b) 50, (c) 100, (d) 250, (e) 500, and (f) 1000 μM of MPP+. The color bar on the right represents the range of τavg. Scale bar: 20 μm.
Figure 3. Summary of the effect of…
Figure 3. Summary of the effect of MPP+ on NADH fluorescence lifetime components.
Average of all the data points (n = 75) from five experimental days of NADH lifetime measurements: (a) short, or free (τ1), (b) long, or protein-bound (τ2), (c) average (τavg) NADH lifetimes, and (d) the ratio of the relative contribution of free, and protein-bound NADH (a1/a2) for cells treated with different concentrations of MPP+. The error bars indicate standard error of the mean (SEM). A one-way ANOVA with LSD-post-hoc analysis was performed to define statistical significance. Statistical significance: **p < 0.001, for control (0 μM MPP+) vs. MPP+ treated cells; #p < 0.05, ##p < 0.001, for 50 μM MPP+ treatment vs. other MPP+ treated cells; Δp < 0.05, ΔΔp < 0.001, for 100 μM MPP+ treatment vs. other MPP+ treated cells; ◊p < 0.05, ◊◊p < 0.001, for 250 μM MPP+ treatment vs. other MPP+ treated cells; ●p < 0.05, ●●p < 0.001, for 500 vs. 1000 μM MPP+ treatment.
Figure 4. Average FAD fluorescence lifetime micrographs.
Figure 4. Average FAD fluorescence lifetime micrographs.
Pseudo-color mapping of FAD average fluorescence lifetime (τavg) at (a) 0, (b) 50, (c) 100, (d) 250, (e) 500, and (f) 1000 μM MPP+ treatment of differentiated PC12 cells. The color bar shows the range of the FAD average lifetime. Scale bar: 20 μm.
Figure 5. Summary of the effect of…
Figure 5. Summary of the effect of MPP+ on FAD fluorescence lifetime components.
Average and SEMs (error bars) of all the 75 data points from all the imaging sessions of five experimental days of fluorescence lifetime mapping: (a) short, or protein-bound (τ1), (b) long, or free (τ2), (c) average (τavg) FAD lifetimes, and (d) the ratio of the relative contribution of free, and protein-bound FAD (a2/a1) as a function of MPP+ concentration. A one-way ANOVA with LSD post-hoc analysis based statistical significance: *p < 0.05, **p < 0.001, for control (0 μM MPP+) vs. MPP+ treated cells; #p < 0.05 for 50 μM MPP+ treatment vs. Other MPP+ treated cells.

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