Novel high-throughput assay to assess cellular manganese levels in a striatal cell line model of Huntington's disease confirms a deficit in manganese accumulation

Abstract

In spite of the essentiality of manganese (Mn) as a trace element necessary for a variety of physiological processes, Mn in excess accumulates in the brain and has been associated with dysfunction and degeneration of the basal ganglia. Despite the high sensitivity, limited chemical interference, and multi-elemental advantages of traditional methods for measuring Mn levels, they lack the feasibility to assess Mn transport dynamics in a high-throughput manner. Our lab has previously reported decreased net Mn accumulation in a mutant striatal cell line model of Huntington's disease (STHdh(Q111/Q111)) relative to wild-type following Mn exposure. To evaluate Mn transport dynamics in these striatal cell lines, we have developed a high-throughput fluorescence-quenching extraction assay (Cellular Fura-2 Manganese Extraction Assay - CFMEA). CFMEA utilizes changes in fura-2 fluorescence upon excitation at 360 nm (Ca(2+) isosbestic point) and emission at 535 nm, as an indirect measurement of total cellular Mn content. Here, we report the establishment, development, and application of CFMEA. Specifically, we evaluate critical extraction and assay conditions (e.g. extraction buffer, temperature, and fura-2 concentration) required for efficient extraction and quantitative detection of cellular Mn from cultured cells. Mn concentrations can be derived from quenching of fura-2 fluorescence with standard curves based on saturation one-site specific binding kinetics. Importantly, we show that extracted calcium and magnesium concentrations below 10 μM have negligible influence on measurements of Mn by fura-2. CFMEA is able to accurately measure extracted Mn levels from cultured striatal cells over a range of at least 0.1-10 μM. We have used two independent Mn supplementation approaches to validate the quantitative accuracy of CFMEA over a 0-200 μM cellular Mn-exposure range. Finally, we have utilized CFMEA to experimentally confirm a deficit in net Mn accumulation in the mutant HD striatal cell line versus wild-type cells. To conclude, we have developed and applied a novel assay to assess Mn transport dynamics in cultured striatal cell lines. CFMEA provides a rapid means of evaluating Mn transport kinetics in cellular toxicity and disease models.

Fig 1. Optimization of buffer, temperature and detergent for CFMEA

(A) No significant difference in two commonly used buffers (PBS and HEPES) on Mn-fura-2 fluorescence quenching. (B) Minimal influence of temperature (15, 28, and 33°C) on Mn-fura-2 fluorescence quenching. Data is represented as RFU (y-axis) versus transformed log₁₀ Mn concentration on a linear scale (x-axis). A one-site specific saturation-binding curve with Hill slope was fit to log₁₀ Mn concentration (x-axis). N=3; 8 wells/experiment. Mean levels are indicated as ± standard deviation for (A) and (B). Fura-2 concentrations for (A) and (B) are 0.05 μM and 0.5 μM respectively. To evaluate the optimal detergent concentration required for intracellular Mn extraction from cultured striatal cells following MnCl₂ exposure, Mn was extracted in different concentrations of (C) Triton X-100 or (D) SDS in PBS containing 0.5 μM fura-2. Arrow (red) indicates the maximum intracellular Mn extracted by each buffer. Data is represented as RFU of baseline control (fura-2 containing buffer with indicated concentration of detergents) and extracts of Mn exposed cells (Mn-extraction). N=3; 4 wells/experiment. Mean levels are indicated as ± standard deviation.

Fig 2. Optimal fura-2 concentration for CFMEA

The detection range for Mn concentration by fura-2 was explored by examining concentration-response curves for Mn at three fura-2 concentrations (0.05, 0.5, and 2 μM) in a cell-free system. Dotted lines represent the estimated Mn detection range of each fura-2 concentration and were chosen based on an arbitrary 10 to 85 % maximal fluorescence intensity range. The width of arrowhead line also indicates fura-2 detection range. Mn-fura-2 concentration-response curves are analyzed by nonlinear regression to fit one-site specific binding curves with Hill slope, and plotted as RFU, top row, or normalized to unbound fura-2 (%_MAX), bottom row, versus transformed log₁₀ Mn concentration on a linear scale for each fura-2 concentration. Approximate optimal Mn detection concentrations (based upon the 10%-85% range) for each fura-2 concentration are provided below each graph. N=3; 8 wells/experiment. Mean levels are indicated as ± standard deviation.

Fig. 3. Mn-fura-2 standard curves

Mn-fura-2 standard curves were generated using a cell-free system with 0.5 μM fura-2 in PTx at Ex₃₆₀/Em₅₃₅. (A) Eight independent fura-2 standard curves were generated over an extended period of time (> 1 year). Data is plotted as RFU versus log₁₀ transformed Mn concentration on a linear scale. (B) The same curves as shown in (A) were plotted instead as %_MAX versus log₁₀ transformed Mn concentration on a linear scale. Data in (A) and (B) were used to fit one-site specific binding curves with Hill slope (dashed lines). Mean values for (A) and (B) are indicated as ± standard deviation, N=3; 8 wells/experiment. (C) A one-site specific binding curve with Hill slope (black, equation indicated on plot) was fit by non-linear regression to %_MAX values obtained from four independent cell-free experiments (blue). The 95% confidence interval of the calculated binding curve constants are indicated on the plot. N=4; 4 wells/exposure condition. Mean levels are indicated as ± standard deviation.

Fig 4. Effect of metal ions on fura-2 fluorescence at Ex₃₆₀/Em₅₃₅

Ex₃₆₀/Em₅₃₅ concentration-response curves were generated for 10 different divalent metal cations with 0.5 μM fura-2 in PTx. The influence of these metal ions on fura-2 fluorescence was measured and fitted by nonlinear regression analysis to a one-site competitive binding curve. Mn concentrations were transformed to log₁₀ Mn concentration versus normalized response curves and represented as transformed log₁₀ Mn concentration on a linear scale. N=3; 8 wells/exposure condition. Mean levels are indicated ± standard deviation.

Fig. 5. Influence of metal ions on CFMEA

To examine the competitive interference of metal ions that have considerable affinity for fura-2 on CFMEA, we utilized 0.5 μM fura-2 in PTx and different concentrations of (A) CaCl₂ (B) MgCl₂, (C) CoCl₂ and (D) FeCl₂ with or without different concentrations of MnCl₂. N=3; 8 wells/exposure condition. Mean levels are indicated ± standard deviation.

Fig. 6. Validation of CFMEA by Mn-supplementation

Mn-spike methods accurately measured supplemented Mn levels in cultured wild-type neuronal cells. (A) Wild-type striatal cells were exposed to MnCl₂ and cellular Mn levels measured by CFMEA. Cell-extracts were quickly supplemented/spiked with Mn (~285 nM final Mn concentration) and changes in fura-2 fluorescence re-measured (post-supplement). (B) The difference in Mn levels in each well between pre and post Mn-spike (difference) was calculated. N=3; 4 wells/exposure condition. Mean levels are indicated ± standard deviation. (*) Indicates a significant concentration-dependent net Mn uptake (p <0.0001, post-hoc t-test) in wild-type striatal cell lines. (C) Wild-type striatal cells were exposed to MnCl₂ and extracted in 0 μM, 250 μM, 500 μM, and 1000 μM MnCl₂ spiked fura-2 containing extraction buffers and total Mn levels assessed by CFMEA. (D) The difference in Mn levels in each well between unspiked and Mn-spiked (measured difference) fura-2 containing extraction buffers was calculated and compared to the known and expected Mn concentration. N=4; 4 wells/exposure condition. Mean levels are indicated ± standard deviation.

Fig. 7. CFMEA confirms net Mn accumulation deficit in mutant HD striatal cell line

Wild-type STHdh^Q7/Q7 (black) and mutant STHdh^Q111/Q111 (white) striatal cell lines were cultured in a 96 well tissue culture plate and exposed to different MnCl₂ concentrations for 27 hours in culture media. Mutant striatal cell lines exhibited statistically significant decrease in net accumulated Mn compared to wild-type striatal cell lines. (*) Indicates a significant difference at the tested MnCl₂ concentrations (post-hoc t-test p <0.0001) in net Mn uptake between wild-type and mutant striatal cell lines. N=3; 6 wells/exposure condition. Mean levels are indicated ± standard deviation.