Fig 1.
Motor neurons show greater acidic vesicle content following treatment with cis-chlordane.
Motor neurons were incubated with 10 μM cis-chlordane, vehicle (DMSO) or the proteasome inhibitor MG132 at 37°C for 1, 2, or 3 hours before staining with LysoTracker dye and subsequent live fluorescent microscopy. (A) Representative live image of motor neurons stained with LysoTracker. Scale bar represents 100 µm. (B) Quantification of LysoTracker intensity compared to in-plate negative control. Bars indicate mean with SD. Statistics were performed by Kruskal-Wallis test with multiple comparisons. **p ≤ 0.01, ***p ≤ 0.001 n = 21-30 (5 biological replicates with at least 4 technical replicates each).
Fig 2.
Cis-chlordane treatment causes increased cellular and mitochondrial reactive oxygen species and disrupts mitochondrial membrane potential in motor neurons.
(A) Differential cellular ROS production in motor neurons treated with 20 μM cis-chlordane for 3 hours. (B) Representative CellRox images. (C) Differential mitochondrial ROS production in motor neurons treated with 20 μM cis-chlordane for 3 hours. (D) Representative MitoSox images. (E) Differential mitochondrial membrane potential in motor neurons treated with 20 μM cis-chlordane. (F) Representative TMRM images. Bars indicate mean with SD. Statistics were performed by unpaired student T-test or one-way ANOVA with multiple comparisons to control, ****p < 0.0001, n = 12 (3 biological replicates with at least 4 technical replicates each). Scale bar represents 100 µm.
Fig 3.
Motor neuron metabolism is highly altered upon treatment with cis-chlordane.
Motor neurons were treated with either 5 μM cis-chlordane, 10 μM cis-chlordane, or vehicle (DMSO) for 3 hours before performing Agilent’s MitoTox XFe Seahorse assay. Values were normalized to total DNA present in subsequent lysates, and treatment values then compared to in-plate control. Bars indicate mean with SD. Statistics performed with Kruskal-Wallis test with multiple comparisons. *p ≤ 0.05, **p ≤ 0.01 ***p ≤ 0.001, ****p < 0.0001, n = 12 (3 biological replicates, 4 technical replicates each).
Fig 4.
Production of metabolic intermediates in motor neurons is altered in response to cis-chlordane.
Motor neurons were treated with either 5 μM cis-chlordane, 10 μM cis-chlordane, or vehicle for 3 hours before the cells were lysed and quantitatively assessed for Pyruvate or Malate. Measured values were compared to in plate control. Statistics performed through one-way ANOVA with multiple comparisons to control. ***p ≤ 0.001, ****p < 0.001, n ≥ 6 (3 biological replicates, 2-3 technical replicates each) except pyruvate with 5 μM cis-chlordane, n = 4 (2 biological replicates and 2 technical replicates each).
Fig 5.
cis-Chlordane is detrimental to motor neuron health.
cis-Chlordane treatment causes motor neurons to reduce their oxygen consumption rate, decreases mitochondrial membrane potential (ΔΨm), and increases lysogenic vesicles and ROS in the cell [41].