Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

<div><p>Purpose</p><p>Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated.</p><p>Methods</p><p>Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD⁺ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy.</p><p>Results</p><p>Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (<i>P</i> < 0.01), 14% increase in ROS production (<i>P</i> < 0.01), and 81% reduction in mitochondrial membrane potential (<i>P</i> < 0.01), compared with untreated cells. Lower concentration of ketamine (100 μM) decreased the ATP level (22%, <i>P</i> < 0.01) and increased the NADH/NAD⁺ ratio (46%, <i>P</i> < 0.05) without caspase activation. Transmission electron microscopy showed enhanced mitochondrial fission and autophagocytosis at the 100 μM ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation.</p><p>Conclusions</p><p>We established an <i>in vitro</i> model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons.</p></div

ROS scavenger Trolox attenuates ROS production and caspase 3/7 activation in ketamine-treated neurons.

<p>To determine whether ROS production mediates activation of caspase 3/7, iPSC-derived dopaminergic neurons were treated for 6 h with ketamine with or without the ROS scavenger, Trolox. (A) Trolox (500 μM) significantly inhibited ROS generation in the 500 μM ketamine-treated neurons (1.24-fold ± 0.14 ketamine alone vs. 1.01-fold ± 0.06 ketamine with Trolox). (B) Trolox also inhibited caspase 3/7 activation in the 500 μM ketamine-treated neurons (1.43-fold ± 0.12 ketamine alone vs. 1.05-fold ± 0.19 ketamine with Trolox). Results are presented as mean ± SD; n = 4 for each experiment. ** <i>P</i> < 0.01, compared with untreated controls.</p

Effect of ketamine on mitochondrial morphology in neurons derived from iPSCs.

<p>Cells were treated with ketamine for 24 h, and observed by transmission electron microscopy. Untreated cells (A) and cells treated with 20 μM ketamine (B) had elongated mitochondria with intact inner and outer membranes. Treatment with 100 μM ketamine (C) resulted in fragmented mitochondria and the presence of autophagosomes (arrow). After treatment with 500 μM ketamine (D), the structure of the mitochondria became discrete and round, and the mitochondrial length was shortened. Autophagosomes (arrow) were detected, and some fragmented mitochondria were degraded by autophagosomes (arrowhead). N: nucleus. Scale bar = 500 nm.</p

Effect of ketamine on mitochondrial membrane potential in cultured neurons derived from iPSCs.

<p>Quantification of mitochondrial membrane potential in ketamine-treated neurons. The cells treated with 500 μM ketamine for 6 and 24 h showed significant reduction in mitochondrial membrane potential. Carbonyl cyanide 3-chlorophenylhydrazone, which disrupts the mitochondrial membrane potential, was used as a positive control. All data were extracted from the fluorescence of 4 μM carbonyl cyanide 3-chlorophenylhydrazone-treated neurons. Results are presented as mean ± SD; n = 4 for each experimental condition. ** <i>P</i> < 0.01 compared with untreated controls. ^##<i>P</i> < 0.01, between the groups.</p

Culture of human iPSC-derived dopaminergic neurons.

<p>(A–D) Differentiation of human iPSC-derived neural progenitor cells into dopaminergic neurons from day 0 to 14 after seeding the cells on 96-well plates at 3 × 10⁴ cells per well. (A) day 0 (B) day 1, (C) day 7, (D) day 14. (E) Human dopaminergic neurons (14 days after seeding) stained with anti-beta III-tubulin (green), a neuronal cell marker. (F) Anti tyrosine hydroxylase (red), a dopaminergic neuronal marker. Cells were counterstained with DAPI (blue). Scale bar = 50 μm.</p

Ketamine induced morphological changes in neurons derived from iPSCs.

<p>Cells were treated with 0 μM (A), 20 μM (B), 100 μM (C) or 500 μM (D) ketamine for 24 h. Lower doses (20, 100 μM) of ketamine treatment did not affect the overall cell morphology (B and C). However, 500 μM ketamine caused neuronal processes to retract and it diminished neuronal networks (D). Scale bar = 50 μm.</p

Effect of ketamine on NADH/NAD⁺ ratio in cultured neurons derived from iPSCs.

<p>The neurons treated with 100 or 500 μM ketamine showed a significant increase in the NADH/NAD⁺ ratio compared with the control cells both after 6 and 24 h treatment. As a positive control, 10 nM rotenone (24 h) was used. Results are presented as mean ± SD; n = 4 for each experiment. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, compared with untreated controls. RLU = relative light units.</p

Effect of Ketamine on caspase 3/7 activity and ROS production, and cell viability in cultured iPSC-derived neurons.

<p>Neurons were exposed to increasing concentrations (20, 100 and 500 μM) of ketamine for 6 and 24 h. (A) Caspase 3/7 activity was used to evaluate ketamine-induced apoptosis in iPSC-derived neurons. Ketamine (500 μM) increased caspase 3/7 activity after 6 and 24 h of treatment. ROS production was used to evaluate ketamine-induced oxidative stress in iPSC-derived neurons. (B) Ketamine (500 μM) increased ROS production both after 6 and 24 h. (C) Cell viability did not change among all groups. Data are presented as mean ± SD; n = 4 in each experimental condition. ** <i>P</i> < 0.01, compared with untreated controls. RLU = relative light units.</p

Effect of ketamine on mitochondrial respiratory complexes from bovine heart mitochondria.

<p>(A) Complex I (NADH dehydrogenase) assay. (B) Complex II (succinate dehydrogenase) assay. (C) Complex IV (cytochrome c oxidase) assay. (D) Complex V (ATP synthase) assay. Ketamine (≥ 125 μM) significantly reduced the activity of complexes I (A) and complex V (D). Results are presented as mean ± SD; n = 3 in each experiment. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, compared with control cells.</p

Ketamine decreases the ATP level and neurotransmitter reuptake activity in cultured iPSC-derived neurons.

<p>(A) Ketamine decreased the ATP level in a time- and dose-dependent manner. In the 6-h treatment, the ATP level significantly decreased to 91% with 100 μM ketamine, and to 66% with 500 μM ketamine, compared with untreated controls. In the 24-h treatment, the ATP level significantly decreased to 78% with 100 μM ketamine, and to 48% with 500 μM ketamine. (B) Treatment with 100 or 500 μM ketamine for 24 h resulted in decreased neurotransmitter reuptake activity to approximately 65% and 51%, respectively, compared with control cells. Results are presented as mean ± SD; n = 4 for each experiment. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, compared with untreated controls. ^##<i>P</i> < 0.01, between the groups. GBR12909 = 1-(2-[bis(4-fluorophenyl)-[methoxy]ethyl)-4-(3-phenylpropyl) piperazine; RLU = relative light units; RFU = relative fluorescence units; AUC = area under the curve.</p