Assessment of ToxCast Phase II for Mitochondrial Liabilities Using a High-Throughput Respirometric Assay

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Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1^G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

<div><p>Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.</p></div

Overexpression of mitochondria-targeted catalase has no significant effect on hSOD1^G93A/mCAT mice survival.

<p>A) Median survival in hSOD1^G93A (G93A) mice (173 days, n = 16) and in hSOD1^G93A/mCAT double transgenic (DTG) mice (170 days, n = 11). Survival curves are not significantly different (χ² = 1.3, p = 0.2). B) Median onset in hSOD1^G93A (G93A) mice (122.5 days, n = 10) and in hSOD1^G93A/mCAT double transgenic (DTG) mice (121, n = 11). Onset curves are not significantly different (χ² = 0.9, p = 0.3). C) Catalase activity in mitochondria isolated from the spinal cord of 30-day-old G93A and DTG animals. Each data bar represents the mean ± SD of at least three animals. D) hSOD1 protein expression in spinal cord extracts from 30-day-old non-transgenic (NTG), hSOD1^G93A (G93A) and hSOD1^G93A/mCAT double transgenic (DTG) animals. No difference was observed in hSOD1 levels between G93A (100±8) and DTG (92±7) mice when quantified and corrected by actin levels.</p

Overexpression of mitochondria-targeted catalase reverts mitochondrial dysfunction in spinal cord hSOD1^G93A astrocytes and reverts their toxicity towards motor neurons.

<p>A) Confluent astrocyte monolayers of the indicated genotype were treated with 200 µM hydrogen peroxide (H₂O₂) and 24 hs later toxicity was assessed by LDH release. Data is expressed as percentage of the respective control. NonTG, Non-transgenic astrocytes; G93A, hSOD1^G93A astrocytes; DTG, hSOD1^G93A/mCAT double transgenic astrocytes. *Significantly different from NonTG control (p<0.05). # Significantly different from NonTG H₂O₂-treated (p<0.05). B) Oxygen consumption rate (OCR) determined for basal conditions or maximal respiration (FCCP 1 µM) in confluent spinal cord astrocyte monolayers of the indicated genotypes. *Significantly different from the respective basal OCR (p<0.05). C) Purified motor neurons from non-transgenic E12.5 mice were co-cultured over spinal cord astrocyte monolayers obtained from NonTG or transgenic mice over-expressing wild-type hSOD1 (WT), hSOD1^G93A (G93A), hSOD1^G93A/mCAT (DTG) or mCAT. Motor neuron survival was assessed 72 hs later. Motor neuron loss observed in co-cultures with G93A astrocytes was prevented by catalase overexpression in DTG astrocytes. * Significantly different from NonTG control (p<0.05). For all panels, data are expressed as the mean ± SD of at least three independent experiments.</p

Overexpression of mitochondria-targeted catalase confers resistance against peroxide toxicity in primary cortical neurons and astrocytes.

<p>A) Cortical neuronal cultures were treated with the indicated concentrations of hydrogen peroxide (H₂O₂) and 24 hs later toxicity was assessed by LDH release. Data is expressed as percentage of the respective control. B) Confluent cortical astrocyte monolayers were treated with the indicated concentrations of H₂O₂ and 24 hs later toxicity was assessed by LDH release. Data is expressed as percentage of the respective control. C) Cortical neurons were treated with H₂O₂ or antimycin A (AA) and mitochondrial reactive oxygen species (MitoROS) production was determined. MitoROS was corrected by mitochondria content. Data is expressed as percentage of the non-transgenic (NonTG) control. D) Confluent astrocyte monolayers were treated with H₂O₂ or antimycin A (AA) followed by MitoROS and mitochondria content determination. Data is expressed as percentage of the NonTG control. For all panels, each data bar represents the mean ± SD of at least three independent experiments. *Significantly different from NonTG control (p<0.05). # Significantly different from NonTG H₂O₂-treated.</p

Increased mitochondrial catalase activity in the central nervous system from mCAT mice.

<p>A) Catalase activity in different regions of the central nervous system and gastrocnemius muscle (Gastroc) from 30-day-old non-transgenic (NonTG) and mCAT animals. Cx, brain cortex; Crb, cerebellum; BS, brain stem; and SC, spinal cord. B) Catalase activity in mitochondria isolated from the spinal cord of 30 days old NonTG and mCAT animals. C) Catalase activity in mitochondria isolated from primary E15 cortical neurons and D) primary cortical astrocytes obtained from NonTG and mCAT mice. For all panels, each data bar represents the mean ± SD of at least three independent experiments. *Significantly different from NonTG control (p<0.05).</p