Cu/Zn Superoxide Dismutase Typical for Familial Amyotrophic Lateral Sclerosis Increases the Vulnerability of Mitochondria and Perturbs Ca²⁺Homeostasis in SOD1^G93AMice

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the selective loss of defined motoneuron populations in the brainstem and spinal cord. Although low cytosolic calcium ([Ca 2ϩ ] i ) buffering and a strong interaction between metabolic mechanisms and [Ca 2ϩ ] i have been associated with selective motoneuron vulnerability, the underlying cellular mechanisms are barely understood. To elucidate the underlying molecular events, we used rapid chargecooled device imaging to evaluate Ca 2ϩ signaling and metabolic signatures in the brainstem slices of SOD1 G93A mice, the mouse model of human ALS, at 8 to 9 and 14 to 15 weeks of age, corresponding to the presymptomatic and symptomatic stages of motor dysfunction, respectively, and compared the results with corresponding age-matched wild-type littermates. We also monitored the mitochondrial membrane potential (⌬ ⌿m ) of brainstem motoneurons, a valuable tool for characterizing the metabolic signature of intrinsic energy profiles and considered to be a good experimental measure for monitoring energy metabolism in cells. We found that different pharmacological interventions substantially disrupt ⌬ ⌿m in SOD1 G93A motoneurons during the symptomatic stage. Furthermore, we investigated the impact of impaired mitochondrial mechanisms on [Ca 2ϩ ] i regulation by using the membrane-permeable indicator fura-acetoxy methyl ester. Taken together, the results indicate that mitochondrial disruptions are critical elements of SOD1G93A -mediated motoneuron degeneration in which selective motoneuron vulnerability results from a synergistic accumulation of risk factors, including the disruption of electrochemical potential, low Ca 2ϩ buffering, and strong mitochondrial control of [Ca 2ϩ ] i . The stabilization of mitochondria-related signal cascades may represent a useful strategy for clinical neuroprotection in ALS.

In vitro profiling of epigenetic modifications underlying heavy metal toxicity of tungsten-alloy and its components

“…The calcium-sensitive fluorescent dye was introduced into C2C12 cells by adding 2 μM of cell membrane-permeable Fluo-4/ AM (Invitrogen) into the media of the culture plates followed by incubation at 37°C, 5% CO 2 for 40 min. Cells were rinsed with DMEM and incubated for 20 min at 37°C to allow for complete deesterification (for details see Jaiswal and Keller, 2009 MicroImaging, Germany). In brief, experiments were performed as previously described (Bergmann and Keller, 2004;Jaiswal and Keller, 2009) using a Zeiss LSM 7 MP system, consisting of an Axio Examiner.Z1 microscope (Carl Zeiss MicroImaging, Germany), Ti:sapphire Chameleon Vision, 2-photon mode-locked laser system operated at 800 nm (680 nm-1080 nm tuning range,~80 MHz; 3.3 W peak power , Coherent, Inc., Auburn, CA) and controlled by Zen 2009 software (Zeiss, Germany).…”

“…Cells were rinsed with DMEM and incubated for 20 min at 37°C to allow for complete deesterification (for details see Jaiswal and Keller, 2009 MicroImaging, Germany). In brief, experiments were performed as previously described (Bergmann and Keller, 2004;Jaiswal and Keller, 2009) using a Zeiss LSM 7 MP system, consisting of an Axio Examiner.Z1 microscope (Carl Zeiss MicroImaging, Germany), Ti:sapphire Chameleon Vision, 2-photon mode-locked laser system operated at 800 nm (680 nm-1080 nm tuning range,~80 MHz; 3.3 W peak power , Coherent, Inc., Auburn, CA) and controlled by Zen 2009 software (Zeiss, Germany). The system was equipped with a X-Cite 120 lamp for reflected light illumination and water immersion objectives (10× achroplan and 20× plan-apochromat, 40× plan-apochromat, 0.30, 1.0 and 0.8 NA respectively, Zeiss; Germany).…”

Oxidative Stress and Altered Mitochondrial Function in Neurodegenerative Diseases: Lessons From Mouse Models

“…Mitochondria from these mouse models of human ALS show increased vulnerability and impaired Ca 2+ homeostasis [180] that precedes the onset of motor symptoms [181]. In fact, while the wild-type protein is anti-apoptotic, mutSOD1 seems to promote the mitochondrial apoptotic pathway.…”

Mitochondrial Pathobiology in Parkinson's Disease and Amyotrophic Lateral Sclerosis

“…high -mSOD1 mouse motor neurons have been seen also by different Ca 2+ detection methods [183,184]. Recent work on a mouse neuromuscular junction preparation has shown that mitochondrial Ca 2+ accumulation is accompanied by greater mitochondrial depolarization, specifically within motor neuron terminals of human mutant SOD1 Tg mice [185].…”