Computational protocol: Genetic inactivation of mitochondria-targeted redox enzyme p66ShcA preserves neuronal viability and mitochondrial integrity in response to oxidative challenges

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Protocol publication

[…] Post-natal neurons were dissociated from hippocampal tissue as described above, and 1–2 million cells were pelleted and resuspended in 100 μL of nucleofection solution with 3 μg each of a plasmid that directs the expression of GFP within the mitochondrial matrix (Pinton et al., ) and a plasmid that directs the expression of mCherry in actin as a neuronal filler (mCherry–β-actin plasmid) (both plasmids generously provided by Dr. Gary Banker at OHSU). The cell/plasmid solution was electroporated following the Amaxa electroporation system protocol (Amaxa, Lonza, Basel, Switzerland) for post-natal neurons using program O-05. Following electroporation, the cells were counted and plated in culture medium at 100,000 cells/well in six well plates, with each well containing a 25 mm glass coverslip coated overnight with 10 μg/mL PDL. After 24 h incubation in a humidified incubator at 37°C and 5% CO2, the medium was replaced with fresh culture medium.Week-old cultures were treated with either the control medium (Neurobasal A medium/0.5 mM glutamine), 25 μM H2O2, or 500 μM DETA-NO for 1 h at 37°C. The cells were then washed twice with Neurobasal A medium/0.5 mM glutamine and fixed with 4% paraformaldehyde for 20 min at 37°C. Following fixation, the cells were washed twice with 1X PBS, and the coverslips were mounted on slides with Prolong Gold Anti-Fade (Invitrogen, Carlsbad, CA). The slides were imaged with a Zeiss LSM710 confocal microscope using a 63X oil objective. For each randomly chosen neuron, a Z-stack of the axon was imaged (0.38 μm sections; 8–10 sections per neurite). Each z-stack was converted into a 3D image and analyzed by Bitplane Imaris™ software (BitPlane Inc., Saint Paul, MN). To quantify mitochondrial morphology changes, mitochondria in each image were selected by thresholding and analyzed using the Ellipsoid Axis C parameter. Mitochondrial length was defined as the Ellipsoid Axis C parameter × 2. All analyses were done blinded to genotype. [...] Post-natal hippocampal neurons were electroporated with mitoGFP, and then plated on 25 mm PDL-coated glass coverslips in six well plates as described above. Week old cells were treated with either the control medium (Neurobasal A medium/0.5 mM glutamine), 25 μM H2O2, or 500 μM DETA-NO for 1 h at 37°C. Afterwards, the cells were washed twice with Neurobasal A medium/0.5 mM glutamine and incubated with 1 μM Mitosox Red, a fluorescent reporter that monitors mitochondrial superoxide levels (Invitrogen, Carlsbad, CA) at 37°C for 10 min. The cells were then washed twice with Neurobasal A/0.5 mM glutamine, fixed with 4% paraformaldehyde for 20 min at 37°C, and washed twice with 1X PBS. The coverslips were mounted onto slides with Prolong Gold Anti-Fade.The slides were imaged with a Zeiss LSM710 confocal microscope using a 63X oil objective. Axons of neurons expressing mito-GFP were imaged in both red and green channels to capture mitochondrial GFP expression and corresponding Mitosox Red staining. Images were analyzed using Metamorph software (Molecular Devices, Sunnyvale, CA) to acquire average intensity measurements of randomly selected mitochondria. Between 20 and 30 axonal mitochondria were analyzed per image. […]

Pipeline specifications

Software tools Imaris, MetaMorph
Application Laser scanning microscopy
Organisms Mus musculus
Diseases Alzheimer Disease, Amyotrophic Lateral Sclerosis, Encephalomyelitis, Autoimmune, Experimental, Huntington Disease, Multiple Sclerosis, Rupture, Neurodegenerative Diseases, Stroke, Mitochondrial Diseases
Chemicals Adenosine Triphosphate, Calcium, Hydrogen Peroxide, Nitric Oxide, Oxygen