Notarianni, T. | bioRxiv Science

bioRxiv2021

Measuring mitochondrial electron transfer complexes in previously frozen cardiac tissue from the offspring of sow: A model to assess exercise induced mitochondrial bioenergetics changes

Measuring the mitochondrial electron transfer complex (ETC) profile from previously frozen heart tissue samples from offspring born to an exercised sow provided descriptive data about exercise-induced mitochondrial biochemical changes in heart tissue from the offspring born to the exercised sow. The hypothesis proposed and tested was that a regular maternal exercise of a sow during pregnancy would increase the mitochondrial efficiency of offspring heart bioenergetics. This hypothesis was tested by isolating mitochondria using a mild-isolation procedure to assess mitochondrial ETC and supercomplex profiles. The procedure described here allowed for the processing of previously frozen archived heart tissues and eliminated the necessity of fresh mitochondria preparation for the assessment of mitochondrial ETC complexes, supercomplexes, and ETC complex activity profiles. This protocol describes the optimal ETC protein complex measurement in multiplexed antibody-based immunoblotting and super complex assessment using blue-native gel electrophoresis. SUMMARYPreparation of mitochondria-enriched samples from previously frozen archived solid tissues allowed the investigators to perform both functional and analytical assessments of mitochondria in various experimental modalities. This study demonstrates how to prepare mitochondria-enriched preparations from frozen heart tissue and perform analytical assessments of mitochondria.

biochemistry↗

bioRxiv2017

Impaired Cu-Zn superoxide dismutase (SOD1) and calcineurin (Cn) interaction in ALS: A presumed consequence for TDP-43 and zinc aggregation in Tg SOD1G93A rodent spinal cord tissue.

Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)-enriched TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1G93A protein does not properly bind zinc (Zn) in animal models; hence, mutant SOD1G93A - Cn interaction weakens. Consequently, unstable Cn fails to dephosphorylate TDP-43 that yields hyperphosphorylated TDP-43 aggregates. Our previous studies had suggested that Cn and SOD1 interaction was necessary to keep Cn enzyme functional. We have observed low Cn level, increased Zn concentrations, and increased TDP-43 protein levels in cervical, thoracic, lumbar, and sacral regions of the spinal cord tissue homogenates. This study further supports our previous published work indicating that Cn stability depends on functional Cn-SOD1 interaction because Zn metal is crucial for maintaining the Cn stability. Less active Cn did not efficiently dephosphorylate TDP-43; hence TDP-43 aggregations appeared in the spinal cord tissue.

neuroscience↗