Isolation and characterization of intact mitochondria from neonatal rat brain

Rajapakse, Nishadi; Shimizu, Katsuyoshi; Payne, M E; Busija, David W.

doi:10.1016/s1385-299x(01)00108-8

Cited by 70 publications

(76 citation statements)

References 22 publications

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“…Mitochondria were isolated by discontinuous Percoll gradient (as described in Ref. 31). With use of this method, citrate synthase activity increased 4.1 Ϯ 0.2-fold (n ϭ 4) in control livers and 3.8 Ϯ 0.3-fold (n ϭ 3) in cirrhotic livers upon mitochondrial isolation.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions

Arduini

Serviddio

Escobar

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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Arduini A, Serviddio G, Escobar J, Tormos AM, Bellanti F, Viña J, Monsalve M, Sastre J. Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions. Am J Physiol Gastrointest Liver Physiol 301: G119 -G127, 2011. First published March 17, 2011 doi:10.1152/ajpgi.00253.2010.-Chronic cholestasis is characterized by mitochondrial dysfunction, associated with loss of mitochondrial membrane potential, decreased activities of respiratory chain complexes, and ATP production. Our aim was to determine the molecular mechanisms that link long-term cholestasis to mitochondrial dysfunction. We studied a model of chronic cholestasis induced by bile duct ligation in rats. Key sensors and regulators of the energetic state and mitochondrial biogenesis, mitochondrial DNA (mtDNA)-to-nuclear DNA (nDNA) ratio (mtDNA/nDNA) relative copy number, mtDNA deletions, and indexes of apoptosis (BAX, BCL-2, and cleaved caspase 3) and cell proliferation (PCNA) were evaluated. Our results show that long-term cholestasis is associated with absence of activation of key sensors of the energetic state, evidenced by decreased SIRT1 and pyruvate dehydrogenase kinase levels and lack of AMPK activation. Key mitochondrial biogenesis regulators (PGC-1␣ and GABP-␣) decreased and NRF-1 was not transcriptionally active. Mitochondrial transcription factor A (TFAM) protein levels increased transiently in liver mitochondria at 2 wk after bile duct ligation, but they dramatically decreased at 4 wk. Reduced TFAM levels at this stage were mirrored by a marked decrease (65%) in mtDNA/nDNA relative copy number. The blockade of mitochondrial biogenesis should not be ascribed to activation of apoptosis or inhibition of cell proliferation. Impaired mitochondrial turnover and loss of the DNA stabilizing effect of TFAM are likely the causative event involved in the genetic instability evidenced by accumulation of mtDNA deletions. In conclusion, the lack of stimulation of mitochondrial biogenesis leads to mtDNA severe depletion and deletions in long-term cholestasis. Hence, long-term cholestasis should be considered a secondary mitochondrial hepatopathy.

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Section: Methodsmentioning

confidence: 99%

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions

Arduini

Serviddio

Escobar

et al. 2011

American Journal of Physiology-Gastrointestinal and Liver Physi

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“…The mitochondrial fraction was separated as described by Rajapakse et al (71). In brief, after removing the nuclear fraction, the homogenates were mixed to achieve a 12% final concentration of Percoll.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Chaperone Activity Is a Shared Property of Several Cu,Zn-Superoxide Dismutase Mutants That Cause Amyotrophic Lateral Sclerosis

Tummala

Jung

Tiwari

et al. 2005

Journal of Biological Chemistry

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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Znsuperoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins.

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“…Citrate synthase activity was measured as described [19,36]. Briefly, in each 1 mL assay reaction mixture containing reaction buffer (50 mM Potassium Phosphate, pH 7.4, 5 mM MgCl 2 , 2 mM EDTA, 0.1% Triton X-100 and 1 mg/ mL BSA) 7-8 μg of sonicated mitochondria (as described above) were added followed by addition of 0.1 mM Acetyl-CoA and 2 mM DTNB.…”

Section: Enzyme Activitiesmentioning

confidence: 99%

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

Choksi

Papaconstantinou

2008

Free Radical Biology and Medicine

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Mitochondrial generated ROS increases with age and is a major factor that damages proteins by oxidative modification. Accumulation of oxidatively damaged proteins has been implicated as a causal factor in the age-associated decline in tissue function. Mitochondrial electron transport chain (ETC) complexes I and III are the principle sites of ROS production, and oxidative modifications to their complex subunits inhibit their in vitro activity. We hypothesize that mitochondrial complex subunits may be primary targets for modification by ROS which may impair normal complex activity. This study of heart mitochondria from young, middle-aged and old mice reveals that there is an agerelated decline in complex I and V activities that correlate with increased oxidative modification to their subunits. The data also show a specificity for modifications of the ETC complex subunits, i.e., several proteins have more than one type of adduct. We postulate that the electron leakage from ETC complexes causes specific damage to their subunits and increased ROS generation as oxidative damage accumulates, leading to further mitochondrial dysfunction, a cyclical process that underlies the progressive decline in physiologic function of aged mouse heart.

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Isolation and characterization of intact mitochondria from neonatal rat brain

Cited by 70 publications

References 22 publications

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions

Mitochondrial biogenesis fails in secondary biliary cirrhosis in rats leading to mitochondrial DNA depletion and deletions

Inhibition of Chaperone Activity Is a Shared Property of Several Cu,Zn-Superoxide Dismutase Mutants That Cause Amyotrophic Lateral Sclerosis

Age-related alterations in oxidatively damaged proteins of mouse heart mitochondrial electron transport chain complexes

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