Limits...
Impairment of mitochondria in adult mouse brain overexpressing predominantly full-length, N-terminally acetylated human α-synuclein.

Sarafian TA, Ryan CM, Souda P, Masliah E, Kar UK, Vinters HV, Mathern GW, Faull KF, Whitelegge JP, Watson JB - PLoS ONE (2013)

Bottom Line: The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria.Oligomers or fibrils were not detected with amyloid conformational antibodies.Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry & Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
While most forms of Parkinson's Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.

Show MeSH

Related in: MedlinePlus

Overexpressed human α-synuclein disrupted brain mitochondrial functions.A. The mitochondrial membrane potential, detected as JC-1 red fluorescence, was reduced in all ASOTg brain region mitochondria (cortex, striatum, cerebellum), and was significantly different from WT values when all brain regions were considered (Two Way ANOVA, P = 0.013)(* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). All values are expressed as mean ± Standard Error of Mean (SEM). However, Bonferroni post-hoc analyses showed no difference in membrane potential between brain regions [F (2,52) = 0.205, P = 0.815]. FCCP (10 µg/ml) decreased greater than 95% of membrane potential on average in separate experiments to confirm authentic mitochondrial membrane potential. B. Mitochondrial reactive oxygen species (ROS) were significantly elevated in all ASOTg brain regions relative to WT brain regions (Two Way ANOVA, P = 0.044) (* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). There was no difference in ROS between brain regions [F (2,30) = 0.156, P = 0.857].
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646806&req=5

pone-0063557-g002: Overexpressed human α-synuclein disrupted brain mitochondrial functions.A. The mitochondrial membrane potential, detected as JC-1 red fluorescence, was reduced in all ASOTg brain region mitochondria (cortex, striatum, cerebellum), and was significantly different from WT values when all brain regions were considered (Two Way ANOVA, P = 0.013)(* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). All values are expressed as mean ± Standard Error of Mean (SEM). However, Bonferroni post-hoc analyses showed no difference in membrane potential between brain regions [F (2,52) = 0.205, P = 0.815]. FCCP (10 µg/ml) decreased greater than 95% of membrane potential on average in separate experiments to confirm authentic mitochondrial membrane potential. B. Mitochondrial reactive oxygen species (ROS) were significantly elevated in all ASOTg brain regions relative to WT brain regions (Two Way ANOVA, P = 0.044) (* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). There was no difference in ROS between brain regions [F (2,30) = 0.156, P = 0.857].

Mentions: Since it has been widely reported that human α-synuclein disrupts brain mitochondrial function [40], we examined the membrane potential in WT and ASOTg brain mitochondria (Fig. 2). The membrane potential (JC-1 red fluorescence alone; assumed little or no cytosol) was significantly reduced in all ASOTg mitochondria relative to WT mitochondria (Two Way ANOVA, P = 0.031) (Fig. 2A). However, Bonferroni post-hoc t-tests showed no difference in membrane potential between brain regions (WT, mean ± Standard Error of Mean (SEM): cortex, 1652.8±90.0, N = 11; striatum, 1706.1±88.5, N = 9; cerebellum, 1706.9±101.4, N = 9)(ASOTg: cortex, 1507.7±67.8, N = 11; striatum, 1451.1±78.5, N = 9; cerebellum, 1566.7±158.0, N = 9 [F(2,52) = 0.205, P = 0.815]. Taken together the data show that mitochondria throughout the brain of adult mice have impaired membrane potential when exposed to overexpressed human α-synuclein.


Impairment of mitochondria in adult mouse brain overexpressing predominantly full-length, N-terminally acetylated human α-synuclein.

Sarafian TA, Ryan CM, Souda P, Masliah E, Kar UK, Vinters HV, Mathern GW, Faull KF, Whitelegge JP, Watson JB - PLoS ONE (2013)

Overexpressed human α-synuclein disrupted brain mitochondrial functions.A. The mitochondrial membrane potential, detected as JC-1 red fluorescence, was reduced in all ASOTg brain region mitochondria (cortex, striatum, cerebellum), and was significantly different from WT values when all brain regions were considered (Two Way ANOVA, P = 0.013)(* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). All values are expressed as mean ± Standard Error of Mean (SEM). However, Bonferroni post-hoc analyses showed no difference in membrane potential between brain regions [F (2,52) = 0.205, P = 0.815]. FCCP (10 µg/ml) decreased greater than 95% of membrane potential on average in separate experiments to confirm authentic mitochondrial membrane potential. B. Mitochondrial reactive oxygen species (ROS) were significantly elevated in all ASOTg brain regions relative to WT brain regions (Two Way ANOVA, P = 0.044) (* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). There was no difference in ROS between brain regions [F (2,30) = 0.156, P = 0.857].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646806&req=5

pone-0063557-g002: Overexpressed human α-synuclein disrupted brain mitochondrial functions.A. The mitochondrial membrane potential, detected as JC-1 red fluorescence, was reduced in all ASOTg brain region mitochondria (cortex, striatum, cerebellum), and was significantly different from WT values when all brain regions were considered (Two Way ANOVA, P = 0.013)(* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). All values are expressed as mean ± Standard Error of Mean (SEM). However, Bonferroni post-hoc analyses showed no difference in membrane potential between brain regions [F (2,52) = 0.205, P = 0.815]. FCCP (10 µg/ml) decreased greater than 95% of membrane potential on average in separate experiments to confirm authentic mitochondrial membrane potential. B. Mitochondrial reactive oxygen species (ROS) were significantly elevated in all ASOTg brain regions relative to WT brain regions (Two Way ANOVA, P = 0.044) (* indicates that all brain regions must be included for significance between genotype, WT vs ASOTg). There was no difference in ROS between brain regions [F (2,30) = 0.156, P = 0.857].
Mentions: Since it has been widely reported that human α-synuclein disrupts brain mitochondrial function [40], we examined the membrane potential in WT and ASOTg brain mitochondria (Fig. 2). The membrane potential (JC-1 red fluorescence alone; assumed little or no cytosol) was significantly reduced in all ASOTg mitochondria relative to WT mitochondria (Two Way ANOVA, P = 0.031) (Fig. 2A). However, Bonferroni post-hoc t-tests showed no difference in membrane potential between brain regions (WT, mean ± Standard Error of Mean (SEM): cortex, 1652.8±90.0, N = 11; striatum, 1706.1±88.5, N = 9; cerebellum, 1706.9±101.4, N = 9)(ASOTg: cortex, 1507.7±67.8, N = 11; striatum, 1451.1±78.5, N = 9; cerebellum, 1566.7±158.0, N = 9 [F(2,52) = 0.205, P = 0.815]. Taken together the data show that mitochondria throughout the brain of adult mice have impaired membrane potential when exposed to overexpressed human α-synuclein.

Bottom Line: The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria.Oligomers or fibrils were not detected with amyloid conformational antibodies.Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry & Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, California, United States of America.

ABSTRACT
While most forms of Parkinson's Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.

Show MeSH
Related in: MedlinePlus