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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.

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Non-specific association of human α-synuclein with cortex mitochondrial complexes by immunocapture.A. SDS-PAGE and Western immunoblotting with a total OXPHOS antibody cocktail detected the appropriate subunit protein equally in each of the five mitochondrial electron transport complexes (cI–cV) complexes from WT and ASOTg mitochondrial fractions. Re-probed blot confirmed elevated human α-synuclein in the ASOTg mitochondrial fraction; minor lower band may correspond to COOH-terminal truncated or alternative spliced forms of α-synuclein. B. Negligible levels of WT mouse α-synuclein were recovered as eluents from antibody-conjugated beads selective for electron transport complexes cI–cV and pyruvate dehydrogenase (PyD) after extensive washing with lauryl maltoside detergent-containing buffer. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein (see labeled bands in bottom panel). Labeling in the PyD fraction represents background immunocapture. C. Higher levels of human α-synuclein were recovered from ASOTg fractions relative to WT (top panel) but were represented equally in all immunocaptured complexes cI–cV, consistent with non-selective association (experiment was repeated 2 times). Human α-synuclein was also observed in PyD, a soluble complex not associated with inner mitochondrial membrane, in amounts roughly similar to those from the cI–cV complexes. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein.
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pone-0063557-g003: Non-specific association of human α-synuclein with cortex mitochondrial complexes by immunocapture.A. SDS-PAGE and Western immunoblotting with a total OXPHOS antibody cocktail detected the appropriate subunit protein equally in each of the five mitochondrial electron transport complexes (cI–cV) complexes from WT and ASOTg mitochondrial fractions. Re-probed blot confirmed elevated human α-synuclein in the ASOTg mitochondrial fraction; minor lower band may correspond to COOH-terminal truncated or alternative spliced forms of α-synuclein. B. Negligible levels of WT mouse α-synuclein were recovered as eluents from antibody-conjugated beads selective for electron transport complexes cI–cV and pyruvate dehydrogenase (PyD) after extensive washing with lauryl maltoside detergent-containing buffer. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein (see labeled bands in bottom panel). Labeling in the PyD fraction represents background immunocapture. C. Higher levels of human α-synuclein were recovered from ASOTg fractions relative to WT (top panel) but were represented equally in all immunocaptured complexes cI–cV, consistent with non-selective association (experiment was repeated 2 times). Human α-synuclein was also observed in PyD, a soluble complex not associated with inner mitochondrial membrane, in amounts roughly similar to those from the cI–cV complexes. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein.

Mentions: Lower membrane potentials and higher ROS in ASOTg brain mitochondria could emanate from a variety of membrane targets compromised by human α-synuclein. One likely candidate is the mitochondrial electron transport complex c1 [18], [19]. Here we examined the association of α-synuclein with cI as well as other electron transport complexes cII-cV in brain mitochondria by immunocapture experiments. Staining with a total OXPHOS antibody cocktail for unique protein subunits in each of the five complexes revealed that the appropriate complex-selective protein was represented equally in WT and ASOTg cortex mitochondrial fractions resolved by SDS-PAGE (Fig. 3A). As expected, the re-probed blot detected a relatively higher amount of human α-synuclein in the ASOTg mitochondrial fraction, while a minor amount of truncated α-synuclein was also evident.


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)

Non-specific association of human α-synuclein with cortex mitochondrial complexes by immunocapture.A. SDS-PAGE and Western immunoblotting with a total OXPHOS antibody cocktail detected the appropriate subunit protein equally in each of the five mitochondrial electron transport complexes (cI–cV) complexes from WT and ASOTg mitochondrial fractions. Re-probed blot confirmed elevated human α-synuclein in the ASOTg mitochondrial fraction; minor lower band may correspond to COOH-terminal truncated or alternative spliced forms of α-synuclein. B. Negligible levels of WT mouse α-synuclein were recovered as eluents from antibody-conjugated beads selective for electron transport complexes cI–cV and pyruvate dehydrogenase (PyD) after extensive washing with lauryl maltoside detergent-containing buffer. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein (see labeled bands in bottom panel). Labeling in the PyD fraction represents background immunocapture. C. Higher levels of human α-synuclein were recovered from ASOTg fractions relative to WT (top panel) but were represented equally in all immunocaptured complexes cI–cV, consistent with non-selective association (experiment was repeated 2 times). Human α-synuclein was also observed in PyD, a soluble complex not associated with inner mitochondrial membrane, in amounts roughly similar to those from the cI–cV complexes. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein.
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Related In: Results  -  Collection

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pone-0063557-g003: Non-specific association of human α-synuclein with cortex mitochondrial complexes by immunocapture.A. SDS-PAGE and Western immunoblotting with a total OXPHOS antibody cocktail detected the appropriate subunit protein equally in each of the five mitochondrial electron transport complexes (cI–cV) complexes from WT and ASOTg mitochondrial fractions. Re-probed blot confirmed elevated human α-synuclein in the ASOTg mitochondrial fraction; minor lower band may correspond to COOH-terminal truncated or alternative spliced forms of α-synuclein. B. Negligible levels of WT mouse α-synuclein were recovered as eluents from antibody-conjugated beads selective for electron transport complexes cI–cV and pyruvate dehydrogenase (PyD) after extensive washing with lauryl maltoside detergent-containing buffer. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein (see labeled bands in bottom panel). Labeling in the PyD fraction represents background immunocapture. C. Higher levels of human α-synuclein were recovered from ASOTg fractions relative to WT (top panel) but were represented equally in all immunocaptured complexes cI–cV, consistent with non-selective association (experiment was repeated 2 times). Human α-synuclein was also observed in PyD, a soluble complex not associated with inner mitochondrial membrane, in amounts roughly similar to those from the cI–cV complexes. Reprobing of blots with the total OXPHOS antibodies confirmed relative enrichment for immunocapture of each representative protein.
Mentions: Lower membrane potentials and higher ROS in ASOTg brain mitochondria could emanate from a variety of membrane targets compromised by human α-synuclein. One likely candidate is the mitochondrial electron transport complex c1 [18], [19]. Here we examined the association of α-synuclein with cI as well as other electron transport complexes cII-cV in brain mitochondria by immunocapture experiments. Staining with a total OXPHOS antibody cocktail for unique protein subunits in each of the five complexes revealed that the appropriate complex-selective protein was represented equally in WT and ASOTg cortex mitochondrial fractions resolved by SDS-PAGE (Fig. 3A). As expected, the re-probed blot detected a relatively higher amount of human α-synuclein in the ASOTg mitochondrial fraction, while a minor amount of truncated α-synuclein was also evident.

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