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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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Isolation of overexpressed human α-synuclein and truncated forms with brain mitochondria.A. The entire amino acid sequence of human α-synuclein (140 amino acid residues) is displayed. Epitope stretches of amino acids used to raise human α-synuclein-selective antibodies are underlined (NH2 terminus, COOH terminus #1, COOH terminus #2). Human substitutions (total of 6) in the α-synuclein amino acid sequence are indicated in red. B. Western immunoblotting detected both endogenous mouse α-synuclein and overexpressed human α-synuclein (αSyn) (15–20 kDa) in synaptoneurosome (SN) fractions (20 µg) from WT and ASOTg littermates respectively using human α-synuclein-selective antibodies (left panel, NH2 terminus; middle panel, COOH terminus #1; right panel, COOH terminus #2). WT and Snca−/−littermates (KO) lacking mouse α-synuclein served as additional controls. Smaller forms (<15 kDa) of human α-synuclein were also detected in ASOTg SNs (left/middle panels) but not with the COOH terminus #2 antibody (right panel), consistent with carboxyl terminal truncation. C. Segments of the same immunoblot with brain (cortex, striatum) mitochondria (mito) and cyotosolic (cyt) fractions (20 µg each) from both WT and ASOTg mice were successively probed with different antibodies based on the size of the target protein. Target proteins included: nuclear pore complex proteins (top panel), subunit proteins for each of the five mitochondrial electron transport complexes (cI–cV) complexes (middle panel), and human α-synuclein (ASOTg) (bottom panel). Since blot segments were not stripped of immunoreactivity between antibody reprobing, electron transport complex cI band was detected as a residual band above α-synuclein. D. Human α-synuclein was also detected in purified cortex mitochondria (20 µg) isolated from ASO transgenic mice by the ISO1 protocol and a subsequent step using ultracentrifugation and density gradients (OptiPrep). cV-ATP synthase subunit (α) served as control for mitochiondria enrichment. E. Truncated forms of human α-synuclein were detected with increasing concentrations (4–20 µg) of ASOTg mitochondria but not with WT mitochondria.
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pone-0063557-g001: Isolation of overexpressed human α-synuclein and truncated forms with brain mitochondria.A. The entire amino acid sequence of human α-synuclein (140 amino acid residues) is displayed. Epitope stretches of amino acids used to raise human α-synuclein-selective antibodies are underlined (NH2 terminus, COOH terminus #1, COOH terminus #2). Human substitutions (total of 6) in the α-synuclein amino acid sequence are indicated in red. B. Western immunoblotting detected both endogenous mouse α-synuclein and overexpressed human α-synuclein (αSyn) (15–20 kDa) in synaptoneurosome (SN) fractions (20 µg) from WT and ASOTg littermates respectively using human α-synuclein-selective antibodies (left panel, NH2 terminus; middle panel, COOH terminus #1; right panel, COOH terminus #2). WT and Snca−/−littermates (KO) lacking mouse α-synuclein served as additional controls. Smaller forms (<15 kDa) of human α-synuclein were also detected in ASOTg SNs (left/middle panels) but not with the COOH terminus #2 antibody (right panel), consistent with carboxyl terminal truncation. C. Segments of the same immunoblot with brain (cortex, striatum) mitochondria (mito) and cyotosolic (cyt) fractions (20 µg each) from both WT and ASOTg mice were successively probed with different antibodies based on the size of the target protein. Target proteins included: nuclear pore complex proteins (top panel), subunit proteins for each of the five mitochondrial electron transport complexes (cI–cV) complexes (middle panel), and human α-synuclein (ASOTg) (bottom panel). Since blot segments were not stripped of immunoreactivity between antibody reprobing, electron transport complex cI band was detected as a residual band above α-synuclein. D. Human α-synuclein was also detected in purified cortex mitochondria (20 µg) isolated from ASO transgenic mice by the ISO1 protocol and a subsequent step using ultracentrifugation and density gradients (OptiPrep). cV-ATP synthase subunit (α) served as control for mitochiondria enrichment. E. Truncated forms of human α-synuclein were detected with increasing concentrations (4–20 µg) of ASOTg mitochondria but not with WT mitochondria.

Mentions: Because α-synuclein is predominantly a presynaptic protein [1], [2], we first used forebrain synaptoneurosomes (SNs) [36] to characterize domain-selective antibodies for human α-synuclein (NH2 terminus, COOH terminus #1, COOH terminus #2) (Fig. 1A). Each antibody detected elevated levels of human α-synuclein (15–20 kDa) in ASOTg relative to WT SNs; each antibody also has some cross-immunoreactivity with endogenous WT mouse α-synuclein (Fig. 1B). No selective immunoreactivity was detected in SN fractions from α-synuclein KO mice used as negative controls. Smaller α-synuclein forms were also detected in ASOTg SNs (left/middle panels). Overall the immunoblotting patterns were consistent with carboxyl terminal truncation, since the COOH terminus #2 antibody (only epitope containing the last 9 amino acids at carboxyl terminus) failed to detect smaller forms (right panel). Truncated forms were not detected in WT SNs, but this may be due to their lower amounts. Larger molecular weight forms (>20 kDa) in the ASOTg lanes appeared to be unrelated cross-immunoreactive bands based on side-by-side comparisons to WT and KO lanes.


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)

Isolation of overexpressed human α-synuclein and truncated forms with brain mitochondria.A. The entire amino acid sequence of human α-synuclein (140 amino acid residues) is displayed. Epitope stretches of amino acids used to raise human α-synuclein-selective antibodies are underlined (NH2 terminus, COOH terminus #1, COOH terminus #2). Human substitutions (total of 6) in the α-synuclein amino acid sequence are indicated in red. B. Western immunoblotting detected both endogenous mouse α-synuclein and overexpressed human α-synuclein (αSyn) (15–20 kDa) in synaptoneurosome (SN) fractions (20 µg) from WT and ASOTg littermates respectively using human α-synuclein-selective antibodies (left panel, NH2 terminus; middle panel, COOH terminus #1; right panel, COOH terminus #2). WT and Snca−/−littermates (KO) lacking mouse α-synuclein served as additional controls. Smaller forms (<15 kDa) of human α-synuclein were also detected in ASOTg SNs (left/middle panels) but not with the COOH terminus #2 antibody (right panel), consistent with carboxyl terminal truncation. C. Segments of the same immunoblot with brain (cortex, striatum) mitochondria (mito) and cyotosolic (cyt) fractions (20 µg each) from both WT and ASOTg mice were successively probed with different antibodies based on the size of the target protein. Target proteins included: nuclear pore complex proteins (top panel), subunit proteins for each of the five mitochondrial electron transport complexes (cI–cV) complexes (middle panel), and human α-synuclein (ASOTg) (bottom panel). Since blot segments were not stripped of immunoreactivity between antibody reprobing, electron transport complex cI band was detected as a residual band above α-synuclein. D. Human α-synuclein was also detected in purified cortex mitochondria (20 µg) isolated from ASO transgenic mice by the ISO1 protocol and a subsequent step using ultracentrifugation and density gradients (OptiPrep). cV-ATP synthase subunit (α) served as control for mitochiondria enrichment. E. Truncated forms of human α-synuclein were detected with increasing concentrations (4–20 µg) of ASOTg mitochondria but not with WT mitochondria.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646806&req=5

pone-0063557-g001: Isolation of overexpressed human α-synuclein and truncated forms with brain mitochondria.A. The entire amino acid sequence of human α-synuclein (140 amino acid residues) is displayed. Epitope stretches of amino acids used to raise human α-synuclein-selective antibodies are underlined (NH2 terminus, COOH terminus #1, COOH terminus #2). Human substitutions (total of 6) in the α-synuclein amino acid sequence are indicated in red. B. Western immunoblotting detected both endogenous mouse α-synuclein and overexpressed human α-synuclein (αSyn) (15–20 kDa) in synaptoneurosome (SN) fractions (20 µg) from WT and ASOTg littermates respectively using human α-synuclein-selective antibodies (left panel, NH2 terminus; middle panel, COOH terminus #1; right panel, COOH terminus #2). WT and Snca−/−littermates (KO) lacking mouse α-synuclein served as additional controls. Smaller forms (<15 kDa) of human α-synuclein were also detected in ASOTg SNs (left/middle panels) but not with the COOH terminus #2 antibody (right panel), consistent with carboxyl terminal truncation. C. Segments of the same immunoblot with brain (cortex, striatum) mitochondria (mito) and cyotosolic (cyt) fractions (20 µg each) from both WT and ASOTg mice were successively probed with different antibodies based on the size of the target protein. Target proteins included: nuclear pore complex proteins (top panel), subunit proteins for each of the five mitochondrial electron transport complexes (cI–cV) complexes (middle panel), and human α-synuclein (ASOTg) (bottom panel). Since blot segments were not stripped of immunoreactivity between antibody reprobing, electron transport complex cI band was detected as a residual band above α-synuclein. D. Human α-synuclein was also detected in purified cortex mitochondria (20 µg) isolated from ASO transgenic mice by the ISO1 protocol and a subsequent step using ultracentrifugation and density gradients (OptiPrep). cV-ATP synthase subunit (α) served as control for mitochiondria enrichment. E. Truncated forms of human α-synuclein were detected with increasing concentrations (4–20 µg) of ASOTg mitochondria but not with WT mitochondria.
Mentions: Because α-synuclein is predominantly a presynaptic protein [1], [2], we first used forebrain synaptoneurosomes (SNs) [36] to characterize domain-selective antibodies for human α-synuclein (NH2 terminus, COOH terminus #1, COOH terminus #2) (Fig. 1A). Each antibody detected elevated levels of human α-synuclein (15–20 kDa) in ASOTg relative to WT SNs; each antibody also has some cross-immunoreactivity with endogenous WT mouse α-synuclein (Fig. 1B). No selective immunoreactivity was detected in SN fractions from α-synuclein KO mice used as negative controls. Smaller α-synuclein forms were also detected in ASOTg SNs (left/middle panels). Overall the immunoblotting patterns were consistent with carboxyl terminal truncation, since the COOH terminus #2 antibody (only epitope containing the last 9 amino acids at carboxyl terminus) failed to detect smaller forms (right panel). Truncated forms were not detected in WT SNs, but this may be due to their lower amounts. Larger molecular weight forms (>20 kDa) in the ASOTg lanes appeared to be unrelated cross-immunoreactive bands based on side-by-side comparisons to WT and KO lanes.

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