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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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Brain synaptic and mitochondrial human α-synuclein were resolved mainly as a monomeric protein with truncated species by Native-PAGE and 2-dimensional PAGE (Native/SDS) immunoblotting.A. An α-synuclein (αSyn)-immunoreactive band of 50–60 kDa plus smaller forms (arrows, bracketed) were detected in sonicated ASOTg (ASO) forebrain SNs (10 µg) by Native-PAGE/immunoblotting. An SN fraction from Snca−/−mice (KO) served as a negative control. B. As a positive control for monomer conformations, sonicated recombinant human α-synuclein (Rec Human αSyn)(0.2–0.4 µg) was run on a Native gel and detected by immunoblotting (last lane). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa amongst a large smear of bands > than 50 kDa half way up the gel (bracketed). WT and ASOTg cortex mitochondria (mito) and cytosolic (cyt) fractions (20 µg) as well as surgically resected human cortex (20 µg) were resolved on the same blot side by side with recombinant α-synuclein. A major band of 50–60 kDa is resolved but smaller bands were also detected mainly in the ASOTg mitochondrial fraction. C. In a separate Native-PAGE/blotting experiment, a major band of 50–60 kDa was also detected in ASOTg striatum mitochondrial/cytosolic fractions, a cerebellum mitochondrial preparation, recombinant α-synuclein, and five human postmortem midbrain tissues [con (control Case #1), con (control Case #2), PD (Case #4), DLBD/PD (Case #3), AD/DLBD (Case #5). D. Cortex mitochondrial and recombinant human α-synuclein were resolved by two-dimensional PAGE (1-D Native/2-D SDS) and immunoblotting. Native-PAGE isoforms (≥50 kDa) of recombinant α-synuclein were resolved mainly as monomers (mono) close to 20 kDa in size on SDS-PAGE. Similar sized Native-PAGE isoforms (≥50 kDa) observed in ASOTg cortex mitochondrial fractions were also resolved as monomers on SDS-PAGE. Smaller mitochondrial forms (trunc) closer to 15 kDa in size were also detected supporting their COOH-terminal truncation. In a separate 2-D experiment, α-synuclein monomers and truncated forms were also observed in an ASOTg striatum mitochondrial fraction (only 2-D SDS-PAGE shown)(bottom panel).
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pone-0063557-g004: Brain synaptic and mitochondrial human α-synuclein were resolved mainly as a monomeric protein with truncated species by Native-PAGE and 2-dimensional PAGE (Native/SDS) immunoblotting.A. An α-synuclein (αSyn)-immunoreactive band of 50–60 kDa plus smaller forms (arrows, bracketed) were detected in sonicated ASOTg (ASO) forebrain SNs (10 µg) by Native-PAGE/immunoblotting. An SN fraction from Snca−/−mice (KO) served as a negative control. B. As a positive control for monomer conformations, sonicated recombinant human α-synuclein (Rec Human αSyn)(0.2–0.4 µg) was run on a Native gel and detected by immunoblotting (last lane). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa amongst a large smear of bands > than 50 kDa half way up the gel (bracketed). WT and ASOTg cortex mitochondria (mito) and cytosolic (cyt) fractions (20 µg) as well as surgically resected human cortex (20 µg) were resolved on the same blot side by side with recombinant α-synuclein. A major band of 50–60 kDa is resolved but smaller bands were also detected mainly in the ASOTg mitochondrial fraction. C. In a separate Native-PAGE/blotting experiment, a major band of 50–60 kDa was also detected in ASOTg striatum mitochondrial/cytosolic fractions, a cerebellum mitochondrial preparation, recombinant α-synuclein, and five human postmortem midbrain tissues [con (control Case #1), con (control Case #2), PD (Case #4), DLBD/PD (Case #3), AD/DLBD (Case #5). D. Cortex mitochondrial and recombinant human α-synuclein were resolved by two-dimensional PAGE (1-D Native/2-D SDS) and immunoblotting. Native-PAGE isoforms (≥50 kDa) of recombinant α-synuclein were resolved mainly as monomers (mono) close to 20 kDa in size on SDS-PAGE. Similar sized Native-PAGE isoforms (≥50 kDa) observed in ASOTg cortex mitochondrial fractions were also resolved as monomers on SDS-PAGE. Smaller mitochondrial forms (trunc) closer to 15 kDa in size were also detected supporting their COOH-terminal truncation. In a separate 2-D experiment, α-synuclein monomers and truncated forms were also observed in an ASOTg striatum mitochondrial fraction (only 2-D SDS-PAGE shown)(bottom panel).

Mentions: Overall the immunocapture data show that α-synuclein does not selectively associate with either inner membrane or soluble complexes localized within ASOTg mitochondria, at least at this adult age (2–4 months). However the use of detergents in the immunocapture and SDS-PAGE/immunoblotting experiments preclude the identification of higher order oligomeric/fibril forms of α-synuclein due to potential detergent-induced structural alterations [42]–[45]. To circumvent this problem, we sonicated WT and ASOTg mitochondria to lyse membranes and resolved proteins by Native-PAGE/immunoblotting (Fig. 4). We first examined SN fractions, where α-synuclein was detected as an immunoreactive band of 50–60 kDa plus minor amounts of smaller forms (arrows) in ASOTg forebrain SNs, but not in WT or KO control samples. As a positive control for unfolded monomer conformations, recombinant human α-synuclein was also examined in side-by-side comparison with WT and ASOTg cortex mitochondria and cytosolic fractions as well as a homogenate prepared from surgically resected human cortex (Fig. 4B). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa with some smaller bands and a smear of larger bands half way up the blot. A similar sized major band of 50–60 kDa and a smear of larger forms were also detected in the WT/ASOTg brain fractions and in resected human cortex. However, smaller bands were only detected in the mouse brain mitochondrial fractions (both WT and ASOTg). In a separate experiment (Fig. 4C), both ASOTg striatum and cerebellum mitochondria also revealed a 50–60 kDa α-synuclein band plus smaller forms; both control and PD-related human postmortem midbrain tissues contained only the 50–60 kDa form and some larger forms.


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)

Brain synaptic and mitochondrial human α-synuclein were resolved mainly as a monomeric protein with truncated species by Native-PAGE and 2-dimensional PAGE (Native/SDS) immunoblotting.A. An α-synuclein (αSyn)-immunoreactive band of 50–60 kDa plus smaller forms (arrows, bracketed) were detected in sonicated ASOTg (ASO) forebrain SNs (10 µg) by Native-PAGE/immunoblotting. An SN fraction from Snca−/−mice (KO) served as a negative control. B. As a positive control for monomer conformations, sonicated recombinant human α-synuclein (Rec Human αSyn)(0.2–0.4 µg) was run on a Native gel and detected by immunoblotting (last lane). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa amongst a large smear of bands > than 50 kDa half way up the gel (bracketed). WT and ASOTg cortex mitochondria (mito) and cytosolic (cyt) fractions (20 µg) as well as surgically resected human cortex (20 µg) were resolved on the same blot side by side with recombinant α-synuclein. A major band of 50–60 kDa is resolved but smaller bands were also detected mainly in the ASOTg mitochondrial fraction. C. In a separate Native-PAGE/blotting experiment, a major band of 50–60 kDa was also detected in ASOTg striatum mitochondrial/cytosolic fractions, a cerebellum mitochondrial preparation, recombinant α-synuclein, and five human postmortem midbrain tissues [con (control Case #1), con (control Case #2), PD (Case #4), DLBD/PD (Case #3), AD/DLBD (Case #5). D. Cortex mitochondrial and recombinant human α-synuclein were resolved by two-dimensional PAGE (1-D Native/2-D SDS) and immunoblotting. Native-PAGE isoforms (≥50 kDa) of recombinant α-synuclein were resolved mainly as monomers (mono) close to 20 kDa in size on SDS-PAGE. Similar sized Native-PAGE isoforms (≥50 kDa) observed in ASOTg cortex mitochondrial fractions were also resolved as monomers on SDS-PAGE. Smaller mitochondrial forms (trunc) closer to 15 kDa in size were also detected supporting their COOH-terminal truncation. In a separate 2-D experiment, α-synuclein monomers and truncated forms were also observed in an ASOTg striatum mitochondrial fraction (only 2-D SDS-PAGE shown)(bottom panel).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646806&req=5

pone-0063557-g004: Brain synaptic and mitochondrial human α-synuclein were resolved mainly as a monomeric protein with truncated species by Native-PAGE and 2-dimensional PAGE (Native/SDS) immunoblotting.A. An α-synuclein (αSyn)-immunoreactive band of 50–60 kDa plus smaller forms (arrows, bracketed) were detected in sonicated ASOTg (ASO) forebrain SNs (10 µg) by Native-PAGE/immunoblotting. An SN fraction from Snca−/−mice (KO) served as a negative control. B. As a positive control for monomer conformations, sonicated recombinant human α-synuclein (Rec Human αSyn)(0.2–0.4 µg) was run on a Native gel and detected by immunoblotting (last lane). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa amongst a large smear of bands > than 50 kDa half way up the gel (bracketed). WT and ASOTg cortex mitochondria (mito) and cytosolic (cyt) fractions (20 µg) as well as surgically resected human cortex (20 µg) were resolved on the same blot side by side with recombinant α-synuclein. A major band of 50–60 kDa is resolved but smaller bands were also detected mainly in the ASOTg mitochondrial fraction. C. In a separate Native-PAGE/blotting experiment, a major band of 50–60 kDa was also detected in ASOTg striatum mitochondrial/cytosolic fractions, a cerebellum mitochondrial preparation, recombinant α-synuclein, and five human postmortem midbrain tissues [con (control Case #1), con (control Case #2), PD (Case #4), DLBD/PD (Case #3), AD/DLBD (Case #5). D. Cortex mitochondrial and recombinant human α-synuclein were resolved by two-dimensional PAGE (1-D Native/2-D SDS) and immunoblotting. Native-PAGE isoforms (≥50 kDa) of recombinant α-synuclein were resolved mainly as monomers (mono) close to 20 kDa in size on SDS-PAGE. Similar sized Native-PAGE isoforms (≥50 kDa) observed in ASOTg cortex mitochondrial fractions were also resolved as monomers on SDS-PAGE. Smaller mitochondrial forms (trunc) closer to 15 kDa in size were also detected supporting their COOH-terminal truncation. In a separate 2-D experiment, α-synuclein monomers and truncated forms were also observed in an ASOTg striatum mitochondrial fraction (only 2-D SDS-PAGE shown)(bottom panel).
Mentions: Overall the immunocapture data show that α-synuclein does not selectively associate with either inner membrane or soluble complexes localized within ASOTg mitochondria, at least at this adult age (2–4 months). However the use of detergents in the immunocapture and SDS-PAGE/immunoblotting experiments preclude the identification of higher order oligomeric/fibril forms of α-synuclein due to potential detergent-induced structural alterations [42]–[45]. To circumvent this problem, we sonicated WT and ASOTg mitochondria to lyse membranes and resolved proteins by Native-PAGE/immunoblotting (Fig. 4). We first examined SN fractions, where α-synuclein was detected as an immunoreactive band of 50–60 kDa plus minor amounts of smaller forms (arrows) in ASOTg forebrain SNs, but not in WT or KO control samples. As a positive control for unfolded monomer conformations, recombinant human α-synuclein was also examined in side-by-side comparison with WT and ASOTg cortex mitochondria and cytosolic fractions as well as a homogenate prepared from surgically resected human cortex (Fig. 4B). Recombinant α-synuclein was resolved as a diffuse band of 50–60 kDa with some smaller bands and a smear of larger bands half way up the blot. A similar sized major band of 50–60 kDa and a smear of larger forms were also detected in the WT/ASOTg brain fractions and in resected human cortex. However, smaller bands were only detected in the mouse brain mitochondrial fractions (both WT and ASOTg). In a separate experiment (Fig. 4C), both ASOTg striatum and cerebellum mitochondria also revealed a 50–60 kDa α-synuclein band plus smaller forms; both control and PD-related human postmortem midbrain tissues contained only the 50–60 kDa form and some larger forms.

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