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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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Amyloid-like soluble oligomers or fibrils of elevated human α-synuclein were not detected in ASOTg brain mitochondria and synaptoneurosome fractions.A. On dots blots, the α-synuclein antibody detected recombinant human α-synuclein in increasing amounts (2.5–100 ng), serving as a monomer positive control (top panel). Aβ42 soluble oligomers and Aβ42 fibrils (200 ng each) served as positive controls for the A11 antibody (middle panel) and the OC antibody (bottom panel) respectively. B. The α-synuclein antibody detected elevated human α-synuclein in ASOTg mitochondrial (mito) and synaptoneurosome (SN) fractions (1 µg) relative to WT fractions (top panel). An SN fraction from Snca−/−mice (KO) served as a negative control. The A11 and OC antibodies failed to detect endogenous mouse (WT) or elevated human α-synuclein (ASOTg) in all fractions when compared to KO negative control (middle/bottom panels).
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pone-0063557-g005: Amyloid-like soluble oligomers or fibrils of elevated human α-synuclein were not detected in ASOTg brain mitochondria and synaptoneurosome fractions.A. On dots blots, the α-synuclein antibody detected recombinant human α-synuclein in increasing amounts (2.5–100 ng), serving as a monomer positive control (top panel). Aβ42 soluble oligomers and Aβ42 fibrils (200 ng each) served as positive controls for the A11 antibody (middle panel) and the OC antibody (bottom panel) respectively. B. The α-synuclein antibody detected elevated human α-synuclein in ASOTg mitochondrial (mito) and synaptoneurosome (SN) fractions (1 µg) relative to WT fractions (top panel). An SN fraction from Snca−/−mice (KO) served as a negative control. The A11 and OC antibodies failed to detect endogenous mouse (WT) or elevated human α-synuclein (ASOTg) in all fractions when compared to KO negative control (middle/bottom panels).

Mentions: It cannot be completely ruled out that the 50–60 kDa band together with larger forms observed on Native-PAGE alone may correspond to soluble amyloid-like oligomeric and more aggregated forms of human α-synuclein. To address this possibility further, we used amyloid conformation-selective antibodies (A11, OC)[46] to ask if overexpressed human α-synuclein forms amyloid-like, β-sheet-rich oligomers in brain mitochondria fractions. Because the amyloid-selective antibodies did not detect specific immunoreactive bands resolved by Native-PAGE/Western blots (unpublished observations), we used dot blots exclusively for these experiments (repeated twice). As a positive control for soluble α-synuclein monomers, the COOH terminus #1 antibody detected recombinant human α-synuclein at increasing amounts (Fig. 5 A, top panel). As a positive control for amyloid conformations, the A11 antibody (middle panel) and the OC antibody (bottom panel) detected amyloid-like oligomers and fibrils of Aβ42 peptides respectively. The α-synuclein antibody detected over-expressed human α-synuclein in both ASOTg brain mitochondrial and forebrain SN fractions (Fig. 5B, top panel). A SN fraction from Snca−/−α-synuclein mice (KO) served as negative control for background staining. However, the amyloid conformational antibodies, both the A11 antibody (oligomers, middle panel) and the OC antibody (fibrils, bottom panel), did not detect overexpressed human α-synuclein in ASOTg fractions or the endogenous mouse protein in WT fractions. These results together with the previous Native-PAGE experiments (Fig. 4) were most consistent with soluble monomeric α-synuclein as the major protein form interacting with brain mitochondria.


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)

Amyloid-like soluble oligomers or fibrils of elevated human α-synuclein were not detected in ASOTg brain mitochondria and synaptoneurosome fractions.A. On dots blots, the α-synuclein antibody detected recombinant human α-synuclein in increasing amounts (2.5–100 ng), serving as a monomer positive control (top panel). Aβ42 soluble oligomers and Aβ42 fibrils (200 ng each) served as positive controls for the A11 antibody (middle panel) and the OC antibody (bottom panel) respectively. B. The α-synuclein antibody detected elevated human α-synuclein in ASOTg mitochondrial (mito) and synaptoneurosome (SN) fractions (1 µg) relative to WT fractions (top panel). An SN fraction from Snca−/−mice (KO) served as a negative control. The A11 and OC antibodies failed to detect endogenous mouse (WT) or elevated human α-synuclein (ASOTg) in all fractions when compared to KO negative control (middle/bottom panels).
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Related In: Results  -  Collection

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pone-0063557-g005: Amyloid-like soluble oligomers or fibrils of elevated human α-synuclein were not detected in ASOTg brain mitochondria and synaptoneurosome fractions.A. On dots blots, the α-synuclein antibody detected recombinant human α-synuclein in increasing amounts (2.5–100 ng), serving as a monomer positive control (top panel). Aβ42 soluble oligomers and Aβ42 fibrils (200 ng each) served as positive controls for the A11 antibody (middle panel) and the OC antibody (bottom panel) respectively. B. The α-synuclein antibody detected elevated human α-synuclein in ASOTg mitochondrial (mito) and synaptoneurosome (SN) fractions (1 µg) relative to WT fractions (top panel). An SN fraction from Snca−/−mice (KO) served as a negative control. The A11 and OC antibodies failed to detect endogenous mouse (WT) or elevated human α-synuclein (ASOTg) in all fractions when compared to KO negative control (middle/bottom panels).
Mentions: It cannot be completely ruled out that the 50–60 kDa band together with larger forms observed on Native-PAGE alone may correspond to soluble amyloid-like oligomeric and more aggregated forms of human α-synuclein. To address this possibility further, we used amyloid conformation-selective antibodies (A11, OC)[46] to ask if overexpressed human α-synuclein forms amyloid-like, β-sheet-rich oligomers in brain mitochondria fractions. Because the amyloid-selective antibodies did not detect specific immunoreactive bands resolved by Native-PAGE/Western blots (unpublished observations), we used dot blots exclusively for these experiments (repeated twice). As a positive control for soluble α-synuclein monomers, the COOH terminus #1 antibody detected recombinant human α-synuclein at increasing amounts (Fig. 5 A, top panel). As a positive control for amyloid conformations, the A11 antibody (middle panel) and the OC antibody (bottom panel) detected amyloid-like oligomers and fibrils of Aβ42 peptides respectively. The α-synuclein antibody detected over-expressed human α-synuclein in both ASOTg brain mitochondrial and forebrain SN fractions (Fig. 5B, top panel). A SN fraction from Snca−/−α-synuclein mice (KO) served as negative control for background staining. However, the amyloid conformational antibodies, both the A11 antibody (oligomers, middle panel) and the OC antibody (fibrils, bottom panel), did not detect overexpressed human α-synuclein in ASOTg fractions or the endogenous mouse protein in WT fractions. These results together with the previous Native-PAGE experiments (Fig. 4) were most consistent with soluble monomeric α-synuclein as the major protein form interacting with brain mitochondria.

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