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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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Unmodified full-length human α-synuclein was the major isoform detected in an ASOTg striatum mitochondrial and cytosolic fractions by combined LC-MS+ and Western immunoblotting.A. The total ion chromatogram of LC-MS+ for an ASOTg striatum mitochondrial fraction (250 µg) is shown. Arrow indicates likely human α-synuclein fraction (∼37–38) based on previous top-down MS with ASOTg cortex mitochondria (see Fig. 7). B. Static nano-electrospray and MSMS resolved single fraction (arrow in panel A, 37.55 minutes retention time from total ion chromatogram, comparable to #38 in panel C) from ASOTg striatum mitochondria (arrow in top panel, 14502.5 Da) and a matching cytosolic fraction (arrow in bottom panel, 14502.8 Da) from the same purification procedure. C. As a positive control, human α-synuclein is detected as a major band of 15–20 kDa and a minor smaller band in whole unfractionated ASOTg striatum mitochondria (5 µg) by SDS-PAGE and Western immunoblotting using an antibody selective for human α-synuclein (left panel). Individual top-down intact fractions (#30–62, yellow underline in panel A) were further examined by Western immunoblotting (right panel). Immunoreactivity for unmodified human α-synuclein is detected in fraction #38, but no additional bands corresponding to covalently modified forms that would change relative migration (e.g. truncation, phosphorylation, ubiquitination) are detected.
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pone-0063557-g008: Unmodified full-length human α-synuclein was the major isoform detected in an ASOTg striatum mitochondrial and cytosolic fractions by combined LC-MS+ and Western immunoblotting.A. The total ion chromatogram of LC-MS+ for an ASOTg striatum mitochondrial fraction (250 µg) is shown. Arrow indicates likely human α-synuclein fraction (∼37–38) based on previous top-down MS with ASOTg cortex mitochondria (see Fig. 7). B. Static nano-electrospray and MSMS resolved single fraction (arrow in panel A, 37.55 minutes retention time from total ion chromatogram, comparable to #38 in panel C) from ASOTg striatum mitochondria (arrow in top panel, 14502.5 Da) and a matching cytosolic fraction (arrow in bottom panel, 14502.8 Da) from the same purification procedure. C. As a positive control, human α-synuclein is detected as a major band of 15–20 kDa and a minor smaller band in whole unfractionated ASOTg striatum mitochondria (5 µg) by SDS-PAGE and Western immunoblotting using an antibody selective for human α-synuclein (left panel). Individual top-down intact fractions (#30–62, yellow underline in panel A) were further examined by Western immunoblotting (right panel). Immunoreactivity for unmodified human α-synuclein is detected in fraction #38, but no additional bands corresponding to covalently modified forms that would change relative migration (e.g. truncation, phosphorylation, ubiquitination) are detected.

Mentions: Based on the previous cortex experiments, ASOTg striatum mitochondria were also resolved by LC-MS+ and a likely fraction (vertical arrow) containing human α-synuclein was isolated (Fig. 8A). The mass spectrum of this mitochondrial fraction and a matching cytosolic fraction are displayed in Fig. 8B (top panel, mitochondria, 14502.5 Da; bottom panel, cytosol, 14502.8 Da). The major peak’s masses (arrows) were consistent with the N-acetylated full-length α-synuclein, while none of the other peaks’ masses corresponded to the mass of human α-synuclein containing PTMs. Individual ASOTg striatum mitochondrial retention fractions (#30–62 minutes, yellow underline)(Fig. 8A) were further examined by Western immunoblotting (Fig. 8C). Only a single band of ∼15 kDa is detected in the 38 minute fraction, corresponding to the approximate fraction containing mostly unmodified human α-synuclein (37.55 minute fraction) (see vertical arrow, Fig. 8A; arrows, Fig. 8B). Although there were peaks of 9–12 kDa in size of potential truncated forms, no smaller bands were detected in fraction #38 nor were larger bands (for example, ubiquitinated) detected in any other fraction on the immunoblot. Thus the combined MS data detect few covalent modifications (except for N-acetylation) of over-expressed human α-synuclein in either mouse brain mitochondrial/cytosolic fractions or in resected human cortex.


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)

Unmodified full-length human α-synuclein was the major isoform detected in an ASOTg striatum mitochondrial and cytosolic fractions by combined LC-MS+ and Western immunoblotting.A. The total ion chromatogram of LC-MS+ for an ASOTg striatum mitochondrial fraction (250 µg) is shown. Arrow indicates likely human α-synuclein fraction (∼37–38) based on previous top-down MS with ASOTg cortex mitochondria (see Fig. 7). B. Static nano-electrospray and MSMS resolved single fraction (arrow in panel A, 37.55 minutes retention time from total ion chromatogram, comparable to #38 in panel C) from ASOTg striatum mitochondria (arrow in top panel, 14502.5 Da) and a matching cytosolic fraction (arrow in bottom panel, 14502.8 Da) from the same purification procedure. C. As a positive control, human α-synuclein is detected as a major band of 15–20 kDa and a minor smaller band in whole unfractionated ASOTg striatum mitochondria (5 µg) by SDS-PAGE and Western immunoblotting using an antibody selective for human α-synuclein (left panel). Individual top-down intact fractions (#30–62, yellow underline in panel A) were further examined by Western immunoblotting (right panel). Immunoreactivity for unmodified human α-synuclein is detected in fraction #38, but no additional bands corresponding to covalently modified forms that would change relative migration (e.g. truncation, phosphorylation, ubiquitination) are detected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646806&req=5

pone-0063557-g008: Unmodified full-length human α-synuclein was the major isoform detected in an ASOTg striatum mitochondrial and cytosolic fractions by combined LC-MS+ and Western immunoblotting.A. The total ion chromatogram of LC-MS+ for an ASOTg striatum mitochondrial fraction (250 µg) is shown. Arrow indicates likely human α-synuclein fraction (∼37–38) based on previous top-down MS with ASOTg cortex mitochondria (see Fig. 7). B. Static nano-electrospray and MSMS resolved single fraction (arrow in panel A, 37.55 minutes retention time from total ion chromatogram, comparable to #38 in panel C) from ASOTg striatum mitochondria (arrow in top panel, 14502.5 Da) and a matching cytosolic fraction (arrow in bottom panel, 14502.8 Da) from the same purification procedure. C. As a positive control, human α-synuclein is detected as a major band of 15–20 kDa and a minor smaller band in whole unfractionated ASOTg striatum mitochondria (5 µg) by SDS-PAGE and Western immunoblotting using an antibody selective for human α-synuclein (left panel). Individual top-down intact fractions (#30–62, yellow underline in panel A) were further examined by Western immunoblotting (right panel). Immunoreactivity for unmodified human α-synuclein is detected in fraction #38, but no additional bands corresponding to covalently modified forms that would change relative migration (e.g. truncation, phosphorylation, ubiquitination) are detected.
Mentions: Based on the previous cortex experiments, ASOTg striatum mitochondria were also resolved by LC-MS+ and a likely fraction (vertical arrow) containing human α-synuclein was isolated (Fig. 8A). The mass spectrum of this mitochondrial fraction and a matching cytosolic fraction are displayed in Fig. 8B (top panel, mitochondria, 14502.5 Da; bottom panel, cytosol, 14502.8 Da). The major peak’s masses (arrows) were consistent with the N-acetylated full-length α-synuclein, while none of the other peaks’ masses corresponded to the mass of human α-synuclein containing PTMs. Individual ASOTg striatum mitochondrial retention fractions (#30–62 minutes, yellow underline)(Fig. 8A) were further examined by Western immunoblotting (Fig. 8C). Only a single band of ∼15 kDa is detected in the 38 minute fraction, corresponding to the approximate fraction containing mostly unmodified human α-synuclein (37.55 minute fraction) (see vertical arrow, Fig. 8A; arrows, Fig. 8B). Although there were peaks of 9–12 kDa in size of potential truncated forms, no smaller bands were detected in fraction #38 nor were larger bands (for example, ubiquitinated) detected in any other fraction on the immunoblot. Thus the combined MS data detect few covalent modifications (except for N-acetylation) of over-expressed human α-synuclein in either mouse brain mitochondrial/cytosolic fractions or in resected human cortex.

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