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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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N-terminally acetylated full-length human α-synuclein was the major isoform detected in a ASOTg cortex mitochondrial fraction and in human brain by top-down high-resolution mass spectrometry.A. Total ion chromatogram is shown for the primary separation and the ion-isolation mass spectrum (m/z 967.76) of a candidate full-length α-synuclein of mass 14503.1 Da found in the 37-minute fraction for a ASOTg cortex mitochondrial fraction (500 µg). B. Static nano-electrospray and MSMS displays peaks of various intensities for the average mass spectrum in the same 37-minute fraction (arrow, unmodified human α-synuclein sequenced in panel C). No masses corresponded to known PTMs for human α-synuclein, e,g, nitration, phosphorylation or ubiquitination. C. Hybrid linear ion-trap FT-MS (LTQ-FT) generates high resolution map of product ions formed upon collisionally activated dissociation (CAD) of the m/z 967 precursor, matched at 10 ppm tolerance for confident assignment of the primary amino acid sequence of full-length human α-synuclein from ASOTg mouse (upper panel). Monoisotopic mass of human α-synuclein from ASOTg mouse was 14493.2591 Da (mean of 4 measurements on 4 different ions). A protein of similar mass was recovered from resected human brain and CAD of the m/z 1209 precursor gave a broadly similar product-ion map (lower panel). Monoisotopic mass of human α-synuclein from human brain was 14493.2592 Da (mean of 2 measurements on 2 different ions). The probability that either species analyzed was incorrectly assigned was calculated to be 9.9×10−31 (ASOTg) and 4.4×10−43 (human brain) using the ProsightPC algorithm at a tolerance of 10 ppm. Note that the top-down approach yields several product ions in the C-terminal region that was poorly covered in the bottom-up experiment (Fig. 6). The species analyzed is shown with N-terminal acetylation of starting methionine (in red) yielding agreement of measured and calculated masses within 10 ppm.
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pone-0063557-g007: N-terminally acetylated full-length human α-synuclein was the major isoform detected in a ASOTg cortex mitochondrial fraction and in human brain by top-down high-resolution mass spectrometry.A. Total ion chromatogram is shown for the primary separation and the ion-isolation mass spectrum (m/z 967.76) of a candidate full-length α-synuclein of mass 14503.1 Da found in the 37-minute fraction for a ASOTg cortex mitochondrial fraction (500 µg). B. Static nano-electrospray and MSMS displays peaks of various intensities for the average mass spectrum in the same 37-minute fraction (arrow, unmodified human α-synuclein sequenced in panel C). No masses corresponded to known PTMs for human α-synuclein, e,g, nitration, phosphorylation or ubiquitination. C. Hybrid linear ion-trap FT-MS (LTQ-FT) generates high resolution map of product ions formed upon collisionally activated dissociation (CAD) of the m/z 967 precursor, matched at 10 ppm tolerance for confident assignment of the primary amino acid sequence of full-length human α-synuclein from ASOTg mouse (upper panel). Monoisotopic mass of human α-synuclein from ASOTg mouse was 14493.2591 Da (mean of 4 measurements on 4 different ions). A protein of similar mass was recovered from resected human brain and CAD of the m/z 1209 precursor gave a broadly similar product-ion map (lower panel). Monoisotopic mass of human α-synuclein from human brain was 14493.2592 Da (mean of 2 measurements on 2 different ions). The probability that either species analyzed was incorrectly assigned was calculated to be 9.9×10−31 (ASOTg) and 4.4×10−43 (human brain) using the ProsightPC algorithm at a tolerance of 10 ppm. Note that the top-down approach yields several product ions in the C-terminal region that was poorly covered in the bottom-up experiment (Fig. 6). The species analyzed is shown with N-terminal acetylation of starting methionine (in red) yielding agreement of measured and calculated masses within 10 ppm.

Mentions: As shown in Figure 6B, the major peptides detected in the total digest covered ∼ 60% of the 140 amino acid sequence of human α-synuclein. The first 10 NH2 terminal amino acids and the last 43 COOH terminal amino acids were not represented due to the tryptic digestion pattern (but see Fig. 7) that resulted in peptides either too small or too large for recovery and analysis by nLC-MSMS. Despite appropriate data mining, no PTMs were detected in each human α-synuclein peptide including previously reported sites of tyrosine nitration, methionine oxidation, serine/threonine phosphorylation, tyrosine phosphorylation, lysine SUMOylation, and lysine ubiquitination.


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)

N-terminally acetylated full-length human α-synuclein was the major isoform detected in a ASOTg cortex mitochondrial fraction and in human brain by top-down high-resolution mass spectrometry.A. Total ion chromatogram is shown for the primary separation and the ion-isolation mass spectrum (m/z 967.76) of a candidate full-length α-synuclein of mass 14503.1 Da found in the 37-minute fraction for a ASOTg cortex mitochondrial fraction (500 µg). B. Static nano-electrospray and MSMS displays peaks of various intensities for the average mass spectrum in the same 37-minute fraction (arrow, unmodified human α-synuclein sequenced in panel C). No masses corresponded to known PTMs for human α-synuclein, e,g, nitration, phosphorylation or ubiquitination. C. Hybrid linear ion-trap FT-MS (LTQ-FT) generates high resolution map of product ions formed upon collisionally activated dissociation (CAD) of the m/z 967 precursor, matched at 10 ppm tolerance for confident assignment of the primary amino acid sequence of full-length human α-synuclein from ASOTg mouse (upper panel). Monoisotopic mass of human α-synuclein from ASOTg mouse was 14493.2591 Da (mean of 4 measurements on 4 different ions). A protein of similar mass was recovered from resected human brain and CAD of the m/z 1209 precursor gave a broadly similar product-ion map (lower panel). Monoisotopic mass of human α-synuclein from human brain was 14493.2592 Da (mean of 2 measurements on 2 different ions). The probability that either species analyzed was incorrectly assigned was calculated to be 9.9×10−31 (ASOTg) and 4.4×10−43 (human brain) using the ProsightPC algorithm at a tolerance of 10 ppm. Note that the top-down approach yields several product ions in the C-terminal region that was poorly covered in the bottom-up experiment (Fig. 6). The species analyzed is shown with N-terminal acetylation of starting methionine (in red) yielding agreement of measured and calculated masses within 10 ppm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646806&req=5

pone-0063557-g007: N-terminally acetylated full-length human α-synuclein was the major isoform detected in a ASOTg cortex mitochondrial fraction and in human brain by top-down high-resolution mass spectrometry.A. Total ion chromatogram is shown for the primary separation and the ion-isolation mass spectrum (m/z 967.76) of a candidate full-length α-synuclein of mass 14503.1 Da found in the 37-minute fraction for a ASOTg cortex mitochondrial fraction (500 µg). B. Static nano-electrospray and MSMS displays peaks of various intensities for the average mass spectrum in the same 37-minute fraction (arrow, unmodified human α-synuclein sequenced in panel C). No masses corresponded to known PTMs for human α-synuclein, e,g, nitration, phosphorylation or ubiquitination. C. Hybrid linear ion-trap FT-MS (LTQ-FT) generates high resolution map of product ions formed upon collisionally activated dissociation (CAD) of the m/z 967 precursor, matched at 10 ppm tolerance for confident assignment of the primary amino acid sequence of full-length human α-synuclein from ASOTg mouse (upper panel). Monoisotopic mass of human α-synuclein from ASOTg mouse was 14493.2591 Da (mean of 4 measurements on 4 different ions). A protein of similar mass was recovered from resected human brain and CAD of the m/z 1209 precursor gave a broadly similar product-ion map (lower panel). Monoisotopic mass of human α-synuclein from human brain was 14493.2592 Da (mean of 2 measurements on 2 different ions). The probability that either species analyzed was incorrectly assigned was calculated to be 9.9×10−31 (ASOTg) and 4.4×10−43 (human brain) using the ProsightPC algorithm at a tolerance of 10 ppm. Note that the top-down approach yields several product ions in the C-terminal region that was poorly covered in the bottom-up experiment (Fig. 6). The species analyzed is shown with N-terminal acetylation of starting methionine (in red) yielding agreement of measured and calculated masses within 10 ppm.
Mentions: As shown in Figure 6B, the major peptides detected in the total digest covered ∼ 60% of the 140 amino acid sequence of human α-synuclein. The first 10 NH2 terminal amino acids and the last 43 COOH terminal amino acids were not represented due to the tryptic digestion pattern (but see Fig. 7) that resulted in peptides either too small or too large for recovery and analysis by nLC-MSMS. Despite appropriate data mining, no PTMs were detected in each human α-synuclein peptide including previously reported sites of tyrosine nitration, methionine oxidation, serine/threonine phosphorylation, tyrosine phosphorylation, lysine SUMOylation, and lysine ubiquitination.

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