Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein
Figure 1
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.