Mitochondrial lipid abnormality and electron transport chain impairment in mice lacking alpha-synuclein

Abstract

The presynaptic protein alpha-synuclein, implicated in Parkinson disease (PD), binds phospholipids and has a role in brain fatty acid (FA) metabolism. In mice lacking alpha-synuclein (Snca-/-), total brain steady-state mass of the mitochondria-specific phospholipid, cardiolipin, is reduced 22% and its acyl side chains show a 51% increase in saturated FAs and a 25% reduction in essential n-6, but not n-3, polyunsaturated FAs. Additionally, 23% reduction in phosphatidylglycerol content, the immediate biosynthetic precursor of cardiolipin, was observed without alterations in the content of other brain phospholipids. Consistent with these changes, more ordered lipid head group and acyl chain packing with enhanced rotational motion of diphenylhexatriene (DPH) about its long axis were demonstrated in time-resolved DPH fluorescence lifetime experiments. These abnormalities in mitochondrial membrane properties were associated with a 15% reduction in linked complex I/III activity of the electron transport chain, without reductions in mitochondrial number, complex II/III activity, or individual complex I, II, III, or IV activity. Reduced complex I activity is thought to be a critical factor in the development of PD. Thus, altered membrane composition and structure and impaired complex I/III function in Snca-/- brain suggest a relationship between alpha-synuclein's role in brain lipid metabolism, mitochondrial function, and PD.

FIG. 1.

The amount of mtDNA is not altered in Snca^−/− mouse brain. Real-time PCR was performed in triplicate on DNA isolated from WT (n = 5) and Snca^−/− (n = 5) mouse brains using primers specific for nuclear and mtDNA as outlined in Materials and Methods. Results shown are plotted as percent mtDNA ± the standard error of the mean in Snca^−/− relative to control (arbitrarily set to 100%). No significant difference was observed between the two genotypes (Student's t test).

FIG. 2.

Complex I/III activity is reduced in Snca^−/− mouse brain in the absence of complex II/III and individual complex activity impairment. Linked (A) and unlinked (B) enzyme assays representing various ETC complex activities were performed on mouse brain lysates as described in Materials and Methods. Relative enzymatic activities observed in Snca^−/− mouse brain are plotted as a percentage of the WT control ± the standard deviation. Each enzyme activity with the corresponding ETC complex(es) assayed is labeled. Significant differences were observed between the two genotypes (Student's t test), as indicated: *, P < 0.02; **, P < 0.01.

FIG. 3.

No alterations in assembly of the ∼950-kDa complex I or dimeric ∼500-kDa complex III were observed in Snca^−/− mouse brain. Mitochondria were isolated as described in Materials and Methods. (A) Fifty micrograms of protein was separated by BN-PAGE (7%) and transferred to PVDF. Western analysis was performed using the 20- (upper panel) and 39-kDa (middle panel) complex I antibodies or the core II (lower panel) complex III antibody as indicated (arrows). (B) A 100-μg aliquot of protein was separated in the first dimension by BN-PAGE (7%) and SDS-PAGE (12%) in the second dimension and transferred to PVDF. Western analysis was performed using the 39-, 30-, 20-, and 17-kDa complex I antibodies as indicated (arrows). No significant differences in complex I or III expression or size were observed in multiple experiments.