Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease - PubMed (original) (raw)

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

Miranda L Bader Lange et al. Neurobiol Dis. 2008 Mar.

Abstract

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

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Figures

Figure 1

Figure 1. NBD-PS assay in synaptosomes from brain of subjects with MCI and AD

HumanMCI, AD, and control synaptosomes were treated with the fluorescent phospholipid NBD-PS. PtdSer exposure to the outer leaflet of the membrane bilayer is measured as the percent decrease in fluorescence signal after Na2S2O4 quenching. The control value was set to 100%, to which experimental values were compared. These data, in arbitrary units on the ordinate axis, are presented as mean ±S.D. AD, N=5, *P<0.0001; MCI, N=5, **P<0.00001.

Figure 2

Figure 2. PMI NBD-PS assay

FVB mouse synaptosomes were treated with the fluorescent phospholipid NBD-PS. PtdSer exposure to the outer leaflet of the plasma membrane is measured as a decrease in fluorescence signal, compared to 0 h, after quenching with Na2S2O4. Since samples were not compared as a percentage of control, negative values are not seen, but graphical interpretation is relatively the same. A fluorescence decrease, compared to 0 h, denotes an increased amount of PtdSer exposed to the lipid bilayer outer leaflet. Data shown in arbitrary fluorescence units on the ordinate axis as mean ±S.D. 0 h, N=3; 2.8 h, N=2, P<0.65; 3.4 h, N=3, P<0.80.

Figure 3

Figure 3. Mg2+ATPase activity assay

FVB mouse synaptosome Mg2+ATPase activity was measured to represent flippase activity, an ATP-dependent membrane bound translocase. Decreased UV-absorbance (810 nm) compared to 0 h denotes decreased Mg2+ATPase activity. Data shown, in arbitrary absorbance units, as mean ±S.D. 0 h, N=3; 2.8 h, N=2, *P<0.04; 3.5 h, N=3, P<0.26.

Figure 4

Figure 4. Bcl-2 levels in brain from subjects with MCI and AD

Bcl-2 levels were measured in IPL of MCI and AD patients by Western blot analysis. (a) MCI and AD gels were run with equal amounts of protein (75 µg/lane). Increased band intensity after normalization with β-tubulin represents an increase in the amount of Bcl-2 protein present. (b) & (c) Graphical analysis of MCI and AD band intensities, respectively. The control value was set to 100%, to which experimental values were compared. Data shown in arbitrary units on the ordinate axis as mean ±S.D. in (a) MCI, N=3, *P<0.006; in (b) AD N=3, *P<0.04.

Figure 4

Figure 4. Bcl-2 levels in brain from subjects with MCI and AD

Bcl-2 levels were measured in IPL of MCI and AD patients by Western blot analysis. (a) MCI and AD gels were run with equal amounts of protein (75 µg/lane). Increased band intensity after normalization with β-tubulin represents an increase in the amount of Bcl-2 protein present. (b) & (c) Graphical analysis of MCI and AD band intensities, respectively. The control value was set to 100%, to which experimental values were compared. Data shown in arbitrary units on the ordinate axis as mean ±S.D. in (a) MCI, N=3, *P<0.006; in (b) AD N=3, *P<0.04.

Figure 5

Figure 5. Bax levels in brain from subjects with MCI and AD

Bax levels were measured in IPL of MCI and AD patients by Western blot analysis. (a) MCI and AD gels were run with equal amounts of protein (75 µg/lane). Increased band intensity after normalization with β-tubulin represents an increase in the amount of Bax protein present. (b) & (c) Graphical analysis of MCI and AD band intensities, respectively. The control value was set to 100%, to which experimental values were compared. Data shown in arbitrary units on the ordinate axis as mean ±S.D. in (a) MCI, N=3, *P<0.03; in (b) AD N=3, *P<0.005.

Figure 5

Figure 5. Bax levels in brain from subjects with MCI and AD

Bax levels were measured in IPL of MCI and AD patients by Western blot analysis. (a) MCI and AD gels were run with equal amounts of protein (75 µg/lane). Increased band intensity after normalization with β-tubulin represents an increase in the amount of Bax protein present. (b) & (c) Graphical analysis of MCI and AD band intensities, respectively. The control value was set to 100%, to which experimental values were compared. Data shown in arbitrary units on the ordinate axis as mean ±S.D. in (a) MCI, N=3, *P<0.03; in (b) AD N=3, *P<0.005.

Figure 6

Figure 6. Caspase-3 levles in brain from subjects with MCI and AD

Caspase-3 levels were measured in IPL of MCI and AD patients by Western blot analysis. (a) MCI and AD gels were run with equal amounts of protein (75 µg/lane). Increased band intensity after normalization with β-tubulin represents an increase in the amount of caspase-3 present. (b) & (c) Graphical analysis of MCI and AD band intensities, respectively. The control value was set to 100%, to which experimental values were compared. Data shown in arbitrary units on the ordinate axis as mean ±S.D. in (a) MCI, N=3, *P<0.02; in (b) AD N=3, *P<0.04.

Figure 6

Figure 6. Caspase-3 levles in brain from subjects with MCI and AD

Caspase-3 levels were measured in IPL of MCI and AD patients by Western blot analysis. (a) MCI and AD gels were run with equal amounts of protein (75 µg/lane). Increased band intensity after normalization with β-tubulin represents an increase in the amount of caspase-3 present. (b) & (c) Graphical analysis of MCI and AD band intensities, respectively. The control value was set to 100%, to which experimental values were compared. Data shown in arbitrary units on the ordinate axis as mean ±S.D. in (a) MCI, N=3, *P<0.02; in (b) AD N=3, *P<0.04.

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