DHA-PC and PSD-95 decrease after loss of synaptophysin and before neuronal loss in patients with Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by senile plaques, neurofibrillary tangles, synaptic disruption, and neuronal loss. Several studies have demonstrated decreases of docosahexaenoic acid-containing phosphatidylcholines (DHA-PCs) in the AD brain. In this study, we used matrix-assisted laser desorption/ionization imaging mass spectrometry in postmortem AD brain to show that PC molecular species containing stearate and DHA, namely PC(18:0/22:6), was selectively depleted in the gray matter of patients with AD. Moreover, in the brain regions with marked amyloid β (Aβ) deposition, the magnitude of the PC(18:0/22:6) reduction significantly correlated with disease duration. Furthermore, at the molecular level, this depletion was associated with reduced levels of the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin. Interestingly, this reduction in PC(18:0/22:6) levels did not correlate with the degrees of Aβ deposition and neuronal loss in AD. The analysis of the correlations of key factors and disease duration showed that their effects on the disease time course were arranged in order as Aβ deposition, presynaptic disruption, postsynaptic disruption coupled with PC(18:0/22:6) reduction, and neuronal loss.

Figure 1. Characterization of PC molecular species in the human brain by Matrix-Assisted Laser Desorption/Ionization-Tandem Mass Spectrometry (MALDI-MS/MS).

(a) The averaged mass spectra from m/z 750 to 900 in non-Alzheimer's Disease (AD; left panel) and AD brains (right panel). The annotations indicate peak assignments to the phosphatidylcholine (PC) molecular species with different fatty acid compositions. (b) The tandem mass spectrum of PC(16:0/18:1) at m/z 798 as an example of the molecular characterization by this technique. The product ions at m/z 739 and m/z 615 (from loss of trimethylamine [NL 59] and phosphocholine [NL 183] residues, respectively), were commonly observed ions formed from the PC species. The product ion at m/z 542 was assigned to a fragment that was formed by the neutral loss of palmitic acid (16:0). (c) The panel shows the structural formula for PC(16:0/18:1) and the assignment of the cleavage positions. NL, neutral loss.

Figure 2. Marked reduction of docosahexaenoic acid (DHA)-PC molecular species in the temporal gray matter in AD.

(a) The pictures show Aβ-immunostained and Kluver-Barrera (KB)-stained coronal sections of non-AD and AD postmortem brains. (b) The distributions of the PC species in coronal sections of non-AD and AD brains that were analyzed by MALDI-imaging mass spectrometry (IMS) with 500-µm raster step sizes. The PCs with identical fatty acid moieties were arranged horizontally. The scale bars show 1 cm. (c–e) The graphs show the histograms of the intensity distributions of PC(16:0/22:6) (c), PC(18:0/22:6) (d), and PC(16:0/16:0) (e), in different brain regions in AD and non-AD brains from the relative intensity values of the MS images.

Figure 3. Quantitative analyses of the PC species in non-AD and AD temporal lobe with liquid chromatography-electrospray ionization (LC-ESI) MS/MS.

(a) The graphs show the concentrations of the PC species in the temporal gray (left panel) and white matter (right panel) of non-AD (blue bar) and AD (red bar) brains. (b) The graphs show the % changes of the PC concentrations between non-AD (blue bar) and AD (red bar) brains in the gray (left panel) and white matter (right panel). (c) The graphs show the % change of the PC composition ratio between the non-AD and AD brains in the gray (left graph) and white matter regions (right graph). The data are shown as mean [standard error (SE)]. n = 9, * P < 0.05.

Figure 4. Correlations between the compositional ratio of PC(18:0/22:6) in the gray matter versus Aβ deposition, disease duration of AD, and age at death.

(a) The compositional ratio of PC(18:0/22:6) in the gray matter plotted against Aβ deposition in non-AD and AD patients. (b) The compositional ratio of PC(18:0/22:6) in the gray matter plotted against the disease duration of AD in non-AD and AD patients. (c) The compositional ratio of PC(18:0/22:6) in the gray matter plotted against age at death in non-AD and AD patients. The left panels show analyses of all of the patients (non-AD and AD). The right panels show analyses of the AD patients only. A Pearson's test was used to determine the correlations between parameters. = non-AD (n = 9); = AD (n = 9). The colors of the markers indicate the Braak stages of each subject as shown in the top-right box.

Figure 5. A decrease in the PC(18:0/22:6) concentration correlates with PSD-95 expression but not synaptophysin expression or neuron density in AD.

(a) The NeuN-positive density in the gray matter plotted against the compositional ratio of PC(18:0/22:6) (left panel) and disease duration (right panel) in AD (n = 9). The colors of the markers indicate the Braak stages of each patient as shown in the top-right box. (b) Shown are the western blot data for PSD-95, synaptophysin, and the internal standard β-tubulin III in the temporal gray matter of non-AD and AD brains. The lanes were arranged in descending order of the compositional ratio of PC(18:0/22:6) in non-AD and AD brains, respectively. (c) The compositional ratio of PC(18:0/22:6) in the gray matter plotted against the protein levels of PSD-95 (left panel) and synaptophysin (right panel) in AD brains (n = 9). (d) Disease duration plotted against the protein levels of PSD-95 (left panel) and synaptophysin (right panel) in AD brains (n = 9). A Pearson's test was used to determine the correlations between the parameters.

Figure 6. The compositional ratio of PC(18:0/22:6) did not decrease in the brain of an Amyloid precursor protein-transgenic (APP-tg) mouse.

(a) Shown are the KB-stained sections and mass spectrometry (MS) images of the PC species in serial coronal sections of APP-tg (J20) and wild-type (WT) mice. The MS images are shown with 50 μm spatial resolution. The scale bars show 1 mm. (b) The graphs show the % change in the PC composition ratio between the APP-tg and WT mice in the gray matter. The data are shown as mean (SE). n = 3.

Figure 7. Hypothetical cascade in AD progression.

A PC(18:0/22:6) reduction parallels postsynaptic disruption when plotted against disease duration, but it does not parallel Aβ deposition, neuronal loss, and presynaptic disruption. It is generally accepted that Aβ deposition becomes abnormal early and before the appearance of clinical symptom, and the abnormal brain morphology resulting from neuronal loss happens late relative to neuronal dysfunction. Because the loss of synaptophysin was not correlated with the disease durations of the patients in this study, presynaptic disruption may occur earlier than postsynaptic disruption. The solid lines show the results of this study, and the broken lines show our speculation.