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. 2010 Dec 8;30(49):16419-28.
doi: 10.1523/JNEUROSCI.3317-10.2010.

Phospholipase d2 ablation ameliorates Alzheimer's disease-linked synaptic dysfunction and cognitive deficits

Tiago Gil Oliveira  1 , Robin B Chan, Huasong Tian, Mikael Laredo, Guanghou Shui, Agnieszka Staniszewski, Hong Zhang, Lili Wang, Tae-Wan Kim, Karen E Duff, Markus R Wenk, Ottavio Arancio, Gilbert Di Paolo
Affiliations

Phospholipase d2 ablation ameliorates Alzheimer's disease-linked synaptic dysfunction and cognitive deficits

Tiago Gil Oliveira et al. J Neurosci. .

Abstract

Growing evidence implicates aberrant lipid signaling in Alzheimer's disease (AD). While phospholipases A2 and C have been recently shown to mediate key actions of amyloid β-peptide (Aβ) through a dysregulation of arachidonic acid and phosphatidylinositol-4,5-bisphosphate metabolism, respectively, the role of phospholipase D (PLD) has so far remained elusive. PLD produces phosphatidic acid (PA), a bioactive lipid involved in multiple aspects of cell physiology, including signaling and membrane trafficking processes. Here we show that oligomeric Aβ enhances PLD activity in cultured neurons and that this stimulatory effect does not occur upon ablation of PLD2 via gene targeting. Aβ fails to suppress long-term potentiation in PLD2-deficient hippocampal slices, suggesting that PLD2 is required for the synaptotoxic action of this peptide. In vivo PLD activity, as assessed by detection of phosphatidylethanol levels using mass spectrometry (MS) following ethanol injection, is also increased in the brain of a transgenic mouse model of AD (SwAPP). Furthermore, Pld2 ablation rescues memory deficits and confers synaptic protection in SwAPP mice despite a significant Aβ load. MS-based lipid analysis of Pld2 mutant brains in the presence or absence of the SwAPP transgene unmasks striking crosstalks between different PA species. This lipid analysis shows an exquisite acyl chain specificity and plasticity in the perturbation of PA metabolism. Collectively, our results point to specific molecular species of PA as key modulators of AD pathogenesis and identify PLD2 as a novel potential target for therapeutics.

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Figures

Figure 1.
Figure 1.
PLD2 lies in the Aβ signaling pathway. A, PC12 cells were transfected with a plasmid expressing GFP-PLD2. Representative examples show internalization of GFP-PLD2 after treatments with 200 nm oAβ42. Pictures show the fluorescence of GFP-PLD2. B, oAβ42 (200 nm) was applied to PC12 cells in cultures for 5, 30, and 60 min, and relocalization of GFP-PLD2 was quantified as the PM/cytosol ratio (0 min, n = 31; 5 min, n = 29; 30 min, n = 32; 60 min, n = 30). C, Effect of 30 min treatments with ionomycin (2 μm) and EGTA (2 mm) on the localization of GFP-PLD2 in the presence or absence 200 nm oAβ42. The number of cells analyzed was as follows: vehicle (n = 37), oAβ42 (n = 33), ionomycin (n = 26), EGTA (n = 33), EGTA + oAβ42 (n = 38). D, Effect of 30 min treatments with pharmacological inhibitors of PLC (U73122, 250 nm) and PLA2 (AACOCF3, 20 μm) on the localization of GFP-PLD2 in the presence or absence of 200 nm oAβ42. The number of cells analyzed was as follows: vehicle (n = 97), oAβ42 (n = 81), U73122 (n = 41), U73122 + oAβ42 (n = 42), AACOCF3 (n = 50), AACOCF3 + oAβ42 (n = 45). For B–D, values denote means ± SEM. *p < 0.05, ***p < 0.001).
Figure 2.
Figure 2.
Ablation of PLD2 reduces basal PLD activity and abolishes the stimulatory effect of Aβ oligomers on PLD activity in cultured neurons. Primary cortical cultures were labeled with [3H]myristic acid at day 12, treatments were performed at day 15, lipids were subsequently extracted and the ratio [3H]phosphatidylbutanol counts/total counts was used as a measure of PLD activity. Four-hour treatments with vehicle or oAβ42 200 nm were performed before PLD activity measurement in Pld2+/+ (n = 19 and 12 for vehicle and oAβ42 treatment, respectively), Pld2+/− (n = 7), and Pld2−/− (n = 5 and 7, respectively). Values denote means ± SEM. ns, Nonsignificant. *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3.
Figure 3.
LTP is unaffected by oAβ42 in Pld2−/− hippocampal slices. There was no difference in LTP between Pld2+/+ slices and Pld2−/− slices in the presence of vehicle (F(1,17) = 0.01, p = 0.9473). Although Pld2+/+ slices showed a reduction of LTP following bath application of 200 nm oAβ42 (F(1,16) = 5.19, p = 0.0367, relative to vehicle), LTP was not reduced by the peptide in Pld2−/− slices (F(1,14) = 0.01, p = 0.9185, relative to vehicle). fEPSP, CA1 field-EPSP. The bar represents the time of bath application of oAβ42. The three arrows represent the θ-burst stimulation used to induce potentiation. Animals were ∼3 months old. Values denote means ± SEM (n = 8–9). Note that the vehicle traces for both Pld2 genotypes (i.e., black filled triangles and circles) are largely overlapping.
Figure 4.
Figure 4.
PLD activity is enhanced in the forebrain of aged SwAPP mice. Mice, with and without SwAPP transgene, were injected with 3 g/kg ethanol, and their forebrain lipids were extracted and subjected to LC-MS analysis. The production and accumulation of PEtOH was used as a reporter of in vivo PLD activity. Relative individual PEtOH species were measured in mutant mice compared with control mice. The nomenclature for fatty acid composition of phospholipids is denoted as total chain length/number of unsaturated bonds. Values denote mean ± SEM (n = 6). **p < 0.01, ***p < 0.001. Absolute amounts of the various molecular species of PEtOH are presented in supplemental Table 1, available at www.jneurosci.org as supplemental material.
Figure 5.
Figure 5.
PLD2 ablation improves learning and memory in SwAPP mice. A, SwAPP mice (Tg2576) were crossed with Pld2 knock-out mice and the resulting offspring [Pld2+/+/no tg (n = 14); Pld2+/−/no tg (n = 14); Pld2−/−/no tg (n = 11); Pld2+/+/SwAPP (n = 10); Pld2+/−/SwAPP (n = 12); Pld2−/−/SwAPP (n = 11)] were subjected to training for contextual fear memory, which was assessed 24 h after the foot shock, using 5- to 6-month-old animals. *p < 0.05 in Student's one-tail t test. B, Twelve-month-old mice were subjected to RAWM testing. Errors were scored in the last 3 d of testing. The n value was 8 for all the genotypes, except for Pld2−/−/SwAPP (n = 7) and Pld2+/+/SwAPP (n = 6). **p < 0.01. Values denote means ± SEM.
Figure 6.
Figure 6.
PLD2 ablation confers synaptic protection in the forebrain of SwAPP mice. After RAWM testing, forebrains from 12-month-old Pld2/SwAPP mice were processed for biochemical analysis. A, Protein levels were evaluated by Western blot analysis of PLD2, PLD1, PSD95, synaptophysin, and tubulin (representative blots are shown). B, Quantification of PSD95 levels by densitometric analysis. C, Quantification of synaptophysin levels by densitometric analysis. Values denote means ± SEM. n = 4, *p < 0.05.
Figure 7.
Figure 7.
Effect of SwAPP overexpression and Pld2 genotypes on PA levels. After RAWM testing, forebrains from 12-month-old Pld2/SwAPP mice were processed for lipid biochemical analysis. Forebrain lipids were extracted from Pld2+/+ and Pld2−/− mice with and without SwAPP transgene and subjected to LC-MS analysis. Relative amounts of PA species were measured in mutant mice compared with control mice (Pld2+/+, no SwAPP). Values denote mean ± SEM (n = 6–8). *p < 0.05, **p < 0.01, ***p < 0.001. The nomenclature for fatty acid composition of phospholipids is denoted as total chain length/number of unsaturated bonds. Absolute amounts of the various molecular species of PA are presented in supplemental Table 2, available at www.jneurosci.org as supplemental material.
Figure 8.
Figure 8.
Effect of SwAPP overexpression and Pld2 genotypes on APP processing. A–D, After RAWM testing, forebrains from 12-month-old Pld2/SwAPP mice were processed for biochemical analysis. A, Protein levels were evaluated by Western blot analysis of APP and tubulin (representative blots are shown). B, Quantification of full-length human APP levels was done by densitometric analysis. Values denote means ± SEM (n = 6). C, D, ELISA analysis of the levels of soluble Aβ40 and Aβ42 (C), and insoluble Aβ40 and Aβ42 (D). Values denote means ± SEM; Pld2+/+/SwAPP (n = 6); Pld2+/−/SwAPP (n = 8); Pld2−/−/SwAPP (n = 7).
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