A Neurotoxic Glycerophosphocholine Impacts PtdIns-4, 5-Bisphosphate and TORC2 Signaling by Altering Ceramide Biosynthesis in Yeast

<div><p>Unbiased lipidomic approaches have identified impairments in glycerophosphocholine second messenger metabolism in patients with Alzheimer's disease. Specifically, we have shown that amyloid-β42 signals the intraneuronal accumulation of PC(<i>O</i>-16:0/2:0) which is associated with neurotoxicity. Similar to neuronal cells, intracellular accumulation of PC(<i>O</i>-16:0/2:0) is also toxic to <i>Saccharomyces cerevisiae</i>, making yeast an excellent model to decipher the pathological effects of this lipid. We previously reported that phospholipase D, a phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂)-binding protein, was relocalized in response to PC(<i>O</i>-16:0/2:0), suggesting that this neurotoxic lipid may remodel lipid signaling networks. Here we show that PC(<i>O</i>-16:0/2:0) regulates the distribution of the PtdIns(4)P 5-kinase Mss4 and its product PtdIns(4,5)P₂ leading to the formation of invaginations at the plasma membrane (PM). We further demonstrate that the effects of PC(<i>O</i>-16:0/2:0) on the distribution of PM PtdIns(4,5)P₂ pools are in part mediated by changes in the biosynthesis of long chain bases (LCBs) and ceramides. A combination of genetic, biochemical and cell imaging approaches revealed that PC(<i>O</i>-16:0/2:0) is also a potent inhibitor of signaling through the Target of rampamycin complex 2 (TORC2). Together, these data provide mechanistic insight into how specific disruptions in phosphocholine second messenger metabolism associated with Alzheimer's disease may trigger larger network-wide disruptions in ceramide and phosphoinositide second messenger biosynthesis and signaling which have been previously implicated in disease progression.</p></div

PC(<i>O</i>-16:0/2:0) inhibits TORC2 signaling.

<p>(<b>A</b>) <b>Phosphorylation of the TORC2 substrate Ypk1 is reduced following treatment.</b> TORC2-dependent Ypk1 (T662) phosphorylation status was assessed in whole cell extracts from vehicle (ethanol, EtOH), PC(<i>O</i>-16:0/2:0) (20 µM) or rapamycin (Rap, 200 ng/ml) treated wild type (YPH500) and <i>spo14</i>Δ (YKB2076) cells. Immunoblots were also probed with anti-sera for total Ypk1 to ensure equal loading. (<b>B</b>) <b><i>tor2-21</i></b><b> mutants display increased sensitivity to PC(</b><b><i>O</i></b><b>-16:0/2:0).</b> Strains expressing plasmid borne wild type <i>TOR2</i> or the temperature sensitive (ts) alleles <i>tor2-21</i> or <i>tor2-30</i> in a <i>tor1</i>Δ, <i>tor2</i>Δ or a combined <i>tor1</i>Δ <i>tor2</i>Δ background were plated in 10-fold serial dilutions on YPD plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml). Plates were incubated for 2 days at a permissive (25 C) or semi-permissive temperature (33 C). (<b>C</b>) <b>Overexpression of hyperactive Ypk2 suppresses sensitivity to PC(</b><b><i>O</i></b><b>-16:0/2:0).</b> Ypk2 wild type (Ypk2), hyperactive (D239A), kinase dead (K373A) and the double mutant (D239A and K373A) were transformed into wild type (SH100) and <i>tor2-21</i> (SH121) expressing cells. Growth was assessed following 2 days at permissive (25 C) and semi-permissive temperature (33 C) on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml).</p

Relocalization of Slm1 by PC(<i>O</i>-16:0/2:0) does not mediate the inhibition of TORC2 signaling.

<p>(<b>A</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment relocalizes Slm1-GFP to foci.</b> The co localization of Slm1-GFP (YKB3035) with Lsp1-mcherry, an eisosome marker, was examined following treatment with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM) for 15 min. Numbers represent the percent of Slm1-GFP foci co-localizing with Lsp1-mcherry foci. (<b>B</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment does not affect TORC2 interactions.</b> The indicated strains were treated with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM) for 15 min. The interaction of Avo3-HA and endogenous Ypk1 with immunopurified (IP) Slm1-GFP was determined by immunoblotting with appropriate antibodies. Total levels of each protein were also examined in whole cell extracts (WCE). (<b>C</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) still reduces Ypk1 phosphorylation in the presence of myriocin.</b> Wild type cells (TB50a) were pretreated with vehicle or myriocin (5 µM, 30 min) prior to adding rapamycin (Rap, 200 ng/ml) or PC(<i>O</i>-16:0/2:0) (20 µM). The ratio of TORC2-dependent Ypk1 phosphorylation to total Ypk1 was determined for each treatment condition and normalized to control. The mean is displayed below the representative blot (n = 2).</p

PC(<i>O</i>-16:0/2:0)-induced changes in PtdIns(4,5)P₂ metabolism.

<p>(<b>A</b>) <b>Mss4 is relocalized upon PC(<i>O</i>-16:0/2:0) treatment.</b> Mss4-GFP expressing cells (YKB2955) were treated with vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) and localization examined. Percentage of cells with relocalized Mss4-GFP are indicated by the figure inset. (<b>B</b>) <b>Mss4 is required for PES formation.</b> Wild type (SEY6210) and <i>mss4-102</i> (AAY202) strains were grown at the indicated temperatures for one hour. Cells were subsequently treated with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) as previously done (20 µM, 15 min). Following treatment cells were collected into ice cold growth media and labeled with FM4-64 in ice cold growth media to visualize the PM. The percentage of cells with PES type structures for each condition are indicated by the figure inset. (<b>C</b>) <b><i>MSS4</i></b><b> and </b><b><i>STT4</i></b><b> are required for buffering against PC(<i>O</i>-16:0/2:0) toxicity.</b> The sensitivity of wild strains (SEY6210) or strains expressing a temperature sensitive alleles of either <i>STT4</i> (<i>stt4-4</i>, AAY102) or <i>MSS4</i> (<i>mss4-102</i>, AAY202) to PC(<i>O</i>-16:0/2:0) was examined by growth on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml or 5.7 µM) for 2 days at permissive (25 C) and semi-permissive (33 C) temperatures. (<b>D</b>) <b>Overexpression of phosphatidylinositol phosphatases increase sensitivity to PC(<i>O</i>-16:0/2:0).</b> The effect of phosphatidylinositol phosphatases upon PC(<i>O</i>-16:0/2:0) sensitivity was examined by spotting 10-fold serial dilutions of wild type strain (BY4741) harboring plasmid borne, GAL-inducible <i>INP51</i>, <i>INP52</i> and <i>INP54</i> on plates containing vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (3 µg/ml) with either dextrose or galactose as the carbon source.</p

PtdIns(4,5)P₂ is redistributed in response to PC(<i>O</i>-16:0/2:0).

<p>Wild type (WT) cells (YPH500) expressing (<b>A</b>) GFP-2×PH^PLCδ (PtdIns(4,5)P₂) (<b>B</b>) GFP-PH^Fapp (PtdIns(4)P) or (<b>C</b>) GFP-FYVE^EEA1 (PtdIns(3)P) were treated with either vehicle (EtOH) or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) and localization of the GFP probe quantified. The percentage of cells displaying a redistribution of the fluorescent reporter is reported in the inset of the figure.</p

PC(<i>O</i>-16:0/2:0) disrupts sphingolipid metabolism leading to changes in Mss4-GFP localization.

<p>(<b>A</b>) <b>PC(</b><b><i>O</i></b><b>-16:0/2:0) treatment disrupts sphingolipid metabolism.</b> Wild type (BY4741) cells were treated with vehicle or PC(<i>O</i>-16:0/2:0) (20 µM) for the indicated times (min). Lipids were extracted and sphingolipid levels were quantified and expressed as a log₂ fold change of PC(<i>O</i>-16:0/2:0) treated from vehicle treated control. LCB, long chain base; IPC, inositol phosphorylceramide; MIPC, mannosyl phosphorylceramide; DHS(-P), dihydrosphingosine (1-phosphate); PHS(-P), phytohydrosphingosine; LC, long chain (acyl chain is equal to or less than 22 carbons); VLC, very long chain (more than 22 carbons). (<b>B</b>) <b>Treatment with ceramide promotes PES formation and inhibits actin cytoskeleton polarization.</b> Wild type (BY4741) cells expressing GFP-2×PH^PLCδ were grown in YPD in the presence of vehicle (EtOH), PC(<i>O</i>-16:0/2:0), Cer(d18:1/2:0) or Cer(d18:0/2:0) (20 µM, 15 min) prior to imaging live or fixing and staining for acting cytoskeleton polarization as described in methods. The percentage of cells displaying a redistribution of the fluorescent reporter or proper actin polarization is reported in the inset of the respective figure. (<b>C</b>) <b>Inhibition of sphingolipid metabolism prevents the relocalization of Mss4.</b> Mss4-GFP (YKB2955) expressing cells were pretreated with vehicle or myriocin (5 µM) for 30 min and subsequently treated with vehicle or PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) as previously done. Pretreatment with myriocin inhibited PC(<i>O</i>-16:0/2:0)-dependent changes in PES formation.</p

Characterization of PM changes in PC(<i>O</i>-16:0/2:0)-treated cells.

<p><b>Large PM invaginations are present in PC(</b><b><i>O</i></b><b>-16:0/2:0)-treated cells.</b> Wild type cells (BY4742) exposed (<b>B, C, D, E and F</b>) or not (<b>A</b>) to PC(<i>O</i>-16:0/2:0) for 15 min were processed for EM as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004010#pgen.1004010-Griffith1" target="_blank">[55]</a>. Panel (<b>C</b>) is an inset of panel (<b>B</b>). Panels (<b>D</b>), (<b>E</b>) <b>and</b> (<b>F</b>) show magnifications of the large PM invaginations induced by PC(<i>O</i>-16:0/2:0), which very likely represent the PES. The asterisks indicate the peripheral ER that is associated with the PM. CW, cell wall; ER, endoplasmic reticulum; M, mitochondrion; V, vacuole. Bars in panels (<b>A</b>) and (<b>B</b>), 500 µm; bars in panels (<b>C</b>), (<b>D</b>), (<b>E</b>) <b>and</b> (<b>F</b>), 100 µm. <b>PES formation still occurs in the presence of depolymerised actin.</b> (<b>G</b>) Wild type cells (YPH500) expressing GFP-2×PH^PLCδ were treated with Latrunculin A (5 µM, 30 min) to induce depolymerization of the actin cytoskeleton prior to treatment with PC(<i>O</i>-16:0/2:0) (20 µM, 15 min) and imaged live. The percentage of cells displaying a redistribution of the fluorescent reporter is reported in the inset of the figure. (<b>H</b>) An aliquot of cells was also fixed following treatment for imaging of the actin cytoskeleton by staining with Rhodamine-conjugated phalloidin. The percentage of small budded cells displaying a polarized actin cytoskeleton is reported in the inset of the figure.</p