Unravelling the Phospholipase D, Bridging Integrator 1/Amphiphysin 2 and Tau

P.1.l.301 Unravelling the phospholipase D, bridging integrator 1/amphiphysin 2 and tau

connections – potential implications for Alzheimer’s disease pathogenesis Bravo FV,1,2 Almeida CG3, Oliveira TG1,2 1Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057, Braga, Portugal; 2ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, 4710-057, Portugal;3 Laboratory of Neuronal trafficking in Aging,Chronic Diseases Research Center (CEDOC), Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa, Portugal

INTRODUCTION Alzheimer’s disease (AD) is the most common form of late-onset dementia. Its pathology is characterized by the accumulation of amyloid-beta and impaired phosphorylation of tau. Also, endocytosis and membrane trafficking are known to be affected in AD pathogenesis. Human genetic studies have identified bridging integrator 1 (Bin1)/amphiphysin 2, an endocytosis modulating protein, as a major risk factor for AD. Although the precise role of Bin1 in the pathogenesis of AD is still unclear, it was shown that its mRNA levels are increased in AD patients, and that it colocalizes and physically interacts with tau. In light of the previous reported finding that Bin1 interacts both with PLD enzymes and with tau, we hypothesized that a potential protein complex comprising PLD/Bin1/tau could play an important role in AD pathogenesis and that it could unravel new therapeutical targets. The lipid modifying phospholipase D (PLD) isoenzymes, PLD1 and PLD2, were shown to modulate the AD related signaling pathways. PLD is responsible for the generation of phosphatidic acid (PA) from phosphatidylcholine. PA is a central signaling lipid with a cone- shaped conformation, which alters membrane curvature. Consequently, the modulation of its levels can affect endocytosis and membrane trafficking and potentially alter synaptic properties with an impact in neuronal functioning. In light of this, membrane trafficking modulating enzymes are considered relevant candidates to modulate the pathological alterations associated with AD. Using cell line models, PLD1 was shown to modulate APP trafficking. Also, amyloid-beta (Aβ) was reported to increase PLD activity both in primary neuronal cultures and in an AD mouse model. Moreover, PLD2 ablation was shown to prevent behavioral deficits in AD mouse models. However the mechanisms underlying the role of these enzymes in AD pathology are poorly understood. Several studies indicate that Aβ pathological signaling has been connected with tau pathology and tau ablated mice were protected from Aβ deleterious effects. Since PLD2 and tau genetic ablation were both shown to be protective in AD mouse models, independently of amyloid precursor protein (APP) processing, it is thus plausible that the protective cellular mechanism could be at least partially shared.

PLD Phosphatidylcholine ? Plasma membrane Figure 1 – Phospholipase D (PLD) is responsible for the hydrolysisPhosphatidic of phosphatidylcholine acid BIN 1 TAU to phosphatidic acid. Few evidence (PA) TAU PLD Chapuis et al.Molecular psyshiatry.2013 BIN 1 Figure 2 – Mammalian PLD1 and PLD2 display a similar structure. Both PLD1 and PLD2 exhibit two catalytic HKD motifs, a phox consensus (PX) and a pleckstrin homology domain (PH), and a PI(4,5)P2 (PIP2) binding site. The main difference in their structure is the presence of a loop region in PLD1. Source: Oliveira and Di Paolo, 2010.

METHODS RESULTS

Figure 3- PLD1/PLD2 overexpression reduces endogenous TAU and BIN1 levels in N2a cells.N2a cells were transiently transfected with PLD1 and PLD2 GFP for 48h. Western Blot analysis of cell lysates from N2a cells overexpressing PLD1 and PLD2 GFP with antibodies against GFP, BIN 1, TAU 5 and GAPDH. Quantification of band intensities of TAU 5 and BIN 1 of the western blot normalized by GAPDH. Results are presented as mean and bars as standard error of the mean.* p ≤ 0,05; ** p ≤ 0,01.(n=3)

Figure 4 - PLD1/PLD2 overexpression reduces human TAU 352 and TAU 441 in N2a cells. (A) N2a cells were transiently co-transfected with PLD1/PLD2 GFP and FLAG TAU 352 for 48h.(B) N2a cells were transiently co-transfected with PLD1/PLD2 GFP and FLAG TAU 441 for 48h.Western Blot analysis of cell lysates from N2a cells overexpressing PLD1 and PLD2 GFP with antibodies against GFP, FLAG and GAPDH. (B) Quantification of band intensities of TAU 352 and TAU 441 of the western blot normalized by GAPDH. Results are presented as mean and bars as standard error of the mean.* p ≤ 0,05; ** p ≤ 0,01.(n=3)

CONCLUSIONS Figure 5 - PLD1/PLD2 genetic ablation has no effect on endogenous TAU and BIN1 in mouse hippocampus.(A) Protein levels were evaluated by Western blot analysis of Tau- 5 and Bin1, actin as a loading control (representative blots are shown).(B) Quantification of band -PLD1 and PLD2 overexpression leads to a intensities of TAU 5 and BIN1 of the western blot normalized by actin. Results are presented as mean decrease in tau endogenous levels. and bars as standard error of the mean.(n=3) -PLD1 and PLD2 overexpression leads to a decrease in levels of human TAU 352 and human TAU 441 upon cotransfection. -The ablation of PLD1 or PLD2 in Figure 6 – Subcellular distribution of PLD1/PLD2 GFP,hTAU 352 and hTAU 441 in N2a hippocampus mice not affect the tau and cells.Representative confocal images of N2a cells trasiently transfected with PLD1 GFP,PLD2,GFP,FLAG hTAU352 and FLAG hTAU441. Scale bar, 10 μm (n=10) BIN1 endogenous levels. - PLD2 colocalizes with TAU 352 and TAU 441.

REFERENCES -Chapuis J, Hansmannel F, Gistelinck M, Mounier A, Van Cauwenberghe C, Kolen KV et al. Increased expression of Figure 7 - PLD2 co-localizes with Tau 352 and Tau 441. Representative confocal images of N2a cells trasiently co-transfected with PLD1/PLD2 GFP with FLAG hTAU352 or PLD1/PLD2 GFP with FLAG BIN1 mediates Alzheimer genetic risk by modulating tau pathology. Molecular psychiatry 2013; 18(11): 1225-1234. hTau441.(B)Quantification of co-localization by Jacop pluggin in Imagej using Pearson’s correlation coefficient (PLD1 GFP and TAU 352 0,5;PLD2 GFP and TAU 352 0,7;PLD1 GFP and TAU 441 0,5;PLD2 GFP and TAU 441 0,6). -Oliveira TG, Di Paolo G. Phospholipase D in brain function and Alzheimer's disease. Biochimica et biophysica acta Scale bar, 10 μm .Results are presented as mean and bars as standard error of the mean.* p ≤ 0,05 (n=10) 2010; 1801(8): 799-805. -Oliveira TG, Chan RB, Tian H, Laredo M, Shui G, Staniszewski A et al. Phospholipase d2 ablation ameliorates Alzheimer's disease-linked synaptic dysfunction and cognitive deficits. The Journal of neuroscience : the official journal of the Society for Neuroscience 2010; 30(49): 16419-16428. -Roberson ED, Scearce-Levie K, Palop JJ, Yan F, Cheng IH, Wu T et al. Reducing endogenous tau ameliorates amyloid beta-induced deficits in an Alzheimer's disease mouse model. Science 2007; 316(5825): 750-754. -Ittner LM, Ke YD, Delerue F, Bi M, Gladbach A, van Eersel J et al. Dendritic function oftau mediates amyloid-beta toxicity in Alzheimer's disease mouse models. Cell 2010; 142(3): 387-397. Acknowledgments: The present work was Neurosciences Research Domain funded by the Portuguese Foundation for University of Minho – Portugal Technology (FCT). www.icvs.uminho.pt There is no potential conflict of interest.