Poster Presentations P2
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P2-279
AFFINITY PULLDOWN OF GAMMA-SECRETASE AND ASSOCIATED PROTEINS FROM HUMAN AND RAT BRAIN
Yasuhiro Teranishi1,2, Ji-Yeun Hur2, Hedvig Welander2, Jenny Fra˚nberg2, Mikio Aoki2,3, Bengt Winblad2, Susanne Frykman2, Lars O. Tjernberg2, 1 Pharmacology Research Laboratories Research Division, Dainippon Sumitomo Pharma Co., Ltd., Suita, Japan; 2Karolinska Institutet and Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), Karolinska Institutet, Huddinge, Sweden; 3Genomic Science Laboratories, Functional Genomics Group, Dainippon Sumitomo Pharma Co., Ltd., Osaka, Japan. Contact e-mail:
[email protected] Background: The deposition of the Ab is a pathogenic event in Alzheimer’s disease and g-secretase is responsible for the final cleavage of amyloid precursor protein to generate Ab. g-Secretase is a transmembrane (TM) aspartyl protease complex which catalyzes the cleavage of type I TM proteins, including APP and Notch. Four proteins: PS, Nct, Aph-1 and Pen-2 are necessary and sufficient for an active g-secretase complex, but little is known about how g-secretase is regulated. Other g-secretase associated proteins may affect g-secretase activity, and studies in cell lines have shown that TMP21, CD147 and many other proteins associate with the g-secretase complex and regulate Ab production. Methods: To investigate whether there are gsecretase associate proteins in brain, we designed and synthesized a g-secretase-inhibitor with hydrophilic long linker and a cleavable biotin group (GCB) for effective and specific pulldown of g-secretase. As a starting material, we used a microsome fraction prepared from rat brain or human brain. The microsomal pellet was dissolved in CHAPSO, the supernatant was incubated with GCB, and streptavidin beads were used for pulldown. The captured proteins from brain samples were digested by trypsin and analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The captured proteins were identified by the MASCOT algorithm using the NCBInr data base. Results: The specificity of the pulldown was confirmed by competition using a non-biotinylated inhibitor. Elution by DTT clearly reduced nonspecific binding compared to elution by SDS sample buffer. All the known g-secretase components were identified by LC-MS/MS, as well as the previously reported g-secretase associated TMP21 and the PS associated syntaxin1 was found to be associated to g-secretase in rat brain. Finally, we prepared and analyzed membranes from human brain, and identified over 50 proteins potentially associated with g-secretase. We are currently evaluating their association to g-secretase and studying their effect on g-secretase activity. Conclusions: We suggest that the present method can be used for further studies on the composition of the g-secretase complex. P2-280
AFFINITY PURIFICATION OF GAMMASECRETASE ASSOCIATED PROTEINS IN SYNAPTIC MEMBRANES AND SYNAPTIC VESICLES.
Susanne Frykman1, Takahiro Kihara1, Natsuko Goto Yamamoto1, Yasuhiro Teranishi2, Bengt Winblad1, Lars O. Tjernberg1, 1Karolinska Institutet, Huddinge, Sweden; 2Dainippon Sumitomo Pharma, Osaka, Japan. Contact e-mail:
[email protected] Background: The g-secretase complex plays a major role in the progression of Alzheimer disease (AD) and many ongoing clinical trials to inhibit or modulate g-secretase are ongoing. In addition to amyloid b-peptide (Ab) production, however, g-secretase cleaves many other substrates, including Notch, and inhibition of g-secretase therefore often results in side effects. In addition, the clinical trials that use g-secretase modulators (which do not affect Notch) still await a positive effect on cognitive decline. Therefore, there is a need for new approaches to modulate g-secretase. Methods: g-Secretase associated proteins (GSAPs) might have an important role in regulation of g-secretase and substrate specificity and to target GSAPs could be a potential new pharmaceutical approach. Therefore, we have recently developed a new method where we use a g-secretase inhibitor with a cleavable biotin moiety (GCB) to affinity-purify the g-secretase complex and identify novel GSAPs with LC-MS/MS (Teranishi et al, 2009, JCMM, EPub ahead of print). Synaptic degeneration is an early event in AD pathology and is be-
lieved to be caused by amyloid b-peptide (Ab). To investigate which putative regulators of g-secretase activity that are present at the synapse, we used GCB to purify g-secretase and its interacting partners in synaptic membranes and synaptic vesicles. Results: Using LC-MS/MS we identified 5 novel GSAPs in synaptic membranes and 5 novel GSAPs in synaptic vesicles. The known components presenilin 1, nicastrin and Aph1b were also identified. The effect of these GSAPs on Ab production and Notch processing is presently investigated using siRNA. Conclusions: We have here idenfied a number synaptic specific GSAPs, which might help to target Ab production specifically at the synapse and thereby prevent synaptic degeneration. P2-281
ALZHEIMER’S DISEASE-ASSOCIATED UBIQUILIN-1 REGULATES PRESENILIN-1 ACCUMULATION AND AGGRESOME FORMATION
Annakaisa Haapasalo1, Jayashree Viswanathan1, Claudia Bo¨ttcher2, Riitta Miettinen1, Kaisa Kurkinen1, Christa J. Maynard2, Alice Lu3, Lars Bertram4, Hilkka Soininen1,5, Rudolph E. Tanzi3, Nico P. Dantuma2, Mikko Hiltunen1, 1University of Eastern Finland, Kuopio, Finland; 2Karolinska Institutet, Stockholm, Sweden; 3Massachusetts General Hospital/ Harvard Medical School, Charlestown, MA, USA; 4Max-Planck-Institute for Molecular Genetics, Berlin, Germany; 5Kuopio University Hospital, Kuopio, Finland. Contact e-mail:
[email protected] Background: Ubiquilin-1 is a ubiquitin-like protein genetically and functionally associated to Alzheimer’s disease (AD). It interacts with the proteasome and binds poly-ubiquitinated proteins. Ubiquilin-1 regulates proteasomal degradation of proteins including the AD-associated presenilin-1 (PS1) and causes PS1 accumulation. Ubiquilin-1 is suggested to play a role also in other neurodegenerative diseases involving abnormal protein accumulation. Here we characterized the effects of different naturally occurring ubiquilin-1 transcript variants (TV), full-length TV1 and TV3 lacking the proteasome-interaction domain, on PS1 accumulation and ubiquitin-proteasome system (UPS). Methods: TV1 or TV3 and PS1 cDNAs were transiently transfected to human embryonic kidney (HEK293) cells overexpressing alkaline phophatase-conjugated amyloid precursor protein (AP-APP) and primary embryonic cortical cells from transgenic mice overexpressing APP and PS1 with the delta-exon-9 mutation. The effects of TV1 and TV3 on PS1 accumulation and beta-amyloid production were assessed by Western blotting, fluorescence and electron microscopy (EM) and enzyme-linked immunosorbent assay. The impact of the TVs on UPS was studied in HEK293T cells overexpressing UbG76V-yellow fluorescent protein proteasomal reporter substrate. Results: Co-expression of especially TV3 with PS1 strongly induced the accumulation of high-molecular-weight PS1 and stabilized full-length PS1 levels. However, these effects were not caused by a general impairment of the UPS in TV1- or TV3-overexpressing cells. The accumulated PS1 co-localized with TV1 and TV3 in juxtanuclear aggresomes in both HEK293-AP-APP and cortical cells. Moreover, aggresome formation was significantly augmented in cells co-expressing PS1 and TV1 or TV3 as compared to control cells. EM confirmed the presence of TV1 and TV3 in the aggresomes and in the aggregates inside autophagosomes. PS1 accumulation and aggresome formation coincided with decreased production of beta-amyloid, particularly in TV3-expressing cells. Conclusions: Our results suggest that specific ubiquilin-1 TVs regulate PS1 accumulation and targeting into the aggresome-autophagosome pathway. PS1 sequestered in the aggresomes is not available for gamma-secretase complex assembly, and subsequently beta-amyloid production is decreased. Our data agree with the suggested function for ubiquilin-1 as a shuttle protein directing proteins to proteasomal degradation or to the aggresome-autophagosome pathway. Thus, ubiquilin-1 alternative splicing may be an important regulator of ubiquilin-1 function also in the context of AD pathogenesis. P2-282
FAD-PRESENILIN-2 MUTANTS: ORGANELLETARGETED RECOMBINANT PROBES REVEAL COMPLEX EFFECTS ON ENDOPLASMIC RETICULUM, GOLGI APPARATUS AND MITOCHONDRIA CALCIUM HANDLING
Enrico Zampese, Lucia Brunello, Valentina Lissandron, Maulilio J. Kipanyula, Paola Capitanio, Tullio Pozzan, Cristina Fasolato,
Poster Presentations P2 Paola Pizzo, University of Padua, Padua, Italy. Contact e-mail: enrico.
[email protected] Background: Calcium (Ca2+) dysfunction is extensively reported in Alzheimer’s Disease (AD). Presenilins’ (PSs) mutations seem to play a key role on this aspect, although their mechanism of action is still debated. FAD-PSs are mainly reported to exaggerate Ca2+ release from intracellular stores: however the vast majority of data is based on the generic estimation of the Ca2+ released into the cytosol upon cell stimulation. Endoplasmic reticulum (ER) is the main Ca2+ store of the cell and PSs’ effect on its Ca2+ handling has been obviously the first issue investigated. Cells are however endowed with many other mutually interacting organelles involved in Ca2+ homeostasis and/or regulated by Ca2+. Mitochondria, cells energy-house and critical crossroads in determining cell fate, are tightly regulated by Ca2+ released by ER or entering across plasma membrane. Moreover, Golgi apparatus (GA), a key point for proteins (including g-secretase and APP) maturation and trafficking, is also functionally influenced by its lumenal Ca2+. Our aim is thus to investigate (wt and FAD) PSs’ role on Ca2+ handling at the level of the single organelle. Methods: We employed genetically encoded Ca2+ probes targeted to different cell compartments (cytosol, ER, mitochondria, GA and nucleus) to investigate quantitatively the effect of PSs on subcellular Ca2+ dynamics at both cell population (by aequorin-based photoprobes) and single-cell (by FRET-based ‘‘cameleon’’ probes) levels, in cell lines as well as in primary neurons. By confocal microscopy we also investigated how PSs influence the subcellular interaction between organelles. Results: We here show that ER and cis-Golgi Ca2+ levels are strongly reduced by (FAD)PS2, while trans-Golgi and mitochondria are not directly affected; however, (FAD-)PS2s modulate the interaction, and thus Ca2+-shuttling, between ER and mitochondria. Conclusions: (FAD-)PS2s inhibit SERCA and thus affect Ca2+ handling by ER and cis-Golgi; trans-Golgi is not affected since its major Ca2+ pump SPCA-1 is not inhibited by (FAD-)PS2s. Finally, (FAD-)PS2s favour the mitochondria Ca2+ uptake upon ER Ca2+ release by increasing the coupling between the two organelles. Altogether these data open new insights in the role of Ca2+ alteration in FAD pathology. P2-283
CHARACTERIZING THE EFFECTS OF BETAAMYLOID ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTOR SUBTYPES FOUND IN THE HIPPOCAMPUS
Sterling Sudweeks, Malia Anderson, Andrew Romney, Gabriel Kelly, Brandon Thompson, Amanda Berbert, Brigham Young University, Provo, UT, USA. Contact e-mail:
[email protected] Background: Hippocampal interneurons express neuronal nicotinic acetylcholine receptors (nAChRs) of various subtypes. These interneurons affect hippocampal processing, and pharmacological activation and blockade of neuronal nAChRs has been shown to affect memory formation. Beta-amyloid has been previously shown to interact with certain subtypes of neuronal nAChRs. Methods: Possible neuronal nAChR subtypes were identified in individual hippocampal CA1 s. radiatum and s. oriens interneurons from acutely prepared rat brain slices using whole-cell patch-clamp electrophysiology and single-cell quantitative real-time RT-PCR. The most common subtypes identified were then reconstituted in X. laevis oocytes and characterized for sensitivity to soluble beta-amyloid using voltage-clamp electrophysiology. Results: Both alpha7 containing and some non-alpha7 containing neuronal nAChR subtypes can be blocked by acute application of soluble betaamyloid. However, some neuronal nAChR subtype are resistant to soluble beta-amyloid blockade. Conclusions: Beta-amyloid in the soluble form can act to modulate neuronal nAChR signaling in hippocampal interneurons, and is therefore likely to affect normal hippocampal processing through this mechanism, as well as others. This modulation could cause alterations in memory formation. P2-284
THE EFFECT OF ESTROGEN RECEPTOR ALPHA AND GLYCOGEN SYNTHASE KINASE-3b IN THE BRAIN OF OVARIECTOMIZED RAT.
Na Qu1, Qing Tian2, Qi Zhang3, 1Institute of Neuroscience, Liyuan Hospital of Tongji Medical College, Huazhong University of Science and Technol-
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ogy., Wuhan, China; 2Institute of Neuroscience, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; 3Institute of Neuroscience, Liyuan Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Contact e-mail:
[email protected] Background: In post-menopausal women have a significantly higher risk of developing Alzheimer’s disease (AD) than men. In this research levels of estrogen receptors (a and b) and GSK-3b were investigated in ovariectomized rat brain. Methods: We ovariectmized (OVX) SD rats to establish animal mode of low estrogen level. Expressions of estrogen receptors (a and b, ERa and ERb) and GSK-3b were detected at 0, 1, 2, 4, 8, 12, 16, 24 week after operation by Immunohistochemistry and Western blots. Results: We found that ERa gradually reduced with time. In particular 4, 8, 12 week afer ovariectomy. Meanwhile phosphorylation of ERa at Ser118 site and the total level of GSK3b was increased. There are no alteration of ERb level in the early stage of ovariectomy. Conclusions: These data indicated that in a particular time increased phosphorylation of ERa might be involved in over-expression of GSK-3b in the brain of OVX rats. ERa might be more important than ERb to effect the increasing of GSK-3b in the early stage of ovariectomy. P2-285
THE ROLE OF LRP IN MEDIATING THE SYNERGISTIC PATHOLOGICAL EFFECTS OF APOE4 AND AMYLOID-b IN VIVO
Moran Frenkel1, Lia Zepa1, Haim Belinson1, Joachim Herz2, Daniel M. Michaelson1, 1Tel-Aviv University, Tel-Aviv, Israel; 2University of Texas, Dallas, TX, USA. Contact e-mail:
[email protected] Background: Activation of the amyloid cascade in apoE4 targeted replacement mice triggers synergistically the degeneration of CA1 neurons. This effect is associated with the accumulation of apoE4 and oligomerized Ab within the affected neurons and with lysosomal activation and subsequent apoptotic neuronal death. Methods: We presently investigated immunohistochemicaly the extent to which the accumulation of Ab and apoE4 in the affected CA1 neurons is mediated via the apoE receptor LRP1. Results: This revealed that the accumulation of Ab and apoE4 in CA1 neurons of apoE4 mice following activation of the amyloid cascade by inhibition of neprilyisn, is associated with up regulation of LRP1. Corresponding experiments with apoE3 mice revealed that inhibition of neprilyisn in these mice, which does not result in either the accumulation of Ab or the degeneration of CA1 neurons, triggers a decrease in the LRP1 levels of these neurons. Additional experiments utilizing apoE deficient mice revealed that under similar conditions (ie up to five days following initiation of the inhibition of neprilyisn) the CA1 neurons of these mice and the levels of their LRP1 receptors do not change and that Ab does not accumulate in these neurons. Further experiments which focused on the apoE receptor apoEr2, revealed that, unlike LRP1, the levels of this receptor in the CA1 neurons decreased in all mice groups following activation of the amyloid cascade. Conclusions: These results show that apoE4 up regulates and apoE3 down regulates specifically the levels of LRP1 in hippocampal CA1 following activation of the amyloid cascade and suggest that this receptor mediates the accumulation of Ab and apoE4 in these neurons. P2-286
PRESERVATION OF CANNABINOID RECEPTOR 1 IN ALZHEIMER’S DISEASE
Jasinda H. Q. Lee1, Nur-Ezan Mohamed1, Esiri M. Margaret2, Mitchell K. P. Lai1, Christopher P. Chen1, 1National University of Singapore, Singapore, Singapore; 2University of Oxfird, United Kingdom, United Kingdom. Contact e-mail:
[email protected] Background: AD is the most common form of dementia. It is characterized by depositions of amyloid plaques (Aß) and neurofibrillary tangles (NFT) in the brain as well as loss of cholinergic markers. Current therapies for AD have shown limited beneficial effect. The cannabinoid system is of interest in AD as it regulates the release of various neurotransmitters. For example, activation of pre-synaptic CB1 receptors (CB1) inhibits the release of glutamate release and may be important in suppressing neurotoxicity. We hypothesize that if CB1 remains intact in AD neocortex, CB1 receptor based
