β-AMYLOID AND SURFACTANT PROTEIN D: POTENTIAL INNATE IMMUNE PARTNERS IN THE BRAIN

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Poster Presentations: P4

recent findings proposing cell surface heparan sulfate proteoglycans (HSPGs) as a unifying uptake mechanism for Ab, tau and a-synuclein, we aimed to investigate whether ApoE interferes with cellular a-synuclein uptake. Methods: Wild-type and heparan sulfate (HS)-deficient CHO cells were exposed to various concentrations of hilyte-488 labeled alphasynuclein in the presence or absence of heparin and recombinant or astrocyte-secreted ApoE in vitro. To investigate whether Ab competes with a-synuclein for cellular uptake, we further co-treated cells with a-synuclein and increasing concentrations of Ab1-42. Cells were harvested using trypsin and a-synuclein uptake was monitored using FACS. Results: a-synuclein uptake was reduced approximately 10-fold in HS-deficient cells compared to wild-type control cells. Heparin also efficiently reduced a-synuclein uptake in both wild-type and HS-deficient cells. Recombinant ApoE2 and ApoE4, but not ApoE3, reduced a-synuclein uptake in both cell lines; however, astrocyte-secreted ApoE reduced a-synuclein uptake only in wild-type CHO cells. Ab did not compete with a-synuclein for cellular uptake at a-synuclein/Ab molecular ratios 1:0.5, 1:1, 1:2 and 1:4. Conclusions: We confirm HS as an important mediator of a-synuclein uptake and propose an ApoE isoform-dependent effect on a-synuclein uptake that is further modulated by ApoE post-translational modification and/or lipidation. We also propose that the HSPG-subclasses mediating Ab and a-synuclein uptake differ as when combined they did not compete for cellular uptake. Our results pave way for future studies aimed at dissecting the cellular mechanisms linking ApoE to not only amyloid pathology but also synucleinopathy.

P4-025

DOES LEUCINE-RICH REPEAT KINASE 2 MEDIATE ALPHA-SYNUCLEIN SEEDING VIA ITS ROLE IN ENDOCYTOSIS?

Lore Delbroek1, Kristof Van Kolen2, Amaia Arranz3, Guy Daneels4, Wim Mandemakers3, Sara Calafate4, Diederik Moechars4, 1Janssen Pharmaceuticals, Beerse, Belgium; 2Janssen PRD, Beerse, Belgium; 3KU Leuven, Leuven, Belgium; 4Janssen Pharmaceuticals, Beerse, Belgium. Contact e-mail: [email protected] Background: The leucine-rich repeat kinase 2 (LRRK2) gene encodes a w250 kDa protein which includes several functional domains, including a kinase domain. At present the physiological function of LRRK2 and its pathogenic mechanism in Parkinson’s disease are poorly understood. Our group recently identified a role for LRRK2 in the endocytosis of synaptic vesicles (Matta et al., Neuron, 2012). The aim of the current study is to investigate whether LRRK2 also mediates endocytosis in mammals. Further, we investigate whether this plays a role in the pathogenic mechanism of LRRK2 in Parkinson’s disease and more specifically could be involved in the seeding and spreading of alpha-synuclein. Methods: Electron microscopy and synaptopHluorin endocytosis assays were used to assess the affect of LRRK2 knockout on endocytosis. To investigate LRRK2-dependent endophilin A1 phosphorylation, phosphoimaging and immunoprecipitation experiments were performed. Primary cultures of wildtype and LRRK2 knockout mice are used to study the potential effect of LRRK2 on uptake and seeding of alpha-synuclein. Uptake of seeded alpha-synuclein is monitored via western blot and specific antibodies are used to distinguish between seeded and endogenous alpha-synuclein. Results: Our data show that LRRK2 knockout causes an impairment in endocytosis and we identify two LRRK2-specific phosphorylation sites in endophilin A1. In this study we also develop an assay to monitor the uptake of alpha-synuclein in neuronal cultures. This assay is currently being used to investigate whether the uptake of alpha-synuclein in neurons is affected by knockout of LRRK2. Preliminary data are suggestive for a potential role of LRRK2-mediated endocytosis in the pathogenic processes driving Parkinson’s disease. Conclusions: We show an important role for LRRK2 in clathrin-mediated endocytosis via phosphorylation of endophilin A1. Albeit too early for firm conclusions, we will be able to contribute to the hypothesis that LRRK2-

mediated endocytosis plays a role in the pathogenesis of Parkinson’s disease with the ongoing study.

P4-026

CALORIC RESTRICTION DIET CAN IMPROVE LEARNING AND MEMORY ABILITY OF C57/BL MICE

Rong Wang, Xuanwu Hospital of Capital Medical University, Beijing, China. Contact e-mail: [email protected] Background: Dietary caloric restriction (CR) is defined as a limitation of food intake below the ad-libitum level without malnutrition. The effect of CR is well known to extend the maximum life span in various organisms, and it is the most robust intervention demonstrated to extend life span and delay the physiological deterioration associated with aging. Because many overlapping and interconnected signaling pathways take part in the process of CR, the mechanisms underlying the beneficial effects of CR is still unclear.This research is to determine the mechanism underlying improved spatial learning ability of CR diet on C57/BL mice. Methods: Male, seven-week-old C57/BL mice were randomly divided into three groups: normal control group(NC group), high-energy group and low-energy group(CR group), each group consisted of 10 mice, body mass and blood sugar were measured every two weeks, and metabolic parameters, serum total cholesterol and seruminsulin-like growth factor 1 (IGF-1) were investigated after six months. Learningand memoryability were tested by Morris Water Maze(MWM). We also determined the expression of brain insulin signaling pathway related proteins IGF-1, insulin receptor (IR), insulin receptor substrate-1 (IRS-1), Akt/PKB, phosphatidylinositol-3 kinase (PI3K) and phosphorylated cAMP-response element binding protein (pCREB) in the brain. Results: Compared to NC group, the CR group had a decrease in body weight and serum glucose, while the high-energy group showed an increasing level of body weight and serum glucose. There was a decreasing tendency in average escape latency and swimming distance of CR group. The serum cholesterol of high energy group was elevated significantly. The expression of IGF-1, IR, IRS-1, PI3K, Akt /protein kinase B (Akt/PKB) and p-CREB protein in CR group were decreased significantly, and the expression of PI3K and Akt/PKB protein in high energy group were decreased. Conclusions: Our findings demonstrate that CR diet improved hippocampus dependent spatial learning ability in C57/BL mice, possibly via regulation of the insulin-PI3K/Akt signaling pathway.

P4-027

b-AMYLOID AND SURFACTANT PROTEIN D: POTENTIAL INNATE IMMUNE PARTNERS IN THE BRAIN

Ruth Kandel1, Mitchell R. White2, Uffe Holmskov3, Grith Lykke Sorensen3, Erika Crouch4, Kevan Hartshorn2, 1HSL, Boston, Massachusetts, United States; 2Boston Medical Center, Boston, Massachusetts, United States; 3University of Southern Denmark, Odense, Denmark; 4Washington University, Saint Louis, Missouri, United States. Contact e-mail: [email protected] Background: Recent studies have shown that b-amyloid has antibacterial properties. We have also found that the peptide inhibits influenza virus. Surfactant protein D (SP-D) is an innate host lectin most highly expressed in the lung that plays important roles in defense against bacterial and viral infections. A recent study demonstrated significant expression of SP-D in the central nervous system and spinal fluid. Methods: We compared viral neutralization and viral and bacterial aggregating activity of various fragments of b-amyloid. Also we tested the ability of these fragments to modify human neutrophil phagocytosis of influenza virus and bacteria (flow cytometry). We tested binding of the SP-D binding domain

Poster Presentations: P4 to b-amyloid (ELISA) and cooperative viral neutralizing activity of SP-D and b-amyloid. Recombinantly modified versions of the SP-D binding domain were also tested for interactions with b-amyloid. Results: Several fragments of b -amyloid (i.e. peptides 1-42, 1-34, 35-42) had viral neutralizing and aggregating activity and also were able to aggregate bacteria and increase uptake of influenza virus and bacteria by human neutrophils. The carbohydrate binding domain of SP-D bound to b -amyloid (1-42) in a dose dependent manner. Some modified versions of the SP-D binding domain had increased binding to b -amyloid. Combining the SP-D binding domain with b -amyloid (1-42) resulted in additive viral neutralization. Conclusions: These findings demonstrate that several fragments of b-amyloid have antiviral activity and also can induce viral and bacterial aggregation and modify neutrophil uptake of pathogens. SP-D is known to have similar activities and we now show that it can bind to b-amyloid. SP-D and b-amyloid separately or together could be important innate immune effectors in the central nervous system. T he SP-D binding domain can be modified to increase binding to b-amyloid. This approach could possibly be exploited to aid clearance of b-amyloid.

P4-028

ZINC-INDUCED DIMERS OF CHEMICALLY MODIFIED A b- ARE POSSIBLE AGGREGATION SEEDS

Sergey A. Kozin1, Philippe Tsvetkov1, Alexandra Kulikova1, Maria Indeikina1, Yuri Mezentsev2, Andrey Istrate1, Igor Popov1, Alexis Ivanov2, Vladimir Polshakov3, Alexander Makarov1, 1Engelhardt Institute of Molecular Biology, Russian Academy of Medical Sciences, Moscow, Russia; 2Orekhovich Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia; 3Faculty of Fundamental Medicine, M. V. Lomonosov Moscow State University, Moscow, Russia. Contact e-mail: [email protected] Background: Zinc ions are necessary for Ab aggregation in vivo. Region 1-16 constitutes the metal-binding domain of human Ab, and amino acids 6-14 form the minimal zinc-binding site. Zinc-dependent Ab dimerization is mediated by a tetrapeptide stretch 11-14. Isomerization of Asp7, the most abundant aging-associated spontaneous chemical modification of Ab, makes the resulting isoAsp7-containing Ab (isoAb) much more susceptible to Zn 2+ -bound dimerization with dimers serving as aggregation seeds. Indeed, it has been shown in experiments on transgenic mice that intravenous injections of isoAb cause a sharp increase in the number (up to 9 times) of congophilic amyloid plaques in the brain compared to their intact littermates. In the present study we show the influence of Ser-8 phosphorilation and H6R mutation on the formation of potentially pathogenic zinc-induced Ab dimers. Methods: We have used synthetic peptide analogs of the metal-binding domains of corresponding Ab isoforms in order to study their zinc-induced complexes. Their thermodynamic, kinetic and structural properties were probed by isothermal titration calorimetry, SPR biosensing, mass-spectrometry and NMR spectroscopy. Results: It has been shown that the Ser-8 phosphorylation as well as H6R mutation result in higher capability of the modified metal-binding domains to form stable zinc-bound dimers. Conclusions: The obtained data show that the Ab species in which the 11-14 stretch is more accessible for intermolecular interactions due to specific chemical modifications or mutations in the region 6-10 can act as triggers for zincinduced aggregation of Ab. These results suggest that the information on three-dimensional structure of the Zn 2+ -binding interface of the dimers can be used for rational drug design, targeting Alzheimer’s disease.

P4-029

ARTIFICIAL STANDARD PROTEIN FOR MULTIMER DETECTION SYSTEM

Seung Chan Kim, Gachon University, Seongnam-si, South Korea. Contact e-mail: [email protected] Background: Protein misfolding-related diseases or proteinopathies are becoming significant in age-related diseases, such as Alzheimer’s disease,

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Parkinson’s disease, CJD, ALS, Type II diabetes, Huntington’s disease, cancer, Marfan syndrome, cystic fibrosis, Fabry disease.Several commercial kits were developed and used to detect their respective oligomer proteins in diseases which are mentioned above. Due to their prone nature of aggregation, these proteins tend to aggregate and oligomerize often, making the standardization of the measurement impossible to compare. Hence, it was difficult to quantify these aggregating proteins without expansive devices in a reasonable time frame. Methods: Amyloid beta 42 is one of those difficult proteins to construct standard proteins. To overcome this obstacle, standard Amyloid-beta 42 protein was constructed by attaching various fragments of amyloid-beta 42 protein onto carrier proteins. The carrier protein would have no immunological relation with anti-target protein antibodies. Three additional artificial standard proteins were constructed for amyloid-beta peptides, which was a biomarker for Alzheimer’s disease. The artificial standard proteins were characterized by ELISA, SDS-PAGE and western blot. Results: These artificial standard proteins were appropriately constructed and identified in the tests. Different kinds of artificial standards were exclusively captured and detected by antibodies which have specific binding sites against to amyloid-beta 42. Conclusions: These artificial standard proteins could be efficiently applied as standard reference in the Multimer Detection System, which was developed for differentiating oligomers in various proteins aggregating disease.

P4-030

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE AGGREGATES ACTS AS A SEED OF AMYLOID-BETA-40 AMYLOIDOGENESIS AND ENHANCES THE CELL DEATH BOTH IN VITRO AND IN VIVO

Hidemitsu Nakajima1, Takeya Kubo1, Masanori Itakura1, Akihiro Kaneshige1, Ayano Fukuhara2, Yasu-Taka Azuma1, Takashi Inui2, Tadayoshi Takeuchi3, 1Osaka Prefecture University, Izumisano, Japan; 2 Osaka Prefecture University, Sakai, Japan; 3Osaka Prefecture University, Izumisano, Japan. Contact e-mail: [email protected] Background: Additional to its glycolytic activity, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple functions in various subcellular components such as nucleus, mitochondria, and extracellular space and suggests a mediator of oxidative-stress-induced cell death. We previously reported that GAPDH aggregates formation induced by oxidative/nitrosative stresses might participate in the cell death both in vitro and in vivo. It has been also indicated that GAPDH aggregates are found in postmortem brains provided by patients suffering Alzheimer’s disease. Here we show that nitric oxide-induced GAPDH aggregates accelerate amyloidogenesis of amyloid-b 40 (A b 40) and promote amyloid-b 40-induced cell death both in PC12 cells and in mice. Methods: GAPDH aggregates were prepared using purified GAPDH treated with 100 mM NOR3 for 3 days, followed by sonication. Structural analysis performed using a Congo Red-birefringence, an atomic force microscopy (AFM), andthe far-UV circular dichroism spectra (FarUV CD). The cell viabilities or mitochondrial membrane potentials were measured by a Cell Titer Glo kit or Rhodamine-123 assay. The cell death in PC12 cells and inflammation of hippocampus of B6 mice injected by amyloids intracerebroventricularly were assessed by H.E.-staining, immunohistochemistry, and Western blotting. Results: When small amounts of GAPDH aggregates were co-incubated with amyloid-b 40, GAPDH aggregates specifically accelerated the amyloidogenesis of amyloid-b 40 in a concentration-dependent manner, whereas native GAPDH did not. Structural analysis used by both Congo Red-birefringence and AFM revealed that amyloid-b 40 incubated with GAPDH aggregates triggered its typical amyloidal fibrils. Also, an increase of b -sheet contents of amyloid-b 40 was confirmed by a Far-UV CD measurement. Furthermore, amyloid-b 40 with GAPDH aggregates led to a significant decrease of the mitochondrial dysfunction, thereby resulted in the cell death in PC12 cells, compared to that of amyloid-b 40 alone. Finally, we found that mice injected by