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Poster Presentations P1 (AD). The cytoplasmic domain of APP is involved in a complex protein network, the specific modulation of which may have a functional significance for the development of AD. The C-terminal region of APP and in particular the motif 682YENPTY687 is a docking site for the PTB domain of Fe65, X11, JIP1 and JIP2, mDab1, Numb, ShcA and Grb2. The signaling activity underlined by these interactions is unknown. Objectives and Methods: We have previously shown that among all these interacting proteins Grb2 requires the specific tyrosine phosphorylation of tyr-682 of APP and that the amount of ShcA is significantly increased in AD brain as compared to control, thus suggesting a pathogenic correlation. To study in detail the interaction between APP and Grb2, and to analyze the signaling beneath this interaction we transfected APP/APLPs null MEF cells, and human H4 cells with vectors encoding for human APP695 wild type or mutated in each tyrosine (Y653F, Y682F, Y687A and double mutant YY682/687FA). Results: Here we show that APP695 and its C-terminal fragments (CTFs) interact with Grb2, that this interaction is dependent on the phosphorylation of tyr-682, and that the activity of MAP Kinase ERK1/2 is modulated by APP695. Evaluating the phosphorylation status of ERK1/2 in cells transfected either with APP wt or mutated in each tyrosine residue we observed that the overexpression of APP correlates with the activation of ERK1/2 and that tyr-682 mutation, which is essential for the interaction between APP and Grb2, diminishes ERK1/2 activation while tyr-653 or tyr-687 mutations do not influence ERK1/2 phosphorylation. Conclusions: In summary, our data suggest that APP may play a role in cell signaling, similarly to that previously reported for other tyrosine kinase receptors (TRKs), through a tightly regulated coupling with intracellular adaptors such as ShcA and Grb2 targeted to the phosphorylation of ERK1/2. P1-421

IMPAIRED IGF-1 RECEPTOR AND INSULIN RECEPTOR SIGNAL TRANSDUCTION IN ALZHEIMER’S DISEASE

Aileen M. Moloney1, Rebecca J. Griffin1, Suzanne Timmons1, Rivka Ravid2, Rosemary O’Connor1, Cora O’Neill1, 1BioSciences Institute, Cork, Ireland; 2The Netherlands Brain Bank, Amsterdam, The Netherlands. Contact e-mail: [email protected] Background: Accumulating evidence indicates that aberrant signalling through the insulin like growth factor-1 receptor (IGF-1R) and related insulin receptor (IR) in the CNS contributes to the development of Alzheimer’s disease (AD). These receptor systems are potential targets for AD therapies. Objective(s): Despite this, detailed analysis of the relationship between IGF-1R/IR proteins and AD pathogenesis is not available, and was the subject of this work. Results: Initial western immunoblot analysis detected significantly increased IGF-1R levels (42%, p ⬍ 0.05) and unchanged IR levels in the AD (n ⫽ 8-9) temporal cortex compared to non-disease matched control (n ⫽ 7-8) temporal cortex. Immunofluorescent microscopic analysis of paraffin embedded sections from control human temporal cortex and cerebellum detected high levels of both IGF-1R and IR within neurons compared to glial cells. Comparative examination of AD cases detected significant changes in IGF-1R selective to areas that degenerate in the disease. These included increased IGF-1R levels in glial cells, markedly increased, IGF-1R levels within and surrounding amyloid plaques, and the colocalization of IGF-1R within neurons with early neurofibrillary tangle pathology, as imaged by AT8 immunoreactivity. Together, these findings link IGF-1R function to the development of AD pathology, which was further substantiated by the detection of decreased IGF-1 (40%, p ⬍ 0.05), and IGF binding protein IGFBP2 (46%, p ⬍ 0.05) levels in the AD cases. On the other hand, the cellular distribution of IR did not noticeably differ when comparing AD and control groups. Despite the preponderance of changes to IGF-1R compared to IR in AD, our results also indicate that downstream signalling, linked to both receptor types is impaired in AD. Thus, highly significant reductions in the levels of IRS1/2, key signalling adaptor proteins for both IR and IGF-1R, and of the IRS responsive PI3-kinase subunits p85␣ and

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p110␣, were detected in AD cases compared to controls, suggesting a diminished level of responsiveness between IGF-1R/IR and the PI3kinase-Akt pathway in AD. Conclusion: Mechanistically, this may be due to hyper-activation of the serine-threonine kinase Akt, which has been previously shown to link to AD pathogenesis. P1-422

FRET ANALYSIS OF AMYLOID PRECURSOR PROTEIN, PRESENILIN 1 AND GRB2 INTERACTIONS

Mario Nizzari1, Valentina Venezia1, Paolo Bianchini1, Valentina Caorsi1, Alberto Diaspro1, Emanuela Repetto1, Stefano Thellung1, Alessandro Corsaro1, Gennaro Schettini1, Carlo Tacchetti1, Cristina Galliani1, Pia Carlo1, Tullio Florio1, Claudio Russo2, 1University of Genoa, Genoa, Italy; 2University of Molise, Campobasso, Italy. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is a neurodegenerative disorder whose causes are unknown. Autosomal dominant familial AD (FAD) is associated with mutations in either amyloid precursor protein (APP), or presenilin 1 and 2 (PS), genes. APP is a transmembrane protein detectable in different intracellular compartments and at the cell surface. Its proteolytic processing generates A␤ peptides, which are considered neurotoxic and responsible for the neurodegeneration. Presenilins are multipass membrane proteins, involved in the enzymatic cleavage of Notch, ErbB4 and APP. Their role in AD development seems related to the formation of A␤, although their physiological function, their reciprocal interaction and the molecular mechanisms leading to the neurodegeneration are largely unclear and widely debated. Both proteins are also involved in multiple interactions with different intracellular proteins, the significance of which for AD development is under investigation. Grb2 is a protein involved in cell signaling related to proliferation, apoptosis and cell differentiation recently identified as APP interactor. Its interaction with APP is modulated by the tyrosine phosphorylation of APP C-terminus and is enhanced in the brain of AD patients, suggesting a pathogenic correlation. Results: In this work we show a new interaction between APP, Grb2 and PS1, in human neuroglioma cells (H4). By coimmunoprecipitation, confocal microscopy and by fluorescence resonance energy transfer (FRET) experiments, we show that Grb2 interacts with both APP and PS1. The confocal microscopic analysis reveals extensive colocalization in different cells compartment of APP, Grb2 and PS1. FRET data suggest a close interaction between them. Conclusions: Thus, Grb2 represents the first adaptor protein known to interact with both gene products responsible for AD development, suggesting that both proteins may be part of a common signaling event. The study of APP-Presenilin-Grb2 interaction might provide a good opportunity to light up on the physio-pathological function of APP and Presenilins. P1-423

FUNCTIONAL STUDIES OF A NOVEL PROTEIN KINASE LRRK2 PROVIDE INSIGHTS INTO PATHOGENESIS OF PARKINSON’S DISEASE

Luxuan Guo, Wen Wang, Payal N. Gandhi, Amy L. Wilson-Delfosse, Shu G. Chen, Case Western Reserve University, Cleveland, OH, USA. Contact e-mail: [email protected] Background: Parkinson’s disease (PD) is a common neurodegenerative disorder characterized clinically by tremor, rigidity, bradykinesia, and postural instability. Pathological features of PD include selective loss of dopaminergic neurons and deposition of cytoplasmic protein aggregates in affected brain regions. Most cases of PD are sporadic, but genetic studies have linked mutations in several genes encoding alphasynuclein, parkin, DJ-1, PINK1 to rare form of familial PD. Recently, mutations in the leucine-rich repeat kinase 2 (LRRK2) have been shown to be a frequent cause of late-onset autosomal dominant and idiopathic PD. LRRK2 is a member of the ROCO family, with a novel Ras-like GTPase domain and a kinase domain with homology to the mitogenactivated protein kinase kinase kinase (MAPKKK). Neither a biological