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Oral O3-02: Disease Mechanisms (Signal Transduction)

mitochondrial dysfunction, energy imbalance, oxidative stress, and activation of pro-death signaling. Recent studies of human postmortem brains linked the molecular and pathological lesions in AD to major impairments in: 1) insulin and insulin-like growth factor (IGF) gene expression; 2) expression and function of the insulin and IGF receptors; 3) neuronal survival signaling; and 4) acetylcholine homeostasis, and showed each of these abnormalities increases with progression of AD. The co-existence of insulin/IGF deficiency and insulin/IGF resistance suggests that AD represents a brain-specific form of diabetes, i.e. Type 3 diabetes. We generated an experimental animal model in which intracerebral Streptozotocin (icSTZ) was used to deplete brain and not pancreatic insulin/IGF, and produce neurodegeneration that is similar to human AD. The ic-STZ-injected rats did not have hyperglycemia, and pancreatic architecture and insulin immunoreactivity were similar to control, yet their brains were reduced in size and exhibited neurodegeneration with cell loss, gliosis, and increased immunoreactivity for p53, activated glycogen synthase kinase 3␤, phospho-tau, ubiquitin, and amyloid-␤. Real time quantitative RT-PCR studies demonstrated that the ic-STZ-treated brains had significantly reduced expression of genes corresponding to neurons, oligodendroglia, and choline acetyltransferase, and increased expression of genes encoding glial fibrillary acidic protein, microglia-specific proteins, acetylcholinesterase, tau, and amyloid precursor protein. These abnormalities were associated with reduced expression of genes encoding insulin, IGF-II, insulin receptor, IGF-I receptor, and insulin receptor substrate-1, and reduced ligand binding to the insulin and IGF-II receptors. Further studies showed that treatment with peroxisome-proliferator activated receptor agonists effectively prevented the ic-STZ-induced Type 3 diabetes and preserved learning and memory. These results demonstrate that many of the characteristic features of AD-type neurodegeneration can be produced experimentally by selectively impairing insulin/IGF functions together with increasing oxidative stress, and support our hypothesis that AD represents a neuro-endocrine disorder associated with brain-specific perturbations in insulin and IGF signaling mechanisms, i.e. Type 3 diabetes. The results also suggest that early treatment with insulin sensitizer agent may prevent or reduce the severity of AD. O3-02-02

EXPRESSION OF PIRS-1 (S312 AND S616) IS ELEVATED IN MCI AND AD AND CORRELATES WITH COGNITIVE IMPAIRMENT AND NEUROFIBRILLARY PATHOLOGY

Konrad Talbot1, LiYing Han1, Julie A. Schneider2, Robert S. Wilson2, David A. Bennett2, Steven E. Arnold1, 1University of Pennsylvania, Philadelphia, PA, USA; 2Rush University, Chicago, IL, USA. Contact e-mail: [email protected] Background: Insulin resistance syndromes (Type II diabetes mellitus, metabolic syndrome) increase risk of Alzheimer’s disease. Impaired insulin signaling can promote pathological events characterizing AD, including neuronal cell loss, decreased glucose metabolism, and hyperphosphorylation of tau. These events may thus stem from development of neuronal insulin resistance in AD. In peripheral tissues, insulin resistance is associated with phosphorylation of insulin receptor substrate-1 (IRS-1) at serine residues 312 and 616, which respectively impair IRS-1 binding to the insulin receptor and PI3K. Both events reduce insulin’s effects on target cells. Objective and Methods: We ran immunohistochemical tests on postmortem tissue from two AD populations with phosphospecific IRS-1 antibodies to test the hypothesis that pIRS (S312) and pIRS-1 (S616) are elevated in vulnerable neurons of AD cases. Results: In matched pairs of 29 AD and 29 control cases from the University of Pennsylvania, we found that pyramidal cell bodies were filled with pIRS-1 (S312) and (S616) in hippocampal field CA1 far more commonly in AD than in control cases, a difference not attributable to neuron loss. We confirmed these findings in a separate sample of 30 elders with no cognitive impairment (NCI), 30 with mild cognitive impairment (MCI), and 30 with mostly early AD from the Religious Orders Study (ROS, Rush University). Controlling for age, sex, and education, regression models showed (1) progressive increases in the

frequency of pIRS-1 filled neurons from NCI to MCI to AD cases and (2) an inverse correlation between that frequency and a composite global cognition score proximate to death. We further found the frequency to be highly correlated with PHFtau-immunoreactive neurofibrillary tangle counts in CA1, but not with beta amyloid deposition. Double immunofluorescence labeling of pIRS-1 and PHFtau in AD cases showed incomplete co-localization of extranuclear pIRS1 and neurofibrillary pathological lesions. Conclusion: These findings support the hypothesis that neuronal insulin resistance develops in AD due at least in part to increased serine phosphorylation of IRS-1 blocking insulin signaling. Ongoing co-localization studies in the ROS cohort will help determine a potential causative contribution of abnormal insulin signaling to the accumulation of neurofibrillary pathology in MCI and AD. O3-02-03

AMYLOID BETA UP REGULATES TOLL LIKE RECEPTOR 3: IMPLICATION IN THE PATHOGENESIS OF ALZHEIMER’S DISEASE

Nurun N. Begum, Toshihisa Tanaka, Kouzin kamino, Golam Sadik, Md. Ashik Bin Ansar, Takashi Kudo, Masatoshi Takeda, Osaka University Graduate School of Medicine, Osaka, Japan. Contact e-mail: [email protected] Introduction: Alzheimer’s disease is characterized by two hallmark lesions: senile plaques, which are predominantly composed of A␤ peptide, and neurofibrillary tangles, composed of filamentous aggregates of hyperphosphorylated tau protein. In addition, immunological and inflammatory changes including microgliosis, atrogliosis, complement activation, cytokine elevation and acute phase protein changes are also common phenomena in AD. These changes are thought to represent, at least in part, a response to the early accumulation of A␤ 1-42 in the brain. However the precise mechanism is still not known. In this study the relevance of mechanisms on A␤ mediated innate immune activation was investigated in relation to Toll like receptor 3(TLR3). TLR3 is a receptor working in innate immune system, and its expression in brain was already reported. Activation of TLR3 can mediate hyperphosphorylation of tau in SH-SY5Y human neuroblastoma cells as reported by our previous experiment (Psychiatry and Clinical Neurosciences, Article in press). Thus we further aimed to focus on TLR3 as a potential innate immune molecule that might be activated by A␤ in the pathogenesis of Alzheimer’s disease. Method and Result: SH-SY5Y cells were treated with A␤ (25-35, 35-25, 1-42) and RNA was isolated and converted to cDNA to perform RT-PCR to observe A␤ induced over expression of TLR3. Further to obtain quantitative data of time dependant and dose dependent increase of TLR3 mRNA by A␤, Taqman quantitative Real time PCR was performed. Finally A␤ induced over expression of TLR3 was confirmed at protein level by means of western blot analysis. Employing human samples, we observed significant increased amount of TLR3 expression in AD brain compared to control brain by Real time PCR and also by immunohistochemistry analysis. Double immunohistochemistry with TLR3 and A␤ was done and TLR3 positive cells were visualized in and around the amyloid plaque in AD brain. Conclusion: A␤ up regulates TLR3 in human neuroblastoma SHSY5Y cells and expression level of TLR3 is significantly higher in AD brain compared to control brain. Our study suggests that TLR3 is involved in the amyloid beta induced activation of innate immunity in the neurodegenerative process of AD. O3-02-04

〈7-NACHR IS A WNT TARGET GENE WHICH PREVENTS A␤ NEURODEGENERATION

Ginny G. Farı´as, Marcela Colombres, Evelyn Isla, Nibaldo C. Inestrosa, Centro de Regulacio´n Celular y Patologı´a “Joaquin V. Luco” (CRCP), MIFAB, Facultad de Ciencias Biolo´gicas, P. Universidad Cato´lica de Chile, Santiago, Chile. Contact e-mail: [email protected] Background: Amyloid-␤-peptide (A␤) deposits are one of the hallmark features of Alzheimer’s disease (AD). Signal transduction alterations may

Oral O3-02: Disease Mechanisms (Signal Transduction) be involved in the neuronal responses to A␤, which may include neurotransmitter systems and pathways involved in maintenance of the nervous system. In this context, we have recently found that A␤ triggers a loss of Wnt signaling. The canonical Wnt signaling is essential for neuronal development and the maintenance of the developing nervous system. On the other hand the nicotinic acetylcholine receptors (nAChR) contribute significantly to hippocampal function and in AD, the expression of ␣7-nAChR is specifically affected. Objectives: To investigate whether the ␣7-nAChR is a Wnt target gene and to evaluate whether the ␣7-nAChR activation prevents A␤ neurodegeneration through the Wnt pathway. Methods: We evaluated by in silico analysis the promoter regions of ␣7-nAChR genes in human, rat and mouse. The ␣7 nAChR levels were studied in hippocampal neurons culture exposed to Wnt-7a or control condition. mRNA and protein was determined by RT-PCR and Western blot, and the localization of ␣7-nAChR was evaluated by immunofluorescence. On the other hand, hippocampal neurons were exposed to A␤ in the presence or absence of nicotine, a nAChR agonist plus or minus ␣-Bungarotoxin, a nAChR antagonist and viability assays and neuronal morphology were evaluated. The effect of the ␣7-nAChR activation in the Wnt pathway was evaluated by Western blot of ␤-catenin, the main effector of such pathway. Results: We report here that ␣7-nAChR is a Wnt target gene; results show the presence of Tcf/LEF binding sites. Moreover, the activation of the Wnt pathway on rat hippocampal neurons leads to increase in the expression of ␣7-nAChR with a concomitant protection from A␤ neurotoxic effects. In addition, activation of ␣7 nAChR with nicotine provides protection against A␤ through the Wnt pathway, since ␣7-nAChR activation prevents the ␤-catenin degradation induced by A␤. Conclusions: We conclude that the modulation by Wnt signaling may be essential for ␣7-nAChR expression and that the cross-talk between the ␣7-nAChR and the Wnt components underlie the neuroprotection observed on hippocampal neurons exposed to A␤. Supported by FONDAP N° 13980001 and MIFAB. O3-02-05

P21-ACTIVATED KINASE PATHWAY DEFECTS AND SYNAPTIC FUNCTION IN ALZHEIMER’S DISEASE

Greg M. Cole, Qiu-Lan Ma, Oliver J. Ubeda, Marni E. Harris-White, Fusheng Yang, Giselle P. Lim, Sally A. Frautschy, UCLA-VA GRECC, Los Angeles, CA, USA. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterized by cognitive deficits. These may stem from a selective attack of soluble beta amyloid peptide aggregates (A␤) on a p21 activated kinase (PAK) pathway that plays a critical role in excitatory synapse formation and function involved in learning and memory. Normal excitatory synapse formation and function requires precise synaptic PAK1 and PAK3 localization in protein complexes coupled to synaptic receptors via appropriate adaptor proteins including PIX and GIT1 to signal through LIMK1 to cofilin and the actin cytoskeleton and to the ERK⬎ CREB pathway for synaptic plasticity/ LTP. We have reported defects in PAK kinases and excitatory synapse markers, notably drebrin, in AD, transgenic mice, and in vitro model systems with elevated soluble A␤ oligomers. Protection was afforded by passive anti-A␤ antibody in vivo or in vitro or PAK over-expression. Objective(s): We investigated whether aberrant activation of PAKs is associated with translocation of both PAK and PIX to membrane receptors with another adaptor protein, Nck, which couples the PAK complex to JNK and a cascade involved in neurodegeneration. Methods: Immunoprecipitation, Western blot and Immunohistochemistry methods were used on AD tissue and model systems. Results: We found Nck, PAK and PIX translocation to the membrane in AD. Nck translocation is highly correlated with loss of soluble PAK, notably PAK3, and predicted to lead to reduced ERK⬎CREB and increased JNK signaling. In vitro, A␤ oligomers resulted in rapid translocation of active PAK and PIX followed by a loss of active PAK. This was partially protected by the anti-oligomer agent curcumin or by inhibition of either calpain or a known A␤ effector, Cdk5, which is

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chronically activated in AD. PAK association with Nck and aberrant translocation to membrane and rac1 activation can also be directly suppressed by neurotrophic or insulin signaling through the PI3-K⬎ Akt pathway which inhibits rac1 and phosphorylates PAK at its Nck binding site, dissociating PAK from Nck and ectopic membrane complexes. Conclusions: This model explains how dietary agents like curcumin and the omega 3 fatty acid DHA that promotes Akt activation may protect against synaptic signaling defects induced by A␤ in AD. O3-02-06

INVOLVEMENT OF PKR/EIF2ALPHA SIGNALLING PATHWAY IN THE NEURONAL DEATH IN A NEW MODEL OF ALZHEIMER’S DISEASE: APPSL/PS-1 KNOCK IN MICE

Guylene Page1, Agnes Rioux Bilan1, Milena Damjanac1, Stephanie Pain1, Laurence Barrier1, Roger Gil1, Thomas Bayer2, Jacques Hugon1, 1University of Poitiers, Poitiers, France; 2University of Saarland, Homburg, Germany. Contact e-mail: [email protected] Background: Neuronal loss is a major feature of the neuropathological lesions in Alzheimer’s disease (AD) associated with amyloid plaques and neurofibrillary tangles. Different activated kinases have been linked to this process such as Gsk-3␤ and cdk 5. We have shown in previous studies that the pro-apoptotic kinase PKR and its substrate eIF2␣ are present in hippocampal degenerating neurons in AD brains and that the knock out of PKR attenuates A␤ neurotoxicity in neural cell cultures. Objective(s): In the present data, we assessed the newly described APPSL/PS-1 knock in mice displaying hippocampal neurodegeneration, in order to evaluate, by western blots and immunohistochemical methods, the involvement of PKR in the cell death process. Results: At 3, 6 and 12 months of age, a marked and progressive apoptosis of hippocampal CA1 neurons was observed (4.0 ⫾ 0.9%, 23.2 ⫾ 1.5%, 35.8 ⫾ 1.6% of apoptotic nuclei, respectively). Western blots revealed the increased levels of activated PKR and the inhibition of eIF2␣ activity in the brains of these double transgenic mice. Hippocampal and cortical neurons displayed a staining of phosphorylated PKR both in nucleus and cytoplasms. Confocal microscopy showed that phospho-PKR and amyloid peptide (A␤) were sparingly co-localized in some neurons and around amyloid deposits. However, almost constantly activated PKR was co-localized with DNA strand breaks (TUNEL) in apoptotic nuclei of CA1 hippocampal and cortical neurons. Conclusions: In summary, PKR is associated with neuronal apoptosis in a transgenic model of AD and could represent a new target for future therapeutic approaches in AD. O3-02-07

EXCESSIVE ACTIVATION OF AKT SIGNALLING LINKS TO NEURONAL CELL DEATH IN ALZHEIMER’S DISEASE

Cora O’ Neill1, Aileen Moloney1, Suzanne Timmons1, Mary Kelliher1, Peter Dockery2, Rosemary O’ Connor1, Janet A. Johnston3, Rivka Ravid4, Rebecca J. Griffin1, 1Department of Biochemistry, Biosciences Institute, University College Cork, Cork, Ireland; 2 Department of Anatomy, Biosciences Institute, University College Cork, Cork, Ireland; 3Division of Psychiatry and Neuroscience. Queen’s University Belfast, Belfast, United Kingdom; 4The Netherlands Brain Bank, Meibergdreef 33, 1105 AZ, Amsterdam, The Netherlands. Contact e-mail: [email protected] The serine threonine kinase Akt is a master regulator of neuronal function controlling survival, growth, metabolism, and polarity, in response to growth factors insulin, cytokines and cell stress. Akt activation is normally transient, and negative control of Akt by the phosphatase PTEN is essential. In vitro studies and some investigations of AD animal models indicate that Akt activity is decreased in AD, and that increasing Akt activity may be a useful therapeutic strategy for the disease. However recent investigations indicate Akt activity may be increased in the AD brain. We thus performed a detailed investigation