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On the mechanisms regulating synaptic vesicle release by endogenous amyloid-beta peptides
S582
ICAD - Abstract Submission P3: Tuesday, July 19, 2011 Poster Presentations P3
Background: Over expression of Amyloid Precursor Protein (APP) and the release of Amyloid ß (Aß) peptides are proposed to be the primary trigger of neurotoxic cascade leading to neurodegeneration in Alzheimer’s disease (AD). However, disturbances of the cell cycle have been also proposed to be important in pathomechanism of AD. Neurons in AD brain can re-enter the cell cycle what may lead to abnormal phosphorylation of MAP tau protein and in consequence to microtubule destabilization, neurodegeneration and apoptosis. The aim of our study was to analyze the role of CDK5 and cyclins in Aß peptides-evoked alteration of cell cycle and in molecular processes leading to degeneration and cell death. Methods: The study was carried out using PC12 cells stably transfected with human APP gene bearing double Swedish mutation. Flow cytometry was used for analysis of cell cycle. Real-time PCR and Western blotting were used for analysis of gene expression and protein phosphorylation, respectively. Results: Our results indicated that APPtransfection leads to significant alterations in the cell cycle. Flow-cytometric DNA analysis showed that APP-transfection enhanced the population of cells at G1-phase and decreased at G2-phase. We found also the significant changes in the level of cyclins A, B, D and cyclin E. However, among tested Cyclin-Dependent Kinases, only CDK5 expression was increased. To evaluate the role of CDK5 in cells over expressing APP, phosphorylation and activity of this kinase was investigated. We found the significant decrease of CDK5 phosphorylation on Tyr15 what decreased its activity and reduced phosphorylation of its substrate, Glycogen Synthase Kinase-3ß (GSK-3ß) on Ser9. This alteration of GSK-3ß phosphorylation leads to its over activation that subsequently may be responsible for hyperphosphorylation of MAP tau. Conclusions: These results indicated the sequence of molecular processes where CDK5 plays a fundamental role. The over expression of APP and enhancement of Aß production, through the alteration of CDK5, may be responsible for the activation of GSK-3ß and in consequence for the impairment of cytoskeleton function, cell degeneration and death. P3-206
APP INTRACELLULAR DOMAIN ENHANCES NEURITE OUTGROWTH THROUGH GaS COUPLING TO ADENYLATE CYCLASE SIGNALING
Carole Deyts1, Kulandaivelu Vetrivel1, Yumiko Shepherd1, Gopal Thinakaran1, Angele Parent1, 1University of Chicago, Chicago, Illinois, United States. Background: APP intracellular domain enhances neurite outgrowth through GaS PROTEIN COUPLING TO adenylate cyclase signaling. Deyts, C., Vetrivel, K.S., Shepherd, M., Thinakaran, G. and Parent, A.T.. Department of Neurobiology, University of Chicago, Chicago, Illinois. Alzheimer’s disease (AD), the most common form of dementia in the elderly, is characterized by the progressive loss of synapses and neurons in the cerebral cortex and certain subcortical regions. Accumulation of b-amyloidpeptides, generated via sequential proteolysis of amyloid precursor protein (APP), is a pathological hallmark of this neurodegenerative disorder. Despite of numerous studies conducted on APP and related b-amyloid pathology, the physiological function of APP still remains poorly understood. Methods: To investigate whether APP mediates intracellular signaling similar to many cell surface receptors, we expressed full-length APP or membrane-tethered APP intracellular domain (mAICD) in primary mouse cortical neurons and mouse N2a neuroblastoma cells. Results: We observed that accumulation of APP C-terminal fragments following inhibition of APP processing by g-secretase produces a significant enhancement of neurite outgrowth in N2a. Similar increases were observed when mAICD was expressed in N2a and in cortical neurons. It is known that neurite outgrowth is intimately linked to cAMP-dependent and PI3K-dependent downstream signaling events such as PKA, CREB and GSK3b pathways. Base on this observation, we examined more in details the signaling events that might be associated with APP-mediated neurite formation using cell signaling inhibitors (PKA: KT5720; PI3K: Wortmannin; and adenylate cyclase: MDL12,300A), and antibodies selective for phospho-
PKA substrate Ser/Thr epitopes, phospho-CREB Ser133 epitope and phospho-GSK3bSer9 epitope. We established that expression of mICD and accumulation of APPCTFs enhance activation of these pathways, which occurs upstream of the adenylate cyclase activation. Furthermore, we demonstrated that Gas-protein coupling to adenylate cyclase is a necessary step in mAICD-induced neurite outgrowth. Taken together, our results indicate that intracellular domain of APP is coupled to adenylate cyclase-dependent receptor-like signaling in neurons. Conclusions: We conclude that accumulation of APP or APP C-terminal fragments at the membrane could impact several brain functions such as neurite out growth, synaptic plasticity and memory formation, as a consequence of Gas-protein coupling to adenylate cyclase activation and subsequent activation of cAMP-dependent signaling cascades.
P3-207
ON THE MECHANISMS REGULATING SYNAPTIC VESICLE RELEASE BY ENDOGENOUS AMYLOID-BETA PEPTIDES
Hilla Fogel1, Yevgeny Berdichevsky1, Inna Slutsky1, 1Tel-Aviv University, Tel-Aviv, Israel. Background: Accumulation of amyloid beta peptides (Abeta) is central to Alzheimer’s disease (AD) pathogenesis. However, physiological functions of Abeta, a normal product of neuronal metabolism, remain largely unknown. Furthermore, the primary mechanisms by which endogenous Abeta initiates synaptic and cognitive impairments have not been identified. Our recent study demonstrates that endogenous Abeta peptides positively modulate release probability on a rapid timescale in hippocampal synapses (Abramov et al., 2009). Amyloid-ß as a positive endogenous regulator of release probability at hippocampal synapses (Nature Neuroscience, 2009). Methods: To identify the cellular and molecular mechanisms underlying Abeta-mediated increase in release probability, we combine optical imaging of vesicle recycling by FM dyes and fluorescence resonance energy transfer (FRET) spectroscopy at the level of single synapses in cultured hippocampal neurons. Results: Our results demonstrate that amyloid precursor protein (APP), in addition to being a source of Abeta, has a signaling function: it is essential for Abeta-induced enhancement of basal vesicle release. To further understand the mechanisms of this regulation, we explored the relationship between neuronal activity, APP structure and extracellular Abeta concentration at the level of presynaptic boutons. We observed FRET between CFP/YFP-tagged APP proteins, suggesting formation of APP homodimers in hippocampal presynaptic boutons. Notably, APP homodimerization depended on the level of neuronal and synaptic activity and exhibited high degree of variability among the boutons. Conclusions: Given a positive relationship between Abeta levels and neuronal activity, we are currently examining whether Abeta is the mediator transducing changes in membrane potential to APP homodimerization. Elucidating the mechanisms underlying the effect of Abeta on synaptic function may help to understand physiological Abeta signaling and to identify primary pathological events initiating synaptic dysfunction in AD.
P3-208
AGE-DEPENDENT CHANGES IN THE GENE EXPRESSION PROFILE OF MICE OVER-EXPRESSING MUTANT APP, PS-1 AND PHOSPHORYLATED TAU
Valentina Gatta1, Valerio Frazzini2, Stefano Sensi2, Liborio Stuppia1, 1 CeSi- Functional Genomics Unit, Chieti, Italy; 2CeSi Molecular Neurology Unit, Chieti, Italy. Background: Changes in gene expression are key to understand the molecular mechanisms and pathways set in motion by Alzheimer’s disease (AD). Methods: In this study, using a microarray approach, we investigated the age-dependent changes in the gene expression profile of hippocampi obtained from young and old AD transgenic and control mice. Employing
ICAD - Abstract Submission P3: Tuesday, July 19, 2011 Poster Presentations P3
Background: Over expression of Amyloid Precursor Protein (APP) and the release of Amyloid ß (Aß) peptides are proposed to be the primary trigger of neurotoxic cascade leading to neurodegeneration in Alzheimer’s disease (AD). However, disturbances of the cell cycle have been also proposed to be important in pathomechanism of AD. Neurons in AD brain can re-enter the cell cycle what may lead to abnormal phosphorylation of MAP tau protein and in consequence to microtubule destabilization, neurodegeneration and apoptosis. The aim of our study was to analyze the role of CDK5 and cyclins in Aß peptides-evoked alteration of cell cycle and in molecular processes leading to degeneration and cell death. Methods: The study was carried out using PC12 cells stably transfected with human APP gene bearing double Swedish mutation. Flow cytometry was used for analysis of cell cycle. Real-time PCR and Western blotting were used for analysis of gene expression and protein phosphorylation, respectively. Results: Our results indicated that APPtransfection leads to significant alterations in the cell cycle. Flow-cytometric DNA analysis showed that APP-transfection enhanced the population of cells at G1-phase and decreased at G2-phase. We found also the significant changes in the level of cyclins A, B, D and cyclin E. However, among tested Cyclin-Dependent Kinases, only CDK5 expression was increased. To evaluate the role of CDK5 in cells over expressing APP, phosphorylation and activity of this kinase was investigated. We found the significant decrease of CDK5 phosphorylation on Tyr15 what decreased its activity and reduced phosphorylation of its substrate, Glycogen Synthase Kinase-3ß (GSK-3ß) on Ser9. This alteration of GSK-3ß phosphorylation leads to its over activation that subsequently may be responsible for hyperphosphorylation of MAP tau. Conclusions: These results indicated the sequence of molecular processes where CDK5 plays a fundamental role. The over expression of APP and enhancement of Aß production, through the alteration of CDK5, may be responsible for the activation of GSK-3ß and in consequence for the impairment of cytoskeleton function, cell degeneration and death. P3-206
APP INTRACELLULAR DOMAIN ENHANCES NEURITE OUTGROWTH THROUGH GaS COUPLING TO ADENYLATE CYCLASE SIGNALING
Carole Deyts1, Kulandaivelu Vetrivel1, Yumiko Shepherd1, Gopal Thinakaran1, Angele Parent1, 1University of Chicago, Chicago, Illinois, United States. Background: APP intracellular domain enhances neurite outgrowth through GaS PROTEIN COUPLING TO adenylate cyclase signaling. Deyts, C., Vetrivel, K.S., Shepherd, M., Thinakaran, G. and Parent, A.T.. Department of Neurobiology, University of Chicago, Chicago, Illinois. Alzheimer’s disease (AD), the most common form of dementia in the elderly, is characterized by the progressive loss of synapses and neurons in the cerebral cortex and certain subcortical regions. Accumulation of b-amyloidpeptides, generated via sequential proteolysis of amyloid precursor protein (APP), is a pathological hallmark of this neurodegenerative disorder. Despite of numerous studies conducted on APP and related b-amyloid pathology, the physiological function of APP still remains poorly understood. Methods: To investigate whether APP mediates intracellular signaling similar to many cell surface receptors, we expressed full-length APP or membrane-tethered APP intracellular domain (mAICD) in primary mouse cortical neurons and mouse N2a neuroblastoma cells. Results: We observed that accumulation of APP C-terminal fragments following inhibition of APP processing by g-secretase produces a significant enhancement of neurite outgrowth in N2a. Similar increases were observed when mAICD was expressed in N2a and in cortical neurons. It is known that neurite outgrowth is intimately linked to cAMP-dependent and PI3K-dependent downstream signaling events such as PKA, CREB and GSK3b pathways. Base on this observation, we examined more in details the signaling events that might be associated with APP-mediated neurite formation using cell signaling inhibitors (PKA: KT5720; PI3K: Wortmannin; and adenylate cyclase: MDL12,300A), and antibodies selective for phospho-
PKA substrate Ser/Thr epitopes, phospho-CREB Ser133 epitope and phospho-GSK3bSer9 epitope. We established that expression of mICD and accumulation of APPCTFs enhance activation of these pathways, which occurs upstream of the adenylate cyclase activation. Furthermore, we demonstrated that Gas-protein coupling to adenylate cyclase is a necessary step in mAICD-induced neurite outgrowth. Taken together, our results indicate that intracellular domain of APP is coupled to adenylate cyclase-dependent receptor-like signaling in neurons. Conclusions: We conclude that accumulation of APP or APP C-terminal fragments at the membrane could impact several brain functions such as neurite out growth, synaptic plasticity and memory formation, as a consequence of Gas-protein coupling to adenylate cyclase activation and subsequent activation of cAMP-dependent signaling cascades.
P3-207
ON THE MECHANISMS REGULATING SYNAPTIC VESICLE RELEASE BY ENDOGENOUS AMYLOID-BETA PEPTIDES
Hilla Fogel1, Yevgeny Berdichevsky1, Inna Slutsky1, 1Tel-Aviv University, Tel-Aviv, Israel. Background: Accumulation of amyloid beta peptides (Abeta) is central to Alzheimer’s disease (AD) pathogenesis. However, physiological functions of Abeta, a normal product of neuronal metabolism, remain largely unknown. Furthermore, the primary mechanisms by which endogenous Abeta initiates synaptic and cognitive impairments have not been identified. Our recent study demonstrates that endogenous Abeta peptides positively modulate release probability on a rapid timescale in hippocampal synapses (Abramov et al., 2009). Amyloid-ß as a positive endogenous regulator of release probability at hippocampal synapses (Nature Neuroscience, 2009). Methods: To identify the cellular and molecular mechanisms underlying Abeta-mediated increase in release probability, we combine optical imaging of vesicle recycling by FM dyes and fluorescence resonance energy transfer (FRET) spectroscopy at the level of single synapses in cultured hippocampal neurons. Results: Our results demonstrate that amyloid precursor protein (APP), in addition to being a source of Abeta, has a signaling function: it is essential for Abeta-induced enhancement of basal vesicle release. To further understand the mechanisms of this regulation, we explored the relationship between neuronal activity, APP structure and extracellular Abeta concentration at the level of presynaptic boutons. We observed FRET between CFP/YFP-tagged APP proteins, suggesting formation of APP homodimers in hippocampal presynaptic boutons. Notably, APP homodimerization depended on the level of neuronal and synaptic activity and exhibited high degree of variability among the boutons. Conclusions: Given a positive relationship between Abeta levels and neuronal activity, we are currently examining whether Abeta is the mediator transducing changes in membrane potential to APP homodimerization. Elucidating the mechanisms underlying the effect of Abeta on synaptic function may help to understand physiological Abeta signaling and to identify primary pathological events initiating synaptic dysfunction in AD.
P3-208
AGE-DEPENDENT CHANGES IN THE GENE EXPRESSION PROFILE OF MICE OVER-EXPRESSING MUTANT APP, PS-1 AND PHOSPHORYLATED TAU
Valentina Gatta1, Valerio Frazzini2, Stefano Sensi2, Liborio Stuppia1, 1 CeSi- Functional Genomics Unit, Chieti, Italy; 2CeSi Molecular Neurology Unit, Chieti, Italy. Background: Changes in gene expression are key to understand the molecular mechanisms and pathways set in motion by Alzheimer’s disease (AD). Methods: In this study, using a microarray approach, we investigated the age-dependent changes in the gene expression profile of hippocampi obtained from young and old AD transgenic and control mice. Employing