- Email: [email protected]
P301L KI AND TAU NULL MICE
Poster Presentations: P1 plaques (p ¼ 0.009). In the AD samples, statistically significant differences in the g-secretase activity were not observed with respect to disease severity (mild, moderate and severe AD according to neurofibrillary pathology). Conversely, b-secretase activity was unaltered in iNPH samples with or without Ab plaques, while it was significantly increased in relation to disease severity in the AD patients. Conclusions: These results show for the first time increased g-secretase but not b -secretase activity in the biopsy samples from the frontal cortex of iNPH patients with ADlike A b pathology. Conversely, the opposite was observed in these secretase activities in AD patients with respect to neurofibrillary pathology. Despite the resemblances in the A b pathology, iNPH and AD patients appear to have marked differences in the cellular mechanisms responsible for the production of Ab.
P1-083
NOREPINEPHRINE PROVIDES SHORT-TERM NEUROPROTECTION AGAINST BETA-AMYLOID BY REDUCING OXIDATIVE STRESS INDEPENDENT OF NRF2 ACTIVATION
Kyoung A. Jhang, Young Hae Chong, Eun Ok Lee, Ewha Womans University, Seoul, South Korea. Contact e-mail: [email protected] Background: Post-mortem studies of alzheimer’s disease (AD)brains have demonstrated that degeneration of the locus ceruleus (LC), the primary source of subcortical norepinephrine (NE), may be the first identifiable AD pathology. Evidence indicates the diverse repertoire for NE neuroprotection. However, further studies are required to obtain more insight into the molecular mechanisms underlying this action. Methods: We were interested to evaluate the effects of NE on beta- amyloid1-42 (A b 1-42)-induced neurotoxicity and determined the mechanisms through which NE exerts its actions in human SK-N-SH neurons. We used protein preparation, MTT assay, t ransient transfection and the luciferase assay, a flow cytometry and western blotting techniques to perform our study. Results: NE protected SK-N-SH cells against A b 1-42 after only a 4 h treatment. The ability of NE to reduce A b 1-42-induced neurotoxicity was independent of the adrenoceptor signaling pathway. Notably, NE downregulated A b 1-42-mediated increases in intracellular reactive oxygen species (ROS) production. However, NE did not affect A b 1-42-induced activation of the nuclear factor erythroid 2 related factor 2 (Nrf2) redox signaling pathway, known to be involved in oxidative stress. Of note, besides isoproterenol and pyrocatechol possessing a catechol moiety of NE, N-acetyl cysteine and glutathione, which are not only ROS scavengers but also thiol reducing agents,mimicked these NE effects. Consistently, keap-1 inhibitors, which activated the Nrf2 pathway, failed to decrease A b 1-42-induced ROS generation and elicited no protection against A b 1-42. Conclusions: Collectively, our findings suggest that NE could exert neuroprotective function against A b 1-42 via redox cycling and reduction of intracellular oxidative stress regardless of downstream activation of the Nrf2 pathway. Thus, NE or NE derivatives as well as thiol reducing antioxidants may have potential value as neuroprotective agents, particularly with regard to oxidative stress related to loss of the LC-NE system as observed in AD.
P1-084
LIGAND-ACTIVATED EPHA4 SIGNALING GOVERNS THE PROTEOSTASIS OF APP-BETA CTF TO CONTROL THE LEVEL OF AMYLOID-BETA
Wei-Bin Lai, Yung-Feng Liao, Bo-Jeng Wang, Academia Sinica, Taipei, Taiwan. Contact e-mail: [email protected] Background: Alzheimer’s disease is the most common dementia afflicting the elderly in the modern society. This disease is a result of the neurotoxicity elicited by abnormal amyloid-beta (Abeta) protein aggregates. Abeta is generated by the gamma-secretase-catalyzed proteolysis of amyloid precursor protein (APP) C-terminal fragment (APP-betaCTF) that is released by the beta-secretase cleavage of APP. Recent evidence suggests that gamma-secreatse’s substrate betaCTF and its metabolite APP intracellular
P333
domain (AICD) both could induce detrimental effects on cells. A recent study demonstrates that expression of EphA4, a member of the receptor tyrosine kinase family, is significantly reduced in the hippocampus of APP transgenic mice and human patients with AD. Methods: The levels of APP-betaCTF, AICD, and Abeta were examined in a stable cell line overexpressing APP-C99 in response to the ligand-dependent EphA4 activation by Western blotting. Mutants of EphA4 and Lyn, along with lentiviral vectors encoding shRNA-targeting EphA4 and Lyn, were employed to determine the mechanism underlying the EphA4-elicited accumulation. Results: We demonstrated that ligand-activated EphA4 signaling could govern the proteostasis of betaCTF, AICD, and Abeta independent of gamma-secretase activity. Consistently, the gamma-secretase cleavage of Notch was not affected by EphA4 signaling, suggesting an EphA4-elicited selective regulation of APP-betaCTF proteostasis without affecting the processing of other gamma-secretase substrates. The inhibition of EphA4 by Dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed the EphA4-induced accumulation of betaCTF and AICD. This EphA4-elicited accumulation of betaCTF and AICD was mediated by a Lyn-dependent pathway. Our data also showed that Lyn can physically interact with EphA4, suggesting that an EphA4-Lyn-dependent positive feedback could form to govern the proteostasis of betaCTF and AICD. Conclusions: Our data delineate an EphA4-Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, providing novel pharmacological targets for the development of anti-Abeta therapeutics for AD.
P1-085
AXONAL TRANSPORT IN TGCRND8/P301L KI AND TAU NULL MICE
Robert Adalbert, Stefan Milde, Claire Harwell, Michael Coleman, The Babraham Instiute, Cambridge, United Kingdom. Contact e-mail: robert. [email protected] Background: In Alzheimer’s disease (AD), many indicators point to a central role for poor axonal transport, but the potential for stimulating axonal transport to alleviate the disease remains untested. As we have enhanced axonal transport of mitochondria without adverse effect in mice by knocking in a mutant sequence of tau (Gilley et al., Neurobiol Aging. 2012 (33): 621.e1-621.e15.), we tested here whether we could boost axonal transport in TgCRND8 mice and thereby alleviate axonal swelling. W e also tested whether removing tau enhances axonal transport by studying mitochondrial transport in tau null mice. Methods: TgCRND8, TgCRND8/P301L knock in and Tau null mice were crossed to Mito-S and YFP-H mice for live imaging of mitochondrial transport and axon morphology. Mito-S mice express CFP-labelled mitochondria in a subset of neurons. Mitochondrial movements were recorded in tibial nerves. The plaques and axonal swellings were quantified on 50mm YFP positive brain slices stained with Thioflavin S. Results: Our preliminary results point towards an enhanced mitochondrial transport in P301Lki homozygotes mice as previously reported and also show for the first time an increase in mitochondrial transport in young P301Lki heterozygous mice. The presence of P301L knockin mutation, does not significantly reduce the number of plaque-associated axonal swellings or amyloid plaques in TgCRND8 brains. However, FAD-APP and P301L ki homozygote mutation combine synergistically to reduce axonal transport of mitochondria in peripheral nerves. Ongoing studies are looking at the effect of P301L ki mutation on tau pathology in TgCRND8 brains and on A €ı¢levels in these nerves.Our data also suggest that complete removal of tau does not significantly affect the axonal transport of mitochondria in vivo. Conclusions: As P301Lki heterozygous mice model FTDP-17T patients, the changes observed in mitochondrial axonal transport could be relevant to understanding the early consequences of tau dysfunction in humans. Our findings suggest that amyloid beta peptide and tau converge in axons to influence axonal transport, which may be useful to study how amyloid beta peptide and tau combine in pathogenesis. The normal function of tau
P334
Poster Presentations: P1
may not involve regulation of mitochondrial trafficking, although we cannot rule out compensatory changes in the null mice.
P1-086
ANALYSIS OF INTRA- AND EXTRACELLULAR CALCIUM IN CORTICAL AND HIPPOCAMPAL NEURON CELL CULTURES TREATED WITH LITHIUM
Marılia Palma Carvalho1, Vanessa de Jesus Rodrigues De Paula2, Orestes Vicente Forlenza3, 1Faculty of Medicine, University of S~ao Paulo, Sao Paulo, Brazil; 2Laboratory of Neuroscience - LIM 27, Department and Institute of Psychiatry, Faculty of Medicine, University of S~ao Paulo, Sao Paulo, Brazil; 3Laboratory of Neuroscience - University of S~ao Paulo, S~ao Paulo, Brazil. Contact e-mail: [email protected] Background: Lithium exerts a number of powerful effects on neuronal signaling pathways, such neuroprotection. Lithium attenuatedcalcium (Ca 2+)release after activation of both metabotropic glutamate receptors as well as muscarinic and cholinergic receptors and another metabolic pathways by kinase and Ca 2+. The Ca 2+ plays an important role in regulating a great variety of neuronal processes. Like other cells, neurons use both extracellular and intracellular sources of Ca 2+. Neurons employ two sources of Ca 2+ signal. There is scarcely a reaction within the brain that is not regulated, directly or indirectly, by Ca 2+. Phenomena ranging in time scale from the sub millisecond triggering of neurotransmitter release at pre synaptic terminals to the essentially permanent changes in gene expression that occur during various forms of brain plasticity all require the second messenger actions of Ca 2+ within neurons. Remarkably, even opposing reactions, such as the growth of neurons during development and the death of these same cells during apoptosis, can be regulated by Ca 2+. The objective is to analyze the intra and extracellular calcium in cortical and hippocampal neurons cell culture with lithium treatment. Methods: Primary cultures of cortical and hippocampal neurons were treated after 4 days in culture (DIC), and were incubated (37 C, 5% CO 2) with different concentrations of lithium chloride until to 10 days in culture. Working concentrations of lithium were 0.02mM, 0.2mM and 2mM.The effects of lithiumon intracellular calcium were determined by Western-blot, using a CaMKII primary antibody, whereas extracellular calcium was measured by electrode in medium cell culture. Results: Preliminary results show intracellular calcium was increased 14%, 10% and 2% in cortical neurons, whereas hippocampal neurons increased 17% on the first treatment and decreased on the others to 11% and 3%, both comparing to the control. The extracellular calcium acted inversely, showing a decrease of 15%, 50% and 35% in cortex and an increase of 50%, 0% and 40% in hippocampus. Conclusions: Treatment with sub therapeutic doses of lithium proved to be dynamic in respect to intra and extracellular calcium, so that their concentrations are inversely proportional.
P1-087
MISFOLDED BRI2 ECTODOMAIN ACTIVATES CRITICAL PATHOLOGICAL PATHWAYS INVOLVED IN EARLY STAGES OF ALZHEIMER’S DISEASE
Marta Del Campo Milan1, Claudia Pereira2, W. Scheper3, R. Zwart4, Jeroen Hoozemans5, Catarina Oliveira6, Charlotte Teunissen7, 1VU Medical Center, Amsterdam, Netherlands; 2CNC, University of Coimbra, Coimbra, Portugal; 3CNCR, VU, Amsterdam, Netherlands; 4VU University, Amsterdam, Netherlands; 5Vrije University Medisch Centrum, Amsterdam, Netherlands; 6University of Coimbra, Coimbra, Portugal; 7 Alzheimer Center, VU Medical Center, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is pathologically characterized by the presence of misfolded proteins such as amyloid beta (A b) in senile plaques and hyperphosphorilated tau (p-tau) in neurofibrillary tangles (NFT). BRI2 is a transmembrane protein that regulates critical proteins involved
in the initial steps of the amyloid cascade, the prevailing hypothesis about AD pathogenesis. We recently found aggregates of misfolded BRI2 ectodomain associated with Ab plaques in early stages of AD. Here we aimed to investigate the effects of misfolded recombinant BRI2 ectodomain (BRI2 76-266) on important molecular pathways involved in early stages of AD, including apoptosis, the unfolded protein response (UPR), and the phosphorylation of glycogen synthase kinase 3 b (GSK3b) and tau (p-tau). Methods: Non-differentiated SH-SY5Y cells were exposed to misfolded recombinant BRI2 76-266. Cell viability was assed by MTT assay. Apoptosis was examined by analysis of the activity of Caspases 3 and 9. Pro- and antiapoptotic proteins Bcl-2 and Bax, and the phosphorylation of GSK3 b (P-GSK3 b) were analysed by western blot. The mRNA of UPR related proteins (BiP, CHOP and Xbp-1) was analysed by qPCR and the levels of p-tau were determined using ELISA. Results: BRI2 76-266 led to a 10% cell death, increased Bax/Bcl-2 ratio and increased activity of caspases 3 and 9, indicating an activation of the apoptosis pathway. UPR mRNA markers were not modified after incubation with BRI2 76-266. Incubation with BRI2 increased P-GSK3 b levels. Conclusions: BRI2 76-266 led to a 10% cell death, increased Bax/Bcl-2 ratio and increased activity of caspases 3 and 9, indicating an activation of the apoptosis pathway. UPR mRNA markers were not modified after incubation with BRI2 76-266. Incubation with BRI2 increased P-GSK3 b levels.
P1-088
MODULATION OF BETA-AMYLOID/APP IN ENDOSOMES
Katarina Ingrid Willen, Gunnar K. Gouras, Lund University, Lund, Sweden. Contact e-mail: [email protected] Background: Alzheimer’s-linked b -amyloid (A b) is generated at synapses and accumulates particularly in endosomal vesicles near synapses with the onset of synaptic dysfunction in AD pathogenesis. Previous evidence has supported that sorting via the multivesicular body (MVB) pathway is impaired by endosomal A baccumulation in cultured AD transgenic primary neurons. Ab dependent translocation into MVBs was particularly affected, suggesting dysfunction of the ESCRT (endosomal sorting complex required for transport) pathway, a key mediator of MVB biogenesis. Our aim is to modulate different steps in endosomal trafficking in neurons to examine how these influence A baccumulation and thereby A b-induced synapse damage. We believe that a better understanding of the cellular and molecular mechanism(s) whereby accumulating Ab at synapses cause synaptic dysfunction will be important for the development of novel treatment for AD. Methods: Chemical and genetic inhibition is used to block various steps in endosomal trafficking in N2a neuroblastoma cellsor primary neurons. Components of the ESCRT pathway are modulated. Alterations in ESCRT components are also analysed in AD transgenic compared to wild type neurons and in primary neurons treated with extracellular A b. Results: The dominant negative form of the ESCRT component VPS4A shows significantly increased intracellular accumulation and decreased secretion of Ab in Swe N2a cells. Lysosomal inhibitor Bafilomycin A1 also markedly increases intracellular Ab while reducing secreted Ab in Swe N2a. Conclusions: We previously reported progressive intraneuronal Ab accumulation and impaired Ab secretion in AD-neurons with time in culture. This is mimicked by inhibition of late ESCRT machinery component VPS4A and consistent with indications of MVB dysfunction in AD. A next step will be to analyse synaptic composition in primary neurons after ESCRT modification.
P1-089
DECREASING PICALM EXPRESSION ALTERS THE AMYLOIDOGENIC PROCESSING OF AMYLOID PRECURSOR PROTEIN
Rhian Thomas, Amy Gerrish, Alex Henson, Lesley Jones, Julie Williams, Emma Kidd, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected]
P1-083
NOREPINEPHRINE PROVIDES SHORT-TERM NEUROPROTECTION AGAINST BETA-AMYLOID BY REDUCING OXIDATIVE STRESS INDEPENDENT OF NRF2 ACTIVATION
Kyoung A. Jhang, Young Hae Chong, Eun Ok Lee, Ewha Womans University, Seoul, South Korea. Contact e-mail: [email protected] Background: Post-mortem studies of alzheimer’s disease (AD)brains have demonstrated that degeneration of the locus ceruleus (LC), the primary source of subcortical norepinephrine (NE), may be the first identifiable AD pathology. Evidence indicates the diverse repertoire for NE neuroprotection. However, further studies are required to obtain more insight into the molecular mechanisms underlying this action. Methods: We were interested to evaluate the effects of NE on beta- amyloid1-42 (A b 1-42)-induced neurotoxicity and determined the mechanisms through which NE exerts its actions in human SK-N-SH neurons. We used protein preparation, MTT assay, t ransient transfection and the luciferase assay, a flow cytometry and western blotting techniques to perform our study. Results: NE protected SK-N-SH cells against A b 1-42 after only a 4 h treatment. The ability of NE to reduce A b 1-42-induced neurotoxicity was independent of the adrenoceptor signaling pathway. Notably, NE downregulated A b 1-42-mediated increases in intracellular reactive oxygen species (ROS) production. However, NE did not affect A b 1-42-induced activation of the nuclear factor erythroid 2 related factor 2 (Nrf2) redox signaling pathway, known to be involved in oxidative stress. Of note, besides isoproterenol and pyrocatechol possessing a catechol moiety of NE, N-acetyl cysteine and glutathione, which are not only ROS scavengers but also thiol reducing agents,mimicked these NE effects. Consistently, keap-1 inhibitors, which activated the Nrf2 pathway, failed to decrease A b 1-42-induced ROS generation and elicited no protection against A b 1-42. Conclusions: Collectively, our findings suggest that NE could exert neuroprotective function against A b 1-42 via redox cycling and reduction of intracellular oxidative stress regardless of downstream activation of the Nrf2 pathway. Thus, NE or NE derivatives as well as thiol reducing antioxidants may have potential value as neuroprotective agents, particularly with regard to oxidative stress related to loss of the LC-NE system as observed in AD.
P1-084
LIGAND-ACTIVATED EPHA4 SIGNALING GOVERNS THE PROTEOSTASIS OF APP-BETA CTF TO CONTROL THE LEVEL OF AMYLOID-BETA
Wei-Bin Lai, Yung-Feng Liao, Bo-Jeng Wang, Academia Sinica, Taipei, Taiwan. Contact e-mail: [email protected] Background: Alzheimer’s disease is the most common dementia afflicting the elderly in the modern society. This disease is a result of the neurotoxicity elicited by abnormal amyloid-beta (Abeta) protein aggregates. Abeta is generated by the gamma-secretase-catalyzed proteolysis of amyloid precursor protein (APP) C-terminal fragment (APP-betaCTF) that is released by the beta-secretase cleavage of APP. Recent evidence suggests that gamma-secreatse’s substrate betaCTF and its metabolite APP intracellular
P333
domain (AICD) both could induce detrimental effects on cells. A recent study demonstrates that expression of EphA4, a member of the receptor tyrosine kinase family, is significantly reduced in the hippocampus of APP transgenic mice and human patients with AD. Methods: The levels of APP-betaCTF, AICD, and Abeta were examined in a stable cell line overexpressing APP-C99 in response to the ligand-dependent EphA4 activation by Western blotting. Mutants of EphA4 and Lyn, along with lentiviral vectors encoding shRNA-targeting EphA4 and Lyn, were employed to determine the mechanism underlying the EphA4-elicited accumulation. Results: We demonstrated that ligand-activated EphA4 signaling could govern the proteostasis of betaCTF, AICD, and Abeta independent of gamma-secretase activity. Consistently, the gamma-secretase cleavage of Notch was not affected by EphA4 signaling, suggesting an EphA4-elicited selective regulation of APP-betaCTF proteostasis without affecting the processing of other gamma-secretase substrates. The inhibition of EphA4 by Dasatinib, a receptor tyrosine kinase inhibitor, effectively suppressed the EphA4-induced accumulation of betaCTF and AICD. This EphA4-elicited accumulation of betaCTF and AICD was mediated by a Lyn-dependent pathway. Our data also showed that Lyn can physically interact with EphA4, suggesting that an EphA4-Lyn-dependent positive feedback could form to govern the proteostasis of betaCTF and AICD. Conclusions: Our data delineate an EphA4-Lyn pathway that is essential for the metabolism of APP and its proteolytic derivatives, providing novel pharmacological targets for the development of anti-Abeta therapeutics for AD.
P1-085
AXONAL TRANSPORT IN TGCRND8/P301L KI AND TAU NULL MICE
Robert Adalbert, Stefan Milde, Claire Harwell, Michael Coleman, The Babraham Instiute, Cambridge, United Kingdom. Contact e-mail: robert. [email protected] Background: In Alzheimer’s disease (AD), many indicators point to a central role for poor axonal transport, but the potential for stimulating axonal transport to alleviate the disease remains untested. As we have enhanced axonal transport of mitochondria without adverse effect in mice by knocking in a mutant sequence of tau (Gilley et al., Neurobiol Aging. 2012 (33): 621.e1-621.e15.), we tested here whether we could boost axonal transport in TgCRND8 mice and thereby alleviate axonal swelling. W e also tested whether removing tau enhances axonal transport by studying mitochondrial transport in tau null mice. Methods: TgCRND8, TgCRND8/P301L knock in and Tau null mice were crossed to Mito-S and YFP-H mice for live imaging of mitochondrial transport and axon morphology. Mito-S mice express CFP-labelled mitochondria in a subset of neurons. Mitochondrial movements were recorded in tibial nerves. The plaques and axonal swellings were quantified on 50mm YFP positive brain slices stained with Thioflavin S. Results: Our preliminary results point towards an enhanced mitochondrial transport in P301Lki homozygotes mice as previously reported and also show for the first time an increase in mitochondrial transport in young P301Lki heterozygous mice. The presence of P301L knockin mutation, does not significantly reduce the number of plaque-associated axonal swellings or amyloid plaques in TgCRND8 brains. However, FAD-APP and P301L ki homozygote mutation combine synergistically to reduce axonal transport of mitochondria in peripheral nerves. Ongoing studies are looking at the effect of P301L ki mutation on tau pathology in TgCRND8 brains and on A €ı¢levels in these nerves.Our data also suggest that complete removal of tau does not significantly affect the axonal transport of mitochondria in vivo. Conclusions: As P301Lki heterozygous mice model FTDP-17T patients, the changes observed in mitochondrial axonal transport could be relevant to understanding the early consequences of tau dysfunction in humans. Our findings suggest that amyloid beta peptide and tau converge in axons to influence axonal transport, which may be useful to study how amyloid beta peptide and tau combine in pathogenesis. The normal function of tau
P334
Poster Presentations: P1
may not involve regulation of mitochondrial trafficking, although we cannot rule out compensatory changes in the null mice.
P1-086
ANALYSIS OF INTRA- AND EXTRACELLULAR CALCIUM IN CORTICAL AND HIPPOCAMPAL NEURON CELL CULTURES TREATED WITH LITHIUM
Marılia Palma Carvalho1, Vanessa de Jesus Rodrigues De Paula2, Orestes Vicente Forlenza3, 1Faculty of Medicine, University of S~ao Paulo, Sao Paulo, Brazil; 2Laboratory of Neuroscience - LIM 27, Department and Institute of Psychiatry, Faculty of Medicine, University of S~ao Paulo, Sao Paulo, Brazil; 3Laboratory of Neuroscience - University of S~ao Paulo, S~ao Paulo, Brazil. Contact e-mail: [email protected] Background: Lithium exerts a number of powerful effects on neuronal signaling pathways, such neuroprotection. Lithium attenuatedcalcium (Ca 2+)release after activation of both metabotropic glutamate receptors as well as muscarinic and cholinergic receptors and another metabolic pathways by kinase and Ca 2+. The Ca 2+ plays an important role in regulating a great variety of neuronal processes. Like other cells, neurons use both extracellular and intracellular sources of Ca 2+. Neurons employ two sources of Ca 2+ signal. There is scarcely a reaction within the brain that is not regulated, directly or indirectly, by Ca 2+. Phenomena ranging in time scale from the sub millisecond triggering of neurotransmitter release at pre synaptic terminals to the essentially permanent changes in gene expression that occur during various forms of brain plasticity all require the second messenger actions of Ca 2+ within neurons. Remarkably, even opposing reactions, such as the growth of neurons during development and the death of these same cells during apoptosis, can be regulated by Ca 2+. The objective is to analyze the intra and extracellular calcium in cortical and hippocampal neurons cell culture with lithium treatment. Methods: Primary cultures of cortical and hippocampal neurons were treated after 4 days in culture (DIC), and were incubated (37 C, 5% CO 2) with different concentrations of lithium chloride until to 10 days in culture. Working concentrations of lithium were 0.02mM, 0.2mM and 2mM.The effects of lithiumon intracellular calcium were determined by Western-blot, using a CaMKII primary antibody, whereas extracellular calcium was measured by electrode in medium cell culture. Results: Preliminary results show intracellular calcium was increased 14%, 10% and 2% in cortical neurons, whereas hippocampal neurons increased 17% on the first treatment and decreased on the others to 11% and 3%, both comparing to the control. The extracellular calcium acted inversely, showing a decrease of 15%, 50% and 35% in cortex and an increase of 50%, 0% and 40% in hippocampus. Conclusions: Treatment with sub therapeutic doses of lithium proved to be dynamic in respect to intra and extracellular calcium, so that their concentrations are inversely proportional.
P1-087
MISFOLDED BRI2 ECTODOMAIN ACTIVATES CRITICAL PATHOLOGICAL PATHWAYS INVOLVED IN EARLY STAGES OF ALZHEIMER’S DISEASE
Marta Del Campo Milan1, Claudia Pereira2, W. Scheper3, R. Zwart4, Jeroen Hoozemans5, Catarina Oliveira6, Charlotte Teunissen7, 1VU Medical Center, Amsterdam, Netherlands; 2CNC, University of Coimbra, Coimbra, Portugal; 3CNCR, VU, Amsterdam, Netherlands; 4VU University, Amsterdam, Netherlands; 5Vrije University Medisch Centrum, Amsterdam, Netherlands; 6University of Coimbra, Coimbra, Portugal; 7 Alzheimer Center, VU Medical Center, Amsterdam, Netherlands. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is pathologically characterized by the presence of misfolded proteins such as amyloid beta (A b) in senile plaques and hyperphosphorilated tau (p-tau) in neurofibrillary tangles (NFT). BRI2 is a transmembrane protein that regulates critical proteins involved
in the initial steps of the amyloid cascade, the prevailing hypothesis about AD pathogenesis. We recently found aggregates of misfolded BRI2 ectodomain associated with Ab plaques in early stages of AD. Here we aimed to investigate the effects of misfolded recombinant BRI2 ectodomain (BRI2 76-266) on important molecular pathways involved in early stages of AD, including apoptosis, the unfolded protein response (UPR), and the phosphorylation of glycogen synthase kinase 3 b (GSK3b) and tau (p-tau). Methods: Non-differentiated SH-SY5Y cells were exposed to misfolded recombinant BRI2 76-266. Cell viability was assed by MTT assay. Apoptosis was examined by analysis of the activity of Caspases 3 and 9. Pro- and antiapoptotic proteins Bcl-2 and Bax, and the phosphorylation of GSK3 b (P-GSK3 b) were analysed by western blot. The mRNA of UPR related proteins (BiP, CHOP and Xbp-1) was analysed by qPCR and the levels of p-tau were determined using ELISA. Results: BRI2 76-266 led to a 10% cell death, increased Bax/Bcl-2 ratio and increased activity of caspases 3 and 9, indicating an activation of the apoptosis pathway. UPR mRNA markers were not modified after incubation with BRI2 76-266. Incubation with BRI2 increased P-GSK3 b levels. Conclusions: BRI2 76-266 led to a 10% cell death, increased Bax/Bcl-2 ratio and increased activity of caspases 3 and 9, indicating an activation of the apoptosis pathway. UPR mRNA markers were not modified after incubation with BRI2 76-266. Incubation with BRI2 increased P-GSK3 b levels.
P1-088
MODULATION OF BETA-AMYLOID/APP IN ENDOSOMES
Katarina Ingrid Willen, Gunnar K. Gouras, Lund University, Lund, Sweden. Contact e-mail: [email protected] Background: Alzheimer’s-linked b -amyloid (A b) is generated at synapses and accumulates particularly in endosomal vesicles near synapses with the onset of synaptic dysfunction in AD pathogenesis. Previous evidence has supported that sorting via the multivesicular body (MVB) pathway is impaired by endosomal A baccumulation in cultured AD transgenic primary neurons. Ab dependent translocation into MVBs was particularly affected, suggesting dysfunction of the ESCRT (endosomal sorting complex required for transport) pathway, a key mediator of MVB biogenesis. Our aim is to modulate different steps in endosomal trafficking in neurons to examine how these influence A baccumulation and thereby A b-induced synapse damage. We believe that a better understanding of the cellular and molecular mechanism(s) whereby accumulating Ab at synapses cause synaptic dysfunction will be important for the development of novel treatment for AD. Methods: Chemical and genetic inhibition is used to block various steps in endosomal trafficking in N2a neuroblastoma cellsor primary neurons. Components of the ESCRT pathway are modulated. Alterations in ESCRT components are also analysed in AD transgenic compared to wild type neurons and in primary neurons treated with extracellular A b. Results: The dominant negative form of the ESCRT component VPS4A shows significantly increased intracellular accumulation and decreased secretion of Ab in Swe N2a cells. Lysosomal inhibitor Bafilomycin A1 also markedly increases intracellular Ab while reducing secreted Ab in Swe N2a. Conclusions: We previously reported progressive intraneuronal Ab accumulation and impaired Ab secretion in AD-neurons with time in culture. This is mimicked by inhibition of late ESCRT machinery component VPS4A and consistent with indications of MVB dysfunction in AD. A next step will be to analyse synaptic composition in primary neurons after ESCRT modification.
P1-089
DECREASING PICALM EXPRESSION ALTERS THE AMYLOIDOGENIC PROCESSING OF AMYLOID PRECURSOR PROTEIN
Rhian Thomas, Amy Gerrish, Alex Henson, Lesley Jones, Julie Williams, Emma Kidd, Cardiff University, Cardiff, United Kingdom. Contact e-mail: [email protected]