- Email: [email protected]
LIGAND-ACTIVATED EPHA4 SIGNALING GOVERNS THE PROTEOSTASIS OF APP-BETA CTF TO CONTROL THE LEVEL OF AMYLOID-BETA
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
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