- Email: [email protected]
P2-133: Okadaic acid–induced tau phosphorylation in rat primary neurons: Involvement of the JNK pathway
Poster Presentations P2: (PTHs) as potent lead structure. Here we report the comparison of PTHs to selected curcumin derivatives and approved tetracyclines for their tau antiaggregatory properties and their affinity to fibrillar beta-amyloid in several assays and cellular models. The most potent fluorescent compounds were evaluated for beta-amyloid/tau PHF selectivity by fluorescence microscopy of human AD probes. Methods: Lead Optimization of PTHs. Several diverse PTH analogues were obtained by variation of the substituents R1-R4 to explore a preliminary SAR. Fluorescent substituents were incorporated without significantly compromising in vitro activity. Results: Tau-biased ligands with enhanced affinity to tau paired helical filaments over beta-amyloid were identified by a panel of protein aggregation assays and fluorescence microscopy of human AD preparations. Conclusions: Phenylthiazolylhydrazides are potent inhibitors of tau-aggregation. The fluorescence microscopy of human AD preparation suggests a binding to growth-relevant sites of tau aggregates. P2-131
P53 FAMILY MEMBERS INDUCE THE PHOSPHORYLATION OF HUMAN TAU IN CULTURED MAMMALIAN CELLS
Claudie Hooper, Richard Killick, Simon Lovestone, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected] Background: Tau is the major microtubule-associated protein in neurones, which functions in the formation and maintenance of axons by influencing microtubule organization. Increased tau phosphorylation is also a salient feature of Alzheimer’s disease (AD) and tauopathies such as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). In these neurodegenerative disorders, normally soluble tau is present as paired-helical filaments (PHFs), which in turn aggregate to form neurofibrillary tangles (NFTs). p53 and p73 are tumor suppressor proteins, which induces cell cycle arrest or apoptosis. Methods: Here we explore the effects of p53 family members (p53 and p73␣) on tau phosphorylation in a mammalian cell culture model by western blotting. We also investigate the expression levels of p53 and p73 in human post-mortem samples from AD and control cases. Results: Normally, p53 and p73 are maintained at low levels inside cells. However, we and others have reported an increase in p53 immunoreactivity, but not p73 immunoreactivity in AD. Furthermore, p53-/- mice display a reduction in tau phosphorylation. Consistent with this, we have demonstrated that p53 induces tau (human 2N4R) phosphorylation at the AT8/tau-1 epitope in HEK293a cells and that p73␣, a related p53 homologue, also induces tau (human 2N4R) phosphorylation at the AT-8/tau-1 epitope as well as the PHF-1 epitope in HEK293a cells. In contrast, we find that the N-terminally truncated transcriptionally inactive or ⌬N isoform of p73␣ fails to induce tau (human 2N4R) phosphorylation. Conclusions: These results suggest that p53 and indeed p73 induce tau phosphorylation in a cell culture model of neurodegeneration and that the effects of p73 and perhaps p53 on tau are dependent on the transcriptional activity of these proteins. Interestingly, p53, but not p73 was upregulated in AD. However, p73 has been reported to be aberrantly expressed in the nucleus of hippocampal neurons in AD - p73 expression co-localizing with NFTs. Therefore, changes in the sub-cellular localization of p73 (without an overall change in expression level) might account for transcription-driven changes in tau phosphorylation in some instances. Collectively, these findings have ramifications for neuronal death associated with AD and other tauopathies. P2-132
POTENTIAL TOXICITY OF TAU CLEAVAGE PRODUCTS IN PROGRESSIVE SUPRANUCLEAR PALSY
Selina Wray, Brian H. Anderton, Diane P. Hanger, MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected]
T409
Background: Hyperphosphorylated, insoluble aggregates of the microtubule associated protein tau are a primary feature of the pathology observed in progressive supranuclear palsy (PSP) and other tauopathies including Alzheimer’s disease (AD). Characterisation of the biochemical properties of PSP-tau, including its precise phosphorylation state, is necessary to fully understand the molecular progression of PSP. Recent reports have demonstrated the importance of tau cleavage in neurodegeneration, and the presence of specific tau fragments might differentiate PSP from other tauopathies. Objective: To identify phosphorylation sites on PSP-tau and to characterise cleavage products of tau identified in PSP-brain. Methods: Phosphorylation sites on PSP-tau were determined by mass spectrometry. Disease specificity and isoform composition of tau35 was analysed by western blotting. The effects of tau35 were examined following overexpression of a cDNA construct encoding tau35 in cells. Results: We report the first direct identification of phosphorylation sites on PSP-tau, including sites not previously reported to be phosphorylated in PSP and residues that have not been detected on insoluble tau from AD. We also identified a 35kDa tau fragment (tau35) that co-enriches with full-length PSP-tau. Preliminary characterisation of tau35 showed that it is comprised of the C-terminal half of tau including an intact C-terminus. Antibody labelling indicated a predominance of 4R, rather than 3R, tau isoforms present in tau35. Furthermore, examination of the disease specificity of tau35 indicated that it was present in tauopathies characterised by an excess of 4R tau isoforms, but was absent from Pick’s disease in which 3R tau predominates. Conclusions: This is the first report of directly identified sites on PSP-tau. Our results indicate differences between PSP-tau and the insoluble tau from AD. Preliminary characterisation of tau35 indicates an association with tauopathies in which an excess of 4R tau is present. These results suggest that cellular mechanisms may exist that process excess amounts of 4R tau. Further characterisation of tau35 may provide important insights into the molecular pathogenesis of tauopathies. Acknowledgments: This work was funded by the PSP (Europe) Association. P2-133
OKADAIC ACID–INDUCED TAU PHOSPHORYLATION IN RAT PRIMARY NEURONS: INVOLVEMENT OF THE JNK PATHWAY
Jennifer Moore, Stan P. Nawoschik, Brian Ludwig, Beal McIlvain, Vasanti Anand, John Dunlop, Steven P. Braithwaite, Wyeth, Princeton, NJ, USA. Contact e-mail: [email protected] Background: The defining neuropathological characteristics of Alzheimer’s disease (AD) are the presence of aggregated amyloid in senile plaques and neurofibrillary tangles (NFT) consisting of hyperphosphorylated tau proteins. This abnormal hyperphosphorylation of tau makes it lose its normal function to stimulate microtubule assembly, gain toxic activity, and aggregate into NFTs. This may be the result of an increase in the activity of kinases involved in tau phosphorylation, or a decrease in the activity of phosphatases involved in the same pathways. One class of kinases showing increased expression and activity in Alzheimer’s disease is the c-Jun N-terminal kinase (JNK), which consists of 3 isoforms. Pathological activation of JNK2 and JNK3 have been associated with plaques and tangles in AD and are thought to be at least partly responsible for neuronal cell death. Methods: In order to study this AD-like pathology in vitro, kinase pathways, including the JNK pathway, have been activated by the addition of a PP2A inhibitor, Okadaic Acid (OA) to primary neurons. Following the addition of OA the intracellular activation of JNK and the phosphorylation of tau have been analyzed using an automated cell-based imaging technique. Results: We have determined the time course and concentration of OA that induces maximal JNK and tau phosphorylation prior to inducing cell death. We have measured the phosphorylation of JNK, c-Jun, and tau (at Ser 422, and Ser202/205 sites) and demonstrated that inhibition of the JNK pathway with a specific JNK inhibitor results in a decrease in tau phosphorylation. Conclusions: In this study we have characterized the role of the JNK pathway in mediating OA induced tau
T410
Poster Presentations P2:
phosphorylation, providing a cellular model for studying this signaling pathway in a model of Alzheimer’s Disease. P2-134
PKB PLAYS A CENTRAL ROLE TO TAU HYPERPHOSPHORYLATION IN INTACT LIVING CELLS
Xin Wen Zhou1, Bengt Winblad2, Jian Zhi Wang1, Jin Jing Pei2, Department of Pathaphysiology of Tongji Medical College, Wuhan, China; 2Karolinska Institutet, KI-Alzheimer Disease Research Center (KI-ADRC), Novum, Stockhelm, Sweden. Contact e-mail: [email protected]
1
Background: Microtubule associated protein tau is abnormally hyperphosphorylated in Alzheimer’s disease (AD) and related tauopathies. In order to address how interactions among PKB, GSK3 and PP2A regulate tau phosphorylation. Methods: Plasmid or siRNA of PKB, GSK and PP2A were tranfected into N2a cell for over-expression or blocking respective enzyme. Results: The results showed that the increased GSK3 phosphorylation at S9, over-expression of GSK dramatically increased PKB phosphorylation at S473 and inhibited the exogenous PKB phosphorylation at T308. Over-expression of GSK3 or PKB could activate PP2A catalytic subunit (PP2AC) through down-regulating the PP2AC phosphorylation at Y307. Reversely partial blocking of PKB or GSK did inhibit PP2A activity by increasing its phosphorylation at Y307. Tau phosphorylation at S396 was increased by over-expression of GSK3 or PKB. Tau phosphorylation at S214 was only induced by over-expression of PKB, While partial blocking of GSK decreased tau phosphorylation at S396, partial blocking of PKB was shown to induce increased tau phosphorylation at both S396 and S214 sites. Blocking PP2AC increased PKB phosphorylation selectively at S473, and GSK phosphorylation selectively at S9, and tau phosphorylation selectively at S396. Conclusions: Results from the intact living cells indicated that initial inhibition of PKB may play a critical role in aberrant phosphorylation of tau in AD brains. P2-135
MECHANISMS OF NEURONAL TRAFFIC INHIBITION BY TAU PROTEIN
Eva-Maria Mandelkow, Edda Thies, Max-Planck Society, Hamburg, Germany. Contact e-mail: [email protected] Background: The missorting of Tau protein from the axonal to the somatodendritic compartment and loss of synapses are hallmarks of Alzheimer’s disease which precede neuronal loss and the pathological aggregation of tau protein into neurofibrillary tangles. We are interested in the mechanisms by which Tau can affect the integrity of synapses. One major factor is the inhibition of movement of mitochondria by Tau which leads to mitochondrial dysfunction and energy deprivation (loss of ATP production). Methods: We have now studied the pathway of Tau’s interference with mitochondrial transport. Primary hippocampal neurons were transfected with CFP-Tau, mitochondrial traffic and synaptic integrity was observed by time-resolved confocal microscopy, followed by thin-sectioning electron microscopy to visualize the state of mitochondria, synapses, and the cytoskeleton. Results: The results show that Tau operates on two distinct levels. One is a direct inhibition mechanism whereby bound Tau covers the microtubule surface and consequently interferes with the attachment of motor proteins. The second is an indirect inhibition mechanism which is based on the higher stability of microtubules when Tau is increased. This leads to a reduced level of tubulin subunits in the cytosol, which in turn causes the upregulation of tubulin synthesis (Cleveland et al., Nature 1983). This causes an increase in microtubule density, the microtubules become tightly bundled so that mitochondria can no longer move freely along axons and dendrites. Conclusions: The example illustrates how an improvement of microtubule stability can cause traffic deficits in neurons, rather than curing them. Research supported by MPG and DFG.
P2-136
HEME OXYGENASE-1 PROMOTES PROTEOSOMAL DEGRADATION OF TAU AND ALPHA-SYNUCLEIN IN HUMAN NEUROBLASTOMA CELLS
Wei Song1, Amar Patel2, Dong Han3, Hemant K. Paudel2, Hyman M. Schipper1,2, 1Centre for Neurotranslational Research, Lady Davis Institute for Medical Research, SMBD Jewish General Hospital, Montreal, QC, Canada; 2Dept. of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada; 3Dept. of Experimental Medicine, McGill University, Montreal, QC, Canada. Contact e-mail: [email protected] Background: HO-1 is a stress-responsive enzyme that degrades heme to free iron, carbon monoxide and biliverdin (bilirubin precursor). HO-1 protein is over-expressed in Alzheimer (AD) and Parkinson (PD) brain and co-localizes, respectively, to tau-containing neurofibrillary tangles (NFT) and ␣-synuclein-laden Lewy bodies in affected neurons.Attenuated proteasomal activity has been implicated in the accumulation of and aggregation of insoluble forms of tau and ␣-synuclein in these conditions. Methods: The effects of transient transfection of human (h) HO-1 cDNA on levels of co-transfected wild-type (WT) and mutant (P301L) tau and WT and mutant (A30P) ␣-synuclein were measured by Western blotting in M17 human neuroblastoma cells in the presence or absence of the HO competitive inhibitor, tin mesoporphyin (SnMP) or proteasome inhibitor, lactacystin. In some experiments, deferoxamine, methylene blue and bilirubin were administered to delineate the role(s) of iron, CO and bilirubin, respectively, in altered tau and ␣-synuclein metabolism. Results: hHO-1 transfection (⬃3-4 fold increase in HO activity) significantly decreased expression levels of both wild type tau and P301L mutant tau. These effects were attenuated by administration of SnMP, lactacystin or deferoxamine (but not methylene blue or bilirubin). hHO-1 transfection similarly diminished endogenous and transgenic WT ␣-synuclein levels, effects reversed by SnMP, lactacystin, deferoxamine or methylene blue (but not bilirubin). HO-1 transfection did not significantly impact A30P levels in these cells. Conclusions: In sporadic AD and PD, induction of neuronal HO-1 may confer neuroprotection by stimulating proteasomal degradation of tau or ␣-synuclein, respectively. Conversely, failure of HO-1 to suppress mutant (A30P) ␣-synuclein may predispose to familial PD. P2-137
INHIBITION OF PROTEASOME INCREASES ACCUMULATION AND INSOLUBILITY OF TAU PROTEINS INDEPENDENT OF TAU HYPERPHOSPHORYLATION IN RAT
Ying-Hua Liu, Tongji Medical College Huazhong University of’ Science, Wuhan, China. Contact e-mail: [email protected] Background: The co-presentation of proteasome deficit and tau inclusions in the Alzheimer’s disease (AD) brain suggests an intrinsic link between proteasome and tau degradation. However, the role of proteasome in the proteolysis of tau is still uncertain. Here, we investigated the influence of proteasome inhibition on the accumulation, phosphorylation, ubiquitination, solubility of tau and as well as the memory retention in rats. Methods: We injected bilaterally lactacystin into the rat hippocampus and employed Western blotting, immunohistochemistry, thioflavin-S staining, biochemical fractionation and Morris water maze to measure the alterations of tau proteins and the spatial memory retention in rats. Results: We observed that lactacystin inhibited prominently both chymotrypsin-like and trypsinlike activities of the proteasome. With inhibition of proteasome, accumulation of different tau species was observed and the increase of tau was more prominent in RIPA-insoluble fractions. The increase of the phosphorylated tau was no longer present and the phosphorylated tau at Ser-214 and Thr-231 was even lower than normal level after normalized to total tau. Inhibition of proteasome resulted in activation of cAMP-dependent protein kinase (PKA), glycogen synthase kinases-3 (GSK-3) and cyclin-dependent kinase-5 (Cdk-5), and inhibition of protein phosphatase-2A (PP-2A) and c-Jun N-terminal kinase (JNK). Proteasome inhibition also induced
P53 FAMILY MEMBERS INDUCE THE PHOSPHORYLATION OF HUMAN TAU IN CULTURED MAMMALIAN CELLS
Claudie Hooper, Richard Killick, Simon Lovestone, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected] Background: Tau is the major microtubule-associated protein in neurones, which functions in the formation and maintenance of axons by influencing microtubule organization. Increased tau phosphorylation is also a salient feature of Alzheimer’s disease (AD) and tauopathies such as frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). In these neurodegenerative disorders, normally soluble tau is present as paired-helical filaments (PHFs), which in turn aggregate to form neurofibrillary tangles (NFTs). p53 and p73 are tumor suppressor proteins, which induces cell cycle arrest or apoptosis. Methods: Here we explore the effects of p53 family members (p53 and p73␣) on tau phosphorylation in a mammalian cell culture model by western blotting. We also investigate the expression levels of p53 and p73 in human post-mortem samples from AD and control cases. Results: Normally, p53 and p73 are maintained at low levels inside cells. However, we and others have reported an increase in p53 immunoreactivity, but not p73 immunoreactivity in AD. Furthermore, p53-/- mice display a reduction in tau phosphorylation. Consistent with this, we have demonstrated that p53 induces tau (human 2N4R) phosphorylation at the AT8/tau-1 epitope in HEK293a cells and that p73␣, a related p53 homologue, also induces tau (human 2N4R) phosphorylation at the AT-8/tau-1 epitope as well as the PHF-1 epitope in HEK293a cells. In contrast, we find that the N-terminally truncated transcriptionally inactive or ⌬N isoform of p73␣ fails to induce tau (human 2N4R) phosphorylation. Conclusions: These results suggest that p53 and indeed p73 induce tau phosphorylation in a cell culture model of neurodegeneration and that the effects of p73 and perhaps p53 on tau are dependent on the transcriptional activity of these proteins. Interestingly, p53, but not p73 was upregulated in AD. However, p73 has been reported to be aberrantly expressed in the nucleus of hippocampal neurons in AD - p73 expression co-localizing with NFTs. Therefore, changes in the sub-cellular localization of p73 (without an overall change in expression level) might account for transcription-driven changes in tau phosphorylation in some instances. Collectively, these findings have ramifications for neuronal death associated with AD and other tauopathies. P2-132
POTENTIAL TOXICITY OF TAU CLEAVAGE PRODUCTS IN PROGRESSIVE SUPRANUCLEAR PALSY
Selina Wray, Brian H. Anderton, Diane P. Hanger, MRC Centre for Neurodegeneration Research, King’s College London, Institute of Psychiatry, London, United Kingdom. Contact e-mail: [email protected]
T409
Background: Hyperphosphorylated, insoluble aggregates of the microtubule associated protein tau are a primary feature of the pathology observed in progressive supranuclear palsy (PSP) and other tauopathies including Alzheimer’s disease (AD). Characterisation of the biochemical properties of PSP-tau, including its precise phosphorylation state, is necessary to fully understand the molecular progression of PSP. Recent reports have demonstrated the importance of tau cleavage in neurodegeneration, and the presence of specific tau fragments might differentiate PSP from other tauopathies. Objective: To identify phosphorylation sites on PSP-tau and to characterise cleavage products of tau identified in PSP-brain. Methods: Phosphorylation sites on PSP-tau were determined by mass spectrometry. Disease specificity and isoform composition of tau35 was analysed by western blotting. The effects of tau35 were examined following overexpression of a cDNA construct encoding tau35 in cells. Results: We report the first direct identification of phosphorylation sites on PSP-tau, including sites not previously reported to be phosphorylated in PSP and residues that have not been detected on insoluble tau from AD. We also identified a 35kDa tau fragment (tau35) that co-enriches with full-length PSP-tau. Preliminary characterisation of tau35 showed that it is comprised of the C-terminal half of tau including an intact C-terminus. Antibody labelling indicated a predominance of 4R, rather than 3R, tau isoforms present in tau35. Furthermore, examination of the disease specificity of tau35 indicated that it was present in tauopathies characterised by an excess of 4R tau isoforms, but was absent from Pick’s disease in which 3R tau predominates. Conclusions: This is the first report of directly identified sites on PSP-tau. Our results indicate differences between PSP-tau and the insoluble tau from AD. Preliminary characterisation of tau35 indicates an association with tauopathies in which an excess of 4R tau is present. These results suggest that cellular mechanisms may exist that process excess amounts of 4R tau. Further characterisation of tau35 may provide important insights into the molecular pathogenesis of tauopathies. Acknowledgments: This work was funded by the PSP (Europe) Association. P2-133
OKADAIC ACID–INDUCED TAU PHOSPHORYLATION IN RAT PRIMARY NEURONS: INVOLVEMENT OF THE JNK PATHWAY
Jennifer Moore, Stan P. Nawoschik, Brian Ludwig, Beal McIlvain, Vasanti Anand, John Dunlop, Steven P. Braithwaite, Wyeth, Princeton, NJ, USA. Contact e-mail: [email protected] Background: The defining neuropathological characteristics of Alzheimer’s disease (AD) are the presence of aggregated amyloid in senile plaques and neurofibrillary tangles (NFT) consisting of hyperphosphorylated tau proteins. This abnormal hyperphosphorylation of tau makes it lose its normal function to stimulate microtubule assembly, gain toxic activity, and aggregate into NFTs. This may be the result of an increase in the activity of kinases involved in tau phosphorylation, or a decrease in the activity of phosphatases involved in the same pathways. One class of kinases showing increased expression and activity in Alzheimer’s disease is the c-Jun N-terminal kinase (JNK), which consists of 3 isoforms. Pathological activation of JNK2 and JNK3 have been associated with plaques and tangles in AD and are thought to be at least partly responsible for neuronal cell death. Methods: In order to study this AD-like pathology in vitro, kinase pathways, including the JNK pathway, have been activated by the addition of a PP2A inhibitor, Okadaic Acid (OA) to primary neurons. Following the addition of OA the intracellular activation of JNK and the phosphorylation of tau have been analyzed using an automated cell-based imaging technique. Results: We have determined the time course and concentration of OA that induces maximal JNK and tau phosphorylation prior to inducing cell death. We have measured the phosphorylation of JNK, c-Jun, and tau (at Ser 422, and Ser202/205 sites) and demonstrated that inhibition of the JNK pathway with a specific JNK inhibitor results in a decrease in tau phosphorylation. Conclusions: In this study we have characterized the role of the JNK pathway in mediating OA induced tau
T410
Poster Presentations P2:
phosphorylation, providing a cellular model for studying this signaling pathway in a model of Alzheimer’s Disease. P2-134
PKB PLAYS A CENTRAL ROLE TO TAU HYPERPHOSPHORYLATION IN INTACT LIVING CELLS
Xin Wen Zhou1, Bengt Winblad2, Jian Zhi Wang1, Jin Jing Pei2, Department of Pathaphysiology of Tongji Medical College, Wuhan, China; 2Karolinska Institutet, KI-Alzheimer Disease Research Center (KI-ADRC), Novum, Stockhelm, Sweden. Contact e-mail: [email protected]
1
Background: Microtubule associated protein tau is abnormally hyperphosphorylated in Alzheimer’s disease (AD) and related tauopathies. In order to address how interactions among PKB, GSK3 and PP2A regulate tau phosphorylation. Methods: Plasmid or siRNA of PKB, GSK and PP2A were tranfected into N2a cell for over-expression or blocking respective enzyme. Results: The results showed that the increased GSK3 phosphorylation at S9, over-expression of GSK dramatically increased PKB phosphorylation at S473 and inhibited the exogenous PKB phosphorylation at T308. Over-expression of GSK3 or PKB could activate PP2A catalytic subunit (PP2AC) through down-regulating the PP2AC phosphorylation at Y307. Reversely partial blocking of PKB or GSK did inhibit PP2A activity by increasing its phosphorylation at Y307. Tau phosphorylation at S396 was increased by over-expression of GSK3 or PKB. Tau phosphorylation at S214 was only induced by over-expression of PKB, While partial blocking of GSK decreased tau phosphorylation at S396, partial blocking of PKB was shown to induce increased tau phosphorylation at both S396 and S214 sites. Blocking PP2AC increased PKB phosphorylation selectively at S473, and GSK phosphorylation selectively at S9, and tau phosphorylation selectively at S396. Conclusions: Results from the intact living cells indicated that initial inhibition of PKB may play a critical role in aberrant phosphorylation of tau in AD brains. P2-135
MECHANISMS OF NEURONAL TRAFFIC INHIBITION BY TAU PROTEIN
Eva-Maria Mandelkow, Edda Thies, Max-Planck Society, Hamburg, Germany. Contact e-mail: [email protected] Background: The missorting of Tau protein from the axonal to the somatodendritic compartment and loss of synapses are hallmarks of Alzheimer’s disease which precede neuronal loss and the pathological aggregation of tau protein into neurofibrillary tangles. We are interested in the mechanisms by which Tau can affect the integrity of synapses. One major factor is the inhibition of movement of mitochondria by Tau which leads to mitochondrial dysfunction and energy deprivation (loss of ATP production). Methods: We have now studied the pathway of Tau’s interference with mitochondrial transport. Primary hippocampal neurons were transfected with CFP-Tau, mitochondrial traffic and synaptic integrity was observed by time-resolved confocal microscopy, followed by thin-sectioning electron microscopy to visualize the state of mitochondria, synapses, and the cytoskeleton. Results: The results show that Tau operates on two distinct levels. One is a direct inhibition mechanism whereby bound Tau covers the microtubule surface and consequently interferes with the attachment of motor proteins. The second is an indirect inhibition mechanism which is based on the higher stability of microtubules when Tau is increased. This leads to a reduced level of tubulin subunits in the cytosol, which in turn causes the upregulation of tubulin synthesis (Cleveland et al., Nature 1983). This causes an increase in microtubule density, the microtubules become tightly bundled so that mitochondria can no longer move freely along axons and dendrites. Conclusions: The example illustrates how an improvement of microtubule stability can cause traffic deficits in neurons, rather than curing them. Research supported by MPG and DFG.
P2-136
HEME OXYGENASE-1 PROMOTES PROTEOSOMAL DEGRADATION OF TAU AND ALPHA-SYNUCLEIN IN HUMAN NEUROBLASTOMA CELLS
Wei Song1, Amar Patel2, Dong Han3, Hemant K. Paudel2, Hyman M. Schipper1,2, 1Centre for Neurotranslational Research, Lady Davis Institute for Medical Research, SMBD Jewish General Hospital, Montreal, QC, Canada; 2Dept. of Neurology and Neurosurgery, McGill University, Montreal, QC, Canada; 3Dept. of Experimental Medicine, McGill University, Montreal, QC, Canada. Contact e-mail: [email protected] Background: HO-1 is a stress-responsive enzyme that degrades heme to free iron, carbon monoxide and biliverdin (bilirubin precursor). HO-1 protein is over-expressed in Alzheimer (AD) and Parkinson (PD) brain and co-localizes, respectively, to tau-containing neurofibrillary tangles (NFT) and ␣-synuclein-laden Lewy bodies in affected neurons.Attenuated proteasomal activity has been implicated in the accumulation of and aggregation of insoluble forms of tau and ␣-synuclein in these conditions. Methods: The effects of transient transfection of human (h) HO-1 cDNA on levels of co-transfected wild-type (WT) and mutant (P301L) tau and WT and mutant (A30P) ␣-synuclein were measured by Western blotting in M17 human neuroblastoma cells in the presence or absence of the HO competitive inhibitor, tin mesoporphyin (SnMP) or proteasome inhibitor, lactacystin. In some experiments, deferoxamine, methylene blue and bilirubin were administered to delineate the role(s) of iron, CO and bilirubin, respectively, in altered tau and ␣-synuclein metabolism. Results: hHO-1 transfection (⬃3-4 fold increase in HO activity) significantly decreased expression levels of both wild type tau and P301L mutant tau. These effects were attenuated by administration of SnMP, lactacystin or deferoxamine (but not methylene blue or bilirubin). hHO-1 transfection similarly diminished endogenous and transgenic WT ␣-synuclein levels, effects reversed by SnMP, lactacystin, deferoxamine or methylene blue (but not bilirubin). HO-1 transfection did not significantly impact A30P levels in these cells. Conclusions: In sporadic AD and PD, induction of neuronal HO-1 may confer neuroprotection by stimulating proteasomal degradation of tau or ␣-synuclein, respectively. Conversely, failure of HO-1 to suppress mutant (A30P) ␣-synuclein may predispose to familial PD. P2-137
INHIBITION OF PROTEASOME INCREASES ACCUMULATION AND INSOLUBILITY OF TAU PROTEINS INDEPENDENT OF TAU HYPERPHOSPHORYLATION IN RAT
Ying-Hua Liu, Tongji Medical College Huazhong University of’ Science, Wuhan, China. Contact e-mail: [email protected] Background: The co-presentation of proteasome deficit and tau inclusions in the Alzheimer’s disease (AD) brain suggests an intrinsic link between proteasome and tau degradation. However, the role of proteasome in the proteolysis of tau is still uncertain. Here, we investigated the influence of proteasome inhibition on the accumulation, phosphorylation, ubiquitination, solubility of tau and as well as the memory retention in rats. Methods: We injected bilaterally lactacystin into the rat hippocampus and employed Western blotting, immunohistochemistry, thioflavin-S staining, biochemical fractionation and Morris water maze to measure the alterations of tau proteins and the spatial memory retention in rats. Results: We observed that lactacystin inhibited prominently both chymotrypsin-like and trypsinlike activities of the proteasome. With inhibition of proteasome, accumulation of different tau species was observed and the increase of tau was more prominent in RIPA-insoluble fractions. The increase of the phosphorylated tau was no longer present and the phosphorylated tau at Ser-214 and Thr-231 was even lower than normal level after normalized to total tau. Inhibition of proteasome resulted in activation of cAMP-dependent protein kinase (PKA), glycogen synthase kinases-3 (GSK-3) and cyclin-dependent kinase-5 (Cdk-5), and inhibition of protein phosphatase-2A (PP-2A) and c-Jun N-terminal kinase (JNK). Proteasome inhibition also induced