- Email: [email protected]
P1-022: Inhibitory mechanism of amyloidgenic APP metabolism by X11 and X11L in brain
T212 P1-022
Poster Presentations P1 INHIBITORY MECHANISM OF AMYLOIDGENIC APP METABOLISM BY X11 AND X11L IN BRAIN
Yuhki Saito, Tadashi Nakaya, Toshiharu Suzuki, Hokkaido University, Sapporo, Japan. Contact e-mail: [email protected] Background: X11 (X11␣) and X11-like (X11L/X11) are an adaptor protein composed of an independent amino terminal half, a conserved central phosphotyrosine interaction (PI) domain, and two carboxyl PDZ domains. X11 and X11L are largely brain-specific, while another family member X11-like 2 (X11L2/X11␥) is expressed ubiquitously. X11L, also X11 and X11L2, binds to the 681GYENPTY687 motif within APP cytoplasmic region through its PI domain and suppress APP metabolism in cultured cells (J. Biol. Chem. [1999] 274, 2243). We previously reported that generation of CTF and A from endogenous mouse APP is facilitated in the brains of X11L-deficient mice (J. Biol. Chem. [2006] 281, 37853), suggesting that X11L suppresses the amyloidogenic metabolism of APP. Although the distinct expression between X11 and X11L in brain neurons is observed, APP metabolism in the brains of X11-deficient mice has not been analyzed. Methods: To reveal the role of X11 in suppressing amyloidogenic cleavage of APP in vivo, we generated and analyzed X11 and X11L genes homozygous doublydeficient (X11-/-, X11L-/-) mice. Results: The double-deficient mutant mice showed normal brain morphology and no compensatory changes in the expression of other X11L2, although 50% of the doubly-deficient mice died just after birth and the weight of surviving double-KO mice is decreased. Using survivors, we analyzed APP metabolism in brain of doubly-deficient mice along with that of X11-deficient mice. The doubly-deficient mice strongly and X11-deficient mice weakly showed enhanced -site cleavage of APP along with increased accumulation of A in brain. Conclusions: Based on the biochemical analysis with X11 and X11L doubly-deficient mice, we present molecular mechanism how X11s suppresses -site cleavage of APP in brain. Our results provide the possibility that dysfunction of X11 proteins might play a role in the pathogenesis of AD. P1-023
IS ALZHEIMER’S DISEASE A CONSEQUENCE OF DYSREGULATED WNT SIGNALING IN THE AGED HUMAN BRAIN?
Amritpal Mudher, University of Southampton, Southampton, United Kingdom. Contact e-mail: [email protected] Background: Alzheimer’s Disease is the commonest cause of dementia in the elderly and it is characterised by the abnormal phosphorylation and aggregation of a microtubule associated protein called tau. Wnt signalling pathway is a signal transduction pathway that is involved in the regulation of cell fate decisions during development. Various lines of evidence implicate this pathway in the pathogenesis of Alzheimer’s Disease. The most significant of these is that stimulation of this pathway culminates in reduced activity of glycogen synthase kinase -3beta (GSK-3b) which is believed to be a major tau kinase. This has led to the hypothesis that activation of this pathway may protect against the generation of the tau pathology in Alzheimer’s Disease and other tauopathies. Methods: We tested this hypothesis in a Drosophila model in which we had previously shown that over-expression of human tau causes disruption of axonal transport and this leads to locomotor impairments. Results: We have now shown that co-expression of Dishevelled (a component of the wnt signalling pathway) in this model results in suppression of the tau mediated axonal transport and locomotor phenotypes. As expected this is accompanied by a reduction in GSK-3b mediated phosphorylation of tau. Conclusions: These findings support the notion that the wnt signalling pathway may control the phosphorylation state of tau in the normal adult brain and that the tau protein abnormalities that characterise AD may arise because of a dysregulation of this pathway.
P1-024
LOSS OF SUT-2 PREVENTS TAU NEUROTOXICITY
Brian C. Kraemer, Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA. Contact e-mail: [email protected] Background: Neurofibrillary tangles composed of insoluble hyperphosphorylated tau are characteristic of Alzheimer’s disease, FTDP-17, and other tauopathies. We previously described a C. elegans model for tauopathy. Human tau expression in worm neurons causes an uncoordinated locomotion (Unc) phenotype. We conducted a forward genetic screen for mutations suppressing the strong Unc phenotype induced by V337M mutant tau. The rationale being that mutation of a gene whose function is required for the formation of neurotoxic tau, should alleviate the tau induced Unc phenotype. Methods: We conducted a forward genetic screen for mutations suppressing the strong Unc phenotype induced by V337M mutant tau. The rationale being that mutation of a gene whose function is required for the formation of neurotoxic tau, should alleviate the tau induced Unc phenotype. Results: We isolated mutant strains resistant to the neurotoxic effects of tau in a classical genetic screens for suppressors of tau pathology. We call these genes suppressor of tau (sut) genes. Loss of sut-2 ameliorates tau induced neuronal dysfunction as seen by an amelioration of the Unc phenotype. Sut-2 mutants also accumulate less insoluble tau and have reduced degeneration of GABAergic neurons. The gene mutated in sut-2 animals was cloned using a microarray assisted positional cloning approach. The sut-2 gene encodes a C-X5-C-X5-C-X3-H / C-X5-C-X4-CX3-H type zinc finger protein. The worm sut-2 gene has a homolog in humans we have named mammalian SUT2 (MSUT2). We have demonstrated that both worm sut-2 and human MSUT2 bind to the hook family of cytoskeletal regulatory proteins in vitro. Conclusions: The worm SUT-2 is a putative zinc finger protein homologous to human MSUT2. Loss of function mutations in sut-2 ameliorate the neuronal dysfunction, accumulation of insoluble tau and neurodegenerative changes induced by the tau transgene. Human and worm SUT-2 proteins bind to Hook proteins possibly mediating cytoskeletal regulation. P1-025
INTRANEURONAL TAU IMMUNOREACTIVITY IS FIXATION DEPENDENT IN YOUNG 3xTg-AD MICE
Kwang-Jin Oh, Cassia R. Overk, Sylvia E. Perez, Elliott J. Mufson, Rush University Medical Center, Chicago, IL, USA. Contact e-mail: [email protected] Background: Triple transgenic (3xTg-AD) mice harboring the human APPSwe, PS1M146V and TauP301L gene mutations display Alzheimer’s Disease (AD) like pathology. Studies using these mutant mice suggest that intraneuronal amyloid (A), visualized by the 6E10 antibody at 6 month of age, precedes the development of tangle-like intraneuronal staining revealed by AT8 at 9 months of age. Interestingly, these findings were primarily derived from hemisected brains immersion fixed in 4% paraformaldehyde prior to immunostaining. Methods: Male and Female 3xTg-AD mice were either transcardially perfused with 4% paraformaldehyde/0.1% glutaraldehyde or immersion fixed in the same solution prior to immunostaining with antibodies A 6E10, Alz50, AT8, AT180, or PHF-1. Additional 2 month old brains were either immersion fixed or transcardially perfused with paraformaldehyde (2%)/Lysine/Periodate (PLP) solution and immunostained for the same antibodies listed above. Results: In 24 day old male and female mice transcardially perfused, we observed 6E10 immunopositive intraneuronal cortical, hippocampal and amygdala neurons and hippocampal Alz50, AT8, PHF-1, and AT180 positive neurons. A few AT8 and Alz50 immunopositive cortical neurons were seen at this age as well. However, at 2 months of age all tau markers appeared in greater numbers the cortex and amygdala. Brain sections from 9 months mice which were perfused continued to show more robust staining in cortex, hippocampus and amygdala as compared to immersion fixed brains. Conversely, A immunopositive intraneuronal staining recognized by 6E10 was not fixation dependent. Two month old brains either immersion or transcardially perfused with PLP solution displayed more robust 6E10 and Alz50 intraneuronal staining, but did not affect the staining characteristics of other
Poster Presentations P1 INHIBITORY MECHANISM OF AMYLOIDGENIC APP METABOLISM BY X11 AND X11L IN BRAIN
Yuhki Saito, Tadashi Nakaya, Toshiharu Suzuki, Hokkaido University, Sapporo, Japan. Contact e-mail: [email protected] Background: X11 (X11␣) and X11-like (X11L/X11) are an adaptor protein composed of an independent amino terminal half, a conserved central phosphotyrosine interaction (PI) domain, and two carboxyl PDZ domains. X11 and X11L are largely brain-specific, while another family member X11-like 2 (X11L2/X11␥) is expressed ubiquitously. X11L, also X11 and X11L2, binds to the 681GYENPTY687 motif within APP cytoplasmic region through its PI domain and suppress APP metabolism in cultured cells (J. Biol. Chem. [1999] 274, 2243). We previously reported that generation of CTF and A from endogenous mouse APP is facilitated in the brains of X11L-deficient mice (J. Biol. Chem. [2006] 281, 37853), suggesting that X11L suppresses the amyloidogenic metabolism of APP. Although the distinct expression between X11 and X11L in brain neurons is observed, APP metabolism in the brains of X11-deficient mice has not been analyzed. Methods: To reveal the role of X11 in suppressing amyloidogenic cleavage of APP in vivo, we generated and analyzed X11 and X11L genes homozygous doublydeficient (X11-/-, X11L-/-) mice. Results: The double-deficient mutant mice showed normal brain morphology and no compensatory changes in the expression of other X11L2, although 50% of the doubly-deficient mice died just after birth and the weight of surviving double-KO mice is decreased. Using survivors, we analyzed APP metabolism in brain of doubly-deficient mice along with that of X11-deficient mice. The doubly-deficient mice strongly and X11-deficient mice weakly showed enhanced -site cleavage of APP along with increased accumulation of A in brain. Conclusions: Based on the biochemical analysis with X11 and X11L doubly-deficient mice, we present molecular mechanism how X11s suppresses -site cleavage of APP in brain. Our results provide the possibility that dysfunction of X11 proteins might play a role in the pathogenesis of AD. P1-023
IS ALZHEIMER’S DISEASE A CONSEQUENCE OF DYSREGULATED WNT SIGNALING IN THE AGED HUMAN BRAIN?
Amritpal Mudher, University of Southampton, Southampton, United Kingdom. Contact e-mail: [email protected] Background: Alzheimer’s Disease is the commonest cause of dementia in the elderly and it is characterised by the abnormal phosphorylation and aggregation of a microtubule associated protein called tau. Wnt signalling pathway is a signal transduction pathway that is involved in the regulation of cell fate decisions during development. Various lines of evidence implicate this pathway in the pathogenesis of Alzheimer’s Disease. The most significant of these is that stimulation of this pathway culminates in reduced activity of glycogen synthase kinase -3beta (GSK-3b) which is believed to be a major tau kinase. This has led to the hypothesis that activation of this pathway may protect against the generation of the tau pathology in Alzheimer’s Disease and other tauopathies. Methods: We tested this hypothesis in a Drosophila model in which we had previously shown that over-expression of human tau causes disruption of axonal transport and this leads to locomotor impairments. Results: We have now shown that co-expression of Dishevelled (a component of the wnt signalling pathway) in this model results in suppression of the tau mediated axonal transport and locomotor phenotypes. As expected this is accompanied by a reduction in GSK-3b mediated phosphorylation of tau. Conclusions: These findings support the notion that the wnt signalling pathway may control the phosphorylation state of tau in the normal adult brain and that the tau protein abnormalities that characterise AD may arise because of a dysregulation of this pathway.
P1-024
LOSS OF SUT-2 PREVENTS TAU NEUROTOXICITY
Brian C. Kraemer, Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA. Contact e-mail: [email protected] Background: Neurofibrillary tangles composed of insoluble hyperphosphorylated tau are characteristic of Alzheimer’s disease, FTDP-17, and other tauopathies. We previously described a C. elegans model for tauopathy. Human tau expression in worm neurons causes an uncoordinated locomotion (Unc) phenotype. We conducted a forward genetic screen for mutations suppressing the strong Unc phenotype induced by V337M mutant tau. The rationale being that mutation of a gene whose function is required for the formation of neurotoxic tau, should alleviate the tau induced Unc phenotype. Methods: We conducted a forward genetic screen for mutations suppressing the strong Unc phenotype induced by V337M mutant tau. The rationale being that mutation of a gene whose function is required for the formation of neurotoxic tau, should alleviate the tau induced Unc phenotype. Results: We isolated mutant strains resistant to the neurotoxic effects of tau in a classical genetic screens for suppressors of tau pathology. We call these genes suppressor of tau (sut) genes. Loss of sut-2 ameliorates tau induced neuronal dysfunction as seen by an amelioration of the Unc phenotype. Sut-2 mutants also accumulate less insoluble tau and have reduced degeneration of GABAergic neurons. The gene mutated in sut-2 animals was cloned using a microarray assisted positional cloning approach. The sut-2 gene encodes a C-X5-C-X5-C-X3-H / C-X5-C-X4-CX3-H type zinc finger protein. The worm sut-2 gene has a homolog in humans we have named mammalian SUT2 (MSUT2). We have demonstrated that both worm sut-2 and human MSUT2 bind to the hook family of cytoskeletal regulatory proteins in vitro. Conclusions: The worm SUT-2 is a putative zinc finger protein homologous to human MSUT2. Loss of function mutations in sut-2 ameliorate the neuronal dysfunction, accumulation of insoluble tau and neurodegenerative changes induced by the tau transgene. Human and worm SUT-2 proteins bind to Hook proteins possibly mediating cytoskeletal regulation. P1-025
INTRANEURONAL TAU IMMUNOREACTIVITY IS FIXATION DEPENDENT IN YOUNG 3xTg-AD MICE
Kwang-Jin Oh, Cassia R. Overk, Sylvia E. Perez, Elliott J. Mufson, Rush University Medical Center, Chicago, IL, USA. Contact e-mail: [email protected] Background: Triple transgenic (3xTg-AD) mice harboring the human APPSwe, PS1M146V and TauP301L gene mutations display Alzheimer’s Disease (AD) like pathology. Studies using these mutant mice suggest that intraneuronal amyloid (A), visualized by the 6E10 antibody at 6 month of age, precedes the development of tangle-like intraneuronal staining revealed by AT8 at 9 months of age. Interestingly, these findings were primarily derived from hemisected brains immersion fixed in 4% paraformaldehyde prior to immunostaining. Methods: Male and Female 3xTg-AD mice were either transcardially perfused with 4% paraformaldehyde/0.1% glutaraldehyde or immersion fixed in the same solution prior to immunostaining with antibodies A 6E10, Alz50, AT8, AT180, or PHF-1. Additional 2 month old brains were either immersion fixed or transcardially perfused with paraformaldehyde (2%)/Lysine/Periodate (PLP) solution and immunostained for the same antibodies listed above. Results: In 24 day old male and female mice transcardially perfused, we observed 6E10 immunopositive intraneuronal cortical, hippocampal and amygdala neurons and hippocampal Alz50, AT8, PHF-1, and AT180 positive neurons. A few AT8 and Alz50 immunopositive cortical neurons were seen at this age as well. However, at 2 months of age all tau markers appeared in greater numbers the cortex and amygdala. Brain sections from 9 months mice which were perfused continued to show more robust staining in cortex, hippocampus and amygdala as compared to immersion fixed brains. Conversely, A immunopositive intraneuronal staining recognized by 6E10 was not fixation dependent. Two month old brains either immersion or transcardially perfused with PLP solution displayed more robust 6E10 and Alz50 intraneuronal staining, but did not affect the staining characteristics of other