- Email: [email protected]
P1-061
S112
Poster Presentations P1
presence of AChE. Ongoing studies will reveal the different interaction of nicotine with amyloid processes in these two transgenic mice models. P1-059
OVEREXPRESSION OF HUMAN ALPHASYNUCLEIN IN TRANSGENIC C. ELEGANS: EFFECTS OF PATHOGENIC MUTATIONS AND PHOSPHORYLATION
Tomoki Kuwahara1, Changhong Ren2, Akihiko Koyama1, Shigeki Arawaka2, Takeo Kato2, Shohei Mitani3, Takeshi Iwatsubo1, 1 Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; 2 Department of Neurology, Hematology, Metabolism, Endocrinology and Diabetology, Yamagata University School of Medicine, Yamagata, Japan; 3Department of Physiology, School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan. Contact e-mail: [email protected] alpha-Synuclein is a component of Lewy bodies in affected neurons of patients with Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and missense mutations or multiplications in alpha-synuclein gene have been identified in pedigrees of familial PD (FPD)/DLB, implicating alpha-synuclein in the pathogenesis of PD and DLB. To gain insights into the mechanism whereby deposition of alpha-synuclein causes neurodegeneration, we established transgenic C. elegans (TG worms) that overexpress wild-type (wt) or FPD-mutant (A53T, A30P) human alpha-synuclein in dopamine neurons. TG worms expressing A53T or A30P FPD mutant alpha-synuclein showed significant defects in dopamine neuron function, the latter being reflected by an adoptive response for feeding (i.e., decrease in bending frequency upon entry into bacterial lawn), whereas those expressing wt alpha-synuclein were normal. We next generated TG worms that overexpress human alpha-synuclein in touch neurons under the control of a mec-7 promoter. These TG worms, especially those expressing A30P FPD mutant alpha-synuclein, showed impaired touch response, whereas those expressing wt alpha-synuclein showed a normal response. To examine the pathogenic significance of alpha-synuclein phosphorylation at Ser129, a characteristic feature of alpha-synuclein deposited in synucleinopathy brains, we next generated TG worms that pan-neuronally overexpress human G protein-coupled receptor kinase 5 (GRK5), previously reported as one of the alpha-synuclein kinases, and crossed them with the alpha-synculein TG worms. Phosphorylation of alpha-synuclein was upregulated in dopamine or touch neurons of double TG worms compared to those in TG worms that singly overexpress alpha-synuclein, as determined by immunohistochemisty. Behavioral examinations showed aggravations in functions of dopamine neurons or touch neurons overexpressing wt alpha-synuclein upon co-expression of GRK5, which were not observed upon co-expression of kinase-inactive mutant GRK5 (K215R). These results support the notion that missense mutations as well as Ser129 phosphorylation of alpha-synuclein contribute to neuronal dysfunction in PD and DLB. P1-060
APP METABOLISM IN THE BRAIN OF X11-LIKE NULL MUTANT MOUSE 1
2
2
Toshiharu Suzuki , Yoshitake Sano , Shigeyoshi Itohara , Tadashi Nakaya1, 1Hokkaido University, Sapporo, Japan; 2RIKEN, Wako, Japan. Contact e-mail: [email protected] Background: The X11-like (X11L), a neuron-specific adaptor protein containing a phosphotyrosine interaction (PI) domain in its central regions and two repeated PDZ domains in its carboxyl-terminal region, was shown to interact with the cytoplasmic domain of amyloid beta-protein precursor (APP). This interaction suppressed the APP metabolism including betaamyloid (A) production when both proteins expressed in cells (J. Biol. Chem. [1999] 274, 2243; J. Biol. Chem. [2003] 278, 49448). Therefore, X11L is one of possible targets for the development of novel therapeutic drugs that suppresses A production. However, it was not obvious whether endogenous X11L suppresses APP metabolism and A generation in vivo
as well as in cells. Objective(s): To elucidate whether X11L has regulated APP metabolism including A production in brain, we generated X11L null mutant mice. The quality and quantity of APP and its fragments including A in brain of X11L null mutant and wild type mice are analyzed and physiological function of X11L in APP metabolism in brain is assayed. Methods: Genome region including the translation site of X11L was replaced by PGK-neo gene cassette and this cassette was finally excised in null mutant mice. Brain proteins of mutant and wild mice were analyzed for APP, CTF and AICD by Western blotting. A40 and A42 were quantified with sELISA system. Results: The APP metabolism was altered in brain of X11L null mutant mice. Conclusions: The function that X11L regulates APP metabolism including A generation was confirmed in mouse brain. P1-061
ACCELERATED NEURODEGENERATION IN MICE EXPRESSING MUTANT TAU AND APP
Karl-Heinz Wiederhold, Simone Danner, Nella Vidotto, Anne-Lise Jaton, Dorothee Abramowski, Peter Frey, Sabine Leuthaeusser, Matthias Staufenbiel, Novartis Institutes for Biomedical Research Basel, Nervous System, CH-4002 Basel, Switzerland. Contact e-mail: [email protected] Background: Neurofibrillary tangles and amyloid deposits are the defining hallmarks for AD. The amyloid cascade hypothesis predicts that tau pathology is secondary to amyloid aggregation. In agreement, an enhancement of tau pathology has been described in higher brain regions of transgenic mice expressing both, mutant tau and APP as compared to mice carrying the tau transgene only. Objective(s): To further study the interaction of the amyloid and tau pathologies we developed bigenic mouse lines and investigated the time course of tau and amyloid deposition. Methods: TAU58/2 mice expressing mutated human (0N4R P301S) under the control of the Thy-1 promoter were generated. Mice were crossbred with APP23 (Thy1-APPSWE) and APP51/16 (Thy1-APP wildtype) mice and analyzed histologically and by Western blotting. Results: At 6 months of age, APP23xTAU58/2 mice showed an at least fivefold increase of Gallyas positive neurons in neocortex, compared to the single transgene TAU58/2 mice. Neurodegeneration was seen in brain sections stained with the phosphorylation-dependent neurofilament antibody SMI310 and ubiquitin. Numerous axonal dilatations (axonal spheroids) and degenerating neurons were visible in neocortex, midbrain, brain stem and spinal cord. Amyloid pathology was not detectable or very minor at this time point. Conclusions: These data indicate that an enhancement of tau deposition does not require A deposition but may be induced by soluble A species or by APP overexpression. P1-062
ALZHEIMER’S DISEASE-LIKE TAU NEUROPATHOLOGY LEADS TO MEMORY DEFICITS AND LOSS OF FUNCTIONAL SYNAPSES IN A NOVEL MUTATED TAU TRANSGENIC MOUSE WITHOUT ANY MOTOR DEFICITS
Katharina Schindowski1, Alexis Bretteville1, Karelle Leroy2, Severine Begard1, Jean-Pierre Brion2, Malika Hamdane1, Luc Buee1, 1 Inserm U815, Universite Droit et Sante de Lille, Lille-Cedex, France; 2 Laboratory of Histology and Neuropathology, Free University of Brussels, School of Medicine, Brussels, Belgium. Contact e-mail: [email protected] Background: Tau transgenic mice are valuable models to investigate the role of tau protein in Alzheimer’s disease (AD) and other tauopathies. However, motor dysfunction and dystonic posture interfering with behavioral testing are the most common late-life undesirable effects of tau transgenic mice such as any motor impairment severely impairs behavior testing and limits the life span that can be investigated. Objective: To generate a novel transgenic model that shows AD-like neuropathology without displaying any motor impairment even in very old mice. Methods:
Poster Presentations P1
presence of AChE. Ongoing studies will reveal the different interaction of nicotine with amyloid processes in these two transgenic mice models. P1-059
OVEREXPRESSION OF HUMAN ALPHASYNUCLEIN IN TRANSGENIC C. ELEGANS: EFFECTS OF PATHOGENIC MUTATIONS AND PHOSPHORYLATION
Tomoki Kuwahara1, Changhong Ren2, Akihiko Koyama1, Shigeki Arawaka2, Takeo Kato2, Shohei Mitani3, Takeshi Iwatsubo1, 1 Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; 2 Department of Neurology, Hematology, Metabolism, Endocrinology and Diabetology, Yamagata University School of Medicine, Yamagata, Japan; 3Department of Physiology, School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan. Contact e-mail: [email protected] alpha-Synuclein is a component of Lewy bodies in affected neurons of patients with Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and missense mutations or multiplications in alpha-synuclein gene have been identified in pedigrees of familial PD (FPD)/DLB, implicating alpha-synuclein in the pathogenesis of PD and DLB. To gain insights into the mechanism whereby deposition of alpha-synuclein causes neurodegeneration, we established transgenic C. elegans (TG worms) that overexpress wild-type (wt) or FPD-mutant (A53T, A30P) human alpha-synuclein in dopamine neurons. TG worms expressing A53T or A30P FPD mutant alpha-synuclein showed significant defects in dopamine neuron function, the latter being reflected by an adoptive response for feeding (i.e., decrease in bending frequency upon entry into bacterial lawn), whereas those expressing wt alpha-synuclein were normal. We next generated TG worms that overexpress human alpha-synuclein in touch neurons under the control of a mec-7 promoter. These TG worms, especially those expressing A30P FPD mutant alpha-synuclein, showed impaired touch response, whereas those expressing wt alpha-synuclein showed a normal response. To examine the pathogenic significance of alpha-synuclein phosphorylation at Ser129, a characteristic feature of alpha-synuclein deposited in synucleinopathy brains, we next generated TG worms that pan-neuronally overexpress human G protein-coupled receptor kinase 5 (GRK5), previously reported as one of the alpha-synuclein kinases, and crossed them with the alpha-synculein TG worms. Phosphorylation of alpha-synuclein was upregulated in dopamine or touch neurons of double TG worms compared to those in TG worms that singly overexpress alpha-synuclein, as determined by immunohistochemisty. Behavioral examinations showed aggravations in functions of dopamine neurons or touch neurons overexpressing wt alpha-synuclein upon co-expression of GRK5, which were not observed upon co-expression of kinase-inactive mutant GRK5 (K215R). These results support the notion that missense mutations as well as Ser129 phosphorylation of alpha-synuclein contribute to neuronal dysfunction in PD and DLB. P1-060
APP METABOLISM IN THE BRAIN OF X11-LIKE NULL MUTANT MOUSE 1
2
2
Toshiharu Suzuki , Yoshitake Sano , Shigeyoshi Itohara , Tadashi Nakaya1, 1Hokkaido University, Sapporo, Japan; 2RIKEN, Wako, Japan. Contact e-mail: [email protected] Background: The X11-like (X11L), a neuron-specific adaptor protein containing a phosphotyrosine interaction (PI) domain in its central regions and two repeated PDZ domains in its carboxyl-terminal region, was shown to interact with the cytoplasmic domain of amyloid beta-protein precursor (APP). This interaction suppressed the APP metabolism including betaamyloid (A) production when both proteins expressed in cells (J. Biol. Chem. [1999] 274, 2243; J. Biol. Chem. [2003] 278, 49448). Therefore, X11L is one of possible targets for the development of novel therapeutic drugs that suppresses A production. However, it was not obvious whether endogenous X11L suppresses APP metabolism and A generation in vivo
as well as in cells. Objective(s): To elucidate whether X11L has regulated APP metabolism including A production in brain, we generated X11L null mutant mice. The quality and quantity of APP and its fragments including A in brain of X11L null mutant and wild type mice are analyzed and physiological function of X11L in APP metabolism in brain is assayed. Methods: Genome region including the translation site of X11L was replaced by PGK-neo gene cassette and this cassette was finally excised in null mutant mice. Brain proteins of mutant and wild mice were analyzed for APP, CTF and AICD by Western blotting. A40 and A42 were quantified with sELISA system. Results: The APP metabolism was altered in brain of X11L null mutant mice. Conclusions: The function that X11L regulates APP metabolism including A generation was confirmed in mouse brain. P1-061
ACCELERATED NEURODEGENERATION IN MICE EXPRESSING MUTANT TAU AND APP
Karl-Heinz Wiederhold, Simone Danner, Nella Vidotto, Anne-Lise Jaton, Dorothee Abramowski, Peter Frey, Sabine Leuthaeusser, Matthias Staufenbiel, Novartis Institutes for Biomedical Research Basel, Nervous System, CH-4002 Basel, Switzerland. Contact e-mail: [email protected] Background: Neurofibrillary tangles and amyloid deposits are the defining hallmarks for AD. The amyloid cascade hypothesis predicts that tau pathology is secondary to amyloid aggregation. In agreement, an enhancement of tau pathology has been described in higher brain regions of transgenic mice expressing both, mutant tau and APP as compared to mice carrying the tau transgene only. Objective(s): To further study the interaction of the amyloid and tau pathologies we developed bigenic mouse lines and investigated the time course of tau and amyloid deposition. Methods: TAU58/2 mice expressing mutated human (0N4R P301S) under the control of the Thy-1 promoter were generated. Mice were crossbred with APP23 (Thy1-APPSWE) and APP51/16 (Thy1-APP wildtype) mice and analyzed histologically and by Western blotting. Results: At 6 months of age, APP23xTAU58/2 mice showed an at least fivefold increase of Gallyas positive neurons in neocortex, compared to the single transgene TAU58/2 mice. Neurodegeneration was seen in brain sections stained with the phosphorylation-dependent neurofilament antibody SMI310 and ubiquitin. Numerous axonal dilatations (axonal spheroids) and degenerating neurons were visible in neocortex, midbrain, brain stem and spinal cord. Amyloid pathology was not detectable or very minor at this time point. Conclusions: These data indicate that an enhancement of tau deposition does not require A deposition but may be induced by soluble A species or by APP overexpression. P1-062
ALZHEIMER’S DISEASE-LIKE TAU NEUROPATHOLOGY LEADS TO MEMORY DEFICITS AND LOSS OF FUNCTIONAL SYNAPSES IN A NOVEL MUTATED TAU TRANSGENIC MOUSE WITHOUT ANY MOTOR DEFICITS
Katharina Schindowski1, Alexis Bretteville1, Karelle Leroy2, Severine Begard1, Jean-Pierre Brion2, Malika Hamdane1, Luc Buee1, 1 Inserm U815, Universite Droit et Sante de Lille, Lille-Cedex, France; 2 Laboratory of Histology and Neuropathology, Free University of Brussels, School of Medicine, Brussels, Belgium. Contact e-mail: [email protected] Background: Tau transgenic mice are valuable models to investigate the role of tau protein in Alzheimer’s disease (AD) and other tauopathies. However, motor dysfunction and dystonic posture interfering with behavioral testing are the most common late-life undesirable effects of tau transgenic mice such as any motor impairment severely impairs behavior testing and limits the life span that can be investigated. Objective: To generate a novel transgenic model that shows AD-like neuropathology without displaying any motor impairment even in very old mice. Methods: