- Email: [email protected]
Carnosine inhibits ß-sheet formation of PrP106-126 and protects against its neurotoxicity
P300
Poster Presentations P2
Background: The frequent co-occurrence of Alzheimer’s disease (AD) pathology in patients with clinically diagnosed normal pressure hydrocephalus suggests a possible link between ventricular dilation and AD. If enlarging ventricles serve as a marker of potentially faulty cerebrospinal fluid (CSF) clearance mechanisms, then a relationship may be demonstrable between increasing ventricular volume and decreasing levels of A-beta in CSF in preclinical and early AD. Methods: CSF biomarker data (A-beta, tau, and phosphorylated tau) as well as direct measurements of whole brain and ventricular volume data were obtained from the Alzheimer’s Disease Neuroimaging Initiative dataset. The ratio of ventricular volume to whole brain volume was derived as a secondary independent measure. Baseline data were used for the group analyses of 819 subjects classified as being either normal (n¼229), having the syndrome of mild cognitive impairment (n¼397), or mild AD (n¼193). Linear regression models were derived for each biomarker as the dependent variable, using the MRI volume measures and age as independent variables. Subjects in each diagnostic group were further stratified by ApoE genotype. Results: For controls, ventricular volume was negatively associated with A-beta in E4 positive subjects, independent of age. Ventricular volume as well as age was negatively associated with tau in E4 negative control subjects. A significant reversed pattern was seen in AD subjects, in whom ventricular volume was negatively associated with tau, but not A-beta, in E4 positive subjects, independent of age. Abeta and tau were not significantly related to whole brain volume in either controls or AD; however, tau was negatively related to whole brain volume in MCI subjects. Conclusions: Increased ventricular volume may be associated with decreased levels of CSF A-beta in preclinical AD. The basis for the apparent effect of ApoE4 genotype on the relationship of ventricular volume to A-beta and tau levels is unknown, but could involve altered CSF-bloodbrain barrier function early in the course of the disease. Further experimental evidence with animal models of hydrocephalus and ApoE may shed light on the exact nature of these relationships. P2-132
DRASTIC IMPACT ON ABETA ACCUMULATION IN APP TG MICE BY GENETIC BACKGROUND
Mikiko Yokokoji, Takashi Morihara, Noriyuki Hayashi, Shinji Tagami, Masatoshi Takeda, Osaka University, Suita City, Japan. Contact e-mail: [email protected] Background: Little attention has been given to the genetic background of APP Tg mice. Each mouse strain has different phenotypes, including hippocampal commissure index, insulin (ng/mL), triglycerides (mg/dL), length of anterior commissure at midsagittal plane (mm). To examine whether genetic background affect on amyloid pathology, APP Tg mice were crossed onto three different strains. Methods: APP Tg mice (Tg2576) which are usually maintained by B6/SJL hybrid mice were crossed onto C57BL6/J, SJL/J and DBA2/J inbred strains.The levels of Abeta40 and Abeta42 in Triton and GuHCl fraction from cortex were measured by ELISA. The mRNA level of APP was measured in APP Tg mice and wild type mice. Results: The shortened life span were observed in APP Tg mice with B6 rich genetic background (B6 75%, SJL 25%), which was similar to previous report (Carlson Hum Mol Genet 1997). No significant life span change, however, was observed in the mice with either SJL or DBA rich genetic background. Analysis of 59 APP Tg mice at the age of 12 months showed that lower Abeta accumulation in mice with DBA rich genetic background compared to SJL or B6 rich genetic background. For example,significant difference (p<0.01, TurkeyKramer) were observed in Abeta40 (-82%)and Abeta42 (-68%) levels in Triton fraction between SJL84% B6 16% mice and DBA75% B6 16% SJL 9%mice. Drastic difference (p<0.01, Turkey-Kramer) were observed in Abeta40 and Abeta42 levels also from GuHCl fraction between SJL 69% B6 31% and DBA 75% B6 16% SJL 9% mice (-85% and -68%) and between SJL 69% B6 31% and DBA 50% B6 31% SJL 19% mice (-75% and -50%). We focus on SJL and DBA, because the difference of Abeta accumulation was most drastic and both genetic backgrounds have no impact on life span. The mRNA level of APP was same in APP Tg mice and wild type mice. Conclusions: Our data demonstrated that genetic background impact on amyloid pathology inAPP Tg mice. These mice can be useful to find the molecular pathways related to amyloid pathology.
P2-133
CARNOSINE INHIBITS ß-SHEET FORMATION OF PRP106-126 AND PROTECTS AGAINST ITS NEUROTOXICITY
Masahiro Kawahara, Tetsuya Nagata, Keiko Konoha, Yutaka Sadakane, Kyushu University of Health and Welfare, Nobeoka-city, MIyazaki, Japan. Contact e-mail: [email protected] Background: Prion diseases, including human Kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy, are associated with conversion of a normal prion protein (PrPC) to an abnormal scrapie isoform (PrPSC). The ß-sheet region of PrPSC is suggested to play a crucial role in its transmissible degenerative processes. A peptide fragment corresponding to residues 106126 (PrP106-126) coincides with the proposed ß-sheet structures and has been reported to cause death in cultured hippocampal neurons. PrP is a copper binding protein, and therefore, it has been hypothesized that dyshomeostasis of trace metals such as zinc or copper may be based on the neurotoxicity of PrP. Methods: We have investigated the molecular mechanism of neurotoxicity of PrP106-126 on cultured rat hippocampal neurons using the LDH method. Conformational changes of PrP106-126 were observed by the thioflavin T method and by the CD-spectroscopy. Results: Aging (incubation at 37 C for several days) increased ß-sheet formation of PrP106-126 and enhanced its neurotoxicity. Co-existence of copper or zinc attenuated PrP106-126 induced neuronal death. Copper-treated PrP106-126 did not exhibit the ß-sheet structures. Carnosine (ß-alanyl histidine) is a dipeptide which exhibits the chelating ability as well as the antioxidant activity. We have already demonstrated that carnosine protects against zinc-induced neurotoxicity and becomes a possible candidate for the treatment of ischemia-induced neurodegeneration. We have demonstrated that co-existence of carnosine inhibited the formation of ß-sheet structure of PrP106-126 and attenuated its neurotoxicity. Conclusions: Our results suggest the implication of trace metals and carnosine in PrP neurotoxicity. Considering the accumulation of carnosine in olfactory neurons, carnosine may play as an endogenous neuronal protective substance with anti-crosslinking ability. It is also suggested that carnosine may become a seed of the new treatment for prion diseases. P2-134
INTERACTION OF ENGULFMENT ADAPTOR PROTEIN (GULP) AND AMYLOID PRECURSOR PROTEIN (APP) DECREASES Ab40/42 SECRETION
Anja-Silke Beyer1, B. von Einem2, D. Schwanzar2, D. Thal2, A. Makarova3, M. Deng3, B. T. Hyman3, C. A. F. von Arnim2, 1Dpt. of Neurology, Ulm University Hospital, Ulm, Germany; 2Ulm University, Ulm, Germany; 3 Alzheimer’s Disease Research Laboratory, Harvard Medical School, Charlestown, MA, USA. Contact e-mail: [email protected] Background: APPs intracellular domain contains a highly conserved YENPTY-motif present in the cytodomains of several receptors that serves as a docking site for phosphotyrosine binding (PTB) domain of adaptor proteins like Fe65, X11/MINT, Numb, JIP1b, and Dab1. Such adaptors play critical roles in tyrosine kinase mediated signal transduction, protein trafficking, phagocytosis, and neuronal development. Most importantly in AD, each of those proteins can impact APP trafficking and processing. Previous studies identified the PTB domain containing engulfment adaptor protein GULP/ hCED6 as an LRP/CD91 NPXY-motif interacting protein. Although there is some information on the function of CED-6 during development in C. elegans and drosophila, there is much less known about GULP in mammalian mature brain. Methods: Using immunohistochemical staining of human hippocampus slices, immunoprecipitation and confocal live cell imaging (CLSM) approaches we analyzed whether GULP and APP are physiologically co-expressed and whether they co-localize within same cellular compartments. The biophysical interaction was analyzed by immunoprecipitation of overexpressed or endogenous proteins. Interaction sites were determined by point mutation within the YENPTY-motif of APP and deletion of GULPs PTB domain. Input upon APP processing was investigated by ELISA (Ab40/42) and cell surface protein biotinylation assay. Results: Here we demonstrate that GULP is expressed in mammalian brain and APP and GULP co-localize in vitro and in vivo. Analysis of the interacting domains of both proteins by point and
Poster Presentations P2
Background: The frequent co-occurrence of Alzheimer’s disease (AD) pathology in patients with clinically diagnosed normal pressure hydrocephalus suggests a possible link between ventricular dilation and AD. If enlarging ventricles serve as a marker of potentially faulty cerebrospinal fluid (CSF) clearance mechanisms, then a relationship may be demonstrable between increasing ventricular volume and decreasing levels of A-beta in CSF in preclinical and early AD. Methods: CSF biomarker data (A-beta, tau, and phosphorylated tau) as well as direct measurements of whole brain and ventricular volume data were obtained from the Alzheimer’s Disease Neuroimaging Initiative dataset. The ratio of ventricular volume to whole brain volume was derived as a secondary independent measure. Baseline data were used for the group analyses of 819 subjects classified as being either normal (n¼229), having the syndrome of mild cognitive impairment (n¼397), or mild AD (n¼193). Linear regression models were derived for each biomarker as the dependent variable, using the MRI volume measures and age as independent variables. Subjects in each diagnostic group were further stratified by ApoE genotype. Results: For controls, ventricular volume was negatively associated with A-beta in E4 positive subjects, independent of age. Ventricular volume as well as age was negatively associated with tau in E4 negative control subjects. A significant reversed pattern was seen in AD subjects, in whom ventricular volume was negatively associated with tau, but not A-beta, in E4 positive subjects, independent of age. Abeta and tau were not significantly related to whole brain volume in either controls or AD; however, tau was negatively related to whole brain volume in MCI subjects. Conclusions: Increased ventricular volume may be associated with decreased levels of CSF A-beta in preclinical AD. The basis for the apparent effect of ApoE4 genotype on the relationship of ventricular volume to A-beta and tau levels is unknown, but could involve altered CSF-bloodbrain barrier function early in the course of the disease. Further experimental evidence with animal models of hydrocephalus and ApoE may shed light on the exact nature of these relationships. P2-132
DRASTIC IMPACT ON ABETA ACCUMULATION IN APP TG MICE BY GENETIC BACKGROUND
Mikiko Yokokoji, Takashi Morihara, Noriyuki Hayashi, Shinji Tagami, Masatoshi Takeda, Osaka University, Suita City, Japan. Contact e-mail: [email protected] Background: Little attention has been given to the genetic background of APP Tg mice. Each mouse strain has different phenotypes, including hippocampal commissure index, insulin (ng/mL), triglycerides (mg/dL), length of anterior commissure at midsagittal plane (mm). To examine whether genetic background affect on amyloid pathology, APP Tg mice were crossed onto three different strains. Methods: APP Tg mice (Tg2576) which are usually maintained by B6/SJL hybrid mice were crossed onto C57BL6/J, SJL/J and DBA2/J inbred strains.The levels of Abeta40 and Abeta42 in Triton and GuHCl fraction from cortex were measured by ELISA. The mRNA level of APP was measured in APP Tg mice and wild type mice. Results: The shortened life span were observed in APP Tg mice with B6 rich genetic background (B6 75%, SJL 25%), which was similar to previous report (Carlson Hum Mol Genet 1997). No significant life span change, however, was observed in the mice with either SJL or DBA rich genetic background. Analysis of 59 APP Tg mice at the age of 12 months showed that lower Abeta accumulation in mice with DBA rich genetic background compared to SJL or B6 rich genetic background. For example,significant difference (p<0.01, TurkeyKramer) were observed in Abeta40 (-82%)and Abeta42 (-68%) levels in Triton fraction between SJL84% B6 16% mice and DBA75% B6 16% SJL 9%mice. Drastic difference (p<0.01, Turkey-Kramer) were observed in Abeta40 and Abeta42 levels also from GuHCl fraction between SJL 69% B6 31% and DBA 75% B6 16% SJL 9% mice (-85% and -68%) and between SJL 69% B6 31% and DBA 50% B6 31% SJL 19% mice (-75% and -50%). We focus on SJL and DBA, because the difference of Abeta accumulation was most drastic and both genetic backgrounds have no impact on life span. The mRNA level of APP was same in APP Tg mice and wild type mice. Conclusions: Our data demonstrated that genetic background impact on amyloid pathology inAPP Tg mice. These mice can be useful to find the molecular pathways related to amyloid pathology.
P2-133
CARNOSINE INHIBITS ß-SHEET FORMATION OF PRP106-126 AND PROTECTS AGAINST ITS NEUROTOXICITY
Masahiro Kawahara, Tetsuya Nagata, Keiko Konoha, Yutaka Sadakane, Kyushu University of Health and Welfare, Nobeoka-city, MIyazaki, Japan. Contact e-mail: [email protected] Background: Prion diseases, including human Kuru, Creutzfeldt-Jakob disease, and bovine spongiform encephalopathy, are associated with conversion of a normal prion protein (PrPC) to an abnormal scrapie isoform (PrPSC). The ß-sheet region of PrPSC is suggested to play a crucial role in its transmissible degenerative processes. A peptide fragment corresponding to residues 106126 (PrP106-126) coincides with the proposed ß-sheet structures and has been reported to cause death in cultured hippocampal neurons. PrP is a copper binding protein, and therefore, it has been hypothesized that dyshomeostasis of trace metals such as zinc or copper may be based on the neurotoxicity of PrP. Methods: We have investigated the molecular mechanism of neurotoxicity of PrP106-126 on cultured rat hippocampal neurons using the LDH method. Conformational changes of PrP106-126 were observed by the thioflavin T method and by the CD-spectroscopy. Results: Aging (incubation at 37 C for several days) increased ß-sheet formation of PrP106-126 and enhanced its neurotoxicity. Co-existence of copper or zinc attenuated PrP106-126 induced neuronal death. Copper-treated PrP106-126 did not exhibit the ß-sheet structures. Carnosine (ß-alanyl histidine) is a dipeptide which exhibits the chelating ability as well as the antioxidant activity. We have already demonstrated that carnosine protects against zinc-induced neurotoxicity and becomes a possible candidate for the treatment of ischemia-induced neurodegeneration. We have demonstrated that co-existence of carnosine inhibited the formation of ß-sheet structure of PrP106-126 and attenuated its neurotoxicity. Conclusions: Our results suggest the implication of trace metals and carnosine in PrP neurotoxicity. Considering the accumulation of carnosine in olfactory neurons, carnosine may play as an endogenous neuronal protective substance with anti-crosslinking ability. It is also suggested that carnosine may become a seed of the new treatment for prion diseases. P2-134
INTERACTION OF ENGULFMENT ADAPTOR PROTEIN (GULP) AND AMYLOID PRECURSOR PROTEIN (APP) DECREASES Ab40/42 SECRETION
Anja-Silke Beyer1, B. von Einem2, D. Schwanzar2, D. Thal2, A. Makarova3, M. Deng3, B. T. Hyman3, C. A. F. von Arnim2, 1Dpt. of Neurology, Ulm University Hospital, Ulm, Germany; 2Ulm University, Ulm, Germany; 3 Alzheimer’s Disease Research Laboratory, Harvard Medical School, Charlestown, MA, USA. Contact e-mail: [email protected] Background: APPs intracellular domain contains a highly conserved YENPTY-motif present in the cytodomains of several receptors that serves as a docking site for phosphotyrosine binding (PTB) domain of adaptor proteins like Fe65, X11/MINT, Numb, JIP1b, and Dab1. Such adaptors play critical roles in tyrosine kinase mediated signal transduction, protein trafficking, phagocytosis, and neuronal development. Most importantly in AD, each of those proteins can impact APP trafficking and processing. Previous studies identified the PTB domain containing engulfment adaptor protein GULP/ hCED6 as an LRP/CD91 NPXY-motif interacting protein. Although there is some information on the function of CED-6 during development in C. elegans and drosophila, there is much less known about GULP in mammalian mature brain. Methods: Using immunohistochemical staining of human hippocampus slices, immunoprecipitation and confocal live cell imaging (CLSM) approaches we analyzed whether GULP and APP are physiologically co-expressed and whether they co-localize within same cellular compartments. The biophysical interaction was analyzed by immunoprecipitation of overexpressed or endogenous proteins. Interaction sites were determined by point mutation within the YENPTY-motif of APP and deletion of GULPs PTB domain. Input upon APP processing was investigated by ELISA (Ab40/42) and cell surface protein biotinylation assay. Results: Here we demonstrate that GULP is expressed in mammalian brain and APP and GULP co-localize in vitro and in vivo. Analysis of the interacting domains of both proteins by point and