- Email: [email protected]
Streptozotocin-induced Alzheimer model in cynomolgus monkeys
S210 P1-123
Poster Presentations P1 STREPTOZOTOCIN-INDUCED ALZHEIMER MODEL IN CYNOMOLGUS MONKEYS
Jae-Hyeok Heo1, Kyoung-Min Lee2, Sang-Rae Lee3, Soon-Tae Lee2, JinHwan Oh4, Nam-Beom Kim4, Kyu-Tae Chang3, Zang-Hee Cho4, 1Seoul Medical Center, Seoul, Korea; 2Seoul National University Hospital, Seoul, Korea; 3National Primate Research Center (NPRC), Chungbuk, Korea; 4 Neuroscience Research Institute, Incheon, Korea. Contact e-mail: [email protected] Background: Intracerebroventricular injection of streptozotocin (ICV-STZ) in rats leads to behavioral and pathological changes mimicking Alzheimer’s disease (AD). In this study, we aimed at developing a primate model of AD using similar technique, and monitored metabolic and structural effects of ICV-STZ in cynomolgus monkeys with serial brain 3T MRI and [18F]FDG micro PET/CT images. Methods: Seven cynomolgus monkeys aged 3 years were randomly assigned to one of the following groups: low dose streptozotocin (2mg/kg, n ¼ 2), high dose streptozotocin (5mg/kg, n ¼ 3), or control (N/S 5mg/kg, n ¼ 2). STZ was injected into cerebrospinal fluid (CSF) via the cerebellomedullary cistern (CM) on day 1, 7, and 14. Brain MRIs and micro PET/CT were performed three times: before, 6 weeks after, and 12 weeks after ICV-STZ. Results: In both STZ groups, significant increase in ventricular size and sulcal markings were observed in MRI, consistent with diffuse brain atrophy. Glucose metabolism was also decreased in the cerebral cortex at 6 and 12 weeks after ICV-STZ. T2-high signal intensities were prominent at the periventricular white matter and hippocampal complex in STZ-injected animals, which were dose-dependent: significantly worse in the higher-dose group than in the lower-dose group. Clinically, convulsive seizures were observed in two weeks of ICV-STZ in two animals with higher-dose ICV-STZ, while none were in the lower dose group. No obvious systemic effects were observed in the latter, and post-mortem analysis of one animal in the higher-dose group showed no evidence of systemic derangements due to ICV-STZ, in particular, in pancreatic beta cells. Histological analyses of the brain are currently under way on the injected animals. Conclusions: Injection of STZ into the CSF space in cynomolgus leads to gross morphological and metabolic changes in the brain consistent with AD. Further pathological and behavioral studies are warranted to validate this method for developing a primate AD model. P1-124
TRANSCRIPTIONAL PROFILE OF THE AGING CANINE BRAIN
Eric M. Blalock1, Amy L. S. Dowling1, Sarah Martin1, Jelena Popovic1, Edward G. Barrett2, Elizabeth Head1, 1University of Kentucky, Lexington, KY, USA; 2Lovelace Respiratory Research Institute, Albuquerque, NM, USA. Contact e-mail: [email protected] Background: The canine is a valuable model of human brain aging, developing similar amyloid b (Ab) pathology and cognitive decline. Here we examined gene transcriptional changes across the canine adult lifespan, and compared these findings to prior reports of human aging. Methods: Parietal cortex was dissected from fifteen beagles (young w 4 years, middle aged w 9 years, and old w 12 years; n ¼ 5/ group). Total RNA was isolated and hybridized to GeneChip Canine Genome 2.0 Arrays (Affymetrix N ¼ 15 chips, one chip per subject). Results: 1,983 genes were significantly affected by age (1-way ANOVA; p 0.05; False Discovery Rate ¼ 0.13). As expected, pairwise post hoc comparisons revealed that the majority (w64%) of these changes occurred between young and aged dogs (537 genes up-regulated with age and 724 genes down-regulated with age). Using the DAVID suite of bioinformatic tools, upregulated genes were associated with transcriptional regulation, translation, RNA processing, DNA metabolic processes, and the ribonucleoprotein complex. Downregulated genes were associated with lipid binding, lipid biosynthesis, nervous system development (e.g. drebrin), neurogenesis, cell communication, and long-term potentiation. These results were contrasted with previously published human brain aging transcriptional studies. There was a significant overlap between the two species, particularly with genes related to inflammatory and neuronal processes, with nearly half of all significant genes in the canine study also identified in human aging. Ongoing studies are assessing protein level changes across age, as
well as the association of transcriptional changes to age-related Ab accumulation. Conclusions: The aged canine brain shows gene expression changes consistent with prior studies in human aging and reveals responses and pathways that may be targets of therapeutic interventions. P1-125
EFFECTS OF TEMPEH ON INFLAMMATION AND ANTI-APOPTOSIS EXPRESSION IN SENESCENSE ACCELERATED-PRONE MICE
Yin-Ching Chan1, Wen-Wen Chen1, Ming-Fu Wang1, Juen-Haur Hwang2, 1 Department of Food and Nutrition, Providence University, Taichung, Taiwan; 2Department of Otolaryngology, Buddhist Dalin Tzu-Chi General Hospital, Chiayi, and School of Medicine, Tzu Chi University, Hualien, Taiwan. Contact e-mail: [email protected] Background: Tempeh is a traditional Indonesian fermented soybean food, which showed many beneficial effects including antioxidantive capacities, lipids reduction, tumor inhibition, gastrointestinal symptoms and iron-deficient anemia improvements. However, the effect of tempeh on aging related expression is unclear. In this study, our aim was to evaluate the effects of tempeh on inflammation and anti-apoptosis expression in senescense accelerated-prone mice (SAMP8). Methods: SAMP8 mice were divided into control and 0.45% tempeh groups, while the SAMR1 mice was used as the normal control. The mice were sacrificed after 12 weeks of treatment, and then the immune related expression of brain were analyzed. Results: The TCR Vb, TNFa and IL-1b mRNA expression of tempeh diet group was significantly downregulated when compared with the SAMP8 control group, and was similar with the SAMR1 group. Tempeh diet supplementation also could significantly inhibit the iNOS, COX-2 mRNA expression, whereas did not affect the expression of Bcl-2 mRNA. Conclusions: Tempeh could enhance the mmune properties in SAMP8 mice. However, the influence of tempeh on apoptosis expression needs further study. P1-126
DEVELOPMENT OF A HUMAN NEURONAL MODEL SYSTEM FOR THE IN VITRO CHARACTERIZATION OF PATHOMECHANISMS RELEVANT TO ALZHEIMER’S DISEASE
Diana Scholz, Marcel Leist, University of Konstanz, Konstanz, Germany. Contact e-mail: [email protected] Background: The two major hallmarks of Alzheimer’s disease (AD) are aggregates of the amyloid beta peptide (Abeta) and neurofibrillary tangles, whose main component is the hyperphosphorylated form of the tau protein. In order to model these AD pathologies, transgenic mice or cancer cell lines have been generated with frequent drawbacks being the difficult transferability to humans or the lack of neuronal properties, respectively. As alternative approach, we here use human neurons, differentiated in vitro from the conditionally-immortalized neuronal cell line LUHMES. Methods: For our experiments we applied state-of-the-art methods like scanning electron microscopy, qPCR, immunoblotting/-cytochemistry and ELISA. Results: Initially, we characterized in detail how these cells assume basal neuronal characteristics upon switching off the immortalizing v-myc oncogene. We demonstrated that during five days of differentiation, axonal growth and the formation of an elaborate neurite network take place and that important pre- and postsynaptic marker proteins are upregulated, localizing to neurite processes. Importantly, also the levels of the AD-relevant proteins amyloid precursor protein (APP), beta-secretase (BACE) and y-secretase increase, leading to an enhanced processing of APP into extracellularly measurable Abeta. Our studies revealed that this enhancement is to a great extent dependent on the presence of cyclic adenosine monophosphate (cAMP) and glialcell line-derived neurotrophic factor (GDNF) in the LUHMES culture medium and that it can be stimulated even when cAMP and GDNF are added only after differentiation day 5. In addition to this approach, we successfully modulated Abeta formation by various inhibitors of the secretases (at IC50 values similar to those for primary neurons), BACE knockdown and redistribution of APP and BACE via cholesterol depletion. Finally, we made the first steps to model AD pathologies. We showed that exposing LUHMES cells to stress conditions like acidic pH or nutrient restriction triggers an increased beta-processing of APP without notably changing APP- or BACE-
Poster Presentations P1 STREPTOZOTOCIN-INDUCED ALZHEIMER MODEL IN CYNOMOLGUS MONKEYS
Jae-Hyeok Heo1, Kyoung-Min Lee2, Sang-Rae Lee3, Soon-Tae Lee2, JinHwan Oh4, Nam-Beom Kim4, Kyu-Tae Chang3, Zang-Hee Cho4, 1Seoul Medical Center, Seoul, Korea; 2Seoul National University Hospital, Seoul, Korea; 3National Primate Research Center (NPRC), Chungbuk, Korea; 4 Neuroscience Research Institute, Incheon, Korea. Contact e-mail: [email protected] Background: Intracerebroventricular injection of streptozotocin (ICV-STZ) in rats leads to behavioral and pathological changes mimicking Alzheimer’s disease (AD). In this study, we aimed at developing a primate model of AD using similar technique, and monitored metabolic and structural effects of ICV-STZ in cynomolgus monkeys with serial brain 3T MRI and [18F]FDG micro PET/CT images. Methods: Seven cynomolgus monkeys aged 3 years were randomly assigned to one of the following groups: low dose streptozotocin (2mg/kg, n ¼ 2), high dose streptozotocin (5mg/kg, n ¼ 3), or control (N/S 5mg/kg, n ¼ 2). STZ was injected into cerebrospinal fluid (CSF) via the cerebellomedullary cistern (CM) on day 1, 7, and 14. Brain MRIs and micro PET/CT were performed three times: before, 6 weeks after, and 12 weeks after ICV-STZ. Results: In both STZ groups, significant increase in ventricular size and sulcal markings were observed in MRI, consistent with diffuse brain atrophy. Glucose metabolism was also decreased in the cerebral cortex at 6 and 12 weeks after ICV-STZ. T2-high signal intensities were prominent at the periventricular white matter and hippocampal complex in STZ-injected animals, which were dose-dependent: significantly worse in the higher-dose group than in the lower-dose group. Clinically, convulsive seizures were observed in two weeks of ICV-STZ in two animals with higher-dose ICV-STZ, while none were in the lower dose group. No obvious systemic effects were observed in the latter, and post-mortem analysis of one animal in the higher-dose group showed no evidence of systemic derangements due to ICV-STZ, in particular, in pancreatic beta cells. Histological analyses of the brain are currently under way on the injected animals. Conclusions: Injection of STZ into the CSF space in cynomolgus leads to gross morphological and metabolic changes in the brain consistent with AD. Further pathological and behavioral studies are warranted to validate this method for developing a primate AD model. P1-124
TRANSCRIPTIONAL PROFILE OF THE AGING CANINE BRAIN
Eric M. Blalock1, Amy L. S. Dowling1, Sarah Martin1, Jelena Popovic1, Edward G. Barrett2, Elizabeth Head1, 1University of Kentucky, Lexington, KY, USA; 2Lovelace Respiratory Research Institute, Albuquerque, NM, USA. Contact e-mail: [email protected] Background: The canine is a valuable model of human brain aging, developing similar amyloid b (Ab) pathology and cognitive decline. Here we examined gene transcriptional changes across the canine adult lifespan, and compared these findings to prior reports of human aging. Methods: Parietal cortex was dissected from fifteen beagles (young w 4 years, middle aged w 9 years, and old w 12 years; n ¼ 5/ group). Total RNA was isolated and hybridized to GeneChip Canine Genome 2.0 Arrays (Affymetrix N ¼ 15 chips, one chip per subject). Results: 1,983 genes were significantly affected by age (1-way ANOVA; p 0.05; False Discovery Rate ¼ 0.13). As expected, pairwise post hoc comparisons revealed that the majority (w64%) of these changes occurred between young and aged dogs (537 genes up-regulated with age and 724 genes down-regulated with age). Using the DAVID suite of bioinformatic tools, upregulated genes were associated with transcriptional regulation, translation, RNA processing, DNA metabolic processes, and the ribonucleoprotein complex. Downregulated genes were associated with lipid binding, lipid biosynthesis, nervous system development (e.g. drebrin), neurogenesis, cell communication, and long-term potentiation. These results were contrasted with previously published human brain aging transcriptional studies. There was a significant overlap between the two species, particularly with genes related to inflammatory and neuronal processes, with nearly half of all significant genes in the canine study also identified in human aging. Ongoing studies are assessing protein level changes across age, as
well as the association of transcriptional changes to age-related Ab accumulation. Conclusions: The aged canine brain shows gene expression changes consistent with prior studies in human aging and reveals responses and pathways that may be targets of therapeutic interventions. P1-125
EFFECTS OF TEMPEH ON INFLAMMATION AND ANTI-APOPTOSIS EXPRESSION IN SENESCENSE ACCELERATED-PRONE MICE
Yin-Ching Chan1, Wen-Wen Chen1, Ming-Fu Wang1, Juen-Haur Hwang2, 1 Department of Food and Nutrition, Providence University, Taichung, Taiwan; 2Department of Otolaryngology, Buddhist Dalin Tzu-Chi General Hospital, Chiayi, and School of Medicine, Tzu Chi University, Hualien, Taiwan. Contact e-mail: [email protected] Background: Tempeh is a traditional Indonesian fermented soybean food, which showed many beneficial effects including antioxidantive capacities, lipids reduction, tumor inhibition, gastrointestinal symptoms and iron-deficient anemia improvements. However, the effect of tempeh on aging related expression is unclear. In this study, our aim was to evaluate the effects of tempeh on inflammation and anti-apoptosis expression in senescense accelerated-prone mice (SAMP8). Methods: SAMP8 mice were divided into control and 0.45% tempeh groups, while the SAMR1 mice was used as the normal control. The mice were sacrificed after 12 weeks of treatment, and then the immune related expression of brain were analyzed. Results: The TCR Vb, TNFa and IL-1b mRNA expression of tempeh diet group was significantly downregulated when compared with the SAMP8 control group, and was similar with the SAMR1 group. Tempeh diet supplementation also could significantly inhibit the iNOS, COX-2 mRNA expression, whereas did not affect the expression of Bcl-2 mRNA. Conclusions: Tempeh could enhance the mmune properties in SAMP8 mice. However, the influence of tempeh on apoptosis expression needs further study. P1-126
DEVELOPMENT OF A HUMAN NEURONAL MODEL SYSTEM FOR THE IN VITRO CHARACTERIZATION OF PATHOMECHANISMS RELEVANT TO ALZHEIMER’S DISEASE
Diana Scholz, Marcel Leist, University of Konstanz, Konstanz, Germany. Contact e-mail: [email protected] Background: The two major hallmarks of Alzheimer’s disease (AD) are aggregates of the amyloid beta peptide (Abeta) and neurofibrillary tangles, whose main component is the hyperphosphorylated form of the tau protein. In order to model these AD pathologies, transgenic mice or cancer cell lines have been generated with frequent drawbacks being the difficult transferability to humans or the lack of neuronal properties, respectively. As alternative approach, we here use human neurons, differentiated in vitro from the conditionally-immortalized neuronal cell line LUHMES. Methods: For our experiments we applied state-of-the-art methods like scanning electron microscopy, qPCR, immunoblotting/-cytochemistry and ELISA. Results: Initially, we characterized in detail how these cells assume basal neuronal characteristics upon switching off the immortalizing v-myc oncogene. We demonstrated that during five days of differentiation, axonal growth and the formation of an elaborate neurite network take place and that important pre- and postsynaptic marker proteins are upregulated, localizing to neurite processes. Importantly, also the levels of the AD-relevant proteins amyloid precursor protein (APP), beta-secretase (BACE) and y-secretase increase, leading to an enhanced processing of APP into extracellularly measurable Abeta. Our studies revealed that this enhancement is to a great extent dependent on the presence of cyclic adenosine monophosphate (cAMP) and glialcell line-derived neurotrophic factor (GDNF) in the LUHMES culture medium and that it can be stimulated even when cAMP and GDNF are added only after differentiation day 5. In addition to this approach, we successfully modulated Abeta formation by various inhibitors of the secretases (at IC50 values similar to those for primary neurons), BACE knockdown and redistribution of APP and BACE via cholesterol depletion. Finally, we made the first steps to model AD pathologies. We showed that exposing LUHMES cells to stress conditions like acidic pH or nutrient restriction triggers an increased beta-processing of APP without notably changing APP- or BACE-