- Email: [email protected]
PS1dE9 mice
Poster Presentations P3 P3-371
STIMULATION OF INNATE IMMUNITY VIA TOLLLIKE RECEPTOR 9 MITIGATES ALZHEIMER PATHOLOGY
Henrieta Scholtzova1, Yong Ji1, Fernando Goni1, Yanjie Sun1, Richard Kascsak2, Pankaj D. Mehta2, Daryl S. Spinner2, Thomas Wisniewski3, 1NYU School of Medicine, Department of Neurology, New York, NY, USA; 2New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA; 3NYU School of Medicine, Department of Neurology, Pathology and Psychiatry, New York, NY, USA. Contact e-mail: [email protected] Background: Immunotherapy has a significant impact on Alzheimer’s disease (AD) related pathology. One limitation with immunotherapeutic approaches is clearance of vascular amyloid and associated hemorrhages. This is an important issue since cerebral amyloid angiopathy (CAA) is present in virtually all AD cases and in about 1/3 of cognitively normal elderly individuals. Our research group postulated stimulation of innate immunity as an alternative approach to overcome these side effects. In addition, the effectiveness of this novel approach for tau related pathology has not been addressed. Recently, stimulation of innate immune responses via the Toll-like receptors (TLRs) has emerged as an effective method both for activating macrophages/microglia, and for inducing specific humoral immune responses. Various CpG DNA drugs that are Tolllike receptor 9 (TLR9) agonists are safe in humans and rodents. Methods: We utilized type B CpG oligodeoxynucleotides (ODNs) to stimulate innate immunity in Tg2576 AD mice. In addition, we tested the effect of the TLR9 agonist CpG ODN on microglial uptake and degradation of Ab42 in culture. Bone marrow-derived perivascular macrophages stimulated with CpG ODN may play an important role in the regulation of CAA. We conducted studies to examine the effect of CpG ODN on perivascular macrophages using rhodamine-conjugated dextran amine Mini-Ruby injected into the cisterna magna and confocal microscopy. Results: We found that stimulation of TLR9 signaling led to a remarkable reduction of total amyloid burden, along with Ab oligomers, which correlated with significant behavioral improvements. CpG ODN treatment reduced the CAA burden without any evidence of increased cerebral microhemorrhages. In vitro results indicate that TLR9 activation enhances microglial uptake and degradation of aggregated Ab42. Confocal microscopy images of dextranpositive perivascular macrophages revealed an increase in cortical perivascular macrophages in CpG ODN injected animals compared to control animals, suggesting that these cells are involved in the reduction of CAA. To better evaluate the efficacy of our approach, studies using mouse model of congophilic angiopathy and triple-transgenic AD model which develops plaques and tangles, are underway. Conclusions: Stimulation of innate immunity through TLR9 signaling represents a novel immunotherapeutic approach for AD. Present results provide essential information prior to any clinical use of CpG ODN. P3-372
VALPROIC ACID DOWNREGULATES HO-1 PROTEIN BY REGULATING AN UBIQUITINPROTEOSOMAL PATHWAY
Seol-Heui Han1, Kyoung Ja Kwon1,2, Chan Young Shin3, 1Konkuk University Hospital, Seoul, Korea; 2Center for Geriatric Neuroscience Research, Institute of Biomedical Science and Technology, School of Medicine, Konkuk University, Seol, Korea; 3Konkuk University School of Medicine, Seoul, Korea. Contact e-mail: [email protected] Background: Valproic acid (VPA) is commonly used in the treatment of bipolar disorder and as an anticonvulsant. In the nervous system, it has been reported that VPA increases neuronal growth and synaptogenesis, improves learning and memory, and has neuroprotective effect in acute neuronal injury, such as cerebral ischemia, via activating ERK and GSK-3/Wnt/b-catenin pathway. VPA induces the generation of ROS which subsequently activates the redox-sensitive transcription factors. The Heme oxygenase-1 (HO-1) is an inducible and redoxregulated enzyme and is believed to be a cytoprotective and anti-inflammatory enzyme. The purpose of current investigation was to assess the effect of VPA on the expression level of HO-1 protein, an antioxidant enzyme. Methods: We investigated the effects of VPA on HO-1 protein in primary cortical neurons and astrocytes: 1) the expression levels of HO-1 mRNA and protein measured by RT-PCR and Western blotting; 2) the effects of VPA on cell viability and
S561
ROS generation by MTT reduction assay and ROS measurement; 3) the signal pathway is regulated by VPA using Western blotting and immunoprecipitation. Results: VPA significantly decreased the expression levels of HO-1 protein in concentration-dependent manner in primary cortical neurons (control: 100 6 2.33%, 10uM: 121.4 6 6.19%, 100uM: 99.3 6 2.75%, 200uM: 79.4 6 6.37%, 500uM: 37.4 6 4.40% and 1000uM: 35.5 6 2.16% for 24 hr) and astrocytes (control: 100 6 1.25%, 10uM: 81.2 6 3.88%, 100uM: 67.2 6 2.74%, 200uM: 53.4 6 3.22%, 500uM: 49.8 6 3.17% and 1000uM: 41.9 6 3.24% for 24 hr). Expression level of HO-1 mRNA was not affected by VPA. VPA did not change the cell viability and the ROS generation in primary cortical neurons and astrocytes. An inhibitor of JNK pathway, SP600125, inhibited VPA mediated changes in HO-1 expression. VPA concentration-dependently increased ERK1/ 2 and JNK activation in primary cortical neurons and astrocytes. Reduction of HO-1 expression by VPA was prevented by MG132, a reversible proteosome inhibitor, and VPA increased HO-1 ubiquitination. Conclusions: Based on these findings, our results suggest VPA inhibits the expression of HO-1 protein in rat primary cortical neurons and astrocytes, which is regulated through ubiquitin-proteosomal degradation by JNK pathway. P3-373
EFFECTS OF CHRONIC AND ACUTE SIMVASTATIN ON NEURONAL EXCITABILITY AND LTP IN APPSWE/PS1DE9 MICE
Caroline E. Herron, Charles Metais, University College Dublin, Dublin, Ireland. Contact e-mail: [email protected] Background: Recent studies have investigated the potential therapeutic properties of statins; agents commonly used to treat hypercholesterolemia. Behavioural studies in Tg2576 mice, demonstrated that chronic treatment with simvastatin improved learning and memory (Li et al., 2006; Ann Neurol 60: 729-739). In cultured neurones, chronic mevastatin treatment however depressed NMDA receptor-mediated current yet enhanced voltage-gated sodium channel current (Kannan et al., Neurobiology of Aging 2008). Here we have investigated the effects of acute and chronic simvastatin treatment on synaptic transmission, action potential regulation and synaptic plasticity in the APPswe/ PS1dE9 mouse model. Methods: Mice were treated chronically by including simvastatin in their diet (0.04%) for periods of 3 to 6 months. Alternatively simvastatin was applied via the perfusion solution. Electrophysiological recordings were performed in transverse hippocampal slices from APPswe/PS1dE9 mice and age matched controls. Antidromic compound action potentials (cAPs) were recorded in the CA1 cell body region by stimulating the alveus (in DNQX). Excitatory post synaptic potentials (EPSPs) were evoked in the CA1 region every 30s and paired pulse facilitation (PPF) examined to assess neurotransmitter release. Stable baseline responses were recorded prior to application of simvastatin or LTP induction. LTP was induced using two trains of stimuli at 100Hz for one second applied 30s apart. Results: In control mice (18month), acute application of simvastatin (35mM) caused a significant increase in the amplitude of the cAP, a shift in the input/put put curve and a decrease in PPF while in age matched APPswe/PS1dE9 mice acute simvastatin had no affect. A lower concentration (10mM) also caused an increased in cAP amplitude which was absent in slices from APPswe/PS1dE9 mice.This increased excitability is blocked by antagonists of PI3 kinase. Acute simvastatin application also had no effect in slices from animals previously treated chronically with simvastatin in their diet. In addition, chronic simvastatin treatment rescued LTP in APPSwe/PS1dE9 mice (18 and 8 months old) however it failed to rescue STP. Conclusions: Chronic simvastatin treatment can therefore rescue LTP in hippocampal CA1 in APPswe/PS1dE9 mice. In addition, acute simvastatin enhances neuronal excitability via a PI3 kinase dependent mechanism which is absent in slices from APPswe/PS1dE9 mice. P3-374
A MEDICAL FOOD SOUVENAIDÒ FOR ALZHEIMER PATIENTS CAN BE USED SAFELY IN COMBINATION WITH AN ACETYLCHOLINESTERASE INHIBITOR IN AN AGED RAT MODEL
Martijn C. de Wilde1, Nick van Wijk1, Almar A. M. Kuipers1, Laus M. Broersen1, Patrick J. Kamphuis1, Jossie A. Garthoff1, Gerrit J. A. Speijers2, 1Nutricia Advanced Medical Nutrition, Danone
STIMULATION OF INNATE IMMUNITY VIA TOLLLIKE RECEPTOR 9 MITIGATES ALZHEIMER PATHOLOGY
Henrieta Scholtzova1, Yong Ji1, Fernando Goni1, Yanjie Sun1, Richard Kascsak2, Pankaj D. Mehta2, Daryl S. Spinner2, Thomas Wisniewski3, 1NYU School of Medicine, Department of Neurology, New York, NY, USA; 2New York State Institute for Basic Research in Developmental Disabilities, New York, NY, USA; 3NYU School of Medicine, Department of Neurology, Pathology and Psychiatry, New York, NY, USA. Contact e-mail: [email protected] Background: Immunotherapy has a significant impact on Alzheimer’s disease (AD) related pathology. One limitation with immunotherapeutic approaches is clearance of vascular amyloid and associated hemorrhages. This is an important issue since cerebral amyloid angiopathy (CAA) is present in virtually all AD cases and in about 1/3 of cognitively normal elderly individuals. Our research group postulated stimulation of innate immunity as an alternative approach to overcome these side effects. In addition, the effectiveness of this novel approach for tau related pathology has not been addressed. Recently, stimulation of innate immune responses via the Toll-like receptors (TLRs) has emerged as an effective method both for activating macrophages/microglia, and for inducing specific humoral immune responses. Various CpG DNA drugs that are Tolllike receptor 9 (TLR9) agonists are safe in humans and rodents. Methods: We utilized type B CpG oligodeoxynucleotides (ODNs) to stimulate innate immunity in Tg2576 AD mice. In addition, we tested the effect of the TLR9 agonist CpG ODN on microglial uptake and degradation of Ab42 in culture. Bone marrow-derived perivascular macrophages stimulated with CpG ODN may play an important role in the regulation of CAA. We conducted studies to examine the effect of CpG ODN on perivascular macrophages using rhodamine-conjugated dextran amine Mini-Ruby injected into the cisterna magna and confocal microscopy. Results: We found that stimulation of TLR9 signaling led to a remarkable reduction of total amyloid burden, along with Ab oligomers, which correlated with significant behavioral improvements. CpG ODN treatment reduced the CAA burden without any evidence of increased cerebral microhemorrhages. In vitro results indicate that TLR9 activation enhances microglial uptake and degradation of aggregated Ab42. Confocal microscopy images of dextranpositive perivascular macrophages revealed an increase in cortical perivascular macrophages in CpG ODN injected animals compared to control animals, suggesting that these cells are involved in the reduction of CAA. To better evaluate the efficacy of our approach, studies using mouse model of congophilic angiopathy and triple-transgenic AD model which develops plaques and tangles, are underway. Conclusions: Stimulation of innate immunity through TLR9 signaling represents a novel immunotherapeutic approach for AD. Present results provide essential information prior to any clinical use of CpG ODN. P3-372
VALPROIC ACID DOWNREGULATES HO-1 PROTEIN BY REGULATING AN UBIQUITINPROTEOSOMAL PATHWAY
Seol-Heui Han1, Kyoung Ja Kwon1,2, Chan Young Shin3, 1Konkuk University Hospital, Seoul, Korea; 2Center for Geriatric Neuroscience Research, Institute of Biomedical Science and Technology, School of Medicine, Konkuk University, Seol, Korea; 3Konkuk University School of Medicine, Seoul, Korea. Contact e-mail: [email protected] Background: Valproic acid (VPA) is commonly used in the treatment of bipolar disorder and as an anticonvulsant. In the nervous system, it has been reported that VPA increases neuronal growth and synaptogenesis, improves learning and memory, and has neuroprotective effect in acute neuronal injury, such as cerebral ischemia, via activating ERK and GSK-3/Wnt/b-catenin pathway. VPA induces the generation of ROS which subsequently activates the redox-sensitive transcription factors. The Heme oxygenase-1 (HO-1) is an inducible and redoxregulated enzyme and is believed to be a cytoprotective and anti-inflammatory enzyme. The purpose of current investigation was to assess the effect of VPA on the expression level of HO-1 protein, an antioxidant enzyme. Methods: We investigated the effects of VPA on HO-1 protein in primary cortical neurons and astrocytes: 1) the expression levels of HO-1 mRNA and protein measured by RT-PCR and Western blotting; 2) the effects of VPA on cell viability and
S561
ROS generation by MTT reduction assay and ROS measurement; 3) the signal pathway is regulated by VPA using Western blotting and immunoprecipitation. Results: VPA significantly decreased the expression levels of HO-1 protein in concentration-dependent manner in primary cortical neurons (control: 100 6 2.33%, 10uM: 121.4 6 6.19%, 100uM: 99.3 6 2.75%, 200uM: 79.4 6 6.37%, 500uM: 37.4 6 4.40% and 1000uM: 35.5 6 2.16% for 24 hr) and astrocytes (control: 100 6 1.25%, 10uM: 81.2 6 3.88%, 100uM: 67.2 6 2.74%, 200uM: 53.4 6 3.22%, 500uM: 49.8 6 3.17% and 1000uM: 41.9 6 3.24% for 24 hr). Expression level of HO-1 mRNA was not affected by VPA. VPA did not change the cell viability and the ROS generation in primary cortical neurons and astrocytes. An inhibitor of JNK pathway, SP600125, inhibited VPA mediated changes in HO-1 expression. VPA concentration-dependently increased ERK1/ 2 and JNK activation in primary cortical neurons and astrocytes. Reduction of HO-1 expression by VPA was prevented by MG132, a reversible proteosome inhibitor, and VPA increased HO-1 ubiquitination. Conclusions: Based on these findings, our results suggest VPA inhibits the expression of HO-1 protein in rat primary cortical neurons and astrocytes, which is regulated through ubiquitin-proteosomal degradation by JNK pathway. P3-373
EFFECTS OF CHRONIC AND ACUTE SIMVASTATIN ON NEURONAL EXCITABILITY AND LTP IN APPSWE/PS1DE9 MICE
Caroline E. Herron, Charles Metais, University College Dublin, Dublin, Ireland. Contact e-mail: [email protected] Background: Recent studies have investigated the potential therapeutic properties of statins; agents commonly used to treat hypercholesterolemia. Behavioural studies in Tg2576 mice, demonstrated that chronic treatment with simvastatin improved learning and memory (Li et al., 2006; Ann Neurol 60: 729-739). In cultured neurones, chronic mevastatin treatment however depressed NMDA receptor-mediated current yet enhanced voltage-gated sodium channel current (Kannan et al., Neurobiology of Aging 2008). Here we have investigated the effects of acute and chronic simvastatin treatment on synaptic transmission, action potential regulation and synaptic plasticity in the APPswe/ PS1dE9 mouse model. Methods: Mice were treated chronically by including simvastatin in their diet (0.04%) for periods of 3 to 6 months. Alternatively simvastatin was applied via the perfusion solution. Electrophysiological recordings were performed in transverse hippocampal slices from APPswe/PS1dE9 mice and age matched controls. Antidromic compound action potentials (cAPs) were recorded in the CA1 cell body region by stimulating the alveus (in DNQX). Excitatory post synaptic potentials (EPSPs) were evoked in the CA1 region every 30s and paired pulse facilitation (PPF) examined to assess neurotransmitter release. Stable baseline responses were recorded prior to application of simvastatin or LTP induction. LTP was induced using two trains of stimuli at 100Hz for one second applied 30s apart. Results: In control mice (18month), acute application of simvastatin (35mM) caused a significant increase in the amplitude of the cAP, a shift in the input/put put curve and a decrease in PPF while in age matched APPswe/PS1dE9 mice acute simvastatin had no affect. A lower concentration (10mM) also caused an increased in cAP amplitude which was absent in slices from APPswe/PS1dE9 mice.This increased excitability is blocked by antagonists of PI3 kinase. Acute simvastatin application also had no effect in slices from animals previously treated chronically with simvastatin in their diet. In addition, chronic simvastatin treatment rescued LTP in APPSwe/PS1dE9 mice (18 and 8 months old) however it failed to rescue STP. Conclusions: Chronic simvastatin treatment can therefore rescue LTP in hippocampal CA1 in APPswe/PS1dE9 mice. In addition, acute simvastatin enhances neuronal excitability via a PI3 kinase dependent mechanism which is absent in slices from APPswe/PS1dE9 mice. P3-374
A MEDICAL FOOD SOUVENAIDÒ FOR ALZHEIMER PATIENTS CAN BE USED SAFELY IN COMBINATION WITH AN ACETYLCHOLINESTERASE INHIBITOR IN AN AGED RAT MODEL
Martijn C. de Wilde1, Nick van Wijk1, Almar A. M. Kuipers1, Laus M. Broersen1, Patrick J. Kamphuis1, Jossie A. Garthoff1, Gerrit J. A. Speijers2, 1Nutricia Advanced Medical Nutrition, Danone