- Email: [email protected]
Microelectrode arrays and Alzheimer's disease: A novel in vitro investigative paradigm
P428
Poster Presentations P3
Ab-40 and Ab-42 monomers. The dissociation constant of anti-amyloid antibodies in the D-IgG fraction was determined by Surface Plasmon Resonance (SPR). As a probe of polyvalency, direct binding and Ab competition ELISAs were performed using thyroglobulin as the competing antigen. Results: Dimeric IgG comprised 8-17% of the total IgG in various IVIG preparations. The Ab-binding specific activity of D-IgG was greater than that of M-IgG by a factor of 10.5. The dissociation constant for D-IgG on the Ab monomer SPR sensor was 1.4 micromolar. Thyroglobulin reduced binding to Ab ELISA monomer plates by 70% suggesting that IVIG contains anti-monomer antibodies that cross-react with non-Ab antigens. Conclusions: The dimer fraction of IVIG possesses significant Ab binding capacity. However, anti-monomer antibodies in the dimer fraction have relatively low affinity for Ab and may be cross-reactive with non-amyloid antigens. Future studies will address the extent to which Ab binding by antibodies in the monomeric and dimeric fractions contributes to IVIG’s therapeutic effects in AD patients.
P3-289
MICROELECTRODE ARRAYS AND ALZHEIMER’S DISEASE: A NOVEL IN VITRO INVESTIGATIVE PARADIGM
Kucku Varghese1, Peter Molnar1, Mainak Das1, Mark S. Kindy2, Bruce C. Wheeler3, James J. Hickman1,2, 1University of Central Florida, Orlando, FL, USA; 2Medical University of South Carolina, Charleston, SC, USA; 3University of Florida, Gainesville, FL, USA. Contact e-mail: [email protected] Background: Soluble oligomers of amyloid beta (Ab) are considered to be one of the major contributing factors to the development of Alzheimer’s disease. Most therapeutic development studies have focused on toxicity directly at the synapse. Using patch clamp studies we have demonstrated that soluble Ab can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of synapses as well, but the effect on function may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs). Methods: Ab oligomers were synthesized using an established protocol and characterized using western blots. Embryonic hippocampal neurons were plated on MEAs and after 14 days in vitro were treated with either 100 nM Ab oligomers alone or a combination of Ab and curcumin, an Ab oligomerization inhibitor depending on the experimental model. Recordings were obtained over this period of time in order to determine the time course of both compounds. The observed results were compared to patch clamp data obtained from similar experiments. Cell death was quantified using a live dead assay. Results: When applied to hippocampal neurons cultured on MEAs, Ab had a pronounced effect on the spontaneous firing of the cells, even at concentrations in the nanomolar range. Treatment with Ab stopped spontaneous activity completely and the time until cessation was concentration dependent. Curcumin was able to partially reverse the loss of spontaneous activity, in accordance with our earlier patch clamp experiments. Conclusions: This study demonstrated that it is possible to develop a high-throughput screen for the measurement of a drug’s effect on functional toxicity of low concentrations of Ab and this model is the first step for an in vitro functional model of the development of Alzheimer’s Disease. This screening method, based on MEA technology, could find important applications in pharmaceutical drug development and could lead to novel drug candidates or therapies for AD.
P3-290
ABCA1 IS REQUIRED FOR THE BENEFICIAL EFFECTS OF THE LIVER-X-RECEPTOR AGONIST GW3965 ON BIOCHEMICAL AND COGNITIVE OUTCOMES IN THE APP/PS1 MOUSE MODEL OF ALZHEIMER’S DISEASE
James Donkin1, Sophie Stukas1, Dhananjay Namjoshi1, Veronica HirschReinshagen1, Jeniffer Chan1, Anna Wilkinson1, Jon Collins2,
Cheryl L. Wellington1, 1UBC, Vancouver, BC, Canada; 2GlaxoSmithKline, Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: Apolipoprotein E (apoE) the major cholesterol carrier in the brain is also the most validated genetic risk factor for late-onset Alzheimer’s Disease (AD). We and others have contributed to a growing body of literature demonstrating that the amount of lipids carried by apoE is a major determinant of Ab and amyloid metabolism. The cholesterol transporter ABCA1 is responsible for moving lipids onto brain apoE. ABCA1 deficiency exacerbates amyloidogenesis, whereas selective overexpression of ABCA1 is sufficient to ameliorate amyloid burden in AD mice. Liver X Receptor (LXR) agonists such as GW3965, which stimulate the expression of several genes including ABCA1 and apoE, reduce Ab levels and protect against cognitive deficits in AD mice. Methods: In this study, we tested the hypothesis that ABCA1 is required for the beneficial effects of GW3965 in AD mice by evaluating behavioural and neuropathogical outcomes in GW3965treated APP/PS1 mice with and without ABCA1. Results: Here we report that both therapeutic and prophylactic treatment with the LXR agonist GW3965 exhibits a significant improvement in cognition as demonstrated in the Novel Object Recognition and Morris Water maze tasks in the presence of ABCA1. Furthermore, ABCA1 is required to increase apoE levels and lipidation in cerebrospinal fluid in response to GW3965. We are currently performing biochemical and neuropathological analysis of the effect of GW3965 on cortical and hippocampal Ab levels and amyloid burden. Conclusions: Our results suggest that ABCA1-mediated lipidation of apoE is a crucial mechanism underlying the beneficial effects of LXR agonists on cognitive behaviour and Ab metabolism and highlights ABCA1 as a potential AD therapeutic target.
P3-291
PUTATIVE GAMMA SECRETASE MODULATORS LOWER Ab42 IN MULTIPLE IN VITRO AND IN VIVO MODELS
Kathryn Rogers1, Richard Chesworth1, Kevin Felsenstein1, Gideon Shapiro1, Faris Albayya1, Zhiming Tu1, Darcie Spaulding1, Florentina Catana1, Lori Hrdlicka1, Scott Nolan1, Melody Wen1, Zhiyong Yang1, Veerle Vulsteke2, Holger Patzke1, Gerhard Koenig1, Bart DeStrooper2, Michael Ahlijanian1, 1EnVivo Pharma, Watertown, MA, USA; 2VIB, Leuven, Belgium. Contact e-mail: [email protected] Background: Inhibition of gamma secretase (GS) has been the focus of multiple drug discovery efforts over the last decade. However, the successful identification of potent gamma secretase inhibitors (GSIs) also led to the discovery of unacceptable side effects associated with this mechanism, i.e., the concomitant inhibition of Notch processing by GS. The observation that some non-steroidal anti-inflammatory drugs (NSAIDs) selectively inhibit the production of toxic Ab42 while sparing Ab and Notch has prompted the development of a class of compounds commonly referred to as gamma secretase modulators (GSMs). Methods: Using solid phase extraction in combination with an ELISA assay that utilized antibodies selective for Ab42, Ab40, Ab38, or total Ab peptides, this study describes presumptive GSMs that lower Ab42 without altering total Ab in a HEKswe_APP cell line, rat cortical cultures, and normal mice or rats. Western blot analysis of HEK293 cells transfected with NotchDE was used to quantify the effect of compounds on Notch processing. Results: IC50 values for Ab42 lowering in the HEKswe_APP cell line were between 100 nM and 50 mM . Cytotoxicity was examined and was separated from the IC50 for Ab42 -lowering by 10-100 fold. In rat cortical cultures the compounds were 3-5 -fold less potent for Ab42 lowering and displayed a wide range of potencies (400 nM-300 mM). The range of potencies for in vitro inhibition of Notch processing were no effect on processing at 30 uM to a large effect at 100 nM. Ab42 lowering in vivo was explored in normal mouse and rat brain. Animals were administered a single dose of 30 mg/kg Of each compound and the effects on brain Ab42 levels were examined in mice or rats at 3 or 4 hours, respectively. The degree of brain penetration and ability to lower Ab42 differed for each of the compounds. Interestingly, the degree of Ab42 lowering did not correlate with the degree of Ab38 production. Conclusions: In conclusion, the pharmacodynamic
Poster Presentations P3
Ab-40 and Ab-42 monomers. The dissociation constant of anti-amyloid antibodies in the D-IgG fraction was determined by Surface Plasmon Resonance (SPR). As a probe of polyvalency, direct binding and Ab competition ELISAs were performed using thyroglobulin as the competing antigen. Results: Dimeric IgG comprised 8-17% of the total IgG in various IVIG preparations. The Ab-binding specific activity of D-IgG was greater than that of M-IgG by a factor of 10.5. The dissociation constant for D-IgG on the Ab monomer SPR sensor was 1.4 micromolar. Thyroglobulin reduced binding to Ab ELISA monomer plates by 70% suggesting that IVIG contains anti-monomer antibodies that cross-react with non-Ab antigens. Conclusions: The dimer fraction of IVIG possesses significant Ab binding capacity. However, anti-monomer antibodies in the dimer fraction have relatively low affinity for Ab and may be cross-reactive with non-amyloid antigens. Future studies will address the extent to which Ab binding by antibodies in the monomeric and dimeric fractions contributes to IVIG’s therapeutic effects in AD patients.
P3-289
MICROELECTRODE ARRAYS AND ALZHEIMER’S DISEASE: A NOVEL IN VITRO INVESTIGATIVE PARADIGM
Kucku Varghese1, Peter Molnar1, Mainak Das1, Mark S. Kindy2, Bruce C. Wheeler3, James J. Hickman1,2, 1University of Central Florida, Orlando, FL, USA; 2Medical University of South Carolina, Charleston, SC, USA; 3University of Florida, Gainesville, FL, USA. Contact e-mail: [email protected] Background: Soluble oligomers of amyloid beta (Ab) are considered to be one of the major contributing factors to the development of Alzheimer’s disease. Most therapeutic development studies have focused on toxicity directly at the synapse. Using patch clamp studies we have demonstrated that soluble Ab can also cause functional toxicity, namely it inhibits spontaneous firing of hippocampal neurons without significant cell death at low concentrations. This toxicity will eventually lead to the loss of synapses as well, but the effect on function may precede this loss by a considerable amount of time. In a key technological advance we have reproduced these results utilizing a fast and simple method based on extracellular electrophysiological recording of electrical activity of cultured hippocampal neurons using multielectrode arrays (MEAs). Methods: Ab oligomers were synthesized using an established protocol and characterized using western blots. Embryonic hippocampal neurons were plated on MEAs and after 14 days in vitro were treated with either 100 nM Ab oligomers alone or a combination of Ab and curcumin, an Ab oligomerization inhibitor depending on the experimental model. Recordings were obtained over this period of time in order to determine the time course of both compounds. The observed results were compared to patch clamp data obtained from similar experiments. Cell death was quantified using a live dead assay. Results: When applied to hippocampal neurons cultured on MEAs, Ab had a pronounced effect on the spontaneous firing of the cells, even at concentrations in the nanomolar range. Treatment with Ab stopped spontaneous activity completely and the time until cessation was concentration dependent. Curcumin was able to partially reverse the loss of spontaneous activity, in accordance with our earlier patch clamp experiments. Conclusions: This study demonstrated that it is possible to develop a high-throughput screen for the measurement of a drug’s effect on functional toxicity of low concentrations of Ab and this model is the first step for an in vitro functional model of the development of Alzheimer’s Disease. This screening method, based on MEA technology, could find important applications in pharmaceutical drug development and could lead to novel drug candidates or therapies for AD.
P3-290
ABCA1 IS REQUIRED FOR THE BENEFICIAL EFFECTS OF THE LIVER-X-RECEPTOR AGONIST GW3965 ON BIOCHEMICAL AND COGNITIVE OUTCOMES IN THE APP/PS1 MOUSE MODEL OF ALZHEIMER’S DISEASE
James Donkin1, Sophie Stukas1, Dhananjay Namjoshi1, Veronica HirschReinshagen1, Jeniffer Chan1, Anna Wilkinson1, Jon Collins2,
Cheryl L. Wellington1, 1UBC, Vancouver, BC, Canada; 2GlaxoSmithKline, Research Triangle Park, NC, USA. Contact e-mail: [email protected] Background: Apolipoprotein E (apoE) the major cholesterol carrier in the brain is also the most validated genetic risk factor for late-onset Alzheimer’s Disease (AD). We and others have contributed to a growing body of literature demonstrating that the amount of lipids carried by apoE is a major determinant of Ab and amyloid metabolism. The cholesterol transporter ABCA1 is responsible for moving lipids onto brain apoE. ABCA1 deficiency exacerbates amyloidogenesis, whereas selective overexpression of ABCA1 is sufficient to ameliorate amyloid burden in AD mice. Liver X Receptor (LXR) agonists such as GW3965, which stimulate the expression of several genes including ABCA1 and apoE, reduce Ab levels and protect against cognitive deficits in AD mice. Methods: In this study, we tested the hypothesis that ABCA1 is required for the beneficial effects of GW3965 in AD mice by evaluating behavioural and neuropathogical outcomes in GW3965treated APP/PS1 mice with and without ABCA1. Results: Here we report that both therapeutic and prophylactic treatment with the LXR agonist GW3965 exhibits a significant improvement in cognition as demonstrated in the Novel Object Recognition and Morris Water maze tasks in the presence of ABCA1. Furthermore, ABCA1 is required to increase apoE levels and lipidation in cerebrospinal fluid in response to GW3965. We are currently performing biochemical and neuropathological analysis of the effect of GW3965 on cortical and hippocampal Ab levels and amyloid burden. Conclusions: Our results suggest that ABCA1-mediated lipidation of apoE is a crucial mechanism underlying the beneficial effects of LXR agonists on cognitive behaviour and Ab metabolism and highlights ABCA1 as a potential AD therapeutic target.
P3-291
PUTATIVE GAMMA SECRETASE MODULATORS LOWER Ab42 IN MULTIPLE IN VITRO AND IN VIVO MODELS
Kathryn Rogers1, Richard Chesworth1, Kevin Felsenstein1, Gideon Shapiro1, Faris Albayya1, Zhiming Tu1, Darcie Spaulding1, Florentina Catana1, Lori Hrdlicka1, Scott Nolan1, Melody Wen1, Zhiyong Yang1, Veerle Vulsteke2, Holger Patzke1, Gerhard Koenig1, Bart DeStrooper2, Michael Ahlijanian1, 1EnVivo Pharma, Watertown, MA, USA; 2VIB, Leuven, Belgium. Contact e-mail: [email protected] Background: Inhibition of gamma secretase (GS) has been the focus of multiple drug discovery efforts over the last decade. However, the successful identification of potent gamma secretase inhibitors (GSIs) also led to the discovery of unacceptable side effects associated with this mechanism, i.e., the concomitant inhibition of Notch processing by GS. The observation that some non-steroidal anti-inflammatory drugs (NSAIDs) selectively inhibit the production of toxic Ab42 while sparing Ab and Notch has prompted the development of a class of compounds commonly referred to as gamma secretase modulators (GSMs). Methods: Using solid phase extraction in combination with an ELISA assay that utilized antibodies selective for Ab42, Ab40, Ab38, or total Ab peptides, this study describes presumptive GSMs that lower Ab42 without altering total Ab in a HEKswe_APP cell line, rat cortical cultures, and normal mice or rats. Western blot analysis of HEK293 cells transfected with NotchDE was used to quantify the effect of compounds on Notch processing. Results: IC50 values for Ab42 lowering in the HEKswe_APP cell line were between 100 nM and 50 mM . Cytotoxicity was examined and was separated from the IC50 for Ab42 -lowering by 10-100 fold. In rat cortical cultures the compounds were 3-5 -fold less potent for Ab42 lowering and displayed a wide range of potencies (400 nM-300 mM). The range of potencies for in vitro inhibition of Notch processing were no effect on processing at 30 uM to a large effect at 100 nM. Ab42 lowering in vivo was explored in normal mouse and rat brain. Animals were administered a single dose of 30 mg/kg Of each compound and the effects on brain Ab42 levels were examined in mice or rats at 3 or 4 hours, respectively. The degree of brain penetration and ability to lower Ab42 differed for each of the compounds. Interestingly, the degree of Ab42 lowering did not correlate with the degree of Ab38 production. Conclusions: In conclusion, the pharmacodynamic