- Email: [email protected]
A novel mechanism that targets misfolded protein assemblies
Monday, July 15, 2013: Poster Presentations: P2 P2-048
P361
PHENOLIC COMPOUNDS PREVENT BETAAMYLOID-PROTEIN OLIGOMERIZATION AND SYNAPTIC DYSFUNCTION BY SITE-SPECIFIC BINDING
Kenjiro Ono1, Lei Li2, Yusaku Takamura3, Yuji Yoshiike4, Tokuhei Ikeda5, Junichi Takasaki6, Hisao Nishijo3, Akihiko Takashima4, David Teplow7, Michael Zagorski2, Masahito Yamada6, 1Kanazawa University, Kanazawa, Japan; 2Case Western Reserve University, Cleveland, Ohio, United States; 3 University of Toyama, Toyama, Japan; 4National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 5Kanazawa University, Kanazawa, Ishikawa, Japan; 6Kanazawa University, Kanazawa, Japan; 7UCLA, Los Angeles, California, United States. Contact e-mail: onoken@med. kanazawa-u.ac.jp Background: Cerebral deposition of amyloid b-protein (Ab) is an invariant feature of Alzheimer’s disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We previously reported that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Ab aggregation in vitro and in vivo. The purpose of this study is to elucidate a mechanistic basis for these results. Methods: We analyzed the assembly of the A b 42 and A b 40 with five phenolic compounds, Myr, ferulic acid, nordihydroguaiaretic acid, curcumin, and RA using several well established techniques for studying amyloid formation, including photo-induced cross-linking of unmodified proteins (PICUP), atomic force microscopy (AFM), and nuclear magnetic resonance (NMR). Next, we examined whether the phenolic compounds reduced A b oligomer-induced cytotoxicity and synaptic dysfunction using 3-(19)-2,5-diphenyltetrazolium bromide (MTT) assays and electrophysiological assays for long term potentiation (LTP) and depression (LTD) in hippocampal slices. Results: The phenolic compounds blocked Aboligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Ab. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Ab oligomers. Conclusions: These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Ab through differently binding.
increased when the catalase overexpressing cells were exposed to 3-Amino1,2,4-triazole (3-AT) in a dose dependent manner, suggesting that the protection against H 2 O 2 requires active catalase. Conclusions: It could be concluded that catalase overexpressing cells are neuroprotective against Ab and H 2 O 2 toxicity. This neuroprotection by catalase overexpressing cells could be reduced by 3-AT, suggesting that activity of catalase is important for the neuroprotection against H 2 O 2 toxicity. The neuroprotective role of catalase overexpressing cells against Ab and H 2 O 2 is novel and suggests that compound that increase catalase expression may also be neuroprotective. P2-050
A NOVEL MECHANISM THAT TARGETS MISFOLDED PROTEIN ASSEMBLIES
Amrutha Chilumuri1, Mark Odell2, Nathaniel Milton2, 1University of Westminster, London, United Kingdom; 2University of Westminster, London, England, United Kingdom. Contact e-mail: amrutha.chilumuri@my. westminster.ac.uk
Rajaraman Krishnan1, Haim Tsubery1, Jonathon Waltho2, Ming Proschitsky1, Michal Lulu1, Eva Asp1, Sharon Gilead1, Peter Davies3, Kimberley Gannon1, Eliezer Masliah4, Jonathan Levenson1, Edward Rockenstein5, Beka Solomon6, Richard Fisher1, 1NeuroPhage Pharmaceuticals, Cambridge, Massachusetts, United States; 2University of Manchester, Manchester, United Kingdom; 3University of Sheffield, Sheffield, United Kingdom; 4University of California San Diego, La Jolla, California, United States; 5University of California San Diego, La Jolla, California, United States; 6Tel Aviv University, Tel Aviv, Israel. Contact e-mail: [email protected]
Background: Amyloid b (Ab) is the major component of the senile plaques in Alzheimer’s disease. The binding interaction between (Ab) and catalase has been previously characterised. This interaction limits the ability of catalase to break down hydrogen peroxide (H 2 O 2), which could lead to an increase in the oxidative stress and decrease in the level of active catalase within a cell. Overexpression of catalase gene in a cell could provide higher levels of active catalase which could bind more Ab and also break down H 2 O 2 in vitro. In this study, we overexpressed catalase in human neuroblastoma SH-SY5Y cells and examined the effects of this catalase overexpression against Ab, H 2 O 2 and cobalt chloride (CoCl 2) toxicity. Methods: The catalase overexpressing model was created by stably transfecting the pcDNA4Ô/TO/myc-His A expression vector containing the human catalase gene (NM_001752.3) into the SH-SY5Y cells. For control experiments SHSY5Y cells stably transfected with pcDNA4Ô/TO/myc-His A expression vector were used. The catalase overexpression was confirmed by western blotting and RT-PCR. The toxicity assays was performed by exposing the cells to different concentrations of Ab, H 2 O 2 or CoCl 2 for various time durations. The cell viability was determined by either MTT or Trypan blue assay. Results: When the catalase overexpressing neurons were exposed to Ab, H 2 O 2 or CoCl 2, the toxicity of Ab and H 2 O 2 but not CoCl 2 was reduced compared to control cells. The control cells did not survive the highest toxic dose of Ab and H 2 O 2. The toxicity of H 2 O 2 was
Background: Misfolded protein aggregates play a central role in the pathobiology of neurodegenerative diseases. Using biophysical and biochemical methods, we established that purified,formulated filamentous bacteriophage M13 (NPT002) directly and potently(binding KD¼4-7 nM) destabilizes and dissociates a broad class of amyloids,including amyloid-b, tau, yeast prion, mammalian prion, andalpha-synuclein. We have shown that NPT002 mediates lowering of misfolded protein aggregates following single intracranial administrations to transgenic mouse models with Ab, a-synuclein and tau deposits, respectively. Recently, we isolated the protein motif from NPT002 responsible for the amyloid interacting activity, which we call the generic amyloid interacting motif or GAIM. Here, we demonstrate that GAIM mediates potent binding to amyloid fibers, effectively blocks misfolded protein oligomer-induced cytotoxicity, and potently inhibits amyloid assembly. Methods: Binding studies employed surface plasmon resonance and an amyloid fiber co-sedimentation assay, utilizing fluorescently-labeled NPT001.Amyloid fiber disaggregation assays performed using thioflavin T (ThT) and filter retardation assays. Fiber assembly inhibition performed using ThT assays and transmission electron microscopy. Nuclear magnetic resonance spectroscopy was used to map binding of GAIM on Ab fibers.Efficacy in an aged Tg2576 AD mouse model was measured by Ab and synaptophysin immunohistochemistry, 7 days after a single bilateral intracranial injection of IgGFc-GAIM.
P2-049
THE NEUROPROTECTIVE ROLE OF CATALASE OVEREXPRESSION IN SH-SY5Y CELLS AGAINST BETA-AMYLOID AND H2O2 TOXICITY
P362
Monday, July 15, 2013: Poster Presentations: P2
Results: GAIM binds to, mediates fiber dissociation of, and blocks assembly of multiple misfolded proteins, including Ab, a-synuclein, and tau. Nuclear magnetic resonance (NMR) spectroscopy shows that GAIM binding to Ab fibers is predominantly through mid-region and C-terminal residues of the Ab1-42 peptide, indicating beta strand-GAIM interactions, which lead to fiber remodeling. Recombinant bivalent GAIM molecules, consisting of immunoglobulin Fc fusions (IgGFc) of GAIM: (1) potently bind amyloid-b fibers (fAb) (KD¼10 nM); (2) block fAb assembly (IC50w5 nM);
(3) dissociate fAb (EC50w800 nM); and (4) block Ab oligomer-mediated neuronal cell cytotoxicity (EC50¼30-100 nM). We show that a single intracranial dose of IgGFc-GAIM effectively lowers Ab plaque loads in brains of aged hAPP transgenic mice. Conclusions: Taken together, the biochemical and animal data presented here suggest a novel general mechanism of amyloid disruption and a potentially broad therapeutic modality. GAIM-based molecules represent a new class of drug candidates for misfolded protein diseases.
P361
PHENOLIC COMPOUNDS PREVENT BETAAMYLOID-PROTEIN OLIGOMERIZATION AND SYNAPTIC DYSFUNCTION BY SITE-SPECIFIC BINDING
Kenjiro Ono1, Lei Li2, Yusaku Takamura3, Yuji Yoshiike4, Tokuhei Ikeda5, Junichi Takasaki6, Hisao Nishijo3, Akihiko Takashima4, David Teplow7, Michael Zagorski2, Masahito Yamada6, 1Kanazawa University, Kanazawa, Japan; 2Case Western Reserve University, Cleveland, Ohio, United States; 3 University of Toyama, Toyama, Japan; 4National Center for Geriatrics and Gerontology, Obu, Aichi, Japan; 5Kanazawa University, Kanazawa, Ishikawa, Japan; 6Kanazawa University, Kanazawa, Japan; 7UCLA, Los Angeles, California, United States. Contact e-mail: onoken@med. kanazawa-u.ac.jp Background: Cerebral deposition of amyloid b-protein (Ab) is an invariant feature of Alzheimer’s disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We previously reported that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Ab aggregation in vitro and in vivo. The purpose of this study is to elucidate a mechanistic basis for these results. Methods: We analyzed the assembly of the A b 42 and A b 40 with five phenolic compounds, Myr, ferulic acid, nordihydroguaiaretic acid, curcumin, and RA using several well established techniques for studying amyloid formation, including photo-induced cross-linking of unmodified proteins (PICUP), atomic force microscopy (AFM), and nuclear magnetic resonance (NMR). Next, we examined whether the phenolic compounds reduced A b oligomer-induced cytotoxicity and synaptic dysfunction using 3-(19)-2,5-diphenyltetrazolium bromide (MTT) assays and electrophysiological assays for long term potentiation (LTP) and depression (LTD) in hippocampal slices. Results: The phenolic compounds blocked Aboligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Ab. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Ab oligomers. Conclusions: These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Ab through differently binding.
increased when the catalase overexpressing cells were exposed to 3-Amino1,2,4-triazole (3-AT) in a dose dependent manner, suggesting that the protection against H 2 O 2 requires active catalase. Conclusions: It could be concluded that catalase overexpressing cells are neuroprotective against Ab and H 2 O 2 toxicity. This neuroprotection by catalase overexpressing cells could be reduced by 3-AT, suggesting that activity of catalase is important for the neuroprotection against H 2 O 2 toxicity. The neuroprotective role of catalase overexpressing cells against Ab and H 2 O 2 is novel and suggests that compound that increase catalase expression may also be neuroprotective. P2-050
A NOVEL MECHANISM THAT TARGETS MISFOLDED PROTEIN ASSEMBLIES
Amrutha Chilumuri1, Mark Odell2, Nathaniel Milton2, 1University of Westminster, London, United Kingdom; 2University of Westminster, London, England, United Kingdom. Contact e-mail: amrutha.chilumuri@my. westminster.ac.uk
Rajaraman Krishnan1, Haim Tsubery1, Jonathon Waltho2, Ming Proschitsky1, Michal Lulu1, Eva Asp1, Sharon Gilead1, Peter Davies3, Kimberley Gannon1, Eliezer Masliah4, Jonathan Levenson1, Edward Rockenstein5, Beka Solomon6, Richard Fisher1, 1NeuroPhage Pharmaceuticals, Cambridge, Massachusetts, United States; 2University of Manchester, Manchester, United Kingdom; 3University of Sheffield, Sheffield, United Kingdom; 4University of California San Diego, La Jolla, California, United States; 5University of California San Diego, La Jolla, California, United States; 6Tel Aviv University, Tel Aviv, Israel. Contact e-mail: [email protected]
Background: Amyloid b (Ab) is the major component of the senile plaques in Alzheimer’s disease. The binding interaction between (Ab) and catalase has been previously characterised. This interaction limits the ability of catalase to break down hydrogen peroxide (H 2 O 2), which could lead to an increase in the oxidative stress and decrease in the level of active catalase within a cell. Overexpression of catalase gene in a cell could provide higher levels of active catalase which could bind more Ab and also break down H 2 O 2 in vitro. In this study, we overexpressed catalase in human neuroblastoma SH-SY5Y cells and examined the effects of this catalase overexpression against Ab, H 2 O 2 and cobalt chloride (CoCl 2) toxicity. Methods: The catalase overexpressing model was created by stably transfecting the pcDNA4Ô/TO/myc-His A expression vector containing the human catalase gene (NM_001752.3) into the SH-SY5Y cells. For control experiments SHSY5Y cells stably transfected with pcDNA4Ô/TO/myc-His A expression vector were used. The catalase overexpression was confirmed by western blotting and RT-PCR. The toxicity assays was performed by exposing the cells to different concentrations of Ab, H 2 O 2 or CoCl 2 for various time durations. The cell viability was determined by either MTT or Trypan blue assay. Results: When the catalase overexpressing neurons were exposed to Ab, H 2 O 2 or CoCl 2, the toxicity of Ab and H 2 O 2 but not CoCl 2 was reduced compared to control cells. The control cells did not survive the highest toxic dose of Ab and H 2 O 2. The toxicity of H 2 O 2 was
Background: Misfolded protein aggregates play a central role in the pathobiology of neurodegenerative diseases. Using biophysical and biochemical methods, we established that purified,formulated filamentous bacteriophage M13 (NPT002) directly and potently(binding KD¼4-7 nM) destabilizes and dissociates a broad class of amyloids,including amyloid-b, tau, yeast prion, mammalian prion, andalpha-synuclein. We have shown that NPT002 mediates lowering of misfolded protein aggregates following single intracranial administrations to transgenic mouse models with Ab, a-synuclein and tau deposits, respectively. Recently, we isolated the protein motif from NPT002 responsible for the amyloid interacting activity, which we call the generic amyloid interacting motif or GAIM. Here, we demonstrate that GAIM mediates potent binding to amyloid fibers, effectively blocks misfolded protein oligomer-induced cytotoxicity, and potently inhibits amyloid assembly. Methods: Binding studies employed surface plasmon resonance and an amyloid fiber co-sedimentation assay, utilizing fluorescently-labeled NPT001.Amyloid fiber disaggregation assays performed using thioflavin T (ThT) and filter retardation assays. Fiber assembly inhibition performed using ThT assays and transmission electron microscopy. Nuclear magnetic resonance spectroscopy was used to map binding of GAIM on Ab fibers.Efficacy in an aged Tg2576 AD mouse model was measured by Ab and synaptophysin immunohistochemistry, 7 days after a single bilateral intracranial injection of IgGFc-GAIM.
P2-049
THE NEUROPROTECTIVE ROLE OF CATALASE OVEREXPRESSION IN SH-SY5Y CELLS AGAINST BETA-AMYLOID AND H2O2 TOXICITY
P362
Monday, July 15, 2013: Poster Presentations: P2
Results: GAIM binds to, mediates fiber dissociation of, and blocks assembly of multiple misfolded proteins, including Ab, a-synuclein, and tau. Nuclear magnetic resonance (NMR) spectroscopy shows that GAIM binding to Ab fibers is predominantly through mid-region and C-terminal residues of the Ab1-42 peptide, indicating beta strand-GAIM interactions, which lead to fiber remodeling. Recombinant bivalent GAIM molecules, consisting of immunoglobulin Fc fusions (IgGFc) of GAIM: (1) potently bind amyloid-b fibers (fAb) (KD¼10 nM); (2) block fAb assembly (IC50w5 nM);
(3) dissociate fAb (EC50w800 nM); and (4) block Ab oligomer-mediated neuronal cell cytotoxicity (EC50¼30-100 nM). We show that a single intracranial dose of IgGFc-GAIM effectively lowers Ab plaque loads in brains of aged hAPP transgenic mice. Conclusions: Taken together, the biochemical and animal data presented here suggest a novel general mechanism of amyloid disruption and a potentially broad therapeutic modality. GAIM-based molecules represent a new class of drug candidates for misfolded protein diseases.