- Email: [email protected]
P4-430
Poster P4:: Wednesday Posters P4-430
NEW GROUP OF POSITIVE MODULATORS OF AMPA-RECEPTORS
Vladimir Grigoriev, Institute of Physiologically Active Compounds Russian Academy of Sciences, Chernogolovka, Russian Federation. Contact e-mail: [email protected] According to the results of 3D-docking of model positive modulators of AMPA receptors on “ampakine’s” binding site of this receptor a radically new group of compounds, which could be characterized as “dimeric ampakines” was designed. We have studied the action of these compounds on AMPA receptors and their behavioral effect in the test of object recognition. In particular, their effect on transmembrane currents evoked application of kainic acid (KA) on neurons from rat brain was studied by patch-clamp method. It was revealed that compounds demonstrate dose-dependent potentiating effect on currents induced by KA acid on Purkinje cells. Comparative investigation of potentiating effect of hit-compound of this group LM-01 and Cyclothiazide permitted to suggest that LM-01 might block desensitization processes induced by AMPA receptors agonists. It was shown that it increased KA-induced currents in so small doses as 0.1- 1.0 pM. In higher doses (about 10-100 nM) its potentiating effect disappeared that pointed on bell-shaped dose-dependence. It was revealed that LM-01 and some of its analogues enhanced memory and cognition in male mice C57/Bl6 in the object recognition test in doses less than 0.01 mg/kg. It was suggested that new “dimeric ampakines” represent new group of positive modulators of AMPA receptors, which was able to block desensitization of AMPA receptors and to enhance of cognition in experimental animals in object recognition test in very small doses.
P4-431
EFFECT OF APP 17-MER PEPTIDE ON HIPPOCAMPAL NEURODEGENERATION IN OVARIECTOMIZED RATS
Yan Meng, Rong Wang, Fang Yang, Shu Li Sheng, Beijing Xuan-Wu Hospital, Capital University of Medical Sciences, Beijing, China. Contact e-mail: [email protected], [email protected] Background: With an aging society, more and more attention is being paid to the health of elderly women. More importantly, estrogen deprivation arising from menopause in association with age-related factors disproportionately increases the risk of many neurodegenerative diseases, such as Alzheimer’s disease (AD). The results of in vitro and animal studies also provide a strong rationale for the use of hormone replacement therapy to prevent dementia and AD. However, the results of the Women’s Health Initiative Memory Study (WHIMS) indicate that estrogen plus progestin or estrogen alone cannot be recommended as a safe and effective strategy to prevent a neurodegenerative disease, especially after the disease has developed. Objective(s): The objective of this study was to investigate whether hippocampal neurodegeneration existed in experimental ovariectomized (OVX) rats, and to study the effect of amyloid precursor protein 17-mer peptide(APP 17-mer peptide) on them. Methods: 1.Creation of animal model: ovariectomized rats(OVX);2.Animals were divided into 3 groups: control, OVX and APP 17-mer peptide-protected group;3. The changes of behavior and histochemistry after ovariectomy and effect of APP 17-mer peptide on OVX rats were observed. Conclusions: These results indicated that APP 17-mer peptide could ameliorate the neurodegeneration due to estrogen deficiency but the mechanism was not through regulation of estrogen level. Our findings suggest that by activating common intracellular signaling pathways and initiating ‘cross talk‘ with neurotrophins, APP 17-mer peptide improves neurodegeneration caused by estrogen deficiency. However, more work will be required to explain the neuroprotection rendered by APP 17-mer peptide in our model.
P4-432
S645 AMYLOID INHIBITION VIA ABETA42 MONOMERIZATION USING SEMI-RANDOM PEPTIDES LIBRARIES
Pharhad Eli, Avijit Chakrabartty, University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected] Background: The amyloid cascade hypothesis is currently the most accepted theory explaining the pathogenesis of Alzheimer’s disease (AD). The major component of AD associated amyloid plaques is the 39- to 42residue - long Abeta Peptide. The spontaneous aggregation of Abeta into soluble oligomers or amyloid fibrils leads to neuronal loss and dementia. Here we propose a novel hypothesis-driven approach to amyloid inhibition in which the monomeric form of Abeta42 is targeted. The strategy involves conversion of aggregation- prone Abbeta42 into a state that is well folded and resembles natively folded natural protein using amphipathic helix. By converting Abeta42 into a soluble native monomeric protein, aggregation will be prevented, thereby facilitating clearance. Objective(s): Use an amphipathic helix as a template for inducing monomeric folding of Abeta. We will employ a rationally designed semi random library of amphipathic helices to identify optimal sequences that will monomerize Abeta42. Methods: The following screening library was genetically constructed by using PCR methods. Amphipathic helix - linker - Abeta42 - linker- YFP. The amphipathic helical segment will consist of a library of degenerate oligonucleotides. When the constructs were expressed in E.coli, bacterial colonies that show typical YFP fluorescence should contain an amphipathic helical segment that reduces the aggregation of Abeta42, thereby permitting proper YFP folding. YFP and Abeta42-YFP were expressed alternatively as a positive and negative control. The selected colonies were sub-cloned without YFP and expressed in E.coli. Subsequently, the solubility and secondary structure of the protein purified from E.coli were tested using biochemical and biophysical methods, such as ThT, dynamic light scattering, analytical ultra centrifugation and circular dichroism. Results: Theoretically, there are 250,000 possible members of the amphipathic helix library. All of the members have been screened and the DNA sequences of twenty colonies that have been selected were determined. Some of them show a higher solubility and less toxic property in cell. Conclusions: Amphipathic helices were designed to stabilize the monomeric structure of Abeta42. Several promising candidates have been detected and are being analyzed. P4-433
THERAPEUTICALLY EFFECTIVE ANTI-PRION PROTEIN MONOCLONAL ANTIBODIES
Joanna Pankiewicz1, Frances Prelli1, Man-Sun Sy2, Richard Kascsak3, Regina Kascsak3, Daryl Spinner3, Richard Carp3, Harry Meeker3, Martin Sadowski1, Thomas Wisniewski1, 1NYU School of Medicine, New York, NY, USA; 2Case Western Reserve University School of Medicine, Cleveland, OH, USA; 3NYS Institute for Basic Research, Staten Island, NY, USA. Contact e-mail: [email protected] Background: Prion diseases are transmissible and invariably fatal, neurodegenerative disorders associated with a conformational transformation of the cellular prion protein PrPC (C-cellular) into a toxic and proteinase K (PK) resistant conformer, PrPSc (Sc-scrapie). Following extracerebral exposure, PrPSc replicates for months to years within the lymphatic system, where it is a potential target for therapeutic interventions, which do not require blood-brain-barrier penetration. Our own studies and those of others have demonstrated that passive immunization may significantly prolong the incubation period and even prevent disease in murine models of prionoses. Objective(s): Since only a minority of anti-PrP Mabs shows therapeutic activity, understanding how Mabs interfere with PrPSc formation and selecting these Mabs, which are therapeutically effective is crucial for the rationale design of passive immunization approaches for prion infection. Methods: Therapeutic efficacy of a large library of Mabs with epitopes that span the majority of structurally important domains of the PrP protein were tested in N2a murine neuroblastoma cells infected with the 22L mouse adapted scrapie strain. Tagging of Mabs with a fluorescent
NEW GROUP OF POSITIVE MODULATORS OF AMPA-RECEPTORS
Vladimir Grigoriev, Institute of Physiologically Active Compounds Russian Academy of Sciences, Chernogolovka, Russian Federation. Contact e-mail: [email protected] According to the results of 3D-docking of model positive modulators of AMPA receptors on “ampakine’s” binding site of this receptor a radically new group of compounds, which could be characterized as “dimeric ampakines” was designed. We have studied the action of these compounds on AMPA receptors and their behavioral effect in the test of object recognition. In particular, their effect on transmembrane currents evoked application of kainic acid (KA) on neurons from rat brain was studied by patch-clamp method. It was revealed that compounds demonstrate dose-dependent potentiating effect on currents induced by KA acid on Purkinje cells. Comparative investigation of potentiating effect of hit-compound of this group LM-01 and Cyclothiazide permitted to suggest that LM-01 might block desensitization processes induced by AMPA receptors agonists. It was shown that it increased KA-induced currents in so small doses as 0.1- 1.0 pM. In higher doses (about 10-100 nM) its potentiating effect disappeared that pointed on bell-shaped dose-dependence. It was revealed that LM-01 and some of its analogues enhanced memory and cognition in male mice C57/Bl6 in the object recognition test in doses less than 0.01 mg/kg. It was suggested that new “dimeric ampakines” represent new group of positive modulators of AMPA receptors, which was able to block desensitization of AMPA receptors and to enhance of cognition in experimental animals in object recognition test in very small doses.
P4-431
EFFECT OF APP 17-MER PEPTIDE ON HIPPOCAMPAL NEURODEGENERATION IN OVARIECTOMIZED RATS
Yan Meng, Rong Wang, Fang Yang, Shu Li Sheng, Beijing Xuan-Wu Hospital, Capital University of Medical Sciences, Beijing, China. Contact e-mail: [email protected], [email protected] Background: With an aging society, more and more attention is being paid to the health of elderly women. More importantly, estrogen deprivation arising from menopause in association with age-related factors disproportionately increases the risk of many neurodegenerative diseases, such as Alzheimer’s disease (AD). The results of in vitro and animal studies also provide a strong rationale for the use of hormone replacement therapy to prevent dementia and AD. However, the results of the Women’s Health Initiative Memory Study (WHIMS) indicate that estrogen plus progestin or estrogen alone cannot be recommended as a safe and effective strategy to prevent a neurodegenerative disease, especially after the disease has developed. Objective(s): The objective of this study was to investigate whether hippocampal neurodegeneration existed in experimental ovariectomized (OVX) rats, and to study the effect of amyloid precursor protein 17-mer peptide(APP 17-mer peptide) on them. Methods: 1.Creation of animal model: ovariectomized rats(OVX);2.Animals were divided into 3 groups: control, OVX and APP 17-mer peptide-protected group;3. The changes of behavior and histochemistry after ovariectomy and effect of APP 17-mer peptide on OVX rats were observed. Conclusions: These results indicated that APP 17-mer peptide could ameliorate the neurodegeneration due to estrogen deficiency but the mechanism was not through regulation of estrogen level. Our findings suggest that by activating common intracellular signaling pathways and initiating ‘cross talk‘ with neurotrophins, APP 17-mer peptide improves neurodegeneration caused by estrogen deficiency. However, more work will be required to explain the neuroprotection rendered by APP 17-mer peptide in our model.
P4-432
S645 AMYLOID INHIBITION VIA ABETA42 MONOMERIZATION USING SEMI-RANDOM PEPTIDES LIBRARIES
Pharhad Eli, Avijit Chakrabartty, University of Toronto, Toronto, ON, Canada. Contact e-mail: [email protected] Background: The amyloid cascade hypothesis is currently the most accepted theory explaining the pathogenesis of Alzheimer’s disease (AD). The major component of AD associated amyloid plaques is the 39- to 42residue - long Abeta Peptide. The spontaneous aggregation of Abeta into soluble oligomers or amyloid fibrils leads to neuronal loss and dementia. Here we propose a novel hypothesis-driven approach to amyloid inhibition in which the monomeric form of Abeta42 is targeted. The strategy involves conversion of aggregation- prone Abbeta42 into a state that is well folded and resembles natively folded natural protein using amphipathic helix. By converting Abeta42 into a soluble native monomeric protein, aggregation will be prevented, thereby facilitating clearance. Objective(s): Use an amphipathic helix as a template for inducing monomeric folding of Abeta. We will employ a rationally designed semi random library of amphipathic helices to identify optimal sequences that will monomerize Abeta42. Methods: The following screening library was genetically constructed by using PCR methods. Amphipathic helix - linker - Abeta42 - linker- YFP. The amphipathic helical segment will consist of a library of degenerate oligonucleotides. When the constructs were expressed in E.coli, bacterial colonies that show typical YFP fluorescence should contain an amphipathic helical segment that reduces the aggregation of Abeta42, thereby permitting proper YFP folding. YFP and Abeta42-YFP were expressed alternatively as a positive and negative control. The selected colonies were sub-cloned without YFP and expressed in E.coli. Subsequently, the solubility and secondary structure of the protein purified from E.coli were tested using biochemical and biophysical methods, such as ThT, dynamic light scattering, analytical ultra centrifugation and circular dichroism. Results: Theoretically, there are 250,000 possible members of the amphipathic helix library. All of the members have been screened and the DNA sequences of twenty colonies that have been selected were determined. Some of them show a higher solubility and less toxic property in cell. Conclusions: Amphipathic helices were designed to stabilize the monomeric structure of Abeta42. Several promising candidates have been detected and are being analyzed. P4-433
THERAPEUTICALLY EFFECTIVE ANTI-PRION PROTEIN MONOCLONAL ANTIBODIES
Joanna Pankiewicz1, Frances Prelli1, Man-Sun Sy2, Richard Kascsak3, Regina Kascsak3, Daryl Spinner3, Richard Carp3, Harry Meeker3, Martin Sadowski1, Thomas Wisniewski1, 1NYU School of Medicine, New York, NY, USA; 2Case Western Reserve University School of Medicine, Cleveland, OH, USA; 3NYS Institute for Basic Research, Staten Island, NY, USA. Contact e-mail: [email protected] Background: Prion diseases are transmissible and invariably fatal, neurodegenerative disorders associated with a conformational transformation of the cellular prion protein PrPC (C-cellular) into a toxic and proteinase K (PK) resistant conformer, PrPSc (Sc-scrapie). Following extracerebral exposure, PrPSc replicates for months to years within the lymphatic system, where it is a potential target for therapeutic interventions, which do not require blood-brain-barrier penetration. Our own studies and those of others have demonstrated that passive immunization may significantly prolong the incubation period and even prevent disease in murine models of prionoses. Objective(s): Since only a minority of anti-PrP Mabs shows therapeutic activity, understanding how Mabs interfere with PrPSc formation and selecting these Mabs, which are therapeutically effective is crucial for the rationale design of passive immunization approaches for prion infection. Methods: Therapeutic efficacy of a large library of Mabs with epitopes that span the majority of structurally important domains of the PrP protein were tested in N2a murine neuroblastoma cells infected with the 22L mouse adapted scrapie strain. Tagging of Mabs with a fluorescent