- Email: [email protected]
P4-336 Manipulation of amyloidogenic APP processing with peptide aptamers
$570
Poster Session P4: Therapeutics and Therapeutic Strategies - Therapeutic Strategies, Amyloid-Based
mice carrying the APP V717F mutation. Objectives: To determine safety, tolerability and effects on A~ concentrations of LY450139. Methods: Volunteer subjects without neuropsychiatric disease over 45 years of age were studied using doses of LY450139 from 5 to 50 mg per day given for 14 days. Plasma concentrations of LY450139, A~1-40 and AI3a_× ("A~total") were assessed at multiple time points, and safety and tolerability were determined. Cerebrospinal fluid (CSF) samples were obtained at baseline and 6 hours after the fourteenth dose of drug or placebo. Results: LY450139 was well tolerated by all subjects taking 5 nag, 20 mg or 40 mg doses. One of seven subjects taking 50 mg per day experienced a transient elevation of serum amylase and lipase, and a second subject, also taking 50 mg per day, experienced brief diarrhea one day after discontinuing study drug; however, neither of these events was clearly drug related. After each dose of LY450139 there was a biphasic change in plasma A~ concentration. Plasma A[~ concentrations decreased in a dose-dependent manner during a 6 hour interval following drug administration. The maximum decrease was approximately 40% relative to baseline levels. The dose-dependent decrease in plasma At3 concentrations was similar after the first and fourteenth doses. After returning to baseline, plasma A[~ concentrations increased above baseline and then returned to near baseline values 24 hours after administration of drug. In CSF, baseline concentrations of A[3 were about 50 fold higher than those in plasma; a trend for a reduction in A[3 was observed in the 40 and 50 nag dose groups that did not reach statistical significance, in part because of a reduction in CSF A[3 in the placebo group. The plasma half-life of LY450139 was approximately 2.5 hours. Conclusions: LY450139 was well tolerated in healthy volunteer subjects treated for 14 days. The reduction and rise in plasma A[3 concentrations temporally corresponded to the increase and decrease in plasma LY450139 concentrations.
•
O M E G A - 3 FATTY ACID E N R I C H E D DIET REDUCES AMYLOID IN AN ALZHEIMER'S DISEASE M O U S E MODEL
Giselle P. Lim* l, Frederic Calon 1, Fusheng yang 1, Takashi Morihara 1, Oliver Ubeda 1, Karen Hsiao Ashe 2, Sally A. Frautsehy 1, Greg M. Cole 1.
1University of California, Los Angeles-VAGLAHS, North Hills, CA, USA; 2Center for Clinical and Molecular Neurobiology, University of Minnesota, Minneapolis, MN, USA. Contact e-mail: [email protected] Background: Epidemiological studies suggest that increased intake of the omega-3 (n-3) polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) is associated with reduced risk of cognitive impairment and Alzheimer's disease (AD). Reduced amounts of DHA in AD may also reflect its vulnerability to oxidative stress while increased DHA may oppose elevated cholesterol levels involved with enhanced amyloidosis by altering membrane fluidity. Objective: We evaluated whether DHA can alter amyloid levels and processing in APPsw (Tg2576) transgenic mice. Methods: Seventeen-month-old animals were placed on control chow (0.09% DHA), diets depleted (0%) or enriched with DHA (0.6%) for 5 months. ELISA, immunoblotting, RT-PCR, and image analysis were used to determine changes in total amyloid, plaque burden, APP processing, and BACE. Results: Total A[3 ELISA showed that diets enriched with DHA significantly reduced detergent-insoluble arnyloid by more than 70% when compared to omega-3 PUFA depleted or control chow diets. Dietary DHA also decreased A~42 levels below those seen with control chow. Image analysis of brain sections with an antibody against A~ (aal-13) revealed that overall plaque burden was significantly reduced by 31.5 %, with the greatest reductions in hippocampus and parietal cortex. Dietary DHA decreased both c~- and [~-secretase CTF products (C83 and C99) and full length APP, but did not alter BACE mRNA, protein, or activity, indicating that DHA can change APP processing independent of BACE. Additional data regarding the effect of DHA on cholesterol will also be presented. Conclusions: Together, these results suggest that adequate levels of DHA could be protective against AD by reducing amyloid production and accumulation.
•
REGULATION OF GINSENOSIDE RG1 ON THE APP AND NEPRILYSIN EXPRESSION INDUCED BY LIPOPOLYSACCHARIDE IN C6 CELL LINE
Huanmin Luo*, Fei Xian, Xiaoguang Li, Qin Gan, Peifen Zhang, Wen Weng. Neuropharmacologieal Research Laboratory, Guangzhou,
China. Contact e-mail: [email protected] Background: At present, there are no specific drugs for Alzheimer's disease (AD). Aggregation and deposition of amyhiid [3-protein (A~) in AD brain may be essential for it. Yet, the increase of A[3 produce is not all facts. Neprilysin (NEP), major enzyme of degrading A[~, is also likely to decline because of aging. So, as a therapeutic strategy it is necessary to study the activator of NEE Objective: Through investigating the regulation of ginsenoside Rgl on the amyhiid [~-protein precursor (APP) and NEP expression induced by lipopolysaccharide (LPS) in C6 cell line, in order to discover and develop new effectual AD-treated drugs. Methods: MTT colorimetric analysis was used to measure the survival rate of C6 cultured with ginsenoside Rgl in different concentrations (2.5, 5, 10 and 20t~mol. L1) and LPS (100 rag.L-1). The expression of APP and NEP was determined by RT-PCR. Results: The survival rate of C6 was obviously inhibited by LPS, furthermore, the increase of APP expression and the decrease of NEP expression were observed. The alteration, however, can be improved by ginsenoside Rgl, including that the survival rate of C6 can be obviously ascended, the expression of APP was decreased and the expression of NEP was increased. Conclusions: This study demonstrates that LPS can cause cell damage, the increase of APP expression and the decrease of NEP expression. Ginsenoside Rg 1 can exert neuroprotective action which protects C6 cell line from the cell toxicity of LPS. And it can reduce the reinforcement of APP expression and the inhibition of NEP expression induced by LPS. [Key words] ginsenoside Rg 1; lipopolysaccharide; C6 glioma cell; Amyloid precursor protein; neprilysin; cell culture; drug development
•
MANIPULATION OF A M Y L O I D O G E N I C APP PROCESSING WITH PEPTIDE APTAMERS
Can M. Zhang*, Trinna L. Cuellar, Aleister J. Sannders. Drexel University, Philadelphia, PA, USA. Contact e-mail: [email protected]
Background: Amyloidogenic amyloid-precursor protein (APP) processing leads to the production of the beta-amyloid peptide (A[3), excessive accumulation of this peptide is thought to directly lead to Alzheimer's disease (AD). ~-secretase (BACE) is one of the key enzymes involved in amyloidogenic APP processing. To find inhibitors and modulators of APP processing, we are targeting BACE and APP using a constrained peptide library displayed from a scaffold protein eglin C. Eglin c is a small naturally thermostable protein from the leech, Hirudo medicinalis, and it is a potent serine proteinase inhibitor with a ten amino acid protease-binding loop. We have randomized this 10 amino acid loop to create a library of 108 unique aptamer sequences. These aptamers are in vivo screened, antibody-like proteins that can bind specific target protein, and can be used as therapeutic and diagnostic agents. Objective: To identify peptide aptamers that inhibit amyloidogenic APP processing. Methods: To construct Eglin c based library, eglin c was cloned into yeast two hybrid (Y2H) prey vector pJG4-5. Using homologous recombination, the 30bp nucleotides coding for the protease-binding loop were replaced with randomized oligos (NNB). BACE and APP were cloned into bait vector pEG202 using homologous recombination. Interaction mating Y2H was used to identify peptide aptamers that bind to the BACE or APP. Results: Aptamers that are identified that bind to APP and BACE will be presented and discussed in a context of sequence homology to known interactors of BACE and APP. The ability of aptamers to inhibit BACE activity and APP processing will be presented. Conclusion: The ability to inhibit and modulate amyloidogenic APP processing holds great promise as a therapeutic avenue for the treatment of AD. These novel reagents may help identify novel inhibitory mechanisms.
Poster Session P4: Therapeutics and Therapeutic Strategies - Therapeutic Strategies, Amyloid-Based
mice carrying the APP V717F mutation. Objectives: To determine safety, tolerability and effects on A~ concentrations of LY450139. Methods: Volunteer subjects without neuropsychiatric disease over 45 years of age were studied using doses of LY450139 from 5 to 50 mg per day given for 14 days. Plasma concentrations of LY450139, A~1-40 and AI3a_× ("A~total") were assessed at multiple time points, and safety and tolerability were determined. Cerebrospinal fluid (CSF) samples were obtained at baseline and 6 hours after the fourteenth dose of drug or placebo. Results: LY450139 was well tolerated by all subjects taking 5 nag, 20 mg or 40 mg doses. One of seven subjects taking 50 mg per day experienced a transient elevation of serum amylase and lipase, and a second subject, also taking 50 mg per day, experienced brief diarrhea one day after discontinuing study drug; however, neither of these events was clearly drug related. After each dose of LY450139 there was a biphasic change in plasma A~ concentration. Plasma A[~ concentrations decreased in a dose-dependent manner during a 6 hour interval following drug administration. The maximum decrease was approximately 40% relative to baseline levels. The dose-dependent decrease in plasma At3 concentrations was similar after the first and fourteenth doses. After returning to baseline, plasma A[~ concentrations increased above baseline and then returned to near baseline values 24 hours after administration of drug. In CSF, baseline concentrations of A[3 were about 50 fold higher than those in plasma; a trend for a reduction in A[3 was observed in the 40 and 50 nag dose groups that did not reach statistical significance, in part because of a reduction in CSF A[3 in the placebo group. The plasma half-life of LY450139 was approximately 2.5 hours. Conclusions: LY450139 was well tolerated in healthy volunteer subjects treated for 14 days. The reduction and rise in plasma A[3 concentrations temporally corresponded to the increase and decrease in plasma LY450139 concentrations.
•
O M E G A - 3 FATTY ACID E N R I C H E D DIET REDUCES AMYLOID IN AN ALZHEIMER'S DISEASE M O U S E MODEL
Giselle P. Lim* l, Frederic Calon 1, Fusheng yang 1, Takashi Morihara 1, Oliver Ubeda 1, Karen Hsiao Ashe 2, Sally A. Frautsehy 1, Greg M. Cole 1.
1University of California, Los Angeles-VAGLAHS, North Hills, CA, USA; 2Center for Clinical and Molecular Neurobiology, University of Minnesota, Minneapolis, MN, USA. Contact e-mail: [email protected] Background: Epidemiological studies suggest that increased intake of the omega-3 (n-3) polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) is associated with reduced risk of cognitive impairment and Alzheimer's disease (AD). Reduced amounts of DHA in AD may also reflect its vulnerability to oxidative stress while increased DHA may oppose elevated cholesterol levels involved with enhanced amyloidosis by altering membrane fluidity. Objective: We evaluated whether DHA can alter amyloid levels and processing in APPsw (Tg2576) transgenic mice. Methods: Seventeen-month-old animals were placed on control chow (0.09% DHA), diets depleted (0%) or enriched with DHA (0.6%) for 5 months. ELISA, immunoblotting, RT-PCR, and image analysis were used to determine changes in total amyloid, plaque burden, APP processing, and BACE. Results: Total A[3 ELISA showed that diets enriched with DHA significantly reduced detergent-insoluble arnyloid by more than 70% when compared to omega-3 PUFA depleted or control chow diets. Dietary DHA also decreased A~42 levels below those seen with control chow. Image analysis of brain sections with an antibody against A~ (aal-13) revealed that overall plaque burden was significantly reduced by 31.5 %, with the greatest reductions in hippocampus and parietal cortex. Dietary DHA decreased both c~- and [~-secretase CTF products (C83 and C99) and full length APP, but did not alter BACE mRNA, protein, or activity, indicating that DHA can change APP processing independent of BACE. Additional data regarding the effect of DHA on cholesterol will also be presented. Conclusions: Together, these results suggest that adequate levels of DHA could be protective against AD by reducing amyloid production and accumulation.
•
REGULATION OF GINSENOSIDE RG1 ON THE APP AND NEPRILYSIN EXPRESSION INDUCED BY LIPOPOLYSACCHARIDE IN C6 CELL LINE
Huanmin Luo*, Fei Xian, Xiaoguang Li, Qin Gan, Peifen Zhang, Wen Weng. Neuropharmacologieal Research Laboratory, Guangzhou,
China. Contact e-mail: [email protected] Background: At present, there are no specific drugs for Alzheimer's disease (AD). Aggregation and deposition of amyhiid [3-protein (A~) in AD brain may be essential for it. Yet, the increase of A[3 produce is not all facts. Neprilysin (NEP), major enzyme of degrading A[~, is also likely to decline because of aging. So, as a therapeutic strategy it is necessary to study the activator of NEE Objective: Through investigating the regulation of ginsenoside Rgl on the amyhiid [~-protein precursor (APP) and NEP expression induced by lipopolysaccharide (LPS) in C6 cell line, in order to discover and develop new effectual AD-treated drugs. Methods: MTT colorimetric analysis was used to measure the survival rate of C6 cultured with ginsenoside Rgl in different concentrations (2.5, 5, 10 and 20t~mol. L1) and LPS (100 rag.L-1). The expression of APP and NEP was determined by RT-PCR. Results: The survival rate of C6 was obviously inhibited by LPS, furthermore, the increase of APP expression and the decrease of NEP expression were observed. The alteration, however, can be improved by ginsenoside Rgl, including that the survival rate of C6 can be obviously ascended, the expression of APP was decreased and the expression of NEP was increased. Conclusions: This study demonstrates that LPS can cause cell damage, the increase of APP expression and the decrease of NEP expression. Ginsenoside Rg 1 can exert neuroprotective action which protects C6 cell line from the cell toxicity of LPS. And it can reduce the reinforcement of APP expression and the inhibition of NEP expression induced by LPS. [Key words] ginsenoside Rg 1; lipopolysaccharide; C6 glioma cell; Amyloid precursor protein; neprilysin; cell culture; drug development
•
MANIPULATION OF A M Y L O I D O G E N I C APP PROCESSING WITH PEPTIDE APTAMERS
Can M. Zhang*, Trinna L. Cuellar, Aleister J. Sannders. Drexel University, Philadelphia, PA, USA. Contact e-mail: [email protected]
Background: Amyloidogenic amyloid-precursor protein (APP) processing leads to the production of the beta-amyloid peptide (A[3), excessive accumulation of this peptide is thought to directly lead to Alzheimer's disease (AD). ~-secretase (BACE) is one of the key enzymes involved in amyloidogenic APP processing. To find inhibitors and modulators of APP processing, we are targeting BACE and APP using a constrained peptide library displayed from a scaffold protein eglin C. Eglin c is a small naturally thermostable protein from the leech, Hirudo medicinalis, and it is a potent serine proteinase inhibitor with a ten amino acid protease-binding loop. We have randomized this 10 amino acid loop to create a library of 108 unique aptamer sequences. These aptamers are in vivo screened, antibody-like proteins that can bind specific target protein, and can be used as therapeutic and diagnostic agents. Objective: To identify peptide aptamers that inhibit amyloidogenic APP processing. Methods: To construct Eglin c based library, eglin c was cloned into yeast two hybrid (Y2H) prey vector pJG4-5. Using homologous recombination, the 30bp nucleotides coding for the protease-binding loop were replaced with randomized oligos (NNB). BACE and APP were cloned into bait vector pEG202 using homologous recombination. Interaction mating Y2H was used to identify peptide aptamers that bind to the BACE or APP. Results: Aptamers that are identified that bind to APP and BACE will be presented and discussed in a context of sequence homology to known interactors of BACE and APP. The ability of aptamers to inhibit BACE activity and APP processing will be presented. Conclusion: The ability to inhibit and modulate amyloidogenic APP processing holds great promise as a therapeutic avenue for the treatment of AD. These novel reagents may help identify novel inhibitory mechanisms.