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Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease.

Dewji NN, Singer SJ, Masliah E, Rockenstein E, Kim M, Harber M, Horwood T - PLoS ONE (2015)

Bottom Line: Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695.Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP.P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Diego, La Jolla, CA, 92093, United States of America; Cenna Biosciences Incorporated, 505 Coast Boulevard, Suite 302, La Jolla, CA, 92037, United States of America.

ABSTRACT
β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

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Dose-dependent Effects Of Peptides P4, P8 and Scrambled P1 On NICD Levels And on BACE-1 Activity In Vitro.A. NICD levels in extracts of Jurkat cells that had been treated with 0–10 μM P4, P8 and SP1 for 24 h determined by ELISA. Absorbance was monitored a 450nm and data are expressed as means ± s.e.m. of percent control without peptide. N = 2–3. B. NICD levels in extracts of Jurkat cells that had been treated with the γ-secretase inhibitor Semagacestat LY450139 for 24 h. Data are expressed as in A, n = 2. C. BACE-1 activity in extracts of IMR-32 cells that were treated with 0–5 μM P4, P8 and SP1 for 24h, determined with the SensiZyme BACE-1 activity assay kit. Absorbance was monitored at 405 nm and data are expressed as means ± s.e.m. of percent control (without peptide) BACE activity. N = 2–3. D. BACE-1 activity in extracts of IMR-32 cells that were treated with the BACE inhibitor LY281137 (0–500 nM) for 24 h, expressed as in C. N = 2.
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pone.0122451.g008: Dose-dependent Effects Of Peptides P4, P8 and Scrambled P1 On NICD Levels And on BACE-1 Activity In Vitro.A. NICD levels in extracts of Jurkat cells that had been treated with 0–10 μM P4, P8 and SP1 for 24 h determined by ELISA. Absorbance was monitored a 450nm and data are expressed as means ± s.e.m. of percent control without peptide. N = 2–3. B. NICD levels in extracts of Jurkat cells that had been treated with the γ-secretase inhibitor Semagacestat LY450139 for 24 h. Data are expressed as in A, n = 2. C. BACE-1 activity in extracts of IMR-32 cells that were treated with 0–5 μM P4, P8 and SP1 for 24h, determined with the SensiZyme BACE-1 activity assay kit. Absorbance was monitored at 405 nm and data are expressed as means ± s.e.m. of percent control (without peptide) BACE activity. N = 2–3. D. BACE-1 activity in extracts of IMR-32 cells that were treated with the BACE inhibitor LY281137 (0–500 nM) for 24 h, expressed as in C. N = 2.

Mentions: Since the NICD levels and BACE activity in mouse brains were measured two weeks after treatment with the peptides had stopped, which theoretically could allow the inhibited β- or γ-secretase activities to return to normal, experiments were carried out in vitro to determine the effect of increasing concentrations of peptides P4, P8 and control scrambled P1 on the activities of the two enzymes. The results in Fig 8A show that there was no appreciable change in the levels of NICD released in extracts of Jurkat cells that were treated with increasing concentrations of P4, P8 and scrambled P1 for 24 h, whereas NICD released in these cells was inhibited in a dose-dependent manner after treatment with the γ-secretase inhibitor Semagacestat under the same conditions (Fig 8B). Jurkat cells were used in these experiments instead of IMR-32 cells as the latter express very low levels of Notch 1 [36]. Since Jurkat cells had not hereto been tested with our peptides, the highest peptide concentration used for these cells was twice that used for IMR-32 cells, to allow for any possible inhibition of γ-secretase activity at the higher peptide concentration. Similarly, BACE-1 activity measured in extracts of IMR-32 cells that were treated with increasing concentrations of peptides P4, P8 and control scrambled P1 for 24h remained essentially unchanged (Fig 8C). On the other hand the activity of the enzyme decreased in a dose-dependent fashion when treated with the β-secretase inhibitor LY281137 under the same conditions (Fig 8D). These results therefore demonstrate the absence of a measurable effect on the catalytic activities of both of the secretases in the P4- or P8-treated mouse brains and in vitro, attesting to a significant degree of specificity of the reduction in Aβ by these peptides.


Peptides of presenilin-1 bind the amyloid precursor protein ectodomain and offer a novel and specific therapeutic approach to reduce ß-amyloid in Alzheimer's disease.

Dewji NN, Singer SJ, Masliah E, Rockenstein E, Kim M, Harber M, Horwood T - PLoS ONE (2015)

Dose-dependent Effects Of Peptides P4, P8 and Scrambled P1 On NICD Levels And on BACE-1 Activity In Vitro.A. NICD levels in extracts of Jurkat cells that had been treated with 0–10 μM P4, P8 and SP1 for 24 h determined by ELISA. Absorbance was monitored a 450nm and data are expressed as means ± s.e.m. of percent control without peptide. N = 2–3. B. NICD levels in extracts of Jurkat cells that had been treated with the γ-secretase inhibitor Semagacestat LY450139 for 24 h. Data are expressed as in A, n = 2. C. BACE-1 activity in extracts of IMR-32 cells that were treated with 0–5 μM P4, P8 and SP1 for 24h, determined with the SensiZyme BACE-1 activity assay kit. Absorbance was monitored at 405 nm and data are expressed as means ± s.e.m. of percent control (without peptide) BACE activity. N = 2–3. D. BACE-1 activity in extracts of IMR-32 cells that were treated with the BACE inhibitor LY281137 (0–500 nM) for 24 h, expressed as in C. N = 2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4414571&req=5

pone.0122451.g008: Dose-dependent Effects Of Peptides P4, P8 and Scrambled P1 On NICD Levels And on BACE-1 Activity In Vitro.A. NICD levels in extracts of Jurkat cells that had been treated with 0–10 μM P4, P8 and SP1 for 24 h determined by ELISA. Absorbance was monitored a 450nm and data are expressed as means ± s.e.m. of percent control without peptide. N = 2–3. B. NICD levels in extracts of Jurkat cells that had been treated with the γ-secretase inhibitor Semagacestat LY450139 for 24 h. Data are expressed as in A, n = 2. C. BACE-1 activity in extracts of IMR-32 cells that were treated with 0–5 μM P4, P8 and SP1 for 24h, determined with the SensiZyme BACE-1 activity assay kit. Absorbance was monitored at 405 nm and data are expressed as means ± s.e.m. of percent control (without peptide) BACE activity. N = 2–3. D. BACE-1 activity in extracts of IMR-32 cells that were treated with the BACE inhibitor LY281137 (0–500 nM) for 24 h, expressed as in C. N = 2.
Mentions: Since the NICD levels and BACE activity in mouse brains were measured two weeks after treatment with the peptides had stopped, which theoretically could allow the inhibited β- or γ-secretase activities to return to normal, experiments were carried out in vitro to determine the effect of increasing concentrations of peptides P4, P8 and control scrambled P1 on the activities of the two enzymes. The results in Fig 8A show that there was no appreciable change in the levels of NICD released in extracts of Jurkat cells that were treated with increasing concentrations of P4, P8 and scrambled P1 for 24 h, whereas NICD released in these cells was inhibited in a dose-dependent manner after treatment with the γ-secretase inhibitor Semagacestat under the same conditions (Fig 8B). Jurkat cells were used in these experiments instead of IMR-32 cells as the latter express very low levels of Notch 1 [36]. Since Jurkat cells had not hereto been tested with our peptides, the highest peptide concentration used for these cells was twice that used for IMR-32 cells, to allow for any possible inhibition of γ-secretase activity at the higher peptide concentration. Similarly, BACE-1 activity measured in extracts of IMR-32 cells that were treated with increasing concentrations of peptides P4, P8 and control scrambled P1 for 24h remained essentially unchanged (Fig 8C). On the other hand the activity of the enzyme decreased in a dose-dependent fashion when treated with the β-secretase inhibitor LY281137 under the same conditions (Fig 8D). These results therefore demonstrate the absence of a measurable effect on the catalytic activities of both of the secretases in the P4- or P8-treated mouse brains and in vitro, attesting to a significant degree of specificity of the reduction in Aβ by these peptides.

Bottom Line: Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695.Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP.P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California San Diego, La Jolla, CA, 92093, United States of America; Cenna Biosciences Incorporated, 505 Coast Boulevard, Suite 302, La Jolla, CA, 92037, United States of America.

ABSTRACT
β-Amyloid (Aβ) accumulation in the brain is widely accepted to be critical to the development of Alzheimer's disease (AD). Current efforts at reducing toxic Aβ40 or 42 have largely focused on modulating γ-secretase activity to produce shorter, less toxic Aβ, while attempting to spare other secretase functions. In this paper we provide data that offer the potential for a new approach for the treatment of AD. The method is based on our previous findings that the production of Aβ from the interaction between the β-amyloid precursor protein (APP) and Presenilin (PS), as part of the γ-secretase complex, in cell culture is largely inhibited if the entire water-soluble NH2-terminal domain of PS is first added to the culture. Here we demonstrate that two small, non-overlapping water-soluble peptides from the PS-1 NH2-terminal domain can substantially and specifically inhibit the production of total Aβ as well as Aβ40 and 42 in vitro and in vivo in the brains of APP transgenic mice. These results suggest that the inhibitory activity of the entire amino terminal domain of PS-1 on Aβ production is largely focused in a few smaller sequences within that domain. Using biolayer interferometry and confocal microscopy we provide evidence that peptides effective in reducing Aβ give a strong, specific and biologically relevant binding with the purified ectodomain of APP 695. Finally, we demonstrate that the reduction of Aβ by the peptides does not affect the catalytic activities of β- or γ-secretase, or the level of APP. P4 and P8 are the first reported protein site-specific small peptides to reduce Aβ production in model systems of AD. These peptides and their derivatives offer new potential drug candidates for the treatment of AD.

Show MeSH
Related in: MedlinePlus