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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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Related in: MedlinePlus

Effect Of Peptides P4 And P8 Treatment On Levels Of Aβ, APP, NICD And BACE-1 In Neocortex Of APP Tg Mice.A. Aβ levels in sections of mouse neocortex that was immunolabeled with a MAb against human Aβ1–16 were quantified as in Fig 2. Data are expressed as mean ±s.e.m. n = 5. ** p <0.005. B. BACE-1 activity in extracts of neocortex of mice treated with peptides P4, P8 and PBS was determined using the SensiZyme BACE1 activity assay kit. Absorbance was monitored at 405 nm. Data are expressed as mean ±s.e.m. of active BACE-1 in ng/ml n = 5. C. Extracts of neocortex of peptide-treated mice were Western-blotted with primary rabbit Ab against NICD followed by HRP-conjugated goat anti-rabbit IgG. Immunoreactive bands were detected by ECL and the signal intensity of the protein bands was quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands. D. After NICD detection, the nitrocellulose membranes were stripped and re-probed with a MAb against APP, followed by HRP-conjugated goat anti-mouse IgG. The signal intensity of the protein bands was then quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands.
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pone.0122451.g007: Effect Of Peptides P4 And P8 Treatment On Levels Of Aβ, APP, NICD And BACE-1 In Neocortex Of APP Tg Mice.A. Aβ levels in sections of mouse neocortex that was immunolabeled with a MAb against human Aβ1–16 were quantified as in Fig 2. Data are expressed as mean ±s.e.m. n = 5. ** p <0.005. B. BACE-1 activity in extracts of neocortex of mice treated with peptides P4, P8 and PBS was determined using the SensiZyme BACE1 activity assay kit. Absorbance was monitored at 405 nm. Data are expressed as mean ±s.e.m. of active BACE-1 in ng/ml n = 5. C. Extracts of neocortex of peptide-treated mice were Western-blotted with primary rabbit Ab against NICD followed by HRP-conjugated goat anti-rabbit IgG. Immunoreactive bands were detected by ECL and the signal intensity of the protein bands was quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands. D. After NICD detection, the nitrocellulose membranes were stripped and re-probed with a MAb against APP, followed by HRP-conjugated goat anti-mouse IgG. The signal intensity of the protein bands was then quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands.

Mentions: An optimal therapeutic approach to AD would avoid producing serious effects on other biochemically and physiologically important processes, as would occur if the cleavages of any of the other substrates of β- or γ-secretase were affected in the process of reducing the levels of Aβ. An especially well-studied example is the γ-secretase activity of PS-1 that is critically involved in the final enzymatic cleavage of Aβ from APP; it also functions in an essential developmental process that generates NICD from Notch [35]. In order to determine whether P4 and P8 could reduce Aβ load without affecting the activities of either β- or γ-secretase, we investigated (Fig 7), as a measure of γ-secretase activity, the production of NICD in the same samples of mThy1-hAPP mouse brains that had been administered P4 and P8 for the analysis of Aβ. Samples were selected that had previously shown a reduction in Aβ load of 50% or more with the two peptides. At the same time we analyzed the same tissue samples for their β-secretase (BACE-1) activity and APP expression. Extracts were prepared of the neocortex of Tg mice that had been treated with P4, P8 or PBS, and the levels of NICD were determined by Western blot analysis with a primary rabbit Ab against NICD. This antibody recognizes only the NICD that is released after cleavage by γ-secretase. The marked reduction in Aβ levels produced by P4 (total Aβ mean reduction ± s.e.m. 58%±4, P<0.001) and P8 (70%±6, P<0.001) compared to PBS controls (n = 5, each treatment) (Fig 7A)) were not accompanied by any significant changes in the levels of NICD (Fig 7C), demonstrating that the γ-secretase activity in these samples was not measurably altered. Similarly, BACE-1 activity in the same samples, measured using an ELISA kit [32], was not significantly changed in the P4- and P8-treated samples (Fig 7B). APP levels in the same samples were also determined by Western blot analysis with a primary mouse MAb against human APP, followed by densitometric scanning of the bands, and did not show significant differences (Fig 7D).


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)

Effect Of Peptides P4 And P8 Treatment On Levels Of Aβ, APP, NICD And BACE-1 In Neocortex Of APP Tg Mice.A. Aβ levels in sections of mouse neocortex that was immunolabeled with a MAb against human Aβ1–16 were quantified as in Fig 2. Data are expressed as mean ±s.e.m. n = 5. ** p <0.005. B. BACE-1 activity in extracts of neocortex of mice treated with peptides P4, P8 and PBS was determined using the SensiZyme BACE1 activity assay kit. Absorbance was monitored at 405 nm. Data are expressed as mean ±s.e.m. of active BACE-1 in ng/ml n = 5. C. Extracts of neocortex of peptide-treated mice were Western-blotted with primary rabbit Ab against NICD followed by HRP-conjugated goat anti-rabbit IgG. Immunoreactive bands were detected by ECL and the signal intensity of the protein bands was quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands. D. After NICD detection, the nitrocellulose membranes were stripped and re-probed with a MAb against APP, followed by HRP-conjugated goat anti-mouse IgG. The signal intensity of the protein bands was then quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0122451.g007: Effect Of Peptides P4 And P8 Treatment On Levels Of Aβ, APP, NICD And BACE-1 In Neocortex Of APP Tg Mice.A. Aβ levels in sections of mouse neocortex that was immunolabeled with a MAb against human Aβ1–16 were quantified as in Fig 2. Data are expressed as mean ±s.e.m. n = 5. ** p <0.005. B. BACE-1 activity in extracts of neocortex of mice treated with peptides P4, P8 and PBS was determined using the SensiZyme BACE1 activity assay kit. Absorbance was monitored at 405 nm. Data are expressed as mean ±s.e.m. of active BACE-1 in ng/ml n = 5. C. Extracts of neocortex of peptide-treated mice were Western-blotted with primary rabbit Ab against NICD followed by HRP-conjugated goat anti-rabbit IgG. Immunoreactive bands were detected by ECL and the signal intensity of the protein bands was quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands. D. After NICD detection, the nitrocellulose membranes were stripped and re-probed with a MAb against APP, followed by HRP-conjugated goat anti-mouse IgG. The signal intensity of the protein bands was then quantified. Top: data are expressed as mean ±s.e.m. n = 5. Bottom: The individual Western blot gel images showing immunoreactive bands.
Mentions: An optimal therapeutic approach to AD would avoid producing serious effects on other biochemically and physiologically important processes, as would occur if the cleavages of any of the other substrates of β- or γ-secretase were affected in the process of reducing the levels of Aβ. An especially well-studied example is the γ-secretase activity of PS-1 that is critically involved in the final enzymatic cleavage of Aβ from APP; it also functions in an essential developmental process that generates NICD from Notch [35]. In order to determine whether P4 and P8 could reduce Aβ load without affecting the activities of either β- or γ-secretase, we investigated (Fig 7), as a measure of γ-secretase activity, the production of NICD in the same samples of mThy1-hAPP mouse brains that had been administered P4 and P8 for the analysis of Aβ. Samples were selected that had previously shown a reduction in Aβ load of 50% or more with the two peptides. At the same time we analyzed the same tissue samples for their β-secretase (BACE-1) activity and APP expression. Extracts were prepared of the neocortex of Tg mice that had been treated with P4, P8 or PBS, and the levels of NICD were determined by Western blot analysis with a primary rabbit Ab against NICD. This antibody recognizes only the NICD that is released after cleavage by γ-secretase. The marked reduction in Aβ levels produced by P4 (total Aβ mean reduction ± s.e.m. 58%±4, P<0.001) and P8 (70%±6, P<0.001) compared to PBS controls (n = 5, each treatment) (Fig 7A)) were not accompanied by any significant changes in the levels of NICD (Fig 7C), demonstrating that the γ-secretase activity in these samples was not measurably altered. Similarly, BACE-1 activity in the same samples, measured using an ELISA kit [32], was not significantly changed in the P4- and P8-treated samples (Fig 7B). APP levels in the same samples were also determined by Western blot analysis with a primary mouse MAb against human APP, followed by densitometric scanning of the bands, and did not show significant differences (Fig 7D).

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