Figures and data in Opposite changes in APP processing and human Aβ levels in rats carrying either a protective or a pathogenic APP mutation

Figures
Tables
Additional files

10 figures, 1 table and 2 additional files

Figures

Figure 1

Download asset Open asset

*App* is mutated to contain protective and humanizing mutations and *App* mRNA expression is normal in *App^p* KI rats.

(A) Alignment of Aβ40 region from wildtype rats (top), *App^h* rats (middle), and *App^p* rats (bottom). 6E10 epitopes are in red, 4G8 epitopes are in blue. Humanizing mutations are in highlighted in black. (B) PCR amplification of *App* gene exon-16 from *App^w/w* and *App^p/h* rats and sequencing of PCR product shows that the humanizing (G to C, T to A and GC to AT substitution) and protective (the G to A substitution) mutations were correctly inserted. Substituted nucleotides are highlighted in gray. The amino acid substitutions introduced by the mutations are highlighted in gray above the DNA sequences (G to T = A to T; G to C = G to R; T to A = Y to F; and GC to AT = R to H). (C) Levels of *App* mRNA were measured at p21 and normalized to *Gapdh* mRNA expression. We used the following male and female animals: *App^h/h*, 3 males and 2 females; *App^p/p*, 3 males and 2 females. Data were analyzed by unpaired student’s t-test, and presented as average (*App*/*Gapdh*)± SEM.

Figure 1—source data 1 Related to Figure 1C.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig1-data1-v2.xlsx
Download elife-52612-fig1-data1-v2.xlsx

Figure 2

Download asset Open asset

The protein encoded by the *App^p* allele contains the humanizing and protective mutations, which reduces β-processing and increases α-processing of APP.

Western blot (WB) analysis of brain lysate isolated from *App^w/w*, *App^δ7/δ7*, *App^h/h*, *App^s/s*, and *App^p/p* rats with: (A) Y188, an antibody that detects mAPP, imAPP, αCTF, and βCTF. Specific APP signals are detected from all animals except the *App^δ7/δ7* rats); (B) M3.2, a mouse monoclonal antibody that detects only rat WT APP and βCTF; (C) 6E10, a mouse monoclonal antibody that detects only APP and βCTF carrying the humanizing mutations. (D) WB analysis with anti-sAPPα and anti-sAPPβ-WT (absent in *App^δ7/δ7* controls, *=non specific signal). (**E–K**) Quantification of APP metabolites in *App^h/h* and *App^p/p* rats normalized to GAPDH; APP levels as detected by either 6E10 (E) or Y188 (F); βCTF levels as detected by either 6E10 (G) or Y188 (H); αCTF levels as detected by Y188 (I); βCTF/αCTF ratio as measured by either 6E10-βCTF (J) or Y188-βCTF (K) quantitation values. Quantification of sAPPβ (L) and sAPPα (M) WB levels. (N) Aβ40 levels in *App^h/h* and *App^p/p* samples measured by Wako ELISA. Overexposed WBs are provided in panels A-B to show CTF levels clearly. Quantitations were performed on non-saturated exposures. Data are represented as mean ± SEM. Statistical analyses are by unpaired student’s t-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Animals were analyzed at p21. We used the following male and female animals: 2E, F, G, H, I, J, K, N; *App^h/h*, 2 males and 2 females; *App^p/p*, 2 males and 2 females: 2L and 2M; *App^h/h*, 3 males and 2 females; *App^p/p*, 3 males and 2 females.

Figure 2—source data 1 Related to Figure 2E,F,G,H,I,J,K,L,M.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig2-data1-v2.xlsx
Download elife-52612-fig2-data1-v2.xlsx

Figure 3

Download asset Open asset

ELISA of APP metabolites in *App^p* rats shows decreased APP processing by β-secretase and increased processing by α-secretase.

To test whether the APP protective mutation results in the expected changes in APP metabolism, we extracted brain tissue from a larger cohort that can identify sex-dependent changes in *App^h/h*, *App^p/h* and *App^p/p* 28 day old rats. (A) Left panel, Amino acid sequence of Aβ and P3 peptides recognized by Meso Scale Discovery kits. Detection antibody epitopes for 6E10 (red) and 4G8 (blue) are shown. Capture antibodies recognize Aβ42 and P3-26 (top), Aβ40 and P3-24 (middle), and Aβ38 and P3-22 (bottom). Right panel, Validation of ELISAs of Aβ/P3-4G8, Aβ−6E10, sAPPα, and sAPPβ using *App^δ7/δ7* and *App^h/h* brain lysates. (B) ELISA of brain lysates for Aβ38 and P3-22 (left) and Aβ38 (right) showed that no significant differences between in *App^h/h*, *App^p/h* and *App^p/p* rats. (C) ELISA of brain lysates for Aβ40 and P3-24 (left) and Aβ40 (right) showed a gene-dose dependent and sex independent decrease as follows: *App^h/h* > *Appp^p/h* > *Appp^p/p*. (D) ELISA of brain lysates for Aβ42 and P3-26 (left) and Aβ42 (right) showed a gene-dose dependent and sex independent decrease as follows: *App^h/h* > *Appp^p/h* > *Appp^p/p*. (E) Aβ42+P3-26/Aβ40+P3-24 ratio and Aβ42/Aβ40 ratio. (F) ELISA of sAPPα and sAPPβ of *App^h/h*, *App^p/h*, and *App^p/p* rat brains separated by sex and (G) with sexes pooled. Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences. Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype. To reduce complexity of the panels, in the graphs with both sexes only intra-sex differences are shown (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 3—source data 1 Related to Figure 3B,C,D,E,F,G.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig3-data1-v2.xlsx
Download elife-52612-fig3-data1-v2.xlsx

Figure 4 with 1 supplement

Download asset Open asset

Western analysis of APP metabolites in *App^p* rats shows decreased APP processing by β-secretase.

Quantitation of WB analysis (**A–G**) with representative blots (**H–J**). (A) Normalized total APP levels in *App^h/h*, *App^p/h* and *App^p/p* male (left) and female (right) rats. (B) Normalized imAPP levels in *App^h/h*, *App^p/h* and *App^p/p* male (left) and female (right) rats. (C) Normalized mAPP levels in *App^h/h*, *App^p/h* and *App^p/p* male (left) and female (right) rats. (D) Ratio of mAPP:imAPP in *App^h/h*, *App^p/h* and *App^p/p* rats. (E) Normalized β-CTF levels in *App^h/h*, *App^p/h* and *App^p/p* male (left) and female (right) rats. (F) Normalized α-CTF levels in *App^h/h*, *App^p/h* and *App^p/p* male (left) and female (right) rats. (G) Ratio of β-CTF: α-CTF. (H) Representative blot against C-terminus of APP (I) Longer exposure of α-APP-C-terminus blot to detect α-CTF and β-CTFs. (J) Anti-GAPDH loading control. Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype.

Figure 4—source data 1 Related to Figure 4A,B,C,D,E,F,G.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig4-data1-v2.xlsx
Download elife-52612-fig4-data1-v2.xlsx

Figure 4—figure supplement 1

Download asset Open asset

Western blots images used for quantitation shown in Figure 4.

(A) Blots used to quantify GAPDH (left), mAPP and imAPP (center), αCTF and βCTF (right) in males. (B) Blots used to quantify GAPDH (left), mAPP and imAPP (center), αCTF and βCTF (right) in females.

Figure 5

Download asset Open asset

ELISA of APP metabolites in *App^s* rats shows increased APP processing by β-secretase and decreased processing by α-secretase.

To confirm the Swedish mutation results in the expected changes in APP metabolism, we extracted brain tissue from a larger cohort that can identify sex-dependent changes in *App^h/h*, *App^s/h*, and *App^s/s* 28 day old rats. (A) ELISA of brain lysates for Aβ38 and P3-22 (left) showed that no significant differences between in *App^h/h*, *App^s/h*, and *App^s/s* when P3-22 is considered, however Aβ38 (right) showed a gene-dose dependent and sex independent increase as follows: *App^h/h* < *Apps^s/h* < *Apps^s/s*. (B) ELISA of brain lysates for Aβ40 and P3-24 (left) and Aβ40 (right) showed a gene-dose dependent and sex independent decrease as follows: *App^h/h* < *Apps^s/h* < *Apps^s/s*. (C) ELISA of brain lysates for Aβ42 and P3-26 (left) and Aβ42 (right) showed a gene-dose-dependent and sex-independent decrease as follows: *App^h/h* < *Apps^s/h* < *Apps^s/s*. (D) Aβ42+P3-26/Aβ40+P3-24 ratio and Aβ42/Aβ40 ratio. (E) ELISA of sAPPα (F) ELISA of sAPPβ-WT (left) sAPPβ-Sw (middle) and the calculated sum of both sAPPβ species (right). Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences. Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype, except for male *App^s/s* (n = 4). To reduce complexity of the panels, in the graphs with both sexes only intra-sex differences are shown (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 5—source data 1 Related to Figure 5A,B,C,D,E,F.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig5-data1-v2.xlsx
Download elife-52612-fig5-data1-v2.xlsx

Figure 6 with 1 supplement

Download asset Open asset

Western analysis of APP metabolites in *App^s* rats shows increased APP processing by β-secretase and decreased levels of mature APP.

Quantitation of WB analysis (**A–G**) with representative blots (**H–J**). (A) Normalized total APP levels in *App^h/h*, *App^s/h*, and *App^s/s* male (left) and female (right) rats, show a gene-dose dependent decrease in *App^s* rats. (B) Normalized imAPP levels in *App^h/h*, *App^s/h*, and *App^s/s* male (left) and female (right) rats. (C) Normalized mAPP levels in *App^h/h*, *App^s/h*, and *App^s/s* male (left) and female (right) rats show a gene-dose dependent decrease in *App^s* rats. (D) Ratio of mAPP:imAPP in *App^h/h*, *App^s/h*, and *App^s/s* rats. (E) Normalized βCTF levels in *App^h/h*, *App^s/h*, and *App^s/s* male (left) and female (right) rats. (F) Normalized αCTF levels in *App^h/h*, *App^s/h*, and *App^s/s* male (left) and female (right) rats. (G) Ratio of βCTF: αCTF. (H) Representative blot against C-terminus of APP (I) Longer exposure of α-APP-C-terminus blot to detect αCTF and βCTFs. (J) Anti-GAPDH loading control. Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.. Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype, except for male *App^s/s* (n = 4).

Figure 6—source data 1 Related to Figure 6A,B,C,D,E,F,G.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig6-data1-v2.xlsx
Download elife-52612-fig6-data1-v2.xlsx

Figure 6—figure supplement 1

Download asset Open asset

Western blots images used for quantitation shown in Figure 6.

(A) Blots used to quantify GAPDH (left), mAPP and imAPP (center), αCTF and βCTF (right) in males. As it is evident from the blots, one *App^s/s* sample was degraded (indicated with *) and therefore that animal was excluded from any analysis. (B) Blots used to quantify GAPDH (left), mAPP and imAPP (center), αCTF and βCTF (right) in females.

Figure 7 with 1 supplement

Download asset Open asset

Western analysis of βCTF shows increased levels of βCTF in *App^s* animals and decreased levels in *App^p* rats.

(A) Representative blot with 6E10 (Long exposure was used to quantify βCTF, Short exposure was used to quantify APP) and anti-GAPDH on *App^h/h*, *App^s/h*, and *App^s/s* female and male brain lysates. The blots used for quantitation are shown in Figure 7—figure supplement 1A and B. (B) Total APP, βCTF and GAPDH were normalized against the average signal of the control *App^h/h* samples and shown as ratio of the normalized APP/GAPDH and βCTF/GAPDH ratios. APP and βCTF are decrease and increased in *App^s* rats, respectively, in a gene-dose-dependent manner. Data are represented as mean ± SEM. Data were analyzed by ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences. Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype, except for male *App^s/s* (n = 4). (C) Representative blot with 6E10 (Long exposure was used to quantify βCTF, Short exposure was used to quantify APP) and anti-GAPDH on *App^h/h* and *App^p/p* female and male brain lysates. The blots used for quantitation are shown in Figure 7—figure supplement 1C and D. Total APP, βCTF and GAPDH were normalized against the average signal of the control *App^h/h* samples. Normalized βCTF (D) and APP (E) values for female, male and female + male samples. (F) Normalized βCTF/APP ratios for female, male and female + male samples. (G) Normalized βCTF/GAPDH ratios for female, male and female + male samples. (H) Normalized βCTF/Ponc (Ponceau stain) ratios for female, male and female + male samples. Overall the data indicate a reduction in βCTF in *App^p/p* rats. Data are represented as mean ± SEM. Statistical analyses are by unpaired student’s t-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype.

Figure 7—source data 1 Related to Figure 7B,C,D,E,F,G,H.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig7-data1-v2.xlsx
Download elife-52612-fig7-data1-v2.xlsx

Figure 7—figure supplement 1

Download asset Open asset

Western blots, ponceau stain and colorimetric images used for quantitation shown in Figure 7 for: (A) βCTF, APP and GAPDH in *App^h/h*, *App^s/h* and *App^s/s* females; (B) βCTF, APP and GAPDH in *App^h/h*, *App^s/h* and *App^s/s* males (as it is evident from the blots, one *App^s/s* sample was degraded and indicated with * and therefore that animal was excluded from any analysis); (C) βCTF, APP and GAPDH in *App^h/h* and *App^p/p* females; (B) βCTF, APP and GAPDH in *App^h/h* and *App^p/p* males.

Figure 8

Download asset Open asset

ELISA of APP metabolites in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* rats.

(A) ELISA of brain lysates for Aβ38 and P3-22 (left) and Aβ38 (right). (B) ELISA of brain lysates for Aβ40 and P3-24 (left) and Aβ40 (right). (C) ELISA of brain lysates for Aβ42 and P3-26 (left) and Aβ42 (right). (D) Aβ42+P3-26/Aβ40+P3-24 ratio and Aβ42/Aβ40 ratio. (E) ELISA of sAPPα (F) ELISA of sAPPβ-WT (left) sAPPβ-Sw (middle) and the calculated sum of both sAPPβ species (right). Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences. Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype. To reduce complexity of the panels, in the graphs with both sexes only intra-sex differences are shown (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Figure 8—source data 1 Related to Figure 8A,B,C,D,E,F.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig8-data1-v2.xlsx
Download elife-52612-fig8-data1-v2.xlsx

Figure 9

Download asset Open asset

Comparison of empirical levels of APP metabolites in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* rats to calculated *App^s/p* levels.

ELISA values from *App^h/h*, *App^s/h*, and *App^p/h* rats in Figure 7 were used to generate a hypothetical value for *App^s/p* rats. One h allele value was calculated as half the value of *App^h/h*. This h allele value was subtracted from *App^s/h* and *App^p/h* values to generate the s and p allele values, respectively, which were then added together to represent a ‘hypothetical’ *App^s/p* rat. (A) Real and calculated ELISA Aβ40 levels. (B) Real and calculated ELISA Aβ42 levels. (C) Real and calculated ELISA sAPPα levels. (D) Real and calculated ELISA total sAPPβ levels. Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype.

Figure 9—source data 1 Related to Figure 9A,B,C,D.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig9-data1-v2.xlsx
Download elife-52612-fig9-data1-v2.xlsx

Figure 10 with 1 supplement

Download asset Open asset

Western analysis of APP metabolites in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* rats.

Quantitation of WB analysis (**A–G**) with representative blots (**H–J**). (A) Normalized total APP levels in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* male (left) and female (right) rats. (B) Normalized imAPP levels in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* male (left) and female (right) rats. (C) Normalized mAPP levels *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* male (left) and female (right) rats. (D) Ratio of mAPP:imAPP in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* rats. (E) Normalized βCTF levels in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* male (left) and female (right) rats. (F) Normalized αCTF levels in *App^h/h*, *App^s/h*, *App^p/h*, and *App^s/p* male (left) and female (right) rats. (G) Ratio of βCTF: αCTF. (H) Representative blot against C-terminus of APP (I) Longer exposure of α-APP-C-terminus blot to detect αCTF and βCTFs. (J) Anti-GAPDH loading control. Data are represented as mean ± SEM. Data were analyzed by Ordinary one-way ANOVA followed by post-hoc Tukey's multiple comparisons test when ANOVA showed statistically significant differences (*p<0.05; **p<0.01; ***p<0.001). Animals were analyzed at p28. We used 5 male and 5 female rats for each genotype.

Figure 10—source data 1 Related to Figure 10A,B,C,D,E,F,G.: https://cdn.elifesciences.org/articles/52612/elife-52612-fig10-data1-v2.xlsx
Download elife-52612-fig10-data1-v2.xlsx

Figure 10—figure supplement 1

Download asset Open asset

Western blots images used for quantitation shown in Figure 10.

(A) Blots used to quantify GAPDH (left), mAPP and imAPP (center), αCTF and βCTF (right) in males. (B) Blots used to quantify GAPDH (left), mAPP and imAPP (center), αCTF and βCTF (right) in females.

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Gene App (Rattus norvegicus)	App, Amyloid beta (A4) precursor protein	http://mar2016.archive.ensembl.org/Rattus_norvegicus/Gene/Summary?g=ENSRNOG00000006997;r=11:24425005-24641858	Location: Chromosome 11: 24,425,005–24,641,858	Mutations were inserted into Exon 16
Genetic reagent (Rattus norvegicus)	App^h	(Tambini et al., 2019)		Rat App allele with humanize Aβ region
Genetic reagent (Rattus norvegicus)	App^s	(Tambini et al., 2019)		Rat App allele with humanize Aβ region and pathogenic Swedish mutations
Genetic reagent (Rattus norvegicus)	App^p	This paper		Rat App allele with humanize Aβ region and protective Icelandic mutation
Recombinant DNA reagent	gRNA1 targeting vector	This paper: http://www.vectorbuilder.com/design/report/733e7a50-b705-45cf-8f8c-8b8206e6f174		Vector expressing gRNA1
Recombinant DNA reagent	gRNA2 targeting vector	This paper http://www.vectorbuilder.com/design/report/2a73df2c-1d5c-4e69-b43a-ee6cfaea80c6		Vector expressing gRNA1
Antibody	Y188 anti APP-C terminus (Rabbit polyclonal)	Abcam Cat# ab32136	RRID:AB_2289606	WB (1:1000)
Antibody	6E10 anti human APP-Aβ_1-16, (Mouse monoclonal)	BioLegend Cat# 803001	RRID:AB_2564653	WB (1:1000)
Antibody	M3.2 anti rodent APP-Aβ_1-16, (Mouse monoclonal)	Biolegend Cat#: 805701	RRID:AB_2564982	WB (1:1000)
Antibody	Anti-sAPPα C-terminus, (Mouse monoclonal)	IBL Cat# 2B3		WB (1:1000)
Antibody	Anti-human sAPPβ WT C-terminus (Rabbit polyclonal)	Covance Cat# SIG-39138	RRID:AB_662870	WB (1:1000)
Antibody	Anti-human sAPPβ Sw C-terminus (Mouse monoclonal)	IBL Cat#: 6A1		WB (1:1000)
Antibody	Anti-GAPDH (Rabbit polyclonal)	Abcam Cat# ab32136	RRID:AB_2289606	WB (1:1000)
Antibody	HRP-Anti-mouse antibodies	Southern Biotech Cat#: 1031–05	RRID:AB_2794307	WB (1:1000)
Antibody	HRP-Anti-rabbit antibodies	Southern Biotech Cat# 4050–05	RRID:AB_2795955	WB (1:1000)
Antibody	HRP-Anti-rabbit antibodies	Cell Signaling Cat# 7074	RRID:AB_2099233	WB (1:1000)
Commercial assay or kit	West Dura ECL	Thermo	Cat# PI34076
Commercial assay or kit	Human β Amyloid (1-42) ELISA Kit – High Sensitive	Wako
Commercial assay or kit	Human β Amyloid (1-40) ELISA Kit II	Wako
Commercial assay or kit	V-PLEX Plus Aβ Peptide Panel 1	Meso Scale Discovery	Cat# K15200G
Commercial assay or kit	V-PLEX Plus Aβ Peptide Panel 1 4G8	Meso Scale Discovery	Cat# K15199G
Commercial assay or kit	sAPPβ-Sw Elisa),	Meso Scale Discovery	Cat# K151BUE
Commercial assay or kit	WT sAPPα/sAPPβ	Meso Scale Discovery	Cat# K15120E
Other	ChemiDoc MP Imaging System	Biorad
Instrument/software, algorithm	MESO QuickPlex SQ 120	Meso Scale Discovery
Other	xMark Spectrophotometer	Biorad
Instrument/software, algorithm	Image Lab software	Biorad	RRID:SCR_014210
Instrument/software, algorithm	GraphPad Prism		RRID:SCR_002798

Additional files

Supplementary file 1 Generation of App KI rats.: https://cdn.elifesciences.org/articles/52612/elife-52612-supp1-v2.docx
Download elife-52612-supp1-v2.docx
Transparent reporting form: https://cdn.elifesciences.org/articles/52612/elife-52612-transrepform-v2.docx
Download elife-52612-transrepform-v2.docx

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Mendeley

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Marc D Tambini
Kelly A Norris
Luciano D'Adamio

(2020)

Opposite changes in APP processing and human Aβ levels in rats carrying either a protective or a pathogenic APP mutation

eLife 9:e52612.

https://doi.org/10.7554/eLife.52612

Figures

App is mutated to contain protective and humanizing mutations and App mRNA expression is normal in App^p KI rats.

Figure 1—source data 1

The protein encoded by the App^p allele contains the humanizing and protective mutations, which reduces β-processing and increases α-processing of APP.

Figure 2—source data 1

ELISA of APP metabolites in App^p rats shows decreased APP processing by β-secretase and increased processing by α-secretase.

Figure 3—source data 1

Western analysis of APP metabolites in App^p rats shows decreased APP processing by β-secretase.

Figure 4—source data 1

Western blots images used for quantitation shown in Figure 4.

ELISA of APP metabolites in App^s rats shows increased APP processing by β-secretase and decreased processing by α-secretase.

Figure 5—source data 1

Western analysis of APP metabolites in App^s rats shows increased APP processing by β-secretase and decreased levels of mature APP.

Figure 6—source data 1

Western blots images used for quantitation shown in Figure 6.

Western analysis of βCTF shows increased levels of βCTF in App^s animals and decreased levels in App^p rats.

Figure 7—source data 1

ELISA of APP metabolites in App^h/h, App^s/h, App^p/h, and App^s/p rats.

Figure 8—source data 1

Comparison of empirical levels of APP metabolites in App^h/h, App^s/h, App^p/h, and App^s/p rats to calculated App^s/p levels.

Figure 9—source data 1

Western analysis of APP metabolites in App^h/h, App^s/h, App^p/h, and App^s/p rats.

Figure 10—source data 1

Western blots images used for quantitation shown in Figure 10.

Tables

Additional files

Supplementary file 1

Transparent reporting form

Download links

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

Be the first to read new articles from eLife

Share this article

Cite this article

App is mutated to contain protective and humanizing mutations and App mRNA expression is normal in Appp KI rats.

Figure 1—source data 1

The protein encoded by the Appp allele contains the humanizing and protective mutations, which reduces β-processing and increases α-processing of APP.

Figure 2—source data 1

ELISA of APP metabolites in Appp rats shows decreased APP processing by β-secretase and increased processing by α-secretase.

Figure 3—source data 1

Western analysis of APP metabolites in Appp rats shows decreased APP processing by β-secretase.

Figure 4—source data 1

Western blots images used for quantitation shown in Figure 4.

ELISA of APP metabolites in Apps rats shows increased APP processing by β-secretase and decreased processing by α-secretase.

Figure 5—source data 1

Western analysis of APP metabolites in Apps rats shows increased APP processing by β-secretase and decreased levels of mature APP.

Figure 6—source data 1

Western blots images used for quantitation shown in Figure 6.

Western analysis of βCTF shows increased levels of βCTF in Apps animals and decreased levels in Appp rats.

Figure 7—source data 1

ELISA of APP metabolites in Apph/h, Apps/h, Appp/h, and Apps/p rats.

Figure 8—source data 1

Comparison of empirical levels of APP metabolites in Apph/h, Apps/h, Appp/h, and Apps/p rats to calculated Apps/p levels.

Figure 9—source data 1

Western analysis of APP metabolites in Apph/h, Apps/h, Appp/h, and Apps/p rats.

Figure 10—source data 1

Western blots images used for quantitation shown in Figure 10.

Supplementary file 1

Transparent reporting form

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

App is mutated to contain protective and humanizing mutations and App mRNA expression is normal in App^p KI rats.

The protein encoded by the App^p allele contains the humanizing and protective mutations, which reduces β-processing and increases α-processing of APP.

ELISA of APP metabolites in App^p rats shows decreased APP processing by β-secretase and increased processing by α-secretase.

Western analysis of APP metabolites in App^p rats shows decreased APP processing by β-secretase.

ELISA of APP metabolites in App^s rats shows increased APP processing by β-secretase and decreased processing by α-secretase.

Western analysis of APP metabolites in App^s rats shows increased APP processing by β-secretase and decreased levels of mature APP.

Western analysis of βCTF shows increased levels of βCTF in App^s animals and decreased levels in App^p rats.

ELISA of APP metabolites in App^h/h, App^s/h, App^p/h, and App^s/p rats.

Comparison of empirical levels of APP metabolites in App^h/h, App^s/h, App^p/h, and App^s/p rats to calculated App^s/p levels.

Western analysis of APP metabolites in App^h/h, App^s/h, App^p/h, and App^s/p rats.