Article Figures & Data
Figures
- Figure 1.
Loss of AP-4 complex enhances amyloid plaque pathology in 3-month-old 5xFAD mice. A, Stitched confocal images of corpus callosum of 3-month-old 5xFAD mice with AP-4 (WT) or lacking AP-4 (KO) stained for LAMP1 (green; lysosomes) and Aβ (red; amyloid aggregates) depicting neuritic plaques. Scale bar, 100 μm. B, C, High-magnification confocal images of neuritic plaques labeled with LAMP1 (green; lysosomes) and Aβ (red; amyloid aggregates) in 5xFAD; AP-4 WT (B) and 5xFAD; AP-4 KO (C). Scale bar, 10 μm. D, Quantification of mean size of amyloid aggregates of neuritic plaques in 3-month-old female 5xFAD; AP-4 WT and KO mice (gray, AP-4 WT; red, AP-4 KO). Mean ± SEM, N = 4 pairs of 5xFAD; AP-4 WT and 5xFAD; AP-4 KO animals; *p < 0.05, unpaired t test. E, Plot of areas of each individual amyloid aggregate present in the hippocampus and corpus callosum of the four animals from each genotype (gray, 5xFAD; AP-4 WT; red, 5xFAD; AP-4 KO). F, Quantification of mean size of lysosome-filled axonal swellings of neuritic plaques in 3-month-old female 5xFAD; AP-4 WT and KO mice (gray, 5xFAD; AP-4 WT; red, 5xFAD; AP-4 KO). Mean ± SEM, N = 4, *p < 0.05, unpaired t test. G, Plot of the areas of individual lysosome-filled axonal swellings present in the hippocampus and corpus callosum of the four animals from each genotype (gray, 5xFAD; AP-4 WT; red, 5xFAD; AP-4 KO). H, I, Plot depicting the correlation between LAMP1 and Aβ areas of all individual neuritic plaques present in the hippocampus and corpus callosum of the four animals from each genotype-5xFAD; AP-4 WT (H) and 5xFAD; AP-4 KO animals (I). See also Extended Data Figures 1-1, 1-2, and 1-3.
- Figure 2.
Loss of AP-4 complex leads to increased gliosis in 3-month-old 5xFAD mice. A, Stitched images of corpus callosum and hippocampus of 3-month-old 5xFAD mice with AP-4 (WT) or lacking AP-4 (KO) depicting neuritic plaques stained with LAMP1 (green; lysosomes) and microglia stained with IBA1 (red). Scale bar, 100 μm. The dashed white boxes in the stitched image are expanded in the inset images on the right of each stitched image. Scale bar, 10 μm. B, C, High-magnification images of neuritic plaques labeled with LAMP1 (green) and IBA1 (red) in 5xFAD; AP-4 WT (B) and 5xFAD; AP-4 KO (C). Scale bar, 10 μm. D, Quantification of percentage of neuritic plaques in 3-month-old female 5xFAD; AP-4 WT animals that have microglia associated with them. Mean ± SEM, N = 4, *p < 0.05, unpaired t test. E, Quantification of percentage of neuritic plaques in 3-month-old female 5xFAD; AP-4 KO animals that have microglia associated with them. Mean ± SEM, N = 4, ****p < 0.0001, unpaired t test. F, Percentage of total neuritic plaques in the hippocampus and corpus callosum of 3-month-old female 5xFAD; AP-4 WT (gray) and 5xFAD; AP-4 KO (red) animals with more than three microglia associated with them. Mean ± SEM, N = 4, **p < 0.01, unpaired t test. G, Percentage of moderately sized neuritic plaques (with areas under 1000 μm2) in the hippocampus and corpus callosum of 3-month-old female 5xFAD; AP-4 WT (gray) and 5xFAD; AP-4 KO (red) animals with more than three microglia associated with them. Mean ± SEM, N = 4, **p < 0.01, unpaired t test. H, I, Plot depicting the correlation between neuritic plaque area and the number of associated microglia for all individual neuritic plaques present in the hippocampus and corpus callosum of the four animals from each genotype: 5xFAD; AP-4 WT (H) and 5xFAD; AP-4 KO mice (I). See also Extended Data Figure 2-1.
- Figure 3.
AP-4 axonal dystrophies observed in 5xFAD; AP-4 KO animals lack amyloid accumulation in their proximity. A, High-magnification confocal images of LAMP1 (green; lysosomes) and Aβ (red; amyloid aggregate) in 5xFAD; AP-4 KO animals depicting both neuritic plaques and AP-4 dystrophies. Scale bar, 10 μm. Yellow arrowheads point to AP-4 dystrophies. B, Plot depicting the areas of all axonal dystrophies obtained from 5xFAD; AP-4 WT and 5xFAD; AP-4 KO animals; N = 4 animals per genotype. Neuritic plaques, circle, and AP-4 dystrophies, triangle. C, Plot depicting the correlation between LAMP1 and Aβ areas of all individual neuritic plaques and AP-4 dystrophies in 5xFAD; AP-4 KO animals; N = 4. Neuritic plaque, red circle; AP-4 dystrophy, blue triangle. D, High-magnification images of LAMP1 (green; lysosomes) and IBA1 (red; microglia) in 5xFAD; AP-4 KO depicting neuritic plaques, AP-4 dystrophies, and association of microglia. Scale bar, 10 μm. Yellow arrowheads point to AP-4 dystrophies. E, Quantification of percentage of AP-4 dystrophies that have microglia associated with them in the 3-month-old female 5xFAD; AP-4 KO animals. Mean ± SEM, N = 4, ****p < 0.0001, unpaired t test. F, Quantification of mean size of AP-4 dystrophies with and without microglia in 3-month-old female 5xFAD; AP-4 KO mice. Mean ± SEM, N = 4, *p < 0.05, unpaired t test. G, Quantification of AP-4 dystrophy burden (fraction of area occupied by AP-4 dystrophies out of total area) in corpus callosum, hippocampus versus cortex of 6–8-month-old AP-4ε KO animals. Mean ± SEM, N = 3, *p < 0.05, unpaired t test. See also Extended Data Figures 3-1, 3-2, and 3-3.
- Figure 4.
Loss of AP-4 complex increases BACE1 enrichment in amyloid plaques. A–C, High-resolution confocal images of LAMP1 (green; lysosomes) and BACE1 (red; BACE1-positive vesicles) depicting neuritic plaques (A, B) and AP-4 dystrophies (C) in the corpus callosum of 3-month-old 5xFAD mice with AP-4 (WT; A) or lacking AP-4 (KO; B, C). Note that AP-4 dystrophies are only observed in mice lacking AP-4. Scale bar, 10 μm. D, Quantification of mean relative enrichment of LAMP1 in axonal dystrophies of neuritic plaques and AP-4 dystrophies compared to neuronal soma in 3-month-old female 5xFAD mice with AP-4 (WT; gray) or lacking AP-4 (KO; red). Mean ± SEM, N = 4, *p < 0.05, one way-ANOVA with Dunnett’s posttest. Neuritic plaques, circle, and AP-4 dystrophies, triangle. E, Plot depicting relative LAMP1 enrichment from individual neuritic plaques and AP-4 dystrophies of the four animals from each genotype (gray, 5xFAD; AP-4 WT; red, 5xFAD; AP-4 KO). Neuritic plaques, circle, and AP-4 dystrophies, triangle. F, Quantification of mean relative enrichment of BACE1 in axonal dystrophies of neuritic plaques and AP-4 dystrophies compared to neuronal soma in 3-month-old female 5xFAD mice with AP-4 (WT; gray) or lacking AP-4 (KO; Red). Mean ± SEM, N = 4, *p < 0.05, one way-ANOVA with Dunnett’s posttest. Neuritic plaques, circle, and AP-4 dystrophies, triangle. G, Plot depicting relative BACE1 enrichment from individual neuritic plaques and AP-4 dystrophies of the four animals from each genotype (gray, 5xFAD; AP-4 WT; red, 5xFAD; AP-4 KO). Neuritic plaques, circle, and AP-4 dystrophies, triangle. See also Extended Data Figures 4-1 and 4-2.
- Figure 5.
Schematic summarizing the exacerbation of neuritic plaque pathology arising from loss of AP-4 complex function in 5xFAD mice. Schematic of neuritic plaque in 5xFAD; AP-4 WT and KO animals exhibiting extracellular amyloid aggregate surrounded by lysosome-filled axonal swellings. Neuritic plaques also have microglia associated with them. AP-4 loss results in increase in size (area of both amyloid/Aβ aggregate and lysosome-filled axonal dystrophies) of neuritic plaques as well as increased glial association with plaques. 5xFAD; AP-4 KO animals also exhibit “AP-4” dystrophies which could be seeds of future neuritic plaques. These are smaller in size, do not yet show Aβ aggregation near them, and exhibit reduced glial association. While lysosomal protein LAMP1 is enriched in all the axonal dystrophies, BACE1 is more enriched in the neuritic plaques of 5xFAD; AP-4 KO animals and lesser in AP-4 dystrophies.
Movies
- Movie 1:
Video of neuritic plaques in the hippocampus of 5xFAD; AP-4 WT labeled with LAMP1 (green) and Aβ (red) visualized following expansion microscopy. Scale bar, 20 µm (on 4× expanded image).
- Movie 2:
Video of neuritic plaques in the hippocampus of 5xFAD; AP-4 KO labeled with LAMP1 (green) and Aβ (red) visualized following expansion microscopy. Scale bar, 20 µm (on 4× expanded image).
- Movie 3:
Video of neuritic plaques and AP-4 dystrophies in the hippocampus of 5xFAD; AP-4 KO labeled with LAMP1 (green) and Aβ (red) visualized following expansion microscopy. Scale bar, 20 µm (on 4× expanded image).
Table 1-1
Antibody Summary. Download Table 1-1, DOCX file.
Figure 1-1
Loss of AP-4 complex increases size of amyloid plaques in individual 3-month-old female 5xFAD mice (A) Plot of areas of every individual amyloid aggregate present in the hippocampus and corpus callosum from a representative section of each of the four animals from each genotype (Grey- 5xFAD; AP-4 WT; Red- 5xFAD; AP-4 KO) of 3-month-old female mice. (B) Plot of the areas of individual lysosome-filled axonal swelling per each animal obtained from representative sections of the four animals from each genotype (Grey- 5xFAD; AP-4 WT; Red- 5xFAD; AP-4 KO) of 3-month-old female mice. Download Figure 1-1, TIF file.
Figure 1-2
Loss of AP-4 complex leads to a trend towards increased size of amyloid plaques in 3-month-old male 5xFAD mice (A) Quantification of mean size of amyloid aggregates of neuritic plaques in 3-mo-old male 5xFAD; AP-4 WT and KO mice (Grey- AP-4 WT; Red- AP-4 KO). Mean ± SEM, N = 3 pairs of 5xFAD; AP-4 WT and 5xFAD; AP-4 KO animals. (B) Plot of areas of each individual amyloid aggregate per each animal present in the hippocampus and corpus callosum of the three animals from each genotype. (C) Quantification of mean size of lysosome-filled axonal swellings of neuritic plaques in 3-mo-old male 5xFAD; AP-4 WT and KO mice (Grey- AP-4 WT; Red- AP-4 KO). Mean ± SEM, N = 3 pairs of 5xFAD; AP-4 WT and 5xFAD; AP-4 KO animals. (D) Plot of the areas of individual lysosome-filled axonal swellings obtained from representative sections of the three animals from each genotype (Grey- 5xFAD; AP-4 WT; Red- 5xFAD; AP-4 KO). (E and F) Plot depicting the correlation between LAMP1 and Aβ areas of all individual neuritic plaques present in the hippocampus and corpus callosum of the three animals from each genotype-5xFAD; AP-4 WT (E) and 5xFAD; AP-4 KO animals (F). Download Figure 1-2, TIF file.
Figure 1-3
Loss of AP-4 complex does not cause an exacerbation of amyloid plaque pathology in the cortex of 3-month-old 5xFAD mice (A) Stitched images of the cortex of 3-month-old female 5xFAD mice with AP-4 (WT) or lacking AP-4 (KO) stained for LAMP1 (green; lysosomes) and Aβ (red; amyloid aggregates) depicting neuritic plaques. Bar, 100μm. (B) Quantification of mean size of amyloid aggregates of neuritic plaques in the cortex of 3-mo-old female 5xFAD; AP-4 WT and KO mice (Grey- AP-4 WT; Red- AP-4 KO). Mean ± SEM, N = 4 pairs of 5xFAD; AP-4 WT and 5xFAD; AP-4 KO animals; *, P < .05, unpaired t test. (C) Plot of areas of each individual amyloid aggregate per each animal present in the cortex of the four animals from each genotype. (D) Quantification of mean size of lysosome-filled axonal swellings of neuritic plaques in the cortex of 3-mo-old female 5xFAD; AP-4 WT and KO mice (Grey- 5xFAD; AP-4 WT; Red- 5xFAD; AP-4 KO). Mean ± SEM, N = 4 pairs of 5xFAD; AP-4 WT and 5xFAD; AP-4 KO animals. (E) Plot of the areas of individual lysosome-filled axonal swellings of neuritic plaques per each animal present in the cortex of the four animals from each genotype. Download Figure 1-3, TIF file.
Figure 2-1
Loss of AP-4 complex leads to increased gliosis in 3-month-old 5xFAD mice (A, B) Greyscale counterparts of the stitched images of corpus callosum and hippocampus of 3-month-old 5xFAD mice with AP-4 (WT; A) or lacking AP-4 (KO; B) from Figure 2A, depicting neuritic plaques stained with LAMP1 (green; lysosomes) and microglia stained with IBA1 (red). Bar, 100μm. Greyscale images highlight increased gliosis in the 5xFAD; AP-4 KO animals. Download Figure 2-1, TIF file.
Figure 3-1
Expansion microscopy images of neuritic plaques and AP-4 dystrophies in 5xFAD; AP-4 KO mice (A, B) Maximum intensity projection (MIP) images showing neuritic plaques and AP-4 dystrophies in the hippocampus of a 3-month-old female 5xFAD mouse lacking AP-4 (KO). The brain slices were stained for LAMP1 (green; lysosomes) and Aβ (red; amyloid aggregates) and expanded about four-fold. The dashed yellow boxes in the third column highlight the AP-4 dystrophies which are also enlarged and depicted in the fourth column. Bar, 5 µm (20 µm); 2.5 µm (10 µm) for the fourth column. Scale bars are provided at the pre-expansion scale (with the corresponding post-expansion size indicated in brackets). Download Figure 3-1, TIF file.
Figure 3-2
Expression of Cathepsin B, Legumain, Progranulin, and Rag C in AP-4 Dystrophies (A) High-resolution confocal images of Progranulin (green), and LAMP1 (red) enrichment in an AP-4 dystrophy in mice lacking AP-4 (AP-4ε KO mice). Bar, 10 µm. (B) High-resolution confocal images of the soluble protease, Cathepsin B (green), and LAMP1 (red) in AP-4 dystrophy in 3-month-old mice lacking AP-4 (AP-4ε KO mice). Bar, 10 µm. (C) High-resolution confocal images of lysosomal protease, Legumain/AEP (Asparaginyl endopeptidase) (green), and LAMP1 (red) in AP-4 dystrophy in mice lacking AP-4 (AP-4ε KO mice). Bar, 10 µm. (D) High-resolution confocal images of small GTPase RagC (green), and LAMP1 (red) in AP-4 dystrophy in mice lacking AP-4 (AP-4ε KO mice). Bar, 10 µm. Download Figure 3-2, TIF file.
Figure 3-3
Loss of AP-4 complex alone (in absence of FAD mutations), does not increase gliosis (A) Stitched images of LAMP1 (green; lysosomes) and IBA1 (red; microglia) in 6–8-month-old AP-4 WT and AP-4ε KO animals. Bar, 100μm. (B) Quantification of microglial burden (number of microglia per 10,000μm2) in the corpus callosum and hippocampus of 6-8- month-old AP-4 WT and AP-4ε KO animals (sex-matched littermates). Mean ± SEM, N = 3, ns, P = 0.5267, unpaired t test. Download Figure 3-3, TIF file.
Figure 4-1
Analysis of LAMP1 and BACE1 intensities in lysosomes of neuronal cell bodies in 5xFAD; AP-4 WT and 5xFAD; AP-4 KO mice (A, B) High-resolution confocal images of LAMP1 (green; lysosomes) and BACE1 (red; BACE1-positive vesicles) in neuronal cell bodies of 3-month-old 5xFAD mice with AP-4 (WT; A) or lacking AP-4 (KO; B), (C) Quantification of fold change in mean intensity of LAMP1 in neuronal cell bodies of 3-month-old female 5xFAD mice with AP-4 (WT; Grey) or lacking AP-4 (KO; Red). Mean ± SEM, N = 4. (D) Quantification of fold change in mean intensity of BACE1 in neuronal cell bodies of 3-month-old female 5xFAD mice with AP-4 (WT; Grey) or lacking AP-4 (KO; Red). Mean ± SEM, N = 4. Download Figure 4-1, TIF file.
Figure 4-2
Arl8b is enriched in AP-4 Dystrophies (A, B) High-resolution confocal images of LAMP1 (green) and Arl8b (red) depicting neuritic plaques (A, B) and AP-4 dystrophies (B; yellow arrowhead) in the corpus callosum of 3-month-old 5xFAD mice with AP-4 (WT; A) or lacking AP-4 (KO; B). Note that AP-4 dystrophies are only observed in mice lacking AP-4. Bar, 10μm. (C) Quantification of mean relative enrichment of Arl8b in axonal dystrophies of neuritic plaques and AP-4 dystrophies compared to neuronal soma in 3-month-old female 5xFAD mice with AP-4 (WT; Grey) or lacking AP-4 (KO; Red). Mean ± SEM, N = 3, *, P < .05, one way-ANOVA with Dunnett’s post-test. Neuritic plaques- circle and AP-4 dystrophies- triangle. (D) Plot depicting relative Arl8b enrichment from individual neuritic plaques and AP-4 dystrophies of the three animals from each genotype (Grey- 5xFAD; AP-4 WT; Red- 5xFAD; AP-4 KO). Neuritic plaques- circle and AP-4 dystrophies- triangle. (E, F) High-resolution confocal images of LAMP1 (green) and Arl8b (red) in neuronal cell bodies of 3-month-old 5xFAD mice with AP-4 (WT; E) or lacking AP-4 (KO; F). Bar, 10μm. (G) Quantification of fold change in mean intensity of Arl8b in neuronal cell bodies of 3-month-old female 5xFAD mice with AP-4 (WT; Grey) or lacking AP-4 (KO; Red). Mean ± SEM, N = 3. Download Figure 4-2, TIF file.
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