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Y682G Mutation of Amyloid Precursor Protein Promotes Endo-Lysosomal Dysfunction by Disrupting APP-SorLA Interaction.

La Rosa LR, Perrone L, Nielsen MS, Calissano P, Andersen OM, Matrone C - Front Cell Neurosci (2015)

Bottom Line: Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA), resulting in endo-lysosomal dysfunctions and neuronal degeneration.These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration.In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular Biology and Neurobiology, National Council of Research of Rome , Rome , Italy.

ABSTRACT
The intracellular transport and localization of amyloid precursor protein (APP) are critical determinants of APP processing and β-amyloid peptide production, thus crucially important for the pathophysiology of Alzheimer's disease (AD). Notably, the C-terminal Y682ENPTY687 domain of APP binds to specific adaptors controlling APP trafficking and sorting in neurons. Mutation on the Y682 residue to glycine (Y682G) leads to altered APP sorting in hippocampal neurons that favors its accumulation in intracellular compartments and the release of soluble APPα. Such alterations induce premature aging and learning and cognitive deficits in APP Y682G mutant mice (APP (YG/YG) ). Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA), resulting in endo-lysosomal dysfunctions and neuronal degeneration. Moreover, disruption of the APP/SorLA complex changes the trafficking pathway of SorLA, with its consequent increase in secretion outside neurons. Mutations in the SorLA gene are a prognostic factor in AD, and changes in SorLA levels in cerebrospinal fluid are predictive of AD in humans. These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration. In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

No MeSH data available.


Related in: MedlinePlus

Y682G mutation prevents binding of APP to SorLA and increases SorLA secretion. (A–H) Confocal microscopy analysis of double-staining for rabbit anti-SorLA [green; (A,E)] and mouse anti-APP [red; (B,F)] in WT and APPYG/YG (YG) hippocampal neurons [(A–C) and (E–G), respectively; 63× objective]. Scale bars = 7 μm. (D,H) high magnification of the (C) and (G), respectively. Co-localization analysis was performed using Zen software (I). The panels are representative of seven different experiments. (G) Quantitative analysis was performed by comparing the R coefficient (Pearson’s coefficient) between WT and APPYG/YG (YG) neurons. n = 7. *p < 0.05. (J,K). Protein samples from hippocampi from either WT or APPYG/YG (YG) mice were immunoprecipitated with mouse anti-α-SorLA antibody (co-IP SorLA) and analyzed with rabbit anti-APP. TL, total lysate; IP, immunoprecipitate. The same membrane was stripped and analyzed with mouse anti-SorLA. (L,M) SorLA WB analysis in the soluble and insoluble fractions from hippocampal tissues from WT and APPYG/YG (YG) mice. As positive and negative controls for the fractionations, we used HSC70 protein and Lamp1, respectively. The optical density analysis is reported below. Data from the soluble (Sol) and insoluble (Unsol) fractions were normalized to the corresponding HSC70 and Lamp1 values and are expressed as a percentage of WT (n = 4). *p < 0.05 vs. WT (Newman–Keuls test). (N) WB analysis of APP, sAPPα, and sSorLA from hippocampal primary neuronal cultures of WT and APPYG/YG (YG) mice (O) Medium collected from APPYG/YG (YG) neurons and the corresponding control (WT) samples were analyzed by WB for sAPPα and sSorLA (see Materials and Methods). WB is representative of three different experiments. (P) Densitometric analysis of (N,O). Black histogram: WT; Gray histogram: YG. (N = 3; Newman–Keuls test) *p < 0.05 vs. WT.
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Figure 4: Y682G mutation prevents binding of APP to SorLA and increases SorLA secretion. (A–H) Confocal microscopy analysis of double-staining for rabbit anti-SorLA [green; (A,E)] and mouse anti-APP [red; (B,F)] in WT and APPYG/YG (YG) hippocampal neurons [(A–C) and (E–G), respectively; 63× objective]. Scale bars = 7 μm. (D,H) high magnification of the (C) and (G), respectively. Co-localization analysis was performed using Zen software (I). The panels are representative of seven different experiments. (G) Quantitative analysis was performed by comparing the R coefficient (Pearson’s coefficient) between WT and APPYG/YG (YG) neurons. n = 7. *p < 0.05. (J,K). Protein samples from hippocampi from either WT or APPYG/YG (YG) mice were immunoprecipitated with mouse anti-α-SorLA antibody (co-IP SorLA) and analyzed with rabbit anti-APP. TL, total lysate; IP, immunoprecipitate. The same membrane was stripped and analyzed with mouse anti-SorLA. (L,M) SorLA WB analysis in the soluble and insoluble fractions from hippocampal tissues from WT and APPYG/YG (YG) mice. As positive and negative controls for the fractionations, we used HSC70 protein and Lamp1, respectively. The optical density analysis is reported below. Data from the soluble (Sol) and insoluble (Unsol) fractions were normalized to the corresponding HSC70 and Lamp1 values and are expressed as a percentage of WT (n = 4). *p < 0.05 vs. WT (Newman–Keuls test). (N) WB analysis of APP, sAPPα, and sSorLA from hippocampal primary neuronal cultures of WT and APPYG/YG (YG) mice (O) Medium collected from APPYG/YG (YG) neurons and the corresponding control (WT) samples were analyzed by WB for sAPPα and sSorLA (see Materials and Methods). WB is representative of three different experiments. (P) Densitometric analysis of (N,O). Black histogram: WT; Gray histogram: YG. (N = 3; Newman–Keuls test) *p < 0.05 vs. WT.

Mentions: Confocal microscopy of hippocampal neurons that were immunostained for APP and SorLA showed a strong co-localization between SorLA and APP in WT cells (Figures 4A–D). However, such co-localization was significantly reduced in APPYG/YG neurons (Figures 4E–H). Interestingly, confocal microscopy analysis indicated a decrease in SorLA in the cell body and an increase along the neurites of Y682G neurons (Figure 4).


Y682G Mutation of Amyloid Precursor Protein Promotes Endo-Lysosomal Dysfunction by Disrupting APP-SorLA Interaction.

La Rosa LR, Perrone L, Nielsen MS, Calissano P, Andersen OM, Matrone C - Front Cell Neurosci (2015)

Y682G mutation prevents binding of APP to SorLA and increases SorLA secretion. (A–H) Confocal microscopy analysis of double-staining for rabbit anti-SorLA [green; (A,E)] and mouse anti-APP [red; (B,F)] in WT and APPYG/YG (YG) hippocampal neurons [(A–C) and (E–G), respectively; 63× objective]. Scale bars = 7 μm. (D,H) high magnification of the (C) and (G), respectively. Co-localization analysis was performed using Zen software (I). The panels are representative of seven different experiments. (G) Quantitative analysis was performed by comparing the R coefficient (Pearson’s coefficient) between WT and APPYG/YG (YG) neurons. n = 7. *p < 0.05. (J,K). Protein samples from hippocampi from either WT or APPYG/YG (YG) mice were immunoprecipitated with mouse anti-α-SorLA antibody (co-IP SorLA) and analyzed with rabbit anti-APP. TL, total lysate; IP, immunoprecipitate. The same membrane was stripped and analyzed with mouse anti-SorLA. (L,M) SorLA WB analysis in the soluble and insoluble fractions from hippocampal tissues from WT and APPYG/YG (YG) mice. As positive and negative controls for the fractionations, we used HSC70 protein and Lamp1, respectively. The optical density analysis is reported below. Data from the soluble (Sol) and insoluble (Unsol) fractions were normalized to the corresponding HSC70 and Lamp1 values and are expressed as a percentage of WT (n = 4). *p < 0.05 vs. WT (Newman–Keuls test). (N) WB analysis of APP, sAPPα, and sSorLA from hippocampal primary neuronal cultures of WT and APPYG/YG (YG) mice (O) Medium collected from APPYG/YG (YG) neurons and the corresponding control (WT) samples were analyzed by WB for sAPPα and sSorLA (see Materials and Methods). WB is representative of three different experiments. (P) Densitometric analysis of (N,O). Black histogram: WT; Gray histogram: YG. (N = 3; Newman–Keuls test) *p < 0.05 vs. WT.
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Related In: Results  -  Collection

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Figure 4: Y682G mutation prevents binding of APP to SorLA and increases SorLA secretion. (A–H) Confocal microscopy analysis of double-staining for rabbit anti-SorLA [green; (A,E)] and mouse anti-APP [red; (B,F)] in WT and APPYG/YG (YG) hippocampal neurons [(A–C) and (E–G), respectively; 63× objective]. Scale bars = 7 μm. (D,H) high magnification of the (C) and (G), respectively. Co-localization analysis was performed using Zen software (I). The panels are representative of seven different experiments. (G) Quantitative analysis was performed by comparing the R coefficient (Pearson’s coefficient) between WT and APPYG/YG (YG) neurons. n = 7. *p < 0.05. (J,K). Protein samples from hippocampi from either WT or APPYG/YG (YG) mice were immunoprecipitated with mouse anti-α-SorLA antibody (co-IP SorLA) and analyzed with rabbit anti-APP. TL, total lysate; IP, immunoprecipitate. The same membrane was stripped and analyzed with mouse anti-SorLA. (L,M) SorLA WB analysis in the soluble and insoluble fractions from hippocampal tissues from WT and APPYG/YG (YG) mice. As positive and negative controls for the fractionations, we used HSC70 protein and Lamp1, respectively. The optical density analysis is reported below. Data from the soluble (Sol) and insoluble (Unsol) fractions were normalized to the corresponding HSC70 and Lamp1 values and are expressed as a percentage of WT (n = 4). *p < 0.05 vs. WT (Newman–Keuls test). (N) WB analysis of APP, sAPPα, and sSorLA from hippocampal primary neuronal cultures of WT and APPYG/YG (YG) mice (O) Medium collected from APPYG/YG (YG) neurons and the corresponding control (WT) samples were analyzed by WB for sAPPα and sSorLA (see Materials and Methods). WB is representative of three different experiments. (P) Densitometric analysis of (N,O). Black histogram: WT; Gray histogram: YG. (N = 3; Newman–Keuls test) *p < 0.05 vs. WT.
Mentions: Confocal microscopy of hippocampal neurons that were immunostained for APP and SorLA showed a strong co-localization between SorLA and APP in WT cells (Figures 4A–D). However, such co-localization was significantly reduced in APPYG/YG neurons (Figures 4E–H). Interestingly, confocal microscopy analysis indicated a decrease in SorLA in the cell body and an increase along the neurites of Y682G neurons (Figure 4).

Bottom Line: Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA), resulting in endo-lysosomal dysfunctions and neuronal degeneration.These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration.In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular Biology and Neurobiology, National Council of Research of Rome , Rome , Italy.

ABSTRACT
The intracellular transport and localization of amyloid precursor protein (APP) are critical determinants of APP processing and β-amyloid peptide production, thus crucially important for the pathophysiology of Alzheimer's disease (AD). Notably, the C-terminal Y682ENPTY687 domain of APP binds to specific adaptors controlling APP trafficking and sorting in neurons. Mutation on the Y682 residue to glycine (Y682G) leads to altered APP sorting in hippocampal neurons that favors its accumulation in intracellular compartments and the release of soluble APPα. Such alterations induce premature aging and learning and cognitive deficits in APP Y682G mutant mice (APP (YG/YG) ). Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA), resulting in endo-lysosomal dysfunctions and neuronal degeneration. Moreover, disruption of the APP/SorLA complex changes the trafficking pathway of SorLA, with its consequent increase in secretion outside neurons. Mutations in the SorLA gene are a prognostic factor in AD, and changes in SorLA levels in cerebrospinal fluid are predictive of AD in humans. These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration. In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

No MeSH data available.


Related in: MedlinePlus