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LRP1 Downregulates the Alzheimer's β-Secretase BACE1 by Modulating Its Intraneuronal Trafficking(1,2,3).

Tanokashira D, Motoki K, Minegishi S, Hosaka A, Mamada N, Tamaoka A, Okada T, Lakshmana MK, Araki W - eNeuro (2015)

Bottom Line: Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons.We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aβ production.Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Demyelinating Disease and Aging, National Institute of Neuroscience , NCNP, Kodaira, Tokyo 187-8502, Japan.

ABSTRACT
The β-secretase called BACE1 is a membrane-associated protease that initiates the generation of amyloid β-protein (Aβ), a key event in Alzheimer's disease (AD). However, the mechanism of intraneuronal regulation of BACE1 is poorly understood. Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons. We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aβ production. Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation. These findings establish that LRP1 specifically downregulates BACE1 by modulating its intraneuronal trafficking and stability through protein interaction and highlight LRP1 as a potential therapeutic target in AD.

No MeSH data available.


Related in: MedlinePlus

LRP1 decreases BACE1 stability and reduces the cell surface expression of BACE1. A, HEK293 cells were transfected with the indicated amounts of BACE1 and either LRP-L4 or vector. Cell-surface biotinylation experiments were performed as described in Materials and Methods. Western blots of total cell lysates and avidin-agarose-precipitated material are shown. Relative BACE1 levels were quantified and graphed. B, HEK293 cells transfected with BACE1 plus LRP-L4 or vector as in A were subjected to cycloheximide chase experiments, as described in Materials and Methods. After incubation with cycloheximide (CHX) for the indicated times, cells were lysed and analyzed by Western blotting. Relative BACE1 levels at 0 and 12 h were quantified and graphed. (A, B: n = 3, *p < 0.05, **p < 0.01). C, HEK293 cells were cotransfected with BACE1, LRP-L4, and/or Dyn2 K44A as indicated. The total amount of DNA was equalized by the addition of vector. Cell lysates were analyzed by Western blotting with appropriate antibodies. Relative BACE1 levels in blots were quantified and graphed (n = 3, *p < 0.05, **p < 0.01).
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Figure 4: LRP1 decreases BACE1 stability and reduces the cell surface expression of BACE1. A, HEK293 cells were transfected with the indicated amounts of BACE1 and either LRP-L4 or vector. Cell-surface biotinylation experiments were performed as described in Materials and Methods. Western blots of total cell lysates and avidin-agarose-precipitated material are shown. Relative BACE1 levels were quantified and graphed. B, HEK293 cells transfected with BACE1 plus LRP-L4 or vector as in A were subjected to cycloheximide chase experiments, as described in Materials and Methods. After incubation with cycloheximide (CHX) for the indicated times, cells were lysed and analyzed by Western blotting. Relative BACE1 levels at 0 and 12 h were quantified and graphed. (A, B: n = 3, *p < 0.05, **p < 0.01). C, HEK293 cells were cotransfected with BACE1, LRP-L4, and/or Dyn2 K44A as indicated. The total amount of DNA was equalized by the addition of vector. Cell lysates were analyzed by Western blotting with appropriate antibodies. Relative BACE1 levels in blots were quantified and graphed (n = 3, *p < 0.05, **p < 0.01).

Mentions: We then performed immunocytochemical analyses to examine whether LRP-L4 influences the intracellular localization of BACE1 in HEK293 cells and primary neurons. In these experiments, comparable levels of BACE1 were expressed in cells coexpressing BACE1 and LRP-L4 and those expressing BACE1 alone by adjusting the amounts of BACE1 cDNA or recombinant BACE1 adenoviruses used for transfection or transduction, respectively (Figs. 3C, 4A). BACE1 and LRP-L4 were visualized using antibodies to the rhodopsin tag (1D4) and hemagglutinin (HA) tag, respectively. Immunostaining for 1D4 revealed positive granular staining in the cytosol in BACE1-expressing HEK293 cells. Interestingly, the size of 1D4-positive granules appeared to be larger in cells coexpressing BACE1 and LRP-L4 compared to those expressing BACE1 alone (Fig. 3B). Double-immunofluorescence staining with anti-1D4 and anti-HA antibodies revealed positive HA immunostaining in the cytosol and nuclear membrane, and showed that HA immunoreactivity partially overlapped with that of 1D4, indicating colocalization of BACE1 and LRP-L4 (Fig. 3B). The HA-immunostaining pattern was comparable between cells expressing BACE1 alone and those expressing BACE1 and LRP-L4. Similar findings were obtained with primary neurons expressing BACE1 and/or LRP-L4 as well. Immunostaining for 1D4 showed that immunopositive granules localized in perikarya and neuritic processes. Immunostaining for HA showed positive reticular immunoreactivity in perikarya and neurites. In neurons coexpressing BACE1 and LRP-L4, 1D4 and HA immunoreactive signals partially overlapped, indicating colocalization of BACE1 and LRP-L4 (Fig. 3D).


LRP1 Downregulates the Alzheimer's β-Secretase BACE1 by Modulating Its Intraneuronal Trafficking(1,2,3).

Tanokashira D, Motoki K, Minegishi S, Hosaka A, Mamada N, Tamaoka A, Okada T, Lakshmana MK, Araki W - eNeuro (2015)

LRP1 decreases BACE1 stability and reduces the cell surface expression of BACE1. A, HEK293 cells were transfected with the indicated amounts of BACE1 and either LRP-L4 or vector. Cell-surface biotinylation experiments were performed as described in Materials and Methods. Western blots of total cell lysates and avidin-agarose-precipitated material are shown. Relative BACE1 levels were quantified and graphed. B, HEK293 cells transfected with BACE1 plus LRP-L4 or vector as in A were subjected to cycloheximide chase experiments, as described in Materials and Methods. After incubation with cycloheximide (CHX) for the indicated times, cells were lysed and analyzed by Western blotting. Relative BACE1 levels at 0 and 12 h were quantified and graphed. (A, B: n = 3, *p < 0.05, **p < 0.01). C, HEK293 cells were cotransfected with BACE1, LRP-L4, and/or Dyn2 K44A as indicated. The total amount of DNA was equalized by the addition of vector. Cell lysates were analyzed by Western blotting with appropriate antibodies. Relative BACE1 levels in blots were quantified and graphed (n = 3, *p < 0.05, **p < 0.01).
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Figure 4: LRP1 decreases BACE1 stability and reduces the cell surface expression of BACE1. A, HEK293 cells were transfected with the indicated amounts of BACE1 and either LRP-L4 or vector. Cell-surface biotinylation experiments were performed as described in Materials and Methods. Western blots of total cell lysates and avidin-agarose-precipitated material are shown. Relative BACE1 levels were quantified and graphed. B, HEK293 cells transfected with BACE1 plus LRP-L4 or vector as in A were subjected to cycloheximide chase experiments, as described in Materials and Methods. After incubation with cycloheximide (CHX) for the indicated times, cells were lysed and analyzed by Western blotting. Relative BACE1 levels at 0 and 12 h were quantified and graphed. (A, B: n = 3, *p < 0.05, **p < 0.01). C, HEK293 cells were cotransfected with BACE1, LRP-L4, and/or Dyn2 K44A as indicated. The total amount of DNA was equalized by the addition of vector. Cell lysates were analyzed by Western blotting with appropriate antibodies. Relative BACE1 levels in blots were quantified and graphed (n = 3, *p < 0.05, **p < 0.01).
Mentions: We then performed immunocytochemical analyses to examine whether LRP-L4 influences the intracellular localization of BACE1 in HEK293 cells and primary neurons. In these experiments, comparable levels of BACE1 were expressed in cells coexpressing BACE1 and LRP-L4 and those expressing BACE1 alone by adjusting the amounts of BACE1 cDNA or recombinant BACE1 adenoviruses used for transfection or transduction, respectively (Figs. 3C, 4A). BACE1 and LRP-L4 were visualized using antibodies to the rhodopsin tag (1D4) and hemagglutinin (HA) tag, respectively. Immunostaining for 1D4 revealed positive granular staining in the cytosol in BACE1-expressing HEK293 cells. Interestingly, the size of 1D4-positive granules appeared to be larger in cells coexpressing BACE1 and LRP-L4 compared to those expressing BACE1 alone (Fig. 3B). Double-immunofluorescence staining with anti-1D4 and anti-HA antibodies revealed positive HA immunostaining in the cytosol and nuclear membrane, and showed that HA immunoreactivity partially overlapped with that of 1D4, indicating colocalization of BACE1 and LRP-L4 (Fig. 3B). The HA-immunostaining pattern was comparable between cells expressing BACE1 alone and those expressing BACE1 and LRP-L4. Similar findings were obtained with primary neurons expressing BACE1 and/or LRP-L4 as well. Immunostaining for 1D4 showed that immunopositive granules localized in perikarya and neuritic processes. Immunostaining for HA showed positive reticular immunoreactivity in perikarya and neurites. In neurons coexpressing BACE1 and LRP-L4, 1D4 and HA immunoreactive signals partially overlapped, indicating colocalization of BACE1 and LRP-L4 (Fig. 3D).

Bottom Line: Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons.We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aβ production.Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Demyelinating Disease and Aging, National Institute of Neuroscience , NCNP, Kodaira, Tokyo 187-8502, Japan.

ABSTRACT
The β-secretase called BACE1 is a membrane-associated protease that initiates the generation of amyloid β-protein (Aβ), a key event in Alzheimer's disease (AD). However, the mechanism of intraneuronal regulation of BACE1 is poorly understood. Here, we present evidence that low-density lipoprotein receptor-related protein 1 (LRP1), a multi-functional receptor, has a previously unrecognized function to regulate BACE1 in neurons. We show that deficiency of LRP1 exerts promotive effects on the protein expression and function of BACE1, whereas expression of LRP-L4, a functional LRP1 mini-receptor, specifically decreases BACE1 levels in both human embryonic kidney (HEK) 293 cells and rat primary neurons, leading to reduced Aβ production. Our subsequent analyses further demonstrate that (1) both endogenous and exogenous BACE1 and LRP1 interact with each other and are colocalized in soma and neurites of primary neurons, (2) LRP1 reduces the protein stability and cell-surface expression of BACE1, and (3) LRP1 facilitates the shift in intracellular localization of BACE1 from early to late endosomes, thereby promoting lysosomal degradation. These findings establish that LRP1 specifically downregulates BACE1 by modulating its intraneuronal trafficking and stability through protein interaction and highlight LRP1 as a potential therapeutic target in AD.

No MeSH data available.


Related in: MedlinePlus