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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

Physical association and colocalization of BACE1 and LRP-L4. A, HEK293 cells were transfected with LRP-L4 and either BACE1 or empty vector. Protein extracts of membrane fractions were immunoprecipitated with 1D4 antibody and the precipitated proteins were analyzed by Western blotting, as described in Materials and Methods. B, HEK293 cells transfected with BACE1 plus LRP-L4, BACE1 only, or LRP-L4 only were analyzed by double-immunofluorescence staining with 1D4 (green) and anti-HA (magenta) antibodies. Overlapping 1D4 and HA immunoreactive signals were observed in cells coexpressing BACE1 and LRP-L4. Scale bar, 10 μm. C, Primary neurons were infected with the indicated amounts of recombinant adenoviruses expressing BACE1, LRP-L4, and/or LacZ. Two days after infection, cell lysates were analyzed by Western blotting with 1D4, anti-LRP1, or anti-β-galactosidase. Comparable BACE1 levels were observed in neurons infected with BACE1 adenoviruses (2.5 moi) only and those infected with BACE1 (5 moi) and LRP-L4 (1.25 moi) adenoviruses. D, Primary neurons grown on coverslips were infected with BACE1 plus LacZ adenoviruses, BACE1 plus LRP-L4 adenoviruses, or LRP-L4 plus LacZ adenoviruses. Cells were analyzed as in B. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4 and HA immunoreactive signals in soma and (continued in page 8). neurites. Scale bar, 20 μm. E, Protein extracts of membrane fractions of primary neurons were immunoprecipiated with anti-BACE1 (MAB9311) or 1D4 (negative control), followed by immunoblotting with anti-LRP1. The blots were reprobed with anti-BACE1 antibodies (AB5832 and D10E5). Images from the same blots were merged in this figure. F, Primary neurons grown on coverslips were analyzed by double-immunofluorescence staining with anti-LRP1 and Alexa568-labeled anti-BACE1. BACE1 and LRP1 immunoreactivities were clearly overlapped in both soma and neurites of neurons. Scale bar, 20 μm.
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Figure 3: Physical association and colocalization of BACE1 and LRP-L4. A, HEK293 cells were transfected with LRP-L4 and either BACE1 or empty vector. Protein extracts of membrane fractions were immunoprecipitated with 1D4 antibody and the precipitated proteins were analyzed by Western blotting, as described in Materials and Methods. B, HEK293 cells transfected with BACE1 plus LRP-L4, BACE1 only, or LRP-L4 only were analyzed by double-immunofluorescence staining with 1D4 (green) and anti-HA (magenta) antibodies. Overlapping 1D4 and HA immunoreactive signals were observed in cells coexpressing BACE1 and LRP-L4. Scale bar, 10 μm. C, Primary neurons were infected with the indicated amounts of recombinant adenoviruses expressing BACE1, LRP-L4, and/or LacZ. Two days after infection, cell lysates were analyzed by Western blotting with 1D4, anti-LRP1, or anti-β-galactosidase. Comparable BACE1 levels were observed in neurons infected with BACE1 adenoviruses (2.5 moi) only and those infected with BACE1 (5 moi) and LRP-L4 (1.25 moi) adenoviruses. D, Primary neurons grown on coverslips were infected with BACE1 plus LacZ adenoviruses, BACE1 plus LRP-L4 adenoviruses, or LRP-L4 plus LacZ adenoviruses. Cells were analyzed as in B. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4 and HA immunoreactive signals in soma and (continued in page 8). neurites. Scale bar, 20 μm. E, Protein extracts of membrane fractions of primary neurons were immunoprecipiated with anti-BACE1 (MAB9311) or 1D4 (negative control), followed by immunoblotting with anti-LRP1. The blots were reprobed with anti-BACE1 antibodies (AB5832 and D10E5). Images from the same blots were merged in this figure. F, Primary neurons grown on coverslips were analyzed by double-immunofluorescence staining with anti-LRP1 and Alexa568-labeled anti-BACE1. BACE1 and LRP1 immunoreactivities were clearly overlapped in both soma and neurites of neurons. Scale bar, 20 μm.

Mentions: We first compared the protein expression level of BACE1 in WT and LRP1-KO cells. Western blot analyses of cell lysates showed that the protein expression levels of endogenous BACE1 and APP in LRP1-KO cells were significantly higher than those in WT cells (Fig. 1A,B). The increase in APP level in LRP1-KO cells is consistent with a previous report (Liu et al., 2007). The specificity of anti-BACE1 antibody (AB5832) was confirmed by comparison with another anti-BACE1 antibody (D10E5), as presented below (see Fig. 3E). Additionally, sandwich ELISAs revealed that the amount of Aβ40 in conditioned media of LRP1-KO cells was higher than that in media conditioned by LRP1-WT cells (Fig. 1C). Overexpression of BACE1 or APP in LRP1-KO and LRP1-WT cells using recombinant adenoviruses resulted in much higher levels of BACE1 or APP in LRP1-KO than in LRP1-WT cells (Fig. 1D).


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)

Physical association and colocalization of BACE1 and LRP-L4. A, HEK293 cells were transfected with LRP-L4 and either BACE1 or empty vector. Protein extracts of membrane fractions were immunoprecipitated with 1D4 antibody and the precipitated proteins were analyzed by Western blotting, as described in Materials and Methods. B, HEK293 cells transfected with BACE1 plus LRP-L4, BACE1 only, or LRP-L4 only were analyzed by double-immunofluorescence staining with 1D4 (green) and anti-HA (magenta) antibodies. Overlapping 1D4 and HA immunoreactive signals were observed in cells coexpressing BACE1 and LRP-L4. Scale bar, 10 μm. C, Primary neurons were infected with the indicated amounts of recombinant adenoviruses expressing BACE1, LRP-L4, and/or LacZ. Two days after infection, cell lysates were analyzed by Western blotting with 1D4, anti-LRP1, or anti-β-galactosidase. Comparable BACE1 levels were observed in neurons infected with BACE1 adenoviruses (2.5 moi) only and those infected with BACE1 (5 moi) and LRP-L4 (1.25 moi) adenoviruses. D, Primary neurons grown on coverslips were infected with BACE1 plus LacZ adenoviruses, BACE1 plus LRP-L4 adenoviruses, or LRP-L4 plus LacZ adenoviruses. Cells were analyzed as in B. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4 and HA immunoreactive signals in soma and (continued in page 8). neurites. Scale bar, 20 μm. E, Protein extracts of membrane fractions of primary neurons were immunoprecipiated with anti-BACE1 (MAB9311) or 1D4 (negative control), followed by immunoblotting with anti-LRP1. The blots were reprobed with anti-BACE1 antibodies (AB5832 and D10E5). Images from the same blots were merged in this figure. F, Primary neurons grown on coverslips were analyzed by double-immunofluorescence staining with anti-LRP1 and Alexa568-labeled anti-BACE1. BACE1 and LRP1 immunoreactivities were clearly overlapped in both soma and neurites of neurons. Scale bar, 20 μm.
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Figure 3: Physical association and colocalization of BACE1 and LRP-L4. A, HEK293 cells were transfected with LRP-L4 and either BACE1 or empty vector. Protein extracts of membrane fractions were immunoprecipitated with 1D4 antibody and the precipitated proteins were analyzed by Western blotting, as described in Materials and Methods. B, HEK293 cells transfected with BACE1 plus LRP-L4, BACE1 only, or LRP-L4 only were analyzed by double-immunofluorescence staining with 1D4 (green) and anti-HA (magenta) antibodies. Overlapping 1D4 and HA immunoreactive signals were observed in cells coexpressing BACE1 and LRP-L4. Scale bar, 10 μm. C, Primary neurons were infected with the indicated amounts of recombinant adenoviruses expressing BACE1, LRP-L4, and/or LacZ. Two days after infection, cell lysates were analyzed by Western blotting with 1D4, anti-LRP1, or anti-β-galactosidase. Comparable BACE1 levels were observed in neurons infected with BACE1 adenoviruses (2.5 moi) only and those infected with BACE1 (5 moi) and LRP-L4 (1.25 moi) adenoviruses. D, Primary neurons grown on coverslips were infected with BACE1 plus LacZ adenoviruses, BACE1 plus LRP-L4 adenoviruses, or LRP-L4 plus LacZ adenoviruses. Cells were analyzed as in B. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4 and HA immunoreactive signals in soma and (continued in page 8). neurites. Scale bar, 20 μm. E, Protein extracts of membrane fractions of primary neurons were immunoprecipiated with anti-BACE1 (MAB9311) or 1D4 (negative control), followed by immunoblotting with anti-LRP1. The blots were reprobed with anti-BACE1 antibodies (AB5832 and D10E5). Images from the same blots were merged in this figure. F, Primary neurons grown on coverslips were analyzed by double-immunofluorescence staining with anti-LRP1 and Alexa568-labeled anti-BACE1. BACE1 and LRP1 immunoreactivities were clearly overlapped in both soma and neurites of neurons. Scale bar, 20 μm.
Mentions: We first compared the protein expression level of BACE1 in WT and LRP1-KO cells. Western blot analyses of cell lysates showed that the protein expression levels of endogenous BACE1 and APP in LRP1-KO cells were significantly higher than those in WT cells (Fig. 1A,B). The increase in APP level in LRP1-KO cells is consistent with a previous report (Liu et al., 2007). The specificity of anti-BACE1 antibody (AB5832) was confirmed by comparison with another anti-BACE1 antibody (D10E5), as presented below (see Fig. 3E). Additionally, sandwich ELISAs revealed that the amount of Aβ40 in conditioned media of LRP1-KO cells was higher than that in media conditioned by LRP1-WT cells (Fig. 1C). Overexpression of BACE1 or APP in LRP1-KO and LRP1-WT cells using recombinant adenoviruses resulted in much higher levels of BACE1 or APP in LRP1-KO than in LRP1-WT cells (Fig. 1D).

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