Limits...
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

BACE1 protein expression is increased in LRP1-KO cells relative to WT cells. A, Cell lysates of LRP1-KO and WT cells were analyzed by Western blotting with antibodies against APP, BACE1, and LRP1. B, Quantification of relative protein levels in A (n = 3, *p < 0.05). C, The amounts of Aβ40 in 24-h-conditioned media from LRP1-KO and WT cells were measured by sandwich ELISA (n = 3, *p < 0.05). D, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ, WT APP, or rho-tagged BACE1 were maintained for 2 d, and cell lysates were analyzed by Western blotting as above. APP CTFs were analyzed by immunoprecipitation−Western blot analysis with anti-APP antibody, as described previously (Motoki et al., 2012). Images from the same blots were merged in the left panel. E−G, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ (E), BACE1 (F), or WT APP (G) were subjected to sucrose density gradient fractionation, as described in Materials and Methods. Each fraction was analyzed by Western blotting with antibodies against the rhodopsin tag (1D4), APP, BACE1, or flotillin-1. The level of BACE1 or APP in each fraction was quantified and expressed as a percentage of the total level in all fractions. For detection of APP CTFs in G, each fraction was additionally analyzed by immunoprecipitation−Western blot analysis with an anti-APP antibody, and the levels of α-CTF and β’-CTF in each fraction were quantified as above (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4596091&req=5

Figure 1: BACE1 protein expression is increased in LRP1-KO cells relative to WT cells. A, Cell lysates of LRP1-KO and WT cells were analyzed by Western blotting with antibodies against APP, BACE1, and LRP1. B, Quantification of relative protein levels in A (n = 3, *p < 0.05). C, The amounts of Aβ40 in 24-h-conditioned media from LRP1-KO and WT cells were measured by sandwich ELISA (n = 3, *p < 0.05). D, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ, WT APP, or rho-tagged BACE1 were maintained for 2 d, and cell lysates were analyzed by Western blotting as above. APP CTFs were analyzed by immunoprecipitation−Western blot analysis with anti-APP antibody, as described previously (Motoki et al., 2012). Images from the same blots were merged in the left panel. E−G, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ (E), BACE1 (F), or WT APP (G) were subjected to sucrose density gradient fractionation, as described in Materials and Methods. Each fraction was analyzed by Western blotting with antibodies against the rhodopsin tag (1D4), APP, BACE1, or flotillin-1. The level of BACE1 or APP in each fraction was quantified and expressed as a percentage of the total level in all fractions. For detection of APP CTFs in G, each fraction was additionally analyzed by immunoprecipitation−Western blot analysis with an anti-APP antibody, and the levels of α-CTF and β’-CTF in each fraction were quantified as above (n = 3).

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)

BACE1 protein expression is increased in LRP1-KO cells relative to WT cells. A, Cell lysates of LRP1-KO and WT cells were analyzed by Western blotting with antibodies against APP, BACE1, and LRP1. B, Quantification of relative protein levels in A (n = 3, *p < 0.05). C, The amounts of Aβ40 in 24-h-conditioned media from LRP1-KO and WT cells were measured by sandwich ELISA (n = 3, *p < 0.05). D, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ, WT APP, or rho-tagged BACE1 were maintained for 2 d, and cell lysates were analyzed by Western blotting as above. APP CTFs were analyzed by immunoprecipitation−Western blot analysis with anti-APP antibody, as described previously (Motoki et al., 2012). Images from the same blots were merged in the left panel. E−G, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ (E), BACE1 (F), or WT APP (G) were subjected to sucrose density gradient fractionation, as described in Materials and Methods. Each fraction was analyzed by Western blotting with antibodies against the rhodopsin tag (1D4), APP, BACE1, or flotillin-1. The level of BACE1 or APP in each fraction was quantified and expressed as a percentage of the total level in all fractions. For detection of APP CTFs in G, each fraction was additionally analyzed by immunoprecipitation−Western blot analysis with an anti-APP antibody, and the levels of α-CTF and β’-CTF in each fraction were quantified as above (n = 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596091&req=5

Figure 1: BACE1 protein expression is increased in LRP1-KO cells relative to WT cells. A, Cell lysates of LRP1-KO and WT cells were analyzed by Western blotting with antibodies against APP, BACE1, and LRP1. B, Quantification of relative protein levels in A (n = 3, *p < 0.05). C, The amounts of Aβ40 in 24-h-conditioned media from LRP1-KO and WT cells were measured by sandwich ELISA (n = 3, *p < 0.05). D, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ, WT APP, or rho-tagged BACE1 were maintained for 2 d, and cell lysates were analyzed by Western blotting as above. APP CTFs were analyzed by immunoprecipitation−Western blot analysis with anti-APP antibody, as described previously (Motoki et al., 2012). Images from the same blots were merged in the left panel. E−G, LRP1-KO and WT cells infected with recombinant adenoviruses expressing LacZ (E), BACE1 (F), or WT APP (G) were subjected to sucrose density gradient fractionation, as described in Materials and Methods. Each fraction was analyzed by Western blotting with antibodies against the rhodopsin tag (1D4), APP, BACE1, or flotillin-1. The level of BACE1 or APP in each fraction was quantified and expressed as a percentage of the total level in all fractions. For detection of APP CTFs in G, each fraction was additionally analyzed by immunoprecipitation−Western blot analysis with an anti-APP antibody, and the levels of α-CTF and β’-CTF in each fraction were quantified as above (n = 3).
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