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

LRP1 induces a shift in the subcellular localization of BACE1 from early to late endosomes in both HEK293 and primary neurons, likely promoting lysosomal degradation. A, HEK293 cells transfected with BACE1 plus LRP-L4 (B+L) or BACE1 only (B only) were analyzed by triple-immunofluorescence staining with anti-EEA1/anti-rab7a (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-EEA1/anti-rab7a (green) and 1D4 (red) antibodies. Note that colocalization of HA, 1D4, and EEA1/rab7a immunoreactive signals was observed in cells expressing BACE1 and LRP-L4, whereas colocalization of signals for 1D4 and EEA1, but not rab7a, was observed in cells expressing BACE1 only. Scale bars, 20 μm. B, HEK293 cells transfected with BACE1 plus LRP-L4 or BACE1 only were analyzed by triple-immunofluorescence staining with anti-β-COP/anti-γ1-adaptin (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-β-COP/anti-γ1- (continued in page 11). adaptin (green) and 1D4 (red) antibodies, as in A. C, Primary neurons grown on coverslips were infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses. Cells were analyzed as in A, except that goat anti-EEA1 and rabbit anti-HA antibodies were used. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4, HA, and EEA1/rab7a immunoreactive signals in soma and neurites, whereas neurons expressing only BACE1 exhibiting overlapping signals of 1D4 and EEA1, but not rab7a. Scale bars, 20 μm. D, Primary neurons infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses were analyzed by triple- or double-immunofluorescence analysis performed with anti-β-COP/anti-γ1-adaptin, anti-HA, and 1D4 antibodies, as in B. Scale bars, 20 μm. E, HEK293 cells transfected with BACE1 plus LRP-L4 were subjected to cycloheximide (CHX) chase experiments, in which cells were coincubated with or without chloroquine (CQ; 50 μM). After 12 h, cells were lysed and analyzed by Western blotting. Relative levels of BACE1 and LRP-L4 were quantified and graphed (n = 3, *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: LRP1 induces a shift in the subcellular localization of BACE1 from early to late endosomes in both HEK293 and primary neurons, likely promoting lysosomal degradation. A, HEK293 cells transfected with BACE1 plus LRP-L4 (B+L) or BACE1 only (B only) were analyzed by triple-immunofluorescence staining with anti-EEA1/anti-rab7a (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-EEA1/anti-rab7a (green) and 1D4 (red) antibodies. Note that colocalization of HA, 1D4, and EEA1/rab7a immunoreactive signals was observed in cells expressing BACE1 and LRP-L4, whereas colocalization of signals for 1D4 and EEA1, but not rab7a, was observed in cells expressing BACE1 only. Scale bars, 20 μm. B, HEK293 cells transfected with BACE1 plus LRP-L4 or BACE1 only were analyzed by triple-immunofluorescence staining with anti-β-COP/anti-γ1-adaptin (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-β-COP/anti-γ1- (continued in page 11). adaptin (green) and 1D4 (red) antibodies, as in A. C, Primary neurons grown on coverslips were infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses. Cells were analyzed as in A, except that goat anti-EEA1 and rabbit anti-HA antibodies were used. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4, HA, and EEA1/rab7a immunoreactive signals in soma and neurites, whereas neurons expressing only BACE1 exhibiting overlapping signals of 1D4 and EEA1, but not rab7a. Scale bars, 20 μm. D, Primary neurons infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses were analyzed by triple- or double-immunofluorescence analysis performed with anti-β-COP/anti-γ1-adaptin, anti-HA, and 1D4 antibodies, as in B. Scale bars, 20 μm. E, HEK293 cells transfected with BACE1 plus LRP-L4 were subjected to cycloheximide (CHX) chase experiments, in which cells were coincubated with or without chloroquine (CQ; 50 μM). After 12 h, cells were lysed and analyzed by Western blotting. Relative levels of BACE1 and LRP-L4 were quantified and graphed (n = 3, *p < 0.05).

Mentions: We hypothesized that LRP-L4 may alter BACE1 subcellular localization to promote its degradation, and therefore investigated the subcellular location of BACE1 using HEK293 cells and primary neurons. To this end, we performed triple immunostaining with anti-HA, 1D4, and antibodies against early endosomal antigen 1 (EEA1), rab7a, β-COP, or γ1-adaptin, markers of early endosomes, late endosomes, the Golgi apparatus, and the trans-Golgi network (TGN), respectively. In HEK293 cells coexpressing BACE1 and LRP-L4, 1D4 and HA immunoreactive signals partially overlapped with those of EEA1 and rab7a (Fig. 5A). In contrast, in HEK293 cells expressing only BACE1, 1D4 immunoreactivity clearly overlapped with that of EEA1, but only marginally with that of rab7a (Fig. 5A). We additionally observed only limited colocalization of 1D4 immunoreactive signals with those of β-COP or γ1-adaptin in both cells expressing only BACE1 and those expressing BACE1 and LRP-L4 (Fig. 5B). The finding that 1D4-positive granules in cells coexpressing BACE1 and LRP-L4 were larger than those in cells expressing only BACE1 seemed to reflect the different subcellular locations of BACE1 in these cells.


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 induces a shift in the subcellular localization of BACE1 from early to late endosomes in both HEK293 and primary neurons, likely promoting lysosomal degradation. A, HEK293 cells transfected with BACE1 plus LRP-L4 (B+L) or BACE1 only (B only) were analyzed by triple-immunofluorescence staining with anti-EEA1/anti-rab7a (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-EEA1/anti-rab7a (green) and 1D4 (red) antibodies. Note that colocalization of HA, 1D4, and EEA1/rab7a immunoreactive signals was observed in cells expressing BACE1 and LRP-L4, whereas colocalization of signals for 1D4 and EEA1, but not rab7a, was observed in cells expressing BACE1 only. Scale bars, 20 μm. B, HEK293 cells transfected with BACE1 plus LRP-L4 or BACE1 only were analyzed by triple-immunofluorescence staining with anti-β-COP/anti-γ1-adaptin (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-β-COP/anti-γ1- (continued in page 11). adaptin (green) and 1D4 (red) antibodies, as in A. C, Primary neurons grown on coverslips were infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses. Cells were analyzed as in A, except that goat anti-EEA1 and rabbit anti-HA antibodies were used. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4, HA, and EEA1/rab7a immunoreactive signals in soma and neurites, whereas neurons expressing only BACE1 exhibiting overlapping signals of 1D4 and EEA1, but not rab7a. Scale bars, 20 μm. D, Primary neurons infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses were analyzed by triple- or double-immunofluorescence analysis performed with anti-β-COP/anti-γ1-adaptin, anti-HA, and 1D4 antibodies, as in B. Scale bars, 20 μm. E, HEK293 cells transfected with BACE1 plus LRP-L4 were subjected to cycloheximide (CHX) chase experiments, in which cells were coincubated with or without chloroquine (CQ; 50 μM). After 12 h, cells were lysed and analyzed by Western blotting. Relative levels of BACE1 and LRP-L4 were quantified and graphed (n = 3, *p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: LRP1 induces a shift in the subcellular localization of BACE1 from early to late endosomes in both HEK293 and primary neurons, likely promoting lysosomal degradation. A, HEK293 cells transfected with BACE1 plus LRP-L4 (B+L) or BACE1 only (B only) were analyzed by triple-immunofluorescence staining with anti-EEA1/anti-rab7a (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-EEA1/anti-rab7a (green) and 1D4 (red) antibodies. Note that colocalization of HA, 1D4, and EEA1/rab7a immunoreactive signals was observed in cells expressing BACE1 and LRP-L4, whereas colocalization of signals for 1D4 and EEA1, but not rab7a, was observed in cells expressing BACE1 only. Scale bars, 20 μm. B, HEK293 cells transfected with BACE1 plus LRP-L4 or BACE1 only were analyzed by triple-immunofluorescence staining with anti-β-COP/anti-γ1-adaptin (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-β-COP/anti-γ1- (continued in page 11). adaptin (green) and 1D4 (red) antibodies, as in A. C, Primary neurons grown on coverslips were infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses. Cells were analyzed as in A, except that goat anti-EEA1 and rabbit anti-HA antibodies were used. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4, HA, and EEA1/rab7a immunoreactive signals in soma and neurites, whereas neurons expressing only BACE1 exhibiting overlapping signals of 1D4 and EEA1, but not rab7a. Scale bars, 20 μm. D, Primary neurons infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses were analyzed by triple- or double-immunofluorescence analysis performed with anti-β-COP/anti-γ1-adaptin, anti-HA, and 1D4 antibodies, as in B. Scale bars, 20 μm. E, HEK293 cells transfected with BACE1 plus LRP-L4 were subjected to cycloheximide (CHX) chase experiments, in which cells were coincubated with or without chloroquine (CQ; 50 μM). After 12 h, cells were lysed and analyzed by Western blotting. Relative levels of BACE1 and LRP-L4 were quantified and graphed (n = 3, *p < 0.05).
Mentions: We hypothesized that LRP-L4 may alter BACE1 subcellular localization to promote its degradation, and therefore investigated the subcellular location of BACE1 using HEK293 cells and primary neurons. To this end, we performed triple immunostaining with anti-HA, 1D4, and antibodies against early endosomal antigen 1 (EEA1), rab7a, β-COP, or γ1-adaptin, markers of early endosomes, late endosomes, the Golgi apparatus, and the trans-Golgi network (TGN), respectively. In HEK293 cells coexpressing BACE1 and LRP-L4, 1D4 and HA immunoreactive signals partially overlapped with those of EEA1 and rab7a (Fig. 5A). In contrast, in HEK293 cells expressing only BACE1, 1D4 immunoreactivity clearly overlapped with that of EEA1, but only marginally with that of rab7a (Fig. 5A). We additionally observed only limited colocalization of 1D4 immunoreactive signals with those of β-COP or γ1-adaptin in both cells expressing only BACE1 and those expressing BACE1 and LRP-L4 (Fig. 5B). The finding that 1D4-positive granules in cells coexpressing BACE1 and LRP-L4 were larger than those in cells expressing only BACE1 seemed to reflect the different subcellular locations of BACE1 in these cells.

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