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: 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.

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

LRP-L4 down-regulates BACE1 in HEK293 cells and primary neurons. A, LRP1 constructs. LRP-L4 is a functional LRP1 mini-receptor; an HA epitope was inserted into LRP-L4 after the signal peptide (SP) at the N-terminus. LRP1 is cleaved by furin to generate an N-terminal α-chain and a C-terminal β-chain. TM, Transmembrane domain. B, HEK293 cells were cotransfected with BACE1 and either LRP-L4 or empty vector. Similarly, cells were cotransfected with APP and LRP-L4 or BACE2 and LRP-L4. Cell lysates were analyzed by Western blotting with the indicated antibodies. C, HEK293 cells were cotransfected with BACE1 and the indicated amounts of LRP-L4 and/or empty vector. Cell lysates were analyzed by Western blotting with 1D4 or anti-LRP1 antibody. Relative BACE1 levels were quantified and graphed. D, Primary cultured neurons were infected with adenoviruses expressing BACE1 (5 moi) plus those expressing the indicated amounts of recombinant LRP-L4 and/or LacZ, and cell lysates were analyzed by Western blotting after 2 d. The graph indicates relative mature BACE1 levels. M, Mature; im, immature. E, Primary neurons were coinfected with the indicated adenoviruses expressing swAPP, BACE1, and/or LRP-L4 (3 moi each), and maintained for 2 d. The total amount of adenovirus infected was equalized by addition of LacZ adenovirus. Cell lysates were analyzed by Western (continued in page 6). blotting as above. The graph indicates relative mature BACE1 levels. Aβ40 levels in 24-h-conditioned media were determined with sandwich ELISA. C−E, n = 3; *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: LRP-L4 down-regulates BACE1 in HEK293 cells and primary neurons. A, LRP1 constructs. LRP-L4 is a functional LRP1 mini-receptor; an HA epitope was inserted into LRP-L4 after the signal peptide (SP) at the N-terminus. LRP1 is cleaved by furin to generate an N-terminal α-chain and a C-terminal β-chain. TM, Transmembrane domain. B, HEK293 cells were cotransfected with BACE1 and either LRP-L4 or empty vector. Similarly, cells were cotransfected with APP and LRP-L4 or BACE2 and LRP-L4. Cell lysates were analyzed by Western blotting with the indicated antibodies. C, HEK293 cells were cotransfected with BACE1 and the indicated amounts of LRP-L4 and/or empty vector. Cell lysates were analyzed by Western blotting with 1D4 or anti-LRP1 antibody. Relative BACE1 levels were quantified and graphed. D, Primary cultured neurons were infected with adenoviruses expressing BACE1 (5 moi) plus those expressing the indicated amounts of recombinant LRP-L4 and/or LacZ, and cell lysates were analyzed by Western blotting after 2 d. The graph indicates relative mature BACE1 levels. M, Mature; im, immature. E, Primary neurons were coinfected with the indicated adenoviruses expressing swAPP, BACE1, and/or LRP-L4 (3 moi each), and maintained for 2 d. The total amount of adenovirus infected was equalized by addition of LacZ adenovirus. Cell lysates were analyzed by Western (continued in page 6). blotting as above. The graph indicates relative mature BACE1 levels. Aβ40 levels in 24-h-conditioned media were determined with sandwich ELISA. C−E, n = 3; *p < 0.05, **p < 0.01.

Mentions: HEK293 cells on a six-well plate were transfected with appropriate cDNAs using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The cDNA constructs used were BACE1 and BACE2 with a C-terminal rhodopsin (rho) tag (generous gifts from Dr. Michael Farzan, The Scripps Research Institute, Jupiter, FL) (Farzan et al., 2000), LRP-L4 (Takeda et al., 2003) (see Fig. 2A), myc-tagged dynamin K44A (a generous gift from Dr. Mark A. McNiven, Mayo Clinic, Rochester, MN), and WT APP695 (Takeda et al., 2004). All cDNAs were subcloned into the pcDNA3.1 vector (Invitrogen). Recombinant adenoviruses expressing rho-tagged BACE1, WT APP, Swedish mutant APP695 (swAPP) (Motoki et al., 2012), LacZ (Araki et al., 2001), or LRP-L4 were prepared using an Adenovirus Dual Expression Vector Kit (Takara Bio) according to manufacturer’s instructions. Rat primary cultured neurons were infected with each recombinant adenovirus at a multiplicity of infection (moi) of 5 at 7-8 d in vitro.


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)

LRP-L4 down-regulates BACE1 in HEK293 cells and primary neurons. A, LRP1 constructs. LRP-L4 is a functional LRP1 mini-receptor; an HA epitope was inserted into LRP-L4 after the signal peptide (SP) at the N-terminus. LRP1 is cleaved by furin to generate an N-terminal α-chain and a C-terminal β-chain. TM, Transmembrane domain. B, HEK293 cells were cotransfected with BACE1 and either LRP-L4 or empty vector. Similarly, cells were cotransfected with APP and LRP-L4 or BACE2 and LRP-L4. Cell lysates were analyzed by Western blotting with the indicated antibodies. C, HEK293 cells were cotransfected with BACE1 and the indicated amounts of LRP-L4 and/or empty vector. Cell lysates were analyzed by Western blotting with 1D4 or anti-LRP1 antibody. Relative BACE1 levels were quantified and graphed. D, Primary cultured neurons were infected with adenoviruses expressing BACE1 (5 moi) plus those expressing the indicated amounts of recombinant LRP-L4 and/or LacZ, and cell lysates were analyzed by Western blotting after 2 d. The graph indicates relative mature BACE1 levels. M, Mature; im, immature. E, Primary neurons were coinfected with the indicated adenoviruses expressing swAPP, BACE1, and/or LRP-L4 (3 moi each), and maintained for 2 d. The total amount of adenovirus infected was equalized by addition of LacZ adenovirus. Cell lysates were analyzed by Western (continued in page 6). blotting as above. The graph indicates relative mature BACE1 levels. Aβ40 levels in 24-h-conditioned media were determined with sandwich ELISA. C−E, n = 3; *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: LRP-L4 down-regulates BACE1 in HEK293 cells and primary neurons. A, LRP1 constructs. LRP-L4 is a functional LRP1 mini-receptor; an HA epitope was inserted into LRP-L4 after the signal peptide (SP) at the N-terminus. LRP1 is cleaved by furin to generate an N-terminal α-chain and a C-terminal β-chain. TM, Transmembrane domain. B, HEK293 cells were cotransfected with BACE1 and either LRP-L4 or empty vector. Similarly, cells were cotransfected with APP and LRP-L4 or BACE2 and LRP-L4. Cell lysates were analyzed by Western blotting with the indicated antibodies. C, HEK293 cells were cotransfected with BACE1 and the indicated amounts of LRP-L4 and/or empty vector. Cell lysates were analyzed by Western blotting with 1D4 or anti-LRP1 antibody. Relative BACE1 levels were quantified and graphed. D, Primary cultured neurons were infected with adenoviruses expressing BACE1 (5 moi) plus those expressing the indicated amounts of recombinant LRP-L4 and/or LacZ, and cell lysates were analyzed by Western blotting after 2 d. The graph indicates relative mature BACE1 levels. M, Mature; im, immature. E, Primary neurons were coinfected with the indicated adenoviruses expressing swAPP, BACE1, and/or LRP-L4 (3 moi each), and maintained for 2 d. The total amount of adenovirus infected was equalized by addition of LacZ adenovirus. Cell lysates were analyzed by Western (continued in page 6). blotting as above. The graph indicates relative mature BACE1 levels. Aβ40 levels in 24-h-conditioned media were determined with sandwich ELISA. C−E, n = 3; *p < 0.05, **p < 0.01.
Mentions: HEK293 cells on a six-well plate were transfected with appropriate cDNAs using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions. The cDNA constructs used were BACE1 and BACE2 with a C-terminal rhodopsin (rho) tag (generous gifts from Dr. Michael Farzan, The Scripps Research Institute, Jupiter, FL) (Farzan et al., 2000), LRP-L4 (Takeda et al., 2003) (see Fig. 2A), myc-tagged dynamin K44A (a generous gift from Dr. Mark A. McNiven, Mayo Clinic, Rochester, MN), and WT APP695 (Takeda et al., 2004). All cDNAs were subcloned into the pcDNA3.1 vector (Invitrogen). Recombinant adenoviruses expressing rho-tagged BACE1, WT APP, Swedish mutant APP695 (swAPP) (Motoki et al., 2012), LacZ (Araki et al., 2001), or LRP-L4 were prepared using an Adenovirus Dual Expression Vector Kit (Takara Bio) according to manufacturer’s instructions. Rat primary cultured neurons were infected with each recombinant adenovirus at a multiplicity of infection (moi) of 5 at 7-8 d in vitro.

Bottom Line: 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.

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