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LRRTM3 interacts with APP and BACE1 and has variants associating with late-onset Alzheimer's disease (LOAD).

Lincoln S, Allen M, Cox CL, Walker LP, Malphrus K, Qiu Y, Nguyen T, Rowley C, Kouri N, Crook J, Pankratz VS, Younkin S, Younkin L, Carrasquillo M, Zou F, Abdul-Hay SO, Springer W, Sando SB, Aasly JO, Barcikowska M, Wszolek ZK, Lewis JM, Dickson D, Graff-Radford NR, Petersen RC, Eckman E, Younkin SG, Ertekin-Taner N - PLoS ONE (2013)

Bottom Line: Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice.These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05).Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.

View Article: PubMed Central - PubMed

Affiliation: Mayo Clinic Florida, Department of Neuroscience, Jacksonville, Florida, USA.

ABSTRACT
Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer's disease (LOAD) risk and plasma amyloid β (Aβ) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5'UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.

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Co-localization of LRRTM3 and a) APP in early endosomes, b) BACE1 in early endosomes, c) APP in primary neurons.For a and b, SH-SY5Y-APP695wt cells were transduced with baculovirus expressing fused early-endosomal protein Rab5a and GFP. These cells were transfected with LRRTM3-V5 (a and b) and also with BACE1-HA (b). Results of staining with GFP fluorescence indicative of Rab5a expression (early endosomes, green); anti-V5 (LRRTM3, red); and either CT20 in a (APP, magenta) or anti-HA in b (BACE1, magenta) are shown with overlay of the three stains in the last panels of a and b. Co-localization of APP, LRRTM3 and early endosomes is visualized as white punctate intracellular structures in the last panel of a(arrow and arrowhead). Co-localization of APP, BACE1 and early endosomes is visualized as white punctate intracellular structures in the last panel of b. (Magnification: ×63). For c, primary neuronal cultures from Tg2576 transgenic mice transduced with rAAV-LRRTM3-V5 were stained with anti-V5 (LRRTM3, green) and CT20 (APP, red). Overlay of the two stains reveals co-localization of LRRTM3 and APP visualized as yellow puncta in the cell body (thin arrow) and neuronal process (thick arrow). (Magnification: ×100).
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pone-0064164-g001: Co-localization of LRRTM3 and a) APP in early endosomes, b) BACE1 in early endosomes, c) APP in primary neurons.For a and b, SH-SY5Y-APP695wt cells were transduced with baculovirus expressing fused early-endosomal protein Rab5a and GFP. These cells were transfected with LRRTM3-V5 (a and b) and also with BACE1-HA (b). Results of staining with GFP fluorescence indicative of Rab5a expression (early endosomes, green); anti-V5 (LRRTM3, red); and either CT20 in a (APP, magenta) or anti-HA in b (BACE1, magenta) are shown with overlay of the three stains in the last panels of a and b. Co-localization of APP, LRRTM3 and early endosomes is visualized as white punctate intracellular structures in the last panel of a(arrow and arrowhead). Co-localization of APP, BACE1 and early endosomes is visualized as white punctate intracellular structures in the last panel of b. (Magnification: ×63). For c, primary neuronal cultures from Tg2576 transgenic mice transduced with rAAV-LRRTM3-V5 were stained with anti-V5 (LRRTM3, green) and CT20 (APP, red). Overlay of the two stains reveals co-localization of LRRTM3 and APP visualized as yellow puncta in the cell body (thin arrow) and neuronal process (thick arrow). (Magnification: ×100).

Mentions: SH-SY5Y-APP695wt cells transiently transfected with LRRTM3 were transduced with baculovirus that expresses a fusion protein of an organelle marker and GFP. LRRTM3 co-localizes with the early endosomal marker (Figure 1, Figure S3), but not with lysosomes (Figure S4) or Golgi apparatus (Figure S5). When these cells were also stained for APP, co-localization of LRRTM3 and APP in punctate intracellular structures (Figure 1a, Figure S6), which also express the early endosomal marker is evident. APP also localizes within early endosomes (Figure 1a, Figures S7–S8), which is consistent with the literature [32].


LRRTM3 interacts with APP and BACE1 and has variants associating with late-onset Alzheimer's disease (LOAD).

Lincoln S, Allen M, Cox CL, Walker LP, Malphrus K, Qiu Y, Nguyen T, Rowley C, Kouri N, Crook J, Pankratz VS, Younkin S, Younkin L, Carrasquillo M, Zou F, Abdul-Hay SO, Springer W, Sando SB, Aasly JO, Barcikowska M, Wszolek ZK, Lewis JM, Dickson D, Graff-Radford NR, Petersen RC, Eckman E, Younkin SG, Ertekin-Taner N - PLoS ONE (2013)

Co-localization of LRRTM3 and a) APP in early endosomes, b) BACE1 in early endosomes, c) APP in primary neurons.For a and b, SH-SY5Y-APP695wt cells were transduced with baculovirus expressing fused early-endosomal protein Rab5a and GFP. These cells were transfected with LRRTM3-V5 (a and b) and also with BACE1-HA (b). Results of staining with GFP fluorescence indicative of Rab5a expression (early endosomes, green); anti-V5 (LRRTM3, red); and either CT20 in a (APP, magenta) or anti-HA in b (BACE1, magenta) are shown with overlay of the three stains in the last panels of a and b. Co-localization of APP, LRRTM3 and early endosomes is visualized as white punctate intracellular structures in the last panel of a(arrow and arrowhead). Co-localization of APP, BACE1 and early endosomes is visualized as white punctate intracellular structures in the last panel of b. (Magnification: ×63). For c, primary neuronal cultures from Tg2576 transgenic mice transduced with rAAV-LRRTM3-V5 were stained with anti-V5 (LRRTM3, green) and CT20 (APP, red). Overlay of the two stains reveals co-localization of LRRTM3 and APP visualized as yellow puncta in the cell body (thin arrow) and neuronal process (thick arrow). (Magnification: ×100).
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Related In: Results  -  Collection

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pone-0064164-g001: Co-localization of LRRTM3 and a) APP in early endosomes, b) BACE1 in early endosomes, c) APP in primary neurons.For a and b, SH-SY5Y-APP695wt cells were transduced with baculovirus expressing fused early-endosomal protein Rab5a and GFP. These cells were transfected with LRRTM3-V5 (a and b) and also with BACE1-HA (b). Results of staining with GFP fluorescence indicative of Rab5a expression (early endosomes, green); anti-V5 (LRRTM3, red); and either CT20 in a (APP, magenta) or anti-HA in b (BACE1, magenta) are shown with overlay of the three stains in the last panels of a and b. Co-localization of APP, LRRTM3 and early endosomes is visualized as white punctate intracellular structures in the last panel of a(arrow and arrowhead). Co-localization of APP, BACE1 and early endosomes is visualized as white punctate intracellular structures in the last panel of b. (Magnification: ×63). For c, primary neuronal cultures from Tg2576 transgenic mice transduced with rAAV-LRRTM3-V5 were stained with anti-V5 (LRRTM3, green) and CT20 (APP, red). Overlay of the two stains reveals co-localization of LRRTM3 and APP visualized as yellow puncta in the cell body (thin arrow) and neuronal process (thick arrow). (Magnification: ×100).
Mentions: SH-SY5Y-APP695wt cells transiently transfected with LRRTM3 were transduced with baculovirus that expresses a fusion protein of an organelle marker and GFP. LRRTM3 co-localizes with the early endosomal marker (Figure 1, Figure S3), but not with lysosomes (Figure S4) or Golgi apparatus (Figure S5). When these cells were also stained for APP, co-localization of LRRTM3 and APP in punctate intracellular structures (Figure 1a, Figure S6), which also express the early endosomal marker is evident. APP also localizes within early endosomes (Figure 1a, Figures S7–S8), which is consistent with the literature [32].

Bottom Line: Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice.These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05).Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.

View Article: PubMed Central - PubMed

Affiliation: Mayo Clinic Florida, Department of Neuroscience, Jacksonville, Florida, USA.

ABSTRACT
Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. LRRTM3 is a nested gene within α-T catenin (CTNNA3) and resides at the linkage peak for late-onset Alzheimer's disease (LOAD) risk and plasma amyloid β (Aβ) levels. In-vitro knock-down of LRRTM3 was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of LRRTM3 had lower BACE1 and higher CTNNA3 mRNA levels, but no change in APP. Brain mRNA levels of LRRTM3 showed significant correlations with BACE1, CTNNA3 and APP in ∼400 humans, but not in LRRTM3 knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking LRRTM3 in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5'UTR-Intron 1 of LRRTM3 that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02-0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of LRRTM3, CTNNA3, APP and BACE1 in humans might influence APP metabolism and ultimately risk of AD.

Show MeSH
Related in: MedlinePlus