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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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Relative expression levels of genes in H4 cells treated with three anti-LRRTM3 and a control siRNA.Bar graphs depicting mean relative gene expression levels and error bars representing the standard deviations obtained from the averages of 2–6 experiments where each experiment is assessed in quadruplicate. Relative expression values are obtained by the delta delta Ct method, where HPRT is utilized as the control gene (delta Ct) and all results are normalized to one of the control wells (delta delta Ct). Relative expression values (2?(-delta delta Ct)) are plotted on the y-axis. The different siRNA treatment groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.
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pone-0064164-g003: Relative expression levels of genes in H4 cells treated with three anti-LRRTM3 and a control siRNA.Bar graphs depicting mean relative gene expression levels and error bars representing the standard deviations obtained from the averages of 2–6 experiments where each experiment is assessed in quadruplicate. Relative expression values are obtained by the delta delta Ct method, where HPRT is utilized as the control gene (delta Ct) and all results are normalized to one of the control wells (delta delta Ct). Relative expression values (2?(-delta delta Ct)) are plotted on the y-axis. The different siRNA treatment groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.

Mentions: To investigate whether the siRNA knock-down of LRRTM3 influenced expression levels of BACE1, APP or its catenin counterpart CTNNA3, we tested the three LRRTM3 siRNAs and the control siRNA in H4 cells that stably overexpress wild type APP and measured expression levels of these genes. LRRTM3 siRNA_1 (SASI_Hs02_00369484) and siRNA_3 (SASI_Hs01_00163676) clearly led to knock-down of this gene. LRRTM3 expression results for siRNA_2 (SASI_Hs01_00163674) treatment experiments had a larger standard deviation precluding this conclusion (Figure 3, Table S2 in File S1). Nonetheless, all three LRRTM3 siRNAs had a knock-down effect observed in the Westerns (Figure S1), although siRNA_3 had the biggest effect (Figure 3, Figures S1–S2, Table S2 in File S1). Testing of differences between the different treatment groups by Kruskall-Wallis non-parametric test revealed significant differences for LRRTM3 gene expression levels (p = 0.016). We observed knock-down of BACE1 and increase in CTNNA3 expression levels, the trends of which were consistent especially for the siRNA_1 and siRNA_3 knocked-down LRRTM3 levels. Significant treatment-group effects were observed for BACE1 (p = 0.006) and CTNNA3 (p = 0.004), but not for APP gene expression (p = 0.9).


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)

Relative expression levels of genes in H4 cells treated with three anti-LRRTM3 and a control siRNA.Bar graphs depicting mean relative gene expression levels and error bars representing the standard deviations obtained from the averages of 2–6 experiments where each experiment is assessed in quadruplicate. Relative expression values are obtained by the delta delta Ct method, where HPRT is utilized as the control gene (delta Ct) and all results are normalized to one of the control wells (delta delta Ct). Relative expression values (2?(-delta delta Ct)) are plotted on the y-axis. The different siRNA treatment groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672107&req=5

pone-0064164-g003: Relative expression levels of genes in H4 cells treated with three anti-LRRTM3 and a control siRNA.Bar graphs depicting mean relative gene expression levels and error bars representing the standard deviations obtained from the averages of 2–6 experiments where each experiment is assessed in quadruplicate. Relative expression values are obtained by the delta delta Ct method, where HPRT is utilized as the control gene (delta Ct) and all results are normalized to one of the control wells (delta delta Ct). Relative expression values (2?(-delta delta Ct)) are plotted on the y-axis. The different siRNA treatment groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.
Mentions: To investigate whether the siRNA knock-down of LRRTM3 influenced expression levels of BACE1, APP or its catenin counterpart CTNNA3, we tested the three LRRTM3 siRNAs and the control siRNA in H4 cells that stably overexpress wild type APP and measured expression levels of these genes. LRRTM3 siRNA_1 (SASI_Hs02_00369484) and siRNA_3 (SASI_Hs01_00163676) clearly led to knock-down of this gene. LRRTM3 expression results for siRNA_2 (SASI_Hs01_00163674) treatment experiments had a larger standard deviation precluding this conclusion (Figure 3, Table S2 in File S1). Nonetheless, all three LRRTM3 siRNAs had a knock-down effect observed in the Westerns (Figure S1), although siRNA_3 had the biggest effect (Figure 3, Figures S1–S2, Table S2 in File S1). Testing of differences between the different treatment groups by Kruskall-Wallis non-parametric test revealed significant differences for LRRTM3 gene expression levels (p = 0.016). We observed knock-down of BACE1 and increase in CTNNA3 expression levels, the trends of which were consistent especially for the siRNA_1 and siRNA_3 knocked-down LRRTM3 levels. Significant treatment-group effects were observed for BACE1 (p = 0.006) and CTNNA3 (p = 0.004), but not for APP gene expression (p = 0.9).

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