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.

Highlights

  • Leucine rich repeat transmembrane protein 3 (LRRTM3) is a member of the synaptic leucine rich repeat transmembrane family with four highly conserved members, each with distinct brain distributions [1]

  • The harvested media was assessed by ELISA measurements of Ab40, Ab42, sAPPa and sAPPb and protein harvested from cells was analyzed for amyloid precursor protein (APP) by Western blot analysis

  • We confirm the prior results of decreased BACE1 cleavage products of APP in the setting of anti-LRRTM3 siRNA treatment of SH-SY5YAPP695wt cells [4]

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Summary

Introduction

LRRTM3 is a member of the synaptic leucine rich repeat transmembrane family with four highly conserved members, each with distinct brain distributions [1]. LRRTM3 exists within the seventh intron of CTNNA3 ( known as VR22), encoding a-T-catenin Both LRRTM3 and CTNNA3 are located at the genetic linkage peaks for plasma levels of amyloid ß (Aß) identified by our group in extended late-onset Alzheimer’s disease (LOAD) families [2] and for LOAD risk detected in an independent sib-pair study [3], making both genes interesting positional candidate LOAD risk genes. In a high-throughput siRNA screening of 15,200 genes by Majercak et al [4], siRNA targeting of LRRTM3 inhibited the secretion of Ab40, Ab42, and sAPPb in HEK293 and SH-SY5Y cells overexpressing amyloid precursor protein (APP), but had no effect on sAPPa or c-secretase cleavage of the 99 amino acid C-terminal fragment of APP. Despite additional scant evidence for promotion of APP processing by LRRTM3 [5,6], we are not aware of any studies to date that investigate the interaction between APP, BACE1 and LRRTM3

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