Impact of RTN3 deficiency on expression of BACE1 and amyloid deposition

Abstract

Reticulon 3 (RTN3) has previously been shown to interact with BACE1 and negatively regulate BACE1 activity. To what extent RTN3 deficiency affects BACE1 activity is an intriguing question. In this study, we aimed to address this by generating RTN3-null mice. Mice with complete deficiency of RTN3 grow normally and have no obviously discernible phenotypes. Morphological analyses of RTN3-null mice showed no significant alterations in cellular structure, although RTN3 is recognized as a protein contributing to the shaping of tubular endoplasmic reticulum. Biochemical analysis revealed that RTN3 deficiency increased protein levels of BACE1. This elevation of BACE1 levels correlated with enhanced processing of amyloid precursor protein at the β-secretase site. We also demonstrated that RTN3 deficiency in Alzheimer's mouse models facilitates amyloid deposition, further supporting an in vivo role of RTN3 in the regulation of BACE1 activity. Since it has been shown that RTN3 monomer is reduced in brains of Alzheimer's patients, our results suggest that long-lasting reduction of RTN3 levels has adverse effects on BACE1 activity and may contribute to Alzheimer's pathogenesis.

Keywords: APP; Alzheimer's disease; BACE1; Nogo; neuritic plaques; reticulon.

Figure 1.

Generation of RTN3-null mice. A, Schematic illustration of rtn3 gene organization and targeted deletion. The targeting RTN3KO vector was generated by insertion of 4 and 5 kb fragments as marked. The position of PCR genotyping and the probe (515) for Southern blot analysis with BstEII digestion is also specified. B, C, Examples of PCR-genotyping results and Southern blot confirmation of RTN3 deficiency (KO), RTN3 heterozygous (Het), and wild type (WT) are shown. Genomic DNA was prepared from mouse G23 BAC clone, mouse tails from C56BL/6 (B6), and BLKSW strains. All control genomic DNA produced the expected fragment of 14.5 kDa after Best EII digestion. RTN3KO mice produced the 9 kb DNA fragment as expected.

Figure 2.

Comparison of neuronal ultrastructure of mouse brains. RTN3-null mice and their WT littermates (2 months old) were fixed for examination by electron microscopy. ER structure is specified with “er,” and no obvious differences were observed between these two different genotype samples. N, nucleus; m, mitochondria; g, Golgi apparatus. Scale bar, 1 μm.

Figure 3.

Expression of RTN3 variants in mouse tissues. A, Extensive in silico searches of the mouse EST database identified multiple spliced transcripts, which are illustrated and specified with a nomenclature consistent with other reticulon members. The transcriptional initiation sites are specified by arrows, and each exon is in a box. RTN3-A1, which contains exon 1 and the common exons 4–9, is the form that is commonly referred to as RTN3. Two transmembrane domains are specified with a boxed line under each relevant exon. B, Tissues were dissected from adult WT and RTN3-null littermates (6 months old in this case) and used for preparing protein lysates. On Western blots, antibody R458 recognizes the C-terminal 16 aa and is expected to recognize all RTN3 variants. Identified RTN3 variants other than RTN3-A1 are marked with arrows. Calnexin antibody was used for load control specification. C, P5 mice were used to prepare protein lysates for evaluation of developmental regulation of RTN3 expression. RTN3 (or RTN3-A1) is a predominant isoform. RTN3-C was only identified in the kidney. Antibody to actin was used to verify sample loading.

Figure 4.

BACE1 levels are elevated in RTN3-null mice. A, Brain lysates were prepared from the indicated genotype and analyzed by Western blotting. BACE1-null mice and Tg-APPswe/PSEN1ΔE9 bitransgenic mice were used for verifying BACE1 expression and APP processing. Antibody R458 was used to verify expression of RTN3 in each mouse. BACE1 levels were elevated in heterozygous RTN3 mice, and this was more pronounced in RTN3-null mice. The nonspecifically reacted band is shown in “>”. Full-length APP and its processing products were detected using antibody A8717. B, Five independent experiments were performed to verify the elevation of BACE1 in RTN3-null mice (6-month-old mice, n = 5 experiments and 2 mice in each experiment, *p < 0.01, ANOVA test).

Figure 5.

Production of Aβ was increased in RTN3-null AD mice. A, Confocal experiments were performed to compare amyloid deposition in Tg-APPswe/PSEN1ΔE9 (PA) versus RTN3^−/−/Tg-APPswe/PSEN1ΔE9 (R3KOPA) mouse brains. The size of amyloid plaques was often larger in R3KOPA mice when compared with PA mice. Scale bar, 100 μm. B, C, Brain sagittal sections were DAB stained with 6E10 antibody and 16 sagittal sections (160 μm apart) from each genotype were examined. The Aβ plaque load was determined by percentage of the area occupied by condensed deposits in the total examined area (*p < 0.05, n = 5, paired t test). Scale bar, 600 μm.

Figure 6.

Increased APP processing in R3KOPA mouse brains. A, Cortical and hippocampal protein lysates from 6-month-old PA and R3KOPA mice were examined by Western blotting. Antibody A8717 was used to detect APP and its BACE1-cleaved APP C-terminal fragment CTF99 and α-secretase-cleaved fragment CTF83. RTN3 deficiency was verified by antibody R458. Calnexin antibody was used to verify protein loading. B, The relative protein levels of full-length APP were normalized by comparing levels of calnexin and are shown in bar graphs. C, Relative BACE1 activity was manifested by the relative ratio of CTF99 to CTF83; higher BACE1 activity tends to produce more APP-C99 (n = 3 experiments, 2 mice in each experiment, *p < 0.05, ANOVA test).