Loss of LR11/SORLA enhances early pathology in a mouse model of amyloidosis: evidence for a proximal role in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is the most prevalent form of dementia, resulting in progressive neuronal death and debilitating damage to brain loci that mediate memory and higher cognitive function. While pathogenic genetic mutations have been implicated in approximately 2% of AD cases, the proximal events that underlie the common, sporadic form of the disease are incompletely understood. Converging lines of evidence from human neuropathology, basic biology, and genetics have implicated loss of the multifunctional receptor LR11 (also known as SORLA and SORL1) in AD pathogenesis. Cell-based studies suggest that LR11 reduces the formation of beta-amyloid (Abeta), the molecule believed to be a primary toxic species in AD. Recently, mutant mice deficient in LR11 were shown to upregulate murine Abeta in mouse brain. In the current study, LR11-deficient mice were crossed with transgenic mice expressing autosomal-dominant human AD genes, presenilin-1 (PS1DeltaE9) and amyloid precursor protein (APPswe). Here, we show that LR11 deficiency in this AD mouse model significantly increases Abeta levels and exacerbates early amyloid pathology in brain, causing a forward shift in disease onset that is LR11 gene dose-dependent. Loss of LR11 increases the processing of the APP holo-molecule into alpha-, beta-, and gamma-secretase derived metabolites. We propose that LR11 regulates APP processing and Abeta accumulation in vivo and is of proximal importance to the cascade of pathological amyloidosis. The results of the current study support the hypothesis that control of LR11 expression may exert critical effects on Alzheimer's disease susceptibility in humans.

Figure 1.

Characterization of LR11 exon 4 deletion variant in Lr11^−/− mice. A, Top, Sequencing results of purified RT-PCR products from LR11 wild-type and LR11 mutant mice were aligned and show a full deletion of exon 4 messenger RNA sequence in Lr11^−/− samples. Bottom, Single stranded cDNA from Lr11^+/+, Lr11^+/−, and Lr11^−/− brain samples was amplified between exon 3 and exon 5 (left) and within exon 4 (right) and visualized by standard agarose gel electrophoresis. The expected size of the reaction products was 300 nucleotides and 160 nucleotides, respectively. In exon 3 to exon 5 RT-PCR, the expected 300 bp band is observed in Lr11^+/+ sample an additional smaller band is observed Lr11^+/− samples and is also evident in Lr11^−/− samples. A fully intact exon 4 reaction product is detected in Lr11^+/+ and Lr11^+/− samples but cannot be detected in Lr11^−/− samples. B, Western blotting for LR11 with a rabbit anti-LR11 C-terminal antibody reveals LR11 protein expression in LR11 wild-type (+/+) and LR11 deficient (−/−) mice (arrow; left panel). This protein migrates at the same molecular weight as overexpressed LR11 in HEK cell lysates (HEK/LR11). The LR11-specific band in these samples can be eliminated by preadsorption of the LR11-CT antibody with free LR11 C-terminal peptide (arrow; right panel). C, Summary of mass spectrometry results collected from Lr11^+/− and Lr11^−/− tissue samples: 14 individual LR11 peptides were identified in +/− brain and 3 LR11 peptides were identified in Lr11^−/− brain. Sequence information for the 3 peptides identified in Lr11^−/− brain is provided. D, Representative spectrum of fully tryptic LR11 peptide ESAPGLIIATGSVGK identified in Lr11^−/− tissue by mass spectrometry.

Figure 2.

LR11^ΔEx4 protein expression in brain and effect on Aβ secretion. A, Immunohistochemistry was used to visualize LR11 protein expression pattern in Lr11^+/+ (top left) and Lr11^−/− (top middle) coronal cortical sections. LR11 immunoreactivity (brown) is observed in both Lr11^+/+ and Lr11^−/− tissue sections, while intensity of immunoreactivity is markedly reduced in Lr11^−/− brain. High magnification (insets) shows punctate somatodendritic LR11 immunoreactivity in both Lr11^+/+ and Lr11^−/− mice. Antibody specificity is demonstrated by eliminating immunoreactivity with preadsorption (PA; top right) of the LR11 C terminus (CT) antibody with free CT peptide before incubation with tissue. Scale bar, 100 μm. B, LR11 protein is detected by Western blot of 4.5-month-old Lr11^+/+, Lr11^+/−, and Lr11^−/− cortex. Calnexin is shown to demonstrate equal loading. Densitometric quantitation of LR11 band intensity reveals significant differences in LR11 protein level across genotypes (p = 0.0154). C, The Lr11^ΔEx4 protein was cloned into pcDNA and transiently transfected at increasing doses into HEK293 cells. Lr11^ΔEx4 overexpression induces a dose-related decrease in Aβ secretion.

Figure 3.

β-amyloid measures in Lr11^+/+ and Lr11^−/− cortex and hippocampus. A, ELISA-quantification of total Aβ levels in Lr11^+/+ (white bars) and Lr11^−/− cortex (black bars) at 3, 4.5, 6, and 12 months of age. Data points are plotted as the natural log raw values (see supplemental Table 1, available at www.jneurosci.org as supplemental material, for raw values). Lr11^−/− mice exhibit significant increases in total Aβ levels at 3 (p = 0.038), 4.5 (p = 0.0079) and 6 months of age (p = 0.048). B, Amyloid plaque density [mean surface area (pixels) of Aβ42-positive immunoreactivity per tissue section] is significantly increased in Lr11^−/− mice at 4.5 (p = 0.0286) and 6 months of age (p = 0.002). C, Total plaque count is significantly increased in Lr11^−/− mice at 3 (p = 0.0379) and 6 months (p = 0.002). D, Qualitative images of Aβ42-stained amyloid deposits (brown) in Lr11^+/+ (left) and Lr11^−/− (right) cortex and hippocampus at 4.5, 6, and 12 months of age. Boxed regions of cortex (a) and hippocampus (b) are magnified and shown as corresponding insets.

Figure 4.

β-amyloid measures in Lr11^+/+ and Lr11^−/− cerebellum. A, Qualitative images of Aβ42-stained sagittal sections of Lr11^+/+ (left) and Lr11^−/− (right) cerebellum at 4.5, 6, and 12 months of age. Boxed regions are magnified and shown as corresponding insets. B, Amyloid plaque density [mean surface area (pixels) of Aβ42-positive immunoreactivity per tissue section] is significantly increased in Lr11^−/− mice at 6 (p = 0.0286) and 12 months of age (p = 0.0357). C, ELISA-quantification of Aβ40 levels (pg/mg tissue) is significantly elevated in Lr11^−/− cerebellum (black bars) compared with Lr11^+/+ littermates (white bars) at 12 months of age (p = 0.0381).

Figure 5.

LR11 gene-dose effects on pathological amyloid measures in 4.5-month-old mice. A, ELISA-measured Aβ42 levels are significantly different across Lr11^+/+, Lr11^+/−, and Lr11^−/− genotypes at 4.5 months of age (p = 0.049). Lr11^−/− mice have significantly higher Aβ42 levels compared with Lr11^+/+ mice (p < 0.05). Lr11^+/− mice are intermediate and not significantly different from Lr11^+/+ or Lr11^+/− littermates. B, Total plaque count is significantly different across Lr11^+/+, Lr11^+/−, and Lr11^−/− genotypes (p = 0.0388). Lr11^−/− mice exhibit more thioflavine-S-positive plaques per tissue section than Lr11^+/+ littermates (p < 0.05). Lr11^+/− mice are intermediate and not significantly different from Lr11^+/+ or Lr11^−/− mice. C, Across all genotypes, amyloid plaque density [mean surface area (pixels) of Aβ42-positive immunoreactivity per tissue section] inversely correlates with LR11 expression levels measured by Western blot (p = 0.0278).

Figure 6.

APP metabolite analysis in cortical tissue and primary cortical neurons from Lr11^+/+ and Lr11^−/− mice. A, Western blot and densitometric quantitation of steady-state APP and relevant APP metabolites in a subset of Lr11^+/+ (n = 7) and Lr11^−/− (n = 6) cortical tissue samples. Membrane proteins and soluble proteins were fractionated by differential centrifugation and blotted with a panel of APP antibodies, including C8 to visualize full-length APP and CTFs, 6E10 to visualize APPsα and CTFβ, and 192swe to specifically detect the Swedish form of APPsβ. For quantitation, APPs levels were normalized to levels of the unrelated soluble protein EF1α and CTF levels were normalized to full-length APP. Lr11^−/− mice exhibit ∼40% increase in APPsα (p = 0.0161) and ∼50% decrease in both CTFα (p = 0.0133) and CTFβ (p = 0.0076). B, Western blot and densitometric quantitation of lentivirally transduced APP and APP metabolites from primary cortical culture lysates and conditioned media (3 independent experiments performed in quadruplicate). Overexpressed wild-type human APP695 is detected by the human specific 6E10 antibody and other metabolites were detected by the same panel of antibodies described above (with the exception of 192wt to visualize wild-type APPsβ). For quantitation, APPs and CTF levels were normalized to over-expressed APP in each sample. Lr11^−/− cortical neurons express equivalent levels of full-length APP695, but secrete ∼2-fold more APPsα (p = 0.0307) and APPsβ (p = 0.0094) into the media. Levels of CTFs are unchanged in Lr11^−/− cortical lysates. EF1α is shown to demonstrate loading consistency.