SORCS1 alters amyloid precursor protein processing and variants may increase Alzheimer's disease risk

Abstract

Objective: Sorting mechanisms that cause the amyloid precursor protein (APP) and the β-secretases and γ-secretases to colocalize in the same compartment play an important role in the regulation of Aβ production in Alzheimer's disease (AD). We and others have reported that genetic variants in the Sortilin-related receptor (SORL1) increased the risk of AD, that SORL1 is involved in trafficking of APP, and that underexpression of SORL1 leads to overproduction of Aβ. Here we explored the role of one of its homologs, the sortilin-related VPS10 domain containing receptor 1 (SORCS1), in AD.

Methods: We analyzed the genetic associations between AD and 16 SORCS1-single nucleotide polymorphisms (SNPs) in 6 independent data sets (2,809 cases and 3,482 controls). In addition, we compared SorCS1 expression levels of affected and unaffected brain regions in AD and control brains in microarray gene expression and real-time polymerase chain reaction (RT-PCR) sets, explored the effects of significant SORCS1-SNPs on SorCS1 brain expression levels, and explored the effect of suppression and overexpression of the common SorCS1 isoforms on APP processing and Aβ generation.

Results: Inherited variants in SORCS1 were associated with AD in all datasets (0.001 < p < 0.049). In addition, SorCS1 influenced APP processing. While overexpression of SorCS1 reduced γ-secretase activity and Aβ levels, the suppression of SorCS1 increased γ-secretase processing of APP and the levels of Aβ.

Interpretations: These data suggest that inherited or acquired changes in SORCS1 expression or function may play a role in the pathogenesis of AD.

FIGURE 1

(A) View of SORCS1 exon expression profile in 19 AD (red triangles) and 10 control (blue squares) amygdala tissue. Each triangle dot represents least squares mean expression of an exon in AD tissue; each square dot represents least squares mean expression of an exon in control tissue. The mean gene expression intensity of AD vs controls was 3.69 ± 0.03 vs 4.35 ± 0.04 (p = 0.00006) across all exons. The top part of the graph shows the structure of the SorCS1b (above) and SorCS1a (below) isoforms in this region retrieved from the UCSC browser. (B) Dot plot showing the 2D distribution of amygdala expression levels of the 19 AD (red) and 10 control (blue) samples. 2D = 2-dimensional; AD = Alzheimer’s disease; UCSC = University of California, Santa Cruz.

FIGURE 2

Overexpression of SorCS1 in HEK293 APPsw cells (XL4 transfected samples). (A) SorCS1 expression was confirmed by 2 different SorCS1 antibodies (R&D and H120). (B) Expression of APP in SorCS1 transfectants. Overexpression of SorCS1 did not have an effect on FL-APP expression or its maturation. (C) Western blot done on the same samples with β-tubulin antibody (loading control). APP = amyloid precursor protein; APPsw = APP Swedish mutation; FL-APP = APP holoprotein; XL4 = empty vector.

FIGURE 3

Measurement of Aβ1–40 and Aβ1–42 from conditioned media of HEK293 APPsw overexpressing SorCS1 isoforms. Aβ levels normalized to total protein were measured. Data shown as percentages of control values (XL4; n = 3 per experiment, 3 independent experiments). APP = amyloid precursor protein; APPsw = amyloid precursor protein Swedish mutation; FL-APP = APP holoprotein; XL4 = empty vector.

FIGURE 4

Overexpression of SorCS1 decreased the secretion of total APPs and sAPPα into culture media. CM and total cell lysate from HEK293 APPsw transfected with SorCS1 isoforms were analyzed by western blot with antibodies against APP (22C11 and 8717), Aβ (6E10), and SorCS1 (R&D). Total sAPP and sAPPα from CM were decreased compared with XL4 in spite of similar FL-APP expression in cell lysate. Western blot was done on the same samples with β -tubulin antibody (loading control). The expression of both SorCS1 isoforms in lysate was similar. APP = amyloid precursor protein; APPsw = amyloid precursor protein Swedish mutation; CM = conditioned media; FL-APP = APP holoprotein; sAPPα = α-secretase cleavage; XL4 = empty vector.

FIGURE 5

Knockdown of SorCS1 using shRNAs (41–45) did not affect the levels of transiently transfected FL-APP, NCT, PS1-NTF, or β-tubulin. (See also Supporting Table 7 for sequence information on shRNAs used and Supporting Fig 2 for western blot confirmation of endogenous expression of SorCS1 protein). APP = APP = amyloid precursor protein; FL-APP = APP holoprotein; NCT = nicastrin; PS1-NTF = pre-senilin1 N-terminal fragment; shRNA = short hairpin RNA.

FIGURE 6

Knockdown of SorCS1 by shRNAs did affect the level of Aβ40. shRNA 41, 42, 43, 44, and 45, or scrambled sequence shRNA were transfected in HEK293 cells and 3 days after transfection, the cells were incubated for additional 48 hours. Data are expressed as percentages of control values. Aβ1–40 secretion was measured in conditioned media and normalized to total protein levels. The data are representative for the Aβassays and the assay has been performed in at least 3 separate experiments in replicates of 8 samples per condition (24-well format), standard deviation error bars are shown, **p < 0.01, ***p < 0.001 as compared to scrambled shRNA condition (ANOVA with Bonferroni correction; GraphPad Software, La Jolla, CA). ANOVA = analysis of variance; shRNA = short hairpin RNA.

FIGURE 7

Gamma secretase activity and nuclear translocation of APP assays with Sorcs1 shRNAs. (A) Both the APP-GV and AICD-GV assay were performed in HEK293 cells. The data from SorCS1a cDNA and the 5 shRNA SorCS1 was normalized to either APP-GV only or AICD-GV with the scrambled sequence shRNA (shRNA-scrambled), which was included as a negative control. (B) APP-GV assay was also performed in the human neuroblastoma cell line, M17 with SorCS1a cDNA or 5 of the Sorcs1 shRNAs along with the shRNA-sc. The data are representative for the APP-GV assays and the assay has been performed in at least 3 separate experiments in replicates of 8 samples per condition (96-well format), standard deviation error bars are shown, *p < 0.05, **p < 0.01 as compared to APP-GV only (ANOVA; GraphPad Software, La Jolla, CA). AICD = APP gene C-terminus; GV = transcription factor composed of the GAL4 DNA binding domain with VP16 transcriptional activator; ANOVA = analysis of variance; APP = APP = amyloid precursor protein; cDNA = complementary DNA; shRNA = short hairpin RNA.

FIGURE 8

Interaction between APP and SorCS1 in HEK293 APPsw cells overexpressing SorCS1 isoforms. HEK293 APPsw cells transfected with SorCS1 isoform or XL4 were lysed using one-third-diluted RIPA buffer and then immunoprecipitated with (A) SorCS1 or (B) APP. (A) Immunoprecipitation for SorCS1 was successful (left). FL-APP was coimmunoprecipitated with SorCS1 (middle). (B) SorCS1 was coimmunoprecipitated with APP. The right immunoblot shows the expression levels of (A) SorCS1 and (B) APP in cell lysate. APP = amyloid precursor protein; APPsw = amyloid precursor protein Swedish mutation; FL-APP = APP holoprotein; XL4 = empty vector.