The Journal of Neuroscience, April 17, 2013 • 33(16):7099–7107 • 7099 Neurobiology of Disease Protein Sorting Motifs in the Cytoplasmic Tail of SorCS1 Control Generation of Alzheimer’s Amyloid-␤ Peptide Rachel F. Lane,1,2 John W. Steele,1,3 Dongming Cai,6 Michelle E. Ehrlich,4 Alan D. Attie,5 and Sam Gandy1,6 1Departments of Neurology and Psychiatry, and the Alzheimer’s Disease Research Center, Icahn School of Medicine at Mount Sinai, New York, New York 10029, 2Alzheimer’s Drug Discovery Foundation, New York, New York 10019, 3Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, 4Departments of Pediatrics and Neurology, Mount Sinai School of Medicine, New York, New York 10029, 5Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, and 6James J. Peters VA Medical Center, Bronx, New York 10468 Endosomal sorting of the Alzheimer amyloid precursor protein (APP) plays a key role in the biogenesis of the amyloid-␤ (A␤) peptide. Genetic lesions underlying Alzheimer’s disease (AD) can act by interfering with this physiological process. Specifically, proteins involved in trafficking between endosomal compartments and the trans-Golgi network (TGN) [including the retromer complex (Vps35, Vps26) and its putative receptors (sortilin, SorL1, SorCS1)] have been implicated in the molecular pathology of late-onset AD. Previously, we demonstrated a role for SorCS1 in APP metabolism and A␤ production and, while we implicated a role for the retromer in this regulation, the underlying mechanism remained poorly understood. Here, we provide evidence for a motif within the SorCS1c cytoplasmic tail that, when manipulated, results in perturbed sorting of APP and/or its fragments to endosomal compartments, decreased retrograde TGN trafficking, and increased A␤ production in H4 neuroglioma cells. These perturbations apparently do not involve turnover of the cell surface APP pool, but rather they involve intracellular APP and/or its fragments, downstream of APP endocytosis. Introduction tases within the endosomal pathway (for review, see Small and Genetic studies of late-onset Alzheimer’s disease (LOAD) point Gandy, 2006). to a number of risk factor genes that encode proteins with known SORL1, SORCS1, and SORCS2, together with sortilin and functions in trafficking to, and within, endocytic compartments SORCS3, belong to the vacuolar protein sorting-10 (Vps10) fam- including: SORL1, SORCS1, SORCS2, BIN1, PICALM-1, and ily of receptors. This receptor family was first identified in yeast as CD2AP (Rogaeva et al., 2007; Harold et al., 2009; Liang et al., trafficking receptors between endosome and trans-Golgi network 2009; Naj et al., 2011). Dysfunction within endocytic compart- (TGN) compartments (for review, see Lane et al., 2012). In mam- ments is evident in the Alzheimer’s disease (AD) brain early in malian systems, while the Vps10 proteins are highly pleiotropic, disease progression and is proposed to contribute to the deposi- SorL1 and SorCS1 are suggested to function in retrograde traf- tion of amyloid-␤ (A␤)(Cataldo et al., 2000; Cataldo et al., 2004). ficking of APP (Small et al., 2005; Lane et al., 2010; Vieira et al., Several of these consistently linked genetic risk factors for LOAD 2010; Fjorback et al., 2012) via their interactions with the retro- are specifically implicated in regulating the intracellular traffick- mer complex (Small et al., 2005; Lane et al., 2010; Fjorback et al., ing of amyloid precursor protein (APP) and/or its cognate secre- 2012). The first member of the Vps10 family of receptors to be im- plicated as a retromer receptor for APP retrograde trafficking was SorL1 (Small et al., 2005; Fjorback et al., 2012). In vitro and in vivo Received Nov. 12, 2012; revised Feb. 4, 2013; accepted Feb. 27, 2013. Author contributions: R.F.L. and S.G. designed research; R.F.L. performed research; J.W.S., D.C., M.E.E., A.D.A., studies have demonstrated that SorL1 exists in a multimeric com- and S.G. contributed unpublished reagents/analytic tools; R.F.L., J.W.S., and S.G. analyzed data; R.F.L. and S.G. plex that includes APP and the retromer, and assembly of this wrote the paper. complex is one mechanism through which SorL1 regulates A␤ ThisresearchwassupportedbyNationalInstitutesofHealth(NIH)GrantNS075685(toS.G.),theCureAlzheimer’s generation (Fjorback et al., 2012). Reduced expression of Vps35 Fund (to S.G.), the U.S. Department of Veterans Affairs (to S.G.), National Institute of Diabetes and Digestive and Kidney Diseases Grants DK58037 and DK66369 (to A.D.A.), and an American Diabetes Association Research Award and/or SorL1 results in increased APP localization to early endo- (to M.E.E). J.W.S. was a trainee in the Integrated Pharmacological Sciences Training Program supported by Grant somes leading to increased A␤42 generation (Offe et al., 2006; T32GM062754fromtheNationalInstituteofGeneralMedicalSciencestoTerryKrulwich(IcahnSchoolofMedicineat Muhammad et al., 2008; Bhalla et al., 2012). Mount Sinai). Confocal laser scanning microscopy was performed at the Icahn School of Medicine at Mount Sinai A second member of the Vps10 family of receptors, SorCS1, Microscopy Shared Resource Facility, supported with funding from NIH-National Cancer Institute Shared Resources ␤ Grant 5R24 CA095823-04, National Science Foundation Major Research Instrumentation Grant DBI-9724504, and has also been reported to regulate A generation from cultured NIH Shared Instrumentation Grant 1 S10 RR0 9145-01. We thank Rudolph E. Tanzi for cDNA constructs. cells (Lane et al., 2010; Reitz et al., 2011b). Furthermore, we have The authors declare no competing financial interests. implicated SorCS1 as a potential retromer receptor, demonstrat- Correspondence should be addressed to either Rachel F. Lane or Sam Gandy, Departments of Neurology and ing in the brains of wild-type (wt) mice that SorCS1 exists in a Psychiatry, and the Alzheimer’s Disease Research Center, Icahn School of Medicine at Mount Sinai, One Gustave L. complex with APP, SorL1, and Vps35, the core component of the Levy Place, New York NY 10029, E-mail: [email protected] or [email protected]. DOI:10.1523/JNEUROSCI.5270-12.2013 retromer complex (Lane et al., 2010). In the brains of female Copyright © 2013 the authors 0270-6474/13/337099-09$15.00/0 Sorcs1-deficient mice, we demonstrated increased endogenous 7100 • J. Neurosci., April 17, 2013 • 33(16):7099–7107 Lane et al. • Mechanisms for SorCS1 Regulation of A␤ Generation levels of A␤ together with decreased levels of Vps35 and SorL1, myc antibody (for immunoprecipitation of SorCS1-myc) according to providing further evidence that Vps35 (and possibly the retro- the manufacturer’s instructions. Empty vector (pCDNA4) transfections mer) is involved in SorCS1-dependent regulation of APP pro- were included as negative controls. cessing and A␤ production (Lane et al., 2010). APP and SorCS1 turnover rates. For a time course of APP metabolism, While evidence suggests a role for SorCS1 in the modulation H4 wt APPs were transiently transfected with cDNA constructs as indi- cated. At 8 h after transfection, cells were divided into six-well plates of APP/A␤ homeostasis, the molecular basis for this regulation is coated with 100 ␮g/ml polyornithine and allowed to recover for 5 h. Cells incompletely understood. We therefore sought to define the roles were subsequently treated with 50 ␮g/ml cycloheximide (CHX) in for sorting motifs in the cytoplasmic tail of the SorCS1c isoform serum-free and antibiotic-free DMEM (Vieira et al., 2010). At the indi- in APP/A␤ homeostasis by investigating potential SorCS1c regu- cated time points, cells were washed in ice-cold 1ϫ PBS and protein lation of APP trafficking into and between endosomal compart- harvested in 1ϫ RIPA buffer as described previously (Lane et al., 2010). ments. Through site-directed mutagenesis of canonical sorting Lysates were subsequently analyzed by SDS PAGE and Western blotting motifs in the SorCS1c cytoplasmic tail, we demonstrated that with immunodetection for holoAPP, APP CTFs, and SorCS1c. SorCS1c does not directly regulate internalization of APP into Cell surface biotinylation experiments. H4 wt APP cells were transfected endocytic compartments, but appears to regulate the exit of APP with the indicated cDNAs. At 48 h after transfection, cells were washed three times in ice-cold 1ϫ PBS (ϩ2.5 mM CaCl ϩ 1mM MgCl, pH 7.4). and/or APP C-terminal fragments (CTFs) out of endosomal ϫ compartments, resulting in increased APP localization to the Cells were then incubated in either ice-cold 1 PBS (negative control) or ice-cold 1ϫ PBS plus 2 mM Sulfo-NHS-SS-biotin and incubated at 4°C TGN and decreased secreted A␤. for 30 min. Cells were washed three times in ice-cold 1ϫ PBS (ϩ100 mM glycine, pH 7.4) to quench the reaction before being lysed in 1ϫ RIPA Materials and Methods buffer as described previously (Lane et al., 2010). A total of 300 ␮gof Antibodies. Anti-Myc (Cell Signaling Technology), anti-EEA1 (Cell protein lysate from each condition was incubated with 100 ␮l streptavi- Signaling Technology), anti-TGN38 (BD Biosciences), anti-Rab7 (Cell din beads (Invitrogen) rotating overnight at 4°C. To isolate the strepta- Signaling Technology), anti-calnexin (Cell Signaling Technology), anti- vidin beads, samples were centrifuged at 5000 rpm at 4°C for 1 min, the syntaxin 6 (Cell Signaling Technology), and anti-mouse, anti-rabbit, and supernatant was retained for analysis of the intracellular fraction (un- anti-goat horseradish peroxidase (HRP) conjugates (Vector Laborato- bound fraction), and the beads were washed five times in ice-cold 1ϫ ries) were purchased from commercial vendors as indicated. Polyclonal RIPA buffer and resuspended in 5ϫ Laemmli buffer. Finally, samples Ab369 (pan-species anti-C-terminal APP antibody) was used to detect were boiled at 95°C for 5 min before analysis by SDS PAGE and immu- human holoAPP and C-terminal fragments (Buxbaum et al., 1990) and nodetection with the indicated antibodies.