GGA1-mediated endocytic traffic of LR11/SorLA alters APP intracellular distribution and amyloid-β production Jeremy H. Herskowitz, Emory University Katrin Offe, Emory University Aniruddha Deshpande, Emory University Richard A Kahn, Emory University Allan I Levey, Emory University James J Lah, Emory University Journal Title: Molecular Biology of the Cell Volume: Volume 23, Number 14 Publisher: The American Society for Cell Biology | 2012-07-15, Pages 2645-2657 Type of Work: Article | Final Publisher PDF Publisher DOI: 10.1091/mbc.E12-01-0014 Permanent URL: http://pid.emory.edu/ark:/25593/cwsw4 Final published version: http://dx.doi.org/10.1091%2Fmbc.E12-01-0014 Copyright information: © 2012 Herskowitz et al. This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). Accessed October 4, 2021 10:12 AM EDT M BoC | ARTICLE GGA1-mediated endocytic traffic of LR11/SorLA alters APP intracellular distribution and amyloid- β production Jeremy H. Herskowitza, Katrin Offea, Aniruddha Deshpandea, Richard A. Kahnb, Allan I. Leveya, and James J. Laha aCenter for Neurodegenerative Disease, Department of Neurology, and bDepartment of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322 ABSTRACT Proteolytic processing of the amyloid-β precursor protein (APP) and generation Monitoring Editor of amyloid-β peptide (Aβ) are key events in Alzheimer’s disease (AD) pathogenesis. Cell bio- Adam Linstedt logical and genetic evidence has implicated the low-density lipoprotein and sorting receptor Carnegie Mellon University LR11/SorLA in AD through mechanisms related to APP and Aβ production. Defining the cel- Received: Jan 9, 2012 lular pathway(s) by which LR11 modulates Aβ production is critical to understanding how Revised: May 9, 2012 changes in LR11 expression affect the development of Aβ pathology in AD progression. We Accepted: May 16, 2012 report that the LR11 ectodomain is required for LR11-mediated reduction of Aβ and that mutagenesis of the LR11 Golgi-localizing, γ-adaptin ear homology domain, ADP-ribosylation factor (GGA)-binding motif affects the endosomal distribution of LR11, as well as LR11’s ef- fects on APP traffic and βA production. Targeted small interfering RNA (siRNA) knockdown studies of GGA1, GGA2, and GGA3 indicate a surprising degree of specificity toward GGA1, suggesting that GGA1 is a candidate regulator of LR11 traffic. Additional siRNA knockdown experiments reveal that GGA1 is necessary for both LR11 and β-site APP-cleaving enzyme-1 (BACE1) modulation of APP processing to Aβ. Mutagenesis of BACE1 serine 498 to alanine enhances BACE1 targeting to LR11-positive compartments and nullifies LR11-mediated re- duction of Aβ. On basis of these results, we propose that GGA1 facilitates LR11 endocytic traffic and that LR11 modulates Aβ levels by promoting APP traffic to the endocytic recycling compartment. INTRODUCTION The amyloid-β precursor protein (APP) and its derivative, amyloid-β altering APP processing and Aβ production (Goate et al., 1991; peptide (Aβ), play central roles in Alzheimer’s disease (AD). Aβ ac- Citron et al., 1992). To generate Aβ, APP is cleaved by β-secretase/β- cumulates in senile plaques, a pathological hallmark of AD (Masters site of APP-cleaving enzyme-1 (BACE1), resulting in secreted APPβ et al., 1985), and single-gene mutations in APP cause familial AD by (APPsβ) and a carboxyl-terminal fragment (CTFβ), and subsequent action of γ-secretase on CTFβ releases Aβ (Hussain et al., 1999; Sinha and Lieberburg, 1999; Vassar et al., 1999; Wolfe et al., 1999; This article was published online ahead of print in MBoC in Press (http://www Yan et al., 1999; Lin et al., 2000; Xia et al., 2000). Cell biological .molbiolcell.org/cgi/doi/10.1091/mbc.E12-01-0014) on May 23, 2012. studies indicate that APP processing is determined by its intracellu- Address correspondence to: James J. Lah ([email protected]). lar traffic and exposure to secretase enzymes and that βA is pro- Abbreviations used: Aβ, amyloid-β peptide; AD, Alzheimer’s disease; APP, duced in the endosomal compartment, as well as in the early secre- amyloid-β precursor protein; BACE1, β-site APP-cleaving enzyme-1; ELISA, en- zyme-linked immunosorbent assay; GGA, Golgi-localizing, γ-adaptin ear homo- tory pathway (Sisodia, 1992; Koo and Squazzo, 1994; Hartmann logy domain, ADP-ribosylation factor; HEK, human embryonic kidney; LDLR, low- et al., 1997; Xu et al., 1997). density lipoprotein receptor; LR11, low-density lipoprotein receptor with 11 class A ligand-binding repeats. Although specific gene mutations can cause familial AD, the © 2012 Herskowitz et al. This article is distributed by The American Society for Cell causes of sporadic AD remain unclear. The best-established genetic Biology under license from the author(s). Two months after publication it is avail- risk factor is the ɛ4 allele of apolipoprotein E (APOE; Corder et al., able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0). 1993; Strittmatter et al., 1993). Moreover, members of the low-den- “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of sity lipoprotein receptor (LDLR) family, which bind ApoE, have been the Cell®” are registered trademarks of The American Society of Cell Biology. implicated in AD (Okuizumi et al., 1995; Kang et al., 1997; Ma et al., Supplemental Material can be found at: Volume 23 July 15, 2012 http://www.molbiolcell.org/content/suppl/2012/05/19/mbc.E12-01-0014.DC1.html 2645 2002). Low-density lipoprotein receptor with 11 class A ligand-bind- role different LR11 domains play in regulating endogenous APP traf- ing repeats (LR11)/SorLA is a multidomain LDLR family member that fic and βA production in a human cell line. Modified LR11 constructs contains structural elements that also place it in the vacuolar protein demonstrate the essential role of the LR11 ectodomain in redistrib- sorting 10 protein (VPS10p) homology domain family of intracellular uting APP to endosomes and reducing Aβ secretion. Single amino sorting receptors (Jacobsen et al., 1996). We first identified LR11 as acid substitutions in the LR11 GGA-binding domain establish the a transcript that was down-regulated in microarray studies examin- requirement for this motif with respect to LR11 traffic and its effects ing gene expression in patients with AD (Scherzer et al., 2004). Sub- on Aβ production and APP distribution. Targeted small interfering sequently, we demonstrated preferential neuronal loss of LR11 pro- RNA (siRNA) knockdown experiments reveal that GGA1 regulates tein in brain regions vulnerable to AD pathology in sporadic but not LR11 endocytic traffic and that GGA1 is critical for BACE1 and LR11’s familial AD patients, suggesting that LR11 loss plays a specific role effects on endogenous Aβ production. On the basis of these stud- in the pathogenesis of sporadic AD (Scherzer et al., 2004; Dodson ies, we propose that LR11 endocytic traffic, facilitated by GGA1, is et al., 2006). More recent studies indicate early loss of neuronal critical for LR11 modulation of APP traffic and the generation of βA . LR11 in individuals with mild cognitive impairment, a condition that often represents prodromal AD (Sager et al., 2007). Single-nucle- RESULTS otide polymorphisms in noncoding regions of SORL1, the gene en- The LR11 ectodomain is required for LR11-mediated coding LR11, have been linked to AD risk, implying that they influ- Aβ reduction ence disease by altering expression levels rather than by producing To assess the affect of the ectodomain or cytoplasmic tail of LR11 on variant forms of LR11 (Rogaeva et al., 2007). Aβ production, we constructed two chimeras in which the ectodo- Several reports revealed insights into the mechanism by which main or cytoplasmic domain of LR11 was replaced with that of hu- LR11 may influence AD pathogenesis. Overexpression of LR11 re- man LDLR, thus generating LDLR-LR11 and LR11-LDLR, respectively sults in a highly consistent, dose-dependent reduction in APP pro- (Figure 1A). LDLR was chosen because it is in the same lipoprotein cessing to Aβ (Andersen et al., 2005; Offe et al., 2006; Schmidt receptor family as LR11 and was reported to have no effect on APP et al., 2007). Conversely, reduced LR11 expression, as occurs in spo- processing or Aβ levels (Fryer et al., 2005). In addition, we gener- radic AD brain, increases Aβ levels in cultured cells and accelerates ated a construct consisting of the LR11 transmembrane and cyto- amyloid pathology in a transgenic mouse model of AD (Rogaeva plasmic domains but lacking the entire ectodomain (LR11ΔED). All et al., 2007; Dodson et al., 2008; Rohe et al., 2008). LR11 interacts constructs were epitope tagged with V5 at the N-termini. with APP, and colocalization between these proteins has been ob- To investigate effects on APP processing and Aβ production, we served in endosomal and Golgi compartments in multiple cell types, transiently expressed LR11 constructs in human embryonic kidney including primary neurons (Andersen et al., 2005; Offe et al., 2006; (HEK) 293 cells. This cell line was chosen due to the ability to detect Spoelgen et al., 2006). LR11 is a large transmembrane protein, and endogenous APP protein by immunoblot and secreted Aβ by en- its domain structure indicates that it may act as an endocytic cell zyme-linked immunosorbent assay (ELISA), and our standard trans- surface receptor and as an intracellular sorting receptor (Jacobsen fection efficiency is ∼60% under these conditions. Cell extracts and et al., 1996, 2001, 2002). The LR11 ectodomain harbors a cluster of conditioned media were assayed for endogenous full-length APP 11 type A ligand-binding repeats and a Vps10p homology domain and α-secretase cleaved APPs, respectively, by immunoblotting, (Jacobsen et al., 1996; Yamazaki et al., 1996), and the LR11 cyto- and secreted Aβ1-40 was detected by sandwich ELISA.