Sortilin-Mediated Endocytosis Determines Levels of the Frontotemporal Dementia Protein, Progranulin

Neuron

Article Sortilin-Mediated Endocytosis Determines Levels of the Frontotemporal Dementia Protein, Progranulin Fenghua Hu,1,2,3,4,8 Thihan Padukkavidana,1,2,3,8 Christian B. Vægter,5 Owen A. Brady,4 Yanqiu Zheng,4 Ian R. Mackenzie,6 Howard H. Feldman,1,2,3,7 Anders Nykjaer,5 and Stephen M. Strittmatter1,2,3,* 1Department

of Neurology of Neurobiology 3Program in Cellular Neuroscience, Neurodegeneration and Repair Yale University School of Medicine, New Haven, CT 06536, USA 4Weill Institute for Cell and Molecular Biology, Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA 5The Lundbeck Foundation Research Center MIND, Department of Medical Biochemistry, Aarhus University, DK-8000C Aarhus, Denmark 6Department of Pathology, University of British Columbia, Vancouver, BC V5Z 1M9, Canada 7Department of Neuroscience, Global Clinical Research, Bristol-Meyers Squibb, Wallingford, CT 06492, USA 8These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.neuron.2010.09.034 2Department

SUMMARY

The most common inherited form of Frontotemporal Lobar Degeneration (FTLD) known stems from Progranulin (GRN) mutation and exhibits TDP-43 plus ubiquitin aggregates. Despite the causative role of GRN haploinsufficiency in FTLD-TDP, the neurobiology of this secreted glycoprotein is unclear. Here, we examined PGRN binding to the cell surface. PGRN binds to cortical neurons via its C terminus, and unbiased expression cloning identifies Sortilin (Sort1) as a binding site. Sort1 / neurons exhibit reduced PGRN binding. In the CNS, Sortilin is expressed by neurons and PGRN is most strongly expressed by activated microglial cells after injury. Sortilin rapidly endocytoses and delivers PGRN to lysosomes. Mice lacking Sortilin have elevations in brain and serum PGRN levels of 2.5- to 5-fold. The 50% PGRN decrease causative in FTLD-TDP cases is mimicked in GRN+/ mice, and is fully normalized by Sort1 ablation. Sortilin-mediated PGRN endocytosis is likely to play a central role in FTLD-TDP pathophysiology.

INTRODUCTION The FTLDs are characterized clinically by dementia with prominent behavioral alterations and distinct syndromes including progressive aphasia and semantic disorders (Cairns et al., 2007; Mackenzie et al., 2009). A subset of cases exhibit Taupositive neurofibrillary tangles, but the majority show TDP-43 and ubiquitin positive inclusions in the brain, termed FTLDTDP. TDP-43 aggregates and TDP-43 mutations are also observed in ALS (Kabashi et al., 2008; Neumann et al., 2006; 654 Neuron 68, 654–667, November 18, 2010 ª2010 Elsevier Inc.

Sreedharan et al., 2008). Mutations in GRN are the most common inherited cause of FTLD known (Baker et al., 2006; Cruts et al., 2006; Gass et al., 2006). Such families exhibit autosomal-dominant inheritance of FTLD-TDP and their GRN mutations result in loss of function. Levels of PGRN protein are reduced by 50% in GRN mutant FTLD-TDP cases (Baker et al., 2006; Cruts et al., 2006; Finch et al., 2009; Gass et al., 2006; Ghidoni et al., 2008; Sleegers et al., 2009). PGRN is an evolutionarily conserved, secreted glycoprotein with 7 granulin (GRN) repeats. It has been implicated in wound healing and is associated with malignancy, although the basis for these effects is not fully defined (He et al., 2003; Zhu et al., 2002). Effects of PGRN on neurite outgrowth or cell survival in different assays have been reported (Van Damme et al., 2008). However, in our preliminary studies, PGRN had no detectable effects on the survival or neurite outgrowth from cerebral cortical or hippocampal neurons (Figure S2 available online). While human haploinsufficiency results in FTLDTDP, mice homozygous for a GRN null mutation exhibit little or no FTLD phenotype (Kayasuga et al., 2007; Yin et al., 2010). As yet, there is no consensus regarding PGRN action in the nervous system or the mechanism whereby 50% reduction might lead to FTLD-TDP. A fraction of PGRN can be proteolytically processed to smaller GRN peptides (Zhu et al., 2002), but the relative role of these fragments in FTLD-TDP is not known. Elucidation of PGRN action and the control of PGRN levels may have broad relevance for both FTLD and ALS. In order to advance understanding of secreted PGRN biology, we searched for high-affinity cell surface binding sites in an unbiased screen. We report that Sortilin is such a site and mediates rapid endocytosis and lysosomal localization of PGRN. Moreover, this mechanism has pronounced effects on brain and serum PGRN levels that are as great as the haploinsufficiency causing FTLD-TDP. Thus, a Sortilin-dependent pathway is likely to play a central role in FTLD-TDP, and possibly in ALS.

Neuron Sortilin-Mediated Endocytosis of Progranulin

RESULTS PGRN Binds with High Affinity to Sortilin We hypothesized that the key step in PGRN action is binding to neurons. We created an alkaline phosphatase (AP)-tagged PGRN ligand to assess binding (Figure S1A). Fusion of the amino terminus of PGRN to AP yields protein with high affinity for 21 DIV cultured neurons (Figure 1A). Binding is saturable with an apparent KD of 15.4 ± 2.5 nM (Figure 1B), and can be displaced by unlabeled PGRN (Figure S1B). Using this ligand binding assay, we searched for the molecular identity of this site in an unbiased fashion by expression cloning in COS-7 cells (Figure 1C). Untransfected COS-7 cells do not bind AP-PGRN. After screening 225,000 clones at low stringency in pools of 100, and 352 transmembrane proteins at high stringency as single clones, we identified only one clone that supported high affinity for AP-PGRN. This cDNA encodes Sortilin (Sort1), a single-pass type I transmembrane protein of the Vps10 family that is localized to the cell surface, secretory, and endocytic compartments of eukaryotic cells (Willnow et al., 2008). The affinity of AP-PGRN binding to Sortilin-expressing COS-7 cells is indistinguishable from AP-PGRN binding to neurons (Figures 1D and 2C). Binding to Sortilin-expressing COS-7 cells is displaced by excess unlabelled PGRN (Figure 2D). The binding of PGRN to Sortilin-expressing cells appears to be directed by several criteria. Bound PGRN localizes to Sortilinexpressing cells and, at high magnification, PGRN colocalizes extensively with cell surface Sortilin (Figure 1E). PGRN and the ectodomain of Sortilin coimmunoprecipitate from the conditioned medium of transfected cells, whereas an unrelated secreted protein (Reg2) does not associate with the ectodomain of Sortilin (Figure 1F). Furthermore, PGRN binding to Sortilinectodomain-coated surface plasmon resonance (SPR) chips exhibits an affinity similar to cellular binding (Figures 1G and 1H). Thus, Sortilin is identified as a direct high-affinity binding site for PGRN. Specificity of the Sortilin Interaction with PGRN Sortilin and related proteins have been reported to bind several ligands (Jansen et al., 2007; Nykjaer et al., 2004; Willnow et al., 2008). SorLA, but not Sortilin, is implicated in APP processing and Ab levels in Alzheimer disease (Andersen et al., 2005; Rogaeva et al., 2007). Therefore, we expressed Sortilin-related proteins, SorLA and SorCS1, in COS-7 cells and examined PGRN binding. PGRN binds only to Sortilin (Figures 2A and S1C). By SPR, SorLA, SorCS1, SorCS2, and SorCS3 exhibit much lower affinity for PGRN than does Sortilin (Figure 2G). Sortilin was originally identified as a low-affinity neurotensin (NT) binding protein and termed NT receptor 3, or NTR3 (Willnow et al., 2008). Two other NT binding sites are G protein-coupled receptors, but NTR1 and NTR2 do not bind PGRN (Figures 2A and S1C). NT has recently been shown to complex with the Sortilin b-propeller domain via its extreme C terminus (Quistgaard et al., 2009), and the last six residues of NT displace PGRN binding to Sortilin (Figures 2D and 2E). We considered a similar model for PGRN and compared the Sortilin association of the C-terminal 100 residues of PGRN, containing the GRN-E domain

plus the extreme C-terminal residues (termed PGRN-E), with that of the N-terminal 80% of the protein (termed PGRNDE). The carboxyl PGRN-E segment fully accounts for PGRN interaction with Sortilin. In fact, PGRN-E displays slightly higher affinity for Sortilin than does full-length PGRN (Figures 1D, 2B, and 2C), whereas the majority of the protein, contained in PGRNDE, has no detectable affinity for Sortilin (Figure 2B). Proneurotrophins, such as proNGF, are also reported ligands of Sortilin, but they do not depend on the C terminus of the ligand for binding to Sortilin, as do NT and PGRN (Domeniconi et al., 2007; Jansen et al., 2007; Nykjaer et al., 2004; Teng et al., 2005). While these ligands can reduce AP-PGRN binding to Sortilin, inhibition requires very high levels of proNGF, in the mM range (Figures 2D and 2E), with no inhibition by nM concentrations (data not shown). In SPR competition assays, proNGF and PGRN show additive binding signals for immobilized Sortilin, consistent with distinct binding sites (Figure 2H). ProNGF is reported to interact with a p75NTR/Sortilin complex with enhanced bipartite affinity (Nykjaer et al., 2004). Therefore, we examined AP-PGRN interaction with cell-expressing p75NTR, Sortilin, or both (Figure 2F). PGRN shows no affinity for p75NTR and the Sortilin/p75NTR coexpressing cells show no enhancement of affinity for PGRN. Thus, the binding of PGRN’s C terminus to Sortilin resembles a high-affinity version of NT binding, but may be distinct from that of proneurotrophin to Sortilin. PGRN Binding to Neuronal Sortilin A key issue is the extent to which Sortilin accounts for neuronal binding sites for PGRN. Several criteria validate Sortilin as a major PGRN binding in brain tissue. First, the development of Sortilin parallels that of AP-PGRN binding sites. For embryonic cortical neuron cultures at 7 DIV, both Sortilin expression and PGRN binding sites are low, but both increase substantially by 14 DIV (Figures 3A and 3B). PGRN-E binds with equal or greater affinity than does full-length PGRN to cortical neurons, whereas PGRNDE does not bind to cortical neurons at 10 nM (Figures 3C and S1D). Recombinant immobilized GST-PGRN-E, but not GST, pulls down Sortilin from whole-brain extracts of wild-type (WT), but not Sort1 / , mice (Figure 3D). Sort1 / mice produce low levels of a misfolded Sortilin deletion mutant that lacks a portion of the beta propeller region and does not bind PGRN-E. Most critically, high-affinity AP-PGRN-E binding to cortical neuron cultures from Sort1 / mice is significantly less than that from WT mice (Figures 3E and 3F). This reduction is prominent even after any potential compensatory upregulation of alternate binding sites in the constitutive Sort1 / knockout strain. Thus, Sortilin is a major high-affinity PGRN binding site in cortical neurons. Sortilin Regulates Extracellular PGRN Level As a first step to explore possible effects of PGRN/Sortilin interaction, we coexpressed the two proteins in HEK293T cells. PGRN is secreted from transfected cells, but when coexpressed with Sortilin, PGRN levels in conditioned medium are dramatically reduced to 15% of control levels (Figures 4A and 4B). It has been reported that Sortilin can be a substrate for regulated intramembranous proteolysis by gamma-secretase (Hermey Neuron 68, 654–667, November 18, 2010 ª2010 Elsevier Inc. 655

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Figure 1. Cellular Progranulin Binding Is Mediated by a Direct High-Affinity Interaction with Sortilin (A) Cultured E18 rat hippocampal neurons at 21 days in vitro (21 DIV) were incubated with conditioned medium containing 50 nM AP-PGRN at 4 C. Binding was visualized with AP substrate BCIP/NBT. (B) AP-PGRN binding to cortical neurons measured as a function of ligand concentration. Inset, Scatchard plot. KD, average ± SEM, n = 4 separate experiments. (C) COS-7 cells transfected with Sortilin or vector control were incubated with conditioned medium containing 50 nM AP-PGRN. (D) AP-PGRN binding to Sortilin expressing COS-7 cells measured as a function of AP-PGRN concentration. (E) Binding of PGRN to Sortilin on COS-7 cell surface. Purified Flag-tagged PGRN was incubated with COS-7 cells transfected with Myc-tagged Sortilin on ice for 2 hr. After washing, live cells were costained with mouse anti-Flag (green) and rabbit anti-Myc (red) antibodies on ice for 1 hr. Cells were then washed, fixed, and stained with secondary antibodies, and counterstained with DAPI (blue). Note that PGRN binds only to Sortilin-expressing cells. In the high-magnification view of a Sortilin-expressing cell in the bottom panels, there is extensive colocalization of the two proteins at the cell surface. (F) Coimmunoprecipitation of Flag-PGRN with Myc-tagged ectodomain of Sortilin (Sort1-ecto). Conditioned media containing Flag-PGRN or Flag-Reg2 were mixed together with conditioned media containing Myc-tagged Sort1-ecto protein. Immunoprecipitation (IP) was then carried out using anti-Myc or anti-Flag antibodies as indicated. The presence of Sort1-ecto or Flag-tagged protein in the IP was detected in the immunoblot using anti-Myc or anti-Flag antibodies, respectively.

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et al., 2006; Nyborg et al., 2006). From HEK cells, there is limited secretion of a Sortilin proteolytic fragment into conditioned medium, and this cleavage is not altered by PGRN coexpression (Figure 4C). The pronounced reduction in extracellular free PGRN caused by Sortilin coexpression might be attributed to impaired secretory trafficking within the cell, sequestration at the cell surface via binding, or endocytosis and clearance from the medium. Sortilin family proteins have been shown to play a role in both trafficking and endocytosis for other ligands (Nielsen et al., 2001; Willnow et al., 2008). To consider these possibilities, we expressed mutants of Sortilin that lack the cytoplasmic tail of the protein but carry an alternative C-terminal domain (pDisplay Sort1) or that carry a mutation disrupting endocytosis and possibly sorting (Sort1 mut) (Nielsen et al., 2001). While these mutants traverse the secretory pathway and reach the cell surface to support binding of AP-PGRN to transfected cells (Figure 6C, Movie S3, and data not shown), they do not alter PGRN levels in conditioned medium (Figures 4A and 4B). A version of Sortilin consisting only of the ectodomain (Sort1 ecto) is secreted efficiently from transfected cells, but does not alter PGRN level in the medium (Figures 4A and 4B). Furthermore, inhibition of endocytosis by addition of 0.45 M sucrose to the culture medium for 12 hr increases the level of PGRN in the medium of Sortilin+PGRN-expressing cells to that of PGRN only cells (data not shown). Thus, these initial studies suggested that the reduced PGRN levels in conditioned medium from Sortilin coexpressing cells are due to endocytosis of PGRN. Localization of PGRN to Activated Microglia and Sortilin to Neurons The potential roles of endocytic versus secretory pathways on PGRN levels depend on whether PGRN and Sortilin are expressed in the same cells (cis) or different cell types (trans) in brain. We examined frontal cortex for Sortilin and PGRN protein localization. Both proteins exhibit intracellular granular immunoreactivity in cortical neuronal soma, but there is little colocalization (Figure 5A). Sortilin and other members of the Vps10 family are known to be enriched in recycling endosomes (Willnow et al., 2008), consistent with this pattern. A vacuolar pattern for PGRN has been reported in neurons and may reflect lysosomal localization (see below). In Sort1 / brain, a similar pattern of PGRN immunoreactivity is observed, but there is a trend toward reduced vacuolar staining and increased diffuse neuropil staining (Figure 5B). Previous studies have documented an unaltered PGRN distribution in FTLD-TDP brain with GRN mutations (Mackenzie et al., 2006). We examined Sortilin histologically in such cases (Baker et al., 2006) (Figure 5C). The granular pattern of Sortilin inmmunoreactivity within frontal cortex neurons in healthy human brain is similar to that seen in mouse brain. This distribution is not altered in FTLD-TDP due to GRN mutation. Thus, these

steady-state localization studies of mouse and human frontal cortex demonstrate that PGRN and Sortilin are present, but do not provide mechanistic insight as to their roles. To provide an experimental model that might mimic CNS stress, repair, and regeneration in a manner relevant to FTLD and ALS, we examined the ventral horn of the lumbar spinal cord of mice after sciatic nerve injury. After axotomy, the protein that aggregates in FTLD-TDP, TDP-43, shifts from a nuclear to a cytoplasmic localization (Moisse et al., 2009; Sato et al., 2009) (Figure S4). Sortilin is strongly expressed by spinal motoneurons (Figure 5D) (Domeniconi et al., 2007), but not by microglia (Figure S7B). In contrast, PGRN is strongly induced in activated microglial cells that surround motor neurons after peripheral axonal injury, but not by astrocytes (Figures 5E, 5F, and S3), consistent with human FTLD-TDP pathology (Baker et al., 2006; Mackenzie et al., 2006). In naive tissue, PGRN expression is much lower and includes a neuronal component (Figures 5E and 5F and Ryan et al., 2009). These histological studies, as well as previous expression surveys (Lein et al., 2007; Su et al., 2004; Wu et al., 2009) (Figure S5), are most supportive of the hypothesis that PGRN is secreted by activated microglial cells and then interacts in trans with Sortilin on motoneurons to be endocytosed. Indeed, the C13-NJ microglial cell line secretes PGRN robustly, but expresses little Sortilin (Figure 5G). Rapid Sortilin-Mediated Endocytosis of PGRN to Lysosomes Given the separate cells of origin, we focused on endocytosis of PGRN by Sortilin in controlled systems. To visualize endocytosis we expressed Sortilin in COS-7 cells and applied mCherryPGRN ligand (Figure 6A). At 4 C, binding of fluorescent PGRN ligand is detected at the cell surface and is extensively colocalized with Sortilin. Cells were then shifted to 37 C and the region of the cell within 400 nm of the cell-attached surface was imaged by total interference fluorescence microscopy (TIRF) (Figures 6B and 6C and Movies S1 and S2). Within 5 min, numerous mobile puncta enriched for both mCherry-PGRN and GFP-Sortilin become apparent, consistent with endocytic vesicles (Figure 6B, inset). Over the ensuing 10 min, nearly all PGRN is removed from the cell surface of Sortilin-expressing cells (Figures 6B, 6C, and 6E, and Movies S1 and S2). By 18 min, no diffuse plasma membrane PGRN signal is visible, and limited signal remaining near the cell surface is concentrated in mobile puncta consistent with endosomes. The half-life for PGRN bound to Sortilin at the cell surface is 4 min (Figure 6E). After 30 min, the mCherryPGRN signal visualized by confocal microscopy further from the cell surface colocalizes extensively with the lysosomal marker Lamp1 (Figure 6F). The clearance of PGRN from the cell surface is dependent on the cytoplasmic domain of Sortilin, because a truncated mutant binds mCherry-PGRN, but there is little change in PGRN TIRF

(G) Binding of PGRN to immobilized Sortilin ectodomain by surface plasmon resonance (SPR) analysis (BIAcore). PGRN (10–100 nM) was applied to a microchip containing immobilized Sortilin (0.0952 pmol/mm2). The calculated KD value is indicated; mean ± SEM. (H) Purity of PGRN ligand for SPR studies. His-PGRN protein was purified by nickel affinity chromatography from transfected cell conditioned medium, and then analyzed by SDS-PAGE with Coomassie Blue staining. Migration of Mr standards, in kDa, at left. Scale bars, 50 mm for all, except 5 mm in the bottom panels of (E).

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Figure 2. Specificity of Progranulin Interaction with Sort1 (A) PGRN does not bind to Sortilin homologs SorLA or SorCS1, or to neurotensin receptor 2 (NTR2) in transfected COS-7 cells. (B) The C terminus of PGRN is the Sortilin binding domain in PGRN. AP-PGRN, AP-PGRN-E (aa 494–593), or AP-PGRNDE (aa 18–493) was allowed to bind to COS-7 cells transfected with a Sortilin expression vector. Whereas PGRN-E exhibits strong binding to Sortilin, PGRNDE does not bind to Sortilin. (C) Scatchard plot of AP-PGRN-E or AP-PGRN binding to Sortilin-expressing COS-7 cells. KD, mean ± SEM, n = 4. (D) Displacement of PGRN binding to Sortilin. COS-7 cells transfected with Sortilin vector were preincubated with 100 nM purified his-tagged PGRN, 100 nM purified Sort1-ectodomain, 20 mM GST, 2 mM NT peptide, 50 mM NT peptide residues 1–6, 2 mM NT peptide residues 8–13, or 20 mM purified GST-tagged Pro domain of NGF before addition of AP-PGRN at 4 nM. Cells were further incubated for another 2 hr before washing, fixation, and visualization of binding using AP substrates. (E) Binding from experiments as in (D) is quantitated. Data are mean ± SEM from 3–4 independent measurements. *p < 0.05 relative to No Addition, one-way ANOVA.

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signal over 60 min (Figure 6C and Movie S3), consistent with a half-life at the cell surface at least five times longer than that for ligand bound to intact Sortilin. A portion of the GFP-Sortilin protein initially colocalizing with PGRN ligand at the cell surface later becomes clustered in mCherry-PGRN-positive puncta (Figure 6B and Movie S1), and there is a net decrease in GFP-Sortilin signal within 400 nm of the plasma membrane. Clearance is not as complete as for mCherry-PGRN (Figure 6B). As the mCherryPGRN ligand signal is cleared over 10 min, the GFP-Sortilin receptor signal stabilizes and there is a recovery of the diffuse plasma membrane signal, consistent with recycling of GFPSortilin from endosomes to plasma membrane (Figure 6B and Movie S1). The decreased TIRF signal for GFP-Sortilin at 37 C after exposure to PGRN requires the presence of bound ligand, since there is little signal decrease in the absence of PGRN (Figure 6D and Movie S4). Thus, there is rapid endocytosis of extracellular PGRN by cell surface Sortilin to COS-7 lysosomes. We considered whether the vacuolar pattern of PGRN staining observed in neurons of the frontal cortex and spinal motoneurons (Figure 5) might reflect lysosomal accumulation of the PGRN protein. Double immunohistochemistry for the lysosomal marker Lamp1 and for PGRN reveals that a substantial fraction of PGRN in frontal cortex is present in Lamp1-positive lysosomes (Figure 6G). Sortilin Controls PGRN Levels In Vivo To consider the impact of this endocytic mechanism in vivo, we examined PGRN levels in Sort1 / mice. PGRN immunoreactivity in brain exhibits two species of 73 and 78 kDa due to differential glycosylation (Figures 7 and S6A), while PGRN in serum is largely of 78 kDa (Figure 7D). In 7-month-old mice lacking Sortilin, PGRN protein levels are strongly upregulated (Figures 7B–7E). The increase in total brain PGRN level is 2.5-fold (Figures 7B and 7C), while the increase of 78 kDa PGRN in brain and in serum is 5-fold (Figures 7D and 7E). Because FTLD derives from PGRN haploinsufficiency with a 50% decrease in PGRN protein levels, the absence of Sortilin may fully normalize PGRN levels. Indeed, GRN+/ mice lacking Sortilin expression exhibit levels of PGRN protein that equal or exceed WT levels (Figures 7F, 7G, S6B, and S6C). The protein changes are specific for PGRN, in that levels of another Sortilin ligand, prosaposin (Lefrancois et al., 2003; Zeng et al., 2009), are not altered (Figure S6E). While Sortilin deficiency increases PGRN level, ablation of PGRN does not alter Sortilin level or proteolysis in brain tissue (Figure S6D). The rapid endocytosis of PGRN by Sortilin (Figure 6) is the likely cause for increased PGRN in Sort1 / mice. To consider an alternative transcriptional mechanism, we assessed the level of PGRN mRNA in brain by quantitative RT-PCR. No difference in PGRN mRNA levels occurs in

Sort1 / versus WT mice (Figure S6F), supporting the hypothesis that Sortilin endocytosis determines PGRN level in brain and serum. DISCUSSION The major finding of this work is that Sortilin is a principal neuronal binding site for the FTLD protein PGRN. In a stressed nervous system, after production by activated microglial cells, PGRN binds to Sortilin expressed on the neuronal cell surface. Binding occurs via the C terminus of PGRN, in a manner that may resemble NT binding to Sortilin (Quistgaard et al., 2009). A dramatic consequence of such binding is the rapid endocytosis of PGRN by Sortilin. In vivo, the absence of Sortilin raises PGRN levels by 2.5- to 5-fold in different compartments. Given that FTLD is caused by a 2-fold reduction of PGRN levels (Baker et al., 2006; Cruts et al., 2006; Finch et al., 2009; Gass et al., 2006; Ghidoni et al., 2008; Sleegers et al., 2009), the magnitude of this change has clear pathophysiological consequences, and PGRN deficiency in GRN+/ mice is fully normalized by deletion of Sortilin expression (Figures 7F, 7G, and S6). There are several mechanisms whereby PGRN/Sortilin interaction might alter neuronal function and contribute to FTLDTDP. Sortilin may function upstream to titrate PGRN levels and/or downstream of PGRN to mediate its effects on cell function. To distinguish the relative importance of Sortilin upstream versus downstream of PGRN will require the creation of FTLDTDP relevant models in which altering PGRN function changes the outcome. Unfortunately, such an experimental model is not yet established. The changes in brain and serum PGRN levels in Sort1 / mice observed here demonstrate that Sortilin can have an upstream effect to regulate levels of PGRN. However, there is ample reason to believe that Sortilin’s primary effect is downstream of PGRN, as a mediator or receptor for functional effects in neurons. Sortilin is reported to possess signaling function in complex with p75NTR for proneurotrophin apoptotic signaling (Domeniconi et al., 2007; Jansen et al., 2007; Nykjaer et al., 2004; Teng et al., 2005). While we did not observe modulation of PGRN binding to Sortilin by p75NTR, proneurotrophin action may be altered in a more complex in vivo setting. Specifically, since proNGF and PGRN can both bind to Sortilin (Figures 2D, 2E, and 2H), PGRN might rescue cells otherwise subject to proneurotrophin-induced cell death through Sortilin/p75NTR. Displacement of PGRN by proNGF is of low potency so it may be indirect or allosteric, and therefore highly dependent on cellular context. Further study of proneurotrophin-related effects under a range of conditions may be revealing. However, it remains at least equally likely that PGRN signals via Sortilin independently of proneurotrophins.

(F) p75NTR does not affect PGRN binding to Sortilin. AP-PGRN at the indicated concentration was allowed to bind COS-7 cells transfected with Sortilin alone, transfected with p75NTR alone, or cotransfected with Sortilin and p75NTR, as indicated. (G) Binding analysis of PGRN to all five Vps10 family members by SPR analysis demonstrated strong PGRN binding to Sortilin, very weak binding to SorLA, and no detectable binding to SorCS1–3. PGRN (200 nM) was included in the perfusate during the interval from 100–550 s. (H) Competitive SPR analysis of PGRN and proNGF to Sortilin. Between time points 1 and 2, Sortilin chips were exposed to saturating proNGF (1000 nM) or buffer, as indicated. Between points 2 and 3, the chips were exposed to PGRN (200 nM) or proNGF (1000 nM). Binding of PGRN during the 2-3 interval is not substantially altered by prior incubation with proNGF. Scale bars, 50 mm.

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Figure 4. Levels of Extracellular Progranulin Are Reduced by Cellular Sortilin

Figure 3. Sortilin Is an Endogenous Brain PGRN Binding Site (A) Sortilin expression in cortical neuron culture lysate by immunoblot. (B) AP-PGRN binding to cortical neuronal cultures grown for indicated times. Scale bar, 50 mm. (C) AP-PGRN-E exhibits strong binding to cortical neurons (21 DIV), while PGRNDE binding is minimal at 10 nM. Scale bar, 50 mm. (D) Brain lysate of WT (+/+) or Sort1 knockout ( / ) mice were incubated with purified GST or GST-PGRN-E (aa 494–593) protein. After further incubation with glutathione beads, the pulldown products were washed and blotted with mouse anti-Sortilin antibodies. The trace immunoreactivity in the lysate of the Sort1 / sample is derived from a truncated, nonfunctional protein (Jansen et al., 2007 and data not shown). Mr markers are shown to the right; 100 kDa in the top panel, and 37 kDa plus 25 kDa in the bottom panel. (E) AP-PGRN-E (1 nM) binding to cultured mouse neurons (21 DIV) from Sort1+/ and Sort1 / mice. Scale bar, 50 mm. (F) Quantification of AP-PGRN-E binding to cultured mouse neurons from Sort1+/ and Sort1 / mice. The gray box represents nonspecific binding assessed in the presence of 200 nM his-PGRN as in Figure S1B. Mean ± SEM, **p < 0.01, one-way ANOVA.

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(A) HEK293T cells were cotransfected with human PGRN expression construct plus pcDNA vector control, SorLA, or various Sortilin expression constructs. The conditioned media (CM) and cell lysates (Cells) were collected 5 days after transfection and immunoblotted for PGRN and Sortilin. (B) Quantification of PGRN level in the CM or in the cell lysate (Cells) for experiments in (A) expressed as percentage of pcDNA control. Mean ± SEM, **p < 0.01, one-way ANOVA. (C) HEK293T cells were cotransfected with human, full-length Sortilin expression vector plus pcDNA control vector or PGRN expression vector. Immunoblot for Sortilin protein shows that the majority is cell associated and not shed, regardless of PGRN coexpression.

Separate from signaling at the cell surface, PGRN may have intracellular functions dependent on endocytosis by neuronal Sortilin and delivery to lysosomes. Importantly, immunologically intact PGRN accumulates within the lysosomal compartment of COS-7 cells and neurons. In healthy or FTLD-TDP human brain, PGRN is most prominent in microglial cells, but is also present in puncta within the cytoplasm of neurons (Mackenzie et al., 2006), and these appear most consistent with lysosomes. These observations raise the possibility that PGRN functions within autophagosomal/lysosomal pathways. Endocytosis and lysosomal delivery of another Sortilin ligand, prosaposin, serves to enhance lysosomal function rather than simply degrading the ligand (Lefrancois et al., 2003; Zeng et al., 2009).

Neuron Sortilin-Mediated Endocytosis of Progranulin

Figure 5. Localization of Sortilin and PGRN in Different CNS Cell Types (A) Double immunohistochemistry for PGRN (green) and Sortilin (red) in adult mouse frontal cortex. The boxed area in the merge is shown at the right. Scale bar, 7 mm. (B) Anti-PGRN staining of mouse frontal cortex from WT and Sort1 / mice. In both cases vacuolar staining is prominent in neurons, and there is a trend to decreased vacuolar localization and increased diffuse neuropil staining in the Sortilin null samples. Scale bar, 7 mm. (C) Anti-Sortilin staining of human frontal cortex from neurological healthy control and an FTLD-TDP case with PGRN mutation. Scale bar, 30 mm for top panels and 10 mm for bottom panels. (D) Sortilin immunohistochemistry of the ventral horn in the mouse L5 spinal cord shows significant expression of Sortilin in motor neurons. Scale bar, 50 mm. (E and F) Immunohistochemistry for PGRN in the ventral horn (VH) of L5 spinal cord transverse sections from mice that had unilateral sciatic nerve resection 7 days previously. Injury-induced PGRN expression is detected on the axotomized side, but not on the uninjured side in (E). Significant colocalization of injury-induced PGRN with the microglial marker (Iba1) is observed on the axotomized side (blue arrows in F), but not on the uninjured side. Less intense vacuolar PGRN immunoreactivity is detected in motoneurons (white arrows in F). Scale bars, 50 mm in (E) and 18 mm in (F). (G) The conditioned medium and cellular fraction of C13-NJ microglial cell line cultures were immunoblotted for PGRN and for Sortilin. PGRN secretion is prominent, and there is little Sortilin immunoreactivity.

Under this hypothesis, Sortilin binding is crucial to provide neuronal competence for TDP-43 clearance via autophagosomal/lysosomal mechanisms. Sortilin is a member of the Vps10 family (Willnow et al., 2008), members of which are required for sorting to the lysosomal vacuole in yeast (Cooper and Stevens, 1996). Sortilin is known to participate in the delivery of extracellular prosaposin and intracellular Golgi-derived cathepsins to

lysosomes (Canuel et al., 2008; Lefrancois et al., 2003; Ni and Morales, 2006; Zeng et al., 2009). PGRN is coregulated with lysosomal genes (see the Supplemental Data in Sardiello et al., 2009). We and others (Ahmed et al., 2010) have observed accelerated brain lipofuscinosis in mice lacking PGRN. Of relevance to FTLD, the clearance of cytoplasmic TDP-43 depends at least in part on autophagy (Caccamo et al., 2009; Ju et al., 2009; Neuron 68, 654–667, November 18, 2010 ª2010 Elsevier Inc. 661

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Figure 6. Rapid Endocytosis of PGRN by Sortilin (A) COS-7 cells expressing Sortilin were incubated with conditioned media containing mCherry-tagged PGRN at 4 C and confocal images were acquired after staining with anti-Sortilin antibodies. A magnified view of the boxed region is provided by the inset. Colocalization at the cell surface was seen at 4 C. (B) Sortilin mediates endocytic trafficking of PGRN. GFP-Sortilin-expressing COS-7 cells were incubated with mCherry-PGRN at 4 C and then shifted to 37 C for live imaging by TIRF microscopy (see Movie S1). The insets provide magnified views of the boxed regions. After 4 min at 37 C, mCherry-PGRN is localized to puncta with GFP-Sortilin (arrow). By 8 min, most mCherry-PGRN diverges into vesicles that lack GFP-Sortilin (arrow). (C) Endocytosis of PGRN by Sortilin requires the cytoplasmic domain of Sortilin. COS-7 cells expressing Sortilin or pDisplay-Sort1 were incubated with media containing mCherry-PGRN washed and imaged by TIRF microscopy for the indicated times. Near complete internalization of mCherry-PGRN is seen in the Sortilin-expressing cells by 18 min at 37 C (Movie S2), whereas pDisplay-Sort1-expressing cells show persistent mCherry-PGRN signal near the cell surface after 60 min (Movie S3).

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Figure 7. PGRN Levels Are Increased in Mice Lacking Sortilin (A) WT and GRN / mouse brain lysate was analyzed by anti-PGRN immunoblot. (B) Tissue lysate collected from the cerebral cortex of 7-month-old mice was subjected to SDS-PAGE and anti-PGRN immunoblot. Two PGRN bands are seen at 78 kDa (band A) and at 73 kDa (band B); both bands increase in the Sort1 / samples compared to those of WT. (C) Quantification of PGRN level (bands A, B, and A+B) in the cortical lysate, normalized to GAPDH immunoreactivity. Sort1 / , n = 4 mice; WT, n = 5. Mean ± SEM, **p < 0.01, one-way ANOVA. (D) Serum samples from 7-month-old Sort1 / and WT mice were collected, stripped of albumin and IgG, and immunoblotted for PGRN and transferrin. Serum levels of PGRN were increased in the Sort1 / mice compared to littermate WT mice. (E) Quantification of PGRN level in the serum normalized to transferrin level. For both genotypes, n = 4 mice. Mean ± SEM, **p < 0.01, one-way ANOVA. (F) Serum samples from 2-month-old mice were obtained from the indicated genotypes and analyzed by anti-PGRN immunoblot, with anti-thrombin III immunoblot as a loading control. (G) Quantification of PGRN level in the serum normalized to anti-thrombin III level. For all genotypes, n = 4 mice. Mean ± SEM; *p < 0.05; **p < 0.01; one-way ANOVA.

Urushitani et al., 2010; Wang et al., 2010). Ubiquitin coaccumulation with TDP-43 aggregates may reflect disrupted balance between proteosomal pathways, macroautophagy, and chaperone-mediated autophagy pathway. In this regard, it may be relevant to consider those mutations in Valosin-Containing Protein (VCP), which are known to cause FTLD plus myopathy and Paget disease (Custer et al., 2010; Ju et al., 2009; Watts et al., 2004). The accumulation of TDP-43 and ubiquitin aggregates in brain of these rare cases is similar to that in more common PGRN mutant cases. Since VCP has a prominent role in autophagy, the PGRN pathway and the VCP pathway may overlap as intracellular constituents are

sorted to lysosomes. Both PGRN and VCP might function to clear TDP-43 aggregates via autophagy. As noted above, PGRN can be converted proteolytically into smaller GRN peptides (Zhu et al., 2002). In the extracellular space, this conversion can be mediated by elastase and inhibited by secreted leukocyte protease inhibitor (SLPI). Lysosomal localization of PGRN may also influence conversion from PGRN to GRN, and therefore the balance between PGRN and GRN function. Such conversion may have downstream effects on PGRN biology and the pathophysiology of FTLD. In order to separate these possibilities further, in vivo neuronal studies of GRN versus PGRN activity will be required.

(D) GFP-Sortilin-expressing cells were incubated without mCherry-PGRN at 4 C and the GFP-Sortilin signal was imaged using TIRF for the indicated times (Movie S4). The GFP-Sortilin distribution near the cell surface is stable compared with cells with ligand in (B). (E) The background-corrected mCherry fluorescence intensity relative to time zero is plotted as a function of time from experiments as in (B)–(D). The mCherryPGRN signal from Sortilin-expressing cells is the mean ± SEM for n = 9, and the apparent half-life is 4 min. (F) Endocytosed PGRN localizes to lysosomes. Sortilin-expressing COS-7 cells were incubated with mCherry-PGRN (red in merge) on ice for 4 hr before being washed and shifted to 37 C. Cells were fixed at indicated times and stained with mouse anti-Lamp1 antibodies (green in merge). Images were obtained with a laser-scanning confocal microscope. (G) Endogenous brain PGRN is localized to lysosomes. Sections of adult frontal cortex were double stained for anti-PGRN (green) and anti-Lamp1 (red). Extensive colocalization demonstrates that many PGRN-reactive puncta are lysosomes in these two examples. Scale bar, 10 mm.

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It is clear from the present data that the interaction of PGRN with Sortilin-mediated endocytosis significantly reduces steadystate PGRN levels. The magnitude of this reduction is at least as great as that of those PGRN mutations causing FTLD-TDP. Thus, Sortilin binding provides a potential therapeutic site to alter PGRN-dependent pathways and alleviate TDP-43 pathology. It is clear that microglial cells are a major source of PGRN production. For example, activated microglia in the vicinity of axotomized motoneurons strongly induce PGRN after sciatic nerve injury. From this observation, it follows that characterizing the mechanisms of microglial PGRN induction or the selectivity of PGRN for subsets of microglia may reveal additional means to modulate the course of FTLD-TDP. Because neither GRN+/ nor GRN / mice exhibit an FTLD-like phenotype (Kayasuga et al., 2007; Yin et al., 2010) (data not shown), this human inherited disease cannot be modeled simply in rodent. Although peripheral axotomy shifts TDP-43 from the nucleus to the cytoplasm of motoneurons (Moisse et al., 2009; Sato et al., 2009) and might sensitize to FTLD/ALS-like pathology, neither GRN / nor Sort1 / mice have detectable motoneuron loss or ventral root axonal degeneration after this perturbation (Figure S4). Future functional studies of Sortilin in PGRN biology will require development of robust rodent models for PGRNdependent neurodegeneration. Nevertheless, our work implicates Sortilin-mediated PGRN endocytosis as a key pathway for further study in FTLD pathophysiology. EXPERIMENTAL PROCEDURES Animals, Cell Lines, Antibodies, and Human Tissue The Sort1 / mouse line has been described (Jansen et al., 2007) and studies here utilized mice backcrossed for more than nine generations to C57BL6. The Sort1 / mice are generated by deletion of a critical exon encoding a portion of the b-propeller domain. Although trace levels of immunoreactivity of smaller size are detectable in the Sort1 / strain, none of the mutant protein folds appropriately or reaches the cell surface (data not shown). GRN mutant mice were obtained from the RIKEN Bioresource Center (Kayasuga et al., 2007). Both littermate controls and C57BL6 mice were used as WT control. COS-7 and HEK293T cells were obtained from ATCC. The following antibodies were used in the study: mouse anti-Sortilin (612101) antibody from BD Biosciences, goat anti-Sortilin (BAF2934) and rabbit anti-Sortilin (ab16640) antibody from Abcam, mouse anti-human PGRN (mAB2420) and sheep anti-mouse PGRN (AF2557) antibodies from R&D Systems, mouse anti-Flag M2 antibodies, rabbit anti-Myc, and mouse anti-GAPDH (G8795) antibodies from Sigma-Aldrich, rat anti-Transferrin (sc-33731) and goat anti-Actin (sc-1616) from Santa Cruz Biotech, rabbit anti-Iba-1 (019-19741) from Wako, mouse anti-NeuN (Mab377) from Millipore, rabbit anti-TDP43 antibody (12892-1-AP) from Protein Tech, mouse anti-Beta III tubulin (G712A) from Promega, mouse anti-Lamp1 antibody from BD biosciences (611043), anti-Prosaposin from Abcam (ab68466), and anti-Prosaposin from Abgent (AP7398c). Human brain tissue from neurologically healthy and FTLD-U cases with PGRN mutations was collected as described (Baker et al., 2006). Paraffinembedded frontal cortex from one affected individual from each of the six families described previously (Baker et al., 2006) was studied for Sortilin localization. Plasmid Constructs and Recombinant Proteins Human PGRN in pCMV-Sport6 vector was obtained from Open Biosystems. To generate AP fusion proteins, human PGRN coding sequence for mature peptides (aa 18–593) and corresponding fragments were amplified and ligated to the pAP5-tag vector (GenHunter, Nashville, TN) digested with restriction enzymes XhoI and PmeI. AP-PGRN fusion proteins were expressed in

664 Neuron 68, 654–667, November 18, 2010 ª2010 Elsevier Inc.

HEK293T cells and purified as described for other proteins (Fournier et al., 2001; Hu et al., 2005; Laure´n et al., 2009). Flag- and his-tagged PGRN were cloned in pSectag2 vector (Invitrogen). An expression vector for mCherrytagged PGRN was generated in the pAP5 vector by replacing AP sequence with mCherry sequence using HindIII and XhoI followed by inserting mature human PGRN (aa 18–593) coding sequence using XhoI and PmeI. Human Sortilin in a mammalian expression vector was obtained from Origene. Myc-his-tagged, full-length, mature human Sortilin (aa 60–831) and Myc-his-tagged Sortilin extracellular domain (ecto, aa 60–725) constructs were generated by replacing AP coding sequence in the pAP5 vector with Myc-his-Sortilin or Myc-his-Sort1-ecto coding sequences after the Pro domain. Myc-his-Sort1-ecto plasmid was then transfected into HEK293T cells and Myc-his-Sort1-ecto protein was purified from the conditioned media using Ni++ beads. To generate fusion protein of Sort1-ectodomain with the platelet derived growth factor receptor (PDGFR) transmembrane domain, Sort1-ectodomain (aa 60–725) was cloned into the pDisplay vector (Invitrogen) using SfiI and XmaI sites. GFP-Sortilin construct was engineered from pEGFP-C1 vector with the signal peptide of IgG followed by GFP and residues 60–831 of Sortilin cloned at XhoI and XmaI sites. The AP-NT8-13 construct was made by cloning sequence of human NT residues 8–13 into pAP5 vector using XhoI and ApaI sites. SorLA expression constructs were a gift from Dr. Wolfgang Hampe at Inst. fu¨r Biochemie und Molekularbiologie and Dr. Thomas E. Willnow at Max-Delbrueck Center for Molecular Medicine. Sort1-mut vector was a gift from Dr. Claus M. Petersen at University of Aarhus (Nielsen et al., 2001). Human NTR1 construct was a gift from Dr. Jean Mazella from Institut de Pharmacologie Mole´culaire et Cellulaire, Sophia Antipolis, Valbonne, France. SorCS1 and NTR expression constructs in pCMV-Sport6 were obtained from Open Biosystems. For soluble protein production, expression plasmids were transfected into HEK293T cells and conditioned media were collected after 5 days. FlagPGRN was purified from the conditioned media using mouse anti-Flag conjugated beads from Sigma-Aldrich and his-PGRN was purified using Ni++ beads from Invitrogen. To generate GST-fusion proteins, a PGRN fragment (aa 494–593, PGRN-E) and the Pro domain of NGF were cloned in the pGEX6P-1 vector (GE Healthcare). GST fusion protein was expressed in BL21 (DE3) E. coli cells and purified using glutathione beads (GE Healthcare).

Binding Assays and Expression Cloning AP-PGRN binding assays and cDNA library screening were performed with transfected COS-7 or mouse cortical neurons, as described for other ligands (Fournier et al., 2001; Hu et al., 2005; Laure´n et al., 2009). Conditioned media with AP ligands were incubated with untransfected or receptor-transfected COS-7 cells for 2 hr at room temperature before fixation and heat inactivation of endogenous AP at 65 C overnight. Bound AP to COS-7 cells was measured using NIH image software. We screened 225,000 clones of an arrayed adult mouse brain cDNA library (Origene) by transfecting COS-7 cells with arrayed pools of 100 clones, and inspecting for those rare cells transfected with a cDNA conferring to APPGRN binding, as for other ligands (Laure´n et al., 2009). DNA preparation from amplified pools of 100 cDNA clones was by the Iowa State University DNA Facility. We also examined individual clones from a 352-member preexisting collection of expression vectors for transmembrane proteins (GFC, Origene) for AP-PGRN binding (Laure´n et al., 2009).

Neuronal Cultures and Neuronal Binding Assays Cortical neurons and hippocampal neurons isolated from E18 rats and E17 mice were cultured as described (Laure´n et al., 2009), omitting laminin. Live neurons were incubated with conditioned media containing AP-fusion proteins at 4 C for 1 hr prior to washes and fixation. Bound AP was measured by obtaining images at 203 and 103 measuring binding using NIH image software as above. Displacement assays were conducted by incubating neurons with the competitor (untagged ligand) for 30 min at 4 C, followed by incubation of AP ligand with the competitor. Neuronal cell lysates were prepared in RIPA buffer. Standard western blot procedures for the Li-Cor Odyssey system were followed.

Neuron Sortilin-Mediated Endocytosis of Progranulin

Brain Lysates, Pulldown, and Immunoblots Immunoblot and affinity chromatography methods have been described (Fournier et al., 2001; Hu et al., 2005; Jansen et al., 2007; Laure´n et al., 2009; Nykjaer et al., 2004). Conditioned media with Flag-PGRN or Myc-his-Sort1-ectodomain were mixed together and incubated with anti-Myc antibody conjugated beads (Santa Cruz) or mouse anti-Flag antibody conjugated beads (Sigma). After incubating at 4 C for 2 hr, the beads were washed four times with PBS buffer. For brain lysate pulldown, adult mouse brain was lysed in 6 ml PBS buffer with 1% Triton. Two milliliters of lysate were then incubated with purified GST or GST-PGRN-E (aa 494–593) fusion protein. The lysates were further incubated with glutathione beads for an additional 2 hr and then the beads were washed with lysis buffer. To determine various protein levels in samples from the various genotypes, 7-month-old or 2-month-old mice were transcardially perfused with cold PBS, cortically dissected, and homogenized in RIPA buffer. Extracts were separated by 4%–20% SDS-PAGE and immunoblotted using sheep anti-PGRN, rabbit anti-Sortilin, rabbit anti-prosaposin, and GAPDH antibodies. Immunoreactive bands were quantified using Li-Cor Odyssey v3.0 software. Serum Analysis Blood was collected from the cardiac right ventricle of deeply anesthetized animals. The collected blood was kept at RT for 15 min to coagulate and was then centrifuged at 1000 3 g for 15 min. Albumin and IgG were removed (Calbiochem #122642 according to manufacturer’s instructions). Equal amounts (35 mg) of protein were then subjected to SDS-PAGE on a 4%– 20% gel and immunoblotted with the appropriate antibodies. RT-PCR Cortical tissue was dissected from 7-month-old mice and was snap frozen in liquid nitrogen. RNA was extracted from 50 mg of tissue using RiboPure Kit (Ambion #AM1924). The extracted RNA was subjected to quantitative PCR using TaqMan RNA-to-CT1 step kit (Applied Biosystems), with two GRNspecific probes (Mm01245914_g1, Mm00433848_m1 from Applied Biosystems) and control probes (GAPDH and actin). Glycosidase Digestion of Brain Tissue Cortical tissue from mice was homogenized in TBS. The resulting lysates (70 mg) were subjected to digestion with PNGaseF (New England Biolabs) or Endo-a-N-Acetylgalactosaminidase Glycosidase (New England Biolabs) treatment for 1 hr at 37 C as directed by the manufacturer. The mixtures were then subjected to methanol/chloroform (4:1) precipitation and the resulting material was resuspended in 23 SDS-PAGE loading buffer and run on a 4%–20% polyacrylamide gel for immunoblots. Sciatic Nerve Injury and Analysis Mice were anesthetized using 4% isofluorane and the sciatic nerve was exposed at the lower hip level. The sciatic nerve was ligated 4 mm below the sciatic foramen of the pelvic girdle with 5-0 vicryl and a 2.5 mm segment was resected distal to the ligation. The mice were given an injection of ampicillin, buprenorphine, and saline subcutaneously postsurgery. Three weeks after surgery, the animals were deeply anesthetized and then perfused with PBS followed by 4% paraformaldehyde in PBS (pH 7.2). The gelatin-embedded L5 spinal cord tissue was processed into 30 mm sections using a vibrating microtome and stained with the indicated primary antibodies using appropriate Alexa secondary antibodies (Invitrogen). For ventral root axon profile analysis, L5 ventral roots were dissected, postfixed in 2.5% glutaraldehyde, and embedded in Epon. Semithin 500 nm sections were stained with toluidine blue. Total intact axon profiles per root were counted, without knowledge of mouse genotype.

DAB staining of paraffin-embedded human frontal cortex samples was conducted using mouse anti-Sortilin (BD biosciences) and the ABC amplification system (Vector Labs), as per manufacturer’s instructions. TIRF Microscopy In brief, COS7-cells were transfected with GFP-Sortilin and cultured for 3 days, or cotransfected with GFP + human Sortilin or GFP + pDisplay-Sortilin and cultured for 16–36 hr prior to conducting experiments. The cells were incubated on ice 10 min prior to addition of ligand, to attenuate endocytosis. Thereafter, mCherry-PGRN medium was added to the cells, which were incubated for 1–2 hr on ice and washed three times with ice cold HBH. The cells were then imaged at 4 s intervals for 30–60 min at 37 C using a 633 objective. The details of the TIRF microscopy were as described (Zoncu et al., 2009). SEC Fractionation The size exclusion chromatography (SEC) data were collected with a 25 ml Superdex 200 SEC column (GE Healthcare) and an AKTA high-performance liquid chromatography system (GE Healthcare) at a flow rate of 0.5 ml min 1 at 4 C. Aliquots of AP fusion protein fractions from the SEC separation were incubated with p-Nitrophenyl Phosphate (PNPP) substrate (Sigma N1891) and the absorbance was measured at 405 nm using Victor 3V multilabel counter (Perkin Elmer). Fractions from the mCherry-PGRN fractionation were assessed for fluorescence (Ex 560/Em 615) using Victor 3V multilabel counter (Perkin Elmer). SPR The expression, purification (>95%), and crystallization of soluble Sortilin has been described (Quistgaard et al., 2009). SPR analysis was performed using a BIAcore 3000 instrument (BIAcore Sweden) as previously described (Munck Petersen et al., 1999). In brief, soluble Sortilin was immobilized at a density of 0.0952 pmol/mm2 on a CM5 sensor chips. PGRN was dialysed against 10 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM CaCl2, 1 mM EGTA, and 0.005% surfactant P20 (running buffer), and 10–100 nM of the ligand was injected (5 ml/min) through the flow cell at 20 C. After 600 s, running buffer containing PGRN was substituted with buffer lacking the ligand, allowing for determination of both the association and dissociation constants. The binding response is expressed in relative response units, i.e., the difference in response between the immobilized protein flow cell and a corresponding control flow cell. Kinetic parameters were determined using the BIAevaluation 3.0 software. To compare alternate Sortilin family proteins, equal amounts of soluble Sortilin, SorLA, SorCS1, SorCS2, or SorCS3 proteins (>95%) were immobilized on a CM5 chip and remaining coupling sites were blocked with 1 M ethanolamine. Sample and running buffer was 10 mM HEPES, 150 mM (NH4)2SO4, 1.5 mM CaCl2, 1 mM EGTA, and 0.005% Tween-20 (pH 7.4). PGRN was applied at 200 nM for all receptors. To test for competitive binding of PGRN and proNGF to Sortilin, the Sortilin chip was initially incubated with 1000 nM proNGF prior to injection with a mixture of PGRN (200 nM) and proNGF (1000 nM), and the signal was compared to binding of PGRN (200 nM) alone. The sensor chip was regenerated in a 10 mM glycine-HCl buffer after each analytic cycle. The SPR signal was expressed in relative response units, as the response obtained in a control flow channel was subtracted. SUPPLEMENTAL INFORMATION Supplemental Information includes six figures and four Supplemental Movies and can be found with this article online at doi:10.1016/j.neuron.2010.09.034. ACKNOWLEDGMENTS

Immunohistochemistry of Mouse and Human Frontal Cortex Seven-month-old mice were deeply anesthetized and then perfused with PBS followed by 4% paraformaldehyde in PBS (pH 7.2). Thereafter, costaining of tissue was conducted using conventional immunohistochemical procedures (block with 10% serum and permeabilize with 0.1% Triton X-100). For immunofluorescent staining, the tissue was imbedded in gelatin and processed into 35 mm sections using a vibrating microtome.

We thank Drs. Wolfgang Hampe and Thomas E. Willnow for SorLA expression construct, Dr. Claus M. Petersen for Sortilin mutant construct, and Dr. Jean Mazella for NTR1 expression plasmid and C13-NJ cells. We also thank Bret Judson for assistance with confocal imaging. S.M.S. is a member of the Kavli Institute for Neuroscience at Yale University. We acknowledge research support to F.H. from Cornell University, Institutional National

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Institutes of Health training grant support to T.P. and to O.A.B., operating grant support to I.R.M. and H.H.M. from the Canadian Institutes of Health, and research support to S.M.S. from the National Institutes of Health, the A.L.S. Association, the Falk Medical Research Trust, an anonymous donor, and GlaxoSmithKline, Inc. Accepted: September 12, 2010 Published: November 17, 2010 REFERENCES

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