NRH2 is a trafficking switch to regulate sortilin localization and permit proneurotrophin-induced cell death
2009; Springer Nature; Volume: 28; Issue: 11 Linguagem: Inglês
10.1038/emboj.2009.118
ISSN1460-2075
AutoresTaeho Kim, Barbara L. Hempstead,
Tópico(s)Signaling Pathways in Disease
ResumoArticle30 April 2009free access NRH2 is a trafficking switch to regulate sortilin localization and permit proneurotrophin-induced cell death Taeho Kim Taeho Kim Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Barbara L Hempstead Corresponding Author Barbara L Hempstead Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY, USA Department of Medicine, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Taeho Kim Taeho Kim Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Barbara L Hempstead Corresponding Author Barbara L Hempstead Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY, USA Department of Medicine, Weill Medical College of Cornell University, New York, NY, USA Search for more papers by this author Author Information Taeho Kim1 and Barbara L Hempstead 1,2 1Graduate Program in Neuroscience, Weill Medical College of Cornell University, New York, NY, USA 2Department of Medicine, Weill Medical College of Cornell University, New York, NY, USA *Corresponding author. Department of Medicine, Weill Medical College of Cornell University, 1300 York Ave, New York, NY 10065, USA. Tel.: +1 212 746 6215; Fax: +1 212 746 8866; E-mail: [email protected] The EMBO Journal (2009)28:1612-1623https://doi.org/10.1038/emboj.2009.118 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Proneurotrophins mediate neuronal apoptosis using a dual receptor complex of sortilin and p75NTR. Although p75NTR is highly expressed on the plasma membrane and accessible to proneurotrophin ligands, sortilin is primarily localized to intracellular membranes, limiting the formation of a cell surface co-receptor complex. Here, we show that the mammalian p75NTR homologue NRH2 critically regulates the expression of sortilin on the neuronal cell surface and promotes p75NTR and sortilin receptor complex formation, rendering cells responsive to proneurotrophins. This is accomplished by interactions between the cytoplasmic domains of NRH2 and sortilin that impair lysosomal degradation of sortilin. In proneurotrophin-responsive neurons, acute silencing of endogenous NRH2 significantly reduces cell surface-expressed sortilin and abolishes proneurotrophin-induced neuronal death. Thus, these data suggest that NRH2 acts as a trafficking switch to impair lysosomal-dependant sortilin degradation and to redistribute sortilin to the cell surface, rendering p75NTR-expressing cells susceptible to proneurotrophin-induced death. Introduction Neurotrophins are growth factors that play crucial roles in the development and maintenance of the vertebrate nervous system through two structurally unrelated receptors, the Trk receptor tyrosine kinases and the neurotrophin receptor p75NTR. Trk receptors regulate neuronal survival, differentiation, and synaptic plasticity through well defined signalling pathways (Patapoutian and Reichardt, 2001; Chao, 2003), whereas p75NTR mediates numerous actions, including neuronal apoptosis or axonal repulsion that are dependant on its co-receptor partners and preferred ligands (Hempstead, 2002; Roux and Barker, 2002; Barker, 2004). Our earlier studies indicate that proforms of neurotrophins (proneurotrophins; i.e. proNGF or proBDNF) selectively bind to p75NTR to induce cell death (Lee et al, 2001; Teng et al, 2005). ProNGF is secreted and can induce cell death under pathologic conditions, including spinal cord injury, CNS lesion, and seizure induction (Beattie et al, 2002; Harrington et al, 2004; Volosin et al, 2006), and is more abundant in Alzheimer's brains, as compared with age-matched controls (Peng et al, 2004), suggesting a pathophysiological role of proNGF in vivo. ProNGF interacts with a dual receptor complex of p75NTR and sortilin, a Vps10p domain-containing transmembrane protein that is highly expressed in the vertebrate CNS (Sarret et al, 2003; Nykjaer et al, 2004; Jansen et al, 2007). Biochemical studies suggest that the prodomains of neurotrophins bind sortilin, whereas the mature domains bind p75NTR on the cell surface to mediate cell death (Nykjaer et al, 2004). Earlier studies, however, indicate that sortilin is predominantly intracellular in location, in the trans-Golgi network (TGN) where it participates in intracellular trafficking, and <10% of the total sortilin pool is expressed on the plasma membrane (Petersen et al, 1997; Nielsen et al, 1999, 2001; Mazella, 2001; Mari et al, 2008). As p75NTR is primarily localized to the plasma membrane, these observations suggest that mechanisms which promote sortilin expression on the cell surface could determine cellular responsiveness to proneurotrophins. Indeed, a recent report indicates that sortilin is co-expressed with p75NTR on the plasma membrane of mouse retinal ganglion cells (RGCs) when p75NTR-dependant developmentally regulated RGC death is robust (embryonic day 13–15.5). In contrast, at later developmental stages of the retina (postnatal day 0–6), sortilin is preferentially expressed in the Golgi complex and p75NTR-dependant RGC death is not observed (Frade and Barde, 1999; Harada et al, 2006; Nakamura et al, 2007). However, the molecular mechanisms that regulate sortilin localization during neuronal development are unknown. A mammalian homologue of p75NTR, NRH2 (also termed as PLAIDD or NRADD) shares some sequence similarity to p75NTR, but contains a unique truncated ectodomain that does not bind to neurotrophins (Frankowski et al, 2002; Kanning et al, 2003; Wang et al, 2003; Murray et al, 2004). Although NRH2 has been suggested to function like p75NTR, the cytoplasmic domains of NRH2 and p75NTR indeed share <40% amino acid identity (Murray et al, 2004), suggesting that NRH2 might not merely mimic p75NTR function, but might perform additional biological actions. NRH2 is developmentally regulated, and is expressed at high levels during embryonic development, but downregulated in adult tissues (Frankowski et al, 2002; Wang et al, 2003). NRH2 is co-expressed with p75NTR in subpopulations of cells in the spinal cord, neonatal retina, dorsal root ganglion, or cultured sympathetic neurons (Kanning et al, 2003; Murray et al, 2004), regions where sortilin is expressed, and proneurotrophin-induced cell death has been reported (Sarret et al, 2003; Nykjaer et al, 2004; Domeniconi et al, 2007; Nakamura et al, 2007), suggesting that NRH2 might play a role in proneurotrophin-mediated cell death. In this study, we investigate the potential actions of NRH2 in regulating proneurotrophin-induced neuronal apoptosis. We find that NRH2 expression is dynamically regulated during development, with increased expression during periods when proneurotrophin-induced apoptosis occurs. NRH2 interacts with both p75NTR and sortilin, facilitating the formation of a p75NTR and sortilin complex. This is achieved by a new mechanism, by which NRH2 acts as a trafficking switch to impair lysosome-dependant sortilin degradation and to redistribute sortilin to the cell surface. These results identify a new process that regulates neuronal responsiveness to pro-apoptotic ligands, in which NRH2 regulates sortilin expression and directs cell surface localization to promote p75NTR–sortilin receptor complex formation and proneurotrophin-induced cell death. Results NRH2 interacts with sortilin as well as p75NTR To assess a potential role for NRH2 in regulating proneurotrophin signalling, we first examined whether NRH2 co-localizes with the proneurotrophin receptors, p75NTR and sortilin in vivo. To this end, immunofluorescence microscopy was carried out using tissues where NRH2 and/or p75NTR are known to be expressed (Kanning et al, 2003; Murray et al, 2004). We detected prominent NRH2 immunoreactivity throughout the dorsal root ganglion (DRG) of P1 mice (Figure 1A, a and b), and co-immunostaining with sortilin and NRH2 antibodies showed that most DRG neurons express both NRH2 and sortilin (Figure 1A, a). In contrast, p75NTR is expressed by a subpopulation of neurons that also express both NRH2 and sortilin (Figure 1A, b and c). Co-expression of NRH2 with sortilin or p75NTR is also observed in a subset of spinal motor neurons of P1 mice (Supplementary Figure S1) and in cultured rat superior cervical ganglion (SCG) or DRG neurons (data not shown). Strong co-immunoreactivity of sortilin and NRH2 is also detected in the ganglion cell layer (GCL) of E15.5 mouse retina (Supplementary Figure S2B) where proNGF is expressed and p75NTR-dependant developmentally regulated apoptosis is robust (Frade and Barde, 1999; Harada et al, 2006; Nakamura et al, 2007). However, NRH2 and sortilin immunoreactivity is markedly reduced in the GCL at a later developmental time when apoptosis is not apparent (Supplementary Figure S2C). To determine if NRH2 interacts with p75NTR and sortilin in vivo, we immunoprecipitated p75NTR or sortilin from rat brain, DRG or spinal cord lysates and observed that NRH2 interacts with p75NTR (Figure 1B and Supplementary Figure S3), consistent with earlier studies of overexpression in HEK-293 cells (Frankowski et al, 2002). Interestingly, we could also detect a new interaction of NRH2 with sortilin in brain lysates (Figure 1B and Supplementary Figure S3). Collectively, these results suggest that NRH2 is expressed by p75NTR- and sortilin-expressing, and proneurotrophin-responsive neurons, and that NRH2 interacts with both p75NTR and sortilin in vivo. Figure 1.NRH2 is co-expressed and interacts with the proneurotrophin receptors, sortilin and p75NTR. (A) Co-expression of NRH2, sortilin and p75NTR in a subpopulation of dorsal root ganglion (DRG) neurons. DRG sections from C57/BL6 mice (P1) were double-immunostained using anti-NRH2 (1074), anti-sortilin (BAF2934) or anti-p75NTR antibodies (BAF1157 or 9992). Scale bars, 20 μm. (B) Endogenous interaction of NRH2 with sortilin or p75NTR was assessed by immunoprecipitation of rat brain lysates from E17 animals with the indicated antibody, followed by probing with anti-NRH2 (juxtamembrane (JM)), anti-sortilin or anti-p75NTR antisera as noted. Lysate, 40 μg. Download figure Download PowerPoint NRH2 enhances p75NTR and sortilin association To identify a potential role for NRH2 in regulating the formation of p75NTR and sortilin receptor complexes, we expressed NRH2, p75NTR, and sortilin in heterologous cells and examined the molecular interactions among them. Immunoprecipitation of NRH2 followed by western blot analysis confirmed interactions of NRH2 with both sortilin and p75NTR (Figure 2A). Surprisingly, we found that the interaction between p75NTR and sortilin is significantly increased when NRH2 is co-expressed (Figure 2B). Quantification of western blots indicated that NRH2 expression induces a 3.2-fold increase in the association of p75NTR with sortilin (Figure 2E, n=3), suggesting that NRH2 positively regulates p75NTR–sortilin association by interacting with sortilin and/or p75NTR. Figure 2.NRH2 promotes the interaction of p75NTR and sortilin. (A) HEK 293 cells were transfected with plasmids encoding sortilin (2 μg), HA–p75NTR (2 μg) or FLAG–NRH2 (2 μg) as indicated, and the association of NRH2 with p75NTR or sortilin was determined by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis. Lysates, 10 μg per lane. (B) HEK 293 cells were transfected with 2 μg of sortilin, HA–p75NTR or FLAG–NRH2, and cell lysates were immunoprecipitated with anti-HA antibody followed by immunoblot analysis. Lysates, 10 μg per lane. (C) Quantitation of western blots for the NRH2 and sortilin association represented in (A), lanes 2 and 3. To determine the relative NRH2–sortilin interaction, sortilin bands co-immunoprecipitated with NRH2 were normalized by the band intensities both in lysates and NRH2 immunoprecipitates, and values in the absence and presence of p75NTR expression were compared (mean±s.e.m., n=3). (D) Quantitation of western blots for the p75NTR and NRH2 association represented in (A), lanes 3 and 4. Values were normalized as described above (n=3). (E) Quantitation of western blots for the sortilin and p75NTR association represented in (B), lanes 2 and 3. Values were normalized as described above (n=3). Download figure Download PowerPoint To determine the domains of NRH2 that selectively promote interaction with p75NTR or sortilin, we generated C-terminal serially deleted NRH2 constructs lacking the entire or part of the intracellular domain (FLAG–NRH2–ΔCT, FLAG–NRH2–ΔDD and FLAG–NRH2–ΔICD; Figure 3A). Deletion of a part or the entire NRH2 death domain markedly reduces its association with p75NTR (Figure 3B and data not shown). Deletion of part of the NRH2 death domain (NRH2–ΔCT) does not impair association with sortilin. However, further deletions in the cytoplasmic domain of NRH2 (NRH2–ΔDD and NRH2–ΔICD) progressively decrease its interaction with sortilin (Figure 3C and Supplementary Figure S4). To test whether the intracellular domain of sortilin is required for this interaction, a deletion mutant of sortilin lacking the cytoplasmic tail (Myc–Sort–ΔICD, Figure 3D) was coexpressed with NRH2. Deletion of the sortilin ICD prevented association with NRH2 (Figure 3E), suggesting that the juxtamembrane region of NRH2 and the sortilin cytoplasmic domain promote their association, whereas the death domain of NRH2 is required for interaction with p75NTR (Figure 3F). Figure 3.Association of NRH2 with p75NTR and sortilin. (A) Diagrammatic representation of full-length or truncated NRH2 constructs. Sequence similarity between murine p75NTR and murine NRH2 is shown as a % identity for each domain. (B) Death domain of NRH2 is necessary for the interaction with p75NTR. HEK 293 cells were co-transfected with HA–p75NTR and FLAG–NRH2–FL or FLAG–NRH2–ΔCT, and cell lysates were subjected to immunoprecipitation of p75NTR. (C) The juxtamembrane domain of NRH2 promotes sortilin binding. Full-length or truncated NRH2 s were co-expressed with sortilin in HEK 293 cells and their associations were assessed by co-immunoprecipitation with NRH2. Asterisks indicate immaturely N-glycosylated NRH2 intermediates verified by N-glycanase reaction (data not shown). (D) Diagrammatic representation of Myc-tagged full-length and truncated sortilin constructs. (E) ICD deletion of sortilin shows impaired NRH2 binding, as assessed by co-immunoprecipitation of NRH2 in HEK 293 cells. (F) Summary of molecular interactions among p75NTR, NRH2 and sortilin. NRH2-DD is required to interact with p75NTR, whereas NRH2-JM and sortilin-ICD promote their association. Download figure Download PowerPoint NRH2 selectively increases cell surface expression of sortilin Recent studies suggest that cell surface expression of sortilin is dynamically regulated during development and correlates with cell apoptosis (Nakamura et al, 2007). Therefore, we speculated that NRH2 might regulate the cell surface expression of sortilin to enhance p75NTR and sortilin co-localization. Using HT-1080 cells that stably express both p75NTR and sortilin (HT-1080P/S), we assessed cellular localization of sortilin and p75NTR in the presence or absence of NRH2 using three complementary techniques. First, using double immunofluorescence microscopy of membrane-permeabilized cells lacking NRH2, sortilin was detected predominantly in the perinuclear region of the cells, reflecting its known localization to the Golgi and ER membranes (verified by co-immunostaining with anti-Golgi-Zone antibody and ER stain, data not shown) and endosomes, whereas p75NTR was detected at the leading edge or plasma membrane of cells as well as in the perinuclear region (Figure 4A). Merged images indicate no obvious co-localization of p75NTR and sortilin at the plasma membrane. However, distinctive and prominent sortilin immunoreactivitiy at the edge of cells was detected in NRH2-coexpressing cells (Figure 4B, arrowheads). To directly test whether NRH2 increases cell surface expression of sortilin, immunofluorescence microscopy was carried out in a membrane non-permeabilized condition (Figure 4D–H and Supplementary Figure S5). We detected a significant increase of sortilin immunoreactivity on the cell surface in NRH2-coexpressing cells (Figure 4D, arrowhead), which was not observed neither in NRH2-nonexpressing (Figure 4D, arrows), nor in GFP-expressing control cells (Figure 4E). Quantification of images indicates a 2.7-fold increase of sortilin immunoreactivity on the cell surface upon NRH2 co-expression as compared with the GFP-expressing cells (Figure 4H, *P<0.05, n=4). By contrast, NRH2 does not alter p75NTR cell surface expression, as comparable levels of p75NTR immunoreactivity are detected in cells with and without NRH2 expression (Figure 4C, F and G). Lastly, cell surface biotinylation of HT-1080P/S cells, transfected with NRH2 or control vector (CTL), corroborate these results that NRH2 expression increases cell surface expression of sortilin (2.1±0.25-fold increase, n=4) but not p75NTR (Figure 4I). Given the modest extent of sequence similarity between NRH2 and p75NTR, we asked whether targeting of sortilin to the cell surface is selectively promoted by NRH2. Unlike NRH2, p75NTR expression in sortilin-expressing cells (HT-1080S) fails to increase the level of sortilin detected on the cell surface (Figure 4J). To further determine whether the association of sortilin with NRH2 is required for sortilin relocalization, we carried out cell surface biotinylation of HT-1080S cells expressing full-length or deletion mutants of NRH2 (Figure 4K). Expression of NRH2–ΔICD, which is impaired in sortilin binding (Figure 3C and Supplementary Figure S4), does not increase cell-surface expression levels of sortilin, whereas sortilin-interacting NRH2–FL and NRH2–ΔCT do (Figure 4K), suggesting that sortilin is selectively redistributed to the plasma membrane on its association with NRH2. Figure 4.NRH2 selectively relocalizes sortilin to the plasma membrane. (A) Immunofluorescence detection of p75NTR and sortilin in membrane permeabilized HT-1080P/S cells. Scale bar, 20 μm. (B, C) NRH2 redistributes sortilin but not p75NTR. HT-1080P/S cells were transfected with FLAG–NRH2, and the effect of NRH2 expression on sortilin (B) or p75NTR localization (C) was monitored by immunofluorescence microscopy in membrane-permeabilized cells. Arrowheads and arrows indicate NRH2-expressing and non-expressing cells, respectively. Insets, higher magnification of co-localization of NRH2 and sortilin at the leading edge of the cell. Scale bars, 20 μm. (D, E) Cell surface expression of sortilin is markedly increased by NRH2 co-expression. HT-1080P/S cells were transfected with FLAG–NRH2 (D) or GFP (E), and sortilin expression on the cell surface was monitored by immunofluorescence microscopy in a membrane non-permeabilized condition. (F, G) NRH2 does not alter p75NTR expression on the cell surface. Immunoreactivity of p75NTR in FLAG–NRH2 (F) or GFP-transfected HT-1080P/S cells (G) was visualized by immunoflurescence microscopy in a membrane non-permeabilized condition. Arrowheads indicate NRH2 or GFP-expressing cells, and arrows indicate non-transfected reference cells. (H) Quantification of p75NTR or sortilin immunoreactivity represented in (D)–(G). Locations containing both transfected and non-transfected cells were chosen and staining intensity of sortilin or p75NTR in NRH2 (+) or GFP (+) cells was normalized by corresponding intensity in NRH2 (−) or GFP (−) cells in the same picture (mean±s.e.m., n=4, *P<0.05). (I) HT-1080P/S cells were transfected with control vector or NRH2 encoding vector, followed by cell surface biotinylation and streptavidin pull-down of cell lysates, showing that NRH2 increases cell surface expression of sortilin but not p75NTR. (J) NRH2 but not p75NTR increases sortilin expression on the cell surface assessed by cell surface biotinylation in transfected HT-1080S. (K) NRH2–ΔICD mutant, which does not interact with sortilin, fails to increase cell surface expression of sortilin in HT-1080S cells. Download figure Download PowerPoint Cell surface expression of sortilin is crucially regulated by endogenous NRH2 in neurons To determine whether endogenous NRH2 in neurons regulates sortilin localization, we delivered short hairpin RNA (shRNA) targeting a sequence in the transmembrane domain of rat NRH2. As DRG neurons express high levels of NRH2, sortilin, and p75NTR (Supplementary Figure S6), we infected DRG neurons with shNRH2-expressing lentivirus (shNRH2). This reduced NRH2 expression by approximately 75%, as compared with the control lentivirus (CTL)-infected cells (Figure 5A and B). Unexpectedly, overall sortilin levels are significantly reduced in NRH2-silenced DRG neurons (69.0±7.2% of control, n=6, P<0.01) (Figure 5A and B). To verify that the reduction in sortilin expression in DRG neurons is mediated by NRH2, we co-expressed an shNRH2-resistant, FLAG-tagged murine NRH2, using the shNRH2-expressing lentiviral vector (shNRH2/FLAG–NRH2R). The reduction in sortilin expression induced by NRH2 knockdown is effectively rescued by co-expression of FLAG–NRH2R (Figure 5C), indicating that the level of sortilin expression is selectively regulated by NRH2, rather than an off target effect of shRNA. This conclusion is also supported by comparable expression levels of other proteins including p75NTR, Trk, and β-actin after silencing or restoring of NRH2 expression in DRG neurons (Figure 5A–C). Figure 5.Sortilin expression levels are selectively reduced in NRH2-depleted DRG neurons. (A) DRG neurons infected with CTL or shNRH2-expressing lentivirus were harvested at DIV7, and expression of NRH2, p75NTR, sortilin or Trk was examined by immunoblot analysis. (B) Quantitation of western blots represented in (A). Mean proportions±s.e.m. were determined from six independently carried out experiments at DIV7-9, **P<0.01. (C) Sortilin levels are rescued upon restoring NRH2 expression. Dissociated DRG cultures were infected with lentiviruses (CTL, shNRH2, or shNRH2/FLAG–NRH2R), and western blot analysis was carried out at DIV7. Bottom, NRH2 Western blot using anti-NRH2 (1074) antibody for the lysates represented in lanes 1, 3 and 5 of upper panels. Download figure Download PowerPoint To assess whether NRH2 regulates sortilin expression on the neuronal cell surface, CTL or shNRH2 expressing DRG neurons were biotinylated, and surface proteins were detected by streptavidin-pull down and western blot analysis (Figure 6A). On acute silencing of NRH2, we observed a reduction of sortilin expression on the cell surface (46.3% reduction compared with control, n=3, **P<0.01) that was more pronounced than the total decrease in sortilin expression (24.4% decrease compared with control, n=3, *P<0.05) (Figure 6B). In addition, augmentation of NRH2 expression in DRG neurons (Figure 6C) or cortical neurons (Figure 6D), where NRH2 is expressed at low levels (Supplementary Figure S5), significantly increases the level of surface-expressed sortilin (2.0±0.32-fold, n=3) with a moderate increase in total sortilin (1.3±0.05-fold, n=3) in cortical neurons (Figure 6E). To determine whether endogenous NRH2 in DRG neurons preferentially regulates cell surface localization of sortilin, DRG neurons were infected with CTL or shNRH2-expressing lentiviruses and immunofluorescence microscopy was carried out in both membrane non-permeabilized (Figure 6F) and permeabilized conditions (Figure 6G), and staining intensity present on the cell surface and in total was compared. We detected a significant decrease of sortilin immunoreactivity in membrane non-permeabilized, NRH2-silenced cells (60% reduction compared with control, **P<0.01, n=5; Figure 6F arrowheads and Figure 6H). In contrast, only a modest decrease was observed in membrane permeabilized, NRH2-silenced cells (22% reduction compared with control, n=4; Figure 6G arrowheads and Figure 6H). Collectively, these studies indicate that NRH2 critically regulates the cell surface localization of endogenous sortilin in two classes of neurons. Figure 6.NRH2 preferentially regulates cell surface expression of sortilin in primary neurons. (A) Reduced cell surface expression of sortilin in NRH2-silenced DRG neurons. Control or NRH2-silenced DRG neurons were biotinylated at DIV7-8, and cellular lysates (300 μg) were subjected to streptavidin-precipitation (surface). Lysates (15 μg) harvested before and after precipitation represented as 'total' and 'intracellular' fractions, respectively. (B) Quantitation of sortilin immunoreactive bands represented in (A). n=3, mean±s.e.m., *P<0.05 and **P<0.01. (C) Control, NRH2-silenced (shNRH2) or NRH2-overexpressed (FLAG–NRH2) DRG neurons were subjected to cell surface biotinylation at DIV7 followed by western blot analysis. (D) Augmentation of NRH2 expression in rat cortical neurons increases sortilin levels on the cell surface, assessed by cell surface biotinylation (DIV7-8). (E) Quantitation of sortilin levels represented in (D). n=3, *P<0.05. (F) DRG cultures infected with CTL (a, c) or shNRH2-expressing lentiviruses (b) were subjected to immunocytochemical staining with anti-sortilin antibody (a, b) or control IgG (c) in a membrane non-permeabilized condition, and surface expression of sortilin in the neuronal cell body was visualized by confocal microscopy. Arrowheads indicate CTL or shNRH2-expressing cells identified by GFP expression, and arrows indicate GFP-negative non-infected cells. Representative images are shown. Scale bars, 10 μm. (G) Total sortilin expression in CTL (a) or NRH2-depleted (b) DRG neuronal cell body was visualized by immunofluorescence confocal microscopy carried out after membrane permeabilization. Scale bars, 10 μm. (H) Quantification of sortilin immunoreactivity represented in (F) and (G). Staining intensity of GFP-positive cells was normalized by the intensity of GFP-negative cells in the same field. Mean±s.e.m. was obtained from four (total) or five (surface) independently conducted experiments, **P<0.01. Download figure Download PowerPoint NRH2 regulates sortilin expression in neurons by altering sortilin degradation As acute silencing of endogenous NRH2 leads to a moderate reduction in total sortilin levels, we carried out pulse-chase experiments to determine whether sortilin degradation was affected. In control neurons, sortilin levels fall significantly at 4 and 6 h of chase conditions (52 and 65% reduction compared with 0 hr of chase). In contrast, in neurons overexpressing NRH2, sortilin levels at these time points are largely maintained (20 and 30% reduction, *P<0.05 and **P<0.01, n=4) (Figure 7A and B), suggesting that NRH2 expression stabilizes sortilin by attenuating its degradation. Recent studies suggest that sorting nexin-1 (SNX-1), a component of retromer, interacts with the sortilin cytoplasmic tail and facilitates endosome–TGN transport of sortilin. Depletion of SNX-1 decreases the sortilin pool in the TGN and increases its lysosomal degradation (Canuel et al, 2008; Mari et al, 2008). Thus, we postulated that the interaction of NRH2 with sortilin might impair the trafficking of sortilin to the lysosome for degradation. To test this hypothesis, DRG neurons infected with CTL or shNRH2-expressing lentiviruses were incubated with lysosomal or proteosomal inhibitors, and sortilin levels in cell lysates were analysed by western blot analysis. Treatment with the lysosomal inhibitor leupeptin restored sortilin levels in NRH2-depleted cells to levels close to those observed in control conditions (98.3±2.6%, **P<0.01, n=3) (Figure 7C and D). However, proteosomal inhibitors, including epoxomycin and MG-132 did not significantly augment sortilin protein levels in NRH2-silenced neurons (Figure 7C and data not shown), demonstrating that a primary effect of NRH2 is to interfere with sortilin degradation in the lysosomal dependant pathway. To determine whether lysosomal inhibition also restores sortilin surface expression in NRH2-silenced cells, control or NRH2-silenced DRG neurons were treated with leupeptin or not, followed by cell surface biotinylation (Figure 7E). Leupeptin treatment rescues total levels of sortilin but not the surface-expressed sortilin in NRH2-depleted neurons (Figure 7E). These studies suggest that enhanced cell surface localization of sortilin is not a direct consequence of increased sortilin stability, and that association of sortilin with NRH2 may be necessary for this process. Figure 7.NRH2 regulates sortilin expression by altering lysosomal degradation of sortilin. (A) Cortical neurons were electroporated (Amaxa) with control or FLAG–NRH2 encoding plasmids and subjected to pulse-chase [35S]Cys/Met labelling at DIV5. Biosynthetically labelled sortilin was immunoprecipitated from cell lysates (300 μg), harvested at the indicated times, followed by autoradiography. (B) Quantitation of autoradiographs in (A). The levels of endogenous biosynthetically labelled sortilin are significantly higher after 4 and 6 h of chase upon NRH2 overexpression (*P<0.05 and **P<0.01, n=4). Inset, NRH2 expression levels in control or FLAG–NRH2-transfected cortical neurons, verified by immunoblotting with NRH2 antibody (1074). (C) Control or NRH2-silenced DRG neurons were treated with vehicle (0.01% DMSO), leupeptin (50 μM) or epoxomycin (1 μM) for 20 h at 37°C (DIV7–8), and subjected to western blot analys
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