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PLoS Pathog. 2017 June; 13(6): e1006470.
Published online 2017 June 30. doi:  10.1371/journal.ppat.1006470
PMCID: PMC5509376

Prions amplify through degradation of the VPS10P sorting receptor sortilin

Keiji Uchiyama, Conceptualization, Formal analysis, Funding acquisition, Investigation, Visualization, Writing – original draft,1,* Mitsuru Tomita, Investigation,1,2 Masashi Yano, Investigation,1 Junji Chida, Investigation,1 Hideyuki Hara, Investigation,1 Nandita Rani Das, Investigation,1 Anders Nykjaer, Resources,3 and Suehiro Sakaguchi, Conceptualization, Funding acquisition, Supervision, Writing – original draft1,*
Neil A. Mabbott, Editor

Abstract

Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.

Author summary

Once prions consisting mainly of PrPSc infect hosts, they constitutively propagate in their brains. Progressive production of PrPSc through the constitutive conformational conversion of PrPC into PrPSc underlies prion propagation. However, the mechanism enabling progressive production of PrPSc in prion-infected cells remains unknown. We here found that the VPS10P sorting receptor sortilin is involved in degradation of PrPC and PrPSc in infected cells by binding to both molecules and subsequently trafficking them to the lysosomal protein degradation pathway. Interestingly, we also found that degradation of sortilin was stimulated in lysosomes in prion-infected cells possibly as a result of the sortilin-PrPC or -PrPSc complexes being trafficked to lysosomes. Our findings indicate that PrPSc itself impairs the sortilin-mediated degradation of PrPC and PrPSc by stimulating degradation of sortilin in lysosomes. This eventually results in progressive production of PrPSc in prion-infected cells by increasing the opportunity of PrPC to convert into PrPSc and by accumulating the already produced PrPSc. This mechanism was confirmed in sortilin-KO mice infected with prions. The mice had exacerbated prion disease with earlier accumulation of PrPSc in their brains.

Introduction

Prion diseases are a group of fatal neurodegenerative disorders, which include Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy and scrapie in animals [1]. They are caused by the infectious agents termed prions, which mainly consist of the abnormally folded, amyloidogenic isoform of prion protein, designated PrPSc. PrPSc is a β-sheet-rich conformer produced by conformational conversion of the cellular counterpart, PrPC [1]. Intermolecular interaction between PrPC and PrPSc is essential for the conversion of PrPC into PrPSc. We and others have shown that mice devoid of PrPC neither developed the disease nor accumulated PrPSc even after prions were inoculated into their brains [25]. These results indicate that the conversion of PrPC into PrPSc plays a pivotal role in the pathogenesis of prion disease, and that depletion of PrPC could be therapeutic by preventing the production of PrPSc.

PrPC is normally located at the cell surface as a glycosylphosphatidylinositol (GPI)-anchored glycoprotein [6]. Some endocytosed PrPC molecules are transported to lysosomes for degradation while others are recycled to the cell surface through the endocytic recycling compartments [7]. PrPSc is also trafficked to lysosomes for degradation [7]. However, the cellular transport mechanism of PrPC and PrPSc to lysosomes remains unknown. Whether prion infection or PrPSc impairs the lysosomal trafficking of PrPC and PrPSc for its progressive propagation is also unknown.

The vacuolar protein sorting-10 protein (VPS10P)-domain receptors, including sortilin, SorLA, SorCS1, SorCS2 and SorCS3, are multi-ligand type I transmembrane proteins abundantly expressed in the brain and involved in neuronal function and viability [8,9]. They function as a cargo receptor to deliver a number of cargo proteins to their subcellular destination through the VPS10P domain in the extracellular luminal N-terminus. Sortilin traffics the amyloid precursor protein (APP)-cleaving enzyme BACE1 [10] and the neurotrophic factor receptors Trks [11]. SorLA directs trafficking of APP into the recycling pathway [12]. SorCS1 also mediates APP transport [13]. Recent lines of evidence indicate that the altered VPS10P receptor-mediated trafficking could be involved in the pathogenesis of neurodegenerative disorders, including Alzheimer’s disease (AD) [1215] and frontotemporal lobar degeneration (FTLD) [16]. However, the role of VPS10P receptors in the trafficking of PrPC or PrPSc and in the pathogenesis of prion disease is little known.

In the present study, we show that sortilin has an inhibitory role in PrPSc accumulation by sorting PrPC and PrPSc to lysosomes for degradation. Interestingly, however, prion infection stimulates lysosomal degradation of sortilin, indicating that prion infection itself could disturb the inhibitory function of sortilin. We also confirm that sortilin-knockout (KO) mice have accelerated prion disease after infection with RML prions, with early accumulation of PrPSc in their brains. These results suggest that PrPSc accumulation may be amplified through PrPSc-induced impairment of the sortilin-mediated lysosomal degradation of PrPC and PrPSc.

Results

Sortilin is a novel PrPC-binding protein regulating the surface levels of PrPC

To investigate the role of VPS10P cargo receptors in the trafficking of PrPC, we first examined whether or not VPS10P molecules could interact with PrPC. Co-immunoprecipitation assay in neuroblastoma N2aC24 cells showed that SAF61 anti-PrP antibody (Ab) precipitated PrPC with sortilin, but not with other VPS10P molecules (Fig 1A, S1 Fig). PrPC was also co-precipitated with sortilin by anti-sortilin Abs (Fig 1B). GST-pulldown assay using purified recombinant proteins revealed that the VPS10P domain of sortilin fused with GST (GST-VPS10P) successfully pulled down His-tagged full-length recombinant PrP, but not PrP with a deletion of 23–88 residues (Fig 1C), suggesting that the residues 23–88 are important for PrPC to interact with sortilin. SAF61 anti-PrP Ab also co-precipitated full-length mycHis-tagged sortilin expressed in sortilin-KO N2aC24 cells, designated ΔSort#1 cells, but not in PrP-KO N2a cells, N2aΔPrP#1 cells (S2A and S2B Fig). Both types of KO cells were established using the CRISPR-Cas genome editing system. This clearly indicates that PrPC expression is required for sortilin to be co-precipitated by SAF61 anti-PrP Ab, further supporting the interaction of sortilin and PrPC. However, sortilin lacking residues 610–753 was not efficiently co-precipitated with the Ab, compared to other deletion mutants of sortilin (S2A and S2B Fig), suggesting that the residues 610–753 of sortilin are involved in interaction with PrPC. Furthermore, co-immunoprecipitation assay using mouse brain homogenates also revealed an interaction between PrPC and sortilin (Fig 1D). Immunofluorescence staining of non-permeabilized N2aC24 cells showed co-localization of sortilin and PrPC on the cell surface (Fig 1E and 1F; S3 Fig). Intracellular co-localization of sortilin and PrPC was also observed in permeabilized cells (Fig 1E and 1F; S3 Fig).

Fig 1
PrPC directly interacts with sortilin on the cell surface and inside cells.

To further investigate PrPC interaction with sortilin on the cell surface, we labeled PrPC on the cell surface of N2aC24 cells with SAF61 anti-PrP Ab, lysed the cells, and incubated the lysate with protein-G-conjugated Dynabeads (S4A Fig). The Ab-labeled PrPC complexes were collected using magnet (S4A Fig). N2aΔPrP#1 cells were used as negative control (S4A Fig). Sortilin was co-collected with PrPC from N2aC24 cells, but not from N2aΔPrP#1 cells (S4B Fig). Sortilin was similarly expressed in N2aC24 and N2aΔPrP#1 cells (S4C Fig). These results further support PrPC interacting with sortilin on the cell surface.

We then knocked down sortilin in N2aC24 cells using two sortilin-specific siRNAs, termed siRNA#1 and #2. Immunostaining of sortilin-knockdown (Sort-KD) cells showed an increase in PrPC expression on the cell surface (Fig 2A). Biotin labeling of surface proteins confirmed the increased surface levels of PrPC in Sort-KD cells (Fig 2B and 2C). Total PrPC levels were also increased in Sort-KD cells (Fig 2B and 2C). However, intracellular PrPC was not increased in Sort-KD cells (Fig 2D), indicating that the surface PrPC is specifically increased in Sort-KD cells. PrP mRNA levels were not increased in Sort-KD cells (Fig 2E), suggesting that the increased surface expression of PrPC might be attributable to the impaired degradation of PrPC in Sort-KD cells. PrPC levels were also significantly increased in the brains of sortilin-KO (Sort1-/-) mice compared to those in wild-type (WT) mice (S5A and S5B Fig).

Fig 2
Knockdown of sortilin increases PrPC on the cell surface.

PrPC undergoes an endopeptidic cleavage by the ADAM family of metalloproteases, with the C-terminal fragment, designated the C1 fragment, being produced [17,18]. Sort-KD cells produced the C1 fragment more abundantly than N2aC24 cells (S6A Fig). This is probably because PrPC was increased on the cell surface in Sort-KD cells. We also investigated PrPC levels in exosomes of N2aC24 and sortilin-KO ΔSort#1 and ΔSort#2 cells. ΔSort#1 and ΔSort#2 cells also showed an increase in total PrPC levels (S6B Fig). PrPC was significantly higher in exosomes from ΔSort#1 and #2 cells than in those from N2aC24 cells (S6B and S6C Fig). Exosomes were verified by the presence of exosome-specific molecules TSG101 and flotillin and the absence of GM130 and Bcl-2, both of which are not normally included in exosomes (S6B Fig) [19,20].

Sortilin sorts surface PrPC to late endosomes/lysosomes

To address whether or not sortilin could sort surface PrPC to lysosomes for degradation, we first investigated the role of sortilin in internalization of surface PrPC using an Ab-labeling technique. Surface PrPC was labeled with SAF61 anti-PrP Ab at 4°C in which internalization of membrane proteins is inhibited, and then allowed to be internalized for 2 h at 37°C. The labeled PrPC was then detected using Alexa Fluoro 488-conjugated anti-mouse IgG Abs. The internalization of the labeled PrPC was slightly but significantly inhibited in Sort-KD cells, compared to that in control N2aC24 cells (Fig 3A and 3B), suggesting that sortilin could be involved in internalization of some portions of PrPC. To further confirm the involvement of sortilin in internalization of PrPC, we biotinylated the surface proteins of N2aC24 cells and sortilin-KO (ΔSort) cells #1 with sulfo-NHS-SS-biotin, whose biotin motif can be removed by reducing agents. We then allowed the biotinylated proteins to be internalized for 2 h, and treated the cells with the membrane-impermeable reducing agent glutathione to remove the biotins only from surface proteins but not from those internalized. The treated cells were lysed, and then biotin-labeled, internalized proteins were purified using avidin-beads, and investigated for internalized PrPC by Western blotting with 6D11 anti-PrP Ab. Strong signals corresponding to the internalized PrPC were detected in N2aC24 cells (Fig 3C and 3D). However, the signals were significantly reduced in ΔSort#1 cells (Fig 3C and 3D). These results reinforce the role of sortilin in internalization of PrPC.

Fig 3
Sortilin is an endocytic receptor for PrPC to late endosomes.

To track the SAF61 anti-PrP Ab-labeled, internalized PrPC in N2aC24 and Sort-KD cells, we immunofluorescently stained both types of cells for internalized PrPC with the late endosome marker Rab9 or the recycling endosome marker Rab11. The labeled PrPC was normally internalized to both late endosomes and recycling endosomes, as observed in N2aC24 cells (Fig 3E and 3F; S7A–S7D Fig). However, in Sort-KD cells, localization of PrPC was markedly shifted from the late endosomes (Fig 3E; S7A and S7B Fig) to the recycling endosomes (Fig 3F; S7C and S7D Fig). These results indicate that internalization of PrPC to the recycling endosomes could be independent of sortilin. Furthermore, sortilin could function to sort surface PrPC to the late endosome/lysosome degradation pathway, thereby regulating levels of surface PrPC. Consistent with the results from Sort-KD cells, sortilin-KO ΔSort#1 cells showed higher expression of PrPC than control N2aC24 cells (Fig 3G and 3H). Inhibition of lysosomal enzymes by NH4Cl increased PrPC markedly in N2aC24 cells, but only slightly in ΔSort#1 cells (Fig 3G and 3H). PrPC was detected in the LAMP1-positive lysosomes in both cell types after NH4Cl treatment (Fig 3I and 3J). However, its lysosomal localization was much less in ΔSort#1 cells than in N2aC24 cells (Fig 3I and 3J). These results confirm that transport of PrPC to lysosomes is disturbed in sortilin-deficient cells, therefore reducing the localization of PrPC in lysosomes and resulting in an increase in PrPC levels in sortilin-deficient cells.

We also investigated localization of PrPC in early endosomes in N2aC24 and Sort-KD cells. PrPC was labeled with SAF61 Ab and spontaneously internalized for 1 h instead of 2 h, which was utilized for detection of internalized PrPC in late endosomes or recycling endosomes, because PrPC may be only transiently located in early endosomes before being trafficked to late endosomes/lysosomes or recycling endosomes. Indeed, only a very small fraction of PrPC was detected in the EAA1-positive early endosomes in N2aC24 cells (S7E and S7F Fig). PrPC was slightly, but not significantly, less localized in early endosomes in Sort-KD cells than in N2aC24 cells (S7E and S7F Fig). This might be consistent with the sortilin-independent trafficking of PrPC being active through early endosomes.

We also investigated the internalization of PrP with a deletion of 23–88 residues, PrPΔ23–88, to lysosomes by establishing PrP-KO N2aΔPrP cells permanently expressing wild-type (WT) mouse PrPC (WT#1 and #2) or PrPΔ23–88 (Δ23–88#1 and #2). Western blotting showed that PrPΔ23–88 was expressed at higher levels than WT PrPC without NH4Cl treatment (S8A and S8B Fig), suggesting that PrPΔ23–88 might be less degraded than WT PrPC. NH4Cl treatment only slightly increased PrPΔ23–88 in Δ23–88#1 and #2 cells, but markedly increased WT PrPC in WT#1 and #2 cells (S8A and S8B Fig). NH4Cl treatment also only slightly increased PrPΔ23–88 in the LAMP1-positive lysosomes in Δ23–88#1 and #2 cells, but markedly increased WT PrPC in the lysosomes in WT#1 and #2 cells (S8C and S8D Fig). These results suggest that residues 23–88 could be involved in trafficking of PrPC to lysosomes for degradation. Sortilin expression was unaffected in Δ23–88#1 and #2 cells (S8E and S8F Fig). No interaction between sortilin and PrPΔ23–88 was detected (S8G Fig). It is thus conceivable that the interaction with sortilin could be important for PrPC transportation to lysosomes for degradation. However, the increased localization of PrPΔ23–88 in lysosomes after NH4Cl treatment in Δ23–88#1 and #2 cells suggests that other molecules are also involved in trafficking of PrPC to lysosomes.

PrPC shifts to raft domains in sortilin-KO cells

To gain insight into the mechanism of sortilin-mediated sorting of PrPC to lysosomes, we investigated membrane microdomain localization of PrPC in N2aC24 and sortilin-KO N2aC24 cells, ΔSort#1 and ΔSort#2 cells, by detergent-based biochemical membrane fractionation. PrPC was detected in both detergent-resistant membrane (DRM) and detergent-soluble membrane fractions, that is raft and non-raft fractions, in N2aC24 cells, with higher amounts of PrPC in raft fractions (63.0%) than in non-raft fractions (37.0%) (Fig 4A and 4B). However, in ΔSort#1 and #2 cells, PrPC in non-raft fractions was reduced to 11.9 and 14.9%, respectively (Fig 4A and 4B). Instead, PrPC was increased in raft fractions (Fig 4A and 4B). These results suggest that sortilin could shift the localization of PrPC to non-raft domains from raft domains. Raft-resident protein flotillin-2 was observed in raft fractions in N2aC24 and ΔSort cells (Fig 4C), ruling out the possibility that lack of sortilin could affect the membrane microdomain integrity leading to the shift in the location of surface PrPC in ΔSort cells. Sortilin was predominantly detected in non-raft fractions in N2aC24 and ΔSort cells (Fig 4D), indicating that sortilin is a non-raft protein. We also performed the membrane fractionation assay for PrPC-expressing WT#1 and #2 cells and PrPΔ23-88-expressing Δ23–88#1 and #2 cells. PrPC was detected in raft and non-raft fractions in WT cells (S9A and S9B Fig). However, PrPΔ23–88 increased its localization at raft fractions (S9A and S9B Fig). These results indicate that residues 23–88 are important for PrPC to be retained at non-raft domains. We then investigated membrane microdomain localization of PrPC in the brains of Sort1-/- and WT mice. Localization of PrPC at raft and non-raft fractions was observed in WT brains (S9C and S9D Fig). However, PrPC was increased in raft fractions in Sort1-/- brains (S9C and S9D Fig). Taken together, these results suggest that sortilin might function to retain surface PrPC in non-raft domains and sort the non-raft PrPC to the late endosome/lysosome degradation pathway through interaction with residues 23–88 of PrPC.

Fig 4
Membrane microdomain distribution of PrPC is changed in sortilin-KO cells.

We also investigated membrane microdomain location of PrP molecules in prion-infected N2aC24L1-3 cells. In contrast to PrPC detected in raft and non-raft domains in uninfected N2aC24 cells (Fig 4A and 4B), total PrP molecules and PK-resistant PrPSc were predominantly detected in raft fractions in prion-infected N2aC24L1-3 cells (S10A and S10B Fig). These results suggest that prion infection accumulates PrPSc and PrPC in raft domains.

Sortilin interacts with PrPSc and facilitates its degradation

We also assessed the role of sortilin in the degradation of PrPSc. Protein interaction assay using protein-G-conjugated magnetic beads in prion-infected N2aC24L1-3 cells showed that PrPSc and sortilin were co-collected by anti-sortilin Abs (Fig 5A), suggesting that sortilin interacts with PrPSc. siRNA-mediated knockdown of sortilin increased PrPSc in N2aC24L1-3 cells (Fig 5B and 5C). In contrast, overexpression of sortilin reduced PrPSc in N2aC24L1-3 cells (Fig 5D and 5E). These results indicate that sortilin could also be involved in PrPSc degradation.

Fig 5
Sortilin regulates PrPSc levels in prion-infected cells.

We then evaluated the degradation kinetics of PrPSc in prion-infected cells with or without sortilin. For this study, it is important to prevent the de novo production of PrPSc from PrPC. PrP siRNA#1 and #2 reduced PrPC in N2aC24 and ΔSort#1 cells from 24 h after transfection, to less than 10% of that in control siRNA-transfected N2aC24 and ΔSort#1 cells (Fig 6A). These results indicate that the de novo production of PrPSc from PrPC could be negligible from 24 h after transfection with PrP siRNAs in these cells even after infection with prions. We thus investigated PrPSc in RML-infected N2aC24 (N2aC24/RML) and ΔSort#1 (ΔSort/RML) cells at 36, 48 and 60 h after transfection with PrP siRNAs. Control siRNA did not affect PrPSc levels in these cells (S11A and S11B Fig). However, PrPSc was decreased after transfection with PrP siRNAs (Fig 6B). In N2aC24/RML cells 60 h after transfection with PrP siRNAs, PrPSc was reduced to less than 20% of that in control siRNA-transfected N2aC24/RML cells (siRNA#1, 16.5%; siRNA#2, 17.1%) (Fig 6B and 6C). However, significantly higher levels of PrPSc were still observed in ΔSort/RML cells 60 h after transfection with PrP siRNAs (siRNA#1, 45.2%; siRNA#2, 38.5%) (Fig 6B and 6C). Similar results were obtained in 22L prion-infected N2aC24 and ΔSort#1 cells (S11C and S11D Fig). These results indicate that sortilin is also involved in the degradation of PrPSc.

Fig 6
PrPSc degradation is delayed in sortilin-knockdown cells infected with RML prions.

Prion infection cell-autonomously reduces sortilin after PrPSc production

We then asked if prion infection could affect sortilin. Interestingly, sortilin was reduced in N2aC24L1-3 cells to 52% of that in N2aC24 cells (Fig 7A and 7B). However, other VPS10P molecules were not reduced (S12A and S12B Fig). The reduced levels of sortilin were recovered in cured N2aC24L1-3 cells, which were cured from prion infection after treatment with SAF32 anti-PrP Ab, to that in N2aC24 cells (Fig 7A and 7B). Sortilin mRNA was similarly expressed between N2aC24 and N2aC24L1-3 cells (S12C Fig). ScN2a cells, N2a cells persistently infected with RML prions, also expressed sortilin less than N2a cells (S12D and S12E Fig). Moreover, sortilin was reduced in the brains of terminally ill mice infected with RML and 22L prions to 46.3 and 45.6%, respectively, compared to uninfected mouse brains (Fig 7C and 7D). These results indicate that prion infection could reduce sortilin.

Fig 7
Prion infection decreases sortilin in a cell-autonomous way.

We then treated uninfected N2aC24 and infected N2aC24L1-3 cells with inhibitors to lysosomes (NH4Cl and concanamycin A) or proteasomes (MG132). Treatment with NH4Cl or concanamycin A increased sortilin in uninfected cells (S13A–S13D Fig). However, sortilin was much more increased in infected cells after treatment with NH4Cl and concanamycin A (S13A–S13D Fig). In contrast, MG132 did not affect sortilin levels (S13A and S13B Fig). These results suggest that sortilin could be degraded in lysosomes, and that the lysosomal degradation of sortilin could be stimulated in prion-infected cells.

We also monitored the levels of sortilin in N2aC24 cells freshly infected with RML prions. There was no significant decrease in sortilin by 3 days post-infection (dpi) while PrPSc was obviously detectable (Fig 7E and 7F). Sortilin was decreased at 6 dpi (Fig 7E and 7F). These results suggest that prion infection reduces sortilin, and that the sortilin reduction is preceded by PrPSc production. We also performed double immunofluorescence staining for sortilin and PrPSc in freshly infected N2aC24 cells at 9 dpi. PrPSc was specifically stained using the mAb132, which was demonstrated to specifically visualize PrPSc under partially denatured conditions [21]. Since the subcellular positions of sortilin and PrPSc might differ vertically in infected cells, 6 horizontally serial images at 1 μm interval were used to detect sortilin and PrPSc. In cells displaying green fluorescence for PrPSc, little or no sortilin (red fluorescence) was detected in any slices (Fig 7G). In contrast, bright red fluorescence for sortilin was observed only in the cells negative for PrPSc (Fig 7G). These results indicate that prion infection could reduce sortilin in a cell-autonomous fashion after PrPSc accumulation.

Sortilin is known to interact with and transport Trk receptors to the cell surface, thereby enhancing nerve growth factor (NGF) signaling leading to activation of MAP kinases [11]. To investigate whether or not prion infection could affect the function of sortilin, we stimulated uninfected N2aC24 and prion-infected N2aC24L1-3 cells with NGF. Phosphorylated ERK1/2 was increased in N2aC24 and N2aC24L1-3 cells after stimulation (S14A–S14C Fig). However, the levels of phosphorylated ERK1/2 were significantly lower in N2aC24L1-3 cells than in N2aC24 cells (S14A–S14C Fig). These results indicate that NGF signaling is impaired in N2aC24L1-3 cells, suggesting that sortilin might be functionally disturbed in prion-infected cells.

Prion disease is aggravated in sortilin-KO mice after infection with prions

We then evaluated the effects of sortilin deficiency on the pathogenesis of prion disease using sortilin-KO (Sort1-/-) mice. Sort1-/- mice were viable and fertile and showed no gross abnormalities [11,22]. Sort1+/+ (n = 19) and Sort1-/- female mice (n = 24) were intracerebrally inoculated with RML prions. Incubation and survival times were significantly shortened in Sort1-/- mice (Fig 8A, S1 Table). Sort1-/- and Sort1+/+ mice developed symptoms at 150.9 ± 7.8 and 171.9 ± 6.0 dpi, respectively (Fig 8A, S1 Table). Western blotting also showed earlier accumulation of PrPSc in the brains of infected Sort1-/- mice. PrPSc was scarcely detectable in the brains of Sort1+/+ mice at 45 dpi (Fig 8B and 8C). However, it was obvious in Sort-/- mice at 45 dpi (Fig 8B and 8C). PrPSc levels were still significantly higher in Sort1-/- mice than in Sort1+/+ mice at 60 and 90 dpi (Fig 8B and 8C). However, no difference in the levels of PrPSc was observed between Sort1+/+ and Sort1-/- mice at terminal stage (Fig 8B and 8C). Immunohistochemical analysis of the brains of infected Sort1+/+ and Sort1-/- mice for PrPSc showed consistent results. PrPSc was detectable in a much larger area of the brains of Sort1-/- mice, compared to that in Sort1+/+ mice, at 60 dpi (Fig 8D). However, PrPSc became indistinguishably accumulated throughout the brains of Sort1-/- and Sort1+/+ mice at terminal stage (Fig 8D). Similar results were also obtained with Sort1-/- and Sort1+/+ male mice inoculated with RML prions (S15A–S15D Fig, S2 Table). These results show that sortilin deficiency accelerates prion disease by causing early accumulation of PrPSc in the brains of mice after infection with prions, reinforcing the inhibitory role of sortilin in the pathogenesis of prion disease.

Fig 8
Sortilin-KO mice have accelerated prion disease with earlier accumulation of PrPSc in their brains.

Discussion

In the present study, we showed that PrPSc accumulation could be enhanced through PrPSc-stimulated degradation of sortilin, a member of the VPS10P sorting receptor family. Sortilin functions as a negative regulator for PrPSc accumulation by sorting PrPC and PrPSc to the late endosome/lysosome protein degradation pathway. However, PrPSc stimulates sortilin to be degraded in lysosomes, thereby disturbing the inhibitory role of sortilin and eventually leading to the further accumulation of PrPSc.

PrPC is a GPI-anchored membrane protein located in raft domains and, to a lesser extent, in non-raft domains. Some of the PrPC molecules internalized are delivered back to the cell surface directly or indirectly via the recycling endosome compartments and others are transported to lysosomes for degradation [7] (Fig 9A). We showed that sortilin could directly interact with PrPC on the cell surface through the VPS10P domain through the residues 610–753 encompassing cysteine rich 10CCs [23] of sortilin and the N-terminal residues 23–88 of PrPC. Sortilin-knockdown increased PrPC on the cell surface and reduced the localization of PrPC to lysosomes, suggesting that the increased surface expression of PrPC in sortilin-knockdown cells might be caused by the decreased trafficking of PrPC to lysosomes for degradation. Sortilin was predominantly located in non-raft domains, and PrPC accumulated at raft domains and decreased in non-raft domains in sortilin-KO cells. It is thus conceivable that sortilin could function to retain PrPC in non-raft domains and be involved in trafficking of non-raft surface PrPC to lysosomes for degradation (Fig 9A). Low-density lipoprotein receptor-related protein 1 has also been reported as a candidate cargo receptor for the non-raft PrPC [24]. A sortilin-independent pathway may also play a role in PrPC internalization (Fig 9A). We showed that sortilin deficiency increased PrPC at raft domains and shifted PrPC internalization into the recycling endosomes from lysosomes in cells. It is thus conceivable that the internalization of raft PrPC to the recycling endosomes could be sortilin-independent (Fig 9A). Taken together, these results suggest that PrPC located in non-raft domains could be internalized to lysosomes for degradation partly via the sortilin-dependent pathway while an internalization pathway to direct PrPC from raft domains to the endocytic recycling pathway could be sortilin-independent (Fig 9A).

Fig 9
A possible amplification model of PrPSc in prion infected cells.

Sortilin-knockdown increased PrPSc in prion-infected cells. In contrast, overexpression of sortilin decreased PrPSc. Moreover, sortilin-KO mice developed the disease earlier than wild-type mice after intracerebral inoculation with RML prions, with earlier accumulation of PrPSc in their brains. These results indicate that sortilin is a negative regulator for PrPSc accumulation. Raft domains may be a site for the conversion of PrPC into PrPSc [25], although the exact site of PrPSc production remains controversial. Sortilin could retain surface PrPC at non-raft domains and transport it to lysosomes for degradation, thereby reducing PrPC located in raft domains. It is thus possible that the reduction of PrPC in raft domains by sortilin could delay the conversion of PrPC into PrPSc, eventually leading to less accumulation of PrPSc. Sortilin also interacts with PrPSc. Kinetics studies for PrPSc showed that knockout of sortilin delayed PrPSc clearance in prion-infected cells. Thus, sortilin also could function to sort PrPSc for degradation, reducing PrPSc accumulation. Some PrPSc molecules are trafficked to lysosomes for degradation via the endolysosomal pathway from the cell surface [21,26,27]. Others are retrogradely transported to the Golgi apparatus where they are subjected to Golgi quality control and trafficked to lysosomes for degradation [28]. Sortilin is expressed on the cell surface and the Golgi apparatus [29]. Therefore, sortilin might be involved in both degradation trafficking pathways of PrPSc. However, a large portion of sortilin and PrPSc molecules differed in their membrane microdomain localization on the cell surface. Sortilin was predominantly detected in non-raft domains. In contrast, PrPSc was exclusively located in raft domains. It is thus likely that the sortilin-mediated lysosomal degradation of PrPSc from the cell surface through their direct interaction might be, if any, a minor event. We previously reported that PrPSc was abundantly detected in the recycling endosomes of prion-infected cells, suggesting that PrPSc molecules accumulated in the recycling endosomes also might not be directly affected by sortilin.

Sortilin was reduced in both prion-infected cultured cells and mouse brains. Sortilin mRNA was not decreased in prion-infected cells. Reduction of sortilin in prion-infected cells was recovered by treatment with lysosomal inhibitors but not proteasomal inhibitor, suggesting that prion infection could stimulate degradation of sortilin in lysosomes. Immunofluorescent staining of freshly infected cells revealed that sortilin was barely detectable in PrPSc-positive cells but abundant in PrPSc-negative cells. PrPSc accumulation preceded the reduction of sortilin. These results suggest that PrPSc accumulated after prion infection could cause sortilin degradation in lysosomes in a cell-autonomous fashion, and that the enhanced degradation of sortilin could abrogate the negative role of sortilin in PrPSc accumulation in prion-infected cells. Thus, sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation could be disturbed in prion-infected cells, causing an increase in PrPC and PrPSc and eventually leading to progressive accumulation of PrPSc (Fig 9B). In prion-infected cells, due to the disturbed function of sortilin, PrPC might also be increasingly located in raft domains, where PrPC is postulated to efficiently convert into PrPSc [25], and might be internalized into the recycling endosomes, where PrPSc was reported to be abundantly detectable [26] (Fig 9B). Increased localization of PrPC in raft domains and in the recycling endosomes in prion-infected cells also could contribute to progressive accumulation of PrPSc by increasing the conversion of PrPC into PrPSc. Elucidation of the mechanism by which PrPSc accumulation stimulates the lysosomal degradation of sortilin would be helpful to further understanding of the mechanism for the progressive accumulation of PrPSc.

Sortilin also regulates neuronal cell viability by controlling the release of pro- and matured-form of neurotrophins (NTs), such as NGF and brain-derived neurotrophic factor, as does the transport of their receptors, TrkA, TrkB, and TrkC, to the plasma membrane [9]. No gross abnormalities were reported in Sort1-/- [11,22], probably due to compensatory mechanisms of other family molecules. However, cultured dorsal root ganglion neurons lacking sortilin showed impaired neurite outgrowth upon NGF stimulation [11]. Loss of sortilin also aggravated neurological phenotypes observed in p75 NT receptor (p75NTR)-KO mice [11]. Sortilin also acts as a co-receptor of p75NTR against proNTs to transduce cell death signals [30]. We found that sortilin was significantly reduced in prion-infected cells and mouse brains, and that the NGF signaling was disturbed in prion-infected cells. It might thus be interesting to investigate whether or not the sortilin-mediated NT signals are involved in the pathogenesis of prion disease.

It remains controversial whether or not degradation of sortilin might be stimulated in lysosomes in other neurodegenerative diseases. Reduced levels of sortilin have been reported in the limbic and occipital regions of AD brains [31]. To the contrary, increased expression of sortilin has been demonstrated in the temporal cortex of AD brains [32]. Other investigators showed no alteration of sortilin levels in the superior frontal and superior temporal cortices of AD brains [33].

In short, we presented a novel accumulation mechanism of PrPSc through degradation of sortilin. Prion infection stimulated degradation of sortilin in lysosomes, reducing sortilin levels in prion-infected cells. The reduction of sortilin disturbs its function to sort PrPC and PrPSc to the late endosomal/lysosomal compartments for degradation. As a result, PrPC is increasingly converted to PrPSc and PrPSc degradation is delayed, and eventually PrPSc progressively accumulates in prion-infected cells. Thus, accelerating the sortilin-mediated lysosomal degradation of PrPC and PrPSc might be therapeutic in prion diseases.

Materials and methods

Ethics statement

All animal experiments complied with Japanese legislation (Act on Welfare and Management of Animals, 1973, revised in 2012). The Ethics Committee of Animal Care and Experimentation of Tokushima University approved the animal experiments in this study (approval number T27-102). Animals were cared for in accordance with The Guiding Principle for Animal Care and Experimentation of Tokushima University and guidelines under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, Japan (Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions, 2006).

Abs

The antibodies used in this study are as follows: rabbit anti-sortilin Abs (12369-1AP, Proteintech, Rosemont, IL), goat anti-sortilin Abs (AF2934, R&D systems, Minneapolis, MN), mouse monoclonal anti-sortilin Ab (clone 48, BD Bioscience, San Jose, CA), sheep anti-SorLA Abs (AF5699, R&D systems), goat anti-SorCS1 Abs (AF3457, R&D systems), sheep anti-SorCS2 Abs (AF4237, R&D systems), rat anti-SorCS3 Abs (MAB3067, R&D systems), rat anti-LAMP1 Ab (ab25245, Abcam, UK), rabbit anti-Rab9 Abs (EPR13272, Abcam), rabbit anti-Rab11 Abs (5589, Cell signaling, MA, USA), rabbit anti-flotillin-2 Abs (3436, Cell signaling), mouse anti-β-actin Ab (A5316, Sigma-Aldrich, St. Louis, MO), 6D11 mouse anti-PrP Ab (SIG-39810, Biolegend, San Diego, CA), SAF61 mouse anti-PrP Ab (A03205, Bertin pharma, Montigny le Bretonneux, France), SAF83 mouse anti-PrP Ab (A03207, Bertin pharma), rabbit anti-PrP Abs (18635, Immuno Biological Laboratories, Gunma, Japan), rabbit anti-TSG101 Ab (14497-1-AP, ProteinTech, IL, USA), rabbit anti-flottilin-1 Ab (Ab 41927, Abcam), mouse anti-GM130 Ab (610823, Transduction Laboratories, CA, USA), rabbit anti-Bcl-2 Ab (#3498, Cell Signaling), mouse anti-EEA1 Ab (610456, Transduction Laboratories), mouse anti-Erk1 Ab (610456, BD Biosciences), mouse anti-pErk1/2 Ab (612358, BD Biosciences), Alexa Fluoro 488-conjugated anti-mouse IgG Abs (Thermo Fisher Scientific, Rockford, IL), Alexa Fluoro 546-conjugated anti-goat IgG Abs (Thermo Fisher Scientific), Alexa Fluoro 546-conjugated anti-rat IgG Abs (Thermo Fisher Scientific), Alexa Fluoro 488-conjugated anti-goat IgG Abs (Thermo Fisher Scientific). Mouse anti-PrP Ab clone 132 was kindly gifted from Prof Horiuchi, Hokkaido University [21].

Plasmid construction and recombinant proteins

A mouse sortilin cDNA fragment was amplified from mouse brain QUICK-clone cDNA (Clontech, California, USA) by using PCR (primers; 5’-cctctcgagatggagcggccccggggagct-3’, 5’-ctcaagcttctattccaggaggtcctcatctga-3’) and the amplified fragment was subcloned into pcDNA3.1(-) (Invitrogen) to make an expression vector for full-length sortilin, designated as pcDNA-Sortilin. DNA fragments encoding the VPS10P domain of sortilin, which corresponds to residues 76–750, (primers; 5’-cctctcgagggcgcgcccgccgaggaccaa-3’, 5’-ctcaagcttctaggaattctgctttgtggg-3’) were also amplified by PCR using pcDNA-Sortilin as a template and subcloned into pGEX4T-2 (GE healthcare, Little Chalfont, UK) to express glutathione S-transferase (GST)-tagged VPS10P domain of sortilin in E coli. GST-tagged VPS10P domain and GST were induced in E. coli with 0.1 mM isopropyl thiogalactoside at 37°C for 3 h and purified with glutathione-beads. His-tagged recombinant full-length mouse PrP and PrP without residues 23–88 were prepared as previously described [34].

For construction of an expression vector, termed pEF1-moPrP(3F4), encoding full-length mouse PrP with a 3F4 tag, the BamH I/Xba I-digested fragment of pcDNA3.1-moPrP(3F4) [35] was inserted into BamH I/Xba I-digested pEF1/Myc-His (Invitrogen). To construct an expression vector, pEF1-moPrP(3F4)Δ23–88, encoding 3F4-tagged mouse PrP with residues 23–88 deleted and the 5’ fragment of moPrP(3F4)Δ23–88 cDNA was amplified by PCR using pcDNA3.1-moPrP(3F4) as a template with a BamHI-PrP(ATG)-S sense primer (5’-tcggatcccgtcatcatggcgaac-3’; underlined, BamH I site; bold, start codon) and a moPrP(3F4)Δ23–88 anti-sense primer (5’-cctccttggccgcagaggccga-3’; underlined, residues 89–90; italic, residues 19–22). Then, full-length moPrP(3F4)Δ23–88 cDNA was amplified by PCR using pcDNA3.1-moPrP(3F4) as a template with the amplified 5’ fragment as a sense primer and a PrP(stop)-XbaI-AS anti-sense primer (5’-cctctagagctcatcccacgatcag-3’; underlined, Xba I site; bold, stop codon). After sequence confirmation, the amplified fragment was inserted into BamH I/Xba I-digested pEF1/Myc-His (Invitrogen).

For the construction of a Sortilin-mycHis expression vector, cDNA encoding full-length sortilin was amplified using pcDNA-Sortilin as a template by PCR with a primer pair of Sort-1 (5’-cctctcgagatggagcggccccgggagct-3’; underlined, Xho I site; bold, start codon) and Sort-12 (5’-ctcaagcttttccaggaggtcctcatctga-3’; underlined, Hind III site) and inserted into Xho I/Hind III digested pcDNA3.1/mycHis(-)A (Invitrogen). Deletion mutants of sortilin were constructed as follows. For construction of sortilinΔ76–177, two DNA fragments with an overlapping DNA segment were amplified using pcDNA-Sortilin as a template by PCR with a primer pair of Sort-1 and Sort-D4 (5’-tatcacctttccgcggcgccaacggccagc-3’; italic, residues 70–75; underlined, residues 178–183) and a primer pair of Sort-D3 (5’-cgttggcgccgcggaaaggtgatactaaca-3’; underlined, residues 72–75; italic, residues 178–183) and Sort-12. These two fragments were hybridized at the overlapping DNA segment and subjected to PCR with a primer pair of Sort-1 and Sort-12, resulting in amplification of a DNA fragment encoding sortilinΔ76–177. The amplified fragment was inserted into Xho I/Hind III digested pcDNA3.1/mycHis(-)A (Invitrogen). Other DNA fragments encoding other sortilin mutants were similarly amplified and inserted into Xho I/Hind III digested pcDNA3.1/mycHis(-)A (Invitrogen). Sort-1 and Sort-D6 (5’-gaaggtttttccagagttctcaggaccaat-3’; italic, residues 172–177; underlined, residues 291–294) and Sort-D5 (5’-cctgagaactctggaaaaaccttcaaaacc-3’; underlined, residues 174–177; italic, residues 291–296) and Sort-12 were used for construction of sortilinΔ178–290. Sort-1 and Sort-D8 (5’-tatatagacccccaagtcggatgttctcca-3’; italic, residues 285–290; underlined, residues 410–413) and Sort-12 and Sort-D7 (5’-acatccgacttgggggtctatataacaagc-3’; underlined, residues 287–290; italic, residues 410–415) were used for construction of sortilinΔ291–409. Sort-1 and Sort-D10 (5’-ggagtaacccccacggagggaagtcacgtt-3’; italic, residues 404–409; underlined, residues 509–612) and Sort-D9 (5’-acttccctccgtgggggttactcctgggcg-3’; underlined, residues 406–409; italic, residues 509–514) and Sort-12 were used for construction of sortilinΔ410–508. Sort-1 and Sort-D12 (5’-atcctcttcacaatcatctgagatgtacac-3’; italic, residues 503–508; underlined, residues 610–613) and Sort-D11 (5’-atctcagatgattgtgaagaggatgactat-3’; underlined, residues 505–508; italic, residues 610–615) and Sort-12 were used for construction of sortilinΔ509–609. Sort-1 and Sort-D14 (5’-aatagggacagaattccgctcaaggatgtc-3’; italic, residues 604–609; underlined, residues 754–757) and Sort-D13 (5’-cttgagcggaattctgtccctattatcctg-3’; underlined, residues 606–609; italic, residues 754–759) and Sort-12 were used for construction of sortilinΔ610–753.

Cell lines and animals

Cells were maintained at 37°C with 5% CO2 in air in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS). N2aC24, N2aC24L1-3, and cured N2aC24L1-3 cells were previously established elsewhere [26]. N2aC24 cells were cloned from mouse neuroblastoma N2a cells overexpressing exogenous mouse PrPC. N2aC24L1-3 cells were cloned from N2aC24 cells persistently infected with 22L scrapie prions. Cured N2aC24L1-3 cells are cured from prion infection by treatment with SAF32 anti-PrP Ab and then maintained in antibody-free DMEM with 10% FBS. ScN2a cells (kindly gifted from Prof Doh-ura, Tohoku University) were N2a cells persistently infected with RML scrapie prions.

Sortilin-KO cells, ΔSort#1 and #2 cells, were established using the CRISPR-Cas genome editing system. N2aC24 cells were transduced with pRGEN-Cas9-CMV (Takara bio, Shiga, Japan) and pRGEN_Mouse-Sort1_U6_SG_1 targeting the sequence (5’-cctgccgccgtcggccaggaccg-3’) (Takara bio), and subjected to limiting dilution cloning. Knockout of sortilin in ΔSort#1 and #2 cells was confirmed by Western blotting.

PrP-KO N2a cells, termed N2aΔPrP cells, were also established using the CRISPR-Cas genome editing system. N2a cells were transduced with pRGEN-Cas9-CMV (Takara bio) and pRGEN_PrP_U6_SG_1 targeting the sequence (5’-accggtggaagccggtatcccgg-3’) (Takara bio), and subjected to limiting dilution cloning. Knockout of PrPC in N2aΔPrP cells was confirmed by Western blotting. To clone N2aΔPrP cells expressing full-length wild-type PrPC or PrPΔ23–88, pEF1-moPrP(3F4) and pEF1-moPrP(3F4)Δ23–88 were linearized by Sca I and transfected into N2aΔPrP cells. The cells were treated with G418 and the G418-resistant cells were cloned by limiting dilution, resulting in establishment WT#1 and #2 cells and Δ23–88#1 and #2 cells.

Sortilin-KO (Sort1-/-) mice used in this study were previously produced elsewhere [11,22]. Sort1-/- mice having been backcrossed for 10 generations into C57BL/6 were intercrossed with C57BL/6 mice (Charles River Laboratories, Kanagawa, Japan), and the resulting heterozygous mice (Sort-/+) were then intercrossed to obtain Sort-/- and wild-type (Sort+/+) mice.

Immunoprecipitation

Cells were lysed in buffer A [20 mM MES-KOH (pH 7.0), 0.15 M KCl, 1 mM DTT, 10% glycerol, 0.2% (w/v) CHAPS] containing protease inhibitor cocktail (Nakalai tesque, Kyoto, Japan). The lysate was cleared by centrifugation for 5 min at 20,000×g at 4°C and the supernatant was transferred to a new tube. 500 μL of supernatant containing 500 μg of total proteins were incubated with 1 μg of indicated Abs for 2 h. 5 μL of protein-G sepharose (GE healthcare) was added and the mixture was rotated for 4 h at 4°C. Thereafter, protein-G sepharose was precipitated and the precipitant was washed with buffer A 5 times. The final precipitate with protein-G sepharose was suspended in 50 μL of Laemmli’s sample buffer, heated, and subjected to Western blotting to detect proteins of interest with appropriate Abs.

Protein interaction assay using Dynabeads protein-G

For detection of interaction of PrPSc and sortilin, 15 μL of Dynabeads protein-G (Thermo Fisher Scientific) was added instead of protein-G sepharose. Immunocomplexes were collected using magnet instead of centrifugation and washed with buffer A. The same procedure was repeated 5 times. The finally collected complexes were suspended in 45 μL of buffer B [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.5% (w/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 1 mM EDTA] containing 100 μg/ml proteinase K (PK) and incubated at 37°C for 60 min with mixing at 1,100 rpm using Thermomixer (Eppendorf, Hamburg, Germany). The PK-treated samples were denatured in Laemmli’s sample buffer and subjected to Western blotting to detect PrPSc.

For detection of interaction of PrPC and sortilin on the cell surface, cells were washed with ice-cold PBS. After blocking of the cells with buffer C [20 mM MES-KOH (pH 7.0), 0.15 M KCl, 0.25 M sucrose] containing 0.5% (w/v) bovine serum albumin (BSA) for 10 min at 4°C, the cell surface PrP was labeled with 1 μg/mL of SAF61 anti-PrP Ab in buffer C containing 0.5% (w/v) BSA for 30 min at 4°C. After washing the cells with ice-cold-PBS, they were suspended in 1 mL of buffer C. To homogenize the cells, the cell suspension was passed through a 27G needle 10 times. The homogenate was centrifuged at 500×g for 10 min at 4°C and the supernatant was incubated with 15 μL of Dynabeads protein-G with gentle agitation for 2 h at 4°C. The beads were collected by magnet and washed with buffer C. The collected immunocomplexes on the beads were washed with buffer A 5 times and finally suspended in 50 μL of Laemmli’s sample buffer and subjected to Western blotting.

GST pulldown assay

2 μg of GST-tagged VPS10P domain of sortilin, which was pre-bound to 5 μL of glutathione beads, was incubated with 2 μg of His-tagged full-length recombinant PrP or His-tagged recombinant PrP lacking residues 23–88 in buffer A containing protease inhibitor cocktail for 2 h with rotation at 4°C. The precipitate was washed with buffer A 5 times, suspended in Laemmli’s sample buffer, heated, and subjected to Western blotting. GST-tagged VPS10P domain and His-tagged PrPs were detected with rabbit polyclonal anti-sortilin Abs and RGS-His Ab, respectively.

Biotinylation of cell surface proteins

Biotinylation of cell surface proteins was carried out as described elsewhere [26]. In brief, cells (85–95% confluent) were washed with PBS and incubated with Sulfo-NHS-LC Biotin (Thermo Fisher Scientific) in PBS for 30 min at room temperature. The cells were then washed with 0.1 M glycine in PBS and lysed in buffer B. The lysate was mixed with NeutrAvidin UltraLink Resin (Thermo Fisher Scientific) for 4 h at 4°C and the biotinylated protein-resin complexes were collected by brief microcentrifugation. The complexes were then washed with the buffer and heated at 99°C for 10 min in Laemmli’s sample buffer to separate the biotinylated proteins from the complexes. The biotinylated proteins in the supernatant were subjected to Western blotting.

Immunofluorescence staining

Cells were stained with indicated Abs as described previously [26]. In brief, cells grown on coverslips were fixed in 3% paraformaldehyde (PF) for 15min and treated with 0.1 M glycine in PBS for 10 min. Permeabilization was carried out using 0.1% Triton X-100 in PBS for 4 min at RT. To detect PrPSc, the cells were treated with 5 M guanidinium thiocyanate for 10 min at RT. After washing with PBS, the cells were incubated with the first Ab in 5% FBS in PBS and then with fluorescent secondary Ab. For detection of PrPSc, mouse anti-PrP Ab clone 132 [21] (kindly gifted from Prof Horiuchi, Hokkaido University) was used as a first Ab. After washing, the coverslips were mounted with Prolong Gold antifade reagent (Invitrogen). Fluorescence images were obtained using BIOREVO BZ-9000 (Keyence, Osaka, Japan), which is equipped with haze reduction function, which enables production of fluorescent images very similar to those taken by a confocal microscope. To assess the co-localization of proteins of interest, Pearson’s correlation coefficient was calculated using Co-localization Plugin (JaCoP) tool in Image J [36].

Internalization assay of antibody-labeled surface PrP

Cells were washed with ice-cold PBS and treated with 1% BSA in PBS for 10 min at 4°C prior to incubation with the indicated anti-PrP Abs (1 μg/mL) for 10 min at 4°C in 1% BSA-containing PBS. The cells were then washed with ice-cold PBS and incubated at 37°C for 2 h. Thereafter, the cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with Alexa Fluoro 488 anti-mouse IgG Ab (Thermo Fisher Scientific). Fluorescent signals were observed using BIOREVO BZ-9000 (Keyence) and their intensities were analyzed using BZ-II analyzer (Keyence).

Internalization assay of biotinylated surface PrP

Cells were washed three times with ice-cold PBS (pH7.4) and incubated with 5 mg/mL sulfo-NHS-SS-biotin (Thermo Fisher Scientific) in PBS (pH 7.4) at 4°C for 10 min to biotinylate cell surface proteins. After washing the cells twice with ice-cold PBS (pH 7.4) and incubation with 50 mM glycine in PBS (pH 7.4), the cells were further washed twice with ice-cold PBS (pH 7.4). Thereafter, the cells were incubated in DMEM medium at 37°C. After 2 h-incubation, the cells were washed three times with ice-cold PBS (pH 7.4), and biotin was removed from the proteins still on the cell surface by incubating the cells with 100 mM reducing glutathione in PBS (pH 7.4) at 37°C for 10 min. After washing the cells with ice-cold PBS (pH 7.4), the cells were lysed in buffer B containing protease inhibitor cocktail (Nakalai tesque). The lysate was cleared by centrifugation for 5 min at 20,000×g at 4°C and the supernatant was transferred to a new tube. 20 mL of NeutrAvidin beads (Thermo Fisher Scientific) was added into the cell lysate containing 300 mg of proteins and the mixture was rotating at 4°C for 2 h. After washing the beads with lysis buffer, the beads was suspended in 50 mL of SDS-PAGE sample buffer and subjected to Western blotting with 6D11 anti-PrP Ab.

Transfection

Cells were transiently transfected with pcDNA-Sortilin and pcDNA-SortilinΔC at the final concentration of 1.6 μg/ml using lipofectamin 2000 (Invitrogen). The expression of sortilin and PrPC was knocked down using Stealth RNAi siRNAs (Invitrogen): Sortilin siRNA #1, 5’-ccaagucaaauucugucccuauuau-3’; Sortilin siRNA #2, 5’-gagaacucuggaaaggugauacuaa-3’; PrP siRNA #1, 5’-gggacaaccucauggugguaguugg-3’; PrP siRNA #2, 5’-ccaguggaucaguacagcaaccaga-3’. Stealth RNAi Negative Control Duplex was purchased from Invitrogen. Each siRNA was transfected into cells at the final concentration of 10 nM using lipofectamin RNAiMax (Invitrogen).

RT-PCR

Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Hilden, Germany) and first-strand cDNA was synthesized using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). The synthesized cDNAs were amplified with the following primer sets: 5’-aagcaggactcccgcccacag-3’ and 5’-ttccaggaggtcctcatctga-3’ for sortilin cDNA, 5’-tacagcaaccagaacaac-3’ and 5’-tcatcccacgatcaggaagat-3’ for PrP cDNA, and 5’-cctgccaagtatgatgacatc-3’ and 5’-gctgtagccgtattcattgtc-3’ for glyceraldehyde-3-phosphate dehydrogenase cDNA.

Fractionation of membrane microdomains

Cells grown to ~80% confluency in a 35 mm tissue culture dish were suspended in 250 μL of MBS buffer [25 mM MES-NaOH (pH 6.5), 0.15 M NaCl] containing 1% (w/v) Triton X-100, and homogenized by being passed through a 21G-needle 15 times. After centrifugation at 500×g for 5 min at 4°C, 220 μL of the supernatant was transferred to a new tube and mixed with 220 μL of MBS buffer containing 80% (w/v) sucrose to make 40% (w/v) sucrose. 200 μL of the sample was placed at the bottom of a discontinuous sucrose gradient consisting of 600 μL of 30% (w/v) sucrose and 200 μL of 5% (w/v) sucrose. The sample was centrifuged at 140,000×g for 24 h at 4°C in an S55S rotor (Hitachi Koki, Tokyo, Japan). Ten fractions (100 μL/fraction) were collected from the top.

Prion infection

Brains were removed from terminally ill wild-type C57BL/6 mice infected with RML prions. A single brain was homogenized (10%, w/v) in PBS using a multi-beads shocker (Yasui Kikai, Osaka, Japan) and then diluted to 1% with PBS. Two 1% (w/v) brain homogenates were mixed to prepare the homogenate of 2 pooled brains and the resulting homogenate was intracerebrally inoculated into a 4–5 week-old mouse with its 20 μL aliquot. The signs for disease-related symptoms were evaluated as previously described [37].

For infection of cells, cells were seeded at a density of 2×105 cells/well in a 6-well tissue culture plate. At 4 h after cell seeding, the clarified RML-infected brain homogenate [34] containing 50 μg proteins was added to the well and cells were subsequently passaged every 3 days.

Immunohistochemistry

Paraffin-embedded samples were sectioned, deparaffinized, and rehydrated. The samples were autoclaved in 1 mM HCl at 121°C for 5 min and subsequently washed with PBS. The samples were then digested with 50 μg/mL PK in PBS at 37°C for 30 min, treated with 3 M guanidine thiocyanate for 10 min, and washed with PBS. After blocking with 5% FBS in PBS for 1 h, the sampled were incubated with 6D11 anti-PrP Ab for 2 h, washed with PBS, and treated with ImmPRESS REAGENT Anti-Mouse IgG (Vector Laboratories, U.S.A) for 30 min. After washing with PBS, the samples were incubated with ImmPACT DAB (Vector Laboratories) for 180 sec for staining.

Western blotting

Western blotting was performed as reported previously [26]. To evaluate protein expression, signals were densitometrically measured using LAS-4000 mini (Fujifilm Co., Tokyo, Japan). The measured intensity of target proteins was normalized against the signal intensity of β-actin used as an internal control protein.

NGF stimulation

Cells were removed from a tissue culture dish by pipetting and transferred to a new tube. After washing with DMEM medium, cells were collected by centrifugation at 500×g for 2 min at 4°C, suspended in 1 mL of DMEM medium containing 2 nM NGF (Thermo Fisher Scientific), transferred into a well of 12 well plate, and incubated for 10 min at 37°C in a 5% CO2 incubator. The cells were collected by centrifugation at 500×g for 5 min at 4°C and washed with ice-cold PBS. The pellet was lysed in 200 μL of buffer B containing protease inhibitor cocktail (Nakalai tesque). The lysate was cleared by centrifugation at 12,000×g for 2 min at 4°C and the supernatant was subjected to Western blotting.

Isolation of exosomes

Cells were cultured in 2 mL of DMEM medium containing 10% exosome-depleted FBS (System Bioscience, CA,USA) in a 6 well tissue culture plate for 72 h and the culture medium was collected. The culture medium was centrifuged at 2,000×g for 10 min at 4°C. The supernatant was passed through 0.22 μm pore filter membrane and the flow-through was centrifuged at 10,000×g for 30 min at 4°C. Exosomes in the supernatant were collected by ultracentrifugation at 100,000×g for 1 h at 4°C and washed with PBS. The exosomes were dissolved in 100 μL of Laemmli’s sample buffer and subjected to Western blotting.

Detection of the C1 fragment

Cell lysate containing 20 μg proteins was incubated in 20 μL of (1×) glycoprotein denaturing buffer (0.5% SDS, 40mM DTT) at 99°C for 10 min. Thereafter, 3μl of 10% NP-40, 3 μL of (10×) Glycobuffer, 3.5 μl of distilled water and 0.5 μL of PNGase (500 units/μl) (New England BioLabs, MA, USA) were added. After 60 min incubation at 37°C, samples were mixed with 10 μL of (4×) Laemmli’s sample buffer and subjected to Western blotting.

Statistical analysis

Survival and incubation times are analyzed using the log-rank test. Other data were analyzed using the one-way ANOVA.

Supporting information

S1 Table

Incubation and survival times of Sort1-/- and Sort1+/+ female mice intracerebrally inoculated with RML prion.

(DOCX)

S2 Table

Incubation and survival times of Sort1-/- and Sort1+/+ male mice intracerebrally inoculated with RML prion.

(DOCX)

S1 Fig

PrPC interacts with sortilin but not with other VPS10P receptors.

Co-immunoprecipitation assay was carried out in N2aC24 cells with SAF61 anti-PrP Ab. The resulting immunoprecipitates were subjected to Western blotting with Abs against each protein. Arrows and arrowheads indicate non-specific signals of the degraded fragment of protein G or the light chain of Abs used in co-immunoprecipitation.

(TIF)

S2 Fig

Sortilin interacts with PrPC through residues 610–753.

(A) Schematic diagrams of full-length (full) sortilin and various deletion mutants of sortilin, all of which are tagged with a mycHis motif. Sortilin is a single-pass transmembrane molecule consisting of a signal peptide (red), a propeptide (yellow), a VPS10P domain (green), and a transmembrane region (blue). Arabic numbers represent the codon numbers. (B) Immunoprecipitation assay of sortilin-KO ΔSort#1 cells expressing full-length (full) sortilin and various deletion mutants of sortilin and of PrP-KO ΔPrP#1 cell expressing full-length (full) sortilin using SAF61 anti-PrP Ab. Immunoprecipitates (IP) and the cell lysates (Lysate) were subjected to Western blotting for sortilin with anti-myc Ab and for PrPC with 6D11 anti-PrP Ab. An arrow indicates light chains of the Ab used in this assay.

(TIF)

S3 Fig

Interaction of PrPC and sortilin.

Orthogonal views of double immunofluorescence staining of PrPC (green) and sortilin (red) in non-permeabilized or permeabilized N2aC24 cells, with SAF83 anti-PrP Ab and goat polyclonal anti-sortilin Abs.

(TIF)

S4 Fig

PrPC interacts with sortilin on the cell surface.

(A) A simple description of the protocol used for detection of interaction of PrPC with sortilin on the cell surface. (B) Western blotting for PrPC and sortilin in the immunocomplexes of SAF61 anti-PrP Ab from N2aC24 and ΔPrP#1 cells. (C) Western blotting for sortilin expressing in N2aC24 and ΔPrP#1 cells.

(TIF)

S5 Fig

PrPC is increased in the brains of Sort1-/- mice.

(A) Western blotting of the brains of WT (Sort1+/+) and Sort1-/- mice for PrPC with 6D11 anti-PrP Ab. Sortilin was detected in Sort1+/+ brains but not in Sort1-/- brains. (B) Quantification of PrPC densities after normalization against β-actin intensities in (A). Data are means ± SD of 3 brains. *** p < 0.001.

(TIF)

S6 Fig

Shading of PrPC and excretion of PrPC in exosomes are increased in sortilin-deficient cells.

(A) Western blotting for deglycosylated PrPC in N2aC24 cells transfected with control and sortilin siRNAs. Full-length deglycosylated PrPC and the C1 fragment were detectable. Quantification of densities for full-length deglycosylated PrPC and the C1 fragment in (A). Data are means ± SD of 3 independent samples. ** p < 0.01. (B) Western blotting of the cell lysates and exosomes from N2aC24 cells and sortilin-KO ΔSort#1 and #2 cells for PrPC with 6D11 anti-PrP Ab. TSG101 and flotillin-1, but not GM130 and Bcl-2, were detectable in exosomes. (C) Quantification of PrPC densities in (B). Data are means ± SD of 3 independent samples. ** p < 0.01, *** p < 0.001.

(TIF)

S7 Fig

Localization of PrPC in late endosomes, recycling endosomes, and early endosomes.

Double immunofluorescence staining of PrPC (green) with the late endosome marker Rab9 (red) (A), the recycling endosome marker Rab11 (red) (C), and the early endosome marker EAA1 (red) (E). Pearson’s correlation coefficient for co-localization of PrPC and Rab9 (B), Rab11 (D) or EAA1 (F). Data are means ± SD of 6 cells. ** p < 0.01, *** p < 0.001.

(TIF)

S8 Fig

Impaired trafficking of PrPΔ23–88 to lysosomes.

(A) Western blotting of full-length wild-type PrPC and PrPΔ23–88 in WT cells and Δ23–88 cells after 12 h-treatment with or without 20 mM NH4Cl. (B) Quantification of wild-type PrPC and PrPΔ23–88 in (A) after normalization against β-actin. Signal intensities in each lane were evaluated against that in NH4Cl-untreated WT#1 cells. Data are means ± SD of 4 independent experiments. * p < 0.05, *** p < 0.001. (C) Double immunofluorescence staining for PrPC and PrPΔ23–88 with the lysosome marker LAMP1 in WT and Δ23–88 cells after 12 h-treatment with or without 20 mM NH4Cl. (D) Pearson’s correlation coefficients for co-localization of PrPC or PrPΔ23–88 and LAMP1 in WT#1 cells untreated (n = 149) or treated (n = 120) with NH4Cl, WT#2 cells treated (n = 121) or untreated (n = 130) with NH4Cl, Δ23–88#1 cells treated (n = 124) or untreated (n = 138) with NH4Cl, and Δ23–88#1 cells treated (n = 122) or untreated (n = 121) with NH4Cl. Data are means ± SD. *** p < 0.001. (E) Western blotting for sortilin in WT and Δ23–88 cells. (F) Quantification of sortilin in (E) after normalization against β-actin. Signal intensities in each lane were evaluated against that in WT#1 cells. Data are means ± SD of 4 independent experiments. (G) Co-immunoprecipitation assay for PrPC or PrPΔ23–88 and sortilin using SAF61 anti-PrP Ab. Arrows and arrowheads indicate non-specific signals of the degraded fragment of protein G or the light chain of Abs used in co-immunoprecipitation.

(TIF)

S9 Fig

Membrane microdomain distribution of PrPΔ23–88 in cells and PrPC in the brains of Sort1-/- mice.

(A) PrP-KO N2aΔPrP cells expressing WT PrPC, designated WT#1 and #2 cells, and those expressing PrPΔ23–88, Δ23–88#1 and #2 cells, were subjected to discontinuous sucrose gradient centrifugation. Each fraction was analyzed by Western blotting with 6D11 anti-PrP Ab. (B) Quantification of PrPC or PrPΔ23–88 in each fraction against the total PrPC or PrPΔ23–88 in (A). The signal density in each lane was evaluated against the total signal density of all lanes. Data are means ± SD of 3 independent experiments. (C) Discontinuous sucrose gradient centrifugation of the brains from Sort1-/- and WT mice. Each fraction was analyzed by Western blotting with 6D11 anti-PrP antibody. (D) Quantification of PrPC in each fraction against the total PrPC in (C). The signal density in each lane was evaluated against the total signal density of all lanes. Data are means ± SD of 3 brains.

(TIF)

S10 Fig

Membrane microdomain distribution of PrP molecules in prion-infected cells.

(A) Discontinuous sucrose gradient centrifugation of prion-infected N2aC24L1-3 cells. Each fraction was treated with or without PK and analyzed by Western blotting with 6D11 anti-PrP Ab. (B) Quantification of PrP in each fraction against the total PrP in (A). The signal density in each lane was evaluated against the total signal density of all lanes. Data are means ± SD of 3 independent experiments.

(TIF)

S11 Fig

PrPSc degradation is delayed in sortilin-KO cells infected with prions.

(A) PrPSc in RML-infected N2aC24 (N2aC24/RML) and ΔSort#1 (ΔSort/RML) cells 36, 48, and 60 h after transfection with control siRNA alone. (B) Quantification of PrPSc in (A) after normalization against β-actin. Signal density in cells at 48 and 60 h was compared with that at 36 h. Data are means ± SD of 3 independent experiments. n.s., not significant. (C) PrPSc in 22L prion-infected N2aC24 (N2aC24/22L) and ΔSort#1 (ΔSort/22L) cells 36, 48, and 60 h after transfection with control siRNA or PrP-specific siRNAs (#1 and 2). (D) Quantification of PrPSc in (C) after normalization against β-actin. Each signal intensity in PrP-knockdown cells was evaluated against that in control siRNA-transfected cells in each blot. Data are means ± SD of 3 independent experiments.

(TIF)

S12 Fig

Sortilin expression is decreased in prion-infected cells.

(A) Western blotting of VPS10P receptors in infected N2aC24L1-3 cells and uninfected N2aC24. (B) Quantification of the VPS10P receptors in (A) after normalization against β-actin. The signal density in infected cells was evaluated against that in uninfected cells. Data are means ± SD of 3 independent experiments. n.s., not significant. (C) RT-PCR for sortilin, PrP, and GAPDH in infected N2aC24L1-3 and uninfected N2aC24 cells. (D) Western blotting of sortilin in N2a and ScN2a cells. (E) Quantification of sortilin in (D) after normalization against β-actin. The signal density in ScN2a was evaluated against that in N2a cells. Data are means ± SD of 4 independent experiments. *** p < 0.001.

(TIF)

S13 Fig

Lysosomal degradation of sortilin is enhanced in prion-infected cells.

(A) Western blotting of sortilin in N2aC24 and N2aC24L1-3 cells 12 h after treatment with 20 mM NH4Cl and 10 μM MG132. (B) Quantification of sortilin in (A) after normalization against β-actin. The signal intensity of sortilin in NH4Cl- or MG132-treated cells was evaluated against that in untreated cells. Data are means ± SD of 3 independent experiments. n.s, not significant; ** p < 0.01, *** p < 0.001. (C) Western blotting for sortilin in N2aC24L1-3 cells 12 h after treatment with 20 mM NH4Cl or 10 nM Concanamycin A. (D) Quantification of sortilin in (C) after normalization against β-actin. The signal intensity of sortilin in NH4Cl- or Concanamycin A-treated cells was evaluated against that in untreated cells. Data are means ± SD of 3 independent experiments. n.s, not significant; *** p < 0.001.

(TIF)

S14 Fig

Impaired activation of ERK1/2 in prion-infected cells.

(A) Western blotting of uninfected N2aC24 and prion-infected N2aC24L1-3 cells for phosphorylated ERK1/2 and total ERK1 after treatment with or without NGF. (B) Quantification of phosphorylated ERK1/2 densities after normalization against β-actin densities in (A). Data are means ± SD of 3 independent experiments. *** p < 0.001. (C) Quantification of total ERK1 intensities in (A) after normalization against β-actin intensities. Data are means ± SD of 3 independent experiments.

(TIF)

S15 Fig

Sortilin-KO male mice have accelerated prion disease with earlier accumulation of PrPSc in their brains.

(A) Kaplan-Meier survival curves for Sort1-/- (n = 21) and Sort1+/+ (n = 23) male mice inoculated with RML prions. *** p < 0.001. (B) Western blotting of PrPSc in the brains of Sort1-/- and Sort1+/+ mice at 45, 60, and 90 dpi and at terminal stage. (C) Quantification of PrPSc in (B) after normalization against β-actin. Signal intensity in Sort1-/- mice was evaluated against that in Sort1+/+ mice. Data are means ± SD of 4–6 independent brains. n.s., not significant; * p < 0.05. (D) Immunohistochemical staining of PrPSc in the brain hippocampus areas of Sort1-/- and Sort1+/+ mice at 60 and 90 dpi and at terminal stage. Bar, 300 μm.

(TIF)

Acknowledgments

We would like to thank Prof. Horiuchi (Hokkaido University) for anti-PrP antibody clone 132 and N2a cells, and Prof. Doh-ura (Tohoku University) for ScN2a cells.

Funding Statement

This work was supported by the following: Pilot Research Support Program in Tokushima University (http://www.tokushima-u.ac.jp/) received by KU; Naito Foundation (https://www.naito-f.or.jp/jp/index.php) received by KU; JSPS KAKENHI grant (grant No. 26460557, https://www.jsps.go.jp/english/e-grants/) received by KU; MEXT KAKENHI grant (grant No, 17H05702, https://www.jsps.go.jp/english/e-grants/) received by KU; JSPS KAKENHI grant (grant No. 26293212, https://www.jsps.go.jp/english/e-grants/) received by SS; MEXT KAKENHI grant (grant No, 15H01560 and 17H05701, https://www.jsps.go.jp/english/e-grants/) received by SS; and Practical Research Project for Rare/Intractable Diseases of the Japan Agency for Medical Research and Development (AMED, http://www.amed.go.jp/) received by SS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

Data Availability

All relevant data are within the paper and its Supporting Information files.

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