Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein
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KeywordsADAM10 Antibody Exosomes Glycosylation Membrane anchor Neurodegeneration Prion protein Proteolytic cleavage Shedding
a disintegrin and metalloproteinase
acquired immunodeficiency syndrome
Amyloid precursor protein
bovine serum albumin
bovine spongiform encephalopathy
fetal bovine serum
full length prion protein
GI254023X (ADAM10 inhibitor)
human immunodeficiency virus
Madin-Darby Canine Kidney (cells)
murine embryonal fibroblasts
phosphate buffered saline
phosphatidylinositol-specific phospholipase C
cellular prion protein
pathological (Scrapie) isoform of the prion protein
antibody against shed PrP
tumor necrosis factor α converting enzyme (ADAM17)
Transcription Activator-like Effector Nuclease
prion protein overexpressing mouse line
Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown.
We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches.
We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell.
With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration.
Proteolytic processing is an essential regulator of protein function and differs from many other posttranslational modifications by its irreversible character. As exemplified decades ago in the case of prohormones (such as the proopiomelanocortin ), differential or subsequent cleavages by endogenous proteases produce fragments with intrinsic biological functions, differing from the ones of the larger precursors. This concept may also help in understanding and explaining the biology of other “multifunctional” proteins, i.e. proteins with more than just one particular function ascribed to them.
One of these proteins is the cellular prion protein (PrPC), for which a multitude of physiological functions has been suggested in different tissues, cells and experimental settings [2, 3], even though not in each case without controversy or questionable reproducibility [4, 5]. For instance, PrPC has been linked to developmental processes [6, 7], cell adhesion [8, 9], neurite outgrowth, axon guidance and synapse formation [10, 11, 12, 13, 14], as well as to neuroprotection [15, 16, 17] and regulation of the circadian rhythm . Among the currently best characterized functions are its contributions to myelin maintenance [4, 19, 20, 21] and cellular homeostasis of divalent ions [22, 23] as well as its involvement in signaling events [24, 25, 26].
Too many functional implications for just one protein? Not necessarily. While transient interactions of PrPC with alternating binding partners in different cellular locations may partially account for this functional diversity [5, 27], so might its proteolytic processing . In fact, different highly conserved cleavage events occur constitutively on a relevant fraction of PrPC [29, 30, 31], yet scientists are just starting to understand their biological relevance.
In contrast to some of the suggested physiological functions, the relevance of PrPC in neurodegenerative proteinopathies is widely accepted. First and foremost, it is the essential substrate for the process of templated misfolding underlying fatal and transmissible prion diseases, such as Creutzfeldt-Jakob disease in humans or BSE in cattle [32, 33, 34]. Once having adopted its pathogenic conformation (PrPSc), the prion protein is the key component of the infectious particles termed prions [32, 35, 36, 37]. Second, binding of toxic oligomeric protein species, such as PrPSc (in prion diseases ), Aβ (in Alzheimer’s disease [39, 40, 41, 42]) or α-synuclein (in Parkinson’s disease [43, 44]), to PrPC at the neuronal surface results in neurotoxic signaling. As for the physiological functions, increasing evidence suggests that proteolytic cleavages also impact on these pathogenic roles of the prion protein [28, 45, 46].
Here, we focus on the most membrane-proximate cleavage of PrPC, i.e. its shedding from the neuronal surface and release into the extracellular space by the metalloprotease ADAM10 [47, 48]. This cleavage not only regulates membrane levels of PrPC and, thus, PrPC-related functions at the neuronal surface . The resulting soluble fragment, shed PrP, likely has intrinsic functions as supported by studies using (recombinant) anchorless analogues, that showed beneficial effects with regard to axon outgrowth and synapse formation [13, 14] or neuroprotection [15, 49]. Focusing on neurodegeneration, we have recently shown a significant impact on the course of prion disease in mice by conditional depletion of the sheddase ADAM10 [50, 51], as have others by overexpression of exogenous ADAM10  or by transgenic expression of anchorless versions of PrP [53, 54]. Moreover, by reducing membrane-bound PrPC as a receptor and by producing anchorless PrP, which can block and detoxify Aβ and other harmful protein species in the extracellular space [55, 56, 57, 58], shedding may also have a protective role in other, more frequent proteinopathies .
Surprisingly, shed PrP has recently been associated with the development of specific tumours in the nervous system, where it correlates with increased cancer cell proliferation . In addition, a recent report shows critical involvement of shed PrP in the neuropathogenesis of HIV/AIDS by recruiting monocytes and aggravating the inflammatory response and the associated cognitive impairment .
Thus, given that shedding of PrPC might provide a promising and potent target for therapy of various pathological conditions, a deeper mechanistic understanding and knowledge of factors influencing this cleavage is required. Here, we first introduce and characterize a novel antibody detecting shed PrP with high specificity and sensitivity. Using this tool, we investigate different structural (i.e. glycosylation state and membrane anchorage) and mechanistic aspects in vitro and in vivo for how they impact on this relevant proteolytic event. Finally, we show that shedding is part of a compensatory cellular network regulating PrPC homeostasis.
The following constructs were used for transient transfection of cells. Detailed descriptions of the constructs can be found in the corresponding references: PrP-WT, PrPC glycomutants PrP-G1, PrP-G2, PrP-G3 and anchor-mutant PrPGPI-Thy1 , PrP-TM (PrP-CD4 [62, 63]). All PrP constructs contained the 3F4 tag . The N-terminally truncated PrP-C1 construct was cloned from the plasmid pcDNA3.1(+)/Zeo containing the murine Prnp gene. The sequence coding for the N-terminal part of PrPC (aa23–110) was deleted by use of the restriction enzymes XbaI and HindIII and the resulting construct (Δaa23–110; i.e. PrP-C1) was verified by DNA sequencing.
Rodent brain samples
Use of animal material in this study was in strict compliance with the Guide for the Care and Use of Laboratory Animals and ethics guidelines of the responsible local authorities. Frozen forebrain samples from wild-type C57BL/6, prion protein deficient (Prnp0/0 ), prion protein overexpressing (tga20 ) mice as well as from mice with conditional knockout of ADAM10 in forebrain neurons (A10 cKO and wild-type littermate controls ), with transgenic overexpression of dominant negative ADAM10 (A10 d.n. and wild-type controls ), with depletion of sortilin-1 (Sort1 KO and wild-type controls ) or with a knock-in of 3F4-tagged PrPC (PrP3F4KI and controls; both had a 192S4 background ), and from a rat and a rabbit were used to prepare 10% (w/v) homogenates in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% NP-40, 0.5% Na-Deoxycholate, 0.1% SDS) freshly supplemented with Complete EDTA-free protease (PI) and PhosStop phosphatase inhibitor cocktails (Roche) on ice. Samples were homogenized with 30 strokes using a dounce homogenizer and incubated on ice for 20 min, shortly vortexed and incubated for another 20 min before centrifugation at 12,000 g at 4 °C for 12 min. Total protein content was assessed by Bradford assay (BioRad). Supernatants were either further processed for SDS-PAGE or stored at − 80 °C.
Cell culture, transfection and treatments
Murine neuroblastoma cells (N2a) and mouse embryonic fibroblasts (MEF; ) were maintained at 37 °C under an atmosphere of 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Scientific Fisher). N2a PrP-KO cells were generated using the TALEN approach and characterized in detail before . N2a PrP-KO cells were transfected using Lipofectamine 2000 (Thermo Fisher Scientific) following the manufacturer’s instructions. For stable overexpression of PrP3F4 in N2a PrP-KO (used for the glycopattern analysis shown in Fig. 1h) cells were kept for 3 weeks in selection media (Zeocin 400 μg/ml; Thermo Fisher Scientific) and single resistant clones were selected for amplification.
Treatments of cells were performed by adding the following compounds (and concentrations) to the cell culture media: Resveratrol (20 μM), Tamibarotene/Am80 (1 μM), GI254023X (3 μM), Tunicamycin (2.5 μg/ml), Swainsonine (5 μg/ml), Leupeptin (200 μg/ml). All compounds were purchased from Merck. These treatments were carried out in 6-well plates with 1 ml OptiMEM for 18 h overnight. In the case of Tunicamycin and Swainsonine cells were pretreated for 8 h. Treatment with GM6001 (25 μM) or Batimastat (10 μM) was for 10 h.
Treatment of cells with PI-PLC
Two days post-transfection cells (grown in 6-well plate format) were incubated with 0.5 U/ml Phospholipase C (PI-PLC; Sigma-Aldrich) in 1 ml OptiMEM for 2 h at 37 °C, 5% CO2 in order to cleave GPI-anchor structures and release GPI-anchored proteins from the cellular surface. Supernatants were subsequently harvested and further processed while cells were lysed as described below.
PNGase F and Endo H digestion
For removal as well as for investigations on processing and maturation of N-linked glycans attached to PrPC, cell lysates and/or supernatants were digested with either PNGase F or Endo H (New England Biolabs) according to the manufacturer’s protocols.
Sample preparation, TCA precipitation, cell surface biotinylation assay, SDS-PAGE and western blot analysis
N2a cells were washed with PBS and lysed with RIPA buffer, incubated on ice for 15 min before centrifugation at 12,000 g for 12 min at 4 °C. The protein content of the resulting supernatant was determined by Bradford assay. Prior to SDS-PAGE, cell lysates or brain homogenates (see above) were mixed with 4× loading buffer (including β-mercaptoethanol) and denatured for 6 min at 96 °C.
For the analysis of cell culture supernatants, experiments were carried out with serum-free media (OptiMEM). Supernatants were precipitated with trichloroacetic acid (TCA). For this, supernatants were collected and immediately incubated with already dissolved protease inhibitor cocktail, cleared from dead cells and debris by mild centrifugations at 500 g and 5.000 g for 5 min each. 1/100 volume of 2% sodium deoxycholate (NaDOC) was then added and each sample was shortly vortexed. After 30 min incubation on ice, samples were mixed with 1/10 volume of 100% TCA and again incubated for 30 min on ice. After centrifugation at 15,000 g for 15 min at 4 °C, the supernatant was aspirated, and the air-dried pellet was dissolved in 1× loading buffer and boiled for 6 min at 96 °C.
For labelling and purification of proteins at the cell surface, a surface biotinylation assay was performed as described earlier  prior to cell lysis.
For SDS-PAGE, denatured samples were loaded on either precast Nu-PAGE 4–12% Bis-Tris protein gels (Thermo Fisher Scientific) or self-made 10% or 12% SDS-gels. After electrophoretic separation, proteins were transferred to nitrocellulose membranes (BioRad) by wet-blotting and membranes were subsequently blocked for 1 h with 5% skimmed dry milk dissolved in TBS-T (containing 0.1% Tween-20) and incubated with primary antibody diluted in 5% skimmed dry milk in TBS-T overnight at 4 °C on a shaking platform. For detection of full length PrPC (fl-PrP), mouse monoclonal antibodies POM1 (1 μg/ml), POM2 (0.6 μg/ml)  or, in the case of the sortilin-1 knockout mouse brains (Fig. 5j), SAF61 (0.2 μg/ml; Bertin Pharma) were used. Proteolytically shed PrPC was detected with our new rabbit polyclonal sPrPG228 antibody (0.2 μg/ml) characterized in detail herein. Moreover, we used anti-ADAM10 (0.4 μg/ml; abcam), anti-mouse β-amyloid antibody for detection of sAPPα (1 μg/ml; BioLegend), anti-actin antibody clone C4 (MAB1501, 1:1000; Merck) and anti-Flotillin-1 clone 18 (0.25 μg/ml; BD Biosciences). Membranes were subsequently washed with TBS-T and incubated for 1 h with respective HRP-conjugated secondary antibodies and subsequently washed 6× with TBS-T. After incubation with Pierce ECL Pico or Super Signal West Femto substrate (Thermo Fisher Scientific), chemiluminescence was detected with a ChemiDoc imaging station (BioRad) and densitometrically quantified using Image Studio Lite software version 5.2 (LI-COR).
Immunofluorescence staining of surface proteins and microscopy
N2a cells were grown on glass coverslips. After washing with PBS, living cells were incubated for 20 min on ice (to avoid endocytosis) with the primary antibody dissolved in 2% BSA/PBS. Surface PrPC was detected with POM1 antibody (10 μg/ml). After several washes with PBS, cells were incubated with suitable secondary antibodies for 20 min on ice, subsequently fixed in 4% paraformaldehyde for 20 min at room temperature and mounted on glass slides with DAPI Fluoromount G (Southern Biotech). Analysis was performed using a TCS SP5 confocal microscope (Leica).
Histological and immunohistochemical stainings
Sampling, formalin fixation, paraffin embedding, hematoxilin and eosin (H&E) staining as well as immunostaining with anti-prion protein antibody SAF84 (Caiman Chemical) of murine brain samples has been described earlier . Immunostaining of shed PrP was likewise performed in one run using a Benchmark XT machine (Ventana) to allow for best comparability. In brief, deparaffinated brain sections were boiled for 1 h in citrate buffer (CC1 Cell Conditioning Solution, Ventana) for antigen retrieval and then incubated for 1 h with the sPrPG228 primary antibody (7 μg/ml; in antibody diluent solution (Zytomed) with 5% goat serum). Detection with anti-rabbit secondary antibody (Nichirei Biosciences) and Ultra View Universal DAB Detection kit (Ventana), as well as blue counterstaining were performed by the machine following standardized protocols.
Exosome isolation and nanoparticle tracking analysis
N2a cells were cultured in OptiMEM for 18 h. For the harvest of extracellular vesicles (here further referred to as exosomes), cell culture supernatants were first complemented with PI and centrifuged for 10 min at 1000 g and further at 7500 g for 15 min at 4 °C, followed by filtration through a 22 μm membrane to clear it from dead cells and debris. Exosomes were then pelleted by ultracentrifugation at 100,000 g for 70 min at 4 °C in an Optima L-100 XP using a SW40Ti rotor (Beckman Coulter, Inc.) and subsequently resuspended in PBS containing PI. For quantification and characterization, a NanoSight LM14 (Malvern Instruments) equipped with a 638 nm laser and a Marlin F033B IRF camera (Allied Vision Technologies) was used. For each sample, 10 videos of 10 s length were recorded with a camera intensity setting of 16 and analysed to assess average size and concentration of exosomes using the batch processing function of the software. For normalized western blot analysis, 5 × 1010 exosomes per sample were used.
For experiments using mouse brain samples, n refers to the number of biological samples (i.e. mice) per experimental group. For cell culture-based data, n stands for the number of independent experiments. Statistical comparison of western blot quantifications was performed using Student’s t-test and significance was considered with p-values as follows: *p < 0.05, **p < 0.005, ***p < 0.001.
A novel antibody specifically detects shed PrP and reveals important insight into the ADAM10-mediated shedding of PrPC in mice and cells
ADAM10 is the relevant sheddase of PrPC releasing a soluble form (shed PrP, sPrP) from the plasma membrane [47, 48]. Since membrane-bound full length (fl) PrPC and its shed form only differ in three amino acids (murine sequence) and the GPI-anchor, it is hard to reliably discriminate between both in most approaches. Based on available sequence information and the previous identification of the cleavage site for ADAM10 , we therefore generated an antibody specific for sPrP (sPrPG228) being directed against the newly generated carboxy-terminus at Glycine 228 (G228) exposed after cleavage (Fig. 1a).
To characterize this antibody in detail, we analyzed forebrain homogenates of age-matched Prnp0/0 mice (as negative control), recently described mice with neuron-specific (CamKIIα-driven) depletion of ADAM10 (A10 cKO; to control for specificity) as well as wild-type littermate controls, and PrPC-overexpressing tga20 mice (as positive controls) by western blot. As expected, detection with our new antibody consistently revealed no signal in Prnp0/0 samples, basal levels in wild-type mice and strongly increased signal intensity in tga20 mice (Fig. 1b). Though we expected significantly reduced levels of shed PrP in A10 cKO mice, to our surprise we could not detect any signal in these samples. Besides supporting the specificity of the antibody, this indicates that no other cell types or proteases contribute to this cleavage in brain. Re-probing the same blot with an antibody against fl-PrP revealed an increase in total PrPC levels in A10 cKO mouse brains (Fig. 1b), a finding that has been made earlier and can be attributed to the lack of shedding . Moreover, while this blotting strategy demonstrated the overlapping banding pattern (as well as the masking of sPrP signals by excess amounts of fl-PrP using common PrP antibodies), it also revealed a small shift in the molecular weight of sPrP corresponding to the lack of three amino acids and the GPI-anchor (Fig. 1b and Additional file 1). We also investigated the species specificity of the new antibody using mouse, rat and rabbit brain samples. As expected for the different C-terminal PrPC sequences, the sPrPG228 antibody only detected sPrP in brain homogenates of mice and rat (Additional file 2).
Though not being in the focus of this study, we were also interested in the applicability of the antibody in morphological analyses and performed immunohistochemical staining of paraffin-embedded mouse brain sections. As exemplified for the hippocampal area in Additional file 3, no signal was obtained in a Prnp0/0 mouse, whereas a diffuse staining was found in wild-type and, with higher intensity, in tga20 brain as could be expected for a soluble fragment distributed in the brain parenchyma.
Although, structurally, all three glycoforms of PrPC can be shed (as demonstrated in tga20 brain (Fig. 1c)), a strong predominance of the diglycosylated form of sPrP was obvious in all of our biochemical analyses. To investigate this in more detail, we analyzed the glycopattern of sPrP compared to cell-associated fl-PrP in brain homogenates of wild-type mice (Fig. 1d) and found a clear shift and a drastic increase in the proportion of diglycosylated sPrP (mean: 97 ± 1%; compared to 60 ± 4% for fl-PrP; n = 3; ±SD) with only little mono- (3 ± 1%; fl-PrP: 33 ± 3%) and almost no unglycosylated sPrP (0.07 ± 0.03%; fl-PrP: 6.8 ± 0.8%).
As a model for downregulation of ADAM10-mediated cleavage events, we investigated PrP shedding in forebrains of mice overexpressing a dominant negative form of ADAM10 (A10 d.n.) in addition to the endogenous protease  (Fig. 1e). When referring the sPrP to the respective fl-PrP signal, we found a ~ 50% reduction (mean sPrP/fl-PrP ratio: 0.51 ± 0.05; n = 4; ±SEM) in A10 d.n. mice compared to matched controls (set to 1.00 ± 0.13).
Since for main parts of this study we used N2a cells transfected with murine versions of PrPC containing the 3F4 tag in the middle of the protein sequence, we first had to show that this modification does not influence the shedding event. This is even more important as it is known, that the course of prion diseases is altered by this tag [70, 74]. We therefore decided to investigate shedding in the best possible model, i.e. in PrP3F4 knock-in (KI) mice expressing levels of 3F4-tagged PrP identical to PrPC levels in wild-type mice (Additional file 1) . No significant differences in sPrP levels were observed between controls (set to 1.00 ± 0.23; n = 3; ±SD) and PrP3F4 KI mice (0.85 ± 0.12) thus ruling out an impact of this modification on PrP shedding as could be expected from its intramolecular distance to the membrane-proximate shedding site.
We next employed the new antibody in cell culture-based experiments. Given that manipulation of PrPC shedding may become a therapeutic option in different pathologies, we investigated how pharmacological stimulation and inhibition of ADAM10 affect sPrP production in N2a cells (Fig. 1f,g). Among others, the stilbenoid resveratrol and the synthetic retinoid tamibarotene (Am80) have been successfully used to increase ADAM10-mediated cleavage events [75, 76]. We also found elevated levels of sPrP in supernatants of N2a cells treated with these substances compared to solvent-treated controls (Fig. 1f). In contrast, shedding was abolished upon treatment with the ADAM10-selective inhibitor GI254023X (GI) . Of note, upon re-probing the “supernatant blot” with another PrP antibody (POM2), a strong signal was obtained under GI-treatment indicative of a release of fl-PrP by alternative routes when shedding is blocked (as discussed later). Fittingly, cell-associated PrPC levels (in lysates) remained rather unaffected by the different treatments further supporting existence of compensatory mechanisms regulating PrPC homeostasis in N2a cells (discussed later). We also assessed the metalloprotease inhibitors GM6001 and batimastat (Fig. 1g). These drugs likewise abolished the shedding of PrPC at the cell surface yet did not significantly alter production of N1 and C1 fragments resulting from the α-cleavage of PrPC. There is controversy regarding the involvement of ADAMs in the α-cleavage (reviewed in [45, 78]). However, due to the lack of membrane permeability of the inhibitors used here, this finding cannot count as an argument against ADAMs as potential “α-PrPases”, given that α-cleavage is thought to occur mainly within the secretory pathway . Again, levels of cell-associated fl-PrP did not appear to be altered by these treatments.
Lastly, consistent with our findings in mouse brain (Fig. 1d), we also observed a changed glycopattern of sPrP compared to fl-PrP in N2a cells (Fig. 1h; diglycosylated: 84.2 ± 4.4% (sPrP) vs. 67.4 ± 0.9% (fl-PrP); monoglycosylated: 15.6 ± 4.2% (sPrP) vs. 29.6 ± 1.0% (fl-PrP); unglycosylated: 0.22 ± 0.18% (sPrP) vs. 2.9 ± 0.3% (fl-PrP); n = 3; ±SD) though relatively more monoglycosylated sPrP is found in N2a cells (15.6 ± 4.2%) than in brain (3 ± 1%; Fig. 1d). To clarify whether our findings indicate a real preference for the shedding of diglycosylated PrP or rather reflect the availability of different glycoforms at the plasma membrane, we performed cell surface biotinylation and glycopattern analysis in N2a cells (Additional file 4). Though relatively more diglycosylated PrP is indeed available at the plasma membrane (compared to total PrP levels in cell lysates; Additional file 4B), our data still argues in favor of a preference for diglycosylated PrP given the strong predominance of this form among shed PrP (Fig. 1d, h). In summary, we have generated a sensitive and highly specific antibody to discriminate between shed and fl-PrP in mouse brains and cell culture supernatants. ADAM10 on neurons seems to be the dominant (if not exclusive) PrP sheddase. ADAM10-mediated shedding of PrPC can be modulated by various means, and our shedding-specific antibody is a useful read-out tool for such experiments. Though all glycoforms can in principle be shed, diglycosylated PrP by far represents the major substrate for ADAM10.
The glycosylation state impacts on PrP shedding
Shedding is also affected by pharmacological modulation of PrPC glycosylation
Membrane anchorage and topology of PrPC determine its shedding efficiency
Immunofluorescent stainings of non-permeabilized cells showed surface expression (Fig. 4b) while western blot analysis revealed comparable expression of all constructs transfected into PrP-KO N2a cells (Fig. 4c). Deglycosylation of samples showed that all PrP mutants are subject to α-cleavage and confirmed an increase in molecular weight for PrP-TM due to its transmembrane domain (Fig. 4d). No alterations in the banding pattern were observed upon treatment with Endo H indicating correct glycosylation and –again– surface transport of all mutants (Fig. 4e). Lack of signal in the media for PrP-TM upon incubation of cells with PI-PLC proved the absence of a GPI-anchor and its attachment via a transmembrane domain (Fig. 4f).
Of note, despite a conserved shedding site in all constructs, shedding was completely abolished for PrP-TM (mean: 0 ± 0.02; n = 3; SEM) and significantly reduced for PrPGPIThy-1 (0.33 ± 0.06) compared to PrP-WT (set to 1.00 ± 0.05) (Fig. 4g,h). In conclusion, altered membrane attachment and, hence, changed membrane localization severely impact on PrPC shedding.
Shedding is part of a compensatory cellular network regulating PrPC homeostasis
Finally, we investigated how degradation, as a third aspect involved in cellular PrPC homeostasis, influences PrPC shedding. We blocked lysosomal degradation by treatment of N2a cells with leupeptin. As expected, cytosolic proteins (e.g. β-actin, β-tubulin, β-catenin) known to be degraded by the proteasome rather than in lysosomes were not accumulated (Fig. 5g). Instead, increased secretion of sAPPα, the proteolytic fragment of APP, indicated successful lysosomal inhibition (Fig. 5g). Of note, cell-associated PrPC levels remained rather stable despite this treatment (Leupt.: 0.97 ± 0.05; untr. Cells set to 1.00 ± 0.05; n = 3; SEM; Fig. 5g,h) yet shedding of PrPC was significantly increased (Leupt.: 2.31 ± 0.24; untr. Cells set to 1.00 ± 0.24; n = 3; SEM; Fig. 5g,i). No obvious differences in alternatively released PrP (Fig. 5g) suggests that, in this condition, shedding is the main contributor avoiding increased cellular PrPC levels.
Impaired lysosomal degradation and increased PrPC levels have recently been shown in mice lacking the sorting receptor sortilin-1 . As a consequence of hindered transport to lysosomes, these mice had shown increased PrPSc conversion and shortened survival when infected with prions. Given the increase in shedding upon lysosomal inhibition with leupeptin in cells shown before, we asked whether shedding of PrPC is likewise affected by the impaired degradation due to lack of sortilin-dependent transport in vivo. In fact, we found an approximately 2-fold increase for sPrP in sortilin1-deficient mice compared to controls (Sort1 KO: 1.85 ± 0.32; Sort1 WT set to 1.00 ± 0.09; n = 4; SD; Fig. 5j). Rather than from up-regulation of sheddase activity, this increase seems to result from elevated cellular PrPC levels caused by impaired degradation (Additional file 9). Nevertheless, this demonstrates the capability of ADAM10 to release increased amounts of substrate.
In summary, these data suggest ADAM10-mediated shedding as a relevant factor regulating PrPC levels. Shedding, exosomal release and degradation of PrPC may be interconnected mechanisms that act in a compensatory manner ensuring PrPC homeostasis and allowing –if at all– only subtle changes thereof.
Evolutionary conserved proteolytic processing of the prion protein has been described a quarter of a century ago [92, 93, 94] (reviewed in ). However, we are just beginning to appreciate the physiological and pathological relevance of such cleavage events, which is partially due to technical difficulties in reliable detection of the respective fragments. We here present a novel antibody that detects shed PrP with high specificity and sensitivity in different applications. Despite the existence of several valuable antibodies against various epitopes in PrPC (e.g. the POM antibodies used in this study ), until now it has only been possible to detect shed PrP upon rather labor-intensive and error-prone immunoprecipitation from or strong concentration of cell culture supernatants [47, 48]. And even that way, contribution of PrPC released from cells by other routes (e.g. via exosomes)  to respective signals has to be considered. In tissue samples it has so far been impossible to specifically detect shed PrP due to the excess amounts of cell- or extracellular vesicle-associated fl-PrPC of similar molecular weight masking any signal coming from the fraction of proteolytically shed PrP. This resulted in a lack of in vivo insight. These problems have been overcome and novel findings have been made with the new antibody.
Despite confirming antibody specificity, the absence of shed PrP in forebrain homogenates from mice with a depletion of ADAM10 in forebrain neurons to our surprise indicates that, at least under physiological conditions, no other cell types in the brain contribute to shedding in a detectable manner. It also questions a shedding of (neuronal) PrPC in trans (e.g. by adjacent glia cells not depleted of the protease), a mechanism that has been shown for the ADAM10 substrate ephrin in HEK cells . Our findings of abolished shedding in the absence of ADAM10 or upon pharmacological inhibition of ADAM10 also indicate that no other protease compensates for these manipulations in vitro or in vivo. Further support comes from mice coexpressing dominant negative ADAM10 with endogenous ADAM10, where we found a comparably strong (~ 50%) reduction in PrP shedding. Instead, previous western blot analyses of sAPPα in ADAM10 d.n. mice only showed a reduction of ~ 25% [96, 97] hinting at the known contribution of ADAM17/TACE in the cleavage of APP . It should be considered that cleavage by another protease at a slightly different cleavage site would prevent detection with our antibody. However, our previous results obtained by pull-down of shed PrP from media of primary ADAM10 knockout neurons with classical PrP antibodies , together with a recent biophysical study , and the lack of any other reported candidate protease linked to the membrane-proximate shedding of PrPC, support the view of ADAM10 as the only relevant sheddase of PrPC. This is in clear contrast to the cleavage of other typical ADAM10 substrates such as APP, which –as mentioned above– to varying degrees and dependent on the experimental paradigm, can also be processed by other proteases [71, 99, 100, 101].
Our analysis suggests that diglycosylated PrPC is the preferred glycoform to be shed by ADAM10, whereas mono- and especially unglycosylated forms seem to be relatively disfavored. Our data also indicates that this finding not simply results from differences in the availability of individual glycoforms as substrates at the plasma membrane under normal conditions (our transfected glycomutants as well as PrPC in cells treated with TM or SWA were by all means localized at the surface). An alternative explanation could be that shed diglycosylated PrP is more protected than the other shed forms from potential cellular uptake and degradation and, thus, more abundant. In any case, among all soluble PrPC fragments released from the cell by the proteolytic cleavages described to date, shed PrP is the only one that is glycosylated. As discussed earlier  this may well impact its binding affinities to both, toxic extracellular oligomers as well as physiological binding partners (e.g. surface signaling receptors), and thus define its specific biological functions. Moreover, by the predominantly diglycosylated state, physiologically shed PrP clearly differs from anchorless, mainly unglycosylated PrP of transgenic mice used in several seminal prion inoculation studies in the past [54, 102, 103, 104]. This difference has to be considered and, in the context of prion diseases, might explain why transgenic anchorless PrP efficiently converts to PrPSc and can even spontaneously form prions [54, 103], whereas shed PrP rather seems to block PrPSc formation in mice . Fittingly, the N-glycans are known to influence transmissibility and conversion to PrPSc [62, 80, 81, 82, 83].
Altered shedding efficiency for different glycoforms, however, might in part also be caused by a different sorting given that the glycans have a significant impact on the polarized trafficking of PrPC in MDCK cells . Despite a role for the N-glycans, we also demonstrated that changes in the type of membrane anchorage and, as a likely consequence, altered membrane topology affects shedding. Shifting PrPC outside of rafts by addition of a transmembrane domain [63, 89] completely abolished the shedding while the assumed re-localization of PrPC within lipid rafts via exchange of the GPI-anchor signal sequence [61, 88] reduced shedding to ~ 30% in N2a cells. The latter is in good agreement with unpublished data obtained in transgenic mice expressing the same PrPGPIThy-1 construct (Puig et al., submitted). Instead of changing the anchorage of the substrate as done here, others have changed membrane attachment of the protease . Lipid raft targeting of ADAM10 by addition of a GPI-anchor in that study severely affected APP processing. Unfortunately, processing of PrPC was not investigated there.
Since ADAM10 is mainly located outside of lipid rafts [105, 106], whereas PrPC is a resident of these microdomains, transient interaction between protease and substrate (presumably regulated by accessory proteins such as tetraspanins [107, 108, 109]) and cleavage is thought to occur at the periphery of rafts. This molecular get-together might further be supported by the capacity of PrPC to leave and re-enter lipid rafts in a constitutive manner [85, 110, 111]. Our findings also suggest an impact of the flexible N-terminal part of PrPC on the shedding efficiency. Whether this unanticipated influence is due to sterical aspects or rather reflects the role of regulatory binding partners known to especially interact with the N-terminal half of PrPC [5, 27, 112], deserves further investigations.
Our data indicate that shedding is a relevant mechanism embedded in a compensatory machinery ensuring homeostasis of PrPC. In neurons and neuronal cells, this system (involving proteolytic and exosomal release as well as trafficking to lysosomes) seems to ensure that cell-associated PrPC levels are kept stable or –at most– increase twofold upon perturbation (as indicated in some experiments of this study and observed in neurons or mice lacking the sheddase ADAM10 [48, 50] or the transport factor sortilin-1 ). Interestingly, a recent study showed that exosomal release is controlled by PrPC membrane levels . Though clearly requiring further investigation, it might be speculated that other cell types, such as fibroblasts studied here, do not possess the system to compensate for such perturbation in one of the mechanisms discussed above, and consequently accumulate PrPC to higher levels.
Manipulation of PrPC shedding is feasible and might be of therapeutic interest. Despite the challenge by possible side effects due to the broad spectrum of ADAM10 substrates, one obvious question then is into which direction to modify PrPC shedding [87, 114].
With regard to neurodegenerative proteinopathies, such as Alzheimer’s or prion diseases, stimulation of this cleavage will likely be beneficial. First, it reduces PrPC levels at the cell surface and may thereby lower neurotoxicity. Moreover, several studies showed that soluble PrP targets toxic oligomers and fibrils in the extracellular space [55, 56, 57, 58, 115]. In prion diseases, shedding efficiency inversely correlates with PrPSc formation [50, 52]. Notably, resveratrol, the drug that was used here to stimulate shedding, reduced PrPSc formation and prion infectivity in a recent study . Whether this anti-prion efficacy is indeed related to shedding, remains to be investigated. Besides proteinopathies, positive effects of stimulated shedding can also be expected given the potential role of this fragment in neurite outgrowth [13, 14] and neuroprotection [15, 49]. In that way, the role of shed PrP is reminiscent of sAPPα, the APP-derived fragment also generated by ADAM10 .
Other pathological conditions, in contrast, may rather require inhibition of PrPC shedding: it is intriguing that both, ADAM10 [118, 119] and PrPC, have been linked with immune signaling and chronic inflammatory processes [120, 121] as well as with tumorigenesis and cancer progression [122, 123, 124], where expression levels of these two proteins generally correlate with poor prognosis. Though this could well be unrelated co-incidence, it might also be speculated that these pathophysiological roles are partially related to the production of shed PrP. Of note, it is precisely shed PrP that was causally linked with chronic inflammatory neuropathology in HIV patients  and development of tumours in the central nervous system  in two recent studies. This further supports the relevance of shed PrP in different pathophysiological conditions and highlights the need for further studies on the ADAM10-mediated shedding of PrPC.
Proteolytic shedding of the prion protein has most recently attracted scientific interest with regard to diverse pathological conditions affecting the brain. Using a novel antibody for the specific detection of shed PrP, we demonstrated structural and regulatory aspects influencing this cleavage and show that it can - in principle - be pharmacologically manipulated. The latter, together with the rather ubiquitous expression of PrPC in several tissues and cell lines, as well as the lack of compensation by other proteases discussed above, also turns (i) PrPC into an ideal “control” substrate, (ii) assessment of PrPC shedding into a reliable “read out”, and (iii) our antibody into a valuable tool for any future studies investigating ADAM10-mediated cleavages and their pharmacological accessibility. With direct regard to the shedding of PrPC, both, therapeutic stimulation as well as inhibition, may be conceivable depending on the pathological context.
We thank Dr. Dirk Kamin (Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany), Kristin Hartmann (Mouse Pathology Core Unit, UKE, Hamburg, Germany) and the UKE Microscopy Imaging Facility (umif) for technical support. We apologize to all colleagues whose important contributions to this field could not be cited due to space limitations.
We are thankful for support by the Deutsche Forschungsgemeinschaft (DFG) Sonderforschungsbereich 877 (projects A12 (to HCA, PS and MG) and A3 (to PS)), the Creutzfeldt-Jakob Disease Foundation, Inc. (to HCA) and the Werner-Otto-Stiftung (to LL, BP and HCA).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. The authors will be happy to provide the sPrPG228 antibody characterized herein as research tool on reasonable request.
Conceptualization and design of the study: LL, MG and HCA; Methodology and investigation: LL, BM, SW, BP, AH, KU, HCA; Providing important research resources, materials and scientific input: WSJ, SS, KE, JT, PS, MG and HCA; Writing the original manuscript draft: LL, MG and HCA; Review and Editing: all authors; Supervision: MG and HCA. All authors read and approved the final manuscript.
No experiments on living animals were conducted for this study. However, housing and sacrification of animals as well as use of animal material in this study was in strict compliance with the Guide for the Care and Use of Laboratory Animals and ethics guidelines of the responsible local authorities.
Consent for publication
The authors declare that they have no competing interests.
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