PrPC enhances neuronal zinc uptake

To investigate whether PrPC is involved in zinc uptake in neuronal cells, we exposed cells to zinc and measured the level of intracellular zinc using fluorescent dyes (Zinpyr-1 and Newport Green), which can be passively loaded into cells and used to detect intracellular-free (weakly bound, rapidly exchangeable) zinc. Untransfected SH-SY5Y cells, which do not endogenously express PrPC (Fig. 1a insert)20, accumulated zinc in a dose-dependent manner as measured with Zinpyr-1 (Fig. 1a). However, SH-SY5Y cells stably expressing PrPC (Fig. 1a insert) showed a significantly enhanced level of zinc-associated fluorescence (Fig. 1a). SH-SY5Y cells expressing PrPC also had a significantly enhanced rate of zinc uptake as measured kinetically using Newport Green as compared with the untransfected cells (Fig. 1b). The specificity of the Zinpyr-1 fluorescence for zinc was determined by incubation of SH-SY5Y cells expressing PrPC with other divalent cations (Mn2+, Fe2+, Ca2+ or Cu2+) before staining (Fig. 1c). Also, there was no competitive effect of either Cu2+ or Mn2+ when present in combination with zinc (Fig. 1c). Treatment with the zinc-specific chelators TPEN (N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine) or 1,10-phenanthroline before Zinpyr-1 staining resulted in a significant decrease in zinc-associated fluorescence, whereas exposure of the cells to the copper chelator bathocuproine sulphonate did not alter the Zinpyr-1 fluorescence (Fig. 1d), confirming the specificity of the Zinpyr-1 staining for zinc. Under the conditions used, zinc (at 32 μM or 100 μM) had no detrimental effect on the viability of the SH-SY5Y cells at any time point as measured by Hoecsht 33342 staining (Supplementary Fig. S1a,b) and the Promega Live/Dead assay (data not shown).

Figure 1: PrPC selectively facilitates zinc uptake. (a) Untransfected SH-SY5Y cells (Un; white bars) or SH-SY5Y cells expressing PrPC (blue bars) were exposed to Zn2+ for 6 h and then stained with 10 μM Zinpyr-1. Kruskal–Wallis, **P<0.01; ***P<0.001. Insert: detection of PrPC in the Un and PrPC-expressing cells with antibody 3F4, with actin as loading control. Molecular weight markers in kDa. (b) Kinetic analysis of the relative increase in zinc uptake using Newport Green in Un SH-SY5Y cells (black symbols) compared with SH-SY5Y cells expressing PrPC (blue symbols), exposed for up to 30 min to 32 μM Zn2+ (n=3). Kruskal–Wallis, P<0.05. The specificity of the Zinpyr-1 staining was determined by exposure of the cells expressing PrPC to (c) alternative divalent cations (100 μM) or the indicated combinations, or (d) specific cation chelators (1,10-phenanthroline (1,10 Phen) 160 μM; N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) 16 μM; bathocuproine sulphonate (BCS) 16 μM). Data shown as mean (±s.e.m.). Zinpyr-1 fluorescence corrected against DNA content (n=8). Kruskal–Wallis, **P<0.01. Full size image

To confirm a role for PrPC in enhancing zinc uptake in neuronal cells, both mouse N2a cells and rat primary hippocampal neurons were utilised in combination with small interfering RNA (siRNA) to knockdown endogenous PrPC and assess the effect of an acute loss of PrPC. Following siRNA treatment, there was a significant reduction (60±2%) in the amount of PrPC that could be detected in the N2a cells (Fig. 2a insert), and zinc uptake was significantly suppressed as evaluated by both kinetic measurements (Fig. 2a) and fluorescence microscopy (Fig. 2c). In contrast, no alteration in zinc uptake was observed in the N2a cells treated with the non-targeting siRNA control (Fig. 2b). Immunofluorescence microscopy was used to validate the knockdown of PrPC in the hippocampal neurons (Fig. 2d). Zinc uptake was also reduced in hippocampal neurons treated with the PrPC-specific siRNA (Fig. 2e; Supplementary Fig. S2), but not in the neurons treated with the non-targeting siRNA control (Fig. 2f). Zinc (32 μM) did not decrease the viability of the N2a cells or the hippocampal neurons (Supplementary Fig. S1c).

Figure 2: PrPC facilitates zinc uptake into neurons. Zinc uptake measured using Newport Green in N2a cells exposed for up to 30 min to (a) 32 μM Zn2+ (blue symbols) or to 32 μM Zn2+ following knockdown of PrPC with siRNA (green symbols; Kruskal–Wallis, P<0.05) or to (b) 32 μM Zn2+ (blue symbols) or 32 μM Zn2+ with non-targeting siRNA control (green symbols; n=6). Insert in a: expression of PrPC in untreated, PrPC-specific siRNA or non-targeting siRNA-treated N2a cells, with actin as loading control. (c) Zinc uptake visualised by fluorescence microscopy using Newport Green in N2a cells exposed to 100 μM Zn2+ (Zn) or to 100 μM Zn2+ following knockdown of PrPC expression with siRNA (Zn+siRNA). Scale bars equal 10 μm. (d) Fluorescence images of primary hippocampal neurons showing the distribution of PrPC (probed with antibody 6H4) in untreated, PrPC-specific siRNA-treated or non-targeting siRNA-treated cells. Scale bar equals 10 μm (n=10). Zinc uptake measured using Newport Green in rat primary hippocampal neurons exposed for up to 30 min to (e) 32 μM Zn2+ (blue symbols) or to 32 μM Zn2+ following knockdown of PrPC expression with siRNA (green symbols; Kruskal–Wallis, P<0.05) or to (f) 32 μM Zn2+ (blue symbols) or 32 μM Zn2+ with non-targeting siRNA control (green symbols; n=3). Data shown as the relative Newport Green fluorescence corrected against DNA content and plotted as mean±s.e.m. (g) Untransfected SH-SY5Y cells (dotted line) or SH-SY5Y cells expressing PrPC (solid line) were pre-loaded with 32 μM Zn, followed by 5 μM Newport Green for a further 30 min, to label the intracellular Zn. The change in fluorescence with time was measured on addition of fresh medium to determine whether PrPC promoted retention of Zn. (h) Cellular localisation of Newport Green fluorescence (green) and PrPC (red) in SH-SY5Y cells expressing PrPC exposed to 32 μM Zn. Scale bars equal 5 μm. Full size image

To exclude the possibility that PrPC was enhancing retention of zinc within the cell rather than increasing its uptake, untransfected SH-SY5Y cells and cells expressing PrPC were preloaded with zinc before the addition of Newport Green, and then the change in fluorescence was measured over time. As the zinc was transported out of the cells, there was a reduction in fluorescence over time; however, there was no difference in the rate by which the fluorescence changed between the untransfected and PrPC-expressing cells (Fig. 2g). Fluorescence microscopy of permeabilised cells revealed virtually no overlap in the distribution of PrPC and the Newport-Green staining for zinc (Fig. 2h). The punctuate staining for zinc reflects the way in which it is stored, as very little zinc exists free in the cytosol. Rather it is stored in at least three pools: tightly bound to a wide variety of proteins, bound to metallothioneins or accumulated in the lumen of cytoplasmic vesicles and organelles27. These data, together with the fact that PrPC is located on the outer surface of the plasma membrane or in the lumen of the secretory pathway, whereas the Newport-Green staining for zinc is predominantly in the cytosol, indicates that PrPC is not acting to directly sequester zinc inside the cell. In contrast to PrPC, the sequence-related proteins Shadoo and Doppel did not enhance the uptake of zinc into the SH-SY5Y cells (Fig. 3a,b). Together, these data, using two different zinc-selective dyes and multiple cell systems, demonstrate for the first time that PrPC selectively enhances zinc uptake into neuronal cells.

Figure 3: Neither Doppel (Dpl) nor Shadoo (Sho) facilitate zinc uptake. Zinc uptake measured using Newport Green in SH-SY5Y cells expressing (a) Dpl or (b) Sho, either not supplemented with Zn2+ (light blue symbols) or supplemented with 32 μM Zn2+ (dark blue symbols). Data shown as the relative Newport Green fluorescence corrected against DNA content and are representative of six independent experiments. Inserts show the expression of (a) Dpl or (b) Sho in the SH-SY5Y cells as detected by western blotting with DDC39 (Dpl) or anti-Sho antibodies (equal protein loading shown by detection of actin). Molecular weight markers in kDa. Un, untransfected. Full size image

The uptake of zinc does not require the endocytosis of PrPC

As zinc promotes the endocytosis of PrPC (ref. 20), we investigated whether the increase in intracellular zinc required the endocytosis of the protein. The copper-mediated endocytosis of PrPC in SH-SY5Y cells is dependent on the transmembrane low-density lipoprotein receptor-related protein-1 (LRP1)28. First, to ascertain whether the zinc-mediated endocytosis of PrPC was also dependent on LRP1, we used soluble receptor-associated protein (RAP) to inhibit the interaction between the two proteins and siRNA knockdown of LRP1 in conjunction with the established endocytosis assay involving surface biotinylation and trypsin digestion28. Co-incubation of the cells with RAP during the zinc incubation period significantly inhibited the endocytosis of PrPC, as did the siRNA-mediated knockdown of LRP1 before the addition of zinc (Fig. 4a and b), indicating that the zinc-mediated endocytosis of PrPC is also dependent on LRP1. However, blocking the endocytosis of PrPC either by siRNA knockdown of LRP1 (Fig. 4c) or with RAP (Fig. 4d) did not reduce the uptake of zinc into the cells, indicating that the endocytosis of PrPC is not the mechanism responsible for the enhanced uptake of zinc. Interestingly, both RAP and siRNA knockdown of LRP1 caused a small but significant increase in the uptake of zinc (Fig. 4c and d). It is possible that blocking the endocytosis of PrPC would cause more PrPC to be available at the cell surface to facilitate zinc uptake.

Figure 4: Zinc uptake does not require the endocytosis of PrPC. (a) SH-SY5Y cells expressing PrPC were surface biotinylated and then either left untreated, exposed to 20 μg ml−1 RAP or 2 μM LRP1 siRNA in the presence or absence of 100 μM Zn2+. Before lysis, cell-surface PrPC was removed by trypsin digestion. PrPC was immunoprecipitated from the cell lysates with antibody 3F4 and analysed by western blot with the biotin-labelled PrPC detected with peroxidase-conjugated streptavidin. (b) Quantification of the inhibitory effect of RAP and LRP1 siRNA treatment on the amount of zinc-stimulated endocytosis across multiple immunoblots from three separate experiments. Kruskal–Wallis, ***P<0.001. (c) Zinc uptake measured using Newport Green in SH-SY5Y cells expressing PrPC exposed to 100 μM Zn2+ (blue symbols) or to 100 μM Zn2+ following knockdown of LRP1 expression with siRNA (green symbols). Kruskal–Wallis, P<0.05. (d) Zinc uptake measured using Newport Green in SH-SY5Y cells expressing PrPC exposed to 100 μM Zn2+ (blue symbols) or to 100 μM Zn2+ in the presence of 20 μg ml−1 RAP (green symbols). Kruskal–Wallis, P<0.05. Data shown as the relative Newport Green fluorescence corrected against DNA content and plotted as mean±s.e.m. (n=3). Full size image

The PrPC-enhanced zinc uptake is mediated by AMPA receptors

Having excluded PrPC endocytosis as the mechanism behind the enhanced uptake of zinc, we investigated alternative routes by which zinc may be entering the cells. It has been shown previously that zinc can enter neurons through AMPA receptors10. To investigate whether AMPA receptors were involved in the PrPC-mediated zinc uptake, the SH-SY5Y cells were incubated with CNQX, a competitive AMPA/kainate receptor antagonist. CNQX significantly reduced the uptake of zinc in the cells expressing PrPC (Fig. 5a), but had no effect on the uptake of zinc in the untransfected cells (Fig. 5b). The ability of CNQX to reduce the PrPC-enhanced uptake of zinc was confirmed by immunofluorescence microscopy of the cells following exposure to Newport Green (Fig. 5c). CNQX also significantly reduced the uptake of zinc into the hippocampal neurons to a level similar to that observed following knockdown of PrPC expression by siRNA (Fig. 5d). Indeed, when the two treatments were combined, addition of CNQX did not further decrease zinc uptake beyond that observed for the siRNA knockdown of PrPC (Fig. 5e). We also explored the effect of direct activation of AMPA receptors on zinc uptake. The siRNA knockdown of PrPC in the hippocampal neurons ablated the AMPA-mediated increase in zinc uptake (Fig. 5f). Together, these data suggest that the PrPC-enhanced zinc uptake is mediated by AMPA receptors.

Figure 5: PrPC-enhanced zinc uptake requires AMPA receptors. Zinc uptake measured using Newport Green in (a) SH-SY5Y cells expressing PrPC or in (b) untransfected SH-SY5Y cells exposed to 32 μM Zn2+ (blue symbols) or to 32 μM Zn2+ and 10 μM CNQX (green symbols). Kruskal–Wallis, P<0.05 in a. (c) Zinc uptake visualised using Newport Green in SH-SY5Y cells expressing PrPC exposed to 32 μM Zn2+ or to 32 μM Zn2+ in the presence of 10 μM CNQX. Scale bars equal 10 μm. (d) Zinc uptake measured using Newport Green in rat primary hippocampal neurons exposed to 32 μM Zn2+ (blue symbols), to 32 μM Zn2+ and 10 μM CNQX (green symbols), or to 32 μM Zn2+ following knockdown of PrPC expression with siRNA (red symbols). (e) Zinc uptake measured using Newport Green in rat primary hippocampal neurons exposed to 32 μM Zn2+ (blue symbols) or to 32 μM Zn2+ following knockdown of PrPC expression with siRNA (green symbols), or 32 μM Zn2+ and 10 μM CNQX following knockdown of PrPC expression with siRNA (red symbols). (f) Zinc uptake measured using Newport Green in rat primary hippocampal neurons exposed to 32 μM Zn2+ in the presence of 100 μM AMPA (green symbols) following knockdown of PrPC expression with siRNA (red symbols), or to 100 μM AMPA following knockdown of PrPC expression with siRNA (purple symbols). Data shown as the relative Newport Green fluorescence corrected against DNA content and plotted as mean±s.e.m. (n=3). Full size image

Mechanism of PrPC-mediated zinc uptake via AMPA receptors

To characterise further the role of AMPA receptors in the PrPC-enhanced zinc uptake, the Newport-Green fluorescence was measured in the SH-SY5Y cells expressing PrPC and in the hippocampal neurons exposed to either IEM-1460 or pentobarbital, which inhibit GluA2-lacking or GluA2-containing AMPA receptors, respectively. Surprisingly, both IEM-1460 (Fig. 6a,b) and pentobarbital (Fig. 6c,d) diminished the PrPC-mediated zinc uptake. Previously, PrPC has been shown to interact with the GluA2 subunit of AMPA receptors29. To confirm this observation and to determine whether PrPC also interacts with the GluA1 subunit, immunoprecipitation from mouse brain homogenate using antibodies against PrPC, GluA1 and GluA2 was performed and the resulting immunoprecipitates western blotted with antibodies against each protein. Both GluA1 and GluA2 co-immunoprecipitated with PrPC, and PrPC co-immunoprecipitated with both GluA1 and GluA2 (Fig. 6e). No co-immunoprecipitation was observed using Sepharose beads only, or with beads coated with a non-specific IgG (Fig. 6e) or using brain homogenate from a PrP-null mouse (Fig. 6f). In addition, PrPC increased the cell surface expression of the GluA1 subunit (Fig. 6g,h).

Figure 6: PrPC interacts with AMPA receptor subunits. Zinc uptake measured using Newport Green in SH-SY5Y cells expressing PrPC (a,c) and in rat primary hippocampal neurons (b,d) exposed to 32 μM Zn2+ (blue symbols) or to 32 μM Zn2+, and (a,b) 10 μM IEM-1460 (green symbols) or (c,d) 100 μM pentobarbital (green symbols). Data shown as the relative Newport Green fluorescence corrected against DNA content and plotted as mean±s.e.m. (n=3). Kruskal–Wallis, P<0.05. Co-immunoprecipitation from (e) wild-type 129/P2 or (f) PrP−/− 129/P2 mouse brain using antibodies against PrPC (6H4), GluA1, GluA2, control rabbit anti-mouse IgG or no antibody for the bead-only control, and then probed with antibodies against PrPC (SAF32), GluA1 or GluA2 as indicated. (g) Cell lysates from either untransfected (Un) SH-SY5Y cells or those expressing PrPC were analysed for their expression of PrPC and GluA1. Actin was included as a loading control. Cell monolayers were treated with cell-impermeant biotin to determine the amount of GluA1 at the cell surface. GluA1 was immunoprecipitated from the lysates and the biotin-labelled GluA1 detected using peroxidase-conjugated streptavidin. (h) Cell surface GluA1 was quantified from multiple immunoblots (n=3). Kruskal–Wallis, *P<0.05. Full size image

To explore further the molecular mechanism underlying the PrPC-mediated zinc uptake via AMPA receptors, we determined the regions of PrPC involved in (i) the uptake of zinc and (ii) the interaction with AMPA receptors. Cells expressing wild-type PrPC were incubated with epitope-specific antibodies before exposure to zinc. Incubation of the cells with antibody SAF32, which recognises an epitope encompassing amino acids 59–89 contained within the octapeptide repeat region, significantly reduced the uptake of zinc (Fig. 7a), whereas incubation of the cells with antibody 8H4, which recognises residues 175–185, had no effect on the PrPC-mediated increase in zinc uptake (Fig. 7a). Incubation of the cells with a non-specific antibody (against the transferrin receptor) had no effect on the uptake of zinc (Fig. 7a). Antibody SAF32, but not the transferrin-receptor antibody, also significantly reduced the uptake of zinc in the hippocampal neurons (Fig. 7b). To explore further the role of the metal-binding octapeptide repeats of PrPC in the uptake of zinc, we utilised cells expressing PrPΔOct, in which the octapeptide repeat sequence has been removed20. The Zinpyr-1 fluorescence in the SH-SY5Y cells expressing PrPΔOct (Fig. 7c insert) following exposure to zinc was similar to that of the untransfected cells (Fig. 7c). Next, we investigated whether the octapeptide repeats were required for the interaction with AMPA receptors. PrPΔOct still co-immunoprecipitated with the GluA2 subunit (Fig. 7d), and wild-type PrPC co-immunoprecipiated with both GluA1 and GluA2 following chelation of zinc (Fig. 7e). To explore further the region of PrPC involved in the interaction with the AMPA receptor, we utilised a construct of PrPC, PrPΔN, which lacks the four amino acids (lysyl-lysyl-arginyl-proline) from the N terminus of the mature protein30. PrPΔN is trafficked to the cell surface, localised to detergent-resistant lipid rafts and undergoes endoproteolytic cleavage to generate the C1 fragment similarly to wild-type PrPC (refs 30, 31, 32). However, PrPΔN failed to enhance the uptake of zinc into cells (Fig. 7c) and also failed to co-immunoprecipitate with the GluA2 subunit (Fig. 7d). These data indicate that the metal-binding octapeptide repeats in PrPC are required for the enhanced zinc uptake but not for the interaction with the AMPA receptor, whereas the N-terminal polybasic region of the protein is critical for the interaction with the AMPA receptor.

Figure 7: The N terminus of PrPC is required for the interaction with AMPA subunits. (a) Zinc uptake measured using Newport Green in SH-SY5Y cells expressing wild-type PrPC exposed to 32 μM Zn2+ (blue symbols), 32 μM Zn2+ and 10 μg ml−1 antibody SAF32 (green symbols), 32 μM Zn2+ and 10 μg ml−1 antibody 8H4 (red symbols) or 32 μM Zn2+ and 10 μg ml−1 anti-transferrin receptor antibody (purple symbols). (b) Zinc uptake measured using Newport Green in rat primary hippocampal neurons exposed to 32 μM Zn2+ (blue symbols), 32 μM Zn2+ and 10 μg ml−1 antibody SAF32 (green symbols) or 32 μM Zn2+ and 10 μg ml−1 anti-transferrin receptor antibody (red symbols). Data shown as the relative Newport Green fluorescence corrected against DNA content and are plotted as mean±s.e.m. (n=3). Kruskal–Wallis, P<0.05 for SAF32 compared with no antibody. (c) Untransfected (Un) SH-SY5Y cells or SH-SY5Y cells expressing either wild-type PrPC, PrPΔOct or PrPΔN were exposed to 100 μM Zn2+ and stained using 10 μM Zinpyr-1. Data shown as mean (±s.e.m.). Zinpyr-1 fluorescence corrected against DNA content (n=8). Insert shows expression of wild-type PrPC, PrPΔOct and PrPΔN with actin as a loading control. Molecular weight markers in kDa. (d) Co-immunoprecipitation from lysates of SH-SY5Y cells expressing PrPΔOct or PrPΔN using antibodies against PrPC (6H4), GluA2, control rabbit anti-mouse IgG or no antibody for the bead-only control, and then probed with antibodies against PrPC (6D11 for PrPΔOct or SAF32 for PrPΔN) or GluA2 as indicated. (e) Co-immunoprecipitation from wild-type Ola/P2 mouse brain using antibodies against PrPC (6H4), GluA1, GluA2, control rabbit anti-mouse IgG or no antibody for the bead-only control with 20 μM TPEN included in all buffers and washes. Samples were probed with antibodies against PrPC (SAF32), GluA1 or GluA2 as indicated. Full size image

Tyrosine phosphatase activity is increased in PrPC-null mice

Intracellular protein tyrosine phosphatase activity is exquisitely sensitive to zinc4,5. Therefore, to determine whether the increased uptake of zinc observed in cells expressing PrPC affected a cellular event, we measured the tyrosine phosphatase activity in the cells. In agreement with previous reports4,5, zinc directly inhibited phosphatase activity when added to the SH-SY5Y cell lysate (Supplementary Fig. S3). In the cells expressing PrPC, there was a significant reduction of phosphatase activity as compared with the untransfected cells (Fig. 8a), indicating that the enhanced uptake of zinc by PrPC has a downstream effect on a cellular process. To confirm that PrPC disrupted zinc homeostasis in vivo, we measured the tyrosine phosphatase activity in the brains of wild-type and PrPC-null mice. Consistent with a role for PrPC in neuronal zinc uptake, there was a higher level of tyrosine phosphatase activity in the brains of the PrPC-null mice compared with the wild-type, age-matched control mice (Fig. 8b). These data, for the first time, indicate that PrPC is involved in the physiological homeostasis of neuronal zinc.

Figure 8: Protein tyrosine phosphatase activity is enhanced in PrPC-null mice and zinc uptake is reduced in prion disease. (a) Lysates from untransfected SH-SY5Y cells (Un) or cells expressing PrPC were incubated with phosphopeptide for 45 min at 37 °C to measure tyrosine phosphatase activity. Data shown as mean (±s.e.m.) (n=3). Independent Student's t-test, **P<0.01. (b) Tyrosine phosphatase activity was measured in whole-brain homogenate from wild-type 129/P2 (WT) or 129/P2 PrP−/− mice. Data shown as mean (±s.e.m.; n=8). Independent Student's t-test, *P<0.05. (c) SH-SY5Y cells expressing the various disease-associated mutants of PrPC were exposed to 100 μM Zn2+ and stained using 10 μM Zinpyr-1. Data shown as mean (±s.e.m.). Zinpyr-1 fluorescence corrected against DNA content (n=8). Kruskal–Wallis, **P<0.01. Insert shows expression of the various disease-associated mutants of PrPC. Lane 1, wild-type PrPC; lane 2, PG14; lane 3, A116V; lane 4, P101L; lane 5, D177N/M128; and lane 6, D117N/V128. Molecular weight marker in kDa. (d) Co-immunoprecipitation from P101 L, D177N (M128), D177N (V128) mouse brain or SH-SY5Y cell lysates expressing PG14 or A116V using antibodies against PrPC (6H4), control rabbit anti-mouse IgG or no antibody for the bead-only control, and then probed with antibodies against PrPC (SAF32), GluA1 or GluA2 as indicated. Zinc uptake measured using Newport Green in (e) uninfected N2a cells and (f) scrapie-infected ScN2a cells, either not supplemented with Zn2+ (light blue symbols) or supplemented with 32 μM Zn2+ (dark blue symbols). Data shown as the relative Newport Green fluorescence corrected against DNA content and plotted as mean±s.e.m. (n=3). Kruskal–Wallis, P<0.05 in the uninfected cells. Full size image

Enhanced uptake of zinc is lost in prion disease

As zinc is reduced in the brain in prion disease33,34, and there is debate whether prion diseases involve a loss of a normal function of PrPC in addition to the toxic gain-of-function14,15, we investigated the effect of a number of prion disease-associated mutations in PrPC on zinc uptake. PG14 has an additional nine octapeptide repeats and results in familial CJD35, whereas A116V and P101L are both single point mutations, which cause the Gerstmann–Straussler–Scheinker syndrome (GSS)36,37. D177N with valine at position 128 (D177N/V128) also results in GSS, whereas with methionine at position 128 (D177N/M128) causes familial fatal insomnia38. All mutants were stably expressed in SH-SY5Y cells to similar levels (Fig. 8c insert). In contrast to wild-type PrPC, none of the five disease-associated mutants of PrPC enhanced zinc uptake above that seen in the untransfected cells (Fig. 8c). To investigate the mechanism underlying the lack of zinc uptake in the disease-associated mutants of PrPC, we determined whether they co-immunoprecipitated with the AMPA receptor. Neither of the D177N mutants nor P101L interacted with the GluA1 or GluA2 subunits (Fig. 8d), indicating that in these disease-associated mutants of PrPC the mechanism underlying the inability to enhance zinc uptake may be due to loss of the interaction with AMPA receptors. Although the PG14 and A116V mutants still co-immunoprecipitated with the GluA2 subunit, in the cells expressing these two mutants we observed that the level of the GluA1 subunit was significantly reduced (Fig. 8d and Supplementary Fig. S4), suggesting that this likely underlies the inability of the PG14 and A116V mutants to increase zinc uptake. Finally, to determine whether zinc uptake was affected upon the conformational conversion of PrPC to PrPSc, zinc uptake was measured in persistently scrapie-infected ScN2a cells. Although a significant increase in zinc uptake was measured in uninfected N2a cells following exposure to exogenous zinc (Fig. 8e), no such increase was measured in the scrapie-infected ScN2a cells (Fig. 8f). These data indicate that the PrPC-mediated zinc uptake is lost in cells expressing prion disease-associated mutants of PrPC and upon conversion of PrPC to PrPSc.