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Mol Biol Cell. Jun 2005; 16(6): 2799–2808.
PMCID: PMC1142425

The Prion Protein and Its Paralogue Doppel Affect Calcium Signaling in Chinese Hamster Ovary Cells

Thomas Fox, Monitoring Editor


The function of the prion protein (PrPc), implicated in transmissible spongiform encephalopathies (TSEs), is largely unknown. We examined the possible influence of PrPc on Ca2+ homeostasis, by analyzing local Ca2+ fluctuations in cells transfected with PrPc and Ca2+-sensitive aequorin chimeras targeted to defined subcellular compartments. In agonist-stimulated cells, the presence of PrPc sharply increases the Ca2+ concentration of subplasma membrane Ca2+ domains, a feature that may explain the impairment of Ca2+-dependent neuronal excitability observed in TSEs. PrPc also limits Ca2+ release from the endoplasmic reticulum and Ca2+ uptake by mitochondria, thus rendering unlikely the triggering of cell death pathways. Instead, cells expressing Doppel, a PrPc paralogue, display opposite effects, which, however, are abolished by the coexpression of PrPc. These findings are consistent with the functional interplay and antagonistic role attributed to the proteins, whereby PrPc protects, and Doppel sensitizes, cells toward stress conditions.


The cellular prion protein (PrPc) is a highly conserved cell surface glycoprotein, particularly expressed in the CNS with a still unrecognized function. A conformationally modified isoform (PrPSc) of PrPc is the major component of prions, the etiological agent at the basis of fatal neurodegenerative disorders, called transmissible spongiform encephalopathies (TSEs). TSEs present as sporadic, genetic, and infectious illnesses, and include Creutzfeldt-Jakob disease (CJD) in humans, and bovine spongiform encephalopathy in cattle (Prusiner, 1998 blue right-pointing triangle).

The mechanism of PrPc conversion into PrPSc is not yet elucidated, nor it is clear if the disease progression relates to PrPSc toxic effect, or to the deprivation of PrPc functionality. The most prominent evidence in support of the former hypothesis is that most PrP-knockout mice remain viable, and do not develop spontaneous neurodegeneration (Bueler et al., 1992 blue right-pointing triangle; Manson et al., 1994 blue right-pointing triangle), even if the PrPc gene is postnatal deleted (Mallucci et al., 2002 blue right-pointing triangle). An essential role of PrPc in cell survival comes, however, from the finding that wild-type PrP transgenes abrogate the cerebellar degeneration and late-onset ataxia developed by some PrP-knockout lines over expressing a protein, named Doppel (Dpl; Sakaguchi et al., 1996 blue right-pointing triangle; Li et al., 2000 blue right-pointing triangle; Moore et al., 1999 blue right-pointing triangle, 2001 blue right-pointing triangle; Rossi et al., 2001 blue right-pointing triangle). Dpl is normally absent in the CNS of adult animals and resembles truncated PrPc; it lacks the copper-binding N-terminus (Brown et al., 1997 blue right-pointing triangle) and is structurally and biochemically similar to PrPc carboxyl end (Silverman et al., 2000 blue right-pointing triangle; Mo et al., 2001 blue right-pointing triangle). Also the role of Dpl in the CNS is obscure, but the capacity of PrPc to restore the normal phenotype of Dpl-over expressing mice has suggested that PrPc and Dpl have antagonistic functions and that PrPc suppresses a death signal triggered by Dpl.

Comparative studies using cells from wild-type or PrP-knockout animals or infected by prions also argue in favor of “the-loss-of-function hypothesis,” suggesting that PrPc protects cells by controlling the copper metabolism governing the cell resistance to oxidative stress (Brown et al., 2002 blue right-pointing triangle) or by playing an antiapoptotic role (reviewed in Hetz et al., 2003a blue right-pointing triangle). Importantly, other data from electrophysiologic studies, and/or cell Ca2+ measurements, converge in supporting that PrPc absence, or its recruitment into prions, leads to a compromised Ca2+ homeostasis and that such a defect ultimately impinges on a few Ca2+-dependent neurophysiologic functions, such as plasma membrane K+ currents, after-hyperpolarization potential (AHP) and depolarizing after-potential (DAP) events (Jefferys et al., 1994 blue right-pointing triangle; Colling et al., 1996 blue right-pointing triangle; Johnston et al., 1998 blue right-pointing triangle; Barrow et al., 1999 blue right-pointing triangle; Herms et al., 2000 blue right-pointing triangle, 2001 blue right-pointing triangle; Mallucci et al., 2002 blue right-pointing triangle).

Yet, if PrPc is involved in the correct handling of cell Ca2+, the above-reported findings are hardly surprising, given that Ca2+ is an ubiquitous intracellular messenger regulating a large amount of cell functions and that subtle alterations of endoplasmic reticulum (ER) and mitochondrial Ca2+ pools control initiation of cell death pathways. For example, both overload and depletion of the ER Ca2+ concentration ([Ca2+]) can impinge on correctly folded protein levels, thereby inducing (ER–) stress responses that may determine apoptosis (Orrenius et al., 2003 blue right-pointing triangle). Accordingly, perturbed ER Ca2+ regulation contributes to neuronal degeneration (reviewed in Mattson et al., 2000 blue right-pointing triangle; Paschen, 2001 blue right-pointing triangle), including Alzheimer's disease and TSEs (Nakagawa et al., 2000 blue right-pointing triangle; Hetz et al., 2003b blue right-pointing triangle). Also mitochondrial Ca2+ fluxes are integrated modulators of the cell Ca2+ signaling and intimately govern the cell fate. Indeed, mitochondria respond to high [Ca2+] microdomains that form in proximity of plasma membrane, and/or sarco/endoplasmic reticulum, Ca2+ channels upon cell stimulation (Brini, 2003 blue right-pointing triangle). By activating the production of ATP, such a relayed signal satisfies immediate physiological energy demands (Jouaville et al., 1999 blue right-pointing triangle). However, it can also turn into a death signal, given that excessive Ca2+ accumulation triggers abnormal mitochondrial membrane permeability (Bernardi, 1999 blue right-pointing triangle) and release of proapoptotic factors to the cytoplasm (Li et al., 1997 blue right-pointing triangle).

In view of the many implications related to the fine tuning of local Ca2+ changes, we have investigated on the involvement of PrPc in Ca2+ homeostasis, using CHO cells transiently transfected with PrPc and the recombinant Ca2+-sensitive photoprotein aequorin (AEQ) targeted to specific cell compartments. Furthermore, within the proposed functional antagonism of PrPc and Dpl, we also monitored the Ca2+ metabolism in AEQ-transfected cells over expressing Dpl, either alone or together with PrPc.


Expression Plasmids

PrPc and Dpl isoforms used in all experiments were the murine (m) and human (h) ones, respectively. The expression vector for eukaryotic cells containing the coding sequence for hDpl was constructed as in Massimino et al. (2004 blue right-pointing triangle), whereas that coding for mPrPc was constructed using a mouse genomic DNA as template, and by Prnp amplification by PCR with primers 5′ GCGCTAGCATGGCGAACCTTGGCTACTGGCTGC 3′; and 5′ CGGAATTCATCCCACGATCAGGAAGATGAG 3′. Amplified products were cut at NheI and EcoRI sites and cloned directly in the phEGFP plasmid (Clontech, Palo Alto, CA) between the same restriction sites to obtain plasmid pcDNAm-PrP. To express mPrPc carrying the epitope specific for monoclonal antibody (mAb) 3F4, L108M, and V111M point mutations were introduced in pcDNAmPrP by inverse PCR, using primers 5′ CATATGGCAGGGGCTGCGGCAGCTGGGC 3′ and 5′ CTTCATGAAGGTTTTTGGTTTGCTGGGCT 3′. The amplified product was itself ligated after T4 polynucleotide kinase treatment, to yield plasmid pmPrP3F4. The expression plasmid for the fusion protein between the green fluorescent protein (GFP) and the glycosylphosphatidylinositol (GPI) attachment signal of bovine PrPc (GFP-GPIPrP) was as described in Cereghetti et al. (2004 blue right-pointing triangle), whereas plasmids for the photoprotein AEQ targeted to the various cell compartments were obtained according to Brini et al. (1995 blue right-pointing triangle) for cytAEQ; Montero et al. (1995 blue right-pointing triangle, 2000 blue right-pointing triangle) for erAEQ; Marsault et al. (1997 blue right-pointing triangle) for pmAEQ; and Rizzuto et al. (1992 blue right-pointing triangle) for mtAEQ. Recombinant (rec) mPrP (23–230; containing the mAb 3F4 epitope) was generated in, and purified from Escherichia coli, following the method described in Negro et al. (2000 blue right-pointing triangle) for the bovine PrP isoform, whereas hDplrec (28–152) was obtained as in Cereghetti et al. (2004 blue right-pointing triangle).

Cell Cultures and Transfection

Experiments were carried out with CHO cells, cultured in 75-cm2 flasks (37°C, 5% CO2 atmosphere), using Ham's F12 medium (EuroClone, Devon, United Kingdom) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml), and streptomycin (100 μg/ml). The cells, seeded onto glass coverslips at 4.5 × 104 and 3.5 × 105 cells/cm2 for fura-2 and aequorin measurements, respectively, were transiently transfected preferentially with the Lipofectamine Plus reagent (Invitrogen, Milano, Italy), following manufacturer's instructions (in double and triple transfection experiments the quantity of each added plasmid was proportionally reduced). To optimize PrPc and Dpl expression, 6 h after transfection the medium was changed, and cells were kept at 30°C for 48 h before fura-2-, or aequorin-based, Ca2+ measurements, or immunolabeling assays. Importantly, identical results were obtained when cells were transfected using the Ca2+ phosphate-based procedure (Rizzuto et al., 1995 blue right-pointing triangle) and a total amount of 3 μg of plasmid DNA (1.5:1.5 μg in the case of double transfection). To generate CHO cells stably expressing mPrPc, the method was as described in Massimino et al. (2004 blue right-pointing triangle).

Immunocytochemistry, Western Blotting, and Densitometric Analysis

Immunocytochemistry and image analysis of intact cells, immunoblotting, and densitometric analyses were carried out as in Negro et al. (2001 blue right-pointing triangle) and Massimino et al. (2004 blue right-pointing triangle), except that 20 μg of cell lysates were loaded in each gel lane.


For immunocytochemistry and immunoblotting of PrPc and Dpl, the following antibodies were used (dilutions are given in parenthesis): anti-PrP mAb 8H4, raised against the human PrP 173–185 sequence (a kind gift of Dr. M.S. Sy, Case Western University, Cleveland, OH; 1/300 for immunolocalization; 1/8000 for immunoblotting), or mAb 3F4 (DAKO, Glostrup, Denmark; 1/100 for immunolocalization; 1/1000 for immunoblotting), which gave the same results as with mAb 8H4; and anti-Dpl mAb Dpl-79 (kindly provided by Dr. J. Grassi, Commissariat a l'Energie Atomique, Saclay, France) raised against hDplrec (28–152; 1/100 for immunolocalization; 1/1000 for immunoblotting). The immunoblotting of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and β-actin was carried out using commercially available mAbs against the ubiquitous isoform (SERCA2; clone IID8, Affinity Bioreagents, Golden, CO; 1/1000), and β-actin (Sigma, St. Louis, MO; 1/4000), respectively, and anti-calreticulin polyclonal antibody (Affinity Bioreagents; 1/1000) was used for calreticulin immunolabelings.

Ca2+ Measurements: Aequorin

Although the cytAEQ plasmid coded for the wild-type AEQ, to reduce the affinity for Ca2+, erAEQ, mtAEQ, and pmAEQ plasmids expressed aequorins carrying a mutation in one Ca2+ binding site (Montero et al., 1995 blue right-pointing triangle). Functional erAEQ was reconstituted with a modified prosthetic group (coelenterazine n, Molecular Probes, Eugene, OR), after depleting ER Ca2+ (Montero et al., 1995 blue right-pointing triangle; Barrero et al., 1997 blue right-pointing triangle). To this end, cells were incubated (1 h, 4°C) in KRB (Krebs-Ringer modified buffer: 125 mM NaCl, 5 mM KCl, 1 mM Na3PO4, 1 mM MgSO4, 5.5 mM glucose, 20 mM HEPES, pH 7.4) supplemented with ionomycin (5 μM), EGTA (600 μM), and coelenterazine n (5 μM). After extensive washings with KRB containing 2% bovine serum albumin and 1 mM EGTA, the coverslip with the cells was transferred to the thermostatted (37°C) chamber of a purpose-built luminometer. Conversely, transfected cytAEQ, mtAEQ, and pmAEQ were reconstituted by incubating cells (1–3 h, 37°C, 5% CO2 atmosphere) with wild-type coelenterazine (5 μM, Molecular Probes) in DMEM containing 1% FCS. Measurements started by perfusing cells with KRB supplemented with 1 mM CaCl2. Alternatively, pmAEQ was reconstituted in cells incubated with coelenterazine in KRB containing EGTA (100 μM). In this case, experiments started with cells in the EGTA-supplemented KRB, which was then replaced by CaCl2 (1 mM)-containing KRB. Other additions were as specified in the figures. Experiments ended by lysing cells with digitonin (100 μM) in a hypotonic Ca2+-rich solution (10 mM CaCl2 in H2O), to discharge the remaining aequorin pool. The light signal was collected and stored in an IBM-compatible computer for further analyses. Aequorin luminescence data were calibrated off-line into [Ca2+] values, using a computer algorithm based on the Ca2+ response curve of wild-type and mutant aequorins, as previously described (Brini et al., 1995 blue right-pointing triangle).


Cells, cotransfected with the phEGFP plasmid (Clontech) and either PrPc or Dpl, were loaded with fura-2 AM (5 μM, Molecular Probes) as in Malgaroli et al. (1987 blue right-pointing triangle), and the coverslip was then placed on the stage of a Zeiss Axiovert 100 epifluorescence microscope (Göttingen, Germany), equipped with a 16-bit digital CCD videocamera (Micromax, Princeton Instruments, Trenton, NJ). Samples were alternately illuminated at 340 and 380 nm, and the emitted light (filtered with an interference filter centered at 510 nm) was collected by the camera. Images were acquired using the Metafluor software (Universal Imaging, West Chester, PA). The ratio values (1 ratio image/s) were calculated off-line, after background subtraction from each single image.

Statistical Analysis

Data are reported as means ± SD; n indicates the number of experiments. Statistical differences were evaluated by Student's two-tailed t test for impaired samples, with a p value lower than 0.05 being considered statistically significant.


Localization and Expression of Transiently Transfected PrPc or Dpl

Before analyzing whether PrPc, or Dpl, affects Ca2+ homeostasis, we ascertained their correct cell distribution by immunostaining CHO cells transiently transfected with either PrPc or Dpl. As evident from the uniform labeling of the plasma membrane (Figure 1A), obtained by adding (4°C) anti-PrP-, or anti-Dpl-, mAbs to intact cells, PrPc and Dpl are exposed to the exoplasmic space, in accord with the presence of a GPI anchor in both proteins, and with similar data reported previously (Massimino et al., 2004 blue right-pointing triangle). Using dual-labeling immunocytochemistry, an identical localization of the proteins to the cell surface was observed in cells expressing PrPc, or Dpl, together with the AEQ probes, and in those cotransfected with both PrPc and Dpl (see also Massimino et al., 2004 blue right-pointing triangle; unpublished data). Figure 1B (second lanes), reporting the immunoblots of PrPc and Dpl in cells housing each protein alone, shows that the two cell types express similar protein amounts. Importantly, by applying immunocytochemistry and Western blot techniques to untransfected CHO cells, we found that no endogenous PrPc, or Dpl, was detectable over the entire experimental time period.

Figure 1.
Localization (A) and expression level (B) of PrPc and Dpl in transiently transfected CHO cells. (A) Distribution of PrPc and Dpl in intact cells expressing each protein alone, after treatment (4°C) with anti-PrP mAb 8H4 (left), or anti-Dpl mAb ...

ER Ca2+ in PrPc- or Dpl-expressing Cells

The ER is the main Ca2+ storage of the cell as it houses Ca2+-binding proteins and SERCA pumps that transport Ca2+ against a high concentration gradient. To measure the ER luminal [Ca2+] ([Ca2+]er), we used erAEQ that targets exclusively to this compartment (Montero et al., 1995 blue right-pointing triangle). To function as a Ca2+ probe, recombinant AEQ needs to be reconstituted in the active complex with its prosthetic group, coelenterazine; added to cells, coelenterazine permeates the cell membranes and combines efficiently with AEQ. However, to avoid the instantaneous consumption of the holophotoprotein by the high luminal [Ca2+]er, erAEQ-containing cells had to be first reduced in the ER Ca2+ content (by adding the Ca2+ ionophore ionomycin in the absence of external Ca2+) before reconstitution (in a Ca2+-free medium) with coelenterazine n, which is modified so as to reduce the Ca2+ affinity of AEQ (Barrero et al., 1997 blue right-pointing triangle).

Figure 2A reports ER Ca2+ measurements in parallel batches of cells transiently expressing erAEQ alone (control cells, light gray trace) or together with PrPc (black trace) or Dpl (dark gray trace). In agreement with previous reports (Brini et al., 2000 blue right-pointing triangle), ~1 min after adding CaCl2 (1 mM), the [Ca2+]er of control cells reached a steady state level close to 400 μM (390 ± 20 μM, n = 15). This value, however, is ~15% lower than that observed in the presence of PrPc, or Dpl, which was 455 ± 20 μM (n = 15) and 460 ± 20 μM (n = 15), respectively. Importantly, unless an ER Ca2+-discharging stimulus was applied, in all cases the shown Ca2+ plateau levels were stably maintained (unpublished data) and thus well represent the uppermost free Ca2+ amount that the three cell types can accumulate in the ER.

Figure 2.
Effects of the presence of PrPc or Dpl (A), and of GFP-GPIPrP (B), on the Ca2+ filling of the ER lumen and on the agonist-stimulated ER Ca2+ discharge, and effects of the presence of PrPc, or/and Dpl, on the ER membrane Ca2+ passive efflux (C), and SERCA ...

Next, we examined cells for the capacity to release Ca2+ after the addition of ATP (100 μM) at the indicated time point (Figure 2A). By activating plasma membrane P2Y receptors coupled to Gq proteins and phospholipase C, ATP induces the generation of inositol-1,4,5-triphosphate (InsP3), which promotes the discharge of the ER Ca2+ stores through the channel activity of InsP3 receptors (InsP3Rs; Streb et al., 1983 blue right-pointing triangle). Obtained results clearly demonstrate that the dynamics of Ca2+ efflux differs in the three cell types. The largest Ca2+ discharge occurs in the presence of Dpl (dark gray trace), so that the remaining [Ca2+]er (130 ± 20 μM, n = 9) is less than in PrPc-containing cells (black trace; 215 ± 20 μM, n = 7), whereas it equals that of controls (light gray trace; 110 ± 20 μM, n = 9; note, however, the lower starting [Ca2+]er in the latter cells). Yet differences regard also the kinetics of the process, in that the fastest value is exhibited by Dpl-expressing cells, whereas the rate rapidly slows down in cells with PrPc (velocity at half maximal Ca2+ discharge [V1/2]: 27 ± 3 μM/s, n = 5, in cells with Dpl; 18 ± 1 μM/s, n = 5, in cells with PrPc; 20 ± 2 μM/s, n = 5, in controls). Intriguingly, however, after ~30–40 s from the maximal Ca2+ depletion and in the continuous presence of the agonist, a partial refilling of ER stores occurred in Dpl-containing cells, whereby the final [Ca2+]er plateau settled to a value (210 ± 30 μM, n = 9), closely approximating that observed with PrPc.

Notably, we could exclude that the peculiar ER Ca2+ handling in cells with PrPc, or Dpl, is the consequence of the double transfection protocol (i.e., PrPc, or Dpl, associated with erAEQ), given that cells coexpressing erAEQ and a non-natural GPI-linked protein (GFP-GPIPrP, made of GFP fused to the GPI-attachment signal of PrPc), have the same behavior of controls (Figure 2B). Nor did PrPc, or Dpl, alter the passive permeability of the ER membrane, at least judging from the similar response (in terms of both kinetics and amplitude) in PrPc- and Dpl-containing cells to the addition of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), an inhibitor of the ER Ca2+ pump (Kass et al., 1989 blue right-pointing triangle; Figure 2C). After the demonstration that IP3-sensitive stores are fully depleted by inhibiting ER Ca2+ ATPases (Vanoevelen et al., 2004 blue right-pointing triangle), these data also suggest that the larger Ca2+ amounts accumulated by cells with PrPc (or Dpl), and the smaller IP3-induced Ca2+ efflux by cells expressing PrPc (shown in Figure 2A), are not due to the recruitment of IP3-insensitive Ca2+ compartments. This conclusion is further corroborated by the finding that, when pretreating control, and PrPc-expressing, cells with thapsigargin (an irreversible SERCA inhibitor; Thastrup et al., 1990 blue right-pointing triangle), in both cases the uptake of Ca2+ in the ER was <10% of the maximal quantity observed in thapsigargin-untreated controls (unpublished data; see also Brini et al., 2000 blue right-pointing triangle). We could also exclude that data with PrPc are biased by the mode of transfection, or the protein quantity, in that the effects of the protein on the ER Ca2+ handling are reproduced in PrPc-stably transfected CHO cells, which express the protein in lower amounts (unpublished data). Unfortunately, analysis of stably expressed Dpl molecules could not be carried out, given the impossibility, under our conditions, to establish viable clones with CHO cells.

We then examined whether the presence of PrPc and Dpl affected the expression of ER proteins such as SERCA or calreticulin. The latter protein is not only crucial for the ER Ca2+-buffering capacity (Krause and Michalak, 1997 blue right-pointing triangle), but can also regulate InsP3Rs (Arnaudeau et al., 2002 blue right-pointing triangle). We could not attribute to calreticulin the different modes of Ca2+ efflux from the ER, because the three cell types expressed the protein at similar levels (unpublished data). However, with respect to controls, we found higher and lower SERCA amounts in the presence of PrPc and Dpl, respectively (Figure 2D, lanes 1–3). Possibly, PrPc and Dpl trigger a signal that interferes with the expression of the protein in an opposed way.

Cytosolic Ca2+ in PrPc- or Dpl-expressing Cells

Having found that the presence of PrPc, or Dpl, affects the Ca2+ handling of the main intracellular Ca2+ store, we then set out to evaluate if and how the proteins influenced cytosolic Ca2+ concentration ([Ca2+]c) under resting conditions, or after cell stimulation. AEQ steep response curve precludes the accurate estimation of [Ca2+] lower than 200–300 nM (Brini et al., 1995 blue right-pointing triangle), a concentration typical of the cytosol of resting cells. This parameter was therefore determined through the image analysis of single cells loaded with the fluorescent Ca2+ indicator fura-2, after identifying successfully transfected cells by the coexpression of GFP (Brini et al., 1995 blue right-pointing triangle). We found that fura-2 signals (given by the ratio of fluorescence emitted by illuminating cells at 340 and 380 nm) detected in PrPc- or Dpl-expressing cells have a magnitude similar to that of the control (Figure 3A), suggesting that no appreciable alteration in basal [Ca2+]c is attributable to these proteins.

Figure 3.
Resting [Ca2+]c (A) and ATP-induced effects on [Ca2+]c (B and C), in CHO cells transiently expressing cytAEQ alone (control), or together with PrPc, or Dpl. (A) Resting [Ca2+]c, monitored by the Ca2+-indicator fura-2, is expressed as the ratio of the ...

Conversely, cytAEQ was used to monitor changes in [Ca2+]c after the cell stimulation by ATP. As shown (Figure 3B), addition of the agonist to cells induced a similar rise of [Ca2+]c in both control cells (light gray trace; 3.9 ± 0.4 μM, n = 19) and in those expressing Dpl (dark gray trace; 3.9 ± 0.3 μM, n = 17), whereas in cells with PrPc a statistically significant reduction of the Ca2+ transient was observed (black trace; 3.4 ± 0.4 μM, n = 18). It is to be noted, however, that under the used conditions two processes contribute to elevating [Ca2+]c; one is the InsP3-induced discharge of ER Ca2+ stores; the other is the Ca2+ influx from the extracellular space through the so-called capacitative Ca2+ entry (CCE), which becomes activated by a retrograde signal arising from the ER Ca2+ depletion (Putney et al., 2001 blue right-pointing triangle). Hence, to dissect the contribution of each individual process, cells were subjected to a time-based protocol. First, adding ATP to cells placed in a Ca2+-free medium induced Ca2+ efflux from the ER (Figure 3C). As shown, we found an identical peak transient in control cells (light gray trace; 2.7 ± 0.6 μM, n = 12) and in those with Dpl (dark gray trace; 2.7 ± 0.3 μM, n = 14), whereas a reduced value was again observed in cells with PrPc (black trace; 2.1 ± 0.3 μM, n = 12). After the transients subsided, cells were exposed to a 1 mM CaCl2-containing medium. This caused a second peak, as expected from CCE through activated store-operated Ca2+ channels (SOCC). In this case, however, no statistically significant difference was evident in the three cell types (0.92 ± 0.13 μM, n = 10 in control cells; 0.80 ± 0.09 μM, n = 7, in cells with Dpl; 0.89 ± 0.14 μM, n = 8, in cells with PrPc). Similar results were obtained with fura-2 (unpublished data).

Subplasma Membrane Ca2+ Pools in PrPc- or Dpl-expressing Cells

pmAEQ, which targets to the cytosolic rim of the plasma membrane (Marsault et al., 1997 blue right-pointing triangle), was used to monitoring the Ca2+ concentration in these restricted cytosolic domains ([Ca2+]pm; Figure 4). ATP addition to cells in the presence of 1 mM CaCl (panel A) resulted in transient [Ca2+]2 pm rises of similar magnitude in controls (light gray trace; 4.7 ± 0.8 μM, n = 12) and in Dpl-expressing cells (dark gray trace; 5.3 ± 1.5 μM, n = 9), in contrast to the almost eightfold higher [Ca2+]pm peak found in those housing PrPc (black trace; 42 ± 12 μM, n = 7). After the reestablishment of resting conditions, cells with PrPc continued to maintain a statistically significant enhanced [Ca2+]pm value (1.9 ± 0.6 μM, n = 6) than in controls (0.90 ± 0.20 μM, n = 10), although also in the presence of Dpl the basal [Ca2+]pm remained at a higher level (1.5 ± 0.4 μM, n = 7; see inset to the figure). As reported in panel B, a similar influence of PrPc and Dpl on [Ca2+]pm was evident when CCE was alternatively activated by adding CaCl2 (1 mM) to Ca2+-depleted cells (peak and steady state values were, respectively, 40 ± 12 μM, n = 11 and 6.2 ± 2.7 μM, n = 7, in cells with PrPc; 24 ± 7 μM, n = 11 and 4.5 ± 0.9 μM, n = 8, in cells with Dpl; and 22 ± 8 μM, n = 7 and 2.9 ± 0.7 μM, n = 6, in controls). Altogether, these results suggest that the recombinant expression of PrPc strongly increases Ca2+ entry from the extracellular space, whereas the attenuated effect by Dpl becomes evident only under steady state conditions.

Figure 4.
Monitoring the subplasma membrane Ca2+ concentration with pmAEQ. (A) Response to ATP (100 μM) of CHO cells transiently expressing pmAEQ alone (control, light gray trace), or together with PrPc (black trace) or Dpl (dark gray trace), and maintained ...

Mitochondrial Ca2+ in PrPc- or Dpl-expressing Cells

Compelling evidence indicates that in several cell types, including CHO cells, mitochondria behave as reporters of localized Ca2+ changes originating from the stimulation of the InsP3Rs, in that the Ca2+ rise at the channels' mouth triggers the activity of the low-affinity Ca2+ uniporter of mitochondria juxtaposed to InsP3Rs (Rizzuto et al., 1998 blue right-pointing triangle). Such a spatial link between the two organelles, which according to recent data may also involve stable physical interactions (Filippin et al., 2003 blue right-pointing triangle), ensures that mitochondria receive ER signals with maximal efficiency. To examine the influence of PrPc and Dpl on such mechanism, we used mtAEQ targeted to the mitochondrial matrix (Rizzuto et al., 1992 blue right-pointing triangle; Montero et al., 2000 blue right-pointing triangle). Intriguingly, ATP addition resulted in a mitochondrial Ca2+ signal ([Ca2+]m) that was specific for each case (Figure 5); cells with PrPc showed the lowest value (black trace; 42 ± 11 μM, n = 9), the control ones the intermediate value (light gray trace; 62 ± 15 μM, n = 7), whereas the highest Ca2+ accumulation was detected in the presence of Dpl (dark gray trace; 110 ± 15 μM, n = 10). In the latter case, the average [Ca2+]m value was more than twice that occurring with PrPc. Therefore, in line with the close coupling between quantity of Ca2+ released from the ER and Ca2+ amounts accumulated by mitochondria, these data emphasize the findings on the ER Ca2+ discharge monitored by erAEQ (see Figure 2A).

Figure 5.
ATP-induced effects on [Ca2+]m in CHO cells transiently expressing mtAEQ alone (control, light gray trace), or together with PrPc (black trace) or Dpl (dark gray trace). mtAEQ was reconstituted by incubating cells with wild-type coelenterazine (5 μM; ...

Coexpression of PrPc and Dpl Abolishes the Divergent Effects on Ca2+ Signaling Observed in Cells Expressing PrPc, or Dpl, Alone

In view of the opposite effects on local Ca2+ fluctuations elicited by PrPc and Dpl reported here and of the functional interplay of the two proteins suggested by genetic approaches (Kuwahara et al., 1999 blue right-pointing triangle; Moore et al., 2001 blue right-pointing triangle; Rossi et al., 2001 blue right-pointing triangle; Yamaguchi et al., 2004 blue right-pointing triangle), one expects that the Ca2+ signals observed in cells expressing PrPc and Dpl separately will be modified by the copresence of the proteins in the same cell. To verify this assumption, we monitored Ca2+ homeostasis in the various compartments of cells transiently cotransfected with PrPc and Dpl, after controlling that the two proteins are expressed at levels comparable to those found in singly transfected cells (Figure 1B, third lanes; note that, irrespective of the used plasmid batch(es), the transfection efficiency was generally ~35%). Indeed, we found that under this condition local Ca2+ signals are extremely close to those of control cells (Figure 6), but for the ER Ca2+ efflux rate (panel A) that after PrPc and Dpl cotransfection is faster than in controls by around 30% (V1/2 being 33 ± 3 μM/s, n = 6, in cotransfected cells; 23 ± 4 μM/s, n = 5, in controls). It is interesting to note that a similar percentage difference is also found when comparing the corresponding V1/2 values of controls and Dpl-expressing cells (Figure 2A, and text), and that cells transfected with Dpl, either alone or together with PrPc, present the lowest amounts of SERCA (third and fourth lanes of Figure 2D).

Figure 6.
Monitoring [Ca2+]er (A), [Ca2+]c (B), [Ca2+]pm (C), and [Ca2+]m (D) in CHO cells transiently expressing the corresponding AEQ alone (control, light gray trace), or together with both PrPc and Dpl (black trace). Experimental conditions were as reported ...


Several reports indicate that reduction of PrPc functionality causes perturbations in Ca2+ signaling and that the provoked alteration in some plasma membrane currents may explain aspects of TSEs, including a few neurologic symptoms (Cathala and Baron, 1987 blue right-pointing triangle) and loss of neurons (Gray et al., 1999 blue right-pointing triangle). On this background, we examined whether PrPc takes part in the complex mechanism that controls Ca2+ homeostasis by analyzing, for the first time, Ca2+ fluctuations in different compartments of cells transfected with PrPc and recombinant targeted aequorins. We found that the way cells with PrPc handle compartmentalized Ca2+ favors the cell protective role attributed to PrPc, in contrast with the effects arising from the presence of Dpl. Such a divergent activity, and the capacity of PrPc—cotransfected with Dpl—to suppress the effects elicited by Dpl alone, highlights the protective function of PrPc N-domain (absent from Dpl), as also deduced from experiments with cells challenged by serum deprivation (Sakudo et al., 2003 blue right-pointing triangle) or over expressing toxic PrP fragments (Shmerling et al., 1998 blue right-pointing triangle), proapoptotic Bax (Bounhar et al., 2001 blue right-pointing triangle), or Dpl itself (Atarashi et al., 2003 blue right-pointing triangle).

The most remarkable features observed in PrPc-containing cells regard [Ca2+]pm pools and the ER-mitochondria Ca2+ coupling. In considering the highest [Ca2+]pm found in the presence of PrPc (Figure 4), the simplest explanation is that PrPc activates SOCCs. This is important, in view of the suggestion that the capacitative entry of Ca2+ in neuronal cells may serve in signaling pathways, not only in the refilling of Ca2+ stores observed in nonexcitable cells, and that subplasma membrane Ca2+-rich domains may trigger key physiological cell events (Putney, 2003 blue right-pointing triangle). For example, SOCCs can be functionally coupled with adenylyl cyclase (Fagan et al., 2000 blue right-pointing triangle); PrPc-elicited [Ca2+]pm rises may thus represent the so-far missing intermediate linking the activation of cytosolic cAMP-dependent protein kinase A by cell surface-attached PrPcs, which was shown to inhibit apoptosis in retinal explants (Chiarini et al., 2002 blue right-pointing triangle). Also, in analogy with SOCCs modulation of transmitter release and synaptic plasticity in certain neurons (Emptage et al., 2001 blue right-pointing triangle), PrPc-elicited Ca2+ hotspots may recruit effectors in the immediate vicinity, e.g., Ca2+-activated K+ channels, thereby explaining why impairment of (Ca2+-dependent) K+ currents in PrP-null cells could not be attributed to specific alterations of voltage-gated Ca2+ channels (Herms et al., 2000 blue right-pointing triangle; but see Whatley et al., 1995 blue right-pointing triangle). In the light of all these considerations, it is thus tempting to speculate that, as observed in the used cell model system, PrPc affects SOCC-dependent subplasma membrane Ca2+ pools also in neurons. If this were the case, impairment of SOCCs could play a major role in TSE neurologic manifestations (Cathala and Baron, 1987 blue right-pointing triangle).

Equally important is the way by which PrPc-expressing cells control ER and mitochondrial Ca2+ homeostases, either of which is crucial in cell survival. Indeed, [Ca2+]er variations may trigger stress responses, whereas substantial ER Ca2+ discharges may increase mitochondrial Ca2+ levels that, by activating caspase-9, a member of the death-specific caspase family (Cryns and Yuan, 1998 blue right-pointing triangle), determine the pathway leading to apoptotic death (Li et al., 1997 blue right-pointing triangle). Hence, despite the potentially dangerous elevated [Ca2+]er (Pinton et al., 2000 blue right-pointing triangle, 2001 blue right-pointing triangle), our demonstration that the presence of PrPc limits the agonist-stimulated ER Ca2+ release (Figures (Figures2A2A and and3C)3C) and, most importantly, the ion accumulation by mitochondria (Figure 5), is not only consistent with a PrPc protective role, but it also predicts an increased cell vulnerability upon reducing PrPc molecules, and/or expression of TSE-associated PrP mutants that accumulate in the ER (Singh et al., 1997 blue right-pointing triangle; Zanusso et al., 1999 blue right-pointing triangle; Jin et al., 2000 blue right-pointing triangle; Negro et al., 2001 blue right-pointing triangle). Accordingly, one of the first events observed in neuronal cells exposed to purified PrPSc is a substantial ER Ca2+ discharge, followed by up-regulation of stress proteins and activation of the ER-resident caspase-12 that acts on other effector caspases to produce apoptosis (Hetz et al., 2003b blue right-pointing triangle).

Conversely, stimulation of Dpl-expressing cells leads to a more pronounced and faster efflux of ER Ca2+ (Figure 2A) and to a large rise in [Ca2+]m (Figure 5). These results, collected in the absence of apoptotic stimuli, disclose a possible mechanism by which Dpl sensitizes cells to environmental insults, whereas the difficulty in obtaining neuroblastoma (Massimino et al., 2004 blue right-pointing triangle), or CHO (this article), clones with Dpl suggests that the intrinsic toxicity of Dpl may manifest in cells other than Purkinje cells, the prime target of Dpl pathogenicity (Sakaguchi et al., 1996 blue right-pointing triangle). Interestingly, biochemical analyses of Dpl-over-expressing brain tissues have proposed that the protein toxicity relates to an increased oxidative damage, possibly linked to impaired copper metabolism (Wong et al., 2001 blue right-pointing triangle). These data well agree with the demonstration that, despite the capacity of Dpl to bind copper like the related PrPc C-terminus, different structural and functional roles pertain to Dpl- and PrPc-bound coppers (Cereghetti et al., 2004 blue right-pointing triangle; see also Sakudo et al., 2004 blue right-pointing triangle). However, it seems also consistent with the influence of Dpl on mitochondrial Ca2+ handling reported here, given that deregulation of the organelle Ca2+ homeostasis can lead to the overproduction of toxic reactive oxygen species (Toescu and Verkhratsky, 2003 blue right-pointing triangle).

As for the divergent ER Ca2+ handling, we found that cells with PrPc and Dpl express higher and lower amounts of SERCA, respectively, than control cells (Figure 2D). This data may explain why, irrespective of an identical Ca2+ load, the balance between InsP3R-mediated Ca2+ efflux from, and SERCA-mediated Ca2+ (re-)loading in, the ER results in a less pronounced net Ca2+ discharge with PrPc than with Dpl (Figure 2A). ER Ca2+ load, however, depends on both the amount and activity of the SERCA pumps. We have shown that in cells with Dpl CCE generates steady state [Ca2+]pm levels higher than in controls (Figure 4). It seems therefore possible to hypothesize that, in line with other observations (Mogami et al., 1997 blue right-pointing triangle), [Ca2+]pm in Dpl-expressing cells stimulates the SERCA activity, thus explaining why, despite lower SERCA amounts, also these cells maintain a higher resting [Ca2+]er than in controls (Figure 2A). Likewise, [Ca2+]pm may also account for the slow refilling of ER stores occurring after the agonist-induced maximal Ca2+ depletion (Figure 2A). An important support to the key role of [Ca2+]pm magnitude in regulating ER Ca2+ handling comes from cells housing PrPc and Dpl together; they display low SERCA levels as well as low [Ca2+]pm and are unable to exhibit the same [Ca2+]er features observed in cells with Dpl alone (Figure 6).

In accord with the suggested functional interrelationship of the proteins, PrPc and Dpl copresence abolishes Dpl effects on local Ca2+ movements (Figure 6). Several hypotheses have been suggested to explain PrPc protection against Dpl (Behrens and Aguzzi, 2002 blue right-pointing triangle): that PrPc and Dpl compete for a common ligand (in the absence of PrPc, the binding of Dpl to the ligand would elicit an apoptotic signal); that PrPc antagonizes an independent action of Dpl that sensitizes neurons to apoptosis (PrP-null cells would no longer control oxidative stress and Dpl deleterious function would predominate); and that PrPc interferes with unselective multimeric pores formed by Dpl in the ER and/or plasma membrane. With respect to the latter hypothesis, the similar passive ER Ca2+ efflux in PrPc- or Dpl-expressing cells (Figure 2C) implies that Dpl is unlikely to form (Ca2+) leak pathways in the ER membrane. On the other hand, the Ca2+ handling in cells expressing PrPc and Dpl together closely matches that of control cells (Figure 6), not that observed in the presence of PrPc alone. A possible interpretation for such a balanced effect entails that, at least in CHO cells, PrPc and Dpl have independent, opposite activities, rather than the capacity to competing for a common ligand.

In conclusion, we have shown that the recombinantly expressed PrPc governs the cell Ca2+ metabolism in a cellprotective manner, by limiting the Ca2+ accumulation into mitochondria. Yet, PrPc consistently increases [Ca2+]pm, suggesting that these elevated Ca2+ pools may contribute to regulating the (Ca2+-dependent) cell excitability and/or PrPc-mediated signaling, either of which is critical for neuronal survival.


M.B. acknowledges funding from the Ministero dell'Istruzione, dell'Università e della Ricerca (MUIR) (PRIN 2003), and M.C.S. from the European Union (BioMed.2 BMH4-CT98–6050), Telethon Onlus (E.0945), MIUR (PRIN 2004 and RBNE01S29H), and the Italian Ministry of Health (1AA/F, 2001).


This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–10–0915) on March 23, 2005.

Abbreviations used: AEQ, aequorin; cytAEQ, erAEQ, mtAEQ, and pmAEQ, aequorin targeted to the cytosol, the endoplasmic reticulum, the mitochondrial matrix and the plasma membrane, respectively; tBuBHQ, 2,5-di-(tert-butyl)-1,4-benzohydroquinone; CCE, capacitative Ca2+ entry; Dpl, Doppel; Dplrec, recombinant Doppel; ER, endoplasmic reticulum; GFP, green fluorescent protein; GPI, glycosylphosphatidylinositol; InsP3, inositol-1,4,5-triphosphate; mAb, monoclonal antibody; PrPc, cellular prion protein; PrPSc, pathological isoform of prion protein; PrPrec, recombinant prion protein; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SOCC, store-operated Ca2+ channels; TSE, transmissible spongiform encephalopathy.


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