The proteasome is a molecular complex that controls intracellular protein homeostasis by degrading misfolded and/or regulatory proteins (Kloetzel 2001; Jung et al. 2009). It is made up of the 20S-Proteasome, a central unit carrying the catalytic activities and several regulatory complexes such as PA700/19S or PA28/11S (Jung et al. 2009). The 26S-Proteasome (19S-20S-19S) is responsible for catalysis of the ATP-dependent degradation of ubiquitinated proteins (Hochstrasser 1995; Rechsteiner and Hill 2005; Jung et al. 2009). Moreover, following IFN-γ stimulation or after lipopolysaccharide injection, the constitutive catalytic subunits β1, β2, and β5 are replaced by the inducible catalytic subunits β1i, β2i, and β5i, leading to immunoproteasome formation that associates with the regulatory complex PA28/11S (Gaczynska et al. 1993; Rivett et al. 2001; Gavilán et al. 2009a). Despite the fact that the exact mechanism responsible for proteasome biogenesis is not fully understood, coordinated assembly of proteasomes requires helper factors such as the mammalian proteasome-assembling chaperones (PACS) (Hirano et al. 2005, 2006) and the proteasome maturation protein (POMP) (Burri et al. 2000; Griffin et al. 2000; Witt et al. 2000; Heink et al. 2005; Fricke et al. 2007). Age-related deficiencies in the ubiquitin proteasome system (UPS) occur in a variety of tissues including the CNS, making aged cells prone to protein accumulation (Keller et al. 2000; Carrard et al. 2002; Paz Gavilán et al. 2006). Moreover, during normal aging, and most notably during progression of neurodegenerative diseases, the proteasome works under cellular stress because of protein accumulation (Squier 2001; Keller et al. 2002). Therefore, regulated biogenesis of proteasomes is critical for adapting to changing proteolytic requirements. Recent evidence suggests that immunoproteasome induction is not only relevant to the immune response but also to the preservation of protein homeostasis under stress situations (Ferrington et al. 2008; Seifert et al. 2010). In this sense, we have previously shown that immunoproteasome content is increased in aged rat hippocampus (Gavilán et al. 2009a), a situation that could be a consequence of the age-related neruoinflammatory processes (Gavilan et al. 2007; Lynch 2010) and/or of the altered protein homeostasis (Paz Gavilán et al. 2006). In this work, we wondered whether proteasome recovery after proteasome inhibition is affected by aging process. Our results point to the existence of quantitative and qualitative differences in the proteasome recovery in aged rat hippocampus. We provide strong evidence supporting a lower rate of de novo proteasome biogenesis with a preferential immunoproteasome synthesis in aged rats under stress situation that is paralleled by a higher and sustained accumulation of ubiquitinated proteins in aged pyramidal neurons. Thus, the age-related differences in the level, composition, and dynamics of proteasome recovery could contribute to neurodegeneration induced by proteasome inhibition observed in aged rats (Gavilán et al. 2009b).
Regulation of proteasome abundance to meet cell needs under stress conditions is critical for maintaining cellular homeostasis. However, the effects of aging on this homeostatic response remain unknown. In this report, we analyzed in young and aged rat hippocampus, the dynamics of proteasome recovery induced by proteasome stress. Proteasome inhibition in young rats leads to an early and coordinate transcriptional and translational up-regulation of both the catalytic subunits of constitutive proteasome and the proteasome maturation protein. By contrast, aged rats up-regulated the inducible catalytic subunits and showed a lower and shorter expression of proteasome maturation protein. This resulted in a faster recovery of proteasome activity in young rats. Importantly, proteasome inhibition highly affected pyramidal cells, leading to the accumulation of ubiquitinated proteins in perinuclear regions of aged, but not young pyramidal neurons. These data strongly suggest that age-dependent differences in proteasome level and composition could contribute to neurodegeneration induced by proteasome dysfunction in normal and pathological aging.
proteasome maturation protein
unfolded protein response
ubiquitin proteasome system
Young (3–4 months) and aged (24–26 months) male Wistar rats were provided by the animal care facility of the University of Seville. All experiments were approved by local ethical committees and complied with international animal welfare guidelines.
Young male Wistar rats (n = 39) and aged male Wistar rats (n = 37) were processed for surgery and drug injection exactly as previously described (Paz Gavilán et al. 2006 and Gavilán et al. 2009b). LT (Sigma-Aldrich, St Louis MO, USA) was dissolved (10 mg/mL) in a solution of sterilized phosphate-buffered saline (PBS) and 1 μL was injected into both hippocampi. Animals were killed at 6, 14, 24, and 72 h. Control animals were processed similarly, but received 1 μL of sterilized PBS in both hippocampi.
Both hippocampi were dissected, frozen in liquid N2, and stored at −80°C until use. Hippocampi were homogenized in 700 μL of ice-cold sucrose buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4), supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Three hundred microliters, were separated and used for RNA isolation (see below). The remaining homogenate (400 μL) was centrifuged at 15 000 g, 60 min at 4°C and the supernatant was recovered and stored at −80°C until use. Protein concentration was determined by the Lowry method.
RNA extraction, reverse transcription, and real-time PCR
Three hundred microliters of each homogenate was used for total RNA extraction and reverse transcription. They performed exactly as previously described (Gavilán et al. 2009a). Real-time PCR were performed in an ABI Prism 7000 sequence detector (Applied Biosystems, Madrid, Spain) using cDNA diluted in sterile water as a template. Analyzed genes were amplified using specific Taqman probes supplied by Applied Biosystems. Threshold cycle (Ct) values were calculated using the software supplied by Applied Biosystems.
Proteasome activity assay
Proteasome activity was determined in hippocampal samples using specific fluorogenic substrates for the postglutamyl, trypsin and chymotrypsin activities, respectively, of the proteasome. Proteasome activity was abolished in the presence of 10 μM MG-132 (see Gavilán et al. 2009b).
Antibodies and Immunoblots
The following primary antibodies were used in this study. Rabbit polyclonal anti-ubiquitin (Dako, Glostrup, Denmark); anti-β5i subunit (Abcam, Cambridge, UK) and anti-POMP (Biomol, Madrid, Spain). Mouse monoclonal anti-β-actin (Sigma-Aldrich). Horseradish-peroxidase-conjugated corresponding secondary antibodies (Dako). Secondary antibodies conjugated to DyLight™ fluorophores (Jackson Inmunoresearch, Madrid, Spain). Immunoblots were performed as we previously described (Paz Gavilán et al. 2006 and Gavilán et al. 2009b).
Immunofluorescence and confocal microscopy
Animals were transcardially perfused with 4% paraformaldehyde and brains were processed as previously described in Gavilan et al. 2007. Sections of 25 μm were cut on a cryostat and mounted on gelatin-coated slides, permeabilized with 0.5% Triton (Sigma-Aldrich) overnight at 20°C, and then incubated with primary antibody anti-ubiquitin for 1 h at 20°C and overnight at 4°C and, finally, with the appropriate DyLight™-conjugated secondary antibodies for 1 h. Nuclei were counter-stained with 4′-6-Diamidino-2-phenylindole at a final concentration of 1 ng/μL, after secondary antibody labeling. Control staining included omission of primary antibodies or irrelevant primary antibodies of the same isotype. Then, sections were washed and coverslipped with 0.01 M PBS containing 50% glycerin and 2.5% triethylenediamine and then examined under a motorized upright wide-field microscope (LEICA DM6000B, Barcelona, Spain). Confocal images were captured using a TCS SP5 Confocal Leica laser scanning microscope equipped with a DMI60000 microscope and an HCX PL APO lambda blue 63x 1.4 oil objective at 22°C. Maximum projection image was obtained.
Statistical analysis was performed using the Statgraphics plus (v 3.1, StatPoint, Warrenton, VA, USA) software. The differences between groups in the time-course experiments were assessed by one-way anova followed by Turkey's test. The data comparison between saline- and lactacystin-injected animals was done using two-tailed t-test. The significance was set at p < 0.05. Significant differences are indicated by * when compared to saline, or by # when comparing young and aged animals.
Proteasome inhibition up-regulates the catalytic subunits of constitutive 20S proteasome in young, but not in aged rat hippocampus
As lactacystin produces an irreversible inhibition of proteasome activity (Fenteany et al. 1995), reposition of proteasome levels is essential for cell survival. Thus, we investigated in young and aged rat hippocampi, the turnover of proteasome induced by proteasome inhibition. We first analyzed the mRNA expression of the catalytic subunits β1, β2, and β5 and two structural subunits α4 and α6 of the proteasome 20S, at different times post lactacystin injection. In young rats, proteasome inhibition induced, early on, a significant and robust mRNA up-regulation of both the catalytic and structural subunits (p < 0.05; Fig 1a). By contrast, aged rats did not significantly increase the mRNA expression of the catalytic subunits, but moderately up-regulated the expression of the constitutive subunits (p < 0.05; Fig 1a). As expected (Gavilán et al. 2009a), at protein level, the basal content of proteasome subunits was significantly increased in aged compared with young saline-injected rats (Fig 1b). According to transcriptional data, young rats significantly increased the content in proteasome subunits from 24 to 72 h post lactacystin injection. However, aged rats showed a slight, but significant decrease in the content of proteasome subunits (p < 0.05; Fig 1b and c).
Proteasome inhibition up-regulates the catalytic subunits of immunoproteasome in aged, but not in young rat hippocampus
We have previously shown that aged rat hippocampus have a higher proportion of immunoproteasome probably because of the age-related neuroinflammatory processes (Gavilan et al. 2007; Gavilán et al. 2009a). Thus, we wondered whether age-dependent neuroinflammation might be modulating proteasome expression under a stress situation. To test this possibility, we analyzed transcriptional expression of the inducible catalytic subunits β1i, β2i, and β5i. As shown in Fig. 2a, proteasome inhibition in young animals did not up-regulate the mRNA expression of none of these inducible subunits. By contrast, aged animals up-regulated the mRNA expression of the inducible catalytic subunits, β1i and β5i, suggesting that age-related neuroinflammation also determines the transcriptional profile of proteasome subunits under a stress situation. At protein level, the mature β5i subunit, a marker for mature proteasomes, was exclusively observed in aged rats under both saline and lactacystin-injected conditions (see also Gavilán et al. 2009b). However, the content of β5i subunit was not increased, compared with saline-injected rats, despite transcriptional up-regulation, suggesting that proteasome biogenesis may be decreased or impaired in aged rats under proteasome stress situation (see below). Taken together, these data indicate that transcriptional regulation of proteasome subunits is quantitatively lower and qualitatively different in aged compared with young rat hippocampus.
POMP protein is strongly up-regulated in young, but weakly in aged rats upon proteasome inhibition
We further investigated whether the transcriptional and translational regulation of proteasome subunits induced by proteasome inhibition resulted in the formation of new proteasomes. For that, we searched for molecular markers of de novo synthesis of proteasome. Because the essential steps in proteasome biogenesis occur at the ER surface by a POMP-mediated mechanism (Fricke et al. 2007), we analyzed the expression of this chaperone, which is involved in de novo biogenesis of both constitutive and inducible proteasomes (Burri et al. 2000; Griffin et al. 2000; Witt et al. 2000; Heink et al. 2005; Fricke et al. 2007). As shown in Fig 3a, the basal expression of the protein POMP was significantly lower in aged than in young saline-injected rats (p < 0.05; Fig 3b), suggesting a lower basal proteasome biogenesis in aged rat hippocampus. Interestingly, proteasome inhibition in young animals induced an early and sustained expression of POMP protein (24–72 h; p < 0.05; Fig 3a and b), that was preceded by a significant transcriptional up-regulation of POMP mRNA (14–24 h; p < 0.05; Fig 3c). These data indicate that proteasome inhibition in young rats induced a coordinate active process of de novo proteasome biogenesis. By contrast, proteasome inhibition in aged rats only produced a moderate although significant increase in the expression of POMP (24 h; p < 0.05; Fig 3a and b) that returned to basal level at 72 h, probably because of the weak and short transcriptional up-regulation of POMP mRNA (just at 14 h; p < 0.05; Fig 3c).
Thus, the lower transcriptional and translational up-regulation of POMP protein observed in aged rats probably indicates a lower activity of de novo proteasome biogenesis under a stress situation.
Recovery of proteasome activity after proteasome inhibition is faster in young than in aged rats
To test whether age-related differences in POMP expression are translated into protease activity, we analyzed the activity of proteasome in young and aged rats. First, we measured the three protease activities in young and aged saline-injected rats. As shown in Fig 4a, the three proteasomal activities were significantly lower in aged than in young rats in a similar manner (p < 0.05). Paradoxically, aged saline-injected rats showed a higher amount of proteasome subunit proteins compared with young animals (see Fig 1b), suggesting that some of these proteasome subunits could correspond to non-functional proteasomes. Proteasome inhibition proportionally produced a similar decrease in the chymotrypsin activity that was recovered in young rats 24 h after lactacystin injection, but not in aged animals (Fig 4b). Taken together, biochemical and activity data strongly support that proteasome biogenesis is limited in aged rats, pointing to POMP disponibility as a potential factor responsible for this dysfunction.
Up-regulation of proteasome subunits induced by proteasome inhibition occur mainly in pyramidal neurons
We next analyzed, at cellular level, the modifications induced by proteasome inhibition. In particular, we tried to elucidate whether neurons are responsive to proteasome inhibition. For that, we investigated, by confocal microscopy, the cellular expression of proteasome subunits after proteasome inhibition by using the same anti-proteasome antibody as above (Fig 1b). As shown in Fig 5a (upper panel), proteasome immunostaining was weakly detected in young saline-injected rats, suggesting a homogeneous distribution throughout the cells. Lactacystin injection increased proteasome immunostaining mostly in the pyramidal layer of both CA1 and CA3 neurons (Fig 5a and data not shown, respectively), where somata of principal neurons are located. Interestingly, proteasome immunostaining was observed in both, cell bodies and projections at 24 h, whereas at 72 h it appears restricted to the somata of pyramidal cells. By contrast, in agreement with biochemical data, aged saline-injected rats showed a more intense proteasome immunostaining that was mainly concentrated in the pyramidal layer (Fig 5a lower panel). Lactacystin injection not only increased the intensity of proteasome immunostaining in the somata of pyramidal cells but also in other cell types distributed throughout the hippocampal parenchymal. Contrary to young animals, aged rats showed a strong labeling even 72 h post injection. As mentioned before, most of the proteasome biogenesis occurs at the ER surface (Fricke et al. 2007). Thus, the higher immunostaining observed in aged rats could correspond to proteasome partially assembled because of POMP limitation. To test this idea, we analyzed by western blot, the presence of proteasome subunits in a rich-membrane fraction that included the ER (Fig 5b upper panel). The amount of proteasome subunits in this rich-membrane fraction was lower in young than in aged saline-injected rats (Fig. 5b middle panel, see also Gavilán et al. 2009a). Importantly, lactacystin injection in young animals transiently increased the content of proteasome subunits in a similar manner as observed in neuronal somata. This could be compatible with a fast process of proteasome biogenesis and neuronal distribution of proteasome. By contrast, lactacystin injection in aged rats also increased the content of proteasome subunits, but levels remained similar from 24 to 72 h, supporting a lower rate of proteasome biogenesis and in turn retention of partially assembled proteasome at the ER surface (Fig 5b lower panel).
Ubiquitinated proteins accumulate in perinuclear regions of pyramidal neurons in aged rats
Finally, we wondered whether age-related differences in the proteasome turnover induced by proteasome inhibition affected the content of neuronal ubiquitinated proteins. As shown in Fig 6a, ubiquitin immunostaining was almost absent in young saline-injected rats (upper panel), whereas an intense ubiquitin labeling was evident in the stratum pyramidale of aged saline-injected rats (lower panel, see also Paz Gavilán et al. 2006). Proteasome inhibition in young rats produced a moderate increase in the ubiquitin immunostaining mostly in pyramidal neurons. However, a stronger labeling was observed in aged rats that did not only affect pyramidal neurons, but also other cell types distributed throughout the hippocampal parenchyma. Higher magnification revealed that accumulation of ubiquitinated proteins in cells from the stratum pyramidale mainly occurred in perinuclear structures that resemble aggresome-like structures (Fig 6b). These structures, only observed in aged neurons, increased in size after proteasome inhibition and highly resembled those previously observed in young rat hippocampus subjected to neuroinflammation and proteasome inhibition (Pintado et al. 2012), supporting the idea that neuroinflammation acts as a synergic risk factor for intracellular protein accumulation. Finally, ubiquitinated proteins and proteasome immunostaining showed a strong codistribution, leading to the formation of bigger aggresome-like structures in aged, but not in young rats (Fig 6c).
Selective degradation of proteins by the UPS is critical for maintaining cellular homeostasis. Reduced proteasome activity is observed during aging in a variety of cell tissues (Chondrogianni and Gonos 2005), indicating that a critical question for cell viability implies a correct reposition of proteasomes under cellular stress situations. Here, we have compared in rat hippocampus the dynamics of de novo proteasome biogenesis induced by irreversible proteasome inhibition in young and aged rats. Our results reveal qualitative and quantitative age-related differences in the homeostatic response induced by proteasome stress. To the best of our knowledge, this is the first study addressing this issue in vivo. One of the main findings of this work is that proteasome inhibition in young rats induced a coordinate response consisting of an early transcriptional and translational up-regulation of both constitutive and catalytic subunits of the constitutive 20S proteasome. Importantly, the increase in proteasome subunits also resulted in the recovery of proteasome activity. By contrast, aged rats did not induce the expression of the constitutive catalytic subunits, but showed a lower and delayed transcriptional and translational up-regulation of the inducible catalytic subunits and POMP that resulted in a sustained inhibition of proteasome activity. Thus, the specific bounce-back response to proteasome inhibition in aged rat hippocampus, characterized by a preferential synthesis of immunoproteasome, could be reflecting an adaptive response to the age-related alterations in cell homeostasis that seems to be conditioned by the neuroinflammatory processes and protein accumulation characteristic of aged hippocampus (Paz Gavilán et al. 2006; Gavilan et al. 2007 and Gavilán et al. 2009a; Lynch 2010; Pintado et al. 2012). This is supported by the fact that the response to proteasome inhibition observed in young rats seems to be a general feature in mammalian cells because of different in vitro studies that have reported similar coordinated up-regulation of both proteasome subunits and POMP, leading to the recovery of proteasome activity (Meiners et al. 2003; Chondrogianni et al. 2005; Radhakrishnan et al. 2010). On the basis of our results, we propose that a lower dynamics of proteasome recovery may represent a novel pathogenic mechanism contributing to the age-related neurodegeneration induced by proteasome inhibition (Gavilán et al. 2009b) that could be also relevant in neurodegenerative diseases such as Alzheimer's disease. Thus, present data add a new view supporting that control of neuroinflammation could be beneficial to limit neurodegeneration as we have previously shown (Pintado et al. 2012).
Moreover, we also provide strong evidence supporting that de novo proteasome biogenesis induced by proteasome stress is impaired in aged rats. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings (Jung et al. 2009). Coordinated assembly of proteasomes requires helper factors as POMP and the mammalian proteasome-assembling chaperones (PACs; Hirano et al. 2005, 2006) among others. POMP, that is present in precursor complexes, but absent from the mature proteasomes (Burri et al. 2000; Witt et al. 2000), facilitates the recruitment and coordinate processing of the β-subunits, thereby becoming the first substrate of the activated constitutive and inducible proteasomes (Griffin et al. 2000; Witt et al. 2000; Hirano et al. 2005). In vitro experiments have demonstrated that over-expression of POMP or PAC proteins increased the content of cellular proteasome and activity in different cell types (Hirano et al. 2005, 2006; Chondrogianni and Gonos 2007). On the contrary, RNA interference for POMP or PAC proteins caused a decrease in proteasome subunit levels and impairment of proteasome function (Hirano et al. 2005, 2006; Chen et al. 2006; Dahlqvist et al. 2012). Thus, these data indicate that levels of both POMP and PAC proteins are critical for modulating proteasome abundance. In this sense, our results revealed that aged rats showed lower levels of POMP, decreased proteasome activity, but higher amount of proteasome subunits, compared with young animals. All these data allow us to speculate that POMP could become a rate-limiting factor in the assembly of proteasomes in aged rat hippocampus. Indeed, proteasome inhibition produced a sustained accumulation of proteasome subunits codistributed with the marker of the ER Grp78/Bip, without an early recovery of proteasome activity and increased accumulation of ubiquitinated proteins (Paz Gavilán et al. 2006 and see below). By contrast, young rats strongly up-regulated POMP expression and showed a transitory codistribution of proteasome subunits with the protein Grp78/Bip. These data support an effective and rapid de novo proteasome formation in young rats, leading to minimal accumulation of ubiquitinated proteins because of the early recovery of proteasome activity (Paz Gavilán et al. 2006 and see below). Thus, unraveling the molecular mechanisms of proteasome assembly under stress situations could be beneficial for situations in which proteasome function is impaired.
On the other hand, we demonstrated that pyramidal neurons respond to proteasome inhibition in young animals. Biochemical and immunohistochemical data indicated that under proteasome stress, principal neurons activate a coordinated homeostatic response designed to promote de novo proteasome biogenesis to protect neurons from intracellular protein accumulation. This homeostatic response seems to be affected in aged neurons. In this sense, young animals displayed an early up-regulation of proteasome subunits that lead to a limited accumulation of ubiquitinated proteins in perinuclear regions. Proteasome subunits were early and simultaneously increased in both somata and neuronal projections, which could be compatible with a high rate of synthesis of proteasomes at the ER surface and its posterior cellular distribution (Fricke et al. 2007). By contrast, aged rats showed a preferential localization of proteasome immuno-staining in perinuclear regions in both basal conditions and after proteasome inhibition. This could partially correspond to immature proteasomes that accumulated because of POMP limitation, as suggested biochemical data. This speculation is also supported by the fact that, as showed before, the proteolytic capacity of proteasome was faster recovered in young than in aged rats, leading to the accumulation of ubiquitinated proteins mostly in aged hippocampal neurons, in addition to other cells (see also Paz Gavilán et al. 2006). This was not observed in young rats. However, we cannot rule out the possibility that accumulated proteasome subunits also correspond to non-functional proteasomes. The presence of aggresome-like structures, like that observed in aged rats, has been extensively described in vitro (Johnston et al. 1998; Kondratyev et al. 2007; Kaganovich et al. 2008). They seem to participate in protein quality control as general centers for cytosolic and secreted protein degradation. However, in vitro experiments also show that protein aggregates bind directly to the proteasome (Höhn et al. 2011) leading to proteasome inhibition (Sitte et al. 2000; Bence et al. 2001; Powell et al. 2005; Höhn et al. 2011). Thus, in addition to a lower transcriptional up-regulation and proteasome assembly, the slow recovery of proteasome activity could be also affected by the presence of these large aggresome-like structures that could act as natural inhibitors of neuronal proteasomes. However, we cannot rule out other possibilities such as differences in the rate of clearance of lactacystin between old and young animals. Thus, further investigations are needed to address these issues.
Finally, as mentioned before, proteasome inhibition produced a higher neurodegeneration in aged rat hippocampus (Gavilán et al. 2009b). Thus, previous and present findings allow us to speculate about the mechanisms underlying age-related neurodegeneration induced by proteasome inhibition. In this sense, perturbations that disrupt ER homeostasis, such as proteasome inhibition produce ER stress and the activation of the unfolded protein response (UPR). The UPR is efficiently activated in young animals, but is compromised in aged rats, leading to a predominant expression of proapoptotic proteins such as caspase-12, CHOP, and caspase-3 in aged rats, but prosurvival proteins in young rats (Paz Gavilán et al. 2006; Gavilán et al. 2009a,b; Naidoo 2009). Moreover, the slow recovery of proteasome activity shown here leads to a neuronal chronic accumulation of ubiquitinated proteins that could sustain the ER-stress situation, giving rise in turn, to a neurodegenerative loop potentiated by defective UPR activation. Thus, determining the mechanisms responsible for the age-related decrease in proteasome activity will be a relevant challenge. In this sense, it has been recently reported that silencing of POMP expression in cell culture resulted in decreased amounts of proteasome subunits and increased ER stress and CHOP expression (Dahlqvist et al. 2012), supporting that deficiencies in both UPR activation and proteasome recovery could have a molecular link.
Authors thank Paloma Dominguez (from CABIMER) for technical assistance in confocal microscopy and Ana Ruano for technical support. This work was supported by grant PS09/00848 (to DR) from the Carlos III Health Institute, Spain. CP was recipient of a fellowship from the Spanish Ministry of Education and Science (MEC), Spain. EG is supported by a fellowship from JA and MPG by a Juan de la Cierva contract from MICINN, Spain. The authors disclose that they have no conflicts of interest.