CB2 Receptors and Neuron–Glia Interactions Modulate Neurotoxicity Generated by MAGL Inhibition

Monoacylglycerol lipase inhibition (MAGL) has emerged as an interesting therapeutic target for neurodegenerative disease treatment due to its ability to modulate the endocannabinoid system and to prevent the production of proinflammatory mediators. To obtain a beneficial response, it is necessary to understand how this inhibition affects the neuron–glia crosstalk and neuron viability. In this study, the effect of MAGL inhibition by KML29 was evaluated in two types of rat cortical primary cultures; mixed cultures, including neuron and glial cells, and neuron-enriched cultures. The risk of neuronal death was estimated by longitudinal survival analysis. The spontaneous neuronal risk of death in culture was higher in the absence of glial cells, a process that was enhanced by KML29 addition. In contrast, neuronal survival was not compromised by MAGL inhibition in the presence of glial cells. Blockade of cannabinoid type 2 (CB2) receptors expressed mainly by microglial cells did not affect the spontaneous neuronal death risk but decreased neuronal survival when KML29 was added. Modulation of cannabinoid type 1 (CB1) receptors did not affect neuronal survival. Our results show that neuron–glia interactions are essential for neuronal survival. CB2 receptors play a key role in these protective interactions when neurons are exposed to toxic conditions.


Lipofectamine Transfection of Rat Cortical Primary Cultures and Drug Treatments
At DIV5, mixed rat cortical primary cultures (-AraC) or neuron-enriched (+AraC), were transfected with plasmids expressing green fluorescent protein (GFP) or mCherry fluorescent proteins (pCAGGs-GFP, pCAGGs-mCherry plasmid [40]) using lipofectamine 2000 transfection reagent (Invitrogen Carlsbad, CA, USA). One hour before transfection, the culture medium was replaced with Neurobasal without FBS. For lipofectamine complex formation, the plasmids (1 µg/well of 24-well plate) and the lipofectamine reagent were diluted in Opti-MEM medium and mixed in equal volumes. After incubating at room temperature (RT) for 20 min, 50 µL of the mixture were added to each well and plates were incubated for 2 h at 37 • C. Then cells were washed and the medium was replaced by Neurobasal supplemented with 1% FBS, 1% GlutaMAX (Gibco), 2% B27 supplement (Gibco), 2 mg/mL gentamicin and 0.25 mg/mL fungizone. KML29 250 nM (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in 1.5% DMSO, 5% PEG, 5% TWEEN 80 and added starting at DIV6 until DIV12 every 48 h. Rimonabant 200 nM (Cayman Chemical, Ann Arbor, MI, USA) and AM630 1 µM (Tocris Bioscience, Bristol, UK) were dissolved in DMSO and added starting at DIV6 until DIV12 every 48 h. The vehicle of each treatment was added as control.

Immunofluorescence
Cells grown in coverslips were fixed 2 days after AraC treatment by replacing the medium with 4% paraformaldehyde (Panreac, Darmstadt, Germany) and 4% sucrose dissolved in phosphate-buffered saline (PBS) for 8 min. Then, cells were washed twice with PBS and kept at 4 • C until immunostaining. For immunofluorescence, cells were first permeabilized with PBS and 0.1% Tx-100 (PBT) for 20 min at RT and followed by incubation in 1 M glycine (Bio-Rad, Hercules, CA, USA) for 20 min at RT. Nonspecific binding was blocked with 3% goat serum and 3% BSA (Millipore) dissolved in PBT for 1 h. Next, primary antibodies were incubated in blocking solution for 2 h at RT (Table 1).
After 3 washes in PBS, the corresponding secondary antibody diluted in blocking solution was added, incubated for 2 h at RT and washed again 3 times. Finally, cells were incubated with DAPI (Gibco; 1:50,000 in PBS) for 5 min and washed 3 times with PBS. Coverslips were removed and placed on microscope slides on an 8 µL drop of mounting medium (25 mg of 1,4-diazabicyclo [2.2.2]octane) (DABCO; Sigma) per milliliter of Permafluor (Thermo Scientific, Waltham, MA, US). The preparations were dried for 30 min at 37 • C and kept at 4 • C until they were visualized.

RNA Extraction and PCR Reaction
Cells were plated at a density of 2 × 10 6 per well in 6-well plates (Corning), at appropriate times the culture medium was replaced by 1 mL of Trizol (Sigma) and RNA was purified following the manufacturer's instructions, including treatment with 2 U of DNase I (Thermo Fisher Scientific, Waltham, MA, USA), 40 U of Recombinant RNase inhibitor (Takara Bio, Shiga, Japan) and 25 mM EDTA in a final volume of 20 µL. When RNA was extracted from sorted cells, the manufacturer's instructions were followed in a Maxwell RSC 48 (Promega). Reverse transcription of RNA (2 µg) was performed with 400 U Superscript IV (Thermo Fisher Scientific), 40 U Recombinant RNase inhibitor (Takara Bio Inc., Shiga, Japan) and 5 µM random oligodeoxyribonucleotides hexamers

Automated Image Acquisition
Transient transfections with fluorescent-tagged proteins allow us to track individual neurons in culture over long periods of time [39,41]. Neuronal survival was studied by automatic longitudinal tracking of neuronal cultures every 24 h after transfection with GFP on a Zeiss Observer Z1 microscope as previously described [40]. Transfected primary neurons were placed on a Zeiss Observer Z1 microscope that maintains stable conditions for temperature and CO 2 (37 • C and 5% CO 2) . Images were acquired automatically at determined positions (designated with particular spatial coordinates) with the 10× long distance objective using the Zen System software (Zeiss). The software enables a sequential and automated repetition of a series of tasks such as locating a particular neuronal field, automatic focusing and image acquisition, and moving forward to the next non overlapping neuronal field. This allows fast and efficient scanning of multiple neuronal fields per plate. Once the full set of images has been acquired, the plate is returned to the incubator until the next scanning. For a typical survival experiment, 10 positions per well and 4 wells per condition were used. Positions were chosen randomly and the selection of neurons to analyze was therefore unbiased. To track the same neuronal fields, a template with the same initial spatial positions was used through the experiment.

Image Processing and Statistics
GraphPad Prism 5 software was used to obtain the graphs. For survival experiments, Matlab-based semi-automated ad hoc programs were developed to estimate the survival times (in hours) of individual neurons in the images acquired. Specific details about how the programs work were previously described [40]. Dead neurons identified along the experiment were categorized as uncensored events. Neurons that survive until the end of the experiment are categorized as censored events. Further survival analysis of the data was performed with STATA 12. The Nelson-Aalen cumulative hazard function was used to estimate and plot cumulative risks of death of different experimental conditions. Differences between groups were analyzed with the log-rank test. In the case of immunofluorescence experiments, 6-10 images per experiment were obtained with a 63× objective on a Zeiss Axiovert 200M fluorescence microscope (Zeiss, Oberkochen, Germany). Image acquisition and processing were performed using MetaMorph Microscopy Automation and Image Analysis Software (Molecular Devices, San Jose, CA, USA). The number of positive cells with respect to the total number of DAPI-stained nuclei or the number of transfected cells with respect to a specific type of cell was calculated.

Glial Cells Are Necessary for Neuronal Survival
We first evaluated whether glial cells influence neuronal survival by longitudinal survival analysis. This methodological approach in primary neurons requires individual identification and tracking of single neurons. A typical experiment involves transient transfection of primary neurons with plasmids expressing fluorescent proteins such as green fluorescent protein (GFP). Fluorescent neurons are then tracked longitudinally with automated microscopy and the survival time (the last time each neuron was observed alive) is estimated for each individual neuron in the experiment ( Figure 1A). Statistics for survival analysis are applied to analyze the data and to quantitatively determine and compare the risk of neuronal death under different experimental conditions [39,40,42].
Here, we compared the risk of death of rat cortical neurons in primary mixed cultures (containing glial cells: astrocytes, oligodendrocytes and microglia) or in enriched neuronal cultures treated with AraC (an inhibitor of glial proliferation). A detailed characterization of mixed and enriched primary cultures was performed using specific antibodies to identify neurons (MAP2), oligodendrocytes (Olig2), astrocytes (GFAP) and microglia (Ox42) by immunofluorescence ( Figure 1B). Quantification of the percentage of each cellular type revealed an enrichment of neurons in primary cultures treated with AraC ( Figure 1C). Furthermore, the efficiency of neuronal transfection in mixed cultures was analyzed. Immunofluorescence experiments of mixed cultures transfected with a red fluorescent protein mCherry (Ch) indicated that most of the transfected cells were MAP2 positive cells or neurons (around 91%) with a small percentage of glial cells (approximately 2% astrocytes and 1% oligodendrocytes) ( Figure 1D). 1B). Quantification of the percentage of each cellular type revealed an enrichment of neurons in primary cultures treated with AraC ( Figure 1C). Furthermore, the efficiency of neuronal transfection in mixed cultures was analyzed. Immunofluorescence experiments of mixed cultures transfected with a red fluorescent protein mCherry (Ch) indicated that most of the transfected cells were MAP2 positive cells or neurons (around 91%) with a small percentage of glial cells (approximately 2% astrocytes and 1% oligodendrocytes) ( Figure 1D). Then, GFP-transfected neurons from mixed (−AraC) and enriched cultures (+AraC) were longitudinally tracked with automated microscopy (Figure 2A,B). We observed that neurons from enriched cultures in which glial cells were removed with AraC exhibited a higher risk of death than neurons in mixed cultures ( Figure 2C). This result indicates that glial cells present in mixed cultures favor neuronal survival.

Glial Cells Counteract the Neurotoxic Effect of MAGL Inhibition through CB2 Receptors.
Next, we evaluated the effect of MAGL inhibition on neuronal survival using KML29, a secondgeneration MAGL inhibitor that shows higher potency (IC50 = 43 nM) than JZL184 (IC50 = 262 nM) for the rat enzyme inhibition [43]. Starting at DIV6, GFP-transfected neurons from mixed and enriched

Glial Cells Counteract the Neurotoxic Effect of MAGL Inhibition through CB2 Receptors.
Next, we evaluated the effect of MAGL inhibition on neuronal survival using KML29, a second-generation MAGL inhibitor that shows higher potency (IC 50 = 43 nM) than JZL184 (IC 50 = 262 nM) for the rat enzyme inhibition [43]. Starting at DIV6, GFP-transfected neurons from mixed and enriched cultures were treated with 250 nM KML29 every 48 h and subjected to longitudinal survival analysis ( Figure 3A). MAGL inhibition significantly increased the risk of death in neurons from enriched primary cultures, but neurons in mixed cultures were not affected. This result strongly suggests that the presence of glial cells counteract the neurotoxic effect of MAGL inhibition with KML29 ( Figure 3B). CB1 and CB2 receptors are potential candidates through which this glial-dependent counteractive effect could be mediated. In particular, CB2 receptors that are preferentially expressed in glial cells.
Biomolecules 2020, 10, x 9 of 16 cultures were treated with 250 nM KML29 every 48 h and subjected to longitudinal survival analysis ( Figure 3A). MAGL inhibition significantly increased the risk of death in neurons from enriched primary cultures, but neurons in mixed cultures were not affected. This result strongly suggests that the presence of glial cells counteract the neurotoxic effect of MAGL inhibition with KML29 ( Figure  3B). CB1 and CB2 receptors are potential candidates through which this glial-dependent counteractive effect could be mediated. In particular, CB2 receptors that are preferentially expressed in glial cells. Thus, we asked whether the activation of CB1 and CB2 receptors was involved in the glialdependent protective effect against MAGL inhibition by KML29. First, we analyzed the mRNA expression of CB1 and CB2 receptors by PCR in mixed and enriched neuronal cultures at DIV6 and DIV12. In accordance with its cellular expression pattern, CB1 receptor transcripts were present in both types of cultures. By contrast, CB2 receptor mRNA expression gradually increased in mixed cultures from DIV6 to DIV12 but was not present in neuronal-enriched cultures ( Figure 4A). We attributed the increase in CB2 receptor expression to the proliferation of glial cells overtime. To further determine the involvement of these receptors in the glial-dependent protective effect against MAGL inhibition, GFP-transfected neurons from mixed and enriched primary cultures were treated with Rimonabant, a CB1 receptor antagonist, or AM630, a CB2 receptor antagonist, and subjected to longitudinal survival analysis ( Figure 4B). In mixed cultures, the risk of neuronal death was not affected by the concomitant use of KML29 and Rimonabant ( Figure 4C). Interestingly, when mixed cultures were treated with KML29 and AM630, the risk of neuronal death was significantly increased ( Figure 4D). Treatment with only AM630 did not affect neuronal survival. This result strongly suggests that the glial-dependent protective effect against MAGL inhibition seems to be mediated by CB2 receptor activation. To evaluate the potential involvement of CB1 receptors in KML29-dependent neurotoxicity of enriched neuronal cultures, treatments with Rimonabant were performed. However, Rimonabant did not enhance KML29-dependent neurotoxicity ( Figure 4E). Altogether, these results Thus, we asked whether the activation of CB1 and CB2 receptors was involved in the glial-dependent protective effect against MAGL inhibition by KML29. First, we analyzed the mRNA expression of CB1 and CB2 receptors by PCR in mixed and enriched neuronal cultures at DIV6 and DIV12. In accordance with its cellular expression pattern, CB1 receptor transcripts were present in both types of cultures. By contrast, CB2 receptor mRNA expression gradually increased in mixed cultures from DIV6 to DIV12 but was not present in neuronal-enriched cultures ( Figure 4A). We attributed the increase in CB2 receptor expression to the proliferation of glial cells overtime. To further determine the involvement of these receptors in the glial-dependent protective effect against MAGL inhibition, GFP-transfected neurons from mixed and enriched primary cultures were treated with Rimonabant, a CB1 receptor antagonist, or AM630, a CB2 receptor antagonist, and subjected to longitudinal survival analysis ( Figure 4B). In mixed cultures, the risk of neuronal death was not affected by the concomitant use of KML29 and Rimonabant ( Figure 4C). Interestingly, when mixed cultures were treated with KML29 and AM630, the risk of neuronal death was significantly increased ( Figure 4D). Treatment with only AM630 did not affect neuronal survival. This result strongly suggests that the glial-dependent protective effect against MAGL inhibition seems to be mediated by CB2 receptor activation. To evaluate the potential involvement of CB1 receptors in KML29-dependent neurotoxicity of enriched neuronal cultures, treatments with Rimonabant were performed. However, Rimonabant did not enhance KML29-dependent neurotoxicity ( Figure 4E). Altogether, these results suggest that the activation of CB2 receptors in glial cells plays a key protective role in the neuronal survival against the toxicity induced by MAGL inhibition.  To determine which cell type contributed to the CB2 receptor protective effect, cells from mixed cultures were collected at DIV12 and separated by flow cytometry based on the cell surface expression of specific molecules. Mature oligodendrocytes were selected as the O4 + population, To determine which cell type contributed to the CB2 receptor protective effect, cells from mixed cultures were collected at DIV12 and separated by flow cytometry based on the cell surface expression of specific molecules. Mature oligodendrocytes were selected as the O4 + population, astrocytes were the GLAST + cells and microglia CD45 + ( Figure 5A). The remaining cells contained mainly neurons and oligodendrocyte precursors. The expression of CB1 receptors was located in neurons and to a lesser extent in oligodendrocytes ( Figure 5B). CB2 receptor expression was predominant in microglial cells and almost undetectable in oligodendrocytes and neurons ( Figure 5B), indicating that microglia are key to establish protective neuron-glia interactions.
Biomolecules 2020, 10, x 11 of 16 neurons and to a lesser extent in oligodendrocytes ( Figure 5B). CB2 receptor expression was predominant in microglial cells and almost undetectable in oligodendrocytes and neurons ( Figure  5B), indicating that microglia are key to establish protective neuron-glia interactions.

Discussion
Our group demonstrated that the neuroprotective effect of JZL184 against MPP + -induced neurotoxicity in a neuroblastoma cell line was mediated by CB2 receptors [38]. In this study, we questioned the effect of MAGL inhibition in a system in which cannabinoid receptors are segregated, CB1 mainly in neurons and CB2 mainly in glial cells. To address this question, we used two types of rat cortical primary cultures: mixed cultures that contain non dividing neurons with a spontaneous death rate together with glial cells that divide until they reach confluence; and neuron-enriched cultures that were obtained by eliminating dividing cells with an AraC treatment. We took advantage of a methodological approach that allows to perform longitudinal survival analysis to estimate the risk of neuronal death in the two types of primary cultures. We found that neuronal survival was highly dependent on glial cells, since the spontaneous risk of neuronal death was higher in neuronalenriched cultures than in mixed cultures. Moreover, MAGL inhibition by KML29 further increased the risk of neuronal death, an effect not observed when glial cells were present in the culture. Interestingly, KML29-dependent toxicity increased in mixed cultures upon blockage of CB2 receptors, indicating that these receptors, expressed mainly in microglial cells, are key players for the protective neuron-glia interactions. Due to this specific location together with their upregulation under pathological conditions [44], our results strongly suggest that CB2 receptors exert selective control over specific neuronal mechanisms that promote neuronal protection and survival. On the contrary, the modulation of CB1 receptors did not affect neuronal survival.
To determine the effect of MAGL inhibition on neuronal survival, we have used a methodological approach based on automated microscopy to track individual primary neurons expressing a fluorescent protein over long periods of time. This methodology has been applied to estimate the risk of death of neurons overexpressing wild type or mutated forms of proteins associated with neurodegenerative diseases [39][40][41][42]. In this study, we applied this powerful approach to evaluate the effect of glial cells and pharmacological modulators of the endocannabinoid system in neuronal survival. The main advantage of this method over traditional assays is the possibility of following the same neuron over a specific period of time, instead of analyzing different neurons at

Discussion
Our group demonstrated that the neuroprotective effect of JZL184 against MPP + -induced neurotoxicity in a neuroblastoma cell line was mediated by CB2 receptors [38]. In this study, we questioned the effect of MAGL inhibition in a system in which cannabinoid receptors are segregated, CB1 mainly in neurons and CB2 mainly in glial cells. To address this question, we used two types of rat cortical primary cultures: mixed cultures that contain non dividing neurons with a spontaneous death rate together with glial cells that divide until they reach confluence; and neuron-enriched cultures that were obtained by eliminating dividing cells with an AraC treatment. We took advantage of a methodological approach that allows to perform longitudinal survival analysis to estimate the risk of neuronal death in the two types of primary cultures. We found that neuronal survival was highly dependent on glial cells, since the spontaneous risk of neuronal death was higher in neuronal-enriched cultures than in mixed cultures. Moreover, MAGL inhibition by KML29 further increased the risk of neuronal death, an effect not observed when glial cells were present in the culture. Interestingly, KML29-dependent toxicity increased in mixed cultures upon blockage of CB2 receptors, indicating that these receptors, expressed mainly in microglial cells, are key players for the protective neuron-glia interactions. Due to this specific location together with their upregulation under pathological conditions [44], our results strongly suggest that CB2 receptors exert selective control over specific neuronal mechanisms that promote neuronal protection and survival. On the contrary, the modulation of CB1 receptors did not affect neuronal survival.
To determine the effect of MAGL inhibition on neuronal survival, we have used a methodological approach based on automated microscopy to track individual primary neurons expressing a fluorescent protein over long periods of time. This methodology has been applied to estimate the risk of death of neurons overexpressing wild type or mutated forms of proteins associated with neurodegenerative diseases [39][40][41][42]. In this study, we applied this powerful approach to evaluate the effect of glial cells and pharmacological modulators of the endocannabinoid system in neuronal survival. The main advantage of this method over traditional assays is the possibility of following the same neuron over a specific period of time, instead of analyzing different neurons at different time points. This procedure allows the identification of risk factors compromising neuronal survival that otherwise, using conventional methodologies could not be revealed. In fact, no differences were detected with the lactate dehydrogenase assay used in our previous study with SH-SY5Y cells [38]. In the case of mixed primary cultures, neurons account for approximately 40% of the total cells, therefore factors affecting specifically neuronal vulnerability would be attenuated when the toxicity of the overall cells in the culture is analyzed.
Here, we have found a differential effect of MAGL inhibition on neuronal survival that depends on neuron-glia interactions. In our previous in vitro study with neuroblastoma cells [38], a unique cell type expressing CB1 and CB2 receptors was present in the culture. MAGL inhibition was protective when cell death was induced with MPP + in a CB2 receptor-mediated manner. In rat cortical primary cultures, CB1 and CB2 receptors are expressed in different cell subsets, neurons and glia, respectively. In this case, the effect of MAGL inhibition was assayed on the basal neuronal survival rate, in the absence of neurotoxic stimuli. Under these conditions, KML29 was neurotoxic for neurons exclusively when glial cells were absent from the culture, suggesting that CB2 receptors could be relevant in promoting neuronal survival when neurons are exposed to damaging insults. The fact that CB2 receptors increase their expression upon microglial activation further supports its role in neuronal survival under pathological conditions. In vivo studies show that MAGL inhibition is neuroprotective and improves motor behavior in different experimental models of Parkinson's disease, an effect mediated by a decrease in inflammation and glial cells [23,36]. However, genetic and pharmacological blockade of MAGL resulted in a concomitant microglial activation and exacerbated a proinflammatory reaction in the cerebellum, leading to motor impairment. This reaction was not observed in other brain regions [37]. Importantly, the cerebellum presents the lowest glia/neuron ratio, 0.23, compared to the rest of the brain with an estimated ratio of 11.35 [45,46], and this fact could explain the differences observed. In our primary cultures, with a glia/neuron ratio of 1.5 in mixed cultures and 0.01 in neuron-enriched cultures, the inhibition of MAGL was innocuous in the presence of glial cells; the risk of neuronal death of neurons remained stable in the presence of KML29. Altogether, these observations suggest that MAGL might exert a protective effect when neurons are exposed to harmful conditions (i.e., MPTP in the experimental models of PD), but does not affect the spontaneous death rate.
Our results indicate that inhibition of MAGL by KML29 constitutes a neurotoxic insult for neurons that disappears in the presence of glia. The protective effect that glial cells exert over neurons seems to be mediated by CB2 receptors expressed by microglial cells. CB2 receptors are almost undetectable in the healthy brain but their expression is upregulated under pathological conditions [47]. In culture, resting microglial cells acquire an amoeboid shape and become highly proliferative resembling microglia found in injured tissue [48,49]. The increase in CB2 receptor expression over time detected in the mixed cultures would reflect this activated state of microglial cells. Under standard culture conditions, the risk of neuronal death was unaffected by CB2 receptor blockage with AM630. However, these receptors were necessary to prevent the toxic effect that KML29 caused directly to neurons. Possible mechanisms for this effect would be the switch in microglial phenotypes after CB2 receptor activation with a shift in the gene expression balance towards a neuroprotective phenotype that releases neurotrophic factors and a different pattern of pro-and anti-inflammatory cytokines [36,50]. In addition, endocannabinoids potentiate the expression of CX 3 CR1 [51] and CD200R [52] that interact with their corresponding ligands in the cell surface of neurons promoting the establishment of protective neuron-glia interactions. Modulation of CB1 receptors did not affect neuronal survival at any condition, suggesting a preferential role in other functions not necessarily related to degeneration or repair. These results show the relevant role of microglial cells in protective neuron-glia interactions mediated by CB2 receptors when neurons are exposed to toxic conditions.
Understanding the molecular pathways that cells in the brain use to communicate with each other will shed light on key features of the physiology and pathology of the nervous system. An unmet need for neurodegenerative diseases is the knowledge of molecular pathways that prevent neuronal death under specific neurotoxic stimuli. MAGL inhibition prolongs 2-AG action in the brain through CB1 and CB2 receptors and reduces de biosynthesis of arachidonic acid, the precursor of proinflammatory prostanoids, for these reasons it has emerged as an interesting target for the treatment of neurodegenerative diseases [53]. In this study, we show that MAGL inhibition is toxic for neurons but glial cells counteract this neuronal damage. No degeneration has been reported when MAGL inhibitors are administered in vivo, probably because of the neuron-glia interactions previously established. Under in vitro conditions, microglial cells representing 15% of the total cells are the main cell type expressing CB2 receptors. Activation of this receptor prevents neuronal death, in this case, caused by KML29 toxicity. In summary, our results demonstrate that neuronal survival depends on successful crosstalk established between neurons and microglia. CB2 receptors play a relevant role in this type of interactions and consequently in neuronal fate. Funding: This work was funded by the Spanish Government (ISCIII-FEDER) PI17/01931, by Navarra Government (060-061), by Navarra Government Health Department 3/2019, and FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación/_Project BFU2017-90043-P. ERB was funded by Colfuturo. IIM, MAB and RVG were funded by the Asociación de Amigos (ADA) de la Universidad de Navarra.

Conflicts of Interest:
The authors declare no conflict of interest.