Dorian Sargent

Dominique Bétemps

Matthieu Drouyer

Jérémy Verchere

Damien Gaillard

Jean-Noël Arsac

Latifa Lakhdar

Anna Salvetti

Thierry Baron
email: thierry.baron@anses.fr

Investigating the neuroprotective effect of AAV-mediated β-synuclein overexpression in a transgenic model of synucleinopathy

Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative diseases characterized by inclusions mainly composed of α-synuclein (α-syn) aggregates. The objective of this study was to investigate if β-synuclein (β-syn) overexpression could have beneficial effects by inhibiting the aggregation of α-syn. The M83 transgenic mouse is a model of synucleinopathy, which develops severe motor symptoms associated with aggregation of α-syn. M83 neonate or adult mice were injected with adeno-associated virus vectors carrying the human β-syn gene (AAVβ-syn) or green fluorescent protein gene (AAVGFP) using different injection sites. The M83 disease was -or not -accelerated using extracts of M83 brains injected with brain extract from mouse (M83) or human (MSA) origins. AAV vectors expression was confirmed using Western blot and ELISA technics.

AAV mediated β-syn overexpression did not delay the disease onset or reduce the α-syn phosphorylated at serine 129 levels detected by ELISA, regardless of the AAV injection route and the inoculation of brain extracts. Instead, a proteinase-K resistant β-syn staining was detected by immunohistochemistry, specifically in sick M83 mice overexpressing β-syn after inoculation of AAVβ-syn. This study indicated for the first time that viral vector-mediated βsyn overexpression could form aggregates in a model of synucleinopathy.

Introduction

Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) are synucleinopathies, characterized by inclusions mainly composed of an aggregated form of α-synuclein (α-syn) in the central nervous system (CNS). As for prions, aggregated forms of α-syn propagate within the CNS during the associated neurodegenerative diseases. This characteristic was initially suggested in humans by Braak's description of PD stages, and was confirmed later in experimental models of synucleinopathies, in particular in the M83 transgenic mouse model [START_REF] Giasson | Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[END_REF] . M83 mice express the human A53T mutated α-syn found in some familial PD forms, under the control of the mouse prion promoter [START_REF] Giasson | Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[END_REF] . These mice spontaneously develop severe motor impairment at 8-16 months of age at the homozygous state. The symptomatology is associated with the accumulation in the CNS, of a pathological form of α-syn (α-syn P ), heavily phosphorylated at serine 129 residue. We previously showed that, in M83 mice, disease onset can be accelerated by the intracerebral inoculation of brain homogenates from sick M83 mice [START_REF] Mougenot | Prion-like acceleration of a synucleinopathy in a transgenic mouse model[END_REF] . This model was further characterized by development of an original ELISA that specifically detects, and allows to easily quantify, the pathological α-syn in sick M83 mice [START_REF] Betemps | Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA[END_REF][START_REF] Betemps | Detection of Disease-associated alpha-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated alpha-synuclein[END_REF][START_REF] Sargent | Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum[END_REF] . This in vivo model may be useful for testing novel therapeutic strategies, particularly targeting progression of the α-syn aggregation.

β-synuclein (β-syn) is another member of the synuclein family, lacking a part of the nonamyloid component, a specific region suggested to be amyloidogenic in α-syn. According to in vitro studies, unlike α-syn, β-syn alone is not able to form aggregates, but could instead interact with α-syn and reduce its capacity to aggregate [START_REF] Janowska | Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein[END_REF] . These anti-aggregative features have been investigated in vivo, and some of these studies indeed reported a neuroprotective effect and reduction of α-syn inclusions after overexpression of the human β-syn mediated by DNA microinjection or lentiviral vectors in the transgenic D mouse model, overexpressing human wild-type α-syn [START_REF] Hashimoto | An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model[END_REF][START_REF] Hashimoto | beta-Synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor[END_REF] . Another study also suggested that crossing mice overexpressing human β-syn with M83 mice delayed the M83 disease onset and reduced the α-syn aggregation [START_REF] Fan | Beta-synuclein modulates alpha-synuclein neurotoxicity by reducing alphasynuclein protein expression[END_REF] . However, two recent studies by Taschenberger et al and Landeck et al which analyzed the impact of human β-syn overexpression mediated by adeno-associated viral vectors (AAV) on nigral dopaminergic neurons in rats, described a neurodegeneration with βsyn aggregates [START_REF] Taschenberger | beta-synuclein aggregates and induces neurodegeneration in dopaminergic neurons[END_REF][START_REF] Landeck | Toxic effects of human and rodent variants of alpha-synuclein in vivo[END_REF] .

Here, in order to assess the effects of β-syn overexpression in the context of these recent and unexpected data suggesting that β-syn produced using AAV may be able to aggregate, we investigated the impact of human β-syn overexpression mediated by AAV on the onset of the synucleinopathy of M83 mice. Two strategies of inoculation of the AAV vector were sequentially tested: (i) an intracerebroventricular (ICV) inoculation at birth, in order to generate a widespread overexpression of the vector in the central nervous system [START_REF] Kim | Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction[END_REF] , and (ii) an inoculation of the AAV vector into the ventral tegmental area (VTA) [START_REF] Cearley | A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease[END_REF] , a region of the mesencephalon in which prominent α-syn aggregation occurs in sick M83 mice [START_REF] Giasson | Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[END_REF] . Our results indicate that, regardless of the AAV inoculation strategy, continuous AAV-mediated β-syn overexpression in neurons did not delay the onset of the disease and did not modify the α-syn P levels measured by ELISA, strongly suggesting that β-syn may not protect against α-syn aggregation and propagation.

Results

Human β-syn is widely expressed after intracerebroventricular injection of AAVβ-syn in neonates

To overexpress β-syn in the M83 CNS, we used self-complementary (sc)AAV9 vectors expressing human β-syn (AAVβ-syn) gene or, as a control, the green fluorescent protein (AAVGFP) gene under the control of the human synapsin1 promoter and with a posttranscriptional WPRE regulatory sequence (Supplementary figure 1A).

To functionally evaluate the AAV vectors, we first inoculated AAVβ-syn in the right and left cerebral ventricles of wild-type B6C3H newborn mice (genetic background of M83 transgenic mice) to obtain a widespread expression of the transgene in the CNS [START_REF] Kim | Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction[END_REF] . One month later, using viral specific primers, vector mRNA was detected in all regions of the brain and in the spinal cord (Supplementary figure 1B), as described [START_REF] Mclean | Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection[END_REF] , with higher levels in rostral regions of the brain (olfactory bulbs, cortex, hippocampus, striatum) and mesencephalon.

Intracerebroventricular injection of AAVβ-syn did not modify the disease of M83 mice

AAV vectors were first tested in M83 mice challenged by inoculation of brain extracts from sick M83 mice, an experimental design which is characterized by a quicker onset of the disease with a lower intragroup variability as compared to unchallenged M83 mice [START_REF] Mougenot | Prion-like acceleration of a synucleinopathy in a transgenic mouse model[END_REF] . AAV vectors were ICV inoculated at birth and two months later challenged with different brain extracts of sick M83 mice, one derived from a second passage of a sick M83 brain extract in M83 mice (M83/M83 inoculum), and the other from a second passage of a human brain extract from a MSA patient in M83 mice (MSA/M83 inoculum) (Figure 1A, Supplementary table 1) [START_REF] Sargent | Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum[END_REF] . M83 mice were euthanized after the detection of the first symptoms of disease, i.e balance disorders or hind limb paralysis, identified by two independent observers.

In both experiments, no delay in the onset of the disease was observed in AAVβ-syn injected mice as compared to control AAVGFP mice, regardless of the type of inoculum (M83/M83 or MSA/M83). Instead, AAVβ-syn injection significantly accelerated the onset of the disease in animals challenged with the M83/M83 extract as compared to control mice (log rank test p<0,05) (Figure 1B). In contrast, no difference was observed in animals challenged with the MSA/M83 extract (Figure 1E). In each experiment, we did not detect any difference concerning the clinical symptoms of sick mice after the injection of AAVβ-syn or AAVGFP.

After dissection of the brains, biochemical analyses were realized. As the vector expression was higher in the rostral regions of the brain than in other CNS regions after ICV injections, we first checked the vector expression by detecting total β-syn in the hippocampus. An ELISA test adapted from Krassnig et al. allowed to detect a significant overexpression of βsyn (both murine β-syn and human β-syn produced by the vector) in the hippocampus, confirming the continuous expression of the AAVβ-syn until the disease onset (Figure 1C, and F). We next focused on the mesencephalon, the brainstem and the spinal cord because these CNS regions are known to be strongly positive in α-syn P in sick M83 mice [START_REF] Betemps | Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA[END_REF][START_REF] Betemps | Detection of Disease-associated alpha-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated alpha-synuclein[END_REF][START_REF] Sargent | Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum[END_REF] . A significant overexpression of β-syn was only detected in the mesencephalon, the brain stem and in the spinal cord of sick M83 mice injected with AAVβ-syn challenged with M83/M83 inoculum, but not in mice challenged with the MSA/M83 inoculum (Figure 1C andF). Since the M83 disease is associated with moderate or high α-syn P levels into different brain regions and in the spinal cord [START_REF] Sargent | Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum[END_REF] , we quantified α-syn P using ELISA in order to detect a potential effect of β-syn on this biochemical marker of the disease. Our analyses did not show any significant difference in the α-syn P levels in any of the examined CNS regions after AAVβ-syn inoculation, regardless of the nature of the inoculum used to accelerate the disease (Figure 1D, and G).

To further study the levels of β-syn protein after AAV injections in M83 mice throughout the CNS, we used Western blot and ELISA which allowed to detect total β-syn in specific brain regions. Comparing total β-syn levels of 5 to 7 months old sick M83 mice inoculated at birth with AAVβ-syn or AAV GFP then challenged with the MSA/M83 inoculum (same mice as in Figure 1E), an overexpression of β-syn protein was detected by ELISA in the olfactory bulbs, cortex, striatum, hippocampus, but not in the other regions (Figure 2A), consistent with the results obtained by qRT-PCR at one month after AAV inoculation (Supplementary figure 1B).

Western blot analyses confirmed an overexpression of β-syn protein in the hippocampus of the same mice after AAVβ-syn inoculation (Figure 2B andC). Most of the animals inoculated with AAVβ-syn showed a β-syn overexpression detectable until the disease onset (Figure 1C,F). Only a few animals did not show significant overexpression of β-syn detected by ELISA or by immunohistochemistry (5/48 animals, in the entire study); these animals were excluded for the study analyzing the onset of disease.

We next examined the possible effects of AAVβ-syn ICV injection on the spontaneous development of the M83 disease during aging, without any challenge to accelerate the disease (Figure 3A). Here again, AAVβ-syn injection neither delayed the disease onset of M83 mice nor modified the α-syn P levels (Figure 3B andD), despite a sustained expression of the transgene, at least in the hippocampus, up to the disease onset (Figure 3C) (ELISA data correspond to 2 mice for the treated group and 3 mice for the control group; consequently, no statistical analysis was done here).

Altogether, these results indicated that AAVβ-syn injection in the ventricles resulted in a widespread expression of the transgene in the CNS, but at a moderate level, especially in the structures that are the most heavily affected by the synucleinopathy lesions. We thus considered another strategy by inoculating the AAV vectors in the ventral tegmental area (VTA), which is located into the mesencephalon, where major brain lesions and accumulation of α-syn P are detected in sick M83 mice and which is also connected to multiple brain areas [START_REF] Cearley | A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease[END_REF] .

β-syn is overexpressed mostly in the mesencephalon after injection of AAVβ-syn in the

VTA of adult mice

In order to validate this injection protocol of AAV vectors into the VTA, two months old wild-type mice were first inoculated with AAVβ-syn in the VTA and sacrificed one month later. After dissection of the brains, vector mRNA was mostly detected in the mesencephalon, but also in the cortex, striatum, hippocampus and brainstem; only traces of vector mRNA were identified in the other regions of the brain (except the olfactory bulbs) and in the spinal cord, as compared to mesencephalon (Supplementary figure 1C).

Injection of AAVβ-syn in the VTA did not modify the disease of M83 mice

The same protocol was then applied to M83 mice. After the inoculation of AAV in the VTA in two months old M83 mice, the disease was accelerated by intracerebrally inoculating the mice one month later with the M83/M83 or the MSA/M83 inoculum (Figure 4A). No significant difference in the survival was observed between the AAVβ-syn and AAVGFP injected mice, between 3 and 8 months (Figure 4B,E). As before, we did not detect any difference concerning the clinical symptoms of sick mice after the injection of AAVβ-syn or AAVGFP. Importantly, analyses by ELISA confirmed the strong overexpression of β-syn in the mesencephalon although no difference was detected in the levels of pathological α-syn P at the disease stage (Figure 4C, D, F, andG).

By immunohistochemistry, using an antibody targeting total β-syn (human and murine β-syn), a specific β-syn staining punctate pattern was detected in the inoculated mesencephalon, as well as in the striatum and brain stem which represent connected regions [START_REF] Cearley | A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease[END_REF] , of all the sick M83 mice inoculated with AAVβ-syn (5/5), but not in sick M83 mice inoculated with AAVGFP (0/2) (Figure 4H). Further analysis indicated that these β-syn immunoreactive dots, which are specifically detected in mice inoculated with AAVβ-syn, mostly co-localize with the presynaptic protein synaptophysin (Supplementary figure 2A). Co-localization of GFP with neuronal marker β-tubulin type 3 in sick M83 mice inoculated with AAVGFP also confirmed that these vectors allowed a neuronal specific expression of the transgene (Supplementary figure 2B). Interestingly, the morphology of the inclusions containing α-syn phosphorylated at serine 129 detected by immunohistochemistry was not modified by β-syn overexpression (Supplementary figure 3A). Furthermore, these inclusions only rarely colocalized with AAV-specific β-syn staining by immunofluorescence (Supplementary figure 3B). We further biochemically characterized vectors expression after injection in the VTA in M83 mice euthanized 4 months later. Total β-syn levels were quantified by ELISA and showed a significant increase of the protein in the mesencephalon of mice injected with AAVβ-syn as well as in the hippocampus and brain stem compared to control mice injected with the AAVGFP (Figure 5A). β-syn protein overexpression was also readily detected by Western blot in the mesencephalon of most of these mice (Figure 5B).

The absence of protective effect was not due to an insufficient dose of AAV vector injected in the VTA

We finally asked whether the absence of any significant effect of AAVβ-syn could be due to an insufficient dose of AAV vector. We thus injected 4-times more vector in the VTA one month before the challenge by intra-cerebral inoculation of the M83/M83 inoculum. This AAV dose allowed to significantly increase the overexpression of β-syn at the disease stage (p<0,05) (Figure 5C,D). However, the M83 disease was still not delayed by β-syn overexpression, but instead, as previously observed after ICV injection of AAV (Figure 1B), the disease appeared significantly earlier in the AAVβ-syn injected mice as compared to control AAVGFP animals (p<0,001) (Figure 5E). As in previous experiments, no impact was found on the α-syn P levels even if β-syn was significantly increased at the stage of clinical signs in all the CNS regions (Figure 5D,F).

By immunohistochemistry, as with the low dose of AAV, a specific β-syn staining punctate pattern was detected in the inoculated mesencephalon, as well as in the striatum and brain stem in two sick M83 mice inoculated with AAVβ-syn, but not in one mouse inoculated with AAVGFP (data not shown).

AAV-mediated overexpression of β-syn might have formed aggregates of β-syn

Proteinase K (PK)-resistant β-syn aggregates have been previously described after inoculation of AAV vectors expressing human β-syn in the substantia nigra of rats [START_REF] Taschenberger | beta-synuclein aggregates and induces neurodegeneration in dopaminergic neurons[END_REF][START_REF] Landeck | Toxic effects of human and rodent variants of alpha-synuclein in vivo[END_REF] . We thus analyzed by immunohistochemistry the brain of several sick M83 mice after inoculation of AAVs in the VTA (Figure 6). After PK digestion, total β-syn antibody revealed a punctate pattern specifically detected after the inoculation of AAVβ-syn, in the mesencephalon of 7/7 sick M83 mice (5 mice injected with low dose of AAV, of which 2 were challenged with M83/M83 inoculum and 3 with MSA/M83 inoculum, as well as 2 mice injected with high dose of AAV and challenged with M83/M83 inoculum). This punctate pattern was also detected in the striatum and the brain stem where the AAV is also expressed, but not in the cerebellum where the AAV is less expressed (Supplementary figure 4). A few cell bodies were also stained with total β-syn antibody after PK digestion, particularly in the mesencephalon of these mice (data not shown). Even if diffuse, PK resistant staining was also detected in the hippocampal region of two sick M83 mice injected with AAVβ-syn and not challenged with brain extract inoculation, but not in one sick M83 control mouse (data not shown). These results confirm that overexpression of β-syn resulted in PK-resistant β-syn staining which could be detected independently of the vector dose, the inoculation site or the age of inoculation. Interestingly, Western blot detection of β-syn in the brain homogenates from sick M83 mice injected with high dose of AAVβ-syn showed the appearance of an additional band, consistent with a dimer, that is not visible in the homogenates from sick M83 mice injected or not with AAVGFP (even after long exposure of the blot) (Supplementary figure 5).

Discussion

β-syn was previously described as a neuroprotective protein, able to inhibit the aggregation process of α-syn in vitro and having benefic effects on synucleinopathies in vivo [START_REF] Janowska | Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein[END_REF][START_REF] Hashimoto | An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model[END_REF][START_REF] Hashimoto | beta-Synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor[END_REF][START_REF] Fan | Beta-synuclein modulates alpha-synuclein neurotoxicity by reducing alphasynuclein protein expression[END_REF][START_REF] Hashimoto | Beta-synuclein regulates Akt activity in neuronal cells. A possible mechanism for neuroprotection in Parkinson's disease[END_REF] . Here, we tried to slow down a synucleinopathy in a transgenic mouse model with dramatic clinical symptoms linked to pathological α-syn aggregation. In particular, we analyzed the effects of the overexpression of β-syn mediated by an AAV vector on α-syn aggregation. We show here that the delivery of the AAVβ-syn vector via two different injection routes (intracerebroventricular or in the ventral tegmental area) in transgenic M83 mice, neither delayed the disease onset nor reduced the levels of pathological α-syn at the disease stage, despite a sustained overexpression of β-syn. A limitation of our study remains the low number of animals that were followed in each of the experimental groups, given the multiple strategies that were studied (different routes and ages of AAV inoculations, different AAV doses, AAV effects during normal aging or after intracerebral experimental challenges of two M83 sources (M83/M83 or MSA/M83) as summarized in supplementary Table 1. This represents a statistical limitation of our study, although it is noteworthy that in the only two experimental groups in which a statistically significant difference was found between the survival of mice injected with AAVβ-syn and AAVGFP vectors, this rather suggested a deleterious effect of β-syn overexpression. Studies on larger cohorts will thus be required to confirm our results. A previous study indeed suggested that constitutive overexpression of βsyn in transgenic M83 mice by generating double transgenic mice delays the M83 disease onset by several months [START_REF] Fan | Beta-synuclein modulates alpha-synuclein neurotoxicity by reducing alphasynuclein protein expression[END_REF] . In our study, interestingly, M83 mice overexpressing β-syn showed a higher spontaneous activity in experiments in which open field was performed and a tendency to have more balance trouble in beam walking test (Supplementary figure 6). These results suggest that β-syn expression might have an impact on mice behavior without affecting the ultimate development of the disease. Importantly, in this study of Fan and colleagues, human β-syn was expressed from the pan-neuronal mouse prion promoter, which is the same promoter used to express human mutated α-syn in the M83 model. It is possible that the strategies used in our study did not provide sufficient expression of β-syn, in the CNS regions targeted by the disease. Indeed, as expected, after the injection of AAV in the VTA, we obtained a very high overexpression of β-syn in the mesencephalon in which the aggregation of α-syn is high at the disease stage, but this region may not be the region where the aggregation process of α-syn begins [START_REF] Emmer | E46K human alpha-synuclein transgenic mice develop Lewy-like and tau pathology associated with age-dependent, detrimental motor impairment[END_REF] . It should be emphasized that the major signs of the clinical disease of M83 mice are in relation with some -still poorly explained-disorders in the spinal cord [START_REF] Sacino | Intramuscular injection of alpha-synuclein induces CNS alpha-synuclein pathology and a rapid-onset motor phenotype in transgenic mice[END_REF] . In an additional series of experiments, we were unable to identify a degeneration of motor neurons in the lumbar spinal cord after the injection of preformed fibrils of human α-syn in M83 mice, as previously reported after intra-muscular injections [START_REF] Sacino | Intramuscular injection of alpha-synuclein induces CNS alpha-synuclein pathology and a rapid-onset motor phenotype in transgenic mice[END_REF] , further interrogating the cause of the appearance of symptoms (Supplementary figure 7). The ICV injection of AAV9 vector in SMNΔ7 mice, a severe model of spinal muscular atrophy, allowed to express a codon-optimized human SMN1 coding sequence in the spinal cord, which as a result, has improved significantly their survival [START_REF] Armbruster | Efficacy and biodistribution analysis of intracerebroventricular administration of an optimized scAAV9-SMN1 vector in a mouse model of spinal muscular atrophy[END_REF] . After the ICV inoculation of AAV β-syn, the overexpression of β-syn was diffuse and lower in caudal regions of the brain and spinal cord, as described [START_REF] Mclean | Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection[END_REF] , but it may have been insufficient to slow down the synucleinopathy process. It must however be noted that an in vitro study using AAV vectors suggests that β-syn overexpression is benefic on α-syn linked toxicity only at a low ratio of βsyn/α-syn expression [START_REF] Taschenberger | beta-synuclein aggregates and induces neurodegeneration in dopaminergic neurons[END_REF] . Even if it was not evaluated here, another study has described transgene expression in the neuromuscular junction after AAV injection into the ventricles at birth; this may be important since the degeneration of the neuromuscular junction was also described as a possible explanation of the appearance of symptoms in M83 mice [START_REF] Mclean | Widespread neuron-specific transgene expression in brain and spinal cord following synapsin promoter-driven AAV9 neonatal intracerebroventricular injection[END_REF][START_REF] Sacino | Intramuscular injection of alpha-synuclein induces CNS alpha-synuclein pathology and a rapid-onset motor phenotype in transgenic mice[END_REF] . At the cellular level, human β-syn seems mostly localized in the presynaptic button and axons in sick M83 mice, which seems appropriate because the aggregation of α-syn was suggested to start at the presynaptic level in synucleinopathies [START_REF] Schulz-Schaeffer | The synaptic pathology of alpha-synuclein aggregation in dementia with Lewy bodies, Parkinson's disease and Parkinson's disease dementia[END_REF][START_REF] Spinelli | Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson's disease[END_REF] . Another study suggested that the benefic effect of β-syn is the result of the down regulation of the expression of α-syn, but we did not detect any decrease of α-syn expression, even in the brain regions overexpressing β-syn (data not shown). In another transgenic mouse model (D line) overexpressing human wild type αsyn, two studies reported the reduction of the synucleinopathy by either generating bigenic mice that also overexpress β-syn, or vehiculating the human β-syn transgene, under the control of cytomegalovirus (CMV) promoter, in a lentiviral vector [START_REF] Hashimoto | An antiaggregation gene therapy strategy for Lewy body disease utilizing beta-synuclein lentivirus in a transgenic model[END_REF][START_REF] Hashimoto | beta-Synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor[END_REF] . As some reports indicate a downregulation of the CMV promoter in the CNS over time [START_REF] Gray | Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors[END_REF] , we chose here to use the human synapsin 1 promoter to obtain a long term expression of β-syn, which was confirmed in the present study, even in old mice. Importantly, as previously described [START_REF] Glover | Adenoviralmediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity[END_REF] , the synapsin 1 promoter also allowed to express human β-syn specifically in neurons, where αsyn aggregates are found in the M83 model [START_REF] Giasson | Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[END_REF] . According to the study by Hashimoto et al., the neuroprotective effect of β-syn was associated to the activation of Akt, a neuroprotective protein which could be indirectly down-regulated by α-syn during the synucleinopathy process [START_REF] Chung | Direct interaction of alpha-synuclein and AKT regulates IGF-1 signaling: implication of Parkinson disease[END_REF] . However, we did not detect any significant increase of Akt activation by Western blot in 6 months-old M83 mice inoculated with AAVβ-syn in the VTA at 2 months, and challenged one month later with the M83/M83 inoculum (Supplementary figure 8). It should be noticed that such Akt changes were not consistently observed, as shown in a study with a lentiviral vector used for β-syn overexpression [START_REF] Krassnig | Influence of Lentiviral beta-Synuclein Overexpression in the Hippocampus of a Transgenic Mouse Model of Alzheimer's Disease on Amyloid Precursor Protein Metabolism and Pathology[END_REF] .

Surprisingly, in the present study, two experiments in mice challenged with a M83/M83 inoculum rather showed a significant acceleration of the disease after AAV β-syn injection (Fig 1B and5E). Interestingly, several studies reported that β-syn could be implicated in the pathological process of synucleinopathies. Indeed, an axonal pathology with β-syn accumulation in axons was reported in patients with Parkinson's disease, DLB or Hallervorden-Spatz syndrome [START_REF] Galvin | Neurodegeneration with brain iron accumulation, type 1 is characterized by alpha-, beta-, and gamma-synuclein neuropathology[END_REF][START_REF] Galvin | Axon pathology in Parkinson's disease and Lewy body dementia hippocampus contains alpha-, beta-, and gamma-synuclein[END_REF] . In addition, two mutations of β-syn, identified in DLB cases, were suggested to be responsible for α-syn aggregation and DLB [START_REF] Ohtake | Beta-synuclein gene alterations in dementia with Lewy bodies[END_REF] . Generating transgenic mice co-expressing P123H mutated β-syn with human α-syn resulted in an enhanced pathology, suggesting that mutated β-syn could potentiate α-syn aggregation [START_REF] Fujita | A beta-synuclein mutation linked to dementia produces neurodegeneration when expressed in mouse brain[END_REF] .

Variable results were also observed after overexpression of β-syn using a lentiviral vector after inoculation into the hippocampus of transgenic mice overexpressing a mutated form of the human β-amyloid precursor protein (APP) involved in Alzheimer's disease, as assessed by examining plaque load, memory deficits, and anxiety [START_REF] Krassnig | Influence of Lentiviral beta-Synuclein Overexpression in the Hippocampus of a Transgenic Mouse Model of Alzheimer's Disease on Amyloid Precursor Protein Metabolism and Pathology[END_REF] . However, it should be noted that the expression level of β-syn was relatively low in this latter study (not detectable at the protein level). Also, it is interesting to notice that a truncated form of human recombinant α-syn lacking the 71-82 residues not able to form aggregates in vitro, like β-syn [START_REF] Giasson | A hydrophobic stretch of 12 amino acid residues in the middle of alpha-synuclein is essential for filament assembly[END_REF] , accelerated the disease of a few M83 mice, after injection in the muscle or in the peritoneal cavity [START_REF] Sacino | Intramuscular injection of alpha-synuclein induces CNS alpha-synuclein pathology and a rapid-onset motor phenotype in transgenic mice[END_REF][START_REF] Ayers | Robust Central Nervous System Pathology in Transgenic Mice following Peripheral Injection of alpha-Synuclein Fibrils[END_REF] . These results raise the possibility that β-syn might be able to accelerate the aggregation of α-syn in M83 mice, although this has not been reported so far.

Further analyzing the effects of β-syn expression in sick M83 mice inoculated with the AAV vector in the VTA allowed detecting a punctate pattern of β-syn staining in the targeted brain region and some connected regions, only in mice inoculated with AAVβ-syn. This staining was more resistant to PK digestion than that endogenous β-syn in control mice. This result suggests that overexpressed β-syn formed aggregates which could explain why β-syn did not act as expected, i.e as an α-syn aggregation inhibitor. Landeck et al. have described the same staining of β-syn resistant to PK digestion referred as "dark punctae", two months after the inoculation of AAV5 carrying human β-syn under the control of chicken beta-actin promoter including a CMV enhancer element (CBA) in the substantia nigra of rats [START_REF] Landeck | Toxic effects of human and rodent variants of alpha-synuclein in vivo[END_REF] . Before this study, Taschenberger et al. have also described the detection of PK-resistant β-syn aggregates in vivo, as early as two weeks following the inoculation of AAV2 carrying human β-syn gene in the substantia nigra of rats [START_REF] Taschenberger | beta-synuclein aggregates and induces neurodegeneration in dopaminergic neurons[END_REF] . This time, the vector design was closer to ours, using the same promoter (human synapsin) to express human β-syn, with a WPRE sequence to enhance transgene expression. It is interesting to notice that, even if the AAV serotype used as well as the vector design can differ between studies, all these studies that used AAV to carry β-syn also reported the detection of β-syn aggregates. Furthermore, our observations in immunohistochemistry are supported biochemically in the present study, by the detection of a specific β-syn pattern in Western blot that could be a specific signature of β-syn aggregation, in mice showing overexpression of β-syn after injection of high dose of AAVβ-syn (Supplementary figure 5). All these observations suggest that the neuroprotective effect of βsyn may vary according to the strategy followed to overexpress it and the animal model. For example, it would be interesting to inject AAV vectors used in this study in transgenic D mice in order to see if we obtain the same β-syn aggregates. The D line is indeed an example of transgenic mice expressing the normal human α-syn, which may thus be more representative of the common situation in humans. The interest of the M83 mouse line, which is well illustrated in our study, is the possibility to easily quantify both survival of mice, ultimately showing a major clinical disease, and levels of pathological α-syn, using an ELISA test. The A53T mutation in the human protein is however associated with a high propensity to aggregation [START_REF] Flagmeier | Mutations associated with familial Parkinson's disease alter the initiation and amplification steps of α-synuclein aggregation[END_REF] and our observations with AAV-mediated β-syn overexpression may not be representative of the situation of humans with non genetic synucleinopathies. Also, it is important to point out that even if most of neurons express the transgene in the cerebral region injected of mice in our study, there is an important variability of the expression of the transgene in transduced cells after AAV vectors injection (Supplementary figure 9). It might explain at least partly the difficulty to detect a benefic effect of β-syn overexpression as it was suggested to depend on a specific α-syn/β-syn ratio in cell culture [START_REF] Taschenberger | beta-synuclein aggregates and induces neurodegeneration in dopaminergic neurons[END_REF] .

The present study suggest for the first time that β-syn overexpression could not be benefic in a synucleinopathy model. This may be due at least partly to the formation of a PK-resistant βsyn species after AAVβ-syn inoculation. According to a very recent study, a mildly acidic pH environment, found in several organelles, could induce β-syn aggregation, highlighting the complexity of β-syn fibrillation mechanisms occurring in vivo [START_REF] Moriarty | A pH-dependent switch promotes beta-synuclein fibril formation via glutamate residues[END_REF] . Further studies are needed to better understand how and to what extent β-syn could play a role in synucleinopathies.

However, even though we could not detect any protective effects of β-syn, our study confirms that an AAV vector is suitable for long-term overexpression of proteins into the CNS.

Notably, after ICV inoculations of AAV in neonates, we confirmed a widespread expression of the protein, as this was also recently described using an α-syn AAV vector [START_REF] Delenclos | Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain[END_REF] . The injection of AAV in the VTA also allowed to express human β-syn in various connected regions, as suggested in a previous study using serotype 9 AAV vector carrying a lysosomal enzyme gene [START_REF] Cearley | A single injection of an adeno-associated virus vector into nuclei with divergent connections results in widespread vector distribution in the brain and global correction of a neurogenetic disease[END_REF] . Our data also illustrate the robustness of the experimental model of M83 mice intracerebrally challenged by inocula containing aggregated α-syn, showing rather short and relatively uniform survival periods before the appearance of characteristics clinical signs, which can be used for the assessment of future therapeutic strategies.

Methods

Animals: M83 transgenic mice were used in this study (B6;C3H-Tg[SNCA]83Vle/J, RRID:MGI:3603036, The Jackson Laboratory, Bar Harbor, ME, USA). These mice express A53T mutated human α-syn protein and spontaneously develop severe motor impairment leading to early death 1 . Homozygous M83 mice develop characteristic motor symptoms between 8 and 16 months of life, beginning with reduced ambulation, balance disorders, partial paralysis of a hind leg and then progressing to prostration, difficulty in feeding, weight loss, hunched back and general paralysis [START_REF] Giasson | Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein[END_REF] AAV vectors: Recombinant self-complementary AAV9 vectors (scAAV9) encoding human βsyn or enhanced GFP (eGFP) were produced by calcium phosphate transfection of HEK-293 cells [START_REF] Gao | Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy[END_REF] . Three plasmids were transfected simultaneously: (i) a vector plasmid containing the human gene of β-syn or eGFP under control of the neuron-specific synapsin 1 gene promoter (Supplementary figure 1A) (ii) a helper plasmid pXX6 [START_REF] Xiao | Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus[END_REF] and (iii) a plasmid carrying rep2 and cap9 genes [START_REF] Gao | Clades of Adeno-associated viruses are widely disseminated in human tissues[END_REF] . Vector particles were extracted and purified on an iodixanol step gradient.

Titers were determined by quantitative polymerase chain reaction (qPCR) and expressed as viral genomes per milliliter (vg/mL).

Inoculations: AAV vectors were injected at birth or at adulthood (two months of age), at different sites of inoculation. Neonates were inoculated using 5 µL Hamilton syringe, with brain sample in M83 mice (MSA/M83 inoculum) (same sample used in a previous publication [START_REF] Sargent | Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum[END_REF] ). These homogenates were injected in the left striatum, using stereotaxic coordinates (AP: +0.14 ML: +2 DV: -2.75). Before each stereotaxic surgery (AAV or brain extract inoculation), mice have been anesthetized with a xylazine (10 mg/kg) and ketamine (100 mg/kg) mixture.

qRT-PCR: B6C3H brains were dissected and RNA was extracted from brain regions using RNeasy lipid tissue Minikit (ref 74804, Qiagen, Courtaboeuf, France). RNA samples were tittered using Nanodrop (Nanodrop 1000, Thermo Fisher scientific, Villebon sur Yvette, France). 500 ng of RNA were reverse-transcripted with Quanta qScript cDNA Super Mix (ref 95048-100, VWR international, Fontenay-sous-Bois, France) on a Biorad iCycler. After a 1:10 dilution, complementary DNA samples were analyzed by qPCR using primers specific for the WPRE region of viral mRNA, and primers specific for GAPDH mRNA. We used the LC 480 SYBR Green 1 Master kit (ref 04887 352 001, Roche, Boulogne-Billancourt, France) on a Roche Light Cycler 480 to perform the qPCR and analyzed the results on the LC 480 software.

ELISA: Brains were dissected as described [START_REF] Betemps | Detection of Disease-associated alpha-synuclein by Enhanced ELISA in the Brain of Transgenic Mice Overexpressing Human A53T Mutated alpha-synuclein[END_REF] and proteins were extracted in high salt buffer (50 mM Tris-HCl, pH 7.5, 750 mM NaCl, 5 mM EDTA, 1 mM DTT, 1% phosphatase and protease inhibitor cocktails), using a mechanical homogenizer (grinding balls, Precellys 24, Bertin Technologies, Montigny-le-Bretonneux , France) to obtain a 10% homogenate (w/v).

Each sample was titered using the DC TM protein assay kit (ref 5000111, Biorad, Marnes-la-Coquette, France).

As already described for α-syn P detection [START_REF] Betemps | Alpha-synuclein spreading in M83 mice brain revealed by detection of pathological alpha-synuclein by enhanced ELISA[END_REF][START_REF] Sargent | Prion-like' propagation of the synucleinopathy of M83 transgenic mice depends on the mouse genotype and type of inoculum[END_REF] , plates were saturated with Superblock T20 (Thermo Scientific, Rockford, IL, USA) for 1 h at 25°C, under agitation (150 rpm). After 5 washes in PBST, 10 µg of protein (for α-syn P detection) diluted in PBST were incubated for 2 h at 25°C, under agitation (150 rpm). α-syn P was detected with a rabbit polyclonal antibody against PSer129 α-syn (ref ab59264, Cambridge, UK) diluted to 1:3,000 in PBST with 1% bovine serum albumin (BSA); plates were incubated for 1 h at 25°C under agitation. After 5 washes, anti-rabbit IgG HRP conjugate (ref 4010-05, SouthernBiotech , Birmingham, AL, USA) was added at 1:2,000 (for α-syn P detection). After washing, 100 μL of 3,3′,5,5′tetramethylbenzidine solution (ref. T0440, Sigma, Saint-Quentin-Fallavier, France) were added to each well and plates were incubated for 15 min with shaking. The reaction was stopped with 100 μL of 1 N HCl, and the absorbance was measured at 450 nm with the microplate reader Model 680 (Clariostar, BMG Labtech, Champigny sur Marne, France).

ELISA allowing total β-syn quantification was adapted based on an ELISA already published [START_REF] Krassnig | Influence of Lentiviral beta-Synuclein Overexpression in the Hippocampus of a Transgenic Mouse Model of Alzheimer's Disease on Amyloid Precursor Protein Metabolism and Pathology[END_REF] . Briefly, 2 µg or 0.2 µg (for Figure 5) of brain homogenate was diluted in carbonatebicarbonate buffer 50mM (pH 9.6) and incubated at 4°C overnight. After 5 washes in PBST, plates were saturated with Superblock T20 for 1 h at 25°C, under agitation (150 rpm). After 5 washes in PBST, plates were incubated with 1:2,000 of an anti-β-syn antibody ab76111 (ref EP1537Y, Abcam, Cambridge, UK). After 5 washes in PBST, plates were incubated with an anti-rabbit IgG HRP conjugate (ref 4010-05, SouthernBiotech , Birmingham, AL, USA) at 1:4,000. After washing, immunoreactivity was revealed with the same protocol as for α-syn PK digestion: After being deparaffinized, sections were incubated with a 10 µg/mL solution of proteinase K (ref EU0090-B, Euromedex, Souffelweyersheim, France) diluted in PBS, 10 minutes at RT (protocol adapted from 10 ). Sections were washed three times in water before blocking the endogenous peroxidase activity and pursuing the immunohistochemistry staining protocol.

Statistical analysis: Survival time was defined as the time from birth until death (Figure 3A, exclusively) or as the time from the inoculation of the brain extract until the appearance of the first specific M83 symptoms and euthanasia of the mouse. The detection of these specific symptoms was done blindly with regard to the initial AAV (β-syn or GFP) treatment. We right-censored mice found dead without M83 disease identification. Survival times were compared using log-rank test. Concerning statistical analysis of ELISA tests results, means were compared using Wilcoxon test. The difference was significant when p<0,05 (*), p<0,01(**), p<0,001(***) .

LEGENDS

  . The animals were housed per group in enriched cages in a temperature-controlled room on a 12h light/dark cycle, and received water and food ad libitum, in our approved facilities (No. C69 387 0801) for breeding and experimental studies, in accordance with EEC Directive 86/609/EEC and French decree No. 2013-118. The experimental studies described in this article were performed in containment level 3 facilities and authorized by the « Comité d'éthique » CE2A -16, ComEth ANSES/ENVA/UPEC and by the « Ministère de l'enseignement supérieur, de la recherche et de l'innovation » (ref 16-006). All experiments were performed in accordance with relevant guidelines and regulations.

9 .

 9 38*10 8 vg of AAVβ-syn or 2.73*10 8 vg of AAVGFP vectors per lateral ventricle of the viral solution with 0.05% Trypan blue 12 . Adults mice were inoculated with 3.75*10 8 vg of AAVβ-syn or 1.09*10 8 vg of AAVGFP (or with high dose: 1.5*10 9 vg of AAVβ-syn or 4.36*10 8 vg of AAVGFP) of viral solution in the ventral tegmental area located in the mesencephalon, using stereotaxic coordinates (anteroposterior: -3.16 mediolateral: +0.25 dorsoventral: -4.50). Two months after the inoculation of the viral solution in neonates or one month after the inoculation of the viral solution in adults, M83 mice were inoculated with brain homogenates from a second passage of a sick M83 brain sample in M83 mice (M83/M83 inoculum) or a second passage of a patient with multiple system atrophy (MSA)

  which sections were treated with a 4 M solution of guanidinium thyocyanate during 20 min. After washing, sections were saturated using a blocking reagent (ref 11096176001, Roche), 1 h at RT and incubated with antibody against β-syn ab76111 in PBST (ref EP1537Y, Abcam, Cambridge, UK) diluted at 1:500, anti-synaptophysin antibody SY38 (ref ab8049, Abcam, Cambridge, UK) diluted at 1:10 or anti-GFP antibody (ref ab290, Abcam, Cambridge, UK) diluted at 1:500 or anti-tubulin β-3 antibody (ref MMS-435P, Biolegend, San diego, USA) diluted at 1:100) at 4°C overnight. For the detection phosphorylated at S129 α-syn we used a rabbit antibody (ref: ab51253, Abcam, Cambridge, GB) diluted at 1:300 in TBST for immunohistochemistry, and a mouse antibody (ref: pSyn#64, FUJIFILM Wako Pure Chemical Corporation, China) diluted at 1:1000 in TBST for immunofluorescence analysis (with the rabbit anti-β-syn antibody, Supplementary figure 3), at 4°C overnight. After another blocking step 30 minutes at RT, for immunohistochemistry, sections were incubated 1 h at RT with anti-rabbit IgG HRP conjugate (ref 4010-05, SouthernBiotech, Birmingham, AL, USA) diluted at 1:250 in PBST or TBST. Antibody binding was detected using DAB peroxidase substrate (ref SK-4100, Vector Laboratories, Burlingame, CA USA) intensified with nickel chloride for β-syn or ImmPACT DAB peroxidase (HRP) substrate (ref SK-4105, Vector Laboratories, Burlingame, CA USA) for phosphorylated α-syn detection. For immunofluorescence, sections were incubated 1 h at RT with anti-rabbit IgG AlexaFluor 488 or 555 (ref A-11008 and ref A21428, respectively, Thermofischer scientific, Villebon sur Yvette, France) or with anti-mouse IgG AlexaFluor 488 or 555 (ref A11001 and A21127 respectively, Thermofischer scientific, Villebon sur Yvette, France) diluted at 1:1000. After washings in PBST then in PBS, sections were treated with an autofluorescence eliminator reagent (ref 2160, Millipore, Temecula, USA), before mounting.

Figure 1 .

 1 Figure 1. Impact of the intracerebroventricular inoculation of AAV on the accelerated disease of M83 mice. (A) M83 mice were injected at birth (Week 0) with AAVβ-syn or AAVGFP vectors per lateral ventricle and challenged two months later (Week 8) by the injection of M83/M83 (B-D) or MSA/M83 (E-G) inoculum in the striatum to accelerate the disease. After the appearance of the first specific symptoms of M83 disease (from 3 months to 8 months after challenge), mice were sacrificed and dissected in order to perform biochemical

Figure 2 .

 2 Figure 2. Characterization of the AAV vectors expression after their intracerebroventricular inoculation in M83 mice. M83 mice were injected with AAV vectors in the ventricles at birth and challenged with MSA/M83 inoculum (same mice than in Figure 1 E-G). (A) Detection of total β-syn synthesis by ELISA in cerebral regions and spinal cord of sick M83 mice 5 to 7 months after ICV injection of AAV. (B) Detection of total β-syn or GFP protein in the hippocampus of the same M83 mice by Western blot. Recombinant βsyn was loaded as a positive control. Numbers refer to each mouse (n=6 in the treated group and 5 in the control group). Full blots are shown in Supplementary figure 10. (C) Quantification of total β-syn levels detected by WB. OB: olfactory bulbs, Cx: cerebral cortex, Str: striatum, Hi: hippocampus, Mes: mesencephalon, BS: brain stem, CSC: cervical spinal cord, TSC: thoracic spinal cord, LSC: lumbar spinal cord. Data are shown as means ± sd. *p<0,05; **p<0,01 according to Wilcoxon test.

Figure 3 .

 3 Figure 3. Impact of the intracerebroventricular inoculation of AAVβ-syn or AAVGFP on the spontaneous M83 disease. (A) M83 mice were injected at birth (Week 0) with AAVβ-syn or AAVGFP vectors per lateral ventricle. After the appearance of the first specific symptoms of M83 disease (from 11 months to 20 months of age), mice were sacrificed and dissected in order to perform biochemical analysis. (B) M83 disease-associated survival after the intracerebroventricular (ICV) inoculation of AAV vectors at birth (no significant difference according to log-rank test). (C, D) Quantification of total β-syn (B) or α-syn P (C) by ELISA in CNS regions of the same sick M83 mice. No statistical analysis was done for ELISA results because the number of mice analyzed was too small (2 mice for the treated group and 3 mice for the control group). Hi: hippocampus, Mes: mesencephalon, BS: brain stem, CSC: cervical spinal cord, TSC: thoracic spinal cord, LSC: lumbar spinal cord. Data are shown as means ± sd (n=2 and 3).

Figure 4 .

 4 Figure 4. Impact of the inoculation of AAV in the ventral tegmental area on M83 disease. (A) Two months old M83 mice (Week 8) were injected with AAVβ-syn or AAVGFP vectors in the ventral tegmental area (VTA) and challenged one month later (Week 12) by the injection of M83/M83 (B-D) or MSA/M83 (E-G) inoculum in the striatum to accelerate the disease. As before, after the appearance of the first specific symptoms of M83 disease (from 3 months to 8 months after challenge), mice were sacrificed and the CNS was dissected in order to realize biochemical analysis and immunohistochemistry /immunofluorescence studies (Figure 4H, Supplementary figure 2, 3, 4). (B) M83 disease-associated survival after the inoculation of the brain extract (no significant difference according to log-rank test). (C, D) Quantification of total β-syn (C) or pathological Ser129-phosphorylated form of α-syn (αsyn P ) (D) by ELISA in CNS regions of the same sick M83 mice groups (n=5 for the treated group, n=5 for the control group). (E) The M83 disease-associated survival after the

Figure 5 .

 5 Figure 5. Characterization and impact of inoculation of AAV in the ventral tegmental area of M83 mice. (A-B) Two months old M83 mice were injected with AAVβ-syn or AAVGFP vectors in the VTA and challenged one month later by the injection of M83/M83 inoculum in the striatum to accelerate the disease. In this experiment, all mice were euthanized 3 months after the challenge for biochemical analysis. (A) Detection of the total βsyn by ELISA in the brain and the spinal cord of M83 mice after inoculation of AAVβ-syn or AAVGFP in the VTA (n=4 and 6, respectively). (B) Detection of β-syn and GFP proteins by Western blot in the mesencephalon of the same sick M83 mice. Recombinant β-syn was loaded as a positive control. Numbers refer to mice. Full blots are shown in Supplementary figure 11. (C) Total β-syn protein quantification by ELISA test using 0,2 µg (instead of 2 µg) of mesencephalon homogenates extracted from sick M83 mice inoculated with 1,5*10 9 vg (high dose, 4-times more than the low dose) or 3,75*10 8 vg (low dose) of AAVβ-syn or AAVGFP. These results were obtained with the same samples used in Figure 4C and 5D, but 10 times diluted. (D-F) Two months old M83 mice (Week 8) were injected with high dose of AAV vectors (4-times higher than in Figure 4 for each vector) in the VTA and challenged one

Figure 6 .

 6 Figure 6. Proteinase K (PK)-resistant β-syn staining in sick M83 mice inoculated with AAV β-syn. (A-D) Immunoreactivity to total β-syn antibody in mesencephalon sections of two sick M83 mice inoculated with low dose of AAVβ-syn or AAVGFP in the VTA and challenged by MSA/M83 inoculum (mice from the study Figure 4E-G) is shown without PK digestion (A, B) or after PK digestion (C, D). After PK digestion, these β-syn immunoreactive dots were detected in all mice analyzed by immunohistochemistry inoculated with the AAVβsyn in the VTA (7/7 mice, comprising 2 mice challenged with M83/M83 inoculum, 3 mice challenged with MSA/M83 inoculum, and also in 2 mice inoculated with high dose of AAV and challenged with M83/M83 inoculum), but not in sick M83 mice inoculated with AAVGFP (3/3, comprising 2 mice challenged with MSA/M83 inoculum and 1 mouse challenged with M83/M83 inoculum). Scale bars 100µm (low magnification) and 25µm (high magnification).

Acknowledgements

We are grateful to Eric Morignat and Habiba Tlili for their help and advices on statistical and immunofluorescence assays, respectively. We also are grateful for Olivier Biondi for its help setting up behavioral test and the method for quantifying motor neurons in the spinal cord.

We thank Ronald Melki and its team for providing us preformed fibrils of recombinant α-syn.

This research was partly funded by the Fondation France Parkinson. D.S was supported by funds from the Région Auvergne-Rhône-Alpes -ARC1 Santé.

Western blot: 50 µg (for phosphorylated Akt detection) or 10 µg of proteins (for total Akt detection) or 2 µg of proteins (for GFP and β-syn detection) were separated in 12% SDSpolyacrylamide gels and electroblotted onto polyvinylidene fluoride (PvF) membranes 0,45 µm (Bio-Rad). The membranes were washed 3 times in Tris-Buffered Saline (TBS) for 5 min at room temperature (RT) under agitation and were saturated 1 h with 5% BSA in TBS 0.1% Tween20 (TBST). Membranes were incubated with rabbit antibody against phosphorylated Akt at Ser473 (ref 9271S, Ozyme, Montigny-le-Bretonneux, France) at 1:1,000 or with rabbit

Conflicts of interest

The authors declare that they have no competing interests.


Rim Aissa

Samia Guezane-Lakoud

Emilie Kolodziej

Martial Toffano

Louisa Aribi-Zouioueche

lipase catalytic promiscuity

Diastereoselective synthesis of bis(α-aminophosphonates) by

Introduction

The α-aminophosphonates are of great biochemical and pharmacological effects 1,2 due to the association of phosphonate and amine. These molecules are phosphorus structural analogues of amino acid 3 and they are interesting for medicinal chemistry. 4 The α-aminophosphonates derivatives have received much attention owing as enzyme inhibitors, pharmacogenic agents, haptens of catalytic antibodies, herbicidals, inhibitors of serine hydrolases, inhibitors of UDP-galactopyranose mutase and also as anticancer agents as reported in the literature. 5 The Kabachnik-Fields reaction is the most efficient method for the synthesis of α-aminophosphonates. 6 We found various synthetic methodologies using different catalysts for the synthesis of α-aminophosphonates derivatives. 7 Recently, we have reported the synthesis of α-aminophosphonates by enzymatic catalytic promiscuity using Candida antarctica lipase B (CAL-B) by Kabachnik-Fields reaction. 8 This is the first example of C-P bond formation involving the principle of enzymatic promiscuity. These findings inspired us to seek lipase that could catalyze the formation of bis(α-aminophosphonates). These multidentate ligands are extremely interesting compounds that can be used in the extraction of metals or as monomers in the preparation of macrocyclic compounds or as polymers carrying phosphonate and amine. 9 Bis(α-aminophosphonates) exhibit significant biological activities (Figure 1), compound 1 shows antifungal activity, compound 2 has antioxidant activity, 10 and compound 3 shows optimal antiproliferative activity to human tumoral cells from colon carcinoma. 11 Figure 1. Bis(α-aminophosphonates) having biological activities Bis(α-aminophosphonates) were synthesized from Schiff bases 12 or diamine 9,13,[START_REF] Milen | [END_REF] under microwave irradiation 10 or with Lewis acid. 9 However, most of these approaches involve longer reaction times, expensive reagents, high temperature, stoichiometric amount of catalysts, low diastereoisomers ratio and metallic contamination due to the use of metal catalyst or Lewis acid. Herein, we applied catalytic promiscuity with lipase as an efficient 'Green' method for the synthesis of new bis (α-aminophosphonates) with excellent diastereocontrol by Kabachnik-Fields multi-component condensation.

Enzymes are well-known for catalyzing specific reactions, and for their ability to perform highly regio-, chemo-and stereo-selective transformations, at high reaction rates in relatively mild conditions. 15 Enzymes have many benefits in the context of green and sustainable chemistry and are integrated to efficient and environmentally benign processes for chiral drug development by kinetic resolution or asymmetric synthesis. 16 Lipases especially, are the most used enzymes in synthetic organic chemistry. 17 They are known for their advantages such as providing products of high purity and good selectivity in one reaction step, fewer byproducts and simple separation, as shown in our previous works. 18 Furthermore, the use of organic solvents in the enzymatic reaction instead of conventional media or aqueousorganic co-solvent mixtures, 19 allowed application of enzymes to expand in catalytic promiscuity, especially in multi-component reaction. Exploiting enzymatic catalytic promiscuity might lead to improvements in existing catalysts and furnish judicious synthesis pathways. Enzymatic promiscuity is the ability of a single enzyme active site to catalyze multiple chemical transformations with different transition states, other than the reactions for which it has been specialized. This innovating approach is the starting point for a divergent evolution of new protein function which is explained by the creation of a new enzymatic activity depending on circumstances. 20, 21 Hult and Berglund 22 defined three types of promiscuity: (i)-condition promiscuity: catalysis of different reactions under different conditions from the native one, (ii)-substrate promiscuity: enzymes with broad substrate specificity and (iii)-catalytic promiscuity based on the ability of a single enzyme active site to catalyze several chemical transformations with different transition states. Recently, the product promiscuity was considered as a fourth kind, in which a single enzyme converts a single substrate to multiple products by different transition states. 20a (Figure 2). 24 Biginelli reaction, 25 Mannich reaction, 26 Morita-Baylis-Hillman reaction, 27 Henry reaction, 28 Michael reaction, 29 Aldolisation reaction 30 and others. 31,32,33 In organophosphorus chemistry, utilization of lipases is also described for the resolution of racemic hydroxyphosphonates by acylation 34 or hydrolysis. 35 To the best of our knowledge, the use of lipase catalytic promiscuity in a diastereoselective synthesis including the simultaneous formation of two P-C bond is novel and has never been reported yet (c est une proposition de modification.

Results and discussion

The multi-component reactions (MCRs) are realized by lipase catalytic promiscuity for the bis(α-aminophosphonates) synthesis. This new benign and clean protocol implies the condensation in one pot of two equivalents of benzaldehyde 1a, one equivalent of benzidine 2 and two equivalents of diethylphosphite 3 which is used as model reaction with lipase in 2 mL of organic solvent. Several parameters have been studied such as: lipases, solvents, temperature and the reaction time. The reactions were monitored by TLC analysis. The products were obtained in two diastereoisomers mixture, and then separated by extraction or by flash chromatography in ethyl acetate and hexane (70/30). All results are summarized in Table 1.

Table 1. Optimization reaction of the bis (α-aminophosphonates).

Entry a

Lipase (mg) Solvent T °C Time /h Yield(%) In all cases the bis (α-aminophosphonates) 4a were obtained with high yield by catalytic promiscuity using CAL-B. Firstly, the multi-component reaction was performed without lipase in THF during 24 hours, the results show that no reaction was observed even by increasing the temperature up to 70°C (Table 1, entry 1). The denatured CAL-B has been tested, there is no advancement. The nature of the lipase has a significant effect on reaction, five lipases were examined (two animals and three microbial): Rabbit gastric Lipase (RGL) lipase, 36 Porcine pancreatic lipase (PPL) and: Candida cylindracea lipase (CCL), Pseudomonas cepacia lipase (PCL) which are free lipases and Candida antarctica B lipase immobilized on acrylic resin (CAL-B). Under these conditions, only CAL-B can promote catalytic promiscuity and allows obtaining 4a compound with 80% yield (Table 1, entry 2), compared with the use of PCL, CCL, PPL and RGL lipases which lead to the bis(α-aminophosphonates) with low yields (< 29%) (Table 1, entries 3, 4, 5 and6). For a better performance, the temperature was increased to 50°C with CAL-B and the product 4a was obtained in 90% yield (Table 1, entry 7). Product 4a was obtained in a similar yield at the same temperature by decreasing the amount of CAL-B lipase from 100 mg to 50 mg (Table 1, entry 8) but no reaction by less than 50 mg of CAL-B or at room temperature. Subsequently, the hydrophobicity solvent effect has been studied, we found that the THF is the best solvent (Table 1, entry 8 vs entries 9, 10, 11 and 12). No progress of the reaction was observed in TBME and toluene (Table 1, entry 9 and 10) and a moderate yield was obtained in Et2O (26%) (Table 1, entry 11), whereas in AcOEt 62% of 4a was isolated (Table 1, entry 12). A study of the time was also carried out; we found that at the 30th minute, a total disappearance of benzidine was observed (90% yield) (Table 1, entry 15).

In addition, a recycling of CAL-B was examined on the model reaction, it showed that the lipase lost its catalytic performance from the fourth cycle (Fig 3). So, in optimized conditions, the bis(α-aminophosphonate) 4a was obtained with high yield using CAL-B (50mg) in THF at 50°C. In order to explore the effectiveness and limitations of the MCRs by biocatalytic promiscuity with CAL-B, we applied optimized reaction conditions on a series of variously substituted aldehydes with electron-withdrawing and electron-donating groups (Table 2). The molecular structure of all bis (α-aminophosphonates) was confirmed by NMR 1 H, 13 C and 31 P spectroscopy and HRMS analysis. 1 H NMR and 31 P NMR experiments were used for diastereomers (meso/dl ratio) quantification in the crude reaction. The results of Table 2 show the efficiency of the MCRs catalyzed by CAL-B for the preparation of bis(α-aminophosphonates) giving a 66-90% yield with high diastereoselectivity control depending on the electronic effect of aldehydes. The best results yields (> 80%) are obtained for: 4a, 4b, 4c, 4d, 4h, 4j and 4k, and with average yields (66-77%) for 4e, 4f, 4g, and 4i compounds.

In addition, five derivatives of bis (α-aminophosphonates) 4a, 4c, 4h, 4i and 4k are obtained with a total diastereoselectivity (100% dr). A high diastereoselectivity is obtained for 4b (96/4% dr), even for 4f and 4j which is obtained with (80/20% and 86/14% dr) respectively, it also remains good for 4d (71/29% dr). On the other hand, the diastereoselectivity decreases sharply for the 4g and 4e, this is probably be due to the high steric hindrance of aldehydes. To the best of our knowledge, all these bis(α-aminophosphonates) derivatives, except the 4h compound, have never been reported in the literature yet. 37 We completed this works by a multi-gram scale reaction. Thus, a scaling up of the procedure was carried out from 0,636 g (6 mmol) of aromatic aldehyde, 0,636 g (3 mmol) benzidine, (0,828 g, 6 mmol) diethylphosphite in THF and an appropriate amount of CAL-B lipase. The quantitative yields were obtained regardless of the scale and the remaining product was isolated with (up on 92%).

In order to be able to propose a reaction mechanism, we proceeded to several experiments. When the multicomponent reaction is carried out with an equimolar quantity of starting materials (1/1/1 ratio of aldehyde / benzidine / diethylphosphite), the results show that the bis(α-aminophosphonates) is the sole and only compound obtained in these reaction conditions. No trace of mono-phosphonated compound is observed. This suggests that the formation of the C-P bond is performed on both sides simultaneously of benzidine or that the second addition of diethylphosphonate is extremely rapid and efficient which is probably favored by the proximity of the monophosphonate/enzyme pair. Anyway, at this stage, we suppose that the enzyme plays a crucial role in the privileged selectivity for the bis-phosphonate compounds formation. Combining the viewpoints developed by Per Berglund et al 22 and on the basis of our observations described above, we proposed the mechanism of the reaction, summarized in Scheme 1. We propose a mechanism involving the coordination to the oxyanion hole by the carbonyl and simultaneous activation of nucleophile by histidine and aspartate from the active site.

Scheme 1. Proposed mechanism of CAL-B-catalyzed bis(α-aminophosphonates) synthesis

The oxyanion hole activates the carbonyl of aldehyde substrate and renders it susceptible to nucleophilic attack by one amine of benzidine to form an imine (Step I). The active-site histidine with aspartate as a base that activates the diethylphosphite as nucleophile for attack of the imine to form the corresponding product (Step II). In a concomitant manner in the same active site of lipase, the oxyanion hole activates the carbonyl of aldehyde which promotes the attack of the second amine of benzidine to form the corresponding imine (Step III). The following nucleophilic attack of the diethylphosphite activated by histidine / aspartate couple, leads to the formation of the bis(α-aminophosphonate) 4.

Conclusion

In conclusion, we describe the first simultaneous formation of two Carbon-Phosphore bonds by catalytic promiscuity using acrylic resin-immobilized lipase CAL-B in a one pot multi-component reaction. This original strategy gives access to a novel series of bis(α-aminophosphonates) with high diastereoselectivity and very satisfactory yields, in a short reaction time and under mild and green chemistry conditions. The lipase-catalyzed Kabachnik-Fields reaction provides a new case of catalytic promiscuity and might be a useful synthetic method for bioorganic synthesis. The CAL-B was easily recovered and reused several times. We have also, devised a multi-gram scale reaction for the preparation of bis(α-aminophosphonates) 4a with replicable quantitative yields . The development of this new easy route opens up very interesting perspectives to easily recoverable applications. We design a straightforward, efficient and utile protocol to create double Carbon-Phosphore bond formation using CAL-B in non-conventional media and metal-free access. These results suggest a new synthetic route toward biological derivatives under mild and green conditions.

Experimental section

General information: All starting materials, reagents and enzymes used in this study were purchase from Sigma-Aldrich or Acros company and were used without purification. Various commercially available lipases were screened: lipase from Porcine pancreas type II (PPL; LA = 100-500 U/mg), Rabbit Gastric Lipase (RGL; LA > 1170 U/mg), Candida Cylindracea lipase (CCL, LA = 3.85 U/mg), Pseudomonas Cepacia lipase (PCL; LA > 30 000 U/mg), and the Candida antarctica lipase fraction B immobilized on acrylic resin (CAL-B; LA > 5 000 U/mg). All reactions were monitored by thin-layer chromatography (TLC) carried out on 0.25-mm Merck silica gel plates (60F-254) using ultraviolet light (254 nm) as the visualizing agent and KMnO4 solution as developing agents. 1 H NMR and 13 C NMR spectra were recorded with Bruker spectrometers (360MHz, 300 MHz and 250 MHz for 1H, 90 MHz, 75 MHz or 63 MHz for 13 C and 101 MHz or 121 MHz for 31 P). Chemical shifts were reported downfield from CDCl3 (δ = 7.26 ppm). For 13 C NMR, chemical shifts were reported in the scale relative to the solvent of CDCl3 (δ = 77 ppm) used as internal reference. Coupling constants (J) are given in hertz. Following abbreviations classify the multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad signal. Melting points were measured using Buchi Melting Point B-545. Mass spectra were recorded with a MicrOTOF-Q Bruker spectrometer using electrospray ionization (ESI) analysis.

General procedure for synthesis of bis(α-aminophosphonates) 4a-4k.

To the stirred mixture of aromatic aldehyde (2 mmol), benzidine (184 mg, 1 mmol) and diethylphosphite (331.2 mg, 2.4 mmol) in THF (2ml), the CAL-B (50 mg) was added. The reaction was stirred at 50°C for 30 minutes. After the reaction proceeded to completion, the lipase was removed by filtration. The two diastereoismer mixtures were separated by column chromatography using ethyl acetate /hexane: 90/10 as eluent. Complete experimental data have been provided (NMR spectra and HRMS). These compounds do not have rotatory power, it means that the diastereomers obtained exist as meso (RS/SR) or racemic (RR/SS) forms mixtures. Tetraethyl [(4,1-phenylene) bis(azanediyl)] bis[(phenyl) methylene] bisphosphonate (4a). Yellow solid, Yield: 90%, mp 210°C. 1 H NMR (250 MHz, CDCl3) δ 7.49 (dd, J = 5.6, 3.8 Hz, 4H, H-Ar), 7.38 -7.17 Tetraethyl [(4,1-phenylene)bis(azanediyl)] bis[(4-nitrophenyl) methylene]bisphosphonate (4b). Orange solid, 90% Yield, mp: 219.5°C. 1 H NMR (250 MHz, CDCl3) δ 8.20 (d, J = 8.3 Hz, 4H), 7.66 (dd, J = 8.9, 2.3 Hz, 4H), 7.27 -7.18 (m, 4H), 6.54 (d, J = 8.6 Hz, 4H), 4.86 (d, J = 25.1 Hz, 2H), 4.22 -3.96 (m, 8H), 3.93 -3.81 (m, 2H), 1.30 (t, J = 7.1 Hz, 6H), 1.18 (t, J = 7.1 Hz, 6H). 13 C NMR (63 MHz, CDCl3) δ 144. 21 (dd, J = 20.4, 8.9 Hz), 132.16 -131.84 (m), 128.63 (d, J = 4.7 Hz), 127.31 (s), 123.78 (s), 114.12 (s), 63.63 (dd, J = 15.8, 7.0 Hz), 57.33 (s), 54.98 (s), 16.33 (dd, J = 10.9, 5.7 Hz). 31 Tetraethyl [(4,1-phenylene)bis(azanediyl)] bis[(4-fluoro-phenyl)methylene] bisphosphonate (4d). Green solid, 84% Yield, mp: 172.3°C. 1 H NMR (250 MHz,CDCl3,4H,7.27 (d,J = 8.5 Hz,4H,, 7.06 (t, J = 8.6 Hz, 4H, H-Ar), 6.62 (d, J = 8.4 Hz, 4H, H-Ar), 4.79 (d, J = 24.2 Hz, 3H, HCP+NH), 4.25 -4.07 (m, 4H, O-CH2-CH3), 4.07 -3.84 (m, 2H, O-CH2-CH3), 3.86 -3.63 (m, 2H, O-CH2-CH3), 1.31 (t, J = 7.1 Hz, 6H, O-CH2-CH3), 1.17 (t, J = 7.0 Hz, 6H, O-CH2-CH3). 13 C NMR (63 MHz, CDCl3): δ 164. 44 (s), 160.47 (s), 144.79 (d, J = 15.0 Hz), 131.64 (d, J = 13.2 Hz), 130.13 -128.79 (m), 127.14 (s), 115.76

Figure 2 .

 2 Figure 2. Different kinds of promiscuity Catalytic promiscuity is described by several reactions for the C-C and C-N bond formation in the presence of different enzymes 23 such as: Knoevenagel reaction,24 Biginelli reaction,25 Mannich reaction,26 Morita-Baylis-Hillman reaction, 27 Henry reaction,28 Michael reaction,29 Aldolisation reaction 30 and others.31,32,33 In organophosphorus chemistry, utilization of lipases is also described for the resolution of racemic hydroxyphosphonates by acylation 34 or hydrolysis.35 To the best of our knowledge, the use of lipase catalytic promiscuity in a diastereoselective synthesis including the simultaneous formation of two P-C bond is novel and has never been reported yet (c est une proposition de modification.

Figure 3

 3 Figure 3 Reusing of the CAL-B catalyst

a

  Reaction conditions: benzaldehyde (2 mmol), benzidine (1 mmol) and diethylphosphite (2.4 mmol), CAL-B (50 mg), room temperature, 30min. b Yield of the pure product, purified by column chromatography or recrystallization from hexane.

  (m, 10H, H-Ar), 6.61(d, J = 8.6 Hz, 4H, H-Ar), 4.78 (d, J = 24.3 Hz, HCP), 4.24 -4.02 (m, 4H, O-CH2-CH3), 4.02 -3.82 (m, 2H, O-CH2-CH3 ), 3.74 -3.57 (m, 2H, O-CH2-CH3), 1.28 (t, J = 7.1 Hz, 6H, O-CH2-CH3), 1.11 (t, J = 7.1 Hz, 6H, O-CH2-CH3). 13 C NMR (91 MHz, CDCl3) δ 144.89 (d, J = 14.7 Hz), 135.92 (s), 131.42 (s), 128.62 (s), 127.90 (dd, J = 9.5, 4.0 Hz), 127.10 (s), 114.10 (s), 63.31 (d, J = 6.0 Hz), 56.96 (s), 55.30 (s), 16.32 (dd, J = 22.2, 5.9 Hz). 31 P NMR (121 MHz, CDCl3, 25°C): δ 22.63 ppm. HRMS (ESI) m/z calcd for C34H42N2O6NaP2 [M +Na + ]: 659.2410; Found 659.2406.

  P NMR (121 MHz, CDCl3, 25°C): δ 20.70 ppm. HRMS (ESI) m/z calcd for C34H40N4O10NaP2 [M+Na + ]: 749.2111; Found 749.2129. Tetraethyl [(4,1-phenylene)bis(azanediyl)] bis[(4-cyano-phenyl) methylene] bisphosphonate (4c). Yellow solid, 85% Yield, mp: 203.7°C. NMR 1 H (250 MHz, CDCl3, 25°C) :δ 7.71 -7.58 (m, 8H, H-Ar), 7.31 -7.18 (m, 4H, H-Ar), 6.57 (d, J = 11.0 Hz, 4H, H-Ar), 4.84 (d, J = 22.0 Hz, 4H, HCP+NH ), 4.24 -4.10 (m, 4H, O-CH2-CH3), 4.09 -3.96 (m, 2H, O-CH2-CH3), 3.95 -3.75 (m, 2H, O-CH2-CH3), 1.31 (t, J = 7.1 Hz, 6H , O-CH2-CH3), 1.19 (t, J = 7.1 Hz, 6H, O-CH2-CH3). NMR 13 C (63 MHz, CDCl3, 25°C): δ 144.42 (d, J 3 C-P = 14.2 Hz), 142.00, 139.17 (s), 132.38 (s), 131.78 (s), 128.55 (d, J 2 C-P = 5.1 Hz), 127.27 (s), 114.09 (s), 111.79 (d, J = 3.4 Hz), 111.13 (s), 63.66 (d, J C-P = 14.8Hz, C-O), 63.42 (d, J C-P = 6.7 Hz, C-O), 57.38(s, C-N), 55.02(s, C-P), 16.43 (d, J 3 C-P = 5.7 Hz, CH3-CH2-O-P), 16.24 (d, J 3 C-P = 5.5 Hz, CH3-CH2-O-P). 31 P NMR (121 MHz, CDCl3, 25°C): δ 22.05 ppm. HRMS (ESI) m/z calcd for C36H40N4NaO6P2 [M +Na + ]: 709.2135; Found 709.2344.

Table 2

 2 Lipase-catalyzed bis (α-aminophosphonates) synthesis a

Acknowledgements

The Algerian Ministry of Higher Education and Scientific Research (DGRSDT, FNR) is gratefully acknowledged for financial support of this work. The authors knowledge Dr. Nadia Grine for English reviewing of this manuscript.

Conflicts of interest

There are no conflicts to declare.