Amina Annane

Mouna Kamel

Cassia Trojahn

Nathalie Aussenac-Gilles

Catherine Comparot

Christophe Baehr
email: christophe.baehr@meteo.fr

Towards the FAIRification of Meteorological Data: a Meteorological Semantic Model

Keywords: Meteorological data, FAIR principles, Semantic metadata

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Meteorology data is essential in many applications, including forecasts, climate change, environmental studies, agriculture, health and risk management. Their production is based on mathematical models that assimilate different data from several sources including weather stations, satellites and weather radars. While this data has been made available as open data through different portals, such as governmental portals (e.g., MeteoFrance3 , worldweather 4 ), or associative or private portals (e.g., infoclimat5 , meteociel 6 ), under open licenses, its exploitation is rather limited: it is described and presented with properties that are relevant for meteorology domain experts (data producers) but that are not properly understood and reusable by other scientific communities. For the latter, one of the challenges is to find relevant data among the increasingly large amount of continuously generated data, by moving from the point of view of data producers to the point of view of users and usages. One way to reach this goal is to guarantee compliance of data to the FAIR principles (Findability, Accessibility, Interoperability, and Reusability) [START_REF] Wilkinson | The FAIR Guiding Principles for scientific data management and stewardship[END_REF]. These principles correspond to a set of 15 recommendations to be followed in order to make data FAIR (Figure 1). A key step toward improving FAIRness of data is the use of semantic models (i.e., ontologies) for data and metadata representation [START_REF] Guizzardi | Ontology, Ontologies and the "I" of FAIR[END_REF].

While most efforts in data FAIRification are limited to describing data with semantic metadata, such a description is not enough to fully address all FAIR principles [START_REF] Koesten | Everything you always wanted to know about a dataset: Studies in data summarisation[END_REF], in particular for promoting reuse of this data by other scientific communities. We propose to overcome this weakness through a richer representation of the meaning of meteorological data in a formal model which allows for sharing the semantic meaning with third-parties [START_REF] Kremen | Improving discoverability of open government data with rich metadata descriptions using semantic government vocabulary[END_REF]. Contrary to existing works involving ontology population [START_REF] Lefort | A linked sensor data cube for a 100 year homogenised daily temperature dataset[END_REF][START_REF] Roussey | Weather data publication on the LOD using SOSA /SSN ontology[END_REF][START_REF] Atemezing | Transforming meteorological data into linked data[END_REF][START_REF] Patroumpas | Exposing points of interest as linked geospatial data[END_REF][START_REF] Arenas | Un modèle pour l'intégration spatiale et temporelle de données géolocalisées[END_REF], and due to the characteristics of meteorological data, we do not transform all data into RDF but rather represent in a fine-grained way the data schema and its distribution structure. The contributions of this paper are as in the following:

-Proposing a semantic model representing both metadata and data schema of meteorological observation data. -Combining existing vocabularies that follow themselves the FAIR principles.

-Instantiating the proposed model with a real meteorological dataset provided by Météo-France -the official weather service in France. -Evaluating the FAIRness degree of this dataset without and with the proposed model showing how the proposed model improves the FAIRness degree.

The paper is organized as follows. §2 introduces the meteorological data specificities and presents the proposed model. §3 shows the instantiation and §4 presents the evaluation. §5 discusses the related work and §6 ends the paper.

Meteorological Semantic Model

In order to develop our model, we have followed the NeOn methodology scenario 3 "Reusing ontological resources" [START_REF] Suárez-Figueroa | The neon methodology framework: A scenario-based methodology for ontology development[END_REF]: those cases in which ontology developers have at their disposal ontological resources useful for their problem and that can be reused in the ontology development. The methodology includes activities related to the reuse of ontologies (ontology search, ontology assessment, ontology comparison, ontology selection and ontology integration) in addition to the main activities (specification, conceptualization, formalization, implementation). For the sake of space, we focus here on the presentation of the model, without describing in detail the different activities of the methodology. In the following, we summarize the result of specification and knowledge acquisition in §2.1, and the result of ontology selection and integration in §2.2.

Meteorological data characteristics

There exist different types of meteorological data: satellite data, model data that are computed using statistical models such as weather forecast data, radar data, etc. We focus on observation data referred to as "in situ" data. They are direct measurements of various parameters (temperature, wind, humidity, etc.) taken by instruments on the ground or at altitude from predefined locations:

-Geospatial data: the measure values must be localised, otherwise it is not fully exploitable. The localisation is usually defined using geospatial coordinates (latitude, longitude and altitude). The interpretation of these coordinates depends on the used coordinate reference system, hence the Coordinate Reference System (CRS) has also to be indicated. -Temporal data. Each measurement is made at a specific time that must be noted with the measurement result (i.e., value). As for the geospatial, the temporal localisation is essential to the right interpretation of measurements. -Large volume data: meteorological data are produced continuously. Within each station, several sensors are installed (thermometer, barometer, etc.). Each sensor generates multiple measure values with a frequency that differs from one measure to another (hourly, trihoral, daily, etc.). -Conform to WMO guidelines: the measurement procedures, the types of sensors to use, the quality standards, and many other specifications are defined by the World Meteorological Organization (WMO). The latter provides detailed guides, such as the guide to meteorological instruments and observation methods 7 where there is a chapter for each measure describing all details about it. Nevertheless, to our knowledge, no semantic version of these guides exists. -Tabular data: observation data are usually published in tabular format where measure values are organized according to spatio-temporal dimension. Ac-cording to a recent study made by Google, the tabular format is the most widespread format for publishing data on the Web (37% of the datasets indexed by Google are in CSV or XLS) [START_REF] Benjelloun | Google dataset search by the numbers[END_REF].

Metadata and data representation

The proposed semantic model (Figure 2) represents both metadata and data schema of meteorological observation data, as described in the following. It has been implemented in OWL 8 and its consistency has been verified with the different reasoners implemented in Protégé (Hermit, ELK, and Pellet).

Metadata representation Making data FAIR requires first and foremost the generation of semantic metadata. Indeed, 12 out of the 15 FAIR principles refer to metadata as described in [START_REF] Wilkinson | The FAIR Guiding Principles for scientific data management and stewardship[END_REF] (Figure 1). This metadata must remain accessible even if the data itself is no longer accessible. These 12 principles provide guidance on the categories of metadata: (i) descriptive metadata for data indexing and discovery (title, keywords, etc.); (ii) metadata about data provenance;

(iii) metadata about access rights and usage licenses. Particularly for publishing data on the web, W3C recommends three other categories of metadata: (i) version history; (ii) quality; (iii) structure. Our goal is therefore to propose metadata model that covers these different categories, thus ensuring adherence to the principle on rich metadata. For metadata representation, our proposition relies on GeoDcat-AP, except structural metadata that are covered by CSVW.

GeoDcat-AP.

It is a specification of the DCAT-AP vocabulary which is a specification of the W3C DCAT (Data CATalog vocabulary) recommendation. The choice of GeoDCAT-AP is motivated by the richness of the vocabulary metadata.

In addition to allowing to describe data catalogs, it offers specific properties required to correctly interpret spatial data such as the geographical area covered by the data (dct:spatial), the reference coordinate system used (dct:conformsTo) to be chosen from a list defined by the OGC9 , as well as the spatial resolution (dcat:spatialResolutionInMeters) of the data. GeoDCAT-AP is also recommended by W3C/OGC to describe geospatial data on the Web [START_REF] Van Den Brink | Best practices for publishing, retrieving, and using spatial data on the web[END_REF].

CSVW. As pointed out in [START_REF] Koesten | Everything you always wanted to know about a dataset: Studies in data summarisation[END_REF], it is essential for data reuse to represent the internal structure of the data. Since observation data are mostly tabular data, CSVW10 is a suitable vocabulary. It resulted from the work of the W3C group on publishing tabular data on the web. It allows to define the different columns csvw:Column of a given csvw:Table (i.e., csv file) via the csvw:Schema concept. Moreover, it represents the interdependence between two tables. Indeed, it allows to represent if a column (or a set of columns) in a given csv file is a foreign key csvw:ForeignKey that references a column (or columns) of another CSV file.

Web Annotation ontology (oa). It is a W3C recommendation for representing data annotations. As discussed in §2.1, all WMO members (i.e., states) use the same guides developed by WMO. It is used here to annotate parts of these guides that describe relevant metadata about measures. We use mainly three classes: (i) oa:Annotation, (ii) oa:SpecificResource to represent the annotated document, and (iii) the document-part annotated via the class oa:RangeSelector.

Data representation

We have made the choice to not transform all data into RDF because it is: (i) expensive: transforming the data archived for decades requires human and physical resources, and (ii) not effective: it would result in a huge RDF graph that would not be effective for querying and accessing the data [START_REF] Karim | Compact representations for efficient storage of semantic sensor data[END_REF]. We rather represent the data schema and the RDF data cube (qb) vocabulary has been considered for that. In addition to qb, we have reused several domain and cross-domains ontologies for making explicit the semantics of measures and dimensions using concepts from the meteorological domain. It is worth to mention that we use CSVW to represent the syntactical structure of a tabular dataset distribution, while we use RDF data cube and domain ontologies to represent the semantics of the dataset independently of any data format.

RDF data cube (qb).

It is a W3C vocabulary [START_REF] Van Den Brink | Best practices for publishing, retrieving, and using spatial data on the web[END_REF] 11 dedicated to the representation of multi-dimensional data. qb is suitable in our case since observation data is multi-dimensional data organized according to spatio-temporal dimensions. It describes the multidimensional data schema using three subclasses of qb:ComponentProperty: (i) measures qb:MeasureProperty, (ii) dimensions qb:DimensionProperty according to which the measures are organized, and (iii) attributes to represent metadata qb:AttributeProperty. The qb:concept property allows to link a qb:ComponentProperty (i.e., measure, dimension or attribute) to the corresponding concept to explicit its semantics. We use this property to associate component properties to domain ontologies.

Domain ontologies. Meteorological data refers to concepts from the meteorological domain such as atmospheric parameters (e.g., temperature, wind speed), sensors (e.g. thermometer, barometer), etc. For expliciting their semantics, the following domain and cross-domain ontologies are used: SOSA [START_REF] Janowicz | Sosa: A lightweight ontology for sensors, observations, samples, and actuators[END_REF] (Sensor, Observation, Sample, and Actuator), the reference ontology to represent observations; ENVO (Environment ontology) [START_REF] Buttigieg | The environment ontology: contextualising biological and biomedical entities[END_REF] and SWEET (Semantic Web Earth and Environment Technology ontology) [START_REF] Raskin | Development of ontologies for earth system science[END_REF], to designate the atmospheric parameters represented with qb as measurements; aws (ontology representing weather sensors) 12 , to designate the type of weather sensor used to measure each atmospheric parameter; and QUDT, to specify the unit of measurement of each measurement.

New entities

In order to be able to fully address the ontology requirements, we have introduced the following entities: -":ObservationDataset" concept: given the continuous production of observation data, it is usually archived as fragments, where each fragment contains data of a given period (day, month, etc.). qb allows to represent the notion of a dataset (qb:Dataset) with multiples fragments (qb:Slice). However, GeoDCAT-AP offers only the possibility to represent a given dataset as a whole (dcat:Dataset). Since it is the same dataset fragment that we describe with GeoDCAT-AP and RDF data cube, we have defined a new concept :ObservationDataset that is a subclass of both qb:Slice and dcat:Dataset. -":CsvDistribution" concept: it represents distributions in csv format. It is a subclass of both csvw:Table and dcat:Distribution. -":requires" property: when a dataset X requires another dataset Y to be exploited, it is essential to enable the reuse of X to represent this dependency relation. As GeoDCAT-AP does not offer the possibility of representing such a relation between two datasets, we have added this new property. -":references" property: it allows to associate each csvw:Column to an qb component property (i.e., qb:MeasureProperty or a qb:DimensionProperty): Thus, we explicit the link between the structural components (i.e., columns) and the data schema components (i.e., measures and dimensions). Moreover, data schema components are associated to domain ontology concepts, thereby explicit the semantic of each column too.

Overview of SYNOP dataset

The SYNOP dataset is an open meteorological datasets provided by Météo-France on its data portal. It includes observation data from international surface observation messages circulating on the Global Telecommunication System (GTS) of the World Meteorological Organization (WMO). The choice of this dataset is motivated by the fact that this data is open and free, and it concerns several atmospheric parameters measured (temperature, humidity, wind direction and force, atmospheric pressure, precipitation height, etc.). These parameters are important for many scientific studies. The dataset 13 is described by seven items: (i) description: natural language summary that describes the content of the dataset, (ii) conditions of access: Etalab license 14 for the data, (iii) means of access: specifies that the data can be accessed via direct download, (iv) download : possibility offered to the user to download the data in csv format for a given date, (v) download archived data: similar to the previous item, but for a given month, (vi) station information: list of stations (station id, name) accompanied by a map displaying the location of these stations, and (vii) documentation, links to a data dictionary, to CSV and JSON files listing the meteorological stations of Météo-France (id station, name, latitude, longitude, altitude). Table 1 shows an extract of the SYNOP data. The file contains 59 columns, the first two correspond to the station number and the date of the measurements made, the other 57 columns to the meteorological measurements.

Model instanciation

The SYNOP data archive consists of a set of monthly files since January 1996, where each file covers only the observations made in one month. The data of each monthly file corresponds to an instance of the :ObservationDataset. :synop dataset feb20 is the instance corresponding to the SYNOP dataset of February 2020 (Table 1). For sake of space, we present a fragment of the instantiated model that show both metadata and data representation. It corresponds to the representation of a distribution of the dataset: :synop distribution feb20 it is subject to an open license, the value of the dct:license property. Finally, the columns (e.g., numer sta and pmer) of this file are characterized by their name (csvw:name), their label (csvw:title), their type (csvw:datatype) from the schema :synop file schema (csvw:tableSchema), etc. Note the representation of the foreign key :fk which connects the column "numer sta" of the SYNOP data, to the column "ID" of the station data (:station distribution) by passing through the instance :tr of csvw:TableReference.

In order to express the fact that all the monthly data are part of the same SYNOP dataset, we represent it as an instance of qb:Dataset (Figure 4). We have defined one spatial dimension :station dimension and three temporal dimensions: :year dimension, :month dimension, and :date dimension. The spatial or temporal nature of a dimension is specified using the concepts of the qb4st vocabulary qb4st:SpatialDimension and qb4st:TemporalProperty, respectively. Note that the year and month dimensions do not refer to existing columns, but are included in the date column. We have added them to instantiate qb:Slice. Indeed, the instantiation of a qb:Slice requires the definition of dimensions with fixed values, which are specified using the qb:SliceKey concept. In our case, the fixed dimensions for a monthly dataset are the year and the month, which have the values month:FEB and year:2020 for the instance :synop dataset feb20. In addition, although the station dimension is not directly a geographic coordinate, it is defined as an instance of qb4st:spatialDimension because it provides access to geospatial coordinates contained in the station file. Each dimension or measure is associated with a concept from domain ontologies via the qb:concept property. Thus, the measure :pmer measure (the only one represented here while all 57 measures have been instantiated) is associated to the concepts sosa:observableProperty and sweet:SeaLevelPressure to explicit its meaning. Similarly, the measure t (see Table 1) is associated to the concept ENVO:ENVO 09200001 which represents the air temperature. We have defined two attributes attached to qb:Measure: (i) :unit of measure attribute associated to qudts:physicalUnit to represent the unit of measurement of each qb:Measure. This makes it possible to specify that the unit of measurement of pmer measure is qudt:Pascal; (ii) :method of measure attribute associated to sosa procedure to represent the procedure of measurement of each measure according to WMO guides that are digital books.

Evaluation

To evaluate the degree of FAIRness, we have chosen the framework FAIR data maturity model (FAIR data maturity model) proposed by the RDA [START_REF] Rda | FAIR Data Maturity Model. Specification and Guidelines[END_REF]. For sake of space, we briefly present the evaluation of the SYNOP dataset using the FAIR maturity model using the 41 indicators that measure the level of a digital resource according to a FAIR principle, and the Fail/Pass evaluation method. Each indicator has a predefined priority: essential, important or recommended. The indicators were applied first considering the original description of the dataset, and then considering the instantiation of the proposed model. The reader car refer to Zenodo 15 for a detailed evaluation report. The first evaluation of the SYNOP dataset consisted in evaluating its original description (without the semantic model). This evaluation resulted in : i) level 0 for principles "F", "A" and "R", because at least one essential indicator was not satisfied for each of them; ii) level 1 for principle "I", because no indicator is essential for this principle (Figure 5 (A)). As it stands, the SYNOP dataset is not FAIR. The data has been re-evaluated after generating the semantic metadata that describes it. This metadata significantly improves the FAIRness level, especially for the "I" and "R" principles (Figure 5 (B)). Although the reevaluation of the "F" principle did not show any improvement, the model allows representing "rich" indexing metadata that satisfy "F2" principle. However, improving the "F" and "A" degree requires satisfying essential indicators that are beyond the abilities of any semantic model e.g., the generation of persistent and unique identifiers ("F1"), persistent metadata ("A2"), publishing metadata on searchable resources ("F4"), etc.

Related work

Hereafter, we present a summary on works related to the main subjects addressed in this work, focusing on works related to geospatial data. Metadata representation. The importance of sharing geospatial data and describing them with rich metadata has been recognized for decades. Indeed, in 1999, Kim has published a comparative analysis of eight already existing metadata schemes for geospatial data. The INSPIRE (2007) directive defined a metadata schema, mainly based on the previous standards for describing the European geospatial data on web portals. Later, with the emergence of semantic web, semantic vocabularies have been developed to describe dataset metadata such as Dublin core, VoID, schema.org and DCAT. DCAT-AP was designed to ensure interoperability between European data portals. GeoDCAT-AP was initially developed to enable interoperability between geospatial data portals implementing the INSPIRE directive, and those implementing DCAT-AP, by developing a set of mappings between the metadata schemes. In December 2020, a new version of GeoDCAT-AP was released, making this vocabulary a fullfledged specification for describing geospatial data catalogs on the Web 16 . Data representation. Several works have focused on the semantic representation of geospatial data [START_REF] Lefort | A linked sensor data cube for a 100 year homogenised daily temperature dataset[END_REF][START_REF] Roussey | Weather data publication on the LOD using SOSA /SSN ontology[END_REF][START_REF] Atemezing | Transforming meteorological data into linked data[END_REF][START_REF] Patroumpas | Exposing points of interest as linked geospatial data[END_REF][START_REF] Arenas | Un modèle pour l'intégration spatiale et temporelle de données géolocalisées[END_REF]. The proposed models are generally a combination of a set of reference ontologies. In [START_REF] Lefort | A linked sensor data cube for a 100 year homogenised daily temperature dataset[END_REF] the authors combined qb and SOSA to represent 100 years of temperature data in RDF. Similarly, in [START_REF] Roussey | Weather data publication on the LOD using SOSA /SSN ontology[END_REF], the ontologies SOSA, GeoSPARQL, LOCN and QUDTS have been reused to represent a meteorological dataset with several measures (temperature, wind speed, etc.). In our case, given the characteristics of the meteorological data ( §2.1), we did not transform Météo-France data into RDF. Representing all the data in RDF generates a huge graph which is not effective for querying the data [START_REF] Karim | Compact representations for efficient storage of semantic sensor data[END_REF]. Moreover, such a choice would require Météo-France to convert all its archives (some of them date back to 1872), which can turn out to be very expensive. Close to ours, [START_REF] Kremen | Improving discoverability of open government data with rich metadata descriptions using semantic government vocabulary[END_REF] propose the Semantic Government Vocabulary, based on the different ontological types of terms occurring in the Open Government Data. They show how vocabularies can be used to annotate Open Government Data on different levels of detail to improve "data discoverability". FAIR principles and FAIRness evaluation. As discussed in [START_REF] Mons | Cloudy, increasingly fair; revisiting the FAIR data guiding principles for the european open science cloud[END_REF][START_REF]FAIR principles: Interpretations and implementation considerations[END_REF], semantic web technologies are the most in compliance to the implementation of FAIR principles. Since the appearance of FAIR principles in 2016, several frameworks have been proposed to evaluate the FAIRness degree of a given digital object. In most cases, the evaluation is performed by answering a set of questions -also called metrics or indicators in some works -or fill in a checklist 17 such as the "FAIR Data Maturity Model" [START_REF] Rda | FAIR Data Maturity Model. Specification and Guidelines[END_REF] or "FAIRshake" [START_REF] Clarke | Fairshake: Toolkit to evaluate the fairness of research digital resources[END_REF]. Other works have proposed automated approaches for FAIRness evaluation [START_REF] Wilkinson | Evaluating FAIR maturity through a scalable, automated, community-governed framework[END_REF][START_REF] Devaraju | Fairsfair data object assessment metrics[END_REF] based on small web applications that test digital resources against some predefined metrics.

Conclusion

This paper has presented an ontological model to represent both metadata and data schema of observational meteorological datasets. We have shown how the proposed model improves the adherence to FAIR principles. This work is part of an approach that aims to make meteorological data FAIR in general, and that of Météo-France in particular. The next step is to study the specifics of the data from the statistical models to enrich the current model if necessary. We plan as well to use the final model to generate the metadata and index it in data portals.

Fig. 2 .

 2 Fig. 2. Proposed modular ontology.

Fig. 3 .

 3 Fig. 3. Representing Synop 20 distribution using GeoDCAT-AP and CSVW.

Fig. 4 .

 4 Fig.[START_REF] Van Den Brink | Best practices for publishing, retrieving, and using spatial data on the web[END_REF]. Representing Synop feb20 data using RDF data cube and domain ontologies.

Fig. 5 .

 5 Fig. 5. Synop data evaluation: (A) without and (B) with semantic metadata.

Table 1 .

 1 Extract from SYNOP data.

	numer sta	date	pmer	ff	t	. . .
	7005	20200201000000 100710 3.200000 285.450000 . . .
	7015	20200201000000 100710 7.700000 284.950000 . . .
	7020	20200201000000 100630 8.400000 284.150000 . . .
	...	...	...	...	...	...
	3 Use case: SYNOP dataset from Météo-France

https://donneespubliques.meteofrance.fr/

http://worldweather.wmo.int/fr/home.html

https://www.infoclimat.fr

http://www.meteociel.fr

https://library.wmo.int/doc_num.php?explnum_id=10179

https://www.irit.fr/recherches/MELODI/ontologies/DMO/core https: //www.w3.org/IRIT/MELODI/Semantics4FAIR/DMO/core/

http://www.opengis.net/def/crs/EPSG/

https://www.w3.org/ns/csvw

https://www.w3.org/TR/eo-qb/

https://www.w3.org/2005/Incubator/ssn/ssnx/meteo/aws


Entry for The Palgrave Dictionary of Transnational History (2009)

United Cities and Local Government

United Cities and Local Government held its first Congress in 2004. Its creation signals the increasing role local authorities are eager to play in the regulation of the world order. Cross-national associations of cities have been in operation since the beginning of the 20th century, reflecting the municipalities' concern to be protagonists on the global scene. The International Union of Local Authorities, one of three groups that created UCLG , was born in 1913. Its history testifies that cities are not bursting in on the international scene in the 1990s.

The Union Internationale des Villes was created during the 'First International Congress of Cities and Compared Exhibition of Cities' (Ghent, Belgium, 1913). After several other attempts to organise municipalities in an international association, UIV was successful because of its solid logistic base, embedded in the socialist European networks and in the fabric of international groups created by Paul Otlet and Henri la Fontaine in Brussels. The Union executive director, Belgian socialist senator Emile Vinck, was the man behind the Union until 1948. He kept the organisation going after WW1, as a mouthpiece for self-government and a documentation network to share municipal policies and technologies information. He lured in the English speaking world municipal organizations (hence the name IULA from 1928). But he was not able to connect the Union and the League of Nations system. Organized on a national sections basis, the Union spent its early life on the knife-edge, its conferences being vital occasions to refurbish its cash flow. This changed in the mid-1930s when the German and US sections notably increased their participation. The latter, powered by Rockefeller philanthropic money and project, wanted to use the Union as a touchstone to establish a transatlantic exchange in public administration practices. The former, imbued with the Nazi project of Neue Europa, was eager to seize power in several international associations. The (soft) clash between those two universal ambitions oriented the Union towards the discussion of technical and administrative subjects, throughout the periodicals and conferences it had been supporting since 1913. WW2, during which the Nazis seized upon the Union, created the conditions for its revamping. Led by its Dutch and US components, the headquarters moved to The Hague (Netherlands) in 1948. The Union began to develop a worldwide strategy to expand beyond its mostly Western domain, and to create strong links with the UN agencies. This was done in a disputed sphere, as other municipal groups were created in Europe to foster intermunicipal links and relations (Council of European Municipalities 1951, United Towns Organization 1957). Very progressively, IULA information work about 'best municipal practices' was coupled with installation, training and demonstration projects. From the 1980s, municipal autonomy and self government recovered their importance in the Union discourse. They echoed favourably the globalization discourse of the 1990s, and espoused the increasing concern for the governance of global problems. IULA played its part in the creation of successive institutions through which local authorities were called to cooperate with UN agencies : International Council for Local Environment Initiative (1990)

  , World Associations of Cities and Local Authorities Coordination (1996), Cities Alliance Program (1999), United Nations Committee of Local Authorities (1999). The creation of UCLG in 2004, merging IULA, United Towns Organization and the 'Metropolis' network, does not end the competition to embody the voice of local governments. Other organizations such as the Arab Towns Organisation, the Eurocities cluster or the Asian Citynet are walking their own way. But the birth of the new organisation clearly points the converging interest of local government leaders and supranational institutions, to bypass national governments in fields such as environment or development. , P.Y. 2001. Selling the idea of cooperation. US Foundations and the European components of the Urban Internationale (1920s-1960s). in Gemelli G. (ed). 2001.American Foundations and large scale research: construction and transfer of knowledge. Bologna: Clueb, 219-246 Saunier, P.Y (ed). 2002. Municipal connections: co-operation, links and transfers among European cities in the Twentieth Century. Contemporary European History, 11, 4.

	Pierre-Yves Saunier
	Cross references : cities in cooperation and competition ; city twinning ;
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	Bautz, I. 1999. Die Auslandsbeziehungen der deutschen Kommunen vor 1945. IULA,
	Städtepatenschaften, Weltstädtebund. Interregiones , 8, 19-58
	Acronyms table :
	IULA : International Union of Local Authorities
	UCLG ; United Cities and Local Government
	UIV : Union Internationale des Villes

Saunier


Matilde Negrini

Giuseppe Tomasello

Marcus Davidsson

Alexis Fenyi

Cécile Adant

Swantje Hauser

Elena Espa

Francesco Gubinelli

Fredric P Manfredsson

Ronald Melki

Cecile Adant

Andreas Heuer
email: andreas.heuer@med.lu.se.

Choi-Lundberg Dl

Chang Yn

Chiang Yl

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Breger Ls

Wan Ow

Mattsson

Paumier Kl

Luk Kc

Manfredsson Fp

Kanaan Nm

Lip- 1358 Jw

Collier Tj

Kemp Cj

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Patel Tp

Tanik Sa

Stieber A Dm

Meaney Df

Trojanowski Jq

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Shrigley S, Mattsson B, Breger LS Hoban Db

Nelander Wahlestedt

KC, Bjorklund J Luk

Sequential or simultaneous injection of preformed fibrils and AAV overexpression of alpha-synuclein are equipotent in producing relevant pathology and behavioral deficits

Keywords: AAV, alpha synuclein, behavioral deficits, dopamine, inflammation, motor deficits, Parkinson's disease, phosphorylated synuclein, preclinical rodent model, preformed fibrils rotameter

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The most common preclinical rodent models of Parkinson's disease (PD) are produced by either injecting neurotoxicants, overexpressing PDrelated genes, or by introducing mutations. Neurotoxicants such as 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) cause a robust and rapid degeneration of the dopaminergic midbrain system, with subsequent behavioral impairments [START_REF] Grealish | Unilateral axonal or terminal injection of 6-hydroxydopamine causes rapid-onset nigrostriatal degeneration and contralateral motor impairments in the rat[END_REF][START_REF] Kirik | Characterization of behavioral and neurodegenerative changes following partial lesions of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat[END_REF][START_REF] Heikkila | Dopaminergic toxicity of rotenone and the 1-methyl-4phenylpyridinium ion after their stereotaxic administration to rats: Implication for the mechanism of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine toxicity[END_REF][START_REF] Sundstrom | Studies on the effect of 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) on central catecholamine neurons in C57BL/6 mice. Comparison with three other strains of mice[END_REF][START_REF] Gerhardt | Dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse: An in vivo electrochemical study[END_REF][START_REF] Ungerstedt | Quantitative recording of rotational behavior in rats after 6-hydroxy-dopamine lesions of the nigrostriatal dopamine system[END_REF]. These approaches, however, neither replicate the alpha synuclein (␣Syn)-linked pathology seen in PD patients [START_REF] Tofaris | Ubiquitination of alpha-synuclein in Lewy bodies is a pathological event not associated with impairment of proteasome function[END_REF][START_REF] Spillantini | Alpha-synuclein in Lewy bodies[END_REF] nor the progressive neurodegeneration [START_REF] Braak | Pathoanatomy of Parkinson's dis-1209 ease[END_REF]. By contrast, genetic-based models, either transgenic animal lines or viral vector overexpression models, exhibit ␣Syn pathology although with variable levels of neurodegeneration and behavioral phenotypes. Viral vector-based overexpression of h-␣Syn is a promising method to trigger the desired PD-like key features.

The field has gone through several generations of viral vectors [START_REF] Lauwers | Neuropathology and neurodegener-1212 ation in rodent brain induced by lentiviral vector-mediated 1213 overexpression of alpha-synuclein[END_REF][START_REF] Kirik | Parkinson-1217 like neurodegeneration induced by targeted overexpression 1218 of alpha-synuclein in the nigrostriatal system[END_REF][START_REF] Klein | 1221 Dopaminergic cell loss induced by human A30P alpha-1222 synuclein gene transfer to the rat substantia nigra[END_REF][START_REF] Bianco | Lentiviral vector delivery 1226 of parkin prevents dopaminergic degeneration in an alpha-1227 synuclein rat model of Parkinson's disease[END_REF] and the most commonly used are recombinant adeno-associated viral (AAV) vectors.

However, there is a great heterogeneity in the results from AAV-based studies in terms of ␣Syn expression levels, neurodegeneration, and behavioral deficits. This may be due to differences in expression systems and transgene cassettes such as promoter-transgene combinations, and posttranscriptional modifying and regulatory sequences (e.g., polyA and WPRE) as well as in the choice of capsid serotype, and in the quality control of the virus itself, resulting in variable success rate [START_REF] Kirik | Parkinson-1217 like neurodegeneration induced by targeted overexpression 1218 of alpha-synuclein in the nigrostriatal system[END_REF][START_REF] Yamada | 1230 Overexpression of alpha-synuclein in rat substantia nigra 1231 results in loss of dopaminergic neurons, phosphorylation 1232 of alpha-synuclein and activation of caspase-9: Resem-1233 blance to pathogenetic changes in Parkinson's disease[END_REF][START_REF] Da Silveira | Phosphorylation does not prompt, nor prevent, the 1238 formation of alpha-synuclein toxic species in a rat model of 1239 Parkinson's disease[END_REF][START_REF] Gorbatyuk | 1242 The phosphorylation state of Ser-129 in human alpha-1243 synuclein determines neurodegeneration in a rat model 1244 of Parkinson disease[END_REF][START_REF] Sanchez-Guajardo | Microglia acquire distinct activation profiles 1248 depending on the degree of alpha-synuclein neuropathol-1249 ogy in a rAAV based model of Parkinson's disease[END_REF][START_REF] Decressac | Compari-1252 son of the behavioural and histological characteristics of 1253 the 6-OHDA and alpha-synuclein rat models of Parkinson's 1254 disease[END_REF][START_REF] Decressac | Progressive neurodegenerative and 1257 behavioural changes induced by AAV-mediated overexpres-1258 sion of alpha-synuclein in midbrain dopamine neurons[END_REF][START_REF] Ulusoy | Viral 1261 vector-mediated overexpression of alpha-synuclein as a pro-1262 gressive model of Parkinson's disease[END_REF][START_REF] Aldrin-Kirk | Novel AAV-based rat model of forebrain synucle-1266 inopathy shows extensive pathologies and progressive loss 1267 of cholinergic interneurons[END_REF][START_REF] Kanaan | Rationally engineered 1271 AAV capsids improve transduction and volumetric spread 1272 in the CNS[END_REF]. Overexpression of clinical mutations such as the A53T variant [START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF][START_REF] Gaugler | Nigrostriatal overabundance of alpha-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity[END_REF][START_REF] Landeck | Two C-terminal sequence variations determine differential neurotoxicity between human and mouse alphasynuclein[END_REF], S129D and S129A [START_REF] Gorbatyuk | 1242 The phosphorylation state of Ser-129 in human alpha-1243 synuclein determines neurodegeneration in a rat model 1244 of Parkinson disease[END_REF][START_REF] Febbraro | Ser129D mutant alpha-synuclein induces earlier motor dysfunction while S129A results in distinctive pathology in a rat model of Parkinson's disease[END_REF], and A30P [START_REF] Gaugler | Nigrostriatal overabundance of alpha-synuclein leads to decreased vesicle density and deficits in dopamine release that correlate with reduced motor activity[END_REF] are an alternative to exacerbate relevant pathology. Moreover, increasing dosing of AAV to elicit a greater effect may confound the experimentation as the use of control vectors (e.g., GFP) can also confer a negative effect on the integrity of nigrostriatal neurons [START_REF] Albert | Downregulation of tyrosine hydroxylase phenotype after AAV injection above substantia nigra: Caution in experimental models of Parkinson's disease[END_REF][START_REF] Landeck | Toxic effects of human and rodent variants of alpha-synuclein in vivo[END_REF][START_REF] Koprich | Progressive neurodegeneration or endogenous compensation in an animal model of Parkinson's disease produced by decreasing doses of alphasynuclein[END_REF][START_REF] Klein | Efficient neuronal gene transfer with AAV8 leads to neurotoxic levels of tau or green fluorescent proteins[END_REF].

Nonetheless, in a typical experimental design, neurodegeneration seen in most AAV-based expression systems has been rather moderate with usually few animals per experimental group displaying a stable 75 behavioral phenotype. [START_REF] Giasson | Initiation and synergistic fibrillization of tau and alpha-synuclein[END_REF] The development of a reliable model is impera- We report that transgene expression along the 116 nigrostriatal pathway in all the AAV-injected groups Fig. 1. Experimental layout. A) Description of the experimental groups and timepoints for behavioral test, electrochemical analysis, perfusions, and dissections. One group was injected with PFFs only (PFFs), two groups received either AAV-h-␣Syn or AAV-GFP (AAV), two groups received AAV-h-␣Syn or AAV-GFP and PFFs simultaneously (Sim.), and two groups received AAV-h-␣Syn or AAV-GFP and PFFs sequentially (Seq.). B) Schematic of the pre-formed fibrils and viral genome expressing h-␣Syn or GFP under the CBA promoter injected into the midbrain of rats. ITR, inverted terminal repeat; CBA, chicken ␤-actin; pA, polyadenylation site; h-␣Syn, human alpha-synuclein; GFP, green fluorescent protein; PFFs, preformed fibrils; MB, midbrain; ST, striatum.

Experimental outline

Naïve Sprague-Dawley rats (n = 63) were randomly assigned into seven experimental groups receiving PFFs and AAV-vector overexpressing h-␣Syn or GFP either alone or in combination (see Fig. 1 and Table 1). In the combination groups, one respectively. The assembly reaction completion was also assessed by sedimentation at 100,000 g at 25 The protocol for immunohistochemistry has been described elsewhere [START_REF] Heuer | Dopamine-rich grafts alleviate deficits in contralateral response space induced by extensive dopamine depletion in rats[END_REF] and a list of the primary and secondary antibodies used can be found in Table 1.

Briefly, tissue sections were washed (3x) in potassium phosphate buffered saline (KPBS, pH = 7.4) and then incubated for 15 min in 3% H 2 O 2 , 10% methanol in PBS to quench endogenous peroxidase activity. After

Brain tissue homogenization

Frozen tissues were weighed in 2 ml Eppendorf tubes. The samples were diluted ten times in 150 mM KCl, 50 mM Tris-HCl pH 7.5 to obtain a homogenate at 10% (weight:volume). The homogenization was performed by sonication using the SFX 150 Cell

Disruptor sonicator with a 3.17 mm microtip probe (Branson) for 15 s, with 1 s pulses followed by 1 s

pauses in a biosafety level 3 environment (BSL-3).

The homogenates were aliquoted and immediately frozen in liquid nitrogen before storage at -80 • C. All contaminated surfaces were cleaned with SDS (1%) [START_REF] Fenyi | Assessment of the efficacy of different procedures that remove and disassemble alpha-synuclein, tau and A-beta fibrils from laboratory material and surfaces[END_REF].

Protein quantification

The Time-resolved FRET was measured after incubation upon excitation at 337 nm using a plate reader (CLARIOstar, BMG Labtech, Germany) as described [START_REF] Degorce | HTRF: A technology tailored for drug discovery -a review of theoretical aspects and recent applications[END_REF]. The HTRF signal was recorded at two different wavelengths (665 nm and 620 nm). The amount of aggregated ␣Syn was derived from the 665/620 nm fluorescence ratio and multiplied by 10000. Three high-resolution 63X z-stacks were taken from 461 central, dorso-medial, and dorso-lateral regions of the 462 striatum as previously described [START_REF] Decressac | Progressive neurodegenerative and 1257 behavioural changes induced by AAV-mediated overexpres-1258 sion of alpha-synuclein in midbrain dopamine neurons[END_REF]. Each z-stack 463 had a z-step size of 1 m for a total of 30 z-steps.

Densitometry

464

The ImageJ 3D object count tool was used to iden-

RESULTS

In the current study we injected PFFs and/or ␣Syn overexpressing AAV either individually or in combination, simultaneous or sequentially after a 4-week delay, to induce a pathologically relevant level of neuronal cell loss in the midbrain of rats. 5.12 (Fig. 3H, Sidak, all p < 0.0001). Surprisingly, we observed similar results when GFP was overexpressed (Fig. 3E-G). Densitometric analysis of the AAV-GFP injected animals revealed a significant reduction by 69.01% ± 9.00 in TH staining intensity in the ipsilateral side (Fig. 3I, Side F 1,13 = 54.10, p < 0.0001, Sidak, all p < 0.01).

Injection of AAV-h-αSyn leads to strong overexpression of αSyn

To quantify the degree of TH+neuron loss in the midbrain we performed unbiased stereological counting of TH immunoreactive cells in the midbrain in the ipsi-and contralateral SN (Fig. 3J).

There was a significant difference in the number of TH immunoreactive cells in the SNpc between the groups and the brain side (Fig. 3J; Group * Side, F 6,24 = 4.96, p < 0.01). All AAV-h-␣Syn injected groups, but not the PFFs group, displayed a difference between the absolute TH + -cell numbers counted between the injected and non-injected side (Sidak, all p < 0.05). In particular, the TH + cell loss in PFFs, h-␣Syn, h-␣Syn/PFFs, and h-␣Syn+PFFs groups was was lower in all the AAV-injected groups compared to the PFFs only group (Fig. 4H). These results are but not in control groups (Supplementary Figure 3E-G ii ). Importantly, the phosphorylated ␣Syn inclusions found in all target groups receiving PFFs were resistant to proteinase K-digestion (pK), a feature that is a key characteristic of the Lewy body (Fig. 5A i , C i , D i ). Although there was a substantial amount of punctate pSer129-immunoreactivity in the AAV-h-␣Syn PFFs-only rats (Supplementary Figure 5A-C). These 773 swellings appear in single fibers and have a beaded 774 chain-like structure as described previously [START_REF] Decressac | Progressive neurodegenerative and 1257 behavioural changes induced by AAV-mediated overexpres-1258 sion of alpha-synuclein in midbrain dopamine neurons[END_REF].

In AAV-h-␣Syn injected animals, this axonopathy 776 could also be detected after immunohistochemistry

782

Volumetric quantification of axonal ␣Syn211 + , GFP + , and TH + swellings further corroborated these observations. At first, we quantified the number of ␣Syn211 + and GFP + swellings in target and control groups, respectively. Overall, AAV-h-␣Syn injected groups had higher numbers of swellings than the AAV-GFP injected groups (Fig. 7M,O). The h-␣Syn groups had 4465 ± 1030 ␣Syn211 + swellings per brain area analyzed, while GFP control groups had 969 ± 306 GFP + swellings. As expected, PFFs only group did not show relevant numbers of ␣Syn211 + swellings (2 ± 2). We then divided the swellings in small (5-10 m 3 ), medium (10-20 m 3 ), and large (> 20 m 3 ) volumes. Overall, the AAV-h-␣Syn injected groups had higher number of swellings, independently from the size, than the AAV-GFP injected groups (Fig. 7N,P). Interestingly there was no difference in the quantity of small, medium, and large swellings within groups. Subsequently, we quantified TH + swellings and obtained a proportionally similar outcome as in the previous quantification. Overall, AAV-h-␣Syn injected groups had higher number of swellings than the AAV-GFP injected groups (Supplementary Figure 5G,I). Target groups had 808 ± 206 TH + swellings per brain area analysed, 807 while control groups had 209 ± 124 TH + swellings.

As above, AAV-h-␣Syn injected groups had higher 809 number of TH + swellings, independently from the 810 size, than the AAV-GFP injected groups (Supplemen- there is a need for a vector expression system that 852 achieves robust expression and subsequent degener-853 ation in the majority of test subjects [START_REF] Decressac | Progressive neurodegenerative and 1257 behavioural changes induced by AAV-mediated overexpres-1258 sion of alpha-synuclein in midbrain dopamine neurons[END_REF][START_REF] Albert | Downregulation of tyrosine hydroxylase phenotype after AAV injection above substantia nigra: Caution in experimental models of Parkinson's disease[END_REF]. In our 854 current study, we injected 2.4x10 10 GC of AAV9 car-rying the CBA-h-␣Syn construct and animals were tested over a time period of 12 weeks. We observed a stable behavioral deficit, loss of TH immunoreactivity in midbrain and striatum, neuronal cell loss in the midbrain, impaired DA release and reuptake kinetics, as well as relevant pathology such as TH + and ␣Syn211 + axonal swellings. Moreover, we report the development of pSer129 + , Ub + , and ThioS + inclusions in midbrain indicating Lewy-like pathology.

The combinatorial effect of h-αSyn and PFFs

In the human condition one of the pathological hallmarks of PD is the formation of Lewy bodies and Lewy neurites in cortical and subcortical structures, with one of the major components being insoluble oligomers of h-␣Syn assemblies [START_REF] Spillantini | Alpha-synuclein in Lewy bodies[END_REF][START_REF] Fares | Reverse engi-1514 neering Lewy bodies: How far have we come and how far 1515 can we go?[END_REF]. It is important to note that the majority of preclinical rodent models of AAV vector based overexpression of ␣Syn has focused mainly on the loss of dopaminergic neurons alone, but axon loss and dysfunction is an early and predominant feature of PD which has been postulated to exhibit retrograde degeneration (as discussed in [START_REF] Tagliaferro | Retrograde axonal degeneration in Parkinson disease[END_REF]). In the present study, AAV-h-␣Syn injected animals, but not AAV-GFP injected groups, displayed an elevated number of axonal swellings in the dorsolateral striatum, the region which underlie motor function in PD.

␣Syn

Behavioral impairments

In humans, about 30 % of SNpc dopamine neurons and about 50-70% of striatal dopaminergic terminals are lost by the time of symptom onset [START_REF] Ma | Correlation between neuromorphometry in the substantia nigra and clinical features in Parkinson's disease using disector counts[END_REF][START_REF] Greffard | Motor score of the Unified Parkinson Disease Rating Scale as a good predictor of Lewy body-associated neuronal loss in the substantia nigra[END_REF][START_REF] Fearnley | Ageing and Parkinson's disease: Substantia nigra regional selectivity[END_REF].

In the present study we observed about 37% of TH loss in SNpc and 53 % in striatum of AAV-h-␣Syn groups with evident dysmorphic axons, and 49% of TH loss in SNpc and 69% in striatum of AAV-GFP groups with low axonal dysmorphia. In our study, both treatment paradigms resulted in behavioral deficits, which remained stable over a period of 12 weeks. For the d-amphetamine induced rotations, most rats reached the behavioral threshold that is considered to be indicative of a well lesioned rat of five rotations per minute (rpm) at 8 weeks [START_REF] Bjorklund | The amphetamine induced rotation test: A re-assessment of its use as a tool to monitor motor impairment and functional recovery in rodent models of Parkinson's disease[END_REF][START_REF] Torres | Amphetamine induced rotation in the assessment of lesions and grafts in the unilateral rat model of Parkinson's disease[END_REF][START_REF] Torres | Increased efficacy of the 6-hydroxydopamine lesion of the median forebrain bundle in small rats, by modification of the stereotaxic coordinates[END_REF]. The threshold of 5 rpm as chosen here is considered quite conservative as similar studies had either lower "success" rates (e.g., 25% [START_REF] Kirik | Parkinson-1217 like neurodegeneration induced by targeted overexpression 1218 of alpha-synuclein in the nigrostriatal system[END_REF]) or used a more lenient threshold of 3 rpm (e.g., [START_REF] Decressac | Progressive neurodegenerative and 1257 behavioural changes induced by AAV-mediated overexpres-1258 sion of alpha-synuclein in midbrain dopamine neurons[END_REF]). It is worth also men- In conclusion, the pathology observed in our model is more likely caused by a combination of axonal dysfunction, TH downregulation and actual neurodegeneration. In our own work we have compared several AAV-vector constructs and observed variable behavioral and electrochemical outcomes, even though the histopathology appeared similar and in agreement with previous studies [START_REF] Decressac | Progressive neurodegenerative and 1257 behavioural changes induced by AAV-mediated overexpres-1258 sion of alpha-synuclein in midbrain dopamine neurons[END_REF]37,[START_REF] Decressac | TFEB-mediated autophagy rescues midbrain dopamine neurons from alpha-synuclein toxicity[END_REF]. Importantly, we did not observe any differences between the simultaneous or sequential addition of PFFs with respect to histopathology and behavioral phenotype.

GFP as a control protein in AAV overexpression

77 tive 86 the 6 - 94 (

 7786694 in order to investigate the mechanisms of disease 78 pathology as well as to test therapeutic interventions. 79 High predictive and face-validity are needed to be 80 able to translate approaches into clinical trials. As an 81 example, glial cell line-derived neurotrophic factor 82 (GDNF) showed great promise in the 6-OHDA model 83 but failed to meet its primary endpoint in double-84 blind placebo-controlled clinical trials [31-35]. A 85 direct comparison of the effects of GDNF between OHDA model and the ␣Syn model demon-87 strated neuroprotection only in the 6-OHDA model 88 [36]. The availability of a robust model that mimics 89 the progressive cell loss, ␣Syn pathology, and stable 90 behavioral deficits is thus currently not widely avail-91 able. In the attempt of accomplishing this aim, a more 92 recent approach combines AAV vector-based overex-93 pression methods with injection of pre-formed fibrils PFFs), which seed endogenous ␣Syn inclusions 95 [37-41]. PFFs have been injected either 4 weeks after 96 an AAV-␣Syn injection [37] or simultaneously [40, 97 42], with the latter approach being more practical and 98 less invasive, requiring only a single surgical inter-99 vention. Finally, the combination of ectopic human 100 ␣Syn together with PFFs derived from the human 101 protein allows for studies focused on studying the 102 human synucleinopathy with minimal interference 103 from endogenous rodent ␣Syn. In the present study 104 we directly compare the effects of the combination of 105 human PFFs inoculation and AAV-h-␣Syn in the mid-106 brain of rats, either individually, combined, or after 107 a 4-week delay. Pathological changes were assessed 108 by immunohistochemical analysis, electrochemical 109 recordings, and behavioral testing. The aims of the 110 present study were i) to establish a model that gen-111 erates stable behavioral phenotypes in the majority 112 of animals, and ii) to investigate whether there is 113 a difference in outcomes between the sequential or 114 simultaneous PFFs injection approach.

  115

  group received both PFFs and AAV simultaneously (h-␣Syn/PFFs and GFP/PFFs) and in the other group PFFs inoculation followed the AAV injection sequentially after a four-week delay (h-␣Syn + PFFs and GFP + PFFs). All rodents were subjected to a battery of behavioral tests at four-weeks intervals (i.e., week 4, 8, and 12 after the first injection). Twelve weeks after the first surgical session we performed electrochemical recordings for dopamine (DA) from three anaesthetized rats per group. After all data was collected, we either perfused the animals with PFA and harvested the brains for immunohistochemistry (5-7 rats per group) or we harvested the brains (three rats per group) for dissections of midbrain and striatum, and snap-froze the tissue for subsequent quantification of phosphorylated ␣Syn protein. A single rat was injected with the neurotoxin 6-OHDA for histological comparison.Preformed fibrils productionHuman wild type ␣Syn was expressed in E. coli BL21 DE3 CodonPlus cells (Stratagene, San Diego, CA, USA) and purified and assembled into the fibrillar polymorph "Fibrils" as described previously [43]. Briefly, the protein (100 M) was incubated in 50 mM Tris-HCl, pH 7.5, 150 mM KCl at 37 • C under continuous shaking in an Eppendorf Thermomixer set at 600 rpm for 5 days while withdrawing aliquots (20 l) at different time intervals, mixing them to Thioflavin T (10 M final) and recording the fluorescence increase on a Cary Eclipse Fluorescence Spectrophotometer (Varian Medical Systems Inc., Palo Alto, CA, USA) using an excitation wavelength = 440 nm, an emission wavelength = 480 nm and excitation and emission slits set at 2 and 5 nm,

232a 10 L 2 L

 102 stereotaxic frame with the incisor bar adjusted to 233 the flathead position (-4.5 mm below the interaural 234 line). Each solution was infused unilaterally into the 235 midbrain using a pulled glass capillary attached to a 236 Hamilton syringe. The following coordinates 237 (from Bregma, in mm) and volumes were utilized: 238 at AP = -5.3, ML = -0.8, DV = -7.5 and 2 uL 239 at AP = -5.6, ML = -2.3, DV = -7.5 with an infusion 240 rate of 0.5 L/min. After injection, the syringe was 241 left in place for additional 3 min and thereafter slowly 242 retracted. 6-OHDA was injected as described previ-243 ously [45]. In brief, 5.41 g/L 6-OHDA (calculated 244 from free-base 6-OHDA-HBr salt, Sigma) was dis-245 solved in 0.2 mg/ml ascorbic acid in 0.9% sterile 246 saline and a volume of 3 L was injected at the fol-247 lowing coordinates AP = -4.0, ML = -1.3, DV = -7.0. 248 The injection speed was set to 1 L/min and the nee-249 dle was left in place for an additional 3 min to allow 250 for diffusion of the toxin.

(

  3x) PBS washes, sections were incubated for 1 h in blocking solution consisting of 5% serum in KPBS and 0.25% Triton X-100 (TPBS, pH = 7.4). The sections were then incubated in primary antibody in 5% serum overnight at room temperature. On the second day, sections were rinsed with (2x) KPBS and subsequently incubated in 5% serum for 1 h before being incubated for 1 h in secondary antibody solution in 5% serum. The secondary antibody was removed by (3x) KPBS washes and sections were incubated in avidin-biotin-peroxidase complex (ABC, Vectorlabs) in KPBS for 1 h. After an additional (3x) KPBS washes sections were incubated for 2-10 min in a peroxidase (HRP) detection system (Vector® DAB,

  quantification by TR-FRET of phosphorylated ␣Syn at Ser-129 or aggregated ␣Syn was performed using a fluorescence resonance energy transfer (FRET) assay (Cisbio, France, cat #6FSYN-PEG and # 6FASYPEG respectively). Briefly, the brain homogenates were diluted to 5% (W:V) in lysis buffer provided in the HTRF kit. For phosphorylated ␣Syn assay, 16 L of each diluted brain homogenates were loaded into a 96 well plate and mixed with 4 L of the FRET donor and acceptor antibodies in the kit. For aggregated ␣Syn assay, 10 L of each diluted brain homogenates were loaded into a 96 well plate and mixed with 10 L of the FRET donor and acceptor antibodies in the kit. The plates were sealed with a film (CmlAB, Denmark, cat #13076-9P-500) and incubated for 20 h at 20 • C without shaking in a Thermomixer comfort (Eppendorf, Montesson, France).

465

  tify the swellings and calculate their total number, as 466 well as their volume, by setting an exclusion threshold 467 for particles < 4 m 3 and circularity between 0.5-1.0.468The average number and volume size of swellings 469 was calculated based on the total swelling number 470 from all the three pictures from one animal and 471 for Fig.7Mand 7O and Supplementary Figure5G

  472and 5I were divided in small (4-10 m 3 ), medium 473 (10-20 m 3 ) and large (> 20 m 3 ).StatisticsAll data were analyzed using the statistical software GraphPad Prism (V 8.4.3) using an alpha of 0.05 as threshold for determining statistical significance. For group and group by side analysis we performed analysis of variance with the factors Group and Side as dependent variables. For time-course analysis we performed repeated measures ANOVAs with the factors Week and Group. Post hoc testing used Sidak's correction were applied to avoid Type 1 errors/inflating alpha due to multiple comparison testing.

  along the nigrostriatal pathway We first assessed the expression of h-␣Syn throughout the nigrostriatal pathway at 12 weeks following vector delivery. The groups injected with AAV-h-␣Syn displayed strong ␣Syn immunoreactivity on the injected side of the midbrain with evident spread along dopaminergic nigrostriatal projections (Fig. 2A-D) with some diffusion into the contralateral hemisphere in the midbrain. The comparison of the staining intensity between the left and right striatum (Fig. 2E) for the four treatment groups showed that there was a significant difference in human synuclein immunoreactivity between the groups and the side of the brain analyzed (Groups * Side, F 3,18 = 6.88, p < 0.01). All groups injected with AAV-h-␣Syn displayed a similar degree of ␣Syn immunoreactivity which was absent in the group that received PFFs only (Sidak, all p < 0.0001). Triple immunofluorescent immunoreactivity for the enzyme tyrosine-hydroxylase (TH), the Vesicular Monoamine Transporter 2 (VMAT2), and ␣Syn211 (human ␣Syn) confirmed that the midbrain dopaminergic neurons of the PFFs-inoculated group did not express h-␣Syn (Fig. 2F-F iii ), whereas all groups receiving the AAV-vector expressed h-␣Syn throughout most of the midbrain dopamine neurons (Fig. 2G-I iii ). The lack of staining in the PFFs treated

Fig. 2 .

 2 Fig. 2. AAV-vector overexpression. A-D) Representative midbrain and striatal sections for each group stained for human ␣Syn (␣Syn211). E) Densitometric analysis comparing human ␣Syn211 staining intensity between contralateral and ipsilateral right striatum. F-I iii ) Triple immunofluorescence demonstrating colocalization between TH, VMAT2, and ␣Syn211 antibodies in SNpc. PFFs, preformed fibrils; h-␣Syn, human alpha-synuclein; contra, contralateral; ipsi, ipsilateral; O.D., optical density. Data are expressed as mean ± SEM. ( * * * * p < 0.0001).

Fig. 3 .

 3 Fig. 3. TH downregulation in striatum and substantia nigra. A-G) TH immunoreactivity from representative midbrain and striatal sections. H-I) Densitometric analysis comparing TH staining intensity between left and right striatum. J) Cell count of TH + cells in the SNpc represented as number of TH + cells between contralateral and ipsilateral SNpc. PFFs, preformed fibrils; h-␣Syn, human alpha-synuclein; GFP, green fluorescent protein; ST, striatum; SN, substantia nigra pars compacta; O.D., optical density. Data are expressed as mean ± SEM. ( * * p < 0.01, * * * p < 0.001, * * * * p < 0.0001).

Fig. 4 .nificantly less pronounced than that in weeks 8 and 645 12 (

 412 Fig.4. Behavioral impairments following h-␣Syn and/or PFFs injection. A-F) Behavioral tests performed to assess motor function at 4-, 8-, and 12-weeks post-injection for Cylinder, Stepping, and Rotation test for the h-␣Syn (circles, A, C, E), GFP (squares, B, D, F), and PFF (circles, A-F) injected groups, respectively. G) Electrochemical recordings (chronoamperometry) for KCl-evoked striatal DA release; black arrow indicates the timepoint of KCl application. H) Average peak amplitude, reflecting the maximum amount of dopamine available, for all the seven experimental groups. PFFs, preformed fibrils; h-␣Syn, human alpha-synuclein; GFP, green fluorescent protein; w, weeks; s, seconds. Data are expressed as mean ± SEM. Note: the behavioral results of the PFFs group are duplicated in each graph for ease of comparison.

Fig. 5 .693

 5 Fig. 5. h-␣Syn overexpression and PFFs treatment results in Lewy-like midbrain pathology. A-D i ) pSer129 immunoreactivity in midbrain before (-pK) and after (+pK) proteinase K treatment. E, F) TR-FRET quantification of pSer129 (E) and aggregated ␣Syn (F) in the ipsilateral midbrain and striatum of rats and cingulate cortex of human samples. Black dots represent single subjects. G-J iii ) Triple immunofluorescence showing co-localization between pSer129 and Thioflavin S. K-N iii) Triple immunofluorescence showing co-localization between 81A (pSer129) and Ubiquitin antibodies. PFFs, preformed fibrils; h-␣Syn, human alpha-synuclein; pK, Proteinase K; Ub, ubiquitin; ThioS, Thioflavin S; ST, striatum; MB, midbrain; CC, cingulate cortex. (A.U. = ratio 665 nm/620 nm multiplied by 10000).

Fig. 6 .

 6 Fig. 6. Inflammatory response. A-G) Overviews of rat midbrain sections stained for Ox42. A i -G ii ) Close-up 20X images for Ox42 immunoreactivity in contralateral (A i -G i ) and ipsilateral (A ii -G ii ) side for target and control groups and additional 63X magnification images (A ii -G ii ) for the ipsilateral side. H) Densitometric analysis showing the increase in percentage for Ox42 staining intensity in the ipsilateral midbrain (vs. contralateral) for target and control groups. PFFs, preformed fibrils; h-␣Syn, human alpha-synuclein; GFP, green fluorescent protein; Ipsi, ipsilateral; Contra, contralateral. Data are expressed as mean ± SEM. ( * p < 0.05, * * p < 0.01).

Fig. 7 .

 7 Fig. 7. Axonal pathology in the ipsilateral striatum. Immunohistochemical analysis to investigate the presence of axonal pathology using antibodies for ␣Syn211 (A-C), GFP (D-F), and TH (G-L). Quantification of ␣Syn211 + (M,N) and GFP + swellings (O,P) in the ipsilateral striatum. The quantified swellings were counted by total number (M,O) and volume size (N,P). PFFs, preformed fibrils; h-␣Syn, human alpha-synuclein; GFP, green fluorescent protein. Data are expressed as mean ± SEM.

  777for TH (Fig.7G-I), while it was not detected for the 778 AAV-GFP injected animals (Fig.7J-L). Again, the 779 contralateral side of the brain and PFFs only group 780 (Supplementary Figure5D-F) did not display any 781 clear axonopathy.

811tary

  Figure 5H, L) with no clear difference in the 812 quantity of small, medium, and large swellings within 813 groups. 814 These findings clearly corroborate the observed 815 functional impairments and are indicative of a stage 816 where the dopaminergic cells might be still alive, but 817 dysfunctional. As can be seen in the representative tions and the striatal fiber density measures reported 820 previously, the difference in the quantity of h-821 ␣Syn211/GFP positive fibers and TH positive fibers 822 suggest a downregulation of TH in the striatum. 823 DISCUSSION 824 In this manuscript we present a direct comparison 825 of AAV-mediated h-␣Syn overexpression and inoc-826 ulation with PFFs either in combination or alone, 827 simultaneous or sequential. In doing so we utilized 828 human protein in a rodent background in order to 829 generate a preclinical rodent model of PD. 830 AAV-vector based overexpression of h-αSyn 831 AAV vectors are a preferred tool for protein over-832 expression in vivo providing spatiotemporal control, 833 and they are widely used to generate preclinical 834 models for PD. AAV-based strategies have gone 835 through several generations of development, using 836 a variety of serotypes, promoters, transgenes, and 837 post-transcriptional elements. Additionally, differ-838 ences between studies in terms of genome copies 839 (GC) injected, volumes and coordinates have intro-840 duced a large variability in AAV-h-␣Syn mediated 841 histopathological and phenotypical outcomes [11, 12, 842 66]. Overall, the overexpression of h-␣Syn by AAV 843 vectors injected into the midbrain of rodents can 844 lead to a loss of TH expressing midbrain dopamine 845 cells and dopaminergic denervation of the striatum 846 [18, 19, 23, 36, 50, 67-70]. Few studies have con-847 firmed that loss of TH immunoreactivity represents 848 overt neurodegeneration and not simply a loss of TH 849 expression [19, 67, 68, 70]. As the levels of degen-850 eration depend on the levels of overexpressed ␣Syn, 851

  tioning that the rotational response to d-amphetamine seen in rats is neither a naturally occurring behavior, nor does it have a linear relationship with cell loss[START_REF] Bjorklund | The amphetamine induced rotation test: A re-assessment of its use as a tool to monitor motor impairment and functional recovery in rodent models of Parkinson's disease[END_REF][START_REF] Torres | Amphetamine induced rotation in the assessment of lesions and grafts in the unilateral rat model of Parkinson's disease[END_REF]. Therefore, of interest are the non-drug induced behavioral tests, where we report distinct and stable lateralized deficits in paw use on the Cylinder test (20-50% bias) and on the Stepping test (20-50% bias) for all the groups except the PFFs only group.Similar deficits have been reported on the Cylinder test when overexpressing the A53T mutant form of ␣Syn[START_REF] Van Der Perren | Longitudinal follow-up and characterization of a robust rat model for Parkinson's disease based on overexpression of alpha-synuclein with adeno-associated viral vectors[END_REF]. An interesting aspect of the AAV vector model compared to the classic neurotoxicant-based models is the fact that the behavioral impairments cannot be easily ascribed to TH-cell numbers alone.In the AAV-based model, depending on the level of transgene expression, the neuron can persist in a dysfunctional state. Many presynaptic and axonal transport proteins are indeed downregulated before overt loss of dopaminergic fibers takes place[START_REF] Chung | 1503 Dynamic changes in presynaptic and axonal transport pro-1504 teins combined with striatal neuroinflammation precede 1505 dopaminergic neuronal loss in a rat model of AAV alpha-1506 synucleinopathy[END_REF] and striatal dopamine levels are significantly reduced as shown by HPLC[START_REF] Kim | Transneuronal propagation of pathologic alpha-synuclein from the gut to the brain models Parkinson's disease[END_REF]. As absolute cell counts do not provide an indication of the level of functional impairment, methods such as electrochemistry are valuable additions to the characterization of the model or intervention and warrant further investigation. On the other hand, in the neurotoxicant-based models neuronal death occurs rapidly and resembles an advanced disease stage. In our study the combination of TH loss, neurodegeneration, and axonal pathology were associated with impairments in DA release-and reuptake kinetics as measured by electrochemical recordings (chronoamperometry) in the striatum. Our results suggest a direct effect of ␣Syn on the functionality of dopaminergic axons, which is further supported by the impaired dopamine transmission reported by us and others[START_REF] Lundblad | Impaired neurotransmission caused by overexpression of alpha-synuclein in nigral dopamine neurons[END_REF][START_REF] Hansen | A novel alpha-synuclein-GFP mouse model displays progressive motor impairment, olfactory dysfunction and accumulation of alpha-synuclein-GFP[END_REF][START_REF] Tozzi | Dopaminedependent early synaptic and motor dysfunctions induced by alpha-synuclein in the nigrostriatal circuit[END_REF].

  commonly used control protein in AAV 1060 overexpression-based studies. However, it has been 1061 reported that GFP can induce in vivo toxicity and 1062 can lead to a decrease in TH immunoreactivity [27, 1063 28, 30]. In order to mitigate this unspecific TH loss, 1064 some studies have been lowering the titer of the 1065 AAV-GFP compared to the AAV-h-␣Syn [11, 36, 1066 91], but this approach might not be ideal in stud-1067 ies where protein overexpression is used to cause 1068 cell loss. The levels at which GFP vectors become 1069 toxic are not clear, and it is difficult to determine 1070 the exact gc titer in which the ␣Syn construct is 1071 inducing pathology while the GFP control vector 1072 has no significant effect. Albert et al. (2019) [27] 1073 reported that the GFP vector becomes significantly 1074 toxic at 3.5x10 10 gc whereas Landeck et al. (2017) 1075 [28] set this threshold above 4x10 11 gc. Differences 1076 in the experimental setup, vector construct and titra-1077 tion method do not allow for a direct comparison 1078 between studies, and it is therefore not possible 1079 to provide a precise estimate. From our own work 1080 we report neurotoxicity of h-␣Syn and GFP at lev-1081 els above 2.4x10 10 gc, with minor dissimilarities 1082 between the two proteins. Differences between the 1083 present work and those reported previously may addi-1084 tionally come from the AAV quantification method 1085 utilized. ddPCR-based quantification is less vari-1086 able than using qPCR, which can produce results 1087 varying of a factor of two or even more [92]. 1088 Therefore, in such studies where a qPCR-based 1089 quantification method was used, the actual titers 1090 might have been lower than those reported. In the 1091 present study, we injected 2.4x10 10 gc of an AAV9 1092 carrying the CBA-GFP construct. We observed a 1093 stable behavioral deficit, TH downregulation in mid-1094 brain and striatum and impaired dopamine release 1095 and reuptake kinetics similarly to what observed in 1096 AAV-h-␣Syn injected animals. However, AAV-GFP 1097 injected animals displayed fewer levels of axonal 1098 swellings and absence of pSer129 + inclusions. Our 1099 results confirm that exogenous protein overexpres-1100 sion can lead to unspecific neuronal pathology when 1101 it exceeds a certain threshold. The use of GFP 1102 as control protein should be taken carefully as it 1103 can complicate the interpretation of results. Alter-1104 natives such as "empty" AAVs, or AVVs carrying 1105 an inducible genome or missing the transgene might 1106 be used instead of the prototypical GFP control 1107 [27, 93, 94]. CONCLUSION 1108 We present here a direct comparison of synucle-1109 inopathy models which result in overt dopaminergic 1110 degeneration and reliably produce a stable behavioral 1111 phenotype. Importantly, the AAV9-CBA construct 1112 utilized in our work does allow for studies to be 1113 conducted within a reasonable timeframe where neu-1114 roprotective and restorative treatments are still a 1115 viable option. However, as stated above, the levels of 1116 protein expression needed to induce a stable behav-1117 ioral phenotype makes the choice of an appropriate 1118 control vector difficult. In the current study, GFP 1119 overexpression was as toxic as h-␣Syn overexpres-1120 sion and it therefore complicated the identification 1121 of the pathological effects specific to ␣Syn. The 1122 transgene overexpression led to stable behavioral 1123 phenotypes, TH loss, and changes in DA kinetics. 1124 However, the presence of h-␣Syn induced relevant 1125 axonal pathology, and the addition of PFFs did gen-1126 erate additional cell body pathology that shares some 1127 of the characteristic of human Lewy bodies. Impor-1128 tantly, the simultaneous injection of PFFs led to 1129 a comparable model which avoids the confound-1130 ing effect of repeated injection mechanical injury of 1131 the sequential approach. This model can represent a 1132 robust platform for the study of PD related h-␣Syn 1133 etiology and the development of novel targets.

Table 1

 1 Experimental groups. Schematic of the experimental groups and sample size for the analyses. Echem, electrochemistry; IHC, immunohistochemistry

		Group Name	Description	AAV	PFFs	N Tot	Behavior	FRET	Echem	IHC
				injection	injection
		PFFs	PFFs only	-	week 0	8	8	3	3	5
		h-␣Syn	AAV only	week 0	-	8	8	3	3	5
		h-␣Syn/PFFs	Simultaneous	week 0	week 0	10	10	3	3	7
		h-␣Syn+PFFs	Sequential	week 0	week 4	9	9	3	3	6
		GFP	AAV only	week 0	-	8	8	3	3	5
		GFP/PFFs	Simultaneous	week 0	week 0	10	10	3	3	7
		GFP+PFFs	Sequential	week 0	week 4	10	10	3	3	7
	131
	132	Lastly, the presence of ectopic h-␣Syn induced strong
	133	axonal pathology, with elevated numbers of striatal
		axonal swellings on the side of injection.

(both AAV-h-␣Syn and AAV-GFP) produces down-118 regulation of tyrosine hydroxylase (TH) expression 119 in the midbrain and striatum, irrespective of whether 120 the AAV-vector was injected alone or in combina-121 tion with PFFs. These animals also displayed altered 122 dopamine release and reuptake kinetics in electro-123 chemical recordings as well as motor impairments 124 which did not recover over time. However, only when 125 AAV-h-␣Syn was combined with PFFs we observed 126 phosphorylated ␣Syn similar to that seen in patient 127 brains. Moreover, the combination of PFFs and 128 AAV-h-␣Syn triggered an enhanced inflammatory 129 response around the area of transduction, which was 130 not observed in the remaining experimental groups. 134 MATERIALS AND METHODS 135 Animals 136 All experimental procedures performed in this 137 study were approved by the Malmö/Lund Ethics 138 Committee on Animal Testing as well as according 139 to Swedish national guidelines (Jordbruksverket) and the EU Directive 2010/63/EU for animal experiments. Sixty-four adult (220-250 g) female Sprague-Dawley rats were purchased from Janvier Labs (France) and were housed under a 12:12 h dark:light cycle with ad libitum access to food and water in a temperature-controlled room in standard laboratory cages. A total of 64 rats were used in the current study and allocated randomly to the seven experimental groups.

Table 2

 2 Antibodies and dyes used in the current study for immunohistochemical analysis

	Antibody	Species	Cat. no	Concentration
	Human ␣Synuclein 211	Mouse SC-12767	1:1000
	GFP	Chicken A10262	1:10000
	␣Synuclein, p-S129	Rabbit	ab51253	1:100000
	␣Synuclein 81A (p-S129) Mouse AB184674	1:10000
	CD11b (Ox42)	Mouse MCA275G	1:500
	TH	Sheep	ab113	1:750-1:1000
	TH	Mouse MAB318	1:1000
	VMAT2	Rabbit	20042	1:10000
	Ubiquitin	Rabbit	AB7780	1:200
	ThioflavinS	-	T1892	1:100
	DAPI	-	D9542	1:2000
	Anti-rabbit Biotinylated	Goat	BA6000	1:200
	Anti-mouse Biotinylated	Horse	BA2001	1:200
	Anti-chicken Biotinylated Goat	BA9010	1:200
	Anti-sheep Alexa 488	Donkey A11015	1:500
	Anti-chicken Alexa 488	Chicken A11039	1:500
	Anti-rabbit Alexa 488	Goat	A11008	1:500
	Anti-rabbit Alexa 568	Goat	A11011	1:500
	Anti-mouse Alexa 568	Goat	A11004	1:500
	Anti-mouse Alexa 647	Donkey A31571	1:500
	Anti-rabbit Alexa 647	Goat	A21245	1:500

Vector Laboratories). The sections were mounted on ondary antibody, washed (3x) in KPBS, mounted onto 364 gelatine-coated glass slides and cover-slipped as soon 365 as dry using PVA/DABCO solution. When DAPI was 366 needed, sections were incubated for 5 min at RT 367 before the last washing step. Thioflavin S coloration 368 was executed as describes elsewhere

[START_REF] Cresto | The C-terminal domain of LRRK2 with the G2019S substitution increases mutant A53T alpha-synuclein toxicity in dopaminergic neurons in vivo[END_REF]

. 369 Proteinase K treatment 370 Coronal sections were mounted on permafrost-371 glass slides and were incubated in proteinase K 372 solution (25 g/mL; QIAGEN) for one hour at room 373 temperature before proceeding with the immunos-374 taining for pSer129 as described above. 375 Dissections 376 Three rats per group were sacrificed by decap-377 itation at 13 weeks post-injection. Brains were 378 extracted, rinsed in 0.9% saline solution, and coro-379 nally sectioned using a rat brain matrix on ice. The 380 striatum and midbrain from each hemisphere were dissected using sterile razor blades. Tissue samples were snap-frozen in isopentane on dry ice and stored in 1.5 mL Eppendorf tubes at -80 • C until further processing.

images in Fig.7, and the volumetric quantifica-

ACKNOWLEDGMENTS

1135

MD would like to thank the Royal Physio-

Crafoord foundation, the Gyllenstiernska Krapperupsstiftelsen, the Thorsten and Elsa Segerfalks Stiftelsen, the Åke Wibergstiftelsen, the Kockskastiftelsen, the Royal Physiographic Society in Lund, the Swedish Parkinson Foundation (Parkinsonsfonden), the Fredrik och Ingrid Thurings Stiftelsen, the Swedish Society of Medicine (SSMF), the Åhlensstiftelsen, the Hedlundsstiftelsen, the Svenska Läkaresällskapet and the Jeansonsstiftelsen.

CONFLICT OF INTEREST

The authors have no conflict of interest to report.

SUPPLEMENTARY MATERIAL

The supplementary material is available in the electronic version of this article: https://dx.doi.org/