come

Twenty years later, Georges Sadoul, another close friend of Moussinac, used the same simile in reference to The Battleship Potemkin, in a text symptomatically entitled 'The Scream Becomes a Hymn': 'in Potemkin, the cradle rolling down the stairs, the blood from the wounded eye, the clenched hands of the woman on her injured belly -all these elements present themselves as screams' . 4 Umberto Barbaro claimed that Eisenstein's treatment of the scream in Potemkin profoundly transformed film history, in the same way that Caravaggio's screams drastically revolutionized art history. In Barbaro's eyes, Potemkin's plastic achievements should be equated with Caravaggio's dramatic and expressive open mouths:

A mere close up of a sailor at work, or of an intellectual woman looking through her glasses, a mere wide open mouth desperately shouting, a mere frame or continuity shot in Potemkin were enough to push into a past era every former cinematographic achievement, even the highest, and to announce new times. Just as four hundred years ago, the silent screaming mouths by Caravaggio […] would weigh heavily on all painting to come. 5 Interestingly, Barbaro drew on a pictorial model to describe Eisenstein's filmic achievements. Moreover, he describes Caravaggio's mouths as paradoxical objects, since they are silent and screaming, exactly like Eisenstein's screams in his silent films -screams that are not literally heard, but which nevertheless roar, and roar powerfully (Figure 9.1).

Screams play a pivotal role in Eisenstein's films, where they are explored in all their polysemy, ambivalence and diversity. The scream is a notion that is hard to define and which, according to Alain Marc in his book Writing the Scream, still awaits theorization. 6 On the one hand, screams can be a merely articulated sound, an emotional and physiological response of the body, often shrill and dissonant. As such, they can be perceived as a visceral, bestial and primitive expression. In that respect Greimas describes the scream as being at the limit of human language. 7 The transposition of this bestial and primitive scream to an open mouth in the visual sphere translates the sound into an abhorrent and formless image, according to the famous statement by Lessing in the Laocoön; hence his advice to not represent it, to avoid offending the viewer with the repulsive spectacle of a distorted and monstrous face. The wide open screaming mouth, which he calls a blot, comes as an intolerable and indecent hole in the representation 'of the worst possible effect' . 8 For these reasons, Bataille and members of the Documents circle, as proponents of 'base materialism' , would On important occasions, human life is still bestially concentrated in the mouth: fury makes men grind their teeth, terror and atrocious suffering transform the mouth into the organ of rending screams. On this theme, it can easily be observed that the overwhelmed individual throws back his head while frenetically stretching his neck so that the mouth becomes, as much as possible, a prolongation of the spinal column; in other words, it assumes the position normally occupied in the constitution of animals. 9

Marking the degradation from human speech to animality, the visual motif of an open mouth blurs the boundaries between inside and outside, giving access to shapeless (informe) flesh, to an intimate space usually unseen that is described by Nathalie Roelens as 'the face seen from inside' . [START_REF] Roelens | The open mouth in painting as a semiotic object: the cry of the gesture[END_REF] These visceral and regressive dimensions make the open mouth an energetic and troubling form of excess, and a powerful source of affect. As such, it is hardly surprising that it caught Eisenstein's attention. In his films, numerous victims and innocent people scream while being molested and executed; as A scream can also be construed as a positive and noble expression when associated with shouted articulate content. The scream functions then as a vehicle of indignation, as the expression of heart and soul. The verbal exclamation works as a powerful and a contagious stimulus, enabling people to join together as a community. In this respect, it is worth remembering that the word 'cry' is etymologically related to the Latin exclamation quirites, uttered by Roman citizens when help was needed. [START_REF] Quirito | A Complete Latin-English Dictionary[END_REF] This political aspect of the scream is quintessential in Eisenstein's poetics of riot and exaltation of the power of the masses, perfectly embodied, for instance, in the exclamations 'Brothers!' and 'One for all / All for one' , which recur throughout the whole of Potemkin.

Eisenstein's intensive use of screaming figures, in all their diversity, is echoed in the numerous promotional posters for his films, which frequently include a wide open mouth. This is especially obvious in posters for Potemkin, which emphasize the role of this motif in the global economy of the film. Eisenstein's films are not only peopled by many screaming characters, but also encapsulate different kinds of 'roaring' cinematic material: expressive titles conveying emotions and, above all, complex and dynamic editing devices, converting the whole film into an extended scream. To create such roaring compositions, Eisenstein used pictorial models, especially in his silent films, where sound had to be suggested by visual means. He carefully studied how painters managed to materialize and express the phenomenon of voice. In the essay in which he conducted a posttheorization and analysis of Potemkin's composition, Eisenstein drew a parallel between the latter and Vasily Surikov's painting Boyaryna Morozova (Figure 9.3), whose elaborate composition, based on the Golden Section, powerfully suggests by visual means what, according to Eisenstein, lies beyond representation -the 'plastic portrayal' of the voice of a woman known for her eloquence. [START_REF] Sergei | Film and the Structure of Things[END_REF] Similarly, when discussing the impact of the stone lions rising in Potemkin ('the roaring lions'), Eisenstein marvelled at Edvard Munch's The Scream, in which the scream is not only represented as an iconographic motif, an open mouth, but also as a visual reverberation that permeates the whole structure and composition of the work, because the painter sought to 'express in the picture's structure the feeling of a scream' . [START_REF] Eisenstein | Sound in Painting[END_REF] Just as Munch's painting screams on every level, the whole of Potemkin is built upon ecstatic jumps from one quality to another, in which screams play a pivotal role as dialectical hinges. Inspired by plastic representations of the scream, Eisenstein's films have in turn inspired painters, in a phenomenon of intermedial circulation which corresponds with Johanne Lamoureux's approach to the scream as an intermedial object. 15 In that respect, Potemkin's screaming nurse, an icon of a revolutionary pathos, functions as a peculiarly haunting image. Severed by the knife of montage, this filmic Gorgon has fascinated and petrified many viewers, not so much with her broken-glassed eyes but with the open gap formed by her screaming mouth. Eisenstein's Gorgon rises again in the work of Francis Bacon and Valerio Adami, who have taken different readings from this image and reworked it in diametrically opposed directions.

Encountering The Battleship Potemkin: The birth of 'The Painter of the Cry'?

Labelled by the French critic Gaëtan Picon as 'the painter of the cry' , Francis Bacon viewed the scream as one of the loftiest objects to be painted. [START_REF] Picon | Le cercle et le cri[END_REF] In a 1963 interview with the art critic David Sylvester, Bacon made a striking statement:

I have always been very moved by the movements of the mouth and the shape of the mouth and the teeth […]. I like, you may say, the glitter and colour that comes from the mouth, and I've always hoped in a sense to be able to paint the mouth like [Claude] Monet painted a sunset. [START_REF] Sylvester | Interview 2[END_REF] Eisenstein's Potemkin played a triggering role in Bacon's sensitivity to this motif, which pervaded all of his painted work. The ground was prepared by 9781350142107_txt_prf.indd 157 21-12-2020 13:18:00 his discovery in 1927 of Poussin's painting The Massacre of the Innocents (Figure 9.4), which made a vivid impression upon him. As he recalls in the same interview with Sylvester: 'I remember I was once with a family for about three months living very near [Chantilly], trying to learn French, and I went a great deal to Chantilly and I remember this picture made a terrific impression on me. ' 18 Poussin's painting creates not only a visual impression but also a powerful auditory effect thanks to its elaborate composition. As Nathalie Roelens notes, it is not enough to depict an open mouth to make the image heard and give it a voice. 19 In this work, described by Louis Marin as 'one of the most outstanding pictorial representations of a scream' , Poussin unfolds an intricate play of correspondences between vision and sound; an elaborate orchestration of visual signs waiting to be deciphered as sounds:

The intensity of the scream, the voice at its extreme range is visually 'notated' by the point of the raised sword and especially by the obelisk behind, above the head: their intersecting point is empty, conveying the scream's 9781350142107_txt_prf.indd 158 21-12-2020 13:18:05 shrillness while the non-realistic elongation of the woman's arm converts into visual terms its unbearable duration. [START_REF] Marin | Aux marges de la peinture: voir la voix[END_REF] Marin also highlights that the trajectory of voice is visually suggested and embodied through the loops formed by the arms of the soldier and the woman, and echoed and enhanced by the movement of the soldier's red cloak. The powerful impact of Poussin on Bacon was reinvigorated when he saw The Battleship Potemkin, probably in Paris during an illegal projection organized by the communist cine-club Les Amis de Spartacus. [START_REF] Bouvard | Pablo Picasso. Francis Bacon. Eléments de chronologie[END_REF] As he explained to Sylvester:

It was a film I saw almost before I started to paint, and it deeply impressed me -I mean the whole film as well as the Odessa Steps sequence and this shot. I did hope at one time to make -it hasn't got any special psychological significance -I did hope one day to make the best painting of the human cry. I was not able to do it and it's much better in the Eisenstein and there it is. I think probably the best human cry in painting was made by Poussin. 22 In his diaries, Bacon further highlights the role of Eisenstein's film as a crucial trigger for his activity as a painter, writing: 'In my youth, I was strongly attracted to painting after having been struck by the remarkable visual imagery of Eisenstein's films, The Strike and The Battleship Potemkin. ' [START_REF]Bacon's autograph note[END_REF] A passionate film viewer, Bacon confessed to the publisher and scholar Michel Archimbaud his amazement at the 'tremendous force of the silent era' and the expressive resources of cinema, and claimed that he would have liked to become a film director if he hadn't been a painter. [START_REF] Archimbaud | Francis Bacon in Conversation with Michel Archimbaud[END_REF] After Bacon's death, several loose blackand-white stills from the Odessa Steps sequence were found in his studio (Figure 9.5a and b). [START_REF] Cappock | Cinema[END_REF] Among these was a close-up of the nurse mounted on cardboard, which Bacon had altered and savagely covered with paint dots and lines, adding a layer of pictorial violence to the violence already encapsulated in the filmic image. This reproduction nurtured his obsession with the scream and the open mouth; as he explained, 'I attempted to use the Potemkin still as a basis on which I could also use these marvellous illustrations of the human mouth. ' 26 Bacon alludes here to a medical volume he owned titled Diseases of the Mouth (1894), which included spectacular and quite repulsive pictures of buccal pathologies.

Another thing that made me think about the human cry was a book that I bought when I was very young from a bookshop in Paris, a second-hand book which had beautiful hand-coloured plates of diseases of the mouth, beautiful plates of the mouth open and of the examination of the inside of the mouth, and they fascinated me, and I was obsessed by them. Bacon also had in his library a copy of Roger Manvell's 1944 book Film, in which a still with the bleeding and screaming nurse was reproduced. [START_REF] Bouvard | Sur-réalité du Massacres des Innocents[END_REF] Long after his striking early encounter with Potemkin, Bacon would acquire Eisenstein's writings, published in English in 1973 and in 1982. 29 Bacon recycled the screaming mouth of the nurse in different paintings, including his variations on Velázquez's portrait of Pope Innocent X (1949, 1953), who, in Bacon's interpretation, wears broken glasses, a marker evidencing Eisenstein as a source, as suggested in a 1949 essay by Robert Melville, which underlined the proximity of Bacon's figures to actors' gestures and the powerful expressivity of silent cinema. [START_REF] Melville | Francis Bacon[END_REF] Interestingly, Bacon painted his first portrait of Pope Innocent in 1949, the year Helen Weigel performed her famous silent scream in Brecht's play Mother Courage. Some of Bacon's studies could also be seen as a condensation of several motifs from the Odessa Steps sequence: a stairstructure, the screaming mouth, the umbrella, with the overtone of red saturating the whole scene with bloody associations. In his 1952 Study for a Portrait, Bacon again associated a screaming mouth with the motif of glasses, like a variation on the broken pince-nez of the nurse.

More explicitly, Bacon dedicated a painting to the nurse in 1957 in the Study for the Nurse in the Battleship Potemkin. In this work, he substitutes a whole body, absolutely naked, for the close-up of the nurse's face, as if he needed to give a surplus of flesh to the screaming mouth. As Gilles Deleuze noted, Bacon portrays not faces but heads, inasmuch as the 'face is a structured, spatial organization that conceals the head, whereas the head is dependent upon the body, even if it is the point of the body' (Figure 9.6). [START_REF] Deleuze | The Logic of Sensation[END_REF] In this operation, the scream plays a crucial role: 'The [open] mouth acquires this power of non-localization that turns all meat into a head without a face. ' 32 Extracted from its original context and placed in an abstract and nonlocalizable space, the screaming nurse appears in Bacon's treatment as a generalized embodiment of human suffering; the blood on the leg echoes the blood in the eye as well as the redness of the mouth, as if the mouth itself were a wound. Crystallized by the medium of painting, which abstracts it from its filmic and dynamic source, the scream of the nurse seems paradoxically suspended in eternity, a never-ending sound. What resonates particularly strongly with Bacon's paintings is Eisenstein's nurse's mouth in its uncanny dimension as a fleshy gulf akin to meat, since in his view, according to Deleuze, 'Every man who suffers is a piece of meat. ' 33 Precisely this equation also appears in Eisenstein, especially in The Strike, in which not only are the strikers literally butchered in the final sequence but the worker who dies by suicide looks like a 9781350142107_txt_prf.indd 162 21-12-2020 13:18:14 hanging piece of meat, cut into pieces by the camera. As if to illustrate Deleuze's point that 'The head-meat is a becoming-animal of man' , in Bacon's aesthetics meat fosters a zone of undecidability between man and animal. 34 The scream enhances this loss of fixed identity and contour, since 'Bacon's scream is the operation through which the entire body escapes through the mouth. ' 35 Bacon's interest in the scream relates to its dynamic process of transformation or, better, 9781350142107_txt_prf.indd 163 21-12-2020 13:18:18 of deformation of the body into animal, into pure meat and flesh. But what is at stake is also the need to capture the energetic movement which overwhelms the subject, to release presences beyond and beneath representation, to express the invisible forces 'that produce the scream, that convulse the body until they emerge at the mouth as a scrubbed zone' . 36 In Bacon's paintings of screaming figures, the characters' 'only function is to render visible these invisible forces that are making [them] scream' . 37 In that respect, while in Eisenstein people scream about and in reaction to, Bacon displaces this militant dimension of the scream and transforms it into a pictorial object per se, into a self-sufficient and absolute motif. works from the 1970s involving a filmic reference, he chose to dedicate one composition to Eisenstein. This was an intertextual gesture typical of his fellow artists from nouvelle figuration; Erro, for instance, would paste, rework and subvert famous images within new narratives. Between 24 September 1970 and 24 February 1971, Adami created a painting entitled The Battleship Potemkin. [START_REF] Valerio | OEuvres récentes et portraits[END_REF] The very fact that he would record and inscribe in the caption 9781350142107_txt_prf.indd 165 21-12-2020 13:18:22 the time spent on the work stresses his concern with time, as if he wanted to convert the temporal flow of the film into the narrative of the process of pictorial creation, with a clear beginning and end. [START_REF] Païni | Le Cuirassé Potemkine. 1970-1971[END_REF] Adami's filmic agenda also appears in one of his diaries, in which he lists different potential titles for the painting, among which is a list sounding almost like a scenario excerpt, or stage directions: 'People licking spits. Hysteria. Inhumane treatments. The mother asks desperately for news of her son.' 42 This list reveals Adami's mental associations with Battleship Potemkin. He is especially interested in moments of pathos, which he combines with images from his memory and imagination (Figure 9.7).

In his Battleship Potemkin, Adami reworked the motif of the screaming nurse in a totally opposite direction to Bacon: where Bacon opens the nurse's mouth wide and for eternity, Adami closes and obturates it in a climax of muting. Where Bacon is interested in the obscenity and organicity of the buccal orifice, Adami enhances the political dimension of Eisenstein's scene. In the film, the nurse's impotent scream symbolizes tsarist repression, which muzzled rioters and was unable to hear any protest. Adami goes further: covering the nurse's mouth -a wound hole -with a bandage; mummifying her, he deprives her of her voice and makes her definitively silent, adding a new layer of violence to the original image. Where Eisenstein's Gorgon would petrify the viewer, Adami gorgonizes the image itself. Representing a gag literally, Adami translates Eisenstein's imagery into a denunciation of contemporary censorship. The violence of the gesture is enhanced by the contrast of the grey with other colours, as a stain of silence invading the whole composition, which gives a peculiar stress to the broken glasses that are painted in acid pink.

Adami claimed that in this painting the use of colour is equivalent to the use of close-ups in cinema. [START_REF] Valerio | Dessiner: la gomme et les crayons[END_REF] In addition, the inhumanity of the scene is reinforced by the smooth cold texture of acrylics and the stylized outlines, as well as by the ironic use of pop and vibrant colours. Everything in this cold, flat, metallic treatment contrasts with Bacon's vibrant, organic flesh. Adami answers Eisenstein's cruel filmic montage with a deconstructing assemblage of elements, a fragmented poetics which enhances the nurse's loss of speech and sight. The fragmenting process in itself is cruel, implying the sacrifice of some elements, as noted by Marc Le Bot: '[Adami's works] are like a scattered puzzle from which some pieces are lost, as if every image by Adami was initially cut in strips, these ones being then glued together at the expense of overlaps and lost fragments' . [START_REF] Marc | La découpe et le vide[END_REF] Adami's aesthetics of 'montage-images' [START_REF] Mourey | Philosophies et pratiques du détail: Hegel, Ingres, Sade et quelques autres[END_REF] relies upon a mutilation of the original image or model, and it never preserves faces' integrity or unity. When he draws, 9781350142107_txt_prf.indd 166 21-12-2020 13:18:22

Adami practises epidermal cuts: arbitrary cuts which echo the way a gaze itself cuts visible reality into arbitrary fragments. 46 It is not accidental that in many works from 1970 and 1971 Adami drew such particular attention to the motif of the glasses, not only in his reworking of The Battleship Potemkin nurse but also in his portraits of Freud, Gandhi and Joyce, where the motif of shattered glasses highlights the cruel operation of the fragmenting gaze, as well as the artist's violent and cruel gesture. Remediated in painting, the scream of Eisenstein's nurse still remains vibrant and resonant, calling for new interpretations and re-elaborations across periods and media. In short, the nurse's voice demands to be heard and continues to reverberate.

Notes 1 I refer here to the screaming woman with broken glasses in the famous Odessa

Steps sequence. While nothing in the film leads us to assume that this woman is a nurse, she has been described as such by many commentators, although sometimes she is also described, more plausibly, as a teacher. There is also another female figure present in the same sequence, a nursemaid, who is shot in the stomach, before her pram careers down the steps. It is, however, the woman with broken glasses who has been referred to as a nurse by the artists I discuss here, Valerio

Figure 9 . 1

 91 Figure 9.1 Caravaggio, Medusa, 1597-8.

Figure 9 . 2 '

 92 Figure 9.2 'The Screaming Nurse' . Still from Sergei Eisenstein's The Battleship Potemkin, 1925.

14

 14

Figure 9 . 3

 93 Figure 9.3 Vasily Surikov, Boyaryna Morozova, 1887.

Figure 9 . 4

 94 Figure 9.4 Nicolas Poussin, The Massacre of the Innocents, 1625.

  27

Figure 9 .

 9 Figure 9.5a and b Materials from Francis Bacon's studio, 'Screaming Nurse' stills from Eisenstein's The Battleship Potemkin (1925). Courtesy of Hugh Lane Gallery, Dublin. © The Estate of Francis Bacon.

Figure 9 . 6

 96 Figure 9.6 Francis Bacon, Study for the Nurse in the film Battleship Potemkin by Eisenstein, 1957. Courtesy of Städel Museum, Frankfurt am Main, U. Edelmann, ARTOTHEK.

Figure 9 . 7

 97 Figure 9.7 Valerio Adami, The Battleship Potemkin, 1971.Karouby collection.

  Valerio Adami's Potemkin: A radicalized political gestureA totally different treatment and appropriation of the screaming nurse is to be found in the work of the painter Valerio Adami. Born in Bologna in 1935, Adami started his career in Milan before settling in Paris in 1970, where he established close contact with the intellectual and artistic avant-garde. His works have been analysed by Hubert Damisch, Jacques Derrida, Jean-François Lyotard and Jean-Luc Nancy, to name only a few, while they have been exhibited in such venues as the Paris Museum of Modern Art and the Centre Georges Pompidou.[START_REF] Adami | s life and career, see[END_REF] During the 1970s, he became one of the major figures of a trend called 'narrative figuration' (nouvelle figuration), which claimed there was a need to return to narrative and figurative art, in opposition to abstraction as well as to the figuration practised in socialist realism. Adami's works are permeated with references to literature, music, pop art and film. He experimented with film in 1971, working with his brother Carlo Romani Adami on the film Vacanze nel deserto. When Adami moved to Paris in 1970, Eisenstein's writings and films were being intensively debated and discussed: Cahiers du Cinéma was publishing many new studies on his work (including Roland Barthes's article 'The Third Meaning'[START_REF] Barthes | The Third Meaning: Research Notes on Some Eisenstein Stills[END_REF] ). Cahiers was also running translations of Eisenstein's texts and dedicating special issues to him. The publisher Christian Bourgois initiated a cycle of translations of Eisenstein's texts including Memoirs, Nonindifferent Nature and others in its 10/18 series. Eisenstein became a key figure in aesthetic and artistic debates at that time, especially in the wake of several protests and uprisings that had shaken European countries at the end of the 1960s. It is not surprising, therefore, that in such a context, among Adami's various

	9781350142107_txt_prf.indd 164	21-12-2020 13:18:18

  Adami and Francis Bacon. 2 Léon Moussinac, 'Poudovkine' , in Le cinéma soviétique (Paris: Gallimard, 1928), 137, translated by the author. 3 Sergei Eisenstein to Moussinac, 15 October 1928, Bibliothèque Nationale de France/Arts du Spectacle/Fonds Léon Moussinac/4° COLIO/35 (1), translated by the author. 4 Georges Sadoul, 'Le cri devenu hymne' , Les lettres françaises, 19 February 1948, quoted by Léon Moussinac, Serge Eisenstein (Paris: Seghers, 1964), 173, translated by the author. 5 Umberto Barbaro, 'Serghei Eisenstein maestro d'arte' , in Servitù e grandezza del cinema (Rome: Editori Riuniti, 1962), 35, translated by the author. 6 Alain Marc, Écrire le cri: Sade, Bataille, Maïakovski (Paris: L'Ecarlate, 2000), 16. 7 Algirdas Julien Greimas, 'Pour une théorie du discours poétique' , in Essais de sémiotique poétique (Paris: Larousse, 1972), 23. 8 Gotthold Ephraim Lessing, Laocoön: An Essay upon the Limits of Painting and Poetry (London: Dent, 1930), 13. 9 Georges Bataille, 'Bouche' (1930), Documents, II (Paris: Jean-Michel Place, 1991), 299.

9781350142107_txt_prf.indd 167 21-12-2020 13:18:22

9781350142107_txt_prf.indd 160 21-12-2020 13:18:09


Adrien Laffont
email: adrien.laffont@isae-supaero.fr

Jean-Pierre Adam

Thierry Callegari

Laurent Liard

Olivier Pascal

Romain Pascaud

Nonlinear Effects of Electromagnetic Pulse on a Plasma-Based Electrically Small VHF Antenna

Keywords: plasma-based antenna, electromagnetic hardening, electrically small antenna, electromagnetic pulse

Demonstration of the electromagnetic hardening properties of a plasma-based electrically small VHF antenna is presented in this paper. It is shown that when illuminated by an electromagnetic pulse, the maximum voltage collected by the antenna grows linearly with the maximum magnitude of the incident electric field, but beyond a certain threshold around 70 kV/m, the maximum voltage collected ceases to grow linearly and seems to saturate around a value of 200 V.

I. INTRODUCTION

Electromagnetic hardening consists in reducing the electromagnetic stresses that an equipment can undergo when it is subjected to an electromagnetic aggression [START_REF] Backstrom | Susceptibility of electronic systems to high-power microwaves: summary of test experience[END_REF]. More specifically, it consists of demonstrating that these constraints are lower than the equipment's susceptibility threshold. The latter is the threshold beyond which we observe non-tolerated events on the equipment, which can range from non-remanent malfunction to destruction. In an electronic warfare context, the transmission chains are priority targets, the main accesses of which being the antennas. In addition to transmitting or receiving the wanted signal, the antennas also collect electromagnetic waves associated with their operating frequency. These unwanted signals are then transformed as a current in the transmit chain which can disturb their operation. Thus, the antenna appears as a central element in the electromagnetic protection of the transmission chains.

Plasma discharges [START_REF] Raizer | Gas Discharge Physics[END_REF] have already shown promising results for the development of radio frequency or microwave systems with electromagnetic hardening properties [START_REF] Goldie | Radioactive (tritium) ignitors for plasma limiters[END_REF]- [START_REF] Simon | Experimental study of microwave power limitation in a microstrip transmission line using a DC plasma discharge for preionization[END_REF]. Indeed, plasma discharges are media for which non-linear electromagnetic properties are observed when a high power electromagnetic excitation is applied to them.

In the case of antenna development, plasma discharges are generally exploited to provide antennas with frequency reconfigurability, beam-forming capabilities, and/or stealth properties [START_REF] Borg | Plasmas as antennas: Theory experiment and applications[END_REF]- [START_REF] Linardakis | Plasma-based lens for microwave beam steering[END_REF]. These antennas mainly exploit two electromagnetic properties of plasma discharges due to its dispervice nature, its electric conductivity when the real part of its relative permittivity is largely negative [START_REF] Jusoh | Performance and radiation patterns of a reconfigurable plasma corner-reflector antenna[END_REF]- [START_REF] Melazzi | Beam-forming capabilities of a plasma circular reflector antenna[END_REF], and its dielectric behavior when the latter is between 0 and 1 [START_REF] Sokoloff | Reconfigurable leaky wave antenne using a gradient index plasma[END_REF], [START_REF] Linardakis | Plasma-based lens for microwave beam steering[END_REF].

Recently, a new type of electrically small plasma-based antenna has been proposed and experimentally studied in VHF band [START_REF] Laquerbe | Towards antenna miniaturization at radio frequencies using plasma discharges[END_REF]. This antenna relies on the theoretical concept proposed in [START_REF] Stuart | Electrically small antenna elements using negative permittivity resonator[END_REF]. In this case, the plasma behaves neither as a good electric conductor nor as a dielectric, but more particularly as a poor electric conductor, i.e. an Epsilon NeGative (ENG) material. More specifically, the operation of the antenna is based on the coupling between a transmitting chain and the plasmonic resonance of electrons within a hemispherical plasma discharge [START_REF] Maier | Plasmonics: Fundamentals and Applications[END_REF], [START_REF] Laffont | A harmonic oscillator model to study the intensification of microwave radiation by a subwavelength uniform plasma discharge[END_REF].

In addition to being electrically small and frequency reconfigurable, the association of the use of a plasma discharge for a resonant phenomenon suggests that this antenna presents potential electromagnetic hardening properties. In this paper, we present first experimental results pointing out electromagnetic hardening properties of such a plasma-based antenna.

II. PLASMA-BASED ANTENNA

Our plasma-based antenna corresponds to the one presented in [START_REF] Laquerbe | Towards antenna miniaturization at radio frequencies using plasma discharges[END_REF]. However, this one having undergone some minor changes and for pedagogical reasons, this part aims at presenting the prototype in detail. Moreover, additional results on the performance of the antenna are given.

Fig. 1 shows a schematic of the plasma-based antenna while Fig. 2 shows two pictures of the antenna with and without plasma. It consists of a hemispherical glass shell positioned on a ground plane. The shell allows confining the plasma discharge. Its external radius is 107.5 mm, its thickness 5.5 mm, and its dielectric constant 5.5. The gas pressure inside the chamber is controlled by two sealed vacuum ports. The plasma discharge is created and maintained with an inductive RF source at 13.56 MHz. This source is a spiral coil with a Faraday shield [START_REF] Godyak | RF discharge diagnostics: Some problems and their resolution[END_REF] and a glass disk ensuring the vacuum seal. A small monopole of 5 cm long is inserted into the plasma through the center of the ground plane and allows the coupling between the transmission chain and the plasma discharge. In order to minimize the electromagnetic losses, the choice of the gas is important. Currently, the gas selected is a mixture of Neon with 1% Xenon at a pressure approximately equal to 10 mTorr (about 1.33 Pa). The electron density of the plasma, and thus its relative permittivity at a given frequency, is controlled by modifying the power injected into the inductive RF source.

Regarding the performance of the antenna presented in [START_REF] Laquerbe | Towards antenna miniaturization at radio frequencies using plasma discharges[END_REF], a reflection coefficient lower than -10 dB was obtained between 290 MHz and 330 MHz. The radiation performance of the antenna was evaluated through the transmission coefficient between the plasma antenna and a coaxial probe positioned at 50 cm from the center of the antenna on the ground plane. Compared to the case without plasma, an increase of 20 dB around 300 MHz was measured on the coefficient of transmission. The frequency reconfigurability of the antenna has also been observed by modifying the RF power supplied to the plasma. Additional measurements were performed to quantify the radiation performance of the antenna more precisely. These results are currently being synthesized and therefore not presented in detail in this document. Nevertheless, it is important to mention that a maximum gain around 0 dBi has been measured at the operating frequency of the antenna for an omnidirectional radiation pattern.

III. EXPERIMENTAL SETUP

To evaluate the potential electromagnetic hardening properties of the antenna, the method consists in experimentally illuminating it with an ElectroMagnetic Pulse (EMP) while collecting its output voltage on a 50 Ω load. Locally similar to a plane wave, the EMP is typically a "double-exponential" but the specific time-domain waveform is classified. However, the frequency spectrum of the EMP is wide enough to induce a significant coupling in the operating band of the antenna. Consequently, the work presented deals only with the peak level of the output voltage of the antenna when it is illuminated with an EMP of a given peak electric field.

In order to cover a wide range of electric field values that can be applied to the antenna, two EMP simulators at CEA, DAM, Gramat have been selected. The first facility is a GTEM cell [START_REF] Nothofer | The use of GTEM cells for EMC measurements[END_REF], which is a strip line expanding pyramidally for guiding an electromagnetic wave similar to a TEM wave through the device under test (DUT), namely the antanna. Associated with swappable pulse generators, the GTEM is a versatile way to deliver a maximum electric field of about 40 kV/m at the DUT location. The hemispherical glass shell containing the plasma discharge is set up in the GTEM cell as shown in Fig. 3.

The second experimental faciity, presented in Fig. 4, delivers an EMP with a maximum field of up to 120 kV/m at the DUT location. Used as a certification tool for various systems, including vehicles, this larger facility provides a stronger electric field but offers less magnitude adjustability.

Note that these two experimental setups allow to illuminate the plasma antenna without disturbing the operation of the equipment required to maintained the plasma discharge. As visible on Fig. 4, the different devices are placed below the metallic ground plane of both facilities. For both EMP systems, the incident electric field illuminating the antenna is also measured locally near the antenna using electric field sensors to evaluate the incoming electric field magnitude.

IV. RESULTS AND DISCUSSION

The results are summarized on Fig. 5. The specific timedomaine waveform of the EMP used in the GTEM was slightly different than the EMP provided by the high voltage pulse generator. Hence, extrapolation on the results of the GTEM cell was performed in order to compare the two configurations. Since the GTEM operates at a lower level, a linear response of the plasma-based antenna has been assumed for this extrapolation. This assumption is supported by the fact that the obtained levels are still below the voltage collected in the highlevel EMP facility. Fig. 5 shows that the results obtained in that second experiment match with the linear fit obtained with the GTEM measurements up to about 70 kV/m. Above this threshold, the output voltage collected by the plasma-based antenna stops growing linearly and seems to be independent of the incident EMP levels. In particular, the collected voltage seems to saturate around a value of 200 V.

To check that this non-linearity is not due to a faulty measurement setup, the high-level EMP coupling was repeated on the plasma-based antenna in the case without plasma. For this configuration, the antenna only consists of a 5 cm long monopole positioned on a ground plane and surrounded by a glass shell. In that case, the high-level EMP no longer couples efficiently with the monopole which theorically behaves like an electric field sensor with a linear reponse. This behavior is well and truly observed in experimentation in Fig. 5. To complete this result, numerical studies have also been performed to simulate the illumination of the plasma-based antenna in the case without plasma by the high-level EMP. As shown in Fig. 5, the numerical results are in good agreement with the experimental ones. Note that the measurement chain performing the acquisition of the voltage collected by the antenna is typically used for applications collecting a much higher voltage than those involved in this study. Thus, we assume that the observed effect does not come from a defect of this equipment.

At present, no explanation of the non-linearly behavior of the plasma-based antenna when illuminated by a high-level EMP is validated. However, a hypothesis may be that the incident EMP provides momentary enough power to change the electron density of the plasma discharge. This modification can lead to a shift of the resonant frequency of the subwavelength ENG resonator, namely the hemispherical plasma discharge. Thus, two phenomena can occur. First, the coupling between the ENG resonator and the small monopole is less efficient reducing the voltage to the transmission line. Second, if the operating point of the antenna is shifted to a higher frequency, the incident spectral content of the high-level EMP is weaker and, therefore, less voltage is able to be collected by the antenna. These two phenomena can tend to stop the progressive rise of the voltage collected by the antenna. Works to confirm this behavior are still ongoing.

V. CONCLUSION

Results on a novel kind of antenna based on plasma discharge have been presented. In addition to be electrically small and frequency reconfigurable, this prototype antenna has also shown a non-linear coupling response to an incident high-level EMP. This feature has been explored experimentally in the present work and may be a promising way to limit the stress on the systems connected to the antenna.

The next step will be to better understand and model the non-linear behavior of the plasma-based antenna in order to optimize this feature and/or extrapolate it to various other electromagnetic threats.

Fig. 1 .

 1 Fig. 1. Schematic of the plasma-based antenna.

Fig. 3 .

 3 Fig. 3. Pictures of the plasma-based antenna in the GTEM cell.

Fig. 4 .

 4 Fig. 4. Pictures of the plasma-based antenna (without plasma) in the high voltage pulse facility. During illumination, the square ground plane is lowered and connected to the facility ground plane.

Fig. 5 .

 5 Fig.5. Antenna output voltage with incident EMP strength. Pink marks for plasma "ON" configurations and gray marks for plasma "OFF" settings.

ACKNOWLEDGMENT

The author would like to thank the Direction Générale de l'Armement (DGA), the Agence de l'Innovation de Défense (AID), and the Région Occitanie for their financial support.


Stefano Espinoza

Margherita Scarpato

Devid Damiani

F Manago

Maddalena Mereu

Andrea Contestabile

Omar Peruzzo

Piero Carninci

Claudio Santoro

Francesco Papaleo

Francesca Managò

Federico Mingozzi

Giuseppe Ronzitti

Silvia Zucchelli

Stefano Gustincich
email: stefano.gustincich@iit.it

SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease

INTRODUCTION

Parkinson's Disease (PD) is one of the most common neurodegenerative disorders, and it is caused by loss of the dopamine (DA) neurons of the substantia nigra pars compacta (SNpc). It manifests its symptomatology mainly as motor-related deficits, such as rigidity, tremor, and bradykinesia. [START_REF] Meissner | Priorities in Parkinson's disease research[END_REF] Although familial PD (about 5% of the total cases) has been important to unveil the molecular pathways involved in neurodegeneration, most of the PD patients are sporadic. [START_REF] Obeso | Past, present, and future of Parkinson's disease: A special essay on the 200th Anniversary of the Shaking Palsy[END_REF] Currently, no treatments are available that can slow or arrest neurodegeneration. The DA precursor L-DOPA is the gold standard for symptomatic treatments, although over time it may lead to several side effects and resistance. [START_REF] Nagatsua | L-dopa therapy for Parkinson's disease: past, present, and future[END_REF] Glial cell-derived neurotrophic factor (GDNF) is the most potent neurotrophic factor for DA neurons with great potentiality for clinical application. [START_REF] Kordower | Trophic factor gene therapy for Parkinson's disease[END_REF][START_REF] Lin | GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons[END_REF] GDNF can protect DA cells and promotes their survival from toxic insults in vitro and in vivo in mice, rats, and non-human primates. [START_REF] Tomac | Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo[END_REF][START_REF] Gash | Functional recovery in parkinsonian monkeys treated with GDNF[END_REF][START_REF] Kirik | Preservation of a functional nigrostriatal dopamine pathway by GDNF in the intrastriatal 6-OHDA lesion model depends on the site of administration of the trophic factor[END_REF] However, clinical trials with recombinant GDNF showed an inconsistency of results, due to the poor spreading of GDNF within the parenchyma and to the unsustainable side effects caused by the high doses of GDNF administered. [START_REF] Nutt | Implanted intracerebroventricular. Glial cell line-derived neurotrophic factor (2003). Randomized, double-blind trial of glial cell line-derived neurotrophic factor (GDNF) in PD[END_REF][START_REF] Gill | Direct brain infusion of glial cell linederived neurotrophic factor in Parkinson disease[END_REF][START_REF] Lang | Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease[END_REF] Similarly, GDNF expression through viral vectors that showed efficacy in PD animal models may result in uncontrolled, ectopic GDNF overexpression. Therefore, GDNF may be the right candidate for PD therapy, provided that a more specific and physiological expression is achieved to increase its efficacy and safety profile in vivo. In this context, a deletion in the 3 0 UTR of the GDNF gene in a transgenic mouse line led to an increase of 2-fold of endogenous GDNF protein levels. [START_REF] Kumar | GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function[END_REF] This was sufficient to induce alterations in the DA system and neuroprotection similar to what was seen with a large overexpression of GDNF, but without the side effects. These data corroborate the hypothesis that a moderate increase in endogenous GDNF could be beneficial for PD.

We have previously identified antisense (AS) Uchl1 as a long noncoding RNA (lncRNA) antisense to mouse Uchl1/Park5 that increases UchL1 protein synthesis at the post-transcriptional level. [START_REF] Carrieri | Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat[END_REF] AS Uchl1 is the representative member of a new functional class of natural and synthetic lncRNAs, named SINEUPs, that promote translation of partially overlapping sense protein-coding mRNAs with no effects on their mRNA levels. [START_REF] Zucchelli | SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation[END_REF] SINEUPs' activity depends on the combination of two domains: the overlapping region or binding domain (BD), which dictates SINEUP specificity, and the embedded inverted SINEB2 element, which acts as the effector domain (ED) controlling the enhancement of mRNA translation. Their modular structure can be employed to artificially engineer their BD and design synthetic SINEUPs to specifically enhance translation of virtually any target gene of interest. miniSINEUP RNAs are exclusively composed by ED and BD sequences.

Synthetic SINEUPs and miniSINEUPs have been proven effective with a number of targets, including GFP, FLAG-tagged proteins, and secreted recombinant antibodies and cytokines, thus showing SINEUP technology is scalable. [START_REF] Patrucco | Engineering mammalian cell factories with SINEUP noncoding RNAs to improve translation of secreted proteins[END_REF][START_REF] Zucchelli | SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells[END_REF][START_REF] Sasso | A long non-coding SINEUP RNA boosts semi-stable production of fully human monoclonal antibodies in HEK293E cells[END_REF] Furthermore, SINEUPs can act on endogenous mRNAs in vitro and in vivo. In a Medaka fish model of microphthalmia with linear skin lesions and in a cellular model of Friedreich ataxia, SINEUPs increased endogenous protein levels of, respectively, Cox7b and frataxin, restoring eye development in vivo and physiological mitochondria activity in patients' cells. [START_REF] Indrieri | Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo[END_REF] These results suggest the use of SINEUP technology in treating haploinsufficiencies where a single functional allele is not sufficient to encode for physiological amounts of the protein. In a parallel strategy, SINEUPs may be used to increase the expression of neuroprotective agents to compensate for a disease phenotype.

Here we have designed and successfully tested miniSINEUP RNAs specific for GDNF (miniSINEUP-GDNF) that selectively induced GDNF protein levels in vitro. We then proved that these non-coding RNAs are able to stably increase GDNF protein levels in the mouse striatum. Finally, miniSINEUP-GDNF was able to ameliorate motor deficits and DA neurodegeneration in mice unilaterally lesioned with 6-hydroxy-dopamine (6-OHDA).

These results may pave the way to a new strategy for increasing endogenous GDNF in PD therapy and to the establishment of a novel, RNA-based therapeutic platform to address genetic and idiopathic diseases.

RESULTS

miniSINEUP-GDNF RNA Selectively Increases GDNF Protein Expression In Vitro

To design a specific SINEUP for endogenous Gdnf mRNA, we first evaluated transcription start site (TSS) usage in cells and tissues relevant for PD. We thus interrogated the FANTOM5 collection of Cap Analysis of Gene Expression (CAGE) datasets, which represents the widest catalog of annotated promoters and TSSs in mammalian sam-ples. [START_REF] Forrest | A promoter-level mammalian expression atlas[END_REF] By using the Zenbu Genome Browser Tool for data visualization, [START_REF] Severin | Interactive visualization and analysis of large-scale sequencing datasets using ZENBU[END_REF] we monitored TSS usage at the mouse GDNF locus and evaluated their expression in a variety of samples. The main TSS was close to the first ATG, suggesting that the first two annotated isoforms (NM_001301332.1 and NM_010275.3) were the most widely expressed (Figure S1).

Based on this TSS analysis, we designed two GDNF-specific miniSINEUPs (miniSINEUP-GDNF) in antisense orientation to Gdnf mRNA around the first AUG, following the rules of pairing at the sense (S)/AS Uchl1 locus [START_REF] Carrieri | Long non-coding antisense RNA controls Uchl1 translation through an embedded SINEB2 repeat[END_REF] and for other natural and synthetic SINEUPs. [START_REF] Zucchelli | SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation[END_REF] While they presented the very same, original ED from the SINEB2 sequence of mouse AS Uchl1 RNA, two different BD sequences were synthesized: a long one (À40/+4) and a short one (À14/+4), giving rise to miniSINEUP-GDNFs of around 250 nt in length (Figure 1A).

To test miniSINEUP-GDNF efficacy, we took advantage of two mouse cell lines that express endogenous Gdnf mRNA, [START_REF] Modarresi | Inhibition of natural antisense transcripts in vivo results in gene-specific transcriptional upregulation[END_REF] the neuroblastoma Neuro2a and the astrocyte C8-D1A cell lines, verifying first that Gdnf mRNA was present (Figure S2). SINEUP activity was evaluated by measuring GDNF protein levels by western blot. Their post-transcriptional control of target gene expression was monitored with qRT-PCR. Both SINEUPs demonstrated a significant activity (Figures 1B and1C), with the one with the short BD (À14/+4) being the more active (2.3-and 1.7-fold increases in Neuro2a and C8, respectively) compared to the miniSINEUP-GDNF with the longest BD (1.9-and 1.4-fold increases in Neuro2a and C8, respectively). Both SINEUPs did not show any effect at the transcriptional level, with no change in Gdnf mRNA (Figures 1B and1C).

Since GDNF is released outside cells and its secretion confers neuroprotection of the DA terminals in the striatum, we measured secreted GDNF protein levels in the medium of C8 astrocytes 2 days from miniSINEUP-GDNF transfection. As shown in Figure 1D, miniSINEUP-GDNF (À14/+4) increased secreted GDNF protein levels by about 40%. We then cloned miniSINEUP-GDNFs in a pLVX-TetOne vector under the control of a doxocycline-inducible promoter. miniSINEUP RNA levels reached a plateau at 250 ng/mL, corresponding to its peak activity (Figure 1E).

SINEUP specificity is given by the BD nucleotide sequence that is unique for the endogenous mRNA target. However, like other nucleic acid-based techniques, off-targets cannot be excluded. Thus, we took advantage of the BLAST to predict potential off-targets, aligning the BD sequence to the mouse mRNA dataset. Three off-target candidates showed the highest sequence similarity: Dad1 (homologous on the 3 0 UTR, 15 nt of similarity), Lrrc14 (homologous in the coding measured by an ELISA (n = 8). (E) Neuro2a cells were transfected with a pLVX-TetOne vector expressing the miniSINEUP-GDNF. After 24 h post-transfection, cells were treated with different concentrations of doxocycline for 24 h to evaluate the correlation between miniSINEUP RNA levels and GDNF protein expression. The induction of the miniSINEUP-GDNF RNA expression by doxocycline (F 4, 29 = 3.45, p < 0.05) provokes an increase in GDNF protein levels (F 4, 20 = 3.52, p < 0.05, n = 3). (F) Off-target effects of miniSINEUP-GDNF were studied. After a BLAST analysis, the protein and mRNA levels of three possible off-targets were measured with western blot and qRT-PCR, namely, DAD1, CBX4, and LRRC14. miniSINEUP-GDNF did not alter protein or mRNA levels of these potential off-targets (n = 3 sequence, 14 nt of similarity), and Cbx4 (homologous on the 3 0 UTR, 17 nt of similarity) (Table S1). As shown in Figure 1F, miniSINEUP-GDNF did not alter the protein or mRNA levels of these genes, proving the specificity of activity. To further demonstrate the ontarget effects of miniSINEUP-GDNF, we produced a miniSINEUP lacking the BD (deltaBD-miniSINEUP-GDNF), to eliminate the specificity for the endogenous Gdnf mRNA. Importantly, deltaBD-miniSINEUP-GDNF had no effects on GDNF protein levels compared to miniSINEUP-GDNF (Figure S3).

AAV9-Mediated Delivery of miniSINEUP-GDNF RNA Increases Endogenous GDNF Expression in the Mouse Striatum

To test the functionality of the miniSINEUP-GDNF in vivo, we designed an AAV9 vector to express SINEUP RNA under the control of the cytomegalovirus (CMV) early enhancer/chicken beta-actin (CAG). Since we couldn't produce an AAV with two CAG promoters, we used another ubiquitous promoter, the phosphoglycerate kinase (PGK), to drive the expression of GFP, although its expected activity in astrocytes is lower (Figure 2A). Using this vector, miniSINEUP-GDNF maintained its GDNF protein upregulation activity when tested in vitro in Neuro2a and C8 astrocytes (Figure 2B). AAV9 vectors expressing miniSINEUP RNA and the empty vector (CTRL) were produced and stereotaxically injected into the dorsal striatum of adult C57BL/6J mice at the dose of 7.0 Â 10 9 vector genomes (vg) per site of injection.

At 5 weeks post-infection, transgenes were widely expressed in the majority of neurons of the dorsal striatum (Figure 2C). As expected, PGK promoter was more effective in expressing GFP in neuronal than in glial cells. We also noted some GFP-positive cells outside the area, especially close to the ventricle, probably due to leaking of the virus. Most importantly, miniSINEUP-GDNF increased GDNF protein levels by about 80% (Figure 2D), an effect comparable to the one seen in vitro in cell lines. RNA expression and SINEUP activity were maintained up to 6 months after vector administration (Figure 2E). These results prove a stable expression of the transgene and a continuous increase in endogenous GDNF protein levels.

Striatal GDNF Is Functionally Active In Vivo When Synthesized through a miniSINEUP-GDNF-Dependent Mechanism

Overexpression of GDNF in the striatum using viruses or the recombinant protein leads to several consequences, including an alteration of the DA system's functions. [START_REF] Ibáñez | Biology of GDNF and its receptors -Relevance for disorders of the central nervous system[END_REF] To test whether the increase in GDNF protein levels achieved by miniSINEUP-GDNF RNA was functionally relevant, we first injected mice with amphetamine, a DA reuptake inhibitor that stimulates locomotion. Interestingly, mice injected with AAV9-miniSINEUP-GDNF were more sensitive to the stimulating effects of amphetamine (Figure 3A). We then used in vivo microdialysis to evaluate DA release in the striatum injected with miniSINEUP-GDNF RNA. As shown in Figure 3B, DA basal levels were not changed. However, when DA release was stimulated by the infusion of potassium or amphetamine, mice with miniSINEUP-GDNF RNA showed an enhanced DA release compared to CTRL animals (Figures 3C and3D). This effect was likely due to an increase in DA storage in the terminals, since we found an increase in DA total content in the striatum of mice injected with AA9-miniSINEUP-GDNF, although a higher number of DA cells' terminals cannot be excluded (Figure 3E).

Uncontrolled ectopic expression of GDNF has led to several side effects in mice and in humans during clinical trials, including an increase in basal locomotor activity and reduced body weight and food intake. [START_REF] Kordower | Trophic factor gene therapy for Parkinson's disease[END_REF][START_REF] Kumar | GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function[END_REF] Here we showed that, 4 months after vector injection, mice expressing miniSINEUP-GDNF RNA did not show any alteration of locomotor parameters (Figure 3F), body weight (Figure 3G), and food intake (Figure 3H). These results prove that the SINEUPmediated increase in GDNF occurs within a physiological range, without triggering any deleterious effects in vivo.

miniSINEUP-GDNF RNA Rescues Neurodegeneration and Motor Phenotype in a PD Neurochemical Mouse Model

We then tested whether miniSINEUP-GDNF RNA was effective as a neuroprotective agent in a PD neurochemical mouse model. To this purpose, we stereotaxically injected AAV9-miniSINEUP-GDNF or the CTRL virus into the striatum and induced 6-OHDA unilateral lesions 4 weeks later (Figure 4A). No evident signs of cortical damage were induced by the two surgeries (Figure S4). 6-OHDA was able to induce lesions of DA terminals, as previously shown by many studies (Figure 4B).

At 6 weeks after the intoxication and 10 weeks after AAV injections, we carried out a battery of behavioral tests. First, we performed the cylinder test to assess the motor functionality of the forepaw, since it is well known to be impaired in lesioned mice. Mice with miniSINEUP-GDNF RNA showed an increase in contralateral touches and a parallel decrease in ipsilateral touches, indicating a recovery of the forepaw that was under the control of the lesioned striatum (Figure 4C). Then, we scored the mice for rotations, a sign of the severity of the lesion. Both spontaneous rotations and those induced by amphetamine were strongly reduced in mice with miniSINEUP-GDNF RNA (Figure 4D), suggesting that miniSINEUP-GDNF was able to reduce striatal neurodegeneration.

To verify this interpretation, we evaluated the quantity of DA terminals in the lesioned striatum by measuring tyrosine hydroxylase (TH) immunoreactivity. As shown in Figure 4E, mice injected with miniSINEUP-GDNF showed an increase in TH staining and a reduction in the lesion induced by 6-OHDA intoxication. Furthermore, mice with miniSINEUP-GDNF RNA showed an increase in TH-positive neurons in the SNpc with a recovery of the lesion of about 50% (Figure 4F). To verify that the enhancement of GDNF protein levels was present under pathological conditions, we carried out the very same protocol of sequential viral injection and lesion with 6-OHDA in another cohort of mice. As shown in Figure S5, mice injected with AAV-miniSINEUP-GDNF presented higher levels of GDNF in the striatum after 1 month from the time of lesion.

DISCUSSION

Nucleic acid-based therapy has been constantly developing in the last years, with several drug candidates in clinical development for human diseases. [START_REF] Sullenger | From the RNA world to the clinic[END_REF] Generally, RNAs act as inhibitory molecules and most of them are small interfering RNAs (siRNAs) and AS oligonucleotides (ASOs). On the other hand, molecules able to increase the expression of selected genes would be of great interest for the treatment of several pathologies. To this purpose, transcriptional activating RNAs and non-degradative ASOs have been recently used in vitro and in in vivo disease models. [START_REF] Li | Therapeutic targeting of microRNAs: current status and future challenges[END_REF][START_REF] Faghihi | RNAi screen indicates widespread biological function for human natural antisense transcripts[END_REF][START_REF] Gustincich | The Yin and Yang of nucleic acid-based therapy in the brain[END_REF] In this study, we demonstrate that SINEUPs, non-coding RNAs that activate the translation of target mRNAs, can be used as a novel tool for gene therapy. As a proof of concept, we designed a SINEUP targeting endogenous Gdnf, and we proved that miniSINEUP-GDNF RNA is effective in increasing GDNF protein levels in vivo and in rescuing motor symptoms and neurodegeneration in a PD mouse model. GDNF was cloned almost 30 years ago, and several experimental studies in rodents and non-human primates have been demonstrating that increasing GDNF levels is beneficial for the survival of DA neurons. [START_REF] Kordower | Trophic factor gene therapy for Parkinson's disease[END_REF][START_REF] Lin | GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons[END_REF][START_REF] Tomac | Protection and repair of the nigrostriatal dopaminergic system by GDNF in vivo[END_REF][START_REF] Gash | Functional recovery in parkinsonian monkeys treated with GDNF[END_REF] However, clinical trials in humans with GDNF recombinant protein did not give straightforward results, showing promising improvements but also a lack of efficacy and unsustainable side effects.

Major problems concern the large excess of the protein, way above physiological levels, and its poor spreading in the tissue. [START_REF] Nutt | Implanted intracerebroventricular. Glial cell line-derived neurotrophic factor (2003). Randomized, double-blind trial of glial cell line-derived neurotrophic factor (GDNF) in PD[END_REF][START_REF] Lang | Randomized controlled trial of intraputamenal glial cell line-derived neurotrophic factor infusion in Parkinson disease[END_REF] Despite a good efficacy in preclinical PD models, GDNF overexpression with viral vectors, either lentivirus or AAV, has showed similar side effects and limitations. [START_REF] Kordower | Trophic factor gene therapy for Parkinson's disease[END_REF] This is possibly due to the uncontrolled overexpression of the transgene occurring above physiological levels. Side effects and poor efficacy also can be caused by unspecific, ectopic expression of the transgene in cells that normally do not present it. While it is sometimes possible to drive transgene expression in the correct cells with specific promoters, the repertory of human neuronal cell typespecific promoters is very limited or they are often too large to be included in a viral vector. GDNF expression in the striatum is restricted to parvalbumin GABAergic interneurons, but the viral vectors used to overexpress GDNF so far always contained ubiquitous promoters.

Here we show that both in vitro and in vivo miniSINEUP-GDNF increases GDNF protein levels by about 2-fold. This is in line with SINEUPs we and others developed, measuring upregulation of the target protein in a range of 1.5-to 4-fold induction. [START_REF] Zucchelli | SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation[END_REF][START_REF] Zucchelli | SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells[END_REF] As previously shown in genetically modified mice, [START_REF] Kumar | GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function[END_REF] this moderate increase is sufficient to physiologically modulate endogenous DA systems preventing side effects.

The active miniSINEUP-GDNF has a BD of 18 nt that specifically pairs with endogenous Gdnf mRNA isoforms. Therefore, SINEUPs act only in cells that express the target mRNA. Since all nucleotidebased approaches, including those based on the CRISPR-Cas9 system, could have potential off-targets effects, [START_REF] Anderson | CRISPR off-target analysis in genetically engineered rats and mice[END_REF] we carried out a BLAST analysis to predict potential off-targets, and we experimentally proved that miniSINEUP-GDNF showed no effects on these mRNAs.

We have also demonstrated that in vivo expression of miniSINEUP-GDNF RNA using an AAV9 is able to alter significantly the DA system's functions. In the striatum, we noted an increase in DA release using in vivo microdialysis after amphetamine infusion or potassium depolarization. Mice were also more sensitive to the locomotor effects induced by the systemic administration of amphetamine. Strikingly, DA storage pools were increased in striatal terminals by about 2-fold. It remains to be determined whether it is due to an increase in DA content in the surviving terminals or in the number of DA terminals in the striatum. Interestingly, we did not observe any of the side effects caused by the GDNF ectopic overexpression, such as decreases in body weight and food intake. This is in line with our hypothesis that a moderate induction of GDNF expression in the correct cells, as mediated by SINEUP RNA activity, could be beneficial as a standard gene therapy approach but with less side effects. It should be noted that a more comprehensive study on the consequences of the ectopic expression of SINEUP-GDNF in other brain areas, including SNpc, will provide additional information on this important issue. More importantly, miniSINEUP-GDNF was able to ameliorate PD symptoms in the 6-OHDA PD mouse model. We used two behavioral assays widely used to assess the motor performance in this PD mouse model, the cylinder test and the rotational behavior. Mice injected with AAV9 expressing miniSINEUP-GDNF showed an improvement in the usage of the contralateral forepaw and significantly decreased the ipsilateral rotations. These data demonstrated an improvement in the motor behavior, the core symptomatology of PD. By analyzing TH immunoreactivity in the striatum and by counting DA neurons in the SNpc, we also demonstrated that miniSINEUP-GDNF could decrease DA loss induced by the 6-OHDA toxin infusion.

A limitation of this study is that there could be a difference in efficacy and in off-targeting of this approach between PD rodent models and humans. Studies in higher species such as non-human primates are, therefore, needed to validate SINEUP efficacy in vivo. Furthermore, it is well known that single PD mouse models are not reflecting the complexity of the human pathology. Therefore, in addition to neurochemical intoxication, miniSINEUP-GDNF should be tested in other PD animal models, including those based on alpha-synuclein dysfunction. Importantly, while in this study RNA was expressed before lesioning the striatum (neuroprotection), for a clinical perspective, miniSINEUP-GDNF activity should be evaluated when the brain is lesioned before the delivery of the neurorestorative agent. There are other technologies that can increase the endogenous expression of selected genes, for example, zinc-finger-based artificial transcription factors [START_REF] Sera | Zinc-finger-based artificial transcription factors and their applications[END_REF] and CRISPR-based activation system (CRISPRa). [START_REF] Matharu | CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency[END_REF] Both methods rely on the ability to stimulate transcription of a selected gene by activating its promoter. Potential advantages of SINEUPs over these technologies comprise SINEUPs work only when the target mRNA is expressed and the induction is within physiological levels.

There are several examples in the literature of long-term expression of transgenes in the human brain with AAV vectors. [START_REF] Deverman | Gene therapy for neurological disorders: progress and prospects[END_REF][START_REF] Evers | AAV5-miHTT Gene Therapy Demonstrates Broad Distribution and Strong Human Mutant Huntingtin Lowering in a Huntington's Disease Minipig Model[END_REF] An alternative strategy should be based on SINEUPs delivered as chemically synthesized nucleic acids by, i.e., intrathecal injection. In this context, we are focusing our efforts in decreasing the length of an active SINEUP. While the original AS Uchl1 RNA was larger than 1 kb, a miniSI-NEUP is around 250 nt long. A recent study of the structure of the invSINEB2 element acting as the ED in AS Uchl1 has shown the importance of Stem Loop 1 for SINEUP activity, prompting the synthesis of shorter active ED variants. [START_REF] Podbev Sek | Structural determinants of the SINE B2 element embedded in the long non-coding RNA activator of translation AS Uchl1[END_REF] In summary, our study has demonstrated that SINEUPs are an innovative technology to selectively increase the expression of target mRNAs in vivo in a physiological and specific manner, representing a new platform for gene therapy of neurodegenerative diseases.

MATERIALS AND METHODS

Construct

miniSINEUP-GDNF was generated by using the SINEUP-DJ1 as backbone. [START_REF] Zucchelli | SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells[END_REF] BDs were designed in antisense orientation to the two most expressed isoforms targeting the first AUG, with a long (À40/+4) and a short (À14/+4) overlapping region. Oligonucleotides were annealed and cloned into recipient pCS2 plasmid. For doxocycline experiments, miniSINEUP-GDNF was cloned into a pLVX-TetOn-puro vector. AAV-miniSINEUP-GDNF was generated modifying an existing pAAV-CAG-EGFP vector (Addgene, 28014) by inserting between the intron and the GFP a sequence coding for the miniSINEUP-GDNF, a polyA signal, and a PGK promoter. This modification led us to have the miniSINEUP-GDNF expressed by the CAG promoter and the GFP expressed under the PGK promoter.

Cell Lines and Transfection

Neuro2a and C8-D1A astrocyte type I clone were obtained from ATCC (Cat. No. CCL-131 and CRL-2541) and maintained in culture with DMEM (Gibco by Life Technologies, Cat. No. 41090-028) supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/ streptomycin). Neuro2a and C8-D1A cells were plated in 6-well plates the day before transfection at 60% confluency (4 Â 10 5 cells/well) and transfected with 1 mg SINEUPs encoding plasmids using Lipofect-amine 2000 (Invitrogen by Life Technologies, Cat. No. 11668019), following the manufacturer's instructions. Cells were collected 48 h after transfection. RNA and protein were obtained from the same transfection in each replica. For doxocycline induction experiments, Neuro2a was transfected with the pLVX-TetOn-puro vector expressing the miniSINEUP-GDNF and after 24 h doxocycline was added to the medium. At 24 h after doxocycline, cells were harvested and RNA and proteins were extracted. For the quantification of the GDNF in the medium, a kit from Abcam (ab171178) was used according to the manufacturer's instructions.

RNA Extraction and qRT-PCR

Total RNA was extracted from cell pellets using TRIzol Reagent (Thermo Fisher Scientific, Cat. No. 15596026) and following the manufacturer's instructions. RNA samples were subjected to DNase (Sigma-Aldrich, Cat. No. AMPD1) treatment to avoid plasmid DNA contamination. A total of 1 mg RNA was subjected to retrotranscription using iScriptcDNA Synthesis Kit (Bio-Rad, Cat. No. 1708890), according to the manufacturer's instructions. qRT-PCR was carried out using SYBR green fluorescent dye (SsoAdvanced Universal SYBR Green Supermix, Bio-Rad, Cat. No. 1725271) and an CFX96 Real time PCR System (Bio-Rad). The reactions were performed on diluted cDNA (1:8). Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the normalizing control in all qRT-PCR experiments. The amplified transcripts were quantified using the comparative Ct method, and the differences in gene expression were presented as normalized fold expression with the DDCt method.

Western Blot

Centrifuged cells or striatum from mice were lysed in radioimmunoprecipitation assay (RIPA) buffer with the addition of protease inhibitor cocktail (Sigma-Aldrich, Cat. No. P83490) buffer, briefly sonicated, and boiled for 5 min at 95 C. 30 mg protein extracts were resolved by 4%-12% SDS-PAGE gels and transferred to 0.2 mM nitrocellulose membrane (Amersham, Cat. No. GEH10600001) for 2 h at 35 V. Membranes were blocked with 5% non-fat dry milk in PBS/ 0.1% Tween 20 and incubated with the following indicated primary and secondary antibodies: anti-b-actin 1:20,000 (Sigma, Cat. No. A2066), anti-GDNF 1:500 (Santa Cruz Biotechnology, Cat. No. sc-328), anti-TH 1:1,000 (Millipore, Cat. No. AB152), anti-DAD1 1:1,000 (Novus Biologicals, Cat. No. NB600-669), anti-LRRC14 1:1,000 (Novus Biologicals, Cat. No. NBP1-80049), and anti-CBX4 1:500 (Santa Cruz Biotechnology, Cat. No. sc-517216). The antibody against GDNF detects the mature form of GDNF, with an apparent molecular weight of approximately 17 kDa. Proteins of interest were visualized with the Amersham ECL Detection Reagents (GE Healthcare by Sigma, Cat. No. RPN2105). Western blotting images were acquired with Image Quant LAS 4000mini system, and band intensity was calculated using Image Quant TL software.

Stereotaxic AAV9 and 6-OHDA Injection

The animals were anesthetized by a mixture of isoflurane/oxygen (1.5% of isoflurane during anesthesia with 1 L/min oxygen) and placed on a stereotaxic apparatus (David Kopf Instrument, Tujunga, CA, USA) with mouse adaptor and lateral ear bars. The skin on the skull was cut and one hole was made on the same side by a surgical drill. AAV9-SINEUP (titer 7.0 Â 10 12 vg/mL) or AAV9-CTRL (titer 7.0 Â 10 12 vg/mL) was injected 1 month before the lesion in order to get the expression of the transgene. 1 mL virus was injected in the right striatum at these cooordinates: anteroposterior (AP) 0.5, dorsoventral (DV) À3.0, and lateral (L) 2.0. The virus was injected using a glass pipette and the rate of infusion was of 1 mL/15 min. After the infusion, the pipette was maintained for another 5 min in the same position and then retracted slowly. At 4 weeks after the virus injection, all the animals received a stereotaxic injection of 6-OHDA or PBS at the same coordinates. 6-OHDA was dissolved in ascorbate-CNS Fluid 0.02% at a concentration of 4 mg/mL and the volume administered was 2 mL. All the animals received a desipramine 35 mg/kg injection (intraperitoneally [i.p.], dissolved in saline) to protect the noradrenergic innervations into the striatum. Sham-lesioned mice were injected with 0.02% ascorbate-saline devoid of 6-OHDA.

Behavioral Testing

All procedures involving animals and their care were carried out in accordance with the guidelines established by the European Community Council (Directive 2010/63/EU of September 22, 2010) and were approved by the Italian Ministry of Health (DL 116/92 and DL 111/ 94-B).

Locomotor Activity

To measure spontaneous locomotor activity, WT mice injected with AAV9 were placed in the locomotor activity chambers (Omnitech Digiscan, Accuscan Instruments, Columbus, PO, USA) for 60 min, and total distance traveled was measured by analyzing infrared beam interruptions. For amphetamine experiments, mice were habituated for 30 min in the apparatus and then injected i.p. with 2.5 mg/kg amphetamine (Sigma-Aldrich), and the distance traveled was measured for another 60 min.

Cylinder Test

During this test, animals were allowed to freely move in a clear glass cylinder for 5 min. A video camera was placed above the cylinder in order to see all the paw placements during the recording. An observer blinded to the animal treatment group viewed the recording and counted all the forepaw touches to the cylinder. The data were expressed as the percentage of contralateral touches.

Rotations

Spontaneous rotations were recorded 7 weeks after the lesion, while amphetamine-induced rotations were recorded 8 weeks after the lesion. Animals were placed in a glass cylinder 20 cm in diameter, and the rotational behavior was video recorded. For spontaneous rotation, mice were monitored for 15 min. For amphetamine-induced rotation, mice were injected i.p. with amphetamine at the dose of 2.5 mg/kg, and then animals were monitored for 45 min. Rotations were expressed as full 360 ipsilateral rotations.

Perfusion and Tissue Processing

Mice were deeply anesthetized with urethane (50 mg/kg, 10 mL/kg body weight, i.p., Sigma-Aldrich) and perfusion fixed by 10 mL 0.1 M phosphate buffer (pH 7.4) followed by 20 mL 4% formaldehyde. Brains were rapidly extracted and post-fixed in the same solution for 12 h at 4 C, and then cryoprotected in 30% sucrose in 0.1 M phosphate buffer (pH 7.4) for 48 h at 4 C. Sections were cut at 40 mm in the coronal plane on a freezing microtome from the front of the striatum to the end of the retrorubral field.

Immunohistochemistry for TH

The sections were rinsed three times in 0.1 M PBS (pH 7.6), contained 0.1% Triton X-100 between each incubation period. All sections were quenched with 3% H 2 O 2 /10% for 10 min, followed by several changes of buffer. As a blocking step, sections were then incubated in 7% normal goat serum and 0.1% Triton X-100 for 2 h at room temperature. This was followed by incubation in primary antibody diluted in 3% normal goat serum and 0.1% Triton X-100 at 4 C for 24 h. The antibody used was an anti-TH diluted 1:500 (AB-152, Millipore). After incubation with the primary antibody, sections were rinsed and then incubated for 2 h at room temperature with biotinylated secondary antibodies (anti-rabbit 1:1,000; Thermo Scientific) in the same buffer solution. The reaction was visualized with avidin-biotin-peroxidase complex (ABC-Elite, Vector Laboratories), using 3,3-diaminobenzidine as a chromogen. Sections were mounted on super-frost ultra plus slides (Thermo Scientific), dehydrated in ascending alcohol concentrations, cleared in xylene, and coverslipped in dibutyl phthalate polystyrene xylen (DPX) mounting medium. All sections were analyzed in the same experiment to keep staining conditions constant.

SNpc Cell Counts

The number of TH-positive cells was determined by counting every fourth 40-mm section, as previously described. [START_REF] Bensadoun | Lentiviral vectors as a gene delivery system in the mouse midbrain: cellular and behavioral improvements in a 6-OHDA model of Parkinson's disease using GDNF[END_REF] The delimitation between the ventral tegmental area and the SN was determined by using the medial terminal nucleus of the accessory optic tract as a landmark. The unlesioned side was used as a control for the percentage of remaining neurons in each group.

Striatal Fiber TH Densitometry

The optical densities of the TH immunoreactive fibers in the striatum were measured using the NIH ImageJ program. For each animal the optical density was measured at four rostocaudal levels over the whole striatum. To estimate TH staining density, the optical density was corrected for non-specific background density, as measured from the corpus callosum in each section. Data were presented as the average of the four rostrocaudal slice levels expressed as a percentage of the intact side.

Statistical Analysis

In all experiments, the significance of differences between groups was evaluated by Student's t test, one-way ANOVA, or repeated-measures ANOVA. p < 0.05 was considered significant. Quantitative data are presented as mean ± SEM.

Figure 1 .

 1 Figure 1. miniSINEUP-GDNF RNA Increases Selectively GDNF Protein Expression In Vitro (A) Schematic representation of miniSINEUP-GDNF structure, with the SINEUP BD overlapping the 5 0 UTR of the Gdnf mRNA. (B and C) Effect of miniSINEUP-GDNF RNA on endogenous GDNF protein levels in two cell lines, the Neuro2a (B) and the C8-D1A astrocytes (C) (F 2, 12 = 8.09, p < 0.001, n = 5 for Neuro2a; and F 2, 9 = 15.69, p < 0.01, n = 4 for astrocytes). miniSINEUP-GDNF transfected in cells increases GDNF proteins levels without altering Gdnf mRNA levels. Both miniSINEUP RNA with two different BD (À40/+4 and the À14/+4, respectively) were expressed in transfected cells, but not in CTRL cells. (D) miniSINEUP-GDNF increases extracellular GDNF secreted by cells, as (legend continued on next page)

Figure 2 .

 2 Figure 2. AAV9-Mediated Delivery Expression of miniSINEUP-GDNF RNA Increases Endogenous GDNF Expression in the Mouse Striatum (A) Schematic representation of the modified AAV9 vector used to express the miniSINEUP-GDNF and GFP as a reporter. (B) AAV9 plasmid was transfected in Neuro2a and astrocytes cell lines to test the expression of miniSINEUP-GDNF. In both cell lines, GDNF protein levels were increased with no alteration of Gdnf mRNA levels (n = 3). (C) AAV9 viral particles were stereotaxically injected in the right dorsal striatum of C57BL/6J mice and GFP expression was studied with a fluorescent microscope. Colocalization immunofluorescence was performed to evaluate GFP expression. NeuN was used to stain neurons and GFAP to stain astrocytes. Most of the GFP (expressed under PGK promoter) was present in neurons. Scale bars, 250 mm upper panel and 50 mm lower panels. (D) At 5 weeks after viral injection, striata were dissected and GDNF protein levels were evaluated with western blot. Mice injected with the AAV9-miniSINEUP-GDNF showed an increase in GDNF protein levels (n = 6 for CTRL mice and 7 for miniSINEUP-GDNF mice). (E, left panel) miniSINEUP RNA expression levels at different time points after viral injection. Mice injected with the virus were dissected at 5, 20, and 30 weeks post-injection. SINEUP RNA was present at high levels up to 30 weeks (n = 7, 5, and 5 for each time point, respectively). (E, right panel) Effect of AAV9-miniSINEUP-GDNF on GDNF protein levels at 30 weeks post-injection. Mice injected with AAV9-miniSINEUP-GDNF showed an increase in GDNF protein levels (n = 6 for CTRL mice and 5 for miniSINEUP-GDNF mice). Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test with Welch's correction was used.

Figure 3 .

 3 Figure3. Striatal GDNF Is Functionally Active In Vivo When Synthesized through a miniSINEUP-GDNF-Dependent Mechanism (A) Mice, 4 months after AAV9-miniSINEUP-GDNF injection, were put in locomotor chambers, injected with amphetamine at 2.5 mg/kg, and locomotor activity was measured. Mice injected with AAV9-miniSINEUP-GDNF showed an increase in locomotor activity (n = 12). (B) Extracellular DA release was measured in the striatum of mice injected with AAV9-miniSINEUP-GDNF, 4 months after injection. In vivo microdialysis was performed to collect samples from the striatum, and DA was measured using a high-performance liquid chromatography (HPLC) system. Basal DA level was not changed between SINEUP and CTRL groups. After depolarization of DA terminals by reverse microdialysis infusion of (C) potassium 120 nM or (D) amphetamine 250 mM in the artificial cerebrospinal fluid (ACSF) solution, mice injected with AAV9-miniSINEUP-GDNF showed an increase in DA extracellular levels (treatment*mouse F 8, 40 = 4.84, p < 0.001, n = 4). (E) Total striatal DA content was measured from animals injected with AAV9-miniSINEUP-GDNF or CTRL virus. Mice injected with AAV9-miniSINEUP-GDNF showed an increase in striatal DA total content (n = 5). (F) Locomotor activity in mice injected with AAV9 vector was measured at different time points after the injection. Total distance traveled by the two groups of animals was evaluated for 3 months (n = 8). (G and H) Two different side effects caused by the ectopic GDNF overexpression were measured, the decrease in body weight (G) and the decrease in food intake (H). Mice injected with AAV9-miniSINEUP-GDNF did not show any side effect (n = 9 for CTRL and 10 for miniSINEUP-GDNF). Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test with Welch's correction or repeated-measured ANOVA with Newman-Keuls test (C and D) was used.

Figure 4 .

 4 Figure 4. miniSINEUP-GDNF RNA Rescues Neurodegeneration and Motor Phenotype in a PD Neurochemical PD Mouse Model (A) Scheme representing the experimental protocol used for the assessment of the neuroprotective effect of miniSINEUP-GDNF. AAV9 expressing miniSINEUP-GDNF was injected 4 weeks before the lesion, then 6, 7, and 8 weeks after the lesion behavioral tests were performed to verify the locomotor performance of mice. (B) Striatal lesion after 6-OHDA infusion was measured by TH quantification by western blot. Lesioned animals revealed a decrease in TH levels of approximately 50% (n = 5). (C) The severity of the lesion was measured by the counting of the ipsilateral rotations of the lesioned mice. Both spontaneous rotations and the amphetamine-induced rotations were scored. For spontaneous rotations, videos were recorded for 15 min, while, for amphetamine-induced rotations, videos were recorded for 45 min (n = 9/group). (D) To assess the severity of the lesion, the cylinder test was carried out, where animals were scored for the contralateral forepaw usage. Ipsilateral touches were also counted (n = 9/group). (E) TH immunoreactivity of the striatum was evaluated by immunohistochemistry in mice injected with AAV9-miniSINEUP-GDNF or CTRL virus. Representative images of the striatum of min-iSINEUP and CTRL animals are shown (upper panel). Quantification of the TH immunoreactivity was calculated for all animals (lower panel) (n = 9/group). (F) TH-positive neurons of the SNpc were evaluated by immunohistochemistry in mice injected with AAV9-miniSINEUP-GDNF or CTRL virus. Representative images of the SNpc of miniSINEUP and CTRL animals are shown (upper panel). Quantification of the TH-positive neurons was calculated for all animals (lower panel) (n = 9/group). Data represent means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired t test with Welch's correction was used.

Please cite this article in press as: Espinoza et al., SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.08.005

Molecular Therapy Vol. 27 No 12 December 2019 Please cite this article in press as: Espinoza et al., SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.08.005

Molecular Therapy Vol. 27 No 12 December 2019

www.moleculartherapy.org Molecular Therapy Vol. 27 No 12 December 2019

Molecular Therapy Vol. 27 No 12 December 2019

ACKNOWLEDGMENTS

We are indebted to all the members of the S.G. lab for thought-provoking discussions. We thank Eva Ferri and Alessandra Sanna for administrative support. We are grateful to Luisa Franco and Ilaria Zanardi for experimental advice and Monica Morini for experimental assistance at the animal facility. This work has been funded by IIT intramural grants to S.G.

Data Availability

All data and materials are available upon request.

SUPPLEMENTAL INFORMATION

Supplemental Information can be found online at https://doi.org/10. 1016/j.ymthe.2019.08.005.

AUTHOR CONTRIBUTIONS

S.E. designed and carried out the experiments, analyzed the data, and composed the manuscript. M.S. carried out experiments. D.D. designed SINEUPs and analyzed experiments. F. Managò analyzed dopamine content. M.M. performed in vivo microdialysis. A.C. constructed the viruses. P.C. analyzed the data and discussed experimental results. C.S. analyzed the data and discussed experimental results. F.P. analyzed in vivo dopamine content and designed behavioral experiments. F. Mingozzi and G.R. designed and produced the AAV vectors used in the study. S.Z. designed SINEUPs, analyzed the data, and discussed experimental results. S.G. conceived the project, designed the experiments, analyzed the data, and composed the manuscript. All authors contributed to this work, read the manuscript, and agreed to its contents.

CONFLICTS OF INTEREST