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Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and autism. FXS is caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene which leads to the lack of the encoded FMRP protein. FMRP is an RNA binding protein involved in protein synthesis regulation at synapses. Many evidences suggest a central role of the Group-I metabotropic glutamate receptor subtype 5 (mGluR5) in the FXS pathophysiology. In particular, an exaggerated signaling response following mGluR5 activation may underlie synaptic dysfunction in this disorder. Although much work has focused on the dysregulation of synaptic protein synthesis as a consequence of this enhanced mGluR5 signaling, it becomes clear that in FXS there is also an altered balance of mGluR5 association with Homer scaffolding proteins, which are postsynaptic density (PSD) partners of mGluR5. Although an extensive literature describes the mGluR5/Homer association, very little is known about the consequences of the disruption of this interaction in the FXS context. Therefore, the goal of my thesis was to study the consequences of mGluR5/Homer crosstalk disruption in the Fmr1 knockout (KO) mouse model of FXS in term of properties and functions of mGluR5, such as expression during development, surface expression and axonal/dendritic targeting, agonist-induced internalization, surface dynamics and mGluR5-mediated modulation of NMDA receptor (NMDAR) currents. In a first set of experiments we investigated the mGluR5 surface expression in cultured hippocampal neurons from WT and Fmr1 KO mice by using immunofluorescence techniques and biotinylation assay. We found that mGluR5 was more expressed on the neuronal surface and was differently distributed in dendrites and axons of Fmr1 KO cultured neurons. We then hypothesized that these alterations were a direct consequence of the mGluR5/Homer crosstalk disruption. We demonstrated that these altered expression and targeting of mGluR5 were critically dependent on mGluR5/Homer crosstalk disruption. We also observed that mGluR5 did not undergo internalization upon sustained mGluR5 activation with DHPG in Fmr1 KO neurons. This latter phenotype, however, was not dependent on the disruption of the mGluR5/Homer crosstalk. Altogether, these results demonstrate that mGluR5/Homer crosstalk disruption contributes to the pathophysiology of FXS altering expression and targeting of mGluR5 on the surface of Fmr1 KO neurons. In the second part of my study we investigated the consequences of the disrupted mGluR5/Homer crosstalk for the mGluR5 surface dynamics, and consequently for NMDAR function in Fmr1 KO neurons. Using a combination of live-cell imaging and single-molecule tracking, we found that mGluR5/Homer crosstalk disruption specifically increased the mGluR5 lateral diffusion at the synapse of cultured Fmr1 KO hippocampal neurons. The higher mGluR5 mobility resulted in an increased probability of transient physical interaction with NMDAR in the PSD of Fmr1 KO. This interaction altered the mGluR5-mediated modulation of NMDAR currents as evidenced by the two following changes. First, using patch-clamp recordings from CA1 pyramidal neurons, we found that NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) evoked by Schaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Second, the postsynaptic expression of mGluR5 mediated long term depression (LTD) of NMDAR-EPSCs was reduced in Fmr1 KO neurons. Finally, we demonstrated that these defects in NMDA currents were strongly dependent on the mGluR5/Homer crosstalk disruption and altered mGluR5 dynamics. Our results show that mGluR5/Homer disruption contributes to the mGluR5 dysregulation in Fmr1 KO neurons. This study might have implication for the treatment of mGluR5 synaptic dysfunctions in FXS by targeting mGluR5/Homer interaction and provide new suggestions to correct the defective signaling underlying cognitive impairment and autism.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and autism. FXS is caused by a mutation in the fragile X mental retardation 1(Fmr1) gene which leads to the lack of the encoded FMRP protein. FMRP is an RNA binding protein involved in protein synthesis regulation at synapses. Many evidences suggest a central role of the Group-I metabotropic glutamate receptor subtype 5(mGluR5) in the FXS pathophysiology. In particular, an exaggerated signaling response following mGluR5 activation may underlie synaptic dysfunction in this disorder. Although much work has focused on the dysregulation of synaptic protein synthesis as aconsequence of this enhanced mGluR5 signaling, it becomes clear that in FXS there is also an altered balance of mGluR5 association with Homer scaffolding proteins, whichare postsynaptic density (PSD) partners of mGluR5. Although an extensive literature describes the mGluR5/Homer association, very little is known about the consequences of the disruption of this interaction in the FXS context. Therefore, the goal of my thesis was to study the consequences of mGluR5/Homer crosstalk disruption in the Fmr1 knockout(KO) mouse model of FXS in terms of properties and functions of mGluR5, such as expression during development, surface expression and axonal/dendritic targeting, agonist-induced internalization, surface dynamics and mGluR5-mediated modulation ofNMDA receptor (NMDAR) currents. In a first set of experiments we investigated the mGluR5 surface expression incultured hippocampal neurons from WT and Fmr1 KO mice by using immunofluorescence techniques and biotinylation assay. We found that mGluR5 was more expressed on the neuronal surface and was differently distributed in dendrites andaxons of Fmr1 KO cultured neurons. We then hypothesized that these alterations were adirect consequence of the mGluR5/Homer crosstalk disruption. We demonstrated that the altered expression and targeting of mGluR5 were critically dependent on mGluR5/Homer crosstalk disruption. We also observed that mGluR5 did not undergo internalization upon sustained mGluR5 activation with DHPG in Fmr1 KO neurons. This latter phenotype, however, was not dependent on the disruption of the mGluR5/Homer crosstalk. Altogether, these results demonstrate that mGluR5/Homer crosstalk disruption contributes to the pathophysiology of FXS altering expression and targeting of mGluR5 on the surface of Fmr1 KO neurons. In the second part of my study we investigated the consequences of the disrupted mGluR5/Homer crosstalk for the mGluR5 surface dynamics, and consequently for NMDAR function in Fmr1 KO neurons. Using a combination of live-cell imaging and single-molecule tracking, we found that mGluR5/Homer crosstalk disruption specifically increased the mGluR5 lateral diffusion at the synapse of cultured Fmr1 KO hippocampal neurons. The higher mGluR5 mobility resulted in an increased probability of transient physical interaction with NMDAR in the PSD of Fmr1 KO. This interaction altered the mGluR5-mediated modulation of NMDAR currents as evidenced by the two following changes. First, using patch-clamp recordings from CA1 pyramidal neurons, we found that NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) evoked by Schaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Second, the postsynaptic expression of mGluR5 mediated long term depression (LTD) of NMDAR-EPSCs was reduced in Fmr1 KO neurons. Finally, we demonstrated that these defects in NMDA currents were strongly dependent on the mGluR5/Homer crosstalk disruption and altered mGluR5 dynamics. Altogether, our results show that mGluR5/Homer disruption contributes to the mGluR5 dysregulation in Fmr1 KO neurons. This study might have implication for the treatment of mGluR5 synaptic dysfunctions in FXS by targeting mGluR5/Homer interaction and provide new suggestions to correct the defective signaling underly ingcognitive impairment and autism., Le Syndrome de l'X Fragile (FXS) est la forme héréditaire majoritaire de déficience intellectuelle et la cause monogénique de l'autisme. Le FXS est causé par une mutation du gène Fragile X Mental Retardation 1 (Fmr1), qui entraîne son inactivation et l'absence d’expression de la protéine codée: Fragile X Mental Retardation Protein(FMRP). FMRP est une protéine de liaison à l’ARN, impliquée dans la régulation de la synthèse protéiques à la synapse. Un rôle central est attribué au sous-type 5 des récepteurs métabotropiques au glutamate du groupe I (mGluR5) dans la physiopathologie du FXS. En effet, une réponse exagérée suite à l'activation de mGluR5 pourrait expliquer le dysfonctionnement synaptique dans ce syndrôme. Bien que de nombreux travaux aient mis l'accent sur la dérégulation de la synthèse des protéines synaptiques comme une conséquence de cette signalisation accrue du mGluR5, il y a aussi un équilibre altéré dans l'association de mGluR5 avec les différentes isoformes des protéines Homer, partenaires de densité post-synaptique (PSD) du mGluR5. Bien qu'une abondante littérature décrit l'association mGluR5/Homer, les conséquences de la perturbation de cette interaction dans le contexte du FXS sont peu connues. Par conséquent, l'objectif de ma thèse était d'étudier les conséquences de la perturbation de l’interaction mGluR5/Homer au niveau des propriétés et des fonctions de mGluR5, telles que l'expression durant le développement, l'expression de surface et le ciblage axonal/dendritique, l’internalisation déclenchée par l'agoniste, les dynamiques de surface, et la modulation des courants NMDAR induite par mGluR5. Dans un premier temps, nous avons étudié l’expression de surface de mGluR5 dans des neurones hippocampiques in vitro issus de souris sauvages et Fmr1 KO, par des techniques d’immunofluorescence et de biotinylation. Nous avons constaté que mGluR5 est plus exprimé à la surface neuronale et est différemment distribué dans les dendrites et les axones des neurones Fmr1 KO. Puis, nous avons démontré que cette altération d’expression et de ciblage est une conséquence directe de l’altération de l’interaction mGluR5/Homer. Nous avons aussi observé que mGluR5, indépendamment de l’altération de l’interaction mGluR5/Homer, ne subit pas d’internalisation suite son activation soutenue par DHPG dans les neurones Fmr1 KO. Dans la seconde partie de mon étude, nous avons étudié les conséquences de la perturbation de l’interaction mGluR5/Homer dans les dynamiques de surface de mGluR5 et par conséquent pour la fonction du NMDAR dans les neurones Fmr1 KO. Par des techniques d'imagerie et de pistage moléculaire, nous avons constaté que l’altération du complexe mGluR5/Homer augmente spécifiquement la diffusion latérale à la synapse des neurones hippocampiques Fmr1 KO in vitro. La mobilité élevée du mGluR5 conduit à une probabilité accrue d'une interaction physique transitoire avec NMDAR dans la PSD du Fmr1 KO. Cette interaction altère la modulation, induite par mGluR5, des courants NMDAR. En effet, en utilisant des enregistrements en patch-clamp de neurones pyramidaux de CA1 sur tranches couplés à la stimulation des fibres collatérales de Schaffer, nous avons constaté que les courants excitateurs post-synaptiques induits par NMDAR (NMDAR-EPSCs) présentent des amplitudes plus faibles dans les neurones Fmr1 KO. De plus, l'expression post-synaptique de mGluR5, induite par la dépression à long-terme de NMDAR-EPSCs est réduite dans les neurones Fmr1 KO. Finalement, nous avons démontré que ces défauts des courants NMDAR sont dépendants de la perturbation de l’interaction mGluR5/Homer et altèrent les dynamiques de mGluR5. Cette étude pourrait avoir des conséquences dans le traitement des dysfonctionnements synaptiques du mGluR5 dans le FXS, en ciblant l’interactionmGluR5/Homer, et offre de nouvelles suggestions pour corriger la signalisation défectueuse sous-jacente aux troubles du spectre autistique.

Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and autism. FXS is caused by a mutation in the fragile X mental retardation 1(Fmr1) gene which leads to the lack of the encoded FMRP protein. FMRP is an RNA binding protein involved in protein synthesis regulation at synapses. Many evidences suggest a central role of the Group-I metabotropic glutamate receptor subtype 5(mGluR5) in the FXS pathophysiology. In particular, an exaggerated signaling response following mGluR5 activation may underlie synaptic dysfunction in this disorder. Although much work has focused on the dysregulation of synaptic protein synthesis as aconsequence of this enhanced mGluR5 signaling, it becomes clear that in FXS there is also an altered balance of mGluR5 association with Homer scaffolding proteins, whichare postsynaptic density (PSD) partners of mGluR5. Although an extensive literature describes the mGluR5/Homer association, very little is known about the consequences of the disruption of this interaction in the FXS context. Therefore, the goal of my thesis was to study the consequences of mGluR5/Homer crosstalk disruption in the Fmr1 knockout(KO) mouse model of FXS in terms of properties and functions of mGluR5, such as expression during development, surface expression and axonal/dendritic targeting, agonist-induced internalization, surface dynamics and mGluR5-mediated modulation ofNMDA receptor (NMDAR) currents. In a first set of experiments we investigated the mGluR5 surface expression incultured hippocampal neurons from WT and Fmr1 KO mice by using immunofluorescence techniques and biotinylation assay. We found that mGluR5 was more expressed on the neuronal surface and was differently distributed in dendrites andaxons of Fmr1 KO cultured neurons. We then hypothesized that these alterations were adirect consequence of the mGluR5/Homer crosstalk disruption. We demonstrated that the altered expression and targeting of mGluR5 were critically dependent on mGluR5/Homer crosstalk disruption. We also observed that mGluR5 did not undergo internalization upon sustained mGluR5 activation with DHPG in Fmr1 KO neurons. This latter phenotype, however, was not dependent on the disruption of the mGluR5/Homer crosstalk. Altogether, these results demonstrate that mGluR5/Homer crosstalk disruption contributes to the pathophysiology of FXS altering expression and targeting of mGluR5 on the surface of Fmr1 KO neurons. In the second part of my study we investigated the consequences of the disrupted mGluR5/Homer crosstalk for the mGluR5 surface dynamics, and consequently for NMDAR function in Fmr1 KO neurons. Using a combination of live-cell imaging and single-molecule tracking, we found that mGluR5/Homer crosstalk disruption specifically increased the mGluR5 lateral diffusion at the synapse of cultured Fmr1 KO hippocampal neurons. The higher mGluR5 mobility resulted in an increased probability of transient physical interaction with NMDAR in the PSD of Fmr1 KO. This interaction altered the mGluR5-mediated modulation of NMDAR currents as evidenced by the two following changes. First, using patch-clamp recordings from CA1 pyramidal neurons, we found that NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) evoked by Schaffer collateral stimulation showed lower amplitudes in Fmr1 KO neurons. Second, the postsynaptic expression of mGluR5 mediated long term depression (LTD) of NMDAR-EPSCs was reduced in Fmr1 KO neurons. Finally, we demonstrated that these defects in NMDA currents were strongly dependent on the mGluR5/Homer crosstalk disruption and altered mGluR5 dynamics. Altogether, our results show that mGluR5/Homer disruption contributes to the mGluR5 dysregulation in Fmr1 KO neurons. This study might have implication for the treatment of mGluR5 synaptic dysfunctions in FXS by targeting mGluR5/Homer interaction and provide new suggestions to correct the defective signaling underly ingcognitive impairment and autism., Le Syndrome de l'X Fragile (FXS) est la forme héréditaire majoritaire de déficience intellectuelle et la cause monogénique de l'autisme. Le FXS est causé par une mutation du gène Fragile X Mental Retardation 1 (Fmr1), qui entraîne son inactivation et l'absence d’expression de la protéine codée: Fragile X Mental Retardation Protein(FMRP). FMRP est une protéine de liaison à l’ARN, impliquée dans la régulation de la synthèse protéiques à la synapse. Un rôle central est attribué au sous-type 5 des récepteurs métabotropiques au glutamate du groupe I (mGluR5) dans la physiopathologie du FXS. En effet, une réponse exagérée suite à l'activation de mGluR5 pourrait expliquer le dysfonctionnement synaptique dans ce syndrôme. Bien que de nombreux travaux aient mis l'accent sur la dérégulation de la synthèse des protéines synaptiques comme une conséquence de cette signalisation accrue du mGluR5, il y a aussi un équilibre altéré dans l'association de mGluR5 avec les différentes isoformes des protéines Homer, partenaires de densité post-synaptique (PSD) du mGluR5. Bien qu'une abondante littérature décrit l'association mGluR5/Homer, les conséquences de la perturbation de cette interaction dans le contexte du FXS sont peu connues. Par conséquent, l'objectif de ma thèse était d'étudier les conséquences de la perturbation de l’interaction mGluR5/Homer au niveau des propriétés et des fonctions de mGluR5, telles que l'expression durant le développement, l'expression de surface et le ciblage axonal/dendritique, l’internalisation déclenchée par l'agoniste, les dynamiques de surface, et la modulation des courants NMDAR induite par mGluR5. Dans un premier temps, nous avons étudié l’expression de surface de mGluR5 dans des neurones hippocampiques in vitro issus de souris sauvages et Fmr1 KO, par des techniques d’immunofluorescence et de biotinylation. Nous avons constaté que mGluR5 est plus exprimé à la surface neuronale et est différemment distribué dans les dendrites et les axones des neurones Fmr1 KO. Puis, nous avons démontré que cette altération d’expression et de ciblage est une conséquence directe de l’altération de l’interaction mGluR5/Homer. Nous avons aussi observé que mGluR5, indépendamment de l’altération de l’interaction mGluR5/Homer, ne subit pas d’internalisation suite son activation soutenue par DHPG dans les neurones Fmr1 KO. Dans la seconde partie de mon étude, nous avons étudié les conséquences de la perturbation de l’interaction mGluR5/Homer dans les dynamiques de surface de mGluR5 et par conséquent pour la fonction du NMDAR dans les neurones Fmr1 KO. Par des techniques d'imagerie et de pistage moléculaire, nous avons constaté que l’altération du complexe mGluR5/Homer augmente spécifiquement la diffusion latérale à la synapse des neurones hippocampiques Fmr1 KO in vitro. La mobilité élevée du mGluR5 conduit à une probabilité accrue d'une interaction physique transitoire avec NMDAR dans la PSD du Fmr1 KO. Cette interaction altère la modulation, induite par mGluR5, des courants NMDAR. En effet, en utilisant des enregistrements en patch-clamp de neurones pyramidaux de CA1 sur tranches couplés à la stimulation des fibres collatérales de Schaffer, nous avons constaté que les courants excitateurs post-synaptiques induits par NMDAR (NMDAR-EPSCs) présentent des amplitudes plus faibles dans les neurones Fmr1 KO. De plus, l'expression post-synaptique de mGluR5, induite par la dépression à long-terme de NMDAR-EPSCs est réduite dans les neurones Fmr1 KO. Finalement, nous avons démontré que ces défauts des courants NMDAR sont dépendants de la perturbation de l’interaction mGluR5/Homer et altèrent les dynamiques de mGluR5. Cette étude pourrait avoir des conséquences dans le traitement des dysfonctionnements synaptiques du mGluR5 dans le FXS, en ciblant l’interactionmGluR5/Homer, et offre de nouvelles suggestions pour corriger la signalisation défectueuse sous-jacente aux troubles du spectre autistique.

Luca Stingeni,1 Giovanna Malara,2,3 Andrea Conti,4 Luisa Di Costanzo,5 Carlo Giovanni Carrera,6 Martina Burlando,7 Piergiorgio Malagoli,8 Maria Letizia Musumeci,9 Federico Bardazzi,10 Valeria Brazzelli,11 Paolo Amerio,12 Clara De Simone,13,14 Sara Trevisini,15 Anna Balato,16 Matteo Megna,17 Francesco Loconsole,18 Catia De Felice,19 Marta Bartezaghi,20 Alice Rausa,20 Elisabetta Aloisi,20 Roberto Orsenigo,20 Antonio Costanzo21,22 On behalf of the SUPREME Study Group1Dermatology Section, Medical and Surgical Department, University of Perugia, Perugia, Italy; 2Dermatology Unit, Hospital “Bianchi Melacrino Morelli”, Reggio Calabria, Italy; 3Department of Dermatology, Papardo Hospital, Messina, Italy; 4Dermatologic Unit, Department of Surgery, Infermi Hospital, AUSL Romagna, Rimini, Italy; 5Department of Dermatology, “Gaetano Rummo” Hospital, Benevento UOC Dermatologia, AO G. Rummo, Benevento, Italy; 6U.O. Dermatologia, Fondazione IRCCS Ca’ Granda - Ospedale Maggiore Policlinico, Milano, Italy; 7IRCCS San Martino Polyclinic Hospital, Di.S.Sal. Section of Dermatology, Genoa, Italy; 8Dermatology Unit IRCCS Policlinico San Donato, Milan, Italy; 9Dermatology Clinic, University of Catania, Catania, Italy; 10Division of Dermatology, University Hospital Policlinico “S. Orsola-Malpighi”, Bologna, Italy; 11Institute of Dermatology, Foundation IRCCS Policlinico San Matteo and University of Pavia, Pavia, Italy; 12Dermatologic Clinic, G. D’Annunzio University, Chieti, Italy; 13Istituto di Dermatologia, Università Cattolica del Sacro Cuore, Rome, Italy; 14Fondazione Policlinico Universitario A. Gemelli – IRCCS, Rome, Italy; 15Dermatology Department, University of Trieste, Trieste, Italy; 16Dermatology Unit, University of Campania “Luigi Vanvitelli”, Naples, Italy; 17Section of Dermatology, Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy; 18Department of Biomedical Sciences and Human Oncology, University of Bari, Bari, Italy; 19Department of Clinical Dermatology, Centre for the Study and Treatment of Psoriasis, San Gallicano Dermatological Institute, IRCCS, Rome, Italy; 20Novartis Farma SpA, Origgio, Italy; 21Unit of Dermatology, IRCCS Humanitas Research Hospital, Milan, Italy; 22Department of Biomedical Sciences, Humanitas University, Milan, ItalyCorrespondence: Luca Stingeni, Dermatology Section, Medical and Surgical Department, University of Perugia, Perugia, Italy, Tel +39075-5783881, Email luca.stingeni@unipg.itPurpose: Psoriasis, a common systemic inflammatory disorder, presents with gender-related differences in the quality of life (QoL) and treatment outcomes. This post hoc analysis from the Phase 3b SUPREME study explored gender-related differences in patient characteristics and efficacy of secukinumab 300 mg on Psoriasis Area and Severity Index (PASI) 75/90/100 and impact on QoL using the Dermatology Life Quality Index (DLQI) in patients with moderate to severe psoriasis through week 24.Patients and Methods: The proportion of patients achieving PASI 75/90/100 was computed using a nonresponder imputation approach. Differences between cohorts were analyzed using a logistic regression model. The mean change from baseline in DLQI was computed using the Wilcoxon test.Results: Among the 433 patients (males: 71.6%), females had a higher DLQI than males at baseline (13.1 vs 9.5; P< 0.0001). Males had a slightly higher response for PASI 90 than females at week 16 (80.7% vs 78.1%; P=0.0779) and 24 (83.2% vs 79.7%; P=0.0319). No differences were observed between genders in PASI 100/75 responses at week 24. Both genders showed an improvement in DLQI with secukinumab at week 24 (− 10.9 vs − 8.1, respectively, in females vs males; P=0.0004).Conclusion: In summary, secukinumab was effective in the treatment of psoriasis, irrespective of gender.Keywords: plaque psoriasis, PASI, patient-reported outcomes, Dermatology Quality of Life Index, Hospital Anxiety and Depression Scale

This study evaluated whether secukinumab treatment for patients with moderate to severe plaque psoriasis correlates with improvements in symptoms of anxiety and depression. SUPREME was a 24-week, phase IIIb, multicentre, prospective study conducted across 50 centres in Italy with an extension period of up to 72 weeks. Assessments used were: Psoriasis Area Sever-ity Index (PASI), Hospital Anxiety and Depression Scale (HADS) - Anxiety (HADS-A), and HADS - Depression (HADS-D) scores and Dermatology Quality Life Index (DLQI). Compared with baseline, a significantly greater proportion of patients who reported moderate to severe clinical symptoms of anxiety or depression (HADS-A or HADS-D ≥ 11) were free of moderate to severe symptoms at weeks 16 and 48. The PASI and DLQI scores reduced over time with secukinumab treatment. Psoriasis treatment with secukinumab for 48 weeks resulted in significantly improved skin clearance and a parallel improvement in symptoms of anxiety and depression, assessed by HADS.