Le mutazioni nel gene Mecp2, sito sul cromosoma X, sono responsabili per la forma “tipica” della sindrome di Rett (RTT), una devastante patologia del neurosviluppo che colpisce 1:10.000 bambine nate vive. Sebbene anche le mutazioni nel gene Cdkl5 erano state inizialmente associate alla forma “atipica” della RTT, oggi la Sindrome da Deficienza di Cdkl5 (CDD) è considerata una patologia a sé stante. Entrambe le patologie presentano una sintomatologia simile e sono caratterizzate perlopiù da un ritardo del neurosviluppo. Ad oggi, purtroppo, non esistono cure efficaci, anche se è stato dimostrato che riattivando i geni responsabili delle malattie in modelli animali può portare al recupero dei sintomi neurologici. Uno dei principali limiti nello sviluppo di una strategia terapeutica è dato dalla mancanza di solidi biomarcatori che possano misurare oggettivamente modificazioni strutturali, funzionali e metaboliche, permettendo quindi di seguire il decorso della patologia ma anche gli effetti benefici di un trattamento. Per ovviare a questa problematica, la mia tesi mira ad identificare biomarcatori per entrambe le patologie. Abbiamo utilizzato diversi approcci di risonanza magnetica (MRI) per investigare le proprietà anatomiche e neurochimiche di diverse regioni cerebrali in modelli murini di RTT e CDD. Nel topo nullo per Cdkl5 non sono state riscontrate differenze anatomiche, tuttavia la spettroscopia di risonanza magnetica ha rivelato una deregolazione in metaboliti coinvolti nell’omeostasi energetica. Studi molecolari hanno confermato un forte decremento di ATP e della proteina AMPK nell’ippocampo nullo per Cdkl5. Abbiamo quindi iniziato un primo studio preclinico che mira a modulare i livelli di AMPK e nei topi KO trattati è stato riscontrato un miglioramento in diversi test comportamentali. Dall’altra parte, abbiamo seguito e comparato longitudinalmente in vivo il topo nullo per Mecp2 e un topo avente una mutazione missenso (Mecp2-Y120D). La MRI strutturale su topi di entrambi i sessi ha mostrato come lo sviluppo cerebrale nei diversi modelli sia comparabile ma non sovrapponibile, evidenziando differenze relative sia al sesso che al genotipo. Allo stesso tempo, tramite la spettroscopia di risonanza magnetica abbiamo riscontrato la deregolazione di metaboliti legati alla neurotrasmissione e al metabolismo energetico. In conclusione, il nostro studio sottolinea il grande potenziale della MRI nel monitorare il decorso di diverse patologie in specifici modelli di RTT e CDD, così come la sua rilevanza nell’identificare pathways importanti nello sviluppo di approcci farmacologici mirati. In più, abbiamo mostrato come la deficienza da Mecp2 affligga diversamente lo sviluppo della patologia a seconda del tipo di mutazione, del sesso e della regione cerebrale.

Mutations in the X-linked Mecp2 gene are responsible for the typical form of Rett syndrome (RTT), a devastating neurodevelopmental disorder that affects almost 1 out of 10,000 females born alive. Although mutations in the Cdkl5 gene have initially been associated with an atypical form of RTT, Cdkl5 Deficiency Disorder is now an independent disease (CDD). Nonetheless, both pathologies share a plethora of symptoms and they are primarily identified by neurodevelopmental delay. To date, no effective cure is available for RTT and CDD, even though it was demonstrated that reactivation of gene-causing disease in animal models leads to the reversal of neurological symptoms. Both diseases lack solid biomarkers capable of quantitatively measuring structural, functional or metabolic changes, permitting the assessment of disease progression and the actual benefits of therapeutic approaches. To fulfil this gap of knowledge, my thesis principally aimed at identifying sensitive measurable outcomes for both diseases. We exploited multiple approaches of ex vivo and in vivo Magnetic Resonance Imaging (MRI) to investigate anatomical and neurochemical properties of different cerebral regions in preclinical models of RTT and CDD. No difference in brain morphology was found in the Cdkl5 KO mouse, whereas a strong deregulation of metabolites related to mitochondrial homeostasis led us to unveil altered ATP and activated AMP-protein kinase (AMPK) levels. To validate those results, we started a pharmacological treatment acting on AMPK, that significantly ameliorated behavioural phenotypes of the Cdkl5 mouse. On the other hand, we longitudinally compared a full Mecp2 knockout mouse to a knock-in mouse harbouring a pathogenic missense mutation (Mecp2-Y120D). In vivo MRI on both genders revealed how developmental trajectory of the brain evolves in a comparable but not overlapping way, thus highlighting relevant gender and genotype discrepancies. Concomitantly, magnetic spectroscopy unveiled dysregulation of metabolites mainly related to energy homeostasis and neurotransmission. Collectively, we revealed the great potential of MRI in monitoring the disease progression in specific models of RTT and CDD and in identifying relevant pathways to pursue ad hoc pharmacological approaches. Further, they have highlighted that Mecp2 deficiency diversely affect the disease progression depending on the genetic lesion, gender and region of the brain.

Identification and validation of novel measurable outcomes for Rett syndrome through MRI and preclinical studies / Sara Carli - : . , 2022 May 13. ((34. ciclo, Anno Accademico 2020/2021.

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Identification and validation of novel measurable outcomes for Rett syndrome through MRI and preclinical studies

CARLI, SARA
2022

Abstract

Mutations in the X-linked Mecp2 gene are responsible for the typical form of Rett syndrome (RTT), a devastating neurodevelopmental disorder that affects almost 1 out of 10,000 females born alive. Although mutations in the Cdkl5 gene have initially been associated with an atypical form of RTT, Cdkl5 Deficiency Disorder is now an independent disease (CDD). Nonetheless, both pathologies share a plethora of symptoms and they are primarily identified by neurodevelopmental delay. To date, no effective cure is available for RTT and CDD, even though it was demonstrated that reactivation of gene-causing disease in animal models leads to the reversal of neurological symptoms. Both diseases lack solid biomarkers capable of quantitatively measuring structural, functional or metabolic changes, permitting the assessment of disease progression and the actual benefits of therapeutic approaches. To fulfil this gap of knowledge, my thesis principally aimed at identifying sensitive measurable outcomes for both diseases. We exploited multiple approaches of ex vivo and in vivo Magnetic Resonance Imaging (MRI) to investigate anatomical and neurochemical properties of different cerebral regions in preclinical models of RTT and CDD. No difference in brain morphology was found in the Cdkl5 KO mouse, whereas a strong deregulation of metabolites related to mitochondrial homeostasis led us to unveil altered ATP and activated AMP-protein kinase (AMPK) levels. To validate those results, we started a pharmacological treatment acting on AMPK, that significantly ameliorated behavioural phenotypes of the Cdkl5 mouse. On the other hand, we longitudinally compared a full Mecp2 knockout mouse to a knock-in mouse harbouring a pathogenic missense mutation (Mecp2-Y120D). In vivo MRI on both genders revealed how developmental trajectory of the brain evolves in a comparable but not overlapping way, thus highlighting relevant gender and genotype discrepancies. Concomitantly, magnetic spectroscopy unveiled dysregulation of metabolites mainly related to energy homeostasis and neurotransmission. Collectively, we revealed the great potential of MRI in monitoring the disease progression in specific models of RTT and CDD and in identifying relevant pathways to pursue ad hoc pharmacological approaches. Further, they have highlighted that Mecp2 deficiency diversely affect the disease progression depending on the genetic lesion, gender and region of the brain.
Identification and validation of novel measurable outcomes for Rett syndrome through MRI and preclinical studies / Sara Carli - : . , 2022 May 13. ((34. ciclo, Anno Accademico 2020/2021.
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/130053
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