La sindrome di Leigh (LS) è una rara malattia neurodegenerativa progressiva che colpisce la normale fisiologia cerebrale, la funzione mobile e il metabolismo del tessuto lipidico. Più prevalentemente, affligge il normale sviluppo e, quindi, i più colpiti sono neonati e bambini, anche se alcuni casi sono stati riportati anche in adulti che mostrano insorgenza tardiva. Segni distintivi tipici della malattia sono la presenza di lesioni simmetriche nei gangli della base, nel tronco cerebrale, con conseguente deterioramento della funzione sia cognitiva che motoria. Con questo in mente, è fondamentale trovare modelli adatti in grado di fornire approfondimenti sui meccanismi molecolari della patologia. Un candidato promettente è La Drosophila melanogaster, ampiamente utilizzata per chiarire patologie umane come il morbo di Alzheimer e altamente trattabile geneticamente. Molte proprietà fisiologiche e neurologiche sono condivise tra gli esseri umani e i moscerini della frutta, ad esempio circa il 75% dei geni umani che causano una patologia presentano un omologo funzionale in Drosophila. È il caso di HIBCH (CG5044 nei moscerini della frutta), un enzima coinvolto nella via catabolica della valina che, se deregolato, può portare a un metabolismo valina difettoso e all'accumulo di metaboliti tossici, responsabili di impedimenti nella normale funzione mitocondriale. Combinando la soppressione genetica dell'espressione genica CG5044 grazie al sistema binario GAL4/UAS con l'osservazione al microscopio confocale di tessuti sezionati, abbiamo analizzato e quantificato il grado di neurodegenerazione, sia dal punto di vista macroscopico che microscopico. Due strumenti genetici sono stati utilizzati in parallelo, CRISPR (knock out) e RNAi (knock down) per sopprimere il gene di interesse. Inoltre, abbiamo eseguito test comportamentali per determinare quando le mosche adulte iniziano a mostrare difficoltà motorie; abbiamo dimostrato che le mosche sperimentali con soppressione genica CG5044 sviluppano la distrofia muscolare a velocità distinte e più velocemente rispetto al controllo. Inoltre, abbiamo analizzato i depositi lipidici utilizzando due tipi di colorante. I risultati indicano che nei campioni sperimentali rispetto al controllo, le goccioline lipidiche sono più grandi e anormalmente distribuite, anche nel cervello. Infine, abbiamo iniziato gli esperimenti di elettroforesi qPCR e gel per capire se il gene viene soppresso nelle mosche sperimentali. Tutti questi risultati confermano l'utilità di D. melanogaster come modello per ricapitolare il fenotipo clinico LS e ottenere nuove informazioni sulla malattia per delineare meglio i suoi meccanismi patologici. Il moscerino della frutta rappresenta un buon punto di partenza per studiare la LS, e da lì sarà possibile estendere lo studio delle alterazioni geniche in modelli molto più complessi. In questo modo, in futuro si potranno sviluppare nuove strategie terapeutiche, dato che, fino ad oggi, non sono disponibili trattamenti contro la LS.
Leigh Syndrome (LS) is a rare progressive neurodegenerative disease affecting the normal brain physiology, the motile function, and the lipid tissue metabolism. Most prevalently, it afflicts the normal development, and therefore, the most affected are newborns and children, even though some cases have been reported also in adults displaying late onset. Typical hallmarks of the disease are the presence of symmetrical lesions in the basal ganglia, in the brainstem, with subsequent deterioration of both cognitive and motor function. With this in mind, it is vital to find suitable models able to give deep insights into the molecular mechanisms of the pathology. A promising candidate is Drosophila melanogaster, widely used to elucidate human pathologies such as Alzheimer disease, and highly genetically tractable. Many physiologic and neurologic properties are shared between humans and fruit flies, for example an estimated 75% of the human genes causing a pathology present a functional homolog in Drosophila. This is the case of HIBCH (CG5044 in fruit flies), an enzyme involved in the catabolic pathway of the valine that, if deregulated, can lead to defective valine metabolism and to the buildup of toxic metabolites, responsible for impediments in the normal mitochondrial function. Combining genetic suppression of the CG5044 gene expression thanks to GAL4/UAS binary system with the observation at the confocal microscope of dissected tissues, we analyzed and quantified the degree of neurodegeneration, both from a macroscopic and microscopic point of view. Two genetic tools have been used in parallel, CRISPR (knock out) and RNAi (knock down) to suppress the gene of interest. Furthermore, we have performed behavioral tests to determine when adult flies start to display motor difficulties; we showed that experimental flies with CG5044 gene suppression do develop muscular dystrophy at distinct rates and faster in comparison with the control. In addition, we analyzed the lipid deposits using two types of dye. The results indicate that in experimental samples in comparison with the control, the lipid droplets are larger and abnormally distributed, also in the brain. Finally, we started qPCR and gel electrophoresis experiments to address whether the gene is suppressed in experimental flies. All these findings confirm D. melanogaster usefulness as a model for recapitulating LS clinical phenotype and getting new insights into the disease to better delineate its pathologic mechanisms. The fruit fly represents a good starting point to study LS, and from there it will be possible to extend the study of gene alterations in much more complex models. In this way, new therapeutic strategies can be developed in the future, given that, up to now, no treatments are available against LS.
Generazione di nuovi modelli di sindrome di Leigh HIBCH-/- affetti da fisiologia cerebrale anormale, distrofia muscolare e metabolismo lipidico alterato
SALAMONE, ALESSANDRO
2021/2022
Abstract
Leigh Syndrome (LS) is a rare progressive neurodegenerative disease affecting the normal brain physiology, the motile function, and the lipid tissue metabolism. Most prevalently, it afflicts the normal development, and therefore, the most affected are newborns and children, even though some cases have been reported also in adults displaying late onset. Typical hallmarks of the disease are the presence of symmetrical lesions in the basal ganglia, in the brainstem, with subsequent deterioration of both cognitive and motor function. With this in mind, it is vital to find suitable models able to give deep insights into the molecular mechanisms of the pathology. A promising candidate is Drosophila melanogaster, widely used to elucidate human pathologies such as Alzheimer disease, and highly genetically tractable. Many physiologic and neurologic properties are shared between humans and fruit flies, for example an estimated 75% of the human genes causing a pathology present a functional homolog in Drosophila. This is the case of HIBCH (CG5044 in fruit flies), an enzyme involved in the catabolic pathway of the valine that, if deregulated, can lead to defective valine metabolism and to the buildup of toxic metabolites, responsible for impediments in the normal mitochondrial function. Combining genetic suppression of the CG5044 gene expression thanks to GAL4/UAS binary system with the observation at the confocal microscope of dissected tissues, we analyzed and quantified the degree of neurodegeneration, both from a macroscopic and microscopic point of view. Two genetic tools have been used in parallel, CRISPR (knock out) and RNAi (knock down) to suppress the gene of interest. Furthermore, we have performed behavioral tests to determine when adult flies start to display motor difficulties; we showed that experimental flies with CG5044 gene suppression do develop muscular dystrophy at distinct rates and faster in comparison with the control. In addition, we analyzed the lipid deposits using two types of dye. The results indicate that in experimental samples in comparison with the control, the lipid droplets are larger and abnormally distributed, also in the brain. Finally, we started qPCR and gel electrophoresis experiments to address whether the gene is suppressed in experimental flies. All these findings confirm D. melanogaster usefulness as a model for recapitulating LS clinical phenotype and getting new insights into the disease to better delineate its pathologic mechanisms. The fruit fly represents a good starting point to study LS, and from there it will be possible to extend the study of gene alterations in much more complex models. In this way, new therapeutic strategies can be developed in the future, given that, up to now, no treatments are available against LS.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14240/51889