Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of neurodegenerative diseases characterized by the eponymous accumulation of iron in the brain parenchyma, especially in the basal ganglia. Mutations in the gene C19orf12 cause NBIA4, a subtype of NBIA, also called Mitochondrial-membrane Protein-associated Neurodegeneration (MPAN). The biological function of the gene is poorly defined so far; available data suggest its involvement in mitochondrial function, autophagy and lipid metabolism. This study aimed to generate cellular and animal models carrying C19orf12 mutant alleles, using a genome editing approach, to investigate the role of the gene and understand the mechanism underpinning the development of neurodegeneration. We evaluated different biological processes including iron homeostasis, mitochondrial respiration, autophagy, and lipid metabolism. The cellular loss-of-function (LOF) model did not display significant differences in mitochondrial respiration, however, it showed impaired autophagy initiation, a high level of labile iron pool and increased total iron content following exposure to iron, as measured by ICP-MS. Preliminary results on C19orf12 knockout (KO) cells displayed changes in the lipidomic profiles, suggesting a possible involvement of the gene in lipid metabolism. The transient downregulation of the zebrafish orthologue of the human C19orf12, c19orf12a, resulted in significant morphological alterations in the early stages of development, confirming the potential of zebrafish KO models to investigate thoroughly the effects of c19orf12a deficiency. We generated KO lines using the CRISPR-Cas9 technology and followed their development, with a particular focus on neurogenesis. According to WISH analysis, we did not find significant defects in the development of the main neuronal regions in embryos. The analysis of the mutant’s behavior at different developmental stages showed differences compared to wild-type siblings with an increase in the number of tail coiling events at 24 hours-post-fertilization (hpf), an increase in the distance traveled at 120 hpf in the light-dark locomotion test and a decrease in velocity and distance covered by adult mutants in a novel-tank diving test. Additionally, preliminary results showed changes in lipid content and profile in mutant larvae. In conclusion, we generated both in vitro and in vivo models for C19orf12 deficiency. Their characterization highlights different phenotypes and altered pathways related to the absence of the gene that could be useful to better understand the mechanisms underlying this disorder and could be used to screen molecules with therapeutic potential.
La categoria Neurodegenerazione con Accumulo di Ferro nel Cervello (NBIA) comprende un gruppo di malattie neurodegenerative caratterizzate dall'accumulo eponimo di ferro nel parenchima cerebrale, specialmente nei gangli della base. Le mutazioni nel gene C19orf12 causano l’NBIA4 nota anche come Neurodegenerazione Associata alla Proteina della Membrana Mitocondriale (MPAN). La funzione biologica del gene è finora scarsamente definita; i dati disponibili suggeriscono il suo coinvolgimento nella funzione mitocondriale, nell'autofagia e nel metabolismo lipidico. Utilizzando un approccio di editing del genoma abbiamo generato modelli cellulari e animali con mutazioni deleterie nel gene C19orf12, per indagare il ruolo del gene e i meccanismi coinvolti nello sviluppo del processo neurodegenerativo. Abbiamo valutato diversi processi biologici tra cui l'omeostasi del ferro, la respirazione mitocondriale, l'autofagia e il metabolismo lipidico. Il modello cellulare knockout (KO) non ha mostrato differenze significative nella respirazione mitocondriale, tuttavia ha evidenziato lievi alterazioni nell’induzione del processo autofagico, un aumento del ferro libero e un aumento del contenuto totale di ferro in seguito all'esposizione al metallo, misurato mediante ICP-MS. Da un’analisi preliminare le cellule KO hanno presentato profili lipidici alterati, suggerendo il possibile coinvolgimento del gene nel metabolismo lipidico. La riduzione dell’espressione dell'ortologo umano in zebrafish, c19orf12a, mediante iniezione di un morfolino specifico, ha indotto significative alterazioni morfologiche nelle fasi iniziali dello sviluppo del pesce, confermando la possibilità di utilizzare tale modello per studiare in modo sistematico gli effetti indotti dal deficit di c19orf12a. Tramite genome editing, abbiamo ottenuto linee KO con mutazioni deleterie in omozigosi nel gene c19orf12a e indagato lo sviluppo neuronale tramite WISH con diversi marcatori specifici senza riscontrare anomalie significative. L'analisi del comportamento dei mutanti a diverse età ha mostrato differenze rispetto agli animali di controllo. In particolare, si è osservato un aumento del numero di avvolgimenti della coda a 24 ore dopo la fecondazione, un aumento della distanza percorsa a 120 ore dopo la fertilizzazione e una diminuzione della velocità e della distanza percorsa nei mutanti adulti. Inoltre, indagini preliminari hanno mostrato cambiamenti nel contenuto totale di lipidi e nel loro profilo di distribuzione nelle larve mutanti. Per concludere, in questo studio sono stati generati modelli KO cellulari e animali per C19orf12, la cui caratterizzazione ha evidenziato la presenza di alterazioni fenotipiche e biologiche che potrebbero essere utili per comprendere meglio il ruolo biologico del gene, i meccanismi alla base di MPAN ed essere utilizzati per screening di molecole con potenziale terapeutico.
Modeling C19orf12 deficiency in mammalian cells and zebrafish / Gnutti, Barbara. - (2024 Apr 17).
Modeling C19orf12 deficiency in mammalian cells and zebrafish
GNUTTI, Barbara
2024-04-17
Abstract
Neurodegeneration with Brain Iron Accumulation (NBIA) is a group of neurodegenerative diseases characterized by the eponymous accumulation of iron in the brain parenchyma, especially in the basal ganglia. Mutations in the gene C19orf12 cause NBIA4, a subtype of NBIA, also called Mitochondrial-membrane Protein-associated Neurodegeneration (MPAN). The biological function of the gene is poorly defined so far; available data suggest its involvement in mitochondrial function, autophagy and lipid metabolism. This study aimed to generate cellular and animal models carrying C19orf12 mutant alleles, using a genome editing approach, to investigate the role of the gene and understand the mechanism underpinning the development of neurodegeneration. We evaluated different biological processes including iron homeostasis, mitochondrial respiration, autophagy, and lipid metabolism. The cellular loss-of-function (LOF) model did not display significant differences in mitochondrial respiration, however, it showed impaired autophagy initiation, a high level of labile iron pool and increased total iron content following exposure to iron, as measured by ICP-MS. Preliminary results on C19orf12 knockout (KO) cells displayed changes in the lipidomic profiles, suggesting a possible involvement of the gene in lipid metabolism. The transient downregulation of the zebrafish orthologue of the human C19orf12, c19orf12a, resulted in significant morphological alterations in the early stages of development, confirming the potential of zebrafish KO models to investigate thoroughly the effects of c19orf12a deficiency. We generated KO lines using the CRISPR-Cas9 technology and followed their development, with a particular focus on neurogenesis. According to WISH analysis, we did not find significant defects in the development of the main neuronal regions in embryos. The analysis of the mutant’s behavior at different developmental stages showed differences compared to wild-type siblings with an increase in the number of tail coiling events at 24 hours-post-fertilization (hpf), an increase in the distance traveled at 120 hpf in the light-dark locomotion test and a decrease in velocity and distance covered by adult mutants in a novel-tank diving test. Additionally, preliminary results showed changes in lipid content and profile in mutant larvae. In conclusion, we generated both in vitro and in vivo models for C19orf12 deficiency. Their characterization highlights different phenotypes and altered pathways related to the absence of the gene that could be useful to better understand the mechanisms underlying this disorder and could be used to screen molecules with therapeutic potential.File | Dimensione | Formato | |
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