De Gennaro, Emanuela (2017) CRISPR-cas9 technology: a new frontier to study granulin gene function in the contest of neuronal ceroid lipofuscinoses 11. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: CRISPR-cas9 technology: a new frontier to study granulin gene function in the contest of neuronal ceroid lipofuscinoses 11
De Gennaro,
Date: 8 April 2017
Number of Pages: 75
Institution: Università degli Studi di Napoli Federico II
Department: Biologia
Scuola di dottorato: Scienze biologiche
Dottorato: Biologia
Ciclo di dottorato: 29
Coordinatore del Corso di dottorato:
Missero, CaterinaUNSPECIFIED
Date: 8 April 2017
Number of Pages: 75
Uncontrolled Keywords: grn; CRISPR
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/11 - Biologia molecolare
Date Deposited: 20 Apr 2017 11:07
Last Modified: 16 Mar 2018 13:04
DOI: 10.6093/UNINA/FEDOA/11482


Progranulin (PGRN) is a growth factor, containing 7.5 tandem repeats of a cysteine-rich motif, encoded by Granulin (GRN) gene. Heterozygous mutations of GRN are the main cause of Frontotemporal Dementia (FTD), while homozygous mutations lead to Neuronal Ceroid Lipofuscinoses type 11 (NCL11), a Lysosomal Storage Disorder (LSD) showing accumulation of un-degraded proteins and lipids (lipofuscin) inside the lysosomes. The role of GRN in regulating lysosomal functions is still unknown and therapies for NCLs are still not available or only aimed to minimize patients’ symptoms rather than to stop the disease progression. One of the main limit in understanding the cellular role of GRN is the absence of a good cellular model system, which can allow functional studies, as well as High Content Screening (HCS) experiments, in order to find drugs suitable for specific NCL11 therapies. For this reason, during my PhD studies I focused my work on the generation of the GRN-/- and GRN+/- Arpe19 cell lines by CRISPR-Cas9 technology and on a deep characterization of the autophagic-lysosomal phenotype in both GRN homozygous and heterozygous conditions. This phenotypic characterization allowed me to highlight differences between these two conditions. Indeed, in absence of PGRN, cells show an enhanced lysosomal and autophagic pathway with impairment of lysosomal function, in contrast to heterozygous condition in which there is a reduction of the autophagic flux with an unchanged number of lysosomes. On the other hand, I found that both in presence of half PGRN amount and in its absence, mTORC1 (mammalian Target Of Rapamycin Complex 1) activity was reduced and that the Transcription Factor EB (TFEB) was located into the nucleus, with a subsequent transcriptional activation of its autophagic-lysosomal target genes. Moreover, due to the low level of variability inside my cell system, I was able to quantify the lysosomal and the autophagic phenotype, by High Content Opera system, demonstrating that this cellular model is a perfect tool to perform screening of Food and Drug Administration (FDA) approved drugs in order to find novel therapeutic strategies for NCL11.


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