Microtubules are highly conserved structures, made of polymers of α and β tubulin organised in a polarised way (Hohmann and Dehghani, 2019). Their function can be fine regulated through post-translational modifications (Gadadhar et al., 2017), such as acetylation of α-tubulin. In mice, this post-translational modification is catalysed by α-tubulin acetyltransferase 1 (Atat1) (Kalebic et al., 2013a). Ablation of the expression of Atat1 and its orthologues in sensory neurons prevents acetylation of α-tubulin and has been associated with deficits in mechanosensation (Akella et al., 2010; Morley et al., 2016; Yan et al., 2018). Nonetheless, it is unknown if this phenotype is a consequence of an absence of Atat1 expression at embryonic stages or if it is possible to achieve this phenotype by ablation/inhibition of Atat1 in fully developed mice. Here I explored the potential of Atat1 ablation in mouse cells, with a gene therapy strategy, using clustered regularly interspaced short-palindromic repeat (CRISPR) and RNA interference (RNAi) technologies delivered by modified versions of recombinant adeno-associated viruses (rAAVs). In-vitro assays in neuroblastoma 2a (N2a) cells demonstrated that both CRISPR (Jinek et al., 2012) and RNAi technologies (Fire et al., 1998) were able to reduce the amount of acetylated α-tubulin, if delivered by lipid based methods. Therefore, in order to address if these constructs were able to reduce acetylated α-tubulin in ex-vivo preparations, I have produced rAAV viruses containing the best CRISPR and RNAi sequences targeting Atat1, identified in the initial screening. Immunofluorescence assays on ex-vivo organotypic cultures of spinal cord and dorsal root ganglia (DRG) neurons infected with the CRISPR virus demonstrated that it is possible to achieve a reduction in acetylated α-tubulin by targeting Atat1 at a genomic level. However, regarding the RNAi virus, ex-vivo organotypic cultures of spinal cord and DRG infected with this virus, did not show an effect in acetylated α-tubulin, comparing to control. Intrathecal administration of CRISPR and RNAi rAAV viruses in in-vivo mouse models failed to successfully achieve viral transduction. In addition to the gene therapy strategies, I evaluated the potential of inhibition of Atat1 at the protein level, using putative chemical inhibitors in in-vivo assays in N2a cells and ex-vivo primary cultures of DRG neurons. The chemical screen did not provide consistent results, which lead to the conclusion that it might be possible to chemically inhibit Atat1, however there is still a need for optimization of putative inhibitors. The overall results obtained provide an effective platform for studies targeting Atat1 in mouse cells. It is well-known that this protein is involved in many diseases and the experimental setting that I developed could be used not only in somatosensory research but also in other research areas, such as neurodegeneration or oncology (Castro-Castro et al., 2012; Dompierre et al., 2007; Govindarajan et al., 2013).

Reduction of α-tubulin acetylation through depletion of Atat1 using genomic, post-transcriptional and post-translational approaches / MONTEIRO SERRAO, JOANA MARIA. - (2021 Nov 24).

Reduction of α-tubulin acetylation through depletion of Atat1 using genomic, post-transcriptional and post-translational approaches

MONTEIRO SERRAO, JOANA MARIA
2021-11-24

Abstract

Microtubules are highly conserved structures, made of polymers of α and β tubulin organised in a polarised way (Hohmann and Dehghani, 2019). Their function can be fine regulated through post-translational modifications (Gadadhar et al., 2017), such as acetylation of α-tubulin. In mice, this post-translational modification is catalysed by α-tubulin acetyltransferase 1 (Atat1) (Kalebic et al., 2013a). Ablation of the expression of Atat1 and its orthologues in sensory neurons prevents acetylation of α-tubulin and has been associated with deficits in mechanosensation (Akella et al., 2010; Morley et al., 2016; Yan et al., 2018). Nonetheless, it is unknown if this phenotype is a consequence of an absence of Atat1 expression at embryonic stages or if it is possible to achieve this phenotype by ablation/inhibition of Atat1 in fully developed mice. Here I explored the potential of Atat1 ablation in mouse cells, with a gene therapy strategy, using clustered regularly interspaced short-palindromic repeat (CRISPR) and RNA interference (RNAi) technologies delivered by modified versions of recombinant adeno-associated viruses (rAAVs). In-vitro assays in neuroblastoma 2a (N2a) cells demonstrated that both CRISPR (Jinek et al., 2012) and RNAi technologies (Fire et al., 1998) were able to reduce the amount of acetylated α-tubulin, if delivered by lipid based methods. Therefore, in order to address if these constructs were able to reduce acetylated α-tubulin in ex-vivo preparations, I have produced rAAV viruses containing the best CRISPR and RNAi sequences targeting Atat1, identified in the initial screening. Immunofluorescence assays on ex-vivo organotypic cultures of spinal cord and dorsal root ganglia (DRG) neurons infected with the CRISPR virus demonstrated that it is possible to achieve a reduction in acetylated α-tubulin by targeting Atat1 at a genomic level. However, regarding the RNAi virus, ex-vivo organotypic cultures of spinal cord and DRG infected with this virus, did not show an effect in acetylated α-tubulin, comparing to control. Intrathecal administration of CRISPR and RNAi rAAV viruses in in-vivo mouse models failed to successfully achieve viral transduction. In addition to the gene therapy strategies, I evaluated the potential of inhibition of Atat1 at the protein level, using putative chemical inhibitors in in-vivo assays in N2a cells and ex-vivo primary cultures of DRG neurons. The chemical screen did not provide consistent results, which lead to the conclusion that it might be possible to chemically inhibit Atat1, however there is still a need for optimization of putative inhibitors. The overall results obtained provide an effective platform for studies targeting Atat1 in mouse cells. It is well-known that this protein is involved in many diseases and the experimental setting that I developed could be used not only in somatosensory research but also in other research areas, such as neurodegeneration or oncology (Castro-Castro et al., 2012; Dompierre et al., 2007; Govindarajan et al., 2013).
24-nov-2021
Heppenstall, Paul Alexander
MONTEIRO SERRAO, JOANA MARIA
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11767/125249
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