INVESTIGATION OF C9ORF72 MOLECULAR HALLMARKS AND DEVELOPMENT OF THERAPEUTIC STRATEGIES
Tesi di Dottorato
Data di Pubblicazione:
2021
Citazione:
INVESTIGATION OF C9ORF72 MOLECULAR HALLMARKS AND DEVELOPMENT OF THERAPEUTIC STRATEGIES / F. Biella ; relatrice: S. P. Corti ; co-relatrice: M.M. Taiana ; coordinatore del corso di dottorato: M. Samaja. Dipartimento di Fisiopatologia Medico-Chirurgica e dei Trapianti, 2021 Mar 26. 33. ciclo, Anno Accademico 2020.
Abstract:
The majority of genetic Amyotrophic Lateral Sclerosis (ALS) cases are
due to the expansion in the number of an hexanucleotidic repeat in a
non-coding site of the locus C9orf72. Many mechanisms for the
C9orf72-ALS are suggested, both loss of function and gain of function
due to RNA foci and Dipeptide Repeats (DPR) toxicity.
Research on C9orf72-ALS is hindered by the lack of satisfactory
models and by the difficult access to the target cell type. Animal
models fail to recreate all the pathological features and the situation is
further complicated by the presence of many disease modifying genes.
To overcome theseissues, this study takes advantages of the induced
Pluripotent Stem Cells (iPSCs) technology to investigate canonical as
well as more recent C9orf72 molecular hallmarks. The iPSCs carry the
same genetic background of patients and can be further differentiated
in motoneurons (MNs) to study neuronal specific alteration and
comparing C9orf72-expanded cell lines with their isogenic corrected
counterpart, the influence of disease-modifying genes can be
eliminated.
We analysed canonical RNA foci and DPR accumulation finding them
increased in C9orf72 samples in both models. DNA damage
accumulation, a recently discovered C9orf72-ALS feature, was also
increased.
Moreover, in iPSC-derived MNs expression of SEPT7, STMN1 and
STMN2 genes, cytoskeletal regulators with important function in
neuronal cells, has been found altered in C9orf72 background.
Taking advantage of these established models we could also evaluate
the efficacy of a morpholine (MO)-based antisense oligonucleotide
(ASO) therapy as a proof of principle for the feasibility of drug
screening in these models. We foundthat both our MO oligomers were
able to rescue DPR accumulation and DNA damage induction.
Global gene expression analysis has also been performed. From this
investigation a subtle alteration in genes related with neuronal function
in iPSC was detected, while C9orf72 MNs showed deregulation in
pathways related to inflammation and cell-to-cell communication
suggesting a non-cell-autonomous mechanism for the disease.
Interestingly, MOs treatment could rescue these alterations.
Moving forward we started the characterization of a 3D organoidbased model of C9orf72-ALS that can reproduce the complexity of
central nervous system. We found in organoids DPR accumulation
and other disease hallmarks which could not be detected in 2D
models, supporting the promising role of this 3D model.
Tipologia IRIS:
Tesi di dottorato
Keywords:
ALS; C9ORF72; iPSCs; iPSC-derived MNs; Organoids
Elenco autori:
F. Biella
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