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Disease modelling of FUS-linked ALS & assessing the risks of Cas9-induced non-homologous end joining as strategy to create gene knockouts

Student thesis: Doctoral ThesisDoctor of Philosophy

Amyotrophic lateral sclerosis (ALS) is the most common adult onset motorneuron (MN) disease with an estimated incidence rate of 1 - 3 in 100’000. ALS is characterised by progressive degeneration of upper and lower MNs, thereby causing denervation of the spinal musculature. It is a devastating disease causing paralysis and eventually death due to respiratory failure 3 – 5 years after symptom onset. Unfortunately, despite decades of research, the underlying pathomechanisms have not yet been elucidated and no efficient treatment is available.
While the majority of ALS cases are sporadic with unknown causes of disease, cases of familial ALS are caused by mutations in high penetrance ALS causing genes. One of these ALS causing genes codes for the RNA-binding protein fused in sarcoma (FUS), which is found in pathologic, cytoplasmic inclusions in the affected tissues of ALS patients harbouring mutation in the FUS gene. Despite FUS being subject of study for more than 20 years, neither its physiological function nor its role in disease has been completely understood. However, one of the known pathways associated with physiological FUS function is splicing. Splicing describes the process of excising introns from a transcript in order to generate translation competent messenger RNA (mRNA) and is executed by the major or the minor intron splicing pathway. Research in mouse and in human cell lines suggest a possible sequestration of spliceosomal components in cytoplasmic FUS aggregates, thereby potentially interfering with proper splicing.
In order to investigate sequestration of spliceosomal components by cytoplasmic FUS in the tissue affected in ALS, I utilised MNs differentiated from isogenic induced pluripotent stem cells (iPSCs) harbouring either a complete FUS knockout (FUSKO) or an either heterozygous (FUSP525Lhet) or homozygous (FUSP525Lhom) ALS associated P525L mutation in the FUS gene. Upon oxidative stress induced condensation of FUS, I could show cytoplasmic colocalisation of FUSP525Lhet and FUSP525Lhom with various spliceosomal small nuclear RNAs (snRNA) and the snRNA import factor snurportin in iPSCs as well as in MNs. Furthermore, I observed a qualitative retardation in the dissolution of the cytoplasmic snurportin foci upon removal of stress in iPSCs expressing FUSP525Lhet or FUSP525Lhom. As these experiments were performed in the cell type affected in ALS, these findings strengthen the argument for a splicing associated pathomechanism in FUS-linked ALS and augment previous studies in less disease relevant models.
Recent evidence suggests a particular involvement of FUS in minor intron splicing and selective downregulation of minor snRNPs in the motor neuron disease SMA. Given this potential link between impaired minor intron splicing and neurodegeneration we used genome wide association studies in order to identify genes coding for minor spliceosomal components which could be causative of ALS when mutated. A C172Y mutation in the ZRSR2 gene (ZRSR2C172Y) was identified in two unrelated ALS patients. ZRSR2 is an essential for the 5’ splice site recognition of minor introns. Using a minor intron containing minigene, I observed a dominant negative effect on minor intron splicing in HeLa cells transiently expressing ZRSR2C172Y. However, further genotyping of the patients revealed that the C172Y mutation in ZRSR2 is probably a somatic mutation in the blood tissue thereby rendering ZRSR2 unlikely to be an ALS associated gene. Nevertheless, the dominant negative function of ZRSR2C172Y could be exploited to strongly reduce minor intron splicing activity to study the impact of reduced minor spliceosome function.
While generating the isogenic iPSCs harbouring ALS relevant mutations our lab became interested in possible pitfalls of targeted genome editing. Introducing frameshifts resulting in premature termination codons (PTCs) as a means of knocking out genes might be problematic as this strategy relies on the degradation of mRNA by the nonsense-mediated mRNA decay (NMD). However, NMD is not always a highly efficient process. Transcripts escaping NMD may give rise to truncated proteins harbouring residual or dominant negative function. Using reporter minigenes commonly used in the NMD field and various detection methods such as slot blot and immunofluorescence, I could show that even excellent NMD substrates can give rise to truncated proteins. Therefore, in order to avoid unwanted effects mediated by truncated target protein, alternative methods such as CRISPR-Trap, which abolishes transcription of the coding sequence of the target gene should be considered.
Original languageEnglish
Awarding Institution
Award date1 May 2020

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