Investigating pathological features of a novel amytrophic lateral schlerosis mouse model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of brain and spinal cord motor neurons. A proportion of ALS patients also develop frontotemporal dementia (FTD). A mutation in the CCNF gene (CCNFS621G) was recently reported in a family with ALS and FTD, offering a new opportunity to model ALS/FTD, including development of pre-clinical animal models. Over-expression of this mutation has been studied in zebrash and in two mouse cell lines, NSC-34 and neuro-2a. However, the effects of endogenously expressed CCNFS621G on the mammalian system remain unclear. To assess this, tissues from a previously generated CRISPR-Cas9 mouse model carrying CCNFS621G in its genome (CRISPR-CCNFS621G), were studied. This thesis aimed to characterise, for the first time, pathological features in the CRISPR- CCNFS621G mouse model, to evaluate its utility for ALS research. Specifically, this study assessed the impact of CCNFS621G on cyclin F expression, and investigated characteristic ALS/FTD histopathologies (mis-localised TDP-43, hyper-phosphorylated and aggregates of TDP-43, and ubiquitinated protein inclusions) and proteomic changes within brain and spinal cord tissues from six-month-old heterozygous and homozygous CRISPR- CCNFS621G mice. Genetics, molecular biology, histology, and proteomic techniques, were employed. It was found that CCNF gene (mRNA) and cyclin F protein levels were not altered in the CRISPR-CCNFS621G mice. No characteristic ALS/FTD histopathologies were identified within brain and spinal cord tissues from the CRISPR-CCNFS621G mice, but a significant loss of spinal cord motor neurons was observed in homozygous CRISPR- CCNFS621G mice when compared to control and heterozygous CRISPR-CCNFS621G mice. Filtering and analysis of proteomics data identified aberrantly expressed proteins in the CRISPR-CCNFS621G mice, which included TBC1D15 and GNAI2 among others, that are implicated in ALS-relevant biological processes. In summary, this project systematically characterised molecular and cellular pathologies present in the CRISPR-CCNFS621G mice, which may represent early features associated with disease pathogenesis prior to the occurrence of significant motor decficits. This was the first CRISPR-CCNF mouse model study and provides insights into cellular and biochemical changes relevant to ALS/FTD linked CCNFS621G. Future studies in an older cohort are required to examine progression of these changes and to gain a full understanding of CCNF-related disease aetiology.