Cellular and molecular characterisation of MFSD8 mutations associated with the variant late-infantile NCL CLN7

Guevara-Ferrer, Marta (2024) Cellular and molecular characterisation of MFSD8 mutations associated with the variant late-infantile NCL CLN7. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Batten disease (BD), also known as neuronal ceroid lipofuscinoses (NCLs), is a collective group of inherited neurodegenerative disorders. NCLs are the most prevalent cause of dementia in children, and they are distinguished by a common symptomatology that includes epileptic seizures, visual impairment, and a progressive decline in cognitive and physical function that results in early mortality. There are currently 12 different NCLs genetically identified in humans (CLN1-CLN8, CLN10-CLN13), with four newly identified genes (CLN9, CLN14, CLCN6, and SGSH). This study specifically focused on the variant late-infantile NCL (vLINCL) CLN7, which is caused primarily by homozygous mutations in CLN7/MFSD8, a major facilitator superfamily gene. MFSD8 encodes a multispanning integral lysosomal membrane protein with 12 transmembrane domains and has recently been described as a potential chloride channel on endosomes and lysosomes. However, there is still no cure or treatment available for CLN7 disease. Additionally, there is strong evidence demonstrating that MFSD8 is involved in the pathogenesis and pathobiology of other adult dementias, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), as well as maculopathies and retinopathies, which share common disease-causing mutations in a heterozygosity manner. Considering the emerging relevance of the use of induced pluripotent stem cells (iPSCs) as a model for the investigation of neurodegenerative diseases, in this thesis, I studied CLN7 disease employing CLN7 patient-derived iPSCs. Specifically, two iPS cell lines were utilised, which were derived from a female patient diagnosed at the age of 2.5 years, who exhibited homozygosity for the common missense mutation p.T294K and a male patient diagnosed at the age of 4.5 years, who was also homozygous for a more severe missense mutation corresponding to p.R465W. These iPS cell lines were further differentiated into Neural Progenitor Cells (NPCs), constituting a novel approach to study the variant late-infantile NCL CLN7 and providing an opportunity to examine the disease using cell types that could more closely resemble those affected in vivo. In light of previous proteomic studies conducted in CLN7 patient-derived NPCs by our group, resulting in a downregulation of several nuclear proteins and, consistent with the observation of other studies, we identify the localisation of MFSD8 in the nucleus. These findings provide evidence of the potential existence of several co-existing MFSD8 variants within the cells, suggesting that MFSD8 might exert different functions depending on the different isoform expressed and its localisation. Additionally, this work also reveals an impairment in the autophagy-lysosomal pathway and mitochondria produced by disease-causing mutations in NPCs and the improvement of these phenotypes with the use of existing compounds. Furthermore, through the study of the post-translational modifications of MFSD8 and different protein stability assays, this work also provides more evidence of the intricacy of this protein. Therefore, further studies on the potential protein binding partners of MFSD8 were conducted to gain a deeper understanding of novel signalling pathways or molecular mechanisms in which MFSD8 might be involved. In summary, this thesis provides significant insights into the cellular and molecular biology of MFSD8 through the use of a clinically relevant model, which sheds new light on future directions for the study of CLN7 disease. These findings can contribute to the development of significant therapeutic strategies to ameliorate CLN7 disease and, as a consequence, improve other adult neurodegenerative diseases which share the same disease-causing mutations as CLN7 disease.