Defining new molecules and biological pathways underlying neurodegenerative disease

Abstract

Motor neurone diseases (MNDs) are a group of disorders characterised by the degeneration of upper and/or lower motor neurones. Unlike communicable diseases in which frequency is decreasing due to improved healthcare provision, the prevalence of neurodegenerative disorders such as MND are increasing proportionally, particularly in Western countries, placing a substantial burden on healthcare services. The hereditary spastic paraplegias (HSPs) are a form of upper motor neurone disease in which the cardinal features involve progressive spasticity of the lower limbs, which may be accompanied by other neurological or non-neurological abnormalities. To date there are >80 distinct genetic causes of HSP, involving genes encoding proteins with a plethora of proposed cellular roles. The overarching aim of the work described in this thesis involves the exploration of the role of disordered lipidomic metabolism in HSP. Chapter three describes studies stemming from an Amish individual that was originally diagnosed with neurodegeneration with brain iron accumulation (NBIA). Genetic studies defined de novo mutation in C19orf12, encoding a protein thought to be involved in lipid metabolism, as the likely cause of disease. Gene variants in C19orf12 have previously been associated with both autosomal dominant as well as recessive forms of NBIA, as well as with HSP. This chapter defines a new mechanistic explanation as to why variants in this gene may be inherited in either a recessive or dominant state, involving C19orf12 isoform specific haploinsufficiency. Mast syndrome is a complicated form of HSP at high frequency amongst the Amish community, due to mutation(s) in the SPG21 gene encoding maspardin. Chapter four involves deeper exploration of the biomolecular role of maspardin, and the cellular consequences that result due to loss of function of the molecule. This work determines that maspardin colocalises with rab7 and localises to endosomes and lysosomes which interact with mitochondria. Using CRISPR-Cas9 gene technology to knockout maspardin, these studies determined that maspardin loss results in altered cell bioenergetics, increased cell death and altered endosomal/lysosomal size. Lipid imbalance is a pathological hallmark among a wide range of neurodegenerative disorders. Similarly, lipid-related proteins associated with HSP entail a notable theme within this heterogeneous group of disorders. To further explore lipid imbalance in HSP, chapter five details the development of a method whereby extremely small amounts of specific lipids can be detected from subcellular fractions obtained from 10ml blood samples, and cultured cell samples. This chapter also describes oxysterol profiles in peripheral blood mononuclear cells (PBMCs) and SH-SY5Y cells, and identifies altered lipid profiles due to maspardin loss. Together, the findings and data presented in this thesis significantly contribute to the understanding of the subcellular role and pathomolecular basis of C19orf12 and maspardin, both responsible for complex forms of HSP-spectrum disorders. Additionally, the new methodologies established in this work to profile lipidomic content in subcellular compartments obtained from blood and cultured cell samples will be applied in future studies to potentially develop revolutionary new approaches to testing in HSPs, and confirm the specific lipid profile imbalances highlighted in this thesis as key lipidomic biomarkers of disease.