Defining the genetic and molecular pathways underlying motor neuron degenerative diseases

Motor neuron degenerative diseases (MNDs) are a large group of genetically and clinically heterogeneous life-limiting disorders. MNDs are characterised by the progressive degeneration of upper and/or lower motor neurons. Unfortunately, there are currently no universal biomarker assays or disease-modifying therapies available for the majority of affected individuals, primarily due to a lack of understanding of the pathomolecular causes of disease. The identification of disease-associated genes provides a direct link between specific molecular pathways and disease pathogenesis. The work described in this thesis entails clinical, genomic and cellular studies which advance scientific knowledge of the genetic and molecular processes important for motor neuron health and survival. Chapter three describes the discovery of biallelic variants in the phospholipid synthesis (Kennedy) pathway gene, CEPT1, as causative of a complex neurological disorder associated with motor and speech delay and spasticity/ataxia. Molecular studies confirmed that disease-associated CEPT1 variants dramatically impair enzymatic function. Additionally, studies using fibroblasts cells derived from affected individuals and a knockout CEPT1 neuroblastoma cell model, showed that loss of CEPT1 altered lipidomic profiles alongside disrupted endoplasmic reticulum (ER) and mitochondrial morphology and function. Chapter four outlines recent findings of two novel candidate disease genes, PLPP1 and C6orf120, as a cause of neurological disorders with motor neuron involvement. While PLPP1 encodes a well-studied enzyme functioning upstream of the Kennedy pathway, little is known about the function of C6orf120. This chapter presents preliminary molecular and cellular studies which identify ERLIN1 as a likely binding partner of C6orf120. ERLIN1 is involved in both cholesterol homeostasis and regulation of IP3R degradation which functions as an ER calcium channel and mitochondria-ER tether. Once confirmed the identification of these two disease genes will add to the growing literature implicating lipidomic/calcium processing pathways and their subcellular hub, mitochondria-ER contact sites, in MND pathology. To explore the relevance and potential of a novel cholesterol metabolite (oxysterol) biomarker blood-based assay for MNDs, chapter five details the development of methods to obtain subcellular (mitochondria and ER) fractions from blood samples and cultured cells for oxysterol quantification. These methods were used to evaluate the oxysterol profiles in peripheral blood mononuclear cells (PBMC) and SH-SY5Y neuroblastoma cells. This work showed that cholesterol metabolites can be identified and quantified in small PBMC subcellular fractions and identified significant changes to oxysterol species due to knockout of an MND-associated molecule (CYP7B1) in cell models. While further work is required this confirms the feasibility and potential for oxysterol analysis in blood samples for a MND diagnostic assay. Together the work defined in this PhD thesis provides important new insights into the pathomolecular molecules and processes crucial for motor neuron health and expands the spectrum of disorders associated with lipidomic dysregulation. Moreover, development of methodologies to quantify oxysterols in small mitochondria-ER subcellular fractions of blood and cell samples lays the foundation for the potential development of a future blood biomarker assay for MNDs, providing new avenues for future research and diagnostic/therapeutic development.<p></p>

Author dc:creator

• Allison Newman (21041420)

• Uncategorised value

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• All rights reserved
• Open Access after 2029-04-01

Identifier

10779/exe.31914612.v1

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oai:figshare.com:article/31914612