Investigating the molecular mechanisms of Hereditary Spastic Paraplegia neuropathies

Hereditary spastic paraplegia (HSP) was first described in the late 1800s and has since become a term used to describe this relatively large, clinically and genetically diverse group of inherited neurodegenerative or neurodevelopmental disorders. HSPs are characterised by progressive lower limb spasticity and pyramidal weakness, caused by genetic mutations. This defining clinical feature is thought to be due to the progressive, length-dependent neuronal degeneration or axonopathy, which predominantly involves the lateral corticospinal tracts. Although unified by their defining clinical feature, HSPs are some of the most genetically diverse diseases. To date, 78 spastic gait disease-loci (SPG1-78) and 61 corresponding spastic paraplegia genes have been identified, and these are likely to increase further still. A critical step towards unravelling the complex molecular relationships in living systems is the mapping of protein-protein interactions. Proteins with similar functions and cellular localisations tend to cluster together within these networks, with the majority sharing at least one function. Proteins that interact with known disease-causing HSP proteins may in some cases also acquire mutations that contribute to specific HSP-related phenotypes. Therefore, identification of proteins that interact with known HSP proteins, or exist in common molecular complexes, may provide new insight into the molecular mechanisms fundamental to the pathogenesis of this group of disorders. The sequence-verified HSP ORFs generated were used to construct a collection of Y2H HSP bait and prey clones. The ‘traditional’ yeast two-hybrid system was then used to test a number of predicted binary interactions, using high-throughput targeted assays, whilst novel HSP interaction partners were identified using high-throughput library screens. Membrane-associated proteins are one of the most biologically important protein classes, with key roles in various cellular processes including cell signalling, molecular transport, metabolism and cell structure maintenance. However, detection of protein-protein interactions (PPIs) of membrane proteins is challenging, due to their hydrophobic nature and non-nuclear localisation, making them difficult to analyse using conventional interaction detection methods. As there is a significant number of membrane-associated HSP proteins, sequence-verified HSP ORFs were used to generate a collection of membrane yeast two-hybrid (MYTH) HSP bait clones. The MYTH system was used to investigate potentially novel interaction profiles for each of the membrane-associated HSP bait constructs, using the optimised high-throughput MYTH library screen approach. In total, 365 novel binary HSP interactions were identified in this study, increasing the complexity of the HSP interactome. This high-density, comprehensive HSP interactome can be used to inform future hypothesis-driven research, looking at the physiological mechanisms and functional relevance of these interactions, providing a greater understanding of the pathogenic mechanisms of HSP, as well as for the development of new strategies for therapeutic intervention