Neuronal pentraxin with chromo domain (NPCD) belongs to a family of neuronally-expressed pentraxin proteins thought to be involved in synaptic refinement and plasticity. One isoform of Npcd, neuronal pentraxin receptor (NPR), is a type-II transmembrane protein responsible for the clustering of related neuronal pentraxins 1 and 2. However, recently identified cytosolic NPCD isoforms with no known function were discovered through their interaction with the intracellular domain of a receptor protein tyrosine phosphatase PTPRO. PTPRO is a signaling molecule known to be involved in the development of the nervous system. Additionally, upregulated expression of neuronal pentraxins has been implicated in neuronal cytotoxicity and associated with neurodegenerative diseases. Here, we demonstrate that a novel cytosolic NPCD isoform interacts with the BTB-Kelch protein Mayven/KLHL2. This interaction was identified through a yeast two-hybrid screen using the C-terminal pentraxin domain region of NPCD and confirmed through mammalian cell colocalization and co-immunoprecipitation studies. Domain truncation analysis suggests that the kelch domains of Mayven/KLHL2 are responsible for this interaction with NPCD. We also show that Mayven/KLHL2 is capable of interacting with Cullin 3, an integral protein in the Cullin-RING ubiquitin ligase complex. An in-vivo ubiquitylation assay demonstrates that overexpression of Mayven/KLHL2 increases NPCD ubiquitylation, and suggests a novel E3 ubiquitin ligase function of Mayven/KLHL2 with NPCD as its substrate. Furthermore, we observed an increased propensity of overexpressed NPCD to form aggresomes with coexpression of Mayven/KLHL2. As the formation of aggresomes is often associated with protein aggregation and deposition diseases, including a multitude of neurodegenerative diseases, we tested NPCD overexpression and the effects of Mayven/KLHL2 coexpression on neuronal cytotoxicity and apoptosis. Overexpressed NPCD in hippocampal neuron cultures resulted in increased cytotoxicity and apoptosis, further exacerbated by Mayven/KLHL2 coexpression. Our findings report an interaction between NPCD and Mayven/KLHL2, demonstrate a novel role of Mayven/KLHL2 as an E3 ubiquitin ligase, and explore a possible intersection between the ubiquitin-proteasome degradation pathway, neuronal pentraxins, and neurodegenerative disease.

The aggregation of alpha-synuclein (αS) protein from soluble monomer into solid amyloid fibrils in the brain is associated with a range of devastating neurodegenerative disorders such as Parkinson’s disease. Soluble oligomers formed during the aggregation process are highly neurotoxic and are thought to play a key role in the onset and spreading of disease. Despite their importance, these species are difficult to study by conventional experimental approaches owing to their transient nature, heterogeneity, low abundance and a remarkable sensitivity of the oligomerisation process to the chosen experimental conditions. In this thesis, well-established single-molecule techniques have been utilised to study the aggregation and oligomerisation of αS in solution.