Bampton, Alexander; (2022) HnRNP K mislocalisation and dysfunction in neurodegenerative disease and ageing. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse, multi-functional family of RNA-binding proteins. Many such proteins, including TDP-43 and FUS, have been strongly implicated in the pathogenesis of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). By contrast hnRNP K, the focus of this thesis, has been underexplored in the context of neurodegenerative disease. The first work to be described here involves a comprehensive pathological assessment of hnRNP K protein’s neuronal localisation profile in FTLD, ALS and control brain tissue. Following pathological examination, hnRNP K mislocalisation from the nucleus to the cytoplasm within pyramidal neurons of the cortex was identified as a novel neuropathological feature that is associated with both neurodegenerative disease and ageing. Double immunofluorescence was used to confirm these neurons were anatomically distinct from those harbouring the classical TDP-43 or Tau proteinaceous inclusions used in the pathological diagnosis of FTLD. Nuclear loss and mislocalisation of hnRNP K to the cytoplasm was then identified to also occur in two further neuronal cell types within the dentate nucleus of the cerebellum and the CA4 region of the hippocampus. As with pyramidal neurons, similar associations were identified between disease, age and hnRNP K mislocalisation in neurons of the dentate nucleus. Hence, neuronal mislocalisation of hnRNP K across the brain has potentially broad relevance to dementia and the ageing process. Almost all hnRNPs have been found to perform essential homeostatic functions in regulating appropriate target gene splicing activity. Recently, several hnRNPs have been found to have important roles in repressing the inclusion of non-conserved, so-called ‘cryptic exons’ within mature mRNA transcripts. Inclusion of cryptic exons following TDP-43 nuclear depletion and subsequent reductions in the functional levels of target transcripts and proteins is an emerging pathogenic theme of several neurodegenerative diseases including FTLD and ALS. To recapitulate the functional implications of the hnRNP K nuclear depletion that is observed in brain tissue, a hnRNP K knockdown neuronal model was developed utilising an iPSC-derived CRISPR-interference based platform. RNA-seq analysis revealed that nuclear hnRNP K protein depletion within cortical neurons is associated with the robust activation of several cryptic exon events in mRNA targets of hnRNP K as well as the upregulation of other abnormal splicing events termed ‘skiptic exons’. Several of these novel splicing events were validated molecularly using three-primer PCRs. Finally, an in situ hybridisation (ISH) based technology (BaseScope™) platform was optimised to visualise novel cryptic events in post-mortem brain tissue. The platform was used to detect a recently discovered cryptic exon within synaptic gene UNC13A and another in the insulin receptor (INSR) gene, two newly described targets of TDP-43. These events were found specifically in FTLD-TDP or ALS brains, validating it as a specific marker of TDP-43-proteinopathy. A methodological pipeline was also developed to delineate the spatial relationship between cryptic exons and associated TDP-43 pathology. Hence, providing a platform for the future detection, validation and analyses of novel cryptic exons associated with hnRNP K protein depletion in pyramidal neurons.

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