HnRNP K mislocalisation and dysfunction in neurodegenerative disease and ageing

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

HnRNP K mislocalisation in neurons of the dentate nucleus is a novel neuropathological feature of neurodegenerative disease and ageing

Nuclear depletion and cytoplasmic mislocalisation of the RNA-binding protein heterogeneous ribonucleoprotein K (hnRNP K) within pyramidal neurons of the frontal cortex have been shown to be a common neuropathological feature in frontotemporal lobar degeneration (FTLD) and elderly control brain. Here, we describe a second neuronal subtype vulnerable to mislocalisation within the dentate nucleus of the cerebellum. In contrast to neurons within the cerebellar cortex that typically exhibited normal, nuclear staining, many neurons of the dentate nucleus exhibited striking mislocalisation of hnRNP K to the cytoplasm within neurodegenerative disease brain. Mislocalisation frequency in this region was found to be significantly higher in both FTLD-TDP A and Alzheimer's disease (AD) brain than in age-matched controls. However, within control (but not disease) subjects, mislocalisation frequency was significantly associated with age-at-death with more elderly controls typically exhibiting greater levels of the pathology. This study provides further evidence for hnRNP K mislocalisation being a more anatomically diverse pathology than previously thought and suggests that potential dysfunction of the protein may be more broadly relevant to the fields of neurodegeneration and ageing

Outcomes and patients’ perspectives of transition from paediatric to adult care in inflammatory bowel disease

This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/ licenses/by-nc/4.0/AIM: To describe the disease and psychosocial outcomes of an inflammatory bowel disease (IBD) transition cohort and their perspectives. METHODS: Patients with IBD, aged > 18 years, who had moved from paediatric to adult care within 10 years were identified through IBD databases at three tertiary hospitals. Participants were surveyed regarding demographic and disease specific data and their perspectives on the transition process. Survey response data were compared to contemporaneously recorded information in paediatric service case notes. Data were compared to a similar age cohort who had never received paediatric IBD care and therefore who had not undergone a transition process. RESULTS: There were 81 returned surveys from 46 transition and 35 non-transition patients. No statistically significant differences were found in disease burden, disease outcomes or adult roles and responsibilities between cohorts. Despite a high prevalence of mood disturbance (35%), there was a very low usage (5%) of psychological services in both cohorts. In the transition cohort, knowledge of their transition plan was reported by only 25/46 patients and the majority (54%) felt they were not strongly prepared. A high rate (78%) of discussion about work/study plans was recorded prior to transition, but a near complete absence of discussion regarding sex (8%), and other adult issues was recorded. Both cohorts agreed that their preferred method of future transition practices (of the options offered) was a shared clinic appointment with all key stakeholders. CONCLUSION: Transition did not appear to adversely affect disease or psychosocial outcomes. Current transition care processes could be optimised, with better psychosocial preparation and agreed transition plans

TDP-43 loss and ALS-risk SNPs drive mis-splicing and depletion of UNC13A

Variants of UNC13A, a critical gene for synapse function, increase the risk of amyotrophic lateral sclerosis and frontotemporal dementia1-3, two related neurodegenerative diseases defined by mislocalization of the RNA-binding protein TDP-434,5. Here we show that TDP-43 depletion induces robust inclusion of a cryptic exon in UNC13A, resulting in nonsense-mediated decay and loss of UNC13A protein. Two common intronic UNC13A polymorphisms strongly associated with amyotrophic lateral sclerosis and frontotemporal dementia risk overlap with TDP-43 binding sites. These polymorphisms potentiate cryptic exon inclusion, both in cultured cells and in brains and spinal cords from patients with these conditions. Our findings, which demonstrate a genetic link between loss of nuclear TDP-43 function and disease, reveal the mechanism by which UNC13A variants exacerbate the effects of decreased TDP-43 function. They further provide a promising therapeutic target for TDP-43 proteinopathies

Markers of cognitive resilience and a framework for investigating clinical heterogeneity in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. Despite the unifying pathological hallmark of TDP-43 proteinopathy, ALS is clinically a highly heterogeneous disease, and little is known about the underlying mechanisms driving this phenotypic diversity. In a recent issue of The Journal of Pathology, Banerjee, Elliott et al use region-specific transcriptomic profiling in postmortem brains from a deeply phenotyped clinical cohort of ALS patients to detect molecular signatures differentiating cognitively affected and unaffected patients. They identified differential expression of specific genes, including upregulation of pro-inflammatory IL-6 in the cognitively affected group and anti-inflammatory IL-1 in the cognitively unaffected group. They then utilised BaseScope™ in situ hybridisation and immunohistochemistry to validate upregulation of NLRP3, an activator of the inflammasome, in the cognitively affected group, and upregulation of SIRT2, an inhibitor of NLRP3, in the cognitively unaffected group. In summary, Banerjee, Elliott et al demonstrate the usefulness of combining a well-curated clinical cohort with transcriptomic analysis of pathological samples to identify a perturbed pathway (e.g., the inflammasome), offering opportunities for novel therapeutic targets in ALS. © 2022 The Pathological Society of Great Britain and Ireland

Many participants in fecal occult blood test population screening have a higher-than-average risk for colorectal cancer

Objectives Population-based colorectal cancer screening by fecal occult blood testing reduces cancer-specific mortality. Current guidelines recommend this strategy for average risk individuals. This study investigated the prevalence of higher-than-average risk characteristics, and rate of prior colonoscopy, in participants in fecal occult blood test screening programs. Methods Randomly selected individuals aged 50–74 years in urban Adelaide were offered free fecal occult blood test screening by mail, without prior knowledge of their medical status. Each invitation included a questionnaire to record the prevalence of higher-than-average risk characteristics related to symptoms, family history or comorbidity, as well as prior colonoscopy. The definition of average risk was taken from updated guidelines published by the US Multisociety Task Force on Colorectal Cancer. Results Of 2538 responses analyzed, 425 individuals had had a colonoscopy within the last 5 years, 106 fulfilled family history criteria for an initial screening colonoscopy, 209 had past polyps and 26 had had colorectal cancer. Eighty-three reported recent rectal bleeding. By current guidelines, 23% of the screened population did not warrant fecal occult blood test, because either prior colonoscopy rendered it unnecessary or particular patient characteristics made colonoscopy a more appropriate initial investigation. Conclusions Fecal occult blood test screening programs capture a sizeable number of higher-than-average risk individuals that may warrant colonoscopic rather than fecal occult blood test screening. Other participants have had a recent colonoscopy and probably warrant a delay in screening. Mass population fecal occult blood test-based screening programs need to more effectively target those at average risk and should divert those of higher or lower risk to more individualized assessment