Cognitive impairment and conversion to dementia in Parkinson's disease: an imaging study of amyloid PET and diffusion MRI

Objective To investigate the association between amyloid deposition or white matter degeneration with cognitive impairment and conversion to dementia in Parkinson’s disease (PD). Background Cognitive decline and dementia are common in Parkinson's disease, however the pathophysiological basis of cognitive impairment in PD is unresolved. The time-course from diagnosis to development of dementia is highly variable, and imaging biomarkers are urgently needed to assist estimation of long-term cognitive outcomes, and enable targeted therapeutic interventions in early disease. Misfolded beta-amyloid protein aggregates, or amyloid plaques, are a significant pathology in Alzheimer's disease, and may play a part in future cognitive decline in PD. Measures of cerebral blood flow and white matter micro- and macro-structural degeneration may correlate more directly with current cognitive impairment, and may interact with amyloid to affect cognitive decline in PD. This thesis comprehensively investigates these measures in PD and aims to differentiate pathology and age-related effects, using both cross-sectional and longitudinal (three-year) study designs. Methods We acquired [18F]-Florbetaben (FBB) amyloid PET, arterial spin labelling perfusion MRI, and structural MRI in 115 patients with Parkinson’s disease, recruited from the Movement Disorders clinic at the New Zealand Brain Research Institute. Movement Disorders Society level II criteria were used to classify PD patients as having normal cognition (PDN, n=23), mild-cognitive impairment (PD-MCI, n=76), or dementia (PDD, n=16), at study baseline and over the course of three-year neuropsychological follow-up. The relationship between amyloid deposition and cognitive classification, global cognitive ability, cerebral blood flow, and conversion to dementia during the three-year follow-up was assessed using both Bayesian regression and whole-brain voxel-wise analysis. High angular resolution diffusion imaging (HARDI) MRI was acquired in a wider cohort of 123 PD participants and 37 controls, for the investigation of white matter integrity in PD across the cognitive spectrum (PDN, n=46; PD-MCI, n=66; PDD, n=11). Cross-sectional fixel-based analysis investigated measures of fibre density, fibre cross-section, and combined fibre-density-cross-section across PD, controls, and PD cognitive subgroups. Clinical measures of global cognitive ability and motor impairment were also assessed for association with these fixel-based metrics. Results We observed significantly higher cortical amyloid accumulation in our PDD group relative to other cognitive subgroups, but model comparison indicated this was due to an effect of age. Cortical amyloid was seen to be present in PD at levels comparable to healthy ageing. Longitudinal assessment identified that, while increased cortical and subcortical amyloid was associated with conversion to dementia within three years, this did not represent a clinically relevant effect. There was no evidence of an interactive effect of amyloid with cerebral blood flow to affect cognition. Reduced fibre density in the substantia nigra correlated with disease, however age exhibited the most widespread association with white matter metrics in this cohort. Conclusions This thesis investigated the effect of amyloid on cognitive impairment within a large, well-characterised, longitudinal PD cohort, and subsequently challenged existing characterisations of regional amyloid deposition relating to cognitive decline. This work also represents the largest application to-date of fixel-based analysis for the investigation of white matter degeneration across the cognitive spectrum in PD

The role of the gut and the gastrointestinal microbiome in Parkinson’s disease

INTRODUCTION: Parkinson’s disease (PD) is a disabling and progressive neurodegenerative disorder that is increasing in prevalence with the aging and urbanisation of the global population. The mechanisms underlying PD pathogenesis and progression are incompletely understood. Improved clinical recognition of early and prodromal non-motor symptoms (NMS), namely gastrointestinal (GI) dysfunction, has focused research over the last two decades on the roles of the gut. More recently, the influences of the microbiota-gut-brain-axis (MGBA) in the development and progression of PD have become an intensive area of research. Studies have demonstrated an association between the GM and a variety of PD-related characteristics, identifying important impacts on levodopa metabolism by certain microbiota. Importantly, the effect of device-assisted therapies (DATs) on the GM and the robustness of microbiota compositional differences between PD patients and household controls (HCs) has not been well defined. The aims of this thesis were to 1) investigate GI dysfunction and nutritional patterns in PD, 2) determine if the GM is a biomarker of PD, and 3) investigate the temporal stability of the GM in PD patients receiving standard therapies and those initiating DATs. METHODS: 103 PD patients and 81 HCs were recruited and participants with PD were considered in two sub-cohorts; 1) PD patients initiating DAT; either Deep Brain Stimulation (DBS) (n=10), or levodopa-carbidopa intestinal gel (LCIG) (n=11), who had GM sampling from stool at -2, 0, 2 and 4 weeks around initiation of DAT and baseline, 6 and 12 months following DAT initiation, 2) 82 PD patients receiving standard PD therapies, who had GM sampling from stool at baseline, 6 and 12 months. Validated PD questionnaire metadata ascertaining motor characteristics and NMS, as well as nutritional data in the form of a Food Frequency Questionnaire, were collected for all participants at baseline, 6 and 12 months. Total DNA was isolated from stool before sequencing the V3-V4 region of 16S rRNA. Relative bacterial abundances, diversity measures, compositional differences and clinical-microbiome associations were determined, as well as developing predictive modelling to identify PD patients and assess disease progression. RESULTS: PD patients reported more prevalent and severe GI dysfunction, especially constipation, which was almost three-times more common compared to HC subjects, (78.6% vs 28.4%, p<0.001). PD patients had a higher intake of total carbohydrates (279 g/day vs 232 g/day; p=0.034), which was largely attributable to an increased daily sugar intake (153 g/day vs 119 g/day; p=0.003), particularly of free sugars (61 g/day vs 41 g/day; p=0.001). Significant GM compositional differences across several taxonomic levels were apparent between PD patients and HCs and associated with a number of PD motor and NMS features, as well as certain therapies. Predictive models to distinguish PD from HCs were developed considering global GM profiles, achieving an area under the curve (AUC) of 0.71, which was improved by addition of data on carbohydrate intake (AUC 0.74). Longitudinal analysis demonstrated persistent underrepresentation of known short-chain fatty acid producing bacteria in PD patients, particularly those concerned with butyrate production; Butyricicoccus, Fusicatenibacter, Lachnospiraceae ND3007 group and Erysipelotrichaceae UCG−003. Taxa differences observed over the short-term (four week) sampling period around DAT (DBS and LCIG) initiation, were not sustained at 6 and 12 months. Despite this, persistent longer-term overrepresentation of Prevotella was observed after DBS initiation, and a trend was found that was suggestive of overrepresentation of Roseburia after LCIG initiation. These results suggest that there may be variable shorter and longer-term DBS and LCIG influences on the GM, which are complex and multifactorial. PD progression analysis did not identify distinct persisting GM compositional differences between faster and slower progressing patients, although predictive modelling was strengthened by the consideration of nutritional data, specifically protein intake, and improved the predictive capacity for PD progression. CONCLUSION: This thesis demonstrates that there are numerous clinically significant associations between the gut, GM and PD. GI dysfunction is common, and carbohydrate nutritional intake appears to be different from the general population in PD. Persistent alterations of GM composition in PD compared to HCs were found. These findings provide support for the existence of disturbances of gut homeostatic pathways, which may disrupt intestinal barrier permeability and lead to gut leakiness, in the pathogenesis of PD. This thesis also highlights the potential to use the GM in the identification of PD and the characterisation of disease progression

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

Glaucoma

This book addresses the basic and clinical science of glaucomas, a group of diseases that affect the optic nerve and visual fields and is usually accompanied by increased intraocular pressure. The book incorporates the latest development as well as future perspectives in glaucoma, since it has expedited publication. It is aimed for specialists in glaucoma, researchers, general ophthalmologists and trainees to increase knowledge and encourage further progress in understanding and managing these complicated diseases