Prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of misfolded prion protein (PrPSc) in the brain. They are caused by the conformational rearrangement of the normal cellular protein, PrPC, to the abnormal isoform PrPSc. The critical relationship between aberrant protein misfolding and neurotoxicity currently remains unclear. The aim of this Thesis was to investigate the role of the ubiquitin-proteasome system (UPS) in prion disease pathogenesis. The UPS is a tightly regulated system for the identification and subsequent degradation of misfolded or redundant proteins by the 26S proteasome. Previous in vitro studies have identified a direct inhibitory interaction between misfolded PrP isoforms and the proteasome. To evaluate whether UPS dysfunction plays a significant role in vivo, a detailed time-course study was carried out in prion-infected UbG76V-GFP proteasome reporter mice. This work revealed a spatiotemporal correlation between the accumulation of PrPSc and the onset of UPS dysfunction in neurons and astrocytes. UPS impairment was shown to occur prior to the onset of behavioural dysfunction and neuronal loss, underlining that upregulation of the UPS may be an important therapeutic approach for the treatment of prion diseases. To test this theory, a novel recombinant adeno-associated viral vector was developed to attempt upregulation of the UPS in vivo by overexpression of the 19S regulatory particle subunit PSMD11. In parallel with in vivo studies, work in this Thesis aimed to develop a neural stem cell line expressing an epitope-tagged PrPC chimera (PrP-224AlaMYC) which can be differentiated into a mixed population of neurons and astrocytes. Since previous cell models of prion infection have utilised immortalised neuroblastoma cells which do not exhibit prion-induced cell death, the development of this post-mitotic cell system could provide important insights into how PrPSc gains access to the cytosolic compartment to interact with the UPS.

Prions accumulate in the lymphoreticular system (LRS) at early stages of prion disease, long before they are detected in the brain. A considerable body of evidence showed that both haematopoietic and stromal cells play a role in prion pathogenesis. However, the contribution of different cell types to the accumulation and the spread of prions in the LRS are not well understood. Taking advantage of a quantitative in-vitro infectivity assay, the Scrapie Cell Assay (SCA) and high density magnetic-activated cell sorting (MACS), we studied the kinetics of prion accumulation in various splenic cell types at early stages of prion disease. The determination of statistically robust infectious titres was achieved by statistical modelling using generalised linear model (GLM) regression. With this novel procedure time-dependent changes of prion titres were monitored in seven distinct splenic cells and identified two cell types that have previously not been associated to prion pathogenesis, plasmacytoid dendritic (pDC) and natural killer (NK) cells. Notably, in Prnp-/- mice, e.g. in absence of prion replication, infectivity was detected in macrophages and dendritic cells (DC) after 3 dpi, but not in lymphocytes, underscoring the importance of prion sequestration by antigen-presenting cells which are among the first cells of the immune system to encounter pathogens. Reports of the secretion of endosome-derived membrane particles, so called exosomes by immune-competent cells and prion-infected cell lines raised the question whether prion secretion could be a potential route for the spread of prions. We here present the first evidence that MACS-isolated lymphocytes and DCs from scrapie-infected mice secrete prions which are associated to the release of exosome-like membrane particles into the cell culture supernatant ex vivo.

Despite intense scientific scrutiny since the outbreak of variant Creutzfeldt-Jakob disease (vCJD) in the mid-1990s, no effective treatment is available for human prion disease. The passive transfer of anti-PrP monoclonal antibodies (mAbs) in prion-infected mice has been demonstrated to extend the murine lifespan significantly. However, prior to considering clinical trials in humans, further understanding the mechanism of action, the optimal dose, and side-effect profile is essential. As vCJD prion infection begins peripherally before CNS invasion, we developed a comparable therapeutic model when peripheral infection is well established before experimental treatments are tested. The in vivo half-life of the therapeutic mAbs used, ICSM18 and ICSM35, was defined and a suitable biweekly treatment was established. To determine the optimal therapeutic dose with least toxicity, the anti-PrP mAbs (or isotype controls) were then titrated in infected FVB/N mice, and the highest dose was also tested in BALB/c and Prnp0/0 mice for the detection of side effects. While phosphate-buffered saline (PBS) control treated RML prion-infected FVB/N mice all succumbed to prion disease at a mean of 194 (+/- 4 SEM) days, >50% of ICSM18 (1-3mg/week) and ICSM35 (4mg/week) treated mice survived >350 days without scrapie signs (p≤0.035 Log-Rank test vs. PBS). Lower doses of either mAb resulted in only a modest increase in survival. Consistent with this outcome, ICSM18 provided maximal early splenic PrPSc clearance at doses of 4mg and 1mg/week whereas ICSM35 was only this effective at 4mg/week. Unexpectedly, all FVB/N mice treated with ICSM18 at 4mg/week developed a fatal ataxic illness at PID134-163. Pathologically, little or no PrPSc was detected in the spleens or brains of these mice, but they all had inflammatory demyelination of the fornix and brainstem with activation of microglial cells. FVB/N mice treated with ICSM35 at 4mg/week, which survived for >450 days were found to have mild focal CNS inflammation without overt demyelination suggesting the effect is related to the specific mAb and dose administered. This side effect is also PrPC and mouse strain dependent, as Prnp0/0 mice and BALB/c mice were unaffected. We have therefore established a safe and effective therapeutic window for using anti-PrP mAb treatment in mice with established peripheral prion replication, and have demonstrated a significant decrease in mortality without side effects in the surviving mice. Future human anti-PrP mAbs trials can be guided by translating these studies to provide a dosing regimen to which provides benefit without deleterious effects. This thesis will discuss the pharmacokinetic properties of the anti-PrP mAbs, the location of the therapeutic action, the competence of the immune system in mAb treated mice, and the relevance of these factors to both anti-PrP effect and CNS inflammation.

Prion diseases are fatal neurodegenerative disorders associated with conformational conversion of normal cellular prion protein (PrPC) to an abnormal disease-associated conformer (PrPSc). The aim of this thesis was to investigate the earliest event in prion infection using a novel cell system. Specifically, it aimed to assess the timescale that PrPC is converted to PrPSc following exposure to RML prions and identify the initial cellular site of PrPSc formation and propagation. The cell biology of the initial events of cellular prion infection are poorly understood since newly formed cellular PrPSc is immunologically indistinguishable from infectious prions in the inocula. As a solution to this problem, an epitope-tag was inserted into the sequence of endogenous PrPC to delineate the formation of de novo PrPSc. A PrP-knock down neuroblastoma cell line was reconstituted with mouse 3F4-, FLAG- and MYC-tagged PrPC. Following identification of cells expressing physiological levels of tagged PrPC, prion-susceptibility was determined by exposure to disease-associated prions. Cells expressing 3F4-tagged PrP, the MYC sequence at position 224 and the FLAG sequence at position 22 or 30 contained PrP resistant to formic acid and proteinase K digestion. A mouse bioassay demonstrated that the PrP-224AlaMYC cell line produce bona-fide infectious epitope-tagged PrPSc on exposure to RML prions. Investigation of de novo tagged PrPSc propagation in this novel cell system demonstrated that cellular prion infection is a dynamic process occurring within one minute of prion exposure and that the plasma membrane is the primary site of prion conversion. It was demonstrated that the late endosomes, lysosomes and endosomal recycling compartments do not appear to be key sites of PrP conversion and prion propagation, whilst the plasma membrane and early endocytic compartments are involved in this key process. The work in this thesis provides new insights into the cell biology of the initial stages of prion conversion and propagation and has implications for neurodegenerative diseases where prion-like mechanisms have been proposed.

A growing body of functional imaging studies provides considerable insight into cortical networks for non-verbal auditory processing. However, determination of the essential cognitive and anatomical components of these networks depends upon the study of damaged brains, and yet, auditory neuropsychology is little studied and poorly understood. Whilst naturally occurring lesions that selectively disrupt auditory processes are rare, increasing evidence suggests that degenerative diseases target functional networks implicated in non-verbal auditory processing. Furthermore, a small but significant auditory neuropsychological literature shows that dementia can lead to impairments of non-verbal sound processing. This thesis comprises a series of studies designed to reveal deficits of non-verbal auditory processing in four distinct dementia syndromes: three variants of primary progressive aphasia (semantic dementia, SD; progressive non-fluent aphasia, PNFA; logopenic aphasia, LPA), and typical Alzheimer’s disease (AD). The first two studies (Chapters 2 and 3) involve the development of two novel non-verbal auditory neuropsychological batteries, including tests to examine perceptual property, apperceptive, and semantic stages of processing; the subsequent use of these batteries reveals syndrome-specific profiles of non-verbal auditory impairment. Next, a detailed psychoacoustic assessment of two single cases (Chapter 4) provides evidence for specific disorders of auditory property and object processing. A further study (Chapter 5) comprises the examination of non-verbal auditory object processing in SD using functional magnetic resonance imaging (fMRI); results suggest that auditory object recognition depends upon a distributed temporo-parietal network involving closely associated mechanisms of perceptual and semantic processing. Finally, novel neuropsychological assessments are used to reveal the selective impairment of auditory scene analysis in AD (Chapter 6). Together, these neuropsychological findings provide novel insights into the organisation of cortical networks for non-verbal auditory cognition.

Mutations in the charged multivesicular body protein 2B gene (CHMP2B) cause frontotemporal dementia termed frontotemporal dementia linked to chromosome 3 (FTD-3) in a large Danish pedigree and also in an unrelated Belgian familial FTD patient. Genetic analyses on the Danish pedigree and the role of CHMP2B mutations in frontotemporal dementia are reported. The clinicopathological spectrum of FTD-3 and other FTD subtypes is also described. CHMP2B is a component of the endosomal sorting complex required for transport (ESCRT-III), which is required for formation and function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. Cellular models of CHMP2B mutations showed an enlarged late endosomal phenotype and an abnormal pattern of ubiquitination. Functional studies demonstrated a specific disruption of endosome-lysosome fusion but not sorting of endocytosed receptors by the MVB. Investigations into the mechanism of impaired fusion suggested impaired recruitment of the GTPase Rab7, known to be necessary for vesicular fusion, onto endosomes in CHMP2B mutant cells. Studies of patient tissue revealed a novel endosomal pathology in CHMP2B mutation-positive patient brains and also abnormal endosomes in patient fibroblasts. These data indicate that defects in endosomal fusion events can lead to neurodegeneration and suggest a potential pathogenic mechanism for CHMP2B mutations.

The central feature of prion disease is the conversion of normal host‐encoded cellular prion protein to an abnormal isoform; designated PrPSc. Current research has shown that PrPSc is the principal, and possibly the sole, component of infectious prions. Despite a wealth of experimental data that supports this protein‐only hypothesis, the goal of systematically producing prions in vitro still remains. The failure to generate high‐titre synthetic mammalian prions from fully defined starting materials has underlined the need to establish the composition of ex vivo purified mammalian prions that retain high titre infectivity. Such characterisation of infectious prions necessitates their purification from brain and this requires an approximate 14,000‐fold enrichment (based upon protein content). The main aim of this thesis therefore, has been to address the problems of purification and to isolate infectious prions. An array of novel enzymatic and biochemical techniques have been used including precipitation with sodium phosphotungstic acid, systematic washing steps with ionic salts, digestion with pronase E, proteinase K, amyloglucosidase and sphingomyelinase. Rapid and accurate measurement of prion infectivity throughout purification has been made possible by the use of the scrapie cell assay. Pronase E has been demonstrated to selectively digest PrPC while preserving both proteinase K‐sensitive and resistant infectious prions. This discovery now facilitates the factionation, isolation and detailed charaterisation of prions with distinct physicochemical properties without being confounded by contaminating PrPC. Alongside purification, high resolution imaging of the infectious prion has been a key unaccomplished challenge in the prion field. The problems of imaging insoluble aggregated proteinaceous material and correlating observed structures with specific infectivity have so far frustrated meaningful studies. Work in this thesis has focused upon solubilising and disaggregating large amorphous protein aggregates obtained after purification for imaging by electron microscopy, and has defined key problems that remain to be addressed. Collectively this thesis describes novel methods for investigating mammalian prions that will improve our understanding and the diagnosis of prion disease.

The aim of this work was to investigate putative biomarkers of neuronal plasticity and repair in the central nervous system. The effects of different disease processes, such as inflammation, demyelination and neurodegeneration were explored. We developed and validated ELISA-based assays for the quantification of neural cell adhesion molecule (NCAM) and growth-associated protein (GAP)-43, two known facilitators of neuronal outgrowth in the nervous system. NCAM isoforms in biological samples were characterised using mass spectrometry. Soluble NCAM was measured in in-vitro and in-vivo models of inflammation/demyelination and neurodegeneration, and across different neurological disorders in the CSF to understand the impact of inflammation and axonal loss on its levels. Recombinant GAP-43 was produced using baculovirus technology and purified in appreciable amounts for use in a new ELISA. Soluble GAP-43 levels were quantified across different neurological disorders in the CSF. Values for CSF NCAM and GAP-43 were correlated with clinical outcome measures. CSF NCAM demonstrated a restricted pattern of expression compared to that of serum whilst GAP-43 is almost exclusively expressed in the CSF, indicating that these biomarkers are intrathecally synthesised. CSF NCAM and GAP-43 levels were lower in neurological disorders with prominent axonal injury; multiple sclerosis, movement disorders, motor neurone disease, Alzheimer’s disease and meningitis. In vitro neuronal cell culture model and in vivo experimental autoimmune encephalomyelitis studies demonstrate that CSF NCAM correlates well with disease progression in multiple sclerosis. A similar relationship was not found with CSF GAP-43. In conclusion, the adult CNS may possess the intrinsic capacity to repair, but this capacity may be dramatically reduced in disease states. Measuring this process may be important in understanding neuronal repair and plasticity.

There is a considerable need for treatments in MS for preventing progressive neurological disability. Assessment of the afferent visual pathway shows potential in investigating new therapies in MS. Mesenchymal Stem Cells exhibit properties of potential therapeutic relevance in progressive MS. A phase I/IIA trial of adult autologous mesenchymal stem cells as a potential therapy for Multiple Sclerosis [MSCIMS] was designed as an open label, pre (up to 20 months) vs. post treatment (up to 10 months) (single intravenous administration of autologous bone marrow derived mesenchymal stem cells) comparison study in ten secondary progressive MS patients. Primary end points were adverse events and secondary end points were efficacy measures. All 10 patients had previous history of clinical optic neuritis: this was in order to enable longitudinal structural and functional assessments of the disease-affected afferent visual pathway. Piecewise linear mixed models were used to assess the change in gradients over time at the point of intervention. All 10 patients tolerated the trial assessments and intervention. No significant or serious adverse events were seen. Improvement after treatment was seen in visual acuity and visual evoked response latency, along with an increase in optic nerve cross-sectional area. The results suggest that autologous mesenchymal stem cells are safe and could possibly promote endogenous repair mechanisms such as remyelination, although a definitive conclusion of this cannot be made from this small study. While MSCIMS was a proof of concept study only, based on the encouraging experience derived from it, there would seem to be potential value in future, larger placebo controlled, double-blinded, randomised therapeutic phase IIb/III trials that could (i) more definitively investigate stem cells as a therapy and (ii) use the visual pathway disease model for investigating the efficacy of potential neuroprotective and reparative therapeutic agents.

Multiple sclerosis (MS) is a disabling neurological disorder that affects young people. Neuroaxonal loss is believed to be the pathological correlate of the secondary progressive phase of this disease and whilst the earlier, relapsing disease is responsive to immunomodulatory treatments, progressive disease has proved resistant. Clinical trials in progressive multiple sclerosis are problematic due the variable nature of disability progression and the insensitivity of currently available disability scales. Volumetric magnetic resonance imaging (MRI) measures of brain and spinal cord atrophy are pathologically non-specific but are likely to reflect destructive processes such as neuroaxonal loss and could be used to assess treatment efficacy in clinical trials of neuroprotective agents in MS. Presented, is work assessing MRI-based atrophy measurements of various regions within the central neuroaxis in secondary progressive MS, in particular assessing their attributes for use as surrogate markers. The clinical and MRI data were acquired as part of a phase II trial of a putative neuroprotective agent, lamotrigine, in subjects with secondary progressive MS, and the treatment effect of this agent on the volumetric MRI measures is also presented. Overall measures of whole brain, grey matter and spinal cord atrophy demonstrated robust correlations with disability, both in cross-sectional and longitudinal analyses. Grey matter and spinal cord volumes were subject to high rates of change. In contrast white matter atrophy was variable and on average did not change significantly over the 2 year follow-up. Of interest was that grey matter volume was highly correlated with T1 hypointense lesion load in the white matter suggesting a possible causative link. No significant beneficial treatment effect of lamotrigine was demonstrated using any of the MRI measures. However, an early “pseudoatrophy” was observed within the white matter compartment in the treatment group suggesting an anti-inflammatory or osmotic effect.