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No description available.

Synucleinopathies are a group of diseases characterised by the presence of insoluble aggregated forms of α-synuclein. The most common of these diseases is Parkinson’s disease (PD) which affects approximately 1% of the UK population over the age of 60. Alpha-synuclein has also been linked to the disease through familial mutations and genome wide association studies as well as by its presence in sporadic cases. Although solid evidence exists for a role of α-synuclein in PD, it remains unclear as to how this protein exerts its toxicity on neurons and exactly how this leads to the cell death characteristic of this neurodegenerative disease. Alpha-synuclein belongs to a family of three proteins which also includes β- and γ-synuclein. These three proteins are highly homologous and evolutionarily conserved, however none of them have a well defined function. Evidence suggests a role for these proteins in synaptic vesicle dynamics but a more specific function remains to be unveiled. However, due to the considerable degree of homology across these three proteins, knockout models have been considered to allow functional compensation of the missing synuclein protein through one of the remaining family members. This has hindered studies from elucidating not only the role of α-synuclein but also β- and γ-synuclein. To overcome this problem triple synuclein knockout mice have been produced and characterised, as described in this thesis. As expected studies of these animals revealed no alterations in the number of dopaminergic neurons in either the substantia nigra pars compacta or ventral tegmental area. Despite this, a significant deficit in striatal dopamine concentrations was detected, regardless of the fact that the levels and function of tyrosine hydroxylase being normal. As well as this triple synuclein null mice were demonstrated to be hyperdopaminergic through various behavioural tests. Work employing physcostimulants and, through a collaboration, using fast scan cyclic voltametry suggested a role for these proteins in normal dopamine release dynamics at the level of the synaptic vesicle. A previous body of work has indicated that the loss of α- and/or γ-synuclein is able to provide a degree of resistance against the toxic affects of the dopaminergic neurotoxin MPTP. It was therefore hypothesised that the triple synuclein null animals would also display resistance to this toxin. However, these animals were shown to be more sensitive than wild type controls. Importantly it was apparent that animals lacking β-synuclein alone or in combination with other synucleins were the most sensitive to this toxin. Further work revealed a significant deficit in the ability of triple synuclein null mice to store dopamine in their synaptic vesicles. This may explain the sensitivity to MPP+, the active metabolite of MPTP, due to the fact it cannot be efficiently stored in synaptic vesicles, which restricts the toxins access to the mitochondria where it normally inhibits complex I, thus leading to cell death. When recombinant β-synuclein was reintroduced the deficit in synaptic vesicle dopamine uptake could be restored. However, β-synuclein can not do this alone and requires incubation with cytosolic factors, suggesting it acts as a chaperone in this role. This may explain why lines of synuclein null mice that specifically have the absence of β-synuclein apparently fair least well when exposed to MPTP. Finally, in order to assess the extent to which a loss of function role of α-synuclein leads to pathological alteration at the synapse an entirely novel conditional α-synuclein knockout mouse model was produced. Currently no ideal model exists to answer this question as conventional knockout models are based on the knockout of the protein in development. This may allow functional compensatory mechanisms to be established which can be overcome with a conditional knockout approach. As well as this it is important to assess this loss in an aged nervous system, as PD is a disease of aging. It is likely that, as α-synuclein forms insoluble Lewy bodies and undergoes abnormal posttranslational modifications, the amount of normally functioning protein at the synapse is depleted, therefore allowing a loss of function effect to develop. It is hoped this model will allow new insight into the early disease process. Overall this work further contributes to a body of evidence that suggests the synucleins play an important role in synaptic dopamine handling, particularly at the synaptic vesicle level. It is hoped that the newly established conditional α-synuclein knockout model will produce a new perspective on the loss of function role of α-synuclein in early disease development, an avenue that has yet to be fully explored.

Throughout this body of work low amplitude high frequency (500 Hz – 5000 Hz) mechanical stimulation and its effect to induce osteogenesis on bone marrow derived MSCs has been investigated. Due to the nanolevel amplitudes of these high frequency vertical vibrations the term nanokicking appeared to be appropriate and was subsequently used throughout this thesis to refer to these high frequency sinusoidal stimulations provided by the bioreactor. In the first instance this work was performed in 2D and biological analyses to determine osteogenesis were carried out at a transcript (mRNA), protein and mineralisation level. Affirmative results for osteogensis were observed from genes and proteins (RUNX2, osteocalin, osteopontin) related to the osteoblast phenotype by qRT PCR, in cell western, and immunostaining. To determine the prescence of inorganic osseous minerals, more specific techniques such as Raman spectroscopy, micro computed tomography and histological stainings (Von Kossa/Alizarin Red) were further employed. The results observed remained in line with previously published material (Gentleman et al., 2009) drawing the conclusion that calcium phosphate (Ca10(PO4)6, through nanokicking,was formed in vitro. The natural progession of this research meant that a novel vibrational bioreactor was conceived and designed, through the use of Lean and Six Sigma principles (Andrew Thomas, 2004; Caldwell, 2006), in order to assess the potential of nanokicking in 3D. Here collagen was employed as a biomimetic scaffold and affirmative results for osteogenesis were observed. The bioreactor was unique in that long term (up to 46 days) sterile culture was achieved, it was easy to use and there was no requirement for osteogenic media, growth factors or complex chemistries (e.g. dexamethasone, rhBMP2) in order to induce osteogenesis. The cost of use and maintence was relatively cheap compared to available commercial bioreactors (Rauh et al., 2011b). It is envisaged that this technology may one day have real world use for ossesous tissue regeneration and care in a GMP and clinical setting, or for the preparation of autologous tissue for medical testing in the burgeoning field of personalised medicine.

Background: The central nervous system is frequently affected in children with inherited metabolic disorders (IMD). The causes of the brain insult are incompletely understood, and novel methods are required for disease diagnosis and monitoring response to novel therapies. Aims & Methods: The study aimed to improve understanding of the pathogenesis of IMD-related neurodegeneration, and to identify potential disease biomarkers in specific IMD, by directly investigating alterations in brain tissue metabolite profiles using non-invasive in vivo magnetic resonance spectroscopy (MRS) in conjunction with conventional MRI brain scans. Results: MRI/MRS studies were performed on over 300 children. Normal brain metabolite profiles were established from a standard comparator cohort. A detailed quality analysis enabled combination of data from different scanner systems. Non-standard brain metabolites were detected in 2.3% of children. Metabolite-based methods of disease progression monitoring were evaluated in Hunter Syndrome. Mechanisms leading to strokes in patients with propionic acidaemia and to learning difficulties and epilepsy in argininosuccinic aciduria were explored using brain tissue metabolite profiling. Conclusions: Non-invasive in vivo brain tissue metabolite profiling is achievable using quantitative magnetic resonance spectroscopy in the routine clinical paediatric setting, and has utility in disease diagnostics, in monitoring disease progression and in investigating disease pathogenesis.