Over the last decade, the search for an alternative to primary fetal material for use in cell replacement therapies for neurodegenerative disorders has led to an increasingly enthusiastic interest in neural stem cells. This thesis attempts to determine the usefulness of cerebellar neural stem cells for the repair of disorders such as the hereditary ataxias, which affect specific cells of the cerebellum, aided by the development of a novel in vitro model of cerebellar damage in the rat.

Background. Over the past decades metabolic profiling has become a valuable tool for the investigation of metabolic changes specific to particular diseases. As this concept considers participants of several metabolic networks and processes within one analyt- ical step, it has been considered suitable for the investigation of cellular mechanisms underlying brain diseases whose pathological backgrounds are assumed to be inher- ently complex. One main technical platform of metabolic profiling is based on in vitro nuclear magnetic resonance (NMR) spectroscopy: a technique that is also available for the non-invasive application in vivo. Hence, it provides a promising mean for the translation from in vitro research to the eventual clinical use. Aim. The studies presented in this thesis applied in vitro NMR spectroscopy based metabolic profiling to extracts of cerebral tissue samples of rodent models of mental and neurodegenerative disorders to evaluate its ability as search tool for biomarkers in these conditions. Method Validation. To verify the comparability between the performance of the in vitro and in vivo application of nuclear magnetic resonance spectroscopy, tissue ex- traction methods were firstly tested for metabolic content, precision and suitability for metabolic profiling studies. Secondly, the in vitro results of the study of socially isolated rats were compared to in vivo results of the same model. This study demonstrated that the two modalities produced similar outcomes with respect to the (patho)physiological research questions.

Acute cerebral ischaemic injury results in increased neurotransmitter release which can result in cell death via receptor and non-receptor mediated mechanisms. Furthermore a delayed apoptotic response has been described which may be attributed to the CD95 death receptor. Using the murine hippocampal HT22 cell line, this study has developed a novel FRET construct to investigate the biochemical pathways associated with two models of stroke induced neurotoxicity; CD95-induced apoptosis and glutamate-induced oxidative toxicity. CD95-induced apoptosis resulted in chromatin condensation and caspase activation which if inhibited rescued cells from death. Mitochondrial involvement was identified by cytochrome c release following CD95 stimulation. Furthermore the pro-apoptotic protein Bax was shown to be conformationally active and translocated to the mitochondrial following CD95 stimulation indicating Bax mediated cytochrome c release. The novel DsRed-EYFP FRET construct which showed caspase activity in living cells and further in vitro analysis confirmed loss of acceptor fluorescence was due to construct cleavage. Oxidative glutamate toxicity was shown to be mediated by calpains (calcium dependent proteases) and furthermore it was shown that the caspases contribute to the survival of the cell. Investigation into the role of the stress associated MAP kinase (SAPK/JNK) resulted in abnormal nuclear morphologies which were shown to be the result of cell cycle arrest. Inhibition of not only JNK but also glycogen synthase kinase (GSK) and calpains resulted in cell cycle arrest in the G2 or pre-mitotic cell cycle phase. In conclusion we have described a model where glutamate induced ROS production damages vital cellular components resulting in the activation of the cell cycle. Upon progression through the cell cycle the damage is identified and the cells enter a caspase independent, calpain mediated cell death programme.

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.