This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease.

This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

This project addressed mechanisms of the neuronal DNA damage response after treatment with the model DNA damaging agent ultraviolet light (UV). The thymidine analogue, 5-bromo-2’-deoxyuridine (BrdU) protected against UV-initiated neuronal apoptosis in a concentration-dependent manner (p<0.001). BrdU did not protect proliferating mouse embryonic fibroblasts from UV-induced apoptosis. We assessed whether the mechanism of BrdU neuroprotection was through a modification in the neuronal DNA damage response. BrdU neuroprotection was independent of BrdU incorporation into DNA, neuronal DNA repair, p53 activation or cell cycle re-entry, a neuronal DNA damage response. Neurons deficient in Cockayne Syndrome B (CSB) or Xeroderma Pigmentosum A (XPA) were paradoxically resistant to UV-initiated apoptosis. Therefore, CSB and XPA play essential roles in the neuronal DNA damage response.

This thesis focuses on the regulation of the ubiquitous second messenger Ca2+ in neuronal physiology and disease. Ca2+ signalling in neurons is regulated by ion channels located in the plasma membrane, as well as in the endoplasmic reticulum (ER) and mitochondrial membranes. Ca2+ signalling is essential for numerous cellular processes, including neuronal excitability, neurotransmitter release, synaptic plasticity, and induction of cell death. Age-related disruptions in Ca2+ signalling may contribute to decline of cognitive function and motor control associated with aging. Furthermore, disruption in neuronal Ca2+ signalling is implicated in several neurodegenerative disorders including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and Amyotrophic Lateral Sclerosis (ALS). In this thesis, I studied neuronal Ca2+ signalling and how it is affected in neurodegenerative disease. First, I examined the role of the ER Ca2+ binding protein Calreticulin (CRT) in AD. CRT is involved in regulation of ER Ca2+ signalling and modulation of susceptibility to cell death. I found that there was an increase in the expression of CRT in in vitro and in vivo models of AD. However, increased levels of CRT did not alter susceptibility of neuronal cells to death induced by AD-related stressors. Second, I examined the role of X-Linked Inhibitor of Apoptosis Protein (XIAP) in the modulation of neuronal Ca2+ signalling. I found that overexpression of XIAP in neuronal cells modified Ca2+ signalling by decreasing Ca2+ flux through multiple plasma membrane and ER channels. These effects appear to be independent of caspase inhibition, which is one of the ways that XIAP can inhibit apoptosis. Third, I examined a compound found in green tea, L-theanine, a glutamate receptor antagonist that is protective in models of excitotoxic neuronal injury. I found that 24 hour L-theanine treatment reduces the amount of Ca2+ released from neuronal intracellular stores in response to both glutamate stimulation and passive leak through ER channels. An acute 30 minute L-theanine treatment had similar effects. In conclusion, these observations further the understanding of the regulation of Ca2+ signalling in neurons and may lead to novel therapeutic strategies in neurodegenerative disease.

The present work investigates the applicability of Genetic Algorithms (GA) to the problem of signal propagation in Native Neuronal Networks (NNNs). These networks are comprised of neurons, some of which receive input signals. The signals propagate though the network by transmission between neurons. The research focuses on the regeneration of the output signal of the network without knowing the original input signal. The computational complexity of the problem is prohibitive for the exact computation. We propose to use a heuristic approach called Genetic Algorithm. Three algorithms are developed, based on the GA technique. The developed algorithms are tested on two different networks with varying input signals. The results obtained from the testing indicate significantly better performance of the developed algorithms compared to the Uniform Random Search (URS) technique, which is used as a control group. The importance of the research is in the demonstration of the ability of GA-based algorithms to successfully solve the problem at hand.

<p>Medulloblastoma is the most common malignant brain tumor in children. The understanding of the genetic alterations in this tumor is emergent, and many such genetic driver events have yet to be functionally-characterized. Our studies have sought to understand the causes and consequences of OTX2 dysregulation in established medulloblastomas and in its putative cellular origins. Using a tumor genetic approach, we have uncovered frequent OTX2 copy number gains driving expression of this oncogene in a subset of medulloblastomas. However, OTX2 is frequently expressed in medulloblastomas independent of genomic copy number gain, and we thus sought to understand the transcriptional regulation of this gene in these tumors. We have found that chromatin accessibility, promoter DNA methylation, and activity of a distal downstream enhancer is distinct between OTX2-expressing and -nonexpressing medulloblastomas. Notably, autoregulation serves to maintain OTX2 expression in some medulloblastomas, whereas DNA methylation actively suppresses OTX2 in tumors not expressing this gene. Finally, we describe the effect of expressing Otx2 (the mouse homolog of OTX2) aberrantly in the developing mouse hindbrain, revealing that Otx2 disrupts spatiotemporal restrictions of neuronal progenitor cell proliferation. The effect of Otx2 in vivo is transient, with ectopically-proliferating cells give way to differentiated neurons. We found that OTX2 expression was not able to give rise to high penetrance medulloblastoma when combined with P53 deletion or double heterozygosity for P53 and PTEN. Thus, although Otx2 alters migration and proliferation dynamics of hindbrain neuronal progenitor cells, further studies are needed to identify the genetic alterations that cooperate with this oncogene to give rise to medulloblastoma.</p> / Dissertation

Glutamate AMPA receptors (AMPARs) are major excitatory receptors in the vertebrate CNS. In many biological systems there are changes in the properties of AMPARs during development that are essential for providing an increase in efficiency of information transfer between neurons and a refinement of motor co-ordination and sensory perception and cognition. It is not surprising that improper development or loss of function of AMPARs can lead to many neurological disorders such as epilepsy and amyotrophic lateral sclerosis. Thus, determining the mechanisms by which AMPARs mature is of particular importance. The objectives of my thesis were to characterize the developmental changes in AMPAR-mediated currents in zebrafish Mauthner cells and to determine the mechanisms underlying any changes. The major findings reported in this thesis are that (1) there are developmental changes in the properties of AMPAR-currents as the Mauthner cell matures; (2) the mechanism underlying these changes is a switch in the composition of AMPA receptor subtypes; and (3) PKC is necessary for the developmental switch in AMPAR subtypes from slow receptors to fast receptors. These findings provide valuable insights into the mechanism underlying the development of AMPARs. In addition, they provide the first instance of a signalling link (PKC) required for the developmental subunit switch and the developmental speeding of AMPAR kinetics. / Physiology, Cell Biology and Developmental Biology

This thesis examines the pathophysiology of motor neurone dysfunction, along with site of disease onset, in amyotrophic lateral sclerosis (ALS). The rationale for this thesis is the "dying forward" hypothesis, which suggests that corticomotoneurons cause anterograde excitotoxic degeneration of motor neurons in ALS. Initially, axonal excitability studies were applied to ALS patients and revealed widespread axonal ion channel dysfunction, with increases in persistent Na+ conductances and reduction in K+ currents. Such changes result in axonal hyperexcitability, thereby resulting in generation of fasciculations and cramps. Subsequently, axonal excitability studies were applied to Kennedy's disease (KD) patients, a pathological control group, revealing similar changes to ALS and suggesting that upregulation of persistent Na+ conductances was responsible for generation of fasciculations. To better understand the mechanisms underlying fatigability and to assess whether Na+/K+ pump dysfunction contributes to neurodegeneration in ALS, activity-dependent changes in axonal excitability were measured after a maximal voluntary contraction. The increase in threshold was more pronounced in ALS patients with predominantly lower motor neuron involvement, suggesting that peripheral factors were responsible for fatigue in ALS and that Na+/K+ pump function was preserved. Having documented abnormalities of axonal excitability, a novel threshold tracking transcranial magnetic stimulation (TMS) technique was developed for assessment of cortical excitability. This technique overcomes the marked variability in the motor evoked potential with consecutive stimuli, a major limitation of the previous "constant stimulus" technique. After establishing normative data, threshold tracking TMS established that cortical hyperexcitability was an early and prominent feature in ALS. Similar changes were found in flail-arm variant ALS, a pure lower motor neuron form of ALS. In KD patients, cortical excitability was normal, thereby suggesting that cortical hyperexcitability is a primary event in ALS rather than a down-regulation of inhibitory control over the motor cortex in order to compensate for anterior horn cell loss. In order to determine whether cortical hyperexcitability underlies motor neurodegeneration, longitudinal studies were undertaken in familial ALS subjects with the copper/zinc superoxide-dismutase-1 gene mutation. These studies established that cortical hyperexcitability precedes the development of clinical ALS, thereby suggesting that cortical hyperexcitability underlies the basis of motor neurodegeneration in familial ALS.

Diss. Naturwiss. Bern (kein Austausch).

Tuning to an optimal stimulus is a widespread property of neurons in cortex. We propose that such tuning is a consequence of normalization or gain control circuits. We also present a biologically plausible neural circuitry of tuning.