Alpha- and gamma-melanocyte stimulating hormones in obesity. A study of the key central areas of metabolic regulation

The melanocortin system is an important regulator of feeding, energy metabolism,and cardiovascular function and it consists of the pro-opiomelanocortin (POMC) derived melanocyte stimulating hormones (α-, β- and γ-MSH) and their endogenous melanocortin receptors, MC1R to MC5R. In the hypothalamus, α-MSH reduces food intake, and increases energy expenditure and sympathetic tone by binding to MC4R. Mutations affecting the MC4R gene lead to obesity in mammals. On the other hand, the metabolic effects of MC3R stimulation using agonists such as the endogenously expressed γ-MSH have been less extensively explored. The main objective of this study was to investigate the long-term effects of increased melanocortin tone in key areas of metabolic regulation in the central nervous system (CNS) in order to investigate the sitespecific roles of both α-MSH and γ-MSH. The aim was to stereotaxically induce local overexpression of single melanocortin peptides using lentiviral vectors expressing α-MSH (LVi-α-MSH-EGFP) and γ-MSH (LVi-γ-MSH-EGFP). The lentiviral vectors were shown to produce a long-term overexpression and biologically active peptides in cell-based assays. The LVi-α-MSHEGFP was targeted to the arcuate nucleus in the hypothalamus of diet induced obese mice where it reduced weight gain and adiposity independently of food intake. When the nucleus tractus solitarus in the brainstem was targeted, the LVi-α-MSH-EGFP treatment was shown to cause a small decrease in adiposity, which did not impact weight development. However, the α-MSH treatment increased heart rate, which was attenuated by adrenergic receptor blockade indicative of increased sympathetic activity. The LVi-γ-MSH-EGFP was targeted to the hypothalamus where it decreased fat mass in mice eating the standard diet, but the effect was abated if animals consumed a high-fat Western type diet. When the diet induced obese mice were subjected again to the standard diet, the LVi-γ-MSH-EGFP treated animals displayed increased weight loss and reduced adiposity. These results indicate that the long-term central anti-obesity effects of α-MSH are independent of food intake. In addition, overexpression of α-MSH in the brain stem efficiently blocked the development of adiposity, but increased sympathetic tone. The evidence presented in this thesis also indicates that selective MC3R agonists such as γ-MSH could be potential therapeutics in combination with low fat diets.

Apolipoprotein E and Recovery from Traumatic Brain Injury

The outcome from traumatic brain injury (TBI) is variable and only partly explained by known prognostic factors. This is especially true for predicting long-term outcome. Genetic factors may influence the brain`s susceptibility to injury or capacity for repair and regeneration. To examine the association of apolipoproteinE (apoE) genotype with long-term outcome, hippocampal volumes and general brain atrophy, we determined the apoE genotype from 61 TBI patients who had been injured over on average 31 years earlier. The long-term outcome was evaluated with repeated neuropsychological testing and by applying various measures of everyday functioning and quality of life. Magnetic resonance imaging (MRI) based volumetric analyses of the hippocampus and lateral ventricles were performed. In the prospective study, the purpose was to examine the association between apoE genotype and visibility of traumatic brain lesions during the first year after TBI and the ability of apoE genotype, the Glasgow Coma Score (GCS), MRI findings and duration of posttraumatic amnesia (PTA) to predict the one-year outcome. Thirty-three patients with TBI were studied and the outcome was evaluated with the Head Injury Symptom Checklist (HISC) and the Glasgow Outcome Scale extended version (GOS-E) scores one year after the injury. MRI and apoE genotyping were carried out. After three decades, neither hippocampal nor lateral ventricle volumes differed significantly in those patients with the apoE ε4 allele vs those without this allele, but the TBI patients with the apoE ε4 allele showed significantly poorer general cognitive level than those without this allele. This decline was wholly accounted for by a subgroup of patients who had developed incident or clinical dementia. In the prospective study the apoE genotype was not associated with visible MRI changes or outcome. The duration of PTA and acute MRI were the best predictors of one-year outcome in TBI. A portion of the TBI patients with the apoE ε4 allele seem to be at risk of long-term cognitive decline. This association may involve mechanisms other than those responsible for the development of brain atrophy. The early MRI and PTA have an important role in assessing the injury severity and prognosis.

Regulation of self-renewal and detection of karyotypic changes of pluripotent human embryonic stem cells

Human embryonic stem cells are pluripotent cells capable of renewing themselves and differentiating to specialized cell types. Because of their unique regenerative potential, pluripotent cells offer new opportunities for disease modeling, development of regenerative therapies, and treating diseases. Before pluripotent cells can be used in any therapeutic applications, there are numerous challenges to overcome. For instance, the key regulators of pluripotency need to be clarified. In addition, long term culture of pluripotent cells is associated with the accumulation of karyotypic abnormalities, which is a concern regarding the safe use of the cells for therapeutic purposes. The goal of the work presented in this thesis was to identify new factors involved in the maintenance of pluripotency, and to further characterize molecular mechanisms of selected candidate genes. Furthermore, we aimed to set up a new method for analyzing genomic integrity of pluripotent cells. The experimental design applied in this study involved a wide range of molecular biology, genome-wide, and computational techniques to study the pluripotency of stem cells and the functions of the target genes. In collaboration with instrument and reagent company Perkin Elmer, KaryoliteTM BoBsTM was implemented for detecting karyotypic changes of pluripotent cells. Novel genes were identified that are highly and specifically expressed in hES cells. Of these genes, L1TD1 and POLR3G were chosen for further investigation. The results revealed that both of these factors are vital for the maintenance of pluripotency and self-renewal of the hESCs. KaryoliteTM BoBsTM was validated as a novel method to detect karyotypic abnormalities in pluripotent stem cells. The results presented in this thesis offer significant new information on the regulatory networks associated with pluripotency. The results will facilitate in understanding developmental and cancer biology, as well as creating stem cell based applications. KaryoliteTM BoBsTM provides rapid, high-throughput, and cost-efficient tool for screening of human pluripotent cell cultures.

The neural processes generating visual phenomenal consciousness: ERP and neuronavigated brain stimulation studies

One of the greatest conundrums to the contemporary science is the relation between consciousness and brain activity, and one of the specifi c questions is how neural activity can generate vivid subjective experiences. Studies focusing on visual consciousness have become essential in solving the empirical questions of consciousness. Th e main aim of this thesis is to clarify the relation between visual consciousness and the neural and electrophysiological processes of the brain. By applying electroencephalography and functional magnetic resonance image-guided transcranial magnetic stimulation (TMS), we investigated the links between conscious perception and attention, the temporal evolution of visual consciousness during stimulus processing, the causal roles of primary visual cortex (V1), visual area 2 (V2) and lateral occipital cortex (LO) in the generation of visual consciousness and also the methodological issues concerning the accuracy of targeting TMS to V1. Th e results showed that the fi rst eff ects of visual consciousness on electrophysiological responses (about 140 ms aft er the stimulus-onset) appeared earlier than the eff ects of selective attention, and also in the unattended condition, suggesting that visual consciousness and selective attention are two independent phenomena which have distinct underlying neural mechanisms. In addition, while it is well known that V1 is necessary for visual awareness, the results of the present thesis suggest that also the abutting visual area V2 is a prerequisite for conscious perception. In our studies, the activation in V2 was necessary for the conscious perception of change in contrast for a shorter period of time than in the case of more detailed conscious perception. We also found that TMS in LO suppressed the conscious perception of object shape when TMS was delivered in two distinct time windows, the latter corresponding with the timing of the ERPs related to the conscious perception of coherent object shape. Th e result supports the view that LO is crucial in conscious perception of object coherency and is likely to be directly involved in the generation of visual consciousness. Furthermore, we found that visual sensations, or phosphenes, elicited by the TMS of V1 were brighter than identically induced phosphenes arising from V2. Th ese fi ndings demonstrate that V1 contributes more to the generation of the sensation of brightness than does V2. Th e results also suggest that top-down activation from V2 to V1 is probably associated with phosphene generation. The results of the methodological study imply that when a commonly used landmark (2 cm above the inion) is used in targeting TMS to V1, the TMS-induced electric fi eld is likely to be highest in dorsal V2. When V1 was targeted according to the individual retinotopic data, the electric fi eld was highest in V1 only in half of the participants. Th is result suggests that if the objective is to study the role of V1 with TMS methodology, at least functional maps of V1 and V2 should be applied with computational model of the TMS-induced electric fi eld in V1 and V2. Finally, the results of this thesis imply that diff erent features of attention contribute diff erently to visual consciousness, and thus, the theoretical model which is built up of the relationship between visual consciousness and attention should acknowledge these diff erences. Future studies should also explore the possibility that visual consciousness consists of several processing stages, each of which have their distinct underlying neural mechanisms.

Natural born weight gainers: The mechanisms of obesity in transgenic mice overexpressing neuropeptide Y

Neuropeptide Y (NPY) is a neurotransmitter promoting energy storage by activating Y-receptors and thus affecting food intake, thermogenesis and adipose tissue metabolism. NPY is expressed both in the central and sympathetic nervous system. Hypothalamic NPY is known to stimulate feeding, but the effects of noradrenergic neuron NPY are more ambiguous. Chronic stress stimulates fat accumulation via NPY release from noradrenergic neurons. Furthermore, polymorphism in the human Npy gene has been associated with metabolic disturbances and increased NPY secretion after sympathetic stimulation. The main objective of this study was to clarify the mechanisms of noradrenergic neuron NPY in the development of obesity. The metabolic phenotype of a homozygous mouse overexpressing NPY in the brain noradrenergic neurons and sympathetic nervous system (OE-NPYDβH mouse) was characterized. OE-NPYDβH mice had an increased fat mass and body weight, which caused impairments of glucose metabolism and hyperinsulinaemia with age. There were no differences in energy intake or expenditure, but the sympathetic tone was down-regulated and the endocannabinoid system activated. Furthermore, peripheral Y2-receptors in energy-rich conditions played an important role in mediating the fat-accumulating effect of NPY. These results indicate that noradrenergic neuron NPY promotes obesity via direct effects in the periphery and by modulating the sympatho-adrenal and endocannabinoid systems. Additionally, NPY in the central noradrenergic neurons is believed to possess many important roles. The phenotype of the OE-NPYDβH mouse resembles the situations of chronic stress and Npy gene polymorphism and thus these mice may be exploited in testing novel drug candidates for the treatment of obesity.