<p>The focus of this thesis is the life and death of adult retinal ganglion cells (RGCs). RGCs are neurons that convey visual information from the retina to higher centers in the brain. If the optic nerve is transected (ONT), adult RGCs die by a form of cell death called apoptosis, and a general hypothesis is that neurotrophic factors can support the survival of injured neurons.</p><p>With the intention to gain knowledge about systems that can be used to decrease RGC death after ONT, we have studied growth factor receptors belonging to the tyrosine kinase family of receptors (RTK), known to mediate important cell survival signals. We found that the RTK Ret and its coreceptor GFRα1 were expressed by RGCs, and to test the above-mentioned hypothesis, we intraocularly administered glial cell-line derived factor, which activates a Ret-GFRα1 complex, and found transiently mediated RGC survival after ONT. </p><p>To identify new, potential neurotrophic factor receptors expressed by RGCs, with the aim to improve RGC survival after ONT, we developed a method for the molecular analysis of acutely isolated RGCs. The method involves retrograde neuronal tracing, mechanical retinal layer-separation, and isolation of individual RGCs under UV-light for RT-PCR analysis. Using this method, in combination with degenerate PCR directed towards the tyrosine kinase domain, several RTKs were identified. Axl, Sky, VEGFR-2, VEGFR-3, CSF-1R, and PDGF-βR are expressed by adult RGCs, and considered to be receptors with potential neurotrophic activity. Other results have shown that RGCs may require depolarization or increase in intracellular cAMP levels in order to fully respond to exogenously added trophic factors. We found that melanocortin receptors (MCRs) were expressed by RGCs, and MCRs can mediate elevation of intracellular AMP. We observed that α-MSH induced neurite outgrowth from embryonic retinal cells, indicating that MCR ligands have direct effects on retinal cells. RTKs and their ligands may be involved in endogenous systems for neuronal repair within the visual system. BDNF, NT-3, FGF2, and HGFR all increased in the retina after ONT and may be a part of an activated system for neuronal repair locally within the retina. </p><p>Adult axotomized RGCs die by apoptosis, therefore we examined the regulation of apoptotic genes after ONT. Bim and Bax increased in the retina after ONT, and may promote death of axotomized RGCs, whereas the increase in Bcl-2 may contribute to limit RGC apoptosis after ONT. </p><p>All in all, this thesis provides insights into the expression and regulation of molecules involved in the death and survival of RGCs. The results have revealed a number of potential neurotrophic receptors expressed by RGCs, and both identified RTKs and MCRs will serve as new targets in therapeutic approaches aiming at counteraction of RGC death after injury.</p>

<p>Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive disorder characterized by degeneration of motor neurons in the cortex, brainstem and spinal cord. The patients usually die within 3-5 years after onset. The full etiology of ALS is unknown and many hypotheses have been proposed to explain the neurodegeneration. However, basic mechanisms of cellular function such as transmethylation and polyamine metabolism have not been extensively studied in ALS. Transmethylation reactions are very important in the synthesis of substrates such as proteins, neurotransmitters, DNA and RNA. The polyamines, putrescine, spermidine and spermine, are involved in essential functions such as cellular growth, proliferation and differentiation.</p><p>An initial study in this thesis concerned the process of neuronal death (apoptosis) in ALS spinal cord. The results showed increased levels of an apoptosis-stimulating protein and increased levels of DNA fragmentation indicative of an apoptotic process in the tissue. A comparative study of MAT-enzyme activity in spinal cord from different mammalian species was undertaken to provide a background for future studies on transmethylation and neurodegeneration. Transmethylation reactions were found altered in erythrocytes from males with ALS but not in spinal cord from ALS patients as compared to controls. An adaptation of previously described polyamine assays was made for the study of polyamines in ALS spinal cord. The method was validated and applied for polyamine analysis in human materials of different characteristics. Determination of polyamines in control and ALS spinal cords showed no major differences. However, in female ALS patients, significantly increased spermidine and spermine levels were observed in ventral horn regions. These gender-related alterations in transmethylation and polyamine metabolism are of interest since there is a male preponderance for the disease.</p><p>The lack of major differences in polyamine levels between ALS and control spinal cord suggests a maintained regulation of polyamines at the end stage of this neurodegenerative disease.</p>

<p>After the acute spell of poliomyelitis, patients commonly suffers from sequelae of weakness. Some of these patients experience new weakness after a time period of stable symptoms.</p><p>The aim of this thesis was to evaluate the possible mechanisms for persistent weakness and development of new weakness in prior polio patients.</p><p>The usefulness of neurophysiologic methods to study prior polio was evaluated. Also two follow up investigations were performed in the attempt to investigate a possible relationship between development of weakness over time and possible failure in neuromuscular function and relation to muscular activity. In another investigation an evaluation of the exceptional finding of a history of paralytic poliomyelitis without neurophysiologic signs of anterior horn cell death was made. The last investigation dealt with reflex pattern in prior polio and it’s relation to weakness and anterior horn cell loss.</p><p>The weakness in prior polio is mainly due to loss of motor neurons with incomplete compensatory mechanisms of reinnervation. The new weakness is mainly due to exaggerated physiological age dependent loss of whole motor neurons, but also fragmentation of the motor unit is likely when these have reached an upper size. Defective neuromuscular transmission and failure in the central drive contribute to a lesser degree to weakness. </p><p>Neurophysiologic method of choice for the assessment of motor unit size and the micro-physiology of the motor unit is Macro EMG.</p><p>Muscular overuse may accelerate motor unit loss over time in prior polio. Extremely large motor units measured with Macro EMG predict new weakness and Macro EMG can be used for prognostication of development of new weakness in prior polio.</p>

<p>Stem cells have the capacity for both self renewal, and to form all cell types in the body. Interestingly, so called neural stem cells (NSCs) are found in the adult human brain, which is of significance both out of a developmental perspective and from a clinical point of view. At the present moment, the regulation of neural stem cell (NSC) proliferation and fate determination is not completely understood.</p><p>The overall aim of this thesis was to study the mechanisms that regulate NSC proliferation and fate determination <i>in vitro</i> and <i>in vivo</i>. In particular, the roles of the female sex hormone estrogen and the testosterone analogue nandrolone, as well as the melanocortin α-melanocyte stimulating hormone (α-MSH), were analyzed in this context. Also, the breast cancer susceptibility gene one (BRCA-1), was studied in the brain with emphasis on regions containing NSCs.</p><p>Our findings show that estrogen and nandrolone have similar effects on NSCs; both decreased NSC proliferation and increased neurogenesis. Estrogen's ability to reduce proliferation was due to increased levels of p21, an inhibitor of cyclin dependent kinases. In contrast, no change in p21 was observed in the case of nandrolone, indicating differential regulation. Adult rats subjected to nandrolone injections had 30% reduced NSC proliferation in the dentate gyrus, indicating profound effects on NSCs <i>in vivo</i>.</p><p>The melanocortin α-MSH acted as a mitogen by increasing levels of cyclinD1 and retinoblastoma protein; as a result NSC proliferation was doubled.</p><p>Finally, BRCA-1 is expressed while NSCs proliferate, but is drastically down regulated upon differentiation, indicating that BRCA-1 could be used as a possible NSC marker.</p><p>In summary, in this thesis estrogen and nandrolone were identified as NSC regulators which decrease proliferation and positively influence neurogenesis. Also, we have identified the hormone α-MSH as a NSC mitogen, and BRCA-1 as a possible NSC marker.</p>

<p>Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of upper and lower motor neurons, resulting in muscle weakness and death from respiratory failure within 3-5 years after onset. The incidence is 1.5-2.7/100,000 inhabitants. 5-10% of all cases are hereditary. The aetiology of sporadic ALS is still unknown. </p><p>The only neuroprotective drug approved for the treatment of ALS is riluzole, a glutamate-antagonist, which has shown to improve survival. We evaluated if riluzole sales statistics can be used as a method for estimating the prevalence of ALS/motor neuron disease in Sweden. We found that this method, which is less time consuming than conventional methods, could be used as a crude marker for the prevalence. </p><p>In a longitudinal study of overall Quality of Life (QoL) in ALS we found that QoL changes only slightly over time despite disease progression. ALS does not necessarily result in a low QoL. </p><p>Growth factors are important for the survival of neurons. In ALS we found increased or normal levels of GDNF mRNA and BDNF mRNA in muscle biopsies, VEGF in serum and spinal cord and FGF-2 in serum and cerebrospinal fluid. There is thus no deficit of these growth factors although there may be a relative lack because of high demands of the motor neurons. Polyamines are small aliphatic molecules that are important for the function of cells. The level of the polyamines spermidine and spermine were increased in red blood cells in both patients with ALS and patients with Parkinson’s disease, suggesting that polyamines may have a role for the neurodegenerative process. Polyamines in spinal cord were of the same level in the patients with ALS and in controls, indicating a maintained regulation of polyamines at the end-stage of the disease.</p>

<p>Human skeletal muscles possess thixotropic, i.e. history-dependent mechanical properties. This means that the degree of passive muscle stiffness and resting tension is dependent on the immediately preceding history of contractions and length changes. Athletes, for instance, reduce passive muscle stiffness by various types of ‘limbering-up’ procedures, whereas muscle stiffness gradually increases during inactivity.</p><p>Passive resistance of antagonistic muscles may significantly add to the total load during voluntary muscle contractions. This resistance may vary from one moment to another, depending on immediately preceding events. This research was conducted to determine whether history-dependent variations in passive muscular forces influence motor control of voluntary joint movements and steady maintenance of joint positions in healthy subjects. </p><p>In study I, the EMG signal revealed motor compensations for history-dependent variations in passive stiffness of the antagonists during slow voluntary wrist joint movements. Studies II and III demonstrated that the voluntary muscle activity required to maintain a certain wrist joint position was highly influenced by previous changes in forearm muscle length and contractions. Study IV showed that rapid voluntary movements varied in speed and onset time depending on the prevailing degree of muscle resistance, and in addition that the central nervous reaction time required to execute rapid movements was highly influenced by immediately preceding muscle-conditioning procedures.</p><p>History-dependent variations in passive muscular forces seem to be effectively compensated by the motor control system. Presumably, voluntary motor commands to the muscles are automatically adjusted in strength to history-dependent changes in passive muscular forces. Such adjustments occur within the central nervous system, which receives information about the mechanical state of the muscles. Several issues in connection with muscle thixotropy remain unaddressed. For instance, do alterations in the normal thixotropic mechanical behaviour of the muscles impose a particular problem in patients with certain neuromuscular diseases? </p>

<p>Headache is the most frequently reported pain in children and is associated with missed schooldays, anxiety, depressive symptoms and various physical symptoms. A secular trend of increasing headache prevalence has been suggested. Few studies have focused on tension-type headache among children from the general population. </p><p>The aims of this thesis were to describe the prevalence, incidence and prognosis of tension-type headache, migraine and overall headache in schoolchildren, to identify medical, psychological and social factors associated with these headache types, and to determine whether the prevalence of headache has increased over the last decades.</p><p>In 1997, 1850 schoolchildren aged 7-15 years from the city of Uppsala participated in a questionnaire study and 1371 (74.1%) responded. Out of these, a randomly selected, stratified sample of 131 children and their parents were interviewed. Three years later, 122 children from the interview sample replied to an identical headache questionnaire.</p><p>Compared with a similar study in 1955, a significantly lower proportion of schoolchildren reported no headache. The prevalence of tension-type headache increased with age and was significantly higher in girls than boys after the age of twelve. Similar age and gender differences were obtained for migraine. A higher proportion of girls reported frequent headache than boys. Children with headache, especially those with migraine, as well as their first-degree relatives suffered from other pains and physical symptoms more frequently than headache-free children and their first-degree relatives. Although the likelihood of experiencing the same headache diagnosis and symptoms at follow-up was high, about one fifth of children with migraine developed tension-type headache and vice versa. Female gender was a predictor of migraine and frequent headache a predictor of overall headache at follow-up. The estimated annual incidence for tension-type headache, migraine and overall headache was 81, 65 and 131 per 1000 children, respectively. </p><p>In conclusion, the results indicate that headache has become increasingly common among schoolchildren over the last decades. Prevention and treatment of headache is particularly important for girls since they have high prevalence of headache, frequent headache episodes and a poor outcome. In children with headache, diagnoses and treatment should be reassessed regularly and other pains should be asked about and treated as well. </p>

<p>Impulses coming from peripheries are modified in the spinal cord and transmitted to the brain. Several neurotransmitters have been involved in the processing of impulses in the spinal dorsal horn. Acetylcholine (ACh) is one of many neurotransmitters involved in the regulation of nociception in the spinal cord. In this study we investigated the role of nicotinic, muscarinic, serotonergic and GABA receptors in the regulation of spinal ACh release since these receptors are reported to be involved in spinal nociceptive processes.</p><p>Different receptor ligands were infused intraspinally via microdialysis and the spinal ACh release was measured by on-line HPLC. Receptor-ligand binding studies were performed with spinal cord homogenates as well as receptors expressed in cells.</p><p>In the first study, we found that nicotine and some of the nicotinic antagonists used increased ACh release suggesting that spinal ACh release is regulated by different nAChRs. Nicotine and nicotinic agonists may act on different types of receptors with different affinity to produce the observed net effect of increased ACh release. We propose the possibility of an involvement of three different nicotinic receptor subtypes in the regulation of spinal ACh release. </p><p>The effect of epibatidine, which is regarded as a nicotinic agonist, on muscarinic receptors was investigated in the second study. We propose that epibatidine, in μM concentrations, is a partial muscarinic receptor agonist that may interact with spinal muscarinic receptors to increase ACh release. The dual action on both nAChRs and mAChRs may explain the potent analgesic effect observed after intra-spinal epibatidine administration.</p><p>In the third study, we investigated the role of serotonin receptor involvement in ACh release control. The results suggest that only 5-HT_1A and 5-HT_2A receptors are involved in spinal ACh release. Considering current knowledge, the most probable location of 5-HT_2A receptors is on cholinergic neurones. On activation of the 5-HT_2A receptors the cellular excitability of cholinergic neurones is increased which results in an increasing ACh release. The 5-HT_1A receptors might be located on cell bodies of GABA neurones which inhibit the firing rate of the GABA neurones when activated by serotonin. </p><p>In the fourth study, we investigated the GABA receptor involvement in the regulation in spinal ACh release. We found that GABA_A receptors are tonically inhibiting spinal ACh release. The results further suggest that GABA_B receptors also are involved in the regulation of spinal ACh release. However, unlike GABA_A antagonists, GABA_B antagonists do not increase ACh release. This suggests that GABA_B receptors are not tonically regulating the spinal ACh release. </p>

<p>Proteins of the band 4.1 superfamily are characterized by their sequence similarity to the ERM proteins ezrin, radixin and moesin, which are involved in cell motility, adhesion of cells, and signal transduction events. Little is however known of the function of ERM proteins in the nervous system, though an essential role for radixin and moesin in neuronal growth cone motility has been suggested. </p><p> This thesis is focused on the cloning, functional characterization and description of the tissue distribution in rat brain of MIR, a novel member of the band 4.1 superfamily. </p><p> The cDNA of MIR encods a protein of 445 amino acids which is composed of an ERM-homology domain and a RING finger, separated by an interregion. To reveal the cellular function of MIR, PC12 cell lines overexpressing MIR was generated and observed to inhibit NGF stimulated neurite outgrowth. </p><p> To elucidate the signal transduction of MIR by which it exerts its physiological activity, the yeast two-hybrid system was employed to screen for proteins that interact with MIR. A number of interactors known to regulate the cytoskeleton was obtained - among them myosin regulatory light chain-B which controls the actomyosin complex - and a novel type 2 membrane protein denoted NSAP for its similarity to saposin A-D. Overexpressed NSAP induced neurite outgrowth in PC12 cells and enhanced cell adhesion in fibroblasts. </p><p> The tissue distribution of MIR in rat brain, as determined by immunohistochemistry studies, showed that MIR is localized especially to neurons in hippocampus and cerebellum. The chromosomal localization of the MIR gene was assessed to 6p22.3-23, a region lost in the 6p23 deletion syndrome.</p><p> These results suggests that MIR is expressed in neurons in discrete regions of rat brain where it may regulate neurite outgrowth by modulating the cytoskeleton.</p>

Proteins of the band 4.1 superfamily are characterized by their sequence similarity to the ERM proteins ezrin, radixin and moesin, which are involved in cell motility, adhesion of cells, and signal transduction events. Little is however known of the function of ERM proteins in the nervous system, though an essential role for radixin and moesin in neuronal growth cone motility has been suggested. This thesis is focused on the cloning, functional characterization and description of the tissue distribution in rat brain of MIR, a novel member of the band 4.1 superfamily. The cDNA of MIR encods a protein of 445 amino acids which is composed of an ERM-homology domain and a RING finger, separated by an interregion. To reveal the cellular function of MIR, PC12 cell lines overexpressing MIR was generated and observed to inhibit NGF stimulated neurite outgrowth. To elucidate the signal transduction of MIR by which it exerts its physiological activity, the yeast two-hybrid system was employed to screen for proteins that interact with MIR. A number of interactors known to regulate the cytoskeleton was obtained - among them myosin regulatory light chain-B which controls the actomyosin complex - and a novel type 2 membrane protein denoted NSAP for its similarity to saposin A-D. Overexpressed NSAP induced neurite outgrowth in PC12 cells and enhanced cell adhesion in fibroblasts. The tissue distribution of MIR in rat brain, as determined by immunohistochemistry studies, showed that MIR is localized especially to neurons in hippocampus and cerebellum. The chromosomal localization of the MIR gene was assessed to 6p22.3-23, a region lost in the 6p23 deletion syndrome. These results suggests that MIR is expressed in neurons in discrete regions of rat brain where it may regulate neurite outgrowth by modulating the cytoskeleton.