GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Death pathways activated in the neurotrophic factor-deprived neurons

Programed cell death (PCD) is a fundamental biological process that is as essential for the development and tissue homeostasis as cell proliferation, differentiation and adaptation. The main mode of PCD - apoptosis - occurs via specifi c pathways, such as mitochondrial or death receptor pathway. In the developing nervous system, programed death broadly occurs, mainly triggered by the defi ciency of different survival-promoting neurotrophic factors, but the respective death pathways are poorly studied. In one of the best-characterized models, sympathetic neurons deprived of nerve growth factor (NGF) die via the classical mitochondrial apoptotic pathway. The main aim of this study was to describe the death programs activated in these and other neuronal populations by using neuronal cultures deprived of other neurotrophic factors. First, this study showed that the cultured sympathetic neurons deprived of glial cell line-derived neurotrophic factor (GDNF) die via a novel non-classical death pathway, in which mitochondria and death receptors are not involved. Indeed, cytochrome c was not released into the cytosol, Bax, caspase-9, and caspase-3 were not involved, and Bcl-xL overexpression did not prevent the death. This pathway involved activation of mixed lineage kinases and c-jun, and crucially requires caspase-2 and -7. Second, it was shown that deprivation of neurotrophin-3 (NT-3) from cultured sensory neurons of the dorsal root ganglia kills them via a dependence receptor pathway, including cleavage of the NT- 3 receptor TrkC and liberation of a pro-apoptotic dependence domain. Indeed, death of NT-3-deprived neurons was blocked by a dominant-negative construct interfering with TrkC cleavage. Also, the uncleavable mutant of TrkC, replacing the siRNA-silenced endogeneous TrkC, was not able to trigger death upon NT-3 removal. Such a pathway was not activated in another subpopulation of sensory neurons deprived of NGF. Third, it was shown that cultured midbrain dopaminergic neurons deprived of GDNF or brainderived neurotrophic factor (BDNF) kills them by still a different pathway, in which death receptors and caspases, but not mitochondria, are activated. Indeed, cytochrome c was not released into the cytosol, Bax was not activated, and Bcl-xL did not block the death, but caspases were necessary for the death of these neurons. Blocking the components of the death receptor pathway - caspase-8, FADD, or Fas - blocked the death, whereas activation of Fas accelerated it. The activity of Fas in the dopaminergic neurons could be controlled by the apoptosis inhibitory molecule FAIML. For these studies we developed a novel assay to study apoptosis in the transfected dopaminergic neurons. Thus, a novel death pathway, characteristic for the dopaminergic neurons was described. The study suggests death receptors as possible targets for the treatment of Parkinson s disease, which is caused by the degeneration of dopaminergic neurons.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Neurotrophins and neuronal plasticity in the action of antidepressants and morphine

Neuronal plasticity is a well characterized phenomenon in the developing and adult brain. It refers to capasity of a single neuron to modify morphology, synaptic connections and activity. Neuronal connections and capacity for plastic events are compromised in several pathological disorders, such as major depression. In addition, neuronal atrophy has been reported in depressive patients. Neurotrophins are a group of secretory proteins functionally classified as neuronal survival factors. Neurotrophins, especially brain derived neurotrophic factor (BDNF), have also been associated with promoting neuronal plasticity in dysfunctional neuronal networks. Chronic antidepressant treatment increases plastic events including neurogenesis and arborization and branching of neurites in distinct brain areas, such as the hippocampus. One suggested mode of action is where the antidepressants elevate the synaptic levels of BDNF thus further activating several signaling cascades via trkB-receptor. In our studies we have tried to clarify the mechanisms of action for antidepressants and to resolve the role of BDNF in this process. We found that chronic antidepressant treatment increases amount of markers of neuronal plasticity in both hippocampus and in the medial prefrontal cortex, both of which are closely linked to the etiology of major depression. Secondary actions of antidepressants include rapid activation of the trkB receptor followed by a phosphorylation of transcription factor CREB. In addition, activation of CREB by phosphorylation appears responsible for the regulation of the expression of the BDNF gene. Using transgenic mice we found that BDNF-induced trkB-mediated signaling proved crucial for the behavioral effects of antidepressants in the forced swimming test and for the survival of newly-born neurons in the adult hippocampus. Antidepressants not only increased neurogenesis in the adult hippocampus but also elevated the turnover of hippocampal neurons. During these studies we also discovered that another trkB ligand, NT-4, is involved in morphine-mediated anti-nociception and tolerance. These results present a novel role for trkB-mediated signaling in plastic events present in the opioid system. This thesis evaluates neuronal plasticity and trkB as a target for future antidepressant treatments.

Differentiation of neural stem cells in fragile X syndrome

Multipotent stem cells can self-renew and give rise to multiple cell types. One type of mammalian multipotent stem cells are neural stem cells (NSC)s, which can generate neurons, astrocytes and oligodendrocytes. NSCs are likely involved in learning and memory, but their exact role in cognitive function in the developing and adult brain is unclear. We have studied properties of NSCs in fragile X syndrome (FXS), which is the most common form of inherited mental retardation. FXS is caused by the lack of functional fragile X mental retardation protein (FMRP). FMRP is involved in the regulation of postsynaptic protein synthesis in a group I metabotropic glutamate receptor 5 (mGluR5)-dependent manner. In the absence of functional FMRP, the formation of functional synapses is impaired in the forebrain which results in alterations in synaptic plasticity. In our studies, we found that FMRP-deficient NSCs generated more neurons and less glia than control NSCs. The newborn neurons derived from FMRP-deficient NSCs showed an abnormally immature morphology. Furthermore, FMRP-deficient NSCs exhibited aberrant oscillatory Ca2+ responses to glutamate, which were specifically abolished by an antagonist of the mGluR5 receptor. The data suggested alterations in glutamatergic differentiation of FMRP-deficient NSCs and were further supported by an accumulation of cells committed to glutamatergic lineage in the subventricular zone of the embryonic Fmr1-knockout (Fmr1-KO) neocortex. Postnatally, the aberrant cells likely contributed to abnormal formation of the neocortex. The findings suggested a defect in the differentiation of distinct glutamatergic mGluR5 responsive cells in the absence of functional FMRP. Furthermore, we found that in the early postnatal Fmr1-KO mouse brain, the expression of mRNA for regulator of G-protein signalling-4 (RGS4) was decreased which was in line with disturbed G-protein signalling in NSCs lacking FMRP. Brain derived neurotrophic factor (BDNF) promotes neuronal differentiation of NSCs as the absence of FMRP was shown to do. This led us to study the effect of impaired BDNF/TrkB receptor signaling on NSCs by overexpression of TrkB.T1 receptor isoform. We showed that changes in the relative expression levels of the full-length and truncated TrkB isoforms influenced the replication capacity of NSCs. After the differentiation, the overexpression of TrkB.T1 increased neuronal turnover. To summarize, FMRP and TrkB signaling are involved in normal differentiation of NSCs in the developing brain. Since NSCs might have potential for therapeutic interventions in a variety of neurological disorders, our findings may be useful in the design of pharmacological interventions in neurological disorders of learning and memory.

Developmental-like responses in injured mature central neurons

Traumatic insults to the central nervous system are frequently followed by profound and irreversible neuronal loss as well as the inability of the damaged neurons to regenerate. One of the major therapeutic challenges is to increase the amount of surviving neurons after trauma. Thus it is crucial to understand how injury affects neuronal responses and which conditions are optimal for survival to prevent neuronal loss. During development neuronal survival is thought to be dependent on the competition for the availability of survival-promoting molecules called neurotrophic factors. Much less is known on the survival mechanisms of mature neurons under traumatic conditions. Increasing amount of evidence points towards the possibility that after injury neuronal responses might aquire some developmental characteristics. One of the important examples is the change in the responses to the neurotransmitter GABA: it is inhibitory in the intact mature neurons, but can induce excitation during development and after trauma. An important step in the maturation of GABAergic transmission in the CNS is the developmental shift in the action of GABAA receptor from depolarization in immature neurons to hyperpolarization in mature neurons. GABAA-mediated responses are tightly linked to the homeostasis of the chloride anion (Cl-), which in neurons is mainly regulated by Na+-K+-2Cl- cotransporter NKCC1 and K+-Cl- cotransporter KCC2. Trauma-induced functional downregulation of KCC2 promotes a shift from hyperpolarizing GABAA-mediated responses to depolarizing. Other important consequences of neuronal trauma are the emergence of dependency of central neurons on brain-derived neuro¬trophic factor (BDNF) for survival, as well as the upregulation of neurotrophin receptor p75NTR. Our aim was to answer the question whether these post-traumatic events are interrelated, and whether the regulation of BDNF and KCC2 expression is different under traumatic conditions and in intact neurons. To study responses of injured mature central neurons, we used an in vitro and in vivo axotomy models. For in vitro studies, we lesioned organotypic hippocampal slices between CA3 and CA1 regions, which resulted in selective axotomy of the CA3 neurons and denervation of the CA1 neurons. Some experiments were repeated in vivo by lesioning the neurons of the corticospinal tract at the internal capsule level, or by lesioning spinal motoneurons at the ventral root. We show that intact mature neurons do not require BDNF for survival, whereas in axotomized neurons apoptosis is induced upon BDNF deprivation. We further show that post-traumatic dependency on BDNF is mediated by injury-induced upregulation of p75NTR. Post-traumatic increase in p75NTR is induced by GABAA-mediated depolarization, consequent opening of voltage-gated Ca2+ channels, and the activation of Rho kinase ROCK. Thus, post-traumatic KCC2 downregulation leads to the dependency on BDNF through the induction of p75NTR upregulation. Neurons that survive after axotomy over longer period of time lose BDNF dependency and regain normal KCC2 levels. This phenomenon is promoted by BDNF itself, since after axotomy contrary to normal conditions KCC2 is upregulated by BDNF. The developmentally important thyroid hormone thyroxin regulates BDNF expression during development. We show that in mature intact neurons thyroxin downregulates BDNF, whereas after axotomy thyroxin upregulates BDNF. The elevation of BDNF expression by thyroxin promoted survival of injured neurons. In addition, thyroxin also enhanced axonal regeneration and promoted the regaining of normal levels of KCC2. Thus we show that this hormone acts at several levels on the axotomy-initiated chain of events described in the present work, and could be a potential therapeutic agent for the injured neurons. We have also characterized a previously unknown downregulatory interaction between thyroxin and KCC2 in intact neurons. In conclusion, we identified several important interactions at the neurotrophin-protein and hormone-neurotrophin level that acquire immature-like characteristics after axotomy and elucidated an important part of the mechanism by which axotomy leads to the requirement of BDNF trophic support. Based on these findings, we propose a new potential therapeutic strategy where developmentally crucial agents could be used to enhance survival and regeneration of axotomized mature central neurons.

Mechanisms of KCC2 upregulation during development

Gamma-aminobutyric acid (GABA) acting through ionotropic GABAA receptors plays a crucial role in the activity of the central nervous system (CNS). It triggers Ca2+ rise providing trophic support in developing neurons and conducts fast inhibitory function in mature neuronal networks. There is a developmental change in the GABAA reversal potential towards more negative levels during the first two postnatal weeks in rodent hippocampus. This change provides the basis for mature GABAergic activity and is attributable to the developmental expression of the neuron-specific potassium chloride cotransporter 2 (KCC2). In this work we have studied the mechanisms responsible for the control of KCC2 developmental expression. As a model system we used hippocampal dissociated cultures plated from embryonic day (E) 17 mice embryos before the onset of KCC2 expression. We showed that KCC2 was significantly up-regulated during the first two weeks of culture development. Interestingly, the level of KCC2 upregulation was not altered by chronic pharmacological blockage of action potentials as well as GABAergic and glutamatergic synaptic transmission. By in silico analysis of the proximal KCC2 promoter region we identified 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. One of these transcription factors, namely early growth response factor 4 (Egr4), had similar developmental profile as KCC2 and considerably increased the activity of mouse KCC2 gene in neuronal cells. Next we investigated the involvement of neurotrophic factors in regulation of Egr4 and KCC2 expression. We found that in immature hippocampal cultures Egr4 and KCC2 levels were strongly up-regulated by brain derived neurotrophic factor (BDNF)and neurturin. The effect of neurotrophic factors was dependent on the activation of a mitogen activated protein kinase (MAPK) signal transduction pathway. Intact Egr4-binding site in proximal KCC2 promoter was required for BDNF-induced KCC2 transcription. In vitro data were confirmed by several in vivo experiments where we detected an upregulation of KCC2 protein levels after intrahippocampal administration of BDNF or neurturin. Importantly, a MAPK-dependent rise in Egr4 and KCC2 expression levels was also observed after a period of kainic acid-induced seizure activity in neonatal rats suggesting that neuronal activity might be involved in Egr4-mediated regulation of KCC2 expression. Finally we demonstrated that the mammalian KCC2 gene (alias Slc12a5) generated two neuron-specific isoforms by using alternative promoters and first exons. A novel isoform of KCC2, termed KCC2a, differed from the previously known KCC2b isoform by 40 unique N-terminal amino acid residues. KCC2a expression was restricted to CNS,remained relatively constant during postnatal development, and contributed 20 50% of total KCC2 mRNA expression in the neonatal mouse brainstem and spinal cord. In summary, our data provide insight into the complex regulation of KCC2 expression during early postnatal development. Although basal KCC2 expression seems to be intrinsically regulated, it can be further augmented by neurotrophic factors or by enhanced activity triggering MAPK phosphorylation and Egr4 induction. Additional KCC2a isoform, regulated by another promoter, provides basal KCC2 level in neonatal brainstem and spinal cord required for survival of KCC2b knockout mice.

Novel CDNF/MANF protein family : Molecular structure, expression and neurotrophic activity

Neurotrophic factors (NTFs) are secreted proteins which promote the survival of neurons, formation and maintenance of neuronal contacts and regulate synaptic plasticity. NTFs are also potential drug candidates for the treatment of neurodegenerative diseases. Parkinson’s disease (PD) is mainly caused by the degeneration of midbrain dopaminergic neurons. Current therapies for PD do not stop the neurodegeneration or repair the affected neurons. Thus, search of novel neurotrophic factors for midbrain dopaminergic neurons, which could also be used as therapeutic proteins, is highly warranted. In the present study, we identified and characterized a novel protein named conserved dopamine neurotrophic factor (CDNF), a homologous protein to mesencephalic astrocyte-derived neurotrophic factor (MANF). Others have shown that MANF supports the survival of embryonic midbrain dopaminergic neurons in vitro, and protects cultured cells against endoplasmic reticulum (ER) stress. CDNF and MANF form a novel evolutionary conserved protein family with characteristic eight conserved cysteine residues in their primary structure. The vertebrates have CDNF and MANF encoding genes, whereas the invertebrates, including Drosophila and Caenorhabditis have a single homologous CDNF/MANF gene. In this study we show that CDNF and MANF are secreted proteins. They are widely expressed in the mammalian brain, including the midbrain and striatum, and in several non-neuronal tissues. We expressed and purified recombinant human CDNF and MANF proteins, and tested the neurotrophic activity of CDNF on midbrain dopaminergic neurons using a 6-hydroxydopamine (6-OHDA) rat model of PD. In this model, a single intrastriatal injection of CDNF protected midbrain dopaminergic neurons and striatal dopaminergic fibers from the 6-OHDA toxicity. Importantly, an intrastriatal injection of CDNF also restored the functional activity of the nigrostriatal dopaminergic system when given after the striatal 6-OHDA lesion. Thus, our study shows that CDNF is a potential novel therapeutic protein for the treatment of PD. In order to elucidate the molecular mechanisms of CDNF and MANF activity, we resolved their crystal structure. CDNF and MANF proteins have two domains; an amino (N)-terminal saposin-like domain and a presumably unfolded carboxy (C)-terminal domain. The saposin-like domain, which is formed by five α-helices and stabilized by three intradomain disulphide bridges, may bind to lipids or membranes. The C-terminal domain contains an internal cysteine bridge in a CXXC motif similar to that of thiol/disulphide oxidoreductases and isomerases, and may thus facilitate protein folding in the ER. Our studies suggest that CDNF and MANF are novel potential therapeutic proteins for the treatment of neurodegenerative diseases. Future studies will reveal the neurotrophic and cytoprotective mechanisms of CDNF and MANF in more detail.

GDNF Receptors: Veterans and Novices

Neurotrophic factors play essential role in the development and functioning of the nervous system and other organs. Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands (GFLs) are of particular interest because they promote the survival of dopaminergic neurons in vitro, in Parkinson s disease animal models and in patients. GDNF is also a potent survival factor for the central motoneurons and thus is considered as a potential lead for the treatment of amyotrophic lateral sclerosis. The survival promoting receptor complex for GFLs consists of a ligand-specific co-receptor, GFRα and a signal transducing module, receptor tyrosine kinase RET. At least GDNF and persephin, a GFL, have established functions outside central nervous system. GDNF is crucial for enteric nervous system and kidney development as well as for spermatogenesis. Persephin controls calcitonin secretion. Communication between cells often occurs in the extracellular matrix (ECM), a meshwork, which is secreted and deposited by the cells and is mainly composed of fibrillar proteins and polymerized sugars. We evaluated the relationship between GFLs and extracellular matrix components and demonstrated that three GFLs - GDNF, neurturin and artemin bind heparan sulfates with nanomolar affinities. The fourth member of the family - persephin binds these polysaccharides thousand times less tightly. GDNF, neurturin and artemin also bind with high affinity to heparan sulfate proteoglycan (HSPG) isolated from the nervous system, syndecan-3. GDNF signals through HSPGs, evoking Src family kinase activation. This signaling induces cell spreading, hippocampal neurite outgrowth in vitro and cellular migration. Specifically, GDNF signaling through syndecan-3 is important for embryonic cortical neuron migration. Syndecan-3-deficient mice, similarly to mice lacking GDNF, have less GABAergic neurons in their cortex, as compared to the wild-type mice. This fact provides indirect evidence that GDNF interaction with syndecan-3 is important for cortical brain development. Noteworthy, in non-neuronal tissues GFLs may signal via other syndecans. We also present the structural model for a GDNF co-receptor, GFRα1. The X-ray structure of the GFRα1 domain 3 was solved with 1.8 Å resolution, revealing a new protein fold. Later we also solved the structure of the truncated GFRα1 in the complex with GDNF and this model was confirmed by site-directed mutagenesis. In summary, our work contributed to the structural characterization of GFRα-based receptor complex and revealed a new receptor for GDNF, neurturin and artemin the HSPG syndecan-3. This information is critically important for the development of GFRα/RET agonists for the treatment of neurodegenerative diseases.

Neurotrophic factors and their receptors

Four GDNF ligands (GDNF, neurturin, artemin and persephin), and mesencephalic astrocyte-derived neurotrophic factor (MANF) and conserved dopamine neurotrophic factor (CDNF) protect midbrain dopaminergic neurons that degenerate in Parkinson's disease. Each GDNF ligand binds a specific coreceptor GDNF family receptor α (GFRα), leading to the formation of a heterotetramer complex, which then interacts with receptor tyrosine kinase RET, the signalling receptor. The present thesis describes the structural and biochemical characterization of the GDNF2-GFRα12 complex and the MANF and CDNF proteins. Previous and current mutation data and comparison between GDNF-GFRα1 and artemin-GFRα3 binding interfaces show that N162GFRα1, I175GFRα1, V230GFRα1, Y120GDNF and L114GDNF are the specificity determinants among different ligand-coreceptor pairs. The structure suggests that sucrose octasulphate, a heparin mimic, interacts with a region R190-K202 within domain 2 of GFRα1. Mutating these residues on the GFRα1 surface, which are not in the GDNF binding region, affected RET phosphorylation, which provides a putative RET binding region in domain 2 and 3 of GFRα1. The structural comparison of the GDNF-GFRα1 and artemin-GFRα3 complexes shows a difference in bend angle between the ligand monomers. This variation in bend angle of the ligand may affect the kinetics of RET phosphorylation. To confirm that the difference is not due to crystallization artefacts, I crystallized the GDNF-GFRα1 complex without SOS in different cell dimensions. The structure of the second GDNF-GFRα1 complex is very similar to the previous one, suggesting that the difference between the artemin-GFRα3 and GDNF-GFRα1 complexes are intrinsic, not due to crystal packing. Finally, MANF and CDNF are bifunctional proteins with extracellular neurotrophic activity and ER resident cytoprotective role. The crystal structures of MANF and CDNF are presented here. Intriguingly, the structures of both the neurotrophic factors do not show structural similarity to any of previously known growth factor superfamilies; instead they are similar to saposins, the lipid-binding proteins. The N-terminal domain of MANF and CDNF contain conserved lysines and arginines on its surface, which may interact with negatively charged head groups of phospholipids, as saposins do. Thus MANF and CDNF may provide neurotrophic activities by interacting with a lipo-receptor. The structure of MANF shows a CXXC motif forming internal disulphide bridge in the natively unfolded C-terminus. This motif is common to reductases and disulphide isomerases. It is thus tempting to speculate that the CXXC motif of MANF and CDNF may be involved in oxidative protein folding, which may explain its cytoprotective role in the ER.

Co-Signaling by Neurotrophic Factors and the Extracellular Matrix for Axonal Growth and Neuronal Survival

Neurotrophic factors (NTFs) and the extracellular matrix (ECM) are important regulators of axonal growth and neuronal survival in mammalian nervous system. Understanding of the mechanisms of this regulation is crucial for the development of posttraumatic therapies and drug intervention in the injured nervous system. NTFs act as soluble, target-derived extracellular regulatory molecules for a wide range of physiological functions including axonal guidance and the regulation of programmed cell death in the nervous system. The ECM determines cell adhesion and regulates multiple physiological functions via short range cell-matrix interactions. The present work focuses on the mechanisms of the action of NTFs and the ECM on axonal growth and survival of cultured sensory neurons from dorsal root ganglia (DRG). We first examined signaling mechanisms of the action of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) on axonal growth. GDNF, neurturin (NRTN) and artemin (ART) but not persephin (PSPN) promoted axonal initiation in cultured DRG neurons from young adult mice. This effect required Src family kinase (SFK) activity. In neurons from GFRalpha2-deficient mice, NRTN did not significantly promote axonal initiation. GDNF and NRTN induced extensive lamellipodia formation on neuronal somata and growth cones. This study suggested that GDNF, NRTN and ARTN may serve as stimulators of nerve regeneration under posttraumatic conditions. Consequently we studied the convergence of signaling pathways induced by NTFs and the ECM molecule laminin in the intracellular signaling network that regulates axonal growth. We demonstrated that co-stimulation of DRG neurons with NTFs (GDNF, NRTN or nerve growth factor (NGF)) and laminin leads to axonal growth that requires activation of SFKs. A different, SFK-independent signaling pathway evoked axonal growth on laminin in the absence of the NTFs. In contrast, axonal branching was regulated by SFKs both in the presence and in the absence of NGF. We proposed and experimentally verified a Boolean model of the signaling network triggered by NTFs and laminin. Our results put forward an approach for predictable, Boolean logics-driven pharmacological manipulation of a complex signaling network. Finally we found that N-syndecan, the receptor for the ECM component HB-GAM was required for the survival of neonatal sensory neurons in vitro. We demonstrated massive cell death of cultured DRG neurons from mice deficient in the N-syndecan gene as compared to wild type controls. Importantly, this cell death could not be prevented by NGF the neurotrophin which activates multiple anti-apoptotic cascades in DRG neurons. The survival deficit was observed during first postnatal week. By contrast, DRG neurons from young adult N-syndecan knock-out mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.

Structure and function of GDNF receptor alpha splice variants

The growth factors of the glial cell line-derived neurotrophic factor (GDNF) family consisting of GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN), are involved in the development, differentiation and maintenance of many types of neurons. They also have important functions outside the nervous system in the development of kidney, testis and thyroid gland. Each of these GFLs preferentially binds to one of the glycosylphosphatidylinositol (GPI)-anchored GDNF family receptors α (GFRα). GDNF binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3 and PSPN to GFRα4. The GFLs in the complex with their cognate GFRα receptors all bind to and signal through the receptor tyrosine kinase RET. Alternative splicing of the mouse GFRα4 gene yields three splice isoforms. These had been described as putative GPI-anchored, transmembrane and soluble forms. My goal was to characterise the function of the different forms of mouse GFRα4. I firstly found that the putative GPI-anchored GFRα4 (GFRα4-GPI) is glycosylated, membrane-bound, GPI-anchored and interacts with PSPN and RET. We also showed that mouse GFRα4-GPI mediates PSPN-induced phosphorylation of RET, promotes PSPN-dependent neuronal differentiation of the rat pheochromocytoma cell line PC6-3 and PSPN-dependent survival of cerebellar granule neurons (CGN). However, although this receptor can mediate PSPN-signalling and activate RET, GFRα4-GPI does not recruit RET into lipid rafts. The recruitment of RET into lipid rafts has previously been thought to be a crucial event for GDNF- and GFL-mediated signalling via RET. I secondly demonstrated that the putative transmembrane GFRα4 (GFRα4-TM) is indeed a real transmembrane GFRα4 protein. Although it has a weak binding capacity for PSPN, it can not mediate PSPN-dependent phosphorylation of RET, neuronal differentiation or survival. These data show that GFRα4-TM is inactive as a receptor for PSPN. Surprisingly, GFRα4-TM can negatively regulate PSPN-mediated signalling via GFRα4-GPI. GFRα4-TM interacts with GFRα4-GPI and blocks PSPN-induced phosphorylation of RET, neuronal differentiation as well as survival. Taken together, our data show that GFRα4-TM may act as a dominant negative inhibitor of PSPN-mediated signaling. The most exciting part of my work was the finding that the putative soluble GFRα4 (GFRα4-sol) can form homodimers and function as an agonist of the RET receptor. In the absence of PSPN, GFRα4-sol can promote the phosphorylation of RET, trigger the activation of the PI-3K/AKT pathway, induce neuronal differentiation and support the survival of CGN. Our findings are in line with a recent publication showing the GFRα4-sol might contribute to the inherited cancer syndrome multiple endocrine neoplasia type 2. Our data provide an explanation to how GFRα4-sol may cause or modify the disease. Mammalian GFRα4 receptors all lack the first Cys-rich domain which is present in other GFRα receptors. In the final part of my work I have studied the function of this particular domain. I created a truncated GFRα1 construct lacking the first Cys-rich domain. Using binding assays in both cellular and cell-free systems, phosphorylation assays with RET, as well as neurite outgrowth assays, we found that the first Cys-rich domain contributes to an optimal function of GFRα1, by stabilizing the interaction between GDNF and GFRα1.

Role of GDNF and its Cross-Talk with Other Growth Factors in the Dopaminergic System

Parkinson´s Disease (PD) is a neurodegenerative movement disorder resulting from loss of dopaminergic (DA) neurons in substantia nigra (SN). Possible causative treatment strategies for PD include neurotrophic factors, which protect and in some cases restore the function of dopaminergic neurons. Glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors have been to date the most promising candidates for treatment of PD, demonstrating both neuroprotective and neurorestorative properties. We have investigated the role of GDNF in the rodent dopaminergic system and its possible crosstalk with other growth factors. We characterized the GDNF-induced gene expression changes by DNA microarray analysis in different neuronal systems, including in vitro cultured Neuro2A cells treated with GDNF, as well as midbrains from GDNF heterozygous (Hz) knockout mice. These microarray experiments, resulted in the identification of GDNF-induced genes, which were also confirmed by other methods. Further analysis of the dopaminergic system of GDNF Hz mice demonstrated about 40% reduction in GDNF levels, revealed increased intracellular dopamine concentrations and FosB/DeltaFosB expression in striatal areas. These animals did not show any significant changes in behavioural analysis of acute and repeated cocaine administration on locomotor activity, nor did they exhibit any changes in dopamine output following treatment with acute cocaine. We further analysed the significance of GDNF receptor RET signalling in dopaminergic system of MEN2B knock-in animals with constitutively active Ret. The MEN2B animals showed a robust increase in extracellular dopamine and its metabolite levels in striatum, increased tyrosine hydroxylase (TH) and dopamine transporter (DAT) protein levels by immunohistochemical staining and Western blotting, as well as increased Th mRNA levels in SN. MEN2B mice had increased number of DA neurons in SN by about 25% and they also exhibited increased sensitivity to the stimulatory effects of cocaine. We also developed a semi-throughput in vitro micro-island assay for the quantification of neuronal survival and TH levels by computer-assisted methodology from limited amounts of tissue. This assay can be applied for the initial screening for dopaminotrophic molecules, as well as chemical drug library screening. It is applicable to any neuronal system for the screening of neurotrophic molecules. Since our microarray experiments revealed possible GDNF-VEGF-C crosstalk we further concentrated on studying the neurotrophic effects of VEGF-C. We showed that VEGF-C acts as a neurotrophic molecule for the DA neurons both in vitro and in vivo, however without additive effect when used together with GDNF. The neuroprotective effect for VEGF-C in vivo in rat 6-OHDA model of PD was demonstrated. The possible signalling mechanisms of VEGF-C in the nervous system were investigated - infusion of VEGF-C to rat brain induced ERK activation, however no direct activation of RET signalling in vitro was found. VEGF-C treatment of rat striatum lead to up-regulation of VEGFR-1-3, indicating that VEGF-C can regulate the expression level of its own receptor. VEGF-C dopaminotrophic activity in vivo was further supported by increased vascular tissue in the neuroprotection experiments.