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.

Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.

Molecular Genetic Studies of Melanocortin Receptors in Morbid Obesity

The prevalence of obesity is increasing at an alarming rate in all age groups worldwide. Obesity is a serious health problem due to increased risk of morbidity and mortality. Although environmental factors play a major role in the development of obesity, the identification of rare monogenic defects in human genes have confirmed that obesity has a strong genetic component. Mutations have been identified in genes encoding proteins of the leptin-melanocortin signaling system, which has an important role in the regulation of appetite and energy balance. The present study aimed at identifying mutations and genetic variations in the melanocortin receptors 2-5 and other genes active on the same signaling pathway accounting for severe early-onset obesity in children and morbid obesity in adults. The main achievement of this thesis was the identification of melanocortin-4 receptor (MC4R) mutations in Finnish patients. Six pathogenic MC4R mutations (308delT, P299H, two S127L and two -439delGC mutations) were identified, corresponding to a prevalence of 3% in severe early-onset obesity. No obesity causing MC4R mutations were found among patients with adult-onset morbid obesity. The MC4R 308delT deletion is predicted to result in a grossly truncated nonfunctional receptor of only 107 amino acids. The C-terminal residues, which are important in MC4R cell surface targeting, are totally absent from the mutant 308delT receptor. In vitro functional studies supported a pathogenic role for the S127L mutation since agonist induced signaling of the receptor was impaired. Cell membrane localization of the S127L receptor did not differ from that of the wild-type receptor, confirming that impaired function of the S127L receptor was due to reduced signaling properties. The P299H mutation leads to intracellular retention of the receptor. The -439delGC deletion is situated at a potential nescient helix-loop-helix 2 (NHLH2) -binding site in the MC4R promoter. It was demonstrated that the transcription factor NHLH2 binds to the consensus sequence at the -439delGC site in vitro, possibly resulting in altered promoter activity. Several genetic variants were identified in the melanocortin-3 receptor (MC3R) and pro-opiomelanocortin (POMC) genes. These polymorphisms do not explain morbid obesity, but the results indicate that some of these genetic variations may be modifying factors in obesity, resulting in subtle changes in obesity-related traits. A risk haplotype for obesity was identified in the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) gene through a candidate gene single nucleotide polymorphism (SNP) genotyping approach. An ENPP1 haplotype, composed of SNPs rs1800949 and rs943003, was shown to be significantly associated with morbid obesity in adults. Accordingly, the MC3R, POMC and ENPP1 genes represent examples of susceptibility genes in which genetic variants predispose to obesity. In conclusion, pathogenic mutations in the MC4R gene were shown to account for 3% of cases with severe early-onset obesity in Finland. This is in line with results from other populations demonstrating that mutations in the MC4R gene underlie 1-6% of morbid obesity worldwide. MC4R deficiency thus represents the most common monogenic defect causing human obesity reported so far. The severity of the MC4-receptor defect appears to be associated with time of onset and the degree of obesity. Classification of MC4R mutations may provide a useful tool when predicting the outcome of the disease. In addition, several other genetic variants conferring susceptibility to obesity were detected in the MC3R, MC4R, POMC and ENPP1 genes.

The function of NR3B and NR4A orphan nuclear receptors in osteoblasts

Nuclear receptors (NRs) comprise a large family of proteins that mediate the effects of small lipophilic molecules such as steroid hormones. In addition, there are a group of NRs which lack identified natural ligands and are referred as orphan NRs. In this thesis, the function of two such orphan NR families, the NR3B (ERRα, ERRβ and ERRγ) and the NR4A family (NGFI-B, Nurr1 and Nor1), was studied. NR3B and NR4A receptors regulate many biological processes such as energy metabolism and carcinogenesis. In addition, NR3B and NR4A receptors are expressed in bone. Therefore, the signaling and function of NR3B and NR4A orphan nuclear receptors was studied specifically in osteoblasts. NR4A receptors were found to be regulated by NR3B receptors and the Wnt/β-catenin signaling pathway as ERRα, ERRγ and β-catenin repressed the transcriptional activity of NR4A receptors in U2-OS cells. NGFI-B was found to repress the transcriptional activity of ERRγ in HeLa cells. The phytoestrogen equol was identified as a new agonist for ERRγ and ERRβ in PC-3, U2-OS, and SaOS-2 cells. Equol increased the transcriptional activity of ERRγ by increasing ERRγ co-activator binding and by inducing a conformational change in the ligand binding pocket of ERRγ. The growth inhibitory effect of equol on PC-3 prostate cancer cells was decreased by blocking ERRγ expression by siRNA. Therefore, ERRγ could mediate some of the beneficial health effects of equol. The Wnt/β-catenin signaling pathway is important for the differentiation and function of osteoblasts. NR3B and NR4A receptors were found to repress the transcriptional activity mediated by β-catenin in U2-OS cells. The mesenchymal stem cells (MSCs) isolated from ERRα knockout (KO) mice showed diminished proliferation and osteoblastic differentiation compared to the wild-type cells. The overexpression of ERRα in osteoblastic MC3T3-E1 cell line increased their mineralization. Bone sialoprotein (BSP) was shown to be a direct target gene for ERRα and ERRγ as the BSP promoter was activated by ERRα or ERRγ and PGC-1α in HeLa cells. The adipogenic differentiation of ERRα KO MSCs was also decreased and they expressed less adipogenic marker genes. In conclusion, the studies described in this thesis demonstrated that the transcriptional activity of NR3B and NR4A receptors can be regulated by other orphan NRs and signaling pathways in osteoblasts. NR3B receptors can also be regulated by ligands and a new agonist, equol, was identified for ERRβ and ERRγ. New roles for NR3B and NR4A were also identified as they were shown to converge with the Wnt signaling pathway in osteoblasts, ERRγ was shown to mediate the growth inhibitory effect of equol in prostate cancer cells, and ERRα was shown to regulate positively MSC proliferation, osteoblastic differentiation and adipogenesis.

Nikotiinireseptoreiden kautta vaikuttavat lääkeaineet nikotiiniriippuvuuden ja neurologisten sairauksien hoidossa

Neuronaaliset nikotiinireseptorit liittyvät tupakkariippuvuuden lisäksi moniin neurologisiin sairauksiin, kuten Alzheimerin tautiin, skitsofreniaan, masennukseen ja tarkkaavaisuus- ja ylivilkkaushäiriöön. Nikotiinireseptorien stimulaation on tutkimuksissa havaittu parantavan kognitiota. Useat lääkeyritykset tutkivat nikotiinireseptoriagonisteja ja -antagonisteja eri neurologisten sairauksien hoidossa. Ongelmana nikotiinireseptori-agonisteja käytettäessä on reseptorissa tapahtuva desensitisaatio. Tällöin reseptori sulkeutuu, eikä aktivoidu vaikka agonistia olisi tarjolla tai sitoutuneena reseptoriin. Varsinkin alfa7-reseptori desensitoituu hyvin nopeasti agonistialtistuksen seurauksena. Reseptorien desensitoituminen voi kliinisessä käytössä aiheuttaa lääkeaineen tehon menetyksen. Perinteisen agonistin sitoutumiskohdan lisäksi nikotiinireseptorissa sijaitsee myös muita sitoutumiskohtia, joita kutsutaan allosteerisiksi sitoutumispaikoiksi. Tutkimuksissa on havaittu, että eräät allosteerisesti sitoutuvat aineet, kuten PNU-120596, voivat vahvistaa agonistin aikaansaamaa vastetta ja/tai estää reseptorin desensitoitumista. Näitä aineita kutsutaan positiivisiksi allosteerisiksi modulaattoreiksi ja niiden ajatellaan olevan vaihtoehto desensitoitumisen aiheuttamaan tehon menetyksen ongelmaan. Nikotiinireseptorien positiivisten allosteeristen modulaattorien tarkkaa vaikutusta ja sitoutumiskohtaa reseptoriin ei vielä tarkkaan tiedetä. Tutkimuksen aiheena oli karakterisoida positiivisten allosteeristen modulaattoreiden vaikutuksia alfa7-nikotiinireseptoriin. Tutkimuksessa tarkoituksena oli käyttää hyväksi laboratoriossa aiemmin tehtyä havaintoa, jonka mukaan alfa7-nikotiinireseptorin transmembraaniosan aminohappoon tehdyn mutaation L247T seurauksena positiiviset allosteeriset modulaattorit muuttuvat agonisteiksi. Haluttiin selvittää, kuinka agonistin sitoutumiskohtaan kohdennettua mutageneesiä käyttäen tehty mutaatio W149M tai W149F vaikuttavat PNU-120596:n kykyyn toimia agonistina alfa7L247T reseptoriin. Asetyylikoliini toimi konventionaalisen agonistin mallina tutkimuksessa. Tutkimuksen toinen tavoite oli tehdä mutaatio M253Lalfa7-reseptorin transmembraaniosaan. Mutaation on todettu estävän allosteeristen potentiaattoreiden kykyä voimistaa agonistin aikaansaamaa vastetta. Tarkoitus oli tutkia millaisia vaikutuksia M253L-mutaatiolla on allosteerisen potentiaattorin kykyyn toimia agonistina L247T-mutaation sisältävään reseptoriin. Mutatoidun reseptorin mRNA mikroinjektoitiin oosyyttiin ja elektrofysiologian avulla tutkittiin ilmennettyjen reseptorien toimintaa käyttäen kahden elektrodin jännitelukitus -menetelmää. Kaikki suunnitellut mutaatiot saatiin tehtyä onnistuneesti alfa7- ja alfa7L247T-reseptoreihin. Ortosteerisen sitoutumiskohdan mutaatio villin tyypin Į7-reseptorissa vaikutti hyvin voimakkaasti joko asetyylikoliinin sitoutumiseen reseptoriin tai reseptorin toimintaan, sillä asetyylikoliinilla ei reseptorista saatu mitattua vasteita. Myöskään PNU-120596 yksinään ei saanut aikaan vasteita alfa7W149M-reseptorissa. Kaksoismutatoidussa alfa7W149M/L247T-reseptorissa puolestaan havaittiin, että asetyylikoliinin annos-vaste -kuvaaja siirtyi huomattavasti enemmän oikealle kuin PNU-120596:n, kun verrattiin annos-vaste –kuvaajia alfa7L247T ja alfa7W149M/L247T–reseptoreiden välillä. Transmembraaniosan mutaatio M253L ei vaikuttanut PNU-120596:n kykyyn toimia agonistina alfa7L247T-reseptoriin, eikä sillä ollut vaikutusta asetyylikoliinin annosvaste-kuvaajiin. Tutkimus tukee aiempia havaintoja siitä, että positiivisten allosteeristen modulaattoreiden sitoutumiskohta nikotiinireseptorissa sijaitsisi transmembraaniosassa. M253L-mutaation osalta tulokset ovat hieman ristiriidassa aiempien tulosten kanssa. L247T-mutaatio vaikuttaa hyvin voimakkaasti nikotiinireseptorin toimintaan sekä sijaitsee aminohapon M253 läheisyydessä. On mahdollista, että se peittää M253L-mutaation vaikutuksen. Toisaalta voi olla, että M253 on aminohappo, joka vaikuttaa vain reseptorivasteiden voimistumiseen eikä allosteeristen potentiaattoreiden sitoutumiseen.

Functional expression and subcellular localization of the Cl- cotransporters KCC2 and NKCC1 in rodent hippocampal and neocortical neurons

The work presented here has focused on the role of cation-chloride cotransporters (CCCs) in (1) the regulation of intracellular chloride concentration within postsynaptic neurons and (2) on the consequent effects on the actions of the neurotransmitter gamma-aminobutyric acid (GABA) mediated by GABAA receptors (GABAARs) during development and in pathophysiological conditions such as epilepsy. In addition, (3) we found that a member of the CCC family, the K-Cl cotransporter isoform 2 (KCC2), has a structural role in the development of dendritic spines during the differentiation of pyramidal neurons. Despite the large number of publications dedicated to regulation of intracellular Cl-, our understanding of the underlying mechanisms is not complete. Experiments on GABA actions under resting steady-state have shown that the effect of GABA shifts from depolarizing to hyperpolarizing during maturation of cortical neurons. However, it remains unclear, whether conclusions from these steady-state measurements can be extrapolated to the highly dynamic situation within an intact and active neuronal network. Indeed, GABAergic signaling in active neuronal networks results in a continuous Cl- load, which must be constantly removed by efficient Cl- extrusion mechanisms. Therefore, it seems plausible to suggest that key parameters are the efficacy and subcellular distribution of Cl- transporters rather than the polarity of steady-state GABA actions. A further related question is: what are the mechanisms of Cl- regulation and homeostasis during pathophysiological conditions such as epilepsy in adults and neonates? Here I present results that were obtained by means of a newly developed method of measurements of the efficacy of a K-Cl cotransport. In Study I, the developmental profile of KCC2 functionality during development was analyzed both in dissociated neuronal cultures and in acute hippocampal slices. A novel method of photolysis of caged GABA in combination with Cl- loading to the somata was used in this study to assess the extrusion efficacy of KCC2. We demonstrated that these two preparations exhibit a different temporal profile of functional KCC2 upregulation. In Study II, we reported an observation of highly distorted dendritic spines in neurons cultured from KCC2-/- embryos. During their development in the culture dish, KCC2-lacking neurons failed to develop mature, mushroom-shaped dendritic spines but instead maintained an immature phenotype of long, branching and extremely motile protrusions. It was shown that the role of KCC2 in spine maturation is not based on its transport activity, but is mediated by interactions with cytoskeletal proteins. Another important player in Cl- regulation, NKCC1 and its role in the induction and maintenance of native Cl- gradients between the axon initial segment (AIS) and soma was the subject of Study III. There we demonstrated that this transporter mediates accumulation of Cl- in the axon initial segment of neocortical and hippocampal principal neurons. The results suggest that the reversal potential of the GABAA response triggered by distinct populations of interneurons show large subcellular variations. Finally, a novel mechanism of fast post-translational upregulation of the membrane-inserted, functionally active KCC2 pool during in-vivo neonatal seizures and epileptiform-like activity in vitro was identified and characterized in Study IV. The seizure-induced KCC2 upregulation may act as an intrinsic antiepileptogenic mechanism.