Browsing by Subject "GDNF"

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  • Ardashov, Oleg V.; Pavlova, Alla V.; Mahato, Arun Kumar; Sidorova, Yulia; Morozova, Ekaterina A.; Korchagina, Dina V.; Salnikov, Georgi E.; Genaev, Alexander M.; Patrusheva, Oksana S.; Li-Zhulanov, Nikolay S.; Tolstikova, Tat'yana G.; Volcho, Konstantin P.; Salakhutdinov, Nariman. F. (2019)
    We previously showed that monoterpenoid (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 alleviates motor manifestations of Parkinson's disease in animal models. In the present study, we designed and synthesized monoepoxides of (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl)cyclohex-3-ene-1,2-diol 1 and evaluated their biological activity in the MPTP mouse model of Parkinson's disease. We also assessed the ability of these compounds to penetrate the blood-brain barrier (BBB). According to these data, we chose epoxide 4, which potently restored the locomotor activity in MPTP-treated mice and efficiently penetrated the BBB, to further explore its potential mechanism of action. Epoxide 4 was found to robustly promote the survival of cultured dopamine neurons, protect dopamine neurons against toxin-induced degeneration, and trigger the mitogen-activated protein kinase (MAPK) signaling cascade in cells of neuronal origin. Meanwhile, neither the survival-promoting effect nor MAPK activation was observed in non-neuronal cells treated with epoxide 4. In the MPTP mouse model of Parkinson's disease, compound 4 increased the density of dopamine neuron fibers in the striatum, which can highlight its potential to stimulate striatal reinnervation and thus halt disease progression. Taken together, these data indicate that epoxide 4 can be a promising compound for further development, not only as a symptomatic but also as a neuroprotective and neurorestorative drug for Parkinson's disease.
  • Albert, Katrina; Voutilainen, Merja H.; Domanskyi, Andrii; Airavaara, Mikko (2017)
    Gene delivery using adeno-associated virus (AAV) vectors is a widely used method to transduce neurons in the brain, especially due to its safety, efficacy, and long-lasting expression. In addition, by varying AAV serotype, promotor, and titer, it is possible to affect the cell specificity of expression or the expression levels of the protein of interest. Dopamine neurons in the substantia nigra projecting to the striatum, comprising the nigrostriatal pathway, are involved in movement control and degenerate in Parkinson's disease. AAV-based gene targeting to the projection area of these neurons in the striatum has been studied extensively to induce the production of neurotrophic factors for disease-modifying therapies for Parkinson's disease. Much less emphasis has been put on AAV-based gene therapy targeting dopamine neurons in substantia nigra. We will review the literature related to targeting striatum and/or substantia nigra dopamine neurons using AAVs in order to express neuroprotective and neurorestorative molecules, as well as produce animal disease models of Parkinson's disease. We discuss difficulties in targeting substantia nigra dopamine neurons and their vulnerability to stress in general. Therefore, choosing a proper control for experimental work is not trivial. Since the axons along the nigrostriatal tract are the first to degenerate in Parkinson's disease, the location to deliver the therapy must be carefully considered. We also review studies using AAV--synuclein (-syn) to target substantia nigra dopamine neurons to produce an -syn overexpression disease model in rats. Though these studies are able to produce mild dopamine system degeneration in the striatum and substantia nigra and some behavioural effects, there are studies pointing to the toxicity of AAV-carrying green fluorescent protein (GFP), which is often used as a control. Therefore, we discuss the potential difficulties in overexpressing proteins in general in the substantia nigra.
  • Huttunen, Henri J.; Saarma, Mart (2019)
    Neurotrophic factors (NTF) are a subgroup of growth factors that promote survival and differentiation of neurons. Due to their neuroprotective and neurorestorative properties, their therapeutic potential has been tested in various neurodegenerative diseases. Bioavailability of NTFs in the target tissue remains a major challenge for NTF-based therapies. Various intracerebral delivery approaches, both protein and gene transfer-based, have been tested with varying outcomes. Three growth factors, glial cell-line derived neurotrophic factor (GDNF), neurturin (NRTN) and platelet-derived growth factor (PDGF-BB) have been tested in clinical trials in Parkinson?s Disease (PD) during the past 20 years. A new protein can now be added to this list, as cerebral dopamine neurotrophic factor (CDNF) has recently entered clinical trials. Despite their misleading names, CDNF, together with its closest relative mesencephalic astrocyte-derived neurotrophic factor (MANF), form a novel family of unconventional NTF that are both structurally and mechanistically distinct from other growth factors. CDNF and MANF are localized mainly to the lumen of endoplasmic reticulum (ER) and their primary function appears to be modulation of the unfolded protein response (UPR) pathway. Prolonged ER stress, via the UPR signaling pathways, contributes to the pathogenesis in a number of chronic degenerative diseases, and is an important target for therapeutic modulation. Intraputamenally administered recombinant human CDNF has shown robust neurorestorative effects in a number of small and large animal models of PD, and had a good safety profile in preclinical toxicology studies. Intermittent monthly bilateral intraputamenal infusions of CDNF are currently being tested in a randomized placebo-controlled phase I?II clinical study in moderately advanced PD patients. Here, we review the history of growth factor-based clinical trials in PD, and discuss how CDNF differs from the previously tested growth factors.
  • Nuotio, Ulpukka (Helsingfors universitet, 2017)
    Neuropathic pain is pain caused by injury or damage to the nervous system. This adverse condition affects millions of people in all parts of the world, and no known cure has been developed. Existing treatments are mainly anti-depressants or opioids that alleviate symptoms instead of repairing damaged neurons. Glial cell line-derived neurotrophic factor (GDNF) and artemin, belonging to GDNF family ligands, have been shown to restore damaged neurons. However due to the poor pharmaceutical properties of these proteins, such as difficult administration and expensive production, their transition to clinics is complicated. That is why we have been developing small molecule GFL-mimetics as an alternative. One of these mimetics is a compound named BT44. Characterization of BT44 began with in vitro experiments, where we tested the compound’s ability to activate luciferase reporter gene in cells expressing GDNF (GFRalpha1 and RET) and artemin (GFRalpha3 and RET) receptors, as well as ability to induce RET phosphorylation and activate intracellular MAPK/ERK and Pi3-K/Akt pathways. Furthermore, we tested stimulation of neurite outgrowth by the compound from cultured dorsal root ganglion neurons. In a similar manner to GDNF and artemin, BT44 was shown to activate GFRalpha1/RET and GFRalpha3/RET receptors and induce RET phosphorylation and intracellular signaling, in addition to stimulating neurite outgrowth from cultured DRG neurons. Because of the promising in vitro results, we moved on to in vivo testing in rat spinal nerve ligation (SNL) model of neuropathic pain. Similarly to artemin, BT44 was able to alleviate mechanical nociception and cold allodynia in SNL rats. In addition, we found that BT44 normalized to a certain degree nociception-related markers influenced by SNL in the tissues of experimental animals, which emulates previously published results for artemin. To summarize, our results indicate that BT44 is effective in neuronal restoration and pain alleviation, suggesting it for further development as innovative neuropathic pain treatment.
  • Turconi, Giorgio; Kopra, Jaakko; Võikar, Vootele; Kulesskaya, Natalia; Vilenius, Carolina; Piepponen, T. Petteri; Andressoo, Jaan-Olle (2020)
    Glial cell line-derived neurotrophic factor (GDNF) supports function and survival of dopamine neurons that degenerate in Parkinson's disease (PD). Ectopic delivery of GDNF in clinical trials to treat PD is safe but lacks significant therapeutic effect. In pre-clinical models, ectopic GDNF is effective but causes adverse effects, including downregulation of tyrosine hydroxylase, only a transient boost in dopamine metabolism, aberrant neuronal sprouting, and hyperactivity. Hindering development of GDNF mimetic increased signaling via GDNF receptor RET by activating mutations results in cancer. Safe and effective mode of action must be defined first in animal models to develop successful GDNF-based therapies. Previously we showed that about a 2-fold increase in endogenous GDNF expression is safe and results in increased motor and dopaminergic function and protection in a PD model in young animals. Recently, similar results were reported using a novel Gdnf mRNA-targeting strategy. Next, it is important to establish the safety of a long-term increase in endogenous GDNF expression. We report behavioral, dopamine system, and cancer analysis of five cohorts of aged mice with a 2-fold increase in endogenous GDNF. We found a sustained increase in dopamine levels, improvement in motor learning, and no side effects or cancer. These results support the rationale for further development of endogenous GDNF-based treatments and GDNF mimetic.
  • Huotarinen, Antti; Penttinen, Anna-Maija; Bäck, Susanne; Voutilainen, Merja H.; Julku, Ulrika; Piepponen, T. Petteri; Männistö, Pekka T.; Saarma, Mart; Tuominen, Raimo; Laakso, Aki; Airavaara, Mikko (2018)
    Several neurotrophic factors ( NTF) are shown to be neuroprotective and neurorestorative in pre-clinical animal models for Parkinson's disease ( PD), particularly in models where striatal dopamine neuron innervation partially exists. The results of clinical trials on late-stage patients have been modest. Subthalamic deep brain stimulation ( STN DBS) is a proven treatment for a selected group of advanced PD patients. The cerebral dopamine neurotrophic factor ( CDNF) is a promising therapeutic protein, but its effects in animal models of late-stage PD have remained under-researched. The interactions of NTF and STN DBS treatments have not been studied before. We found that a nigral CDNF protein alone had only a marginal effect on the behavioral deficits in a late-stage hemiparkinsonian rat model ( 6-OHDA MFB). However, CDNF improved the effect of acute STN DBS on front limb use asymmetry at 2 and 3 weeks after CDNF injection. STN lesion-modeling chronic stimulation-had an additive effect in reducing front limb use in the cylinder test and apomorphine-induced rotation. The combination of CDNF and acute STN DBS had a favorable effect on striatal tyrosine hydroxylase. This study presents a novel additive beneficial effect of NTF and STN DBS, which might be explained by the interaction of DBS-induced endogenous NTFs and exogenously injected CDNF. SNpc can be reached via similar trajectories used in clinical STN DBS, and this interaction is an important area for future studies. (C) 2018 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/).
  • Kopra, Jaakko; Villarta-Aguilera, Marian; Savolainen, Mari; Weingerl, Samo; Myohänen, Timo T.; Rannanpää, Saara; Salvatore, Michael E.; Andressoo, Jaan-Olle; Piepponen, T. Petteri (2018)
    Addictive drugs enhance dopamine release in the striatum, which can lead to compulsive drug-seeking after repeated exposure. Glial cell line-derived neurotrophic factor (GDNF) is an important regulator of midbrain dopamine neurons, and may play a mechanistic role in addiction-related behaviors. To elucidate the components of GDNF-signaling that contribute to addiction-related behaviors of place preference and its extinction, we utilized two genetically modified GDNF mouse models in an amphetamine induced conditioned place preference (CPP) paradigm and evaluated how the behavioral findings correlate with dopamine signaling in the dorsal and ventral striatum. We utilized two knock-in mouse strains to delineate contributions of GDNF and Ret signaling using MEN2B mice (constitutively active GDNF receptor Ret), and GDNF hypermorphic mice (enhanced endogenous GDNF expression). The duration of amphetamine-induced CPP was greatly enhanced in MEN2B mice, but not in the GDNF hypermorphic mice. The enhanced duration of CPP was correlated with increased tyrosine hydroxylase (TH) expression and dopamine content in the ventral striatum. Together, our results suggest that downstream components of GDNF signaling, in this case Ret, may mediate persistent drug-seeking behavior through increased TH expression and dopamine levels in the mesolimbic dopamine neurons. (C) 2017 Elsevier Ltd. All rights reserved.
  • Hakosalo, Vili (Helsingin yliopisto, 2021)
    Parkinson’s disease (PD) is the second most common neurogenerative disease. There are no drugs available to halt the progression of PD. The glial cell line-derived neurotrophic factor (GDNF) has been identified as a potential drug candidate against PD because of its protective properties on dopaminergic neurons, which are an especially vulnerable cell population in PD. It has been recently shown that GDNF can also attenuate aggregation of phosphorylated α-synuclein in dopaminergic neurons, which is one of the most important pathologies of PD. Phosphorylated α-synuclein is a primary component of Lewy bodies, which in turn, are vastly studied intracellular inclusions with a high correlation towards neurodegenerative diseases. GDNF signals through its main receptor RET and activates downstream signalling cascades. RET is indispensable for the effect of GDNF against α-synuclein aggregation. Importance of the downstream molecules Src, AKT and PI3K have been also pharmacologically demonstrated. However, complete mechanism of GNDF’s action and individual importance of downstream signalling molecules has been yet to establish. CRISPR/Cas9 gene editing tool has revolutionized the gene manipulation in biological research. In this thesis work, CRISPR/Cas9 guides were designed to target and mutate the c-Src, Akt1 and NURR1, which are important proteins of the GDNF/RET pathway. As a delivery system for the Cas9 enzyme and individual guides, lentiviral vectors were produced according to the protocols previously established in our laboratory and proved to be high efficiency. Modelling of α-synuclein aggregation in neurons was performed with pre-formed fibrils of α-synuclein, which induce the formation of intracellular Lewy body-like inclusions with the phosphorylation of α-synuclein at serine 129. In this study, primary dopaminergic neuron cultures from E13.5 mouse embryos were cultured in 96-well plates. For each of the target genes, I designed two guide variants, cloned them in lentiviral transfer vectors and produced lentiviral particles for neuronal transduction. My data shows that targeting Akt1 and c-Src impaired the protective mechanism of GDNF against Lewy body-like inclusions. For the importance of NURR1 more studies are needed for coherent conclusions. I also showed that targeting of NURR1 impaired the GDNF/RET signalling at least in one guide construct. The 15-day long cultivation did not affect to the dopaminergic cell numbers in any of the groups. Still the confirmation of successful CRISPR-induced genetic mutations by sequencing as well as the detailed mechanism of how GDNF prevents the formation of Lewy body-like inclusions will be a subject of future studies. This thesis provides important information for the molecular mechanism of attenuation of α-synuclein aggregation by GDNF through its main receptor RET.
  • Runeberg-Roos, Pia; Piccinini, Elisa; Penttinen, Anna-Maija; Matlik, Kert; Heikkinen, Hanna; Kuure, Satu; Bespalov, Maxim M.; Peranen, Johan; Garea-Rodriguez, Enrique; Fuchs, Eberhard; Airavaara, Mikko; Kalkkinen, Nisse; Penn, Richard; Saarma, Mart (2016)
    In Parkinson's disease midbrain dopaminergic neurons degenerate and die. Oral medications and deep brain stimulation can relieve the initial symptoms, but the disease continues to progress. Growth factors that might support the survival, enhance the activity, or even regenerate degenerating dopamine neurons have been tried with mixed results in patients. As growth factors do not pass the blood-brain barrier, they have to be delivered intracranially. Therefore their efficient diffusion in brain tissue is of crucial importance. To improve the diffusion of the growth factor neurturin (NRTN), we modified its capacity to attach to heparan sulfates in the extracellular matrix. We present four new, biologically fully active variants with reduced heparin binding. Two of these variants are more stable than WT NRTN in vitro and diffuse better in rat brains. We also show that one of the NRTN variants diffuses better than its close homolog GDNF in monkey brains. The variant with the highest stability and widest diffusion regenerates dopamine fibers and improves the conditions of rats in a 6-hydroxydopamine model of Parkinson's disease more potently than GDNF, which previously showed modest efficacy in clinical trials. The new NRTN variants may help solve the major problem of inadequate distribution of NRTN in human brain tissue. (C) 2016 Elsevier Inc. All rights reserved.
  • Montonen, Heidi (Helsingfors universitet, 2013)
    Literature review: The plasma membrane DA transporter (DAT) belongs to the family of Na+/ClÙÄÉ≠ dependent neurotransmitter transporters. DAT is the primary mechanism for clearance of dopamine from the extracellular space and transporting it back to the presynaptic nerve terminals. There's a great interest in the DAT and its regulation as its substrate, dopamine, mediates a wide array of physiological functions e.g. locomotor activity, cognition and the control of motivated behaviors. With selective transport DAT limits the intensity and the duration of dopaminergic signal. Its function is regulated by several kinases, phosphatase and protein-protein interactions. The altered expression of DAT may be related to several neurological diseases such as Parkinson's disease, addiction and ADHD. To study DAT's function, several genetically modified mouse lines including DAT knockout mice, DAT knockdown mice and DAT knock in mice with elevated DAT levels have been generated. Experimental part: Glial cell line-derived neurotrophic factor (GDNF) plays important role in the survival and function of dopaminergic neurons, learning, memory and synaptic plasticity. More recently, several studies have shown that GDNF can also negatively regulate the actions of abused drugs. The aim of this study was to investigate GDNF's role and mechanism of action in plasticity and function of the dopaminergic neurons projecting to striatum. For that purpose, we used in vivo microdialysis in freely moving mice. We chose two different mouse lines: MEN2B mice with constitutive active Ret-signaling and elevated striatal dopamine concentrations, and GDND-cKO mice that lack GDND in the central nervous system. Microdialysis guide cannula was implanted in the dorsal striatum in the stereotaxic surgery and the mice were allowed to recover for 5-7 days. The concentrations of dopamine and its metabolites DOPAC and HVA and also 5-HIAA were determined from the samples by highperformance liquid chromatography. Microdialysis was performed twice for every mouse on days 1 and 4. Between microdialysis days, the mice were given amphetamine 1 mg/kg i.p. on days 2 and 3. In the microdialysis experiment, the mice received amphetamine stimulation (100 µM/60 min) via microdialysis probe. The placements of microdialysis probes were verified from fixed brain sections after the experiments. Amphetamine increased the dopamine output in both mouse lines, but there were no statistically significant differences in striatal dopamine concentrations between genotypes neither after acute nor chronic administration. However, there was a difference between the dopamine outputs in days 1 and 4 in both MEN2B and GDNF-cKO mice: The striatal dopamine concentrations were significantly lower on the second microdialysis day. This may be a sing from tolerance to the drug. However, without more research, it is not possible, by this experiment, to draw direct conclusions of GDNF's role in addiction and in plasticity in striatum. It is possible that the differences between genotypes are too small to be seen with microdialysis. Development of compensatory mechanisms in mice cannot be ruled out either. Effects may also vary between different brain areas.
  • Poyhonen, Suvi; Er, Safak; Domanskyi, Andrii; Airavaara, Mikko (2019)
    Astrocytes, oligodendrocytes, and microglia are abundant cell types found in the central nervous system and have been shown to play crucial roles in regulating both normal and disease states. An increasing amount of evidence points to the critical importance of glia in mediating neurodegeneration in Alzheimer's and Parkinson's diseases (AD, PD), and in ischemic stroke, where microglia are involved in initial tissue clearance, and astrocytes in the subsequent formation of a glial scar. The importance of these cells for neuronal survival has previously been studied in co-culture experiments and the search for neurotrophic factors (NTFs) initiated after finding that the addition of conditioned media from astrocyte cultures could support the survival of primary neurons in vitro. This led to the discovery of the potent dopamine neurotrophic factor, glial cell line-derived neurotrophic factor (GDNF). In this review, we focus on the relationship between glia and NTFs including neurotrophins, GDNF-family ligands, CNTF family, and CDNF/MANF-family proteins. We describe their expression in astrocytes, oligodendrocytes and their precursors (NG2-positive cells, OPCs), and microglia during development and in the adult brain. Furthermore, we review existing data on the glial phenotypes of NTF knockout mice and follow NTF expression patterns and their effects on glia in disease models such as AD, PD, stroke, and retinal degeneration.
  • Virtanen, Heikki (Helsingfors universitet, 2019)
    Literature review part: The enteric nervous system (ENS) often called “the second brain” is considered its own autonomic division that can independently regulate gut function. The ENS is derived from enteric neural crest-derived cells (ENCCs), which colonize the gut during development. Development of the ENS is a complex process, and many signalling pathways are required for a properly functioning ENS, especially GDNF/Gfrα1/RET signalling controlling survival, proliferation, migration, and differentiation of ENCCs. Hirschsprung’s disease (HSCR) is the most common congenital disease affecting gut motility. The prevalence of HSCR is 1:5000, and it is characterized by a complete lack of enteric neurons (aganglionosis) in the distal colon. Due to impaired intestinal motility, infants may have constipation, emesis, abdominal pain or distention, and, in some cases, diarrhea. The most life-threatening symptom is HSCR-associated enterocolitis (HAEC), which occurs in 30-50% of patients. Routine treatment for HSCR is a surgical operation called “pull through” in which the aganglionic segment is removed, and the remaining ganglionic segment is joined to the anus. However, the risk of developing HAEC after successful surgery still exists. Histopathological analysis has revealed that HAEC is accompanied by various changes in the gut epithelium, especially in mucin-producing goblet cells. These changes include hyperplasia of the goblet cells, altered mucin profile, retention of mucin, damaged and disorganized epithelium structure, inflammation, and bacterial adherence to the epithelium. However, a lack of suitable postnatal HSCR mouse models has partially hindered the progress of pinpointing the exact order of these events. A RET mutation found in half of the patients is overwhelmingly the biggest risk factor for HSCR. RET is a receptor on the cell membrane that mediates the effects in GDNF/Gfrα1/RET signaling pathway. of Knock-out mice of Gdnf, Gfra1 and Ret all have intestinal aganglionosis, resembling HSCR. However, to date, no mouse models of HSCR affecting GDNF/Gfrα1/RET signalling exist because pups are born without kidneys and die soon after birth. Experimental part: The GFRa1 hypomorphic mouse line (Gfra1hypo/hypo) created by Dr. Jaan-Olle Andressoo is the first successful model that survives past birth while manipulating GDNF-Gfrα1-RET signalling and phenocopying HSCR. These mice have 70-80% reduction in the expression of Gfrα1 in the developing gut and kidneys, which is sufficient to cause aganglionosis in the distal colon, yet not enough to impair kidney development.These mice are sacrificed between P7-P25 because of welfare problems yet giving a time window for analysis of the development of HAEC. Histological analyses revealed that Gfra1hypo/hypo mice had goblet cell hyperplasia and a shift away from acidic mucin production in the distal colon. Goblet cell hyperplasia was first observed at P10, but the shift in mucin profile already appeared at P5. It is not known what causes goblet cells to change their mucin production, but it seems to be the earliest histopathological change in HAEC preceding goblet cell hyperplasia. qPCR-analysis revealed that Muc2, the main secreted mucin that protects epithelium from invading pathogens, was upregulated at both P5 and P10. mRNA levels of Tnfa were also upregulated at P10. The aforementioned changes were not observed in the duodenum where the ENS had developed normally despite the reduction in Gfra1 expression. This indicates that the changes observed in the colon are likely due to the lack of ENS innervation, rather than a direct effect from GDNF-GFRa1-RET signalling itself. Finally, serum analysis indicated that systemic inflammation did not occur from P10-P16, although one Gfra1hypo/hypo animal had high levels of IL6 and TNFa at P14-16. This indicates that inflammation is not an early stage event and it is preceded by goblet cells related changes. In conclusion, changes in goblet cells seems to be earliest histopathological findings preceeding HAEC.
  • Pulkkinen, Nita (Helsingfors universitet, 2013)
    Amphetamine and its derivatives are widely used as medicines but also abused as psychostimulant drugs. The most important action of amphetamine in the central nervous system is to release dopamine to the extracellular space which leads to enhanced dopaminergic neurotransmission. Amphetamine also releases serotonin and norepinephrine by similar mechanisms and it affects indirectly other neurotransmitter systems too. It still remains partly unsolved how amphetamine exactly releases monoamines but it is known to have multiple sites of action. Amphetamine is a substrate for dopamine transporter (DAT) and it acts as a competitive inhibitor of the transporter reducing uptake of dopamine. Amphetamine enters the cell mainly through DAT and partly by diffusing through the cell membrane. The drug induces changes in DAT leading to reverse transport of dopamine from the cytoplasm into the synaptic cleft through DAT. Amphetamine is also substrate for vesicular monoamine transporter 2 (VMAT2) preventing the uptake of dopamine into storage vesicles and promoting its release from the vesicles to cytoplasm. Additionally, amphetamine inhibits monoamine oxidase (MAO), enzyme which degrades monoamines. It also enhances dopamine synthesis and according to recent studies amphetamine augments exocytotic dopamine release. Drug addiction is a chronic disorder related to structural and functional adaptive changes of neurons, called neuronal plasticity. GDNF (glial cell line-derived neurotrophic factor) is one of the many molecules regulating plasticity. It is especially important to the dopaminergic system and some investigations have suggested that it has potential as a protective agent against addiction. The aim of this study was to investigate how the overexpression of endogenic GDNF affects dopaminergic system and how it changes drug responses. A hypermorphic mouse strain (GDNFh), which is overexpressing physiological GDNF, was used. Their wild-type littermates were used as controls. Using brain microdialysis it was measured how the extracellular dopamine concentration changes in striatum and nucleus accumbens (NAcc) after amphetamine stimulation. Amphetamine was administered straight to the brain through the microdialysis probe. Microdialysis was performed on days 1 and 4, and on days 2 and 3 the mice were given amphetamine intraperitoneally. This was done to find out if the response to amphetamine changed after repeated dosing. In addition to these experiments, the biological activity of three small-molecule GDNF mimetics in intact brains was tested by microdialysis. On the first day amphetamine increased striatal dopamine output more in the heterozygous GDNFh mouse than in the wild-type mice. This stronger reaction to amphetamine may be explained by the enhanced activity of DAT in the GDNFh-het mice leading to higher intracellular amphetamine concentration. Also the striatal dopamine levels are increased in the GDNFh-het. On the fourth day no differences were detected between the genotypes. In the NAcc no significant difference was found between the genotypes. Instead in NAcc amphetamine caused a smaller increase in the dopamine output on day 4 than on day 1 in both genotypes suggesting that tolerance was developed. These results confirm that endogenic GDNF has a remarkable role in the regulation of the dopamine system and hence addiction but further investigations are needed to clarify its versatile actions. The small-molecule GDNF mimetics increased striatal dopamine output thus showing biological activity and encouraging to further investigations.
  • Liu, Yixin; Ribeiro, Orquidea De Castro; Robinson, James; Goldman, Adrian (2020)
    The receptor tyrosine kinase RET is essential in a variety of cellular processes. RET gain-of-function is strongly associated with several cancers, notably multiple endocrine neoplasia type 2A (MEN 2A), while RET loss-of-function causes Hirschsprung's disease and Parkinson's disease. To investigate the activation mechanism of RET as well as to enable drug development, over-expressed recombinant protein is needed for in vitro functional and structural studies. By comparing insect and mammalian cells expression of the RET extracellular domain (RETECD), we showed that the expression yields of RETECD using both systems were comparable, but mammalian cells produced monomeric functional RETECD, whereas RETECD expressed in insect cells was non-functional and multimeric. This was most likely due to incorrect disulfide formation. By fusing an Fc tag to the C-terminus of RETECD, we were able to produce, in HEK293T cells, dimeric oncogenic RETECD (C634R) for the first time. The protein remained dimeric even after cleavage of the tag via the cysteine disulfide, as in full-length RET in the context of MEN 2A and related pathologies. Our work thus provides valuable tools for functional and structural studies of the RET signaling system and its oncogenic activation mechanisms. (C) 2020 Published by Elsevier B.V.
  • Barker, Roger A.; Björklund, Anders; Gash, Don M.; Whone, Alan; Laar, Amber Van; Kordower, Jeffrey H.; Bankiewicz, Krystof; Kieburtz, Karl; Saarma, Mart; Booms, Sigrid; Huttunen, Henri J.; Kells, Adrian P.; Fiandaca, Massimo S.; Stoessl, A. Jon; Eidelberg, David; Federoff, Howard; Voutilainen, Merja H.; Dexter, David T.; Eberling, Jamie; Brundin, Patrik; Isaacs, Lyndsey; Mursaleen, Leah; Bresolin, Eros; Carroll, Camille; Coles, Alasdair; Fiske, Brian; Matthews, Helen; Lungu, Codrin; Wyse, Richard K.; Stott, Simon; Lang, Anthony E. (2020)
    The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.
  • Chmielarz, Piotr; Er, Safak; Konovalova, Julia; Bandres, Laura; Hlushchuk, Irena; Albert, Katrina; Panhelainen, Anne; Luk, Kelvin; Airavaara, Mikko; Domanskyi, Andrii (2020)
    Background Parkinson's disease (PD) is associated with proteostasis disturbances and accumulation of misfolded alpha-synuclein (alpha-syn), a cytosolic protein present in high concentrations at pre-synaptic neuronal terminals. It is a primary constituent of intracellular protein aggregates known as Lewy neurites or Lewy bodies. Progression of Lewy pathology caused by the prion-like self-templating properties of misfolded alpha-syn is a characteristic feature in the brains of PD patients. Glial cell line-derived neurotrophic factor (GDNF) promotes survival of mature dopamine (DA) neurons in vitro and in vivo. However, the data on its effect on Lewy pathology is controversial. Objectives We studied the effects of GDNF on misfolded alpha-syn accumulation in DA neurons. Methods Lewy pathology progression was modeled by the application of alpha-syn preformed fibrils in cultured DA neurons and in the adult mice. Results We discovered that GDNF prevented accumulation of misfolded alpha-syn in DA neurons in culture and in vivo. These effects were abolished by deletion of receptor tyrosine kinase rearranged during transfection (RET) or by inhibitors of corresponding signaling pathway. Expression of constitutively active RET protected DA neurons from fibril-induced alpha-syn accumulation. Conclusions For the first time, we have shown the neurotrophic factor-mediated protection against the misfolded alpha-syn propagation in DA neurons, uncovered underlying receptors, and investigated the involved signaling pathways. These results demonstrate that activation of GDNF/RET signaling can be an effective therapeutic approach to prevent Lewy pathology spread at early stages of PD. (c) 2020 International Parkinson and Movement Disorder Society
  • Porokuokka, L. Lauriina; Virtanen, Heikki T.; Linden, Jere; Sidorova, Yulia; Danilova, Tatiana; Lindahl, Maria; Saarma, Mart; Andressoo, Jaan-Olle (2019)
    BACKGROUND & AIMS: RET, the receptor for the glial cell line-derived neurotrophic factor (GDNF) family ligands, is the most frequently mutated gene in congenital aganglionic megacolon or Hirschsprung's disease (HSCR). The leading cause of mortality in HSCR is HSCR-associated enterocolitis (HAEC), which is characterized by altered mucin composition, mucin retention, bacterial adhesion to enterocytes, and epithelial damage, although the order of these events is obscure. In mice, loss of GDNF signaling leads to a severely underdeveloped enteric nervous system and neonatally fatal kidney agenesis, thereby precluding the use of these mice for modeling postnatal HSCR and HAEC. Our aim was to generate a postnatally viable mouse model for HSCR/HAEC and analyze HAEC etiology. METHODS: GDNF family receptor alpha-1 (GFRa1) hypomorphic mice were generated by placing a selectable marker gene in the sixth intron of the Gfra1 locus using gene targeting in mouse embryonic stem cells. RESULTS: We report that 70%-80% reduction in GDNF co-receptor GFRa1 expression levels in mice results in HSCR and HAEC, leading to death within the first 25 postnatal days. These mice mirror the disease progression and histopathologic findings in children with untreated HSCR/HAEC. CONCLUSIONS: In GFRa1 hypomorphic mice, HAEC proceeds from goblet cell dysplasia, with abnormal mucin production and retention, to epithelial damage. Microbial enterocyte adherence and tissue invasion are late events and therefore unlikely to be the primary cause of HAEC. These results suggest that goblet cells may be a potential target for preventative treatment and that reduced expression of GFRa1 may contribute to HSCR susceptibility.
  • Renko, Juho-Matti (Helsingfors universitet, 2012)
    Review of the literature: The purpose of the review is to go through what is known about mechanisms of actions of different neurotrophic factors (GDNF, neurturin, CDNF and MANF) and how they are transported within the brain. Neurotrophic factors are endogenous and secreted proteins which have a pivotal role in the development and maintenance of neurons. They support the survival of neurons and they can help them to recover from different injuries. Due to these functions neurotrophic factors might be beneficial for the treatment of neurodegenerative disorders like Parkinson's disease. There are a great deal of studies that clearly show the neuroprotective and neurorestrorative function of GDNF and neurturin on dopaminergic neurons. They are also studied in clinical studies with Parkinson's patients but the results have been partly contradictory. The signalling route of GDNF and neurturin via RET tyrosinekinasereceptor is fairly well known but the other mechanisms of action of these factors needs to be studied further. CDNF and MANF constitute a novel, evolutionarily conserved family of neurotrophic factors. They are shown to have neuroprotective and neurorestrorative actions on dopaminergic neurons both in vitro and in vivo in a rodent model of Parkinson's disease. The mechanisms of action of CDNF and MANF are not quite clear at the moment. There are two different domains in their structure both of which are likely to carry different functions. The N-terminal domains of these proteins are close to saposins, lipid and membrane binding proteins, some of which are shown to have neurotrophic and anti-apoptotic effects. The C-terminal domain of MANF, in turn, is structurally close to the SAP-domain of Ku70-protein which binds Bax in the cytoplasm and thus inhibits apoptosis mediated by Bax. CDNF and MANF might protect neurons both via intracellular mechanisms and extracellularly acting like a secreted neurotrophic factor. CDNF and GDNF are transported retrogradially from striatum to substantia nigra. MANF, unlike the others, is transported from striatum to the frontal cortex. MANF and CDNF are shown to have better diffusion properties in the brain parenchyma than GDNF. Experimental part: We studied, by means of microdialysis, the effects of CDNF, MANF and GDNF on the dopaminergic neurotransmission of naive rats within the striatum. Neurotrophic factors (10 µg) and PBS as a negative control were injected into the left striatum in stereotaxic surgery. After this rats recovered one week before the first mircodialysis. The second mircodialysis was performed three weeks after the surgery. The samples were collected from the left striatum of freely moving rats. During the microdialysis neurotransmission was stimulated by replacing the perfusion solution with hypertonic potassium solution and with amphetamine solution. The concentration of dopamine, DOPAC, HVA and 5-HIAA was measured from the dialysate samples. In vivo TH-activity experiment was carried out for three rats in each group. NSD1015 was injected i.p.after which rats were decapitated and their striatums were dissected. The concentration of L-DOPA, dopamine and metabolites on the treated and untreated hemisphere were analyzed from the tissue samples. The amount of L-DOPA in the striatum after NSD1015-treatment indicates how active TH-enzyme is. There were no significant differences in the concentrations of dopamine and metabolites during the baseline. MANF and CDNF increased the release of dopamine from the nerve terminals compared to GDNF and PBS one week after the surgery. Three weeks after the surgery there was still significant increase in the release of dopamine in MANF group compared to GDNF group. Also the dopamine-DOPAC-turnover was increased significantly in MANF group compared to GDNF and PBS groups one week after the surgery. DOPAC/HVA -ratio was significantly smaller in GDNF group than in other groups one week after the surgery. These findings suggest that MANF potentiates dopaminergic neurotransmission most drasticly. The effects of MANF seem to last longer time than the effects of other neurotrophic factors. CDNF seems to increase the release of dopamine from the nerve terminals as well. The potentiation of dopaminergic neurotransmission could be due to increased biosynthesis of dopamine or due to the potentiation of the function of nerve terminals. In the results of the TH-activity experiment there was a trend according to which L-DOPA is synthesized less after the neurotrophic factor treatment that after the PBS treatment. This suggests that neurotrophic factors might decrease the activity of TH-enzyme.
  • Huynh, Thi Le Hang (Helsingfors universitet, 2010)
    In the written part of my master -thesis I discuss about GDNF signalling and more specifically how the changes in the GDNF/GFRα1/Ret signaling affect the nigrostriatal dopaminergic neurons in different mutant mice. In the animal models of Parkinson's disease the neuroprotective and neurorestorative effects of exogenous GDNF are very clear which raises hope for use of GDNF in treatment of Parkinson's disease. In intact animals GDNF stimulates the function of nigrostriatal dopaminergic system. Revealing the role of GDNF/GFRα1/Ret signaling in development, maintenance and protection of nigrostriatal dopaminergic system will certainly help in search for treatment of neurodegeneration in Parkinson's disease. In knockout mouse models GDNF/GFRα1/Ret signaling is not crucial for prenatal nigrostriatal dopaminergic neuron development, but it has been shown that it plays an important role in the early postnatal development. Also, it was shown that reduced GDNF/GFRα1/Ret signaling compromises nigrotriatal dopaminergic system in heterozygous GDNF/GFRα1/Ret knockout mice. However the physiological roles of endogenous GDNF and its signalling in the nigrostriatal dopaminergic neurons are not very well understood. In the experimental part of my master -thesis I studied how reduced endogenous GDNF signaling affects the dopaminergic system after 6-OHDA induced neurotoxicity in the conventional heterozygous GDNF mice. Besides that I examined the effects of elevated endogenous GDNF on dopaminergic system of 7 days old so-called GDNF hypermorphs mice. The effects of reduced endogenous GFRα1 levels on dopaminergic system of 20 days old GFRα1 hypomorphs have also been studied. The obtained date showed that mice with the reduced levels of endogenous GDNF are not more susceptible to the 6-OHDA induced neurotoxicity than the wild type littermates. Elevated endogenous GDNF levels did not affect early postnatal development of the nigrostriatal dopaminergic system in GDNF hypermorphs mice as revealed by normal intensity of TH staining in striatum and normal number of TH-positive cells in the substantia nigra pars compacta. Reduced levels of endogenous GFRα1 levels did not affect monoamine levels in the striatum of GFRα1 hypomorph mice.