Endogeeninen GDNF keskiaivojen dopamiinihermosolujen säätelijänä

Midbrain dopamine neurons exert a powerful influence on behavior and their dysfunction is associated with many neurological and neuropsychiatric diseases, including Parkinson s disease (PD). Dopamine neurons are large, complex and sensitive cells. Hence, their survival and correct function requires coordinated action of various transcription and regulatory factors both during development and aging. Potentially, one such factor is glial cell line-derived neurotrophic factor (GDNF). Ectopically applied GDNF is best known for its potent ability to protect and restore damaged dopaminergic neurons both in vitro and in vivo. GDNF-based therapies have been tested in clinical trials with PD patients with variable success. However, the function of endogenous GDNF in brain dopamine system development, aging and disease is poorly understood. Improvement in GDNF-based therapies requires better understanding of the physiological functions of GDNF in the brain. The current knowledge of endogenous GDNF function remains obscure, mainly due to the lack of proper animal models. The present study investigated the regulatory role of endogenous GDNF in the development, maintenance and function of midbrain dopamine neurons utilizing novel mouse models: GDNF conditional knock-out (cKO) mice and GDNF hypermorphic (GDNFh) mice over-expressing GDNF from the endogenous locus. GDNF cKO mice enable GDNF deletion solely from the central nervous system during embryonic development or later in adulthood, preserving its vital role in kidney development. Midbrain dopamine systems of these new mouse strains were studied with immunohistochemical, neurochemical, pharmacological, behavioral and molecular biology methods. We found more substantia nigra dopaminergic cells and elevated striatal dopamine levels in immature and adult GDNFh mice. In cKO mice, dopamine levels and cell numbers were unaltered, even upon aging, and regardless of the timing of GDNF deletion. Both mouse strains exhibited enhanced dopamine uptake, while responses to amphetamine were augmented in GDNFh mice and reduced in cKO mice. GDNFh mice also released more dopamine and GDNF elevation protected them in a lactacystin-based model of PD. Overall, dopamine neurons were more sensitive to moderate elevation than complete absence of endogenous GDNF, which suggests that they can adaptively compensate for GDNF loss. This highlights the limitation of broadly utilized gene deletion approaches in analyzing gene function. Our results indicate a clear role for endogenous GDNF in midbrain dopamine neuron development and function, but also demonstrate that GDNF is not required for their maintenance during aging. Furthermore, the ability of endogenous GDNF to protect animals in a PD model without the side effects associated with ectopic GDNF application suggests that elevation in endogenous GDNF levels may be an important future route for PD therapy.Aivojen dopamiinihermosoluilla on voimakas vaikutus käyttäytymiseemme ja niiden toimintahäiriö onkin liitetty moniin neurologisiin ja psykiatrisiin sairauksiin, kuten Parkinsonin tautiin. Dopamiinineuronit ovat suuria, monimutkaisia ja herkkiä soluja. Tämän vuoksi niiden selviytyminen ja oikeanlainen toiminta niin yksilönkehityksen kuin koko elinkaaren ajan on riippuvaista useiden erilaisten säätelytekijöiden oikeanlaisesta yhteistoiminnasta. Mahdollisesti eräs tällainen säätelytekijä on gliasolulinjaperäinen hermokasvutekijä eli GDNF. GDNF:llä on osoitettu olevan hyvin poikkeuksellinen kyky suojella ja korjata vaurioituneita dopamiinihermosoluja sekä solu- että eläinmalleissa. GDNF-peräisiä lääkehoitoja onkin tutkittu kliinisissä kokeissa Parkinsonintautipotilailla, vaihtelevin tuloksin. Tästä huolimatta endogeenisen, eli aivojemme itse valmistaman, GDNF:n toiminta yksilönkehityksen, vanhenemisen ja sairauksien yhteydessä tunnetaan yhä huonosti. Tehokkaampien GDNF-pohjaisten hoitojen kehittäminen edellyttää parempaa ymmärrystä GDNF:n fysiologisista toiminnoista aivoissa. Endogeenisen GDNF:n toimintojen heikko tuntemus johtuu ensisijaisesti kunnollisten eläinmallien puuttumisesta. Tässä työssä tutkimme endogeenisen GDNF:n roolia keskiaivojen dopamiinihermosolujen kehityksessä, ylläpidossa ja toiminnassa käyttäen uusia eläinmalleja: konditionaalisesti poistogeenisiä (conditional knock-out; cKO) GDNF hiiriä sekä GDNF hypermorfisia (GDNFh) hiiriä, jotka tuottavat normaalia enemmän endogeenistä GDNF:ää. cKO hiiriltä GDNF voidaan sikiövaiheessa poistaa täysin ainoastaan keskushermostosta tai vaihtoehtoisesti vasta myöhemmin aikuisilta eläimiltä. Näin säilytetään GDNF:n elintärkeä rooli munuaisten kehityksessä. Tutkimme näiden uusien hiirikantojen keskiaivojen dopamiinijärjestelmiä immunohistokemiallisten, aivokemiallisten, farmakologisten, molekyylibiologisten sekä erilaisten käyttäytymismenetelmien avulla. Havaitsimme sekä hyvin nuorten että aikuisten GDNFh hiirten aivoissa kohonneen määrän dopamiinia sekä dopamiinihermosoluja. Toisaalta GDNF cKO hiirillä dopamiinipitoisuudet ja -solumäärät säilyivät muuttumattomia, jopa hyvin vanhoilla hiirillä, ja riippumatta GDNF:n poistamisen ajankohdasta. Molemmilla hiirikannoilla dopamiinin takaisinotto oli voimistunut, kun taas amfetamiinivasteet olivat vahvistuneet GDNFh hiirillä ja heikentyneet GDNF cKO hiirillä. GDNFh hiirillä dopamiinia myös vapautui enemmän, minkä lisäksi kohonnet GDNF-pitoisuudet suojasivat niitä kemiallisesti aiheutetulta Parkinsonismilta. Kaiken kaikkiaan aivojen dopamiinihermosolut näyttivät olevan herkempiä GDNF:n määrän kohtuulliselle lisääntymiselle kuin sen täydelliselle puuttumiselle. Dopamiinihermosolut kykenevät siis ilmeisesti jollain tavalla kompensoimaan GDNF:n puuttumisen. Tämä osoittaa selvän puutteen hyvin yleisesti käytetyissä geeninpoistomenetelmissä. Tuloksemme viittaavat siihen että endogeenisella GDNF:llä on selvä rooli aivojen dopamiinihermosolujen kehityksessä ja toiminnassa. Toisaalta tuloksemme myös osoittavat, ettei GDNF:ää välttämättä tarvita ylläpitämään niitä yksilön vanhetessa. Lisäksi endogeenisen GDNF:n kyky suojella eläimiä Parkinsonin tautimallissa ilman GDNF-annosteluun tavallisesti liittyviä sivuvaikutuksia merkitsee, että endogeenisen GDNF:n lisääminen saattaisi joskus tulevaisuudessa olla tehokas tapa hoitaa Parkinsonin tautia

Sub-100 mu m Spatial Resolution Ambient Mass Spectrometry Imaging of Rodent Brain with Laser Ablation Atmospheric Pressure Photoionization (LAAPPI) and Laser Ablation Electrospray Ionization (LAESI)

In this study, we applied a new IR laser-beam-focusing technique to enable sub-100 μm spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 μm produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.In this study, we applied a new IR laser-beamfocusing technique to enable sub-100 mu m spatial resolution in laser ablation atmospheric pressure photoionization (LAAPPI) and laser ablation electrospray ionization (LAESI) mass spectrometry imaging (MSI). After optimization of operational parameters, both LAAPPI- and LAESI-MSI with a spatial resolution of 70 mu m produced high-quality MS images, which allowed accurate localization of metabolites and lipids in the mouse and rat brain. Negative and positive ion LAAPPI- and LAESI-MS detected many of the same metabolites and lipids in the brain. Many compounds were also detected either by LAAPPI- or LAESI-MS, indicating that LAAPPI and LAESI are more complementary than alternative methods.Peer reviewe

Chronic 2-Fold Elevation of Endogenous GDNF Levels Is Safe and Enhances Motor and Dopaminergic Function in Aged Mice

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.Peer reviewe

Implementation of deep neural networks to count dopamine neurons in substantia nigra

Unbiased estimates of neuron numbers within substantia nigra are crucial for experimental Parkinson's disease models and gene-function studies. Unbiased stereological counting techniques with optical fractionation are successfully implemented, but are extremely laborious and time-consuming. The development of neural networks and deep learning has opened a new way to teach computers to count neurons. Implementation of a programming paradigm enables a computer to learn from the data and development of an automated cell counting method. The advantages of computerized counting are reproducibility, elimination of human error and fast high-capacity analysis. We implemented whole-slide digital imaging and deep convolutional neural networks (CNN) to count substantia nigra dopamine neurons. We compared the results of the developed method against independent manual counting by human observers and validated the CNN algorithm against previously published data in rats and mice, where tyrosine hydroxylase (TH)-immunoreactive neurons were counted using unbiased stereology. The developed CNN algorithm and fully cloud-embedded Aiforia (TM) platform provide robust and fast analysis of dopamine neurons in rat and mouse substantia nigra.Peer reviewe

Constitutive Ret signaling leads to long-lasting expression of amphetamine-induced place conditioning via elevation of mesolimbic dopamine

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.Peer reviewe

Glial cell line-derived neurotrophic factor receptor REarranged during transfection agonist supports dopamine neurons in Vitro and enhances dopamine release In Vivo

Background Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood-brain-barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. Methods We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin-induced cell death, to activate intracellular signaling pathways both in vitro and in vivo, and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. Results BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild-type but not from RET-knockout mice. BT13 protects cultured dopamine neurons from 6-Hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+)-induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. Conclusion The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease-modifying treatments against PD. (c) 2019 International Parkinson and Movement Disorder SocietyPeer reviewe

Cerebral dopamine neurotrophic factor-deficiency leads to degeneration of enteric neurons and altered brain dopamine neuronal function in mice

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf(-/-)), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf(-/-) mouse are much greater than in those of Cdnf(+/+) mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf(-/-) mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf(-/-) male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf(-/-) mouse brain is altered. The deficiencies of Cdnf(-/-) mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.Peer reviewe

Elevated endogenous GDNF induces altered dopamine signalling in mice and correlates with clinical severity in schizophrenia

Presynaptic increase in striatal dopamine is the primary dopaminergic abnormality in schizophrenia, but the underlying mechanisms are not understood. Here, we hypothesized that increased expression of endogenous GDNF could induce dopaminergic abnormalities that resemble those seen in schizophrenia. To test the impact of GDNF elevation, without inducing adverse effects caused by ectopic overexpression, we developed a novel in vivo approach to conditionally increase endogenous GDNF expression. We found that a 2-3-fold increase in endogenous GDNF in the brain was sufficient to induce molecular, cellular, and functional changes in dopamine signalling in the striatum and prefrontal cortex, including increased striatal presynaptic dopamine levels and reduction of dopamine in prefrontal cortex. Mechanistically, we identified adenosine A2a receptor (A(2A)R), a G-protein coupled receptor that modulates dopaminergic signalling, as a possible mediator of GDNF-driven dopaminergic abnormalities. We further showed that pharmacological inhibition of A(2A)R with istradefylline partially normalised striatal GDNF and striatal and cortical dopamine levels in mice. Lastly, we found that GDNF levels are increased in the cerebrospinal fluid of first episode psychosis patients, and in post-mortem striatum of schizophrenia patients. Our results reveal a possible contributor for increased striatal dopamine signalling in a subgroup of schizophrenia patients and suggest that GDNF-A(2A)R crosstalk may regulate dopamine function in a therapeutically targetable manner.</p

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function

Peer reviewe