Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons

Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons

Experimental Neurology 219 (2009) 499–506 Contents lists available at ScienceDirect Experimental Neurology j o u r n a l h o m e p a g e : w w w. e ...

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Experimental Neurology 219 (2009) 499–506

Contents lists available at ScienceDirect

Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r

Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons Marjo Piltonen a,⁎, Maxim M. Bespalov b, Dagmar Ervasti a, Tero Matilainen a, Yulia A. Sidorova b, Heikki Rauvala c, Mart Saarma b, Pekka T. Männistö a a b c

Division of Pharmacology and Toxicology, Faculty of Pharmacy, P.O. Box 56, FIN-00014 University of Helsinki, Finland Institute of Biotechnology, Viikki Biocenter, P.O. Box 56, FIN-00014 University of Helsinki, Finland Neuroscience Center, Viikki Biocenter, P.O. Box 56, FIN-00014 University of Helsinki, Finland

a r t i c l e

i n f o

Article history: Received 22 October 2008 Revised 15 June 2009 Accepted 7 July 2009 Available online 15 July 2009 Keywords: GDNF HB-GAM Heparan sulphates Neuroprotection Pleiotrophin Tissue distribution 6-OHDA

a b s t r a c t Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor α1 (GFRα1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinson's disease. We utilized a truncated, non-heparin-binding Δ38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF ± HB-GAM and Δ38N-GDNF was also compared. A protective effect against ipsilateral D-amphetamine-induced turning was seen with 10 μg wtGDNF, 17 μg HB-GAM + 10 μg wtGDNF or 10 μg Δ38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 μg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Δ38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect. © 2009 Elsevier Inc. All rights reserved.

Introduction Glial cell line-derived neurotrophic factor (GDNF) belongs to the transforming growth factor-β superfamily. It signals mainly through a glycosylphosphatidyl inositol (GPI)-linked GDNF family receptor α1 (GFRα1) and a transmembrane receptor tyrosine kinase Ret (Lin et al., 1993; Jing et al., 1996; Treanor et al., 1996; Trupp et al., 1996). GDNF has strong neuroprotective and regenerative properties in animal models of Parkinson's disease (Tomac et al., 1995; Gash et al., 1996; Winkler et al., 1996). It is crucial in the maintenance of catecholaminergic neurons (Pascual et al., 2008) and involved in the development of enteric nervous system, kidneys and sperm (Moore et al., 1996; Pichel et al., 1996; Sanchez et al., 1996; Meng et al., 2000). Neural cell adhesion molecule (NCAM) is an alternative Ret-independent signalling route that nevertheless requires GFRα1 (Paratcha et al., 2003). GDNF binds to heparin with a substantial affinity (Lin et al., 1994; Rickard et al., 2003), as does heparin-binding growth-associated molecule (HB-GAM, pleiotrophin) (Rauvala, 1989). It is very likely that GDNF, like HB-GAM, also binds to heparan sulphates (HS) found in

⁎ Corresponding author. Fax: +358 9 191 59471. E-mail address: marjo.piltonen@helsinki.fi (M. Piltonen). 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.07.002

plasma membranes and extracellular matrix as components of HS proteoglycans (HSPGs) [for review, see (Sariola and Saarma, 2003)]. HSPGs participate in cell adhesion, cell–cell interactions, chemokine presentation, ligand clustering, neurite outgrowth and survival as well as mast cell function [for review, see (Bishop et al., 2007)]. Interactions between growth factors and heparin-related molecules have important consequences. Growth factors are secreted in very small amounts, but HSPGs may keep them concentrated near the receptors [for review, see (Sariola and Saarma, 2003; Proudfoot, 2006)]. The interaction between HSPGs and fibroblast growth factor (FGF) is very well known and it is described in detail how HSPGs are needed to bring together the FGF-receptor and the FGF-molecule and to stabilize the complex to activate the receptor [for review, see (Schlessinger et al., 1995)]. HSPGs also protect ligands from cleavage and thus prolong their action (Rickard et al., 2003; Gospodarowicz and Cheng, 1986). The role of HSs and heparin-binding determinants of GDNF in neuroprotection is not known. Our aim was to investigate the effect of GDNF-HSPG interactions in a unilateral rat model of Parkinson's disease and judge the importance of heparin-binding properties of GDNF. We also assessed, for the first time, the neuroprotective effect of a secretory matrix protein HB-GAM in this model. HB-GAM promotes neurite extension and axonal guidance [for review, see

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(Rauvala and Peng, 1997)], acts as a survival promoting neurotrophic factor for spinal motor neurons (Mi et al., 2007) and enhances survival of dopaminergic cells in culture (Hida et al., 2003; Marchionini et al., 2007). We also combined HB-GAM with GDNF, since their coadministration may reveal additive neurotrophic properties and saturation of brain HSPGs by HB-GAM may allow improved tissue diffusion of GDNF. In addition, we studied the neuroprotective efficacy of an N-terminally truncated Δ38N-GDNF that does not bind to heparin or HSs, but is still able to activate GFRα1/Ret pathway. Our data suggest that intact HS binding domain is needed for the optimal neuroprotective effect of GDNF. A widespread striatal diffusion of Δ38N-GDNF did not improve its efficacy. Materials and methods Animals A total of 142 young adult male Wistar rats (Harlan, Netherlands) weighing 220–280 g at the start of the experiments were used. Rats were housed four per cage under 12:12 h light–dark cycle at 22 °C temperature and had free access to food and water. The animals were housed separately through the surgery days and overnight from the last session before putting them back to home cages. After each surgery session the animals received tramadol 1 mg/kg (Tramal®, Orion Pharma, Finland) for post-operative pain relief. The experiments were carried out according to the European Community guidelines for the use of experimental animals and approved by the Committee for Animal Experiments of the University of Helsinki and the county board veterinarian. Surgical procedures and rotational behaviour studies Sterotaxic surgery and injections were performed in two or three sessions under isoflurane anaesthesia. All coordinates were calculated relative to bregma and dura surface. 6-OHDA model with a four-site striatal lesion Our method is slightly modified from that described by Kirik et al. (1998), who studied 6-hydroxydopamine (6-OHDA)-induced lesions. They concluded that a 4-site striatal lesion mimics well the pathology of symptomatic Parkinson's disease, causes minimal unspecific damage and could be useful in neuroprotection studies. We performed stereotaxic operations in three sessions. In the first operation (18 h before 6-OHDA) a guide cannula was inserted into the striatum through a burr hole (A/P 0.0, M/L −3.5 and D/V − 2.0, 3 mm above the injection site) and fixed on the scull with dental cement. Injections were done at 0.5 μl/min through a 29 G needle reaching the final coordinate D/V −5.0. The needle was attached to PE10-tubing, which was connected with Exmire microsyringe run by a syringe pump (AgnTho's, Lidingö, Sweden). In this first session the animals received either 17 μg/5 μl or 50 μg/10 μl of HB-GAM or a corresponding volume of vehicle (phosphate buffered saline, PBS). The needle was retracted after 4 min from the cessation of injection. Doses of HB-GAM were higher than those of neurotrophic factors used here, since also the expression of HB-GAM is generally much higher than that of any growth factor (Rauvala, 1989). In the second session (6 h before 6-OHDA) the animals were administered 10 μg/4 μl of GDNF (wild-type or wtGDNF produced in Escherichia coli, Amgen Inc., Thousands Oaks, CA, USA) in 10 mM citric acid in saline (pH 2), 3 or 10 μg/4 μl of Δ38N-GDNF in 10 mM HEPES (pH 7.2) or PBS (pH 7.4) or a corresponding volume of vehicle at 0.5 μl/ min. The needle was retracted after 4 min from the cessation of injection. The pretreatments are summarized in Table 1. In the third session, the guide cannula was carefully removed, after which 6-OHDA (Sigma, St. Louis, MO, USA) was injected with a custom made infusion system consisting of 4 needles fixed together at certain

Table 1 Summary of pretreatments in the four-site striatal 6-OHDA model. First injection

Second injection

PBS HB-GAM HB-GAM HB-GAM HB-GAM PBS PBS PBS

PBS pH 7.4/10 mM citric acid pH 2/10 mM HEPES pH 7.2 10 mM citrate wtGDNF 10 μg 10 mM citrate wtGDNF 10 μg wtGDNF 10 μg Δ38N-GDNF 3 μg Δ38N-GDNF 10 μg

17 μg 17 μg 50 μg 50 μg

positions to form a single unit. The coordinates for the needles were A/ P +1.3 / +0.4 / −0.4 / −1.3, M/L − 2.6 / − 3.2 / − 4.2 / − 4.5, D/V −5.0. Each of the needles was connected to a microsyringe with PE10 tubing and run by a syringe pump. Through each needle 7 μg of 6-OHDA in 3.5 μl was infused at a flow rate 0.5 μl/min, and the infusion system was retracted 4 min after the injection was stopped. 6-OHDA model with a single-site striatal lesion In the first session the animals were administered 3 or 10 μg/4 μl of Δ38N-GDNF in 10 mM HEPES, 10 μg of wtGDNF in 10 mM citrate buffer or a corresponding volume of either vehicle. The injections were performed with a 10-μl Hamilton syringe at 1 μl/min to a striatal location A/P +1.0, M/L −3.0, D/V − 5.0. In the second session, 6 h later, the animals received 16 μg/4 μl of 6-OHDA into the same coordinate. Rotational behaviour D-amphetamine (2.5 mg/kg, i.p., University Pharmacy, Helsinki, Finland) was used for inducing rotational behaviour. Full ipsilateral and contralateral turns were registered for 120 min with an automatic rotometer connected to a computer system (Coulbourn Instruments, Allentown, PA, USA; or MedAssociates, St. Albans, VT, USA). Testing was done 2, 3, 7 and 8 weeks post lesion for animals with four-site striatal lesions and 2, 4 and 6 weeks post lesion for animals with single-site striatal lesions. Preparation of truncated Δ38N-GDNF and HB-GAM Wild-type GDNF and Δ38N-GDNF expression in insect cells and purification A construct encoding full-length (or residues 39–134 for cloning of Δ38N-GDNF) mature human GDNF and N-terminal FLAG tag was subcloned into the pFASTBAC1 vector variant (Invitrogen, Carlsbad, CA, USA) containing the secretion signal sequence from Autographa californica nucleopolyhedrovirus ecdysteroid UDP-glucosyl transferase pK503.9 (Keinänen et al., 1998). The baculovirus was generated by the transformation of D10Bac bacterial strain and subsequent transfection of the resulting bacmid into Sf9 insect cells with Cellfectin reagent (Invitrogen). Soluble secreted wtGDNF and Δ38N-GDNF were expressed by infection of Sf9 insect cells with baculovirus. Cells were grown in serum-free SF900II (Invitrogen) medium supplemented with 50 mg/ml gentamycin (Sigma) at 27 °C. At 3 days postinfection, proteins were purified from culture supernatants by affinity purification with M1-FLAG agarose (Sigma). The column was eluted with glycine–HCl buffer, pH 3.5, followed by immediate normalization of pH by Tris–HCl buffer, pH 8.0. The proteins were dialyzed against HEPES buffer, pH 7.2 supplemented with NaCl to 200 mM overnight at +4 °C and then concentrated using Amicon's concentrators. Protein concentrations were measured with BCA assay (Pierce, Rockford, IL 61105 USA) and validated with commercial GDNF protein (Amgen Inc., Thousands Oaks, CA, USA). Insect cell-expressed wtGDNF and Δ38N-GDNF are represented by three or two differently glycosylated variants, respectively, and are otherwise homogenous according to SDS-PAGE analysis (Fig. 1).

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gator program (equipment described above). Pictures of each treatment were taken on a Nikon stereomicroscope equipped with a DS-Fi1 camera head and DS-L2 camera control unit (Nikon, Japan) and representative examples are shown in Fig. 5B. Biochemistry Fig. 1. 15% SDS-PAGE of GDNF variants and HB-GAM. Bands from the left: 1. wtGDNF (E. coli), 2. HB-GAM (baculo), 3. wtGDNF (baculo), 4. Δ38N-GDNF (baculo). Molecular weight markers at the left and right.

HB-GAM expression in insect cells and purification was carried out as earlier described (Raulo et al., 1992). Immunohistochemistry Tyrosine hydroxylase (TH) immunohistochemistry One week after the last rotational behaviour experiment the animals were anaesthetized with 100 mg/kg pentobarbital and transcardially perfused with PBS, followed by 4% paraformaldehyde (PAF) in phosphate buffer, pH 7.4. The brains were postfixed in 4% PAF for 4 h, then immersed in 20% sucrose solution and kept at 4 °C until sunken, whereafter they were frozen at −84 °C. Coronal sections of 40 μm were cut on a cryomicrotome and collected in series of six into a cryoprotectant buffer. The sections were kept at −20 °C until staining. This was done on free-floating sections as described earlier (Mijatovic et al., 2007) with the following exceptions: 2% normal horse serum (Vector laboratories, Burlingame, CA, USA) was used for blocking, primary antibody was changed to mouse monoclonal antibody (MAB#318, Chemicon, Temecula, CA, USA) and secondary antibody for biotinylated horse anti-mouse antibody (BA 2001, Vector laboratories, Burlingame, CA, USA). Stereological analysis of TH-positive cells in substantia nigra The number of TH-positive neurons in substantia nigra (SN) pars compacta was estimated from three TH-immunostained sections from each animal around the medial terminal nucleus of the accessory optic tract according to Sauer et al. (1995), and as earlier described (Kääriäinen et al., 2008). The cells were counted using 60× oil objective on a microscope (Olympus BX51, Olympus Optical, Tokyo, Japan) equipped with an Optronics digital camera (Goleta, CA, USA). Stereo Investigator program (MBF Bioscience, Williston, VT, USA) was used for counting the cells with optical fractionator, which was optimized so that the coefficient of error (CE) value was under 0.15 for all three sections of the intact hemisphere. GDNF immunohistochemistry To study the diffusion of GDNF variants that were used in the behavioural experiments altogether 28 rats were injected with 10 μg of wtGDNF (E. coli or baculoviral-insect cell origin to confirm that the expression system does not influence the distribution of wtGDNF) or Δ38N-GDNF according to either of the surgery protocols described above into both hemispheres. Each animal received one injection of either wtGDNF variant and one injection of Δ38N-GDNF. The animals were transcardially perfused for 10 min (n = 5–9 per group) or 6 h (n = 7–11 per group) after GDNF-variant injection without lesioning procedure. We also performed a pilot study combining HB-GAM and wtGDNF, similarly to the behavioural experiments, but pretreatment with HB-GAM only slightly increased the distribution of GDNF (data not shown). The brains were processed in the same way as for THimmunohistochemistry. GDNF-staining was done as described earlier (Kirik et al., 2000). The goat anti-GDNF antibody (R&D systems, Minneapolis, MN, USA) used also in our experiments recognizes both wtGDNF and Δ38N-GDNF. The volumes of GDNF-immunoreactivity were measured with the Cavalieri estimator-probe in Stereo Investi-

GDNF stability assay Extracts of brain extracellular matrix (ECM) in which stability of wtGDNF and Δ38N-GDNF was tested were prepared as follows. Adult mice brain was dissociated on ice first using a 20 G syringe needle followed by a 25 G needle in 2 ml of Dulbecco's buffer. The cellular suspension was centrifuged at 750 g for 10 min at +4 °C and the supernatant collected. It was further cleared by centrifugation at 14,000 g for 10 min. One microgram of GDNF variant was dissolved in 0.5 ml of the cleared ECM extract and incubated for up to 48 h at 37 °C. Following the incubation the samples were boiled in Laemmli buffer, separated by 15% SDS-PAGE and transferred to the nitrocellulose membrane. The membrane was then stained with anti-GDNF antibodies D-20 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Baculovirally expressed wtGDNF was also used to control so that the expression system itself has no detrimental effect on the protein stability. Mitogen-activated protein kinase (MAPK) activity using luciferase assays AlphaLUC cells (MG87RET murine fibroblasts stably transfected with rGFRα1 and MAPK activation detection system, a kind gift from Dr. J. Milbrandt, described in details by Baloh et al., 2000) were plated on 96-well plates in the cell density of 20,000 cells per well in DMEM, 10% FBS, 100 μg/ml normocin, 2 μg/ml puromycin, 500 μg/ml of geneticin, and 15 mM HEPES pH 7.2 a day before assay. Next day either wild-type or Δ38N-GDNF was added to the cells in the final concentrations ranging from 1 to 100 ng/ml in the absence or presence of 1 or 10 μg/ml of low molecular weight heparin (Sigma, St. Louis, MO, USA). Cells were left in the CO2 incubator to produce luciferase for 24 h, then lysed in 20 μl of 1× cell culture lysis reagent (Promega, Madison, WI, USA) and freeze-thawed once to ensure complete lysis. Then 5 μl of the lysate was mixed with 20 μl of luciferase assay substrate (Promega) on ice. Luminescence was counted on MicroBeta 2 counter (Perkin Elmer) twice. Since the reaction requires some time to achieve maximal velocity, results of the second measurement were used for future calculations and generation of graphs. Data handling and statistics All values are expressed as mean ± SEM, except the results from the luciferase assay, which are expressed as mean ± SD. In behavioural studies, the data of all treated groups were compared to their corresponding control groups but, for clarity, all the control animals were pooled together for graphs. The results were tested with oneway analysis of variance (ANOVA) followed by Tukey's post hoc test, except that the TH-positive cell data from HB-GAM + GDNF-experiments was handled with two-way ANOVA. All analyses were done with GraphPad Prism 4.0 software (GraphPad Software, San Diego, CA, USA). Results Rotational behaviour Effect of GDNF combined with HB-GAM on rotational behaviour D-amphetamine (2.5 mg/kg, i.p.) induced strong ipsilateral rotational behaviour in all treatment groups at two weeks post lesion (data not shown), but no significant differences between groups were observed. At three weeks post lesion the rats treated with a combi-

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nation of 50 μg of HB-GAM and 10 μg of wtGDNF showed a statistically significant (p b 0.05) reduction in ipsilateral turns when compared to the corresponding vehicle control group (data not shown), but either cytokine alone had no significant effect on turning behaviour. At seven weeks post lesion a more robust effect was seen (Fig. 2A), and groups treated with 50 μg of HB-GAM alone or HBGAM in either dose combined with wtGDNF 10 μg had a significantly (p b 0.05) reduced turning response when compared to the control animals. However, at 8 weeks post lesion (Fig. 2B) the most pronounced reduction in ipsilateral turning was seen in animals treated with vehicle + wtGDNF 10 μg (p b 0.01 vs. control). Also the rats that received HB-GAM 17 μg + wtGDNF 10 μg rotated significantly less than the control animals (p b 0.05). Effect of Δ38N-GDNF on rotational behaviour All groups with a 4-site striatal lesion showed a strong ipsilateral turning behaviour when challenged with D-amphetamine (2.5 mg/kg, i.p.). No differences were observed between groups at 2, 3 (data not shown) or 7 weeks post lesion (Fig. 2C). At 8 weeks post lesion (Fig. 2D), there was a significant difference in ipsilateral turns between vehicle + wtGDNF 10 μg (p b 0.001) or Δ38N-GDNF 10 μg (p b 0.05) and their vehicle control group. A significant difference was observed also between vehicle + wtGDNF 10 μg and vehicle + Δ38N-GDNF 3 μg (p b 0.01). Groups with a single-site striatal lesion showed no statistically significant reduction in ipsilateral turning behaviour 2 or 4 weeks post lesion (data not shown). At 6 weeks post lesion, however, ipsilateral turnings are almost fully abolished in all groups treated with neurotrophic factors (Fig. 3A). One animal from Δ38N-GDNF 10 μg-group was discarded from the results at 6 weeks post lesion, since it deviated notably from the rest of the group (1917 ipsilateral rotations in 120 min, whereas the rest of the group rotated −1–184 ipsilateral turns).

Survival of TH-positive neurons in substantia nigra Effect of GDNF combined with HB-GAM on TH-positive cell survival All groups treated with wtGDNF, irrespective of whether it was combined with HB-GAM or vehicle, had almost double the amount of TH-positive cells left in the pars compacta of SN (SNpc) when compared to groups treated with vehicle or only with HB-GAM (Fig. 4A.). Two-way ANOVA revealed a significant effect of GDNFtreatment (p b 0,001), but no interaction with pretreatment was found. HB-GAM alone was not able to protect TH-positive neurons against 6-OHDA toxicity. Effect of Δ38N-GDNF on TH-positive cell survival Pretreatment with Δ38N-GDNF did not significantly increase the survival of dopaminergic neurons of SNpc at either of the doses against 4-site striatal lesion, whereas wtGDNF had a significant protective effect when compared to control (p b 0.01) or Δ38N-GDNF 3 μg (p b 0.05) (Fig. 4B.). No significant differences were observed in the numbers of TH-positive cells between groups with a single-site lesion (Fig. 3B). Diffusion of wtGDNF and Δ38N-GDNF visualized by GDNF immunohistochemistry We found that Δ38N-GDNF diffuses significantly better than wtGDNF in the rat brain tissue (Figs. 5A and B; p b 0.001). The difference was obvious already 10 min after injection and persisted also at 6 h after injection, which would be the time point for lesioning in the behavioural experiments. All the GDNF variants were clearly concentrated with a minimal diffusion zone after 10 min from the injection. After 6 h the diffusion zone had almost doubled in wtGDNF-treated striata, and Δ38N-GDNF had spread at least to the whole striatum or even further, producing a diluted

Fig. 2. Ipsilateral rotational behaviour induced with D-amphetamine (2.5 mg/kg, i.p.) in groups with four-site striatal lesions, pretreated with wtGDNF, a combination of HB-GAM and wtGDNF, or Δ38N-GDNF 7 weeks post lesion (A and C) or 8 weeks post lesion (B and D). Statistics: ⁎p b 0.05 vs. VEH+VEH, ⁎⁎p b 0.01 vs. VEH+VEH, ⁎⁎⁎p b 0.001 vs. VEH+VEH, ## p b 0.01 vs. Δ38N-GDNF 3 μg (one-way ANOVA with Tukey's post hoc test). n = 6–9.

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Fig. 3. Effects of Δ38N-GDNF and wtGDNF in a single-site lesion model. (A) Ipsilateral rotational behaviour 6 weeks post lesion and (B) survival of TH-positive cells in SNpc. Statistics: ⁎p b 0.05 vs. vehicle (one-way ANOVA with Tukey's post hoc test). n = 7–8.

colour signal. Furthermore, the wtGDNF variants had a very similar diffusion pattern, which confirms that structural differences brought by the expression systems do not affect distribution in the brain.

demonstrated by Western blotting of the incubated samples and staining with anti-GDNF antibodies (Fig. 6). Activation of MAPK by wtGDNF and Δ38N-GDNF using luciferase reporter assay

GDNF stability test ECM is enriched with metalloproteases and it was possible that observed differences in our 4-site 6-OHDA model were due to the compromised stability of Δ38N-GDNF. However, incubation of the GDNF variants in the brain ECM extract revealed no significant degradation of Δ38N-GDNF even after 48 h incubation. This was

We compared the abilities of wtGDNF and Δ38N-GDNF to activate RET down-stream target mitogen-activated protein kinases (MAPK) using a sensitive assay utilizing luciferase as a reporter gene. In all tested concentrations, Δ38N-GDNF was more active than wtGDNF in terms of luciferase expression stimulation (Fig. 7). We also studied the effect of the low molecular weight heparin on MAPK-inducing properties of wt and Δ38N-GDNF. Quite expectedly, addition of heparin had no influence on Δ38N-GDNF ability to activate luciferase expression. Administration of 1 or 10 μg/ml of heparin together with wtGDNF significantly improved its MAPK-inducing properties and resulted in levels of luciferase expression comparable with those produced by Δ38N-GDNF. Discussion GDNF has so far been the most potent neurotrophic factor in rescuing dopaminergic function in animal models of Parkinson's disease. However, results from the clinical trials were controversial (Gill et al., 2003, Slevin et al., 2005, Lang et al., 2006). There may still be important aspects unknown about the effects and signalling of GDNF, discovery of which could help understand its therapeutic potential more deeply. For example, the significance of heparan sulphates in GDNF's effects has not been extensively studied in in vivo models. Neuroprotective effects of HB-GAM and Δ38N-GDNF

Fig. 4. Survival of TH-positive cells in SNpc. (A) Groups with four-site striatal lesions and pretreated with wtGDNF or a combination of HB-GAM and GDNF (n = 6–9); (B) groups with four-site striatal lesions and pretreated with wtGDNF or Δ38N-GDNF (n = 8–13). Statistics: (A) ⁎⁎⁎p b 0.001 for wtGDNF treatment effect (two-way ANOVA); (B) ⁎⁎p b 0.01 vs. VEH+VEH, #p b 0.05 vs. Δ38N-GDNF 3 μg (one-way ANOVA with Tukey's post hoc test).

To our knowledge these are the first experiments describing the neuroprotective effects of HB-GAM alone or combined with GDNF, as well as the effects of a truncated GDNF variant (Δ38N-GDNF) in an in vivo model of Parkinson's disease. According to our results, it seems that heparin-binding determinants of GDNF are needed to keep it concentrated at the injection area, which leads to a better therapeutic response. Hida et al. (2003) have earlier described an enhancement of THpositive cell survival in cultured cells in vitro when GDNF and HBGAM are combined, in comparison to either substance alone. A later study showed, that pretreating dopaminergic cell grafts with HBGAM or GDNF improves survival of the grafts as well as the following functional recovery in 6-OHDA-lesioned rats. The effects were additive (Hida et al., 2007). However, the situation seems to be more complicated when looking at direct effects of GDNF and

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Fig. 5. (A) Diffusion volumes of GDNF variants 10 min and 6 h after injection. ⁎⁎⁎p b 0.001 vs. both wtGDNF variants within the corresponding time point (one-way ANOVA with Tukey's post hoc test). (B) Representative sections showing diffusion patterns of wtGDNF (E. coli or baculoviral) and Δ38N-GDNF in the rat striatum. n = 5–11.

HB-GAM on nigro-striatal dopaminergic cells in vivo, as the data from our behavioural experiments show. Pretreatment with wtGDNF alone more effectively abolished ipsilateral turns than when combined with HB-GAM. When the dose of HB-GAM was increased, the effects of wtGDNF were reduced more effectively. A possible explanation is that as more HS containing structures in the brain become occupied by HB-GAM, the effects of wtGDNF are interrupted. On the other hand, there were no differences in the survival of TH-positive neurons in the SN between any of the groups that received wtGDNF alone or combined with HB-GAM. Thus, if the dopaminergic cell bodies remain, but the ipsilateral turning bias stays unaffected, it is possible that the nerve terminals have atrophied and the neuritogenic effects of GDNF were obstructed by HB-GAM. HB-GAM itself, however, reduced ipsilateral turning behaviour in the rats at both doses used. The number of TH-positive neurons in SN on the other hand was at the same level with control animals. Thus, it is possible that the dying neurons were not rescued, but the remaining terminals had either an enhanced function or HB-GAM had induced neuritogenic effects, which would be in line with earlier in vitro data (Rauvala 1989; Hida et al., 2003; Kinnunen et al., 1999). On the other hand, a single administration may not reveal the full potential of HB-GAM in neuroprotection and restoration of neuronal function, but it may require even higher or continuous dosage. It cannot be excluded either, that HB-GAM and GDNF could have some opposing effects, perhaps through competitive binding to their targets. It even seems likely, since the combination of HB-GAM and wtGDNF was less effective in inducing functional recovery than wtGDNF alone. Nevertheless, it seems that HB-GAM may be associated with Parkinson's disease, since it was shown that the number of HBGAM-immunoreactive cells is increased in the SN of Parkinson patients (Marchionini et al., 2007) and that HB-GAM is upregulated in 6-OHDA-lesioned rats (Hida et al., 2003; Ferrario et al., 2008). Also, long term levodopa treatment upregulates HB-GAM and its receptor RPTPζ/β (Ferrario et al., 2004, 2008). The effects of N-terminally truncated Δ38N-GDNF were inferior to those of wtGDNF in the 4-site lesion model. The animals treated with Δ38N-GDNF did not recover from the ipsilateral turning bias to the

same extent as the wtGDNF-treated animals, and even the survival of TH-positive neurons in the SNpc was reduced. For better understanding of the mechanisms behind this phenomenon, we performed some additional experiments studying the distribution and activity of Δ38N-GDNF. Δ38N-GDNF diffuses better than wtGDNF in the brain GDNF immunohistochemistry revealed a considerably larger diffusion area of Δ38N-GDNF than wtGDNF. We also confirmed that the diffusion of wtGDNF is similar regardless of the expression system it was produced in. Since Δ38N-GDNF seems to spread further in the brain tissue, it may cause reduced local concentrations of Δ38N-GDNF, and thus lead to an insufficient concentration for neuroprotection. This view was supported by our additional experiments in the one-site lesion model. Here, Δ38N-GDNF was as efficient as wild-type GDNF, even in a lower dose (3 vs. 10 μg). The lesion was created in the middle of the protein diffusion area, where the concentration of the growth factor is the highest. This was sufficient to protect the dopaminergic neurons from 6-OHDA-induced toxicity. In support of our distribution data, Hamilton et al. (2001) showed that simultaneous administration of heparin significantly increases the distribution volume of GDNF in the rat brain, but the functional consequences of this have not been studied. The distribution of infused GDNF has also been studied in the monkey brain, with the diffusion pattern being very unpredictable (Gash et al., 2005; Salvatore et al., 2006). Nevertheless, Gash et al. (2005) showed a positive correlation between GDNF diffusion volume and functional recovery. It was difficult to define, to which extent the lack of efficacy with Δ38N-GDNF was due to insufficient concentrations caused by diffusion or the actual absence of heparin-binding domain. Therefore, we wanted to rule out the possibilities that the lack of efficacy of Δ38NGDNF could have been due to unstability or inability to activate Retmediated signalling pathways. The stability test proved that Δ38NGDNF was very stable in ECM extract up to 48 h. It was also capable of activating RET down-stream target MAPK, and was even more active both alone and in the presence of heparin than wtGDNF in our assay. Our results with wtGDNF are in accordance with that of Tanaka et al.

Fig. 6. Stability of GDNF variants in the ECM extract during a 48-h incubation period, blotted on 15% SDS-PAGE. No significant degradation can be seen.

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Fig. 7. Activation of MAPK-pathway by wtGDNF and Δ38N-GDNF alone or in the presence of heparin (1 or 10 μg/ml). Luminescence was measured after a 24 h incubation period.

(2002), who showed that addition of heparin to the cell culture medium dose dependently increases TH-promoter activity. These results, however, contradict those by Davies et al. (2003) and Barnett et al. (2002), who described an inhibition of GDNF-induced neurite formation in PC12-cells and Ret phosphorylation when exogenous heparin was present. Some studies have also demonstrated that normal signalling of GDNF at low, physiologically relevant concentrations clearly requires cell surface HSPGs, but at experimentally high concentrations the lack of HS glycosaminoglycans does not abolish the effects of GDNF (Barnett et al., 2002; Sainio et al., 1997). Thus, especially at low concentrations of GDNF, interactions with exogenous heparin-like molecules could significantly reduce GDNF availability for GFRα1/RET receptor complex and decrease its signalling. It cannot be excluded that exogenous heparin may specifically change GDNF signalling in some cell cultures as was shown in our experiments and by Tanaka et al. (2002), although these systems do not thoroughly represent the natural in vivo environment. Although several experiments have proved the importance of GDNF binding to heparin, there are also conflicting data. Alfano et al. (2007) have shown that some N-terminally truncated GDNFs, which bind to heparin only with a low affinity, can still bind to GFRα1 and stimulate neurite outgrowth similarly to wild-type GDNF. Also Hu (2001) reported that truncated GDNF variants (including Δ38NGDNF) promoted the survival and differentiation of dopaminergic neurons. In another study, however, an N-terminally truncated GDNF form showed a significantly lower affinity to GFRα1, but still retained complete ability to activate Ret (Eketjäll et al., 1999). Thus, it is likely that the ligand–receptor interaction of GDNF itself does not fully depend on the presence of heparin or HSs. In fact, Alfano et al. (2007) concluded that heparin or HSs are not bridging GDNF and its receptor, since the recombinant GFRα1 does not bind to heparin. It must be taken into account, however, that all the data from nonheparin-binding forms of GDNF was generated in in vitro conditions, and therefore it does not reveal the true importance of cell surface HSPGs in the effects of GDNF. All in all, these in vitro data support our view, that in vivo Δ38N-GDNF is inferior to the wild-type protein most likely because it simply diffuses from the HS anchors and the concentration drops to a suboptimal level, rather than Δ38N-GDNF lacking efficacy. One could assume that at higher doses or continuous administration, causing higher local concentrations, Δ38N-GDNF could be as effective as wtGDNF. However, since Δ38N-GDNF seems to diffuse easily in the brain, it might be difficult to control the spreading of the protein and prevent it from diffusing to unwanted areas of the brain. The increase in dosing could increase the

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risk of toxicity and immunological adverse effects and thus may not be rational from a therapeutical point of view. When interpreting our results one must also bear in mind that some of the differences between Δ38N-GDNF and wtGDNF here may still arise through other factors than just affinity for heparin and heparan sulphates. For example, quite a substantial proportion of the GDNF protein is truncated in our Δ38N-GDNF, and although this region is associated with heparin binding, it may also have some yet unknown functions. Furthermore, Δ38N-GDNF is glycosylated and FLAG-tagged, whereas our commercial wtGDNF that is produced in E. coli is not. Although Lin et al. (1993) have concluded that E. coli-derived GDNF retains full biological activity, and Hu (2001) demonstrated the activity of various truncated GDNF variants, our knowledge of the missing structures and their effects on the activity of the protein remains limited. In conclusion, our results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect of GDNF. It seems that GDNF signalling is very sensitive to changes in concentration when administered as an injection to the brain. HSs are most likely not needed for GDNF–receptor interaction and triggering the signalling itself, but rather to keep GDNF concentrated around its targets. Acknowledgments This study was financially supported by grants from Sigrid Juselius Foundation, the Academy of Finland, University of Helsinki and EU FP6 CancerGRID (LSCH-CT-2006-037559). Amgen Inc. is acknowledged for supplying wtGDNF. The authors also wish to thank Anna Niemi and Elisa Piranen for their assistance. References Alfano, I., Vora, P., Mummery, R.S., Mulloy, B., Rider, C.C., 2007. The major determinant of the heparin binding of glial cell-line-derived neurotrophic factor is near the N-terminus and is dispensable for receptor binding. Biochem. J. 404, 131–140. Baloh, R.H., Tansey, M.G., Johnson Jr., E.M., Milbrandt, J., 2000. Functional mapping of receptor specificity domains of glial cell line-derived neurotrophic factor (GDNF) family ligands and production of GFRalpha1 RET-specific agonists. J. Biol. Chem. 275, 3412–3420. Barnett, M.W., Fisher, C.E., Perona-Wright, G., Davies, J.A., 2002. Signalling by glial cell line-derived neurotrophic factor (GDNF) requires heparan sulphate glycosaminoglycan. J. Cell. Sci. 115, 4495–4503. Bishop, J.R., Schuksz, M., Esko, J.D., 2007. Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446, 1030–1037. Davies, J.A., Yates, E.A., Turnbull, J.E., 2003. Structural determinants of heparan sulphate modulation of GDNF signalling. Growth Factors 21, 109–119. Eketjäll, S., Fainzilber, M., Murray-Rust, J., Ibanez, C.F., 1999. Distinct structural elements in GDNF mediate binding to GFRalpha1 and activation of the GFRalpha1-c-Ret receptor complex. EMBO J. 18, 5901–5910. Ferrario, J.E., Taravini, I.R., Mourlevat, S., Stefano, A., Delfino, M.A., Raisman-Vozari, R., Murer, M.G., Ruberg, M., Gershanik, O., 2004. Differential gene expression induced by chronic levodopa treatment in the striatum of rats with lesions of the nigrostriatal system. J. Neurochem. 90, 1348–1358. Ferrario, J.E., Rojas-Mayorquin, A.E., Saldana-Ortega, M., Salum, C., Gomes, M.Z., Hunot, S., Raisman-Vozari, R., 2008. Pleiotrophin receptor RPTP-zeta/beta expression is up-regulated by L-DOPA in striatal medium spiny neurons of parkinsonian rats. J. Neurochem. 107, 443–452. Gash, D.M., Zhang, Z., Ovadia, A., Cass, W.A., Yi, A., Simmerman, L., Russell, D., Martin, D., Lapchak, P.A., Collins, F., Hoffer, B.J., Gerhardt, G.A., 1996. Functional recovery in parkinsonian monkeys treated with GDNF. Nature 380, 252–255. Gash, D.M., Zhang, Z., Ai, Y., Grondin, R., Coffey, R., Gerhardt, G.A., 2005. Trophic factor distribution predicts functional recovery in parkinsonian monkeys. Ann. Neurol. 58, 224–233. Gill, S.S., Patel, N.K., Hotton, G.R., O'Sullivan, K., McCarter, R., Bunnage, M., Brooks, D.J., Svendsen, C.N., Heywood, P., 2003. Direct brain infusion of glial cell line-derived neurotrophic factor in Parkinson disease. Nat. Med. 9, 589–595. Gospodarowicz, D., Cheng, J., 1986. Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol. 128, 475–484. Hamilton, J.F., Morrison, P.F., Chen, M.Y., Harvey-White, J., Pernaute, R.S., Phillips, H., Oldfield, E., Bankiewicz, K.S., 2001. Heparin coinfusion during convection-enhanced delivery (CED) increases the distribution of the glial-derived neurotrophic factor (GDNF) ligand family in rat striatum and enhances the pharmacological activity of neurturin. Exp. Neurol. 168, 155–161. Hida, H., Jung, C.G., Wu, C.Z., Kim, H.J., Kodama, Y., Masuda, T., Nishino, H., 2003. Pleiotrophin exhibits a trophic effect on survival of dopaminergic neurons in vitro. Eur. J. Neurosci. 17, 2127–2134.

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