Glial cell line-derived neurotrophic factor (GDNF) gene ... - Springer Link

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Pellegrin, Bordeaux, France. Accepted June 2, 1996 ..... Bowenkamp KE, Hoffman AF, Gerhardt GA, Henry MA, Biddle PT, Hoffer B J,. Granholm A-CE (1995) ...

J Neural Transm (1996) 103:1043-1052

__Journal o f _ Neural Transmission 9 Springer-Verlag 1996 Printed in Austria

Glial cell line-derived neurotrophic factor ( G D N F ) gene expression in the human brain: a post mortem in situ hybridization study with special reference to Parkinson's disease S. Hunot z, V. Bernard ~,2, B. Faucheux 1, F. Boissi~re 1, E. Leguern ~, C. Brana 2, P. P. Gautris 3, J. Gu~rin 3, B. Bloch 2, Y. Agid a, and E. C. Hirsch ~ IINSERM U289, H6pital de la Salp~tri~re, Paris, 2Laboratoire d'histologie et d'embryologie, EP CNRS 14, and 3Service de Neurochirurgie, H6pital Pellegrin, Bordeaux, France Accepted June 2, 1996

Summary. Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for dopaminergic neurons. Since dopaminergic neurons degenerate in Parkinson's disease, this factor is a potential therapeutical tool that may save dopaminergic neurons during the pathological process. Moreover, a reduced GDNF expression may be involved in the pathophysiology of the disease. In this study, we tested whether altered G D N F production may participate in the mechanism of cell death in this disease. G D N F gene expression was analyzed by in situ hybridization using riboprobes corresponding to a sequence of the exon 2 human GDNF gene. Experiments were performed on tissue sections of the mesencephalon and the striatum from 8 patients with Parkinson's disease and 6 control subjects matched for age at death and for post mortem delay. No labelling was observed in either group of patients. This absence of detectable expression could not be attributed to methodological problems as a positive staining was observed using the same probes for sections of astroglioma biopsies from human adults and for sections of a newborn infant brain obtained at post-mortem. These data suggest that G D N F is probably expressed at a very low level in the adult human brain and its involvement in the pathophysiology of Parkinson's disease remains to be demonstrated. GDNF may represent a powerful new therapeutic agent for Parkinson's disease, however.

Keywords: GDNF, in situ hybridization, cell death, Parkinson's disease, adult, newborn infant.

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S. Hunot et al. Introduction

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the nigrostriatal pathway, the origin and mechanism of which are as yet unknown. Parkinsonian symptoms become apparent only when a given threshold of dopaminergic denervation is reached, which suggests the existence of compensatory mechanisms (Agid et al., 1993). Indeed, surviving nigrostriatal dopaminergic neurons are considered to be hyperactive (Bernheimer et al., 1973) and postsynaptic dopamine receptors are hypersensitive at least during early stages of the disease (Brooks et al., 1992). From a theoretical point of view, changes in the activity and/or expression of neurotrophic factors may explain both the loss of neurons and the compensatory mechanisms (Unsicker, 1994). The reduced expression of a compound vital for cell survival may explain neuronal death, whereas the increased expression of a neurotrophic factor may account for the compensatory mechanisms by stimulating the sprouting of the surviving neurons. A role of a decreased expression of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) or nerve growth factor in the mechanism of nerve cell death has already been suggested in Alzheimer's disease (Phillips et al., 1991; Murray et al., 1994; Scott et al., 1995). Several neurotrophic factors have been shown to exert growth-promoting or survival effects on dopaminergic neurons, including BDNF (Altar et al., 1994; Beck et al., 1993), epidermal growth factor (Hadjiconstantinou et al., 1991; Kntisel et al., 1990), basic fibroblast growth factor (Kn~isel et al., 1990; Otto and Unsicker, 1993, 1990; Mayer et al., 1993), neurotrophin 3 (Altar et al., 1994; Hyman et al., 1994; Studer et al., 1995), neurotrophin 4/5 (Studer et al., 1995), transforming growth factor-a and -[3 (Alexi et al., 1993; Krieglstein et al., 1995), ciliary neurotrophic factor (Hagg and Varon, 1993) and the more recently described glial cell line-derived neurotrophic factor (GDNF) (Lin et al., 1993). Due to its particularly strong effects on dopaminergic neurons, GDNF has recently attracted considerable interest in relation to the pathophysiology of PD. Indeed, GDNF represents a new potential therapeutical tool for the rescue of dopaminergic neurons which degenerate in PD. In addition, like for any other neurotrophic factor acting on dopaminergic neurons, its altered expression may participate in the mechanism of nerve cell death in PD. GDNF is a glycosylated, disulfite-bonded homodimer that is distantly related to the transforming growth factor-[3 superfamily (Lin et al., 1993). It has been shown to promote the survival and morphological differentiation of dopaminergic neurons and to increase their high-affinity dopamine uptake (Lin et al., 1993). In animal models of PD including MPTP-intoxicated mice and medial forebrain bundle transection in rat, GDNF protects dopaminergic neurons against degeneration (Beck et al., 1995; Tomac et al., 1995) which suggests that this factor is indeed a potential therapeutical tool. By contrast, little is known about GDNF expression during the pathological process of neuronal death in PD. In order to determine whether GDNF could also play a role in dopaminergic degeneration or in the compensatory mechanisms, we analyzed

GDNF mRNA expression in control and parkinsonian brains

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t h e g e n e e x p r e s s i o n of G D N F in t h e m e s e n c e p h a l o n and s t r i a t u m of p a t i e n t s with P D a n d m a t c h e d c o n t r o l subjects.

Materials and methods

Patients and tissue preparation Brains were obtained post mortem from 8 parkinsonian patients, and from 6 control subjects who died with no known neurological disorders. These two groups did not differ significantly either for age at death (mean + S.E.M.; PD patients, 78 _+ 3yrs; controls, 82 _+ 3 yrs; Student's t-test, p = 0.4) or for delay between death and tissue freezing (mean + S.E.M.; PD patients, 20 _+ 3h; controls, 18 _ 3h; Student's t-test; p = 0.7). All parkinsonian patients were responsive to L-DOPA therapy and suffered from akinesia, rigidity and tremor. The diagnosis of PD was confirmed on neuropathological examination of one brain hemisphere (loss of melanized neurons and presence of Lewy bodies in the substantia nigra, in the absence of other histopathological features suggesting another neurodegenerative disease). Within 2 hours of autopsy, blocks containing the striatum or the substantia nigra were dissected from the other hemisphere, rapidly frozen, and stored at -80~ Serial 15-~tm sections were cut using a cryostat. Positive controls of the staining were performed on 1) whole brain sections obtained from a human male newborn infant who died as a result of respiratory distress due to a massive diaphragmatic hernia (postmortem delay: 10h) and 2) sections of glioma biopsies obtained at surgery from four different patients (grades: 1 to 3; localization: cerebellum, temporal cortex, occipital cortex).

Probes preparation GDNF cRNA antisense and sense probes were synthesized by in vitro transcription using 35S-UTP (>1000Ci/mmol; Amersham, UK), from a 250 basepairs DNA sequence. This D N A sequence was obtained from the human GDNF exon 2 by polymerase chain reaction using two primers containing specific sequences, corresponding to the basepairs 151 to 170 ( 5 ' - A G A A T T C C A G A G G A A A A G G T - 3 ' ) and 381 to 400 (5'A A G G C G A T G G G T C T G C A A C A - 3 ' ; Genbank entry accession number L19063 and L15306), and the T3 or T7 polymerase promoting sequences, and human genomic DNA. Unincorporated nucleotides were separated from the labeled probes by Sephadex G50 gel filtration (Pharmacia, Sweden). The probes were then resuspended in the hybridization buffer (50% formamide, 300raM NaC1, 1.25ram E D T A pH = 8, 20mM Tris-HC1, 0.02% Denhardt's solution, 10% dextran sulfate, 0.016% denaturated salmon sperm DNA, 0.064% yeast tRNA, 100mM DTT, 0.1% Sodium Dodecyl Sulfate, 0.l% sodium thiosulfate), to a specific activity of 2 • 104cpm/~tl, and stored at -20~ until use. The PCR products obtained were characterized by migration on a 1% agarose gel. As shown in Fig. 1, a single band of 280bp corresponding to the expected size of the PCR product was detectable in these experiments. Furthermore, the PCR products were sequenced using the PRISM AmpliTaq FS Ready Reaction Dye Primer sequencing kits (Applied Biosystems, Perkin Elmer) and an ABI Prism model 377 automated D N A sequencer (Perkin Elmer). The sequence obtained was similar to the published sequence of human GDNF with less than 0.5% missmatch (data not shown).

In situ hybridization In situ hybridization was performed in triplicate, on slide-mounted sections as described by Brana et al. (1995). Briefly, slide-mounted sections were brought to room temperature, dried under a stream of cold air and post-fixed in 4% (w/v) paraformadehyde/0.1M phosphate buffer pH 7.4. There were then treated with proteinase K (0.01 ~tg/ml in 10• Tris/EDTA buffer) at 37~ In order to acetylate the amino groups and reduce non-

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Fig. 1. Agarose gel electrophoresis of products obtained from PCR reaction using total human genomic DNA as template and specific primers from human GDNF exon 2 (lines 1 and 2), shows a single band of 280pb (arrow) corresponding to the expected lenght

specific binding, sections were incubated in 0.25 % acetic anhydride in 0.1 M ethanolamine (pH 8), followed by dehydratation in increasing concentrations of ethanol (70, 80, 95%) and dried. Sections were then incubated for 4hrs at 55~ in a humid chamber with 50 btl of hybridization mixture containing either the antisense or sense 3sS-labelled RNA probe and sealed under a coverslip. Then, the coverslips were removed in 4• standard sodium citrate (SSC). Post-hybridization treatments were performed as follows, in order to limit non-specific labelling: sections were treated with RNAse A (Boehringer; 20~tg/ml in NaC1/Tris-EDTA) at 37~ to digest the non hybridized probe, washed in 2• SSC at room temperature (RT), in 2• SSC/50% formamide at 60~ in 2• SSC (60~ in 0.1• SSC (60~ and in 0.1 • SSC (RT). All sections were then dehydrated in increasing concentrations of ethanol and dried. After hybridization, the sections were dipped into NTB-2 emulsion (Kodak), diluted 1 : 1 and apposed to Hyperfilm ~-max (Amersham, U.K.) for exposure at 4~ in lightproof boxes. The autoradiographic films and emulsions were developed after 2 and 3 months of exposure, respectively. The sections were counterstained with 0.1% hematoxylin (w/v). This counterstaining allowed cell nuclei to be visualized with bright-field illumination. Results

Control experiments Characteristic and r e p r o d u c i b l e patterns of hybridization were o b s e r v e d with the antisense G D N F p r o b e on film a u t o r a d i o g r a m s of control tissue sections: i.e. glioma and n e o n a t e striatum and c e r e b e l l u m (Fig. 2 A and B). Within t h e striatum, the staining was m o r e intense in the gray m a t t e r (caudate nucleus, p u t a m e n and ventral striatum) t h a n in the white m a t t e r (internal capsule and corpus callosum). Similarly, in t h e c e r e b e l l u m the staining was m o r e pron o u n c e d in the gray matter, with the highest staining intensity in the granular cell layer. No staining was observed w h e n the e x p e r i m e n t s were p e r f o r m e d using the c o r r e s p o n d i n g sense probes (Fig. 2C). Microscopically in the glioma sections, silver grains were observed clustered t o g e t h e r over all the counterstained cells (Fig. 3A), while in the n e o n a t e striatum and cerebellum, only small cells with a dark c o u n t e r s t a i n e d nucleus were labeled (Fig. 3B).

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Fig. 2. Reverse contrast photographs of film autoradiograms of GDNF mRNA in situ hybridization in the post mortem human brain. The hybridization signal was detected in the striatum (A) and the cerebellum (B) of a newborn infant using an antisense cRNA probe. Experiments performed with the corresponding sense probe did not show any staining in the neonate striatum (C). No hybridization signal was detected in the mesencephalon (D, E) and the striatum (F, G) of adult control subjects (D, F) and parkinsonian patients (E, G). CN caudate nucleus; GCL granular cell layer; IC internal capsule; MCL molecular cell layer; PUputamen. Scale bars = 2ram (B); 8ram (A,C,D,E) and 10ram (F,G)

G D N F m R N A in Parkinson's disease

In the striatum (Fig. 2F and G) and m e s e n c e p h a l o n (Fig. 2D and E) of both control subjects and P D patients, no staining was observed on film autoradiograms. Similarly, the silver grain density observed at the cellular level in the striatum and the substantia nigra was very low, with no difference being detected between the antisense and the sense probes (Fig. 3C and

D).

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Fig. 3. Bright-field photomicrographs of GDNF mRNA in situ hybridization obtained with a GDNF antisense cRNA probe in a human astroglioma (A), in a human neonate striatum (B; arrows show examples of GDNF-positive glial cells and arrowheads indicate large neuronal nuclei) and in the striatum of an adult control subject (C) and a parkinsonian patient (D). Scale bar = 10 ~tm Discussion

The specificity of G D N F in situ hybridization was tested on transformed glial cells (glioma) and was taken as a positive control because this neurotrophic factor was first identified in a rat glial cell line (B49 cells) grown in vitro (Lin et al., 1993). Several lines of evidence support the specificity of the observed staining: 1) when the tissue was incubated with the corresponding sense probe, no staining was observed; 2) with the antisense probe, silver grains clustered together over the soma of the glioma cells; 3) a reproducible pattern of staining was observed from one experiment to another and between the different gliomas. Furthermore, in the newborn infant brain the silver grains were concentrated over small cells presenting a morphology compatible with that of glial cells (i.e. cells with a small and dark nucleus on hematoxylin staining) but not of neurons. G D N F m R N A was detected in human gliomas and neonate cerebellum and striatum but not in the adult human striatum and mesencephalon. The absence of staining in the brains of control subjects cannot be attributed to the probe used since it effectively labeled G D N F m R N A in h u m a n astrogliomas. Because gliomas were obtained from adult human brains, this result suggests that G D N F m R N A can be detected in adult human transformed cells. The time interval between the death of the patients and conservation of the brain tissues did not affect G D N F m R N A , since, under similar conditions, the probe allowed detection of G D N F m R N A in a newborn infant striatum also obtained post mortem. In addition, the absence of staining observed in the control and parkinsonian brains used in this study cannot be attributed to a

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global mRNA degradation since glutamic acid decarboxylase (isoform 67 KD) mRNAs (Levy et al., 1995a; Vila et al., 1996), several glutamate receptors mRNAs (Bernard et al., 1996), substance P and methionin-enkephalin mRNAs (Levy et al., 1995b) have been successfully detected by in situ hybridization in the studied brains. It cannot be excluded however that GDNF mRNA is particularly instable post mortem. Yet, this is unlikely given the staining that we observed post mortem in the brain of a young infant. Our data suggest that GDNF might be transiently expressed during development in the human brain, as previously reported in the rat (Olson et al., 1993; Poulsen et al., 1994; Str6mberg et al., 1993). It may also be expressed during adulthood but at levels below the sensitivity of the in situ hybridization technique. In addition, only very low GDNF expression levels have been observed by reverse transcriptase polymerase chain reaction of homogenates from the adult human striatum, hippocampus, cortex or spinal cord (Springer et al., 1994). This result suggests that GDNF is probably not a major neurotrophic factor during adulthood in comparison to TGF[32 or TGF[33 (Poulsen et al., 1994). Moreover, the lack of staining cannot be explained by the sequence of the probe that we used in this study. Several transcripts of GDNF have been extensively described in the rat (Cristina et al., 1995; Schaar et al., 1993; Springer et al., 1994, 1995; Suter-Crazzolara and Unsicker, 1994) and in the human brain (Springer et al., 1994). In the rat, one transcript has been characterized by a 78 basepairs deletion that begins at basepair 123 (Genbank entry L15305) (Cristina et al., 1995) and corresponds to basepair 74 of the human GDNF exon I (Genbank entry L19063 and L15306). The human homologue of this sequence presenting a deletion in rat was carefully excluded from our probe. Accordingly, taken as a whole our results suggest that a lack of GDNF expression in PD does not contribute to nerve cell death. In PD patients, GDNF mRNA was also undetectable under our experimental conditions in both the substantia nigra and the striatum. This result suggests that GDNF is not upregulated under these pathological conditions. In the rat, however, in pilocarpine-induced status epilepticus or kainateinduced seizures, GDNF mRNA expression has been shown to be markedly increased in the hippocampus (Humpel et al., 1994; Schmidt-Kastner et al., 1994). Such an increase of GDNF expression seems also to occur under abnormal conditions since in the weaver mouse, where nigral dopaminergic neurons degenerate, GDNF levels have been reported to increase when dopaminergic axonal fibers are lost (Blum and Weickert, 1995). As a glial proliferation is observed both in PD and under these pathological conditions (Damier et al., 1993; Represa et al., 1995), these two series of data suggest that the glial reaction associated with lesions of the central nervous system does not always stimulate GDNF expression. Moreover, the lack of detectable increase in GDNF expression in PD brains suggests that GDNF is not associated with the plastic changes that are observed in the mesostriatal system of PD patients (Anglade et al., 1996). By contrast, recent preliminary studies indicate that an increase of GDNF-binding in the substantia nigra could be induced by medial forebrain bundle transection suggesting an upregulation of

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a putative G D N F receptor after nigral dopaminergic loss (Treanor et al., 1995). However, this remains to be investigated in PD. Finally, although G D N F expression does not seem to be greatly increased in the pathophysiology of PD, in view of the considerable neurotrophic capacities that G D N F exerts on dopaminergic neurons (Beck et al., 1995; B o w e n k a m p et al., 1995; Lin et al., 1993; T o m a c et al., 1995), it may represent a useful therapeutic agent. On condition that the necessary specific receptor and transduction systems are present and that no side effects are observed, the administration of G D N F or related compounds may greatly improve striatal functions or slow the neuronal degeneration in PD.

Acknowledgements We are grateful to Drs J.-J. Hauw and C. Duyckaerts for providing post mortem human brain samples. This study was supported by INSERM, French Ministry of Research and Education (SH), Association Claude Bernard pour le ddveloppement des recherches biologiques et mEdicales dans les h6pitaux de l'Assistance Publique ~ Paris (BF) and Rh6ne Poulenc-Rorer (FB).

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