- Email: [email protected]
GDNF protects nigral dopamine neurons against 6-hydroxydopamine in vivo
BRAIN RESEARCH ELSEVIER
Brain Research 672 (1995) 104-111
Research report
GDNF protects nigral dopamine neurons against 6-hydroxydopamine in vivo Cecilia M. Kearns *, Don M. Gash Department of Anatomy and Neurobiology, University of Kentucky Chandler Medical Center MN 224, Lexington, KY 40536 USA Accepted 16 November 1994
Abstract Giial cell line-derived neurotrophic factor (GDNF), a novel member of the TGF-/3 superfamity, has been shown to promote the survival and morphological differentiation of fetal dopamine neurons in culture and increase dopamine levels and metabolism in adult rats. Since several other trophic factors are able to rescue specific populations of mature CNS neurons following injury, the present study was designed to investigate a possible neuroprotective role by G D N F for midbrain dopamine neurons in rats exposed to the neurotoxin 6-hydroxydopamine (6-OHDA). Prior to surgery, young adult male Fisher 344 rats were divided into the following groups (n = 7-8/group): (1) intranigral saline + intranigral 6-OHDA; (2) intranigral G D N F + intranigral 6-OHDA; (3) intranigral saline + intrastriatal 6-OHDA; and (4) intranigral G D N F + intrastriatal 6-OHDA. The saline treated groups received a single 2/xl intranigral injection of phosphate buffered saline (PBS) while the G D N F treated rats received 10/~g/2 tzl G D N F in PBS. Twenty-four hours later, the animals received a unilateral 4/xg//zl 6-OHDA infusion either into the substantia nigra or striatum. The rats were sacrificed two weeks postsurgery and the brains processed for tyrosine hydroxylase (TH) immunocytochemistry. Representative TH immunoreactive (TH-IR) sections were also counterstained with hematoxylin and eosin to determine the total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area. In the nigral lesion groups, there was significantly less loss of TH-IR neurons in the substantia nigra pars compacta of G D N F (47% survival) vs. PBS (9% survival) treated animals. The same was true in the ventral tegmental area, where there was a 90% survival of TH-IR neurons in the G D N F treated animals as compared to a 68% survival in PBS treated animals. In the striatal lesion groups, there was significant sparing of TH-IR neurons in the substantia nigra pars compacta of the G D N F (40% survival) compared to the PBS (16% survival) treated animals. However, in the ventral tegmental area, the protection seen in the G D N F treated animals (69% survival) was not statistically significant when compared to the PBS treated rats (48% survival). In sections counter stained with hematoxytin and eosin, the percentage of neurons surviving in G D N F treated hosts was higher suggesting that the 6-OHDA toxicity may reduce TH expression in some dopamine neurons without inducing cell death. Therefore, in both lesion models, our results demonstrate a substantial neuroprotective effect in rats pretreated with G D N F when compared to the vehicle treated groups.
Keywords: Glial cell line-derived neurotrophic factor; Dopamine; Tyrosine hydroxylase; Substantia nigra; Striatum; Neuroprotection
1. Introduction P a r k i n s o n ' s d i s e a s e results from a r e l e n t l e s s progressive loss o f d o p a m i n e n e u r o n s in the m i d b r a i n . C u r r e n t available t h e r a p y relieves m a n y of the symptoms in t h e early to m i d d l e stages o f the d i s e a s e b u t d o e s not a r r e s t or a t t e n u a t e t h e p r o g r e s s i o n of the disease. T h e r e f o r e , identifying i n t e r v e n t i o n s which slow
* Corresponding author. Fax: (1) (606) 323-5946. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 3 6 6 - 7
or inhibit the p r o g r e s s i o n of the n e u r o n a l d e g e n e r a t i o n could be of significant b e n e f i t [16]. C o n s i d e r a b l e e v i d e n c e suggests t h a t n e u r o t r o p h i c factors which p r o m o t e t h e survival o r d i f f e r e n t i a t i o n of d e v e l o p i n g n e u r o n s also m a y p r o t e c t m a t u r e n e u r o n s from axotomy a n d n e u r o t o x i c d a m a g e in vivo. T h e ability of nerve growth factor ( N G F ) [9,24] a n d a rel a t e d n e u r o t r o p h i n , b r a i n - d e r i v e d n e u r o t r o p h i c factor ( B D N F ) [12], to rescue cholinergic n e u r o n s in the b a s a l f o r e b r a i n in s e p t o - h i p p o c a m p a l l e s i o n e d rats has b e e n extensively d o c u m e n t e d . F u r t h e r m o r e , n e u r o t r o p h i n - 3
C.M. Kearns, D.M. Gash~Brain Research 672 (1995) 104-111
(NT-3) has recently been shown to prevent the degeneration of adult rat central noradrenergic neurons of the locus coeruleus in a 6-hydroxydopamine lesion model in vivo [1]. Less is known about the ability of trophic factors to protect midbrain dopamine neurons. The continuous intranigral infusion of ciliary neurotrophic factor (CNTF) recently has been shown to prevent degeneration of axotomized adult rat substantia nigra dopamine neurons in vivo [7]. However, rescued neurons did not express immunoreactivity for tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. A similar administration of BDNF, which supports the survival of fetal dopamine neurons in culture [21], failed to attenuate the decline of striatal dopaminergic function following medial forebrain bundle transection in adult rats [13]. Thus, while CNTF has some effect, it is important to determine if there are other factors that can contribute to the survival and maintenance of a dopaminergic phenotype of injured midbrain dopamine neurons. Glial cell line-derived neurotrophic factor (GDNF) which enhances the survival and neuritogenesis of cultured embryonic midbrain dopaminergic neurons was recently purified and cloned [14]. GDNF is a glycosylated disulfide-bonded homodimer related to the transforming growth factor-/3 superfamily [14]. In culture, GDNF specifically promotes the survival and morphological differentiation of dopamine neurons without effecting the high-affinity uptake of y-aminobutyric acid or serotonin by GABAergic and serotoninergic neurons in fetal midbrain cell cultures. Furthermore, GDNF promotes extensive neurite outgrowth and increased cell body size in TH-positive neurons [14]. Polymerase chain reaction analysis of GDNF mRNA expression in the adult rat and human brain has revealed detectable levels in the striatum of both species [22]. Significant long-lasting effects of GDNF on adult rat midbrain dopaminergic neurons in vivo have been found after intranigral administration [10]. A single injection of GDNF into the substantia nigra initiated sprouting of TH-positive neurites towards the injection site and increased TH immunoreactivity of the ipsilateral striatum. It also produced a significant and dosedependent increase in dopamine turnover in the sutrstantia nigra and striatum and increased ipsilateral dopamine levels in the substantia nigra. The present study was designed to investigate a possible neuroprotective role for GDNF in rats receiving 6-hydroxydopamine (6-OHDA) administered either into the ipsilateral substantia nigra or striatum. Both intranigral and intrastriatal administrations of 6-OHDA result in neurotoxic injury and cell death of midbrain dopamine neurons [19,23]. Control rats received an intranigral injection of phosphate buffered saline (PBS)
105
twenty-four hours prior to either an intranigral or intrastriatal 6-OHDA neurotoxic lesion. Trophic factor treated rats received 10/~g of GDNF in buffer in the same site and time frame as the control groups. THpositive neurons and TH-positive plus hematoxylin and eosin (H and E) counterstained neurons in the ventral tegmental area and substantia nigra of the midbrain were counted. The reason for the latter group was that the 6-OHDA lesion might reduce TH expression without actually killing the neuron. In both lesion models, our results demonstrate a significant neuroprotective effect in rats pretreated with GDNF when compared to the PBS treated groups.
2. Materials and methods
2.1. Animals Thirty-two young adult male Fisher 344 rats weighing 200-260 g at the start of the experiment were used. One died from anesthesia during surgery; 31 animals completed the experimental protocol and were analyzed. The animals were housed one per cage in a temperature-controlled room with a 12 h light/12 h dark cycle and were given free access to food and water ad libitum. Animals were maintained according to the NIH Guide for the Care and Use of Laboratory
Animals. 2.2. Surgery The animals were anesthetized with sodium-pentobarbitol (50 mg/kg, i.p.; Butler Co., Columbus, OH) and placed in a stereotaxic frame (David Kopf Instuments, Tujunga, CA) with the mouth bar set at -3.3 mm. The skull was exposed and burr holes were made using a high-speed dental drill. All control animals were administered 2 /zl of PBS in the right substantia nigra pars compacta using the following coordinates: A / P -5.4 mm, LAT - 2 . 2 mm and Depth -7.5 mm from dura [18]. Trophic factor treated animals received 10/zg of human recombinant GDNF (Synergen, Boulder, CO) in 2 /~1 buffer delivered to the same site. Twenty-four hours later all animals were subjected to a 6-hydroxydopamine hydrobromide (6-OHDA; Sigma, St. Louis, MO) neurotoxic challenge. For half of the animals, the 6-OHDA (8/zg in 2 ~1 saline containing 0.2% ascorbic acid) was injected into the right substantia nigra pars compacta using the same coordinates used for PBS or GDNF administration. The other rats received 6-OHDA administrations into the right striatum using the following coordinates: A / P 0.8 mm, LAT -2.5 mm and Depth -5.5 mm from dura [18]. Therefore, the study consisted of four groups: PBS + intranigral lesion (n = 8),
106
C.M. Kearns, D.M. Gash/Brain Research672 (1995) 104-111
PBS + intrastriatal lesion (n = 7), G D N F + intranigral lesion (n = 8) and G D N F + intrastriatal lesion (n = 8). All injections were performed using a Hamilton 10 gl syringe with a 26-gauge blunt tapered needle, at a rate of 0.2 /~l/min. At the completion of each injection, the needle was left in place for 5 min and then withdrawn at a rate of 1 m m / m i n . All surgeries were carried out under sterile conditions. 2.3. Tissue preparation
Two weeks following surgery, the rats were anesthetized with an overdose of sodium-pentobarbital (50 mg/kg, i.p.; Butler Co., Columbus, OH) and perfused with heparinized saline followed by 4% paraformaldehyde solution in 50 mM potassium phosphate buffered saline (KPBS). Brains were removed and postfixed for 24 h in a 30% sucrose-fixative at 4°C and later transferred to 30% sucrose in 50 mM KPBS at 4°C. Serial coronal frozen sections of 2 0 / x m thickness were cut on a sliding microtome. Six sets of sections were collected in cryoprotectant solution (100 mM KPBS, 30% sucrose, PVP-40, 30% ethylene glycol) and stored at - 2 0 ° C until immunocytochemical and histochemical processing. A series using every sixth section was stained for the primary antibody against tyrosine hydroxylase (TH, 1:500; Boehringer-Mannheim, Germany; 1:500 Chemicon, Temecula, CA). Sections exposed to the primary anti-TH antibodies were incubated in biotinylated horse anti-mouse secondary antibody (1:500 Vector, Burlingame, CA). Sections were then incubated in the avidin-biotin-peroxidase complex using the Elite ABC Vectastain Kit (Vector, Burlingame, CA). TH-immunoreactivity (IR) was visualized using 3,3'-diaminobenzidine as the chromogen with nickel enhancement [4]. Additional T H reacted sections were also counterstained with H and E using procedures previously described [4]. 2.4. Cell counts
The total number of T H - I R and T H - I R plus H and E stained neurons in the substantia nigra pars compacta and ventral tegmental area were counted on both sides at three levels: - 5 . 3 mm, - 5 . 4 mm and - 5 . 5 mm with respect to bregma [18]. The number of cells in the substantia nigra pars compacta and ventral tegmental area were counted in every case. The criteria for delineating the substantia nigra pars compacta from the ventral tegmental area was the localization of the oculomotor nerve root. The ventral tegmental area is medial to the root whereas the substantia nigra pars compacta is laterally located. The degree of dopamine depletion was determined by the loss of T H - I R substantia nigra pars compacta neurons on the lesion side
with respect to the control side of the brain. The total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area on both the lesion and control side of the brain were determined by counting H and E counterstained sections. 2.5. Statistical analysis
The experimental data were statistically analyzed by means of an analysis of variance (ANOVA). Results were provided as the mean _+ S.E.M.
3. Results 3.1. General appearance o f the lesions
In the two PBS treated groups, loss of substantia nigra pars compacta neurons appeared to be quite extensive (Fig. 1A and C). Both T H - I R and H and E stained neurons were absent near the injection sites. Macrophages were evident in the needle tracts leading down to the substantia nigra pars compacta and around the injection sites. Damage to substantia nigra pars compacta neurons in both G D N F treated groups (Fig. 1B and D) appeared to be less severe than observed in the PBS treated animals. Macrophages were still evident in the injection sites. Neurons of each treated group in the substantia nigra pars compacta could be identified by T H immunocytochemistry as well as staining by H and E staining. The morphology of surviving neurons was similar in all four test groups (Fig. 1E-H). In the striatal lesion of both the PBS and G D N F treated groups, the needle tract was delineated by surrounding macrophages. The injury was confined to a diameter of approximately 100/zm around the needle tract except for occasional focal areas of astrocytic reaction. The cytoarchitecture of the surrounding striatal parenchyma appeared normal (Fig. 1I). Based on the results of this study, for the ventral tegmental area there was significant three factor ( P B S / G D N F treated groups by lesioned/control side of brain by type of stain) interaction: F = 3.50 based on 3 and 27 degrees of freedom, P = 0.029. For the substantia nigra pars compacta the three factor interaction was marginally significant: F = 2.56 based on 3 and 27 degrees of freedom, P = 0.075. Also for the substantia nigra pars compacta, two of the two factor interactions were significant: P B S / G D N F treated groups by lesioned/control side of brain ( F = 5.34 based on 3 and 27 degrees of freedom, P = 0.0051) and lesioned/control side of brain by type of stain ( F = 8.92 based on 1 and 27 degrees of freedom, P = 0.0059). For these reasons, between group comparisons were done separately for each lesioned/control side of brain by type of stain combination.
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111
~
~ ~
~
~iiii!i!~ ~.......
~
@i! ~
107
....
Fig. 1. Representative sections are shown through the midbrain of animals in all four groups processed for T H immunocytochemistry and counterstained with H and E. A: the mesencephalon of animals receiving a PBS + nigral lesion revealed significant loss ( < 10% survival) of substantia nigra pars compacta neurons on the lesioned side. B: in contrast, there was marked sparing ( > 45% survival) of substantia nigra pars compacta neurons in the mesencephalon of animals administered G D N F before the nigral lesion. C: extensive loss of snbstantia nigra pars compacta neurons ( < 20% survival) was also evident in the mesencephalon of animals which received PBS + striatal lesion. D: significant sparing of substantia nigra pars compacta neurons ( > 35% survival) was seen in the mesencephalon of G D N F + striatal lesion animals. A needle tract (arrow) can be identified above the substantia nigra in all figures. Macrophage infiltration was evident around all injection sites. A - D are at the same magnification. T h e Scale bar = 500 p.m. E, F, G and H show close-ups of cell morphology within the substantia nigra on the lesioned side. E, F, G and H are higher power magnifications from A, B, C and D, respectively. E - H are at the same magnification. Scale bar in F ffi 10/zm. I shows a needle tract (arrows) for a 6 - O H D A injection in the striatum. Dark macrophages mark the tract while the surrounding H and E stained neurons and glia appear normal. Scale bar in I ~ 50 ~ m .
108
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111 110
• PBS [] QDNF
3.2. Nigral lesions 100
On the lesion side of the brain (Fig. 2), in rats administered intranigral PBS followed by a 6 - O H D A intranigral lesion, a 9% survival of T H - I R neurons in the substantia nigra pars compacta and a 68% survival in the ventral tegmental area was seen. Rats treated with G D N F prior to a 6 - O H D A intranigral lesion had a 47% survival level of T H - I R neurons in the substantia nigra pars compacta and a 90% survival level in the ventral tegmental area. The differences in dopamine neuron survival between the PBS and G D N F treated groups were highly significant for the ventral tegmental area ( P < 0.001) and substantia nigra pars compacta ( P < 0.001) T H - I R neurons. To observe the total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area, an additional set of T H - I R sections were counterstained with H and E. In the 6 - O H D A intranigral lesion groups, rats treated with PBS showed a 19% survival of neurons in the substantia nigra pars compacta and a 63% survival in the ventral tegmental area with H and E counterstained sections. In the G D N F treated rats, there was a 54% survival of neurons in the substantia nigra pars compacta and a 96% survival in the ventral tegmental area. As with the T H - I R cell counts, H and E neuronal counts revealed a significant sparing of neurons in both the ventral tegmental area ( P < 0.001) and substantia nigra pars compacta ( P < 0.001) of G D N F treated animals. 110 • PB8 [] (3DNF
100 90 80 -
70
i,o ©
50
Z
40 30 20 10 0 VTA
SN TH
VTA
SN
TH end H&E
Fig. 2. Percent survival following 6-OHDA nigral lesions. The number of TH-IR and TH-IR plus H and E neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted at 3 levels on both the lesioned and control side of brain. Values are represented as the m e a n s + S.E.M. * * * P < 0.001, significantly different from the PBS group.
90
I
J
80
E 7O E= eo (e) i=
50
=,. o o.
40
g
30 20 10 0 VTA
SN TH
VTA
SN
TH and HIkE
Fig. 3. Percent survival following 6-OHDA striatal lesions. The number of TH-IR and TH-IR plus H and E neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted at 3 levels on both the lesioned and control side of brain. Values are represented as the means_+S.E.M. * P < 0.05 and * * * P < 0.001, significantly different from the PBS group.
3.3. Striatal lesions In the striatal lesion groups (Fig. 3), a 16% survival of T H - I R neurons in the substantia nigra pars compacta and a 48% survival in the ventral tegmental area was found in PBS treated rats. In the G D N F treated animals, survival of T H - I R neurons in the substantia nigra pars compacta increased to 40% and 69% in the ventral tegmental area. Based on the T H - I R plus H and E staining, with PBS pretreatment there was a 36% survival of neurons in the substantia nigra pars compacta and a 42% survival in the ventral tegmental area. In the G D N F treated rats, survival of neurons in the substantia nigra pars compacta was 68% with a 91% survival rate in the ventral tegmental area. In this lesion model, the survival seen in T H - I R neurons demonstrated that G D N F does confer some protection in the substantia nigra pars compacta ( P < 0.05); however, the protection observed in the vental tegmental area was not significant when compared to the PBS treated animals. With respect to the T H plus H and E staining, G D N F also protected neurons in the substantia nigra pars compacta ( P < 0.001) and the ventral tegmental area ( P < 0.001) when compared to the PBS.
3. 4. Control side of brain In all groups, the number of neurons in the substantia nigra pars compacta and ventral tegmental area on
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111 500
• []
VTA
SN
400
o
3O0
I= "6
200
E
m z
100
PB8 GDNF PB$ GDNF PBS GDNF PB$ GDNF TH
TH + H&E
TH
TH + H&E
Fig. 4. T H - I R and TH-IR plus H and E nigral cell counts for the control side of brain. No changes in T H - I R neurons or total cell number were observed after either striatal or nigral lesions on the contralateral side. The number of T H - I R and T H - I R plus H and E neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted at 3 levels. Values were represented as the mean _+S.E.M.
the control side of the brain was counted to determine whether there was any change in the number of TH-IR neurons or neuronal cell death following the lesions on the contralateral side. In the nigral lesion groups, based on the TH and TH plus H and E cell counts, no significant difference was found between the PBS and GDNF treatments on the control side of the brain. Similarly, results from TH and TH plus H and E cell counts also did not reveal significant differences between the PBS and GDNF treatments in the striatal lesion groups (Fig. 4). Therefore, no changes in TH-IR neurons or the total number of neurons identified by H and E staining were observed on the contralateral side in either lesion model.
4. Discussion
Although there is little evidence that trophic factor deficiencies are intimately involved in the etiology of Parkinson's disease, it seems clear thaf trophic factors play a key role in the development, maintenance and repair of dopamine neurons [6]. Based on the effects of neurotrophins in ameliorating the loss of forebrain cholinergic neurons following axotomy [9,12,24], it is reasonable to postulate that trophic factors active in the development of dopamine neurons may also help adult neurons survive when injured. Lin et al. have recently shown that GDNF is a
109
specific neurotrophic factor for developing midbrain dopamine neurons in vitro [14]. Furthermore, GDNF significantly and dose-dependently increases dopamine turnover and ipsilateral dopamine levels in the adult substantia nigra in vivo following a single intranigral injection of GDNF [10]. The present study demonstrates that in addition to enhancing the survival and morphological differentiation of fetal dopamine neurons in vitro and influencing adult rat dopamine neurons in vivo, G D N F protects mature midbrain dopamine neurons from the neurotoxic effects of 6OHDA in vivo. With 6-OHDA administered to two sites (substantia nigra and striatum), GDNF provided significant protection of TH-IR neurons within the substantia nigra. However, only a clear protective effect was observed in the ventral tegmental area following the intranigral lesion. Based on T H plus H and E counterstain, total neuronal counts revealed that ventral tegmental area neurons were being protected though they were not TH-IR. The higher cell counts observed in the T H plus H and E counterstained sections in both the GDNF and PBS treated animals suggest that the remaining neurons in the substantia nigra pars compacta and ventral tegrnental area which do not label with T H alone are producing low levels of TH. The production of T H in these neurons may have been effected by 6-OHDA neurotoxicity with the higher number of surviving cells in the substantia nigra and ventral tegrnental area of GDNF treated animals reflecting the ability of the trophic factor to maintain injured neurons. On the control side of the brain, there were no significant changes in TH-IR cell numbers or the total number of H and E neurons in either lesion model. Therefore, in this study, the effects of GDNF administration observed in the nigral and striatal lesion models were primarily seen on the ipsilateral side of the brain. Based on the TH-IR cell counts, the protective effect of GDNF was somewhat more robust in the nigral lesion model. One explanation for this is that 6-OHDA infused into the substantia nigra produces an acute lesion with neuronal death evident within ten minutes [23]. In contrast, the midbrain neuronal loss produced by 6-OHDA infused into the striatum appears to take place over a number of weeks. Sauer and Oertel [20] have shown progressive degeneration of nigral dopamine neurons starting one week post striatal 6-OHDA administration and continuing over 8 to 16 weeks. GDNF, like other proteins in the intercellular space has a short half-life in the brain and exogenously administered GDNF may be most effective in protecting against the acute effects of 6-OHDA. Therefore, repeated administrations of GDNF may be needed to improve the survival of dopamine neurons subjected to a progressive striatal 6-OHDA insult as opposed to an acute nigral lesion.
110
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111
Recent studies have identified a number of trophic factors which influence dopamine neuron viability in culture [6]. Most of these trophic effects on dopamine neurons appear to be indirect and mediated by glial cells since inhibition of glial proliferation in culture, prevents their neurotrophic effects [2,6]. Only four trophic factors appear to exert specific effects on fetal dopamine neurons in culture: BDNF, NT-3, NT-4/5 and GDNF [11,14]. Less is known about the trophic factors influencing mature dopamine neurons in vivo. Epidermal growth factor (EGF) [8], acidic fibroblast growth factor (aFGF) [5] and basic fibroblast growth factor (bFGF) [17] have been shown to promote recovery from 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridinetreated (MPTP) toxicity in the adult mouse striatum. Intrastriatal administration of aFGF or bFGF increases the number of TH-IR fibers and striatal dopamine levels in MPTP-treated mice [5,17]. In addition, EGF has been reported to increase striatal dopamine levels and TH activity [8]. There is also evidence that direct infusion of ciliary neurotrophic factor (CNTF) can rescue axotomized nigral dopamine neurons in the rat, although these neurons do not express immunoreactivity for TH [7]. The present study shows that in addition to ameliorating neuronal loss, GDNF maintains TH-IR in the substantia nigra and to some extent in the ventral tegrnental area following a neurotoxic insult such as 6-OHDA. The precise mechanisms of GDNF neuroprotection still remain to be determined; however, there are several hypotheses. First, since 6-OHDA enters the dopamine neuron via its specific uptake carrier, GDNF may protect neurons by blocking its uptake [21]. Another possibility is that a GDNF-receptor interaction may activate a second messenger signal transduction pathway capable of activating immediate early genes, the products of which are transcriptional factors capable of mediating biological responses to growth factors (i.e. stabilization of Ca 2+ homeostasis, enhanced free radical defence mechanism) [15]. Once 6-OHDA enters the neuron and undergoes nonenzymatic oxidation to produce hydrogen peroxide (H202), superoxide radical ( 0 2 - ) and hydroxyl radical (.OH), these active oxygen species initiate cell destruction by damaging a variety of essential proteins, DNA and membrane lipids [3]. H 2 0 2 can be detoxified by the enzyme, glutathione peroxidase. A glutathione peroxidase/glutathione reductase system has been shown to detoxify H202 generated by monoamine oxidase in brain [21]. GDNF may elicit its protective effects by interacting with this enzymatic system to prevent against oxidative stress events and consequent cell death. The evidence presented here suggests that GDNF has significant protective properties for dopamine neurons. The exact mechanisms will need to be deter-
mined in future studies. The present study provides a preliminary basis for defining GDNF as a potential neuroprotective agent and opens up new opportunities for a better understanding of the degenerative processes underlying neurotoxic damage and loss of central dopamine neurons.
Acknowledgements We thank Dr. Deborah Russell and Dr. David Martin of Synergen (Boulder, CO) for their helpful discussions on this study. We also thank Dr. Richard Kryscio for assistance with statistical analysis. Technical assistance was provided by Linda Simmerman. Supported by NIH Grant P01-25778.
References [1] Arenas, E. and Persson, H., Neurotrophin-3 prevents the death of adult central noradrenergic neurons in vivo, Nature, 367 (1994) 368-371. [2] Casper, D., Mytilineou, C. and Blum, M., EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture, Z Neurosci. Res., 30 (1991) 372-381. [3] Cohen, G. and Heikkila, R., The generation of hydrogen peroxide, superoxide radical and hydroxyl radical by 6-OHDA, dialuric acid and related cytotoxic agents, J. Biol. Chem., 249 (1974) 2447-2452. [4] Date, I., Felten, S.Y. and Feiten, D.L., Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice, Brain Res., 537 (1990) 33-39. [5] Date, I., Notter, M.F.D., Felten S.Y. and Felten D.L., MPTPtreated young mice but not aging mice show partial recovery of the nigrostriatal dopaminergic system by stereotaxic injection of acidic fibroblast growth factor (aFGF), Brain Res., 526 6-160. [6] Gash, D.M., Bresjanac, M., Junn, F. and Zhang, Z., Trophic mechanisms mediating functional recovery following intrastriatal implantation. In Functional Neural Transplantation, Raven Press, New York, 1994, pp. 139-154. [7] Hagg, T. and Varon, S., Ciliary neurotrophic factor prevents degeneration of adult rat substantia nigra dopaminergic neurons, Proc. Natl. Acad. Sci. USA, 90 (1993) 6315-6319. [8] Hadjiconstantinou, M., Fitkin, J.G., Dalia, A. and Neff, N.H., Epidermal growth factor enhances striatal dopaminergic parameters in the l-methyl-4-phenyt-l,2,3,6-tetrahydropyridine-treated mouse, 3. Neurochem. 57 (1991) 479-482. [9] Hefti, F., Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections, J. Neurosci., 6 (1986) 2155-2162. [10] Hudson, J., Granholm, A., Gerhardt, G.A., Henry, M.A., Hoffman, A., Biddle, P., Leela, N.S., Mackerlova L., Lile, J.D., Collins, F. and Hoffer, B., GDNF, a glial cell line-derived novel member of the TGFfl superfamily, manifests neurotrophic activity on midbrain dopaminergic neurons in vivo, Brain Res. Bull., in press. [11] Hyman, C., Juhasz, M., Jackson, C., Wright, P., Ip, N.Y. and Lindsay, R.M., Overlapping and distinct actions of the neurotrophins BDNF, NT-3 and NT-4/5 on cultured dopaminergic
C.M. Kearns, D.M. Gash~Brain Research 672 (1995) 104-111
and GABAergic neurons of the ventral mesencephalon, .L Neurosci., 14(1X1994) 335-347. [12] Knusel, B., Beck, K.D., Winslow, J.W., Rosenthal, A., Burton, L.E., Widmer, H.R., Nikolics, K. and Hefti, F., Brain-derived neurotrophic factor administration protects basal forebrain cholinergic but not nigral dopaminergic neurons from degenerative changes after axotomy in the adult rat brain, Z Neurosci., 12(11) (1992) 4391-4402. [13] Lapchak, P.A., Beck, K.D., Araujo, D.M., Irwin, I., Langston, J.W. and Hefti, F., Chronic intranigral administration of brainderived neurotrophic factor produces striatal dopaminergic hypofunction in unlesioned adult rats and fails to attenuate the decline of striatal dopaminergic function following medial forebrain bundle transection, Neuroscience, 53 (1993) 639-650. [14] Lin, L.H., Doherty, D.H., Lile, J.D., Bektesh, S. and Collins, F., GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons, Science, 260 (1993) 1130-1132. [15] Mattson, M.P., Cheng, B. and Smith-Swintosky, V.L., Neurotrophic factor mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism, Neurosciences, 5 (1993) 295-307. [16] Olanow, C.W., A scientific rationale for protective therapy in parkinson's disease, J. Neural Transm., 91 (1993) 161-180. [17] Otto, D. and Unsicker, K., Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTPtreated mice, J. Neurosci., 10 (1990) 1912-1921. [18] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Australia, 1986.
111
[19] Przedborski, S., Levivier, M., Kostic, V., Jackson-Lewis, V., Dollison, A., Gash, D.M., Fahn, S. and Cadet, J., Sham transplantation protects against 6-hydroxydopamine-induced dopaminergic toxicity in rats: behavioral and morphological evidence, Brain Res., 550 (1991) 231-238. [20] Sauer, H. and Oertel, W.H., Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-OHDA: a combined retrograde tracing and immunocytochemical study in the rat, Neuroscience, 59 (1994) 401-415. [21] Spina, M., Squinto, S.P., Miller. J., Lindsay, R.M. and Hyman, C., Brain-derived neurotrophic factor protects dopamine neurons against 6-hydroxydopamine and N-methyl-4-phenylpyridinium ion toxicity : involvement of the glutathione system, J. Neurochem., 59 (1992) 99-106. [22] Springer, J.E., Mu, X., Bergmann, L.W. and Trojanowski, J.Q., Expression of GDNF mRNA in rat and human nervous tissue, Exp. Neurol., 127 (1994) 167-170. [23] Ungerstedt, U. and Arbuthnott, G.W., Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigrostriatal dopamine system, Brain Res., 24 (1970) 485-493. [24] Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bjorklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc. Natl. Acad. Sci. USA, 83 (1986) 9231-9235.
Brain Research 672 (1995) 104-111
Research report
GDNF protects nigral dopamine neurons against 6-hydroxydopamine in vivo Cecilia M. Kearns *, Don M. Gash Department of Anatomy and Neurobiology, University of Kentucky Chandler Medical Center MN 224, Lexington, KY 40536 USA Accepted 16 November 1994
Abstract Giial cell line-derived neurotrophic factor (GDNF), a novel member of the TGF-/3 superfamity, has been shown to promote the survival and morphological differentiation of fetal dopamine neurons in culture and increase dopamine levels and metabolism in adult rats. Since several other trophic factors are able to rescue specific populations of mature CNS neurons following injury, the present study was designed to investigate a possible neuroprotective role by G D N F for midbrain dopamine neurons in rats exposed to the neurotoxin 6-hydroxydopamine (6-OHDA). Prior to surgery, young adult male Fisher 344 rats were divided into the following groups (n = 7-8/group): (1) intranigral saline + intranigral 6-OHDA; (2) intranigral G D N F + intranigral 6-OHDA; (3) intranigral saline + intrastriatal 6-OHDA; and (4) intranigral G D N F + intrastriatal 6-OHDA. The saline treated groups received a single 2/xl intranigral injection of phosphate buffered saline (PBS) while the G D N F treated rats received 10/~g/2 tzl G D N F in PBS. Twenty-four hours later, the animals received a unilateral 4/xg//zl 6-OHDA infusion either into the substantia nigra or striatum. The rats were sacrificed two weeks postsurgery and the brains processed for tyrosine hydroxylase (TH) immunocytochemistry. Representative TH immunoreactive (TH-IR) sections were also counterstained with hematoxylin and eosin to determine the total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area. In the nigral lesion groups, there was significantly less loss of TH-IR neurons in the substantia nigra pars compacta of G D N F (47% survival) vs. PBS (9% survival) treated animals. The same was true in the ventral tegmental area, where there was a 90% survival of TH-IR neurons in the G D N F treated animals as compared to a 68% survival in PBS treated animals. In the striatal lesion groups, there was significant sparing of TH-IR neurons in the substantia nigra pars compacta of the G D N F (40% survival) compared to the PBS (16% survival) treated animals. However, in the ventral tegmental area, the protection seen in the G D N F treated animals (69% survival) was not statistically significant when compared to the PBS treated rats (48% survival). In sections counter stained with hematoxytin and eosin, the percentage of neurons surviving in G D N F treated hosts was higher suggesting that the 6-OHDA toxicity may reduce TH expression in some dopamine neurons without inducing cell death. Therefore, in both lesion models, our results demonstrate a substantial neuroprotective effect in rats pretreated with G D N F when compared to the vehicle treated groups.
Keywords: Glial cell line-derived neurotrophic factor; Dopamine; Tyrosine hydroxylase; Substantia nigra; Striatum; Neuroprotection
1. Introduction P a r k i n s o n ' s d i s e a s e results from a r e l e n t l e s s progressive loss o f d o p a m i n e n e u r o n s in the m i d b r a i n . C u r r e n t available t h e r a p y relieves m a n y of the symptoms in t h e early to m i d d l e stages o f the d i s e a s e b u t d o e s not a r r e s t or a t t e n u a t e t h e p r o g r e s s i o n of the disease. T h e r e f o r e , identifying i n t e r v e n t i o n s which slow
* Corresponding author. Fax: (1) (606) 323-5946. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 3 6 6 - 7
or inhibit the p r o g r e s s i o n of the n e u r o n a l d e g e n e r a t i o n could be of significant b e n e f i t [16]. C o n s i d e r a b l e e v i d e n c e suggests t h a t n e u r o t r o p h i c factors which p r o m o t e t h e survival o r d i f f e r e n t i a t i o n of d e v e l o p i n g n e u r o n s also m a y p r o t e c t m a t u r e n e u r o n s from axotomy a n d n e u r o t o x i c d a m a g e in vivo. T h e ability of nerve growth factor ( N G F ) [9,24] a n d a rel a t e d n e u r o t r o p h i n , b r a i n - d e r i v e d n e u r o t r o p h i c factor ( B D N F ) [12], to rescue cholinergic n e u r o n s in the b a s a l f o r e b r a i n in s e p t o - h i p p o c a m p a l l e s i o n e d rats has b e e n extensively d o c u m e n t e d . F u r t h e r m o r e , n e u r o t r o p h i n - 3
C.M. Kearns, D.M. Gash~Brain Research 672 (1995) 104-111
(NT-3) has recently been shown to prevent the degeneration of adult rat central noradrenergic neurons of the locus coeruleus in a 6-hydroxydopamine lesion model in vivo [1]. Less is known about the ability of trophic factors to protect midbrain dopamine neurons. The continuous intranigral infusion of ciliary neurotrophic factor (CNTF) recently has been shown to prevent degeneration of axotomized adult rat substantia nigra dopamine neurons in vivo [7]. However, rescued neurons did not express immunoreactivity for tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. A similar administration of BDNF, which supports the survival of fetal dopamine neurons in culture [21], failed to attenuate the decline of striatal dopaminergic function following medial forebrain bundle transection in adult rats [13]. Thus, while CNTF has some effect, it is important to determine if there are other factors that can contribute to the survival and maintenance of a dopaminergic phenotype of injured midbrain dopamine neurons. Glial cell line-derived neurotrophic factor (GDNF) which enhances the survival and neuritogenesis of cultured embryonic midbrain dopaminergic neurons was recently purified and cloned [14]. GDNF is a glycosylated disulfide-bonded homodimer related to the transforming growth factor-/3 superfamily [14]. In culture, GDNF specifically promotes the survival and morphological differentiation of dopamine neurons without effecting the high-affinity uptake of y-aminobutyric acid or serotonin by GABAergic and serotoninergic neurons in fetal midbrain cell cultures. Furthermore, GDNF promotes extensive neurite outgrowth and increased cell body size in TH-positive neurons [14]. Polymerase chain reaction analysis of GDNF mRNA expression in the adult rat and human brain has revealed detectable levels in the striatum of both species [22]. Significant long-lasting effects of GDNF on adult rat midbrain dopaminergic neurons in vivo have been found after intranigral administration [10]. A single injection of GDNF into the substantia nigra initiated sprouting of TH-positive neurites towards the injection site and increased TH immunoreactivity of the ipsilateral striatum. It also produced a significant and dosedependent increase in dopamine turnover in the sutrstantia nigra and striatum and increased ipsilateral dopamine levels in the substantia nigra. The present study was designed to investigate a possible neuroprotective role for GDNF in rats receiving 6-hydroxydopamine (6-OHDA) administered either into the ipsilateral substantia nigra or striatum. Both intranigral and intrastriatal administrations of 6-OHDA result in neurotoxic injury and cell death of midbrain dopamine neurons [19,23]. Control rats received an intranigral injection of phosphate buffered saline (PBS)
105
twenty-four hours prior to either an intranigral or intrastriatal 6-OHDA neurotoxic lesion. Trophic factor treated rats received 10/~g of GDNF in buffer in the same site and time frame as the control groups. THpositive neurons and TH-positive plus hematoxylin and eosin (H and E) counterstained neurons in the ventral tegmental area and substantia nigra of the midbrain were counted. The reason for the latter group was that the 6-OHDA lesion might reduce TH expression without actually killing the neuron. In both lesion models, our results demonstrate a significant neuroprotective effect in rats pretreated with GDNF when compared to the PBS treated groups.
2. Materials and methods
2.1. Animals Thirty-two young adult male Fisher 344 rats weighing 200-260 g at the start of the experiment were used. One died from anesthesia during surgery; 31 animals completed the experimental protocol and were analyzed. The animals were housed one per cage in a temperature-controlled room with a 12 h light/12 h dark cycle and were given free access to food and water ad libitum. Animals were maintained according to the NIH Guide for the Care and Use of Laboratory
Animals. 2.2. Surgery The animals were anesthetized with sodium-pentobarbitol (50 mg/kg, i.p.; Butler Co., Columbus, OH) and placed in a stereotaxic frame (David Kopf Instuments, Tujunga, CA) with the mouth bar set at -3.3 mm. The skull was exposed and burr holes were made using a high-speed dental drill. All control animals were administered 2 /zl of PBS in the right substantia nigra pars compacta using the following coordinates: A / P -5.4 mm, LAT - 2 . 2 mm and Depth -7.5 mm from dura [18]. Trophic factor treated animals received 10/zg of human recombinant GDNF (Synergen, Boulder, CO) in 2 /~1 buffer delivered to the same site. Twenty-four hours later all animals were subjected to a 6-hydroxydopamine hydrobromide (6-OHDA; Sigma, St. Louis, MO) neurotoxic challenge. For half of the animals, the 6-OHDA (8/zg in 2 ~1 saline containing 0.2% ascorbic acid) was injected into the right substantia nigra pars compacta using the same coordinates used for PBS or GDNF administration. The other rats received 6-OHDA administrations into the right striatum using the following coordinates: A / P 0.8 mm, LAT -2.5 mm and Depth -5.5 mm from dura [18]. Therefore, the study consisted of four groups: PBS + intranigral lesion (n = 8),
106
C.M. Kearns, D.M. Gash/Brain Research672 (1995) 104-111
PBS + intrastriatal lesion (n = 7), G D N F + intranigral lesion (n = 8) and G D N F + intrastriatal lesion (n = 8). All injections were performed using a Hamilton 10 gl syringe with a 26-gauge blunt tapered needle, at a rate of 0.2 /~l/min. At the completion of each injection, the needle was left in place for 5 min and then withdrawn at a rate of 1 m m / m i n . All surgeries were carried out under sterile conditions. 2.3. Tissue preparation
Two weeks following surgery, the rats were anesthetized with an overdose of sodium-pentobarbital (50 mg/kg, i.p.; Butler Co., Columbus, OH) and perfused with heparinized saline followed by 4% paraformaldehyde solution in 50 mM potassium phosphate buffered saline (KPBS). Brains were removed and postfixed for 24 h in a 30% sucrose-fixative at 4°C and later transferred to 30% sucrose in 50 mM KPBS at 4°C. Serial coronal frozen sections of 2 0 / x m thickness were cut on a sliding microtome. Six sets of sections were collected in cryoprotectant solution (100 mM KPBS, 30% sucrose, PVP-40, 30% ethylene glycol) and stored at - 2 0 ° C until immunocytochemical and histochemical processing. A series using every sixth section was stained for the primary antibody against tyrosine hydroxylase (TH, 1:500; Boehringer-Mannheim, Germany; 1:500 Chemicon, Temecula, CA). Sections exposed to the primary anti-TH antibodies were incubated in biotinylated horse anti-mouse secondary antibody (1:500 Vector, Burlingame, CA). Sections were then incubated in the avidin-biotin-peroxidase complex using the Elite ABC Vectastain Kit (Vector, Burlingame, CA). TH-immunoreactivity (IR) was visualized using 3,3'-diaminobenzidine as the chromogen with nickel enhancement [4]. Additional T H reacted sections were also counterstained with H and E using procedures previously described [4]. 2.4. Cell counts
The total number of T H - I R and T H - I R plus H and E stained neurons in the substantia nigra pars compacta and ventral tegmental area were counted on both sides at three levels: - 5 . 3 mm, - 5 . 4 mm and - 5 . 5 mm with respect to bregma [18]. The number of cells in the substantia nigra pars compacta and ventral tegmental area were counted in every case. The criteria for delineating the substantia nigra pars compacta from the ventral tegmental area was the localization of the oculomotor nerve root. The ventral tegmental area is medial to the root whereas the substantia nigra pars compacta is laterally located. The degree of dopamine depletion was determined by the loss of T H - I R substantia nigra pars compacta neurons on the lesion side
with respect to the control side of the brain. The total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area on both the lesion and control side of the brain were determined by counting H and E counterstained sections. 2.5. Statistical analysis
The experimental data were statistically analyzed by means of an analysis of variance (ANOVA). Results were provided as the mean _+ S.E.M.
3. Results 3.1. General appearance o f the lesions
In the two PBS treated groups, loss of substantia nigra pars compacta neurons appeared to be quite extensive (Fig. 1A and C). Both T H - I R and H and E stained neurons were absent near the injection sites. Macrophages were evident in the needle tracts leading down to the substantia nigra pars compacta and around the injection sites. Damage to substantia nigra pars compacta neurons in both G D N F treated groups (Fig. 1B and D) appeared to be less severe than observed in the PBS treated animals. Macrophages were still evident in the injection sites. Neurons of each treated group in the substantia nigra pars compacta could be identified by T H immunocytochemistry as well as staining by H and E staining. The morphology of surviving neurons was similar in all four test groups (Fig. 1E-H). In the striatal lesion of both the PBS and G D N F treated groups, the needle tract was delineated by surrounding macrophages. The injury was confined to a diameter of approximately 100/zm around the needle tract except for occasional focal areas of astrocytic reaction. The cytoarchitecture of the surrounding striatal parenchyma appeared normal (Fig. 1I). Based on the results of this study, for the ventral tegmental area there was significant three factor ( P B S / G D N F treated groups by lesioned/control side of brain by type of stain) interaction: F = 3.50 based on 3 and 27 degrees of freedom, P = 0.029. For the substantia nigra pars compacta the three factor interaction was marginally significant: F = 2.56 based on 3 and 27 degrees of freedom, P = 0.075. Also for the substantia nigra pars compacta, two of the two factor interactions were significant: P B S / G D N F treated groups by lesioned/control side of brain ( F = 5.34 based on 3 and 27 degrees of freedom, P = 0.0051) and lesioned/control side of brain by type of stain ( F = 8.92 based on 1 and 27 degrees of freedom, P = 0.0059). For these reasons, between group comparisons were done separately for each lesioned/control side of brain by type of stain combination.
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111
~
~ ~
~
~iiii!i!~ ~.......
~
@i! ~
107
....
Fig. 1. Representative sections are shown through the midbrain of animals in all four groups processed for T H immunocytochemistry and counterstained with H and E. A: the mesencephalon of animals receiving a PBS + nigral lesion revealed significant loss ( < 10% survival) of substantia nigra pars compacta neurons on the lesioned side. B: in contrast, there was marked sparing ( > 45% survival) of substantia nigra pars compacta neurons in the mesencephalon of animals administered G D N F before the nigral lesion. C: extensive loss of snbstantia nigra pars compacta neurons ( < 20% survival) was also evident in the mesencephalon of animals which received PBS + striatal lesion. D: significant sparing of substantia nigra pars compacta neurons ( > 35% survival) was seen in the mesencephalon of G D N F + striatal lesion animals. A needle tract (arrow) can be identified above the substantia nigra in all figures. Macrophage infiltration was evident around all injection sites. A - D are at the same magnification. T h e Scale bar = 500 p.m. E, F, G and H show close-ups of cell morphology within the substantia nigra on the lesioned side. E, F, G and H are higher power magnifications from A, B, C and D, respectively. E - H are at the same magnification. Scale bar in F ffi 10/zm. I shows a needle tract (arrows) for a 6 - O H D A injection in the striatum. Dark macrophages mark the tract while the surrounding H and E stained neurons and glia appear normal. Scale bar in I ~ 50 ~ m .
108
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111 110
• PBS [] QDNF
3.2. Nigral lesions 100
On the lesion side of the brain (Fig. 2), in rats administered intranigral PBS followed by a 6 - O H D A intranigral lesion, a 9% survival of T H - I R neurons in the substantia nigra pars compacta and a 68% survival in the ventral tegmental area was seen. Rats treated with G D N F prior to a 6 - O H D A intranigral lesion had a 47% survival level of T H - I R neurons in the substantia nigra pars compacta and a 90% survival level in the ventral tegmental area. The differences in dopamine neuron survival between the PBS and G D N F treated groups were highly significant for the ventral tegmental area ( P < 0.001) and substantia nigra pars compacta ( P < 0.001) T H - I R neurons. To observe the total number of neurons remaining in the substantia nigra pars compacta and ventral tegmental area, an additional set of T H - I R sections were counterstained with H and E. In the 6 - O H D A intranigral lesion groups, rats treated with PBS showed a 19% survival of neurons in the substantia nigra pars compacta and a 63% survival in the ventral tegmental area with H and E counterstained sections. In the G D N F treated rats, there was a 54% survival of neurons in the substantia nigra pars compacta and a 96% survival in the ventral tegmental area. As with the T H - I R cell counts, H and E neuronal counts revealed a significant sparing of neurons in both the ventral tegmental area ( P < 0.001) and substantia nigra pars compacta ( P < 0.001) of G D N F treated animals. 110 • PB8 [] (3DNF
100 90 80 -
70
i,o ©
50
Z
40 30 20 10 0 VTA
SN TH
VTA
SN
TH end H&E
Fig. 2. Percent survival following 6-OHDA nigral lesions. The number of TH-IR and TH-IR plus H and E neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted at 3 levels on both the lesioned and control side of brain. Values are represented as the m e a n s + S.E.M. * * * P < 0.001, significantly different from the PBS group.
90
I
J
80
E 7O E= eo (e) i=
50
=,. o o.
40
g
30 20 10 0 VTA
SN TH
VTA
SN
TH and HIkE
Fig. 3. Percent survival following 6-OHDA striatal lesions. The number of TH-IR and TH-IR plus H and E neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted at 3 levels on both the lesioned and control side of brain. Values are represented as the means_+S.E.M. * P < 0.05 and * * * P < 0.001, significantly different from the PBS group.
3.3. Striatal lesions In the striatal lesion groups (Fig. 3), a 16% survival of T H - I R neurons in the substantia nigra pars compacta and a 48% survival in the ventral tegmental area was found in PBS treated rats. In the G D N F treated animals, survival of T H - I R neurons in the substantia nigra pars compacta increased to 40% and 69% in the ventral tegmental area. Based on the T H - I R plus H and E staining, with PBS pretreatment there was a 36% survival of neurons in the substantia nigra pars compacta and a 42% survival in the ventral tegmental area. In the G D N F treated rats, survival of neurons in the substantia nigra pars compacta was 68% with a 91% survival rate in the ventral tegmental area. In this lesion model, the survival seen in T H - I R neurons demonstrated that G D N F does confer some protection in the substantia nigra pars compacta ( P < 0.05); however, the protection observed in the vental tegmental area was not significant when compared to the PBS treated animals. With respect to the T H plus H and E staining, G D N F also protected neurons in the substantia nigra pars compacta ( P < 0.001) and the ventral tegmental area ( P < 0.001) when compared to the PBS.
3. 4. Control side of brain In all groups, the number of neurons in the substantia nigra pars compacta and ventral tegmental area on
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111 500
• []
VTA
SN
400
o
3O0
I= "6
200
E
m z
100
PB8 GDNF PB$ GDNF PBS GDNF PB$ GDNF TH
TH + H&E
TH
TH + H&E
Fig. 4. T H - I R and TH-IR plus H and E nigral cell counts for the control side of brain. No changes in T H - I R neurons or total cell number were observed after either striatal or nigral lesions on the contralateral side. The number of T H - I R and T H - I R plus H and E neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted at 3 levels. Values were represented as the mean _+S.E.M.
the control side of the brain was counted to determine whether there was any change in the number of TH-IR neurons or neuronal cell death following the lesions on the contralateral side. In the nigral lesion groups, based on the TH and TH plus H and E cell counts, no significant difference was found between the PBS and GDNF treatments on the control side of the brain. Similarly, results from TH and TH plus H and E cell counts also did not reveal significant differences between the PBS and GDNF treatments in the striatal lesion groups (Fig. 4). Therefore, no changes in TH-IR neurons or the total number of neurons identified by H and E staining were observed on the contralateral side in either lesion model.
4. Discussion
Although there is little evidence that trophic factor deficiencies are intimately involved in the etiology of Parkinson's disease, it seems clear thaf trophic factors play a key role in the development, maintenance and repair of dopamine neurons [6]. Based on the effects of neurotrophins in ameliorating the loss of forebrain cholinergic neurons following axotomy [9,12,24], it is reasonable to postulate that trophic factors active in the development of dopamine neurons may also help adult neurons survive when injured. Lin et al. have recently shown that GDNF is a
109
specific neurotrophic factor for developing midbrain dopamine neurons in vitro [14]. Furthermore, GDNF significantly and dose-dependently increases dopamine turnover and ipsilateral dopamine levels in the adult substantia nigra in vivo following a single intranigral injection of GDNF [10]. The present study demonstrates that in addition to enhancing the survival and morphological differentiation of fetal dopamine neurons in vitro and influencing adult rat dopamine neurons in vivo, G D N F protects mature midbrain dopamine neurons from the neurotoxic effects of 6OHDA in vivo. With 6-OHDA administered to two sites (substantia nigra and striatum), GDNF provided significant protection of TH-IR neurons within the substantia nigra. However, only a clear protective effect was observed in the ventral tegmental area following the intranigral lesion. Based on T H plus H and E counterstain, total neuronal counts revealed that ventral tegmental area neurons were being protected though they were not TH-IR. The higher cell counts observed in the T H plus H and E counterstained sections in both the GDNF and PBS treated animals suggest that the remaining neurons in the substantia nigra pars compacta and ventral tegrnental area which do not label with T H alone are producing low levels of TH. The production of T H in these neurons may have been effected by 6-OHDA neurotoxicity with the higher number of surviving cells in the substantia nigra and ventral tegrnental area of GDNF treated animals reflecting the ability of the trophic factor to maintain injured neurons. On the control side of the brain, there were no significant changes in TH-IR cell numbers or the total number of H and E neurons in either lesion model. Therefore, in this study, the effects of GDNF administration observed in the nigral and striatal lesion models were primarily seen on the ipsilateral side of the brain. Based on the TH-IR cell counts, the protective effect of GDNF was somewhat more robust in the nigral lesion model. One explanation for this is that 6-OHDA infused into the substantia nigra produces an acute lesion with neuronal death evident within ten minutes [23]. In contrast, the midbrain neuronal loss produced by 6-OHDA infused into the striatum appears to take place over a number of weeks. Sauer and Oertel [20] have shown progressive degeneration of nigral dopamine neurons starting one week post striatal 6-OHDA administration and continuing over 8 to 16 weeks. GDNF, like other proteins in the intercellular space has a short half-life in the brain and exogenously administered GDNF may be most effective in protecting against the acute effects of 6-OHDA. Therefore, repeated administrations of GDNF may be needed to improve the survival of dopamine neurons subjected to a progressive striatal 6-OHDA insult as opposed to an acute nigral lesion.
110
C.M. Kearns, D.M. Gash/Brain Research 672 (1995) 104-111
Recent studies have identified a number of trophic factors which influence dopamine neuron viability in culture [6]. Most of these trophic effects on dopamine neurons appear to be indirect and mediated by glial cells since inhibition of glial proliferation in culture, prevents their neurotrophic effects [2,6]. Only four trophic factors appear to exert specific effects on fetal dopamine neurons in culture: BDNF, NT-3, NT-4/5 and GDNF [11,14]. Less is known about the trophic factors influencing mature dopamine neurons in vivo. Epidermal growth factor (EGF) [8], acidic fibroblast growth factor (aFGF) [5] and basic fibroblast growth factor (bFGF) [17] have been shown to promote recovery from 1-methyl-4-phenyl-l,2,3,6-tetrahydropyridinetreated (MPTP) toxicity in the adult mouse striatum. Intrastriatal administration of aFGF or bFGF increases the number of TH-IR fibers and striatal dopamine levels in MPTP-treated mice [5,17]. In addition, EGF has been reported to increase striatal dopamine levels and TH activity [8]. There is also evidence that direct infusion of ciliary neurotrophic factor (CNTF) can rescue axotomized nigral dopamine neurons in the rat, although these neurons do not express immunoreactivity for TH [7]. The present study shows that in addition to ameliorating neuronal loss, GDNF maintains TH-IR in the substantia nigra and to some extent in the ventral tegrnental area following a neurotoxic insult such as 6-OHDA. The precise mechanisms of GDNF neuroprotection still remain to be determined; however, there are several hypotheses. First, since 6-OHDA enters the dopamine neuron via its specific uptake carrier, GDNF may protect neurons by blocking its uptake [21]. Another possibility is that a GDNF-receptor interaction may activate a second messenger signal transduction pathway capable of activating immediate early genes, the products of which are transcriptional factors capable of mediating biological responses to growth factors (i.e. stabilization of Ca 2+ homeostasis, enhanced free radical defence mechanism) [15]. Once 6-OHDA enters the neuron and undergoes nonenzymatic oxidation to produce hydrogen peroxide (H202), superoxide radical ( 0 2 - ) and hydroxyl radical (.OH), these active oxygen species initiate cell destruction by damaging a variety of essential proteins, DNA and membrane lipids [3]. H 2 0 2 can be detoxified by the enzyme, glutathione peroxidase. A glutathione peroxidase/glutathione reductase system has been shown to detoxify H202 generated by monoamine oxidase in brain [21]. GDNF may elicit its protective effects by interacting with this enzymatic system to prevent against oxidative stress events and consequent cell death. The evidence presented here suggests that GDNF has significant protective properties for dopamine neurons. The exact mechanisms will need to be deter-
mined in future studies. The present study provides a preliminary basis for defining GDNF as a potential neuroprotective agent and opens up new opportunities for a better understanding of the degenerative processes underlying neurotoxic damage and loss of central dopamine neurons.
Acknowledgements We thank Dr. Deborah Russell and Dr. David Martin of Synergen (Boulder, CO) for their helpful discussions on this study. We also thank Dr. Richard Kryscio for assistance with statistical analysis. Technical assistance was provided by Linda Simmerman. Supported by NIH Grant P01-25778.
References [1] Arenas, E. and Persson, H., Neurotrophin-3 prevents the death of adult central noradrenergic neurons in vivo, Nature, 367 (1994) 368-371. [2] Casper, D., Mytilineou, C. and Blum, M., EGF enhances the survival of dopamine neurons in rat embryonic mesencephalon primary cell culture, Z Neurosci. Res., 30 (1991) 372-381. [3] Cohen, G. and Heikkila, R., The generation of hydrogen peroxide, superoxide radical and hydroxyl radical by 6-OHDA, dialuric acid and related cytotoxic agents, J. Biol. Chem., 249 (1974) 2447-2452. [4] Date, I., Felten, S.Y. and Feiten, D.L., Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice, Brain Res., 537 (1990) 33-39. [5] Date, I., Notter, M.F.D., Felten S.Y. and Felten D.L., MPTPtreated young mice but not aging mice show partial recovery of the nigrostriatal dopaminergic system by stereotaxic injection of acidic fibroblast growth factor (aFGF), Brain Res., 526 6-160. [6] Gash, D.M., Bresjanac, M., Junn, F. and Zhang, Z., Trophic mechanisms mediating functional recovery following intrastriatal implantation. In Functional Neural Transplantation, Raven Press, New York, 1994, pp. 139-154. [7] Hagg, T. and Varon, S., Ciliary neurotrophic factor prevents degeneration of adult rat substantia nigra dopaminergic neurons, Proc. Natl. Acad. Sci. USA, 90 (1993) 6315-6319. [8] Hadjiconstantinou, M., Fitkin, J.G., Dalia, A. and Neff, N.H., Epidermal growth factor enhances striatal dopaminergic parameters in the l-methyl-4-phenyt-l,2,3,6-tetrahydropyridine-treated mouse, 3. Neurochem. 57 (1991) 479-482. [9] Hefti, F., Nerve growth factor promotes survival of septal cholinergic neurons after fimbrial transections, J. Neurosci., 6 (1986) 2155-2162. [10] Hudson, J., Granholm, A., Gerhardt, G.A., Henry, M.A., Hoffman, A., Biddle, P., Leela, N.S., Mackerlova L., Lile, J.D., Collins, F. and Hoffer, B., GDNF, a glial cell line-derived novel member of the TGFfl superfamily, manifests neurotrophic activity on midbrain dopaminergic neurons in vivo, Brain Res. Bull., in press. [11] Hyman, C., Juhasz, M., Jackson, C., Wright, P., Ip, N.Y. and Lindsay, R.M., Overlapping and distinct actions of the neurotrophins BDNF, NT-3 and NT-4/5 on cultured dopaminergic
C.M. Kearns, D.M. Gash~Brain Research 672 (1995) 104-111
and GABAergic neurons of the ventral mesencephalon, .L Neurosci., 14(1X1994) 335-347. [12] Knusel, B., Beck, K.D., Winslow, J.W., Rosenthal, A., Burton, L.E., Widmer, H.R., Nikolics, K. and Hefti, F., Brain-derived neurotrophic factor administration protects basal forebrain cholinergic but not nigral dopaminergic neurons from degenerative changes after axotomy in the adult rat brain, Z Neurosci., 12(11) (1992) 4391-4402. [13] Lapchak, P.A., Beck, K.D., Araujo, D.M., Irwin, I., Langston, J.W. and Hefti, F., Chronic intranigral administration of brainderived neurotrophic factor produces striatal dopaminergic hypofunction in unlesioned adult rats and fails to attenuate the decline of striatal dopaminergic function following medial forebrain bundle transection, Neuroscience, 53 (1993) 639-650. [14] Lin, L.H., Doherty, D.H., Lile, J.D., Bektesh, S. and Collins, F., GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons, Science, 260 (1993) 1130-1132. [15] Mattson, M.P., Cheng, B. and Smith-Swintosky, V.L., Neurotrophic factor mediated protection from excitotoxicity and disturbances in calcium and free radical metabolism, Neurosciences, 5 (1993) 295-307. [16] Olanow, C.W., A scientific rationale for protective therapy in parkinson's disease, J. Neural Transm., 91 (1993) 161-180. [17] Otto, D. and Unsicker, K., Basic FGF reverses chemical and morphological deficits in the nigrostriatal system of MPTPtreated mice, J. Neurosci., 10 (1990) 1912-1921. [18] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, 2nd edn., Academic Press, Australia, 1986.
111
[19] Przedborski, S., Levivier, M., Kostic, V., Jackson-Lewis, V., Dollison, A., Gash, D.M., Fahn, S. and Cadet, J., Sham transplantation protects against 6-hydroxydopamine-induced dopaminergic toxicity in rats: behavioral and morphological evidence, Brain Res., 550 (1991) 231-238. [20] Sauer, H. and Oertel, W.H., Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-OHDA: a combined retrograde tracing and immunocytochemical study in the rat, Neuroscience, 59 (1994) 401-415. [21] Spina, M., Squinto, S.P., Miller. J., Lindsay, R.M. and Hyman, C., Brain-derived neurotrophic factor protects dopamine neurons against 6-hydroxydopamine and N-methyl-4-phenylpyridinium ion toxicity : involvement of the glutathione system, J. Neurochem., 59 (1992) 99-106. [22] Springer, J.E., Mu, X., Bergmann, L.W. and Trojanowski, J.Q., Expression of GDNF mRNA in rat and human nervous tissue, Exp. Neurol., 127 (1994) 167-170. [23] Ungerstedt, U. and Arbuthnott, G.W., Quantitative recording of rotational behavior in rats after 6-hydroxydopamine lesions of the nigrostriatal dopamine system, Brain Res., 24 (1970) 485-493. [24] Williams, L.R., Varon, S., Peterson, G.M., Wictorin, K., Fischer, W., Bjorklund, A. and Gage, F.H., Continuous infusion of nerve growth factor prevents basal forebrain neuronal death after fimbria fornix transection, Proc. Natl. Acad. Sci. USA, 83 (1986) 9231-9235.