GM1 ganglioside treatment promotes recovery of striatal dopamine concentrations in the mouse model of MPTP-induced Parkinsonism

EXPERIMENTALNEUROLOGY

105,177-183

(1989)

GM, Ganglioside Treatment Promotes Recovery of Striatal Dopamine Concentrations in the Mouse Model of MPTP-Induced Parkinsonism J.S. SCHNEIDER*AND

A. YUWILER~

*Center for Neurological Research, Department of Neurology, and Institute of Neuroscience, Philadelphia, Pennsylvania 19102; and TBrentwood VA Hospital, Neurobiochemistry

GM1 ganglioside (GM,) has in the past been reported to promote regenerative sprouting and functional recovery in both central and peripheral nervous systems. The present experiments were performed in order to investigate whether GM1 might have any therapeutic effect on young mice who had been exposed to the Parkinson-producing neurotoxin MPTP. GM1 caused moderate to dramatic increases in striatal dopamine levels, depending upon duration of exposure to GM1, in animals previously exposed to MPTP. Furthermore, the effects of GM1 on enhancing striatal dopamine levels were apparent when GM1 administration was delayed until 3 days after the last MPTP injection was given and these effects were not reversed when GM1 was withdrawn. Tyrosine hydroxylase (TH) immunohistochemistry of the striatum demonstrated increased numbers of TH-positive fibers and TH-positive terminal fields in GM1-treated animals as compared to animals that received only MPTP. TH immunohistochemistry of the substantia nigra revealed little or no loss of pars compacta neurons in the MPTP-treated mice. On the basis of these observations, GM1 appears to increase the dopamine content of the striatum by promoting or stimulating regenerative sprouting of dopaminergic terminals and perhaps collateral sprouting from remaining intact fibers in the MPTP model of Parkinsonism in the young mouse. We suggest that GM1 ganglioside may hold some promise as a potential adjunct in the treatment of Parkinson’s Disease. d 1999 Academic Press, Inc.

INTRODUCTION

Gangliosides, complex sphingolipids found in fairly high concentrations in brain and neuronal membranes (ll), have been thought to play an important role in neuronal development and differentiation (12,26). More recently, the administration of exogenous gangliosides (specifically, GM, ganglioside) has been shown to promote neuronal regeneration and axonal sprouting in damaged central and peripheral nervous systems (5, 15, 25), and GM1 ganglioside (GM,) has been suggested to

Hahnemann University Laboratory, Los Angeles,

School of Medicine, California 90018

act as a neuronotrophic factor in noradrenergic, serotonergic, cholinergic, and dopaminergic systems (2, 5, 15, 25). Regenerative sprouting and functional recovery has been particularly impressive within the nigrostriatal dopamine system. In most studies to date, the effect of GM, ganglioside on the recovery of nigrostriatal dopaminergic neurons has been examined in rats following unilateral hemitransections of the nigrostriatal fibers (2,3,10, 17). After partial lesions of ascending dopaminergic fibers, the chronic administration of GM, ganglioside (up to 56 days) significantly increased the tyrosine hydroxylase (TH) activity and dopamine content in the striatum ipsilateral to the lesion (24). Furthermore, chronic GM, treatment promoted the survival of axotomized nigral dopaminergic cell bodies and increased the length of dopaminergic dendrites in the substantia nigra pars reticulata on the lesioned side (1,2). Lesion-induced dopamine receptor supersensitivity in the striatum was likewise reversed by GM1 treatment, as was apomorphine-induced rotational behavior (1, 2). In contrast, after an almost total unilateral lesion of the ascending dopamine system, GM, had no effect on striatal TH activity (24). These results are compatible with the view that chronic GM, treatment increases the density of striatal dopaminergic terminals via collateral sprouting from fibers of intact dopamine cells, leading to recovery of striatal dopaminergic synaptic function (1,2). If this hypothesis is correct, GM1 ganglioside could be an important clinical tool in the treatment of disorders of the dopaminergic system such as Parkinson’s disease. Since a reasonable case exists for exogenous GM, ganglioside as a neuronotrophic factor within the dopamine system, the present experiments were conducted to determine whether the administration of exogenous GM, ganglioside could enhance striatal dopamine levels in an animal model of MPTP-induced Parkinsonism. METHODS

In the first experiment, MPTP.HCl (30 mg/kg) was dissolved in physiological saline and administered subcutaneously to young adult, male C-57 black mice (8-12

177 All

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0014.4886/89 $3.00 by Academic Press, Inc. in any form reserved.

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weeks of age, n = 17) and given twice a day (4-6 h apart) for 5 consecutive days. After the final MPTP injections, some mice (n = 7) were left alone for 4 weeks, while others (n = 10) were sacrificed after 2 weeks. These animals constituted the MPTP control groups. Other mice were given identical MPTP injections but received an intraperitoneal GM, ganglioside injection within 15 min of each MPTP injection. Ganglioside (obtained from Fidia Research Laboratories) was administered intraperitoneally in a dose of 30 mg/kg (n = 15). GM, treatment continued daily for either 2 (n = 9) or 4 weeks (n = 6) following cessation of MPTP administration. An additional four animals served as saline-treated normal control animals and three animals received saline injections and GM, ganglioside (30 mg/kg) daily for 2 weeks. In an effort to test whether GM, might have some protective effects against MPTP toxicity, we coadministered MPTP (30 mg/kg, two injections per day for 5 days) and GM, (30 mg/kg) to an additional five animals and sacrificed these animals 2 days following the cessation of injections. These animals were compared with mice who received only MPTP on the same injection schedule (n = 3). In order to assess whether GM1 might have beneficial effects on the dopamine system once damage has already occurred, we administered 5 days of MPTP, as described above, to 14 mice and began administering daily GM1 injections (30 mg/kg) on the third day following cessation of MPTP administration. Daily GM, injections continued for 3 weeks. At the end of the 3 weeks of GM1 treatment, nine mice were sacrificed. The five remaining mice were sacrificed 2 weeks later, having received no additional GM, treatments. At the end of all experimental periods, animals were killed by decapitation and the brains were rapidly removed, chilled, and dissected over ice to remove the dorsal neostriatum (caudate-putamen) on one side. This tissue was then assayed to determine concentrations of dopamine and its metabolite dihydroxyphenylacetic acid by high-pressure liquid chromatography (HPLC) with electrochemical detection. Catecholamines were quantitated by peak height relative to a 3,4-dihydroxybenzylamine standard after separation on an Axion ODS 15 CM, 5-pm column using a pH 2.8 phosphate buffer with sodium octane sulfonate and EDTA as mobile phase. Means and standard errors were calculated for HPLC data. HPLC data were further analyzed by analysis of variance with multiple comparisons (22) and the Bonferri t test. After dissection, brain stems and remaining striatal tissues were submersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for subsequent tyrosine hydroxylase (TH) immunohistochemical processing. The avidin-biotin peroxidase method (20) was used to visualize TH. Following fixation, brains were im-

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mersed in 30% sucrose for 1 week and then cut frozen (50 pm thickness). Sections were collected in cold PBS and subjected to a methanolic-peroxide quenching of endogenous peroxidase activity. Blocking sera was applied to the tissue followed by incubation in the primary antibody (1:2000 dilution, rabbit anti-TH, Eugene Tech., Inc.). The tissue was then processed using the Vectastain avidin-biotin method, with DAB as the chromogen. Alternate sections through the brain stem were counter stained with cresyl violet. RESULTS

GM, ganglioside administration significantly increased the measurable dopamine in the striatum of treated mice (Table 1). The duration of ganglioside treatment seemed to be an important factor in the recovery process. Animals given MPTP and 2 weeks of GM1 (n = 9) had a mean striatal dopamine level of 1.91 lug/g wet tissue (SEM = 0.33), whereas animals receiving only MPTP and left alone for 2 weeks had a mean of only 0.34 pg/g (f0.05) (P < 0.001). MPTP-treated animals given GM1 for 4 weeks (n = 6) had a mean striatal dopamine level of 8.76 pg/g (kO.58) as compared to animals given only MPTP (X = 0.67 + 0.18; P < .OOl). Animals that received five injections of MPTP and GM1 coadministered showed no evidence of a GM, protective effect against MPTP toxicity. Animals with MPTP and GM, coadministered for a week and sacrificed 3 days later had a mean striatal dopamine content of 0.74 pug/g (+0.12), which was not significantly different from the mean levels observed in animals given only 5 days of MPTP (0.90 pg/g f 0.12). GM1 ganglioside appears to have had beneficial effects on striatal dopamine levels even when GM1 administration was delayed until after MPTP-induced damage had occurred. Animals that received 5 days of MPTP and then received 3 weeks of GM, beginning on the third day following cessation of MPTP had mean striatal dopamine levels of 3.05 fig/g (f0.23). This is a significant increase compared to animals that had MPTP only (0.67 pg/g + 0.18). Likewise, mice that had 3 weeks of delayed onset GM, treatments (as described above) and that were taken off GM1 for 2 weeks prior to sacrifice had mean striatal dopamine levels that were not significantly different from those measured in the above-mentioned delayed onset GM1 mice that received continuous GM1 treatment. Last, we found that GM1 administered for 2 weeks to normal, saline-injected control mice had no significant effect on striatal dopamine levels. Other investigators have claimed similar results with even longer duration GM1 administration (8, 25). There were also no significant differences between any of the experimental or control groups in regard to the amount of striatal tissue

GM,

GANGLIOSIDE

TABLE

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1

Effects of GM1 Ganglioside on Striatal Dopamine Concentrations

in Mice Exposed to MPTP Striatal dopamine

Group 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Normal-saline Normal-saline + GM1 for 2 weeks MPTP only-2 week survival MPTP only-4 week survival MPTP + GM, for 2 weeks MPTP + GMl, for 4 weeks MPTP only-5 days-2 day survival MPTP/GM1 coadministration-5 days-2 day survival MPTP + delayed GM, for 3 weeks MPTP + delayed GM, for 3 weeks, off GM, for 2 weeks

N 4 3 10 7 9 6 3 5 9 5

k/d 12.70 14.64 0.34 0.67 1.91 8.76 0.90 0.74 3.05 2.90

f 1.9 f 1.74t Lt 0.05 k 0.18 + 0.33* f 0.58** k 0.12 i 0.12tt f 0.23 f 0.46t’l-f

Striatal

DOPAC bg/d

2.24 2.46 0.20 0.26 0.61 2.72

f zk k t f k 1.73 * 0.97 f

0.50 0.56 0.06 0.18 0.23 0.30

0.59 0.19

Note. Values represent means + standard errors. Dopamine and DOPAC are expressed as rg/g wet tissue. All animals received the same dose of MPTP (30 mg/kg, 2X daily for 5 consecutive days) and GM, ganglioside (30 mg/kg). MPTP-only animals were sacrificed either 2 days or 2 or 4 weeks following cessation of MPTP injections. MPTP t GM1 animals received daily MPTP and GM, injections for 5 days and continued to receive GM, for 2 or 4 weeks. MPTP/GM, animals were coadministered MPTP and GM, for 5 days and were sacrificed 2 days following cessation of injections. MPTP + delayed GM, animals were given 5 days of MPTP, started on GM, on the third day following cessation of MPTP injections, continued on GM, for 3 weeks, and sacrificed either immediately or 2 weeks following cessation of GM, injections. t No-significant difference compared to Group 1. tt No significant difference compared to Group 7. ttt No significant difference compared to Group 9. * Significant (P < 0.01) compared to Group 3. ** Significant (P < 0.001) compared to Group 4.

sampled. For example, normal control, ganglioside controls, MPTP only animals, and MPTP plus 4 weeks of ganglioside animals had mean striatal tissue weights of 0.014 g (+O.O02), 0.013 g (+0.003), 0.013 g (f0.002), and 0.014 g (+O.OOl), respectively. Striatal levels of the major dopamine metabolite in the mouse, dihydroxyphenylacetic acid (DOPAC), were markedly decreased in the animals that received only MPTP but recovered to normal levels in mice that received 4 weeks of GM, treatment (Table 1). Some recovery of DOPAC levels was also seen in animals that received only 2 weeks of GM1 or delayed onset GM1 treatment (Table 1). TH immunocytochemistry of the striatum revealed an increase in TH staining in GM,-treated animals as compared to MPTP controls (Fig. 1). In the normal mouse striatum, TH appears as diffuse, intense staining of terminal fields with some fiber staining apparent in the background (Fig. 1A). After MPTP exposure, the TH terminal field staining in the striatum is greatly reduced, most probably reflecting MPTP-induced damage to striatal dopaminergic terminals. Many residual TH-containing fibers are still visible in the striatum at this time (Fig. 1B). MPTP-induced decreases in TH terminal field staining in the nucleus accumbens were less severe than those observed in the dorsal striatum. Following GM1 administration, the diffuse TH-positive terminal field staining was increased somewhat after 2 weeks of GM, treatment and increased greatly after 4 weeks of GM,

treatment (Fig. 1C). This terminal field staining was likewise greatly increased following 3 weeks of delayed onset GM, and showed no significant reduction in staining following a 2-week withdrawal of GM1. In contrast to effects at the st+iatal level, neither MPTP nor GM, appeared to have significant effects on brain stem dopaminergic neurons. The number of THpositive neurons in the substantia nigra pars compacta did not appear significantly diminished, as compared to normal control tissue, in MPTP-exposed mice, with or without GM1 treatment (Figs. lD-1F). GM, administration alone also had no gross effect on striatal dopaminergic terminal fields or nigral dopamine-containing neurons. MPTP caused a transient akinesia/catatonia that abated shortly after termination of the MPTP injections. Due to the transient nature of the motor impairment produced by MPTP, we did not assess the effects of GM1 on motor functions in these animals. DISCUSSION The present results suggest that exogenously administered GM, ganglioside may at least partially restore dopamine levels in the striatum of the young, mature MPTP-exposed mouse. While it is true that MPTP-treated mice can show substantial recovery of striatal dopamine levels 2-5 months after MPTP administration (18), we have not observed any spontaneous recovery within the

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FIG. 1. Mouse caudate/putamen (striatum) stained to visualize tyrosine hydroxylase (TH) immunoreactivity. (A) Normal distribution of TH in the mouse dorsal striatum, which appears as diffuse, heavy staining indicative of TH-positive terminals and fibers. (B) After 5 days of MPTP (30 mg/kg, SC, twice daily, 4-6 h apart) and no further treatment for 4 weeks, TH immunoreactivity in the dorsal striatum is greatly reduced. Although a number of TH-containing fibers are still visible within the striatum, the diffuse terminal field-like staining is virtually absent. (C) In a mouse given the same MPTP dosage as above but which also received GM, injections within 15 min of each MPTP injection and which continued to receive GM, daily for 4 weeks, there is a dramatic increase in TH immunoreactivity in the striatum. (D) TH-positive neurons in the normal mouse substantia nigra pars compacta @NC). (E) Four weeks after MPTP administration (same dose as stated above), there was no evidence of significant cell loss in the SNc. (F) After MPTP administration and 4 weeks of GM1 treatment, there again was no evidence of any significant changes in the SNc cell population. The presence or the absence of cells in the vental mesencephalon was corroborated in all animals by Nissl staining of alternate sections. Calibration = 100 pm (A-C) and 500 pm (D-F).

3-5 weeks that we have examined these animals. If we assume that our MPTP-treated mice would have shown at least some recovery of striatal dopamine levels given a long enough post-MPTP survival period, at the very least then, we can assume that administration of GM1 has accelerated that recovery process. While the precise

mechanisms responsible for the presently observed increase in striatal dopamine levels are not known, at least one possibility is that GM1 may in some way have acted to stimulate or promote the sprouting of TH-containing terminals and/or the sprouting of TH-containing axon collaterals from remaining intact dopaminergic fibers.

GM, SN dopamine

neuron

GANGLIOSIDE

Striatum

FIG. 2. This figure illustrates some of the possible mechanisms which may be contributing to the recovery of striatal dopamine levels in young mice exposed to MPTP. (A) Normal substantia nigra (SN) dopamine neurons project to the striatum and have intact terminals. (B) After administration of MPTP, the toxic metabolite of MPTP, MPP+ causes severe destruction of dopaminergic terminals (dotted arrow) in the striatum, but most SN dopamine-containing cell bodies survive. (C) As long as SN (and ventral tegmental area and retrorubral) dopamine neurons are relatively intact, GM, might stimulate either regeneration of lost terminals (single arrow) or might stimulate collaterals to sprout from intact fibers (double arrow). These collaterals from intact dopaminergic fibers may provide functional release of dopamine in the striatum.

Both types of these processes have been reported in the past to be stimulated by exogenous GM, ganglioside (1, 25). Alternatively, GM1 may have caused a significant up-regulation of TH and dopamine synthesis in existing nigral neurons, resulting in the observed increase in striatal dopamine and TH levels. It is of note that in the mouse MPTP model, as we have used it, we found there to be little degeneration of substantia nigra dopaminergic neurons. This is consistent with our previous results using this model (18) and with the results of others (4, 17, 27), which have suggested that MPTP may cause primarily terminal damage when administered to young, mature mice. Therefore, since most of the cells of origin of striatal dopaminergic fibers were left intact, it is possible that terminal regrowth occurred from damaged fibers in the striatum and that undamaged fibers sprouted axon collaterals (Fig. 2). It is possible that GM1 might exert different effects on the MPTP-damaged nervous system when degeneration occurs at both the dopamine cell body and the terminal. In preliminary studies, we have observed a greatly diminished response to GM1 in aged MPTPtreated mice (21). Since we have observed considerable dopamine cell loss in these aged mice (Schneider, unpublished observation) it is possible that either the aged

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brain has a diminished capacity for the regenerative growth stimulated by GM, or that under conditions of more extensive degeneration, GM, has either reduced effectiveness or requires a longer time period to see evidence of its effects. The possible mechanism(s) responsible for GMi-induced terminal or collateral sprouting is presently unknown. Exogenous gangliosides can be incorporated into normal peripheral nerve but more extensively into damaged nerve (5,6). Gorio and Carmignoto (6) have postulated that neural regeneration may be regulated by at least two signals: one that determines the sprouting site and one that signals the cell body to increase protein synthesis. Gangliosides may have a primary influence on the first process, perhaps by incorporating themselves into membranes and altering membrane ion permeability, thus possibly increasing calcium ion influx. This could then help to initiate sprout formation via alterations induced in the cell membrane/cytoskeleton (6, 12). Alternatively, GM,, through its incorporation into neuronal membranes, may exert its regenerative or growth-promoting effects indirectly by causing some change (perhaps proliferation) of receptor sites for some other growth factor which might be circulating in the damaged CNS. Recently, Hadjiconstantinou and Neff (8) have also reported that GM1 ganglioside treatment can restore striatal dopamine levels in MPTP-treated mice. These researchers found a greater magnitude increase in striatal dopamine than reported in the present paper. This was perhaps due to the fact that MPTP-treated mice in their study had initial dopamine decreases of only approximately 50%, while the mice in our study had dopamine depletions in excess of 90%. While there are several similarities between our present data and previously published results (8), there is disagreement concerning the permanence of the GMi-induced striatal dopamine increases. It was previously reported that dopamine concentrations fell to levels observed in mice which only received MPTP within 30 days of discontinuing GM1 treatment (8). We have not observed decreases in dopamine levels after a 2-week withdrawal of GM1 . It is possible that a longer delay between discontinuation of GM1 and sacrifice of the animals may also result in decreases in dopamine levels in our model. This possibility is presently being examined. The present findings, while rather preliminary, could have important implications for the treatment of Parkinson’s Disease. GM1 ganglioside can cross the bloodbrain barrier (16), can be administered peripherally, is actively taken up into neuronal cell membranes (2) and apparently has no toxic side effects (9). If GM, ganglioside were administered to Parkinson’s patients at the first signs of impairment (when presumably, many nigral dopaminergic neurons are still intact), collateral sprouting (if it occurs) from remaining intact neurons

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might at the very least delay the onset of severe motor disability. Ganglioside administered to patients with established disability might promote enough repair to permit decreased drug therapy or improve drug-related fluctuations in performance. It has been suggested that adrenal medullary transplants into the ventricular surface of one caudate nucleus of Parkinson’s patients may make or stimulate production of a growth-promoting trophic substance(s) which may provoke the patient’s own surviving dopaminergic axons to regenerate and innervate the dopamine-deprived striatum. Such atrophic substance could diffuse through the cerebrospinal fluid and cause bilateral improvement. Interestingly, the patients claimed to have the most benefit from adrenal transplantation to date have been young and without long-standing Parkinson’s Disease (13), further strengthening the possibility that neuronal growth may have been stimulated by transplant-induced release of trophic factors (14). Clearly, many questions need to be answered concerning the mechanisms of GM1-mediated effects. If GM, can improve function by sprouting or some other means in MPTP-exposed animals, it may have alternative treatment implications for human Parkinson’s Disease. But, before this can be considered, much more work is required at the animal experimental level in order to more clearly define the conditions under which GM, ganglioside may or may not be effective. ACKNOWLEDGMENTS The authors thank Grace Unguez, Leslie Roth, and Ray Wallace for their expert technical assistance and Setsuko Kashitani for preparing the manuscript. We thank Dr. G. Toffano and Fidia Laboratories for their very generous gift of GM1 ganglioside. This research was supported by gifts from Dr. Harry Kaufman and Mrs. Heidi Fisher. REFERENCES AGNATI, L. F., K. FUXE, L. CALZA, F. BENFENATI, L. CAVICCHIOLI, G. TOFFANO, AND M. GOLDSTEIN. 1983. Gangliosides increase the survival of lesioned nigral dopamine neurons and favour the recovery of dopaminergic synaptic function in striatum of rats by collateral sprouting. Actu Physiol. Stand. 119: 347363. AGNATI, L. F., K. FUXE, I. ZINI, P. DAVILLI, A. CORTI, L. CALZA, G. TOFFANO, M. ZOLI, G. PICCININI, AND M. GOLDSTEIN. 1985. Effects of lesions and ganglioside GM1 treatment on striatal polyamine levels and nigral DA neurons. A role of putrescine in the neurotrophic activity of gangliosides. Actu Physiol. Stand. 124: 499-506. AGNATI, L. F., F. BENFENATI, N. BATTISTINI, L. CAVICCHIOLI, K. FUXE, AND G. TOFFANO. 1983. Selective modulation of 3Hspiperone labelled 5-HT receptors by subchronic treatment with the ganglioside GM1 in the rat. Actu Physiol. Stand. 117: 311314. DONNAN, G. A., G. L. WILLIS, S. J. KACZMARCZYK, AND P. ROWE. 1987. Motor function in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse. J. Neural. Sci. 77: 185-191.

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