Implantation of genetically modified mesencephalic fetal cells into the rat striatum

Bruin Rwurch Bdk/in. Vol. 29. pp. 81-93, Printed in the USA. All rights reserved.

1992 Copyright

0361-9230/92 $5.00 + .OO 0 1992 Pergamon Press Ltd.

RAPID COMMUNICATION

Implantation of Genetically Modified Mesencephalic Fetal Cells Into the Rat Striatum SHU MING ZHU,* KYOKO KUJIRAI,* ALFRED DOLLISON,* JESUS ANGULO,? STANLEY FAHN* AND JEAN LUD CADET*’ *Department c?f’Neurology, Columbia University, New York, NY 10032 fDepartment of Neuroendocrinology, Rockefeller Center. New York, NY 10021

Received 16 December 199 1 ZHU, S. M., K. KUJIRAI, A. DOLLISON, J. ANGULO, S. FAHN AND J. L. CADET. Implantation ol‘geneticull~~ mod$ed mesencepha/i~,/&a/ ce//.~ into /kc> ra/ striatzrm BRAIN RES BULL 29(I) 8 l-93, 1992.-Transplantation of dopamine (DA) cells into the rat model of hemiparkinsonism induced by intranigral 6-hydroxydopamine (6-OHDA) injections has so far focused mainly on DA replacement via a pump-like mechanism. In the present study, we employed a model of hemiparkinsonism that uses an intrastriatal approach to lesioning the nigrostriatal DA pathway to assess the possibility of using cell transplantation to cause regeneration of that system. Toward that end, we transplanted two types of cells on the side of the 6-OHDA-induced lesions: I) nonmodified fetal mesencephalic cells and 2) fetal mesencephalic cells that have been infected with a retrovirus vector containing a PKC,, cDNA. Both types of cells cause behavioral improvement although the changes were more prominent and occurred earlier in the PKC-modified groups. Tyrosine hydroxylase (TH) immunocytochemistry revealed significantly cell survival in both groups of animals; in situ hybridization studies confirmed the continuous expression of TH mRNA in both groups. Interestingly, long TH-positive axons were observed only in the striata of animals implanted with PKC-modified cells. More importantly, surviving endogenous nigral TH-positive cell bodies were found only on the lesioned side in the latter group. The observations in these animals were associated with significantly smaller decreases in [3H]mazindol-labeled DA uptake sites in both the striata and substantia nigra pars compacta on the side ipsilateral to the 6-OHDA-induced lesions. Furthermore, immunohistochemical studies revealed increased gliosis in the striata of animals grafted with the PKC-modified cells. When taken together, these results indicate that transplantation of normal fetal mesencephalic cells can cause behavioral improvement by providing DA to the host striata whereas PKC-modified cells can, in addition, prevent the progressive degeneration of or cause regeneration of the dying nigrostriatal DA neurons in this model of hemiparkinsonism. These results are discussed in terms of their support for a role for second messenger systems and glial cells, as well as extracellular matrix molecules in the regeneration of the CNS. Transplantation

Striatum

Parkinson’s disease

PARKINSON’S Disease (PD) is a common neurodegenerative disease characterized by extensive loss ofdopamine (DA) neurons in the substantia nigra pars compacta (SNpc) (1). This is associated with DA depletion in the human basal ganglia ( 158). To date, the main therapeutic approach to this disorder has been to replace dopamine by either the DA precursor levodopa or by a directly acting DA receptor agonist. In general, the beneficial effects of the drug treatment subside within a few years and, often, major motor and psychiatric side effects complicate the use of these drugs (59,70). From a theoretical perspective, the more parsimonious approach would be to cause regeneration of the dying nigral neurons through the use of exogenous trophic factors. However, this approach may entail a more thorough understanding of the developmental history of DA neurons and of the factors that led to their demise in PD patients. Another

Retrovirus

approach that has met with some degree of success in a number of animal studies is the replacement of the degenerated neurons with exogenous DA-producing cells (29,35,86). These have included adrenal medullary cells (25,35,37), fetal mesencephalic cells (13,36,56,67). and fibroblasts modified to produce levodopa (34,47,48,86). It is, however, not completely clear if all of the beneficial actions of these transplantation procedures are secondary to DA replacement (37). For example, intrastriatal transplantation of medullary cells in I-methyl-4-phenyl1,2,3,6tetrahydropyridine (MPTP)-treated mice has been reported to cause regeneration of endogenous DA fibers even in the absence of surviving medullary cells ( 14). Moreover, the infusion of nerve growth factor (NGF), after implantation of medullary cells, also causes behavioral improvement, although no tyrosine hydroxylase (TH)-positive cells could be identified in the striata ofthese

I Requests for reprints should be addressed to Jean Lud Cadet, M.D., Department of Neurology, Columbia University, College of Physicians and Surgeons, 630 West 168th Street, New York, NY 10032.

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ZHU ET AL. q6-OHDA

alone

h96-OHDA/FC I6-OHDA/FC/PKC

model of intrastriatal injection of 6-OHDA that we recently characterized ( 19). In that model, there is progressive retrograde degeneration of the nigrostriatal DA system accompanied by persistent and marked loss of DA terminals and cell bodies (9,18,19). METHOD

6-OHDA-Induced Lesions and Behavioral Testing

4w

6W

6W

[after transplantation) FIG. I. Amphetamine-induced circling behavior in rats treated with intrastriatal injections of 6-OHDA. The graph shows rotational behavior as a percentage of baseline in 6-OHDA-treated animals that received intrastriatal injection of the culture media alone (6-OHDA alone), implanted with nonmodified mesencephahc cells (6-OHDA/FC), or PKC@,modified fetal cells (6_OHDA/FC/PKC). The results shown were those obtained 4,6, and 8 weeks after the grafting procedures. The dotted line represents the pregraft rotational behavior. *p < 0.05, **I, < 0.01. in comparison to pregraft circling behavior.

(68). These data have indicated that DA released by medullary cells might not be the only factor responsible for the improvement in the rotational behavior observed in the rat model of hemiparkinsonism induced by 6-hydroxydopamine (6-OHDA) injections into the SNpc. The use of fetal cells in that model have indicated that these cells survive and cause marked behavioral improvement in circling behavior (13,15,16,67). Although synaptic contact with the host by axonal outgrowth from these cells has been found (38,56), functional recovery in these animals could, in addition, be secondary to diffusion of DA released from these cells (36). This assertion is supported by the reports that providing DA via TH-modified fibroblasts is enough to cause behavioral improvement (34,47,48,86). If both the release of DA and the use of trophic factors can cause improvement in circling behavior, the possibility exists that they might do so through common molecular mechanisms that could involve putative second messenger systems. Protein kinase C (PKC) is a good candidate for such a role (23,5 1,54,65,83). Phosphorylation by PKC of the rate-limiting enzyme in DA synthesis, TH, causes increase in TH enzymatic activity (85). Moreover, increases in PKC activity correlate with impulse flow-induced DA release following the injection of various drugs (40). Thus, since recent immunohistochemical studies have demonstrated the localization of PKC subspecies in the substantia nigra and the striatum (87), these data indicate that PKC may indeed play an important role in modulating the function of the nigrostriatal DA system. Furthermore, the reports that the functions of several trophic factors are mediated via PKC activation (5) provide more support for a possible role of the phosphoinositide (PI) second messenger in the effects of transplantation. Thus, we thought it likely that implantation of mesencephlic cells modified to produce PKC might survive, cause behavioral improvement, and stimulate regeneration of endogenous DA fibers. To test these possibilities, we used the animals

Young adult, male Sprage-Dawley rats (Charles River) weighing 220-250 g at the beginning of the experiment were used. They were housed five per cage under a 12 L: 12 D cycle and were given ad lib access to food and water. On the day of lesion, animals were given an intraperitoneal injection of the anesthetic agent chloral hydrate (400 mg/kg) and then placed into a stereotactic instrument for the performance of surgery. Animals received two infusions of 6-OHDA (20 pg in 2 ~1 normal saline containing 0.02% ascorbic acid) at the following stereotaxic coordinates: AP 1.6, ML 2.4, and DV 4.2 and AP 0.02, ML 2.6, and DV 7.0 from bregma as previously described (18-2 1). The drug was infused at a rate of I pl/min through a Hamilton syringe. At the end of the infusion, the needle was left in place for another 3 min to allow for diffusion of the drug away from the tip of the needle. Two weeks postoperatively, animals were tested for circling behavior with amphetamine (3 mg/kg. SC); rotational behavior was monitored at lo-min intervals for 120 min. Rats that turned ipsilateral to the lesion side for at least five rotations/min were used in the transplantation experiments. We had previously shown that animals that meet these criteria show marked loss of DA terminals and cell bodies (19). Those rats were divided into three groups: sham grafts, fetal cells (FC) grafts, and PKCmodified fetal cells (FC/PKC) grafts. Cell Line and Retrovirul Vectors The amphotropic packaging cell line CRIP (24) (obtained from Dr. R. Mulligan’s laboratory, MIT, Cambridge, MA) was grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum, 50 U/ml penicillin, and 50 PLgstreptomycin/ml. Conditioned media generated from the psi2-RP58 (49) (obtained from Dr. I. B. Weinstein’s Laboratory, Columbia University, New York) that contain the PKCBl cDNA were used to infect the packaging cell, CRIP, in the presence of 8 pg/ml polybrene. After 4 h of incubation, the viral supernatant was replaced with new media. Twenty-four hours later, the cells were put into selection with genticin (G4 18) ( 1 mg/ml). G4 18-resistant CRIP-RP58 were cloned and then expanded. Conditioned media containing the retroviral vector were collected from confluent CRIP-RP58 for use in the infections of the fetal mesencephalic cells as described below. Transplantation Procedwes Cells for transplantation

were obtained as described previIn brief, we used female Sprague-Dawley rats, 14 days in gestation. Embryos were removed and collected in phosphate-buffered saline (PBS). DArich tissues were dissected from the ventral mesencephalon of each fetus under sterile conditions. To dissociate the cells, the tissues were incubated in a solution of 0.1% trypsin (GIBCO, Grand Island. NY) in PBS at 37°C for 30 min. After trypsinization, the tissue was mechanically triturated by repeatedly passing them first through l8-ga and then through 25-ga needles. The cell suspension was then filtered through polyamide nylon mesh l50/5 I (Tetko Inc., Elmsford, NY) and centrifuged at 1500 rpm for 5 min. The pellets were resuspended in DMEM supously (13) with some minor modifications.

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FIG. 2. Photomicrographs of (A) nonmodified and (B) PKC-modified mesencephalic cells in culture. The cells were stained for TH immunohistochemistry as described in the Method section. These cells were stained after an overnight incubation of an aliquot obtained from the single cell suspension used in the implantation procedures. Note the long processes put out by some of the TH-positive neurons found in the mixed cell population that have been infected with the retrovirus vector containing the PKCP, cDNA.

plemented with 7% fetal bovine serum (FBS), 2 mM L-glutamine. I mM sodium pyruvate, and 5 U/ml penicillin. 5 &ml streptomycin. The dissociated cells were then plated at a density of 2.5 X lo6 cells per 75 cm’ on a tissue culture flask that had previously been coated with DL-polyornithine (40 pg/ml, Sigma Chemical Co., St. Louis, MO) and laminin (IO pg/ml. Sigma). Cells were cultured in a humidified environment of 95% air and 5% CO*. Twenty-four hours after plating, some of the fetal cells were infected with retrovirus supernatant generated from CRIPRP58 that contains the PKC,,, cDNA (49) in the presence of 2 pg/ml polybrene incubated at 37°C for 4-5 h. At the end of the infection. the viral supernatant was replaced with new culture media. This procedure was repeated at 48 and 72 h after initial plating. All the fetal cells were kept in culture for a period of 5 days before transplantation. Control cells were processed similarly with medium not containing the retroviral vector. The repeated infection procedures were used to increase the infection rate since the cells were not being put in selected with the antibiotic genticin. Prior to the implantation procedure, cell counts and viability were assessed by trypan blue exclusion. Twenty microliters of suspension containing approximately 2.5 X IO4 cells/PI were injected into four different sites corresponding to the following coordinates: AP I .6, ML 2.4, and DV 3.7 and 4.7: and AP 0.2, ML 2.6, DV 6.5 and 7.5. An aliquot of these cells was replated for the performance of TH immunohistochemistry. The sham group received a similar injection of DMEM without

any cells. Rats were retested for amphetamine-induced after the transplantation procedure.

rotation

TH immunocytochemistry was carried out on cells in culture and on slide-mounted sections of both striatal and substantia nigral levels to identify the implanted cells and the endogenous nigral TH-positive neurons. Fetal cells grown on polyornithinecoated 35-mm dishes were fixed with 2% paraformaldehyde and washed several times with PBS. The cells were permeabilized with 0.2%’ Triton X-100 in PBS and then incubated with TH antiserum (Eugene Tech. Allendale. NJ) diluted I: 1000 in PBS/ bovine serum albumin (BSA) for 48 h at 4°C. Cells were processed using the procedures described in the Vectastain ABC kit. The presence of peroxidase was revealed using glucose oxidase and diaminobenzidene (DAB, Sigma). To obtain slide-mounted sections, animals were sacrificed at the end of the behavioral studies. Their brains were removed, rapidly frozen on dry ice, and kept at -70°C until further use. Sections (I6 pm thick) were cut at -20°C and then thaw mounted onto gelatin-coated glass slides. TH immunohistochemistry was processed as described (82). Prior to fixation, the slides were warmed to room temperature for 30 min. The mounted sections were then fixed with methanol for I5 min followed by 100% ethyl alcohol for another I5 min. The sections

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FIG. 3. Photomicrograph of a section showing the effects of 6-OHDA on the TH-positive terminals in the striatum. TH immunohistochemistry revealed complete loss of TH-positive fibers on the side of the 6-OHDA injections into the striatum.

were then rinsed in 0.1 M PBS for 5 min and preincubated for 1 h in a solution of PBS containing Triton X-100 (0.4%) and BSA (0.5%). The sections were then incubated with TH antiserum (Eugene Tech) diluted I:1000 in PBS/BSA for 48 h at 4’C. After rinsing in PBS/BSA, the sections were treated with biotinylated antibody (antirabbit IgG) 1: 100 dilution in 0. I M PBS for 1 h at room temperature. Sections were then processed with the ABC Kit (Vector Laboratories) and DAB, as described above for the cells. Similar techniques were used for the detection of glial fibrillary acid protein (GFAP). The rabbit anti-GFAP (diluted 1:1500) was a gift of Dr. J. Goldman (Department of Neuropathology. Columbia University, NY). Biotinylated protein A diluted I:200 was used as the second antibody (DAK0 Corporation, Carpenteria, CA) in this procedure.

Expression of TH mRNA was investigated by using in situ hybridization histochemistry on slide-mounted sections obtained at the level of the grafts. The procedures were carried out according to modifications of methods used in our previous studies (3,17,21). In brief, sections were prepared as described above for receptor autoradiogmphy. After fixation in 4% formaldehyde in PBS for 30 min at room temperature, the sections were washed twice in PBS. The slides were then acetylated by incubating in 0.25% acetic anhydride in 0.1% triethanolamine/0.9% NaCl for 10 min at 37°C. After acetylation, the slides were washed in 0.5 X SSC (1 X SSC = 0.15 sodium chloride/O.01 5 sodium citrate, pH 7.0) for 2 min and then air dried. Sections were hybridized

with 2 X IO6cpm/ml of a f3sS]-labeled TH oligonucleotide probe (S’dTCAAAGGCTCGGACCTCAGG~TC~TCTGAC) that corresponds to nucleotides 1226- 1256 of the rat cDNA (4 1) in a buffer that consisted of 50% formamide, 4 X SSC, 0.5 X Denhardt’s solution, 0.0 I % single-stranded DNA, and 10 mM DTT at 37°C overnight. Following hybridization, the sections were washed first with a solution consisting of 1 X SSC and I mM DTT at room temperature with three changes followed by another wash in a similar solution at 40°C. The shdes were then dipped in nuclear emulsion and exposed for 6 weeks. After development, the slides were lightly stained with cresyl violet and coverslipped.

Slide-mounted sections (16 pm) were obtained as described above. Dopamine uptake sites were labeled with [3H]mazindol (24.7 Ci/mmol, New England Nuclear, Boston, MA) according to published protocols from our laboratory ( 19). For autoradiographic assays, the slides were warmed to room temperature for 30 min and then preincubated at 4°C for 5 min in a buffer consisting 50 mM Tris-HCL, 120 mM NaCl, 5 mM KCI, pH 7.9. The slide-mounted sections were then incubated with 15 nM [3H]mazindol in 50 mM Tris-HCL containing 300 mM NaCl, 5 mM KCI, pH 7.9, for 40 min at 4°C. Desmethylimipramine (DMI) was used (0.3 KM) to block binding to norepinephrine uptake sites. Nonspecific binding was generated with 30 PM mazindol and represented about 15% of total binding. At the end of the incubation period, the slides were put

FIG. 4. Photomicrographs of TH-positive cells implanted within the striatum. TH-positive cells were observed in the group that received the (A, B) FC and (C, D) FC/PKC cells. Note that there were lengthy processes that appear to emanate from the grafts in animals that received the implantation of(D) PKC-modified cells. The arrows in that figure follows one such process. The other processes that do not appear to connect to the graft may be of endogenous origin secondary to the rescuing effects of the PKC-modified cells on the SNpc. Magnification: objective = X IO in A and C; objective = X20 in B and D.

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FIG. 5. In situ hybridization for TH mRNA in an animal transplanted with (a) FC and (b) FC/PKC mesencephalic cells. These brightfield photomicrographs indicate cells specifically labeled with the TH probe within the striatum. Magnification: objective = X20 in both a and b.

through two 3-min washes in ice-cold buffer, dipped in deionized water, and then dried under a stream of cold air. Dried sections were placed in an X-ray cassette with plastic standards ([3H]microscales, Amersham Corp., Arlington Heights, IL) and apposed to tritium-sensitive Hyperfilm (Amersham) for 3 weeks at 4°C. The developed autoradiograms were quantitated using a computer-based analysis system (Loats, Amersham). The film optical densities were converted to fmol/mg of tissue using a standard curve generated by the [3H]microscales. Data Analyses Statistical analyses of the behavioral and biochemical data were performed by one-way analysis of variance (ANOVA). The least significant difference (LSD) was used for posthoc comparisons. The null hypothesis was rejected at the 0.05 level. All values are expressed as means * SEM. RESULTS

Rotational Behavior Amphetamine (3 mg/kg) caused significant ipsilateral rotation in rats with unilateral 6-OHDA-induced lesions of the striatal DA terminals as previously described by us (19). The effects of transplantation on amphetamine-induced circling are illustrated in Fig. 1. Sham transplanted animals who had received only intrastriatal injection of culture media showed no significant differences between baseline values and those obtained on retesting (Fig. 1). Rats transplanted with only the fetal cells showed a gradual improvement that reached statistical significance (-38.5 t- 3.6%, p < 0.05) at 8 weeks posttransplantation (Fig. 1) while those transplanted with PKC-modified fetal cells showed decreases in rotation that were significant at both 6 weeks (-5 1.4

f 2.8%, p < 0.05) and at 8 weeks (-63.9 posttransplantation (Fig. 1). In?mllnohistochemistry

f 4.3%, p < 0.01)

and In Situ Hybridization

Cells cultzue. Dishes that contained either PKC-modified or nonmodified mesencephalic cells showed TH-like immunoreactivity (Fig. 2). However, only a minority of the cells were strongly TH positive (TH+) in both sets of cultures. Interestingly, PKC-modified cells put out long processes after an overnight in culture whereas the nonmodified cells did not (compare Fig. 2A to 2B). Slide-mounted sections. On the side ipsilateral to the lesion, intrastriatal 6-OHDA injection caused marked reduction of THlike immunoreactivity to background levels (Fig. 3). Animals transplanted with nonmodified or PKC-modified cells showed surviving grafts that contained TH+ cell bodies (Fig. 4). Nevertheless, while the PKC-modified cells expressed long processes that extended long distances into the host striatum, no such processes were observed in the group of rats implanted with the nonmodified cells (compare Fig. 4B to Fig. 4D). The in situ hybridization studies confirmed that the TH immunostaining observed in the striatum was due to continuous expression of TH in the surviving transplanted cells (Fig. 5). As expected, intrastriatal injections of 6-OHDA caused complete disappearance of TH-like immunoreactivity in the SNpc on the side of the lesions (Fig. 6A). Similar observations were made in the group of animals that received nonmodified mesencephalic cells (Fig. 6B). Interestingly, animals implanted with the PKC-modified cells showed a significant proportion of surviving cells on the side of the lesion (Fig. 6C). The results of the GFAP immunostaining revealed more differences between the two groups of transplanted animals (Fig. 7). There was more intense gliosis in the striata of rats implanted

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AND DOPAMINE

FIG. 6. TH immunohistochemistry at the level of the SNpc in a (A) sham-, (B) FC-, and (C) FC/PKCgrafted animal. Note the complete loss of TH-like immunoreactivity in both animals that received only (A) the cell media or (B) the nonmodified fetal cells. Note, on the other hand, the remaining THpositive cell bodies on the side of the 6-OHDA-induced lesions in the animal grafted with the PKCmodified cells.

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with the PKC-modified cells (compare Fig. 7A to 7D, 7B to 7E, and 7C to 7F). [‘H]Mazindol

Autoradiography

The autoradiographic distribution of [3H]mazindol is similar to what has been described previously in rats (9,19). DMI-insensitive [3H]mazindol binding sites, which represent dopamine uptake sites, are found in the caudate-putamen (CPU) and the SNpc of the rats. As shown in Fig. 8B, there was almost total disappearance of specific DMI-insensitive [3H]mazindol sites in the CPU ipsilateral to the side of 6-OHDA injection. These values are similar to those obtained previously in animals treated with intrastriatal 6-OHDA (I 8,19). The quantitative data from these experiments are shown in Table 1. Animals transplanted with nonmodified cells had values similar to the sham group (Table I). However, the percentage of changes was smaller in the group with grafts of PKC-modified cells (-59%) in comparison to both sham (-91%) and fetal cell alone groups (-82%) (compare Fig. 8C to 8D). In the SNpc, 6-OHDA caused marked decreases (-87%) in [3H]mazindol binding sites on the side of injection (Table 1) as previously reported by us (18,19). The group with implantation of nonmodified cells also showed significant decreases (-78%) whereas the PKC-modified group showed values that represent much smaller decreases (-42%) on the side of lesion and grafting (compare data in Table 1). DISCUSSION

The implantation of both nonmodified and PKC-modified cells resulted in significant, gradual reduction in amphetamineinduced rotation although the reduction observed in the PKCmodified group was of somewhat greater magnitude and reached statistical significance earlier than the group with nonmoditied embryonic cells. These results indicate that fetal cells grown in vitro can be used successfully in transplantation experiments. These findings are similar to those of Brundin et al. (16), who reported that culturing mesencephalic cells for 2.5 days before implantation caused decreases in rotational behavior whereas culturing these cells for 7 days rendered them ineffective. Since our study was done after 5 days in culture, there seems to be a time limit beyond which primary cells grown in vitro may not be capable of restoring DA levels within the host’s striatum. This is supported by the report that suspension of cells of later embryonic age (ED-20) are not as effective as cells from ED 1415 in causing behavioral improvement in the rat model of hemiparkinsonism induced by intranigral injection of 6-OHDA (79). The lack of process formation by the nonmodified cells implanted into the striatum also suggests that they have grown to a developmental stage when they are no longer able to respond to tropic cues from the striatum. The present results suggest a number of possible differences between the two types of cells used in the present experiments. First, there might have been better survival of the PKC-modified cells in comparison to the nonmodified cells. This interpretation is not consistent with the histological data, which showed the survival of a large number of TH+ cells, especially along the needle tracts in both groups. Second, the PKC-modified cells might have made better synaptic contact with the host striatum, thus causing release of DA at more distant sites than the nonmodified cells. This possibility is supported by the histological results, which showed that the PKC-modified cells extended axons deep into the host striata whereas the nonmodified cells did not. Third, the modified cells might have prevented the progressive degeneration or might have triggered the regeneration

of endogenous nigrostriatal DA neurons, with subsequent increases in striatal DA levels. This assertion is supported by the results ofthe binding studies, which showed significant increases in [3H]mazindol binding in both the CPU and SNpc ofthe group with grafts of PKC-modified cells, and by the TH immunohistochemistry, which showed surviving cells in the SNpc of animals with the PKC-modified grafts but not in the other groups. The latter two possibilities hint at the intriguing suggestions that agents that activate the phosphoinositide (PI) second messenger cascade might play a role in DA release, as well as in the rescue or regeneration of the nigrostriatal DA pathway. Because we used an intrasttiatal instead of a nigral approach to lesioning the nigrostriatal pathway, it is very likely that the FC/PKCmodified cells are preventing cell death as well as rescuing damaged cells. In what follows, we discuss these suggestions in view of the known striatal substances that use the PI second messenger system. PKC activation modulates a number of metabolic and molecular events in the CNS (4,11,6 1). These include calcium-mediated neurotransmitter release (83) and cell proliferation and differentiation (4) as well as gene expression (65). Because implantation of both nonmodified and PKC-modified cells show a similar degree of surviving TH-positive cells in the host striata, it could be argued that increased PKC activity in the modified cells might have induced a proportionally greater DA synthesis and release from these cells in comparison to the nonmodified cells. This interpretation is consistent with the fact that phosphorylation of tyrosine hydroxylase increased TH activity (85). The report that activation of PKC causes increase in striatal DA release (40) provides further support for this idea. Nevertheless, this proposition does not take into consideration the increase in striatal and nigral [3H]mazindol binding sites observed in the group with PKC-modified grafts (see Table 1) and the observation that PKC-modified TH-positive neurons extended long processes into the host brain whereas the nonmodified cells did not; nor does it account for the presence of the TH-positive nigral cell bodies on the side of the lesion in animals implanted with the PKC-modified cells. When taken together, the biochemical and histological data thus suggest that implantation of PKC-modified cells might have potentiated the effects of molecular mechanisms that are involved in axonal outgrowth, regeneration of nigral DA neurons, or both. These two types of PKC-dependent events could occur in a number of ways. For instance, the PKC substrate GAP-43, which is involved in memory consolidation, development, and regeneration (8) is highly concentrated in the nigrostriatal system (7), where it is thought to stimulate axon outgrowth. Thus, increased concentration of PKC secreted by the PKC-modified grafted cells might have stimulated the regenerative potential of GAP-43 by increasing its phosphorylation (8,80). Our observations that TH-positive cells infected with the PKCP, retroviral vector put out long processes in vitro (see Fig. 2B) and in vivo (see Fig. 4D) are consistent with the report that PKC activation in neuronal cell cultures causes these cells to differentiate and to put out long neurites (4). In addition to its influence on the neuronal elements used in the implantation procedure, PKC might have stimulated a number of biochemical and molecular reactions within the glial cells that are also present among the cells used in this type of study. This assumption is supported by our findings of more intense GFAP staining in the striata of rats transplanted with the PKC-modified cells (Fig. 8). The report that PKC activation by the phorbol ester, phorbol12-myristate- 13-acetate (PMA), causes increases in GFAP mRNA and that continuous application of PMA, which causes down regulation of PKC, led to subsequent decreases in GFAP mRNA (76) suggests that, once incorporated, the PKC& remains

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FIG. 7. GFAP immunostaining at the level of the striatum in an animal transplanted with (A, B, C) FC and (D, E, F) FC/PKC cells within the striatum. (A, D) Very low magnification photomicrographs of the brain at the level ofthe grafts, objective = X4. (B, E) Higher magnification showing the more intense gliosis in the striatum grafted with the FC/PKC cells (E) in comparison to the reaction in the other striatum with the nonmodified FC (B), objective = X 10. v corresponds to the ventricle. (C, F) Still higher magnification reveals glial cell bodies and fibers that were stained darker in the FC/PKC group (F), objective = X20.

activated; this statement is in agreement with previous demonstrations in a rat fibroblast cell line (49). In addition to GFAP, glial cells elaborate a number of substances that have been demonstrated to have trophic influence on the survival (5) and differentiation (22,28,32) of neurons in culture. These include nerve growth factor, epidermal growth factor, and fibroblast growth factors, among others (5). Since

most of these trophic factors exert their influence on neurons via the stimulation of PKC (5,23,33), it is possible that PKCinduced augmentation in the secretion of these glial factors could have potentiated the cascade of stimulatory events that might have led to the expression of long axons by the PKC-modified cells into the host striata. It is also important to note that, in addition to secreting differentiating or mitotic factors that influ-

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FIG. 8. Brightfield photomicrographs showing the autoradiographic distribution of [‘H]mazindol binding in the striatum of(a) controls, (b) sham-, (c) FC-, and (d) FC/PKC-grafted animals. There were no differences between the nonlesioned side of the 6-OHDA-injected animals and the controls. Note the almost complete disappearance of the DA terminals on the lesioned side of the (b) shamand (c) FC-grafted animals. Note, on the other hand. the remaining ~3H~mazindol binding sites on the lesioned side of the FC/PKC-grafted animals. The quantitative data, given in Table 1, revealed significant differences between the FC/PKC and the FC group, as well as between the FC/PKC and the sham group. There were no differences between the FC and the sham group.

ence neurons, glial cells may synthesize both membrane-associated and diffusible factors that might provide a supporting network for the migration of neurons or the extention of neurites (2,6,30,42,43,45,46,55,66,69,77). For instance, chick retinal neurons extend unfasciculated neurites on astrocytic but not on fibroblast surfaces (31). Although the exact manner by which these substances affect neuronal migration or differentiation has not been completely elucidated, several molecules are known to play such a role. These include ~mentin, laminin, fibrone~tin, and cytotactin (42,44,52,69,74,75). Of these, Iaminin may be the most effective (43,55,81); it has, indeed, been shown to be a powerful promoter of neurite outgrowth in vitro (6,57,84). It has even been suggested that the ability of axons to regenerate is related to their close association with astrocytes that continuously expressed laminin (558 1). The fact that the nonmodified cell population used as controls also included glial cells suggests that phosphorylation of these molecules by PKC that is found in glia (60,62) may be necessary for these substances to exert their influence on axonal outgrowth. Recent evidence has indeed shown that activation of PKC by phorbol esters can potentiate neurite outgrowth on suboptimal concentration of laminin (11,12), as well as cell growth in the presence of optimal concentrations of N-cadherin (I 2). When taken together, the accumulated data suggest that stimulation of PKC activity in glia and neurons, both of which are known to contain high concentrations of the enzyme (60-62). could have led to the phosphorylation of both neuronal and glial PKC-de~ndent substrates. These processes, which include the expression of integrins, neuronal receptors with which laminin and fibronectin interact (63,84), could have worked in concert to stimulate and provide a scaffold for the growth of axons (45,46). Support for this suggestion is provided by the fact that phospho~lation of receptors can affect their affinity for specific ligands (53,78).

The increase in [3H]mazindol binding in both the CPU and SNpc in rats transplanted with the PKC-modified cells is consistent with the notion that glial cells may support the survival of transplanted neurons (26,39,8 1). Previous studies have reported that gliaf cells promote neuronal recovery after cortical ablation (64). More recently, it was reported that coimplantation of C6 glioma cells with adrenal medullary cells promoted better survival of medullary cells implanted into denervated striata and caused them to express axon-like processes into the host’s striata (IO). Moreover, the implantation of these C6 glioma cells by themselves also caused increases in striatal DA levels on the side ipsilateral to the 6-OHDA-induced lesion, thus suggesting a certain degree of nigral DA cell regeneration since C6 glioma cells do not secrete DA (I 0). The present data, in which we have demonstrated increases in DA uptake sites in both the striata and SNpc of rats implanted with PKC-modified cells, are consistent with these results and support the view that, under certain circumstances, some glia cells may play an impo~nt role in the regenerative capacity of the brain (26,3 1,57,66) and call into question the idea that regeneration is prevented by glia-induced scar formation in the CNS (71) [see also (46) for further discussion of that point]. Our data further implicate PKC activation and the phospho~lation of a number of PKC-dependent substrates in successful regenerative events that take place after lesion of the CNS. The fact that the transplanted neurons were able to extend very long axons instead of short dendritic-like processes into the host striata may indicate that the host’s factors were also playing a significant role in the results obtained in our studies. This statement is stimulated by the reports that mesencephalic neurons grown in the presence of nigral glia put out multiple short processes whereas those that were cocultured with striatal glia extended two processes, namely, a short dendriticlike and a longer axon-like extension (22,28,72,73). Nevertheless,

FETAL CELLS, TRANSPLANTATION

91

AND DOPAMINE

TABLE 1 EFFECTS OF FETAL CELL IMPLANTATION ON [‘H]MAZINDOL BINDING IN THE CAUDATE-PUTAMEN AND SUBSTANTIA NIGRA SUBSEQUENT TO INTRASTRIATAL 6-OHDA INJECTION I’HlMazindol Binding (fmol/mg tissue)

Control Brain Areas Striatum Total

Dorsolateral Dorsomedial Ventrolater~ Ventromedial SNpc

L

713 * 18 IO7 ?z 44 737 f 25 689 ? 29 727 f 38 379 f 19

R

137 733 733 698 744 327

+ 30 i 45 z? 31 rt 33 rt 37 f 25

L

81 40 53 81 76 44

_t + i t zt +

17****t lo***? 1i**t 17***? 14***? 4***t

Lesion/FC/PKC

Lesion/FC

Lesion

R

R

L

696 +_31 164 +: 12 711&24 687 i 10 671 F I8 332 + 12

126 * 15****t IO I -+ 20***t 126 * 20**t 114iI 12***t 121 -t If***+ 54 f 12***t

697 734 636 745 689 241

f * ? t + +

L

19 21 28 13 15 16

213 + 388 + 267 + 353 + 252 + 164 f

R

14* 13* 60* 56* 44* 19*

673 738 633 677 635 284

-f: 12 + 11 r 20 r 13 t 16 + II

Values represent means f SEM of five animals. All animals were lesioned on the left side. * p < 0.05, **p < 0.005, ***p < 0.0005, and ****p < 0.00001, in comparison to the contralateral side. fp < 0.05, in comparison to the same side of the animals that received transplants of PKC-modified fetal cells (LSD test). There were no differences between the right side of any of the groups.

the proof of this contention will depend upon future experiments aimed at identifying specific host and graft factors that are essential to the stimulation of the regenerative process in the CNS DA system. The results of the present study indicate that several mechanisms may lead to the behavioral recovery observed after transplantation within the DA-depleted striata of rats. First, grafted tissues may behave as a minipump (27,37) that provides DA in a fashion similar to the administration of exogenous levodopa (70). This may explain the partial beneficial results obtained with the nonmodified fetal cells that did not extend long axons into the host brain (present study) and of TH-m~ified fibroblasts (34,47,48). Second, implanted tissues may make synaptic contact with striatal cell bodies just as is observed in the group with PKC-modified cells and in previous reports of grafts of embryonic cell suspensions (29,35,38). Third, transplantation may stimulate the recovery of the host’s nigrostriatal DA system by stimulating the production of both survival and scaffold factors by glial cells of both nigral and striatal origins. The latter point is consistent with studies that showed the importance of astrocytes in the sprouting that occurs in the septohippocampal circuit (39) and in modifying the restrictive effects of the adult CNS (26). In summary, our study has demonstrated for the first time that activation of the PI second messenger cascade by insertion of a subspecies of PKC into mesencephalic fetal cells can promote rescue or regeneration of nigral cell bodies as represented by the

smaller decreases in nigral dopamine uptake sites and by the presence of surviving TH-positive cell bodies in the SNpc on the side of the GOI-IDA-induced lesions in FC/PKC grafted animals in comparison to FC-grafted rats. These results also indicate that, despite the lack of a thorou~ unde~tanding of the molecular mechanisms that control the differentiation of mesencephalic into specialized DA cells that extend long axons from the SNpc to the striatum, we might have been able to create, in animals implanted with PKC-modified cells, a situation that was, possibly, reminiscent of a stage in the development of the nigrostriatal DA system. That stage may be associated with substantial activation of specific PKC subspecies. Thus, further studies are needed to identify the specific PKC-dependent glial scaffolding substrates and neuronal receptors that might have interacted with each other to rescue the degenerating nigrostriatal DA system after intrastriatal injection of 6-OHDA and tissue transplantation. These substances may include the brain-derived trophic factor (BDNF), which has been demonstrated to influence the survival of mesencephalic DA cells and provide protection against the toxic effects of the dopaminotoxic agent, MPTP, in vitro (50). ACKNOWLEDGEMENTS

This work was supported by the Parkinson’s Disease Foundation (PDF). K.K. is a visiting fellow from Yamagata University School of Medicine (Japan). J.L.C. is the recipient of a PHS Award from the NIMH (R29 MH 47509).

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