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Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats
Neuroscience Research 35 (1999) 101 – 112 www.elsevier.com/locate/neures
Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats Hideki Hida a, Mitsuhiro Hashimoto b, Ichiro Fujimoto c, Keiya Nakajima a, Yasunobu Shimano a, Toshiharu Nagatsu d, Katsuhiko Mikoshiba b,e, Hitoo Nishino a,* a
Department of Physiology, Nagoya City Uni6ersity Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467 -8601, Japan b De6elopmental Neurobiology Laboratory, RIKEN Brain Science Institute, Wako, Japan c Department of Information Physiology, National Institute of Physiological Sciences, Okazaki, Japan d Molecular Genetics Laboratory, Institute of Integrati6e Medical Sciences, Fujita Health Uni6ersity School of Medicine, Toyoake, Japan e Department of Molecular Neurobiology, Institute of Medical Science, Uni6ersity of Tokyo, Tokyo, Japan Received 8 March 1999; accepted 29 July 1999
Abstract Astrocytes secreting a large amount of 3,4-dihydroxyphenylalanine (dopa) were generated by adenoviral transduction of the human tyrosine hydroxylase (TH) gene. After characterizing in vitro, the effect of transplantation of these astrocytes to the striatum of hemiparkinsonian model rats was investigated. Subconfluent cortical astrocytes were infected by replication-defect adenovirus type 5 carrying the human TH-1 gene or the LacZ reporter gene under the promoter of the glial fibrillary acidic protein (AdexGFAP-HTH-1, AdexGFAP-NL-LacZ). Dopa secretion was not evident at 3 days after the transduction of the HTH-1 gene but it increased from 7 days up to at least 4 months. The secretion was substrate (tyrosine)-dependent, and was enhanced by loading tetrahydrobioputerin (BH4) concentration-dependently. One-third of the hemiparkinsonian model rats, that were transplanted the HTH-1 gene-transduced astrocytes or introduced the direct injection of the viral vector to the striatum, showed a reduction of methamphetamine-induced rotations for at least 6 weeks. Apomorphine-induced rotation was decreased to the 50% level of the control’s, but the reduction was obtained equally by the transplantation of HTH-1 gene-transduced or LacZ reporter gene-transduced astrocytes, or by the introduction of HTH-1 or LacZ gene carrying adenovirus. Treatment with FK506 for 3 weeks improved the late-phase apomorphine-induced rotations following the introduction of the HTH-1 gene carrying adenovirus. Histological examination revealed that, in animals that showed a reduction of methamphetamine-rotation, the TH positive astrocytes-like cells were distributed widely in the host striatum for at least 4 weeks. The number of TH positive astrocytes-like cells and their immunoreactivity decreased after 6 weeks when OX-41 positive microglias/macrophages were infiltrated. Data indicate that the adenoviral transduction of the human TH gene to astrocytes and its introduction to the striatum is a promising approach for the treatment of Parkinson’s disease. However, the further technical improvements are required to optimize the adenoviral gene delivery, such as the control of viral toxicity and the regulation of the immune response. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Parkinson’s disease; Tyrosine hydroxylase; Adenoviral vector; Gene transduction; Astrocytes; Dopamine; L-dopa; Transplantation
1. Introduction In the latest two decades extensive studies have been made in the field of experimental neural transplantation * Corresponding author. Tel.: + 81-52-853-8134; fax: + 81 52-8423069. E-mail address: [email protected] (H. Nishino)
(Bjo¨rklund and Stenevi, 1979; Perlow et al., 1979) and neural transplantation is now evaluated to be one of the most promising approaches that can ameliorate damaged brain function (Nishino et al., 1990; Nishino, 1993; Martı´nez-Serrano and Bjo¨rklund, 1997; Studer et al., 1998) However, in the clinic there still remains a big problem in this field, that is the source of donor cells (where could donor cells be obtained from?). Recently,
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it has become rather easy to develop various kinds of donor cells by gene transduction using viral vectors (Davidson et al., 1993; Le Gal La Salle et al., 1993; Hashimoto et al., 1996; Karpati et al., 1996). The gene delivery of NGF, bFGF, CNTF, BDNF or TH to fibroblasts, astrocytes and other dividing cells using retroviral vectors and others was attempted, and the transplantation of these transduced cells demonstrated the promotion of cell survival and the amelioration of motor function (Fisher et al., 1991; Jiao et al., 1993; During et al., 1994; Kaplitt et al., 1994; Levivier et al., 1995; Takayama et al., 1995; Bankiewicz et al., 1997; Emerich et al., 1997; Mandel et al., 1997). Among viral vectors the adenovirus has several advantages for gene delivery (Karpati et al., 1996): intermediate (up to 7 kb) insert size, high attainable titer, broad infectivity and low serious side effects for humans. So far, by using the adenoviral vector extensive studies have been made to transduce TH- (Horellou et al., 1994), NGF- (CastelBarthe et al., 1996), CNTF-, BDNF-, (Gravel et al., 1997), GDNF- (Choi-Lundberg et al., 1997; Lapchak et al., 1997), NT-3 (Haase et al., 1997) or superoxide dismutase-genes (Barkats et al., 1997) to neurons, glias and fibroblasts. However, the survival of the transduced cells, the interaction with the host, and the functional outcome after introduction to the brain have not been well understood yet (Akli et al., 1993; During et al., 1994; Isacson, 1995; Bencsics et al., 1996; Tornatore et al., 1996). In the present study, we constructed a replication-defect adenoviral vector carrying the human TH-1 gene under the promoter of GFAP. Using this vector we generated astrocytes secreting a large amount of dopa. After characterizing the nature of the transfected astrocytes in vitro we transplanted them or introduced the viral vector to the striatum of hemiparkinsonian model rats and investigated the functional outcome.
2. Materials and methods
2.1. Construction of the adeno6iral 6ector 2.1.1. AdexGFAP-NL-LacZ The HindIII 256 bp fragment from pGF8L (Miura et al., 1990), the promoter region of glial fibrillary acidic protein (GFAP), was subcloned into pBluescript II KS(− ) (pL8). The HindIII 5.5 kb fragment which included a nuclear localization signal (NL1), LacZ, and the second intron and the polyadenylation site of the rabbit b-globin gene (rabbit b-globin IVS2 and pA) (Pa¨a¨bo et al., 1983) was inserted into the SalI site of pL8. The fragment including the GFAP promoter linked nuclear targeted b-galactosidase gene and rabbit b-globin IVS2 and pA was inserted into the SwaI site of pAdex1cw (Kanegae et al., 1995; Hashimoto et al., 1996), this was termed pAdexGFAP-NL-LacZ. The structure of recombinant adenovirus, AdexGFAP-NLLacZ (titer: 1× 1011 p.f.u./ml), which was constructed with pAdexGFAP-NL-LacZ is shown in Fig. 1. 2.1.2. AdexGFAP-HTH-1 The EcoRI 1.9 kbp fragment from pHTH-1 which included cDNA of human tyrosine hydroxylase type 1 (HTH-1) was conjugated with rabbit b-globin IVS2 and pA in pL8. The fragment of the HTH-1 linked rabbit b-globin IVS2 and pA (HTH-1 pA) was inserted into the SalI site of pL8. Then the GFAP promoter linked HTH-1 pA unit (GFAP HTH-1 pA) was inserted into the SwaI site of pAdex1cw, this was termed pAdexGFAP-HTH-1. The structure of the recombinant adenovirus, AdexGFAP-HTH-1 (titer: 1× 108 p.f.u./ml), which was constructed with pAdexGFAP-HTH-1 is shown in Fig. 1.
Fig. 1. Structures of adenoviral vectors. E1A, E1B (1.3–9.3 map units) and E3 (79.6 – 84.8 map units) coding regions were deleted from type 5 adenovirus (Ad 5, 36 kb). A reporter gene, LacZ, with nuclear localization signal (NL 1) and human tyrosine hydroxylase-1 (HTH-1) gene were inserted under the promoter of GFAP (256 bp). They were named AdexGFAP-NL-LacZ and AdexGFAP-HTH-1, respectively.
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2.1.3. AdexDP-NL-LacZ This contained an NL LacZ IVSpA unit inserted into the SwaI site of the pAdex1cw vector as a control. 2.1.4. Adex1w This did not have an expression unit (Miyake et al., 1996)
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2.4. b-Galactosidase (b-Gal) and TH histochemical staining of astrocytes in primary culture Cerebral astrocytes in primary culture were infected with AdexGFAP-NL-LacZ, AdexDP-NL-LacZ, AdexGFAP-HTH-1, and Adex1w. Two days later the cells were fix with 4% paraformaldehyde and stained for b-Gal and TH.
2.2. Preparation of recombinant adeno6irus
2.5. Dopa assay
Recombinant adenoviruses were generated by the method described previously (Miyake et al., 1996) with a minor modification. Briefly, expression cosmid vectors and the EcoT22I digested DNA-terminal protein complex of Ad5-dlx (Chang et al., 1995), which is the human type 5 adenovirus lacking the E3 region, were co-transfected into HEK293 cells (ATCC; CRL 1573) by calcium phosphate co-precipitation. EcoT22I digests the Ad5-dlx genome at seven sites between 0 and 29 m.u.(including the E1A and E1B regions) and this procedure prevents the generation of the parent adenovirus (Ad5-dlx). The recombinant adenovirus was generated through a homologous recombination in HEK293 cells between the expression cosmid vectors (pAdexGFAP-NL-LacZ or pAdexGFAP-HTH-1) and the EcoT22I digested Ad5-dlx. For each vector a single batch of high titer recombinant adenoviral stock was prepared by double cesium step gradient purification (Kanegae et al., 1994) and purified recombinant adenoviruses were dialyzed in 10% sucrose/PBS( − ). The titers of the viral stocks were determined by plaque assay on HEK293 cells. To confirm that the wild-type adenovirus was not included in every stock of recombinant adenovirus, PCR of E1A was performed (Zhao et al., 1998). Negative PCR amplification of the E1A gene was observed in every stock of recombinant adenovirus.
The infected astrocytes were cultured in DMEM with 10% FBS or in RPMI 1640 with 5% FBS, and the secretion of dopa in culture media was assayed using a HPLC with ECD (Eicom EP-10, ECD-100) at different times after the transduction. The culture medium (1 ml) was put into an Ependorff tube and mixed/shaken with aluminum oxide 90 powder (30–40 mg). The powder was washed twice with Tris buffer (pH 7.5), then 100 ml 0.5 N HCl solution was added. After a light centrifuge a certain volume of the supernatant (HCl solution) was injected into the HPLC column to measure the concentration of dopa and DA in the medium.
2.3. Infection of cultured astrocytes Astrocytes were prepared from the cortex of neonatal ICR mice and Wistar rats by a modification of the methods described previously (McCarthy and DeVellis, 1980). The cortical tissue was dissected, minced and treated with 0.05% trypsin solution. The mixed glial cells were cultured in 60 cm2 non-coated flasks in DMEM with 10% fetal bovine serum (FBS). After confluence, the flasks were shaken for 12 h. The cells were replated in 24 cm2 flasks for the second confluence. For the infection of adenovirus, the astrocytes were washed with PBS(×2) and DMEM (× 2), then immersed with a small amount of DMEM with viruses for at 37°C. After 30 min the virus solution was aspirated, new medium (DMEM with 10% FBS) was added and kept for the culture.
2.6. Hemiparkinsonian model rats and drug-induced rotation Under pentobarbital anesthesia, 4 ml 6-OHDA solution (2 mg/ml 6-OHDA in saline containing 0.5 mg/ml ascorbic acid) was injected stereotaxically into the left substantia nigra (SN, 2 mm anterior to interaural line; 1.6 mm lateral to midline and 7.2 mm below the dura) of young female Wistar rats to make chemical lesions in the unilateral nigrostriatal DAergic pathway (Paxinos and Watson, 1982; Nishino et al., 1990). For the detection of motor imbalance and its improvement after transplantation, methamphetamine (3 mg/kg, i.p.)- or apomorphine (0.1 mg/kg, i.p.)-induced rotations (Ungerstedt and Arbuthnott, 1970) were assessed by counting the rotations per min every 10 min for 30–60 min. The rats that made more than eight full turns/min in two tests at 1 and 2 weeks after 6-OHDA lesions, were regarded as hemiparkinsonian model rats. The rotations were evaluated at 1, 2, 3, 4, 6 and 8 weeks after the transplantation of the transduced astrocytes or the introduction of the virus.
2.7. Transplantation or 6iral introduction into the striatum One week after the infection, the transduced rat astrocytes were harvested and made into cell suspensions by trypsinization and trituration. A 5 ml amount of the cell suspension (5 × 107 cells/ml) was stereotaxically injected into two separate sites of the striatum ipsilateral to the lesion (A, 0.5 mm anterior to the
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bregma; L, 2.5 mm; V, 5 mm below the dura; and A, 0.5 mm posterior to the bregma; L, 3.2 mm; V, 5 mm below the dura) at 3 weeks after the 6-OHDA lesion (Nishino et al., 1990) (n =17). For direct infection, the adenovirus vector (1 ml, 1 ×108 p.f.u./ml in 10% sucrose) was introduced to the striatum in a similar way (n=11).
2.8. Treatment with an immunosuppressant FK506 To suppress the immune responses against the virus, in one series of the experiments (n= 6, each), FK506 (Fujisawa Pharmaceutical) was administered every day for 3 weeks after the introduction of the adenovirus carrying HTH-1 gene.
2.9. b-Gal staining, immunocytochemistry and histology The animals were anesthetized and perfusion-fixed transcardially with a fixative (4% paraformaldehyde in 0.1 M phosphate buffer) at 4, 6 and 8 weeks after the transplantation. The brains were removed and cryosections (50 mm thickness) were made. For b-Gal staining, the sections were stained with 5 mM K4(Fe(CN)6), 5 mM K3(Fe(CN)6), 1 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and 0.1% halogenated indoyl-b-D-galactoside (Bluo-gal; Gibco) in PBS for 12 h at 37°C. For the immunocytochemistry, the sections were processed by a standard ABC method using a monoclonal antibody of human TH, antibodies of rabbit anti-cow glial fibrillary acidic protein (anti-GFAP antibody, Dakopatts) and mouse anti-rat OX-41 (antiOX41 antibody, Serotec). Alternate sections were processed for a standard hematoxylin – eosin (H-E) staining.
3. Results
3.1. Detection of b-Gal and TH in infected astrocytes Two days after the infection, the cultured astrocytes were stained for b-Gal and TH. The nucleus of most cells were b-Gal positive after the infection of AdexGFAP-NL-LacZ (Fig. 2A). b-Gal was negative in almost all cells with the infection of AdexDP-NL-LacZ (Fig. 2B). The TH positive astrocytes were detected after the infection of AdexGFAP-HTH-1 but not after the infection of Adex1w (Fig. 2C and D).
3.2. Dopa release in 6itro The release of dopa and DA into the culture medium was measured using a HPLC-ECD. As shown in Fig.
3A the dopa release from non-transduced control astrocytes in the culture medium of DMEM+ 10% FBS was about 100 nM (n= 6). The dopa release increased up to about double (180–190 nM) of the control’s at 1 week after the transduction of the HTH-1 gene (n= 10). This increase was further enhanced by loading BH4 concentration-dependently (more than 300 or 500 nM). In culture medium of RPMI 1640+ 5% FBS, the basal level of the dopa release was low, and it was not increased at 1 week after the transduction (n=6). The release of dopa into the culture medium of DMEM+ 10% FBS was investigated for 4 months. As shown in Fig. 3B, at 3 days after the transduction, there was no difference in the dopa release between the control (n=10) and tranfected astrocytes (n=10). However, at 1 week after the transduction, the dopa release from the transfected astrocytes increased to 180–200 nM, and there was a significant difference between control and transfected ones. Though the levels decreased day by day, the dopa release was still higher from the transfected astrocytes at 4 months after the transduction.
3.3. Impro6ement of motor imbalance in apomorphine or methamphetamine rotations The motor imbalance before and after the transplantation of HTH-1 gene transduced astrocytes or the introduction of the HTH-1 gene carrying virus in the striatum of hemiparkinsonian model rats was assessed by loading apomorphine or methamphetamine. As shown in Fig. 4A apomorphine rotations decreased to the 50% level of control’s (n= 6) after the transplantation of transduced astrocytes (n= 17) or the direct introduction of adenovirus (n=11). The control rats were introduced with saline. In contrast to apomorphine rotations, the variation in methamphetamine rotations after the transplantation of astrocytes or the introduction of the virus was quite large among each of the animals (Fig. 4B). One-third of the animals decreased the rotations, but the others did not show changes or rather increased the rotations.
3.4. FK506 treatment impro6ed the late-phase apomorphine rotation In order to suppress the activation of the immune response following the introduction of the adenovirus to the striatum, we administered an immunosuppressant, FK506, every day for 3 weeks in one group of animals, and monitored the behavioral outcome for up to 8 weeks. Compared to the control group (saline introduced) (n= 6), rotations during the first 30 min after loading apomorphine decreased similarly in all three groups: animals introduced with AdexGFAP-NLLac Z (LacZ) (n= 6), AdexGFAP-HTH-1 (TH) (n=6)
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Fig. 2. The b-Gal and TH histochemical staining of mouse cortical astrocytes in primary culture 2 days after infection. The cells were infected with AdexGFAP-NL-LacZ (A), AdexDP-NL LacZ (B), AdexGFAP-HTH-1 (C), and Adex1w (D) at a moi of 100 (A, B) and 10 (C, D). Two days later the cells were stained for b-Gal (A, B) and TH (C, D). (A) Almost all cells were b-Gal positive. (B) No b-Gal positive cells after the infection with AdexDP-NL LacZ (phase-contrasted picture). (C) TH positive cells (arrow heads) were detected after the infection of AdexGFAP-HTH-1 but not after the infection of the insertless vector (D). (E) Non-infected intact astrocytes. Scales, 100 mm.
or AdexGFAP-HTH-1 with FK506 (TH+ FK506) (n = 6) (Fig. 5). However, the decrease in rotations over 50 min after apomorphine (late-phase rotation) was more consistent in the TH+FK506 group than in others (Fig. 5).
3.5. Sur6i6al and rejection of transduced astrocytes in the striatum The survival of transduced astrocytes in the striatum after the transplantation or the introduction of the viral vector was investigated by TH-immunohistochemistry at 4, 6 and 8 weeks. In animals that showed a reduction in methamphetamine rotations at 4 weeks after transplantation or the introduction of the vector, a strong TH-immunoreaction was detected around the track in the DA-depleted striatum where the intensity of the TH immunoreaction was basically lower compared to the intact (opposite-side) striatum. A typical example is shown in Fig. 6A and B. In this specific animal, mixed viral solutions of 1 ml AdexGFAP-HTH-1 and 1 ml AdexGFAP-NL-LacZ were introduced together (coinjection) to the striatum, thus b-Gal positive cells distributed widely in the track, suggesting a high
efficiency of the transduction (Fig. 6C and D). A mild gliosis was detected around the track (Fig. 6E). Another example is shown in Fig. 7. The TH positive astrocytes-like cells distributed widely in the striatum especially around the track (Fig. 7A, C, D), again with the expression of b-Gal and slight gliosis (Fig. 7B–E). Similar results were obtained in all animals (n = 4) that exhibited a decrease in methamphetamine rotations after 4 weeks. In animals that showed no changes or an increase in methamphetamine rotations, no TH positive astrocytes-like cells were detected (not shown). At 6 weeks after the transplantation or the introduction of the vector, the number of TH immunoreactive cells decreased even in the animals that showed a reduction in methamphetamine rotations. Fig. 8 shows the histology of such an animal transplanted with HTH-1 gene-transduced astrocytes. The H-E staining demonstrated the transplantation tracks (Fig. 8A and B). The TH immunoreactivity has already disappeared in one track but it still remains in the other (Fig. 8C and D). The intensity of the TH immunoreactivity is negatively correlated with the invasion of the OX-41 immunoreactive cells (Fig. 8C–E). A mild gliosis was detected around the grafting tracks (Fig. 8F).
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At 8 weeks after the transplantation, the TH immunoreactive cells could not be detected and only scar tracks remained in the striatum (not shown).
4. Discussion It was found that the astrocytes that were transduced by the HTH-1 gene in an adenoviral vector released dopa at least for 4 months in vitro. After the transplan-
tation of these astrocytes or the direct introduction of the virus into the striatum of hemiparkinsonian model rats, the methamphetamine-induced rotations were decreased in one-third of the animals at least for 6 weeks. The adenoviral vector has more advantages than other viral vectors for gene transduction. It can attain a high titer, can incorporate rather a big size of cDNA (up to 7 kb), has a broad infection spectrum covering even the post-mitotic cells, and has no serious side effects for humans. In the present study, we made an
Fig. 3. L-Dopa concentration in culture medium detected by HPLC-ECD. (A) 35 – 40 nM dopa was detected in the medium (DMEM + 10% FBS) without cells. Dopa was released into the culture medium at the concentration of 100 nM and 180 – 190 nM from control (non-transduced) astrocytes and transduced astrocytes, respectively. Dopa release from the transduced astrocytes was increased by loading BH4 concentration-dependently. In RPMI 1640 (5% FBS) medium the dopa level was low (20 nM), and no difference was observed in dopa release from control and transduced astrocytes at 1 week after the infection. (B) No difference in dopa release from control and transduced astrocytes at 3 days after infection, but thereafter the transduced ones released significantly more dopa than control at least for 4 months. Ordinate, concentration of dopa in culture medium; abscissa, week or month after transfection.
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Fig. 4. Apomorphine- and methamphetamine-induced rotations before and after transplantation of HTH-1 gene transduced astrocytes or introduction of HTH-1 gene carrying viral vector in the striatum of hemiparkinsonian model rats. (A) Apomorphine-induced rotations decreased consistently to 50% level of control’s (saline introduced) after transplantation of the transduced astrocytes or introduction of the viral vector. (B) Variation in methamphetamine-induced rotations was quite large. One-third of the animals decreased the rotations after transplantation of the transduced astrocytes or introduction of the viral vector. Ordinates, rotation (%); abscissas, week before and after transplantation of astrocytes or introduction of viral vector.
adenoviral vector carrying the HTH-1 gene under the promoter of GFAP. For the over-expression of the transduced exogenous genes, a strong and ubiquitous promoter such as the
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human cytomegalovirus (HCMV) major immediate early promoter is often used. However, previous studies (Scharfmann et al., 1991; Kaplitt et al., 1994) have indicated that long-term expression may be limited by the use of HCMV major immediate early promoter, but a promoter for an endogenous cellular gene would facilitate it. We chose the GFAP promoter, a housekeeping cell specific promoter, for the expression of the HTH-1 gene, expecting not a robust but rather a continuous and long-term expression of the target gene. The promoter region required for the expression of GFAP is located within only the 256 bp 5%-flanking region (Miura et al., 1990). Astrocyte-specific expression was observed by means of an analysis using a retrovirus vector including the same promoter region (Ikenaka et al., 1992). In the present study, using a recombinant adenovirus (AdexGFAP-NL-LacZ) astrocyte-specific expression was observed in vitro and in vivo. After the infection with AdexGFAP-HTH-1, cultured astrocytes became TH positive with no significant changes in morphology, suggesting an efficient and safe transduction of the TH gene by the present vector. In Parkinson’s disease, and other neurodegenerative diseases, neurons are damaged to various extent while often accompanied by reactive gliosis, thus the activation of GFAP promoter may enhance the HTH-1 gene expression in response to physiological demand. Subconfluent astrocytes that were introduced to the HTH-1 gene released dopa for at least 4 months in vitro. The release was very stable and was enhanced by loading BH4 (coenzyme of TH) concentration-dependently. Further, the most release was obtained in DMEM+10% FBS but not in RPMI 1640 + 5% FBS, suggesting tyrosine (substrate, 0.46 versus 0.13 mM), Ca2 + (2.4 versus 0.42 mM), and other factors in FBS (10 versus 5%) either singly or in combination promote the dopa release from the transduced astrocytes. Since the level of extracellular Ca++ has not affected the release of dopa much (Lundberg et al., 1996), the difference in the concentration of tyrosine in two culture media or other differences in culture conditions might influence the dopa release. We could not detect DA in the culture medium though the message of aromatic amino acid decarboxylase, protein (Li et al., 1992) and its enzymatic activity (Juorio et al., 1993), and BH4 (Sakai et al., 1995) have been reported to be present in the astrocytes. Thus it is suggested that the level or activity of these enzymes might be rather low in the present culture condition of pure astrocytes. In previous studies the TH gene was transferred directly into the striatum of the rodent model of Parkinson’s disease with an adenovirus vector (Horellou et al., 1994), an adeno-associated virus (AAV) vector (Kaplitt et al., 1994) or a herpes simplex virus type 1 (HSV-1) vector (During et al., 1994). The behavioral recovery was observed after in vivo infection of
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these vectors. However, there is a big discrepancy in the behavioral recovery after these viral introductions, and the final outcome is not clear yet (Akli et al., 1993; During et al., 1994; Isacson, 1995; Bencsics et al., 1996). Apomorphine-induced rotations that mainly reflect the extent of the overexpression (supersensitivity) of the postsynaptic DA receptor could be reduced by the release of DA from the grafts, by mechanical or other damage owing to the transplantation or, by a cytotoxic effect of the virus on the postsynaptic neurons. To get some clues for the underlying mechanism of the functional recovery, we compared the functional outcomes obtained by loading apomorphine (DA agonist) or methamphetamine (DA releaser). Apomorphine directly stimulates the DA receptor, thus the contralateral rotations induced by apomorphine reflect the grade of the extension of the receptor. The bigger the number of apomorphine-induced rotations, the bigger the extension of the receptor which may parallel the extent of DA denervation. Methamphetamine stimulates the release of DA, thus the ipsilateral rotations induced by methamphetamine mainly reflect the extent of DA depletion. The bigger the number of methamphetamineinduced rotations, the bigger the loss of DA innervation (loss of DAergic terminals). We also compared the results of apomorphine-induced rotations after introducing saline, adenovirus carrying LacZ reporter gene or adenovirus carrying HTH-1 gene to the striatum, with or without treatment with FK506. In contrast to an uniformly good reduction (about 50% of the control) in apomorphine rotations in each animal in each stage after the transplantation of HTH-1 gene-transduced astrocytes or the direct introduction of the adenovirus carrying HTH-1 gene, big variations in
methamphetamine rotations were observed in each animal at each stage (Fig. 4). One-third of the animals exhibited the reduction but others showed no changes or even an increase in methamphetamine rotations. This big discrepancy (uniform good reduction in apomorphine rotations versus partial reduction or increase in methamphetamine rotations) may suggest the involvement of the background cytotoxicity of the adenovirus (Byrnes et al., 1995, 1996; Wood et al., 1996) to neurons of the host striatum including the DAergic receptor, since a uniformly good reduction in apomorphine rotations was observed similarly even to the introduction of viral vector carrying the LacZ reporter gene (Fig. 5, 30 min). In other words, the reduction in apomorphine rotation may mainly reflect the damage in the postsynaptic DAergic receptor due to the cytotoxicity of the adenovirus. In this sense the reduction in methamphetamine rotations observed in one third of the animals in the present study reflects a certain restoration of the presynaptic (DA supplying) mechanism. This was clearly confirmed by the TH-immunohistochemistry; a considerable number of TH positive astrocytes-like cells survived in the striatum of the animals that showed a decrease in methamphetamineinduced rotation but no TH positive astrocytes-like cells were detected in the animals that showed an increase or no changes in methamphetamine-induced rotation. The measurement of dopa or DA level in the striatum in future studies in relation to behavioral outcome may offer more information for understanding the subserving mechanisms. In any case, the present study showed that for evaluation of cell survival and behavioral outcome, methamphetamine-induced rota-
Fig. 5. FK506 treatment improved the late-phase apomorphine-induced rotations after introduction of AdexGFAP-HTH-1. FK506 was administered every day for 3 weeks after introduction of AdexGFAP-HTH-1. No difference in rotations during the first 30 min after administration of apomorphine in LacZ, TH, TH + FK506 groups, but the decrease in rotations over 50 min after administration of apomorphine was consistently evident (up to 8 weeks) by the treatment with FK506, suggesting the improvement of late-phase rotations. Ordinates, apomorphine-induced rotation; abscissas, week before and after viral introduction.
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Fig. 6. TH immunoreactive and b-Gal positive cells in an animal that showed the decrease in methamphetamine rotation at 4 weeks after co-introduction of AdexGFAP-HTH-1 and AdexGFAP-NL-LacZ. (A) TH immunoreaction was strongly and moderately positive in and around the track, respectively, compared to the remaining part of the striatum. (B) Higher magnification of (A). (C) b-Gal positive cells around the track. (D) Higher magnification of (C) counterstained with H-E. (E) Mild gliosis around the track (GFAP immunostaining). Scales: (A) 1 mm, (B, C) 250 mm, (D) 50 mm, (E) 100 mm. Fig. 7. Survival of TH positive cells in the striatum at 4 weeks after co-introduction of AdexGFAP-HTH-1 and AdexGFAP-NL-LacZ. (A) Strong and mild TH immunoreaction in the center of the striatum. (B) b-Gal positive cells localized densely in the track and distributed diffusely in the striatum. Many b-Gal positive cells were detected on the wall of the lateral ventricle. (C, D) Higher magnification of (A). TH positive astrocytes-like cells survived diffusely in the striatum. (E) Mild gliosis around the track (GFAP immunostaining). Scales: (A) 1 mm, (B, C) 250 mm, (D, E) 100 mm.
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Fig. 8. Decrease of TH immunoreactivity and increase of OX-41 positive cells at 6 weeks after transplantation of transduced astrocytes. (A, B) (Higher magnification of (A)), H-E staining. (C, D) (Higher magnification of (C)), TH immunostaining. (E) OX-41 immunostaining. (F) GFAP immunostaining. Two tracks of the graft are visible (A, B). TH positive cells still remained in one (lateral) track but they have disappeared in the other (medial) track (C, D). (E) In parallel with the loss of TH immunoreactivity, the invasion of OX-41 positive cells was observed. (F) Gliosis around the tracks. Scales: (A, C) 1 mm, (B, D, E) 250 mm, (F) 100 mm.
tion is more reliable than apomorphine-induced rotation in the case of gene delivery using adenoviral vector. We used a replication-defect adenovirus that deleted the E1A, E1B and E3 from the wild adenovirus. Despite of these deletions this vector still has the elements (E2, E4) that would induce immune reactions. A good survival up to 4 weeks and gradual late rejection over 6–8 weeks after the transplantation in accordance with the infiltration of OX-41 positive microglias and macrophages suggest the activation of a late immune response (Wood et al., 1996). Thus, in order to obtain a more stable and better functional outcome, an additional design or improvement of the vector is needed. The treatment with an immunosuppressant, FK506, for 3 weeks after the introduction of the HTH-1 gene carrying virus reduced the apomorphine rotations profoundly for up to 8 weeks. The reason why the treatment reduced the rotations most profoundly during the 50 min (late-phase) but not during the 30 min (early-phase) is not clear yet. It may reflect a real downregulation (normalization) of the receptor upregulation after protection against cytotoxicity, which could be detected when the effect of apomorphine decreased relatively in the late-phase. This behavioral amelioration after the treatment with FK506 may suggest one means of adenoviral gene delivery in future studies. In summary, the transplantation of the astrocytes that were transduced by the human TH gene by the adenoviral vector or the direct introduction of the viral vector
itself is a promising approach for the treatment of motor deficits in hemiparkinsonian model rats. However, further technical improvements (Krougliak and Graham, 1995; Wood et al., 1996) are required to control the cytotoxicity or immunogeneity of the vector before clinical application.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research, No. 09558104, Joint Research No. 10044311 (HN) from the Ministry of Education, Science, Sports and Culture, and by the SPSBS, Japan.
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Dopa-producing astrocytes generated by adenoviral transduction of human tyrosine hydroxylase gene: in vitro study and transplantation to hemiparkinsonian model rats Hideki Hida a, Mitsuhiro Hashimoto b, Ichiro Fujimoto c, Keiya Nakajima a, Yasunobu Shimano a, Toshiharu Nagatsu d, Katsuhiko Mikoshiba b,e, Hitoo Nishino a,* a
Department of Physiology, Nagoya City Uni6ersity Medical School, Mizuho-cho, Mizuho-ku, Nagoya 467 -8601, Japan b De6elopmental Neurobiology Laboratory, RIKEN Brain Science Institute, Wako, Japan c Department of Information Physiology, National Institute of Physiological Sciences, Okazaki, Japan d Molecular Genetics Laboratory, Institute of Integrati6e Medical Sciences, Fujita Health Uni6ersity School of Medicine, Toyoake, Japan e Department of Molecular Neurobiology, Institute of Medical Science, Uni6ersity of Tokyo, Tokyo, Japan Received 8 March 1999; accepted 29 July 1999
Abstract Astrocytes secreting a large amount of 3,4-dihydroxyphenylalanine (dopa) were generated by adenoviral transduction of the human tyrosine hydroxylase (TH) gene. After characterizing in vitro, the effect of transplantation of these astrocytes to the striatum of hemiparkinsonian model rats was investigated. Subconfluent cortical astrocytes were infected by replication-defect adenovirus type 5 carrying the human TH-1 gene or the LacZ reporter gene under the promoter of the glial fibrillary acidic protein (AdexGFAP-HTH-1, AdexGFAP-NL-LacZ). Dopa secretion was not evident at 3 days after the transduction of the HTH-1 gene but it increased from 7 days up to at least 4 months. The secretion was substrate (tyrosine)-dependent, and was enhanced by loading tetrahydrobioputerin (BH4) concentration-dependently. One-third of the hemiparkinsonian model rats, that were transplanted the HTH-1 gene-transduced astrocytes or introduced the direct injection of the viral vector to the striatum, showed a reduction of methamphetamine-induced rotations for at least 6 weeks. Apomorphine-induced rotation was decreased to the 50% level of the control’s, but the reduction was obtained equally by the transplantation of HTH-1 gene-transduced or LacZ reporter gene-transduced astrocytes, or by the introduction of HTH-1 or LacZ gene carrying adenovirus. Treatment with FK506 for 3 weeks improved the late-phase apomorphine-induced rotations following the introduction of the HTH-1 gene carrying adenovirus. Histological examination revealed that, in animals that showed a reduction of methamphetamine-rotation, the TH positive astrocytes-like cells were distributed widely in the host striatum for at least 4 weeks. The number of TH positive astrocytes-like cells and their immunoreactivity decreased after 6 weeks when OX-41 positive microglias/macrophages were infiltrated. Data indicate that the adenoviral transduction of the human TH gene to astrocytes and its introduction to the striatum is a promising approach for the treatment of Parkinson’s disease. However, the further technical improvements are required to optimize the adenoviral gene delivery, such as the control of viral toxicity and the regulation of the immune response. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Parkinson’s disease; Tyrosine hydroxylase; Adenoviral vector; Gene transduction; Astrocytes; Dopamine; L-dopa; Transplantation
1. Introduction In the latest two decades extensive studies have been made in the field of experimental neural transplantation * Corresponding author. Tel.: + 81-52-853-8134; fax: + 81 52-8423069. E-mail address: [email protected] (H. Nishino)
(Bjo¨rklund and Stenevi, 1979; Perlow et al., 1979) and neural transplantation is now evaluated to be one of the most promising approaches that can ameliorate damaged brain function (Nishino et al., 1990; Nishino, 1993; Martı´nez-Serrano and Bjo¨rklund, 1997; Studer et al., 1998) However, in the clinic there still remains a big problem in this field, that is the source of donor cells (where could donor cells be obtained from?). Recently,
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it has become rather easy to develop various kinds of donor cells by gene transduction using viral vectors (Davidson et al., 1993; Le Gal La Salle et al., 1993; Hashimoto et al., 1996; Karpati et al., 1996). The gene delivery of NGF, bFGF, CNTF, BDNF or TH to fibroblasts, astrocytes and other dividing cells using retroviral vectors and others was attempted, and the transplantation of these transduced cells demonstrated the promotion of cell survival and the amelioration of motor function (Fisher et al., 1991; Jiao et al., 1993; During et al., 1994; Kaplitt et al., 1994; Levivier et al., 1995; Takayama et al., 1995; Bankiewicz et al., 1997; Emerich et al., 1997; Mandel et al., 1997). Among viral vectors the adenovirus has several advantages for gene delivery (Karpati et al., 1996): intermediate (up to 7 kb) insert size, high attainable titer, broad infectivity and low serious side effects for humans. So far, by using the adenoviral vector extensive studies have been made to transduce TH- (Horellou et al., 1994), NGF- (CastelBarthe et al., 1996), CNTF-, BDNF-, (Gravel et al., 1997), GDNF- (Choi-Lundberg et al., 1997; Lapchak et al., 1997), NT-3 (Haase et al., 1997) or superoxide dismutase-genes (Barkats et al., 1997) to neurons, glias and fibroblasts. However, the survival of the transduced cells, the interaction with the host, and the functional outcome after introduction to the brain have not been well understood yet (Akli et al., 1993; During et al., 1994; Isacson, 1995; Bencsics et al., 1996; Tornatore et al., 1996). In the present study, we constructed a replication-defect adenoviral vector carrying the human TH-1 gene under the promoter of GFAP. Using this vector we generated astrocytes secreting a large amount of dopa. After characterizing the nature of the transfected astrocytes in vitro we transplanted them or introduced the viral vector to the striatum of hemiparkinsonian model rats and investigated the functional outcome.
2. Materials and methods
2.1. Construction of the adeno6iral 6ector 2.1.1. AdexGFAP-NL-LacZ The HindIII 256 bp fragment from pGF8L (Miura et al., 1990), the promoter region of glial fibrillary acidic protein (GFAP), was subcloned into pBluescript II KS(− ) (pL8). The HindIII 5.5 kb fragment which included a nuclear localization signal (NL1), LacZ, and the second intron and the polyadenylation site of the rabbit b-globin gene (rabbit b-globin IVS2 and pA) (Pa¨a¨bo et al., 1983) was inserted into the SalI site of pL8. The fragment including the GFAP promoter linked nuclear targeted b-galactosidase gene and rabbit b-globin IVS2 and pA was inserted into the SwaI site of pAdex1cw (Kanegae et al., 1995; Hashimoto et al., 1996), this was termed pAdexGFAP-NL-LacZ. The structure of recombinant adenovirus, AdexGFAP-NLLacZ (titer: 1× 1011 p.f.u./ml), which was constructed with pAdexGFAP-NL-LacZ is shown in Fig. 1. 2.1.2. AdexGFAP-HTH-1 The EcoRI 1.9 kbp fragment from pHTH-1 which included cDNA of human tyrosine hydroxylase type 1 (HTH-1) was conjugated with rabbit b-globin IVS2 and pA in pL8. The fragment of the HTH-1 linked rabbit b-globin IVS2 and pA (HTH-1 pA) was inserted into the SalI site of pL8. Then the GFAP promoter linked HTH-1 pA unit (GFAP HTH-1 pA) was inserted into the SwaI site of pAdex1cw, this was termed pAdexGFAP-HTH-1. The structure of the recombinant adenovirus, AdexGFAP-HTH-1 (titer: 1× 108 p.f.u./ml), which was constructed with pAdexGFAP-HTH-1 is shown in Fig. 1.
Fig. 1. Structures of adenoviral vectors. E1A, E1B (1.3–9.3 map units) and E3 (79.6 – 84.8 map units) coding regions were deleted from type 5 adenovirus (Ad 5, 36 kb). A reporter gene, LacZ, with nuclear localization signal (NL 1) and human tyrosine hydroxylase-1 (HTH-1) gene were inserted under the promoter of GFAP (256 bp). They were named AdexGFAP-NL-LacZ and AdexGFAP-HTH-1, respectively.
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2.1.3. AdexDP-NL-LacZ This contained an NL LacZ IVSpA unit inserted into the SwaI site of the pAdex1cw vector as a control. 2.1.4. Adex1w This did not have an expression unit (Miyake et al., 1996)
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2.4. b-Galactosidase (b-Gal) and TH histochemical staining of astrocytes in primary culture Cerebral astrocytes in primary culture were infected with AdexGFAP-NL-LacZ, AdexDP-NL-LacZ, AdexGFAP-HTH-1, and Adex1w. Two days later the cells were fix with 4% paraformaldehyde and stained for b-Gal and TH.
2.2. Preparation of recombinant adeno6irus
2.5. Dopa assay
Recombinant adenoviruses were generated by the method described previously (Miyake et al., 1996) with a minor modification. Briefly, expression cosmid vectors and the EcoT22I digested DNA-terminal protein complex of Ad5-dlx (Chang et al., 1995), which is the human type 5 adenovirus lacking the E3 region, were co-transfected into HEK293 cells (ATCC; CRL 1573) by calcium phosphate co-precipitation. EcoT22I digests the Ad5-dlx genome at seven sites between 0 and 29 m.u.(including the E1A and E1B regions) and this procedure prevents the generation of the parent adenovirus (Ad5-dlx). The recombinant adenovirus was generated through a homologous recombination in HEK293 cells between the expression cosmid vectors (pAdexGFAP-NL-LacZ or pAdexGFAP-HTH-1) and the EcoT22I digested Ad5-dlx. For each vector a single batch of high titer recombinant adenoviral stock was prepared by double cesium step gradient purification (Kanegae et al., 1994) and purified recombinant adenoviruses were dialyzed in 10% sucrose/PBS( − ). The titers of the viral stocks were determined by plaque assay on HEK293 cells. To confirm that the wild-type adenovirus was not included in every stock of recombinant adenovirus, PCR of E1A was performed (Zhao et al., 1998). Negative PCR amplification of the E1A gene was observed in every stock of recombinant adenovirus.
The infected astrocytes were cultured in DMEM with 10% FBS or in RPMI 1640 with 5% FBS, and the secretion of dopa in culture media was assayed using a HPLC with ECD (Eicom EP-10, ECD-100) at different times after the transduction. The culture medium (1 ml) was put into an Ependorff tube and mixed/shaken with aluminum oxide 90 powder (30–40 mg). The powder was washed twice with Tris buffer (pH 7.5), then 100 ml 0.5 N HCl solution was added. After a light centrifuge a certain volume of the supernatant (HCl solution) was injected into the HPLC column to measure the concentration of dopa and DA in the medium.
2.3. Infection of cultured astrocytes Astrocytes were prepared from the cortex of neonatal ICR mice and Wistar rats by a modification of the methods described previously (McCarthy and DeVellis, 1980). The cortical tissue was dissected, minced and treated with 0.05% trypsin solution. The mixed glial cells were cultured in 60 cm2 non-coated flasks in DMEM with 10% fetal bovine serum (FBS). After confluence, the flasks were shaken for 12 h. The cells were replated in 24 cm2 flasks for the second confluence. For the infection of adenovirus, the astrocytes were washed with PBS(×2) and DMEM (× 2), then immersed with a small amount of DMEM with viruses for at 37°C. After 30 min the virus solution was aspirated, new medium (DMEM with 10% FBS) was added and kept for the culture.
2.6. Hemiparkinsonian model rats and drug-induced rotation Under pentobarbital anesthesia, 4 ml 6-OHDA solution (2 mg/ml 6-OHDA in saline containing 0.5 mg/ml ascorbic acid) was injected stereotaxically into the left substantia nigra (SN, 2 mm anterior to interaural line; 1.6 mm lateral to midline and 7.2 mm below the dura) of young female Wistar rats to make chemical lesions in the unilateral nigrostriatal DAergic pathway (Paxinos and Watson, 1982; Nishino et al., 1990). For the detection of motor imbalance and its improvement after transplantation, methamphetamine (3 mg/kg, i.p.)- or apomorphine (0.1 mg/kg, i.p.)-induced rotations (Ungerstedt and Arbuthnott, 1970) were assessed by counting the rotations per min every 10 min for 30–60 min. The rats that made more than eight full turns/min in two tests at 1 and 2 weeks after 6-OHDA lesions, were regarded as hemiparkinsonian model rats. The rotations were evaluated at 1, 2, 3, 4, 6 and 8 weeks after the transplantation of the transduced astrocytes or the introduction of the virus.
2.7. Transplantation or 6iral introduction into the striatum One week after the infection, the transduced rat astrocytes were harvested and made into cell suspensions by trypsinization and trituration. A 5 ml amount of the cell suspension (5 × 107 cells/ml) was stereotaxically injected into two separate sites of the striatum ipsilateral to the lesion (A, 0.5 mm anterior to the
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bregma; L, 2.5 mm; V, 5 mm below the dura; and A, 0.5 mm posterior to the bregma; L, 3.2 mm; V, 5 mm below the dura) at 3 weeks after the 6-OHDA lesion (Nishino et al., 1990) (n =17). For direct infection, the adenovirus vector (1 ml, 1 ×108 p.f.u./ml in 10% sucrose) was introduced to the striatum in a similar way (n=11).
2.8. Treatment with an immunosuppressant FK506 To suppress the immune responses against the virus, in one series of the experiments (n= 6, each), FK506 (Fujisawa Pharmaceutical) was administered every day for 3 weeks after the introduction of the adenovirus carrying HTH-1 gene.
2.9. b-Gal staining, immunocytochemistry and histology The animals were anesthetized and perfusion-fixed transcardially with a fixative (4% paraformaldehyde in 0.1 M phosphate buffer) at 4, 6 and 8 weeks after the transplantation. The brains were removed and cryosections (50 mm thickness) were made. For b-Gal staining, the sections were stained with 5 mM K4(Fe(CN)6), 5 mM K3(Fe(CN)6), 1 mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet P-40, and 0.1% halogenated indoyl-b-D-galactoside (Bluo-gal; Gibco) in PBS for 12 h at 37°C. For the immunocytochemistry, the sections were processed by a standard ABC method using a monoclonal antibody of human TH, antibodies of rabbit anti-cow glial fibrillary acidic protein (anti-GFAP antibody, Dakopatts) and mouse anti-rat OX-41 (antiOX41 antibody, Serotec). Alternate sections were processed for a standard hematoxylin – eosin (H-E) staining.
3. Results
3.1. Detection of b-Gal and TH in infected astrocytes Two days after the infection, the cultured astrocytes were stained for b-Gal and TH. The nucleus of most cells were b-Gal positive after the infection of AdexGFAP-NL-LacZ (Fig. 2A). b-Gal was negative in almost all cells with the infection of AdexDP-NL-LacZ (Fig. 2B). The TH positive astrocytes were detected after the infection of AdexGFAP-HTH-1 but not after the infection of Adex1w (Fig. 2C and D).
3.2. Dopa release in 6itro The release of dopa and DA into the culture medium was measured using a HPLC-ECD. As shown in Fig.
3A the dopa release from non-transduced control astrocytes in the culture medium of DMEM+ 10% FBS was about 100 nM (n= 6). The dopa release increased up to about double (180–190 nM) of the control’s at 1 week after the transduction of the HTH-1 gene (n= 10). This increase was further enhanced by loading BH4 concentration-dependently (more than 300 or 500 nM). In culture medium of RPMI 1640+ 5% FBS, the basal level of the dopa release was low, and it was not increased at 1 week after the transduction (n=6). The release of dopa into the culture medium of DMEM+ 10% FBS was investigated for 4 months. As shown in Fig. 3B, at 3 days after the transduction, there was no difference in the dopa release between the control (n=10) and tranfected astrocytes (n=10). However, at 1 week after the transduction, the dopa release from the transfected astrocytes increased to 180–200 nM, and there was a significant difference between control and transfected ones. Though the levels decreased day by day, the dopa release was still higher from the transfected astrocytes at 4 months after the transduction.
3.3. Impro6ement of motor imbalance in apomorphine or methamphetamine rotations The motor imbalance before and after the transplantation of HTH-1 gene transduced astrocytes or the introduction of the HTH-1 gene carrying virus in the striatum of hemiparkinsonian model rats was assessed by loading apomorphine or methamphetamine. As shown in Fig. 4A apomorphine rotations decreased to the 50% level of control’s (n= 6) after the transplantation of transduced astrocytes (n= 17) or the direct introduction of adenovirus (n=11). The control rats were introduced with saline. In contrast to apomorphine rotations, the variation in methamphetamine rotations after the transplantation of astrocytes or the introduction of the virus was quite large among each of the animals (Fig. 4B). One-third of the animals decreased the rotations, but the others did not show changes or rather increased the rotations.
3.4. FK506 treatment impro6ed the late-phase apomorphine rotation In order to suppress the activation of the immune response following the introduction of the adenovirus to the striatum, we administered an immunosuppressant, FK506, every day for 3 weeks in one group of animals, and monitored the behavioral outcome for up to 8 weeks. Compared to the control group (saline introduced) (n= 6), rotations during the first 30 min after loading apomorphine decreased similarly in all three groups: animals introduced with AdexGFAP-NLLac Z (LacZ) (n= 6), AdexGFAP-HTH-1 (TH) (n=6)
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Fig. 2. The b-Gal and TH histochemical staining of mouse cortical astrocytes in primary culture 2 days after infection. The cells were infected with AdexGFAP-NL-LacZ (A), AdexDP-NL LacZ (B), AdexGFAP-HTH-1 (C), and Adex1w (D) at a moi of 100 (A, B) and 10 (C, D). Two days later the cells were stained for b-Gal (A, B) and TH (C, D). (A) Almost all cells were b-Gal positive. (B) No b-Gal positive cells after the infection with AdexDP-NL LacZ (phase-contrasted picture). (C) TH positive cells (arrow heads) were detected after the infection of AdexGFAP-HTH-1 but not after the infection of the insertless vector (D). (E) Non-infected intact astrocytes. Scales, 100 mm.
or AdexGFAP-HTH-1 with FK506 (TH+ FK506) (n = 6) (Fig. 5). However, the decrease in rotations over 50 min after apomorphine (late-phase rotation) was more consistent in the TH+FK506 group than in others (Fig. 5).
3.5. Sur6i6al and rejection of transduced astrocytes in the striatum The survival of transduced astrocytes in the striatum after the transplantation or the introduction of the viral vector was investigated by TH-immunohistochemistry at 4, 6 and 8 weeks. In animals that showed a reduction in methamphetamine rotations at 4 weeks after transplantation or the introduction of the vector, a strong TH-immunoreaction was detected around the track in the DA-depleted striatum where the intensity of the TH immunoreaction was basically lower compared to the intact (opposite-side) striatum. A typical example is shown in Fig. 6A and B. In this specific animal, mixed viral solutions of 1 ml AdexGFAP-HTH-1 and 1 ml AdexGFAP-NL-LacZ were introduced together (coinjection) to the striatum, thus b-Gal positive cells distributed widely in the track, suggesting a high
efficiency of the transduction (Fig. 6C and D). A mild gliosis was detected around the track (Fig. 6E). Another example is shown in Fig. 7. The TH positive astrocytes-like cells distributed widely in the striatum especially around the track (Fig. 7A, C, D), again with the expression of b-Gal and slight gliosis (Fig. 7B–E). Similar results were obtained in all animals (n = 4) that exhibited a decrease in methamphetamine rotations after 4 weeks. In animals that showed no changes or an increase in methamphetamine rotations, no TH positive astrocytes-like cells were detected (not shown). At 6 weeks after the transplantation or the introduction of the vector, the number of TH immunoreactive cells decreased even in the animals that showed a reduction in methamphetamine rotations. Fig. 8 shows the histology of such an animal transplanted with HTH-1 gene-transduced astrocytes. The H-E staining demonstrated the transplantation tracks (Fig. 8A and B). The TH immunoreactivity has already disappeared in one track but it still remains in the other (Fig. 8C and D). The intensity of the TH immunoreactivity is negatively correlated with the invasion of the OX-41 immunoreactive cells (Fig. 8C–E). A mild gliosis was detected around the grafting tracks (Fig. 8F).
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At 8 weeks after the transplantation, the TH immunoreactive cells could not be detected and only scar tracks remained in the striatum (not shown).
4. Discussion It was found that the astrocytes that were transduced by the HTH-1 gene in an adenoviral vector released dopa at least for 4 months in vitro. After the transplan-
tation of these astrocytes or the direct introduction of the virus into the striatum of hemiparkinsonian model rats, the methamphetamine-induced rotations were decreased in one-third of the animals at least for 6 weeks. The adenoviral vector has more advantages than other viral vectors for gene transduction. It can attain a high titer, can incorporate rather a big size of cDNA (up to 7 kb), has a broad infection spectrum covering even the post-mitotic cells, and has no serious side effects for humans. In the present study, we made an
Fig. 3. L-Dopa concentration in culture medium detected by HPLC-ECD. (A) 35 – 40 nM dopa was detected in the medium (DMEM + 10% FBS) without cells. Dopa was released into the culture medium at the concentration of 100 nM and 180 – 190 nM from control (non-transduced) astrocytes and transduced astrocytes, respectively. Dopa release from the transduced astrocytes was increased by loading BH4 concentration-dependently. In RPMI 1640 (5% FBS) medium the dopa level was low (20 nM), and no difference was observed in dopa release from control and transduced astrocytes at 1 week after the infection. (B) No difference in dopa release from control and transduced astrocytes at 3 days after infection, but thereafter the transduced ones released significantly more dopa than control at least for 4 months. Ordinate, concentration of dopa in culture medium; abscissa, week or month after transfection.
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Fig. 4. Apomorphine- and methamphetamine-induced rotations before and after transplantation of HTH-1 gene transduced astrocytes or introduction of HTH-1 gene carrying viral vector in the striatum of hemiparkinsonian model rats. (A) Apomorphine-induced rotations decreased consistently to 50% level of control’s (saline introduced) after transplantation of the transduced astrocytes or introduction of the viral vector. (B) Variation in methamphetamine-induced rotations was quite large. One-third of the animals decreased the rotations after transplantation of the transduced astrocytes or introduction of the viral vector. Ordinates, rotation (%); abscissas, week before and after transplantation of astrocytes or introduction of viral vector.
adenoviral vector carrying the HTH-1 gene under the promoter of GFAP. For the over-expression of the transduced exogenous genes, a strong and ubiquitous promoter such as the
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human cytomegalovirus (HCMV) major immediate early promoter is often used. However, previous studies (Scharfmann et al., 1991; Kaplitt et al., 1994) have indicated that long-term expression may be limited by the use of HCMV major immediate early promoter, but a promoter for an endogenous cellular gene would facilitate it. We chose the GFAP promoter, a housekeeping cell specific promoter, for the expression of the HTH-1 gene, expecting not a robust but rather a continuous and long-term expression of the target gene. The promoter region required for the expression of GFAP is located within only the 256 bp 5%-flanking region (Miura et al., 1990). Astrocyte-specific expression was observed by means of an analysis using a retrovirus vector including the same promoter region (Ikenaka et al., 1992). In the present study, using a recombinant adenovirus (AdexGFAP-NL-LacZ) astrocyte-specific expression was observed in vitro and in vivo. After the infection with AdexGFAP-HTH-1, cultured astrocytes became TH positive with no significant changes in morphology, suggesting an efficient and safe transduction of the TH gene by the present vector. In Parkinson’s disease, and other neurodegenerative diseases, neurons are damaged to various extent while often accompanied by reactive gliosis, thus the activation of GFAP promoter may enhance the HTH-1 gene expression in response to physiological demand. Subconfluent astrocytes that were introduced to the HTH-1 gene released dopa for at least 4 months in vitro. The release was very stable and was enhanced by loading BH4 (coenzyme of TH) concentration-dependently. Further, the most release was obtained in DMEM+10% FBS but not in RPMI 1640 + 5% FBS, suggesting tyrosine (substrate, 0.46 versus 0.13 mM), Ca2 + (2.4 versus 0.42 mM), and other factors in FBS (10 versus 5%) either singly or in combination promote the dopa release from the transduced astrocytes. Since the level of extracellular Ca++ has not affected the release of dopa much (Lundberg et al., 1996), the difference in the concentration of tyrosine in two culture media or other differences in culture conditions might influence the dopa release. We could not detect DA in the culture medium though the message of aromatic amino acid decarboxylase, protein (Li et al., 1992) and its enzymatic activity (Juorio et al., 1993), and BH4 (Sakai et al., 1995) have been reported to be present in the astrocytes. Thus it is suggested that the level or activity of these enzymes might be rather low in the present culture condition of pure astrocytes. In previous studies the TH gene was transferred directly into the striatum of the rodent model of Parkinson’s disease with an adenovirus vector (Horellou et al., 1994), an adeno-associated virus (AAV) vector (Kaplitt et al., 1994) or a herpes simplex virus type 1 (HSV-1) vector (During et al., 1994). The behavioral recovery was observed after in vivo infection of
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these vectors. However, there is a big discrepancy in the behavioral recovery after these viral introductions, and the final outcome is not clear yet (Akli et al., 1993; During et al., 1994; Isacson, 1995; Bencsics et al., 1996). Apomorphine-induced rotations that mainly reflect the extent of the overexpression (supersensitivity) of the postsynaptic DA receptor could be reduced by the release of DA from the grafts, by mechanical or other damage owing to the transplantation or, by a cytotoxic effect of the virus on the postsynaptic neurons. To get some clues for the underlying mechanism of the functional recovery, we compared the functional outcomes obtained by loading apomorphine (DA agonist) or methamphetamine (DA releaser). Apomorphine directly stimulates the DA receptor, thus the contralateral rotations induced by apomorphine reflect the grade of the extension of the receptor. The bigger the number of apomorphine-induced rotations, the bigger the extension of the receptor which may parallel the extent of DA denervation. Methamphetamine stimulates the release of DA, thus the ipsilateral rotations induced by methamphetamine mainly reflect the extent of DA depletion. The bigger the number of methamphetamineinduced rotations, the bigger the loss of DA innervation (loss of DAergic terminals). We also compared the results of apomorphine-induced rotations after introducing saline, adenovirus carrying LacZ reporter gene or adenovirus carrying HTH-1 gene to the striatum, with or without treatment with FK506. In contrast to an uniformly good reduction (about 50% of the control) in apomorphine rotations in each animal in each stage after the transplantation of HTH-1 gene-transduced astrocytes or the direct introduction of the adenovirus carrying HTH-1 gene, big variations in
methamphetamine rotations were observed in each animal at each stage (Fig. 4). One-third of the animals exhibited the reduction but others showed no changes or even an increase in methamphetamine rotations. This big discrepancy (uniform good reduction in apomorphine rotations versus partial reduction or increase in methamphetamine rotations) may suggest the involvement of the background cytotoxicity of the adenovirus (Byrnes et al., 1995, 1996; Wood et al., 1996) to neurons of the host striatum including the DAergic receptor, since a uniformly good reduction in apomorphine rotations was observed similarly even to the introduction of viral vector carrying the LacZ reporter gene (Fig. 5, 30 min). In other words, the reduction in apomorphine rotation may mainly reflect the damage in the postsynaptic DAergic receptor due to the cytotoxicity of the adenovirus. In this sense the reduction in methamphetamine rotations observed in one third of the animals in the present study reflects a certain restoration of the presynaptic (DA supplying) mechanism. This was clearly confirmed by the TH-immunohistochemistry; a considerable number of TH positive astrocytes-like cells survived in the striatum of the animals that showed a decrease in methamphetamineinduced rotation but no TH positive astrocytes-like cells were detected in the animals that showed an increase or no changes in methamphetamine-induced rotation. The measurement of dopa or DA level in the striatum in future studies in relation to behavioral outcome may offer more information for understanding the subserving mechanisms. In any case, the present study showed that for evaluation of cell survival and behavioral outcome, methamphetamine-induced rota-
Fig. 5. FK506 treatment improved the late-phase apomorphine-induced rotations after introduction of AdexGFAP-HTH-1. FK506 was administered every day for 3 weeks after introduction of AdexGFAP-HTH-1. No difference in rotations during the first 30 min after administration of apomorphine in LacZ, TH, TH + FK506 groups, but the decrease in rotations over 50 min after administration of apomorphine was consistently evident (up to 8 weeks) by the treatment with FK506, suggesting the improvement of late-phase rotations. Ordinates, apomorphine-induced rotation; abscissas, week before and after viral introduction.
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Fig. 6. TH immunoreactive and b-Gal positive cells in an animal that showed the decrease in methamphetamine rotation at 4 weeks after co-introduction of AdexGFAP-HTH-1 and AdexGFAP-NL-LacZ. (A) TH immunoreaction was strongly and moderately positive in and around the track, respectively, compared to the remaining part of the striatum. (B) Higher magnification of (A). (C) b-Gal positive cells around the track. (D) Higher magnification of (C) counterstained with H-E. (E) Mild gliosis around the track (GFAP immunostaining). Scales: (A) 1 mm, (B, C) 250 mm, (D) 50 mm, (E) 100 mm. Fig. 7. Survival of TH positive cells in the striatum at 4 weeks after co-introduction of AdexGFAP-HTH-1 and AdexGFAP-NL-LacZ. (A) Strong and mild TH immunoreaction in the center of the striatum. (B) b-Gal positive cells localized densely in the track and distributed diffusely in the striatum. Many b-Gal positive cells were detected on the wall of the lateral ventricle. (C, D) Higher magnification of (A). TH positive astrocytes-like cells survived diffusely in the striatum. (E) Mild gliosis around the track (GFAP immunostaining). Scales: (A) 1 mm, (B, C) 250 mm, (D, E) 100 mm.
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Fig. 8. Decrease of TH immunoreactivity and increase of OX-41 positive cells at 6 weeks after transplantation of transduced astrocytes. (A, B) (Higher magnification of (A)), H-E staining. (C, D) (Higher magnification of (C)), TH immunostaining. (E) OX-41 immunostaining. (F) GFAP immunostaining. Two tracks of the graft are visible (A, B). TH positive cells still remained in one (lateral) track but they have disappeared in the other (medial) track (C, D). (E) In parallel with the loss of TH immunoreactivity, the invasion of OX-41 positive cells was observed. (F) Gliosis around the tracks. Scales: (A, C) 1 mm, (B, D, E) 250 mm, (F) 100 mm.
tion is more reliable than apomorphine-induced rotation in the case of gene delivery using adenoviral vector. We used a replication-defect adenovirus that deleted the E1A, E1B and E3 from the wild adenovirus. Despite of these deletions this vector still has the elements (E2, E4) that would induce immune reactions. A good survival up to 4 weeks and gradual late rejection over 6–8 weeks after the transplantation in accordance with the infiltration of OX-41 positive microglias and macrophages suggest the activation of a late immune response (Wood et al., 1996). Thus, in order to obtain a more stable and better functional outcome, an additional design or improvement of the vector is needed. The treatment with an immunosuppressant, FK506, for 3 weeks after the introduction of the HTH-1 gene carrying virus reduced the apomorphine rotations profoundly for up to 8 weeks. The reason why the treatment reduced the rotations most profoundly during the 50 min (late-phase) but not during the 30 min (early-phase) is not clear yet. It may reflect a real downregulation (normalization) of the receptor upregulation after protection against cytotoxicity, which could be detected when the effect of apomorphine decreased relatively in the late-phase. This behavioral amelioration after the treatment with FK506 may suggest one means of adenoviral gene delivery in future studies. In summary, the transplantation of the astrocytes that were transduced by the human TH gene by the adenoviral vector or the direct introduction of the viral vector
itself is a promising approach for the treatment of motor deficits in hemiparkinsonian model rats. However, further technical improvements (Krougliak and Graham, 1995; Wood et al., 1996) are required to control the cytotoxicity or immunogeneity of the vector before clinical application.
Acknowledgements This work was supported by a Grant-in-Aid for Scientific Research, No. 09558104, Joint Research No. 10044311 (HN) from the Ministry of Education, Science, Sports and Culture, and by the SPSBS, Japan.
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