Gene Therapy (1998) 5, 1650–1655  1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt Astrocyte-specific expression of hydroxylase after intracerebral gene transfer induces behavioral recovery in experimental Parkinsonism

J Segovia1, P Vergara1 and M Brenner2 1Departamento de Fisiologı´a, Biofı´sica y Neurociencias, Centro de Investigacio´n y de Estudios Avanzados del IPN, Me´xico; and 2Stroke Branch, National Institute of Neurological Disorders and Stroke, NIH, Bethesda, MD, USA

Parkinson’s disease is a neurodegenerative disorder in a rat model of Parkinson’s disease produced by lesion- characterized by the depletion of in the caudate ing the striatum with 6-hydroxydopamine. Following putamen. Dopamine replacement with levodopa, a precur- microinjection of the transgene into the denervated stria- sor of the neurotransmitter, is presently the most common tum as a DNA–liposome complex, expression of the trans- treatment for this disease. However, in an effort to obtain gene was detected by RT-PCR and TH protein was better therapeutic results, tissue or cells that synthesize observed specifically in astrocytes by using double-labeling have been grafted into experimental ani- immunofluorescence for GFAP and TH coupled with laser mals and human patients. In this paper, we present a novel confocal microscopy. Efficacy was demonstrated by sig- technique to express (TH) in the nificant behavioral recovery, as assessed by a decrease in host’s own astrocytes. This procedure uses a transgene in the pharmacologically induced turning behavior generated which the expression of a TH cDNA is under the control of by the unilateral denervation of the rat striatum. These a glial fibrillary acidic protein (GFAP) promoter, which con- results suggest this is a valuable technique to express fers astrocyte-specific expression and also increases its molecules of therapeutic interest in the brain. activity in response to brain injury. The method was tested

Keywords: tyrosine hydroxylase; glial fibrillary acidic protein; promoter; intracerebral gene transfer; Parkinson’s disease; 6-hydroxydopamine (6-OHDA)

Introduction in response to dopaminergic agonists. This model was used to demonstrate the potential of -pro- Parkinson’s disease is a neurodegenerative disorder ducing grafts in 1979,3,4 and since then the amount of caused by the death of the dopaminergic neurons of the experimental work in this area has grown at an extraordi- substantia nigra. It is generally accepted that levodopa (L nary pace in attempts to produce transplants that are 3–4-dihydroxyphenylalanine), a precursor of dopamine safer, more readily available, longer lasting and express (DA) that can readily cross the blood–brain barrier, is the more strongly. Transplants used include fetal nigral most effective agent to control the symptoms of Parkin- tissue and adrenal medulla, as well as primary cells and son’s disease. Most Parkinson’s patients have a good cell lines genetically engineered to express tyrosine initial response to levodopa, but after a few years become hydroxylase (TH), the limiting enzyme in catecholamine subject to adverse effects, which include dyskinesia, fluc- synthesis (for reviews see Refs 5 and 6). Various methods tuations of efficacy (on–off effect), freezing, mental of gene transfer have also been explored to engineer the 1 changes and loss of efficacy. Transplantation of cat- cultured cells, from classical transfections to the use of echolamine-producing tissue has received considerable retroviral, herpes virus and adenoviral vectors (for attention as an alternative therapy that delivers DA reviews see Refs 7 and 8). More recently, direct transfer directly to the striatum, sparing other tissues from of TH transgenes into the brain has been accomplished adverse effects of DA stimulation and metabolism, and using viral delivery systems,9–11 or liposome complexes.12 avoiding the drug peaks and valleys of pharmacological In general, these experiments have demonstrated that administration by providing a relatively constant source. intracerebral grafts survive within the adult brain, and The most used rodent model of Parkinson’s disease is that they induce behavioral recovery that is consistently the destruction of nigral dopaminergic neurons by the associated with increases in dopaminergic markers, such 2 stereotactic injection of 6-hydroxydopamine (6-OHDA). as TH immunoreactivity and synthesis and release of DA. When this lesion is performed unilaterally, rats will turn These results also provide further evidence that DA replacement is sufficient to ameliorate some of the symp- toms of the disease. The success in experimental animals Correspondence: J Segovia, Departamento de Fisiologı´a, Biofı´sica y Neuro- ciencias, Centro de Investigacio´n y de Estudios Avanzados del IPN, Av has led to the use of brain transplants to treat human Instituto Polite´cnico Nacional 2508, Me´xico, 07300, D.F. patients. Both adrenal tissue, from the same patient, and Received 18 February 1998; accepted 8 July 1998 fetal tissue have been transplanted into Parkinson’s Astrocyte-specific expression of TH in experimental Parkinsonism J Segovia et al 1651 patients, with varied degrees of success.13–16 Never- theless, safety, availability and duration and level of DA production remain paramount issues. In this article, we present a new system which addresses these issues. The method involves engineering the host’s own astrocytes to produce DA by introducing a transgene consisting of a TH cDNA under the control of an astrocyte-specific promoter derived from the glial fibrillary acidic protein (GFAP) gene. Using the host’s cells to produce DA avoids complications of rejection and of obtaining tissues for implant. The GFAP promoter also confers several advantages. By confining expression to astrocytes, it targets a presumably long-lived cell popu- lation, and one that can be expected to be more resistant than neurons to the oxidative stress that is a byproduct of DA metabolism.17 The GFAP promoter also has a built- in regulatory system that responds to reactive gliosis by increasing its transcriptional activity.18 Glial scars are observed in the substantia nigra of Parkinson’s patients, as well as in the caudate putamen and globus pallidus in advanced cases of Parkinson’s and in autosomal domi- Figure 1 Effect of gene transfer on turning behavior. The graph shows nant parkinsonism.19–21 Thus it is a reasonable expec- the turns per minute in response to apomorphine as a percentage of that tation that the activity of a GFAP promoter-driven TH observed before gene transfer (Pre). Rats were tested at 1, 2 and 3 weeks transgene will be increased in response to the progression after lipofection with either pGfa2-cLac or pGfa2-TH. Data presented are means; bars are s.e.m. *P Ͻ 0.05 as compared with Pre (ANOVA, fol- of the disease. lowed by Dunnett’s multiple comparison test). The efficacy of this system was tested by lipofection of the GFAP-TH transgene (pGfa2-TH) into the striatum of the 6-OHDA lesioned rat. The lipofection procedure was weeks after the gene transfer procedure. This result indi- used for this feasibility test as it avoids the possible toxic cates that the TH-containing plasmid induces behavioral effects of the more commonly used viral transfection sys- recovery in an experimental model of Parkinson’s tems.12,22,23 We report that the lipofected TH transgene is disease. expressed in the lesioned striatum, and that its expression is restricted to astrocytes. Furthermore, there is a signifi- Vector expression cant behavioral recovery of the lesioned rats. In order to demonstrate that the behavioral recovery can be attributed to the expression of the gfa2-TH transgene, Results we used RT-PCR to assay for the presence of TH mRNA in rats that underwent the gene transfer procedure. Three Behavioral effect days after the the third behavioral test (3 weeks after in The potential of the gfa2-TH transgene for treatment of vivo lipofection), rats were killed and total cellular RNA Parkinson’s disease was tested using the rat model in was separately isolated from each striatum. As can be which striatal lesions are produced by 6-OHDA (see observed in Figure 2, a signal was obtained that corre- Materials and methods). Following the lesioning, rats sponds to TH mRNA in the side ipsilateral to the 6- that turned at least five times per minute in response to OHDA lesion, where the pGfa2-TH vector–liposome apomorphine and seven times per minute in response to complex was injected, whereas there was no signal from amphetamine were selected for the gene transfer experi- the non-lesioned side. No TH mRNA was detected either ments, as we have previously shown that these responses in the lesioned or control striata of rats that received the indicate a greater than 95% decrease in striatal dopamine pGfa2-cLac vector (data not shown). and an almost complete disappearance of TH in the stria- tum ipsilateral to the lesion.24,25 One to 2 weeks after the Tyrosine hydroxylase and ␤-galactosidase protein behavioral testing, rats were subjected to the gene trans- expression fer procedure. Rats were randomly assigned to two dif- Immunohistochemistry was used to ascertain if the TH ferent experimental groups. The test group received mRNA was translated into protein and if so, if its 10 ␮g of the pGfa2-TH plasmid and the control group expression was localized to astrocytes. Coronal sections received 10 ␮g of plasmid pGfa2-cLac, in which the lacZ close to the injection sites were immunostained for TH gene replaces the TH cDNA. To increase the volume of and GFAP using secondary antibodies tagged with striata subjected to the gene transfer procedure, the fluorescein and with rhodamine, respectively. This dou- DNA–liposome complexes were injected in two different ble labeling technique allowed us to determine the co- positions separated by 1.5 mm in the antero-posterior localization of both markers by merging the images axis. Rats were tested again for their response to apo- obtained for each chromophore (Figure 3). As expected, morphine, 1, 2 and 3 weeks after the second surgical pro- staining of a control striatum (neither lesioned nor cedure. As can be seen in Figure 1, the control group lipofected) showed clear separation of the TH (green) and (pGfa2-cLac) did not change their turning behavior at GFAP (red) signals (panel g), as these should emanate any time-point, whereas rats that received the gfa2-TH from the nigro-striatal nerve terminals and astrocytes, transgene showed a significant (P Ͻ 0.05) decrease in respectively. In a lesioned striatum (panel h), the TH sig- turning behavior, that was maintained for as long as 3 nal disappears due to loss of the nigral neurons, leaving Astrocyte-specific expression of TH in experimental Parkinsonism J Segovia et al 1652 cally in astrocytes, the cells targeted by the gfa2 promoter, rather than in neurons. Secondly, when the gfa2-TH transgene was replaced by gfa2-cLac, there was no pro- duction of either TH mRNA or protein and no behavioral recovery. Our interest in testing the GFAP promoter for Parkin- son’s disease therapy was predicated on both the promis- ing properties of the promoter itself and of the astrocytes in which it is expressed. Astrocytes are known to be able to synthesize DA from l-dopa and to release it into the medium,29,30 so that expression of TH in these cells is suf- ficient for DA production and release. These cells are also relatively resistant to oxidative damage, which may be important as DA synthesis and metabolism produce reactive oxidative species.31 The close apposition of astro- cytic processes to neurons, particularly in the regions of the Nodes of Ranvier and synapses, also recommends these cells for the delivery of neuroactive substances. Genetically engineered cultured astrocytes, modified to express TH or growth factors under control of viral pro- moters, have in fact been implanted in adult rat brains to ameliorate disease.32–35 Use of the GFAP promoter per- mits these cells to be specifically targeted in vivo. Another Figure 2 TH and ␤-actin mRNA detection by RT-PCR. (a) Detection of attribute of the GFAP promoter is that its activity TH mRNA. Lanes 1, 3 and 5 show RT-PCR of RNA from the 6-OHDA increases in response to extracellular signals that induce lesioned striata of three different rats after pGfa2-TH gene transfer; lanes reactive gliosis,18,36,37 or increase intracellular cAMP.38,39 2, 4 and 6 show the RT-PCR from the corresponding contralateral striata of each rat; lane 7 is the PCR product of the pGfa2-TH plasmid. No signal It is thus likely that transcription from a gfa2-regulated was obtained if the RT was omitted. (b) Detection of ␤-actin mRNA from transgene will be stimulated in response to gliosis, gener- the same samples as 1–6 from panel a; lane 7, RT-PCR with no cDNA. ating a feedback mechanism in which an increase in the Left lanes in both panels are DNA size markers. Arrows indicate size damage induced by a disease will up-regulate the tran- markers of 298 and 220 bp in panel a, and of 517 bp in panel b. scription of the ameliorating transgene. Although we have not tested it here, it is also likely that transfected transgenes driven by the GFAP promoter will be tran- the red GFAP signal on a dark background. When the scribed for an extended period, as both its endogenous lesioned striatum is lipofected with the pGfa2-TH plas- counterpart and germline-transmitted gfa2-driven trans- mid, the TH signal reappears (panel i), but is now coinci- genes remain active throughout postnatal life.40 dent with that for GFAP (producing yellow), in accord- Lipofection was used to deliver the gfa2-TH transgene ance with the astrocytic specificity of the gfa2 promoter. to the striata of test animals as this method is simple to Finally, histochemistry for ␤-galactosidase demonstrated perform and avoids possible toxicity and immunological expression of the lacZ transgene in the lesioned striatum complications that have been associated with viral vec- lipofected with pGfa2-cLac (panel j). tors. However, the gfa2-TH construct should be readily adapted to viral vectors or other gene delivery methods Discussion if they prove superior to lipofection. Finally, we note that most of the attributes that rec- In this article we have tested the efficacy of an astrocyte- ommend the GFAP promoter for genetic therapy for Par- targeted TH transgene in an experimental model of Park- kinson’s disease are also relevant for treatment of other inson’s disease. The 6-OHDA lesioned rat model was neurological disorders. Consequently, the GFAP pro- chosen for these studies because there is a good corre- moter, and the astrocytes in which it is expressed, should lation between the turning behavior induced by the provide a versatile platform to express transgenes in lesion and the level of striatal dopaminergic denervation, the brain. and because it has been widely used to assess the degree of the reinnervation and functional recovery induced by Materials and methods other therapies for Parkinson’s disease.2 Immunohistoch- emistry showed the virtual elimination of TH protein Plasmids from the striata of the 6-OHDA lesioned animals, and its The pGfa2-TH and pGfa2-cLac plasmids have been pre- reappearance following in vivo lipofection of the gfa2-TH viously described.38 Briefly, each contains the gfa2 pro- transgene. More importantly, there was a significant moter,41 a 2.2 kb section of the human GFAP gene that (P Ͻ 0.05) recovery of function, with about a 50% is sufficient to direct expression to astrocytes in trans- reduction in turning frequency in response to apomorph- genic mice and is up-regulated by gliosis.18 The TH ine. This outcome is comparable to those that have been coding sequence was obtained from a rat cDNA clone,42 achieved using tissue transplants,4 or gene delivery with and the lacZ coding sequence is the standard, cytoplasmic viral vectors.9,26–28 Two observations lead to the con- version of the E. coli gene. Both the TH and lacZ coding clusion that these effects are directly attributable to regions are followed by a segment of the mouse prota- expression of the gfa2-TH transgene. Firstly, the TH pro- mine-1 gene,43 which supplies an intron and polyadenyl- tein in the lesioned lipofected rats was detected specifi- ation signal. Astrocyte-specific expression of TH in experimental Parkinsonism J Segovia et al 1653

Figure 3 Expression of TH and GFAP proteins determined by immunohistochemistry, and ␤-galactosidase by histochemistry. Panels a, b and c show staining for TH using a fluorescein-labeled (green) second antibody. Tissues labeled were (a) a non-lesioned control striatum, (b) a lesioned striatum lipofected with the pGfa2-cLac plasmid, and (c) a lesioned striatum lipofected with the pGfa2-TH plasmid. Panels d, e and f show the same fields as panels a, b and c respectively, except immunostaining was for GFAP using a rhodamine-labeled second antibody (red). Panels g, h and i show the merged images of the fluorescein and rhodamine channels for each field. Note that in the non-lesioned control striatum (g) the red and green signals are clearly separated; that in the lesioned striatum lipofected with the pGfa2-cLac vector (h) there is an absence of TH immunoreactivity (green) due to the lesioning; and that in the lesioned striatum that received the pGfa2-TH plasmid (i) the two images superimpose (generating a yellow signal) as the TH is being synthesized in astrocytes. Panel j shows lesioned striatum lipofected with pGfa2-cLac stained by histochemistry for ␤-galactosidase. Positive cells are identified by the blue staining. Bar is 20 ␮m. Astrocyte-specific expression of TH in experimental Parkinsonism J Segovia et al 1654 Surgical procedures and behavioral testing sected on a cold glass plate and immediately frozen at Experiments were conducted in accordance with the −70°C. Total cellular RNA was isolated from each stria- Guide for the Care and Use of Laboratory Animals (1996, tum using the acid-guanidinium thiocyanate-phenol- National Academy of Sciences, Washington DC) and chloroform method.45 RT-PCR for TH and ␤-actin was with the approval of the Internal Review Committee performed as previously described. The sizes of the PCR (CINVESTAV-IPN). Male Wistar rats weighing 200–225 g products are 257 bp for TH, and 517 bp for ␤-actin.38,39 at the start of the experiment were used for this work. After PCR, the DNA products for TH and ␤-actin were The animals were housed under standard conditions electrophoresced on 2.0% and 1.5% agarose gels, respect- with ad libitum access to food and water. For the 6-OHDA ively, and stained with ethidium bromide. lesion, rats were anesthetized with chloral hydrate (350 mg/kg) and placed in a stereotactic frame (Stoelting, Wood Dale, IL, USA). Unilateral (left) lesions of the Immunohistochemistry ascending medial forebrain bundle were made by infus- Five to 7 days after the post-gene transfer behavioral test- ing 4 ␮l of saline (0.2% ascorbic acid) containing 8 ␮gof ing (3 weeks after the gene transfer procedure), both 6-OHDA (Sigma, St Louis, MO, USA) through a Hamil- pGfa2-TH and pGfa-cLac lipofected rats were deeply ton microsyringe at a rate of 1 ␮l/min, as previously anesthetized with sodium pentobarbital and perfused described.44 To select for successfully lesioned rats, the through the heart with a saline rinse (0.9% NaCl) fol- turning behavior in response to apomorphine-HCl (0.5 lowed by fixation solution (4% paraformaldehyde in mg/kg, s.c., Sigma) and amphetamine sulphate (5.0 phosphate-buffered saline (PBS), pH = 8.0). Brains were mg/kg, i.p, Sigma) was determined 1 to 2 weeks after postfixed for 24 h in paraformaldehyde and in 20% surgery. Rats that turned at least five times per minute sucrose in PBS, pH = 7.4, for an additional 24–72 h, as in the directional contralateral to the lesion in response previously described.25 Serial coronal sections were cut to apomorphine, and seven times per minute in the direc- at 50 ␮m and collected into wells containing PBS. Sec- tion ipsilateral to the lesion in response to amphetamine tions were permeabilized with 0.2% Triton X-100 in PBS were selected. We have previously demonstrated that for 30 min, blocked with 1% bovine serum albumin these behavioral responses to the pharmacological chal- (Sigma) for 20 min at room temperature, and incubated lenges indicate a greater than 95% decrease in striatal for 24 h at 4°C in the presence of a monoclonal antibody dopamine and an almost complete disappearance of TH 24,25 against TH (Boehringer Mannheim, Mannheim, Ger- in the striatum ipsilateral to the lesion. One week after many, Cat No. 1017381) at a final dilution of 1:40. Sec- the behavioral testing, the gene transfer procedure was tions were washed three times with PBS and incubated performed in the denervated striatum of successfully for 1 h at room temperature with a secondary fluorescin- lesioned rats. Liposomes were formed in aliquots follow- ated antibody (Vector, Burlingame, CA, USA) against ing the manufacturer’s instructions (Gibco BRL, Gaithers- mouse IgG, at a final dilution of 1:100. Sections were thor- burg, MD, USA) by mixing 5 ␮g of the plasmid (pGfa2- oughly washed with PBS and incubated for 24 h at 4°C TH or pGfa2-cLac) in 5 ␮l sterile water with 15 ␮l of lipo- with a polyclonal antibody against GFAP (Dako, Carpin- fectin and incubating for 10–15 min at room temperature teria, CA, USA; code No Z0334) at a final dilution of before injecting stereotactically into the lesioned striata. 1:375. Sections were washed again with PBS and incu- One aliquot was injected in each of two different locations in a lesioned striatum, for a total of 10 ␮gof bated for 1 h at room temperature with a secondary rho- DNA. The first injection site was 1.0 mm rostral to damine labeled antibody (Pierce, Rockford, IL, USA) bregma, 2.5 mm lateral to the sagittal suture and 4.0 mm against rabbit IgG, at a final dilution of 1:60. Sections ventral to dura; and the second injection site was 0.5 mm were washed and mounted with Vectashield (Vector). caudal to bregma, 3.3 mm lateral to the sagittal suture Incubations in the absence of TH and GFAP antibodies and 4.0 mm ventral to dura. The DNA–liposome complex were performed to ensure specificity. A Nikon Diaphot was injected through a Hamilton microsyringe over a 20- microscope (Nikon, Tokyo, Japan) with the appropriate min period, the needle left in place for another 5 min and filters to detect both channels of fluorescence was used then slowly withdrawn. One, 2 and 3 weeks after gene in conjunction with a BioRad MRC-600 (BioRad, Her- transfer, rats were tested again for their turning behavior cules, CA, USA) confocal system. Optical sections that in response to apomorphine. Two independent experi- encompassed the whole vertical cross-section of the sam- ments were performed. In the first experiment, 10 rats ple were collected to generate the projection images. receiving the pGfa2-TH plasmid and six rats receiving Images from the same fields were obtained for each chan- the pGfa2-cLac plasmid were tested for their turning nel, and then were merged using the COMOS 7.2 behavior in response to apomorphine 2 weeks after the software to generate the double-labeled images. gene transfer procedure. In the second experiment, seven rats receiving the pGfa2-TH plasmid and three rats receiving the pGfa2-cLac plasmid were tested for their LacZ histochemistry turning behavior at 1, 2 and 3 weeks after gene transfer. Rats lipofected with the pGfa-cLac–liposome complex For statistical analysis, an analysis of variance (ANOVA) were killed as previously described, 5 to 7 days after the test was performed followed by a Dunnett’s multiple post-gene transfer behavioral testing. Brains were fixed comparison test. with 2% paraformaldehyde and 0.2% glutaraldehyde in PBS, postfixed in the same solution for 24 h, then in 20% mRNA determination sucrose PBS for another 24 h. Coronal sections 50 ␮m Three days after the third post-gene transfer behavioral thick were cut and stained at 37°C for 24 h in the dark evaluation, rats were deeply anesthetized with sodium as previously described,38 and then mounted with 50% pentobarbital, decapitated and the striata rapidly dis- glycerol/50% PBS. Astrocyte-specific expression of TH in experimental Parkinsonism J Segovia et al 1655 Acknowledgements 22 Ono T, Fujino Y, Tsuchiya T, Tsuda M. Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporateed This work was supported by a CONACYT (Me´xico) grant and expressed by brain cells. Neurosci Lett 1990; 117: 259–263. 4852-N (JS), and the Intramural Research Program of the 23 Sahenk Z, Seharaseyon J, Mendell JR, Burghes AHM. 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