Gene Therapy (2011), 1–5 & 2011 Macmillan Publishers Limited All rights reserved 0969-7128/11 www.nature.com/gt
SHORT COMMUNICATION
Intracisternal rSV40 administration provides effective pan-CNS transgene expression J-P Louboutin1, BAS Reyes2, L Agrawal1, EJ Van Bockstaele2 and DS Strayer1 Potential genetic treatments for many generalized central nervous system (CNS) diseases require transgene expression throughout the CNS. Using oxidant stress and apoptosis caused by HIV-1 envelope gp120 as a model, we studied pan-CNS neuroprotective gene delivery into the cisterna magna (CM). Recombinant SV40 vectors carrying Cu/Zn superoxide dismutase or glutathione peroxidase were injected into rat CMs following intraperitoneal administration of mannitol. Sustained transgene expression was seen in neurons throughout the CNS. On challenge, 8 weeks later with gp120 injected into the caudate putamen, significant neuroprotection was documented. Thus, intracisternal administration of antioxidant-carrying rSV40 vectors may be useful in treating widespread CNS diseases such as HIV-1-associated neurocognitive disorders characterized by oxidative stress. Gene Therapy advance online publication, 26 May 2011; doi:10.1038/gt.2011.75 Keywords: gp120; HIV-1; gene transfer; cisterna magna; Cu/Zn superoxide dismutase; neuroAIDS
INTRODUCTION Gene delivery to the central nervous system (CNS) is of great interest for many experimental and therapeutic applications, but presents particular challenges. Each delivery system has limitations1–5 and the effectiveness of gene therapy for CNS diseases has so far been very limited.6–8 Pan-CNS gene delivery to treat such widespread CNS diseases as Alzheimer’s diseases and HIV-1-associated neurocognitive disorders (HAND) has been particularly problematic. We previously demonstrated that SV40-derived vectors deliver long-term transgene expression to brain neurons and microglia, whether administered locally by intraparenchymal administration9,10 or diffusely by intravenous11 or intraventricular routes, the latter with or without previous intraperitoneal injection of mannitol.12 Intracerebral injection of rSV40s carrying antioxidant enzymes, Cu/Zn superoxide dismutase (SOD1) or glutathione peroxidase (GPx1), SV(SOD1) or SV(GPx1), into the rat caudate putamen (CP), significantly protected neurons from apoptosis caused by injection of recombinant HIV-1 envelope glycoprotein, gp120 or Tat at the same location.12–14 Moreover, intra-CP SV40-mediated gene delivery of antioxidant enzymes largely protected against several deleterious consequences elicited by injecting gp120 into the CP.15–17 Vector administration into the lateral ventricle, particularly if preceded by mannitol intraperitoneal, protected from intra-CP gp120-induced neurotoxicity comparably to intra-CP vector administration.12 We studied here whether a simpler and less-traumatic approach to panCNS gene transfer of antioxidant enzymes into the cisterna magna (CM) can also protect against HIV-1 gp120.
RESULTS The distribution of transgene expression was assessed in different areas of the brain, including the CP, sensory and motor cortex, hippocampus,
cingulate cortex and lateral septum. Eight weeks after vector administration into the CM with previous mannitol injection, transgene expression was detected in the striatum and different areas of the brain. Many cells, most of them stained by NeuroTrace or immunostained by neuN (not shown, both neuronal markers), expressed the transgenes, SOD1 and GPx1, either in the CP (Figure 1a), or in the cortex (Figure 1b). Ependymal cells and cells of the choroid plexus expressed the transgenes SOD1 and GPx1 (Figure 1c). A few microglial cells, which were identified by immunopositivity for Iba-1, expressed the transgenes (Figure 1d). However, astrocytes, which we visualized by immunostaining for glial fibrillary acidic protein, were negative for the transgenes (Figure 1e). Transgene expression was seen in both cerebral hemispheres. Expression of SOD1 and GPx1 was not detected after injection of the control vector, SV(BUGT), into the CM (Figure 1a). The distribution of transgene-expressing cells in different areas of the brain is shown in Figure 2a. We also tested transgene expression at 4 weeks after vector injection. The percentages of transgene-positive cells enumerated 4 weeks after vector administration into the CM were statistically identical to those at 8 weeks after injection, indicating that transgene expression was stable in this time frame (Figure 2b). This is consistent with our results from intracerebral SV(SOD1) and SV(GPx1) injection, in which antioxidant transgene expression was steady over the 6-month experimental period.9 Eight weeks after injection of SV(SOD1), SV(GPx1) or SV(BUGT) into the CM with previous admistration of mannitol, we injected an oxidant neurotoxic challenge intra-CP, 500 ng gp120. Apoptotic cells were enumerated by TUNEL one day later. Previous injection of SV(SOD1) and SV(GPx1) into the CM reduced the number of TUNEL-positive cells in the CP after gp120 challenge by B70% (SV(GPx1)) to 75% (SV(SOD1)), Po0.01 for both, compared with the control vector SV(BUGT) (Figure 3).
1Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA and 2Department of Neurosurgery, Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA, USA Correspondence: Dr J-P Louboutin, Department of Pathology, Jefferson Medical College, 1020 Locust Street, Room 251, Philadelphia, PA 19107, USA. E-mail:
[email protected] Received 13 December 2010; revised 8 March 2011; accepted 11 April 2011
Intracisternal gene transfer J-P Louboutin et al 2
Figure 1 rSV40 delivery of antioxidant enzymes into the cisterna magna (CM) results in transgene expression in neurons. Eight weeks after injection of SV(SOD1) or SV(GPx1) into the CM with previous intraperitoneal injection of mannitol, SOD1 and GPx1 expression in the CP (a) and the motor cortex (b) was assessed by immunostaining. Sections were stained by NeuroTrace (NT), a neuronal marker. Many neurons expressed the transgenes (arrows). Injection of the CM with SV(BUGT), a control vector, was used as a negative control. (c) Transgene expression was detected in ependymal cells and in cells of the choroid plexus (Ch. P). (d) A few microglial cells, which were identified by immunopositivity for Iba-1, expressed the transgenes (SOD1 is shown here; similar results were observed for GPx1). (e) Astrocytes, which were visualized by immunostaining for glial fibrillary acidic protein (GFAP), were negative for the transgenes. Bar: a, b: 100 mm; c: 60 mm; d: 10 mm; e: 20 mm.
DISCUSSION Abnormalities in oxidative metabolism have been reported in many CNS diseases. Oxidative stress probably plays a role in the development of HIV-associated neurocognitive disorder, and other neurodegenerative diseases.18–20 A potential therapeutic strategy for treatment of HIV-1-associated neurocognitive disorders would be to limit oxidative stress-induced neurotoxicity, but requires an approach to transgene delivery that allows expression throughout the CNS. Gene Therapy
rSV40s were employed in the current study for several reasons: they transduce cells in G0 with high efficiency; they infect a wide range of cell types, particularly neurons from humans and other mammals; and they deliver genes to nondividing cells efficiently, to achieve long-term transgene expression in vitro and in vivo.21 These vectors transduce 495% of cultured human NT2-derived neurons, primary human neurons22,23 and microglia24 without detectable toxicity.
Intracisternal gene transfer J-P Louboutin et al 3
Figure 2 (a) Distribution of transgene expression in different areas of the brain. Percentages of neurons (stained by NT) expressing the transgenes in the brain 8 weeks after injection of SV(SOD1) and SV(GPx1) into the CM. Somato. Cortex, Somatosensory Cortex; Ventral Hippo., Ventral hippocampus; Lat. Septum, Lateral Septum. (b) The percentages of transgene-expressing cells enumerated 4 weeks after vector inoculation were statistically identical from the ones measured 8 weeks after injection, indicating that there was no decrease in transgene expression with time.
CNS locations, and relatively atraumatically, that is, without necessitating penetrating the skull or brain substance. Gene transfer in the CM has previously been performed using adenoviruses30–32 and hemagglutinin virus of Japan envelope vector.33 However, evaluation of transgene expression in the brain parenchyma was not described in those studies, and the analyses of functional benefit from transgene expression in those animal models gave conflicting results. When AAVs were injected in the CM after administration of mannitol, broad dispersion of transgene expression was observed but only sporadic cells were transgene positive.26 In conclusion, these present findings suggest the potential utility of intracisternal gene delivery in treating diffusely distributed CNS diseases characterized by oxidative stress.
Figure 3 Delivery of antioxidant enzymes into the cisterna magna (CM) protects neurons from gp120-induced apoptosis. Quantitation of apoptotic cells and protection from apoptosis by SV(SOD1) and SV(GPx1) injected into the CM 8 weeks before gp120 challenge. Apoptosis was assessed by TUNEL one day after injection of 500 ng gp120 into the CP. **Po0.01.
We previously reported that systemic mannitol-induced hyperosmolarity can improve transgene expression after injection of SV(SOD1) and SV(GPx1) in the lateral ventricle, most likely by increasing the number of cells exposed to the administered vector.9,12 Few studies have reported the effects of previous injection of mannitol on CNS transgene expression; some were based on injection of adenovirus into the lateral ventricle,25 or of adeno-associated virus vectors (AAVs) into the CM or intraparenchimally into the striatum.26 Combining systemic mannitol-induced hyperosmolarity with injection of rAAV27 or adenovirus28 enhances gene expression in the rat brain and provides greater rAAV2-mediated striatal transduction than does local coinfusion.29 Little is known about the therapeutic effects of vectors injected intra-CM. Intracisternal administration of rSV40 vectors shortly after systemic injection of mannitol resulted in a sustained transgene expression in the brain. It also decreased the number of apoptotic cells in the CP following gp120 injection into the CP. This reduction in apoptosis in the CP achieved by intra-CM vector delivery was comparable in magnitude to neuroprotection provided by localized intra-CP vector injection.12 These data thus suggest that intracisternal administration of SV40 vectors encoding antioxidant molecules may provide efficient neuroprotection from gp120 oxidant injury in diverse
MATERIALS AND METHODS Animals Female Sprague-Dawley rats (200–250 g) were purchased from Charles River Laboratories (Wilmington, MA, USA). Protocols for injecting and euthanizing animals were approved by the Thomas Jefferson University Institutional Animal Care and Use Committee, and are consistent with Association for Assessment and Accreditation of Laboratory Animal Care standards. Animals were maintained in the animal facility according to standards and diet previously reported.9
Antibodies Different primary antibodies were used for immunocytochemistry on tissue sections: rabbit anti-ionized calcium-binding adaptor molecule 1 (Iba1; IgG; 1:100; Waco Chemicals, Osaka, Japan), a marker of quiescent and active microglia, goat anti-glial fibrillary acidic protein (IgG; 1:100; Santa-Cruz, Santa-Cruz, CA, USA), rabbit anti-SOD1 (IgG; 1:100; AssayDesigns, Ann Arbor, MI, USA), mouse anti-GPx1 (IgG1 kappa; 1:100; Abnova, Taipei City, Taiwan) and mouse anti-NeuN (IgG1; 1:100; Chemicon International, Temecula, CA, USA). Secondary antibodies were used at 1:100 dilution: fluorescein isothiocyanate and tetramethyl rhodamine isothiocyanate-conjugated goat anti-mouse, tetramethyl rhodamine isothiocyanate-conjugated goat anti-rabbit (Sigma, Saint-Louis, MO, USA), fluorescein isothiocyanate and tetramethyl rhodamine isothiocyanate-conjugated donkey anti-mouse and anti-rabbit, fluorescein isothiocyanate- and Cy3-conjugated donkey anti-goat antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA).
Vector production The general principles for making recombinant, Tag-deleted, replicationdefective SV40 viral vectors have been previously reported.21 SOD1 and GPx1 transgenes were subcloned in to pT7[RSVLTR], in which transgene expression is controlled by the Rous sarcoma virus long terminal repeat (RSVLTR) as a promoter. The cloned rSV40 genome was excised from its carrier Gene Therapy
Intracisternal gene transfer J-P Louboutin et al 4 considered in one cryosection; at least five frontal cryosections passing through the CPs were examined).12
plasmid, gel purified and recircularized, then transfected into COS-7 cells. These cells supply in trans large T-antigen and SV40 capsid proteins, which are needed to produce recombinant replication-defective SV40 viral vectors.34 Crude virus stocks were prepared as cell lysates, then band purified by discontinuous sucrose density gradient ultracentrifugation and titered by quantitative PCR (Stratagene, Santa Clara, CA, USA).35 SV(BUGT), carrying the cDNA for human bilirubin-uridine 5¢-diphosphate-glucuronosyl-transferase (BUGT), has been reported and was used here as negative control vector.36
Statistical analysis
Experimental design and sample processing
CONFLICT OF INTEREST
Transgene expression study. A volume of 3 ml of sterile 25% mannitol in 0.9% saline per 100 g body weight was injected intraperitoneally 10 min before injection of 50 ml of SV(SOD1), SV(GPx1) or the control vector SV(BUGT) into the CM of rats (5 rats per experimental group), whose brains were harvested 4 and 8 weeks after injection.
The authors declare no conflict of interest.
Gp120 challenge study. To study the protective effect of SV-mediated overexpression of SOD1 and GPx1 on gp120-related apoptosis, the right CP of rats given SV(SOD1) and SV(GPx1) into the CM 8 weeks earlier (see above) was injected with 500 ng ml1 gp120. Brains were harvested one day after the injection of 500 ng gp120 in the CP. Apoptotic cells were assessed by TUNEL assay (n¼5 in each experimental group). In all cases, controls received SV(BUGT) into the CM instead of SV(SOD1) and SV(GPx1) (n¼5).
Brains processing for morphology analysis Brains were processed as previously reported.9,12
Intracisternal injection The head of each rat was fixed in the prone position and the atlanto-occipital membrane was exposed through an occipitocerebral midline incision. 5108 IU of SV(SOD1), SV(GPx1), or the control vector SV(BUGT), in 50 ml PBS was injected into the CM using a 50 ml Hamilton syringe (22 gauge; Hamilton Co., Reno, NV, USA) after removal of 50 ml of cerebrospinal fluid. Then, the animals were placed head down for 30 min.
Immunocytochemistry For immunofluorescence, 10 mm cryostat sections were processed for indirect immunofluorescence as previously reported.9,11 Mounting media contained 4¢,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) to stain nuclei. Specimens were finally examined under a Leica DMRBE microscope (Leica Microsystems, Wetzlar, Germany). As a negative control, nonimmune isotype-matched control antibody was substituted for primary antibody, and/or the primary antibody was omitted. Data presented are representative of at least three independent experiments.
Staining of neurons using NeuroTrace NeuroTrace staining has been used as a neuronal marker in studies focusing on the characterization of neurons37 and NT staining has been performed as previously reported.12,13 Combination NT+antibody staining was performed using primary and secondary antibodies staining first (see above), followed by staining with the NT fluorescent stain. All experiments were repeated three times and test and control slides were stained the same day.
Morphometric studies Transduction was assessed for each injected brain by serial cryo sectioning of the whole brain (10 mm thick coronal sections), each slide being numbered, then by immunostaining of every fifth section for GPx1 or SOD1 and finally by using a computerized imaging system (Image-Pro Plus, MediaCybernetics, Bethesda, MD, USA). The total number (and not the number in random areas) of GPx1- or SOD1-positive cells for every fifth section was counted and summed. The final number presented for a structure was an average of the results measured in the different sections examined. This procedure already described for assessment of numbers of transgene-positive cells in the brain9,38 allows quantitative comparisons. We followed a similar procedure for enumerating the number of apoptotic cells after gp120 injection (the whole CPs were Gene Therapy
Results were expressed as average±s.e.m. For comparison between two groups we used the Mann–Whitney test (with a two-tail P-value). Comparisons among more than two groups employed the Kruskall–Wallis test. Comparisons of two percentages were done using Fisher’s exact test (with a two-sided P-value).
ACKNOWLEDGEMENTS This work was supported by NIH grants MH69122 and MH70287.
1 Akli S, Caillaud C, Vigne E, Stratford-Perricaudet LD, Poenaru L, Perricaudet M et al. Transfer of a foreign gene into the brain using adenovirus vectors. Nat Genet 1993; 3: 224–228. 2 Betz AL, Shakui P, Davidson BL. Gene transfer to rodent brain with recombinant adenoviral vectors: effects of infusion parameters, infectious titer, and virus concentration on transduction volume. Exp Neurol 1998; 150: 136–142. 3 Wu P, Phillips MI, Bui J, Terwilliger EF. Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets. J Virol 1998; 72: 5919–5926. 4 McCown TJ, Xiao X, Li J, Breese GR, Samulski RJ. Differential and persistent expression patterns of CNS gene transfer by an adeno-associated virus (AAV) vector. Brain Res 1996; 713: 99–107. 5 Naldini L, Blomer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc Natl Acad Sci USA 1996; 93: 11382–11388. 6 Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 2000; 290: 767–773. 7 Bosch A, Perret E, Desmaris N, Trono D, Heard JM. Reversal of pathology in the entire brain of mucopolysaccharidosis type VII mice after lentivirus-mediated gene transfer. Hum Gene Ther 2000; 11: 1139–1150. 8 Skorupa AF, Fisher KJ, Wilson JM, Parente MK, Wolfe JH. Sustained production of betaglucuronidase from localized sites after AAV vector gene transfer results in widespread distribution of enzyme and reversal of lysosomal storage lesions in a large volume of brain in mucopolysaccharidosis VII mice. Exp Neurol 1999; 160: 17–27. 9 Louboutin JP, Reyes BAS, Agrawal L, van Bockstaele EJ, Strayer DS. Strategies for CNS-directed gene delivery: in vivo gene transfer to the brain using SV40-derived vectors. Gene Therapy 2007; 14: 939–949. 10 Louboutin JP, Marusich E, Fisher-Perkins J, Dufour J, Bunnell BA, Strayer DS. Gene transfer to the Rhesus monkey brain using SV40-derived vectors is durable and safe. Gene Therapy 2011; e-pub ahead of print 3 March 2011; doi:10.1038/gt.2011.13. 11 Louboutin JP, Chekmasova AA, Marusich E, Chowdhury JR, Strayer DS. Efficient CNS gene delivery by intravenous injection. Nat Methods 2010; 7: 905–907. 12 Louboutin JP, Agrawal L, Reyes BAS, van Bockstaele EJ, Strayer DS. Protecting neurons from HIV-1 gp120-induced oxidant stress using both localized intracerebral and generalized intraventricular administration of antioxidant enzymes delivered by SV40-derived vectors. Gene Therapy 2007; 14: 1650–1661. 13 Agrawal L, Louboutin JP, Reyes BAS, van Bockstaele EJ, Strayer DS. Antioxidant enzyme gene delivery to protect from HIV-1 gp120-induced neuronal apoptosis. Gene Therapy 2006; 13: 1645–1656. 14 Agrawal L, Louboutin JP, Strayer DS. Preventing HIV-1 Tat-induced neuronal apoptosis using antioxidant enzymes: mechanistic and therapeutic implications. Virology 2007; 363: 462–472. 15 Louboutin JP, Reyes BAS, Agrawal L, van Bockstaele EJ, Strayer DS. HIV-1 gp120induced neuroinflammation: relationship to neuron loss and protection by rSV40delivered antioxidant enzymes. Exp Neurol 2010; 221: 231–245. 16 Louboutin JP, Reyes BAS, Agrawal L, van Bockstaele EJ, Strayer DS. Blood-brain barrier abnormalities caused by exposure to HIV-1 gp120-protection by gene delivery of antioxidant enzymes. Neurobiol Dis 2010; 38: 313–325. 17 Louboutin JP, Agrawal L, Reyes BAS, van Bockstaele EJ, Strayer DS. HIV-1 gp120induced injury to the blood-brain barrier: role of metalloproteinases 2 and 9 and relationship to oxidative stress. J Neuropathol Exp Neurol 2010; 69: 801–816. 18 Turchan J, Pocernich CB, Gairola C, Chauhan A, Schifitto G, Butterfield DA et al. Oxidative stress in HIV demented patients and protection ex vivo with novel antioxidants. Neurology 2003; 60: 307–314. 19 McArthur JC, Brew BJ, Nath A. Neurological complications of HIV infection. Lancet Neurol 2005; 4: 543–555. 20 Mattson MP, Haughey NJ, Nath A. Cell death in HIV dementia. Cell Death Diff 2005; 12: 893–904. 21 Strayer DS. Gene therapy using SV40-derived vectors: what does the future hold? J Cell Physiol 1999; 181: 375–384.
Intracisternal gene transfer J-P Louboutin et al 5 22 Cordelier P, Calarota SA, Pomerantz RJ, Xiaoshan J, Strayer DS. Inhibition of HIV-1 in the central nervous system by IFN-alpha2 delivered by an SV40 vector. J Interferon Cytokine Res 2003; 23: 477–488. 23 Cordelier P, Van Bockstaele E, Calarota SA, Strayer DS. Inhibiting AIDS in the central nervous system: gene delivery to protect neurons from HIV. Mol Ther 2003; 7: 801–810. 24 Cordelier P, Strayer DS. Using gene delivery to protect HIV-susceptible CNS cells: inhibiting HIV replication in microglia. Virus Res 2006; 118: 87–97. 25 Ghodsi A, Stein C, Derksen T, Martins I, Anderson RD, Davidson BL. Systemic hyperosmolarity improves beta-glucuronidase distribution and pathology in murine MPS VII brain following intraventricular gene transfer. Exp Neurol 1999; 160: 109–116. 26 Fu H, Muenzer J, Samulski RJ, Breese G, Sifford J, Zeng X et al. Self-complementary adeno-associated virus serotype 2 vector: global distribution and broad dispersion of AAV-mediated transgene expression in mouse brain. Mol Ther 2003; 6: 911–917. 27 Mastakov MY, Baer K, Xu R, Fitzsimons H, During MJ. Combined injection of rAAV with mannitol enhances gene expression in the rat brain. Mol Ther 2001; 3: 225–232. 28 Bourgoin C, Emiliani C, Kremer EJ, Gelot A, Tancini B, Gravel RA et al. Widespread distribution of b-hexosaminidase activity in the brain of a Sandhoff mouse model after coinjection of adenoviral vector and mannitol. Gene Ther 2003; 10: 1841–1849. 29 Burger C, Nguyen FN, Deng J, Mandel RJ. Systemic mannitol-induced hyperosmolarity amplifies rAAV2-mediated striatal transduction to a greater extent than local coinfusion. Mol Ther 2005; 11: 327–331. 30 Nakane H, Chu Y, Faraci FM, Oberley LW, Heistad DD. Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebrospinal fluid. Stroke 2001; 32: 184–189.
31 Watanabe Y, Chu Y, Andresen JJ, Nakane H, Faraci FM, Heistad DD. Gene transfer of extracellular superoxide dismutase reduces cerebral vasospasm after subarachnoid hemorrhage. Stroke 2003; 34: 434–440. 32 Yamaguchi M, Zhou C, Heistad DD, Watanabe Y, Zhang JH. Gene transfer of extracellular superoxide dismutase failed to prevent cerebral vasospasm after experimental subarachnoid hemorrhage. Stroke 2004; 35: 2512–2517. 33 Shimamura M, Sato N, Oshima K, Aoki M, Kurinami H, Waguri S et al. Novel therapeutic strategy to treat brain ischemia. Overexpression of hepatocyte growth factor gene reduced ischemic injury without cerebral edema in rat model. Circulation 2004; 109: 424–431. 34 Strayer DS, Kondo R, Milano J, Duan LX. Use of SV40-based vectors to transduce foreign genes to normal human peripheral blood mononuclear cells. Gene Ther 1997; 4: 219–225. 35 Strayer DS, Lamothe M, Wei D, Milano J, Kondo R. Generation of recombinant SV40 vectors for gene transfer. SV40 protocols. In: Raptis L (ed). Methods in Molecular Biology, vol. 165. Humana Press: Totowa, NJ, 2001, pp 103–117. 36 Sauter BV, Parashar B, Chowdhury NR, Kadakol A, Ilan Y, Singh H et al. A replicationdeficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats. Gastroenterology 2000; 119: 1348–1357. 37 Nosheny RL, Bachis A, Acquas E, Mocchetti I. Human immunodeficiency virus type 1 glycoprotein gp120 reduces the levels of brain-derived neurotrophic factor in vivo: potential implication for neuronal cell death. Eur J Neurosci 2004; 20: 2857–2864. 38 Mandel RJ, Rendahl KG, Spratt SK, Snyder RO, Cohen LK, Leff SE. Characterization of intrastriatal recombinant adeno-associated virus-mediated gene transfer of human tyrosine hydroxylase and human GTP-cyclohydrolase I in a rat model of Parkinson’s disease. J Neurosci 1998; 18: 4271–4284.
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