A tyrosine hydroxylase–neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors

Molecular Brain Research 84 (2000) 17–31 www.elsevier.com / locate / bres

Research report

A tyrosine hydroxylase–neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors Guo-rong Zhang a , Xiaodan Wang a , Tianzhong Yang a , Mei Sun a , Wei Zhang a , Yaming Wang a , Alfred I. Geller a,b , * a

Division of Endocrinology, Children’ s Hospital, Boston, MA 02115, USA b Program in Neuroscience, Harvard Medical School, Boston, MA, USA Accepted 8 August 2000

Abstract Helper virus-free herpes simplex virus (HSV-1) plasmid vectors are attractive for neural gene transfer, but a promoter that supports neuronal-specific, long-term expression is required. Although expression from many promoters is unstable, a 6.8-kb, but not a 766-bp, fragment of the tyrosine hydroxylase (TH) promoter supports long-term expression. Thus, 59 upstream sequences in this promoter may enhance expression. In this study, we evaluated expression from vectors that contain 59 upstream sequences from this promoter (20.5 to 26.8 kb) inserted at the 59 end of either a neurofilament heavy subunit (NF-H) promoter or the cytomegalovirus (CMV) immediate early promoter. The TH-NFH promoter supported expression for 6 months in the striatum, 2 months in the hippocampus, and for 1 month in both perirhinal and postrhinal cortex (the longest time points examined). Expression was targeted to neurons. The enhanced expression may require specific sequences in the TH promoter fragment because replacing this fragment with a similar sized fragment of bacteriophage l DNA did not enhance expression. The reverse orientation of the TH promoter fragment also enhanced expression. Insertion of insulators from the chicken b-globin locus between the TH-NFHlac transcription unit and the vector backbone may support a modest additional enhancement in expression. Other eucaryotic sequences may also enhance expression; a S. cerevisiae (40-kb fragment)-NFH promoter enhanced expression. In contrast, the TH-CMV promoter did not enhance expression. Thus, the TH-NFH promoter may support some physiological studies that require long-term expression in forebrain neurons.  2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Long-term expression; Tyrosine hydroxylase promoter; Neurofilament promoter; Herpes simplex virus vector; Gene transfer

1. Introduction Gene transfer into neural cells may support both gene therapy of neurological disorders and analyses of neuronal physiology. Virus vector systems that are under development include herpes simplex virus type one (HSV-1) plasmid vectors or amplicons [16], recombinant HSV-1 vectors [11], adenovirus vectors [23], adeno-associated virus vectors [21], and defective lentivirus vectors [30]. *Corresponding author. Tel.: 11-617-355-6185; fax: 11-617-3553741. E-mail address: geller [email protected] (A.I. Geller). ]

HSV-1 vectors are attractive due to specific properties of the virus, and a growing number of scientists have used HSV-1 plasmid vectors to modify neuronal physiology by expressing a wide range of genes (reviewed in Ref. [15]). To reduce the cytopathic effects and inflammatory response previously associated with gene transfer, we developed a helper virus-free packaging system for these vectors [12]. Nonetheless, the lack of a promoter that supports long-term expression in forebrain neurons has limited the utility of this system. A number of viral promoters that support long-term expression in other systems do not support significant levels of long-term expression from HSV-1 vectors. At 1

0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00197-2

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month after gene transfer into either the midbrain or striatum, HSV-1 plasmid vectors that contain either the HSV-1 immediate early (IE) 4 / 5 promoter or the cytomegalovirus (CMV) IE promoter support expression in only a small percentage of the number of cells observed at 4 days (helper virus or helper virus-free system [9,12,38]). The decline in expression from the CMV IE promoter is even more rapid in the hippocampus (helper virus system [17]). The latency associated transcript (LAT) promoter can be active during HSV-1 latency [40], and in recombinant HSV-1 vectors, the LAT promoter supports some long-term expression in facial and hypoglossal nerve neurons [22], but similar results have not been reported in forebrain areas. The Maloney murine leukemia virus longterminal-repeat (LTR) promoter supports long-term expression in retrovirus vectors; but in recombinant HSV-1 vectors, this promoter supports some long-term expression in primary sensory neurons, but only low levels of longterm expression in hypoglossal nerve neurons or forebrain neurons [4,8]. A number of neuronal-specific cellular promoters have been examined, and the results are similar to those obtained using viral promoters. A HSV-1 plasmid vector that contains the neurofilament heavy subunit (NF-H) promoter supports high-level expression at 4 days, but expression is absent at 1 month (helper virus-free system [44]). Although a neuron-specific enolase (NSE) promoter in a recombinant HSV-1 vector has been reported to support some expression for 1 month [1], interpretation of these results is complicated because this thymidine kinasedeficient vector supports a low level of reactivation, repackaging, and reinfection. Moreover, HSV-1 plasmid vectors that contain either a NSE promoter or a voltagegated sodium channel promoter support only low levels of expression in cultured neuronal cells and no detectable expression in the striatum (helper virus-free system [44]). In contrast to the results with viral and neuronal-specific promoters, two promoters that are active only in specific types of neurons support significant levels of long-term expression from HSV-1 plasmid vectors [19,20,38,44]. A vector that contains the preproenkephalin promoter supports expression for 2 months in both the amygdala and the ventromedial hypothalamus (helper virus system [20]). Vectors that contain either a 6.8- or a 9-kb fragment of the tyrosine hydroxylase (TH) promoter support expression for 2.0 or 2.5 months, respectively, in dopaminergic neurons in the substantia nigra pars compacta or locus ceruleus (helper virus system [19,38]; helper virus-free system [44]). These results suggest that HSV-1 plasmid vectors might be capable of supporting long-term, neuronal-specific expression. In one attempt to derive a neuronal-specific promoter that supports long-term expression, we examined a 766-bp fragment of the TH promoter [44]. In transgenic mice, small (#3.6 kb) fragments of rodent TH promoters support ectopic expression in non-catecholaminergic cells with

neuronal morphology in many brain areas (larger fragments (4.8–9.0 kb) target expression to catecholaminergic neurons) [2,25,27,28]. As expected, at 4 days after gene transfer, this vector (766-bp TH promoter) supported expression in both catecholaminergic and non-catecholaminergic brain areas, but expression was absent at 2 weeks (helper virus-free system [44]). Because 6.8- and 9.0-kb fragments, but not a 766-bp fragment, of the TH promoter support long-term expression, these results raised the possibility that 59 upstream sequences in the TH promoter might enhance expression from HSV-1 vectors. In this study, we show that addition of 59 upstream sequences from the TH promoter to the 59 end of a NF-H promoter, but not the CMV IE promoter, can enhance long-term expression in forebrain neurons from helper virus-free HSV-1 vectors. This enhancement appears to require specific sequences in the TH promoter fragment because addition of a similar sized fragment of bacteriophage l to the NF-H promoter did not enhance expression. The reverse orientation of the TH promoter fragment retained some activity. Other eucaryotic sequences (from S. cerevisiae) may also enhance expression from the NF-H promoter.

2. Materials and methods

2.1. Materials Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs and Boehringer Mannheim. Dulbecco’s modified minimal essential medium, fetal bovine serum, G418, lipofectamine, and OPTI-MEM I were obtained from Gibco-BRL. 5-Bromo4-chloro-3-indoyl-b-D-galactopyranoside (X-Gal) was obtained from Sigma. Rabbit anti-E. coli b-galactosidase antibody was obtained from ICN, and mouse monoclonal anti-NeuN antibody was obtained from Chemicon. Alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin (Ig) G, biotinylated goat anti-mouse IgG, and the avidin–biotinylated peroxidase complex (ABC) reagent were obtained from Vector Laboratories. The BCIP/ NBT substrate and levamisole were obtained from Sigma. Fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine isothiocyanate-conjugated goat anti-mouse IgG were obtained from Jackson ImmunoResearch Laboratories.

2.2. Cells Baby hamster kidney fibroblast (BHK21) cells and 2-2 cells [36] were maintained in Dulbecco’s modified minimal essential medium supplemented with 10% fetal bovine serum, penicillin / streptomycin, and 4 mM glutamine at 378C in humidified incubators containing 5% CO 2 . G418

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(0.5 mg / ml) was present during the growth of 2-2 cells but was removed before use in vector packaging.

2.3. HSV-1 Vectors Vectors are diagrammed in Fig. 1. This vector backbone has been previously described [38,43,44]. To attempt to reduce interactions between the vector backbone and the

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transcription unit, the HSV-1 IE 4 / 5 promoter in the HSV-1 ori s fragment is followed by a cassette of three SV40 early region polyadenylation sites [26]. pNFHlac [44] contains a 0.6-kb fragment of the mouse NF-H promoter (from plasmid pH-615 [35]). pTH-NFHlac contains a 6.3-kb BamHI fragment from the rat TH promoter (20.5 to 26.8 kb [5]) inserted at the 59 end (BamHI site) of the NF-H promoter in pNFHlac. pTH(R)-NFHlac

Fig. 1. Schematic diagrams of vectors that contain 59 upstream sequences from the TH promoter fused to either the NF-H promoter or the CMV IE promoter and control vectors. (A) The overall structure of the vectors used in this study. Each vector contains a HSV-1 a sequence (contains the DNA cleavage / packaging sites; vertical line segment), the E. coli amp r gene and the col E1 origin of DNA replication (Amp r), the HSV-1 ori s (shaded circle), and the HSV-1 IE 4 / 5 promoter (arrow) followed by three SV40 early region polyadenylation sites (black segment) [38,43,44]. The transcription unit in each vector contains a promoter (arrow), the LacZ gene (horizontal line segment), the second intron from the mouse a-globin gene, and the SV40 early region polyadenylation site (shaded segment). In addition, most vectors also contain a 59 upstream element (diagonal line segment), and in one vector, the transcription unit is flanked on each end by two copies of an insulator from the chicken b-globin locus [7]. (B) The 59 upstream elements and promoters contained in these vectors. The 59 upstream elements were a 6.3-kb fragment of the TH promoter (20.5 to 26.8 kb; segment with diagonal lines from left to right [5]), this fragment in the reverse orientation (segment with diagonal lines from right to left; TH(R)), a 5.6-kb fragment of bacteriophage l DNA (stippled segment), a 40-kb fragment of S. cerevisiae DNA (wavy line segment [43]), or two copies of an insulator (block segment; INS) from the chicken b-globin locus at the 59 end of the TH fragment. The 59 upstream elements were fused to either the NF-H promoter (black segment [35]) or the CMV IE promoter (white segment). pNFHlac [44] and pNFHlac-Sac [43] have been previously described.

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contains the TH promoter fragment in the reverse orientation. pl-NFHlac contains a 5.6-kb BamHI fragment from bacteriophage l (nucleotides 22 346–27 972 [32]) inserted at the 59 end (BamHI site) of the NF-H promoter in pNFHlac. pNFHlac-Sac [43] contains a 40-kb fragment of S. cerevisiae DNA (nucleotides 176–40 114 of cosmid C8179 (ATCC)) inserted at the 59 end of the NF-H promoter. pINS-TH-NFHlac contains two copies of an insulator (INS; 1.2 kb) from the chicken b-globin locus [7] at each end of the TH-NFHlac transcription unit. pINSTH-NFHlac was constructed in several stages: First, pHSVpUCINS was constructed by inserting the 5.1-kb EcoRI and SalI fragment from pJC5-4 (contains INS [7]) into pHSVpUClinker I (contains HSV-1 vector sequences [43]) that had been digested with the same enzymes. Next, pINS-NFHlac was constructed by inserting a 4.8-kb BamHI and XhoII fragment from pNFHlac (contains NFHlac transcription unit [44]) into the 7.5-kb BamHI fragment from pHSVpUCINS. Next, pINS-TH-NFHlac was constructed by inserting a 6.3-kb BamHI fragment from the rat TH promoter (20.5 to 26.8 kb [5]) at the 59 end of the NF-H promoter (BamHI site) in pINS-NFHlac. To construct pCMVlac, the CMV IE promoter in a pBluescript plasmid was excised using XbaI and PstI and inserted into pSP73 (Promega) that had been digested with the same enzymes (pSP73cmv). The CMV IE promoter was excised from pSP73cmv using XbaI and HindIII and inserted into pNFHlac that had been digested with the same enzymes (pCMVlac). pTH-CMVlac contains the 6.3kb BamHI fragment from the rat TH promoter inserted at the 59 end (BamHI site) of the CMV IE promoter in pCMVlac. Each of these vectors expresses the E. coli LacZ gene. The pCMVlac described above was used for the microinjections into the hippocampus, and a previously described version of pCMVlac (pIE1bgalori [17]), which contains a different vector backbone, was used for the microinjections into the striatum.

2.4. Packaging vectors into HSV-1 particles Vectors were packaged into HSV-1 particles using the helper virus-free packaging system [12] and a modified protocol [41] that improves the efficiency. Vector stocks were purified and concentrated as described [24]. Vector

stocks were titered by counting the number X-gal positive cells obtained at 1 day after infection of BHK cells. The titers obtained on BHK cells were higher than those obtained on PC12 cells (not shown; see Ref. [45]), even though expression from the NF-H promoter in BHK fibroblast cells represents ectopic expression. Use of this ectopic expression may result in an underestimation of the titers, and the titer of these vectors were 5- to 10-fold lower than the titer of pHSVlac (contains the HSV-1 IE 4 / 5 promoter which is active in fibroblast cells).

2.5. Stereotactic injection of HSV-1 vectors into the brain Male Sprague Dawley rats (150–175 g) were used for these experiments. Vector stocks were delivered by stereotactic injection (two sites, 3 ml / site) into the striatum (anterior–posterior (AP) 10.8, medial–lateral (ML) 12.5, dorsal–ventral (DV) 25.5; AP 10.8, ML 22.5, DV 25.5), hippocampus (AP 23.4, ML 12.2, DV 23.8; AP 23.4, ML 22.2, DV 23.8), perirhinal cortex (AP 22.0, ML 16.2, DV 27.0; AP 22.0, ML 26.2, DV 27.0), or postrhinal cortex (AP 28.0, ML 16.0, DV 25.2; AP 28.0, ML 26.0, DV 25.2). AP is relative to bregma, ML is relative to the sagittal suture, and DV is relative to the bregma–lambda plane [31]. These studies were approved by the Children’s Hospital IACUC.

2.6. Histological analyses Four days to 6 months after gene transfer, the rats were anesthetized with chloral hydrate (300 mg / kg, intraperitoneal) and then perfused with 50 ml phosphate-buffered saline (PBS) followed by 200 ml of 4% paraformaldehyde in PBS. The brains were postfixed in 4% paraformaldehyde in PBS (4 h, 48C), cryoprotected in 25% sucrose in PBS (2 days, 48C), and 25-mm coronal sections were cut on a freezing microtome. Enzymatic staining and immunohistochemistry were performed on free-floating sections. Expression of b-galactosidase was detected using X-gal [10]; the X-gal reaction was carried out for 3 h at room temperature at pH 7.9. Alternatively, E. coli b-galactosidase-immunoreactivity (IR) and NeuN-IR were detected in the same sections.

Fig. 2. X-gal positive cells from rats sacrificed at 4 days (A,B), 2 weeks (C,D), 1 month (E,F), 2 months (G,H), 4 months (I,J), or 6 months (K,L) after microinjection of pTH-NFHlac into the striatum. After the rats were sacrificed, the brains were sectioned, and X-gal staining was performed. (A) Numerous X-gal positive cells and processes are visible in a low power view of the striatum from a rat sacrificed at 4 days after gene transfer. The arrows indicate an X-gal positive cell in a low power photomicrograph that is also shown in the corresponding high power photomicrograph. (B) A high power photomicrograph reveals a number of X-gal positive cell bodies and proximal processes. (C) A low power view of X-gal positive cell bodies from a rat sacrificed at 2 weeks after gene transfer. The number of X-gal positive cells is less than that observed at 4 days after gene transfer. (D) A high power view shows a number of X-gal positive cell bodies, and many of the positive cell bodies are large (characteristic of neurons). (E,G,I,K) Low power views show X-gal positive cells and processes in sections from rats that were sacrificed at 1, 2, 4, or 6 months after gene transfer. The numbers of positive cells are similar to that observed at 2 weeks after gene transfer. (F,H,J,L) High power views show X-gal positive cell bodies and processes. Many of these cells display neuronal morphology. The staining fills the cell body in many of the positive cells and some processes are visible, indicative of a high level of expression. Panels (F) and (H) each contain an insert in the top right; the insert in panel (F) shows two X-gal positive cell bodies and the insert in panel (H) shows one positive cell body and proximal process. Scale bars: (A,C,E,G,I,K) 500 mm; (B,D,F,H,J,L) 83 mm.

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Fig. 3. X-Gal positive cells from rats sacrificed at 4 days (A,B), 2 weeks (C,D), 1 month (E,F), or 2 months (G–I) after microinjection of pTH-NFHlac into the hippocampus. (A) Numerous X-gal positive cell bodies and proximal processes are visible in a low power view of the hippocampus from a rat sacrificed at 4 days after gene transfer. Many of the positive cell bodies are in the dentate granule cell layer, and many of their proximal processes are also stained. Some of the X-gal positive cell bodies are located in the hilar region. The arrows indicate an X-gal positive cell in a low power photomicrograph that is also shown in the corresponding high power photomicrograph. (B) High power reveals large X-gal positive cells, and some proximal processes are also shown. These cell bodies are located in the granule cell layer. (C,E,G) Low power views of sections from rats sacrificed at 2 weeks, 1 month or 2 months after gene transfer contain X-gal positive cells and processes, but the numbers of positive cells are less than that observed at 4 days. (D,F,H,I) High power views shows X-gal positive cell bodies and processes at each of these time points. The large X-gal positive cell bodies are characteristic of neurons, and some stained processes are also visible. Panel (F) contains an insert in the top right that shows two X-gal positive cell bodies and proximal processes. Scale bars: (A,C,E,G) 500 mm; (B,D,F,H,I) 83 mm.

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Sections were preincubated in PBS, 0.3% H 2 O 2 (10 min, room temperature) and then rinsed with PBS (3 times, 5 min each). Sections were permeabilized by incubation for 30 min at 378C in PBS, 2% normal goat serum, 0.2% Triton X-100 (buffer A), and then incubated overnight at 48C in buffer A with rabbit anti-E. coli b-galactosidase antibody (1:500 dilution). The sections were rinsed at room temperature with PBS, 0.2% Triton X-100 (buffer B; 3 times, 10 min each), and then incubated for 2 h at room temperature in buffer B with an alkaline phosphataseconjugated goat anti-rabbit immunoglobulin IgG (1:1000 dilution). The sections were rinsed with 100 mM Tris– HCl, pH 9.6 (3 times, 10 min each), and alkaline phosphatase activity was visualized using the BCIP/ NBT substrate in the presence of levamisole to inhibit endogenous alkaline phosphatase activity. To detect NeuN-IR, the sections were washed with PBS (3 times, 5 min each) and then incubated overnight at 48C in buffer A with a mouse monoclonal anti-NeuN antibody (1:50 dilution [29]). The sections were rinsed at room temperature with buffer B (3 times, 10 min each), and then incubated for 2 h at room temperature in buffer B with biotinylated goat anti-mouse IgG (1:200 dilution). The sections were rinsed at room temperature with buffer B (3 times, 10 min each), incu-

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bated (room temperature, 1 h) with the ABC reagent, and rinsed. Immunoreactivity was visualized with diaminobenzidene according to the manufacturer’s instructions. E. coli b-galactosidase-IR and NeuN-IR were also detected using immunofluorescent visualization. Sections were permeabilized as just described and then incubated with both a rabbit anti-E. coli b-galactosidase antibody (1:500 dilution) and a mouse monoclonal anti-NeuN antibody (1:50 dilution [29]) in buffer A (overnight at 48C, then 1 h at 378C). Sections were washed with PBS (3 times, 5 min each) and then incubated with both fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and rhodamine isothiocyanate-conjugated goat anti-mouse IgG (1:150 dilutions) in buffer A (3 h at room temperature). Sections were washed with PBS (3 times, 5 min each), and then mounted in PBS and immediately examined under the microscope.

2.7. Cell counts Twenty-five-mm coronal sections were prepared from the relevant area of each brain. Every fourth section was analyzed for expression of b-galactosidase and |12 of these sections contained either the X-gal positive cells or

Fig. 4. X-Gal positive cells from rats sacrificed at 4 days (A,B) or 1 month (C,D) after microinjection of pTH-NFHlac into the perirhinal cortex. (A) Numerous X-gal positive cells are visible in a low power view of the perirhinal cortex from a rat sacrificed at 4 days after gene transfer. The arrows indicate an X-gal positive cell in a low power photomicrograph that is also shown in the corresponding high power photomicrograph. A high power view (B) of this section shows a large, central X-gal positive area, in which it is difficult to distinguish individual cells, and individual X-gal positive cells and processes on the periphery of the area. Many of these cells have large cell bodies and proximal processes that are characteristic of neurons. (C) A low power view of X-gal positive cells from a rat sacrificed at 1 month after gene transfer. The number of X-gal positive cells is less than that observed at 4 days. A high power view (D) of this section reveals individual X-gal positive cell bodies. Some of the staining fills the cell body, but much of the staining is punctate. Scale bars: (A,C) 500 mm; (B,D) 83 mm.

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the b-galactosidase-IR positive cells. Positive cells were identified under low power (310), and cell counts were performed under 340 magnification. To verify the accuracy of the cell counts, each section was counted at least two times, on different days; the two values differed by ,10% for each section. The statistical significance of the differences in the numbers of X-gal positive cells were analyzed using Student’s unpaired t-test.

3. Results

3.1. Insertion of 59 upstream sequences from the rat TH promoter into a vector that contains the NF-H promoter enhances long-term expression 59 Upstream sequences (6.3 kb) from the TH promoter were inserted at the 59 end of a NF-H promoter in a HSV-1 vector (pTH-NFHlac; Fig. 1). To distinguish between sequence-specific effects and sequence-independent spacing effects, a 5.6-kb fragment from bacteriophage l was inserted at the 59 end of the NF-H promoter (pl-NFHlac). To investigate if the 59 upstream sequences in the TH promoter can function in an orientation-independent manner, the TH promoter fragment was inserted in the reverse orientation (pTH(R)-NFHlac). Vector stocks were prepared using a helper virus-free packaging system [12,41].

Vector stocks were microinjected into the rat striatum, hippocampus, perirhinal cortex, or postrhinal cortex. In rats, each of these areas essentially lacks TH-IR positive neurons [18]. The rats were sacrificed at 4 days, 2 weeks, or 1, 2, 4, or 6 months after gene transfer, and X-gal staining was performed. Rats that received PBS lacked X-gal positive cells in each of the brain areas under study, but faintly positive cells were occasionally observed in brain vasculature endothelium (not shown). At 4 days after gene transfer with pTH-NFHlac, X-gal positive cells were observed proximal to each injection site, and many of these cells displayed neuronal morphology (striatum, Fig. 2A,B; hippocampus, Fig. 3A,B; perirhinal cortex, Fig. 4A,B; postrhinal cortex, Fig. 5A,B). X-Gal positive cells were also observed at 4 days after gene transfer of the control vectors (pl-NFHlac and pTH(R)-NFHlac; not shown). Using each of these vectors, X-gal positive cells were also observed at distant site(s) that project to a specific injection site (not shown). The numbers of X-gal positive cells at these distant sites were much smaller than the numbers of positive cells at the injection sites, and subsequent analyses focused on the positive cells proximal to the injection sites. The efficiency of gene transfer was quantitated as the number of X-gal positive cells at 4 days after gene transfer divided by the amount of vector that was injected. The amount of vector that was injected was determined from the titer (infectious vector particles / ml, assayed as the

Fig. 5. X-Gal positive cells from rats sacrificed at 4 days (A,B) or 1 month (C,D) after microinjection of pTH-NFHlac into the postrhinal cortex. (A) A low power view of the postrhinal cortex from a rat sacrificed at 4 days after gene transfer shows numerous X-gal positive cells. A high power view (B) of this section shows a large X-gal positive cell with several processes. (C) A low power view of X-gal positive cells from a rat sacrificed at 1 month after gene transfer. The level of staining is less than that observed at 4 days. A high power view (D) of this section reveals several positive cell bodies and extensive punctate staining. Scale bars: (A,C) 500 mm; (B,D) 83 mm.

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number of X-gal positive cells obtained at 1 day after infection of cultured BHK cells). The efficiencies of gene transfer for the specific vectors and injection sites ranged between 0.3 and 4.8%. These modest variations may reflect different efficiencies of gene transfer in different brain areas. Also, these vector stocks had different titers, and the numbers of X-gal positive cells does not increase linearly with increasing amounts of vector injected, presumably both because of multiple gene transfer events into the same cells and because of the limited diffusion of vector particles in the extracellular space. These efficiencies of gene transfer are similar to those we have observed using herpesvirus IE promoters [12]. We investigated if the TH-NFH promoter can target expression to neurons. Sections from rats that received either striatal or hippocampal microinjections of pTHNFHlac (and sacrificed at 4 days after gene transfer), were costained with antibodies directed against either E. coli b-galactosidase or a neuronal marker, NeuN. The antibodies were visualized using either the horseradish peroxidase and alkaline phosphatase reactions or immunofluorescence. Using either method of visualization, the results (Figs. 6 and 7) revealed double labeled cells in each brain area. Cell counts (Table 1) demonstrated that 89% of the cells in either brain area that contained b-galactosidase-IR also contained NeuN-IR.

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We investigated the long-term expression supported by pTH-NFHlac. X-Gal positive cells were observed in sections from rats sacrificed at 2 weeks or 1, 2, 4, or 6 months after microinjection of pTH-NFHlac into the striatum (Fig. 2C–L), and many of these positive cells displayed neuronal morphology. Positive cells were also observed in sections from rats sacrificed at 2 weeks or 1 or 2 months after microinjection of pTH-NFHlac into the hippocampus (Fig. 3C–I), and at 1 month after microinjection into either the perirhinal cortex (Fig. 4C,D) or postrhinal cortex (Fig. 5C,D). The X-gal staining in rhinal cortex was not as strong as that observed in either the striatum or the hippocampus; although much of the staining was punctate, some cell bodies were visible. Punctate X-gal staining is also observed in some neural cells in transgenic mice that use four different promoters, including the TH promoter, to express the LacZ gene [13,25]. Thus, the punctate staining observed in rhinal cortex may be partially due to properties of the specific cortical cell types under study. Cell counts were performed to quantitate the stability of long-term expression that was supported by each vector (Table 2 and Fig. 8). We compared the numbers of X-gal positive cells at 4 days to those obtained at longer time points. Because this comparison uses the number of X-gal positive cells at 4 days as the initial value, this comparison

Fig. 6. b-Galactosidase-IR positive cells that also contain NeuN-IR from rats sacrificed at 4 days after microinjection of pTH-NFHlac into either the striatum or the hippocampus. b-galactosidase-IR was detected using a rabbit anti-E. coli b-galactosidase antibody, and the staining was visualized using an alkaline phosphatase-conjugated secondary antibody and the alkaline phosphatase reaction (black reaction product). NeuN-IR was detected using a mouse monoclonal anti-NeuN antibody, and NeuN-IR was visualized using a biotinylated secondary antibody followed by the ABC reagent and the peroxidase reaction (yellow–brown reaction product). (A) A low power view of the striatum shows a central area of b-galactosidase-IR positive cells, and NeuN-IR positive cells are visible throughout the section. (B) A high power view shows several areas of b-galactosidase-IR proximal to NeuN-IR (arrow). (C) A low power view of the hippocampus shows b-galactosidase-IR positive cells in the dentate granule cell layer and in the hilar region. (D) A high power view of the granule cell layer shows a number of b-galactosidase-IR positive cells that contain NeuN-IR. One cell (arrow) contains b-galactosidase-IR (presumably in the cytoplasm) surrounding the NeuN-IR. Scale bars: (A,C) 500 mm; (B,D) 83 mm.

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Fig. 7. Immunofluorescent colocalization of b-galactosidase-IR (A,C) and NeuN-IR (B,D) in either hippocampal (A,B) or striatal (C,D) cells from rats sacrificed at 4 days after microinjection of pTH-NFHlac. b-galactosidase-IR was visualized using a fluorescein-conjugated secondary antibody and NeuN-IR was visualized using a rhodamine-conjugated secondary antibody. In (C,D), the arrows point to one cell that contains both b-galactosidase-IR and NeuN-IR. (E,F) Photomicrographs of a striatal section that was treated as in panels (C) and (D), except the primary antibodies were omitted. Scale bars: (A,B) 300 mm; (C–F) 175 mm.

Table 1 The numbers of b-galactosidase-IR positive cells that contain or lack NeuN-IR from rats sacrificed at 4 days after microinjection of pTH-NFHlac into either the striatum or the hippocampus a Injection site

b-Galactosidase-IR and NeuN-IR positive cells

b-Galactosidase-IR positive, NeuN-IR negative cells

% of double labeled cells b

Striatum Hippocampus

2296 3785

253 464

89 89

a

Three rats received microinjections of pTH-NFHlac into the striatum, and three rats received microinjections of pTH-NFHlac into the hippocampus. Every fourth section was analyzed by double staining for b-galactosidase-IR and NeuN-IR. The total numbers of b-galactosidase-IR positive cells from the three rats in each brain area are shown. b The percentage of double labeled cells was calculated as the number of double labeled cells divided by the total number of b-galactosidase-IR cells (3100).

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Table 2 The numbers of X-gal positive cells from rats sacrificed at various times after microinjection of pTH-NFHlac, pINS-TH-NFHlac, or control vectors into the striatum, hippocampus, or rhinal cortices a Vector

Injection site

Average X-gal positive cells per rat Time after gene transfer: 4 days

pTH-NFHlac pTH-NFHlac pTH-NFHlac

Striatum 17196384 Hippocampus 748161073 Perirhinal 7846151 cortex pTH-NFHlac Postrhinal 9996221 cortex pTH(R)-NFHlac Striatum 5326114 pINS-TH-NFHlac Striatum 7856158 pNFHlac-Sac Striatum 6886130 pl-NFHlac Striatum 4876190 pNFHlac Striatum 192630 pNFHlac Hippocampus 93639 b

2 weeks

1 month †

2 months

†,[[

†,[[

315644 260695 230635 11376138 ** 6736106 ** ,[[ 7896215 ** ,[[ 123626 **

4 months 3086128 **

6 months ,[[

229627 ** ,[[

165625 **

219631 ** 113618 * 22613 * 25613 †

3766 ** 3086128 * ,[[ 73610 ** ,[ 060 060 060 b

060 b

a

The titers of the vector stocks following purification and concentration were: pTH-NFHlac, 3.2310 7 infectious vector particles (IVP) / ml; pTH(R)NFHlac, 2.0310 7 IVP/ ml; pINS-TH-NFHlac, 4.8310 7 IVP/ ml; pNFHlac-Sac 2.4310 6 IVP/ ml; pl-NFHlac 1.0310 7 IVP/ ml; and pNFHlac, 2.0310 6 IVP/ ml. For each vector, three rats were analyzed for each injection site and each time point. The values shown are the average numbers of X-gal positive cells per rat6standard deviations. b Data previously reported in Ref. [44]. These data areincluded in this table only to enable comparisons. * P,0.05, ** P,0.01, or † P,0.001 compared to 4 days; [ P,0.05 or [[ P.0.05 compared to 2 weeks; Student’s unpaired t-test.

is designed to be independent of any variability in either the titering or the gene transfer process. By 2 weeks after microinjection of pTH-NFHlac into either the striatum or the hippocampus, the numbers of positive cells had declined to 18 and 15%, respectively, of those observed at 4 days, and these declines were statistically significant (Table 2). An initial large decline in expression was also observed at 2 weeks after microinjection of either plNFHlac or pNFHlac into the striatum (Table 2 and Fig. 8), and similar declines have been observed with other vectors [9,12,38,44].

Of note, using pTH-NFHlac, the percent stability of expression showed minimal changes either from 2 weeks to 6 months after gene transfer into the striatum or from 2 weeks to 2 months after gene transfer into the hippocampus (Table 2 and Fig. 8). These minimal changes were not statistically significant (Table 2). Also, similar stabilities of expression were observed at 1 month after gene transfer into either area of rhinal cortex (Table 2). In contrast, no positive cells were observed at 1 month after gene transfer of either pl-NFHlac or pNFHlac into the striatum or at 1 month after gene transfer of pNFHlac into the hippocam-

Fig. 8. The stabilities of expression in either the striatum or the hippocampus supported by pTH-NFHlac and control vectors. For each vector, injection site, and time point, the percentage of X-gal positive cells at 4 days is shown (the average number of X-gal positive cells at 2 weeks, 1 month, or 2 months after gene transfer divided by the average number of X-gal positive cells at 4 days (3100); calculated using the data in Table 2). The data at 2 months after microinjection of pNFHlac into the striatum was previously reported [44] and is included here only to enable comparisons.

G.-r. Zhang et al. / Molecular Brain Research 84 (2000) 17 – 31

28

Table 3 The numbers of X-gal positive cells from rats sacrificed at various times after microinjection of pTH-CMVlac or pCMVlac into either the striatum or the hippocampus a Vector

Injection site

Average X-gal positive cells per rat Time after gene transfer: 4 days

pTH-CMVlac pTH-CMVlac pCMVlac b pCMVlac

Striatum Hippocampus Striatum Hippocampus

22616317 15536517 194657 9396215

2 weeks

1 month †

515694 237683 *

†

34621 060 2866 ** 060

2 months 060 261 **

a

The titers of the vector stocks following purification and concentration were: pTH-CMVlac, 2.0310 7 IVP/ ml; pCMVlac (pIE1bgalori), 2.0310 6 IVP/ ml (striatum); and pCMVlac, 1.2310 7 IVP/ ml (hippocampus). For each vector, three rats were analyzed for each injection site and each time point. The values shown are the average numbers of X-gal positive cells per rat6standard deviations. b This version of pCMVlac has been previously described (pIE1bgalori [17]) and contains a vector backbone that differs from that of the other vectors used in this study. * P,0.05, ** P,0.01, or † P,0.001 compared to 4 days; Student’s unpaired t-test.

pus (Table 2 and Fig. 8). Using pTH(R)-NFHlac, some X-gal positive cells were observed at 1 month after gene transfer into the striatum (Table 2), but the percent stability of expression (7%) was |2-fold lower than that supported by pTH-NFHlac (15%).

3.2. Insertion of sequences from S. cerevisiae into a vector that contains the NF-H promoter also enhances long-term expression We reasoned that the TH promoter may not be the only eucaryotic promoter that contains elements that can enhance expression from the NF-H promoter in a HSV-1 vector. Therefore, we examined the expression supported by a vector that contains a 40-kb fragment of S. cerevisiae DNA inserted at the 59 end of the NF-H promoter (Fig. 1; pNFHlac-Sac [43]). pNFHlac-Sac was microinjected into the striatum, and the rats were sacrificed at 4 days, 2 weeks, or 1 month after gene transfer. Of note, X-gal positive cells were still present at 1 month after gene transfer, and the stability of long-term expression was similar to that observed using pTH-NFHlac (Table 2 and Fig. 8).

respectively, and this represents a modest increase compared to those observed using pTH-NFHlac (18 or 15%).

3.4. Insertion of 59 upstream sequences from the TH promoter into a vector that contains the CMV IE promoter does not enhance long-term expression To determine if 59 upstream sequences in the TH promoter can enhance expression from a herpesvirus promoter, we inserted these TH promoter sequences at the 59 end of the CMV IE promoter (pTH-CMVlac; Fig. 1). pTH-CMVlac, and pCMVlac as a control, were microinjected into either the striatum or the hippocampus, and the rats were sacrificed at 4 days, 2 weeks, 1 month, or 2 months. Using pTH-CMVlac, the numbers of X-gal positive cells declined rapidly over time, and no positive cells were observed at either 2 months after gene transfer into the striatum or 1 month after gene transfer into the hippocampus (Table 3). The declines in expression obtained using pTH-CMVlac are similar to those observed using pCMVlac.

4. Discussion

3.3. Insertion of an insulator between the TH-NFHlac transcription unit and the HSV-1 sequences may support a modest additional enhancement of long-term expression Genetic regulatory elements in the HSV-1 sequences in the vectors might influence expression from the TH-NFH promoter. Therefore, we inserted an insulator from the chicken b-globin locus (1.2 kb [7]) at both ends of the TH-NFHlac transcription unit (pINS-TH-NFHlac; Fig. 1). pINS-TH-NFHlac was microinjected into the striatum, and the rats were sacrificed at 4 days, 2 weeks, or 1 month after gene transfer. The stabilities of expression at 2 weeks or 1 month were 28 or 39% (Table 2 and Fig. 8),

4.1. The TH-NFH promoter enhances long-term expression and targets expression to neurons pTH-NFHlac supports expression for 6 months in the striatum, 2 months in the hippocampus, and 1 month in two areas of rhinal cortex. These are the longest time points we have evaluated. Similarly, we recently used this promoter to express an HA tagged TH, and we detected positive striatal cells at both 1 and 2 months (Sun et al., in preparation). In contrast, using pNFHlac, no expression was observed at 1 month in either the striatum or the hippocampus. The enhanced expression appears to depend upon specific sequences in TH promoter fragment because

G.-r. Zhang et al. / Molecular Brain Research 84 (2000) 17 – 31

pl-NFHlac, which contains a similar sized fragment of bacteriophage l DNA, did not enhance expression. pTH-NFHlac targeted expression to neurons, consistent with the known properties of the NF-H promoter [35]. Although the transcription start site(s) of the TH-NFH promoter were not examined, it seems plausible that the predominant start site may be the transcription start site in the NF-H promoter because the TH promoter fragment (20.6 to 26.8 kb) lacks the transcription start site, TATA box, and other elements proximal to the transcription start site, and because expression was also observed with the TH promoter fragment in the reverse orientation. Although neurofilaments are found in essentially all neurons [39], the complete cell type specificity of pTHNFHlac remains to be determined. pTH-NFHlac supported expression in each of the four forebrain areas that were examined, and, in rats, each of these areas essentially lacks TH-IR positive neurons [18]. In particular, most striatal neurons are GABAergic medium spiny neurons, and most hippocampal dentate granule neurons are glutamatergic. Thus, the TH-NFH promoter appears to have activity in multiple types of neurons. Insertion of insulators between the TH-NFHlac transcription unit and the vector backbone sequences supports a modest additional improvement in expression. This suggests that interactions between specific sequences in the vector backbone and the transcription unit may affect expression. However, the large initial decline in expression appears to be caused primarily by specific HSV-1 proteins that affect the virion (U S 3, U L 13, and VP16 [45]).

4.2. The TH-CMV promoter does not enhance expression In transgenic mice, expression from the CMV IE promoter is restricted to limited types of neurons and other neural cells [3,14]. Thus, at times shortly after gene transfer, pCMVlac may support expression in many types of neural cells, but at longer times, the cell type specificity may be reduced to that obtained in transgenic mice. Addition of the TH promoter fragment may not broaden the cell type specificity of this IE promoter. Moreover, this herpesvirus promoter may be affected by the processes that shut off most HSV-1 gene expression as HSV-1 enters the latent state.

4.3. The critical elements in the TH promoter fragment for enhancing expression are not known This fragment of the TH promoter lacks both the basal promoter elements and a number of genetic regulatory elements that mediate short-term changes in promoter activity in response to specific signaling pathways. However, this fragment contains both positive and negative regulatory elements that support catecholaminergic cellspecific expression in the central nervous system [2,25,27,28]. Interestingly, in transgenic mice, both 6.0-

29

and 9.0-kb fragments of the rat TH promoter support limited ectopic expression in specific types of neurons that lack expression of the endogenous TH gene [25,28]. This fragment retained some activity in the reverse orientation; some genetic regulatory elements, such as enhancers, have activity in both orientations, while other elements, such as locus control regions, have activity in only one orientation [42]. A 40-kb fragment of S. cerevisiae DNA also enhanced long-term expression from the NF-H promoter. This DNA fragment contains at least 26 genes and open reading frames as well as their genetic regulatory elements. It is not clear if the TH promoter fragment and the S. cerevisiae DNA fragment enhance expression by similar mechanisms. Deletion analyses of both the TH promoter and S. cerevisiae fragments will be required to isolate the critical sequence(s) that enhance expression. The capability of the TH-NFH promoter to enhance expression raises the possibility that other cellular promoters might contain activities similar to those found in either the TH or NF-H promoter fragments. HSV-1 vectors that contain either a preproenkephalin promoter or large fragments of the TH promoter support long-term expression [19,20,38,44]. Thus, specific sequences in the preproenkephalin promoter might also enhance expression from the NF-H promoter. Many neuronal-specific promoters, including specific NF promoters, contain the neuronal silencer element [6,33,34], but the NF-H promoter is the only cellular promoter that we have tested in combination with the TH promoter fragment.

4.4. Implications for physiological studies Most of the published physiological studies with HSV-1 vectors have relied upon only short-term expression [15]. One of the principal limitations on using HSV-1 vectors for physiological studies has been the lack of a promoter that supports long-term expression in forebrain neurons. Thus, as HSV-1 vectors that contain the TH promoter have been used in a long-term physiological study [37], HSV-1 vectors that contain the TH-NFH promoter may support such studies in forebrain neurons.

Acknowledgements We gratefully thank Dr. A. Davison for HSV-1 cosmid set C, Dr. R. Sandri-Goldin for 2-2 cells, Dr. K. O’Malley for the TH promoter, Dr. W. Schlaepfer for the NF-H promoter, Dr. G. Felsenfeld for the chicken b-globin insulator, Drs. D. Ho and E. Sapolsky for an initial version of pCMVlac (pIE1bgalori), and Dr. X. Langlois for constructing pCMVlac. This work was supported by F32NS10805 (MS), R01AG16777, and R01NS34025 (AG).

30

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