- Email: [email protected]
Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders
Methods 28 (2002) 248–252 www.academicpress.com
Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders Hideki Mochizuki,a,* Masauki Miura,b Takashi Shimada,c and Yoshikuni Mizunoa a Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Laboratory of Cell Recovery Mechanisms, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan b
c
Accepted 15 July 2002
Abstract Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of ParkinsonÕs disease. Our results suggest that AAV–Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as ParkinsonÕs disease. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Adeno-associated virus vector; Apoptosis; Gene therapy; Apaf-1; ParkinsonÕs disease
1. Introduction Apoptosis is a programmed cell death resulting from activation of a genetically determined cell suicide cascade. Cells undergoing apoptosis show characteristic morphological features such as condensation of cytoplasmic and nuclear contents, blebbing of plasma membranes, and nuclear fragmentation and ultimately breakdown into membrane-bound apoptotic bodies that are rapidly phagocytosed [1]. Caspases, which are involved in the execution of apoptosis, are present in living cells as inactive zymogens and become activated through intracellular caspase cascades. Several caspase cascades have been described. One pathway is initiated by the activation of cell-surface death receptors, such as Fas and tissue necrosis factor, leading to caspase-8 activa*
Corresponding author. Fax: +81-3-5684-0476. E-mail address: [email protected] (H. Mochizuki).
tion, which in turn cleaves and activates downstream caspases such as caspase-3, -6, and -7. Another pathway is triggered by cytochrome c released from mitochondria, which promotes the activation of caspase-9 through apoptotic protease activating factor-1 (Apaf-1) [2]. The cascade of caspase-1 and -11 is also an independent and important pathway for apoptosis. Caspase-11 is a member of the caspase-1 subfamily and is required for caspase-1-induced apoptosis and interleukin (IL)-1b secretion. Physical interaction between caspase-11 and caspase-1 is a critical step in the activation of caspase-1. The substrate specificity of caspase-11 is similar to that of caspase-9, and caspase-11 can promote the processing of caspase-3, suggesting that caspase-11 works as the apical caspase in the cascade that activates downstream executioner caspases under pathological conditions. In this review, we analyze the potential application of antiapoptotic gene therapy for neurological disorders including stroke, amyotrophic lateral sclerosis (ALS),
1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 2 2 9 - 3
H. Mochizuki et al. / Methods 28 (2002) 248–252
multiple sclerosis, and ParkinsonÕs disease. We also discuss the strategies and methods that we have developed and applied successfully to study the dominant negative inhibitor of caspase in regulating mitochondrial caspase cascade using the adeno-associated virus vector (AAV) for the treatment of neurodegenerative disorders [3].
2. Natural antiapoptotic proteins The regulation of caspase cascades rescues neuronal cells from apoptotic death in neurodegenerative disorders [4]. Caspases are inhibited by natural antiapoptotic proteins such as p35 protein of Autographica californica nuclear polyhedrosis virus [5] or members of the inhibitors of apoptosis (IAP) family [6]. Transgenic mice that express the antiapoptotic protein p35 in oligodendrocytes (OLG) through the Cre-loxP system are resistant to experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin OLG glycoprotein, which is used as a disease model of multiple sclerosis [7]. These transgenic mice show a significant reduction of the number of infiltrating T cells and macrophages/microglia in the EAE lesions and apoptotic OLG expressing the activated form of caspase-3. These mice also confer significant resistance to hypoxiainduced neuronal injury [8]. IAPs were originally identified in mutant baculovirus lacking p35 based on their ability to rescue cells from apoptosis. One such member, X-chromosome-linked IAP (XIAP), specifically inhibits caspase-3, -7, and -9. Adenoviral gene transfer of XIAP promoted survival of potassium-deprived cerebellar granule neurons [9]. Eberhardt et al. [10] recently reported that adenoviral gene transfer of XIAP prevented cell death of dopaminergic substantia pars compacta neurons in a mouse model of ParkinsonÕs disease induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP). Virus vector mediating the pan-caspase inhibitor baculovirus p35 and a specific class of caspase-inhibitory proteins such as cytokine response modifier A and XIAP have also been shown to be effective in vivo, especially under pathological conditions [10,11].
3. Antiapoptotic function of Bcl-2 and Bcl-xL The human oncoprotein Bcl-2 was the first identified member of a large family of proapoptotic (Bax, Bad, Bcl-xs) and antiapoptotic (Bcl-2, Bcl-xL) molecules, active in neuronal and nonneuronal cells, which form homodimers and heterodimers with each other. The Bcl2 inhibits neuronal apoptosis induced by a variety of noxious stimuli and preserves the functional integrity of injured cells [12,13]. As a gene therapy for ParkinsonÕs disease, herpes simplex virus vector-mediated expression
249
of Bcl-2 has been applied to prevent 6-hydroxydopamine-induced degeneration of neurons [14]. Azzouz et al. [15] also investigated the potential of recombinant AAV (rAAV) to transfer neuroprotective molecules in an animal model of ALS. They demonstrated that injection of a rAAV encoding the antiapoptotic protein Bcl-2 into the lumbar spinal cord in SOD1G93A mice resulted in sustained Bcl-2 expression in motoneurons and significantly increased the number of surviving motoneurons at the end stage of the disease. These results suggested that the use of rAAV for delivery of Bcl2 to spinal cord motoneurons is potentially useful for enhancing motoneuron survival and repair. In another study, AAV vector-mediated Bcl-2 gene transfer also prevented DNA fragmentation and ischemia-induced cell death of CA1 forebrain neurons [16]. Bcl-xL, one of three isoforms of Bcl-x, protects cells from the damaging effect of reactive oxygen species, e.g., lipid peroxidation, which has been shown to induce apoptotic cell death in vitro. Bl€ omer et al. [17] reported that intracellular delivery of lentiviral vectors expressing Bcl-xL prevented apoptotic death of axotomized cholinergic neurons. Thus, virus vector-mediated Bcl-2 or Bcl-x expression in vivo can prevent neuronal death under certain pathological conditions.
4. Ribozyme and dominant negative inhibitors of caspase There are several approaches to downregulate particular caspases by ribozyme targeting or dominant negative inhibitors of caspases. Ribozymes are enzymatic RNA molecules that suppress the expression of specific genes by selective cleavage of other RNA species. Eldadah et al. [18] reported that a hammerhead ribozyme directed against caspase-3 mRNA protected cerebellar granule cells against apoptosis induced by serum potassium deprivation. In their study, maximal protection by this ribozyme was observed after 24 h of deprivation, at which time apoptosis was 18% compared with 32% in control cells. This study suggests that AAVderived ribozyme-directed cleavage of mutant mRNAs can regulate the apoptotic pathway in vivo. In the mitochondrial pathway, the formation of Apaf-1–caspase-9 complex is accomplished by the heteromeric CARD/CARD (caspase recruitment domain) interaction, an apical step. In addition, homophilic CARD/CARD interactions are also found in an array of other proteins involved in apoptotic regulation, including the majority of the initiator procaspases, adapter proteins, and cellular apoptosis inhibitors. Extensive in vitro binding assays have shown that these homophilic interactions are highly specific. The recombinant wildtype CARD domain inhibits proteolytic cleavage of procaspase-9. Overexpression of Apaf-1 CARD can act as a dominant negative inhibitor. Therefore, for inhibi-
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tion of the mitochondrial apoptotic cascade in vivo, we generated a rAAV vector that contained the CARD of Apaf-1 to block the Apaf-1/caspase-9 pathway of cell death via dominant negative interference with the formation of a functional Apaf-1–caspase 9 complex [3]. However, the use of Apaf-1 CARD as an antiapoptotic molecule requires caution because it can lead to caspase9 activation at least under certain in vitro experimental conditions [19]. Active site mutants of each caspase had been generated and reported to act in a dominant negative manner [20–23]. AAV vectors incorporating dominant negative inhibitors, antisense DNA or ribozymes, could operate to regulate the caspase cascade.
5. Construction of rAAV vector with double promoter We established an AAV vector that could express both the dominant negative truncated Apaf-1 transgene (Apaf-1-DN) and the enhanced green fluorescent protein (EGFP) as a marker gene simultaneously with two promoters. To construct a plasmid bearing Apaf1-DN, the DNA fragment corresponding to the CARD of mouse Apaf-1 (1–97) was obtained by reverse transcription-PCR. After digesting the amplified fragment, the resulting fragment was ligated into a multiple cloning site of the pCDNA3–FLAG expression vector. Plasmid containing the complete AAV genome (psub201) and the AAV packaging plasmid (pAAV/Ad) were constructed by Samulski et al. [24,25]. A pAAV–CAG–CARD–B19–EGFP was constructed with a cassette containing a chicken b-actin promoter/cytomegalovirus enhancer (CAG promoter), CARD cDNA as an Apaf-1-dominant negative inhibitor, a B19 promoter, EGFP, and b-globin poly(A) inserted between the AAV inverted terminal repeats of psub201. We confirmed the efficient expressions of both transgenes using this virus vector both in vitro and in vivo.
6. In vivo detection of antiapoptotic effect Long-term administration of MPTP causes apoptotic cell death in the substantia nigra of C57/BL mice [26]. To identify the antiapoptotic effect of gene therapy in vivo, we injected the AAV vector encoding the caspase dominant negative inhibitor stereotactically into the striatum of 8-week-old mice, which were then treated with MPTP 2 weeks later to model ParkinsonÕs disease. Three weeks after the first MPTP treatment, dopaminergic neurons profiles were assessed by immunohistochemistry for tyrosine hydroxylase (TH). The methods used in these experiments were as follows. (1) Stereotactic surgical procedures were per-
formed aseptically under anesthesia with pentobarbital (10 mg/kg) and ketamine hydrochloride (6 mg/kg). (2) After placing each mouse in a stereotactic frame, 4 ll of the vector suspended in phosphate-buffered saline (PBS) was injected into the striatum (AP +1.0, medial lateral 2.0, DV )3.0) over a period of 5 min by using a 25-ll Hamilton syringe. (3) Two weeks after vector injection, to allow for expression of these molecules, the mice received four intraperitoneal injections of MPTP–HCl (30 mg/kg; Research Biochemicals, Natick, MA) in saline at 24-h intervals (chronic model). (4) Each mouse was perfused via the aorta with PBS followed by ice-cold 4% paraformaldehyde while under deep pentobarbital anesthesia. (5) Then, 30-lm nigral sections were cut and immunostained with a monoclonal antibody (mAb) for TH (Chemicon International, Temecula, CA) or with a mAb for GFP (Sigma, St. Louis, MO). In addition, sections were stained for anti-FLAG or anti-activated caspase-3 (PharMingen, San Diego, CA) using polyclonal antibodies. (6) The corresponding secondary antibodies [donkey antimouse fluorescein isothiocyanate (FITC) or Texas red and donkey anti-rabbit FITC or Texas red (Jackson Immunoresearch Laboratories, West Grove, PA); dilution 1:250] were pooled, and sections were incubated with these antibodies for 2 h at room temperature, followed by washing in PBS. (7) The sections were then examined by confocal scanning laser microscopy, with the collected signals undergoing digital color enhancement before superimposition. (8) For cell counting, the substantia nigra was cut into serial sections, and every third section was subjected to immunostaining for TH using a polyclonal antibody (a kind gift from I. Nagatsu, Fujita Health University, Aichi, Japan). (9) The number of viable TH-positive neurons was determined by manual counting in a blinded fashion using coded slides. The results showed that delivery of AAV–Apaf-1-DN prevented MPTP-induced dopaminergic neuronal cell death in the left substantia nigra (injected side) compared with the right side (untreated side) (Fig. 1A). The number of TH-positive dopaminergic neurons in the substantia nigra of the AAV–Apaf-1-DN–EGFP-injected mouse was significantly higher than that on the untreated side (Fig. 1B).
7. Summary Several strategies have been applied for gene delivery into neurons using virus vectors including lentivirus vector [27,28], adenovirus vector [9,10], and AAV. AAV is nonpathogenic and infects neurons. Immune responses to vector-corrected cells have historically limited the application of gene therapy for neurological disorders such as stroke, ALS, multiple sclerosis, and
H. Mochizuki et al. / Methods 28 (2002) 248–252
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Fig. 1. (A) Photomicrographs of TH immunostaining of the substantia nigra of a MPTP-treated AAV–Apaf-1-DN-injected (arrow) mouse. Note the extensive neuronal loss on the noninjected side. Note also the significantly greater number of neurons on the injected side compared with that on the noninjected side. (B) Comparison of proportion of TH-positive cells on the noninjected and injected sides. The total number of TH-positive neurons was counted in three sections each from four different mice. Statistical analysis was performed using ANOVA followed by ScheffeÕs post hoc test.
ParkinsonÕs disease. AAV efficiently transfers genes into the brain without activation of cellular and humoral immunity, which differs from the experience with recombinant adenovirus. A considerable number of agents capable of arresting apoptosis are available at present and have been extensively examined experimentally. It is hoped that these agents could be used therapeutically in neurological disorders, including those affecting the central nervous system involving apoptosis of neuronal cells. Mitochondrial dysfunction occurs not only in ParkinsonÕs disease but is also present in the brains of patients with HuntingtonÕs disease and in mouse models of ALS. Therefore, our AAV system with a dominant negative inhibitor could possibly regulate the mitochondrial apoptotic neuronal cell death in several neurological disorders. However, attention has to be paid to possible adverse side effects, especially oncogenic changes, in antiapoptotic gene therapy.
Acknowledgments This study was supported in part by a High Technology Research Center Grant and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Adeno-associated virus-mediated antiapoptotic gene delivery: in vivo gene therapy for neurological disorders Hideki Mochizuki,a,* Masauki Miura,b Takashi Shimada,c and Yoshikuni Mizunoa a Department of Neurology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan Laboratory of Cell Recovery Mechanisms, RIKEN Brain Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan b
c
Accepted 15 July 2002
Abstract Apoptosis is an important mechanism of physiological and pathological cell death and is known to occur in various neurological disorders. Apoptosis is associated with activation of genetic programs in which apoptosis-effector genes promote cell death, thereby opposing repressor genes that enhance cell survival. In this review, we describe various apoptotic pathways, with a special reference to the caspase cascade and discuss the role of individual antiapoptotic factors in various target diseases. Apoptosis could be suppressed by in vivo gene delivery of antiapoptotic factors directly into the central nervous system. The adeno-associated virus (AAV) vector is a good candidate for such gene therapy because it can infect postmitotic neurons. We also describe our in vivo system for overexpression of apoptotic protease activating factor-1 (Apaf-1) caspase recruitment domain as an Apaf1-dominant negative inhibitor (Apaf-1-DN) to regulate the mitochondrial caspase cascade. Apaf-1-DN delivery using an AAV vector system inhibited mitochondrial apoptotic signaling pathway and prevented dopaminergic cell death in a mouse model of ParkinsonÕs disease. Our results suggest that AAV–Apaf-1-DN is potentially useful as an antimitochondrial apoptotic gene therapy for neurodegenerative disorders such as ParkinsonÕs disease. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Adeno-associated virus vector; Apoptosis; Gene therapy; Apaf-1; ParkinsonÕs disease
1. Introduction Apoptosis is a programmed cell death resulting from activation of a genetically determined cell suicide cascade. Cells undergoing apoptosis show characteristic morphological features such as condensation of cytoplasmic and nuclear contents, blebbing of plasma membranes, and nuclear fragmentation and ultimately breakdown into membrane-bound apoptotic bodies that are rapidly phagocytosed [1]. Caspases, which are involved in the execution of apoptosis, are present in living cells as inactive zymogens and become activated through intracellular caspase cascades. Several caspase cascades have been described. One pathway is initiated by the activation of cell-surface death receptors, such as Fas and tissue necrosis factor, leading to caspase-8 activa*
Corresponding author. Fax: +81-3-5684-0476. E-mail address: [email protected] (H. Mochizuki).
tion, which in turn cleaves and activates downstream caspases such as caspase-3, -6, and -7. Another pathway is triggered by cytochrome c released from mitochondria, which promotes the activation of caspase-9 through apoptotic protease activating factor-1 (Apaf-1) [2]. The cascade of caspase-1 and -11 is also an independent and important pathway for apoptosis. Caspase-11 is a member of the caspase-1 subfamily and is required for caspase-1-induced apoptosis and interleukin (IL)-1b secretion. Physical interaction between caspase-11 and caspase-1 is a critical step in the activation of caspase-1. The substrate specificity of caspase-11 is similar to that of caspase-9, and caspase-11 can promote the processing of caspase-3, suggesting that caspase-11 works as the apical caspase in the cascade that activates downstream executioner caspases under pathological conditions. In this review, we analyze the potential application of antiapoptotic gene therapy for neurological disorders including stroke, amyotrophic lateral sclerosis (ALS),
1046-2023/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 6 - 2 0 2 3 ( 0 2 ) 0 0 2 2 9 - 3
H. Mochizuki et al. / Methods 28 (2002) 248–252
multiple sclerosis, and ParkinsonÕs disease. We also discuss the strategies and methods that we have developed and applied successfully to study the dominant negative inhibitor of caspase in regulating mitochondrial caspase cascade using the adeno-associated virus vector (AAV) for the treatment of neurodegenerative disorders [3].
2. Natural antiapoptotic proteins The regulation of caspase cascades rescues neuronal cells from apoptotic death in neurodegenerative disorders [4]. Caspases are inhibited by natural antiapoptotic proteins such as p35 protein of Autographica californica nuclear polyhedrosis virus [5] or members of the inhibitors of apoptosis (IAP) family [6]. Transgenic mice that express the antiapoptotic protein p35 in oligodendrocytes (OLG) through the Cre-loxP system are resistant to experimental autoimmune encephalomyelitis (EAE) induced by immunization with myelin OLG glycoprotein, which is used as a disease model of multiple sclerosis [7]. These transgenic mice show a significant reduction of the number of infiltrating T cells and macrophages/microglia in the EAE lesions and apoptotic OLG expressing the activated form of caspase-3. These mice also confer significant resistance to hypoxiainduced neuronal injury [8]. IAPs were originally identified in mutant baculovirus lacking p35 based on their ability to rescue cells from apoptosis. One such member, X-chromosome-linked IAP (XIAP), specifically inhibits caspase-3, -7, and -9. Adenoviral gene transfer of XIAP promoted survival of potassium-deprived cerebellar granule neurons [9]. Eberhardt et al. [10] recently reported that adenoviral gene transfer of XIAP prevented cell death of dopaminergic substantia pars compacta neurons in a mouse model of ParkinsonÕs disease induced by 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP). Virus vector mediating the pan-caspase inhibitor baculovirus p35 and a specific class of caspase-inhibitory proteins such as cytokine response modifier A and XIAP have also been shown to be effective in vivo, especially under pathological conditions [10,11].
3. Antiapoptotic function of Bcl-2 and Bcl-xL The human oncoprotein Bcl-2 was the first identified member of a large family of proapoptotic (Bax, Bad, Bcl-xs) and antiapoptotic (Bcl-2, Bcl-xL) molecules, active in neuronal and nonneuronal cells, which form homodimers and heterodimers with each other. The Bcl2 inhibits neuronal apoptosis induced by a variety of noxious stimuli and preserves the functional integrity of injured cells [12,13]. As a gene therapy for ParkinsonÕs disease, herpes simplex virus vector-mediated expression
249
of Bcl-2 has been applied to prevent 6-hydroxydopamine-induced degeneration of neurons [14]. Azzouz et al. [15] also investigated the potential of recombinant AAV (rAAV) to transfer neuroprotective molecules in an animal model of ALS. They demonstrated that injection of a rAAV encoding the antiapoptotic protein Bcl-2 into the lumbar spinal cord in SOD1G93A mice resulted in sustained Bcl-2 expression in motoneurons and significantly increased the number of surviving motoneurons at the end stage of the disease. These results suggested that the use of rAAV for delivery of Bcl2 to spinal cord motoneurons is potentially useful for enhancing motoneuron survival and repair. In another study, AAV vector-mediated Bcl-2 gene transfer also prevented DNA fragmentation and ischemia-induced cell death of CA1 forebrain neurons [16]. Bcl-xL, one of three isoforms of Bcl-x, protects cells from the damaging effect of reactive oxygen species, e.g., lipid peroxidation, which has been shown to induce apoptotic cell death in vitro. Bl€ omer et al. [17] reported that intracellular delivery of lentiviral vectors expressing Bcl-xL prevented apoptotic death of axotomized cholinergic neurons. Thus, virus vector-mediated Bcl-2 or Bcl-x expression in vivo can prevent neuronal death under certain pathological conditions.
4. Ribozyme and dominant negative inhibitors of caspase There are several approaches to downregulate particular caspases by ribozyme targeting or dominant negative inhibitors of caspases. Ribozymes are enzymatic RNA molecules that suppress the expression of specific genes by selective cleavage of other RNA species. Eldadah et al. [18] reported that a hammerhead ribozyme directed against caspase-3 mRNA protected cerebellar granule cells against apoptosis induced by serum potassium deprivation. In their study, maximal protection by this ribozyme was observed after 24 h of deprivation, at which time apoptosis was 18% compared with 32% in control cells. This study suggests that AAVderived ribozyme-directed cleavage of mutant mRNAs can regulate the apoptotic pathway in vivo. In the mitochondrial pathway, the formation of Apaf-1–caspase-9 complex is accomplished by the heteromeric CARD/CARD (caspase recruitment domain) interaction, an apical step. In addition, homophilic CARD/CARD interactions are also found in an array of other proteins involved in apoptotic regulation, including the majority of the initiator procaspases, adapter proteins, and cellular apoptosis inhibitors. Extensive in vitro binding assays have shown that these homophilic interactions are highly specific. The recombinant wildtype CARD domain inhibits proteolytic cleavage of procaspase-9. Overexpression of Apaf-1 CARD can act as a dominant negative inhibitor. Therefore, for inhibi-
250
H. Mochizuki et al. / Methods 28 (2002) 248–252
tion of the mitochondrial apoptotic cascade in vivo, we generated a rAAV vector that contained the CARD of Apaf-1 to block the Apaf-1/caspase-9 pathway of cell death via dominant negative interference with the formation of a functional Apaf-1–caspase 9 complex [3]. However, the use of Apaf-1 CARD as an antiapoptotic molecule requires caution because it can lead to caspase9 activation at least under certain in vitro experimental conditions [19]. Active site mutants of each caspase had been generated and reported to act in a dominant negative manner [20–23]. AAV vectors incorporating dominant negative inhibitors, antisense DNA or ribozymes, could operate to regulate the caspase cascade.
5. Construction of rAAV vector with double promoter We established an AAV vector that could express both the dominant negative truncated Apaf-1 transgene (Apaf-1-DN) and the enhanced green fluorescent protein (EGFP) as a marker gene simultaneously with two promoters. To construct a plasmid bearing Apaf1-DN, the DNA fragment corresponding to the CARD of mouse Apaf-1 (1–97) was obtained by reverse transcription-PCR. After digesting the amplified fragment, the resulting fragment was ligated into a multiple cloning site of the pCDNA3–FLAG expression vector. Plasmid containing the complete AAV genome (psub201) and the AAV packaging plasmid (pAAV/Ad) were constructed by Samulski et al. [24,25]. A pAAV–CAG–CARD–B19–EGFP was constructed with a cassette containing a chicken b-actin promoter/cytomegalovirus enhancer (CAG promoter), CARD cDNA as an Apaf-1-dominant negative inhibitor, a B19 promoter, EGFP, and b-globin poly(A) inserted between the AAV inverted terminal repeats of psub201. We confirmed the efficient expressions of both transgenes using this virus vector both in vitro and in vivo.
6. In vivo detection of antiapoptotic effect Long-term administration of MPTP causes apoptotic cell death in the substantia nigra of C57/BL mice [26]. To identify the antiapoptotic effect of gene therapy in vivo, we injected the AAV vector encoding the caspase dominant negative inhibitor stereotactically into the striatum of 8-week-old mice, which were then treated with MPTP 2 weeks later to model ParkinsonÕs disease. Three weeks after the first MPTP treatment, dopaminergic neurons profiles were assessed by immunohistochemistry for tyrosine hydroxylase (TH). The methods used in these experiments were as follows. (1) Stereotactic surgical procedures were per-
formed aseptically under anesthesia with pentobarbital (10 mg/kg) and ketamine hydrochloride (6 mg/kg). (2) After placing each mouse in a stereotactic frame, 4 ll of the vector suspended in phosphate-buffered saline (PBS) was injected into the striatum (AP +1.0, medial lateral 2.0, DV )3.0) over a period of 5 min by using a 25-ll Hamilton syringe. (3) Two weeks after vector injection, to allow for expression of these molecules, the mice received four intraperitoneal injections of MPTP–HCl (30 mg/kg; Research Biochemicals, Natick, MA) in saline at 24-h intervals (chronic model). (4) Each mouse was perfused via the aorta with PBS followed by ice-cold 4% paraformaldehyde while under deep pentobarbital anesthesia. (5) Then, 30-lm nigral sections were cut and immunostained with a monoclonal antibody (mAb) for TH (Chemicon International, Temecula, CA) or with a mAb for GFP (Sigma, St. Louis, MO). In addition, sections were stained for anti-FLAG or anti-activated caspase-3 (PharMingen, San Diego, CA) using polyclonal antibodies. (6) The corresponding secondary antibodies [donkey antimouse fluorescein isothiocyanate (FITC) or Texas red and donkey anti-rabbit FITC or Texas red (Jackson Immunoresearch Laboratories, West Grove, PA); dilution 1:250] were pooled, and sections were incubated with these antibodies for 2 h at room temperature, followed by washing in PBS. (7) The sections were then examined by confocal scanning laser microscopy, with the collected signals undergoing digital color enhancement before superimposition. (8) For cell counting, the substantia nigra was cut into serial sections, and every third section was subjected to immunostaining for TH using a polyclonal antibody (a kind gift from I. Nagatsu, Fujita Health University, Aichi, Japan). (9) The number of viable TH-positive neurons was determined by manual counting in a blinded fashion using coded slides. The results showed that delivery of AAV–Apaf-1-DN prevented MPTP-induced dopaminergic neuronal cell death in the left substantia nigra (injected side) compared with the right side (untreated side) (Fig. 1A). The number of TH-positive dopaminergic neurons in the substantia nigra of the AAV–Apaf-1-DN–EGFP-injected mouse was significantly higher than that on the untreated side (Fig. 1B).
7. Summary Several strategies have been applied for gene delivery into neurons using virus vectors including lentivirus vector [27,28], adenovirus vector [9,10], and AAV. AAV is nonpathogenic and infects neurons. Immune responses to vector-corrected cells have historically limited the application of gene therapy for neurological disorders such as stroke, ALS, multiple sclerosis, and
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Fig. 1. (A) Photomicrographs of TH immunostaining of the substantia nigra of a MPTP-treated AAV–Apaf-1-DN-injected (arrow) mouse. Note the extensive neuronal loss on the noninjected side. Note also the significantly greater number of neurons on the injected side compared with that on the noninjected side. (B) Comparison of proportion of TH-positive cells on the noninjected and injected sides. The total number of TH-positive neurons was counted in three sections each from four different mice. Statistical analysis was performed using ANOVA followed by ScheffeÕs post hoc test.
ParkinsonÕs disease. AAV efficiently transfers genes into the brain without activation of cellular and humoral immunity, which differs from the experience with recombinant adenovirus. A considerable number of agents capable of arresting apoptosis are available at present and have been extensively examined experimentally. It is hoped that these agents could be used therapeutically in neurological disorders, including those affecting the central nervous system involving apoptosis of neuronal cells. Mitochondrial dysfunction occurs not only in ParkinsonÕs disease but is also present in the brains of patients with HuntingtonÕs disease and in mouse models of ALS. Therefore, our AAV system with a dominant negative inhibitor could possibly regulate the mitochondrial apoptotic neuronal cell death in several neurological disorders. However, attention has to be paid to possible adverse side effects, especially oncogenic changes, in antiapoptotic gene therapy.
Acknowledgments This study was supported in part by a High Technology Research Center Grant and a Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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