Overexpression of netrin-1 improves neurological outcomes in mice following transient middle cerebral artery occlusion

Abstract

Netrin-1 (NT-1) is one of the axon-guiding molecules that are critical for neuronal development. Because of its structural homology to the endothelial mitogens, NT-1 may have similar effects on vascular network formation. NT-1 was shown to be able to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and also promote focal neovascularization in adult brain in vivo. In the present study, we reported the delivery of NT-1 using an adeno-associated virus (AAV) vector (AAV-NT-1) into mouse brain followed by transient middle cerebral artery occlusion (tMCAO). We found that AAV vectors did not elicit a detectable inflammatory response, cell loss or neuronal damage after brain transduction. The level of NT-1 was increased in the AAV-NT-1-transduced tMCAO mice compared with the control mice. Furthermore, the neurobehavioral outcomes were significantly improved in AAV-NT-1-transduced mice compared with the control animals (P<0.05) 7 days after tMCAO. Our data suggests that NT-1 plays a neuronal function recovery role in ischemic brain and that NT-1 gene transfer might present a valuable approach to treat brain ischemic disorders.

Keywords

adeno-associated virus / angiogenesis / gene transfer / ischemia / middle cerebral artery occlusion / netrin-1

Introduction

Netrin-1(NT-1) is one of the major factors that guide growing axons. It was first isolated from chick brain [1,2]. NT-1 belongs to a family of homolog that includes netrin-1, -2, -G1, -G2, and-4/b. NT-1 receptors include deleted in colorectal cancer (DCC) and UNC family members [3-5]. By binding with these receptors, NT-1 functions as a chemotropic or repulsive factor that mediates axon outgrowth, axon orientation and neuronal migration during development [6-8]. NT-1 is also crucial to the survival of UNC5H- or DCC-expressing neurons [9,10]. NT-1 and its homologs have been extensively studied in the neuronal development. However, the effects of netrins on vascular development are not clear. Netrins are structurally homologous to the endothelial mitogens and may share their effects on vascular network formation. But the roles netrins play in the vascular system are still under debate [11,12]. It was recently shown that NT-1 was able to stimulate the proliferation and migration of human cerebral endothelial cells in vitro and promote focal neovascularization in adult brain in vivo [13].

Recombinant AAV (rAAV) contains no viral genomes and elicits no or less inflammatory response compared to other viral vectors [14]. rAAV can infect both dividing and non-dividing cells and mediate long-term gene expression up to months or several years in vivo [15,16]. rAAV has a range of applications in experimental gene therapy including tumor, genetic disorders, neurodegenerative disorders, cardinal and cerebral vascular diseases [17]. Our previous study demonstrated that rAAV transferred vascular endothelial growth factor (VEGF) had neuroprotective effect on ischemic injury both in vitro and in vivo.

AAV mediated gene transfer has been studied in many organs, and showed that the overexpression of target gene can induce specific functions. For example, AAV-VEGF gene transfer reduces ischemia induced brain injury. However, it appears difficult for a long time stable induction of neuroprotection because of the short half-life of VEGF protein and the limitation of delivery approach. It is necessary to establish a reproducible and stable intermediate target gene expression. Based on the application of AAV mediated gene transfer technique and the function of NT-1, we constructed a novel AAV-NT-1 vector. We investigated the overexpression induced by AAV mediated NT-1 gene transfer in the mouse brain. We also examined the neurobehavioral outcomes of NT-1 transduced mice following transient middle cerebral artery occlusion. Our result showed that the neurobehavioral outcomes were significantly improved in AAV-NT-1-transduced mice compared with control animals (P<0.05) 7 days after transient middle cerebral artery occlusion (tMCAO).

Methods and materials

AAV-NT-1 production, purification, and titration

We constructed AAV-NT-1 vector which contained NT-1 gene and the inverted terminal repeat (ITR) sequences. The pAAV-NT-1 vector was generated by inserting the chicken NT-1 cDNA between two ITRs of pAAV-MCS plasmid (Invitrogen, Carlsbad, CA). Cytomegalovirus (CMV) promoter was used to control the gene expression in this vector. pAAV-NT-1 was co-transfected with pHelper and pAAV-RC plasmids into AAV293 cells with calcium phosphate precipitation method. Cells were harvested 48 h after transfection. After three freeze–thaw cycles, cell debris was removed by centrifugation and the adeno-associated virus in the supernatant was pelleted with calcium chloride and was further purified by CsCl density gradient ultra-centrifugation. Viral titers were determined by RT-PCR analysis of the gene content. Adeno-associated virus-IRES-hrGFP (AAV-GFP) was simultaneously prepared as a control.

Animal experiments

All animal procedures were carried out according to a protocol approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University, China. The first experiment was designed to examine the transduction efficiency. Three groups of adult male CD-1 mice weighing 30-35 g were injected with AAV-NT-1, AAV-GFP or saline, respectively (n = 6 per group). Animals were sacrificed 7 days after AAV virus or saline injection, and the brains were used for immunostaining and Western blot examination. The second experiment was designed to study the effect of AAV-NT-1 in promoting angiogenesis after cerebral ischemia. Three groups of CD-1 mice were injected with AAV-NT-1, AAV-GFP or saline (n = 6 per group). Seven days after AAV vector injection, these mice underwent 60 min of tMCAO followed by 7 days of reperfusion. The animals were sacrificed at 7 days after tMCAO following the measurement of regional cerebral blood flow (CBF).

Recombinant AAV-NT-1 transfer in the mouse brain

Mice were anesthetized with 100mg/kg bodyweight Ketamine and 10mg/kg xylazine (Sigma, San Louis, MO) intraperitoneally. Following the induction of anesthesia, the mice were placed in a stereotactic frame with a mouse adaptor (David Kopf Instruments, Tujunga, CA). A burr hole was drilled to the pericranium 2 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture. A 10 µL syringe (WPI Inc., Sarasota, FL) was inserted into the basal ganglia 3 mm under the meninges. AAV suspension (2.5 µL) with 2 ×10⁹ virus particles was injected stereotactically at a rate of 0.2 μL/min. The needle was then withdrawn over a course of 15 min. The hole was sealed with bone wax, and the wounds were closed with suture. After waking up from anesthesia, the animals were put back to their cages for recovery.

tMCAO

Animals were anesthetized with 1.5% isoflurane in 70/30 nitrogen/oxygen gas. Body temperature was maintained at 37.0±0.5°C. The tMCAO method was as described previously [18]. One week after the injection of virus or saline, the animals were anesthetized for surgery. The intra carotid artery (ICA) was isolated and the pterygopalatine artery was ligated. The left middle cerebral artery (MCA) of mice was occluded by a 6-0 suture. The suture was coated with silicon and doused in heparin 1 h before use. Reperfusion was performed by partially withdrawing the suture from the ICA to the common carotid artery (CCA). The occlusion lasted for 60 min and reperfusion was maintained up to the 7th day.

CBF measurement using a laser Doppler flowmetry (LDF)

The regional CBF was monitored by a laser Doppler flowmeter (Vasamedics, St. Paul, MN) with a small caliber probe of 0.7 mm diameter. The laser Doppler probe, held in a micromanipulator, was lowered until it was in contact with the surface of skull. The point at 3.5 mm lateral to the sagittal suture and 1 mm posterior to the coronal suture was monitored as the ischemic core. Continuous digital displays of LDF values were averaged over 5 s intervals and recorded during the surgical procedure. The CBF was re-measured before euthanizing the animals. CBF values were calculated and expressed as percentage of the baseline value [18,19].

Western blot analysis

The brain tissues were homogenized in a lysis buffer. The protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kits (Pierce, Rockford, IL). An equal amount of the total proteins (30 μg) was loaded on 5%-10% gradient gel for electrophoresis. Subsequently, proteins were trans-blotted onto a nitrocellulose filter membrane (Bio-Rad, Hercules, CA) in a transfer buffer. The membrane was placed in 5% non-fat milk in 0.1% (tris-buffered saline with Tween-20) TBST for 1 h to block non-specific binding, and immuno-probed with specific primary antibodies at 4°C overnight. After washing, the blots were incubated with (Peroxidase, Horseradish) HRP-conjugated secondary antibodies and then reacted with an enhanced electrogenerated chemiluminescence (ECL) substrate (Pierce, Rockford, IL). The resulted chemiluminescence was recorded with an imaging system (ChemiDoc, Bio-Rad, Hercules, CA).

Behavioral tests

Animals underwent two behavioral tests before tMCAO, and 7 days following tMCAO. Mice were trained for 3 days before AAV injection with 3 consecutive trials to generate stable baseline values.

Beam walk test

Mice were trained to traverse a series of elevated beam, which was 7 mm in diameter to reach an escape platform placed 1 meter away. Mice were placed on one end of the beam and the latency to traverse the central 80% of the beam toward the escape platform was recorded. Motor test data were analyzed as mean latency to cross the beam (3 trials).

Rotor-rod test

The task required the mice to balance on a rotating rod. The animals were allowed a 1 min adaptation period on the rod at rest, after which the rod was continuously accelerated at 40 rpm for 4 min and time spending on the rod (fall latency) was recorded. Motor test data were analyzed as mean duration (3 trials) on the rotor-rod.

Immunohistochemistry

Brain sections were incubated with 10% serum for 1 h to block non-specific binding, washed with phosphate buffered saline (PBS), and then incubated with anti-NT-1 primary antibody (1∶100 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) in PBS at 4°C overnight. Biotin-conjugated secondary antibody, avidin-biotin enzyme reagent (Vector Laboratories, Inc., Burlingame, CA), and diaminobenzidine (DAB) were used to visualize the signal. After counterstaining with hematoxylin, the sections were dehydrated for further microscopic study.

Vascular morphology assessment

The microvessel density was assessed by lectin staining, which was a simple and reproducible way to observe morphological evidence that angiogenesis occurred after a biological stimulus [20]. Lectin staining was performed on frozen sections [21]. Briefly, frozen sections were fixed with 100% ethanol (ETOH) at 20°C, and then incubated with fluorescein-lycopersicin esculentum lectin, 2 g/mL, at 4°C overnight. Our previous studies demonstrated that this method was feasible and reproducible [22,23].

Statistical analysis

Parametric data in different groups were compared using a one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls protected least significant difference test, as appropriate. All data were presented as mean±SD. A probability value of less than 5% was considered to represent statistical significance.

Results

NT-1 expression following tMCAO

To examine if tMCAO can induce NT-1 expression in the mouse brain, we performed NT-1 immunostaining in mice underwent 1 h tMCAO followed by 1 or 7 days of reperfusion. We found that NT-1 positive cells were increased in the ischemic hemisphere following ischemia compared with the control animals (Fig. 1). The NT-1 positive cells were mostly located in the ischemic boundary zone, suggesting that ischemia could stimulate focal NT-1 expression and that NT-1 may play a role in response to focal ischemia in the brain.

AAV vector mediated netrin-1 expression in vitro and in vivo

In general, no visible neurological deficits, bodyweight loss or hyperthermia were observed following virus injection in AAV-NT-1 treated mice. To determine whether AAV-NT-1 was capable of mediating NT-1 expression, we examined NT-1 expression in both HEK293 cells infected with AAV-NT-1 in vitro and mouse brains in vivo. After the infection of HEK293 cells with AAV-NT-1, NT-1 was detected using RT-PCR and Western blot (data not shown). In in vivo experiment, a significant amount of NT-1 positive cells were detected around the needle track 3 and 7 days after the injection of AAV-NT-1. No NT-1 positive staining cells were detected in the AAV-GFP-transduced brain (Fig. 2). Western blot result further demonstrated that NT-1 expression was greatly increased in the ipsilateral hemisphere of AAV-NT-1 transduced mouse brain compared with the same location of brain in the saline control and AAV-GFP-transduced mouse brain (Fig. 3).

NT-1 overexpression improved focal CBF after tMCAO

To examine whether CBF was better recovered following AAV-NT-1 gene transfer compared with the control animals, we measured CBF using a laser Doppler flowmeter. No difference was observed in regional CBF in the ischemic region before, during, and after 60 min of tMCAO among the AAV-NT-1or AAV-GFP transduced, and saline-treated mice (Fig. 4). Surface CBF in the ischemic region was decreased in all the three groups of animals during 60 min of tMCAO. The surface CBF in the ischemic region was better recovered in the AAV-NT-1-transduced mice compared with the AAV-GFP and saline-treated mice 1 week after tMCAO (P<0.05).

NT-1 promotes focal angiogenesis following tMCAO in AAV-NT-1-transduced mice

To further determine whether the neuroprotection of NT-1 was via stimulating focal angiogenesis following tMCAO, we examined the number of microvessels in the AAV-NT-1, AAV-GFP, and saline injected mouse brain. The lectin straining result showed that the number of microvessels in the ischemic region was increased in the AAV-NT-1-transduced mice compared to the AAV-GFP-transduced, and saline injected mice (Fig. 5), suggesting the neuroprotection effect of netrin-1 may be via the promotion of focal angiogenesis.

NT-1 overexpression improved neurobehavioral outcome after tMCAO

We further examined the neurobehavioral functions 7 days after tMCAO. Beam walk and rotor-rod test showed that there was no difference in performance before AAV-NT-1 injection and before tMCAO among the three groups. tMCAO mice with AAV-NT-1 treatment showed significant improvements in performance during both beam walk and rotor-rod test after 7 days of tMCAO, compared with the control animals (P<0.05, Fig. 6).

Discussion

This study provides evidence that NT-1 expression is induced in the mouse brain by tMCAO. The expression of NT-1 can be further enhanced via AAV-NT-1 gene transfer. NT-1 overexpression improves focal CBF recovery after tMCAO in the AAV-NT-1-transduced mice, which may be related to the promotion of focal angiogenesis. Furthermore, neurobehavioral outcomes are improved in the AAV-NT-1-tranduced mice compared with the control. These results indicate that NT-1 not only plays a key role in neuronal development as reported before, but also participates in neovascularization and vessel remodeling in adult brains.

Since the recombinant AAV vector is a reliable tool for the gene transfer in vivo, we choose AAV1 vector for the NT-1 gene delivery. The advantage of using recombinant AAV vector includes, but not limits to, eliciting little or no inflammatory responses, infecting both dividing and non-dividing cells, and mediating long-term gene expression for months to years in vivo [24,25]. After infecting HEK293 cells with AAV-NT-1, NT-1 expression increased significantly at both mRNA and protein levels [13].

Endogenous NT-1, predominantly expressed by the floor plate cells in the central nervous system (CNS), induces axonal outgrowth, axonal orientation, and neuronal migration during neuronal development [26-28]. Recent studies have found NT-1 can stimulate the proliferation and migration of human aortic endothelial cells (HAEC) and human microvascular endothelial cells (HMVEC), and induce angiogenesis on chorioallantoic membrane and murine cornea in vivo [12]. Our group confirmed that AAV-mediated gene transfer induced the overexpression of NT-1 in HEK293 cells in vitro. We further demonstrated that the AAV-mediated overexpression of NT-1 induced angiogenesis in the normal brain [13] and ischemic penumbra region in the mouse brain in vivo. These newly developed vessels are distributed within the AAV-NT-1 transduced hemisphere, including cortex, subventricular zone, corpus callosom, and caudate putaman although the cell types expressing NT-1 need to be further explored. These findings suggest that NT-1, a neuronal chemotrophic factor, can also participate in the neovascularization and vessel remodeling process after ischemic brain injury.

We monitor regional CBF using LDF measurement, which is noninvasive, simple and fast. Two-dimensional spatiotemporal dynamics of CBF changes can be monitored during ischemia and reperfusion in mice through an intact skull. By setting the baseline value as reference group, CBF changes in percentage can be reliably compared at different time points. We found that AAV-NT-1 improved CBF recovery in the ischemic region 1 week after tMCAO. This result suggests that NT-1 may increase collateral blood supply or focal angiogenesis, although further study is needed.

Finally, we demonstrated that the neurobehavioral outcomes were improved in AAV-NT-1 transduced mice compared to the control. We chose beam walk and rotor-rod tests because both tests could detect motor function injury in the CNS. We performed beam walk and rotor-rod tests before AAV-NT-1 gene transfer and before MCAO for baseline establishment. We found no differences in baseline level among groups, suggesting that AAV brain injection did not affect neurobehavioral function. The improvement in performance in beam walk and rotor-rod in NT-1 overexpressed group indicated that NT-1 could promote neurobehavioral function recovery.

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