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Mol Ther. Aug 2010; 18(8): 1536–1544.
Published online Jun 8, 2010. doi:  10.1038/mt.2010.107
PMCID: PMC2927058

Macrophage-mediated GDNF Delivery Protects Against Dopaminergic Neurodegeneration: A Therapeutic Strategy for Parkinson's Disease

Abstract

Glial cell line–derived neurotrophic factor (GDNF) has emerged as the most potent neuroprotective agent tested in experimental models for the treatment of Parkinson's disease (PD). However, its use is hindered by difficulties in delivery to the brain due to the presence of the blood–brain barrier (BBB). In order to circumvent this problem, we took advantage of the fact that bone marrow stem cell–derived macrophages are able to pass the BBB and home to sites of neuronal degeneration. Here, we report the development of a method for brain delivery of GDNF by genetically modified macrophages. Bone marrow stem cells were transduced ex vivo with lentivirus expressing a GDNF gene driven by a synthetic macrophage-specific promoter and then transplanted into recipient mice. Eight weeks after transplantation, the mice were injected with the neurotoxin, MPTP, for 7 days to induce dopaminergic neurodegeneration. Macrophage-mediated GDNF treatment dramatically ameliorated MPTP-induced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH+ terminals in the striatum, stimulated axon regeneration, and reversed hypoactivity in the open field test. These results indicate that macrophage-mediated GDNF delivery is a promising strategy for developing a neuroprotective therapy for PD.

Introduction

Parkinson's disease (PD) is an increasingly common neurodegenerative disease resulting from the death of dopaminergic neurons in the substantia nigra.1 Current PD treatments are primarily based on pharmacological replacement of lost striatal dopamine, providing only symptomatic relief without retarding the ongoing loss of dopaminergic neurons. Moreover, the effectiveness of dopamine replacement therapy diminishes with time, and most patients develop progressive dyskinesia and other adverse side effects.2,3 As the symptoms of PD become evident only after the loss of at least half of the dopaminergic neurons in the substantia nigra over an extended period of time, it has been suggested that a strategy that protects neurons from cell death and promotes regeneration would be of great benefit for treating the disease.

Although glial cell line–derived neurotrophic factor (GDNF) is the most potent survival factor for the nigrostriatal dopaminergic neurons that degenerate in PD,4,5,6,7,8 attempts to develop GDNF-based therapies have been seriously compromised by the difficulty of delivering it to the brain. Moreover, the progressive nature of PD requires continuous and sustained delivery of GDNF over months or years in order to maintain dopamine neuron survival and function. Although conjugation of GDNF with other molecules, enabling blood–brain barrier (BBB) penetration, may at least partially overcome these difficulties, the feasibility of this approach for long-term sustained delivery of GDNF remains uncertain. Viral-mediated GDNF gene transfer may ensure long-term expression, but it requires vector delivery by invasive surgery, along with potentially adverse effects associated with high-level expression of GDNF. Therefore, the development of a cell-based gene therapy approach that can be applied systemically and can achieve sustained release of moderate amounts of GDNF would represent a major therapeutic advance in the PD field. In this context, the use of macrophages to deliver GDNF is particularly attractive, given the fact that these cells are capable of crossing the BBB, following which they differentiate into microglia. Microglial cells originate in the bone marrow from hematopoietic stem cells (HSCs), sharing a close relationship with the monocyte/macrophage lineage.9,10 Previous studies have demonstrated that bone marrow–derived microglial engraftment was enhanced by neuropathology and that cells were recruited preferentially to the site of brain insult.11,12,13,14,15,16,17,18,19 Subsequently, bone marrow–derived microglia were used successfully for gene therapy strategies in animal models of metachromatic leukodystrophy20 and multiple sclerosis.21 Although the feasibility of using bone marrow–derived cells as vehicles for delivery of neurotropic factors for the treatment of PD has been established,22 its translation to a clinical setting presents potential problems due to ubiquitous expression of the transgene in marrow cells.

The goal of this study was to enhance both the therapeutic efficacy and safety of GDNF gene therapy for PD by restricting the expression of the transgene to microglial precursor cells (monocyte/macrophage). We developed a highly active macrophage-specific synthetic promoter (MSP) that restricts transgene expression to this lineage. The GDNF gene driven by this promoter was transduced ex vivo into mouse bone marrow stem cells using lentiviral vectors. The transduced cells were then transplanted into mice in the context of a neurotoxin-based model of PD.

Bone marrow–derived microglia expressing the transgene engrafted in large numbers at the sites of MPTP-induced dopaminergic neurodegeneration. This treatment approach dramatically reduced degeneration of tyrosine hydroxylase (TH)-positive neurons of the substantia nigra and TH+ terminals in the striatum of MPTP-treated mice. No major side effects, such as weight loss and allodynia, were observed in the recipient mice. Our results thus offer a proof-of-principle for the therapeutic use of bone marrow stem cell–derived macrophages for sustained delivery of GDNF to selective brain lesion sites.

Results

MSP restricts transgene expression in macrophages following bone marrow transplantation

We developed a highly active MSP that restricts transgene expression to this lineage (Figure 1a and Supplementary Figure S1). Bone marrow stem cells from donor mice were genetically modified using lentiviral vectors encoding either GDNF or GFP cDNA driven by the MSP. Male recipient mice 7–8 weeks of age were lethally irradiated and then transplanted with bone marrow–derived stem cells transduced with either GDNF (MSP-GDNF mice) or GFP (MSP-GFP mice) vector. After 3 weeks, peripheral blood from the recipient mice was analyzed for the tissue specificity of the MSP and its efficiency for driving synthesis and secretion of GDNF. In the MSP-GFP mice, ~66% of the CD11b (monocyte/macrophage marker) -positive leukocytes expressed GFP (Figure 1b), whereas only 5–7% of CD11b leukocytes expressed low levels (Figure 1b,c) of GFP, suggesting that MSP was driving the expression of the transgene selectively in monocytes/macrophages. In the MSP-GDNF mice, a significant quantity (1.723 ± 0.622 ng/ml) of GDNF protein was detected in the plasma (Figure 1d), indicating that the genetically modified cells are capable of synthesizing and secreting GDNF. GDNF protein was detected in the plasma over the entire experimental period of 6 months, whereas no GDNF was detectable in the plasma of MSP-GFP mice. In addition, GDNF levels in the substantia nigra and striatum were measured to make certain that gene silencing did not occur following the migration of macrophages into the brain and their subsequent differentiation into microglia. In MSP-GDNF mice 4 months after transplantation, the mean substantia nigra GDNF protein level was 36.42 ± 6.10 pg/mg of tissue, whereas the level of endogenous nigral GDNF in MSP-GFP mice was 8.38 ± 1.34 pg/mg of tissue (Figure 1e). A significant increase in the striatal GDNF level was observed for MSP-GDNF mice (21.56 ± 1.19 pg/mg tissue) compared with that of MSP-GFP mice (13.53 ± 0.63 pg/mg tissue; Supplementary Figure S2)

Figure 1
Lentiviral vector with MSP restricts transgene expression to macrophages. (a) Schematics of lentiviral vector expressing rat GDNF or GFP driven by MSP with a CD68 mini-promoter. (b) Peripheral blood flow cytometry analysis of MSP-GFP mice showing GFP ...

Monocytes/macrophages differentiate into microglia and their recruitment to substantia nigra is enhanced during neurodegeneration

Eight weeks after transplantation, recipient mice were injected with MPTP for 7 days to induce dopaminergic neurodegeneration. Control mice were similarly injected with saline. Nine weeks after the last injection of MPTP or saline, MSP-GFP mice were killed to evaluate the differentiation of gene-modified macrophages into microglia and their recruitment to substantia nigra. In the brain, gene-modified macrophages strongly expressed GFP, displayed the ramified morphology characteristic of microglia, and expressed Iba1, a marker for microglia (Figure 2a). In the saline-treated MSP-GFP mice, a few GFP-expressing cells were observed in the substantia nigra, whereas there were threefold more GFP-expressing cells in the substantia nigra of MPTP-treated mice (Figure 2b,c).

Figure 2
Gene-modified macrophages are recruited in large numbers to substantia nigra following MPTP-induced neurodegeneration. (a) Midbrain sections of MSP-GFP mice treated with MPTP showing GFP+ cells expressing microglial marker Iba1 in the nigra. ( ...

Macrophage-mediated GDNF delivery protects nigral dopaminergic neurons and its terminals in the striatum

Three or nine weeks after the last MPTP or saline injection, recipient mice were killed, and the neuroprotective effect of macrophage-mediated GDNF delivery on the nigrostriatal dopaminergic system was assessed by quantitative analysis of TH+ neurons in the substantia nigra pars compacta (SNpc), as well as the density of TH+ terminals in the striatum. The organization and intensity of TH-immunoreactive neurons were essentially similar in the saline-treated MSP-GFP and MSP-GDNF animal groups (Figure 3a). Stereological analysis demonstrated up to a 50–55% loss of TH+ neurons in the SNpc of MSP-GFP mice following MPTP treatment, compared with saline-treated animals (Figure 3b,c). The TH+ dendritic fiber networks in the substantia nigra pars reticulata (SNpr) were also dramatically reduced after MPTP treatment in MSP-GFP mice (Figure 3a). In contrast, there was only a 15–20% MPTP-induced loss of TH+ neurons in the SNpc of MSP-GDNF mice (Figure 3). Moreover, the density of the SNpr TH+ dendritic fiber network in the MSP-GDNF mice was largely preserved in the face of MPTP treatment, relative to saline treatment.

Figure 3
Macrophage-mediated GDNF delivery protects nigral dopaminergic neurons from MPTP-induced degeneration. (a) Midbrain sections of MSP-GFP and MSP-GDNF mice showing TH-immunostained cell bodies and their processes in the substantia nigra 3 weeks after MPTP ...

Parallel results were observed for striatal dopamine fiber terminals. In order to quantify the intensity of TH staining, optical density measurements were performed on the dorsolateral aspects of the striatum, which receive the largest share of innervation from dopamine neurons of the SNpc. By this method, TH immunoreactivity within the striatum was similar between saline groups of MSP-GFP and MSP-GDNF mice (Figure 4a). Relative to the controls, there was an average 70% loss of the TH staining intensity in MSP-GFP mice killed 3 weeks after MPTP treatment, whereas the loss in MSP-GDNF mice was only 35% (Figure 4b). Interestingly, TH staining intensity in MSP-GDNF mice improved over time. In MSP-GDNF mice killed later at 9 weeks after the last dose of MPTP, the reduction in the intensity of TH staining was only 15% (Figure 4c), suggesting an ongoing regenerative process within the nigrostriatal pathway. Indeed, microscopic examination of the striatum of MPTP-treated MSP-GDNF mice revealed numerous long and thick TH+ fibers (Figure 4d) that were often branched with irregular swellings, suggesting sprouting or regenerating axons of the nigral dopamine neurons. Substantially, fewer fibers of this type were observed in the striatum of MPTP-treated MSP-GFP mice.

Figure 4
Macrophage-mediated GDNF delivery protects dopaminergic terminals in the striatum from MPTP-induced degeneration. (a) Coronal sections of forebrain showing immunostaining of TH+ terminals in the striatum of MSP-GFP and MSP-GDNF mice 3 weeks after ...

Striatal levels of dopamine and its metabolites

For further confirmation of our findings, tissue levels of dopamine and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were determined biochemically. Compared with MPTP-treated MSP-GFP mice, the striatum of MPTP-treated MSP-GDNF mice exhibited a significantly higher level of dopamine (38.8% increase, Figure 5a), DOPAC (27.7% increase, Figure 5b), and HVA (40.3% increase, Figure 5c). Striatal levels of serotonin (5-HT), another monoamine neurotransmitter, and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), were also measured to assess whether the relative preservation in levels of dopamine and its metabolites in MPTP-treated MSP-GDNF versus MSP-GFP mice was selective or possibly a generalized effect on monoamine neurotransmitters. These analyses demonstrated similar levels of 5-HT (Figure 5d) and 5-HIAA (Figure 5e) in MPTP-treated group MSP-GFP versus MSP-GDNF mice.

Figure 5
Macrophage-mediated GDNF delivery enhanced striatal levels of dopamine and its metabolites. HPLC analysis revealed a significantly higher level of (a) dopamine (n = 5, **P < 0.0275), and its metabolites (b) DOPAC (n = 5, *** ...

Behavioral tests

General activity levels assessed by the open field test demonstrated that, relative to control mice, MPTP treatment significantly reduced the activity levels of MSP-GFP mice. In contrast, the activity of MSP-GDNF mice was preserved at levels similar to those of control mice (Figure 6a). Moreover, MSP-GFP mice exhibited reduced food intake normalized for body weight, and this effect was reversed in MSP-GDNF mice (Figure 6b).

Figure 6
Macrophage-mediated GDNF delivery improved functional recovery without major side effects. (a) Total activity assessed by open field test. Macrophage-mediated GDNF delivery reversed MPTP-induced hypoactivity (*P < 0.05). The number of ...

Assessment of side effects of macrophage-mediated GDNF therapy

Direct brain infusion of GDNF is associated with significant adverse effects, including allodynia and weight loss.23 In our study, none of the animal showed signs of allodynia, as determined by paw withdrawal frequency (Figure 6c) or duration (Figure 6d) in response to the application of acetone on the mid-plantar surface of the hindpaw. Body weight was recorded every 2 days throughout the duration of the experiment and expressed as mean change from initial body weight. Although both the MSP-GFP and MSP-GDNF groups lost weight acutely after whole body irradiation and transplantation, both groups regained weight quickly and then continued to gain additional weight (Figure 6e). Over time, MSP-GDNF mice gained significantly less weight than did MSP-GFP mice, a trend that continued even after MPTP administration (Supplementary Figure S3). Although the exact mechanism underlying this effect is not known, it may be attributable to effects of GDNF on the hypothalamus. Similar patterns of reduced weight gain were observed in young rats with hypothalamic GDNF overexpression,24 as well as in adult rats with nigral GDNF overexpression using a recombinant adeno-associated viral vector tet-regulated expression system.25

GDNF exerts biological effects outside of the central nervous system, acting as a kidney morphogen during embryonic development and regulating the differentiation of spermatogonia in the testis.26 Accordingly, testes from MSP-GFP and MSP-GDNF mice were analyzed for variations possibly attributable to the differences in levels of circulating GDNF. No structural or morphological changes were observed in hematoxylin- and eosin-stained sections of testes at the light microscopic level (Supplementary Figure S4)

Discussion

We demonstrate here that macrophages offer a powerful tool for delivery and expression of therapeutic transgenes at the sites of neurodegeneration, suggesting that macrophage-mediated GDNF delivery may have promise as a means for developing neuroprotective strategies for PD. Current therapies provide symptomatic relief for the cardinal motor features of PD, but over time, the patients' quality of life invariably deteriorates due to progressive gait and equilibrium difficulties, autonomic dysfunction, and cognitive impairment. These nondopaminergic clinical manifestations are probably caused by the loss of neurons in many cortical, subcortical, brainstem, and peripheral autonomic sites as a result of a neurodegenerative process similar to that which affects the nigrostriatal system.27 Therefore, therapies that are directed solely at restoration of striatal dopamine are insufficient for arresting disease progression. Because bone marrow–derived microglia are recruited specifically to the sites of neurodegeneration wherever they occur in the central nervous system, their use as a delivery vehicle for neurotrophic factors, for example, GDNF as in this study, offers potential avenues for protection of dopaminergic, as well as nondopaminergic systems that ultimately undergo degeneration in PD. Moreover, using bone marrow–derived microglia, neuroprotection can be achieved with relatively low and apparently safe levels of tissue exposure to GDNF, thereby reducing dose-related side effects. Notably, brain tissue levels of GDNF in MSP-GDNF mice in the present study were about 36 pg/mg of tissue, whereas viral-mediated gene transfer resulted in up to 4,200 pg/mg of tissue.28

Microglial activation is a crucial step in the pathogenesis of PD. Evidence suggests that microglia can function as components of both survival-promoting and cell death–promoting pathways.10,29 Survival-promoting functions of microglia are associated with the expression of neurotrophic factors, such as GDNF and BDNF.10 Because the majority of microglia in the MSP-GDNF mice are derived from gene-modified macrophages expressing GDNF, they may act directly to protect SN neurons. Significant reduction in MPTP-induced neurodegeneration supports this view; though the exact mechanisms for the neuroprotective action of microglia cannot be established from the current experiments. In a recent clinical gene therapy trial, HSCs transduced with a lentiviral vector encoding an adenosine triphosphate–binding cassette transporter gene was used to treat X-linked adrenoleukodystrophy, a severe neurodegenerative disease.30 Some of the gene-corrected HSCs replaced diseased microglia in the brain and prevented progressive cerebral demyelination, underscoring the potential of microglia to serve as a cellular vehicle for central nervous system gene therapy.

In our proof-of-principle experiments, we used whole-body irradiation to provide niches for transplanted bone marrow stem cells. Of note, total body irradiation at 1,200 cGy was used successfully in a conditioning regimen for HSC transplantation in patients with multiple sclerosis.31 However, irradiation-based myeloablative regimens may cause problems in humans, as they are known to affect the integrity of BBB.32 In this scenario, myeloablative conditioning regimens with cyclophosphamide and busulfan30 or antibody-based clearance of HSC niches33,34 may provide a more attractive transplantation strategy in humans. Additionally, the influence of whole-body irradiation on BBB integrity and the possibility that such damage permitted enhanced recruitment of bone marrow–derived microglia to the brain cannot be excluded, although the radiation dose used in the study was somewhat less than standard doses used in previous studies.11,32 We used a lentiviral vector to transduce bone marrow stem cells, recognizing that random genome insertion of such vectors presents potential problems for their use in humans. However, after years of skepticism, recently Cartier et al. successfully demonstrated clinical feasibility of lentiviral vector–based gene therapy.30 Use of macrophages derived from induced pluripotent stem cells may provide an alternative approach with an enhanced safety profile. Because induced pluripotent stem cells can be expanded in vitro, they can be cloned and screened extensively to define insertion site, quality of clone, and efficiency and biological activity of the therapeutic transgene before transplantation into the recipient. Nonetheless, several major challenges, including the risk of developing teratocarcinoma in recipients, must be resolved before macrophages derived from induced pluripotent stem cell technology can become a clinical reality.

Symptoms of PD start to appear when about 70–80% of striatal dopamine and about half of nigral dopamine neurons have been lost,35,36,37 generally over the course of a preclinical period of several years.38 Therefore, implementation of strategies for primary prevention will require the identification of markers that would permit early diagnosis prior to the onset of symptoms. Indeed, recent imaging data indicate that it may be possible to predict decline in striatal dopamine function before the onset of obvious clinical symptoms.39 Meanwhile, prevention of ongoing neuronal loss in symptomatic patients, as well as the induction of nerve regeneration, represent high priority objectives that would be immediately feasible if an effective and well-tolerated intervention could be developed. In this context, the prevention of nigrostriatal dopaminergic neurodegeneration and hypokinesia, plus regeneration of axons, observed in the current study indicate that macrophage-mediated GDNF delivery may provide effective clinical benefit in PD.

Finally, in addition to GDNF, both neurturin40,41 and conserved dopamine neurotrophic factor42 are known to protect dopaminergic neurons. Thus, the macrophage-mediated system that we have described herein for GDNF could be exploited for the delivery of a number of alternative therapeutic transgenes for PD, as well as for other common neurodegenerative diseases, such as Alzheimer's disease and amyotrophic lateral sclerosis, underscoring the future potential for this strategy.

Materials and Methods

Construction of GDNF lentiviral plasmid and MSP. A lentiviral vector containing the MSP (Supplementary Figure S1) is based on the design previously described in detail.43 The MSP consists of a sequence containing two cis elements, C/EBPα and AML-1. The p47phox mini-promoter gene in the original design was replaced with a CD68 mini-promoter gene to improve the specificity. The reporter gene (GFP) in the original design was then replaced with a rat GDNF gene (gene bank no. NM019139, STS 50-685) using standard molecular procedures. The resulting construct was sequenced to verify the site of insertion, as well as the integrity of the GDNF gene (DNA sequence will be provided upon request). A similar lentiviral vector carrying the gene that encodes GFP driven by the macrophage-specific promoter was also generated and used as a control. Virus stocks were generated by transient transfection of 293T cells with the lentiviral vector together with three separate packaging plasmids (pMDLg/pRRE, pRSV Rev, and pMD.G; PlasmidFactory, Bielefeld, Germany). One liter of virus was concentrated by ultracentrifugation at 50,000g for 90 minutes at 4 °C, and the viral pellet was resuspended in 400 µl of StemPro-34-SFM medium (GIBCO Invitrogen, Carlsbad, CA).

Mice. Male C57BL/6J mice, 6–8 weeks old at the time of transplantation, were used. The animals were group-housed in a 12/12-hour light/dark cycle and in accordance with institutional requirements for animal care, National Institutes of Health Guide for the Care and Use of Laboratory Animals, and Society for Neuroscience guidelines. About 19 recipient mice were used per experiment (transplantation) and repeated three times (total 57 recipient mice).

Lentiviral transduction and transplantation of bone marrow cells. The procedure was described previously.44 Briefly, donor mice were injected 4 days before bone marrow harvest with 150 mg/kg of 5-fluorouracil. Bone marrow cells were harvested from the tibias and femurs of donor mice by flushing the bones with StemPro-34-SFM complete medium (GIBCO Invitrogen) supplemented with 200 mmol/l -glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 5 units/ml heparin, and then passed through a 70 µm nylon mesh cell strainer (BD Biosciences, San Jose, CA) to make a single cell suspension. Bone marrow–derived stem cells were enriched in the lymphocyte medium using density-gradient separation. After washing, enriched bone marrow–derived stem cells were prestimulated overnight in StemPro-34-SFM supplemented with 200 mmol/l -glutamine, 6 ng/ml of murine interleukin-3, 10 ng/ml of human interleukin-6, 10 ng/ml of murine interleukin-1, and 100 ng/ml of murine stem cell factor (PeproTech, Rocky Hill, NJ). The next day, harvested cells were pelleted and resuspended in 400 µl of concentrated viral supernatant, which was supplemented with the aforementioned growth factors. Infections were performed on RetroNectin- (Takara, Otsu, Japan) coated plates for 6 hours in the presence of 4 µg/ml of protamine sulfate (Sigma, St Louis, MO). Following infection, cells were harvested and 1–2 × 106 cells were injected, without selection, via tail vein into recipient mice previously treated with 950 cGy of total body irradiation. Mice that received bone marrow stem cells transduced with a lentiviral vector in which the expression of GDNF was driven by the MSP are designated MSP-GDNF mice, whereas control mice that received bone marrow stem cells transduced with the lentiviral vector in which the expression of GFP was driven by the same promoter are designated MSP-GFP mice.

Flow cytometry. Three weeks after bone marrow stem cell transplantation, peripheral blood from MSP-GFP mice was analyzed by flow cytometry (BD FACSCalibur system; BD Biosciences, San Jose, CA). After erythrocyte lysis with red blood cell lysing buffer (Sigma), cells labeled with APC-conjugated CD11b monoclonal antibodies (2 µl prediluted antibodies per 106 cells; BD Pharmingen, San Jose, CA) were analyzed for GFP expression. Peripheral blood samples collected from normal and GFP transgenic mice were used as negative and positive controls, respectively. Because both GDNF and GFP lentiviral vectors were driven by the same promoter and GFP expression was analyzed without further processing, peripheral blood from only MSP-GFP mice was subjected to FACS analysis.

MPTP treatment. Eight weeks after transplantation, MPTP-HCl (Sigma) dissolved in physiological saline was injected subcutaneously into MSP-GDNF and MSP-GFP mice as follows: 15 mg/kg free base MPTP on day 1, 25 mg/kg on day 2, and 30 mg/kg on days 3–7. By using lower doses on the first 2 days, we were able to avoid completely animal death due to peripheral toxicity. Control mice were treated with saline following the same regimen. In the first experiment, the animals were killed 3 weeks after the last dose of MPTP, whereas in the second and third experiments, the animals were killed 9 weeks after the last dose of MPTP. A subset of animals in the third experiment was kept until 6 months after transplantation to assess the long-term expression of GDNF.

Open field test. The open field test was performed as described before.45 Briefly, animals were acclimatized to the testing room for at least 1 hour prior to the session, and the test was performed between 12 noon and 5 . The floor of the white 60 × 60 cm open field was subdivided into 25 12 × 12 cm squares by blue lines. On each test, mice were placed individually into the open field, and the behavior was recorded on videotape over 10 minutes. The open field was wiped with ethanol solution and dried between each test to remove odor trails. Locomotor activity was defined as the total number of squares entered in 10 minutes. The open field test was performed only on animals (n = 13) in the third set of experiments. The tests were performed 4 weeks after the last dose of MPTP to avoid any potential influence on behavior of peripheral toxicity associated with MPTP intoxication.

Allodynia. Mice were placed in a clear plastic cage with wire mesh bottom, and a small drop of 100% acetone was gently applied to the mid-plantar surface of the right hindpaw with a syringe connected to a thin polyethylene tube. The frequency and duration of brisk foot withdrawal in response to acetone application was measured as previously described.46 Acetone was applied eight times with an interval of 5 minutes between each application. The frequency of foot withdrawal was expressed as a percent: [(number of trials accompanied by brisk foot withdrawal) × 100/(total number of trials)]. The cumulative time (duration) the paw was held above the wire mesh during the first 60 seconds after applying acetone was also recorded.

Body weight. Body weight was measured between 10  and 12 noon every 2 days. For the purpose of clarity, cumulative change in body weight on every fourth day is represented in Figure 6e and Supplementary Figure S3.

Food intake. Twenty-four-hour food intake was measured manually as the difference between the pelleted chow put into the cage and that remaining at the end of 24 hours. Measurements were taken between 5 and 6 , and the chow crumbs that had fallen into the bedding were accounted for.

Enzyme-linked immunosorbent assay. Animals were deeply anesthetized with an overdose of ketamine HCl/xylazine HCl solution (Sigma) and decapitated after collecting peripheral blood in EDTA for separation of plasma. Midbrain regions containing substantia nigra were quickly dissected out and stored at ‐80 °C until analysis. The frozen tissue samples were sonicated in a homogenization buffer (137 mmol/l NaCl, 20 mmol/l Tris, pH 8.0, 1% NP-40, 10% glycerol, 1 mmol/l phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 0.5 mmol/l sodium vanadate) at a tissue concentration of 30 mg/ml and centrifuged at 20,000 g for 10 minutes at 4 °C. GDNF levels in the blood plasma and midbrain region containing substantia nigra were measured using a commercially available GDNF Emax ImmunoAsssay System (Promega, Madison, WI) according to the manufacturer's protocol.

High performance liquid chromatography. Striata of MPTP-treated MSP-GFP and MSP-GDNF mice were collected as described for enzyme-linked immunosorbent assay. The concentrations of DA, DOPAC, HVA, serotonin (5-HT), and 5-HIAA were determined by high performance liquid chromatography combined with electrochemical detection as described.47 Briefly, frozen tissues were sonicated in 0.2 mol/l perchloric acid (20% wt/vol) containing 100 ng/ml 3,4-dihydroxybenzylamine (internal standard) and centrifuged at 15,000g for 7 minutes. The supernatant was filtered using a 0.2 µm Nylon-66 filter and a 25 µl aliquot of the filtrate, equivalent to 2.5 mg of tissue, was injected directly into the high performance liquid chromatography/electrochemical system. The mobile phase consisted of 0.07 mol/l potassium phosphate, pH 3.0, 8% methanol, and 1.02 mmol/l 1-heptane sulfonic acid. Concentrations of DA, DOPAC, HVA, 5-HT, and 5-HIAA were calculated using a curve generated with authentic standards.

Tissue processing and immunohistochemistry. The mice were anesthetized with an overdose of ketamine HCl/xylazine HCl solution (Sigma) and perfused transcardially with 10–20 ml phosphate-buffered saline (pH 7.4) followed by an equal volume of 4% paraformaldehyde in 0.1 mol/l phosphate buffer (pH 7.4). Brain, liver, kidney, and testis were dissected out and post-fixed overnight in the same fixative at 4 °C. The tissues were cryoprotected in sequential 10% (2 hours), 20% (2 hous), and 30% (overnight) solutions of sucrose, and then embedded in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA). Although brains were processed for cryosectioning at 30 µm thickness in the coronal plane, other tissues were cut at 10 µm thickness. Four series of slides containing every fourth section were prepared for substantia nigra, whereas six series of every sixth section were prepared for striatum. Anatomical landmarks were determined according to Paxinos and Franklin.48 The standard avidin–biotin complex (ABC) method was employed to immunostain brain sections. Briefly, sections were treated with 1% bovine serum albumin in phosphate-buffered saline containing 0.3% Triton X-100 for 30 minutes and then incubated with rabbit anti-TH (Chemicon International, Billerica, MA) at 1:2,000 dilution for 48 hours at 4 °C. Sections were rinsed in phosphate-buffered saline and incubated with biotinylated goat anti-rabbit secondary antibody (1:200) for 1 hour, followed by avidin–biotin peroxidase complex (ABC Elite Kit; Vector Laboratories, Burlingame, CA) at room temperature for 1 hour. The chromogen used was either 3-amino-9-ethyl carbazole (AEC Chromogen Kit; Sigma) or 3,3′-diaminobenzidine tetrahydrochloride (Liquid DAB Substrate Kit; Zymed, Carlsbad, CA). DAB-stained midbrain sections were counterstained with cresyl violet and used for stereology. For double-color immunofluorescence, the sections were first incubated with TH antibody followed by anti-Iba1 antibody (1:2,000; Wako, Osaka, Japan). Alexa Fluor 488 and 568 (1:200; Molecular Probes, Carlsbad, CA) were used as secondary antibodies. Sections were coverslipped with Vectashield (Vector Laboratories), and fluorescent images were analyzed with a regular fluorescent microscope (Nikon Eclipse TE2000-U; Nikon Instruments, Melville, NY) or confocal microscope (Zeiss LSM 510 NLO; Carl Zeiss Microimaging, Thornwood, NY). Stringent control procedures were employed to ensure specificity of immunoreactions.

Stereology. The total number of Nissl+/TH-immunoreactive neurons in SNpc was estimated, as described before,49 using the optical fractionator method in combination with unbiased counting rules, an approach that is not affected by either the volume of SNpc or the size of the neurons.50 Briefly, the reference space (SNpc) in each 30 µm thick midbrain section was outlined at ×10 magnification using Stereo Investigator workstation (MicroBrightField, Williston, VT) attached to an Axioplan 2 imaging microscope (Carl Zeiss), fitted with a DEI-750 CE video camera (Optronics, Goleta, CA) and a LEP MAC5000 motorized stage controller (Ludl Electronic Products, Hawthorne, NY). Anatomical landmarks were determined according to Paxinos and Franklin.48 Then at random start, Nissl+/TH-immunoreactive neurons were counted from every fourth serial section throughout the entire extent of the SNpc using a ×63 oil immersion objective (numerical aperture 1.4). Cells were counted only when their nuclei were optimally visualized, which occurred only in one focal plane.

Optical density. Optical densities (OD) of the TH+ fibers in the striatum were measured from digitized images of every sixth section using NIH ImageJ software (NIH, Bethesda, MD). Conditions for tissue processing, immunostaining, and image capturing were kept constant for all animals. The measurements were taken from dorsolateral aspects of the striatum that receive the majority of innervation from dopamine neurons of SNpc. Relative OD of TH+ fibers in the striatum was calculated by subtracting the background OD from the measured OD of the dorsolateral aspects of the striatum.

Quantification of microglial engraftment to substantia nigra. To demarcate the substantia nigra, midbrain sections of saline- and MPTP-treated MSP-GFP mice were first immunostained with TH antibody followed by Alexa Fluor 568–conjugated secondary antibody. Images of TH-stained neurons (red channel) and GFP+ microglia (green channel) were captured separately and merged using Adobe Photoshop CS2 (Adobe, San Jose, CA). All the GFP-expressing cells within the substantia nigra were counted from five representative sections per animal.

Statistical analysis. Data analysis was performed with GraphPad InStat (GraphPad Software, La Jolla, CA) or Minitab 15 (Minitab, State College PA). Statistical significance was determined using Student's t-test or one-way analysis of variance at the 95% confidence level and was followed by pairwise multiple comparison tests (Tukey–Kramer multiple comparison test). Results are expressed as mean ± SEM and considered significant at P < 0.05. The body weight data at different time points were analyzed by repeated measures analysis of variance.

SUPPLEMENTARY MATERIAL Figure S1. Design of lentiviral vector expressing rat GDNF driven by macrophage-specific synthetic promoter. Figure S2. GDNF levels by ELISA in the striatum. Figure S3. Mean change from initial body weight after MPTP treatment. Figure S4. Histology of testis showing no structural differences between control and treatment groups.

Acknowledgments

We thank Jessica Han for excellent laboratory assistance, Syed F. Ali for help with HPLC analysis, Rene Santacruz for help with statistical analysis, and Fabio Jimenez for technical support with flow cytometry analysis. The work was supported by NIH grants (NS046004 and AG024579) and a Merit Review grant from the Research Division of the Department of Veterans Affairs awarded to S.L. Confocal microscopy images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (Cancer Therapy and Research Center), NIH-NIA P30 AG013319 (Nathan Shock Center), and NIH-NIA P01AG19316.

Supplementary Material

Figure S1.

Design of lentiviral vector expressing rat GDNF driven by macrophage-specific synthetic promoter.

Figure S2.

GDNF levels by ELISA in the striatum.

Figure S3.

Mean change from initial body weight after MPTP treatment.

Figure S4.

Histology of testis showing no structural differences between control and treatment groups.

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