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,
,,,,1
Departments of
* Microbiology,
Biochemistry and Molecular Biology, and
Pharmacology, College of Medicine, and
Institute of Mental Health, Hanyang University, Seoul, Korea;
|| Department of Physiology, College of Medicine, Yonsei University, Seoul, Korea;
¶ Department of Physiology, College of Dentistry and Dental Research Institute, Seoul National University, Seoul, Korea; and
** Laboratory of Stem Cell and Tumor Biology, Neurosurgery and Developmental Biology, Sloan Kettering Cancer Institute, New York, New York, USA
1Correspondence: Department of Biochemistry and Molecular Biology, College of Medicine, Hanyang University, #17 Haengdang-dong, Sungdong-gu, Seoul, 133–791 Korea. E-mail: leesh{at}hanyang.ac.kr
SPECIFIC AIMS
The derivation of functional dopamine (DA) neurons from proliferating neural precursor or stem cell populations has been an important goal in the development of cell-based therapies for the treatment of Parkinson’s Disease (PD). The primary goal of the present study was the identification of a set of genetic factors sufficient for directing the differentiation of naive neural precursors into fully functional DA neurons that can reverse behavioral deficits in Parkinsonian rats.
PRINCIPAL FINDINGS
1. Sonic hedgehog (SHH), Bcl-XL, and Mash1 induce morphological differentiation in Nurr1-induced DA cells
Neural precursor cells were isolated from the developing rat cortex, and proliferated in vitro by basic fibroblast growth factor (bFGF). Cultured cortical precursors were retrovirally tranduced with Nurr1, an orphan nuclear receptor specific to midbrain DA neuron development. Cells were differentiated for several days prior to analysis. As reported previously, ectopic expression of Nurr1 efficiently induced the expression of the DA neuron marker tyrosine hydroxylase (TH) but resulted in poor morphological and functional differentiation of the cells (Fig. 1
A). We tried to define additional genetic factors for the neuronal differentiation and functional acquisition of Nurr1-induced DA cells.
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We initiated this study by comparing morphologic differentiation of Nurr1-DA cells in various cultures such as passaged (P1) vs. unpassaged (P) cultures, and cultures derived from different developmental stages. Our comparative analyses of the different culture conditions suggested SHH and Bcl-XL as candidate genes for the morphologic differentiation of Nurr1-DA cells. Co-expression of SHH along with Nurr1 (Bicistronic Nurr1-IRES-SHH, NH) extensively increased TH fiber length in Nurr1-induced DA cells, compared with cell transduced with Nurr1 alone (N). Previous studies have demonstrated Bcl-XL-mediated morphologic maturation and synaptic function of neuronal cells. Expectedly, cells transduced with Nurr1-IRES-Bcl-XL (NB) exhibited a marked increase in TH fibers of Nurr1-DA cells. Additional studies showed that SHH and Bcl-XL have additive effects in the morphologic differentiation (TH fiber length per Nurr1-DA cell were Nurr1-IRES-SHH-IRES-Bcl-XL (NHB): 82.5 ± 1.5 µm, NH: 38.0 ± 5.8 µm, NB: 62.2 ± 6.1 µm, N: 10.3 ± 0.6 µm, Fig. 1B, D
).
Neuronal basic-helix-loop-helix (bHLH) transcriptional factors are known to play an essential role in the neuronal specification of precursor cells and to promote the morphological and functional maturation of postmitotic neurons. Forced expression of the bHLH factor Mash1 (Nurr1-IRES-Mash1 retrovirus (NM)), caused a dramatic increase in TH+ fiber length of the Nurr1-DA cells (NM: 147.4±50.9 µm vs. N: 9.0±1.8 µm, Fig. 1C, D
).
2. Analysis of in vitro presynaptic neuronal function in Nurr1-induced DA cells
We next examined whether enhanced morphological maturation is correlated with the expression of mature neuronal and dopaminergic markers and the acquisition of presynaptic DA neuron function. Coexpression of SHH+Bcl-XL (NHB) or Mash1 (NM) greatly enhanced the percentage of Nurr1-induced DA cells expressing MAP2, a mature neuronal marker. The percentages of all TH+ cells expressing MAP2 were NHB: 67.5 ± 4.1%; NM: 71.0 ± 2.4%; N: 11.8 ± 2.1% (Fig. 1)
. Measurements of DA release by HPLC showed significant differences among the various group of Nurr1 induced DA cells. Levels of DA on depolarization (56 mM KCl) were NHB: 2558.2 ± 152.4 pg/ml; NM: 3049.9 ± 377.6 pg/ml; and N: 561.2 ± 50.2 pg/ml. Functional differences in DA metabolism were further illustrated by measuring dopamine transporter (DAT)-mediated high-affinity reuptake of DA. While levels of reuptake in Nurr1 cultures were at the limit of detection (N: 0.23±0.01 fmol/min/well), NHB- and NM-transduced cells exhibited robust DA uptake (NHB: 4.59±0.50 fmol/min/well; NM: 2.90±0.61 fmol/min/well). Electrophysiological analyses of Nurr1-induced DA cells in NHB- and NM-transduced cultures demonstrated well-developed sodium and potassium channels, and the generation of action potentials on injection of depolarizing currents. In addition to general neuronal features, electrophysiological analyses also revealed properties characteristic for mesolimbic and nigrostriatal DA neurons. DA characteristic features included anomalous rectification, the time-dependent reduction in the membrane deflection, which was observed on injection of hyperpolarizing currents at increasing intensities.
3. In vivo transplantation studies
The final set of experiments was directed at addressing the capacity of transplanted Nurr1-induced DA cells for in vivo survival, integration, and function in Parkinsonian rats.
At 4 and 8 wk after intrastriatal transplantation, grafts derived from NHB- and NM-transduced precursors showed a dramatic increase in the number of surviving TH+ cells compared with grafts of Nurr1-only (N) transduced cells (Fig. 2
A–C and G). In addition to effects on cell survival, there were also marked differences in the degree of morphological maturation in grafted TH+ cells. NM- or NHB-derived TH+ cells within the graft exhibited mature neuronal morphologies with multiple long processes extending into the host striatum, while Nurr1-transduced TH+ cells (N) were morphologically immature without significant neurite extension (Fig. 2A
–F).
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In vivo graft function was assessed by measuring striatal DA levels. HPLC analysis revealed significantly increased levels of DA in NHB or NM grafts vs. N grafts: DA levels in the grafted tissues were NHB, 316.3 ± 28.6 µg/mg protein of graft; NM, 286.8 ± 32.5 µg/mg; N, 49.6 ± 14.7 µg/mg (n=3; P<0.05 for both NHB and NM vs. N; Fig. 2H
).
Behavioral analysis was performed by measuring amphetamine-induced rotation behavior (Fig. 2I
) and step adjustment tests (Fig. 2J
). Animals grafted with NHB or NM-tranduced cells demonstrated significant improvement in both parameters. Consistent with our published work, transplantation of precursors transduced with Nurr1 only (N) did not lead to significant behavioral restoration. These data suggest that expression of SHH and Bcl-XL or expression of Mash1 potentiates the in vitro and in vivo function of Nurr1-induced DA cells and that genetic manipulation of these genes serves as a powerful strategy toward the development of donor cell sources suitable for cell replacement in PD.
CONCLUSIONS AND SIGNIFICANCE
Our study demonstrates that combinatorial genetic manipulations acting in concert with Nurr1 provide an efficient strategy to generate functional DA neurons from non-dopaminergic neural precursors (Fig. 3
). Extensive functional characterizations in vitro and in vivo suggest these DA neurons generated in vitro from dividing forebrain precursors adopt biochemical and physiological properties characteristic of midbrain type DA neurons. Finally, our study provides the first example for achieving functional restoration in Parkinsonian rats using DA neurons derived from naive nonmidbrain-derived neural precursors. The use of genetically manipulated forebrain precursors could provide an interesting alternative to the use of human fetal midbrain tissue given the availability and scalability of human forebrain progenitors. However, it remains to be determined whether our approach is applicable to human cells and whether the risk of introducing multiple transgenes is manageable in a therapeutic setting.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6159fje
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  J.-W. Shim, C.-H. Park, Y.-C. Bae, J.-Y. Bae, S. Chung, M.-Y. Chang, H.-C. Koh, H.-S. Lee, S.-J. Hwang, K.-H. Lee, et al. Generation of Functional Dopamine Neurons from Neural Precursor Cells Isolated from the Subventricular Zone and White Matter of the Adult Rat Brain Using Nurr1 Overexpression Stem Cells, May 1, 2007; 25(5): 1252 - 1262. [Abstract] [Full Text] [PDF]
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