Midbrain dopaminergic neuron fate specification: Of mice and embryonic stem cells

The midbrain dopaminergic (mDA) neurons of the substantia nigra and the ventral tegmental area play a fundamental role in the control of voluntary movement and the regulation of emotion, and are severely affected in Parkinson's disease. Recent advances in mouse genetics and vertebrate development have provided us with insight into the genetic cascades involved in the development of mDA neurons, including the induction of mDA neuron progenitors in the ventral mesencephalon, the specification of the mDA neuronal fate and the maintenance of postmitotic mDA neurons. In parallel, rapid progress has been made in the generation of DA neurons from pluripotent stem cells and the development of stem cell-based therapies for Parkinson's disease. Here, we summarize the new findings via the developmental progression of mDA neurons and outline how this knowledge has been exploited to develop novel paradigms for the in vitro generation of these neurons from embryonic stem cells.     

Panax notoginseng saponins influence on transplantation of neural stem cell-derived dopaminergic neurons in a rat model of Parkinson’s disease *☆

BACKGROUND： Dopaminergic neurons differentiated from neural stem cells have been successfully used in the treatment of rat models of Parkinson＇s disease; however, the survival rate of transplanted cells has been low. Most cells die by apoptosis as a result of overloaded intracellular calcium and the formation of oxygen free radicals.
OBJECTIVE： To observe whether survival of transplanted cells, transplantation efficacy, and dopaminergic differentiation from neural stem cells is altered by Panax notoginseng saponins （PNS） in a rat model of Parkinson＇s disease.
DESIGN, TIME AND SETTING： Cellular and molecular biology experiments with randomized group design. The experiment was performed at the Animal Experimental Center, First Hospital of Sun Yat-sen University from April to October 2007.
MATERIALS： Thirty-two adult, healthy, male Sprague Dawley rats, and four healthy Sprague Dawley rat embryos at gestational days 14-15 were selected. The right ventral mesencephalon was injected with 6-hydroxydopamine to establish a model of Parkinson＇s disease. 6-hydroxydopamine and apomorphine were purchased from Sigma, USA.
METHODS： Neural stem cells derived from the mesencephalon of embryonic rats were cultivated and passaged in serum-free culture medium. Lesioned animals were randomly divided into four groups （n = 8）： dopaminergic neuron, dopaminergic neuron ＋ PNS, PNS, and control. The dopaminergic neuron group was injected with 3 μL cell suspension containing dopaminergic neurons differentiated from neural stem cells. The dopaminergic neurons ＋ PNS group received 3 μ L dopaminergic cell suspension combined with PNS （250 mg/L）. The PNS group received 3 μL PNS （250 mg/L）, and the control group received 3 μL DMEM/F12 culture medium.
MAIN OUTCOME MEASURES： The rats were transcardially perfused with 4% paraformaldehyde at 60 days post-grafting for immunohistochemistry. The rats were intraperitoneally injected with apomorphine （0.5 mg/kg） to induce rotational behavior. RESU     

Primary tissue for cellular brain repair in Parkinson's disease: Promise,problems and the potential of biomaterials

The dopamine precursor, levodopa, remains the “gold standard” treatment for Parkinson's disease, and, although it provides superlative efficacy in the early stages of the disease, its long‐term use is limited by the development of severe motor side effects and a significant abating of therapeutic efficacy. Therefore, there remains a major unmet clinical need for the development of effective neuroprotective, neurorestorative or neuroreparatory therapies for this condition. The relatively selective loss of dopaminergic neurons from the nigrostriatal pathway makes Parkinson's disease an ideal candidate for reparative cell therapies, wherein the dopaminergic neurons that are lost in the condition are replaced through direct cell transplantation into the brain. To date, this approach has been developed, validated and clinically assessed using dopamine neuron‐rich foetal ventral mesencephalon grafts which have been shown to survive and reinnervate the denervated brain after transplantation, and to restore motor function. However, despite long‐term symptomatic relief in some patients, significant limitations, including poor graft survival and the impact this has on the number of foetal donors required, have prevented this therapy being more widely adopted as a restorative approach for Parkinson's disease. Injectable biomaterial scaffolds have the potential to improve the delivery, engraftment and survival of these grafts in the brain through provision of a supportive microenvironment for cell adhesion, growth and immune shielding. This article will briefly review the development of primary cell therapies for brain repair in Parkinson's disease and will consider the emerging literature which highlights the potential of using injectable biomaterial hydrogels in this context.     

Neural grafting for Parkinson's disease: challenges and prospects

Parkinson's disease (PD) is a neurodegenerative condition which causes a characteristic movement disorder secondary to loss of dopaminergic neurons in the substanitia nigra. The motor disorder responds well to dopamine-replacement therapies, though these result in significant adverse effects due to non-physiolog-ical release of dopamine in the striatum, and off-target effects. Cell-based regenerative treatments offer a potential means for targeted replacement of dopamine, in a physiological manner. Dopaminergic neurons for cell-based therapies can be obtained from several sources. Fetal ventral mesencephalon tissue contains dopaminergic neuron progenitors, and has been transplanted into the striatum of PD patients with good results in a number of cases. However, the ethical implications and logistical challenges of using fetal tissue mean that fetal ventral mesencephalon is unlikely to be used in a widespread clinical setting. Induced plu-ripotent stem cells can be used to generate dopaminergic neurons for transplantation, providing a source of autologous tissue for grafting. This approach means that challenges associated with allografts, such as the potential for immune rejection, can be circumvented. However, the associated cost and difficulty in producing a standardized product from different cell lines means that, at present, this approach is not com-mercially viable as a cell-based therapy. Dopaminergic neurons derived from embryonic stem cells offer the most promising basis for a cell-based therapy for Parkinson's disease, with trials due to commence in the next few years. Though there are ethical considerations to take into account when using embryonic tissue, the possibility of producing a standardized, optimized cell product means that this approach can be both effective, and commercially viable.     

Neurogenin2 identifies a transplantable dopamine neuron precursor in the developing ventral mesencephalon

In neural transplantation studies, there is an interest in identifying and isolating mesencephalic dopamine (mesDA) neuron precursors that have the capacity to differentiate into fully mature mesDA neurons after transplantation. We report here that in the developing ventral mesencephalon (VM) the proneural gene Neurogenin2 (Ngn2) is expressed exclusively in the part of the ventricular zone that gives rise to the migrating mesDA neuroblasts, but not in the differentiated mesDA neurons. From other studies, we know that Ngn2 is involved in the generation of mesDA neurons and that the development of mesDA neurons is severely compromised in Ngn2-null mutant mice. We show here that cells isolated by FACS from the developing VM of Ngn2-GFP knock-in mice are capable of generating mesDA neurons, both in vitro and after transplantation to the striatum of neonatal rats. All mesDA neuron precursors, but not the serotonergic or GABAergic neuron precursors, are contained in the Ngn2-GFP-expressing population. Moreover, all glial cells were generated from cells contained in the GFP-negative cell fraction. The results show that surviving mesDA neurons in VM grafts are derived from early postmitotic, probably Nurr1-expressing precursors before they have acquired their fully differentiated neuronal phenotype. The Ngn2-GFP reporter construct used here thus provides a tool for the identification of mesDA neuron precursors in the VM and selective isolation of transplantable mesDA neuron precursors for transplantation.     

Trophic and protective effects of growth/differentiation factor 5, a member of the transforming growth factor-β superfamily,on midbrain dopaminergic neurons

Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor-β (TGF-β) superfamily of multifunctional cytokines. We show here that GDF5 is expressed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum-free conditions, to almost the same extent as TGF-β, and is as effective as glial cell line-derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF-β and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF-β and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N-methylpyridinium ion (MPP⁺), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD. © 1995 Wiley-Liss, Inc.     

Doxycycline restrains glia and confers neuroprotection in a 6‐OHDA Parkinson model

Neuron–glia interactions play a key role in maintaining and regulating the central nervous system. Glial cells are implicated in the function of dopamine neurons and regulate their survival and resistance to injury. Parkinson's disease is characterized by the loss of dopamine neurons in the substantia nigra pars compacta, decreased striatal dopamine levels and consequent onset of extrapyramidal motor dysfunction. Parkinson's disease is a common chronic, neurodegenerative disorder with no effective protective treatment. In the 6‐OHDA mouse model of Parkinson's disease, doxycycline administered at a dose that both induces/represses conditional transgene expression in the tetracycline system, mitigates the loss of dopaminergic neurons in the substantia nigra compacta and nerve terminals in the striatum. This protective effect was associated with: (1) a reduction of microglia in normal mice as a result of doxycycline administration per se; (2) a decrease in the astrocyte and microglia response to the neurotoxin 6‐OHDA in the globus pallidus and substantia nigra compacta, and (3) the astrocyte reaction in the striatum. Our results suggest that doxycycline blocks 6‐OHDA neurotoxicity in vivo by inhibiting microglial and astrocyte expression. This action of doxycycline in nigrostriatal dopaminergic neuron protection is consistent with a role of glial cells in Parkinson's disease neurodegeneration. The neuroprotective effect of doxycycline may be useful in preventing or slowing the progression of Parkinson's disease and other neurodegenerative diseases linked to glia function.     

Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone

An understanding of the regulators of neurogenesis in the normal and diseased brain is necessary in order to recruit endogenously produced neural precursors for cell replacement in neurodegenerative disorders such as Parkinson's disease. The location of dopaminergic projections from the midbrain to the neostriatum and nucleus accumbens overlaps with the most active region of neurogenesis in the adult brain, the subventricular zone of the anterior lateral ventricle. This suggests that dopamine may contribute to regulation of the subventricular niche of adult neurogenesis. Here, we show in adult mice that destruction of the dopaminergic neurons in the substantia nigra and ventral tegmental area in a 6-hydroxydopamine model of Parkinson's disease reduced the number of proliferating neural precursors in the subventricular zone of the anterior lateral ventricle by approximately 40%. The effect on neural precursor proliferation correlated with the extent of dopaminergic denervation in the neighboring neostriatum. This identifies dopamine as one of the few known endogenous regulators of adult neurogenesis with implications for the potential use of endogenous neural precursors in cell replacement strategies for Parkinson's disease.     

Lentivirus-mediated Persephin over-expression in Parkinson's disease rats

Persephin, together with glial cell line-derived neurotrophic factor and neurturin, has a neurotrophic effect and promotes the survival of motor neurons cultured in vitro. In this study, dopaminergic neurons in the substantia nigra of rats were transfected with the Persephin gene. One week later 6-hydroxydopamine was injected into the anterior medial bundle to establish a Parkinson''s disease model in the rats. Results found that the number of dopaminergic neurons in the substantia nigra increased, tyrosine hydroxylase expression was upregulated and concentrations of dopamine and its metabolites in corpus striatum were increased after pretreatment with Persephin gene. In addition, the rotating effect of the induced Parkinson''s disease rats was much less in the group pretreated with the Persephin gene. Persephin has a neuroprotective effect on the 6-hydroxydopamine-induced Parkinson''s disease through protecting dopaminergic neurons.     

Estradiol promotes proliferation of dopaminergic precursors resulting in a higher proportion of dopamine neurons derived from mouse embryonic stem cells

Estradiol protects dopamine neurons of the substantia nigra from toxic insults. Such neurons succumb in Parkinson's disease; one strategy for restoring dopamine deficiency is cell therapy with neurons differentiated from embryonic stem cells. We investigated the effects of 17β-estradiol on dopaminergic induction of embryonic stem cells using the 5-stage protocol. Cells were incubated with different steroid concentrations during the proliferation (stage 4) or differentiation (stage 5) phases. Estradiol added at nM concentrations only during stage 4 increases the proliferation of dopaminergic precursors expressing Lmx1a, inducing a higher proportion of dopamine neurons at stage 5. These actions were mediated by activation of estrogen receptors, because co-incubation of cells with estradiol and ICI 182,780 completely abolished the positive effect on both proliferation of committed precursors, and subsequent differentiation to dopaminergic neurons. Our results suggest that estradiol should be useful in producing higher proportions of dopamine neurons from embryonic stem cells aimed for treating Parkinson's disease.     

Identification of transplantable dopamine neuron precursors at different stages of midbrain neurogenesis

Protocols used for generation of mesencephalic dopamine (mesDA) neurons from stem cells, or fetal brain tissue, invariably result in cell preparations that are highly mixed in composition, containing mesDA neuron precursors in various states of fate commitment and differentiation. For further optimisation and refinement of these procedures it is essential to determine the optimal stage of development and phenotypic characteristics of cells used for grafting. We have used fluorescence-activated cell sorting procedures to isolate mesDA precursors in defined stages of differentiation from mouse ventral mesencephalon (VM), at embryonic day 10.5 (E10.5), when the mesDA neuron domain consists of proliferative radial glia-like cells expressing the mesDA neuron determinant Lmx1a and the floorplate marker Corin, and at E12.5, when the VM has expanded to comprise a mixture of proliferative progenitors, neuroblasts and young neurons. The sorted cells were transplanted to the striatum of 6-hydroxydopamine-lesioned rats. Results show that the Lmx1a/Corin-expressing ventricular zone progenitors, which are the source of mesDA neurons in grafts from E10.5 VM, had lost this capacity at E12.5. At this later stage all transplantable mesDA precursors resided in the intermediate zone as postmitotic Nurr1-expressing neuroblasts. The more differentiated, TH-expressing cells survived sorting and transplantation poorly. We also provide evidence that, during early mesDA neurogenesis, the progenitors for nigral mesDA neurons segregate to lateral parts of the Lmx1a-expressing domain and can be selectively isolated based on their level of Corin expression. These results have implications for current efforts to develop well-characterized stem cell-derived mesDA progenitor cell preparations for cell therapy.     

Zhichan decoction induces differentiation of dopaminergic neurons in Parkinson’s disease rats after neural stem cell transplantation

The goal of this study was to increase the dopamine content and reduce dopaminergic metabolites in the brain of Parkinson’s disease rats. Using high-performance liquid chromatography, we found that dopamine and dopaminergic metabolite（dihydroxyphenylacetic acid and homovanillic acid） content in the midbrain of Parkinson’s disease rats was increased after neural stem cell transplantation ＋ Zhichan decoction, compared with neural stem cell transplantation alone. Our genetic algorithm results show that dihydroxyphenylacetic acid and homovanillic acid levels achieve global optimization. Neural stem cell transplantation ＋ Zhichan decoction increased dihydroxyphenylacetic acid levels up to 10-fold, while transplantation alone resulted in a 3-fold increment. Homovanillic acid levels showed no apparent change. Our experimental findings show that after neural stem cell transplantation in Parkinson’s disease rats, Zhichan decoction can promote differentiation of neural stem cells into dopaminergic neurons.     

Expression of N-Methyl-D-Aspartate receptor subunit mRNA in the human brain: Mesencephalic dopaminergic neurons

Evidence is accumulating that glutamate-mediated excitotoxicity plays an important role in neuronal degeneration in Parkinson's disease (PD). In addition, alterations in excitatory amino acid neurotransmission in the basal ganglia contribute to the clinical manifestations of motor dysfunction. However, detailed knowledge of the anatomical distribution and subtype specificity of glutamate receptors in the dopamine neurons of human substantia nigra (SN) has been lacking. In order to test the hypothesis that selective expression of particular N-methyl-D-aspartate receptor (NR) subunit mRNA contributes to the differential vulnerability of specific neuronal populations to excitotoxic injury in PD, we have used a quantitative dual label, in situ hybridization technique with ribonucleotide probes to examine the cellular distribution of NR subunit mRNA in postmortem human mesencephalic dopaminergic neurons from subjects with no known neurological disorder. Analysis of both film autoradiograms and emulsion-dipped sections demonstrated significant labeling of nigral neurons for each NR subunit. Neuronal labeling was most intense for the NR1 and NR2D subunits, with low level labeling for the remaining subunits. In addition, we examined four subregions of the ventral mesencephalon for differential expression of NR subunit mRNA. For all NR subunits, the pars lateralis (PL) exhibited the most intense signal, while neurons of the ventral tier substantia nigra pars compacta (SNpc) failed to demonstrate a preponderance of a particular subunit. These results demonstrate that NRs are expressed to a significant degree in dopaminergic neurons of the SN and that their distribution does not correlate with the characteristic pattern of neuronal degeneration in PD. J. Comp. Neurol. 390:91–101, 1998. © 1998 Wiley-Liss, Inc.     

Conditioned media derived from glial cell lines promote survival and differentiation of dopaminergic neurons in vitro: role of mesencephalic glia.

Neuronal differentiation is influenced by extracellular factors; however, only a few such factors have been identified for central neurons. To address this issue, we have screened media conditioned (CM) by several glial cell lines for neurotrophic effects on dopaminergic neurons in dissociated cell cultures of the E14.5 rat mesencephalon grown in serum-free conditions. To establish culture conditions under which dopaminergic cell survival depends on the exogenous support from neurotrophic factors, cell suspensions were seeded at varying densities and the number of tyrosine hydroxylase-immunoreactive (TH-IR) neurons was determined. This number was maximal at plating densities greater than 175,000 cells/cm2 and was 10-fold lower at the plating density of 80,000 cells/cm2. Cell density had only a minimal effect on [3H]dopamine uptake per TH-IR neuron. Treatment of cultures plated at 80,000 cells/cm2 with CM derived from the glial cell line, B49, the neural retina glial cell line, R33, and the Schwannoma cell line JS1, increased the number of surviving TH-IR neurons 160-330%. These effects were dose dependent and heat sensitive. All CM stimulated neurite elongation of TH-IR neurons, while only the B49-CM increased [3H]dopamine uptake. The neurotrophic effects of these media were not confined to dopaminergic neurons but increased overall neuronal density in culture by 50-100%. Moreover, all three CM were mitogenic for mesencephalic glia as demonstrated by glial fibrillary acidic protein (GFAP)-immunocytochemistry in combination with [3H]thymidine-autoradiography. By contrast, medium conditioned by the pheochromocytoma cell line, PC12, did not increase the number of astrocytes or promote the survival of dopaminergic neurons. Inhibition of glial proliferation reduced the neurotrophic effects of the B49-, R33-, and JS1-CM by 40-80%. These observations suggest that the glial cell lines B49, R33, and JS1 secrete factors that promote the survival of dopaminergic neurons and induce proliferation of glial precursors. The partial decrease of the survival-promoting effects of these CM on dopaminergic neurons in glial-free mesencephalic cultures further suggests that the observed neurotrophic effects result from the combined action of cell line-derived substances directly on neurons and indirectly via effects on mesencephalic astrocytes or astrocyte precursors.     

Altered distribution of dopaminergic neurons in the brain of L1 null mice

Dopaminergic neurons of the mouse mesencephalon originate in the ventricular zone and migrate radially along radial glia then tangentially along nerve fibers that express the neural cell adhesion molecule L1 to form the substantia nigra (A9 group) and ventral tegmental area (VTA) (A10 group). The role of L1 in migration of dopaminergic neuronal precursors was investigated in L1 knockout mice by tyrosine hydroxylase (TH) immunostaining. An altered rostrocaudal distribution of dopaminergic neurons was observed within the substantia nigra and VTA of L1-minus mice. In L1-minus mice at postnatal day 0, TH-positive cells were present abnormally in the dorsomedial mesencephalon, suggesting impaired migration. Axons projecting from the substantia nigra to the caudate putamen also exhibited an abnormal targeting pattern. There was no evidence of dopaminergic cell loss in the mutant SN. Abnormal localization of dopaminergic neurons in L1-minus mice was also evident in the zona incerta of the thalamus (A13 group), and the arcuate (A12) and periventricular nucleus (A14) of the hypothalamus. Cell bodies and axons in the substantia nigra, VTA, and hypothalamus of wild type mouse embryos expressed L1. These results suggested that L1 plays an important developmental role in the organization of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.     

Glial cell line‐derived neurotrophic factor enhances in vitro differentiation of mid‐/hindbrain neural progenitor cells to dopaminergic‐like neurons

Parkinson's disease (PD) affects the motor system through the degeneration of the dopaminergic neurons of the substantia nigra. The use of human embryonic stem cell (hESC)‐derived human neural progenitor (hNP) cells provides a potential cell source for cell therapies and drug screens for future treatments. Glial cell line‐derived neurotrophic factor (GDNF) is a known dopaminergic neuroprotectant agent; however, its potential role in neural differentiation remains largely unknown. Addition of 25 ng/ml GDNF to hNP cell differentiation media, over a 21‐day period, induced a significantly (P < 0.05) greater portion of hNP cells to differentiate into dopaminergic neurons than non‐GDNF cultures, 50% compared with 2.9% of cells expressing tyrosine hydroxylase (TH), respectively. The hNP cells exposed to GDNF selectively expressed dopamine receptors 1, 4, and 5 and were evoked to release dopamine with KCl. This is the first report of GDNF and leukemia inhibitory factor enriching hESC‐derived hNP cells toward dopaminergic‐like neurons. © 2010 Wiley‐Liss, Inc.     

Chondroitinase improves midbrain pathway reconstruction by transplanted dopamine progenitors in Parkinsonian mice

Within the adult central nervous system the lack of guidance cues together with the presence of inhibitory molecules produces an environment that is restrictive to axonal growth following injury. Consequently, while clinical trials in Parkinson's disease (PD) patients have demonstrated the capacity of fetal-derived dopamine neurons to survive, integrate and alleviate symptoms, the non-permissive host environment has contributed to the incomplete re-innervation of the target tissue by ectopic grafts, and even more noticeable, the poor reconstruction of the midbrain dopamine pathways following homotopic midbrain grafting. One such inhibitory molecule is the chondroitin sulfate proteoglycan (CSPG), a protein that has been shown to impede axonal growth during development and after injury. Digestion of CSPGs, by delivery of the bacterial enzyme chondroitinase ABC (ChABC), can improve axonal regrowth following a number of neural injuries. Here we examined whether ChABC could similarly improve axonal growth of transplanted dopamine neurons in an animal model of PD. Acute delivery of ChABC, into the medial forebrain bundle, degraded CSPGs along the nigrostriatal pathway. Simultaneous homotopic transplantation of dopaminergic progenitors, into the ventral midbrain of ChABC treated PD mice, had no effect on graft survival but resulted in enhanced axonal growth along the nigrostriatal pathway and reinnervation of the striatum, compared to control grafted mice. This study demonstrates that removal of axonal growth inhibitory molecules could significantly enhance dopaminergic graft integration, thereby holding implications for future approaches in the development of cell replacement therapies for Parkinsonian patients.     

The search for a curative cell therapy in Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder, characterised by the progressive loss of dopaminergic neurons in the substantia nigra, and typically treated by dopamine replacement. This treatment, although very effective in the early stages of the disease, is not curative and has side-effects. As such there has been a search for a more definitive treatment for this condition, which has mainly concentrated on replacing the lost neurons with neural grafts. Possible cell sources for replacement range from autologous grafts of dopamine secreting cells to allografts of fetal ventral mesencephalon and neural precursor cells derived from fetal tissue or embryonic stem cells. Some of these cells have been the subject of clinical trials, which to date have produced disparate outcomes. Therefore, whilst cell therapies remain a promising treatment for PD, there is need for further refinement of the techniques involved in this experimental procedure, before any new trials in patients are undertaken.