Molecular mechanisms controlling the development of dopaminergic neurons

Seminars in Cell & Developmental Biology 14 (2003) 175–180

Molecular mechanisms controlling the development of dopaminergic neurons John C. Lin, Arnon Rosenthal∗ Rinat Neuroscience Corp., Palo Alto, CA 94304, USA

Abstract Dopaminergic (DA) neurons in the midbrain are critically involved in several neurological-psychiatric illnesses and are specifically lost in Parkinson’s disease. The DA neurons are generated through the interactions of multiple extrinsic and intrinsic factors during the embryogenesis. The identities and mechanisms of actions of a subset of these factors have recently been elucidated. The same factors have also been successfully used to induce efficient differentiation of DA neurons in vitro from embryonic stem cells or neural progenitors. These advances have far reaching scientific and medical implications. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Dopamine; Development; Neuron; Stem cells

1. Introduction Dopamine (DA) is a major catecholamine neurotransmitter of the vertebrate central nervous system (CNS). It is also synthesized in noradrenergic and adrenergic neurons where DA is a precursor to noradernaline and adrenaline. There are several major populations of DA neurons in the CNS: a subset of interneurons in the olfactory bulb, a subset of amacrine interneurons of the retina, distinct neuronal groups in the hypothalamus, and the ventral mesencephalon which includes substantia nigra (SN, so-called nigrostriatal system) and ventral tegmental area (VTA, so-called mesolimbic system). The largest and the most important group of DA neurons in the mammalian CNS are in the mesecephalon. The nigrostriatal DA neurons are affected in Parkinson’s disease (PD), whereas the mesolimbic DA neurons are implicated prominently in obsessive–compulsive disorder, schizophrenia and substance abuse. Since the hallmark of PD is a progressive loss of the nigrostriatal DA neurons, “DA supplementation” therapy such as L-dopa and DA receptor agonists has traditionally been the mainstay of the symptomatic management of this most common movement disorder [1]. However, the standard DA supplementation therapies are effective only for the 2–4 years. Even when effective, current therapies are frequently accompanied by dyskinetic and psychotic side ∗ Corresponding

author. Fax: +1-650-813-9268. E-mail addresses: [email protected] (J.C. Lin), [email protected] (A. Rosenthal).

effects, which can be attributed at least in part to unregulated and ectopic DA transmission, respectively. As a result, “DA cell replacement” therapy emerges as a potential alternative. Transplantation of DA neurons or their precursors in the striatum or in the substantia nigra may restore physiologically regulated and anatomically precise DA release [2]. Unfortunately, there are both technical and ethical constrains on the use of human tissue for transplantation since DA neurons from up to seven fetal human donors are required for transplantation into a single PD patient. As a result, increased attention has being directed toward deriving DA neurons from the embryonic stem (ES) cells as well as from the neural stem (NS) cells. Understanding the mechanism by which normal DA neurons develop, therefore, has become important not only for its relevance to the basic knowledge of neural development, but also for the enormous medical implications.

2. Mesencephalic DA neuron development in vivo Mesencephalic DA neurons are generated in the mid-hindbrain boundary (MHB) under the influence of two major signaling centers, the floor plate and the isthmus, of the developing embryos. Explant culture studies revealed that the secreted factors Shh and Fgf8 are the principal molecular entities underlying the activity of each of these signaling centers, respectively [3–5]. Shh determines the location of DA neurons in the dorsal–ventral (D–V) axis, whereas Fgf8 positions the mesencephalic DA neurons in

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the anterior–posterior (A–P) axis of the neural tube. In vivo evidence that DA neuronal development is indeed controlled by these signals came from genetic experiments in mouse. The Gli proteins are a conserved family of zinc finger transcription factors that function both as activators and repressors in transducing the Shh signal inside the cells. Forced Gli-1 expression in transgenic mice induced ectopic mesencephalic DA neurons [6] while Gli-2 deficient mouse embryos failed to develop the mesencephalic DA neurons [7]. Shh expression in the floor plate was shown to be controlled by the forkhead transcription factor FoxA1/HNF3␤ in mouse and zebrafish [8,9], bHLH-PAS family transcription factors Sim-1 and -2 in the mouse [10], as well as directly by TGF-␤ family factor Cyclop in the zebrafish [11]. Likewise, Fgf8 expression in the MHB is controlled by a complex network of regulatory genes including Pax-2 [12]. These early developmental events that initiate, refine and maintain the pattern and signaling centers along the A–P and D–V axes in the MHB have been reviewed elsewhere [13–15], and are not further elaborated in this article. One important and yet unsolved puzzle is that, despite the evolutionary conservation of these signaling centers around MHB in virtually all vertebrates, the teleost fish generally do not possess the equivalent DA neurons in the mesencephalon [16], while the cartilaginous fish like the sharks do. Once the DA neuronal fate is induced by the extrinsic signals in the ventral MHB region, several regulatory genes are required for initiating and maintaining the expression of DA-specific genes as well as for survival of DA neurons. Here we discuss a few such genes or candidate genes that have been characterized. One candidate gene that was thought to regulate the differentiation or maintenance of DA neurons is Pitx-3/Ptx-3; a paired type homeodomain transcription factor that is expressed almost exclusively in the DA neurons [17]. However, ectopic over-expression of the rat Pitx-3 in the mouse or chick embryos does not induce ectopic DA neurons (JL and AR, unpublished observation). As the time of this writing the phenotypes of Pitx-3 null mice have not been reported but the result of Lmx-1b knock out mice suggested that Pitx-3 is probably not necessary for tyrosine hydroxylase (TH) expression in the mesencephalic DA neurons (see below). It remains to be determined whether Pitx-3 is required for at least some later aspects of DA differentiation or survival. Another gene that attracted significant attention is Nurr-1, an orphan nuclear receptor that is widely expressed in the CNS including in the mesencephalic DA neurons [18]. In the absence of Nurr-1, the mice initially still develop a group of Pitx-3, Lmx-1b and HNF3␤ expressing cells in the ventral MHB region but fail to initiate TH expression [19–22]. These “prospective” DA cells eventually degenerate and disappear, resulting in a complete loss of mature DA neurons. Conversely, forced expression of Nurr-1 in the adult hippocampus-derived progenitors can induce TH expression without affecting other DA-specific genes, suggesting a direct regulation of TH expression by Nurr-1

[23]. Furthermore, over-expression of Nurr-1 can induce the differentiation of mature DA neuronal phenotype by a cerebellum-derived immortalized cell line in the presence of the type 1 astrocytes [24]. Therefore Nurr-1 appear to be a transcriptional regulator of TH expression and is essential for DA neuronal differentiation and survival. A third gene that may be involved in the regulation of DA neurons is Lmx-1b, a LIM homeodomain transcription factor that is initially highly expressed in the MHB and the dorsal neural tube, and is later expressed in developing and adult mesencephalic DA neurons. The early expression of Lmx-1b is induced and/or maintained by Fgf8, and retroviral-mediated Lmx-1b expression is able to induce ectopic Wnt-1 expression, another secreted signal of the MHB [25]. In the absence of Lmx-1b, the ventral mesencephalic DA neurons retain expression of Nurr-1 and TH, but most of them lose Pitx-3 expression [26]. These results suggest that Lmx-1b and Pitx-3 form a regulatory cascade independent of the expression of Nurr-1 or TH in DA neurons. Two homeodomain transcription factors En-1 and En-2 are also highly expressed in the developing and mature mesencephalic DA neurons as well as other cell types in the MHB. Although the midbrain DA neurons are initially specified in single- or double-En knock out mice, their survival requires the En-1 and -2 genes in a dosage-dependent manner [27]. In the En-1/En-2-double knock out mice a very small population of TH-positive mesencephalic DA neurons remains, but the expression of ␣-synuclein, the gene that is mutated in familial forms of PD, is completely lost in the ventral midbrain, suggesting that En genes may directly regulate ␣-synuclein expression in the DA neurons. The loss of ␣-synuclein expression, however, is unlikely to be responsible for the loss of DA neurons in the En-1/En-2-double knock out mice since the ␣-synuclein knock out mice maintain a full complement of midbrain TH neurons [28]. The expression of Pitx-3, Nurr-1, Lmx-1b or ␥-synuclein was not examined in the En knock out mice. GDNF is the most potent trophic factor for mesencephalic DA neurons in vitro. The GDNF−/− mice initially develop a full complement of midbrain DA neurons, but these mice die soon after birth, precluding the analysis of later survival and function of DA neurons in the absence of GDNF [29]. When transplanted into the striatum of an MPTP-lesioned wild type mouse, the GDNF mutant (−/−) donor tissues exhibit a greatly reduced survival of DA neurons compared to the wild type (+/+) donor tissues. Moreover, such a defect can be rescued by immersion of the GDNF−/− donor tissues with high concentration of GDNF prior to transplantation [30]. These results can be attributed to a low level of endogenous GDNF expression in the wild type adult striatum, limited diffusion of endogenous GDNF into the donor tissue, and the long half life of the exogenous GDNF in the brain. Thus GDNF is needed for continued survival of mesencephalic DA neurons in the adult. This is particularly significant in view of the recent clinical trials where continuous and localized delivery

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of recombinant GDNF into the SN by a pump led to a rapid and significant rescue of the DA neurons of PD patients.

3. Distinction of subtypes of DA neurons The mesencephalic DA neurons are mainly distributed in two distinct anatomical structures, SN DA neurons that innervate the striatum and VTA DA neurons that innervate the nucleus accumbens and the prefrontal cortex. Little is known about how these two groups of DA neurons develop and make distinct connections since they largely express a common set of genes (J. Grimm and A. Rosenthal, unpublished data). Loss of TGF-␣, a secreted growth factor, leads to a selective reduction of DA neurons in SN, but not in VTA [31]. This appears to reflect an early, possibly a developmental, requirement of TGF-␣ since such a defect was already present in the newborns. The site of expression and the mechanism of TGF-␣ action in a subset of the SN DA neurons are currently unknown. Raldh-1 (class 1 aldehyde dehydrogenase) is also expressed in a subset of mesencephalic DA neurons [19,32]. The Paldh-1 expressing axonal terminals form a gradient in the striatum and nucleus accumbens such that its density is highest in the rostral and dorsal region of the basal forebrain. Such a graded distribution suggests that Raldh-1 is preferentially expressed in the DA neurons of SN instead of those of the VTA. Given the importance of retinoic acid in early embryogenesis, hindbrain patterning and spinal motoneuron development, such a striking expression pattern of a retinoic acid synthesizing enzyme suggested that retinoic acid and/or its derivatives may play a role in the development of subsets of DA neurons.

4. DA axonal development DA neurons project their axons in the rostral direction to form the medial forebrain bundle (MFB) before reaching the final targets in the forebrain, which include the corpus striatum, nucleus accumbens and the prefrontal cortex (Fig. 1). MHB and DA neurons co-culture experiments have shown that the MHB tissue alone is not repulsive to the DA axons suggesting that no localized repulsive signal from the MHB and rostral hindbrain region prohibits the DA axons from entering the hindbrain [33]. In contrast the DA axons do turn abruptly when forced to grow into a stratum of the reverse polarity, suggesting that locally distributed directional cues guide the rostral projection of DA axons to their precise positions in their physiological targets. The absence of repulsive signals in the hindbrain suggest that the DA axons may turn rostrally in response to attractive guidance cue(s) distributed in the vicinity of ventral midbrain. In this context it is interesting to note that, in the Sey/Sey (i.e. loss of Pax-6 function) mutant mice, the DA axons fail to turn rostrally but instead continue to grow dorsally [34]. It remains

Fig. 1. The schematic diagram of the axonal projections of the mesencephalic dopamineric neurons in the mouse embryo. Rostral is to the left, caudal to the right. Please refer to the text for a description of the DA axonal trajectory and topographic mapping. Abbreviations: DT, dorsal thalamus; H, hypothalamus; Med, medulla; MFB, medial forebrain bundle; NA, nucleus accumbens; PFC, prefrontal cortex; PTec, pretectum; SN, substantia nigra; Tec, tectum; VTA, ventral tegmental area.

to be elucidated exactly how Pax-6 control the guidance of DA axons. Unlike many ascending axons originating from the spinal cord and elsewhere, the mesencephalic DA axons never cross the midline. Once turned rostrally, the DA axons join additional ascending axons to form the medial forebrain bundle (MFB), which are a symmetrical pair of axonal bundles running in parallel to the ventral midline of the forebrain. Studies in the Drosophila embryo showed that longitudinal axonal pathways are kept from crossing the midline and maintained at a constant distance from the midline by the repulsive signal Slit [35]. Similarly, mammalian Slit-1 and Slit-2 are both expressed in the ventral midline of the CNS. The TH-positive axons as well as other axons in the MFB are displaced ventrally in Slit-2−/− mutant mice and frequently enter the hypothalamus in Slit-1−/−;Slit-2−/− double mutants [36]. Therefore, the repulsive Slit signals contribute to the maintenance of DA axonal trajectory in the ventral forebrain. Finally DA axons invade selected targets in the forebrain, i.e. the striatum and the prefrontal cortex in a topographic manner. The SN DA axons preferentially innervate the dorsal striatum, while the VTA DA axons the ventral striatum (mainly nucleus accumbens) and the prefrontal cortex. The Eph receptor–ligand system has been implicated in the formation of DA topographical map. EphB1 is preferentially expressed in the SN DA neurons, while its ligand ephrin-B2 shows a graded expression in the DA axonal targets with the highest level in the ventral striatum [37]. Furthermore, ephrin-B2 specifically inhibits neurite outgrowth of SN but not VTA, suggesting that repulsive interaction between EphB1 and ephrin-B2 may underlie at least in part the formation of DA topographic map. This proposal awaits confirmation by gain and loss of function experiments in vivo.

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5. DA neuron differentiation in vitro Our understanding of the mechanisms by which the mesencephalic DA neurons are generated during normal development has been successfully extended to useful methods of promoting DA neuronal differentiation from the totipotent embryonic stem cells (ES). Addition of Shh and Fgf8 to the ES cell culture increases the efficiency of DA cell differentiation from ∼5 to 20–30% [38]. Similar results were also obtained with clonal ES cell lines derived from nuclear transplantation of adult donor cells (ntES cells). Interestingly, different ntES cell lines produced DA neurons at highly variable frequencies with one line C15 generating 50% DA neurons, suggesting that inherent potential of DA neuron differentiation is ES cell line dependent [39]. Recently, one such intrinsic factor which can influence DA neuron differentiation has been identified as Nurr-1, the orphan nuclear receptor required for TH expression (see above). It was found that the efficiency of DA differentiation can be increased up to 80% by adding Shh and Fgf8 to an ES cell line forced to express Nurr-1 [40]. The DA cells derived from these experimental protocols not only express DA-synthesizing enzymes, DA receptor and transporter but also molecular characteristics that are specific to the differentiating and mature mesencepahlic DA neurons (e.g. cRet, Pitx-3, En-1). When transplanted into the striatum, these cells can correct the amphetamine-induced ipsilateral rotation of rats whose mesencephalic DA neurons were ablated by 6-hydroxydopamine in one side. Moreover, animals grafted with Nurr-1 expressing ES cells not only exhibited contralateral turning upon amphetamine stimulation, but also manifested spontaneous contralateral rotations. This phenomenon, which results from over-production of dopamine in one side of the brain, demonstrates a robust differentiation of Nurr-1 ES cells into functional dopamine producing DA neurons in vivo. It also highlights the current difficulty to achieve regulated differentiation and balanced transmitter release with transplanted cells. In addition, an independent group reported a definite risk of teratoma-like tumor formation in the ES cell-grafted animals [41]. These safety concerns need to be addressed before the ES cell-derived DA neuron transplants can become a realistic therapy for PD patients. Another method to promote DA cell differentiation from ES cells relied on an activity produced by a bone marrow-derived stromal cell line PA3, so-called stromal cell-derived inducing activity (SDIA) [42,43]. SDIA has shown activity on both murine and primate ES cells, and was able to increase the efficiency of DA differentiation to 30–35% [44]. Although the biochemical nature of SDIA remains unknown, it is resistant to fixation by cross-linking agent, para-formaldehyde, cannot be transferred from the culture supernatant, and is permeable to a 0.4 ␮m membrane barrier. Importantly SDIA can modify the response of ES cells to BMP’s, which are general inhibitors of neural induction and are potent inducers of mesodermal fates in the early embryo. Mesodermal markers are normally induced

when ES cells are exposed to BMP-4 alone. In contrast, ES cells give rise to epidermal cells when exposed to both BMP-4 and SDIA [42]. Furthermore, sequential exposure of murine ES cells to SDIA, retinoid acid (RA, a caudalizing activity) and Shh (a ventralizing activity) promote their differentiation into spinal motoneurons and selected interneurons in lieu of the mesencephalic DA neurons [45]. These results together suggest that SDIA probably represents a general neural promoting factor rather than a specific DA neuron inducer. Given the success in inducing ES cells to the DA lineage, promoting the differentiation of adult or fetal mesencephalic neural progenitor cells would seem conceptually straightforward. This approach, however, generally achieved a lower percentage of DA neuron production as compared to ES cells [46–49]. One possible explanation to this discrepancy relates to the fact that the tissue oxygen pressure in the developing and adult brain ranges from 1 to 5% whereas the standard techniques expose the neural progenitor cells to the ambient 20% of oxygen pressure [50,51]. Lowering the tissue culture condition to 3% oxygen significantly increased the proliferation of mesencepahlic progenitor cells, decreased their death, and most importantly enhanced efficiency of their differentiation into DA neurons up to 56% (compared to 18% at the regular 20% oxygen condition). Nurr-1 and Shh expression levels were not altered by low oxygen but Fgf8, VEGF and erythropoietin (EPO) mRNA levels are significantly increased by low oxygen tension. Addition or blocking of VEGF did not affect DA differentiation. However, Fgf8 exposure enhanced mesencephalic progenitor proliferation and delayed the differentiation of TH+ cells. Interestingly, recombinant EPO protein increased the yield of DA neurons from mesencephalic progenitors at 20% oxygen environment in a dose-dependent manner and an EPO-neutralizing antibody partially abolished the efficiency of DA differentiation at the 3% oxygen condition [50]. It will be interesting to learn whether and how EPO acts as an inducer of DA neuron in vivo as well as in the ES cell culture system.

6. Conclusion In the past few years considerable advances have been made in our understanding of the molecular mechanisms controlling the development of DA neurons both in vivo and in vitro. This progress was partly fueled by the expectation that production of DA neurons in vitro will be useful in cell replacement therapy for PD. Indeed the extrinsic and intrinsic signals underlying the native DA neuron development turned out to be highly relevant and useful in efforts of directing DA differentiation from the ES cells in vitro. Conversely, new observations made with the in vitro culture systems revealed unexpected molecular mechanisms influencing DA neuron differentiation, which will inspire studies of their roles in vivo. Recent technological explosion in genome sequencing, transcript expression profiling,

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proteomics, as well as improved methods of DA neuronal cell isolation [52] promise to uncover the entire molecular repertoire of the general mesencephalic and subset-specific DA neurons in health and in disease. The scientific community, physicians and the patients can all expect to benefit from the more exciting developments to come.

Acknowledgements We thank former members of Rosenthal lab as well as members of Rinat Neuroscience Corp. for discussion and unpublished data.

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