mDA Neurons Research Articles

Drug discovery toward successful cell transplantation therapy for Parkinson's disease using human pluripotent stem cells

Cell transplantation therapy using human pluripotent stem cell (PSC)–derived midbrain dopaminergic (mDA) neurons is soon expected to be available for patients with Parkinson’s disease (PD). Highly efficient and reproducible protocols for the induction of mDA neurons for clinical application have already been reported, and the therapeutic potential and safety of these cells have been studied in parkinsonian animal models as preclinical trials. However, a new strategy that improves the survival and functional quality of the grafted mDA neurons is needed to achieve maximal efficacy of the cell transplantation therapy. One strategy would definitively be to adapt the brain’s microenvironment with the use of small compounds, such as soluble factors and clinical drugs, in addition to current pharmacotherapies for PD. In this mini review, we focus on recent findings regarding the induction of mDA neurons from human PSCs toward clinical application and on a complementary strategy of drug treatment toward more efficient cell transplantation therapy for PD patients. Keywords: Parkinson’s disease; human pluripotent stem cells; cell transplantation therapy; drug discovery; clinical application (Published: 9 August 2016) Citation: Advances in Regenerative Biology 2016, 3 : 31772 - http://dx.doi.org/10.3402/arb.v3.31772

Definition of a critical spatiotemporal window within which primary cilia control midbrain dopaminergic neurogenesis.

Midbrain dopaminergic (mDA) neurons are generated in the ventral midbrain floor plate depending on Sonic Hedgehog (SHH) signaling for induction. Primary cilia transduce canonical SHH signals. Loss of intraflagellar transport protein IFT88, essential for ciliary function, disrupts SHH signaling in the ventral midbrain and results in the reduction in mDA progenitors and neurons. We investigate whether conditional inactivation of the kinesin motor protein KIF3A recapitulates phenotypes observed in conditional Ift88 mutants. Conditional Kif3a inactivation reduced the mDA progenitor domain size, but did not result in mDA neuron reduction, most likely because of a delayed loss of cilia and delayed inactivation of SHH signaling. We thereby define a precise spatiotemporal window within which primary cilia-dependent SHH signaling determines mDA fate.

Neuroprotective Transcription Factors in Animal Models of Parkinson Disease.

A number of transcription factors, including En1/2, Foxa1/2, Lmx1a/b, Nurr1, Otx2, and Pitx3, with key roles in midbrain dopaminergic (mDA) neuron development, also regulate adult mDA neuron survival and physiology. Mouse models with targeted disruption of some of these genes display several features reminiscent of Parkinson disease (PD), in particular the selective and progressive loss of mDA neurons in the substantia nigra pars compacta (SNpc). The characterization of these animal models has provided valuable insights into various mechanisms of PD pathogenesis. Therefore, the dissection of the mechanisms and survival signalling pathways engaged by these transcription factors to protect mDA neuron from degeneration can suggest novel therapeutic strategies. The work on En1/2-mediated neuroprotection also highlights the potential of protein transduction technology for neuroprotective approaches in PD.

A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells.

Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-βRI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs.

Primary cilia are critical for Sonic hedgehog-mediated dopaminergic neurogenesis in the embryonic midbrain

Midbrain dopaminergic (mDA) neurons modulate various motor and cognitive functions, and their dysfunction or degeneration has been implicated in several psychiatric diseases. Both Sonic Hedgehog (Shh) and Wnt signaling pathways have been shown to be essential for normal development of mDA neurons. Primary cilia are critical for the development of a number of structures in the brain by serving as a hub for essential developmental signaling cascades, but their role in the generation of mDA neurons has not been examined. We analyzed mutant mouse lines deficient in the intraflagellar transport protein IFT88, which is critical for primary cilia function. Conditional inactivation of Ift88 in the midbrain after E9.0 results in progressive loss of primary cilia, a decreased size of the mDA progenitor domain, and a reduction in mDA neurons. We identified Shh signaling as the primary cause of these defects, since conditional inactivation of the Shh signaling pathway after E9.0, through genetic ablation of Gli2 and Gli3 in the midbrain, results in a phenotype basically identical to the one seen in Ift88 conditional mutants. Moreover, the expansion of the mDA progenitor domain observed when Shh signaling is constitutively activated does not occur in absence of Ift88. In contrast, clusters of Shh-responding progenitors are maintained in the ventral midbrain of the hypomorphic Ift88 mouse mutant, cobblestone. Despite the residual Shh signaling, the integrity of the mDA progenitor domain is severely disturbed, and consequently very few mDA neurons are generated in cobblestone mutants. Our results identify for the first time a crucial role of primary cilia in the induction of mDA progenitors, define a narrow time window in which Shh-mediated signaling is dependent upon normal primary cilia function for this purpose, and suggest that later Wnt signaling-dependent events act independently of primary cilia.

Transcription factors FOXA1 and FOXA2 maintain dopaminergic neuronal properties and control feeding behavior in adult mice

Midbrain dopaminergic (mDA) neurons are implicated in cognitive functions, neuropsychiatric disorders, and pathological conditions; hence understanding genes regulating their homeostasis has medical relevance. Transcription factors FOXA1 and FOXA2 (FOXA1/2) are key determinants of mDA neuronal identity during development, but their roles in adult mDA neurons are unknown. We used a conditional knockout strategy to specifically ablate FOXA1/2 in mDA neurons of adult mice. We show that deletion of Foxa1/2 results in down-regulation of tyrosine hydroxylase, the rate-limiting enzyme of dopamine (DA) biosynthesis, specifically in dopaminergic neurons of the substantia nigra pars compacta (SNc). In addition, DA synthesis and striatal DA transmission were reduced after Foxa1/2 deletion. Furthermore, the burst-firing activity characteristic of SNc mDA neurons was drastically reduced in the absence of FOXA1/2. These molecular and functional alterations lead to a severe feeding deficit in adult Foxa1/2 mutant mice, independently of motor control, which could be rescued by L-DOPA treatment. FOXA1/2 therefore control the maintenance of molecular and physiological properties of SNc mDA neurons and impact on feeding behavior in adult mice.

Excessive Wnt/beta-catenin signaling promotes midbrain floor plate neurogenesis, but results in vacillating dopamine progenitors.

The floor plate (FP), a ventral midline structure of the developing neural tube, has differential neurogenic capabilities along the anterior-posterior axis. The midbrain FP, unlike the hindbrain and spinal cord floor plate, is highly neurogenic and produces midbrain dopaminergic (mDA) neurons. Canonical Wnt/beta-catenin signaling, at least in part, is thought to account for the difference in neurogenic capability. Removal of beta-catenin results in mDA progenitor specification defects as well as a profound reduction of neurogenesis. To examine the effects of excessive Wnt/beta-catenin signaling on mDA specification and neurogenesis, we have analyzed a model wherein beta-catenin is conditionally stabilized in the Shh+domain. Here, we show that the Foxa2+/Lmx1a+ domain is extended rostrally in mutant embryos, suggesting that canonical Wnt/beta-catenin signaling can drive FP expansion along the rostrocaudal axis. Although excess canonical Wnt/beta-catenin signaling generally promotes neurogenesis at midbrain levels, less tyrosine hydroxylase (Th)+, mDA neurons are generated, particularly impacting the Substantia Nigra pars compacta. This is likely because of improper progenitor specification. Excess canonical Wnt/beta-catenin signaling causes downregulation of net Lmx1b, Shh and Foxa2 levels in mDA progenitors. Moreover, these progenitors assume a mixed identity to that of Lmx1a+/Lmx1b+/Nkx6-1+/Neurog1+ progenitors. We also show by lineage tracing analysis that normally, Neurog1+ progenitors predominantly give rise to Pou4f1+ neurons, but not Th+ neurons. Accordingly, in the mutant embryos, Neurog1+ progenitors at the midline generate ectopic Pou4f1+ neurons at the expense of Th+ mDA neurons. Our study suggests that an optimal dose of Wnt/beta-catenin signaling is critical for proper establishment of the mDA progenitor character. Our findings will impact embryonic stem cell protocols that utilize Wnt pathway reagents to derive mDA neuron models and therapeutics for Parkinson's disease.

Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease

Parkinson's disease (PD), primarily caused by selective degeneration of midbrain dopamine (mDA) neurons, is the most prevalent movement disorder, affecting 1-2% of the global population over the age of 65. Currently available pharmacological treatments are largely symptomatic and lose their efficacy over time with accompanying severe side effects such as dyskinesia. Thus, there is an unmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nuclear orphan receptor Nurr1 for development and maintenance of mDA neurons and their protection from inflammation-induced death, we hypothesize that Nurr1 can be a molecular target for neuroprotective therapeutic development for PD. Here we show successful identification of Nurr1 agonists sharing an identical chemical scaffold, 4-amino-7-chloroquinoline, suggesting a critical structure-activity relationship. In particular, we found that two antimalarial drugs, amodiaquine and chloroquine stimulate the transcriptional function of Nurr1 through physical interaction with its ligand binding domain (LBD). Remarkably, these compounds were able to enhance the contrasting dual functions of Nurr1 by further increasing transcriptional activation of mDA-specific genes and further enhancing transrepression of neurotoxic proinflammatory gene expression in microglia. Importantly, these compounds significantly improved behavioral deficits in 6-hydroxydopamine lesioned rat model of PD without any detectable signs of dyskinesia-like behavior. These findings offer proof of principle that small molecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

WNT signaling in midbrain dopaminergic neuron development and cell replacement therapies for Parkinson's disease.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of midbrain dopamine (mDA) neurons. Clinical trials using human embryonic midbrain tissue for transplantation have provided proof of concept that cell replacement therapy (CRT) can lead to not only symptomatic relief, but also changes in the course of disease and withdrawal of medication. Human pluripotent stem cells are currently regarded as the main candidate cell type for CRT because they are readily available, expandable, and can be standardized and differentiated into mDA neurons capable of inducing functional recovery in animal models of PD. However, protocols for mDA differentiation are still far from optimal and require further improvement. We previously found that members of the Wnt family of morphogens regulate multiple aspects of mDA neuron development [1]. Different branches of the Wnt signaling pathway, such as Wnt/β-catenin, activated by Wnt1, and Wnt/PCP, activated by Wnt5a, have been thought to regulate separate or opposing functions. However, we found that Wnt5a cooperates with Wnt1 to promote mDA neurogenesis and that Wnt1 cooperates with Wnt5a to promote the differentiation of postmitotic mDA neuroblasts [2]. We are currently applying this knowledge to improve protocols for the differentiation of human stem cells into mDA neurons suitable for transplantation and functional recovery in animal models for PD [3].

How to make a midbrain dopaminergic neuron.

Midbrain dopaminergic (mDA) neuron development has been an intense area of research during recent years. This is due in part to a growing interest in regenerative medicine and the hope that treatment for diseases affecting mDA neurons, such as Parkinson's disease (PD), might be facilitated by a better understanding of how these neurons are specified, differentiated and maintained in vivo. This knowledge might help to instruct efforts to generate mDA neurons in vitro, which holds promise not only for cell replacement therapy, but also for disease modeling and drug discovery. In this Primer, we will focus on recent developments in understanding the molecular mechanisms that regulate the development of mDA neurons in vivo, and how they have been used to generate human mDA neurons in vitro from pluripotent stem cells or from somatic cells via direct reprogramming. Current challenges and future avenues in the development of a regenerative medicine for PD will be identified and discussed.

Dopaminergic control of autophagic-lysosomal function implicates Lmx1b in Parkinson's disease.

The role of developmental transcription factors in maintenance of neuronal properties and in disease remains poorly understood. Lmx1a and Lmx1b are key transcription factors required for the early specification of ventral midbrain dopamine (mDA) neurons. Here we show that conditional ablation of Lmx1a and Lmx1b after mDA neuron specification resulted in abnormalities that show striking resemblance to early cellular abnormalities seen in Parkinson's disease. We found that Lmx1b was required for the normal execution of the autophagic-lysosomal pathway and for the integrity of dopaminergic nerve terminals and long-term mDA neuronal survival. Notably, human LMX1B expression was decreased in mDA neurons in brain tissue affected by Parkinson's disease. Thus, these results reveal a sustained and essential requirement of Lmx1b for the function of midbrain mDA neurons and suggest that its dysfunction is associated with Parkinson's disease pathogenesis.

Epigenetic regulation contributes to urocortin-enhanced midbrain dopaminergic neuron differentiation.

The production of midbrain dopaminergic (mDA) neurons requires precise extrinsic inductive signals and intrinsic transcriptional cascade at a specific time point in development. Urocortin (UCN) is a peptide of the corticotropin-releasing hormone family that mediates various responses to stress. UCN was first cloned from adult rat midbrain. However, the contribution of UCN to the development of mDA neurons is poorly understood. Here, we show that UCN is endogenously expressed in the developing ventral midbrain (VM) and its receptors are exhibited in Nurr1(+) postmitotic mDA precursors and TH(+) neurons, suggesting possible roles in regulating their terminal differentiation. UCN treatment increased DA cell numbers in rat VM precursor cultures by promoting the conversion of Nurr1(+) precursors into DA neurons. Furthermore, neutralization of secreted UCN with anti-UCN antibody resulted in a reduction in the number of DA neurons. UCN induced an abundance of acetylated histone H3 and enhanced late DA regulator Nurr1, Foxa2, and Pitx3 expressions. Using pharmacological and RNA interference approaches, we further demonstrated that histone deacetylase (HDAC) inhibition and late transcriptional factors upregulation contribute to UCN-mediated DA neuron differentiation. Chromatin immunoprecipitation analyses revealed that UCN promoted histone acetylation of chromatin surrounding the TH promoter by directly inhibiting HDAC and releasing of methyl CpG binding protein 2-CoREST-HDAC1 repressor complex from the promoter, ultimately leading to an increase in Nurr1/coactivators-mediated transcription of TH gene. Moreover, UCN treatment in vivo also resulted in increased DA neuron differentiation. These findings suggest that UCN might contribute to regulate late mDA neuron differentiation during VM development.

TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor β1 (TGFβ1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFβ1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFβ1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFβ1 treatment. Finally, we demonstrate that TGFβ1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFβ1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor β1 (TGFβ1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFβ1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFβ1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.

Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells

Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the forkhead/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.

Human pluripotent stem cell derived midbrain PITX3(eGFP/w) neurons: a versatile tool for pharmacological screening and neurodegenerative modeling.

PITX3 expression is confined to adult midbrain dopaminergic (mDA) neurons. In this study we describe the generation and basic functional characteristics of mDA neurons derived from a human pluripotent stem cell (hPSC) line expressing eGFP under the control of the PITX3 promoter. Flow cytometry showed that eGFP was evident in 15% of the neuron population at day 12 of differentiation and this level was maintained until at least day 80. From days 20 to 80 of differentiation intracellular chloride decreased and throughout this period around ∼20% of PITX3eGFP/w neurons exhibited spontaneous Ca2+ transients (from 3.3 ± 0.3 to 5.0 ± 0.1 min-1, respectively). These neurons also responded to any of ATP, glutamate, acetylcholine, or noradrenaline with elevations of intracellular calcium. As neuronal cultures matured more dopamine was released and single PITX3eGFP/w neurons began to respond to more than one neurotransmitter. MPP+ and tumor necrosis factor (TNF), but not prostaglandin E2, caused death of the ∼50% of PITX3eGFP/w neurons (day 80). Tracking eGFP using time lapse confocal microscopy over 24 h demonstrated significant TNF-mediated neurite retraction over time. This work now shows that these PITX3eGFP/w neurons are amenable to flow cytometry, release dopamine and respond to multiple neurotransmitters with elevations of intracellular calcium, we believe that they represent a versatile system for neuropharmacological and neurotoxicological studies.

Vitamin C facilitates dopamine neuron differentiation in fetal midbrain through TET1- and JMJD3-dependent epigenetic control manner.

Intracellular Vitamin C (VC) is maintained at high levels in the developing brain by the activity of sodium-dependent VC transporter 2 (Svct2), suggesting specific VC functions in brain development. A role of VC as a cofactor for Fe(II)-2-oxoglutarate-dependent dioxygenases has recently been suggested. We show that VC supplementation in neural stem cell cultures derived from embryonic midbrains greatly enhanced differentiation toward midbrain-type dopamine (mDA) neurons, the neuronal subtype associated with Parkinson's disease. VC induced gain of 5-hydroxymethylcytosine (5hmC) and loss of H3K27m3 in DA phenotype gene promoters, which are catalyzed by Tet1 and Jmjd3, respectively. Consequently, VC enhanced DA phenotype gene transcriptions in the progenitors by Nurr1, a transcription factor critical for mDA neuron development, to be more accessible to the gene promoters. Further mechanism studies including Tet1 and Jmjd3 knockdown/inhibition experiments revealed that both the 5hmC and H3K27m3 changes, specifically in the progenitor cells, are indispensible for the VC-mediated mDA neuron differentiation. We finally show that in Svct2 knockout mouse embryos, mDA neuron formation in the developing midbrain decreased along with the 5hmC/H3k27m3 changes. These findings together indicate an epigenetic role of VC in midbrain DA neuron development.

Genome-wide characterisation of Foxa1 binding sites reveals several mechanisms for regulating neuronal differentiation in midbrain dopamine cells.

Midbrain dopamine neuronal progenitors develop into heterogeneous subgroups of neurons, such as substantia nigra pars compacta, ventral tegmental area and retrorubal field, that regulate motor control, motivated and addictive behaviours. The development of midbrain dopamine neurons has been extensively studied, and these studies indicate that complex cross-regulatory interactions between extrinsic and intrinsic molecules regulate a precise temporal and spatial programme of neurogenesis in midbrain dopamine progenitors. To elucidate direct molecular interactions between multiple regulatory factors during neuronal differentiation in mice, we characterised genome-wide binding sites of the forkhead/winged helix transcription factor Foxa1, which functions redundantly with Foxa2 to regulate the differentiation of mDA neurons. Interestingly, our studies identified a rostral brain floor plate Neurog2 enhancer that requires direct input from Otx2, Foxa1, Foxa2 and an E-box transcription factor for its transcriptional activity. Furthermore, the chromatin remodelling factor Smarca1 was shown to function downstream of Foxa1 and Foxa2 to regulate differentiation from immature to mature midbrain dopaminergic neurons. Our genome-wide Foxa1-bound cis-regulatory sequences from ChIP-Seq and Foxa1/2 candidate target genes from RNA-Seq analyses of embryonic midbrain dopamine cells also provide an excellent resource for probing mechanistic insights into gene regulatory networks involved in the differentiation of midbrain dopamine neurons.

Combined Nurr1 and Foxa2 roles in the therapy of Parkinson's disease.

Use of the physiological mechanisms promoting midbrain DA (mDA) neuron survival seems an appropriate option for developing treatments for Parkinson's disease (PD). mDA neurons are specifically marked by expression of the transcription factors Nurr1 and Foxa2. We show herein that Nurr1 and Foxa2 interact to protect mDA neurons against various toxic insults, but their expression is lost during aging and degenerative processes. In addition to their proposed cell-autonomous actions in mDA neurons, forced expression of these factors in neighboring glia synergistically protects degenerating mDA neurons in a paracrine mode. As a consequence of these bimodal actions, adeno-associated virus (AAV)-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model markedly protected mDA neurons and motor behaviors associated with nigrostriatal DA neurotransmission. The effects of the combined gene delivery were dramatic, highly reproducible, and sustained for at least 1 year, suggesting that expression of these factors is a promising approach in PD therapy.

Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

An important challenge for the continued development of cell therapy for Parkinson's disease (PD) is the establishment of procedures that better standardize cell preparations for use in transplantation. Although cell sorting has been an anticipated strategy, its application has been limited by lack of knowledge regarding transmembrane proteins that can be used to target and isolate progenitors for midbrain dopamine (mDA) neurons. We used a "FACS-array" approach to identify 18 genes for transmembrane proteins with high expression in mDA progenitors and describe the utility of four of these targets (Alcam, Chl1, Gfra1, and Igsf8) for isolating mDA progenitors from rat primary ventral mesencephalon through flow cytometry. Alcam and Chl1 facilitated a significant enrichment of mDA neurons following transplantation, while targeting of Gfra1 allowed for robust separation of dopamine and serotonin neurons. Importantly, we also show that mDA progenitors isolated on the basis of transmembrane proteins are capable of extensive, functional innervation of the host striatum and correction of motor impairment in a unilateral model of PD. These results are highly relevant for current efforts to establish safe and effective stem cell-based procedures for PD, where clinical translation will almost certainly require safety and standardization measures in order to deliver well-characterized cell preparations.

Interaction between Oc-1 and Lmx1a promotes ventral midbrain dopamine neural stem cells differentiation into dopamine neurons

Recent studies have shown that Onecut (Oc) transcription factors may be involved in the early development of midbrain dopaminergic neurons (mdDA). The expression profile of Oc factors matches that of Lmx1a, an important intrinsic transcription factor in the development of mDA neuron. Moreover, the Wnt1-Lmx1a pathway controls the mdDA differentiation. However, their expression dynamics and molecular mechanisms remain to be determined. To address these issues, we hypothesize that cross-talk between Oc-1 and Lmx1a regulates the mdDA specification and differentiation through the canonical Wnt-β-catenin pathway. We found that Oc-1 and Lmx1a displayed a very similar expression profile from embryonic to adult ventral midbrain (VM) tissues. Oc-1 regulated the proliferation and differentiation of ventral midbrain neural stem cells (vmNSCs). Downregulation of Oc-1 decreased both transcript and protein level of Lmx1a. Oc-1 interacted with lmx1a in vmNSCs in vitro and in VM tissues in vivo. Knockdown of Lmx1a reduced the expression of Oc-1 and Wnt1 in vmNSCs. Inhibiting Wnt1 signaling in vmNSCs provoked similar responses. Our data suggested that Oc-1 interacts with Lmx1a to promote vmNSCs differentiation into dopamine neuron through Wnt1-Lmx1a pathway.