Sunghoi Hong

Wnt1-lmx1a Forms a Novel Autoregulatory Loop and Controls Midbrain Dopaminergic Differentiation Synergistically with the SHH-FoxA2 Pathway

Selective degeneration of midbrain dopaminergic (mDA) neurons is associated with Parkinsons disease (PD), and thus an in-depth understanding of molecular pathways underlying mDA development will be crucial for optimal bioassays and cell replacement therapy for PD. In this study, we identified a novel Wnt1-Lmx1a autoregulatory loop during mDA differentiation of ESCs and confirmed its in vivo presence during embryonic development. We found that the Wnt1-Lmx1a autoregulatory loop directly regulates Otx2 through the beta-catenin complex and Nurr1 and Pitx3 through Lmx1a. We also found that Lmx1a and Lmx1b cooperatively regulate mDA differentiation with overlapping and cross-regulatory functions. Furthermore, coactivation of both Wnt1 and SHH pathways by exogenous expression of Lmx1a, Otx2, and FoxA2 synergistically enhanced the differentiation of ESCs to mDA neurons. Together with previous works, this study shows that two regulatory loops (Wnt1-Lmx1a and SHH-FoxA2) critically link extrinsic signals to cell-intrinsic factors and cooperatively regulate mDA neuron development.

Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons

Human embryonic stem (hES) cells have the ability to renew themselves and differentiate into multiple cell types upon exposure to appropriate signals. In particular, the ability of hES cells to differentiate into defined neural lineages, such as neurons, astrocytes, and oligodendrocytes, is fundamental to developing cell‐based therapies for neurodegenerative disorders and studying developmental mechanisms. However, the utilization of hES cells for basic and applied research is hampered by the lack of well‐defined methods to maintain their self‐renewal and direct their differentiation. Recently we reported that neural precursor (NP) cells derived from mouse ES cells maintained their potential to differentiate into dopaminergic (DA) neurons after significant expansion in vitro. We hypothesized that NP cells derived from hES cells (hES‐NP) could also undergo the same in vitro expansion and differentiation. To test this hypothesis, we passaged hES‐NP cells and analyzed their proliferative and developmental properties. We found that hES‐NP cells can proliferate approximately 380 000‐fold after in vitro expansion for 12 weeks and maintain their potential to generate Tuj1+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes as well as tyrosine hydroxylase‐positive (TH+) DA neurons. Furthermore, TH+ neurons originating from hES‐NP cells expressed other midbrain DA markers, including Nurr1, Pitx3, Engrail‐1, and aromatic l‐amino acid decarboxylase, and released significant amounts of DA. In addition, hES‐NP cells maintained their developmental potential through long‐term storage (over 2 years) in liquid nitrogen and multiple freeze–thaw cycles. These results demonstrate that hES‐NP cells have the ability to provide an expandable and unlimited human cell source that can develop into specific neuronal and glial subtypes.

Selection of Embryonic Stem Cell-Derived Enhanced Green Fluorescent Protein-Positive Dopamine Neurons Using the Tyrosine Hydroxylase Promoter Is Confounded by Reporter Gene Expression in Immature Cell Populations

Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence‐activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5‐kilobase (kb) and 9‐kb TH promoter fragments and showed that clones generated using the 9‐kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9‐kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage‐specific embryonic antigen‐positive (SSEA‐1+) and that the genetically engineered clones contained more SSEA‐1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA‐1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell‐derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression.

Vesicular monoamine transporter 2 and dopamine transporter are molecular targets of Pitx3 in the ventral midbrain dopamine neurons

Midbrain dopamine (mDA) neurons play critical roles in the regulation of voluntary movement and their dysfunction is associated with Parkinson’s disease. Pitx3 has been implicated in the proper development of mDA neurons in the substantia nigra pars compacta, which are selectively lost in Parkinson’s disease. However, the basic mechanisms underlying its role in mDA neuron development and/or survival are poorly understood. Toward this goal, we sought to identify downstream target genes of Pitx3 by comparing gene expression profiles in mDA neurons of wild‐type and Pitx3‐deficient aphakia mice. This global gene expression analysis revealed many potential target genes of Pitx3; in particular, the expression of vesicular monoamine transporter 2 and dopamine transporter, responsible for dopamine storage and reuptake, respectively, is greatly reduced in mDA neurons by Pitx3 ablation. In addition, gain‐of‐function analyses and chromatin immunoprecipitation strongly indicate that Pitx3 may directly activate transcription of vesicular monoamine transporter 2 and dopamine transporter genes, critically contributing to neurotransmission and/or survival of mDA neurons. As the two genes have been known to be regulated by Nurr1, another key dopaminergic transcription factor, we propose that Pitx3 and Nurr1 may coordinately regulate mDA specification and survival, at least in part, through a merging and overlapping downstream pathway.

GATA‐3 regulates the transcriptional activity of tyrosine hydroxylase by interacting with CREB

The zinc finger transcription factor GATA‐3 is a master regulator of type 2 T‐helper cell development. Interestingly, in GATA‐3–/– mice, noradrenaline (NA) deficiency is a proximal cause of embryonic lethality. However, neither the role of GATA‐3 nor its target gene(s) in the nervous system were known. Here, we report that forced expression of GATA‐3 resulted in an increased number of tyrosine hydroxylase (TH) expressing neurons in primary neural crest stem cell (NCSC) culture. We also found that GATA‐3 transactivates the promoter function of TH via specific upstream sequences, a domain of the TH promoter residing at −61 to −39 bp. Surprisingly, this domain does not contain GATA‐3 binding sites but possesses a binding motif, a cAMP response element (CRE), for the transcription factor, CREB. In addition, we found that site‐directed mutation of this CRE almost completely abolished transactivation of the TH promoter by GATA‐3. Furthermore, protein–protein interaction assays showed that GATA‐3 is able to physically interact with CREB in vitro as well as in vivo. Based on these results, we propose that GATA‐3 may regulate TH gene transcription via a novel and distinct protein–protein interaction, and directly contributes to NA phenotype specification.

UbcH6 interacts with and ubiquitinates the SCA1 gene product ataxin-1

UbcH6 is a member of an evolutionally conserved subfamily of E2 ubiquitin-conjugating enzymes. In this study, we report that UbcH6 interacts with and ubiquitinates ataxin-1, the spinocerebellar ataxia type 1 gene product. UbcH6 was identified as an ataxin-1-interacting protein using a yeast two-hybrid screen. UbcH6 co-immunoprecipitates and co-localizes with the ataxin-1 protein in the nucleus. Our binding assays showed that ataxin-1 interacts with UbcH6 through its AXH domain. Interestingly, UbcH6 could ubiquitinate ataxin-1 in the absence of an E3 ligase. The expression level of UbcH6 regulated the rate of ataxin-1 degradation. This study demonstrates that UbcH6 and ataxin-1 are E2-substrate cognate pairs in the ubiquitin-proteasome system.

Trim11 increases expression of dopamine β-hydroxylase gene by interacting with Phox2b

The homeodomain transcription factor Phox2b is one of the key determinants involved in the development of noradrenergic (NA) neurons in both the central nervous system (CNS) and the peripheral nervous system (PNS). Using yeast two-hybrid screening, we isolated a Phox2b interacting protein, Trim11, which belongs to TRIM (Tripartite motif) or RBCC proteins family, and contains a RING domain, B-boxes, a coiled-coil domain, and the B30.2/SPRY domain. Protein-protein interaction assays showed that Phox2b was able to physically interact with Trim11. The B30.2/SPRY domain of Trim11 was required for the interaction with Phox2b. Expression of Phox2b and Trim11 was detected in the sympathetic ganglia (SG) of mouse embryos. Forced expression of Trim11 with Phox2b further increased mRNA levels of dopamine beta-hydroxylase (DBH) gene in primary avian neural crest stem cell (NCSC) culture. This study suggests a potential role for Trim11 in the specification of NA phenotype by interaction with Phox2b.

Stem cell grafting improves both motor and cognitive impairments in a genetic model of parkinson'S disease, the aphakia (ak) mouse

Stem cell-based cell replacement of lost midbrain dopamine (mDA) neurons is a potential therapy for Parkinsons disease (PD). Toward this goal, it is critical to optimize various aspects of cell transplantation and to assess functional recovery through behavioral tests in validated animal model(s) of PD. At present, cell transplantation studies are being done almost exclusively in neurotoxin-based animal models, because few genetic models of PD exhibit robust mDA neuronal loss. Here we used a genetic model of PD, the aphakia mouse, which demonstrates selective degeneration of mDA neurons in the substantia nigra. We systematically investigated the functional effects of transplanting embryonic stem cell-derived cells at different stages of in vitro differentiation: embryoid body (EB), neural progenitor (NP), and neuronal differentiated (ND) stages. We found that transplantation of NP cells yielded the best outcomes for both survival and behavioral improvement, while transplantation of EB and ND cells resulted in high teratoma-like tumor formation and poor survival, respectively. In behavioral paradigms specific to basal ganglia, the NP cells group prominently improved motor behavioral defects 1 and 2 months posttransplantation. Furthermore, we found that NP cell transplantation also improved cognitive impairments of aphakia mice, as examined by the passive avoidance task. Importantly, these graft-induced functional improvements well correlated with survival of tyrosine hydroxylase-positive DA neurons. Taken together, we propose that the aphakia mouse can serve as a novel and useful platform for cell transplantation studies to assess both neurological and cognitive improvements and that NP stage cells represent an optimal stage for transplantation.