Shanzheng Yang

Wnt5a cooperates with canonical Wnts to generate midbrain dopaminergic neurons in vivo and in stem cells

Wnts are a family of secreted proteins that regulate multiple steps of neural development and stem cell differentiation. Two of them, Wnt1 and Wnt5a, activate distinct branches of Wnt signaling and individually regulate different aspects of midbrain dopaminergic (DA) neuron development. However, several of their functions and interactions remain to be elucidated. Here, we report that loss of Wnt1 results in loss of Lmx1a and Ngn2 expression, as well as agenesis of DA neurons in the midbrain floor plate. Remarkably, a few ectopic DA neurons still emerge in the basal plate of Wnt1−/− mice, where Lmx1a is ectopically expressed. These results indicate that Wnt1 orchestrates DA specification and neurogenesis in vivo. Analysis of Wnt1−/−;Wnt5a−/− mice revealed a greater loss of Nurr1+ cells and DA neurons than in single mutants, indicating that Wnt1 and Wnt5a interact genetically and cooperate to promote midbrain DA neuron development in vivo. Our results unravel a functional interaction between Wnt1 and Wnt5a resulting in enhanced DA neurogenesis. Taking advantage of these findings, we have developed an application of Wnts to improve the generation of midbrain DA neurons from neural and embryonic stem cells. We thus show that coordinated Wnt actions promote DA neuron development in vivo and in stem cells and suggest that coordinated Wnt administration can be used to improve DA differentiation of stem cells and the development of stem cell-based therapies for Parkinson’s disease.

Cholestenoic acids regulate motor neuron survival via liver X receptors

Cholestenoic acids are formed as intermediates in metabolism of cholesterol to bile acids, and the biosynthetic enzymes that generate cholestenoic acids are expressed in the mammalian CNS. Here, we evaluated the cholestenoic acid profile of mammalian cerebrospinal fluid (CSF) and determined that specific cholestenoic acids activate the liver X receptors (LXRs), enhance islet-1 expression in zebrafish, and increase the number of oculomotor neurons in the developing mouse in vitro and in vivo. While 3β,7α-dihydroxycholest-5-en-26-oic acid (3β,7α-diHCA) promoted motor neuron survival in an LXR-dependent manner, 3β-hydroxy-7-oxocholest-5-en-26-oic acid (3βH,7O-CA) promoted maturation of precursors into islet-1+ cells. Unlike 3β,7α-diHCA and 3βH,7O-CA, 3β-hydroxycholest-5-en-26-oic acid (3β-HCA) caused motor neuron cell loss in mice. Mutations in CYP7B1 or CYP27A1, which encode enzymes involved in cholestenoic acid metabolism, result in different neurological diseases, hereditary spastic paresis type 5 (SPG5) and cerebrotendinous xanthomatosis (CTX), respectively. SPG5 is characterized by spastic paresis, and similar symptoms may occur in CTX. Analysis of CSF and plasma from patients with SPG5 revealed an excess of the toxic LXR ligand, 3β-HCA, while patients with CTX and SPG5 exhibited low levels of the survival-promoting LXR ligand 3β,7α-diHCA. Moreover, 3β,7α-diHCA prevented the loss of motor neurons induced by 3β-HCA in the developing mouse midbrain in vivo.Our results indicate that specific cholestenoic acids selectively work on motor neurons, via LXR, to regulate the balance between survival and death.

Efficient expansion and dopaminergic differentiation of human fetal ventral midbrain neural stem cells by midbrain morphogens

Human fetal midbrain tissue grafting has provided proof-of-concept for dopamine cell replacement therapy (CRT) in Parkinsons disease (PD). However, limited tissue availability has hindered the development and widespread use of this experimental therapy. Here we present a method for generating large numbers of midbrain dopaminergic (DA) neurons based on expanding and differentiating neural stem/progenitor cells present in the human ventral midbrain (hVM) tissue. Our results show that hVM neurospheres (hVMN) with low cell numbers, unlike their rodent counterparts, expand the total number of cells 3-fold, whilst retaining their capacity to differentiate into midbrain DA neurons. Moreover, Wnt5a promoted DA differentiation of expanded cells resulting in improved morphological maturation, midbrain DA marker expression, DA release and electrophysiological properties. This method results in cell preparations that, after expansion and differentiation, can contain 6-fold more midbrain DA neurons than the starting VM preparation. Thus, our results provide evidence that by improving expansion and differentiation of progenitors present in the hVM it is possible to greatly enrich cell preparations for DA neurons. This method could substantially reduce the amount of human fetal midbrain tissue necessary for CRT in patients with PD, which could have major implications for the widespread adoption of this approach.

A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease

Pre‐B‐cell leukemia homeobox (PBX) transcription factors are known to regulate organogenesis, but their molecular targets and function in midbrain dopaminergic neurons (mDAn) as well as their role in neurodegenerative diseases are unknown. Here, we show that PBX1 controls a novel transcriptional network required for mDAn specification and survival, which is sufficient to generate mDAn from human stem cells. Mechanistically, PBX1 plays a dual role in transcription by directly repressing or activating genes, such as Onecut2 to inhibit lateral fates during embryogenesis, Pitx3 to promote mDAn development, and Nfe2l1 to protect from oxidative stress. Notably, PBX1 and NFE2L1 levels are severely reduced in dopaminergic neurons of the substantia nigra of Parkinsons disease (PD) patients and decreased NFE2L1 levels increases damage by oxidative stress in human midbrain cells. Thus, our results reveal novel roles for PBX1 and its transcriptional network in mDAn development and PD, opening the door for new therapeutic interventions.

Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP

Activation of the transcription factor YAP by an extracellular laminin promotes the differentiation and survival of dopaminergic neurons. YAP supports dopaminergic neurons Parkinson’s disease (PD) is a neurodegenerative disorder marked by progressive loss of dopaminergic neurons and motor control. Various factors promote or inhibit neuronal survival. Zhang et al. found that a prosurvival signal was mediated by the transcription cofactor YAP. YAP was activated in midbrain dopaminergic neurons in culture and in mice through an interaction between an integrin and the extracellular matrix protein laminin-511. YAP then transcriptionally activated dopaminergic neuron differentiation factors and a microRNA that decreased the synthesis of the apoptotic protein PTEN. The findings uncover a new role for YAP in neurons and a pathway that might be explored for the purpose of promoting dopaminergic neuron survival in PD patients. Parkinson’s disease (PD) is a neurodegenerative disorder in which the loss of dopaminergic neurons in the midbrain (mDA neurons) causes progressive loss of motor control and function. Using embryonic and mDA neurons, midbrain tissue from mice, and differentiated human neural stem cells, we investigated the mechanisms controlling the survival of mDA neurons. We found that the extracellular matrix protein laminin-511 (LM511) promoted the survival and differentiation of mDA neurons. LM511 bound to integrin α3β1 and activated the transcriptional cofactor YAP. LM511-YAP signaling enhanced cell survival by inducing the expression of the microRNA miR-130a, which suppressed the synthesis of the cell death–associated protein PTEN. In addition, LM511-YAP signaling increased the expression of transcription factors critical for mDA identity, such as LMX1A and PITX3, and prevented the loss of mDA neurons in response to oxidative stress, a finding that warrants further investigation to assess therapeutic potential for PD patients. We propose that by enhancing LM511-YAP signaling, it may be possible to prevent mDA neuron degeneration in PD or enhance the survival of mDA neurons in cell replacement therapies.

The Matricellular Protein R-Spondin 2 Promotes Midbrain Dopaminergic Neurogenesis and Differentiation

Summary The development of midbrain dopaminergic (mDA) neurons is controlled by multiple morphogens and transcription factors. However, little is known about the role of extracellular matrix proteins in this process. Here we examined the function of roof plate-specific spondins (RSPO1-4) and the floor plate-specific, spondin 1 (SPON1). Only RSPO2 and SPON1 were expressed at high levels during mDA neurogenesis, and the receptor LGR5 was expressed by midbrain floor plate progenitors. Surprisingly, RSPO2, but not SPON1, specifically promoted the differentiation of mDA neuroblasts into mDA neurons in mouse primary cultures and embryonic stem cells (ESCs). In addition, RSPO2 was found to promote not only mDA differentiation, but also mDA neurogenesis in human ESCs. Our results thus uncover an unexpected function of the matricellular protein RSPO2 and suggest an application to improve mDA neurogenesis and differentiation in human stem cell preparations destined to cell replacement therapy or drug discovery for Parkinson disease.

Transcriptional synergy as an emergent property defining cell subpopulation identity enables population shift

Single-cell RNA sequencing allows defining molecularly distinct cell subpopulations. However, the identification of specific sets of transcription factors (TFs) that define the identity of these subpopulations remains a challenge. Here we propose that subpopulation identity emerges from the synergistic activity of multiple TFs. Based on this concept, we develop a computational platform (TransSyn) for identifying synergistic transcriptional cores that determine cell subpopulation identities. TransSyn leverages single-cell RNA-seq data, and performs a dynamic search for an optimal synergistic transcriptional core using an information theoretic measure of synergy. A large-scale TransSyn analysis identifies transcriptional cores for 186 subpopulations, and predicts identity conversion TFs between 3786 pairs of cell subpopulations. Finally, TransSyn predictions enable experimental conversion of human hindbrain neuroepithelial cells into medial floor plate midbrain progenitors, capable of rapidly differentiating into dopaminergic neurons. Thus, TransSyn can facilitate designing strategies for conversion of cell subpopulation identities with potential applications in regenerative medicine.Gaining insight into cell identities from single cell RNA-seq data remains a challenge. Here, the authors introduce an approach to identify transcription factors (TFs) that synergistically determine cellular identities, and demonstrate its ability to identify TFs that can induce cellular conversion.

A Zeb2-miR-200c loop controls midbrain dopaminergic neuron neurogenesis and migration

Zeb2 is a homeodomain transcription factor that plays pleiotropic functions during embryogenesis, but its role for midbrain dopaminergic (mDA) neuron development is unknown. Here we report that Zeb2 is highly expressed in progenitor cells in the ventricular zone of the midbrain floor plate and downregulated in postmitotic neuroblasts. Functional experiments show that Zeb2 expression in the embryonic ventral midbrain is dynamically regulated by a negative feedback loop that involves miR-200c. We also find that Zeb2 overexpression reduces the levels of CXCR4, NR4A2, and PITX3 in the developing ventral midbrain in vivo, resulting in migration and mDA differentiation defects. This phenotype was recapitulated by miR-200c knockdown, suggesting that the Zeb2-miR-200c loop prevents the premature differentiation of mDA progenitors into postmitotic cells and their migration. Together, our study establishes Zeb2 and miR-200c as critical regulators that maintain the balance between mDA progenitor proliferation and neurogenesis.Shanzheng Yang et al. show that Zeb2 expression in the embryonic ventral midbrain is regulated by a negative feedback loop that involves miR-200c. This Zeb2-miR-200c loop provides novel insight into the mechanisms that properly cue the differentiation of midbrain dopaminergic neuronal progenitors.