Understanding human embryonic ventral midbrain is of major interest for Parkinson’s disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent ...models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.
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•Species differences in developmental timing and cell proliferation•Multiple radial glia subtypes biased toward distinct fates•Adult dopaminergic neuron subtypes emerge postnatally•A machine learning method to score dopaminergic differentiation of stem cells
Analyzing the time course of ventral midbrain development in mouse, human, and stem cells by single-cell RNA-sequencing provides insight into dopaminergic neuron development and offers a strategy to assess the composition of stem-cell-derived preparations for clinical applications.
Cell replacement therapies for neurodegenerative disease have focused on transplantation of the cell types affected by the pathological process. Here we describe an alternative strategy for ...Parkinson's disease in which dopamine neurons are generated by direct conversion of astrocytes. Using three transcription factors, NEUROD1, ASCL1 and LMX1A, and the microRNA miR218, collectively designated NeAL218, we reprogram human astrocytes in vitro, and mouse astrocytes in vivo, into induced dopamine neurons (iDANs). Reprogramming efficiency in vitro is improved by small molecules that promote chromatin remodeling and activate the TGFβ, Shh and Wnt signaling pathways. The reprogramming efficiency of human astrocytes reaches up to 16%, resulting in iDANs with appropriate midbrain markers and excitability. In a mouse model of Parkinson's disease, NeAL218 alone reprograms adult striatal astrocytes into iDANs that are excitable and correct some aspects of motor behavior in vivo, including gait impairments. With further optimization, this approach may enable clinical therapies for Parkinson's disease by delivery of genes rather than cells.
Recent evidence supports a neuroprotective role for Wnt signaling in neurodegenerative disorders such as Alzheimer's Disease (AD). In fact, a relationship between amyloid-beta-peptide (Abeta)-induced ...neurotoxicity and a decrease in the cytoplasmic levels of beta-catenin has been observed. Apparently Abeta binds to the extracellular cysteine-rich domain of the Frizzled receptor (Fz) inhibiting Wnt/beta-catenin signaling. Cross-talk with other signaling cascades that regulate Wnt/beta-catenin signaling, including the activation of M1 muscarinic receptor and PKC, the use of Ibuprofen-ChE bi-functional compounds, PPAR alpha, gamma agonists, nicotine and some antioxidants, results in neuroprotection against Abeta. These studies indicate that a sustained loss of Wnt signaling function may be involved in the Abeta-dependent neurodegeneration observed in Alzheimer's brain. In conclusion the activation of the Wnt signaling pathway could be proposed as a therapeutic target for the treatment of AD.
Recent advances in single-cell RNA sequencing have allowed researchers to explore transcriptional function at a cellular level. In particular, single-cell RNA sequencing reveals that there exist ...clusters of cells with similar gene expression profiles, representing different transcriptional states.
In this study, we present SCPPIN, a method for integrating single-cell RNA sequencing data with protein-protein interaction networks that detects active modules in cells of different transcriptional states. We achieve this by clustering RNA-sequencing data, identifying differentially expressed genes, constructing node-weighted protein-protein interaction networks, and finding the maximum-weight connected subgraphs with an exact Steiner-tree approach. As case studies, we investigate two RNA-sequencing data sets from human liver spheroids and human adipose tissue, respectively. With SCPPIN we expand the output of differential expressed genes analysis with information from protein interactions. We find that different transcriptional states have different subnetworks of the protein-protein interaction networks significantly enriched which represent biological pathways. In these pathways, SCPPIN identifies proteins that are not differentially expressed but have a crucial biological function (e.g., as receptors) and therefore reveals biology beyond a standard differential expressed gene analysis.
The introduced SCPPIN method can be used to systematically analyse differentially expressed genes in single-cell RNA sequencing data by integrating it with protein interaction data. The detected modules that characterise each cluster help to identify and hypothesise a biological function associated to those cells. Our analysis suggests the participation of unexpected proteins in these pathways that are undetectable from the single-cell RNA sequencing data alone. The techniques described here are applicable to other organisms and tissues.
In the past several years, we postulated that the loss of Wnt signaling was implicated in the pathology of Alzheimer's disease (AD). Since then, our lab and other groups have confirmed the ...involvement of the Wnt signaling in some aspects of AD. So far, we have demonstrated that activation of Wnt signaling protects neurons against neurotoxic injuries, including both amyloid-beta (Abeta) fibrils and Abeta oligomers by using either lithium, an inhibitor of the glycogen-synthase-kinase-3beta (GSK-3beta), or different Wnt ligands. Also, we have found that several molecules which activate well known neurotransmitter systems and other signaling system, are able by crosstalk to activate Wnt/beta-catenin signaling in order to protect neurons against both Abeta fibrils or Abeta oligomers. In particular, the activation of non-canonical Wnt signaling was able to protect postsynaptic regions and dendritic spines against Abeta oligomers. Furthermore Wnt signaling ligands also affect stem cells, and they are also involved in cell fate decision during neurogenesis and embryonic development as well as in adult stem cells differentiation in the nervous system. The Wnt signaling plays a key role modulating their cell differentiation or proliferation states. Altogether, these findings in both stem cell biology and neuroprotection, may introduce new approaches in the treatment of neurodegenerative diseases, including drug screening and therapies against neurodegenerative diseases which activates the Wnt signaling pathway.
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 Parkinson's 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.
Synopsis
PBX1 controls a novel transcriptional network required for midbrain dopaminergic specification and survival. PBX1 and its target NFE2L1 protect dopaminergic neurons from oxidative stress, and their levels are severely reduced in the substantia nigra of Parkinson's disease patients.
Pbx1 is expressed in a subpopulation of dopaminergic neuroblasts and controls the specification and survival of midbrain dopaminergic neurons.
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.
PBX1 and NFE2L1 protect dopaminergic neurons from oxidative stress.
Nuclear PBX1 and NFE2L1 levels are severely reduced in midbrain dopaminergic neurons of Parkinson's disease patients.
The homeobox factor PBX1 directly represses or activates key genes involved in specification, survival, and oxidative stress protection of midbrain dopaminergic neurons.
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 α
β
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.
Wnt signalling is a highly conserved pathway across species that is critical for normal development and is deregulated in multiple disorders including cancer and neurodegenerative diseases. Wnt ...signalling is critically required for midbrain dopaminergic (mDA) neuron development and maintenance. Understanding the molecular processes controlled by Wnt signalling may thus hold the key to understand the physiopathology and to develop novel therapies aimed at preventing the loss of mDA neurons in Parkinson's disease (PD). Pharmacological tools to activate Wnt signalling have been used to translate in vivo developmental processes into protocols for the generation of bona fide mDA neurons from human pluripotent stem cells. Moreover, these protocols are currently being fine‐tuned to generate mDA neurons for clinical trials in PD. At the same time, a vast amount of molecular details of Wnt signalling continues to emerge and remains to be implemented into new protocols. We hereby review novel pharmacological tools to activate Wnt signalling and how single‐cell RNA‐sequencing is contributing to unravel the complexity of this pathway in the developing human ventral midbrain, generating novel hypotheses and identifying new players and opportunities to further improve cell replacement therapy for PD.
Linked Articles
This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc
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.
Objective
To define whether anti‐ribosomal P (anti‐P) autoantibodies from patients with neuropsychiatric systemic lupus erythematosus (NPSLE) impair the function of hippocampal neurons that express ...the neuronal surface P antigen (NSPA) when accessing the brain via circulating blood.
Methods
We used anti‐P antibodies from patients with NPSLE and rabbit‐generated anti‐P and anti‐NSPA antibodies. Primary hippocampal neurons from mice were analyzed to determine antibody cell surface binding (double immunofluorescence), intracellular calcium variations (Fura 2 AM), and apoptosis (caspase 3 activation). Hippocampal‐dependent spatial flexible memory was assessed in mice subjected to a water maze test 24 hours after an intravenous injection of anti‐P or anti‐NSPA, using lipopolysaccharide (LPS) to permeate the blood–brain barrier. Presence of antibodies and apoptosis in the hippocampus was studied using immunohistochemistry and TUNEL assays.
Results
Hippocampal neurons expressed NSPA on the cell surface, as revealed by anti‐P and anti‐NSPA staining colocalization, and responded to both anti‐P and anti‐NSPA by exhibiting increased intracellular calcium levels. Neuronal apoptosis was induced when anti‐P was directly injected by stereotaxis into the hippocampus or added to primary cultures. Upon LPS treatment, intravenously injected anti‐P impaired memory but did not elicit neuronal apoptosis in the hippocampus, where it was detectable in low amounts. Anti‐NSPA antibodies also impaired memory.
Conclusion
Anti‐P antibodies interact with NSPA on the surface of hippocampal neurons leading to apoptotic death or to functional perturbations, results that are likely dependent on the concentration of these antibodies. Circulating anti‐P can access the hippocampus and impair memory without requiring neuronal death when the blood–brain barrier is disrupted. NSPA can mediate antibody‐driven diffuse brain dysfunction, and anti‐P might contribute to the cognitive impairment that is frequently observed in SLE.
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