Gonçalo Castelo-Branco

Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq

Cellular diversity in the brain revealed The mammalian brain has an extraordinarily large number of cells. Although there are quite a few different cell types, many cells in any one category tend to look alike. Zeisel et al. analyzed the transcriptomes of mouse brain cells to reveal more than meets the eye. Interneurons of similar type were found in dissimilar regions of the brain. Oligodendrocytes that seemed to be all of one class were differentiated by their molecular signatures into a half-dozen classes. Microglia associated with blood vessels were distinguished from look-alike perivascular macrophages. Thus, the complex microanatomy of the brain can be revealed by the RNAs expressed in its cells. Science, this issue p. 1138 A close look at the genes expressed by cells in the brain reveals hidden and coordinated cellular complexity. The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

Differential regulation of midbrain dopaminergic neuron development by Wnt-1, Wnt-3a, and Wnt-5a

The Wnts are a family of glycoproteins that regulate cell proliferation, fate decisions, and differentiation. In our study, we examined the contribution of Wnts to the development of ventral midbrain (VM) dopaminergic (DA) neurons. Our results show that β-catenin is expressed in DA precursor cells and that β-catenin signaling takes place in these cells, as assessed in TOPGAL [Tcf optimal-promoter β-galactosidase] reporter mice. We also found that Wnt-1, -3a, and -5a expression is differentially regulated during development and that partially purified Wnts distinctively regulate VM development. Wnt-3a promoted the proliferation of precursor cells expressing the orphan nuclear receptor-related factor 1 (Nurr1) but did not increase the number of tyrosine hydroxylase-positive neurons. Instead, Wnt-1 and -5a increased the number of rat midbrain DA neurons in rat embryonic day 14.5 precursor cultures by two distinct mechanisms. Wnt-1 predominantly increased the proliferation of Nurr1+ precursors, up-regulated cyclins D1 and D3, and down-regulated p27 and p57 mRNAs. In contrast, Wnt-5a primarily increased the proportion of Nurr1+ precursors that acquired a neuronal DA phenotype and up-regulated the expression of Ptx3 and c-ret mRNA. Moreover, the soluble cysteine-rich domain of Frizzled-8 (a Wnt inhibitor) blocked endogenous Wnts and the effects of Wnt-1 and -5a on proliferation and the acquisition of a DA phenotype in precursor cultures. These findings indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and that different Wnts have specific and unique activity profiles.

Histone H2AX-dependent GABA A receptor regulation of stem cell proliferation

Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in numbers of stem cells may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1 phase, which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1 checkpoint. Regulation of proliferation in the ‘DNA damage’ S/G2 cell cycle checkpoint pathway is known for its role in the maintenance of chromatin structural integrity. Here we show that autocrine/paracrine γ-aminobutyric acid (GABA) signalling by means of GABAA receptors negatively controls ES cell and peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth and proliferation in the boundary-cap stem cell niche, resulting in an attenuation of neuronal progenies from this stem cell niche. Activation of GABAA receptors leads to hyperpolarization, increased cell volume and accumulation of stem cells in S phase, thereby causing a rapid decrease in cell proliferation. GABAA receptors signal through S-phase checkpoint kinases of the phosphatidylinositol-3-OH kinase-related kinase family and the histone variant H2AX. This signalling pathway critically regulates proliferation independently of differentiation, apoptosis and overt damage to DNA. These results indicate the presence of a fundamentally different mechanism of proliferation control in these stem cells, in comparison with most somatic cells, involving proteins in the DNA damage checkpoint pathway.

Citrullination regulates pluripotency and histone H1 binding to chromatin

Citrullination is the post-translational conversion of an arginine residue within a protein to the non-coded amino acid citrulline. This modification leads to the loss of a positive charge and reduction in hydrogen-bonding ability. It is carried out by a small family of tissue-specific vertebrate enzymes called peptidylarginine deiminases (PADIs) and is associated with the development of diverse pathological states such as autoimmunity, cancer, neurodegenerative disorders, prion diseases and thrombosis. Nevertheless, the physiological functions of citrullination remain ill-defined, although citrullination of core histones has been linked to transcriptional regulation and the DNA damage response. PADI4 (also called PAD4 or PADV), the only PADI with a nuclear localization signal, was previously shown to act in myeloid cells where it mediates profound chromatin decondensation during the innate immune response to infection. Here we show that the expression and enzymatic activity of Padi4 are also induced under conditions of ground-state pluripotency and during reprogramming in mouse. Padi4 is part of the pluripotency transcriptional network, binding to regulatory elements of key stem-cell genes and activating their expression. Its inhibition lowers the percentage of pluripotent cells in the early mouse embryo and significantly reduces reprogramming efficiency. Using an unbiased proteomic approach we identify linker histone H1 variants, which are involved in the generation of compact chromatin, as novel PADI4 substrates. Citrullination of a single arginine residue within the DNA-binding site of H1 results in its displacement from chromatin and global chromatin decondensation. Together, these results uncover a role for citrullination in the regulation of pluripotency and provide new mechanistic insights into how citrullination regulates chromatin compaction.

Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice.

Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell-derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.

Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system

One size does not fit all Oligodendrocytes are best known for their ability to myelinate brain neurons, thus increasing the speed of signal transmission. Marques et al. surveyed oligodendrocytes of developing mice and found unexpected heterogeneity. Transcriptional analysis identified 12 populations, ranging from precursors to mature oligodendrocytes. Transcriptional profiles diverged as the oligodendrocytes matured, building distinct populations. One population was responsive to motor learning, and another, with a different transcriptome, traveled along blood vessels. Science, this issue p. 1326 Brain oligodendrocytes express transcriptional heterogeneity between brain regions and age of development. Oligodendrocytes have been considered as a functionally homogeneous population in the central nervous system (CNS). We performed single-cell RNA sequencing on 5072 cells of the oligodendrocyte lineage from 10 regions of the mouse juvenile and adult CNS. Thirteen distinct populations were identified, 12 of which represent a continuum from Pdgfra+ oligodendrocyte precursor cells (OPCs) to distinct mature oligodendrocytes. Initial stages of differentiation were similar across the juvenile CNS, whereas subsets of mature oligodendrocytes were enriched in specific regions in the adult brain. Newly formed oligodendrocytes were detected in the adult CNS and were responsive to complex motor learning. A second Pdgfra+ population, distinct from OPCs, was found along vessels. Our study reveals the dynamics of oligodendrocyte differentiation and maturation, uncoupling them at a transcriptional level and highlighting oligodendrocyte heterogeneity in the CNS.

GSK-3β inhibition/β-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons

Wnts are important regulators of dopamine (DA) neuron differentiation in the developing ventral mesencephalon and could thus serve as potential tools in the treatment of Parkinsons disease. In this study, we investigate whether established intracellular Wnt signalling components could modulate the development of DA neurons. Two chemical inhibitors of glycogen synthase kinase (GSK)-3β, indirubin-3-monoxime and kenpaullone, were found to increase neuronal differentiation in ventral mesencephalon precursor cultures. In addition, the GSK-3β-specific inhibitor kenpaullone increased the size of the DA neuron population through conversion of precursors expressing the orphan nuclear receptor-related factor 1 into tyrosine hydroxylase positive neurons, thereby mimicking an effect of Wnts. We show that GSK-3β inhibitors stabilized β-catenin and that overexpression of β-catenin in ventral mesencephalic precursors resulted in increased DA differentiation. The three- to fivefold increase in DA differentiation of precursor cells by GSK-3β inhibitors suggests that such compounds could be used to improve stem/precursor cell therapy approaches in Parkinsons disease.

Ventral midbrain glia express region-specific transcription factors and regulate dopaminergic neurogenesis through Wnt-5a secretion

Glial cells have been classically described as supporting cells for neurons. Recently, additional roles during neural development have begun to emerge. Here, we report that ventral midbrain glia, including astrocytes and radial glia, are the source of signals required by neural precursors to acquire a dopaminergic phenotype. We found that ventral midbrain glia, but not cortical glia, secrete high levels of the glycolipoprotein Wnt-5a, express region-specific transcription factors such as Pax-2, En-1 and Otx-2 and increase the differentiation of cortical or ventral midbrain Nurr1 precursors into tyrosine hydroxylase-positive neurons. Moreover, blocking experiments using a Wnt-5a blocking antibody indicated that the effects of ventral midbrain glia on Nurr1-positive neural precursors are partially mediated by Wnt-5a. Thus, our results identify Wnt-5a as an important component of the dopaminergic inductive activity of the ventral midbrain glia.

Persephin-Overexpressing Neural Stem Cells Regulate the Function of Nigral Dopaminergic Neurons and Prevent Their Degeneration in a Model of Parkinson's Disease

Persephin (PSP) is a neurotrophic factor of the GDNF family that has been found to promote the survival of multiple populations of neurons. In the present study we have examined: (1) the mechanism of action and the function of PSP on nigrostriatal dopamine neurons and (2) the therapeutic potential of PSP, delivered by neural stem cells (NSCs) in a model of Parkinsons disease. Interestingly we found that the prenatal ventral mesencephalon and the newborn striatum express high levels of PSP mRNA. Moreover, midbrain dopamine neurons express its preferred receptor GFRalpha4, allowing a cis type of action of PSP on dopamine neurons. Primary culture studies showed that PSP is as potent and efficacious as GDNF at promoting both survival and neuritogenesis of midbrain dopamine neurons. To study the function and therapeutic potential of PSP in vivo we engineered NSCs to overexpress PSP. PSP-c17.2 cells were found to stably express PSP mRNA and protein for at least 3 months in vivo, to disperse within the striatum, and to give rise to neurons, astrocytes, and a large proportion of oligodendrocytes that integrated within white matter tracts in the striatum. Moreover, PSP-c17.2 cells enhanced dopamine-dependent behavioral parameters in unlesioned mice and prevented the loss of dopamine neurons and the behavioral impairment of mice receiving intrastriatal 6-OHDA injections. Thus, our findings are consistent with a direct action of PSP on developing and adult midbrain dopamine neurons and suggest that the delivery of PSP by NSCs may constitute a very useful strategy in the treatment of Parkinsons disease.

Function of Wnts in dopaminergic neuron development.

Dopaminergic (DA) neurons in the ventral midbrain (VM) are one of the major cell types lost in Parkinson’s disease (PD). Proof of principle exists for cell replacement therapies for PD, but wider application is halted by the unavailability of abundant sources of DA neurons. Stem cells might constitute one of these sources. However, efficient protocols promoting their specific differentiation into a DA neuronal phenotype are required. In this review, we summarize the latest findings concerning the contribution of the Wnt family of glycolipoproteins in the development of VM DA neurons. Regulators of Wnt signaling are involved in several neurodevelopmental processes. Recent results indicate that Wnts are key regulators of proliferation and differentiation of DA precursors during VM neurogenesis and different Wnts have specific and unique activity profiles. Interestingly, chemical inhibitors of glycogen synthase kinase-3β stabilize β-catenin and increase DA differentiation in VM precursor cultures. We hereby propose that Wnts are likely to contribute in the future to improve stem/precursor cell replacement therapy approaches to PD.