A cross-species proteomic map reveals neoteny of human synapse development.

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

Prdm16 and Vcam1 regulate the postnatal disappearance of embryonic radial glia and the ending of cortical neurogenesis.

Embryonic neural stem cells (NSCs, i.e., radial glia) in the ventricular-subventricular zone (V-SVZ) generate the majority of neurons and glia in the forebrain. Postnatally, embryonic radial glia disappear and a subpopulation of radial glia transition into adult NSCs. As this transition occurs, widespread neurogenesis in brain regions such as the cerebral cortex ends. The mechanisms that regulate the postnatal disappearance of radial glia and the ending of embryonic neurogenesis remain poorly understood. Here, we show that PR domain-containing 16 (Prdm16) promotes the disappearance of radial glia and the ending of neurogenesis in the cerebral cortex. Genetic deletion of Prdm16 from NSCs leads to the persistence of radial glia in the adult V-SVZ and prolonged postnatal cortical neurogenesis. Mechanistically, Prdm16 induces the postnatal reduction in Vascular Cell Adhesion Molecule 1 (Vcam1). The postnatal disappearance of radial glia and the ending of cortical neurogenesis occur normally in Prdm16-Vcam1 double conditional knockout mice. These observations reveal novel molecular regulators of the postnatal disappearance of radial glia and the ending of embryonic neurogenesis, filling a key knowledge gap in NSC biology.

Ensembles of endothelial and mural cells promote angiogenesis in prenatal human brain.

Interactions between angiogenesis and neurogenesis regulate embryonic brain development. However, a comprehensive understanding of the stages of vascular cell maturation is lacking, especially in the prenatal human brain. Using fluorescence-activated cell sorting, single-cell transcriptomics, and histological and ultrastructural analyses, we show that an ensemble of endothelial and mural cell subtypes tile the brain vasculature during the second trimester. These vascular cells follow distinct developmental trajectories and utilize diverse signaling mechanisms, including collagen, laminin, and midkine, to facilitate cell-cell communication and maturation. Interestingly, our results reveal that tip cells, a subtype of endothelial cells, are highly enriched near the ventricular zone, the site of active neurogenesis. Consistent with these observations, prenatal vascular cells transplanted into cortical organoids exhibit restricted lineage potential that favors tip cells, promotes neurogenesis, and reduces cellular stress. Together, our results uncover important mechanisms into vascular maturation during this critical period of human brain development.

Comment on "Impact of neurodegenerative diseases on human adult hippocampal neurogenesis".

Terreros-Roncal et al. investigated the impacts of human neurodegeneration on immunostainings assumed to be associated with neurogenesis. However, the study provides no evidence that putative proliferating cells are linked to neurogenesis, that multipolar nestin+ astrocytes are progenitors, or that mature-looking doublecortin+ neurons are adult-born. Their histology-marker expression differs from what is observed in species where adult hippocampal neurogenesis is well documented.

Nests of dividing neuroblasts sustain interneuron production for the developing human brain.

The human cortex contains inhibitory interneurons derived from the medial ganglionic eminence (MGE), a germinal zone in the embryonic ventral forebrain. How this germinal zone generates sufficient interneurons for the human brain remains unclear. We found that the human MGE (hMGE) contains nests of proliferative neuroblasts with ultrastructural and transcriptomic features that distinguish them from other progenitors in the hMGE. When dissociated hMGE cells are transplanted into the neonatal mouse brain, they reform into nests containing proliferating neuroblasts that generate young neurons that migrate extensively into the mouse forebrain and mature into different subtypes of functional interneurons. Together, these results indicate that the nest organization and sustained proliferation of neuroblasts in the hMGE provide a mechanism for the extended production of interneurons for the human forebrain.

Single-cell analysis of the ventricular-subventricular zone reveals signatures of dorsal and ventral adult neurogenesis.

The ventricular-subventricular zone (V-SVZ), on the walls of the lateral ventricles, harbors the largest neurogenic niche in the adult mouse brain. Previous work has shown that neural stem/progenitor cells (NSPCs) in different locations within the V-SVZ produce different subtypes of new neurons for the olfactory bulb. The molecular signatures that underlie this regional heterogeneity remain largely unknown. Here, we present a single-cell RNA-sequencing dataset of the adult mouse V-SVZ revealing two populations of NSPCs that reside in largely non-overlapping domains in either the dorsal or ventral V-SVZ. These regional differences in gene expression were further validated using a single-nucleus RNA-sequencing reference dataset of regionally microdissected domains of the V-SVZ and by immunocytochemistry and RNAscope localization. We also identify two subpopulations of young neurons that have gene expression profiles consistent with a dorsal or ventral origin. Interestingly, a subset of genes are dynamically expressed, but maintained, in the ventral or dorsal lineages. The study provides novel markers and territories to understand the region-specific regulation of adult neurogenesis.

Nerve cells, or neurons, are the central building blocks of brain circuits. Their damage, death or loss of function leads to cognitive decline. Neural stem/progenitor cells (NSPCs) first appear during embryo development, generating most of the neurons found in the nervous system. However, the adult brain retains a small subpopulation of NSPCs, which in some species are an important source of new neurons throughout life. In the adult mouse brain the largest population of NSPCs, known as B cells, is found in an area called the ventricular-subventricular zone (V-SVZ). These V-SVZ B cells have properties of specialized support cells known as astrocytes, but they can also divide and generate intermediate 'progenitor cells' called C cells. These, in turn, divide to generate large numbers of young 'A cells' neurons that undertake a long and complex migration from V-SVZ to the olfactory bulb, the first relay in the central nervous system for the processing of smells. Depending on their location in the V-SVZ, B cells can generate different kinds of neurons, leading to at least ten subtypes of neurons. Why this is the case is still poorly understood. To examine this question, Cebrián-Silla, Nascimento, Redmond, Mansky et al. determined which genes were expressed in B, C and A cells from different parts of the V-SVZ. While cells within each of these populations had different expression patterns, those that originated in the same V-SVZ locations shared a set of genes, many of which associated with regional specification in the developing brain. Some, however, were intriguingly linked to hormonal regulation. Salient differences between B cells depended on whether the cells originated closer to the top ('dorsal' position) or to the bottom of the brain ('ventral' position). This information was used to stain slices of mouse brains for the RNA and proteins produced by these genes in different regions. These experiments revealed dorsal and ventral territories containing B cells with distinct 'gene expression'. This study highlights the heterogeneity of NSPCs, revealing key molecular differences among B cells in dorsal and ventral areas of the V-SVZ and reinforcing the concept that the location of NSPCs determines the types of neuron they generate. Furthermore, the birth of specific types of neurons from B cells that are so strictly localized highlights the importance of neuronal migration to ensure that young neurons with specific properties reach their appropriate destination in the olfactory bulb. The work by Cebrián-Silla, Nascimento, Redmond, Mansky et al. has identified sets of genes that are differentially expressed in dorsal and ventral regions which may contribute to regional regulation. Furthering the understanding of how adult NSPCs differ according to their location will help determine how various neuron types emerge in the adult brain.

Cellular response to spinal cord injury in regenerative and non-regenerative stages in Xenopus laevis.

BACKGROUND: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. METHODS: We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP+ cells for RNAseq analysis. RESULTS: In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. CONCLUSIONS: The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.

Cellular composition and organization of the spinal cord central canal during metamorphosis of the frog Xenopus laevis.

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.

Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus. This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease. In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day, whereas other studies find many fewer putative new neurons. Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Adult Neurogenesis Is Sustained by Symmetric Self-Renewal and Differentiation.

Somatic stem cells have been identified in multiple adult tissues. Whether self-renewal occurs symmetrically or asymmetrically is key to understanding long-term stem cell maintenance and generation of progeny for cell replacement. In the adult mouse brain, neural stem cells (NSCs) (B1 cells) are retained in the walls of the lateral ventricles (ventricular-subventricular zone [V-SVZ]). The mechanism of B1 cell retention into adulthood for lifelong neurogenesis is unknown. Using multiple clonal labeling techniques, we show that the vast majority of B1 cells divide symmetrically. Whereas 20%-30% symmetrically self-renew and can remain in the niche for several months before generating neurons, 70%-80% undergo consuming divisions generating progeny, resulting in the depletion of B1 cells over time. This cellular mechanism decouples self-renewal from the generation of progeny. Limited rounds of symmetric self-renewal and consuming symmetric differentiation divisions can explain the levels of neurogenesis observed throughout life.

Unique Organization of the Nuclear Envelope in the Post-natal Quiescent Neural Stem Cells.

Neural stem cells (B1 astrocytes; NSCs) in the adult ventricular-subventricular-zone (V-SVZ) originate in the embryo. Surprisingly, recent work has shown that B1 cells remain largely quiescent. They are reactivated postnatally to function as primary progenitors for neurons destined for the olfactory bulb and some corpus callosum oligodendrocytes. The cellular and molecular properties of quiescent B1 cells remain unknown. Here we found that a subpopulation of B1 cells has a unique nuclear envelope invagination specialization similar to envelope-limited chromatin sheets (ELCS), reported in certain lymphocytes and some cancer cells. Using molecular markers, [3H]thymidine birth-dating, and Ara-C, we found that B1 cells with ELCS correspond to quiescent NSCs. ELCS begin forming in embryonic radial glia cells and represent a specific nuclear compartment containing particular epigenetic modifications and telomeres. These results reveal a unique nuclear compartment in quiescent NSCs, which is useful for identifying these primary progenitors and study their gene regulation.

Netrin-1 receptor antibodies in thymoma-associated neuromyotonia with myasthenia gravis.

OBJECTIVE: To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications. METHODS: Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers). RESULTS: Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma (p < 0.05). Coexisting Caspr2 and Netrin-1 receptor antibodies were associated with concurrent thymoma, myasthenia gravis, and neuromyotonia, often with Morvan syndrome (p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus. CONCLUSIONS: Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%).

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain.

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc.

Temporal dynamics of hippocampal neurogenesis in chronic neurodegeneration.

The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour γ-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer's disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases.

The adult macaque spinal cord central canal zone contains proliferative cells and closely resembles the human.

The persistence of proliferative cells, which could correspond to progenitor populations or potential cells of origin for tumors, has been extensively studied in the adult mammalian forebrain, including human and nonhuman primates. Proliferating cells have been found along the entire ventricular system, including around the central canal, of rodents, but little is known about the primate spinal cord. Here we describe the central canal cellular composition of the Old World primate Macaca fascicularis via scanning and transmission electron microscopy and immunohistochemistry and identify central canal proliferating cells with Ki67 and newly generated cells with bromodeoxyuridine incorporation 3 months after the injection. The central canal is composed of uniciliated, biciliated, and multiciliated ependymal cells, astrocytes, and neurons. Multiciliated ependymal cells show morphological characteristics similar to multiciliated ependymal cells from the lateral ventricles, and uniciliated and biciliated ependymal cells display cilia with large, star-shaped basal bodies, similar to the Ecc cells described for the rodent central canal. Here we show that ependymal cells with one or two cilia, but not multiciliated ependymal cells, proliferate and give rise to new ependymal cells that presumably remain in the macaque central canal. We found that the infant and adult human spinal cord contains ependymal cell types that resemble those present in the macaque. Interestingly, a wide hypocellular layer formed by bundles of intermediate filaments surrounded the central canal both in the monkey and in the human, being more prominent in the stenosed adult human central canal.

Age-related changes in astrocytic and ependymal cells of the subventricular zone.

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence.

Axonal control of the adult neural stem cell niche.

The ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain's neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C.

Axons take a dive: Specialized contacts of serotonergic axons with cells in the walls of the lateral ventricles in mice and humans.

In the walls of the lateral ventricles of the adult mammalian brain, neural stem cells (NSCs) and ependymal (E1) cells share the apical surface of the ventricular-subventricular zone (V-SVZ). In a recent article, we show that supraependymal serotonergic (5HT) axons originating from the raphe nuclei in mice form an extensive plexus on the walls of the lateral ventricles where they contact E1 cells and NSCs. Here we further characterize the contacts between 5HT supraependymal axons and E1 cells in mice, and show that suprependymal axons tightly associated to E1 cells are also present in the walls of the human lateral ventricles. These observations raise interesting questions about the function of supraependymal axons in the regulation of E1 cells.