Anju Vasudevan

Compartment-specific transcription factors orchestrate angiogenesis gradients in the embryonic brain.

Prevailing notions of cerebral vascularization imply that blood vessels sprout passively into the brain parenchyma from pial vascular plexuses to meet metabolic needs of growing neuronal populations. Endothelial cells, building blocks of blood vessels, are thought to be homogeneous in the brain with respect to their origins, gene expression patterns and developmental mechanisms. These current notions that cerebral angiogenesis is regulated by local environmental signals contrast with current models of cell-autonomous regulation of neuronal development. Here we demonstrate that telencephalic angiogenesis in mice progresses in an orderly, ventral-to-dorsal gradient regulated by compartment-specific homeobox transcription factors. Our data offer new perspectives on intrinsic regulation of angiogenesis in the embryonic telencephalon, call for a revision of the current models of telencephalic angiogenesis and support novel roles for endothelial cells in brain development.

Indomethacin-induced mitochondrial dysfunction and oxidative stress in villus enterocytes

Nonsteroidal anti-inflammatory drugs (NSAIDs) are known to cause small intestinal damage but the pathogenesis of this toxicity is not well established. Intestinal epithelial cells are thought to be affected by these drugs in the course of their absorption. These cells are of different types, viz. villus, middle and crypt cells. There is little information on which of these cells, if any, are particularly vulnerable to the effects of NSAIDs. This paper aimed to study the effects of indomethacin, an NSAID commonly used in toxicity studies, on different populations of enterocytes. Effects of the drug were assessed in terms of oxidative damage, mitotic activity, mitochondrial function and lipid composition in enterocytes isolated from the small intestine of rats that had been orally administered indomethacin. In addition, the effects of arginine and zinc in protecting against such changes were assessed. Cell viability, tetrazolium dye (MTT) reduction and oxygen uptake were significantly reduced in villus tip cells from rats dosed with the drug. Thymidine uptake was higher in the crypt cell fraction from these rats. Similarly, products of lipid peroxidation were elevated in the villus tip cells with a corresponding decrease in the level of the anti-oxidant, alpha-tocopherol. In isolated mitochondrial preparations from various enterocyte fractions, significant functional impairment and altered lipid composition were seen mainly in mitochondria from villus cells. Arginine and zinc pre-treatment were found to protect against these effects. These results suggest for the first time that the villus tip cells are more vulnerable to the damaging effects of indomethacin and that oxidative stress is possibly involved in this damage.

Developmental patterns of torsinA and torsinB expression.

Early onset torsion dystonia is characterized by involuntary movements and distorted postures and is usually caused by a 3-bp (GAG) deletion in the DYT1 (TOR1A) gene. DYT1 codes for torsinA, a member of the AAA+ family of proteins, implicated in membrane recycling and chaperone functions. A close relative, torsinB may be involved in similar cellular functions. We investigated torsinA and torsinB message and protein levels in the developing mouse brain. TorsinA expression was highest during prenatal and early postnatal development (until postnatal day 14; P14), whereas torsinB expression was highest during late postnatal periods (from P14 onwards) and in the adult. In addition, significant regional variation in the expression of the two torsins was seen within the developing brain. Thus, torsinA expression was highest in the cerebral cortex from embryonic day 15 (E15)-E17 and in the striatum from E17-P7, while torsinB was highest in the cerebral cortex between P7-P14 and in the striatum from P7-P30. TorsinA was also highly expressed in the thalamus from P0-P7 and in the cerebellum from P7-P14. Although functional significance of the patterns of torsinA and B expression in the developing brain remains to be established, our findings provide a basis for investigating the role of torsins in specific processes such as neurogenesis, neuronal migration, axon/dendrite development, and synaptogenesis.

Efficient Specification of Interneurons from Human Pluripotent Stem Cells by Dorsoventral and Rostrocaudal Modulation

GABAergic interneurons regulate cortical neural networks by providing inhibitory inputs, and their malfunction, resulting in failure to intricately regulate neural circuit balance, is implicated in brain diseases such as Schizophrenia, Autism, and Epilepsy. During early development, GABAergic interneuron progenitors arise from the ventral telencephalic area such as medial ganglionic eminence (MGE) and caudal ganglionic eminence (CGE) by the actions of secreted signaling molecules from nearby organizers, and migrate to their target sites where they form local synaptic connections. In this study, using combinatorial and temporal modulation of developmentally relevant dorsoventral and rostrocaudal signaling pathways (SHH, Wnt, and FGF8), we efficiently generated MGE cells from multiple human pluripotent stem cells. Most importantly, modulation of FGF8/FGF19 signaling efficiently directed MGE versus CGE differentiation. Human MGE cells spontaneously differentiated into Lhx6‐expressing GABAergic interneurons and showed migratory properties. These human MGE‐derived neurons generated GABA, fired action potentials, and displayed robust GABAergic postsynaptic activity. Transplantation into rodent brains results in well‐contained neural grafts enriched with GABAergic interneurons that migrate in the host and mature to express somatostatin or parvalbumin. Thus, we propose that signaling modulation recapitulating normal developmental patterns efficiently generate human GABAergic interneurons. This strategy represents a novel tool in regenerative medicine, developmental studies, disease modeling, bioassay, and drug screening. Stem Cells 2014;32:1789–1804

Autonomous vascular networks synchronize GABA neuron migration in the embryonic forebrain

GABA neurons, born in remote germinative zones in the ventral forebrain (telencephalon), migrate tangentially in two spatially distinct streams to adopt their specific positions in the developing cortex. The cell types and molecular cues that regulate this divided migratory route remains to be elucidated. Here we show that embryonic vascular networks are strategically positioned to fulfill the task of providing support as well as critical guidance cues that regulate the divided migratory routes of GABA neurons in the telencephalon. Interestingly, endothelial cells of the telencephalon are not homogeneous in their gene expression profiles. Endothelial cells of the periventricular vascular network have molecular identities distinct from those of the pial network. Our data suggest that periventricular endothelial cells have intrinsic programs that can significantly mold neuronal development and uncovers new insights into concepts and mechanisms of CNS angiogenesis from both developmental and disease perspectives.

Angiogenesis in the embryonic CNS: a new twist on an old tale.

The central nervous system (CNS) acquires its vasculature by angiogenesis, a process consisting of proliferation of endothelial cells in existing blood vessels or vascular plexuses, and leading to formation of new blood vessels. Angiogenesis begins early in CNS development and continues throughout life. Prevailing notions depict CNS angiogenesis as a passive process driven primarily by demands for oxygen and other nutrients by the growing neuronal populations. Thus, although the CNS vasculature develops concomitant with neuronal identities, which emerge under the influence of compartment-specific cell autonomous factors, cell autonomous patterning signals are not considered to instruct vascular development. We have challenged this prevailing notion by showing that angiogenesis in the mouse telencephalon progresses in an orderly, ventral-to-dorsal gradient regulated in a cell-autonomous manner by compartment-specific homeobox transcription factors. These are the same transcription factors that confer compartmental identities on telencephalic neurons and progenitor populations. Thus, the same cell-autonomous, regional patterning signals that regulate development of telencephalic neuronal networks also regulate development of telencephalic vascular networks, underscoring shared mechanisms in CNS vascular and neuronal development. These novel concepts represent a new twist in the intriguing tale of CNS angiogenesis and offer new perspectives on telencephalic regionalization and histogenesis principles.

NAD+ protects against EAE by regulating CD4+ T-cell differentiation.

CD4+ T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD+) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4+IFNγ+IL-10+ T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD+ regulates CD4+ T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD+, the frequency of T-bet−/− CD4+IFNγ+ T cells was twofold higher than wild-type CD4+ T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4+ T-cell differentiation and demonstrate that NAD+ may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases.

Basement membrane protein nidogen-1 shapes hippocampal synaptic plasticity and excitability.

The basement membrane (BM) is a specialized form of extracellular matrix (ECM) underlying epithelia and endothelia and surrounding many types of mesenchymal cells. Nidogen, along with collagen IV and laminin, is a major component of BMs. Although certain ECM proteins such as laminin or reelin influence neuronal function via interactions with cell‐surface receptors such as integrins, behavioral neurological impairments due to deficits of BM components have been recognized only recently. Here, alterations in neuronal network function underlying these behavioral changes are revealed. Using nidogen‐1 knockout mice, with or without additional heterozygous nidogen‐2 knockout (NID1−/−/NID2+/+ or NID1−/−/NID2±), we demonstrate that nidogen is essential for normal neuronal network excitability and plasticity. In nidogen‐1 knockouts, seizurelike behavior occurs, and epileptiform spiking was seen in hippocampal in vivo EEG recordings. In vitro, hippocampal field potential recordings revealed that lack of nidogen‐1, while not causing conspicuous morphological changes, led to the appearance of spontaneous and evoked epileptiform activity, significant increase of the input/output ratio of synaptically evoked responses in CA1 and dentate gyrus, as well as of paired pulse accentuation, and loss of perforant‐path long‐term synaptic potentiation. Nidogen‐1 is thus essential for normal network excitability and plasticity.

Nidogen and Nidogen-Associated Basement Membrane Proteins and Neuronal Plasticity

Extracellular matrix (ECM) proteins are thought to subserve structural functions as, for example, tissue barriers as well as guidance structures during cell growth, differentiation and tissue repair. Deletion of basement membrane (BM) components results in malformations of different organs, including the brain. Recent data, however, suggest that interference with cellular membrane-associated proteins interacting with ECM can alter neuronal excitability and synaptic plasticity without obvious underlying structural damage. This does not only apply to classical ECM proteins such as laminin, reelin and tenascin, but also to molecules of a rather specialized ECM, the BM. Here, nidogen (also termed entactin) appears to subserve a function in neuronal plasticity. Nidogen ablation leads to epileptic activity in vivo and the appearance of spontaneous epileptiform activity in vitro. This raises the intriguing question whether the BM protein nidogen may directly influence neuronal function in the CNS, opening the possibility of modulatory mechanisms of synaptic plasticity and excitability reaching beyond classical processes confined to cellular interactions.

Endothelial VEGF Sculpts Cortical Cytoarchitecture

Current models of brain development support the view that VEGF, a signaling protein secreted by neuronal cells, regulates angiogenesis and neuronal development. Here we demonstrate an autonomous and pivotal role for endothelial cell-derived VEGF that has far-reaching consequences for mouse brain development. Selective deletion of Vegf from endothelial cells resulted in impaired angiogenesis and marked perturbation of cortical cytoarchitecture. Abnormal cell clusters or heterotopias were detected in the marginal zone, and disorganization of cortical cells induced several malformations, including aberrant cortical lamination. Critical events during brain development-neuronal proliferation, differentiation, and migration were significantly affected. In addition, axonal tracts in the telencephalon were severely defective in the absence of endothelial VEGF. The unique roles of endothelial VEGF cannot be compensated by neuronal VEGF and underscores the high functional significance of endothelial VEGF for cerebral cortex development and from disease perspectives.