YAP and TAZ differentially regulate postnatal cortical progenitor proliferation and astrocyte differentiation.

WW domain-containing transcription regulator 1 (WWTR1, referred to here as TAZ) and Yes-associated protein (YAP, also known as YAP1) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway, and are best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In this study, we show in mouse neural stem and progenitor cells (NPCs) that TAZ regulates astrocytic differentiation and maturation, and that TAZ mediates some, but not all, of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, both TAZ and YAP mediate the effects on NPC fate of ß1-integrin (ITGB1) and integrin-linked kinase signaling, and these effects are dependent on extracellular matrix cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes.

YAP and TAZ differentially regulate postnatal cortical progenitor proliferation and astrocyte differentiation.

WW domain-containing transcription regulator 1 (WWTR1, referred to here as TAZ) and Yes-associated protein (YAP, also known as YAP1) are transcriptional co-activators traditionally studied together as a part of the Hippo pathway, and are best known for their roles in stem cell proliferation and differentiation. Despite their similarities, TAZ and YAP can exert divergent cellular effects by differentially interacting with other signaling pathways that regulate stem cell maintenance or differentiation. In this study, we show in mouse neural stem and progenitor cells (NPCs) that TAZ regulates astrocytic differentiation and maturation, and that TAZ mediates some, but not all, of the effects of bone morphogenetic protein (BMP) signaling on astrocytic development. By contrast, both TAZ and YAP mediate the effects on NPC fate of ß1-integrin (ITGB1) and integrin-linked kinase signaling, and these effects are dependent on extracellular matrix cues. These findings demonstrate that TAZ and YAP perform divergent functions in the regulation of astrocyte differentiation, where YAP regulates cell cycle states of astrocytic progenitors and TAZ regulates differentiation and maturation from astrocytic progenitors into astrocytes.

Ketamine's rapid and sustained antidepressant effects are driven by distinct mechanisms.

Administration of multiple subanesthetic doses of ketamine increases the duration of antidepressant effects relative to a single ketamine dose, but the mechanisms mediating this sustained effect are unclear. Here, we demonstrate that ketamine's rapid and sustained effects on affective behavior are mediated by separate and temporally distinct mechanisms. The rapid effects of a single dose of ketamine result from increased activity of immature neurons in the hippocampal dentate gyrus without an increase in neurogenesis. Treatment with six doses of ketamine over two weeks doubled the duration of behavioral effects after the final ketamine injection. However, unlike ketamine's rapid effects, this more sustained behavioral effect did not correlate with increased immature neuron activity but instead correlated with increased numbers of calretinin-positive and doublecortin-positive immature neurons. This increase in neurogenesis was associated with a decrease in bone morphogenetic protein (BMP) signaling, a known inhibitor of neurogenesis. Injection of a BMP4-expressing lentivirus into the dentate gyrus maintained BMP signaling in the niche and blocked the sustained - but not the rapid - behavioral effects of ketamine, indicating that decreased BMP signaling is necessary for ketamine's sustained effects. Thus, although the rapid effects of ketamine result from increased activity of immature neurons in the dentate gyrus without requiring an increase in neurogenesis, ketamine's sustained effects require a decrease in BMP signaling and increased neurogenesis along with increased neuron activity. Understanding ketamine's dual mechanisms of action should help with the development of new rapid-acting therapies that also have safe, reliable, and sustained effects.

miR-182 integrates apoptosis, growth, and differentiation programs in glioblastoma.

Glioblastoma multiforme (GBM) is a lethal, therapy-resistant brain cancer consisting of numerous tumor cell subpopulations, including stem-like glioma-initiating cells (GICs), which contribute to tumor recurrence following initial response to therapy. Here, we identified miR-182 as a regulator of apoptosis, growth, and differentiation programs whose expression level is correlated with GBM patient survival. Repression of Bcl2-like12 (Bcl2L12), c-Met, and hypoxia-inducible factor 2α (HIF2A) is of central importance to miR-182 anti-tumor activity, as it results in enhanced therapy susceptibility, decreased GIC sphere size, expansion, and stemness in vitro. To evaluate the tumor-suppressive function of miR-182 in vivo, we synthesized miR-182-based spherical nucleic acids (182-SNAs); i.e., gold nanoparticles covalently functionalized with mature miR-182 duplexes. Intravenously administered 182-SNAs penetrated the blood-brain/blood-tumor barriers (BBB/BTB) in orthotopic GBM xenografts and selectively disseminated throughout extravascular glioma parenchyma, causing reduced tumor burden and increased animal survival. Our results indicate that harnessing the anti-tumor activities of miR-182 via safe and robust delivery of 182-SNAs represents a novel strategy for therapeutic intervention in GBM.

Hippocampal BMP signaling as a common pathway for antidepressant action.

The benefits of current treatments for depression are limited by low response rates, delayed therapeutic effects, and multiple side effects. Antidepressants affect a variety of neurotransmitter systems in different areas of the brain, and the mechanisms underlying their convergent effects on behavior have been unclear. Here we identify hippocampal bone morphogenetic protein (BMP) signaling as a common downstream pathway that mediates the behavioral effects of five different antidepressant classes (fluoxetine, bupropion, duloxetine, vilazodone, trazodone) and of electroconvulsive therapy. All of these therapies decrease BMP signaling and enhance neurogenesis in the hippocampus. Preventing the decrease in BMP signaling blocks the effect of antidepressant treatment on behavioral phenotypes. Further, inhibition of BMP signaling in hippocampal newborn neurons is sufficient to produce an antidepressant effect, while chemogenetic silencing of newborn neurons prevents the antidepressant effect. Thus, inhibition of hippocampal BMP signaling is both necessary and sufficient to mediate the effects of multiple classes of antidepressants.

Nanofountain Probe Electroporation Enables Versatile Single-Cell Intracellular Delivery and Investigation of Postpulse Electropore Dynamics.

Introducing exogenous molecules into cells with high efficiency and dosage control is a crucial step in basic research as well as clinical applications. Here, the capability of the nanofountain probe electroporation (NFP-E) system to deliver proteins and plasmids in a variety of continuous and primary cell types with appropriate dosage control is reported. It is shown that the NFP-E can achieve fine control over the relative expression of two cotransfected plasmids. Finally, the dynamics of electropore closure after the pulsing ends with the NFP-E is investigated. Localized electroporation has recently been utilized to demonstrate the converse process of delivery (sampling), in which a small volume of the cytosol is retrieved during electroporation without causing cell lysis. Single-cell temporal sampling confers the benefit of monitoring the same cell over time and can provide valuable insights into the mechanisms underlying processes such as stem cell differentiation and disease progression. NFP-E parameters that maximize the membrane resealing time, which is essential for increasing the sampled volume and in meeting the challenge of monitoring low copy number biomarkers, are identified. Its application in CRISPR/Cas9 gene editing, stem cell reprogramming, and single-cell sampling studies is envisioned.

BMP-Responsive Protease HtrA1 Is Differentially Expressed in Astrocytes and Regulates Astrocytic Development and Injury Response.

Astrocytes perform a wide array of physiological functions, including structural support, ion exchange, and neurotransmitter uptake. Despite this diversity, molecular markers that label subpopulations of astrocytes are limited, and mechanisms that generate distinct astrocyte subtypes remain unclear. Here we identified serine protease high temperature requirement A 1 (HtrA1), a bone morphogenetic protein 4 signaling regulated protein, as a novel marker of forebrain astrocytes, but not of neural stem cells, in adult mice of both sexes. Genetic deletion of HtrA1 during gliogenesis accelerates astrocyte differentiation. In addition, ablation of HtrA1 in cultured astrocytes leads to altered chondroitin sulfate proteoglycan expression and inhibition of neurite extension, along with elevated levels of transforming growth factor-ß family proteins. Brain injury induces HtrA1 expression in reactive astrocytes, and loss of HtrA1 leads to an impairment in wound closure accompanied by increased proliferation of endothelial and immune cells. Our findings demonstrate that HtrA1 is differentially expressed in adult mouse forebrain astrocytes, and that HtrA1 plays important roles in astrocytic development and injury response.SIGNIFICANCE STATEMENT Astrocytes, an abundant cell type in the brain, perform a wide array of physiological functions. Although characterized as morphologically and functionally diverse, molecular markers that label astrocyte subtypes or signaling pathways that lead to their diversity remain limited. Here, after examining the expression profile of astrocytes generated in response to bone morphogenetic protein signaling, we identify high temperature requirement A 1 (HtrA1) as an astrocyte-specific marker that is differentially expressed in distinct adult mouse brain regions. HtrA1 is a serine protease that has been linked to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy, a small blood vessel disease in humans. Understanding the role of HtrA1 during development and after injury will provide insights into how distinct astrocyte populations are generated and their unique roles in injury and disease.

ß1-integrin restricts astrocytic differentiation of adult hippocampal neural stem cells.

Integrins are transmembrane receptors that mediate cell-extracellular matrix and cell-cell interactions. The ß1-integrin subunit is highly expressed by embryonic neural stem cells (NSCs) and is critical for NSC maintenance in the developing nervous system, but its role in the adult hippocampal niche remains unexplored. We show that ß1-integrin expression in the adult mouse dentate gyrus (DG) is localized to radial NSCs and early progenitors, but is lost in more mature progeny. Although NSCs in the hippocampal subgranular zone (SGZ) normally only infrequently differentiate into astrocytes, deletion of ß1-integrin significantly enhanced astrocyte differentiation. Ablation of ß1-integrin also led to reduced neurogenesis as well as depletion of the radial NSC population. Activation of integrin-linked kinase (ILK) in cultured adult NSCs from ß1-integrin knockout mice reduced astrocyte differentiation, suggesting that at least some of the inhibitory effects of ß1-integrin on astrocytic differentiation are mediated through ILK. In addition, ß1-integrin conditional knockout also resulted in extensive cellular disorganization of the SGZ as well as non-neurogenic regions of the DG. The effects of ß1-integrin ablation on DG structure and astrogliogenesis show sex-specific differences, with the effects following a substantially slower time-course in males. ß1-integrin thus plays a dual role in maintaining the adult hippocampal NSC population by supporting the structural integrity of the NSC niche and by inhibiting astrocytic lineage commitment. GLIA 2016;64:1235-1251.

BMP signaling regulates the tempo of adult hippocampal progenitor maturation at multiple stages of the lineage.

Novel environmental stimuli, such as running and learning, increase proliferation of adult hippocampal neural stem cells (NSCs) and enlarge the population of new neurons. However, it remains unclear how increased numbers of new neurons can be generated in a time frame far shorter than the time required for proliferating stem cells to generate these neurons. Here, we show that bone morphogenetic protein (BMP) signaling in the subgranular zone regulates the tempo of neural progenitor cell (NPC) maturation by directing their transition between states of quiescence and activation at multiple stages along the lineage. Virally mediated overexpression of BMP4 caused NPC cell cycle exit and slowed the normal maturation of NPCs, resulting in a long-term reduction in neurogenesis. Conversely, overexpression of the BMP inhibitor noggin promoted NPC cell cycle entry and accelerated NPC maturation. Similarly, BMP receptor type 2 (BMPRII) ablation in Ascl1(+) intermediate NPCs accelerated their maturation into neurons. Importantly, ablation of BMPRII in GFAP(+) stem cells accelerated maturation without depleting the NSC pool, indicating that an increased rate of neurogenesis does not necessarily diminish the stem cell population. Thus, inhibition of BMP signaling is a mechanism for rapidly expanding the pool of new neurons in the adult hippocampus by tipping the balance between quiescence/activation of NPCs and accelerating the rate at which they mature into neurons.

BMP receptor 1A regulates development of hypothalamic circuits critical for feeding behavior.

Hypothalamic neural circuits are known to regulate energy homeostasis and feeding behavior, but how these circuits are established during development is not well understood. Here we report that embryonic neural progenitors that express the transcription factor OLIG1 contribute neurons to the ventral hypothalamus including the arcuate nucleus (ARH), a center that regulates feeding behavior. Ablation of bone morphogenetic protein receptor 1a (BMPR1A) in the OLIG1 lineage resulted in hypophagia, hypoglycemia, and weight loss after the second postnatal week with death by week 4. Differentiation and specification of inhibitory hypothalamic neurons contributing to melanocortin and dopaminergic systems were abnormal in the BMPR1A-deficient ARH. Although the hypophagia promoted expression of the orexigenic neuropeptide agouti related protein (AgRP) in the BMPR1A-deficient ARH, there was a profound decrease of AgRP(+) axonal terminals in the mutant ARH targets including dorsomedial and paraventricular hypothalamic nuclei. Projection of AgRP(+) neurons to these nuclei is known to be regulated by leptin. Leptin injection in neonatal mice increased bone morphogenic protein (BMP) signaling in the ventral hypothalamus, and blocking BMP signaling prevented leptin-induced neurite outgrowth in ARH explant cultures. These findings suggest that BMPR1A signaling is critical for postnatal establishment of leptin-responsive orexigenic fibers from ARH to multiple hypothalamic nuclei. More generally these observations indicate that BMPR1A signaling regulates postnatal establishment of OLIG1 lineage-derived ARH neuronal circuits that are critical for leptin-mediated feeding behavior.

Why Some Mice Are Smarter than Others: The Impact of Bone Morphogenetic Protein Signaling on Cognition.

Inbred mice (C57Bl/6) display wide variability in performance on hippocampal-dependent cognitive tasks. Examination of microdissected dentate gyrus (DG) after cognitive testing showed a highly significant negative correlation between levels of bone morphogenetic protein (BMP) signaling and recognition memory. Cognitive performance decline during the aging process, and the degree of cognitive decline is strongly correlated with aging-related increases in BMP signaling. Further, cognitive performance was impaired when the BMP inhibitor, noggin, was knocked down in the DG. Infusion of noggin into the lateral ventricles enhanced DG-dependent cognition while BMP4 infusion led to significant impairments. Embryonic overexpression of noggin resulted in lifelong enhancement of recognition and spatial memory while overexpression of BMP4 resulted in lifelong impairment, substantiating the importance of differences in BMP signaling in wild-type mice. These findings indicate that performance in DG-dependent cognitive tasks is largely determined by differences in levels BMP signaling in the dentate gyrus.

Single cell transcriptomics reveals reduced stress response in stem cells manipulated using localized electric fields.

Membrane disruption using Bulk Electroporation (BEP) is a widely used non-viral method for delivering biomolecules into cells. Recently, its microfluidic counterpart, Localized Electroporation (LEP), has been successfully used for several applications ranging from reprogramming and engineering cells for therapeutic purposes to non-destructive sampling from live cells for temporal analysis. However, the side effects of these processes on gene expression, that can affect the physiology of sensitive stem cells are not well understood. Here, we use single cell RNA sequencing (scRNA-seq) to investigate the effects of BEP and LEP on murine neural stem cell (NSC) gene expression. Our results indicate that unlike BEP, LEP does not lead to extensive cell death or activation of cell stress response pathways that may affect their long-term physiology. Additionally, our demonstrations show that LEP is suitable for multi-day delivery protocols as it enables better preservation of cell viability and integrity as compared to BEP.

Ketamine activates adult-born immature granule neurons to rapidly alleviate depression-like behaviors in mice.

Ketamine treatment decreases depressive symptoms within hours, but the mechanisms mediating these rapid antidepressant effects are unclear. Here, we demonstrate that activity of adult-born immature granule neurons (ABINs) in the mouse hippocampal dentate gyrus is both necessary and sufficient for the rapid antidepressant effects of ketamine. Ketamine treatment activates ABINs in parallel with its behavioral effects in both stressed and unstressed mice. Chemogenetic inhibition of ABIN activity blocks the antidepressant effects of ketamine, indicating that this activity is necessary for the behavioral effects. Conversely, chemogenetic activation of ABINs without any change in neuron numbers mimics both the cellular and the behavioral effects of ketamine, indicating that increased activity of ABINs is sufficient for rapid antidepressant effects. These findings thus identify a specific cell population that mediates the antidepressant actions of ketamine, indicating that ABINs can potentially be targeted to limit ketamine's side effects while preserving its therapeutic efficacy.

Notch and MAML signaling drives Scl-dependent interneuron diversity in the spinal cord.

The ventral spinal cord generates multiple inhibitory and excitatory interneuron subtypes from four cardinal progenitor domains (p0, p1, p2, p3). Here we show that cell-cell interactions mediated by the Notch receptor play a critical evolutionarily conserved role in the generation of excitatory v2aIN and inhibitory v2bIN interneurons. Lineage-tracing experiments show that the v2aIN and v2bIN develop from genetically identical p2 progenitors. The p2 daughter cell fate is controlled by Delta4 activation of Notch receptors together with MAML factors. Cells receiving Notch signals activate a transcription factor code that specifies the v2bIN fate, whereas cells deprived of Notch signaling express another code for v2aIN formation. Thus, our study provides insight into the cell-extrinsic signaling that controls combinatorial transcription factor profiles involved in regulating the process of interneuron subtype diversification.

Pluripotent stem cell-derived endometrial stromal fibroblasts in a cyclic, hormone-responsive, coculture model of human decidua.

Various human diseases and pregnancy-related disorders reflect endometrial dysfunction. However, rodent models do not share fundamental biological processes with the human endometrium, such as spontaneous decidualization, and no existing human cell cultures recapitulate the cyclic interactions between endometrial stromal and epithelial compartments necessary for decidualization and implantation. Here we report a protocol differentiating human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs) that are highly pure and able to decidualize. Coculture of PSC-ESFs with placenta-derived endometrial epithelial cells generated organoids used to examine stromal-epithelial interactions. Cocultures exhibited specific endometrial markers in the appropriate compartments, organization with cell polarity, and hormone responsiveness of both cell types. Furthermore, cocultures recapitulate a central feature of the human decidua by cyclically responding to hormone withdrawal followed by hormone retreatment. This advance enables mechanistic studies of the cyclic responses that characterize the human endometrium.

Establishment of human induced trophoblast stem-like cells from term villous cytotrophoblasts.

Human trophoblast stem cells (hTSC) can be isolated from first trimester placenta but not from term placenta. Here we demonstrate that villous cytotrophoblasts (vCTB) from term placenta can be reprogrammed into induced trophoblastic stem-like cells (iTSC) by introducing sets of transcription factors. The iTSCs express TSC markers such as GATA3, TEAD4 and ELF5, and are multipotent, validated by their differentiation into both extravillous trophoblasts (EVT) and syncytiotrophoblasts (STB) in vitro and in vivo. The iTSC can be passaged indefinitely in vitro without slowing of growth. The transcriptome profile of these cells closely resembles the profile of hTSC isolated from first trimester placentae but different from the term placental vCTB from which they originated. The ability to reprogram cells from term placenta into iTSC will allow study of early gestation events which impact placental function later in gestation, including preeclampsia and spontaneous preterm birth.

Noggin expands neural stem cells in the adult hippocampus.

New neurons are added to the adult hippocampus throughout life and contribute to cognitive functions, including learning and memory. It remains unclear whether ongoing neurogenesis arises from self-renewing neural stem cells (NSCs) or from multipotential progenitor cells that cannot self-renew in the hippocampus. This is primarily based on observations that neural precursors derived from the subventricular zone (SVZ) can be passaged long term, whereas hippocampal subgranular zone (SGZ) precursors are rapidly depleted by passaging. We demonstrate here that high levels of bone morphogenetic protein (BMP) signaling occur in hippocampal but not SVZ precursors in vitro, and blocking BMP signaling with Noggin is sufficient to foster hippocampal cell self-renewal, proliferation, and multipotentiality using single-cell clonal analysis. Moreover, NSC maintenance requires continual Noggin exposure, which implicates BMPs as crucial regulators of NSC aging. In vivo, Noggin is expressed in the adult dentate gyrus and limits BMP signaling in proliferative cells of the SGZ. Transgenic Noggin overexpression in the SGZ increases multiple precursor cell populations but proportionally increases the glial fibrillary acidic protein-positive cell population at the expense of other precursors, suggesting that Noggin acts on NSCs in vivo. To confirm this, we used a dual thymidine analog paradigm to repeatedly label slowly dividing cells over a long duration. We find that small populations of label-retaining cells exist in the SGZ and that Noggin overexpression increases their numbers. Thus, we propose that the adult hippocampus contains a population of NSCs, which can be expanded both in vitro and in vivo by blocking BMP signaling.

Activating newborn neurons suppresses depression and anxiety-like behaviors.

The etiology of major depressive disorder (MDD), the leading cause of worldwide disability, is unknown. The neurogenic hypothesis proposes that MDD is linked to impairments of adult neurogenesis in the hippocampal dentate gyrus (DG), while the effects of antidepressants are mediated by increased neurogenesis. However, alterations in neurogenesis and endophenotypes are not always causally linked, and the relationship between increased neurogenesis and altered behavior is controversial. To address causality, we used chemogenetics in transgenic mice to selectively manipulate activity of newborn DG neurons. Suppressing excitability of newborn neurons without altering neurogenesis abolish the antidepressant effects of fluoxetine. Remarkably, activating these neurons is sufficient to alleviate depression-like behavior and reverse the adverse effects of unpredictable chronic mild stress. Our results demonstrate a direct causal relationship between newborn neuronal activity and affective behavior. Thus, strategies that target not only neurogenesis but also activity of newborn neurons may lead to more effective antidepressants.

Increased bone morphogenetic protein signaling contributes to age-related declines in neurogenesis and cognition.

Aging is associated with decreased neurogenesis in the hippocampus and diminished hippocampus-dependent cognitive functions. Expression of bone morphogenetic protein 4 (BMP4) increases with age by more than 10-fold in the mouse dentate gyrus while levels of the BMP inhibitor, noggin, decrease. This results in a profound 30-fold increase in phosphorylated-SMAD1/5/8, the effector of canonical BMP signaling. Just as observed in mice, a profound increase in expression of BMP4 is observed in the dentate gyrus of humans with no known cognitive abnormalities. Inhibition of BMP signaling either by overexpression of noggin or transgenic manipulation not only increases neurogenesis in aging mice, but remarkably, is associated with a rescue of cognitive deficits to levels comparable to young mice. Additive benefits are observed when combining inhibition of BMP signaling and environmental enrichment. These findings indicate that increased BMP signaling contributes significantly to impairments in neurogenesis and to cognitive decline associated with aging, and identify this pathway as a potential druggable target for reversing age-related changes in cognition.