Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia-cell cycle dual reporter.

Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring ("non-Warburg") cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic "non-Warburg" cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity.

Seamless reconstruction of intact adult-born neurons by serial end-block imaging reveals complex axonal guidance and development in the adult hippocampus.

In the adult mammalian hippocampus, newborn dentate granule cells are continuously integrated into the existing circuitry and contribute to specific brain functions. Little is known about the axonal development of these newborn neurons in the adult brain due to technological challenges that have prohibited large-scale reconstruction of long, thin, and complex axonal processes within the mature nervous system. Here, using a new serial end-block imaging (SEBI) technique, we seamlessly reconstructed axonal and dendritic processes of intact individual retrovirus-labeled newborn granule cells at different developmental stages in the young adult mouse hippocampus. We found that adult-born dentate granule cells exhibit tortuous, yet highly stereotyped, axonal projections to CA3 hippocampal subregions. Primary axonal projections of cohorts of new neurons born around the same time organize into laminar patterns with staggered terminations that stack along the septo-temporal hippocampal axis. Analysis of individual newborn neuron development further defined an initial phase of rapid axonal and dendritic growth within 21 d after newborn neuron birth, followed by minimal growth of primary axonal and whole dendritic processes through the last time point examined at 77 d. Our results suggest that axonal development and targeting is a highly orchestrated, precise process in the adult brain. These findings demonstrate a striking regenerative capacity of the mature CNS to support long-distance growth and guidance of neuronal axons. Our SEBI approach can be broadly applied for analysis of intact, complex neuronal projections in limitless tissue volume.

Subventricular zone localized irradiation affects the generation of proliferating neural precursor cells and the migration of neuroblasts.

Radiation therapy is a part of the standard treatment for brain tumor patients, often resulting in irreversible neuropsychological deficits. These deficits may be due to permanent damage to the neural stem cell (NSC) niche, damage to local neural progenitors, or neurotoxicity. Using a computed tomography-guided localized radiation technique, we studied the effects of radiation on NSC proliferation and neuroblast migration in the mouse brain. Localized irradiation of the subventricular zone (SVZ) eliminated the proliferating neural precursor cells and migrating neuroblasts. After irradiation, type B cells in the SVZ lacked the ability to generate migrating neuroblasts. Neuroblasts from the unirradiated posterior SVZ did not follow their normal migratory path through the irradiated anterior SVZ. Our results indicate that the migrating neuroblasts were not replenished, despite the presence of type B cells in the SVZ post-irradiation. This study provides novel insights into the effects of localized SVZ radiation on neurogenesis and cell migration that may potentially lead to the development of new radiotherapy strategies to minimize damage to NSCs and neuroblast migration.

GABA regulates synaptic integration of newly generated neurons in the adult brain.

Adult neurogenesis, the birth and integration of new neurons from adult neural stem cells, is a striking form of structural plasticity and highlights the regenerative capacity of the adult mammalian brain. Accumulating evidence suggests that neuronal activity regulates adult neurogenesis and that new neurons contribute to specific brain functions. The mechanism that regulates the integration of newly generated neurons into the pre-existing functional circuitry in the adult brain is unknown. Here we show that newborn granule cells in the dentate gyrus of the adult hippocampus are tonically activated by ambient GABA (gamma-aminobutyric acid) before being sequentially innervated by GABA- and glutamate-mediated synaptic inputs. GABA, the major inhibitory neurotransmitter in the adult brain, initially exerts an excitatory action on newborn neurons owing to their high cytoplasmic chloride ion content. Conversion of GABA-induced depolarization (excitation) into hyperpolarization (inhibition) in newborn neurons leads to marked defects in their synapse formation and dendritic development in vivo. Our study identifies an essential role for GABA in the synaptic integration of newly generated neurons in the adult brain, and suggests an unexpected mechanism for activity-dependent regulation of adult neurogenesis, in which newborn neurons may sense neuronal network activity through tonic and phasic GABA activation.

The neuropeptide VIP potentiates intestinal innate type 2 and type 3 immunity in response to feeding.

The nervous system and the immune system both rely on an extensive set of modalities to perceive and act on perturbations in the internal and external environments. During feeding, the intestine is exposed to nutrients that may contain noxious substances and pathogens. Here we show that Vasoactive Intestinal Peptide (VIP), produced by the nervous system in response to feeding, potentiates the production of effector cytokines by intestinal type 2 and type 3 innate lymphoid cells (ILC2s and ILC3s). Exposure to VIP alone leads to modest activation of ILCs, but strongly potentiates ILCs to concomitant or subsequent activation by the inducer cytokines IL-33 or IL-23, via mobilization of cAMP and energy by glycolysis. Consequently, VIP increases resistance to intestinal infection by the helminth Trichuris muris and the enterobacteria Citrobacter rodentium. These findings uncover a functional neuro-immune crosstalk unfolding during feeding that increases the reactivity of innate immunity necessary to face potential threats associated with food intake.

Hematopoietic stem cell transplantation chemotherapy causes microglia senescence and peripheral macrophage engraftment in the brain.

Hematopoietic stem cell transplantation (HSCT) is a therapy used for multiple malignant and nonmalignant diseases, with chemotherapy used for pretransplantation myeloablation. The post-HSCT brain contains peripheral engrafted parenchymal macrophages, despite their absence in the normal brain, with the engraftment mechanism still undefined. Here we show that HSCT chemotherapy broadly disrupts mouse brain regenerative populations, including a permanent loss of adult neurogenesis. Microglial density was halved, causing microglial process expansion, coinciding with indicators of broad senescence. Although microglia expressed cell proliferation markers, they underwent cell cycle arrest in S phase with a majority expressing the senescence and antiapoptotic marker p21. In vivo single-cell tracking of microglia after recovery from chemical depletion showed loss of their regenerative capacity, subsequently replaced with donor macrophages. We propose that HSCT chemotherapy causes microglial senescence with a gradual decrease to a critical microglial density, providing a permissive niche for peripheral macrophage engraftment of the brain.

Development of hippocampal mossy fiber synaptic outputs by new neurons in the adult brain.

New neurons are continuously generated in restricted regions of the adult mammalian brain. Although these adult-born neurons have been shown to receive synaptic inputs, little is known about their synaptic outputs. Using retrovirus-mediated birth-dating and labeling in combination with serial section electron microscopic reconstruction, we report that mossy fiber en passant boutons of adult-born dentate granule cells form initial synaptic contacts with CA3 pyramidal cells within 2 weeks after their birth and reach morphologic maturity within 8 weeks in the adult hippocampus. Knockdown of Disrupted-in-Schizophrenia-1 (DISC1) in newborn granule cells leads to defects in axonal targeting and development of synaptic outputs in the adult brain. Together with previous reports of synaptic inputs, these results demonstrate that adult-born neurons are fully integrated into the existing neuronal circuitry. Our results also indicate a role for DISC1 in presynaptic development and may have implications for the etiology of schizophrenia and related mental disorders.

New Approach to Accelerated Image Annotation by Leveraging Virtual Reality and Cloud Computing.

Three-dimensional imaging is at the core of medical imaging and is becoming a standard in biological research. As a result, there is an increasing need to visualize, analyze and interact with data in a natural three-dimensional context. By combining stereoscopy and motion tracking, commercial virtual reality (VR) headsets provide a solution to this critical visualization challenge by allowing users to view volumetric image stacks in a highly intuitive fashion. While optimizing the visualization and interaction process in VR remains an active topic, one of the most pressing issue is how to utilize VR for annotation and analysis of data. Annotating data is often a required step for training machine learning algorithms. For example, enhancing the ability to annotate complex three-dimensional data in biological research as newly acquired data may come in limited quantities. Similarly, medical data annotation is often time-consuming and requires expert knowledge to identify structures of interest correctly. Moreover, simultaneous data analysis and visualization in VR is computationally demanding. Here, we introduce a new procedure to visualize, interact, annotate and analyze data by combining VR with cloud computing. VR is leveraged to provide natural interactions with volumetric representations of experimental imaging data. In parallel, cloud computing performs costly computations to accelerate the data annotation with minimal input required from the user. We demonstrate multiple proof-of-concept applications of our approach on volumetric fluorescent microscopy images of mouse neurons and tumor or organ annotations in medical images.

XTRPC1-dependent chemotropic guidance of neuronal growth cones.

Calcium arising through release from intracellular stores and from influx across the plasma membrane is essential for signalling by specific guidance cues and by factors that inhibit axon regeneration. The mediators of calcium influx in these cases are largely unknown. Transient receptor potential channels (TRPCs) belong to a superfamily of Ca2+-permeable, receptor-operated channels that have important roles in sensing and responding to changes in the local environment. Here we report that XTRPC1, a Xenopus homolog of mammalian TRPC1, is required for proper growth cone turning responses of Xenopus spinal neurons to microscopic gradients of netrin-1, brain-derived neurotrophic factor and myelin-associated glycoprotein, but not to semaphorin 3A. Furthermore, XTRPC1 is required for midline guidance of axons of commissural interneurons in the developing Xenopus spinal cord. Thus, members of the TRPC family may serve as a key mediator for the Ca2+ influx that regulates axon guidance during development and inhibits axon regeneration in adulthood.

Synaptic integration and plasticity of new neurons in the adult hippocampus.

Adult neurogenesis, a developmental process encompassing the birth of new neurons from adult neural stem cells and their integration into the existing neuronal circuitry, highlights the plasticity and regenerative capacity of the adult mammalian brain. Substantial evidence suggests essential roles of newborn neurons in specific brain functions; yet it remains unclear how these new neurons make their unique contribution. Recently, a series of studies have delineated the basic steps of the adult neurogenesis process and shown that many of the distinct steps are dynamically regulated by the activity of the existing circuitry. Here we review recent findings on the synaptic integration and plasticity of newborn neurons in the adult hippocampus, including the basic biological process, unique characteristics, critical periods, and activity-dependent regulation by the neurotransmitters GABA and glutamate. We propose that adult neurogenesis represents not merely a replacement mechanism for lost neurons, but also an ongoing developmental process in the adult brain that offers an expanded capacity for plasticity for shaping the existing circuitry in response to experience throughout life.

Youth Comes But Once in a Lifetime for Adult-Born Neurons.

Using new methods to functionally dissect circuits, two papers from 2015 found enhanced synaptic properties of the inputs and outputs of hippocampal adult-born neurons specifically during a critical period of their development. These studies provided a circuit-level view of unique roles for new neurons and how they cope with the ever-changing environment.

Monocyte chemoattractant protein-1 plays a critical role in neuroblast migration after focal cerebral ischemia.

Transient focal ischemia is known to induce proliferation of neural progenitors in adult rodent brain. We presently report that doublecortin positive neuroblasts formed in the subventricular zone (SVZ) and the posterior peri-ventricle region migrate towards the cortical and striatal penumbra after transient middle cerebral artery occlusion (MCAO) in adult rodents. Cultured neural progenitor cells grafted into the non-infarcted area of the ipsilateral cortex migrated preferentially towards the infarct. As chemokines are known to induce cell migration, we investigated if monocyte chemoattractant protein-1 (MCP-1) has a role in post-ischemic neuroblast migration. Transient MCAO induced an increased expression of MCP-1 mRNA in the ipsilateral cortex and striatum. Immunostaining showed that the expression of MCP-1 was localized in the activated microglia and astrocytes present in the ischemic areas between days 1 and 3 of reperfusion. Furthermore, infusion of MCP-1 into the normal striatum induced neuroblast migration to the infusion site. The migrating neuroblasts expressed the MCP-1 receptor CCR2. In knockout mice that lacked either MCP-1 or its receptor CCR2, there was a significant decrease in the number of migrating neuroblasts from the ipsilateral SVZ to the ischemic striatum. These results show that MCP-1 is one of the factors that attract the migration of newly formed neuroblasts from neurogenic regions to the damaged regions of brain after focal ischemia.

Adult neurogenesis and hippocampal memory function: new cells, more plasticity, new memories?

The discovery of active adult neurogenesis in mammals, a process of generating functional neurons from neural stem cells, suggests that the adult brain is more dynamic than once imagined. The coincidence of this phenomenon occurring in the hippocampus, a region critical to the learning process, begs the question of whether adult neurogenesis is involved in memory formation. Here, the authors review rapidly accumulating evidence showing a strong correlation between certain types of memory functions and adult neurogenesis in the hippocampus. Establishment of the potential link between memory formation and adult neurogenesis is instrumental, at a basic science level, to understand the function of neural networks and is essential, at a clinical level, to develop effective therapies for various cognitive dysfunctions.

Adult neurogenesis beyond the niche: its potential for driving brain plasticity.

Adult neurogenesis emerges as a tremendous form of plasticity with the continuous addition and loss of neurons in the adult brain. It is unclear how preexisting adult circuits generated during development are capable of modifying existing connections to accommodate the thousands of new synapses formed and exchanged each day. Here we first make parallels with sensory deprivation studies and its ability to induce preexisting non-neurogenic adult circuits to undergo massive reorganization. We then review recent studies that show high structural and synaptic plasticity in circuits directly connected to adult-born neurons. Finally, we propose future directions in the field to decipher how host circuits can accommodate new neuron integration and to determine the impact of adult neurogenesis on global brain plasticity.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by nicotinic acetylcholine receptor (nAChR) activation by cholinergic inputs. PFC spontaneous default activity is altered in neuropsychiatric disorders, including schizophrenia-a disorder that can be accompanied by heavy smoking. Recently, genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, encoding the α5 nAChR subunit, that increase the risks for both smoking and schizophrenia. Mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but it is unknown how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying behavior. Here we show that mice expressing a human α5 SNP exhibit neurocognitive behavioral deficits in social interaction and sensorimotor gating tasks. Two-photon calcium imaging in awake mouse models showed that nicotine can differentially influence PFC pyramidal cell activity by nAChR modulation of layer II/III hierarchical inhibitory circuits. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive over layer II/III pyramidal neurons. The decreased activity observed in α5-SNP-expressing mice resembles the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine administration reversed this hypofrontality, suggesting that administration of nicotine may represent a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking.

Persistent Structural Plasticity Optimizes Sensory Information Processing in the Olfactory Bulb.

In the mammalian brain, the anatomical structure of neural circuits changes little during adulthood. As a result, adult learning and memory are thought to result from specific changes in synaptic strength. A possible exception is the olfactory bulb (OB), where activity guides interneuron turnover throughout adulthood. These adult-born granule cell (GC) interneurons form new GABAergic synapses that have little synaptic strength plasticity. In the face of persistent neuronal and synaptic turnover, how does the OB balance flexibility, as is required for adapting to changing sensory environments, with perceptual stability? Here we show that high dendritic spine turnover is a universal feature of GCs, regardless of their developmental origin and age. We find matching dynamics among postsynaptic sites on the principal neurons receiving the new synaptic inputs. We further demonstrate in silico that this coordinated structural plasticity is consistent with stable, yet flexible, decorrelated sensory representations. Together, our study reveals that persistent, coordinated synaptic structural plasticity between interneurons and principal neurons is a major mode of functional plasticity in the OB.

Preconditioning-induced ischemic tolerance stimulates growth factor expression and neurogenesis in adult rat hippocampus.

Preconditioning (PC) is a phenomenon in which a brief ischemic insult induces tolerance against a subsequent severe ischemic insult. Recent studies showed that cerebral ischemia in adult rat upregulates progenitor cell proliferation in the hippocampal dentate gyrus. We presently evaluated whether PC can also stimulate progenitor cell proliferation in rat brain. Middle cerebral artery was transiently occluded in spontaneously hypertensive rats for 10 min to induce PC and 1h to induce focal ischemia. Progenitor cell proliferation (defined as BrdU(+) cell number) significantly increased after focal ischemia (by 3.9-fold; p<0.05) as well as PC (by 2.7-fold; p<0.05) compared to sham. PC 3 days prior had neither an inhibitory nor an additive effect on focal ischemia-induced progenitor cell proliferation. In both ischemia and PC groups, approximately 45% of the progenitor cells proliferated in week 1 survived to the end of week 3 and approximately 21% of those matured into NeuN(+) neurons. Furthermore, cerebral mRNA expression of the growth factors IGF1, FGF2, TGFbeta1, EGF and PDGF-A was significantly elevated after PC. Thus, we show that the beneficial effects of PC extend beyond providing neuroprotection during the acute phase after ischemia. Induction of growth factor expression and neurogenesis by PC might be a positive adaptation for an efficient repair and plasticity in the event of an ischemic insult.

Infarct volume quantification in mouse focal cerebral ischemia: a comparison of triphenyltetrazolium chloride and cresyl violet staining techniques.

Triphenyltetrazolium chloride (TTC) and cresyl violet (CV) staining are routinely used methods to determine cerebral infarct volume and area. In this study, we compared these staining techniques using the mouse middle cerebral artery occlusion (MCAO) model of focal ischemia. Male C57BL6 mice were subjected to a 90 min transient MCAO and sacrificed at 24 h reperfusion. Sham operated mice served as controls. Two millimeters coronal brain slices were cut at +1.3, -0.7, -2.7 and -4.7 mm from bregma. The sections were stained with 2% TTC for 20 min and the caudal face of each slice was scanned with a flatbed scanner. The sections were kept in 4% paraformaldehyde solution for 4 weeks (the solution was changed every week). The slices were cryosectioned (40 microm thick), mounted on slides and stained with CV and scanned. The infarct volume and area were measured by the image-J program for both the staining techniques. There was no significant difference in either infarct area or volume between the TTC and CV stained sections (P > 0.05). TTC and CV staining showed a high degree of correlation in infarct area and volume indicating that both methods are suitable for producing accurate measurements of cerebral experimental infarcts.

Polyamines and central nervous system injury: spermine and spermidine decrease following transient focal cerebral ischemia in spontaneously hypertensive rats.

Polyamines (putrescine, spermidine and spermine) are ubiquitous cellular components, but their specific role in central nervous system (CNS) injury has yet to be characterized. CNS injury results in increased activities of ornithine decarboxylase and spermidine/spermine-N(1)-acetyltransferase, and accumulation of putrescine. The present study determined the polyamine profile in three models of CNS injury, in two different species (gerbil and rat) and two strains of rats (Sprague-Dawley and spontaneously hypertensive): (1) transient focal cerebral ischemia in spontaneously hypertensive rats (SHR); (2) traumatic brain injury in Sprague-Dawley rats; and (3) transient forebrain ischemia in gerbils. While there was a significant increase in putrescine in all three models, spermine and spermidine levels were unaltered in forebrain ischemia and traumatic brain injury. However, transient focal cerebral ischemia shows depletion of spermine and spermidine levels in injured hemisphere compared to contralateral region. Exogenous spermine significantly restored the spermine as well as spermidine levels in the ipsilateral hemisphere after transient focal cerebral ischemia, but did not alter putrescine levels or the ratio of spermidine to spermine. The loss of spermine in particular, may have several consequences that contribute to ischemic injury, including destabilization of chromatin, decreased mitochondrial Ca(2+) buffering capacity, and increased susceptibility to oxidative stress. Based on our and other studies, we propose a tentative antioxidant mechanism of spermine neuroprotection.