Astrocyte-conditioned Medium Research Articles

Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against Aβ Oligomers in Alzheimer's Disease Model.

Alzheimer's disease (AD) is characterized by progressive cognitive decline, increasingly attributed to neuronal dysfunction induced by amyloid-β oligomers (AβOs). Although the impact of AβOs on neurons has been extensively studied, only recently have the possible effects of AβOs on astrocytes begun to be investigated. Given the key roles of astrocytes in synapse formation, plasticity, and function, we sought to investigate the impact of AβOs on astrocytes, and to determine whether this impact is related to the deleterious actions of AβOs on synapses. We found that AβOs interact with astrocytes, cause astrocyte activation and trigger abnormal generation of reactive oxygen species, which is accompanied by impairment of astrocyte neuroprotective potential in vitro We further show that both murine and human astrocyte conditioned media (CM) increase synapse density, reduce AβOs binding, and prevent AβO-induced synapse loss in cultured hippocampal neurons. Both a neutralizing anti-transforming growth factor-β1 (TGF-β1) antibody and siRNA-mediated knockdown of TGF-β1, previously identified as an important synaptogenic factor secreted by astrocytes, abrogated the protective action of astrocyte CM against AβO-induced synapse loss. Notably, TGF-β1 prevented hippocampal dendritic spine loss and memory impairment in mice that received an intracerebroventricular infusion of AβOs. Results suggest that astrocyte-derived TGF-β1 is part of an endogenous mechanism that protects synapses against AβOs. By demonstrating that AβOs decrease astrocyte ability to protect synapses, our results unravel a new mechanism underlying the synaptotoxic action of AβOs in AD.SIGNIFICANCE STATEMENT Alzheimer's disease is characterized by progressive cognitive decline, mainly attributed to synaptotoxicity of the amyloid-β oligomers (AβOs). Here, we investigated the impact of AβOs in astrocytes, a less known subject. We show that astrocytes prevent synapse loss induced by AβOs, via production of transforming growth factor-β1 (TGF-β1). We found that AβOs trigger morphological and functional alterations in astrocytes, and impair their neuroprotective potential. Notably, TGF-β1 reduced hippocampal dendritic spine loss and memory impairment in mice that received intracerebroventricular infusions of AβOs. Our results describe a new mechanism underlying the toxicity of AβOs and indicate novel therapeutic targets for Alzheimer's disease, mainly focused on TGF-β1 and astrocytes.

Estrogen and Progesterone Integration in an in vitro Model of RP3V Kisspeptin Neurons

Positive feedback on gonadotropin release requires not only estrogen but also progesterone to activate neural circuits. In rodents, ovarian estradiol (E2) stimulates progesterone synthesis in hypothalamic astrocytes (neuroP), needed for the luteinizing hormone (LH) surge. Kisspeptin (kiss) neurons are the principal stimulators of gonadotropin-releasing hormone neurons, and disruption of kiss signaling abrogates the LH surge. Similarly, blocking steroid synthesis in the hypothalamus or deleting classical progesterone receptor (PGR) selectively in kiss neurons prevents the LH surge. These results suggest a synergistic action of E2 and progesterone in kiss neurons to affect gonadotropin release. The mHypoA51, immortalized kiss-expressing neuronal cell line derived from adult female mice, is a tractable model for examining integration of steroid signaling underlying estrogen positive feedback. Here, we report that kiss neurons in vitro integrate E2 and progesterone signaling to increase levels of kiss translation and release. mHypoA51 neurons expressed nonclassical membrane progesterone receptors (mPRα and mPRβ) and E2-inducible PGR, required for progesterone-augmentation of E2-induced kiss expression. With astrocyte-conditioned media or in mHypoA51-astrocyte co-culture, neuroP augmented stimulatory effects of E2 on kiss protein. Progesterone activation of classical, membrane-localized PGR led to activation of MAPK and Src kinases. Importantly, progesterone or Src activation induced release of kiss from E2-primed mHypoA51 neurons. Consistent with previous studies, the present results provide compelling evidence that the interaction of E2 and progesterone stimulates kiss expression and release. Further, these results demonstrate a mechanism though which peripheral E2 may prime kiss neurons to respond to neuroP, mediating estrogen positive feedback.

CD38 positively regulates postnatal development of astrocytes cell-autonomously and oligodendrocytes non-cell-autonomously.

Glial development is critical for the function of the central nervous system. CD38 is a multifunctional molecule with ADP-ribosyl cyclase activity. While critical roles of CD38 in the adult brain such as oxytocin release and social behavior have been reported, those in the developing brain remain largely unknown. Here we demonstrate that deletion of Cd38 leads to impaired development of astrocytes and oligodendrocytes in mice. CD38 is highly expressed in the developing brains between postnatal day 14 (P14) and day 28 (P28). In situ hybridization and FACS analysis revealed that CD38 is expressed predominantly in astrocytes in these periods. Analyses of the cortex of Cd38 knockout (Cd38-/- ) mice revealed delayed development of astrocytes and subsequently delayed differentiation of oligodendrocytes (OLs) at postnatal stages. In vitro experiments using primary OL cultures, mixed glial cultures, and astrocytic conditioned medium showed that astrocytic CD38 regulates the development of astrocytes in a cell-autonomous manner and the differentiation of OLs in a non-cell-autonomous manner. Further experiments revealed that connexin43 (Cx43) in astrocytes plays a promotive role for CD38-mediated OL differentiation. Finally, increased levels of NAD+ , caused by CD38 deficiency, are likely to be responsible for the suppression of astrocytic Cx43 expression and OL differentiation. Our data indicate that CD38 is a positive regulator of astrocyte and OL development.

Abstract WP103: ABCA1/HDL Signaling Pathway Mediates White Matter Remodeling and Improves Functional Outcome After Stroke

The ATP-binding cassette transporter A1 (ABCA1) plays a critical role in the formation of brain HDL cholesterol, mainly from astrocytes. Specific brain ABCA1 deficient (ABCA1 -B/-B ) mice exhibit reduced HDL levels in brain tissue and cerebrospinal fluid, as well as increases in white matter (WM) damage and neurological deficits after stroke. We tested the hypothesis, that treatment of stroke in ABCA1 -B/-B mice with intraventricularly infused HDL increases WM remodeling in the ischemic brain and attenuates ABCA1 -B/-B induced deficits after stroke. Method: Adult male ABCA1 -B/-B mice were subjected to permanent right distal middle cerebral artery occlusion (dMCAo), and were intraventricularly infused with PBS or recombinant human HDL3 (rhHDL3, 25μg in 100μl PBS) by transplanting a micro-osmotic pump (D1002, 0.25 μl/h for 14 days, Alzet) into the right lateral ventricle starting 24h after dMCAo. Animals were sacrificed 21 days after dMCAo. In vitro primary cortical neuron (PCN) culture derived from C57BL/6 and ABCA1 -B/-B embryos and axonal outgrowth measurements were also employed. Results: 1) There were no ischemic lesion volume changes between the two groups. 2) rhHDL3-infused ABCA1 -B/-B stroke mice exhibited significantly increased WM-remodeling identified by increased level of myelin basic protein, increased densities of myelin and axons, increased number of oligodendrocytes and oligodendrocyte progenitor cells in the ischemic boundary zone, as well as improved functional outcome compared with PBS-infused ABCA1 -B/-B stroke mice (p<0.05, n=8/group). 3) HDL (40 or 80μg/ml) treatment in ABCA1 -B/-B -PCNs significantly increased axonal outgrowth compared to non-treatment control. Treatment of C57BL/6-PCNs with astrocyte-conditioned media derived from ABCA1 -B/-B embryos significantly decreased axonal outgrowth compared to treatment with astrocyte-conditioned media derived from ABCA1 floxed-control embryos. However, ABCA1 -B/-B -astrocyte-conditioned media combination with HDL treatment significantly increased C57BL/6-PCN axonal outgrowth compared to treatment with ABCA1 -B/-B -astrocyte-conditioned media alone. Conclusion: ABCA1/HDL pathway may contribute to WM-remodeling as well as functional recovery after stroke.

An improved method for growing neurons: Comparison with standard protocols

BackgroundSince different culturing parameters – such as media composition or cell density – lead to different experimental results, it is important to define the protocol used for neuronal cultures. The vital role of astrocytes in maintaining homeostasis of neurons – both in vivo and in vitro – is well established: the majority of improved culturing conditions for primary dissociated neuronal cultures rely on astrocytes. New methodOur culturing protocol is based on a novel serum-free preparation of astrocyte – conditioned medium (ACM). We compared the proposed ACM culturing method with other two commonly used methods Neurobasal/B27- and FBS- based media. We performed morphometric characterization by immunocytochemistry and functional analysis by calcium imaging for all three culture methods at 1, 7, 14 and 60days in vitro (DIV). ResultsACM-based cultures gave the best results for all tested criteria, i.e. growth cone’s size and shape, neuronal outgrowth and branching, network activity and synchronization, maturation and long-term survival. The differences were more pronounced when compared with FBS-based medium. Neurobasal/B27 cultures were comparable to ACM for young cultures (DIV1), but not for culturing times longer than DIV7. Comparison with existing method(s)ACM-based cultures showed more robust neuronal outgrowth at DIV1. At DIV7 and 60, the activity of neuronal network grown in ACM had a more vigorous spontaneous electrical activity and a higher degree of synchronization. ConclusionsWe propose our ACM-based culture protocol as an improved and more suitable method for both short- and long-term neuronal cultures.

Heterogeneity in Synaptogenic Profile of Astrocytes from Different Brain Regions.

Astrocytes, the most abundant glial cells in the central nervous system (CNS), comprise a heterogeneous population of cells. However, how this heterogeneity impacts their function within brain homeostasis and response to injury and disease is still largely unknown. Recently, astrocytes have been recognized as important regulators of synapse formation and maturation. Here, we analyzed the synaptogenic property of astrocytes from different regions of the CNS. The effect of conditioned medium derived from astrocytes (astrocyte-conditioned medium (ACM)) from cerebral cortex, hippocampus, midbrain and cerebellum, in synapse formation, was evaluated. Synapse formation was analyzed by quantification of pre- and postsynaptic proteins, synaptophysin, and postsynaptic density protein 95 (PSD-95). ACM from the four regions increased significantly the number of synaptophysin/PSD-95 puncta on neurons from the same and different brain regions. Differences on astrocytic synaptogenic potential between the regions were observed according to ACM protein concentration. Thus, cerebellar astrocytes have higher synaptogenic effect when ACM is less concentrated. Also, heterotypical co-culture assays revealed that neurons from cerebral cortex and midbrain equally respond to ACM, indicating that differences in synapse effect are unlike to be neuron-autonomous. The expression profile of the synaptogenic molecules secreted by astrocytes from distinct brain regions was analyzed by qPCR. Gene expression of glypicans 4 and 6, hevin, and secreted protein-acidic and rich in cysteine (SPARC) greatly varies between astrocytes from different brain regions. Furthermore, in vivo analysis of hevin protein confirmed that variance. These findings highlight the heterogeneity of astrocytes and suggest that their synaptogenic potential may be different in each brain region, mainly due to distinct gene expression profiles.

Vulnerability of primary neurons derived from Tg2576 Alzheimer mice to oxygen and glucose deprivation: role of intraneuronal amyloid-β accumulation and astrocytes.

ABSTRACTMicrovascular dysfunction is considered an integral part of Alzheimer disease (AD) pathogenesis, but the possible relationship between amyloid pathology, microvascular dysfunction and cell death is still unclear. In order to investigate the influence of intraneuronal amyloid-β (Aβ) accumulation on vulnerability to hypoxia, we isolated primary cortical neurons from Tg2576 (carrying the amyloid precursor protein APPSwe mutation) and wild-type fetal mice. We first demonstrated that neurons isolated from Tg2576 newborn mice show an increase in VEGFa mRNA expression and a decrease in the expression of the two VEGF receptors, Flt1 and Kdr, compared with wild-type cells. Moreover, APPSwe primary neurons displayed higher spontaneous and glutamate-induced cell death. We then deprived the cultures of oxygen and glucose (OGD) as an in vitro model of hypoxia. After OGD, APPSwe neurons display higher levels of cell death in terms of percentage of pyknotic/fragmented nuclei and mitochondrial depolarization, accompanied by an increase in the intraneuronal Aβ content. To explore the influence of intraneuronal Aβ peptide accumulation, we used the γ-secretase inhibitor LY450139, which showed that the reduction of the intracellular amyloid fully protects APPSwe neurons from OGD-induced degeneration. Conditioned medium from OGD-exposed APPSwe or wild-type astrocytes protected APPswe neurons but not wild-type neurons, during OGD. In conclusion, the presence of the mutated human APP gene, leading to the intracellular accumulation of APP and Aβ fragments, worsens OGD toxicity. Protection of APPSwe neurons can be obtained either using a γ-secretase inhibitor or astrocyte conditioned medium.

PI3K Pathway Inhibition Achieves Potent Antitumor Activity in Melanoma Brain Metastases In Vitro and In Vivo.

Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for "brain-specific" therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases. Buparlisib inhibited AKT activity, decreased proliferation, and induced apoptosis in metastatic melanoma cell lines and short-term brain melanoma cells, irrespective of their BRAF and NRAS mutation status. In addition, buparlisib inhibited hyperactivated AKT and induced apoptosis in melanoma cells that were stimulated with astrocyte-conditioned medium. The growth of tumors induced by injecting human BRAF- and NRAS-mutant metastatic melanoma cells into the brain of mice was significantly inhibited by buparlisib. These results emphasize the value of targeting the PI3K pathway as a strategy to develop drugs for melanoma brain metastases. Clin Cancer Res; 22(23); 5818-28. ©2016 AACR.

Cell autonomous and noncell‐autonomous role of NF‐κB p50 in astrocyte‐mediated fate specification of adult neural progenitor cells

In previous work, we demonstrated that NF-κB p50 acts as crucial regulator of adult hippocampal neural progenitor cells (ahNPC). Indeed, NF-κB p50 knockout (KO) mice are characterized by remarkably reduced hippocampal neurogenesis. As a follow up to that work, herein we show that when cultured in vitro, ahNPC from wild type (WT) and p50KO mice are not significantly different in their neurogenic potential. This observation prompted us to investigate cell-autonomous and noncell-autonomous consequences of p50 absence on neuronal fate specification of ahNPC. In particular, we focused our attention on astrocytes, known to provide soluble proneurogenic signals, and investigated the influence of WT and p50KO astrocyte conditioned media (ACM) on WT and p50KO ahNPC differentiation. Interestingly, while WT ACM promoted both neuronal and astroglial differentiations, p50KO ACM only supported astroglial differentiation of WT ahNPC. By using a LC-MS/MS approach, we identified some proteins, which are significantly upregulated in p50KO compared with WT astrocytes. Among them, lipocalin-2 (LCN-2) was recognized as a novel astroglial-derived signal regulating neuronal fate specification of ahNPC. Interestingly, LCN-2 proneurogenic effect was greatly reduced in p50KO NPC, where LCN-2 receptor gene expression appeared downregulated. In addition to that, we demonstrated p50KO NPC unresponsiveness to both neuronal and astroglial fate specification signals from WT and p50KO ACM, and we identified a reduced expression of α2δ1, a thrombospondin-1 receptor, as another phenotypic change occurring in ahNPC in the absence of p50. Altogether, our data suggest that dysregulated NPC-astrocyte communication may contribute to a reduced hippocampal neurogenesis in p50KO mice in vivo. GLIA 2016 GLIA 2017;65:169-181.

Astroglioma conditioned medium increases synaptic elimination and correlates with major histocompatibility complex of class I (MHC I) upregulation in PC12Cells

Astrocytes are multifunctional glial cells that actively participate in synaptic plasticity in health and disease. Little is known about molecular interactions between neurons and glial cells that result in synaptic stability or elimination. In this sense, the main histocompatibility complex of class I (MHC I) has been shown to play a role in the synaptic plasticity process during development and after lesion of the CNS. MHC I levels in neurons appear to be influenced by astrocyte secreted molecules, which may generate endoplasmic reticulum stress. In vitro studies are of relevance since cell contact can be avoided by the use of astrocyte conditioned medium, allowing investigation of soluble factors isolated from cell direct interaction. Thus, we investigated synaptic preservation by synaptophysin and MHC I immunolabeling in PC12 neuron-like cells exposed to NG97 astroglioma conditioned medium (CM). For that, PC12 cells were cultured and differentiated into neuron-like profile with nerve growth factor. MHC I was induced with interferon beta treatment (IFN), and the effects were compared to PC12 exposure to NG97 CM. Overall, the results show that NG97 CM increases, more than IFN alone, the expression of MHC I, negatively influencing synaptic stability. This indicates that glial soluble factors influence synapse elimination, compatible to in vivo synaptic stripping process, in a cell contact independent fashion. In turn, our results indicate that deleterious effects of astroglioma are not only restricted to rapid growth ratio of the tumor, but also correlated with secretion of stress-related molecules that directly affect neuronal networks.

CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p<0.05), (ii) elevation in ΔΨm (p<0.05), (iii) increased OCR and ATP formation (p<0.05), (iv) increased intracellular NO levels (p<0.05), (v) increased mitochondrial ROS production (p<0.05), and (vi) increased susceptibility to rotenone (p<0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p<0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development.

Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment.

Astrocyte-secreted thrombospondin-1 modulates synapse and spine defects in the fragile X mouse model.

Astrocytes are key participants in various aspects of brain development and function, many of which are executed via secreted proteins. Defects in astrocyte signaling are implicated in neurodevelopmental disorders characterized by abnormal neural circuitry such as Fragile X syndrome (FXS). In animal models of FXS, the loss in expression of the Fragile X mental retardation 1 protein (FMRP) from astrocytes is associated with delayed dendrite maturation and improper synapse formation; however, the effect of astrocyte-derived factors on the development of neurons is not known. Thrombospondin-1 (TSP-1) is an important astrocyte-secreted protein that is involved in the regulation of spine development and synaptogenesis. In this study, we found that cultured astrocytes isolated from an Fmr1 knockout (Fmr1 KO) mouse model of FXS displayed a significant decrease in TSP-1 protein expression compared to the wildtype (WT) astrocytes. Correspondingly, Fmr1 KO hippocampal neurons exhibited morphological deficits in dendritic spines and alterations in excitatory synapse formation following long-term culture. All spine and synaptic abnormalities were prevented in the presence of either astrocyte-conditioned media or a feeder layer derived from FMRP-expressing astrocytes, or following the application of exogenous TSP-1. Importantly, this work demonstrates the integral role of astrocyte-secreted signals in the establishment of neuronal communication and identifies soluble TSP-1 as a potential therapeutic target for Fragile X syndrome.Electronic supplementary materialThe online version of this article (doi:10.1186/s13041-016-0256-9) contains supplementary material, which is available to authorized users.

Astrocytes donate mitochondria

Astrocytes release mitochondria that help neurons survive stroke.

Astrocyte-Secreted Factors Selectively Alter Neural Stem and Progenitor Cell Proliferation in the Fragile X Mouse.

An increasing body of evidence indicates that astrocytes contribute to the governance and fine tuning of stem and progenitor cell production during brain development. The effect of astrocyte function in cell production in neurodevelopmental disorders is unknown. We used the Neural Colony Forming Cell assay to determine the effect of astrocyte conditioned media (ACM) on the generation of neurospheres originating from either progenitor cells or functional stem cells in the knock out (KO) Fragile X mouse model. ACM from both normal and Fmr1-KO mice generated higher percentages of smaller neurospheres indicative of restricted proliferation of the progenitor cell population in Fmr1-KO brains. Wild type (WT) neurospheres, but not KO neurospheres, showed enhanced responses to ACM from the Fmr1-KO mice. In particular, Fmr1-KO ACM increased the percentage of large neurospheres generated, representative of spheres produced from neural stem cells. We also used 2D DIGE to initiate identification of the astrocyte-secreted proteins with differential expression between Fmr1-KO and WT cortices and hippocampi. The results further support the critical role of astrocytes in governing neural cell production in brain development and point to significant alterations in neural cell proliferation due to astrocyte secreted factors from the Fragile X brain.Highlights:• We studied the proliferation of neural stem and progenitor cells in Fragile X.• We examined the role of astrocyte-secreted factors in neural precursor cell biology.• Astrocyte-secreted factors with differential expression in Fragile X identified.

Differentiation of Spiral Ganglion-Derived Neural Stem Cells into Functional Synaptogenetic Neurons.

Spiral ganglion neurons (SGNs) are usually damaged in sensorineural hearing loss. SGN-derived neural stem cells (NSCs) have been identified and proposed to differentiate into neurons to replace damaged SGNs. However, it remains obscure whether SGN-NSC-derived neurons (ScNs) are electrophysiologically functional and possess the capability to form neural connections. Here, we found that SGN-derived cells demonstrated NSC characteristics and differentiated into SGN-like glutamatergic neurons. Neurotrophins significantly increased neuronal differentiation and neurite length of ScNs. Patch clamp recording revealed that ScNs possessed SGN-like NaV and HCN channels, suggesting electrophysiological function. FM1-43 staining and synaptic protein immunofluorescence showed ScNs possess the ability to form neural connections. Astrocyte-conditioned medium was able to stimulate ScNs to express synaptic proteins. These data suggested that neurotrophins are able to stimulate postnatal SGN-NSCs to differentiate into functional glutamatergic ScNs with the capability to form synaptic connections in vitro.

CNTF-Treated Astrocyte Conditioned Medium Enhances Large-Conductance Calcium-Activated Potassium Channel Activity in Rat Cortical Neurons.

Seizure activity is linked to astrocyte activation as well as dysfunctional cortical neuron excitability produced from changes in calcium-activated potassium (KCa) channel function. Ciliary neurotrophic factor-treated astrocyte conditioned medium (CNTF-ACM) can be used to investigate the peripheral effects of activated astrocytes upon cortical neurons. However, CNTF-ACM's effect upon KCa channel activity in cultured cortical neurons has not yet been investigated. Whole-cell patch clamp recordings were performed in rat cortical neurons to evaluate CNTF-ACM's effects upon charybdotoxin-sensitive large-conductance KCa (BK) channel currents and apamin-sensitive small-conductance KCa (SK) channel current. Biotinylation and RT-PCR were applied to assess CNTF-ACM's effects upon the protein and mRNA expression, respectively, of the SK channel subunits SK2 and SK3 and the BK channel subunits BKα1 and BKβ3. An anti-fibroblast growth factor-2 (FGF-2) monoclonal neutralizing antibody was used to assess the effects of the FGF-2 component of CNTF-ACM. CNTF-ACM significantly increased KCa channel current density, which was predominantly attributable to gains in BK channel activity (p<0.05). CNTF-ACM produced a significant increase in BKα1 and BKβ3 expression (p<0.05) but had no significant effect upon SK2 or SK3 expression (p>0.05). Blocking FGF-2 produced significant reductions in KCa channel current density (p>0.05) as well as BKα1 and BKβ3 expression in CNTF-ACM-treated neurons (p>0.05). CNTF-ACM significantly enhances BK channel activity in rat cortical neurons and that FGF-2 is partially responsible for these effects. CNTF-induced astrocyte activation results in secretion of neuroactive factors which may affect neuronal excitability and resultant seizure activity in mammalian cortical neurons.

Abstract A03: The effects of the cellular microenvironment on medulloblastoma metastasis

Abstract Medulloblastoma (MB) is the most common pediatric brain tumor, known to be highly malignant and invasive within the brain as well as extraneural locations. The mechanisms inducing MB metastasis and dissemination are not completely understood. Astrocytes, the most abundant cell type in the brain, have been shown in our lab to secrete factors that induce breast cancer brain metastasis (Wang et al., 2013). In addition to astrocytes, many brain cell types have also been implicated in the malignancy of brain tumors, including microglia, pericytes, and endothelial cells as part of the neurovascular unit. The overall goal of this study is to determine the effects of cells in the tumor microenvironment on MB metastasis. Our hypothesis is that astrocytes and/or other cell types in the neurovascular unit and microenvironment secrete factors that bring upon phenotypic changes in MB cells, leading to increased metastasis. To investigate this hypothesis, astrocyte conditioned media (ACM) from neonatal rat astrocyte cultures were used in in vitro assays to determine functional changes to Daoy MB cells. We observed that ACM (both serum free and serum depleted) in the lower chamber of a Boyden chamber Matrigel invasion assay induced invasion of Daoy cells more than 50-fold in comparison to DMEM in the lower well. Brain endothelial cell conditioned media also increased invasion of Daoy cells, which was comparable to ACM. ACM did not significantly affect proliferation or apoptosis of Daoy cells, as tested by BrdU incorporation and Caspase-3/-7 activity, respectively. When cultured appropriately in serum free ACM (+bFGF and EGF), Daoy cells were able to form larger and more proliferative neurospheres in comparison to DMEM, indicative of an increase in neural or cancer stem cell characteristics. To determine the effects of ACM on MB metastasis in vivo, we developed a zebrafish xenotransplant model. Daoy-GFP cells were injected into the hindbrain of 48 hour post fertilization Absolut FLK-mCherry zebrafish embryos. Using time-lapse confocal microscopy, we are able to image the cells and observe their movement within the microenvironment after injection. With this method we are working to determine changes in cell morphology and movement in Daoy-GFP cells that were cultured in ACM prior to injection. In order to further elucidate the mechanism in which ACM increases invasion and neurosphere formation, microarray studies are underway to pin-point a factor(s) or pathway(s) involved. Together, these data indicate that cells comprising the neurovascular unit, namely endothelial cells and astrocytes, contribute in a paracrine manner to MB metastasis and dissemination. Citation Format: Emily Gronseth, Ling Wang, Chris Koceja, Ramani Ramchandran. The effects of the cellular microenvironment on medulloblastoma metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A03.

Ciliary neurotrophic factor-treated astrocyte-conditioned medium increases the intracellular free calcium concentration in rat cortical neurons.

Ciliary neurotrophic factor (CNTF) is involved in the activation of astrocytes. A previous study showed that CNTF-treated astrocyte-conditioned medium (CNTF-ACM) contributed to the increase of the calcium current and the elevation of corresponding ion channels in cortical neurons. On this basis, it is reasonable to assume that CNTF-ACM may increase the intracellular free calcium concentration ([Ca2+]i) in neurons. In the present study, the effects of CNTF-ACM on [Ca2+]i in rat cortical neurons were determined, and on this basis, the aim was to investigate the potential active ingredients in ACM that are responsible for this biological process. As expected, the data indicated that CNTF-ACM resulted in a clear elevation of [Ca2+]i in neurons. Additionally, the fibroblast growth factor-2 (FGF-2) contained in the CNTF-ACM was found to participate in the upregulation of [Ca2+]i. Taken together, CNTF induces the production of active factors (at least including FGF-2) released from astrocytes, which finally potentiate the increase of [Ca2+]i in cortical neurons.

Astrocyte-derived phosphatidic acid promotes dendritic branching.

Astrocytes play critical roles in neural circuit formation and function. Recent studies have revealed several secreted and contact-mediated signals from astrocytes which are essential for neurite outgrowth and synapse formation. However, the mechanisms underlying the regulation of dendritic branching by astrocytes remain elusive. Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline, has been implicated in the regulation of neurite outgrowth. Here we showed that knockdown of PLD1 selectively in astrocytes reduced dendritic branching of neurons in neuron-glia mixed culture. Further studies from sandwich-like cocultures and astrocyte conditioned medium suggested that astrocyte PLD1 regulated dendritic branching through secreted signals. We later demonstrated that PA was the key mediator for astrocyte PLD1 to regulate dendritic branching. Moreover, PA itself was sufficient to promote dendritic branching of neurons. Lastly, we showed that PA could activate protein kinase A (PKA) in neurons and promote dendritic branching through PKA signaling. Taken together, our results demonstrate that astrocyte PLD1 and its lipid product PA are essential regulators of dendritic branching in neurons. These results may provide new insight into mechanisms underlying how astrocytes regulate dendrite growth of neurons.