搜索结果: 'NeuroCult Proliferation'

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets,the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay,a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053% per hour) in the cortex,consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally,brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary,our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets. View Publication

The PI3K/AKT/mTOR pathway is commonly over activated in glioblastoma (GBM),and Rictor was shown to be an important regulator downstream of this pathway. EGFR overexpression is also frequently found in GBM tumors,and both EGFR and Rictor are associated with increased proliferation,invasion,metastasis and poor prognosis. This research evaluated in vitro and in vivo whether the combined silencing of EGFR and Rictor would result in therapeutic benefits. The therapeutic potential of targeting these proteins in combination with conventional agents with proven activity in GBM patients was also assessed. In vitro validation studies were carried out using siRNA-based gene silencing methods in a panel of three commercially available human GBM cell lines,including two PTEN mutant lines (U251MG and U118MG) and one PTEN-wild type line (LN229). The impact of EGFR and/or Rictor silencing on cell migration and sensitivity to chemotherapeutic drugs in vitro was determined. In vivo validation of these studies was focused on EGFR and/or Rictor silencing achieved using doxycycline-inducible shRNA-expressing U251MG cells implanted orthotopically in Rag2M mice brains. Target silencing,tumor size and tumor cell proliferation were assessed by quantification of immunohistofluorescence-stained markers. siRNA-mediated silencing of EGFR and Rictor reduced U251MG cell migration and increased sensitivity of the cells to irinotecan,temozolomide and vincristine. In LN229,co-silencing of EGFR and Rictor resulted in reduced cell migration,and increased sensitivity to vincristine and temozolomide. In U118MG,silencing of Rictor alone was sufficient to increase this line's sensitivity to vincristine and temozolomide. In vivo,while the silencing of EGFR or Rictor alone had no significant effect on U251MG tumor growth,silencing of EGFR and Rictor together resulted in a complete eradication of tumors. These data suggest that the combined silencing of EGFR and Rictor should be an effective means of treating GBM. View Publication

The multifunctional signaling protein p75 neurotrophin receptor (p75(NTR)) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here,we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75(NTR) is required for p75(NTR)-mediated glioma invasion,and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75(NTR) or treatment of animals bearing p75(NTR)-positive intracranial tumors with clinically applicable gamma-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly,proteolytic processing of p75(NTR) was observed in p75(NTR)-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75(NTR) as a therapeutic target,suggesting that gamma-secretase inhibitors may have direct clinical application for the treatment of malignant glioma. View Publication

The polarization and migration of neural progenitor cells (NPCs) are critical for embryonic brain development and neurogenesis after brain injury. Although stromal-derived factor-1α (SDF-1α,CXCL12) and its receptor CXCR4 are well-known to mediate the migration of NPCs in the developing brain,the dynamic cellular processes and structure-related molecular events remain elusive. Transwell and microfluidic-based assays are classical assays to effectively study cellular migration. However,both of them have limitations in the analysis of a single cell. In this study,we modified the stripe assay and extended its applications in the study of NPC polarization and intracellular molecular events associated with CXCL12-mediated migration. In response to localized CXCL12,NPCs formed lamellipodia in the stripe assay. Furthermore,CXCR4 and Rac1 quickly re-distributed to the area of lamellipodia,indicating their roles in NPC polarization upon CXCL12 stimulation. Although the chemokine stripes in the assay provided concentration gradients that can be best used to study cellular polarization and migration through immunocytochemistry,they can also generate live imaging data with comparable quality. In conclusion,stripe assay is a visual,dynamic and economical tool to study cellular mobility and its related molecule mechanisms. View Publication

Neurotrophin-3 (NT-3) and its high-affinity receptor TrkC play crucial trophic roles in neuronal differentiation,axon outgrowth,and synapse development and plasticity in the nervous system. We demonstrated previously that postsynaptic TrkC functions as a glutamatergic synapse-inducing (synaptogenic) cell adhesion molecule trans-interacting with presynaptic protein tyrosine phosphatase σ (PTPσ). Given that NT-3 and PTPσ bind distinct domains of the TrkC extracellular region,here we tested the hypothesis that NT-3 modulates TrkC/PTPσ binding and synaptogenic activity. NT-3 enhanced PTPσ binding to cell surface-expressed TrkC and facilitated the presynapse-inducing activity of TrkC in rat hippocampal neurons. Imaging of recycling presynaptic vesicles combined with TrkC knockdown and rescue approaches demonstrated that NT-3 rapidly potentiates presynaptic function via binding endogenous postsynaptic TrkC in a tyrosine kinase-independent manner. Thus,NT-3 positively modulates the TrkC-PTPσ complex for glutamatergic presynaptic assembly and function independently from TrkC kinase activation. Our findings provide new insight into synaptic roles of neurotrophin signaling and mechanisms controlling synaptic organizing complexes. Significance statement: Although many synaptogenic adhesion complexes have been identified in recent years,little is known about modulatory mechanisms. Here,we demonstrate a novel role of neurotrophin-3 in synaptic assembly and function as a positive modulator of the TrkC-protein tyrosine phosphatase σ complex. This study provides new insight into the involvement of neurotrophin signaling in synapse development and plasticity,presenting a molecular mechanism that may underlie previous observations of short- and long-term enhancement of presynaptic function by neurotrophin. Given the links of synaptogenic adhesion molecules to autism and schizophrenia,this study might also contribute to a better understanding of the pathogenesis of these disorders and provide a new direction for ameliorating imbalances in synaptic signaling networks. View Publication

BACKGROUND AND PURPOSE HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo.HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems,we examined the protective effects of the CB receptor agonist AM1241 on Gp120-mediated insults on neurogenesis. EXPERIMENTAL APPROACH We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry. KEY RESULTS AM1241 inhibited in vitroGp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis,as indicated by reduction in BrdU cells and doublecortin (DCX) cells,and a decrease in cells with proliferating cell nuclear antigen (PCNA),administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts,neuronal cells,BrdU cells and PCNA cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241,compared with those treated with vehicle. CONCLUSIONS AND IMPLICATIONS The CB receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus,CB receptor agonists may act as neuroprotective agents,restoring impaired neurogenesis in patients with HAD. View Publication

BACKGROUND Tumor cells present high levels of oxidative stress. Cancer therapeutics exploiting such biochemical changes by increasing reactive oxygen species (ROS) production or decreasing intracellular ROS scavengers could provide a powerful treatment strategy. METHODS To test the effect of our compound,obtusaquinone (OBT),we used several cell viability assays on seven different glioblastoma (GBM) cell lines and primary cells and on 12 different cell lines representing various cancer types in culture as well as on subcutaneous (n = 7 mice per group) and two intracranial GBM (n = 6-8 mice per group) and breast cancer (n = 6 mice per group) tumor models in vivo. Immunoblotting,immunostaining,flow cytometry,and biochemical assays were used to investigate the OBT mechanism of action. Histopathological analysis (n = 2 mice per group) and blood chemistry (n = 2 mice per group) were used to test for any compound-related toxicity. Statistical tests were two-sided. RESULTS OBT induced rapid increase in intracellular ROS levels,downregulation of cellular glutathione levels and increase in its oxidized form,and activation of cellular stress pathways and DNA damage,subsequently leading to apoptosis. Oxidative stress is believed to be the main mechanism through which this compounds targets cancer cells. OBT was well tolerated in mice,slowed tumor growth,and statistically prolonged survival in GBM tumor models. The ratio of median survival in U251 intracranial model in OBT vs control was 1.367 (95% confidence interval [CI] of ratio = 1.031 to 1.367,P = .008). Tumor growth inhibition was also observed in a mouse breast cancer model (average tumor volume per mouse,OBT vs control: 36.3 vs 200.4mm(3),difference = 164.1mm(3),95% CI =72.6 to 255.6mm(3),P = .005). CONCLUSIONS Given its properties and efficacy in cancer killing,our results suggest that OBT is a promising cancer therapeutic. View Publication

Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1),we studied paired tumor samples from 41 patients,comparing higher-grade,progressed samples to their lower-grade counterparts. Integrated genomic analyses,including whole-exome sequencing and copy number,gene expression and DNA methylation profiling,demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions,as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach. View Publication

The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors,and also determined the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knockdown of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012,and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase-9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase-8 inhibitor c-FLIP-s,or knockdown of death receptor CD95 or the death receptor caspase-8 linker protein FADD,suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knockdown of the autophagy regulatory proteins Beclin1 or ATG5 protected the cells from OSU-03012 and from [OSU-03012 + PDE5 inhibitor] toxicity. Knockdown of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor]-induced JNK activation,and inhibition of JNK suppressed the elevated killing caused by IRE1 knockdown. Knockdown of CD95 blunted JNK activation. Collectively,our data demonstrate that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in glioblastoma multiforme (GBM) cells. View Publication

Neural progenitor cell (NPC) migration is an essential process for brain development,adult neurogenesis,and neuroregeneration after brain injury. Stromal cell-derived factor-1 (SDF-1,CXCL12) and its traditional receptor CXCR4 are well known to regulate NPC migration. However,the discovery of CXCR7,a newly identified CXCL12 receptor,adds to the dynamics of the existing CXCL12/CXCR4 pair. Antagonists for either CXCR4 or CXCR7 blocked CXCL12-mediated NPC migration in a transwell chemotaxis assay,suggesting that both receptors are required for CXCL12 action. We derived NPC cultures from Cxcr4 knockout (KO) mice and used transwell and stripe assays to determine the cell migration. NPCs derived from Cxcr4 KO mice polarized and migrated in response to CXCL12 gradient,suggesting that CXCR7 could serve as an independent migration receptor. Furthermore,Cxcr4 KO NPCs transplanted into the adult mouse striatum migrated in response to the adjacent injection of CXCL12,an effect that was blocked by a CXCR7 antagonist,suggesting that CXCR7 also mediates NPC migration in vivo. Molecular mechanism studies revealed that CXCR7 interact with Rac1 in the leading edge of the polarized NPCs in the absence of CXCR4. Both CXCR7 and Rac1 are required for extracellular signal-regulated kinases (ERK) 1/2 activation and subsequent NPC migration,indicating that CXCR7 could serve as a functional receptor in CXCL12-mediated NPC migration independent of CXCR4. Together these results reveal an essential role of CXCR7 for CXCL12-mediated NPC migration that will be important to understand neurogenesis during development and in adulthood. View Publication

Adeno-associated virus (AAV) vectors expressing tumoricidal genes injected directly into brain tumors have shown some promise,however,invasive tumor cells are relatively unaffected. Systemic injection of AAV9 vectors provides widespread delivery to the brain and potentially the tumor/microenvironment. Here we assessed AAV9 for potential glioblastoma therapy using two different promoters driving the expression of the secreted anti-cancer agent sTRAIL as a transgene model; the ubiquitously active chicken β-actin (CBA) promoter and the neuron-specific enolase (NSE) promoter to restrict expression in brain. Intravenous injection of AAV9 vectors encoding a bioluminescent reporter showed similar distribution patterns,although the NSE promoter yielded 100-fold lower expression in the abdomen (liver),with the brain-to-liver expression ratio remaining the same. The main cell types targeted by the CBA promoter were astrocytes,neurons and endothelial cells,while expression by NSE promoter mostly occurred in neurons. Intravenous administration of either AAV9-CBA-sTRAIL or AAV9-NSE-sTRAIL vectors to mice bearing intracranial patient-derived glioblastoma xenografts led to a slower tumor growth and significantly increased survival,with the CBA promoter having higher efficacy. To our knowledge,this is the first report showing the potential of systemic injection of AAV9 vector encoding a therapeutic gene for the treatment of brain tumors. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. View Publication