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Supplementary Data from The Telomerase Antagonist, Imetelstat, Efficiently Targets Glioblastoma Tumor-Initiating Cells Leading to Decreased Proliferation and Tumor Growth

Purpose: Telomerase activity is one of the hallmarks of cancer and is a highly relevant therapeutic target. The effects of a novel human telomerase antagonist, imetelstat, on primary human glioblastoma (GBM) tumor-initiating cells were investigated in vitro and in vivo.Experimental Design: Tumor-initiating cells were isolated from primary GBM tumors and expanded as neurospheres in vitro. The GBM tumor-initiating cells were treated with imetelstat and examined for the effects on telomerase activity levels, telomere length, proliferation, clonogenicity, and differentiation. Subsequently, mouse orthotopic and subcutaneous xenografts were used to assess the in vivo efficacy of imetelstat.Results: Imetelstat treatment produced a dose-dependent inhibition of telomerase (IC50 0.45 μmol/L). Long-term imetelstat treatment led to progressive telomere shortening, reduced rates of proliferation, and eventually cell death in GBM tumor-initiating cells. Imetelstat in combination with radiation and temozolomide had a dramatic effect on cell survival and activated the DNA damage response pathway. Imetelstat is able to cross the blood-brain barrier in orthotopic GBM xenograft tumors. Fluorescently labeled GBM tumor cells isolated from orthotopic tumors, following systemic administration of imetelstat (30 mg/kg every day for three days), showed ∼70% inhibition of telomerase activity. Chronic systemic treatment produced a marked decrease in the rate of xenograft subcutaneous tumor growth.Conclusion: This preclinical study supports the feasibility of testing imetelstat in the treatment of GBM patients, alone or in combination with standard therapies. Clin Cancer Res; 16(1); 154–63

Nuclear factor-κB (NF-κB) activation may play an important role in the pathogenesis of cancer and also in resistance to treatment. Inactivation of the p53 tumor suppressor is a key component of the multistep evolution of most cancers. Links between the NF-κB and p53 pathways are under intense investigation. In this study, we show that the receptor interacting protein 1 (RIP1), a central component of the NF-κB signaling network, negatively regulates p53 tumor suppressor signaling. Loss of RIP1 from cells results in augmented induction of p53 in response to DNA damage, whereas increased RIP1 level leads to a complete shutdown of DNA damage–induced p53 induction by enhancing levels of cellular mdm2. The key signal generated by RIP1 to up-regulate mdm2 and inhibit p53 is activation of NF-κB. The clinical implication of this finding is shown in glioblastoma, the most common primary malignant brain tumor in adults. We show that RIP1 is commonly overexpressed in glioblastoma, but not in grades II and III glioma, and increased expression of RIP1 confers a worse prognosis in glioblastoma. Importantly, RIP1 levels correlate strongly with mdm2 levels in glioblastoma. Our results show a key interaction between the NF-κB and p53 pathways that may have implications for the targeted treatment of glioblastoma. [Cancer Res 2009;69(7):2809–16]

Glioblastomas (GBM) are lethal brain tumors that are highly resistant to therapy. The only meaningful improvement in therapeutic response came from use of the SN1-type alkylating agent temozolomide in combination with ionizing radiation. However, no genetic markers that might predict a better response to DNA alkylating agents have been identified in GBMs, except for loss of O6-methylguanine-DNA methyltransferase via promoter methylation. In this study, using genetically defined primary murine astrocytes as well as human glioma lines, we show that loss of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) confers sensitivity to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a functional analogue of temozolomide. We find that MNNG induces replication-associated DNA double-strand breaks (DSB), which are inefficiently repaired in PTEN-deficient astrocytes and trigger apoptosis. Mechanistically, this is because PTEN-null astrocytes are compromised in homologous recombination (HR), which is important for the repair of replication-associated DSBs. Our results suggest that reduced levels of Rad51 paralogs in PTEN-null astrocytes might underlie the HR deficiency of these cells. Importantly, the HR deficiency of PTEN-null cells renders them sensitive to the poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 due to synthetic lethality. In sum, our results tentatively suggest that patients with PTEN-null GBMs (about 36%) may especially benefit from treatment with DNA alkylating agents such as temozolomide. Significantly, our results also provide a rational basis for treating the subgroup of patients who are PTEN deficient with PARP inhibitors in addition to the current treatment regimen of radiation and temozolomide. Cancer Res; 70(13); 5457–64. ©2010 AACR.

Supplementary Methods, Figures 1-8 from PTEN Loss Compromises Homologous Recombination Repair in Astrocytes: Implications for Glioblastoma Therapy with Temozolomide or Poly(ADP-Ribose) Polymerase Inhibitors

The blood-spinal cord barrier (BSCB) tightly regulates molecular transport from the blood to the spinal cord. Herein, we present a novel approach for transient modulation of BSCB permeability and localized delivery of peptides into the spinal cord for behavior modulation with high spatial resolution. This approach utilizes optical stimulation of vasculature-targeted nanoparticles and allows delivery of BSCB-nonpermeable molecules into the spinal cord without significant glial activation or impact on animal locomotor behavior. We demonstrate minimally invasive light delivery into the spinal cord using an optical fiber and BSCB permeability modulation in the lumbar region. Our method of BSCB modulation allows delivery of bombesin, a centrally-acting and itch-inducing peptide, into the spinal cord and induces a rapid and transient increase in itching behaviors in mice. This minimally invasive approach enables behavior modulation without genetic modifications and is promising for delivering a wide range of biologics into the spinal cord for behavior modulation and potentially therapy.Significance StatementSpinal cord diseases and disorders are common and cause significant disability, including chronic pain, paralysis, cognitive impairment, and mortality. The blood-spinal cord barrier is a considerable challenge for delivery by systemic therapeutic administration. We developed an optical approach for effectively and safely delivering molecules to the spinal cord to overcome this barrier. The fiberoptic method is minimally invasive and overcomes challenges that previous technologies face, including the complicated bone structure and standing waves that complicate BSCB opening using ultrasound. Optical stimulation offers unprecedented spatial resolution for the precise delivery in intricate spinal cord structures. Significantly, our approach modulates animal behavior (i.e., itch) without genetic modifications and demonstrates the potential for delivery of biologics such as peptides into the spinal cord.

Transient disruption of the blood-brain barrier (BBB) with focused ultrasound (FUS) is an emerging clinical method to facilitate targeted drug delivery to the brain. The focal noninvasive disruption of the BBB can be applied to promote local delivery of hyperpolarized substrates. In this study, we investigated effects of FUS on imaging brain metabolism using two hyperpolarized (13)C-labeled substrates in rodents: [1-(13)C]pyruvate and [1-(13)C]glycerate. The BBB is a rate-limiting factor for pyruvate delivery to the brain and glycerate minimally passes through the BBB. First, cerebral imaging with hyperpolarized [1-(13)C]pyruvate resulted in an increase in total (13)C signals (p = 0.05) after disrupting the BBB with FUS. Significantly higher levels of both [1-(13)C]lactate (lactate/total (13)C signals, p = 0.01) and [(13)C]bicarbonate (p = 0.008) were detected in the FUS-applied brain region as compared to the contralateral FUS-unaffected normal-appearing brain region. Application of FUS without opening the BBB in a separate group of rodents resulted in comparable lactate and bicarbonate productions between the FUS-applied and the contralateral brain regions. Second, (13)C imaging with hyperpolarized [1-(13)C]glycerate after opening the BBB showed increased [1-(13)C]glycerate delivery to the FUS-applied region (p = 0.04) relative to the contralateral side, and [1-(13)C]lactate production was consistently detected from the FUS-applied region. Our findings suggest that FUS accelerates the delivery of hyperpolarized molecules across the BBB and provides enhanced sensitivity to detect metabolic products in the brain; therefore, hyperpolarized (13)C imaging with FUS may provide new opportunities to study cerebral metabolic pathways as well as various neurological pathologies.

Malignant brain tumors are known to utilize acetate as an alternate carbon source in the citric acid cycle for their bioenergetics. 13C NMR based isotopomer analysis has been used to measure turnover of 13C-acetate carbons into glutamate and glutamine pools in tumors. Plasma from the patients infused with [1,2-13C]acetate further revealed the presence of 13C isotopomers of glutamine, glucose and lactate in the circulation that were generated due to metabolism of [1,2-13C]acetate by peripheral organs. In the tumor cells, [4-13C] and [3,4-13C] glutamate and glutamine isotopomers were generated from blood-borne 13C labeled glucose and lactate which were formed due to [1,2-13C[acetate metabolism of peripheral tissues. [4,5-13C] and [3,4,5-13C] glutamate and glutamine isotopomers were produced from [1,2-13C]acetyl-CoA that were derived from direct oxidation of [1,2-13C]acetate in the tumor.

Oncogenes influence nutrient metabolism and nutrient dependence. The oncogene c-Myc stimulates glutamine metabolism and renders cells dependent on glutamine to sustain viability (“glutamine addiction”), suggesting that treatments targeting glutamine metabolism might selectively kill c-Myc–transformed tumor cells. However, many current or proposed cancer therapies interfere with the metabolism of glucose, not glutamine. Here, we studied how c-Myc–transformed cells maintained viability when glucose metabolism was impaired. In SF188 glioblastoma cells, glucose deprivation did not affect net glutamine utilization but elicited a switch in the pathways used to deliver glutamine carbon to the tricarboxylic acid cycle, with a large increase in the activity of glutamate dehydrogenase (GDH). The effect on GDH resulted from the loss of glycolysis because it could be mimicked with the glycolytic inhibitor 2-deoxyglucose and reversed with a pyruvate analogue. Furthermore, inhibition of Akt signaling, which facilitates glycolysis, increased GDH activity whereas overexpression of Akt suppressed it, suggesting that Akt indirectly regulates GDH through its effects on glucose metabolism. Suppression of GDH activity with RNA interference or an inhibitor showed that the enzyme is dispensable in cells able to metabolize glucose but is required for cells to survive impairments of glycolysis brought about by glucose deprivation, 2-deoxyglucose, or Akt inhibition. Thus, inhibition of GDH converted these glutamine-addicted cells to glucose-addicted cells. The findings emphasize the integration of glucose metabolism, glutamine metabolism, and oncogenic signaling in glioblastoma cells and suggest that exploiting compensatory pathways of glutamine metabolism can improve the efficacy of cancer treatments that impair glucose utilization. [Cancer Res 2009;69(20):7986–93]

Glioblastoma multiforme (GBM) is the most lethal of brain tumors and is highly resistant to ionizing radiation (IR) and chemotherapy. Here, we report on a molecular mechanism by which a key glioma-specific mutation, epidermal growth factor receptor variant III (EGFRvIII), confers radiation resistance. Using Ink4a/Arf-deficient primary mouse astrocytes, primary astrocytes immortalized by p53/Rb suppression, as well as human U87 glioma cells, we show that EGFRvIII expression enhances clonogenic survival following IR. This enhanced radioresistance is due to accelerated repair of DNA double-strand breaks (DSB), the most lethal lesion inflicted by IR. The EGFR inhibitor gefitinib (Iressa) and the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 attenuate the rate of DSB repair. Importantly, expression of constitutively active, myristylated Akt-1 accelerates repair, implicating the PI3K/Akt-1 pathway in radioresistance. Most notably, EGFRvIII-expressing U87 glioma cells show elevated activation of a key DSB repair enzyme, DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Enhanced radioresistance is abrogated by the DNA-PKcs–specific inhibitor NU7026, and EGFRvIII fails to confer radioresistance in DNA-PKcs–deficient cells. In vivo, orthotopic U87-EGFRvIII–derived tumors display faster rates of DSB repair following whole-brain radiotherapy compared with U87-derived tumors. Consequently, EGFRvIII-expressing tumors are radioresistant and continue to grow following whole-brain radiotherapy with little effect on overall survival. These in vitro and in vivo data support our hypothesis that EGFRvIII expression promotes DNA-PKcs activation and DSB repair, perhaps as a consequence of hyperactivated PI3K/Akt-1 signaling. Taken together, our results raise the possibility that EGFR and/or DNA-PKcs inhibition concurrent with radiation may be an effective therapeutic strategy for radiosensitizing high-grade gliomas. [Cancer Res 2009;69(10):4252–9]

Malignant astrocytic gliomas such as glioblastoma are the most common and lethal intracranial tumors. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches, destruction of normal brain tissue, and certain death. The recent confluence of advances in stem cell biology, cell signaling, genome and computational science and genetic model systems have revolutionized our understanding of the mechanisms underlying the genetics, biology and clinical behavior of glioblastoma. This progress is fueling new opportunities for understanding the fundamental basis for development of this devastating disease and also novel therapies that, for the first time, portend meaningful clinical responses.

Glioblastoma (GBM) is an astrocytic brain tumor characterized by an aggressive clinical course and intense resistance to all therapeutic modalities. Here, we report the identification and functional characterization of Bcl2L12 (Bcl2-like-12) that is robustly expressed in nearly all human primary GBMs examined. Enforced Bcl2L12 expression confers marked apoptosis resistance in primary cortical astrocytes, and, conversely, its RNA interference (RNAi)-mediated knockdown sensitizes human glioma cell lines toward apoptosis in vitro and impairs tumor growth with increased intratumoral apoptosis in vivo. Mechanistically, Bcl2L12 expression does not affect cytochrome c release or apoptosome-driven caspase-9 activation, but instead inhibits post-mitochondrial apoptosis signaling at the level of effector caspase activation. One of Bcl2L12’s mechanisms of action stems from its ability to interact with and neutralize caspase-7. Notably, while enforced Bcl2L12 expression inhibits apoptosis, it also engenders a pronecrotic state, which mirrors the cellular phenotype elicited by genetic or pharmacologic inhibition of post-mitochondrial apoptosis molecules. Thus, Bcl2L12 contributes to the classical tumor biological features of GBM such as intense apoptosis resistance and florid necrosis, and may provide a target for enhanced therapeutic responsiveness of this lethal cancer.

The PTEN protein has a single catalytic domain possessing both lipid phosphoinositol and protein phosphatase activities. The lipid phosphoinositol phosphatase activity is essential for PTEN to block the cell cycle in the G1 phase and thereby to suppress tumor formation and progression (Cantley, L. C., and Neel, B. G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 4240-4245), although the mechanisms governing PTEN activity under normal and neoplastic growth conditions remain unclear. Here, we report that PTEN interacts physically and functionally with PCAF, a histone acetyltransferase that regulates gene transcription through interaction with p300/CBP and various sequence-specific transcription factors (Nakatani, Y. (2001) Genes Cells 6, 79-86). Expression of PCAF results in increased acetylation of lysine residues (Lys125 and Lys128) within the catalytic cleft of PTEN, a structure essential for phosphatidylinositol 3,4,5-trisphosphate specificity (Lee, J. O., Yang, H., Georgescu, M. M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J. E., Pandolfi, P., and Pavletich, N. P. (1999) Cell 99, 323-334). The acetylation of PTEN caused by PCAF expression depends on the presence of growth factors. Reduction of endogenous PCAF activity using shRNA results in a loss of PTEN acetylation in response to growth factors and restores the ability of PTEN to down-regulate phosphatidylinositol 3-kinase signaling and to induce G1 cell cycle arrest. The retention of phosphatidylinositol 3-kinase/AKT signaling and cell cycle regulatory activities of acetylation-resistant PTEN K125R and K128R mutants in the presence of enforced PCAF expression suggest a causal relationship. Together, these findings indicate a mechanism of PTEN regulation that forges a link between distinct cancer-relevant pathways central to the control of growth factor signaling and gene expression.

The three-dimensional architecture of the nascent microvascular network is a critical determinant of vascular perfusion in the setting of regenerative growth, vasculopathies and cancer. Current methods for microvessel visualization are limited by insufficient penetration and instability of endothelial immunolabels, inadequate vascular perfusion by the high-viscosity polymers used for vascular casting, and destruction of tissue stroma during the processing required for scanning electron microscopy. The aim of this study was to develop whole-mount tissue processing methods for 3D in situ visualization of the microvasculature that were also compatible with supplementary labeling for other structures of interest in the tissue microenvironment. Here, we present techniques that allow imaging of the microvasculature by confocal microscopy, to depths of up to 1500 mum below the specimen surface. Our approach includes labeling luminal surfaces of endothelial cells by i.v. injection of fluorescently conjugated lectin and filling the microvasculature with carbon or fluorescent nanoparticles/Mercox, followed by optical clearing of thick tissue sections to reduce light scatter and permit 3D visualization of microvessel morphology deep into the sample. Notably, tissue stroma is preserved, allowing simultaneous labeling of other structures by immunohistochemistry or nuclear dyes. Results are presented for various murine tissues including fat, muscle, heart and brain under conditions of normal health, as well as in the setting of a glioma model growing in the subcutaneous space or orthotopically in the brain parenchyma.

The catalytic subunit of telomerase reverse transcriptase (TERT) protects dividing cells from replicative senescencein vitro. Here, we show that expression of TERT mRNA is induced in the ipsilateral cortical neurons after occlusion of the middle cerebral artery in adult mice. Transgenic mice that overexpress TERT showed significant resistance to ischemic brain injury. Among excitotoxicity, oxidative stress, and apoptosis comprising of routes of ischemic neuronal death, NMDA receptor-mediated excitotoxicity was reduced in forebrain cell cultures overexpressing TERT. NMDA-induced accumulation of cytosolic free Ca2+([Ca2+]c) was reduced in forebrain neurons from TERT transgenic mice, which was attributable to the rapid flow of [Ca2+]cinto the mitochondria from the cytosol without change in Ca2+influx and efflux through the plasma membrane. The present study provides evidence that TERT is inducible in postmitotic neurons after ischemic brain injury and prevents NMDA neurotoxicity through shift of the cytosolic free Ca2+into the mitochondria, and thus plays a protective role in ameliorating ischemic neuronal cell death.

To evaluate the T2 relaxation time of lactate (Lac) in brain tumors and the correlation of the T2 and concentration with tumor grades.Eight pairs of the subecho time sets of point-resolved spectroscopy were selected between 58 and 268 ms, with numerical and phantom analyses, for Lac T2 measurement. In vivo spectra were acquired from 24 subjects with gliomas (13 low grade and 11 high grade) and analyzed with LCModel using numerically-calculated basis spectra. The metabolite T2 relaxation time was obtained from monoexponential fitting of the multi-echo time (TE) signal estimates versus TE. The metabolite concentration was estimated from the zero-TE extrapolation of the T2 fits.The Lac T2 was estimated to be approximately 240 ms, without a significant difference between low and high grade tumors. The Lac concentration was estimated to be 4.1 ± 3.4 and 7.0 ± 4.7 mM for low and high grades respectively, but the difference was not significant.The Lac T2 was similar among gliomas regardless of their tumor grades. This suggests that the T2 value from this study may be applicable to obtain the T2 relaxation-free estimates of Lac in a subset of brain tumors.

Glioblastoma (GBM), the most common primary brain tumor, is resistant to currently available treatments. The development of mouse models of human GBM has provided a tool for studying mechanisms involved in tumor initiation and growth as well as a platform for preclinical investigation of new drugs. In this study we used (1) H MR spectroscopy to study the neurochemical profile of a human orthotopic tumor (HOT) mouse model of human GBM. The goal of this study was to evaluate differences in metabolite concentrations in the GBM HOT mice when compared with normal mouse brain in order to determine if MRS could reliably differentiate tumor from normal brain. A TE =19 ms PRESS sequence at 9.4 T was used for measuring metabolite levels in 12 GBM mice and 8 healthy mice. Levels for 12 metabolites and for lipids/macromolecules at 0.9 ppm and at 1.3 ppm were reliably detected in all mouse spectra. The tumors had significantly lower concentrations of total creatine, GABA, glutamate, total N-acetylaspartate, aspartate, lipids/macromolecules at 0.9 ppm, and lipids/macromolecules at 1.3 ppm than did the brains of normal mice. The concentrations of glycine and lactate, however, were significantly higher in tumors than in normal brain.

Malignant brain tumors strike deep into the psyche of those receiving and those delivering the diagnosis. Malignant gliomas, the most common subtype of primary brain tumors, are aggressive, highly invasive, and neurologically destructive tumors considered to be among the deadliest of human cancers. In its most aggressive manifestation, glioblastoma (GBM), median survival ranges from 9 to 12 months, despite maximum treatment efforts—a statistical fact that has changed little over several decades of technological advances in neurosurgery, radiation therapy, and clinical trials of conventional and novel therapeutics. Over the same time period, there has been an explosion of knowledge in cancer biology and basic science discovery that has fueled meaningful progress in the treatment of many common human cancers, including those of the breast, lung, and prostate. It is perplexing that therapies used effectively in the treatment of these solid tumors are overwhelmingly ineffective in the treatment of GBM, perhaps reflecting the eccentric biology and cellular origin of this neoplasm. To date, only one new agent has been documented to have modest activity against intermediate-grade gliomas, whereas no effective agents have emerged for the treatment of GBM, despite 20 years of enrolling patients in clinical trials. It is ironic that although a comprehensive view of the genetic lesions encountered in malignant gliomas has been compiled, substantive conceptual and practical barriers remain in assigning functional significance to these genetic changes and in harnessing this basic information into the development of drugs that make a difference in patient care. The history of treating malignant gliomas dates back to the middle of the 19th century and parallels landmark advances in modern surgical technique and the clinical discipline of neurology. The first brain tumor surgery of the modern era was performed in 1884 by Rickman

Large changes in neuronal gene expression occur in adult peripheral neurons after axonal transection. In the rat superior cervical ganglion, for example, neurons that do not normally express vasoactive intestinal peptide (VIP) or galanin do so after postganglionic nerve transection. These effects of axotomy could result from a number of aspects of the surgical procedure. To test the idea that the important variable might be the disconnection of axotomized neuronal cell bodies from their target tissues, we examined the effects of producing such a disconnection by means of the compound 6-hydroxydopamine (6-OHDA), a neurotoxin that causes degeneration of sympathetic varicosities and avoids many of the complications of surgery. Two days after 6-OHDA treatment, VIP and galanin immunoreactivities had increased two- and 40-fold, respectively. Nevertheless, these increases were substantially smaller than the 30- and 300-fold changes seen after surgical axotomy. When expression of VIP and galanin was examined at the mRNA level, however, comparable increases were found after either procedure. The results indicate that chemical destruction of sympathetic varicosities produces an equivalent signal for increasing VIP and galanin mRNA as does axonal transection. The differences in the neuropeptide levels achieved suggests that peptide expression after nerve transection is regulated both at the mRNA and protein levels.

In vagotomized, midcollicular decerebrate, non-anesthetized cats, in which the preganglionic input to the stellate ganglion was intact and the right forelimb was autoperfused at constant flow, the muscarinic receptor antagonist pirenzepine (PZP, 50 micrograms/kg i.v.), which does not cross the blood-brain barrier, produced a decrease in forelimb perfusion pressure (FLPP), heart rate (HR) and systemic arterial pressure (SAP). Administration of the nicotinic receptor antagonist hexamethonium (30 mg/kg i.v.) after PZP produced a further, larger drop in FLPP, HR and SAP. The dose of PZP used blocked the increase in FLPP and HR evoked by the muscarinic receptor agonist McN-A-343, but not the increase evoked by the nicotinic receptor agonist DMPP, when these drugs were injected into the arterial supply of the ganglion. Following administration of phentolamine and propranolol (2 mg/kg i.v. of each) which caused bradycardia and forelimb vasodilation, PZP had no effect on FLPP and HR. This finding suggests that PZP decreases sympathetic tone in resistance vessels and in the heart. Pirenzepine did not depress the forelimb vasoconstriction and the cardioacceleration evoked by electrical stimulation of the postganglionic vertebral and inferior cardiac nerves, respectively, suggesting that PZP does not act at the neuro-effector junctions. On the other hand, PZP blocked the forelimb vasoconstriction and the cardioacceleration produced by stimulating the preganglionic input of the stellate ganglion in presence of hexamethonium (30 mg/kg i.v.), indicating the stellate ganglion as the likely site of action of the drug. These findings suggest that the ganglion cell firing that underlies the sympathetic tone of cardiovascular effector cells is generated, in part, by a muscarinic, PZP-sensitive, synaptic mechanism.

A low-frequency stimulus train to the preganglionic input inhibits synaptic transmission in the superior cervical ganglion (SCG) of the cat. The inhibition is blocked by naloxone as well as by selective antagonists at mu and delta opiate receptors, which suggests that the mediator is an endogenous opioid [27,29]. Exogenous opioid peptides, including methionine-enkephalin (Met-Enk), which is present in preganglionic axons of the SCG, inhibit ganglionic transmission by a naloxone-sensitive mechanism. In the present study we test, in the anesthetized cat, whether the naloxone-sensitive synaptic inhibition is mediated by a pre- and/or post-synaptic mechanism. As a test of presynaptic inhibition, we measured the acetylcholine (ACh) released by preganglionic stimulation into the venous effluent of the perfused SCG. As a test of post-synaptic inhibition, we measured the effect of a preganglionic conditioning train on the ganglion cell firing evoked by ganglion-stimulant drugs injected into the arterial supply of the ganglion. In presence of naloxone (3 microM), which blocked the synaptic inhibition, the amount of ACh released by stimulated preganglionic axons did not change. Thus, the endogenous opioid which mediates the naloxone-sensitive inhibition does not act by depressing ACh release. In contrast, the ganglion cell firing evoked by ganglion-stimulant drugs was markedly depressed by a conditioning train, and naloxone blocked the depression, which suggests that the endogenous mediator of the naloxone-sensitive inhibition acts postsynaptically to decrease the excitability of ganglion cells. Exogenous Met-Enk depressed both ACh release by preganglionic stimulation and the firing of ganglion cells evoked by ganglion-stimulant drugs.(ABSTRACT TRUNCATED AT 250 WORDS)

In anesthetized cats, in which a nodose-superior cervical ganglion (SCG) anastomosis had been performed 6-9 months earlier, the nictitating membrane contraction evoked by electrical stimulation of the cervical vagus nerve ipsilateral to the anastomosis was recorded. The competence of nodose neurons in regulation the multiple synaptic mechanisms of the sympathetic ganglion was tested by comparing this response with the responses to stimulation of (self-reinnervated SCG). The response of the nictitating membrane ipsilateral to the anastomosis was smaller and had a lower EC50 for hexamethonium (C6) than the responses of the nictitating membrane ipsilateral to the intact or sutured CST. A 40 Hz 10s stimulus train to the intact or sutured CST produced potentiation of ganglionic transmission lasting 1 hour or longer, while a similar stimulus train to the anastomosed cervical vagus nerve produced no potentiation. During block of ganglionic nicotinic transmission with C6, CST or vagus nerve stimulation evoked responses which increased in amplitude with increasing stimulus frequency and were blocked by the selective muscarinic receptor antagonist pirenzepine. When the anticholinesterase eserine was added, the responses evoked by preganglionic stimulation decreased in amplitude in the intact SCG, as previously shown [7], and in the self-reinnervated SCG. This effect, which is due to inhibition mediated by muscarinic receptors selectively blocked by AF-DX116, was absent in the anastomosed SCG. During block of ganglionic transmission with C6 and atropine, a 40 Hz stimulus train to the intact or to the sutured CST evoked a slow, small amplitude contraction that was enhanced by naloxone. This response, most likely mediated by peptides [6], was absent in the anastomosed SCG.(ABSTRACT TRUNCATED AT 250 WORDS)

1. In anesthetized, atropine-treated cats we measured the acetylcholine (ACh) release into the venous effluent of the superior cervical ganglion (SCG) and the nictitating membrane (NM) contraction evoked by a 2-Hz, 20-s test train to the ipsilateral cervical sympathetic trunk (CST). We also measured NM contraction produced by injection of ACh (50 micrograms) or 1,1-dimethyl-4-phenylpiperazinium (DMPP, 5 micrograms) into the arterial supply of the ipsilateral SCG. 2. After a 10- to 30-s, 40-Hz conditioning train to CST these responses were all potentiated. The potentiation of the NM response evoked by the test train or by the exogenous agonists was long lasting (90% decay in 64 +/- 10 min, mean +/- SE, for the train-evoked response; 42 +/- 9 min for the response to injected ACh; 61 +/- 18 min for the response to injected DMPP), whereas the potentiation of the ACh release lasted only for 9 min. 3. On the assumption that ACh and DMPP injected into the ganglionic arterial supply acted postsynaptically, these data suggest that the main mechanism of the long-term potentiation (LTP) of nicotinic transmission in SCG is an increase in postsynaptic responsiveness. Because the response to KCl (250 micrograms) injected into the ganglionic arterial supply was not potentiated after the conditioning train, a posttrain increase in excitability of the postsynaptic membrane is not likely to be the cause of the postsynaptic increased responsiveness to ACh and DMPP.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been hypothesized that increased flux through the pentose phosphate pathway (PPP) is required to support the metabolic demands of rapid malignant cell growth. Using orthotopic mouse models of human glioblastoma (GBM) and renal cell carcinoma metastatic to brain, we estimated the activity of the PPP relative to glycolysis by infusing [1,2-(13) C(2) ]glucose. The [3-(13) C]lactate/[2,3-(13) C(2) ]lactate ratio was similar for both the GBM and brain metastasis and their respective surrounding brains (GBM, 0.197 ± 0.011 and 0.195 ± 0.033, respectively (p = 1); metastasis: 0.126 and 0.119 ± 0.033, respectively). This suggests that the rate of glycolysis is significantly greater than the PPP flux in these tumors, and that the PPP flux into the lactate pool is similar in both tumors. Remarkably, (13) C-(13) C coupling was observed in molecules derived from Krebs cycle intermediates in both tumor types, denoting glucose oxidation. In the renal cell carcinoma, in contrast with GBM, (13) C multiplets of γ-aminobutyric acid (GABA) differed from its precursor glutamate, suggesting that GABA did not derive from a common glutamate precursor pool. In addition, the orthotopic renal tumor, the patient's primary renal mass and brain metastasis were all strongly immunopositive for the 67-kDa isoform of glutamate decarboxylase, as were 84% of tumors on a renal cell carcinoma tissue microarray of the same histology, suggesting that GABA synthesis is cell autonomous in at least a subset of renal cell carcinomas. Taken together, these data demonstrate that (13) C-labeled glucose can be used in orthotopic mouse models to study tumor metabolism in vivo and to ascertain new metabolic targets for cancer diagnosis and therapy.

Nicotinic transmission in the superior cervical ganglion of the cat was studied in vivo and in vitro by recording the postganglionic compound action potential evoked, under partial block with hexamethonium, by supramaximal 0.2 Hz stimulation of the cervical sympathetic trunk. The compound action potential was depressed following a stimulus train (5 Hz, 40 s) applied to the same set of axons. The inhibition was antagonized by naloxone, suggesting mediation by endogenous opioids. Agonists selective for mu-, delta- and kappa-opioid receptors, injected into the arterial supply of the in situ ganglion or added to the perfusion fluid in the in vitro ganglion, also produced a naloxone-sensitive inhibition of the compound action potential. The synaptic inhibition was antagonized by the delta-selective antagonist ICI 174,864 and by the mu-selective antagonist CTAP, but not by the kappa-selective antagonist Nor-BNI. These results suggest that all three main types of opioid receptors are present in the ganglion but only mu- and delta-receptors are involved in the inhibition of ganglionic transmission mediated by the endogenous opioids.

We have shown previously that stimulation of the cat cervical sympathetic trunk for 2 h at 40 Hz Depletes the large Dense-cored vesicle Store in synaptic boutons of the superior cervical ganglion and that post-depletion recovery of the store takes several days. In the present study, we examine the properties of the depletion and recovery mechanisms. Invaginations of the plasmalemma suggestive of the exocytosis of dense cores were seen frequently in boutons from stimulated ganglia. The depletion process is calcium dependent: in ganglia perfused with calcium-free Krebs solution no depletion was produced by 40 Hz preganglionic stimulation. The depletion process is rapid: during continuous stimulation of the cervical sympathetic trunk with 40 Hz, depletion observed by the end of 2 h was similar to depletion by the end of the initial 5 min of stimulation. The depletion process is frequency dependent: when the cervical sympathetic trunk was stimulated with a constant number of stimuli, no depletion occurred at the frequency of 2 or 10 Hz, while the frequencies of 20 and 40 Hz produced depletion, which was greater at 40 Hz. Recovery of the large dense-cored vesicle store during the initial 24 h after 10 min of 40 Hz stimulation was faster, and of approximately the same magnitude, than during the succeeding five days. Recovery of the store after stimulus-evoked depletion was prevented by application of colchicine to the cervical sympathetic trunk, which suggests dependence of recovery on fast axonal transport. Large dense-cored vesicles accumulated in the colchicine-treated segment of cervical sympathetic trunk axons. In conclusion, these observations suggest that the stimulus-evoked depletion of large dense-cored vesicle stores in synaptic boutons of the cat superior cervical ganglion is the result of calcium-dependent exocytosis of the large dense-cored vesicle core and that the post-stimulus recovery is critically dependent on microtubule-mediated axonal transport.

The effect of the muscarinic receptor antagonist AF-DX 116 on the inhibitory action of muscarinic agonists and on responses mediated by nicotinic or muscarinic ganglionic transmission was studied in the superior cervical ganglion of the anesthetized cat. The postganglionic compound action potential evoked by cervical sympathetic trunk stimulation was depressed by methacholine or acetylcholine (ACh) injected into the ganglionic arterial supply. The depression was blocked by AF-DX 116. The compound action potentials evoked by preganglionic stimulus trains were also depressed when the intratrain frequency was 2 Hz or greater. This intratrain depression was, however, insensitive to AF-DX 116. The anticholinesterase drug physostigmine markedly enhanced the intratrain depression of the compound action potential. This effect was reversed by AF-DX 116. During nicotinic receptor block with hexamethonium, preganglionic stimulus trains with intratrain frequencies of 5 Hz or greater produced nictitating membrane contractions that could be blocked by the M1 muscarinic receptor antagonist pirenzepine. The amplitude of the contractions increased with frequency and reached a maximum at 20–40 Hz. AF-DX 116 had no effect on these responses. After administration of physostigmine, the amplitude of the nictitating membrane responses decreased with increasing intratrain frequency. AF-DX 116 reversed this effect. The data suggest that, in the superior cervical ganglion, AF-DX 116 sensitive muscarinic receptors which depress synaptic transmission are activated by exogenous agonists but not by the ACh released by the preganglionic axon terminals unless cholinesterase activity is inhibited. This may result from the fact that these receptors, although accessible to agonists injected into the ganglionic arterial supply, are remote from synaptic release sites and that, under normal conditions, cholinesterase activity prevents sufficient activation of these receptors by the nerve-released ACh to result in modulation of synaptic transmission.Key words: muscarinic inhibition, M2 receptors, sympathetic ganglion modulation.

The superior cervical ganglion of rats was perfused with Ringer solution containing hexamethonium to produce a steady, partial, nicotinic block. The compound action potential (CAP) evoked by supramaximal single shock stimulation of the cervical sympathetic trunk (CST) was recorded from the internal carotid nerve. Bolus injection of the protein kinase C (PKC) activators 4 beta-phorbol-12,13-dibutyrate (PDBu) or 4 beta-phorbol-12,13-diacetate (PDAc) produced a marked, prolonged, dose-dependent potentiation of the CAP amplitude (e.g. 90% decay 2 h). A non-PKC activating phorbol ester (PE), 4 alpha-phorbol-12,13-didecanoate, produced no potentiation. The PE-induced potentiation was antagonized by the PKC inhibitor H-7. In addition, after 1 h exposure to PDBu (3 microM) and recovery from the potentiation (e.g. 2-4 h), a second exposure to PDBu or PDAc produced no potentiation. A 5 s 40 Hz supramaximal train to the CST produced a long lasting potentiation of the CAP (long-term potentiation, LTP) as described previously. However, a similar train did not evoke LTP after perfusion for 1 h with PDBu. The train-evoked LTP was depressed by the PKC inhibitor H-7 at a concentration which antagonized the PE-evoked potentiation. These data suggest that (i) PKC activation potentiates nicotinic transmission, and (ii) a component of the train-evoked LTP is mediated by PKC.

A stimulus train to preganglionic axons produces long-term potentiation (LTP) of population responses of sympathetic ganglion cells evoked by the same or by other converging axons. The present study shows that preganglionic axons emerging from the spinal cord in different thoracic rami, and converging onto a common pool of ganglion cells that innervate a single target, differ in their ability to induce LTP. In anesthetized cats under partial nicotinic block with hexamethonium, the nictitating-membrane (NM) contraction evoked by stimulation of the first (T1) and third (T3) thoracic white rami (WR) was recorded. Each ramus produced a contraction of similar amplitude. In contrast, the homosynaptic potentiation produced by a 40-Hz 10-s train differed markedly. T1WR produced a potentiation of duration comparable to that produced by stimulation of the cervical sympathetic trunk, while T3WR produced either no potentiation or a potentiation of much shorter duration. The NM response evoked by T3WR, however, was potentiated by similar extent and duration as was the response evoked by T1WR when the train was applied to T1WR (heterosynaptic LTP). This suggests that the ganglionic synapses made by T3WR possess the mechanism for expressing LTP. Conversely, T3WR was ineffective at potentiating heterosynaptically the NM response evoked by T1WR. These results suggest that the ability to produce or release the LTP inducer varies markedly among sympathetic preganglionic neurons.Key words: preganglionic neuron heterogeneity, long-term potentiation induction, synaptic transmission, neuropeptides.

Neurotensin (NT) within the stellate ganglion (SG) of the cat is present in axon terminals of extrinsic, presumably preganglionic, neurons and may play a role in ganglionic transmission. NT content of the SG, and of the preganglionic axons which innervate it, was determined by radioimmunoassay in the anesthetized cat under various experimental conditions in order to understand the factors determining the size of the ganglionic NT stores. The immunoreactive NT (iNT) from extracts of SG and its preganglionic inputs (white rami T2 and T3, sympathetic trunk between SG and T4 white ramus) coeluted with synthetic NT(1-13) on RP-HPLC. NT accumulated proximal to ligatures on the preganglionic inputs of the SG. The daily rate of axonal transport of NT, estimated from the accumulation, represents 28.7% of the ganglionic stores of NT. Preganglionic stimulation at 2 Hz for 100 min did not change ganglionic NT content. Preganglionic stimulation at 40 Hz reduced the NT content to 70.4 +/- 1.8% of control in 10 min and to 34.7 +/- 4.2% of control in 20 min. Additional 100 min of 40 Hz stimulation produced no further depletion. The residual iNT, which coeluted with NT1-13, presumably represents a pool of unreleasable NT. Post-depletion recovery was complete in 7 days and showed an initial rapid phase over the first 24 h followed by a slower phase over the remaining 6 days. Pepsin treatment, which has previously been shown to generate iNT from NT precursor in liver and other tissues, provided no evidence of the NT precursor in extracts of SG and its preganglionic input.(ABSTRACT TRUNCATED AT 250 WORDS)

In anesthetized, artificially ventilated cats, pretreated with the muscarinic antahonist scopolamine, the effect of naloxone on the efficacy of nicotinic transmission in the superior cervical ganglion was used as a test of endogenous opioid release by the preganglionic axons. The cervical sympathetic trunk (CST) was split into two bundles. The compound action potential (CAP), evoked by supramaximal low-frequency stimulation (0.25 Hz) of one CST bundle, was recorded from a postganglionic nerve of the superior cervical ganglion. Partial block with hexamethonium was used to reduce the ‘safety factor’ of nicotinic transmission. A conditioning train (5 Hz, 40 s, supramaximal) to the other CST bundle (heterosynaptic conditioning) inhibited the CAP. At the peak of the inhibition the CAP was attenuated by 51 ± 4%. Recovery was 90% complete in 201 ± 28s. The inhibition was antagonized in a dose-dependent manner by i.v. naloxone with an apparent IC 50 of 60 ± 12 μg/kg. The maximum effect obtained with naloxone was an 80% decrease in the magnitude of the inhibition. Magnitude and duration of the heterosynaptic inhibition were related to frequency, duration and intensity of the conditioning train. Naloxone-sensitive inhibition was observed with frequencies of the conditioning train as low as 0.5 Hz. A train (5 Hz, 40 s) to the postganglionic nerves produced a naloxone-insensitive depression of the orthodromic test CAP which was of smaller amplitude and duration than when the conditioning train was applied to the preganglionic axons. The endopeptidase inhibitors thiorphan (0.3–0.5 mg/kg i.v.) and captopril (0.2 mg/kg i.v.) increased magnitude and duration of the depression produced by CST stimulation, but not of that produced by postganglionic nerve stimulation. The aminopeptidase inhibitor bestatin (0.2 mg/kg i.v.) had no effect. These observations suggest that preganglionic axon terminals in the superior cervical ganglion release opioid peptides when stimulated electrically. Very low frequencies of preganglionic stimulation are effective, suggesting that endogenous opioids may also be released during the natural discharge of the preganglionic neurons. Endopeptidases provide a significant modulation of the actions of the endogenous opioids.

1. In the anesthetized, scopolamine-treated cat, the compound action potential (CAP) evoked by a single supramaximal shock to the third thoracic white ramus (T3WR) was recorded in the inferior cardiac nerve (ICN). The CAP was depressed in a dose-dependent manner by the intravenous administration of the nicotinic antagonist hexamethonium (C6). 2. During steady intravenous infusion of C6, which reduced the amplitude of the CAP by 80-90%, a short train of stimuli (few seconds, 10-40 Hz) to the sympathetic trunk just below T4WR potentiated the CAP for periods of tens of minutes to 1-2 h (heterosynaptic long-term potentiation, LTP). An LTP of similar time course was obtained when both train and single shock were applied to T3WR (homosynaptic LTP). Magnitude and duration of the heterosynaptic LTP were dependent on number, frequency, and intensity of the stimuli. No LTP was produced by a train to the ICN. Heterosynaptic LTP was also observed in the absence of C6. Because of the limited subliminal fringe of the test input under this condition, the LTP was of small magnitude. Heterosynaptic LTP also of the heart rate (HR) response to a test stimulus was observed after a conditioning train. 3. The conditioning train produced a displacement to the right of the dose-response curve for C6. The intravenous dose of C6 required for 50% attenuation of the test CAP increased from 0.84 +/- 0.15 (SE) mg/kg pretrain to 2.56 +/- 0.46 mg/kg posttrain (n = 5, P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the transcription factor STAT3 is thought to potently promote oncogenesis in a variety of tissues, leading to intense efforts to develop STAT3 inhibitors for many tumors, including the highly malignant brain tumor glioblastoma. However, the function of STAT3 in glioblastoma pathogenesis has remained unknown. Here, we report that STAT3 plays a pro-oncogenic or tumor-suppressive role depending on the mutational profile of the tumor. Deficiency of the tumor suppressor PTEN triggers a cascade that inhibits STAT3 signaling in murine astrocytes and human glioblastoma tumors. Specifically, we forge a direct link between the PTEN–Akt–FOXO axis and the leukemia inhibitory factor receptor β (LIFRβ)–STAT3 signaling pathway. Accordingly, PTEN knockdown induces efficient malignant transformation of astrocytes upon knockout of the STAT3 gene. Remarkably, in contrast to the tumor-suppressive function of STAT3 in the PTEN pathway, STAT3 forms a complex with the oncoprotein epidermal growth factor receptor type III variant (EGFRvIII) in the nucleus and thereby mediates EGFRvIII-induced glial transformation. These findings indicate that STAT3 plays opposing roles in glial transformation depending on the genetic background of the tumor, providing the rationale for tailored therapeutic intervention in glioblastoma.

In cats anesthetized with sodium pentobarbital, a supramaximal 40-Hz, 30-s train to the cervical sympathetic trunk, during block of ganglionic cholinergic transmission with hexamethonium and scopolamine, produced a delayed, slow, small amplitude contraction of the nictitating membrane that persisted for several minutes after the end of the stimulus train. The post-stimulus component of the response was due to afterdischarge of the ganglion cells, since section of the post-ganglionic axons at the end of the train resulted in elimination of this component. The amplitude of the slow nictitating membrane response was enhanced in a dose-dependent manner by i.v. injection of naloxone. The enhancement was detectable at a dose as low as 1 μg/kg and was maximal at 10 μg/kg. During continuous preganglionic stimulation at 40 Hz, the amplitude of the slow nictitating membrane response reached a peak in 2–4 min and then faded with time until it became undetectable. Time for 90% decay was 82 ± 5 min (n = 18). The nictitating membrane response to postganglionic nerve stimulation was not modified by prolonged preganglionic stimulation. In three cats, the cervical sympathetic trunk was split into two bundles and one bundle was stimulated continuously at 40 Hz until the slow response disappeared. At this time stimulation of the unconditioned bundle evoked a slow response of normal appearance. This suggests that the process underlying the fade involves only the conditioned axons. Recovery from the fade was slow, the response approaching control by 24 h post-stimulus. In contrast, the nictitating membrane response mediated by ganglionic nicotinic transmission was of amplitude similar to control within the 1st min after the end of a 2-h period of continuous 40 Hz stimulation. The fade and recovery of the slow response may be the consequence of presynaptic events and reflect the exhaustion followed by slow replenishment of the releasable stores of the noncholinergic transmitter. The enhancement of the slow response by naloxone suggests that an inhibitory noncholinergic transmitter, presumably an opioid, is also released by the preganglionic axon terminals.Key words: peptides, opioids, ganglionic transmission, cotransmitters, synapse.

Large dense core vesicles (LDCV), a possible site for neuropeptide storage, were counted within synaptic boutons of the superior cervical ganglion of anaesthetized cats in the unstimulated condition and 2 h, 1 day or 10 days after 40 Hz 2 h stimulation of the cervical sympathetic trunk. The number of LDCV decreased markedly after stimulation. It was lowest at 2 h and then recovered slowly. Ten days post-train the number was close to, but still significantly lower than, control. This time course of recovery is much longer than for agranular vesicles number, which is known to be back to normal within 2 h of the end of a comparable stimulus train. The post-stimulus loss of LDCVs is consistent with the hypothesis that LDCVs are released by the nerve terminal. The slow recovery is consistent with the hypothesis of replenishment by axoplasmic transport.

The effect of brief superior laryngeal nerve stimulation on the respiratory rhythm was investigated in midcollicular decerebrate, unanesthetized, artificially ventilated, paralysed, vagotomized and debuffered cats. Stimulus trains (50 ms, 200 Hz) delivered during inspiration (I) with intensities exceeding a threshold value, that was inversely related to the phase of the cycle, terminated I and shortened the following expiration (E) (irreversible I termination). Stimulus trains given during I with intensities just below this threshold value produced a transient suppression of I followed by resumption of activity, resulting in a slight prolongation of both I and the following E (reversible I termination). Stimulation during E produced a phase-dependent prolongation of E, but did not affect the next I. Phase-resetting curves were constructed by measuring the changes in respiratory cycle duration produced by stimuli given at phases throughout the cycle. A single stimulus produced aftereffects that lasted several cycles. The aftereffects were investigated by delivering stimuli at a fixed delay from cycle onset every n cycles (n is an integer). Certain combinations of delay, stimulus intensity, and n, resulted in (1) a variable combination of reversible and irreversible I terminations, rather than a consistent response, and (2) an increase in cycle duration. The stimulus aftereffects, that can last up to 9 cycles, may account for previously described unpredictability in the response of the respiratory oscillator to a given stimulus.

Little progress has been made toward the use of embryonic stem (ES) cells to study and isolate skeletal muscle progenitors. This is due to the paucity of paraxial mesoderm formation during embryoid body (EB) in vitro differentiation and to the lack of reliable identification and isolation criteria for skeletal muscle precursors. Here we show that expression of the transcription factor Pax3 during embryoid body differentiation enhances both paraxial mesoderm formation and the myogenic potential of the cells within this population. Transplantation of Pax3-induced cells results in teratomas, however, indicating the presence of residual undifferentiated cells. By sorting for the PDGF-alpha receptor, a marker of paraxial mesoderm, and for the absence of Flk-1, a marker of lateral plate mesoderm, we derive a cell population from differentiating ES cell cultures that has substantial muscle regeneration potential. Intramuscular and systemic transplantation of these cells into dystrophic mice results in extensive engraftment of adult myofibers with enhanced contractile function without the formation of teratomas. These data demonstrate the therapeutic potential of ES cells in muscular dystrophy.

SummaryRecent studies have identified stem cells in brain cancer. However, their relationship to normal CNS progenitors, including dependence on common lineage-restricted pathways, is unclear. We observe expression of the CNS-restricted transcription factor, OLIG2, in human glioma stem and progenitor cells reminiscent of type C transit-amplifying cells in germinal zones of the adult brain. Olig2 function is required for proliferation of neural progenitors and for glioma formation in a genetically relevant murine model. Moreover, we show p21WAF1/CIP1, a tumor suppressor and inhibitor of stem cell proliferation, is directly repressed by OLIG2 in neural progenitors and gliomas. Our findings identify an Olig2-regulated lineage-restricted pathway critical for proliferation of normal and tumorigenic CNS stem cells.

The astrocyte represents the most abundant yet least understood cell type of the CNS. Here, we use a stringent experimental strategy to molecularly define the astrocyte lineage by integrating microarray datasets across several in vitro model systems of astrocyte differentiation, primary astrocyte cultures, and various astrocyterich CNS structures. The intersection of astrocyte data sets, coupled with the application of nonastrocytic exclusion filters, yielded many astrocyte-specific genes possessing strikingly varied patterns of regional CNS expression. Annotation of these astrocyte-specific genes provides direct molecular documentation of the diverse physiological roles of the astrocyte lineage. This global perspective in the normal brain also provides a framework for how astrocytes may participate in the pathogenesis of common neurological disorders like Alzheimer's disease, Parkinson's disease, stroke, epilepsy, and primary brain tumors.

Ink4a/Arf inactivation and epidermal growth factor receptor (EGFR) activation are signature lesions in high-grade gliomas. How these mutations mediate the biological features of these tumors is poorly understood. Here, we demonstrate that combined loss of p16(INK4a) and p19(ARF), but not of p53, p16(INK4a), or p19(ARF), enables astrocyte dedifferentiation in response to EGFR activation. Moreover, transduction of Ink4a/Arf(-/-) neural stem cells (NSCs) or astrocytes with constitutively active EGFR induces a common high-grade glioma phenotype. These findings identify NSCs and astrocytes as equally permissive compartments for gliomagenesis and provide evidence that p16(INK4a) and p19(ARF) synergize to maintain terminal astrocyte differentiation. These data support the view that dysregulation of specific genetic pathways, rather than cell-of-origin, dictates the emergence and phenotype of high-grade gliomas.

Object. The authors evaluated the effectiveness of microsurgical C-2 ganglionectomy in 39 patients with medically refractory chronic occipital pain. In this procedure the neurons transmitting sensory inputs from the occiput are removed and, unlike peripheral nerve ablation, axonal regeneration is not possible. Methods. The patients in this series had symptoms for 1 to 43 years. In 22 patients the occipital pain was caused by trauma; in 17 patients the pain was spontaneous. Pain relief failed in 17 patients who had undergone a previous occipital neurectomy or C-2 rhizolysis. Twenty-three patients experienced pain that was described as shocklike, electric, shooting, jabbing, stabbing, sharp, or exploding (Group I). Eight patients described their pain as dull, pounding, aching, throbbing, or pressurelike (Group II). The patients underwent unilateral or bilateral C-2 open microsurgical ganglionectomies. The postoperative follow-up period ranged from 19 to 48 months. Nineteen patients experienced an excellent result (> 90% reduction in pain). Pain caused by trauma or that described using Group I terms responded best to ganglionectomy (80% good or excellent response). In contrast, the majority of the patients with nontraumatic pain or those described using Group II descriptors did not achieve favorable results. Conclusions. The authors conclude that: 1) patients who suffer from chronic occipital pain after having sustained injury obtain worthwhile benefit from microsurgical C-2 ganglionectomy; 2) patients suffering from migraine, tension, and vascular headaches involving the occipital area are most often not helped by this operation; and 3) terms such as “shock,” “electric,” “shooting,” “jabbing,” and “sharp” used to describe occipital pain predict a favorable pain outcome following a C-2 ganglionectomy.