Angiogenic Factors Stimulate Growth of Adult Neural Stem Cells

The ability to grow a uniform cell type from the adult central nervous system (CNS) is valuable for developing cell therapies and new strategies for drug discovery. The adult mammalian brain is a source of neural stem cells (NSC) found in both neurogenic and non-neurogenic zones but difficulties in culturing these hinders their use as research tools.Here we show that NSCs can be efficiently grown in adherent cell cultures when angiogenic signals are included in the medium. These signals include both anti-angiogenic factors (the soluble form of the Notch receptor ligand, Dll4) and pro-angiogenic factors (the Tie-2 receptor ligand, Angiopoietin 2). These treatments support the self renewal state of cultured NSCs and expression of the transcription factor Hes3, which also identifies the cancer stem cell population in human tumors. In an organotypic slice model, angiogenic factors maintain vascular structure and increase the density of dopamine neuron processes.We demonstrate new properties of adult NSCs and a method to generate efficient adult NSC cultures from various central nervous system areas. These findings will help establish cellular models relevant to cancer and regeneration

Cholera Toxin Regulates a Signaling Pathway Critical for the Expansion of Neural Stem Cell Cultures from the Fetal and Adult Rodent Brains

Background: New mechanisms that regulate neural stem cell (NSC) expansion will contribute to improved assay systems and the emerging regenerative approach that targets endogenous stem cells. Expanding knowledge on the control of stem cell self renewal will also lead to new approaches for targeting the stem cell population of cancers. Methodology/Principal Findings: Here we show that Cholera toxin regulates two recently characterized NSC markers, the Tie2 receptor and the transcription factor Hes3, and promotes the expansion of NSCs in culture. Cholera toxin increases immunoreactivity for the Tie2 receptor and rapidly induces the nuclear localization of Hes3. This is followed by powerful cultured NSC expansion and induction of proliferation both in the presence and absence of mitogen. Conclusions/Significance: Our data suggest a new cell biological mechanism that regulates the self renewal and differentiation properties of stem cells, providing a new logic to manipulate NSCs in the context of regenerative disease and cancer

Enhanced 5-ALA Induced Fluorescence in Hormone Secreting Pituitary Adenomas

Introduction Cushing’s Disease (CD) is caused by millimeter-sized corticotropinomas (microadenomas) that lead to supraphysiological levels of glucocorticoid. Up to 40% of microadenomas are not visualized on gold-standard MR imaging. Pituitary adenomas metabolize exogenous 5-ALA (an endogenous metabolite) to protoporphyrin IX (PpIX) at rates 20-50 times higher compared with normal tissues. PpIX intensely fluoresces red (635nm) when excited with blue light (375-440nm), enabling its use as an intraoperative fluorescence imaging agent. 5-ALA is now an FDA approved prodrug. We examined the efficacy of ALA-induced-PpIX fluorescence in human derived adenomatous and normal pituitary samples. We explored the modulation of PpIX conversion with CRH or dexamethasone (DEX), and subcellular localization of PpIX. Methods We used flow cytometry for PpIX intensity analysis. A human-derived corticotropinoma, it’s adjacent normal gland, murine normal pituitary cells, and AtT20 cells were incubated with 5-ALA (300 nM) with/without DEX (1µM) or CRH (50nM). For confocal microscopy, live cells imaged for PpIX (405nm/615nm) and mitochondrial (550nm/615nm) fluorescence. Results We found a 10-fold-increase in 5-ALA induced PpIX fluorescence intensity in human-derived adenomatous compared to adjacent normal pituitary tissue (n=1, p\u3c0.05). AtT-20 cell lines (n=6, p\u3c0.05) fluoresced 7-fold more intensely compared to normal murine pituitary tissue (n=3, p\u3c0.05). The addition of DEX, before or after 5-ALA exposure, increased the fluorescence intensity by 31% (n=4, p\u3c0.05). The addition of CRH did not have a significant effect on 5-ALA fluorescence (n=3, p\u3c0.05). We saw localization of 5-ALA to mitochondria, and mitochondrial disruption in 5-ALA treated At-T20s. Conclusions Our results support the use of 5-ALA for fluorescence guided resection in hormone secreting microadenomas. The supraphysiological levels of glucocorticoids, as seen in CD, may enhance the 5-ALA fluorescence in corticotropinomas. We confirm the mitochondrial localization and disruption by 5-ALA, a basis for photodynamic therapy

Convection perfusion of glucocerebrosidase for neuronopathic Gaucher's disease

Systemic enzyme replacement for Gaucher's disease has not prevented premature death or severe morbidity in patients with a neuronopathic phenotype, because the enzyme does not cross the blood-brain barrier. We used convection-enhanced delivery for regional distribution of glucocerebrosidase in rat and primate brains and examined its safety and feasibility for neuronopathic Gaucher's disease. Rats underwent intrastriatal infusion and were observed and then sacrificed at 14 hours, 4 days, or 6 weeks. Primates underwent serial magnetic resonance imaging during enzyme perfusion of the right frontal lobe or brainstem, were observed and then sacrificed after infusion completion. Animals underwent histologic and enzymatic tissue analyses. Magnetic resonance imaging revealed perfusion of the primate right frontal lobe or Pons with infitsate. Enzyme activity was substantially and significantly (p <0.05) increased in cortex and white matter of the infused frontal lobe and pons compared to control. Immunohistochemistry demonstrated intraneuronal glucocerebrosidase. There was no toxicity. Convection-enhanced delivery can be used to safely perfuse large regions of the brain and brainstem with therapeutic levels of glucocerebrosidase. Patients with neuronopathic Gaucher's disease and similar central nervous system disorders may benefit from this treatmen

Targeting protein arginine methyltransferase 5 sensitizes glioblastoma to trametinib

Background: The prognosis of glioblastoma (GBM) remains dismal because therapeutic approaches have limited effectiveness. A new targeted treatment using MEK inhibitors, including trametinib, has been proposed to improve GBM therapy. Trametinib had a promising preclinical effect against several cancers, but its adaptive treatment resistance precluded its clinical translation in GBM. Previously, we have demonstrated that protein arginine methyltransferase 5 (PRMT5) is upregulated in GBM and its inhibition promotes apoptosis and senescence in differentiated and stem-like tumor cells, respectively. We tested whether inhibition of PRMT5 can enhance the efficacy of trametinib against GBM. Methods: Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, ELISA, and western blot were analyzed. In vivo, NSG mice were intracranially implanted with PRMT5-intact and -depleted GBMNS, treated with trametinib by daily oral gavage, and observed for tumor progression and mice survival rate. Results: PRMT5 depletion enhanced trametinib-induced cytotoxicity in GBMNS. PRMT5 knockdown significantly decreased trametinib-induced AKT and ERBB3 escape pathways. However, ERBB3 inhibition alone failed to block trametinib-induced AKT activity suggesting that the enhanced antitumor effect imparted by PRMT5 knockdown in trametinib-treated GBMNS resulted from AKT inhibition and not ERBB3 inhibition. In orthotopic murine xenograft models, PRMT5-depletion extended the survival of tumor-bearing mice, and combination with trametinib further increased survival. Conclusion: Combined PRMT5/MEK inhibition synergistically inhibited GBM in animal models and is a promising strategy for GBM therapy

Human endogenous retrovirus K contributes to a stem cell niche in glioblastoma

Human endogenous retroviruses (HERVs) are ancestral viral relics that constitute nearly 8% of the human genome. Although normally silenced, the most recently integrated provirus HERV-K (HML-2) can be reactivated in certain cancers. Here, we report pathological expression of HML-2 in malignant gliomas in both cerebrospinal fluid and tumor tissue that was associated with a cancer stem cell phenotype and poor outcomes. Using single-cell RNA-Seq, we identified glioblastoma cellular populations with elevated HML-2 transcripts in neural progenitor–like cells (NPC-like) that drive cellular plasticity. Using CRISPR interference, we demonstrate that HML-2 critically maintained glioblastoma stemness and tumorigenesis in both glioblastoma neurospheres and intracranial orthotopic murine models. Additionally, we demonstrate that HML-2 critically regulated embryonic stem cell programs in NPC-derived astroglia and altered their 3D cellular morphology by activating the nuclear transcription factor OCT4, which binds to an HML-2–specific long-terminal repeat (LTR5Hs). Moreover, we discovered that some glioblastoma cells formed immature retroviral virions, and inhibiting HML-2 expression with antiretroviral drugs reduced reverse transcriptase activity in the extracellular compartment, tumor viability, and pluripotency. Our results suggest that HML-2 fundamentally contributes to the glioblastoma stem cell niche. Because persistence of glioblastoma stem cells is considered responsible for treatment resistance and recurrence, HML-2 may serve as a unique therapeutic target