BACKGROUND: Although both the alkylating agent temozolomide (TMZ) and oncolytic viruses hold promise for treating glioblastoma, which remains uniformly lethal, the effectiveness of combining the two treatments and the mechanism of their interaction on cancer stem cells are unknown. METHODS: We investigated the efficacy of combining TMZ and the oncolytic herpes simplex virus (oHSV) G47Δ in killing glioblastoma stem cells (GSCs), using Chou-Talalay combination index analysis, immunocytochemistry and fluorescence microscopy, and neutral comet assay. The role of treatment-induced DNA double-strand breaks, activation of DNA damage responses, and virus replication in the cytotoxic interaction between G47Δ and TMZ was examined with a panel of pharmacological inhibitors and short-hairpin RNA (shRNA)-mediated knockdown of DNA repair pathways. Comparisons of cell survival and virus replication were performed using a two-sided t test (unpaired). The survival of athymic mice (n = 6-8 mice per group) bearing GSC-derived glioblastoma tumors treated with the combination of G47Δ and TMZ was analyzed by the Kaplan-Meier method and evaluated with a two-sided log-rank test. RESULTS: The combination of G47Δ and TMZ acted synergistically in killing GSCs but not neurons, with associated robust induction of DNA damage. Pharmacological and shRNA-mediated knockdown studies suggested that activated ataxia telangiectasia mutated (ATM) is a crucial mediator of synergy. Activated ATM relocalized to HSV DNA replication compartments where it likely enhanced oHSV replication and could not participate in repairing TMZ-induced DNA damage. Sensitivity to TMZ and synergy with G47Δ decreased with O(6)-methylguanine-DNA-methyltransferase (MGMT) expression and MSH6 knockdown. Combined G47Δ and TMZ treatment extended survival of mice bearing GSC-derived intracranial tumors, achieving long-term remission in four of eight mice (median survival = 228 days; G47Δ alone vs G47Δ + TMZ, hazard ratio of survival = 7.1, 95% confidence interval = 1.9 to 26.1, P = .003) at TMZ doses attainable in patients. CONCLUSIONS: The combination of G47Δ and TMZ acts synergistically in killing GSCs through oHSV-mediated manipulation of DNA damage responses. This strategy is highly efficacious in representative preclinical models and warrants clinical translation. View Publication

PURPOSE: To develop a new oncolytic herpes simplex virus (oHSV) for glioblastoma (GBM) therapy that will be effective in glioblastoma stem cells (GSC), an important and untargeted component of GBM. One approach to enhance oHSV efficacy is by combination with other therapeutic modalities. EXPERIMENTAL DESIGN: MG18L, containing a U(S)3 deletion and an inactivating LacZ insertion in U(L)39, was constructed for the treatment of brain tumors. Safety was evaluated after intracerebral injection in HSV-susceptible mice. The efficacy of MG18L in human GSCs and glioma cell lines in vitro was compared with other oHSVs, alone or in combination with phosphoinositide-3-kinase (PI3K)/Akt inhibitors (LY294002, triciribine, GDC-0941, and BEZ235). Cytotoxic interactions between MG18L and PI3K/Akt inhibitors were determined using Chou-Talalay analysis. In vivo efficacy studies were conducted using a clinically relevant mouse model of GSC-derived GBM. RESULTS: MG18L was severely neuroattenuated in mice, replicated well in GSCs, and had anti-GBM activity in vivo. PI3K/Akt inhibitors displayed significant but variable antiproliferative activities in GSCs, whereas their combination with MG18L synergized in killing GSCs and glioma cell lines, but not human astrocytes, through enhanced induction of apoptosis. Importantly, synergy was independent of inhibitor sensitivity. In vivo, the combination of MG18L and LY294002 significantly prolonged survival of mice, as compared with either agent alone, achieving 50% long-term survival in GBM-bearing mice. CONCLUSIONS: This study establishes a novel therapeutic strategy: oHSV manipulation of critical oncogenic pathways to sensitize cancer cells to molecularly targeted drugs. MG18L is a promising agent for the treatment of GBM, being especially effective when combined with PI3K/Akt pathway-targeted agents. View Publication

p73, a member of the p53 family, is a transcription factor that plays a key role in many biological processes. In the present study, we show that TAp73 is expressed in neural stem cells (NSC) and its expression increases following their differentiation. NSC from p73 null mice have a reduced proliferative potential, together with reduced expression of members of the Sox-2 and Notch gene families known to be important for NSC proliferation. In parallel with this in vitro data, the width of the neurogenic areas was reduced in the brains of embryonic and adult p73-/- mice. These data suggest that p73, and in particular TAp73, is important for maintenance of the NSC pool. View Publication

Current data suggests an association between elevations in interleukin 1 (IL-1)α, IL-1β, and IL-6 and the proliferation of neural progenitor cells (NPCs) following brain injury. A limited amount of work implicates changes in these pro-inflammatory responses with diminished NPC proliferation observed as a function of aging. In the current study, adolescent (21day-old) and 1year-old CD-1 male mice were injected with trimethyltin (TMT, 2.3mg/kg, i.p.) to produce acute apoptosis of hippocampal dentate granule cells. In this model, fewer 5-bromo-2'-deoxyuridine (BrdU)+ NPC were observed in both naive and injured adult hippocampus as compared to the corresponding number seen in adolescent mice. At 48h post-TMT, a similar level of neuronal death was observed across ages, yet activated ameboid microglia were observed in the adolescent and hypertrophic process-bearing microglia in the adult. IL-1α mRNA levels were elevated in the adolescent hippocampus; IL-6 mRNA levels were elevated in the adult. In subgranular zone (SGZ) isolated by laser-capture microdissection, IL-1β was detected but not elevated by TMT, IL-1a was elevated at both ages, while IL-6 was elevated only in the adult. Naïve NPCs isolated from the hippocampus expressed transcripts for IL-1R1, IL-6Rα, and gp130 with significantly higher levels of IL-6Rα mRNA in the adult. In vitro, IL-1α (150pg/ml) stimulated proliferation of adolescent NPCs; IL-6 (10ng/ml) inhibited proliferation of adolescent and adult NPCs. Microarray analysis of SGZ post-TMT indicated a prominence of IL-1a/IL-1R1 signaling in the adolescent and IL-6/gp130 signaling in the adult. View Publication

Monoamine neurotransmitters play major roles in regulating a range of brain functions in adults and increasing evidence suggests roles for monoamines in brain development. Here we show that mice lacking the monoamine metabolic enzymes MAO A and MAO B (MAO AB-deficient mice) exhibit diminished proliferation of neural stem cells (NSC) in the developing telencephalon beginning in late gestation [embryonic day (E) 17.5], a deficit that persists in neonatal and adult mice. These mice showed significantly increased monoamine levels and anxiety-like behaviors as adults. Assessments of markers of intermediate progenitor cells (IPC) and mitosis showed that NSC in the subventricular zone (SVZ), but not in the ventricular zone, are reduced in MAO AB-deficient mice. A developmental time course of monoamines in frontal cortical tissues revealed increased serotonin levels as early as E14.5, and a further large increase was found between E17.5 and postnatal day 2. Administration of an inhibitor of serotonin synthesis (parachlorophenylalanine) between E14.5 and E19.5 restored the IPC numbers and SVZ thickness, suggesting the role of serotonin in the suppression of IPC proliferation. Studies of neurosphere cultures prepared from the telencephalon at different embryonic and postnatal ages showed that serotonin stimulates proliferation in wild-type, but not in MAO AB-deficient, NSC. Together, these results suggest that a MAO-dependent long-lasting alteration in the proliferation capacity of NSC occurs late in embryonic development and is mediated by serotonin. Our findings reveal novel roles for MAOs and serotonin in the regulation of IPC proliferation in the developing brain. View Publication

The vast majority of pedigrees with familial Alzheimer's disease (FAD) are caused by inheritance of mutations in the PSEN1 1 gene. While genetic ablation studies have revealed a role for presenilin 1 (PS1) in embryonic neurogenesis, little information has emerged regarding the potential effects of FAD-linked PS1 variants on proliferation, self-renewal and differentiation, key events that control cell fate commitment of adult brain neural progenitors (NPCs). We used adult brain subventricular zone (SVZ)-derived NPC cultures transduced with recombinant lentivirus as a means to investigate the effects of various PS1 mutants on self-renewal and differentiation properties. We now show that viral expression of several PS1 mutants in NPCs leads to impaired self-renewal and altered differentiation toward neuronal lineage, in vitro. In line with these observations, diminished constitutive proliferation and steady-state SVZ progenitor pool size was observed in vivo in transgenic mice expressing the PS1DeltaE9 variant. Moreover, NPC cultures established from the SVZ of adult mice expressing PS1DeltaE9 exhibit reduced self-renewal capacity and premature exit toward neuronal fates. To these findings, we show that both the levels of endogenous Notch/CBF-1-transcriptional activity and transcripts encoding Notch target genes are diminished in SVZ NPCs expressing PS1DeltaE9. The deficits in self-renewal and multipotency are restored by expression of Notch1-ICD or a downstream target of the Notch pathway, Hes1. Hence, we argue that a partial reduction in PS-dependent gamma-secretase processing of the Notch, at least in part, accounts for the impairments observed in SVZ NPCs expressing the FAD-linked PS1DeltaE9 variant. View Publication

The tyrosine kinase Fyn plays a key role in oligodendrocyte differentiation and myelination in the central nervous system, but the molecules responsible for regulating Fyn activation in these processes remain poorly defined. Here we show that receptor-like protein-tyrosine phosphatase alpha (PTPalpha) is an important positive regulator of Fyn activation and signaling that is required for the differentiation of oligodendrocyte progenitor cells (OPCs). PTPalpha is expressed in OPCs and is up-regulated during differentiation. We used two model systems to investigate the role of PTPalpha in OPC differentiation: the rat CG4 cell line where PTPalpha expression was silenced by small interfering RNA, and oligosphere-derived primary OPCs isolated from wild-type and PTPalpha-null mouse embryos. In both cell systems, the ablation of PTPalpha inhibited differentiation and morphological changes that accompany this process. Although Fyn was activated upon induction of differentiation, the level of activation was severely reduced in cells lacking PTPalpha, as was the activation of Fyn effector molecules focal adhesion kinase, Rac1, and Cdc42, and inactivation of Rho. Interestingly, another downstream effector of Fyn, p190RhoGAP, which is responsible for Rho inactivation during differentiation, was not affected by PTPalpha ablation. In vivo studies revealed defective myelination in the PTPalpha(-/-) mouse brain. Together, our findings demonstrate that PTPalpha is a critical regulator of Fyn activation and of specific Fyn signaling events during differentiation, and is essential for promoting OPC differentiation and central nervous system myelination. View Publication

Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis. View Publication

Toll-like receptors (TLRs) play important roles in innate immunity. Several TLR family members have recently been shown to be expressed by neurons and glial cells in the adult brain, and may mediate responses of these cells to injury and infection. To address the possibility that TLRs play a functional role in development of the nervous system, we analyzed the expression of TLRs during different stages of mouse brain development and assessed the role of TLRs in cell proliferation. TLR3 protein is present in brain cells in early embryonic stages of development, and in cultured neural stem/progenitor cells (NPC). NPC from TLR3-deficient embryos formed greater numbers of neurospheres compared with neurospheres from wild-type embryos. Numbers of proliferating cells, as assessed by phospho histone H3 and proliferating cell nuclear antigen labeling, were also increased in the developing cortex of TLR3-deficient mice compared with wild-type mice in vivo. Treatment of cultured embryonic cortical neurospheres with a TLR3 ligand (polyIC) significantly reduced proliferating (BrdU-labeled) cells and neurosphere formation in wild type but not TLR3(-/-)-derived NPCs. Our findings reveal a novel role for TLR3 in the negative regulation of NPC proliferation in the developing brain. View Publication

The mechanisms underlying the decision of a stem or progenitor cell to either self-renew or differentiate are incompletely understood. To address the role of Myc in this process, we expressed different forms of the proto-oncogene Myc in multipotent neural progenitor cells (NPCs) using retroviral transduction. Expression of Myc in neurospheres increased the proportion of self-renewing cells fivefold, and 1% of the Myc-overexpressing cells, but none of the control cells, retained self-renewal capacity even under differentiation-inducing conditions. A Myc mutant (MycV394D) deficient in binding to Miz-1, did not increase the percentage of self-renewing cells but was able to stimulate proliferation of NPCs as efficiently as wild-type Myc, indicating that these two cellular phenomena are regulated by at least partially different pathways. Our results suggest that Myc, through Miz-1, enhances self-renewal of NPCs and influences the way progenitor cells react to the environmental cues that normally dictate the cellular identity of tissues containing self-renewing cells. View Publication

The relatively new field of stem cell biology is hampered by a lack of sufficient means to accurately determine the phenotype of cells. Cell-type-specific markers, such as cell surface proteins used for flow cytometry or fluorescence-activated cell sorting, are limited and often recognize multiple members of a stem cell lineage. We sought to develop a complementary approach that would be less dependent on the identification of particular markers for the subpopulations of cells and would instead measure their overall character. We tested whether a microfluidic system using dielectrophoresis (DEP), which induces a frequency-dependent dipole in cells, would be useful for characterizing stem cells and their differentiated progeny. We found that populations of mouse neural stem/precursor cells (NSPCs), differentiated neurons, and differentiated astrocytes had different dielectric properties revealed by DEP. By isolating NSPCs from developmental ages at which they are more likely to generate neurons, or astrocytes, we were able to show that a shift in dielectric property reflecting their fate bias precedes detectable marker expression in these cells and identifies specific progenitor populations. In addition, experimental data and mathematical modeling suggest that DEP curve parameters can indicate cell heterogeneity in mixed cultures. These findings provide evidence for a whole cell property that reflects stem cell fate bias and establish DEP as a tool with unique capabilities for interrogating, characterizing, and sorting stem cells. View Publication