Winston M. Walters

Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage.

Proliferating populations of undifferentiated neural stem cells were isolated from the embryonic day 14 rat cerebral cortex or the adult rat subventricular zone. These cells were pluripotent through multiple passages, retaining the ability to differentiate in vitro into neurons, astrocytes, and oligodendrocytes. Two weeks to 2 months after engraftment of undifferentiated, BrdU-labeled stem cells into the normal adult spinal cord, large numbers of surviving cells were seen. The majority of the cells differentiated with astrocytic phenotype, although some oligodendrocytes and undifferentiated, nestin-positive cells were detected; NeuN-positive neurons were not seen. Labeled cells were also engrafted into the contused adult rat spinal cord (moderate NYU Impactor injury), either into the lesion cavity or into the white or gray matter both rostral and caudal to the injury epicenter. Up to 2 months postgrafting, the majority of cells either differentiated into GFAP-positive astrocytes or remained nestin positive. No BrdU-positive neurons or oligodendrocytes were observed. These results show robust survival of engrafted stem cells, but a differentiated phenotype restricted to glial lineages. We suggest that in vitro induction prior to transplantation will be necessary for these cells to differentiate into neurons or large numbers of oligodendrocytes.

Lineage restriction of neuroepithelial precursor cells from fetal human spinal cord

In the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF2), neuroepithelial precursor cells from dissociated fetal human spinal cord are mitotically active and form free‐floating spheres of undifferentiated cells. Proliferating cells were obtained in approximately 40% of preparations with each mitogen, were immunoreactive for the intermediate filament nestin, and did not express neuronal‐ or glial‐specific markers. Early passage neuroepithelial precursor cells were pluripotent and differentiated into neurons expressing MAP2a,b, NF‐M, and TuJ1, and GFAP‐positive astrocytes; however, oligodendrocytes were never seen. As the cells were passaged from P0 to P4, the percentage of differentiating neurons significantly decreased and the prevalence of astrocytes significantly increased. While the majority of cell populations from individual preparations stopped proliferating between 3 and 6 passages, two expanding cell lines have been successfully expanded in EGF and FGF2 for over 25 passages and have been maintained in culture for over one year. These cells express nestin and not other cell‐specific lineage markers. When differentiated, these neuroepithelial cell lines differentiate only into astrocytes, showing no expression of any neuronal marker. These data suggest that continued passage under these conditions preferentially selects for spinal cord neural precursors that are restricted to the astrocytic lineage. Despite the lineage restriction of later passage cell populations, these results provide a rationale for future investigation into the lineage potential of these cells in vivo following transplantation into the adult CNS, potentially as a therapeutic approach for traumatic injury and neurodegenerative disease. J. Neurosci. Res. 57:590–602, 1999.

Neuronal glutamate transporter EAAT4 is expressed in astrocytes.

High‐affinity excitatory amino acid transporters (EAATs) are essential to terminate glutamatergic neurotransmission and to prevent excitotoxicity. To date, five distinct EAATs have been cloned from animal and human tissues: GLAST (EAAT1), GLT‐1 (EAAT2), EAAC1 (EAAT3), EAAT4, and EAAT5. EAAT1 and EAAT2 are commonly known as glial glutamate transporters, whereas EAAT3, EAAT4, and EAAT5 are neuronal. EAAT4 is largely expressed in cerebellar Purkinje cells. In this study, using immunohistochemistry and Western blotting, we found that EAAT4‐like immunoreactivity (ir) is enriched in the spinal cord and forebrain. Double‐labeled fluorescent immunostaining and confocal image analysis indicated that EAAT4‐like ir colocalizes with an astrocytic marker, glial fibrillary acidic protein (GFAP). The astrocytic localization of EAAT4 was further confirmed in astrocyte cultures by double‐labeled fluorescent immunocytochemistry and Western blotting. Reverse transcriptase‐polymerase chain reaction analysis demonstrated mRNA expression of EAAT4 in astrocyte cultures. Sequencing confirmed the specificity of the amplified fragment. These results demonstrate that EAAT4 is expressed in astrocytes. This astrocytic localization of neuronal EAAT4 may reveal a new function of EAAT4 in the central nervous system.

Neuropathic pain-induced depressive-like behavior and hippocampal neurogenesis and plasticity are dependent on TNFR1 signaling.

Patients suffering from neuropathic pain have a higher incidence of mood disorders such as depression. Increased expression of tumor necrosis factor (TNF) has been reported in neuropathic pain and depressive-like conditions and most of the pro-inflammatory effects of TNF are mediated by the TNF receptor 1 (TNFR1). Here we sought to investigate: (1) the occurrence of depressive-like behavior in chronic neuropathic pain and the associated forms of hippocampal plasticity, and (2) the involvement of TNFR1-mediated TNF signaling as a possible regulator of such events. Neuropathic pain was induced by chronic constriction injury of the sciatic nerve in wild-type and TNFR1(-/-) mice. Anhedonia, weight loss and physical state were measured as symptoms of depression. Hippocampal neurogenesis, neuroplasticity, myelin remodeling and TNF/TNFRs expression were analyzed by immunohistochemical analysis and western blot assay. We found that neuropathic pain resulted in the development of depressive symptoms in a time dependent manner and was associated with profound hippocampal alterations such as impaired neurogenesis, reduced expression of neuroplasticity markers and myelin proteins. The onset of depressive-like behavior also coincided with increased hippocampal levels of TNF, and decreased expression of TNF receptor 2 (TNFR2), which were all fully restored after mice spontaneously recovered from pain. Notably, TNFR1(-/-) mice did not develop depressive-like symptoms after injury, nor were there changes in hippocampal neurogenesis and plasticity. Our data show that neuropathic pain induces a cluster of depressive-like symptoms and profound hippocampal plasticity that are dependent on TNF signaling through TNFR1.

Identification and characterization of a novel Nogo‐interacting mitochondrial protein (NIMP)

Nogo is a potent inhibitor of regeneration following spinal cord injury. To develop a better understanding of the mechanisms responsible for regenerative failure we used a yeast two‐ hybrid approach to try and identify proteins that interact with Nogo. We identified a novel mitochondrial protein designated Nogo‐interacting mitochondrial protein (NIMP) in a screen of an adult human brain cDNA library. This interaction was confirmed by co‐immunoprecipitation in both brain tissue (endogenous) and transfected HEK293T cells (overexpressed). In support of these studies we demonstrate that Nogo interacts with the UQCRC1 and UQCRC2 components of complex III, within the mitochondrial respiratory chain. The mitochondrial localization of NIMP was evidenced by confocal image analysis and western blot analysis of isolated mitochondria. NIMP is highly conserved and ubiquitously expressed in mitochondria‐enriched tissues. Within the CNS, NIMP‐like immunoreactivity is present in neurons and astrocytes. These data suggest that NIMP is a novel mitochondrial protein that interacts with Nogo. The interaction of Nogo with mitochondrial proteins may provide insight into the mechanisms for Nogo‐induced inhibition of neurite growth.

TNAP, a Novel Repressor of NF-κB-inducing Kinase, Suppresses NF-κB Activation

NF-κB-inducing kinase (NIK) has been implicated as an essential component of NF-κB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-κB activation induced by tumor necrosis factor (TNF)-α, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-κB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-α-induced NF-κB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IκBα degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-κB signaling pathways.

Activation of NF-κB in Schwann Cells Is Dispensable for Myelination In Vivo

Peripheral myelination is a dynamic process orchestrated by axons and Schwann cells. Although the signaling mechanisms governing myelination are not fully understood, NF-κB activation in Schwann cells has been implicated as a key regulator in vitro. Using a mouse model, we show that nuclear factor κB activation in Schwann cells is not required for myelination in vivo.

Abstract B39: Targeting cancer stem cells via inhibition of PI3K/AKT pathway alone and in combination with autophagy blockade

Background: Glioblastoma Multiforme (GBM) is the most aggressive form of malignant primary brain tumor. Despite an aggressive treatment regimen consisting of surgery, radiation and chemotherapy, individuals with GBM survive on average 15 months. A subset of glioblastoma cells, known as glioblastoma stem-like cells (GSCs), are chemotherapy and radiotherapy-resistent and are hypothesized to be responsible for tumor formation, maintenance, and recurrence. The PI3K/AKT pathway is overactive in GBM and has been shown to play an important role in cancer stem cell maintenance. Therefore we sought to determine the efficacy of drugs targeting the AKT pathway in multiple patient-derived GSC lines. In addition, we evaluated the cytotxicity of the most promising therapy on medulloblastoma and neuroblastoma stem cells. Methods: GSC lines were generated from patient9s tumors, propagated in neurosphere media and analyzed for stem cell markers by immunocytochemistry. Effect of growth factor receptor inhibitors (erlotinib, gefetinib, sunitinib), the intracellular PI3K/AKT pathway inhibitors (GDC-0941, OSU-03012, perifosine, NVP-BEZ235, KU-0063794 and everolimus) and autophagy inhibitor (chloroquine) on cell viability was determined with MTS assay. Caspase 3/7 activity was determined using CellEvent caspase reagent. The effect of the inhibitors on cell signaling pathways was evaluated by western blot analysis. Medulloblastoma and neuroblastoma stem cells were derived from Daoy and NB1691 cells respectively via propagation in neurosphere media. Results: GSC9s expressed nestin, CD133, GFAP, musashi, BMi1, and SOX2. In addition, transplantation of GSC9s into nude mice generated tumors. Of 11 GSC lines examined, all 11 demonstrated activation of the AKT pathway as indicated by robust phosphorylation of AKT (S473) and BAD (S136) and 10/11 demonstrated phosphorylation of mTOR (S2481). Growth factor receptor inhibition failed to induce significant reduction in cell viability. Intracellular inhibitors produced variable results except for dual PI3K/mTOR inhibitor NVB-BEZ235 (100nM) which consistantly induced approximately 50% reduction in cell viability. While the inhibition of autophagy with chloroquine significantly increased inhibitor induced cell death in some cell lines, combined PDK1 and autophagy inhibition induced robust caspase activity and significantly reduced viability in all six GSCs examined to an average of 6.1±2.7% viability compared to non-treated controls (100±1.9%). Similarly the combination significantly reduced both DAOY and NB1691 stem cell viability. In contrast the combination had no effect on primary neuronal cultures. Conclusion: The AKT/PI3K pathway is overactive in GSCs and may contribute to stem cell maintenance as well as inhibition of apoptosis. Our data indicates that inhibiting autophagy concommitantly with PI3K/AKT pathway inhibition can potentiate their cytotoxic effect. Furthermore combined chloroquine and OSU-03012 resulted in a substantial decrease cell viability accross multiple GSC lines and in medulloblastoma and neuroblastoma stem cells suggesting that this combination may represent a potential adjuvant therapy these cancers. Citation Format: Regina M. Graham, Andrew Middleton, Daniel A. Benito, Raisa Uddin, Baoyu Zhang, Winston Walters, Amade Bregy, Steven Vanni, Ricardo J. Komotar. Targeting cancer stem cells via inhibition of PI3K/AKT pathway alone and in combination with autophagy blockade. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B39.