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Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation,migration,and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme,a highly aggressive brain cancer,suggesting that ion channel expression may be perturbed in this population. However,little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing,we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance,expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally,genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes,gene mutations,survival outcomes,regional tumor expression,and experimental responses to loss-of-function. Together,the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. View Publication

BACKGROUND Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia,although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer's disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. METHODS We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling,insulin regulation of the major AD proteins Abeta$ and/or tau,and/or calcium fluxes is altered by the PSEN2 N141I mutation. RESULTS We report herein that wildtype,PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Abeta$42/40 ratio. Unexpectedly,we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux,which could be prevented by chronic exposure of BFCNs to insulin. CONCLUSIONS Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Abeta$42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence,our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD. View Publication

Global cerebral ischemia induces selective acute neuronal injury of the CA1 region of the hippocampus. The type of cell death that ensues may include different programmed cell death mechanisms namely apoptosis and necroptosis,a recently described type of programmed necrosis. We investigated whether necroptosis contributes to hippocampal neuronal death following oxygen-glucose deprivation (OGD),an in vitro model of global ischemia. We observed that OGD induced a death receptor (DR)-dependent component of necroptotic cell death in primary cultures of hippocampal neurons. Additionally,we found that this ischemic challenge upregulated the receptor-interacting protein kinase 3 (RIP3) mRNA and protein levels,with a concomitant increase of the RIP1 protein. Together,these two related proteins form the necrosome,the complex responsible for induction of necroptotic cell death. Interestingly,we found that caspase-8 mRNA,a known negative regulator of necroptosis,was transiently decreased following OGD. Importantly,we observed that the OGD-induced increase in the RIP3 protein was paralleled in an in vivo model of transient global cerebral ischemia,specifically in the CA1 area of the hippocampus. Moreover,we show that the induction of endogenous RIP3 protein levels influenced neuronal toxicity since we found that RIP3 knock-down (KD) abrogated the component of OGD-induced necrotic neuronal death while RIP3 overexpression exacerbated neuronal death following OGD. Overexpression of RIP1 also had deleterious effects following the OGD challenge. Taken together,our results highlight that cerebral ischemia activates transcriptional changes that lead to an increase in the endogenous RIP3 protein level which might contribute to the formation of the necrosome complex and to the subsequent component of necroptotic neuronal death that follows ischemic injury. View Publication

General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However,the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines,a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D,drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus,isoflurane destabilizes spine F-actin,resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus,a locus for anesthetic-induced amnesia,and have important implications for anesthetic effects on synaptic plasticity. View Publication

BACKGROUND Transplanting autologous patient-derived enteric neuronal stem/progenitor cells (ENSCs) is an innovative approach to replacing missing enteric neurons in patients with Hirschsprung disease (HSCR). Using autologous cells eliminates immunologic and ethical concerns raised by other cell sources. However,whether postnatal aganglionic bowel is permissive for transplanted ENSCs and whether ENSCs from HSCR patients can be successfully isolated,cultured,and transplanted in vivo remains unknown. METHODS ENSCs isolated from the ganglionic intestine of Ednrb(-/-) mice (HSCR-ENSCs) were characterized immunohistochemically and evaluated for their capacity to proliferate and differentiate in vitro. Fluorescently labeled ENSCs were co-cultured ex vivo with aganglionic Ednrb(-/-) colon. For in vivo transplantation,HSCR-ENSCs were labeled with lentivirus expressing green fluorescent protein (GFP) and implanted into aganglionic embryonic chick gut in ovo and postnatal aganglionic Ednrb(-/-) rectum in vivo. KEY RESULTS HSCR-ENSCs maintain normal capacity self-renewal and neuronal differentiation. Moreover,the Ednrb(-/-) aganglionic environment is permissive to engraftment by wild-type ENSCs ex vivo and supports migratrion and neuroglial differentiation of these cells following transplantation in vivo. Lentiviral GFP-labeled HSCR-ENSCs populated embryonic chick hindgut and postnatal colon of Ednrb(-/-) HSCR,with cells populating the intermuscular layer and forming enteric neurons and glia. CONCLUSIONS & INFERENCES ENSCs can be isolated and cultured from mice with HSCR,and transplanted into the aganglionic bowel of HSCR littermates to generate enteric neuronal networks. These results in an isogenic model establish the potential of using autologous-derived stem cells to treat HSCR and other intestinal neuropathies. View Publication

Stem-like cells have been isolated in tumors such as breast,lung,colon,prostate and brain. A critical issue in all these tumors,especially in glioblastoma mutliforme (GBM),is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation,progression,and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue,and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days,primary neurospheres of 150-200 μm in diameter can be observed and are ready for further passaging and expansion. View Publication

Adult neural stem/precursor cells (NPCs) play a pivotal role in neuronal plasticity throughout life. Among ion channels identified in adult NPCs,voltage-gated delayed rectifier K(+) (KDR) channels are dominantly expressed. However,the KDR channel subtype and its physiological role are still undefined. We used real-time quantitative RT-PCR and gene knockdown techniques to identify a major functional KDR channel subtype in adult NPCs. Dominant mRNA expression of Kv3.1,a high voltage-gated KDR channel,was quantitatively confirmed. Kv3.1 gene knockdown with specific small interfering RNAs (siRNA) for Kv3.1 significantly inhibited Kv3.1 mRNA expression by 63.9% (P < 0.001) and KDR channel currents by 52.2% (P < 0.001). This indicates that Kv3.1 is the subtype responsible for producing KDR channel outward currents. Resting membrane properties,such as resting membrane potential,of NPCs were not affected by Kv3.1 expression. Kv3.1 knockdown with 300 nm siRNA inhibited NPC growth (increase in cell numbers) by 52.9% (P < 0.01). This inhibition was attributed to decreased cell proliferation,not increased cell apoptosis. We also established a convenient in vitro imaging assay system to evaluate NPC differentiation using NPCs from doublecortin-green fluorescent protein transgenic mice. Kv3.1 knockdown also significantly reduced neuronal differentiation by 31.4% (P < 0.01). We have demonstrated that Kv3.1 is a dominant functional KDR channel subtype expressed in adult NPCs and plays key roles in NPC proliferation and neuronal lineage commitment during differentiation. View Publication

The functional significance of the electrophysiological properties of neural precursor cells (NPCs) was investigated using dissociated neurosphere-derived NPCs from the forebrain subventricular zone (SVZ) of adult mice. NPCs exhibited hyperpolarized resting membrane potentials,which were depolarized by the K(+) channel inhibitor,Ba(2+). Pharmacological analysis revealed two distinct K(+) channel families: Ba(2+)-sensitive K(ir) channels and tetraethylammonium (TEA)-sensitive K(v) (primarily K(DR)) channels. Ba(2+) promoted mitogen-stimulated NPC proliferation,which was mimicked by high extracellular K(+),whereas TEA inhibited proliferation. Based on gene and protein levels in vitro,we identified K(ir)4.1,K(ir)5.1 and K(v)3.1 channels as the functional K(+) channel candidates. Expression of these K(+) channels was immunohistochemically found in NPCs of the adult mouse SVZ,but was negligible in neuroblasts. It therefore appears that expression of K(ir) and K(v) (K(DR)) channels in NPCs and related changes in the resting membrane potential could contribute to NPC proliferation and neuronal lineage commitment in the neurogenic microenvironment. View Publication

The transforming growth factor-$\$(TGF-$\$) family member activin A exerts multiple neurotrophic and protective effects in the brain. Activin also modulates cognitive functions and affective behavior and is a presumed target of antidepressant therapy. Despite its important role in the injured and intact brain,the mechanisms underlying activin effects in the CNS are still largely unknown. Our goal was to identify the first target genes of activin signaling in the hippocampus in vivo. Electroconvulsive seizures,a rodent model of electroconvulsive therapy in humans,were applied to C57BL/6J mice to elicit a strong increase in activin A signaling. Chromatin immunoprecipitation experiments with hippocampal lysates subsequently revealed that binding of SMAD2/3,the intracellular effectors of activin signaling,was significantly enriched at the Pmepa1 gene,which encodes a negative feedback regulator of TGF-$\$ in cancer cells,and at the Kdm6b gene,which encodes an epigenetic regulator promoting transcriptional plasticity. Underlining the significance of these findings,activin treatment also induced PMEPA1 and KDM6B expression in human forebrain neurons generated from embryonic stem cells suggesting interspecies conservation of activin effects in mammalian neurons. Importantly,physiological stimuli such as provided by environmental enrichment proved already sufficient to engender a rapid and significant induction of activin signaling concomitant with an upregulation of Pmepa1 and Kdm6b expression. Taken together,our study identified the first target genes of activin signaling in the brain. With the induction of Kdm6b expression,activin is likely to gain impact on a presumed epigenetic regulator of activity-dependent neuronal plasticity. View Publication