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Cellular damage by reactive oxygen species and altered neurogenesis are implicated in the etiology of AD and the pathogenic actions of amyloid β-peptide (Aβ); the underlying mechanisms and the early oxidative intracellular events triggered by Aβ are not established. In the present study,we found that mouse embryonic cortical neural progenitor cells exhibit intermittent spontaneous mitochondrial superoxide (SO) flashes that require transient opening of mitochondrial permeability transition pores (mPTPs). The incidence of mitochondria SO flash activity in neural progenitor cells (NPCs) increased during the first 6-24 hours of exposure to aggregating amyloid β-peptide (Aβ1-42),indicating an increase in transient mPTP opening. Subsequently,the SO flash frequency progressively decreased and ceased between 48 and 72 hours of exposure to Aβ1-42,during which time global cellular reactive oxygen species increased,mitochondrial membrane potential decreased,cytochrome C was released from mitochondria and the cells degenerated. Inhibition of mPTPs and selective reduction in mitochondrial SO flashes significantly ameliorated the negative effects of Aβ1-42 on NPC proliferation and survival. Our findings suggest that mPTP-mediated bursts of mitochondrial SO production is a relatively early and pivotal event in the adverse effects of Aβ1-42 on NPCs. If Aβ inhibits NPC proliferation in the brains of AD patients by a similar mechanism,then interventions that inhibit mPTP-mediated superoxide flashes would be expected to protect NPCs against the adverse effects of Aβ. 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

Cell therapy offers an innovative approach for treating enteric neuropathies. Postnatal gut-derived enteric neural stem/progenitor cells (ENSCs) represent a potential autologous source,but have a limited capacity for proliferation and neuronal differentiation. Since serotonin (5-HT) promotes enteric neuronal growth during embryonic development,we hypothesized that serotonin receptor agonism would augment growth of neurons from transplanted ENSCs. Postnatal ENSCs were isolated from 2 to 4 week-old mouse colon and cultured with 5-HT4 receptor agonist (RS67506)-loaded liposomal nanoparticles. ENSCs were co-cultured with mouse colon explants in the presence of RS67506-loaded (n = 3) or empty nanoparticles (n = 3). ENSCs were also transplanted into mouse rectum in vivo with RS67506-loaded (n = 8) or blank nanoparticles (n = 4) confined in a thermosensitive hydrogel,Pluronic F-127. Neuronal density and proliferation were analyzed immunohistochemically. Cultured ENSCs gave rise to significantly more neurons in the presence of RS67506-loaded nanoparticles. Similarly,colon explants had significantly increased neuronal density when RS67506-loaded nanoparticles were present. Finally,following in vivo cell delivery,co-transplantation of ENSCs with 5-HT4 receptor agonist-loaded nanoparticles led to significantly increased neuronal density and proliferation. We conclude that optimization of postnatal ENSCs can support their use in cell-based therapies for neurointestinal diseases. View Publication

Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind,we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation,of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin,deguelin,patulin and phenethyl caffeate) exhibited high cytotoxicity,with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular,the FDA approved drug for the treatment of alcoholism,disulfiram (DSF),was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu),which markedly increased GSC death. DSF-Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs,consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier,we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM. View Publication

Prominin-1 (CD133) is a commonly used cancer stem cell marker in central nervous system (CNS) tumors including glioblastoma (GBM). Expression of Prom1 in cancer is thought to parallel expression and function in normal stem cells. Using RNA in situ hybridization and antibody tools capable of detecting multiple isoforms of Prom1,we find evidence for two distinct Prom1 cell populations in mouse brain. Prom1 RNA is first expressed in stem/progenitor cells of the ventricular zone in embryonic brain. Conversely,in adult mouse brain Prom1 RNA is low in SVZ/SGZ stem cell zones but high in a rare but widely distributed cell population (Prom1(hi)). Lineage marker analysis reveals Prom1(hi) cells are Olig2+Sox2+ glia but Olig1/2 knockout mice lacking oligodendroglia retain Prom1(hi) cells. Bromodeoxyuridine labeling identifies Prom1(hi) as slow-dividing distributed progenitors distinct from NG2+Olig2+ oligodendrocyte progenitors. In adult human brain,PROM1 cells are rarely positive for OLIG2,but express astroglial markers GFAP and SOX2. Variability of PROM1 expression levels in human GBM and patient-derived xenografts (PDX) - from no expression to strong,uniform expression--highlights that PROM1 may not always be associated with or restricted to cancer stem cells. TCGA and PDX data show that high expression of PROM1 correlates with poor overall survival. Within proneural subclass tumors,high PROM1 expression correlates inversely with IDH1 (R132H) mutation. These findings support PROM1 as a tumor cell-intrinsic marker related to GBM survival,independent of its stem cell properties,and highlight potentially divergent roles for this protein in normal mouse and human glia. View Publication

Multiple research groups have observed neuropathological phenotypes and molecular symptoms in vitro using induced pluripotent stem cell (iPSC)-derived neural cell cultures (i.e. patient-specific neurons and glia). However,the global differences/similarities that may exist between in vitro neural cells and their tissue-derived counterparts remain largely unknown. In this study,we compared temporal series of iPSC-derived in vitro neural cell cultures to endogenous brain tissue from the same autopsy donor. Specifically,we utilized RNA sequencing (RNA-Seq) to evaluate the transcriptional progression of in vitro-differentiated neural cells (over a timecourse of 0,35,70,105 and 140 days),and compared this with donor-identical temporal lobe tissue. We observed in vitro progression towards the reference brain tissue,and the following three results support this conclusion: (i) there was a significant increasing monotonic correlation between the days of our timecourse and the number of actively transcribed protein-coding genes and long intergenic non-coding RNAs (lincRNAs) (P < 0.05),consistent with the transcriptional complexity of the brain; (ii) there was an increase in CpG methylation after neural differentiation that resembled the epigenomic signature of the endogenous tissue; and (iii) there was a significant decreasing monotonic correlation between the days of our timecourse and the percent of in vitro to brain-tissue differences (P < 0.05) for tissue-specific protein-coding genes and all putative lincRNAs. Taken together,these results are consistent with in vitro neural development and physiological progression occurring predominantly by transcriptional activation of downregulated genes rather than deactivation of upregulated genes. View Publication

Stress bears a negative impact on adult neurogenesis. High levels of corticoids have been shown to inhibit neural stem cell proliferation,and are considered responsible for the loss of neural precursors. Their effects on the differentiation of the glial and neuronal lineages have been less studied. We examined the effect of dexamethasone (Dex),a synthetic glucocorticoid,on the differentiation of rat neural stem cells in vitro. Dex had no effect on the differentiation of cells cultured under standard conditions. Since we previously determined that NSC,when cultured under classical conditions,were deprived of polyunsaturated fatty acids (PUFA),and displayed phospholipid compositions very different from the in vivo figures [1],we examined the effect of Dex under PUFA supplementation. Dex impaired neuron and oligodendrocyte maturation in PUFA-supplemented cells,demonstrated by the reduction of neurite lengths and oligodendrocyte sizes. This effect was mediated by the glucocorticoid receptor (GR),since it was eliminated by mifepristone,a GR antagonist,and could be relayed by a reduction of ERK phosphorylation. We determined that GR was associated with PPAR β and α under basal conditions,and that this association was disrupted when PUFA were added in combination with Dex. We assumed that this effect on the receptor status enabled the effect of Dex on PUFA supplemented cells,since we determined that the binding to the glucocorticoid response element was higher in cells incubated with PUFA and Dex. In conclusion,corticoids can impair NSC differentiation,and consequently impact the entire process of neurogenesis. View Publication