技术资料

Defects in the control of Wnt signaling have emerged as a recurrent mechanism involved in cancer pathogenesis and acute myeloid leukaemia (AML),including the hematopoietic regeneration-associated WNT10B in AC133bright leukaemia cells,although the existence of a specific mechanism remains unproven. We have obtained evidences for a recurrent rearrangement,which involved the WNT10B locus (WNT10BR) within intron 1 (IVS1) and flanked at the 5' by non-human sequences whose origin remains to be elucidated; it also expressed a transcript variant (WNT10BIVS1) which was mainly detected in a cohort of patients with intermediate/unfavorable risk AML. We also identified in two separate cases,affected by AML and breast cancer respectively,a genomic transposable short form of human WNT10B (ht-WNT10B). The intronless ht-WNT10B resembles a long non-coding RNA (lncRNA),which suggests its involvement in a non-random microhomology-mediated recombination generating the rearranged WNT10BR. Furthermore,our studies supports an autocrine activation primed by the formation of WNT10B-FZD4/5 complexes in the breast cancer MCF7 cells that express the WNT10BIVS1. Chemical interference of WNT-ligands production by the porcupine inhibitor IWP-2 achieved a dose-dependent suppression of the WNT10B-FZD4/5 interactions. These results present the first evidence for a recurrent rearrangement promoted by a mobile ht-WNT10B oncogene,as a relevant mechanism for Wnt involvement in human cancer. View Publication

The present studies examined the toxic interaction between the non-coxib celecoxib derivative OSU-03012 and phosphodiesterase 5 (PDE5) inhibitors,and also determined the roles of endoplasmic reticulum stress response regulators in cell survival. PDE5 inhibitors interacted in a greater than additive fashion with OSU-03012 to kill parental glioma and stem-like glioma cells. Knockdown of the endoplasmic reticulum stress response proteins IRE1 or XBP1 enhanced the lethality of OSU-03012,and of [OSU-03012 + PDE5 inhibitor] treatment. Pan-caspase and caspase-9 inhibition did not alter OSU-03012 lethality but did abolish enhanced killing in the absence of IRE1 or XBP1. Expression of the mitochondrial protective protein BCL-XL or the caspase-8 inhibitor c-FLIP-s,or knockdown of death receptor CD95 or the death receptor caspase-8 linker protein FADD,suppressed killing by [OSU-03012 + PDE5 inhibitor] treatment. CD95 activation was blocked by the nitric oxide synthase inhibitor L-NAME. Knockdown of the autophagy regulatory proteins Beclin1 or ATG5 protected the cells from OSU-03012 and from [OSU-03012 + PDE5 inhibitor] toxicity. Knockdown of IRE1 enhanced OSU-03012/[OSU-03012 + PDE5 inhibitor]-induced JNK activation,and inhibition of JNK suppressed the elevated killing caused by IRE1 knockdown. Knockdown of CD95 blunted JNK activation. Collectively,our data demonstrate that PDE5 inhibitors recruit death receptor signaling to enhance OSU-03012 toxicity in glioblastoma multiforme (GBM) cells. View Publication

Network activity homeostatically alters synaptic efficacy to constrain neuronal output. However,it is unclear how such compensatory adaptations coexist with synaptic information storage,especially in established networks. Here,we report that in mature hippocampal neurons in vitro,network activity preferentially regulated excitatory synapses within the proximal dendrites of CA3 neurons. These homeostatic synapses exhibited morphological,functional,and molecular signatures of the specialized contacts between mossy fibers of dentate granule cells and thorny excrescences (TEs) of CA3 pyramidal neurons. In vivo TEs were also selectively and bidirectionally altered by chronic activity changes. TE formation required presynaptic synaptoporin and was suppressed by the activity-inducible kinase,Plk2. These results implicate the mossy fiber-TE synapse as an independently tunable gain control locus that permits efficacious homeostatic adjustment of mossy fiber-CA3 synapses,while preserving synaptic weights that may encode information elsewhere within the mature hippocampal circuit. View Publication

A number of genes regulate regeneration of peripheral axons,but their ability to drive axon growth and regeneration in the central nervous system (CNS) remains largely untested. To address this question we overexpressed eight transcription factors and one small GTPase alone and in pairwise combinations to test whether combinatorial overexpression would have a synergistic impact on CNS neuron neurite growth. The Jun oncogene/signal transducer and activator of transcription 6 (JUN/STAT6) combination increased neurite growth in dissociated cortical neurons and in injured cortical slices. In injured cortical slices,JUN overexpression increased axon growth to a similar extent as JUN and STAT6 together. Interestingly,JUN overexpression was not associated with increased growth associated protein 43 (GAP43) or integrin alpha 7 (ITGA7) expression,though these are predicted transcriptional targets. This study demonstrates that JUN overexpression in cortical neurons stimulates axon growth,but does so independently of changes in expression of genes thought to be critical for JUNs effects on axon growth. We conclude that JUN activity underlies this CNS axonal growth response,and that it is mechanistically distinct from peripheral regeneration responses,in which increases in JUN expression coincide with increases in GAP43 expression. View Publication

Giant cell tumor of bone (GCTB) is a very rare tumor entity,which is little examined owing to the lack of established cell lines and mouse models and the restriction of available primary cell lines. The stromal cells of GCTB have been made responsible for the aggressive growth and metastasis,emphasizing the presence of a cancer stem cell population. To identify and target such tumor-initiating cells,stromal cells were isolated from eight freshly resected GCTB tissues. Tumorigenic properties were examined by colony and spheroid formation,differentiation,migration,MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,immunohistochemistry,antibody protein array,Alu in situ hybridization,FACS analysis and xenotransplantation into fertilized chicken eggs and mice. A sub-population of the neoplastic stromal cells formed spheroids and colonies,differentiated to osteoblasts,migrated to wounded regions and expressed the metastasis marker CXC-chemokine receptor type 4,indicating self-renewal,invasion and differentiation potential. Compared with adherent-growing cells,markers for pluripotency,stemness and cancer progression,including the CSC surface marker c-Met,were enhanced in spheroidal cells. This c-Met-enriched sub-population formed xenograft tumors in fertilized chicken eggs and mice. Cabozantinib,an inhibitor of c-Met in phase II trials,eliminated CSC features with a higher therapeutic effect than standard chemotherapy. This study identifies a c-Met(+) tumorigenic sub-population within stromal GCTB cells and suggests the c-Met inhibitor cabozantinib as a new therapeutic option for targeted elimination of unresectable or recurrent GCTB. View Publication

Aging and oxidative stress are two prominent pathological mechanisms for Parkinson's disease (PD) that are strongly associated with the degeneration of dopamine (DA) neurons in the midbrain. DA and other catechols readily oxidize into highly reactive o-quinone species that are precursors of neuromelanin (NM) pigment and under pathological conditions can modify and damage macromolecules. The role of DA oxidation in PD pathogenesis remains unclear in part due to the lack of appropriate disease models and the absence of a simple method for the quantification of DA-derived oxidants. Here,we describe a rapid,simple,and reproducible method for the quantification of o-quinones in cells and tissues that relies on the near-infrared fluorescent properties of these species. Importantly,we demonstrate that catechol-derived oxidants can be quantified in human neuroblastoma cells and midbrain dopamine neurons derived from induced pluripotent stem cells,providing a novel model to study the downstream actions of o-quinones. This method should facilitate further study of oxidative stress and DA oxidation in PD and related diseases that affect the dopaminergic system. View Publication

UNLABELLED This pilot feasibility study aimed to determine the outcome of canine epidermal neural crest stem cell (cEPI-NCSC) grafts in the normal spinal cords of healthy bred-for-research dogs. This included developing novel protocols for (a) the ex vivo expansion of cEPI-NCSCs,(b) the delivery of cEPI-NCSCs into the spinal cord,and (c) the labeling of the cells and subsequent tracing of the graft in the live animal by magnetic resonance imaging. A total of four million cEPI-NCSCs were injected into the spinal cord divided in two locations. Differences in locomotion at baseline and post-treatment were evaluated by gait analysis and compared with neurological outcome and behavioral exams. Histopathological analyses of the spinal cords and cEPI-NCSC grafts were performed at 3 weeks post-transplantation. Neurological and gait parameters were minimally affected by the stem cell injection. cEPI-NCSCs survived in the canine spinal cord for the entire period of investigation and did not migrate or proliferate. Subsets of cEPI-NCSCs expressed the neural crest stem cell marker Sox10. There was no detectable expression of markers for glial cells or neurons. The tissue reaction to the cell graft was predominantly vascular in addition to a degree of reactive astrogliosis and microglial activation. In the present study,we demonstrated that cEPI-NCSC grafts survive in the spinal cords of healthy dogs without major adverse effects. They persist locally in the normal spinal cord,may promote angiogenesis and tissue remodeling,and elicit a tissue response that may be beneficial in patients with spinal cord injury. SIGNIFICANCE It has been established that mouse and human epidermal neural crest stem cells are somatic multipotent stem cells with proved innovative potential in a mouse model of spinal cord injury (SCI) offering promise of a valid treatment for SCI. Traumatic SCI is a common neurological problem in dogs with marked similarities,clinically and pathologically,to the syndrome in people. For this reason,dogs provide a readily accessible,clinically realistic,spontaneous model for evaluation of epidermal neural crest stem cells therapeutic intervention. The results of this study are expected to give the baseline data for a future clinical trial in dogs with traumatic SCI. View Publication

Alzheimer's disease (AD) involves the progressive degeneration of neurons critical for learning and memory. In addition,patients with AD typically exhibit impaired olfaction associated with neuronal degeneration in the olfactory bulb (OB). Because DNA base excision repair (BER) is reduced in brain cells during normal aging and AD,we determined whether inefficient BER due to reduced DNA polymerase-β (Polβ) levels renders OB neurons vulnerable to degeneration in the 3xTgAD mouse model of AD. We interrogated OB histopathology and olfactory function in wild-type and 3xTgAD mice with normal or reduced Polβ levels. Compared to wild-type control mice,Polβ heterozygous (Polβ+/- ),and 3xTgAD mice,3xTgAD/Polβ+/- mice exhibited impaired performance in a buried food test of olfaction. Polβ deficiency did not affect the proliferation of OB neural progenitor cells in the subventricular zone. However,numbers of newly generated neurons were reduced by approximately 25% in Polβ+/- and 3xTgAD mice,and by over 60% in the 3xTgAD/Polβ+/- mice compared to wild-type control mice. Analyses of DNA damage and apoptosis revealed significantly greater degeneration of OB neurons in 3xTgAD/Polβ+/- mice compared to 3xTgAD mice. Levels of amyloid β-peptide (Aβ) accumulation in the OB were similar in 3xTgAD and 3xTgAD/Polβ+/- mice,and cultured Polβ-deficient neurons exhibited increased vulnerability to Aβ-induced death. Olfactory deficit is an early sign in human AD,but the mechanism is not yet understood. Our findings in a new AD mouse model demonstrate that diminution of BER can endanger OB neurons,and suggest a mechanism underlying early olfactory impairment in AD. View Publication

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets,the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood-brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay,a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053% per hour) in the cortex,consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally,brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary,our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

Slow-onset adaptive changes that arise from sustained antidepressant treatment,such as enhanced adult hippocampal neurogenesis and increased trophic factor expression,play a key role in the behavioral effects of antidepressants. alpha(2)-Adrenoceptors contribute to the modulation of mood and are potential targets for the development of faster acting antidepressants. We investigated the influence of alpha(2)-adrenoceptors on adult hippocampal neurogenesis. Our results indicate that alpha(2)-adrenoceptor agonists,clonidine and guanabenz,decrease adult hippocampal neurogenesis through a selective effect on the proliferation,but not the survival or differentiation,of progenitors. These effects persist in dopamine beta-hydroxylase knock-out (Dbh(-/-)) mice lacking norepinephrine,supporting a role for alpha(2)-heteroceptors on progenitor cells,rather than alpha(2)-autoreceptors on noradrenergic neurons that inhibit norepinephrine release. Adult hippocampal progenitors in vitro express all the alpha(2)-adrenoceptor subtypes,and decreased neurosphere frequency and BrdU incorporation indicate direct effects of alpha(2)-adrenoceptor stimulation on progenitors. Furthermore,coadministration of the alpha(2)-adrenoceptor antagonist yohimbine with the antidepressant imipramine significantly accelerates effects on hippocampal progenitor proliferation,the morphological maturation of newborn neurons,and the increase in expression of brain derived neurotrophic factor and vascular endothelial growth factor implicated in the neurogenic and behavioral effects of antidepressants. Finally,short-duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test,which normally requires 3 weeks of treatment with classical antidepressants. Our results demonstrate that alpha(2)-adrenoceptors,expressed by progenitor cells,decrease adult hippocampal neurogenesis,while their blockade speeds up antidepressant action,highlighting their importance as targets for faster acting antidepressants. View Publication