技术资料

Azidothymidine (AZT) is a synthetic,chain-terminating nucleoside analog used to treat HIV-1 infection. While AZT is not actively transported across the blood brain barrier,it does accumulate at high levels in cerebrospinal fluid,and subsequently diffuses into the overlying parenchyma. Due to the close anatomical proximity of the neurogenic niches to the ventricular system,we hypothesize that diffusion from CSF exposes neural stem/progenitor cells and their progeny to biologically relevant levels of AZT sufficient to perturb normal cell functions. We employed in vitro and in vivo models of mouse neurogenesis in order to assess the effects of AZT on developing and adult neurogenesis. Using in vitro assays we show that AZT reduces the population expansion potential of neural stem/progenitor cells by inducing senescence. Additionally,in a model of in vitro neurogenesis AZT severely attenuates neuroblast production. These effects are mirrored in vivo by clinically-relevant animal models. We show that in utero AZT exposure perturbs both population expansion and neurogenesis among neural stem/progenitor cells. Additionally,a short-term AZT regimen in adult mice suppresses subependymal zone neurogenesis. These data reveal novel negative effects of AZT on neural stem cell biology. Given that the sequelae of HIV infection often include neurologic deficits-subsumed under AIDS Dementia Complex (Brew,1999)-it is important to determine to what extent AZT negatively affects neurological function in ways that contribute to,or exacerbate,ADC in order to avoid attributing iatrogenic drug effects to the underlying disease process,and thereby skewing the risk/benefit analysis of AZT therapy. View Publication

Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD),dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However,whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end,progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels,increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits. View Publication

Glioblastoma multiforme (GBM) is the most common and lethal adult brain tumor. Resistance to standard radiation and chemotherapy is thought to involve survival of GBM cancer stem cells (CSCs). To date,no single marker for identifying GBM CSCs has been able to capture the diversity of CSC populations,justifying the needs for additional CSC markers for better characterization. Employing targeted mass spectrometry,here we present five cell-surface markers HMOX1,SLC16A1,CADM1,SCAMP3,and CLCC1 which were found to be elevated in CSCs relative to healthy neural stem cells (NSCs). Transcriptomic analyses of REMBRANDT and TCGA compendiums also indicated elevated expression of these markers in GBM relative to controls and non-GBM diseases. Two markers SLC16A1 and HMOX1 were found to be expressed among pseudopalisading cells that reside in the hypoxic region of GBM,substantiating the histopathological hallmarks of GBM. In a prospective study (N%=%8) we confirmed the surface expression of HMOX1 on freshly isolated primary GBM cells (P0). Employing functional assays that are known to evaluate stemness,we demonstrate that elevated HMOX1 expression is associated with stemness in GBM and can be modulated through TGFβ. siRNA-mediated silencing of HMOX1 impaired GBM invasion-a phenomenon related to poor prognosis. In addition,surgical resection of GBM tumors caused declines (18%%±%5.1SEM) in the level of plasma HMOX1 as measured by ELISA,in 8/10 GBM patients. These findings indicate that HMOX1 is a robust predictor of GBM CSC stemness and pathogenesis. Further understanding of the role of HMOX1 in GBM may uncover novel therapeutic approaches. Stem Cells 2016;34:2276-2289. View Publication

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury,as well as neurodegenerative conditions such as Parkinson's disease,Alzheimer's disease,and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However,the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly,in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore,we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically,we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly,we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs,indicating that Puma induction is required for NPC death. Consistent with this,we demonstrate that Puma-deficient NPCs exhibit an 13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary,we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions. View Publication

Many neuropsychiatric disorders are thought to result from subtle changes in neural circuit formation. We used human embryonic stem cells and induced pluripotent stem cells (hiPSCs) to model mature,post-mitotic excitatory neurons and examine effects of fibroblast growth factor 2 (FGF2). FGF2 gene expression is known to be altered in brain regions of major depressive disorder (MDD) patients and FGF2 has anti-depressive effects in animal models of depression. We generated stable inducible neurons (siNeurons) conditionally expressing human neurogenin-2 (NEUROG2) to generate a homogenous population of post-mitotic excitatory neurons and study the functional as well as the transcriptional effects of FGF2. Upon induction of NEUROG2 with doxycycline,the vast majority of cells are post-mitotic,and the gene expression profile recapitulates that of excitatory neurons within 6 days. Using hES cell lines that inducibly express NEUROG2 as well as GCaMP6f,we were able to characterize spontaneous calcium activity in these neurons and show that calcium transients increase in the presence of FGF2. The FGF2-responsive genes were determined by RNA-Seq. FGF2-regulated genes previously identified in non-neuronal cell types were up-regulated (EGR1,ETV4,SPRY4,and DUSP6) as a result of chronic FGF2 treatment of siNeurons. Novel neuron-specific genes were also identified that may mediate FGF2-dependent increases in synaptic efficacy including NRXN3,SYT2,and GALR1. Since several of these genes have been implicated in MDD previously,these results will provide the basis for more mechanistic studies of the role of FGF2 in MDD. 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

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

Retinal ganglion cells (RGCs) are the projection neurons of the retina and transmit visual information to postsynaptic targets in the brain. While this function is shared among nearly all RGCs,this class of cell is remarkably diverse,comprised of multiple subtypes. Previous efforts have identified numerous RGC subtypes in animal models,but less attention has been paid to human RGCs. Thus,efforts of this study examined the diversity of RGCs differentiated from human pluripotent stem cells (hPSCs) and characterized defined subtypes through the expression of subtype-specific markers. Further investigation of these subtypes was achieved using single-cell transcriptomics,confirming the combinatorial expression of molecular markers associated with these subtypes,and also provided insight into more subtype-specific markers. Thus,the results of this study describe the derivation of RGC subtypes from hPSCs and will support the future exploration of phenotypic and functional diversity within human RGCs. View Publication

Neurotoxicity testing still relies on ethically debated,expensive and time consuming in vivo experiments,which are unsuitable for high-throughput toxicity screening. There is thus a clear need for a rapid in vitro screening strategy that is preferably based on human-derived neurons to circumvent interspecies translation. Recent availability of commercially obtainable human induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes holds great promise in assisting the transition from the current standard of rat primary cortical cultures to an animal-free alternative. We therefore composed several hiPSC-derived neuronal models with different ratios of excitatory and inhibitory neurons in the presence or absence of astrocytes. Using immunofluorescent stainings and multi-well micro-electrode array (mwMEA) recordings we demonstrate that these models form functional neuronal networks that become spontaneously active. The differences in development of spontaneous neuronal activity and bursting behavior as well as spiking patterns between our models confirm the importance of the presence of astrocytes. Preliminary neurotoxicity assessment demonstrates that these cultures can be modulated with known seizurogenic compounds,such as picrotoxin (PTX) and endosulfan,and the neurotoxicant methylmercury (MeHg). However,the chemical-induced effects on different parameters for neuronal activity,such as mean spike rate (MSR) and mean burst rate (MBR),may depend on the ratio of inhibitory and excitatory neurons. Our results thus indicate that hiPSC-derived neuronal models must be carefully designed and characterized prior to large-scale use in neurotoxicity screening. View Publication