技术资料

Glioblastoma multiforme (GBM) is a grade IV malignant brain tumor with high mortality and has been well known to involve many molecular pathways,including G-protein coupled receptor (GPCR)-mediated signaling (such as epithelial growth factor receptor [EGFR] and platelet derived growth factor receptor [PDGFR]). G protein-coupled receptor kinases (GRK) directly regulate GPCR activity by phosphorylating activated agonist-bound receptors to desensitize signaling and internalize receptors through beta-arrestins. Recent studies in various cancers,including prostate and breast cancer,have highlighted the role of change in GRK expression to oncogenesis and tumor proliferation. In this study,we evaluated the expression of GRK5 in grade II to grade IV glioma specimens using immunohistochemistry and found that GRK5 expression levels are highly correlated with aggressiveness of glioma. We used culture conditions to selectively promote the growth of either glioblastoma cells with stem cell markers (GSC) or differentiated glioblastoma cells (DGC) from fresh GBM specimens. GSC are known to be highly invasive and mobile,and have the capacity to self-renew and are more resistant to chemotherapy and radiation compared to differentiated populations of GBM. We examined the expression of GRK5 in these two sets of culturing conditions for GBM cells and found that GRK5 expression is upregulated in GSC compared to differentiated GBM cells. To better understand the role of GRK5 in GBM-derived stem cells,we created stable GRK5 knockdown and evaluated the proliferation rate. Using an ATP chemiluminescence assay,we show,for the first time,that knocking down the expression of GRK5 decreased the proliferation rate of GSC in contrast to control. View Publication

Human corneal endothelial cells are derived from neural crest and because of postmitotic arrest lack competence to repair cell loss from trauma,aging,and degenerative disorders such as Fuchs endothelial corneal dystrophy (FECD). Herein,we identified a rapidly proliferating subpopulation of cells from the corneal endothelium of adult normal and FECD donors that exhibited features of neural crest-derived progenitor (NCDP) cells by showing absence of senescence with passaging,propensity to form spheres,and increased colony forming efficacy compared with the primary cells. The collective expression of stem cell-related genes SOX2,OCT4,LGR5,TP63 (p63),as well as neural crest marker genes PSIP1 (p75(NTR)),PAX3,SOX9,AP2B1 (AP-2β),and NES,generated a phenotypic footprint of endothelial NCDPs. NCDPs displayed multipotency by differentiating into microtubule-associated protein 2,β-III tubulin,and glial fibrillary acidic protein positive neurons and into p75(NTR)-positive human corneal endothelial cells that exhibited transendothelial resistance of functional endothelium. In conclusion,we found that mitotically incompetent ocular tissue cells contain adult NCDPs that exhibit a profile of transcription factors regulating multipotency and neural crest progenitor characteristics. Identification of normal NCDPs in FECD-affected endothelium holds promise for potential autologous cell therapies. View Publication

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However,ADSCs require invasive procedures,and has potential complications. Recently,urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study,we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization,and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation,colony formation,cell surface markers,immune modulation,chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3,5,and 7. USCs showed high cell proliferation rate,enhanced colony forming ability,strong positive for stem cell markers expression,high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3,5,and 7. In chromosome stability analysis,both cells showed normal karyotype through all passages. In analysis of multi-lineage capability,USCs showed higher myogenic,neurogenic,and endogenic differentiation rate,and lower osteogenic,adipogenic,and chondrogenic differentiation rate compared to ADSCs. Therefore,we expect that USC can be an alternative autologous stem cell source for muscle,neuron and endothelial tissue reconstruction instead of ADSCs. View Publication

BACKGROUND The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects,including a reduction in cancer risk. METHODS We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations. RESULTS In vitro studies showed that cerivastatin,pitavastatin,and fluvastatin were the most potent anti-proliferative,autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently,pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment,tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells. CONCLUSIONS Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies. View Publication

Surgery is an essential component in the treatment of brain tumors. However,delineating tumor from normal brain remains a major challenge. We describe the use of stimulated Raman scattering (SRS) microscopy for differentiating healthy human and mouse brain tissue from tumor-infiltrated brain based on histoarchitectural and biochemical differences. Unlike traditional histopathology,SRS is a label-free technique that can be rapidly performed in situ. SRS microscopy was able to differentiate tumor from nonneoplastic tissue in an infiltrative human glioblastoma xenograft mouse model based on their different Raman spectra. We further demonstrated a correlation between SRS and hematoxylin and eosin microscopy for detection of glioma infiltration (κ = 0.98). Finally,we applied SRS microscopy in vivo in mice during surgery to reveal tumor margins that were undetectable under standard operative conditions. By providing rapid intraoperative assessment of brain tissue,SRS microscopy may ultimately improve the safety and accuracy of surgeries where tumor boundaries are visually indistinct. View Publication

The activity of neural precursor cells in the adult hippocampus is regulated by various stimuli; however,whether these stimuli regulate the same or different precursor populations remains unknown. Here,we developed a novel cell-sorting protocol that allows the purification to homogeneity of neurosphere-forming neural precursors from the adult mouse hippocampus and examined the responsiveness of individual precursors to various stimuli using a clonal assay. We show that within the Hes5-GFP(+)/Nestin-GFP(+)/EGFR(+) cell population,which comprises the majority of neurosphere-forming precursors,there are two distinct subpopulations of quiescent precursor cells,one directly activated by high-KCl depolarization,and the other activated by norepinephrine (NE). We then demonstrate that these two populations are differentially distributed along the septotemporal axis of the hippocampus,and show that the NE-responsive precursors are selectively regulated by GABA,whereas the KCl-responsive precursors are selectively modulated by corticosterone. Finally,based on RNAseq analysis by deep sequencing,we show that the progeny generated by activating NE-responsive versus KCl-responsive quiescent precursors are molecularly different. These results demonstrate that the adult hippocampus contains phenotypically similar but stimulus-specific populations of quiescent precursors,which may give rise to neural progeny with different functional capacity. View Publication

Neuronal morphology is highly sensitive to ischemia,although some re-organization may promote neuroprotection. In this study we investigate the role of actin regulating proteins (ARP2,ARP3 and WAVE-1) and their role in rapid ischemic tolerance. Using an established in vitro model of rapid ischemic tolerance,we show that WAVE-1 protein levels are stabilized following brief tolerance inducing ischemia (preconditioning). The stabilization appears to be due to a reduction in the ubiquitination of WAVE-1. Levels of ARP2,ARP3 and N-WASP were not affected by ischemic preconditioning. Immunocytochemical studies show a relocalization of ARP2 and ARP3 proteins in neurons following preconditioning ischemia,as well as a re-organization of actin. Blocking the protein kinase CK2 using emodin blocks ischemic tolerance,and our data suggests CK2 binds to WAVE-1 in neurons. We observe an increase in binding of the ARP2 subunit with WAVE-1. The neuroprotection observed following preconditioning is inhibited when cells are transduced with an N-WASP CA domain that blocks the activation of ARP2/3. Together these data show that ischemia affects actin regulating enzymes,and that the ARP2/3 pathway plays a role in rapid ischemic tolerance induced neuroprotection. View Publication

Although many previous investigations have studied how mercury compounds cause cell death,sub-cytotoxic levels may affect mechanisms essential for the proper development of the nervous system. The present study investigates whether low doses of methylmercury (MeHg) and mercury chloride (HgCl2) can modulate the activity of JAK/STAT signaling,a pathway that promotes gliogenesis. We report that sub-cytotoxic doses of MeHg enhance ciliary neurotrophic factor (CNTF) evoked STAT3 phosphorylation in human SH-SY5Y neuroblastoma and mouse cortical neural progenitor cells (NPCs). This effect is specific for MeHg,since HgCl2 fails to enhance JAK/STAT signaling. Exposing NPCs to these low doses of MeHg (30-300nM) enhances CNTF-induced expression of STAT3-target genes such as glial fibrillary acidic protein (GFAP) and suppressors of cytokine signaling 3 (SOCS3),and increases the proportion of cells expressing GFAP following 2 days of differentiation. Higher,near-cytotoxic concentrations of MeHg and HgCl2 inhibit STAT3 phosphorylation and lead to increased production of superoxide. Lower concentrations of MeHg effective in enhancing JAK/STAT signaling (30nM) do not result in a detectable increase in superoxide nor increased expression of the oxidant-responsive genes,heme oxygenase 1,heat shock protein A5 and sirtuin 1. These findings suggest that low concentrations of MeHg inappropriately enhance STAT3 phosphorylation and glial differentiation,and that the mechanism causing this enhancement is distinct from the reactive oxygen species-associated cell death observed at higher concentrations of MeHg and HgCl2. View Publication

BACKGROUND Glioblastoma (GBM),being a highly vascularised and locally invasive tumour,is an attractive target for anti-angiogenic and anti-invasive therapies. The GBM/endothelial cell response to gossypol/temozolomide (TMZ) treatment was investigated with a particular aim to assess treatment effects on cancer hallmarks. METHODS Cell viability,endothelial tube formation and GBM tumour cell invasion were variously assessed following combined treatment in vitro. The U87MG-luc2 subcutaneous xenograft model was used to investigate therapeutic response in vivo. Viable tumour response to treatment was interrogated using immunohistochemistry. Combined treatment protocols were also tested in primary GBM patient-derived cultures. RESULTS An endothelial/GBM cell viability inhibitory effect,as well as an anti-angiogenic and anti-invasive response,to combined treatment have been demonstrated in vitro. A significantly greater anti-proliferative (P=0.020,P=0.030),anti-angiogenic (P=0.040,P<0.0001) and pro-apoptotic (P=0.0083,P=0.0149) response was observed when combined treatment was compared with single gossypol/TMZ treatment response,respectively. GBM cell line and patient-specific response to gossypol/TMZ treatment was observed. CONCLUSIONS Our results indicate that response to a combined gossypol/TMZ treatment is related to inhibition of tumour-associated angiogenesis,invasion and proliferation and warrants further investigation as a novel targeted GBM treatment strategy. View Publication

Neurobasal®/B27 is a gold standard culture media used to study primary neurons in vitro. An alternative media (BrainPhys®/SM1) was recently developed which robustly enhances neuronal activity vs. Neurobasal® or DMEM. To the best of our knowledge BrainPhys® has not been explored in the setting of neuronal injury. Here we characterized the utility of BrainPhys® in a model of in vitro mechanical-stretch injury. METHODS/RESULTSPrimary rat cortical neurons were maintained in classic Neurobasal®,or sequentially maintained in Neurocult® followed by BrainPhys® (hereafter simply referred to as BrainPhys® maintained neurons?). The levels of axonal markers and proteins involved in neurotransmission were compared on day in vitro 10 (DIV10). BrainPhys® maintained neurons had higher levels of GluN2B,GluR1,Neurofilament light/heavy chain (NF-L & NF-H),and protein phosphatase 2 A (PP2A) vs. neurons in Neurobasal®. Mechanical stretch-injury (50ms/54% biaxial stretch) to BrainPhys® maintained neurons modestly (albeit significantly) increased 24h lactate dehydrogenase (LDH) levels but markedly decreased axonal NF-L levels post-injury vs. uninjured controls or neurons given a milder 38% stretch-injury. Furthermore,two 54% stretch-injuries (in tandem) exacerbated 24h LDH release,increased α-spectrin breakdown products (SBDPs),and decreased Tau levels. Also,BrainPhys® maintained cultures had decreased markers of cell damage 24h after a single 54% stretch-injury vs. neurons in Neurobasal®. Finally,we tested the hypothesis that lentivirus mediated overexpression of the pro-death protein RBM5 exacerbates neuronal and/or axonal injury in primary CNS cultures. RBM5 overexpression vs. empty-vector controls increased 24h LDH release,and SBDP levels,after a single 54% stretch-injury but did not affect NF-L levels or Tau. CONCLUSIONBrainPhys® is a promising new reagent which facilities the investigation of molecular targets involved in axonal and/or neuronal injury in vitro. View Publication

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4,which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [(3)H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [(3)H]ERGO uptake. On the other hand,exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin,but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP),with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly,edaravone and ascorbic acid did not affect such differentiation of NPCs,in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP,but decreased the number immunoreactive for βIII-tubulin,with concomitant down-regulation of Math1 in P19-NPCs. Thus,OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress,and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action. View Publication

UNLABELLED Among the known genetic risk factors for Parkinson disease,mutations in GBA1,the gene responsible for the lysosomal disorder Gaucher disease,are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics,we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease,two with and two without parkinsonism,and one patient with Type 2 (acute neuronopathic) Gaucher disease,and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine,demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein,a protein present as aggregates in Parkinson disease and related synucleinopathies,were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607,a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme,restored glucocerebrosidase activity and protein levels,and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons,indicating its potential for treating neuronopathic Gaucher disease. In addition,NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism,suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease,dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity,reduced lysosomal glucocerebrosidase levels,and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype,the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone,which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition,the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons,indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease. View Publication