技术资料

Glioblastoma multiforme (GBM) is the most common and lethal form of intracranial tumor. We have established a lentivirus-induced mouse model of malignant gliomas,which faithfully captures the pathophysiology and molecular signature of mesenchymal human GBM. RNA-Seq analysis of these tumors revealed high nuclear factor κB (NF-κB) activation showing enrichment of known NF-κB target genes. Inhibition of NF-κB by either depletion of IκB kinase 2 (IKK2),expression of a IκBαM super repressor,or using a NEMO (NF-κB essential modifier)-binding domain (NBD) peptide in tumor-derived cell lines attenuated tumor proliferation and prolonged mouse survival. Timp1,one of the NF-κB target genes significantly up-regulated in GBM,was identified to play a role in tumor proliferation and growth. Inhibition of NF-κB activity or silencing of Timp1 resulted in slower tumor growth in both mouse and human GBM models. Our results suggest that inhibition of NF-κB activity or targeting of inducible NF-κB genes is an attractive therapeutic approach for GBM. View Publication

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival,these regimens are highly toxic and are associated with significant morbidity. Here,we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas,independent of their subtype. Moreover,high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo,resulting in medulloblastoma regression,decreased metastasis,and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh,PlGF,and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway. View Publication

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS),as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear,with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed,and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased,leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α,suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6,and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. View Publication

In culture conditions,human induced-pluripotent stem cells (hiPSC)-derived neurons form synaptic connections with other cells and establish neuronal networks,which are expected to be an in vitro model system for drug discovery screening and toxicity testing. While early studies demonstrated effects of co-culture of hiPSC-derived neurons with astroglial cells on survival and maturation of hiPSC-derived neurons,the population spiking patterns of such hiPSC-derived neurons have not been fully characterized. In this study,we analyzed temporal spiking patterns of hiPSC-derived neurons recorded by a multi-electrode array system. We discovered that specific sets of hiPSC-derived neurons co-cultured with astrocytes showed more frequent and highly coherent non-random synchronized spike trains and more dynamic changes in overall spike patterns over time. These temporally coordinated spiking patterns are physiological signs of organized circuits of hiPSC-derived neurons and suggest benefits of co-culture of hiPSC-derived neurons with astrocytes. View Publication

Glioblastoma multiforme (GBM) is a highly aggressive brain tumor,with dismal survival outcomes. Recently,cancer stem cells (CSCs) have been demonstrated to play a role in therapeutic resistance and are considered to be the most likely cause of cancer relapse. The identification of CSCs is an important step toward finding new and effective ways to treat GBM. Tenascin-C (TNC) protein has been identified as a potential marker for CSCs in gliomas based on previous work. Here,we have investigated the expression of TNC in tissue microarrays including 17 GBMs,18 WHO grade III astrocytomas,15 WHO grade II astrocytomas,4 WHO grade I astrocytomas,and 7 normal brain tissue samples by immunohistochemical staining. TNC expression was found to be highly associated with the grade of astrocytoma. It has a high expression level in most of the grade III astrocytomas and GBMs analyzed and a very low expression in most grade II astrocytomas,whereas it is undetectable in grade I astrocytomas and normal brain tissues. Double-immunofluorescence staining for TNC and CD133 in GBM tissues revealed that there was a high overlap between theses two positive populations. The results were further confirmed by flow cytometry analysis of TNC and CD133 in GBM-derived stem-like neurospheres in vitro. A limiting dilution assay demonstrated that the sphere formation ability of CD133(+)/TNC(+) and CD133(-)/TNC(+) cell populations is much higher than that of the CD133(+)/TNC(-) and CD133(-)/TNC(-) populations. These results suggest that TNC is not only a potential prognostic marker for GBM but also a potential marker for glioma CSCs,where the TNC(+) population is identified as a CSC population overlapping with part of the CD133(-) cell population. View Publication

Neurogenesis decreases during aging causing a progressive cognitive decline but it is still controversial whether proliferation defects in neurogenic niches result from a loss of neural stem cells or from an impairment of their progression through the cell cycle. Using an accurate fluorescence-activated cell sorting technique,we show that the pool of neural stem cells is maintained in the subventricular zone of middle-aged mice while they have a reduced proliferative potential eventually leading to the subsequent decrease of their progeny. In addition,we demonstrate that the G1 phase is lengthened during aging specifically in activated stem cells,but not in transit-amplifying cells,and directly impacts on neurogenesis. Finally,we report that inhibition of TGFβ signaling restores cell cycle progression defects in stem cells. Our data highlight the significance of cell cycle dysregulation in stem cells in the aged brain and provide an attractive foundation for the development of anti-TGFβ regenerative therapies based on stimulating endogenous neural stem cells. 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

Microtubules (MTs),key cytoskeletal elements in living cells,are critical for axonal transport,synaptic transmission,and maintenance of neuronal morphology. NAP (NAPVSIPQ) is a neuroprotective peptide derived from the essential activity-dependent neuroprotective protein (ADNP). In Alzheimer's disease models,NAP protects against tauopathy and cognitive decline. Here,we show that NAP treatment significantly affected the alpha tubulin tyrosination cycle in the neuronal differentiation model,rat pheochromocytoma (PC12) and in rat cortical astrocytes. The effect on tubulin tyrosination/detyrosination was coupled to increased MT network area (measured in PC12 cells),which is directly related to neurite outgrowth. Tubulin beta3,a marker for neurite outgrowth/neuronal differentiation significantly increased after NAP treatment. In rat cortical neurons,NAP doubled the area of dynamic MT invasion (Tyr-tubulin) into the neuronal growth cone periphery. NAP was previously shown to protect against zinc-induced MT/neurite destruction and neuronal death,here,in PC12 cells,NAP treatment reversed zinc-decreased tau-tubulin-MT interaction and protected against death. NAP effects on the MT pool,coupled with increased tau engagement on compromised MTs imply an important role in neuronal plasticity,protecting against free tau accumulation leading to tauopathy. With tauopathy representing a major pathological hallmark in Alzheimer's disease and related disorders,the current findings provide a mechanistic basis for further development. NAP (davunetide) is in phase 2/3 clinical trial in progressive supranuclear palsy,a disease presenting MT deficiency and tau pathology. View Publication

The aim of this investigation was to characterize the proliferative precursor cells in the adult mouse hippocampal region. Given that a very large number of new hippocampal cells are generated over the lifetime of an animal,it is predicted that a neural stem cell is ultimately responsible for maintaining this genesis. Although it is generally accepted that a proliferative precursor resides within the hippocampus,contradictory reports exist regarding the classification of this cell. Is it a true stem cell or a more limited progenitor? Using a strict functional definition of a neural stem cell and a number of in vitro assays,we report that the resident hippocampal precursor is a progenitor capable of proliferation and multipotential differentiation but is unable to self-renew and thus proliferate indefinitely. Furthermore,the mitogen FGF-2 stimulates proliferation of these cells to a greater extent than epidermal growth factor (EGF). In addition,we found that BDNF was essential for the production of neurons from the hippocampal progenitor cells,being required during proliferation to trigger neuronal fate. In contrast,a bona fide neural stem cell was identified in the lateral wall of the lateral ventricle surrounding the hippocampus. Interestingly,EGF proved to be the stronger mitogenic factor for this cell,which was clearly a different precursor from the resident hippocampal progenitor. These results suggest that the stem cell ultimately responsible for adult hippocampal neurogenesis resides outside the hippocampus,producing progenitor cells that migrate into the neurogenic zones and proliferate to produce new neurons and glia. View Publication

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM,a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells,GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However,the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature,which defines aggressiveness and invasion,and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1,which encode a key regulator of selective autophagy,p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs,DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation,thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast,autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally,DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism,in particular reduced ATP and lactate levels. Taken together,these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells. View Publication

BACKGROUND AND PURPOSE HIV-1 glycoprotein Gp120 induces apoptosis in rodent and human neurons in vitro and in vivo.HIV-1/Gp120 is involved in the pathogenesis of HIV-associated dementia (HAD) and inhibits proliferation of adult neural progenitor cells (NPCs) in glial fibrillary acidic protein (GFAP)/Gp120 transgenic (Tg) mice. As cannabinoids exert neuroprotective effects in several model systems,we examined the protective effects of the CB receptor agonist AM1241 on Gp120-mediated insults on neurogenesis. EXPERIMENTAL APPROACH We assessed the effects of AM1241 on survival and apoptosis in cultures of human and murine NPCs with immunohistochemical and TUNEL techniques. Neurogenesis in the hippocampus of GFAP/Gp120 transgenic mice in vivo was also assessed by immunohistochemistry. KEY RESULTS AM1241 inhibited in vitroGp120-mediated neurotoxicity and apoptosis of primary human and murine NPCs and increased their survival. AM1241 also promoted differentiation of NPCs to neuronal cells. While GFAP/Gp120 Tg mice exhibited impaired neurogenesis,as indicated by reduction in BrdU cells and doublecortin (DCX) cells,and a decrease in cells with proliferating cell nuclear antigen (PCNA),administration of AM1241 to GFAP/Gp120 Tg mice resulted in enhanced in vivo neurogenesis in the hippocampus as indicated by increase in neuroblasts,neuronal cells,BrdU cells and PCNA cells. Astrogliosis and gliogenesis were decreased in GFAP/Gp120 Tg mice treated with AM1241,compared with those treated with vehicle. CONCLUSIONS AND IMPLICATIONS The CB receptor agonist rescued impaired neurogenesis caused by HIV-1/Gp120 insult. Thus,CB receptor agonists may act as neuroprotective agents,restoring impaired neurogenesis in patients with HAD. View Publication

BACKGROUND Glioblastomas (GBMs) are a heterogeneous group of primary brain tumors. These tumors are resistant to therapeutic interventions and invariably recur after surgical resection. The multifunctional protein transglutaminase 2 (TG2) has been shown to promote cell survival in a number of different tumors. There is also evidence that TG2 may be a pro-survival factor in GBMs. However,the roles that TG2 plays in facilitating GBM survival and proliferation have not yet been clearly delineated . METHODS The functions of TG2 are often cell- and context-specific. Therefore,in this study we examined the ability of TG2 to facilitate GBM proliferation using colony formation assays and 5-ethynyl-2'-deoxyuridine (EdU) incorporation in several different GBM cell lines as well as neurospheres derived from patient tumors representing the 3 major subtypes of GBM tumors (mesenchymal,proneural,and classical) and maintained in the absence of serum. TG2 knockdown or selective TG2 inhibitors were used to modulate TG2 expression and activity. RESULTS We show that TG2 plays differential roles in the proliferative process depending on the cell type. In most,but not all,GBM models TG2 plays a crucial role in the proliferative process,and some but not all TG2 inhibitors were highly effective at reducing proliferation in a large subset of the GBM models. CONCLUSION Our results show that TG2 plays an important-but notoriously context-specific-role in GBM cell biology. Nonetheless,as future studies unravel the genetic fingerprints" that make TG2 inhibitors effective this information could be exploited to develop TG2 inhibitors into personalized GBM therapies. View Publication