技术资料

AIM Bevacizumab has been reported to result in increased tumor invasion when used to treat malignant glioma. We hypothesized that BMP4 would prevent diffuse tumor infiltration induced by bevacizumab for malignant glioma in a xenograft model. METHODS Human glioblastoma (GBM) tumor cells were implanted in the striatum of immunocompromised mice. The animals were treated with bevacizumab and BMP4. Tumor growth and invasion were measured. RESULTS The bevacizumab-treated mice had increased survival compared with control animals (p = 0.02). BMP4 alone did not result in improved survival (p = 1.0). The bevacizumab (p = 0.006) and bevacizumab plus BMP4 (p = 0.006) groups demonstrated significantly decreased total tumor size compared with control. Tumor invasion was significantly decreased in the bevacizumab (p = 0.005),BMP4 (p = 0.04) alone and bevacizumab plus BMP4 (p = 0.002) groups compared with control. No synergistic effect between bevacizumab and BMP4 was observed. CONCLUSION Bevacizumab treatment did not result in diffuse infiltration of human GBM in a mouse xenograft model. BMP4 did have an independent favorable effect on GBM that was not synergistic with bevacizumab treatment. View Publication

Somatic cell reprogramming may become a powerful approach to generate specific human cell types for cell-fate determination studies and potential transplantation therapies of neurological diseases. Here we report a reprogramming methodology with which human adipose stem cells (hADSCs) can be differentiated into neural cells. After being reprogrammed with polycistronic plasmid carrying defined factor OCT3/4,SOX2,KLF4 and c-MYC,and further treated with neural induce medium,the hADSCs switched to differentiate toward neural cell lineages. The generated cells had normal karyotypes and exogenous vector sequences were not inserted in the genomes. Therefore,this cell lineage conversion methodology bypasses the risk of mutation and gene instability,and provides a novel strategy to obtain patient-specific neural cells for basic research and therapeutic application. View Publication

Glioblastoma multiforme (GBM) comprises several molecular subtypes,including proneural GBM. Most therapeutic approaches targeting glioma cells have failed. An alternative strategy is to target cells in the glioma microenvironment,such as tumor-associated macrophages and microglia (TAMs). Macrophages depend on colony stimulating factor-1 (CSF-1) for differentiation and survival. We used an inhibitor of the CSF-1 receptor (CSF-1R) to target TAMs in a mouse proneural GBM model,which significantly increased survival and regressed established tumors. CSF-1R blockade additionally slowed intracranial growth of patient-derived glioma xenografts. Surprisingly,TAMs were not depleted in treated mice. Instead,glioma-secreted factors,including granulocyte-macrophage CSF (GM-CSF) and interferon-γ (IFN-γ),facilitated TAM survival in the context of CSF-1R inhibition. Expression of alternatively activated M2 markers decreased in surviving TAMs,which is consistent with impaired tumor-promoting functions. These gene signatures were associated with enhanced survival in patients with proneural GBM. Our results identify TAMs as a promising therapeutic target for proneural gliomas and establish the translational potential of CSF-1R inhibition for GBM. View Publication

The potential ability to differentiate dedifferentiated adipocytes into a neural lineage is attracting strong interest as an emerging method of producing model cells for the treatment of a variety of neurological diseases. Here,we describe the efficient conversion of dedifferentiated adipocytes into a neural-like cell population. These cells grew in neurosphere-like structures and expressed a high level of the early neuroectodermal marker Nestin. These neurospheres could proliferate and express stemness genes,suggesting that these cells could be committed to the neural lineage. After neural induction,NeuroD1,Sox1,Double Cortin,and Eno2 were not expressed. Patch clamp data did not reveal different electrophysiological properties,indicating the inability of these cells to differentiate into mature neurons. In contrast,the differentiated cells expressed a high level of CLDN11,as demonstrated using molecular method,and stained positively for the glial cell markers CLDN11 and GFAP,as demonstrated using immunocytochemistry. These data were confirmed by quantitative results for glial cell line-derived neurotrophic factor production,which showed a higher secretion level in neurospheres and the differentiated cells compared with the untreated cells. In conclusion,our data demonstrate morphological,molecular,and immunocytochemical evidence of initial neural differentiation of mature adipocytes,committing to a glial lineage. View Publication

General anesthetics produce a reversible coma-like state through modulation of excitatory and inhibitory synaptic transmission. Recent evidence suggests that anesthetic exposure can also lead to sustained cognitive dysfunction. However,the subcellular effects of anesthetics on the structure of established synapses are not known. We investigated effects of the widely used volatile anesthetic isoflurane on the structural stability of hippocampal dendritic spines,a postsynaptic structure critical to excitatory synaptic transmission in learning and memory. Exposure to clinical concentrations of isoflurane induced rapid and non-uniform shrinkage and loss of dendritic spines in mature cultured rat hippocampal neurons. Spine shrinkage was associated with a reduction in spine F-actin concentration. Spine loss was prevented by either jasplakinolide or cytochalasin D,drugs that prevent F-actin disassembly. Isoflurane-induced spine shrinkage and loss were reversible upon isoflurane elimination. Thus,isoflurane destabilizes spine F-actin,resulting in changes to dendritic spine morphology and number. These findings support an actin-based mechanism for isoflurane-induced alterations of synaptic structure in the hippocampus. These reversible alterations in dendritic spine structure have important implications for acute anesthetic effects on excitatory synaptic transmission and synaptic stability in the hippocampus,a locus for anesthetic-induced amnesia,and have important implications for anesthetic effects on synaptic plasticity. View Publication

Although inhibition of p16(INK4a) expression is critical to preserve the proliferative capacity of stem cells,the molecular mechanisms responsible for silencing p16(INK4a) expression remain poorly characterized. Here,we show that the histone acetyltransferase (HAT) monocytic leukemia zinc finger protein (MOZ) controls the proliferation of both hematopoietic and neural stem cells by modulating the transcriptional repression of p16(INK4a) . In the absence of the HAT activity of MOZ,expression of p16(INK4a) is upregulated in progenitor and stem cells,inducing an early entrance into replicative senescence. Genetic deletion of p16(INK4a) reverses the proliferative defect in both Moz(HAT) (-) (/) (-) hematopoietic and neural progenitors. Our results suggest a critical requirement for MOZ HAT activity to silence p16(INK4a) expression and to protect stem cells from early entrance into replicative senescence. View Publication

OBJECTIVE Oxidative stress in the brain is highly prevalent in many neurodegenerative disorders including lysosomal storage disorders,in which neurodegeneration is a devastating manifestation. Despite intense studies,a precise mechanism linking oxidative stress to neuropathology in specific neurodegenerative diseases remains largely unclear. METHODS Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease caused by mutations in the ceroid lipofuscinosis neuronal-1 (CLN1) gene encoding palmitoyl-protein thioesterase-1. Previously,we reported that in the brain of Cln1 (-/-) mice,which mimic INCL,and in postmortem brain tissues from INCL patients,increased oxidative stress is readily detectable. We used molecular,biochemical,immunohistological,and electrophysiological analyses of brain tissues of Cln1 (-/-) mice to study the role(s) of oxidative stress in mediating neuropathology. RESULTS Our results show that in Cln1 (-/-) mice oxidative stress in the brain via upregulation of the transcription factor,CCAAT/enhancer-binding protein-δ,stimulated expression of serpina1,which is an inhibitor of a serine protease,neurotrypsin. Moreover,in the Cln1 (-/-) mice,suppression of neurotrypsin activity by serpina1 inhibited the cleavage of agrin (a large proteoglycan),which substantially reduced the production of agrin-22,essential for synaptic homeostasis. Direct whole-cell recordings at the nerve terminals of Cln1 (-/-) mice showed inhibition of Ca(2+) currents attesting to synaptic dysfunction. Treatment of these mice with a thioesterase-mimetic small molecule,N-tert (Butyl) hydroxylamine (NtBuHA),increased agrin-22 levels. INTERPRETATION Our findings provide insight into a novel pathway linking oxidative stress with synaptic pathology in Cln1 (-/-) mice and suggest that NtBuHA,which increased agrin-22 levels,may ameliorate synaptic dysfunction in this devastating neurodegenerative disease. View Publication

Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy,many patients succumb to the disease,and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro,in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB. View Publication

While no effective therapy is available for the treatment of methamphetamine (METH)-induced neurotoxicity,aerobic exercise is being proposed to improve depressive symptoms and substance abuse outcomes. The present study focuses on the effect of exercise on METH-induced aberrant neurogenesis in the hippocampal dentate gyrus in the context of the blood-brain barrier (BBB) pathology. Mice were administered with METH or saline by i.p. injections for 5 days with an escalating dose regimen. One set of mice was sacrificed 24 h post last injection of METH,and the remaining animals were either subjected to voluntary wheel running (exercised mice) or remained in sedentary housing (sedentary mice). METH administration decreased expression of tight junction (TJ) proteins and increased BBB permeability in the hippocampus. These changes were preserved post METH administration in sedentary mice and were associated with the development of significant aberrations of neural differentiation. Exercise protected against these effects by enhancing the protein expression of TJ proteins,stabilizing the BBB integrity,and enhancing the neural differentiation. In addition,exercise protected against METH-induced systemic increase in inflammatory cytokine levels. These results suggest that exercise can attenuate METH-induced neurotoxicity by protecting against the BBB disruption and related microenvironmental changes in the hippocampus. View Publication

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases,for example,in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency,which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions,deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template,such as an introduced single-stranded oligo DNA nucleotide (ssODN),allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ,editing by HDR remains inefficient and can be corrupted by additional indels,preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore,targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations,and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB,we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation,whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach,we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in amyloid precursor protein (APP(Swe)) and presenilin 1 (PSEN1(M146V)) and derived cortical neurons,which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9,facilitating study of human disease. View Publication

A critical challenge to deciphering the pathophysiology of neurodevelopmental disease is identifying which of the myriad abnormalities that emerge during CNS maturation persist to contribute to long-term brain dysfunction. Childhood-onset dystonia caused by a loss-of-function mutation in the AAA+ protein torsinA exemplifies this challenge. Neurons lacking torsinA develop transient nuclear envelope (NE) malformations during CNS maturation,but no NE defects are described in mature torsinA null neurons. We find that during postnatal CNS maturation torsinA null neurons develop mislocalized and dysfunctional nuclear pore complexes (NPC) that lack NUP358,normally added late in NPC biogenesis. SUN1,a torsinA-related molecule implicated in interphase NPC biogenesis,also exhibits localization abnormalities. Whereas SUN1 and associated nuclear membrane abnormalities resolve in juvenile mice,NPC defects persist into adulthood. These findings support a role for torsinA function in NPC biogenesis during neuronal maturation and implicate altered NPC function in dystonia pathophysiology. View Publication

The generation of differentiated and functional neurons is a complex process,which requires coordinated expression of several proteins and microRNAs (miRNAs). The present study using nerve growth factor (NGF)-differentiated PC12 cells led to the identification of miR-200,miR-221/222 and miR-34 families as major up-regulated miRNAs in fully differentiated neurons. Similar to PC12 cells,induction of miR-200 family was observed in differentiating neural stem cells,demonstrating a direct role of miR-200 family in neuronal differentiation. Over-expression of miR-200 induced neurite formation in PC12 cells and regulated neuronal markers in favour of differentiation. However,inhibition of miR-200 induced proliferation of PC12 cells. In differentiating PC12 cells and neural stem cells,an inverse relationship was observed between expression of reprogramming transcription factors (SOX2,KLF4,NANOG,OCT4 and PAX6) and miR-200. Over-expression of miR-200 in PC12 cells significantly down-regulated mRNA and protein levels of SOX2 and KLF4. Moreover,we observed two phases of dramatic down-regulation of miR-200 expression in developing rat brains correlating with periods of neuronal proliferation. In conclusion,our results indicate that increased expression of the miR-200 family promotes neuronal differentiation,while decreased expression of the miR-200 family promotes neuronal proliferation by targeting SOX2 and KLF4. View Publication