技术资料

It is generally believed that cerebellar granule neurons originate exclusively from granule neuron precursors (GNPs) in the external germinal layer (EGL). Here we identified a rare population of neuronal progenitors in mouse developing cerebellum that expresses Nestin. Although Nestin is widely considered a marker for multipotent stem cells,these Nestin-expressing progenitors (NEPs) are committed to the granule neuron lineage. Unlike conventional GNPs,which reside in the outer EGL and proliferate extensively,NEPs reside in the deep part of the EGL and are quiescent. Expression profiling revealed that NEPs are distinct from GNPs and,in particular,express markedly reduced levels of genes associated with DNA repair. Consistent with this,upon aberrant activation of Sonic hedgehog (Shh) signaling,NEPs exhibited more severe genomic instability and gave rise to tumors more efficiently than GNPs. These studies revealed a previously unidentified progenitor for cerebellar granule neurons and a cell of origin for medulloblastoma. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However,clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy,in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression,we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells,we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs,modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type,expressing pro-angiogenic factors. In contrast,phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data. View Publication

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs),cancers occurring increasingly in children to young adults,is poorly understood. We present a transplantable tumour line,maintained in immune-compromised mice,and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays,spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model,with parallel studies on 27 primary hFL-HCC tumours,provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple,normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells--newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies. 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 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

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

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

Neural stem and progenitor cells (NSCs/NPCs) are distinct groups of cells found in the mammalian central nervous system (CNS). Previously we determined that members of the High Mobility Group (HMG) B family of chromatin structural proteins modulate NSC proliferation and self-renewal. Among them HMGB2 was found to be dynamically expressed in proliferating and differentiating NSCs,suggesting that it may regulate NSC maintenance. We report now that Hmgb2(-/-) mice exhibit SVZ hyperproliferation,increased numbers of SVZ NSCs,and a trend towards aberrant increases in newly born neurons in the olfactory bulb (OB) granule cell layer. Increases in the levels of the transcription factor p21 and the Neural cell adhesion molecule (NCAM),along with down-regulation of the transcription/pluripotency factor Oct4 in the Hmgb2-/- SVZ point to a possible pathway for this increased proliferation/differentiation. Our findings suggest that HMGB2 functions as a modulator of neurogenesis in young adult mice through regulation of NSC proliferation,and identify a potential target via which CNS repair could be amplified following trauma or disease-based neuronal degeneration. 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

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX+) cells more stringently,these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic,early postnatal,and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX(high)) in the embryonic brain coexpressed the neuronal marker betaIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formation in vitro. However,the DCX+ population from the early postnatal brain and the adult subventricular zone and hippocampus,which expressed low levels of DCX (DCX(low)),was enriched for neurosphere-forming cells,with only a small subpopulation of these cells coexpressing the neuronal markers betaIII-tubulin or microtubule-associated protein 2. Similarly,the DCX(low) population from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX(high) cells,which were shown to be similar to the E14 DCX(high) cells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX+ cells,with some cells displaying characteristics of immature or mature neurons,whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX(high) cells,regardless of location,are restricted to the neuronal lineage or are bone fide neurons,whereas some DCX(low) cells retain their multipotentiality. View Publication

Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction,vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification,these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1),a lysosomal enzyme,cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus,inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL),a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL,the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL,we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further,we demonstrate that the level of growth-associated protein-43 (GAP-43),a palmitoylated neuronal protein,is elevated in the brains of PPT1-KO mice. Moreover,forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results,we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor,eIF2alpha,increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12,a cysteine proteinase in the ER,mediating caspase-3 activation and apoptosis. Our results,for the first time,link PPT1 deficiency with the activation of UPR,apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease. View Publication

The low success rate of animal cloning by somatic cell nuclear transfer (SCNT) is believed to be associated with epigenetic errors including abnormal DNA hypermethylation. Recently,we elucidated by using round spermatids that,after nuclear transfer,treatment of zygotes with trichostatin A (TSA),an inhibitor of histone deacetylase,can remarkably reduce abnormal DNA hypermethylation depending on the origins of transferred nuclei and their genomic regions [S. Kishigami,N. Van Thuan,T. Hikichi,H. Ohta,S. Wakayama. E. Mizutani,T. Wakayama,Epigenetic abnormalities of the mouse paternal zygotic genome associated with microinsemination of round spermatids,Dev. Biol. (2005) in press]. Here,we found that 5-50 nM TSA-treatment for 10 h following oocyte activation resulted in more efficient in vitro development of somatic cloned embryos to the blastocyst stage from 2- to 5-fold depending on the donor cells including tail tip cells,spleen cells,neural stem cells,and cumulus cells. This TSA-treatment also led to more than 5-fold increase in success rate of mouse cloning from cumulus cells without obvious abnormality but failed to improve ES cloning success. Further,we succeeded in establishment of nuclear transfer-embryonic stem (NT-ES) cells from TSA-treated cloned blastocyst at a rate three times higher than those from untreated cloned blastocysts. Thus,our data indicate that TSA-treatment after SCNT in mice can dramatically improve the practical application of current cloning techniques. View Publication