技术资料

Stochastic processes and imprinting,along with genetic factors,lead to monoallelic or allele-biased gene expression. Stochastic monoallelic expression fine-tunes information processing in immune cells and the olfactory system,and imprinting plays an important role in development. Recent studies suggest that both stochastic events and imprinting may be more widespread than previously considered. We are interested in allele-biased gene expression occurring in the brain because parent-of-origin effects suggestive of imprinting appear to play a role in the transmission of schizophrenia (SZ) and autism spectrum disorders (ASD) in some families. In addition,allele-biased expression could help explain monozygotic (MZ) twin discordance and reduced penetrance. The ability to study allele-biased expression in human neurons has been transformed with the advent of induced pluripotent stem cell (iPSC) technology and next generation sequencing. Using transcriptome sequencing (RNA-Seq) we identified 801 genes in differentiating neurons that were expressed in an allele-biased manner. These included a number of putative SZ and ASD candidates,such as A2BP1 (RBFOX1),ERBB4,NLGN4X,NRG1,NRG3,NRXN1,and NLGN1. Overall,there was a modest enrichment for SZ and ASD candidate genes among those that showed evidence for allele-biased expression (chi-square,p = 0.02). In addition to helping explain MZ twin discordance and reduced penetrance,the capacity to group many candidate genes affecting a variety of molecular and cellular pathways under a common regulatory process - allele-biased expression - could have therapeutic implications. View Publication

TDP-43 is found in cytoplasmic inclusions in 95% of amyotrophic lateral sclerosis (ALS) and 60% of frontotemporal lobar degeneration (FTLD). Approximately 4% of familial ALS is caused by mutations in TDP-43. The majority of these mutations are found in the glycine-rich domain,including the variant M337V,which is one of the most common mutations in TDP-43. In order to investigate the use of allele-specific RNA interference (RNAi) as a potential therapeutic tool,we designed and screened a set of siRNAs that specifically target TDP-43(M337V) mutation. Two siRNA specifically silenced the M337V mutation in HEK293T cells transfected with GFP-TDP-43(wt) or GFP-TDP-43(M337V) or TDP-43 C-terminal fragments counterparts. C-terminal TDP-43 transfected cells show an increase of cytosolic inclusions,which are decreased after allele-specific siRNA in M337V cells. We then investigated the effects of one of these allele-specific siRNAs in induced pluripotent stem cells (iPSCs) derived from an ALS patient carrying the M337V mutation. These lines showed a two-fold increase in cytosolic TDP-43 compared to the control. Following transfection with the allele-specific siRNA,cytosolic TDP-43 was reduced by 30% compared to cells transfected with a scrambled siRNA. We conclude that RNA interference can be used to selectively target the TDP-43(M337V) allele in mammalian and patient cells,thus demonstrating the potential for using RNA interference as a therapeutic tool for ALS. View Publication

Friedreich's ataxia (FRDA) is the most common autosomal recessive ataxia. This severe neurodegenerative disease is caused by an expansion of guanine-adenine-adenine (GAA) repeats located in the first intron of the frataxin (FXN) gene,which represses its transcription. Although transcriptional silencing is associated with heterochromatin-like changes in the vicinity of the expanded GAAs,the exact mechanism and pathways involved in transcriptional inhibition are largely unknown. As major remodeling of the epigenome is associated with somatic cell reprogramming,modulating chromatin modification pathways during the cellular transition from a somatic to a pluripotent state is likely to generate permanent changes to the epigenetic landscape. We hypothesize that the epigenetic modifications in the vicinity of the GAA repeats can be reversed by pharmacological modulation during somatic cell reprogramming. We reprogrammed FRDA fibroblasts into induced pluripotent stem cells (iPSCs) in the presence of various small molecules that target DNA methylation and histone acetylation and methylation. Treatment of FRDA iPSCs with two compounds,sodium butyrate (NaB) and Parnate,led to an increase in FXN expression and correction of repressive marks at the FXN locus,which persisted for several passages. However,prolonged culture of the epigenetically modified FRDA iPSCs led to progressive expansions of the GAA repeats and a corresponding decrease in FXN expression. Furthermore,we uncovered that differentiation of these iPSCs into neurons also results in resilencing of the FXN gene. Taken together,these results demonstrate that transcriptional repression caused by long GAA repeat tracts can be partially or transiently reversed by altering particular epigenetic modifications,thus revealing possibilities for detailed analyses of silencing mechanism and development of new therapeutic approaches for FRDA. View Publication

mTOR inhibition is beneficial in neurodegenerative disease models and its effects are often attributable to the modulation of autophagy and anti-apoptosis. Here,we report a neglected but important bioenergetic effect of mTOR inhibition in neurons. mTOR inhibition by rapamycin significantly preserves neuronal ATP levels,particularly when oxidative phosphorylation is impaired,such as in neurons treated with mitochondrial inhibitors,or in neurons derived from maternally inherited Leigh syndrome (MILS) patient iPS cells with ATP synthase deficiency. Rapamycin treatment significantly improves the resistance of MILS neurons to glutamate toxicity. Surprisingly,in mitochondrially defective neurons,but not neuroprogenitor cells,ribosomal S6 and S6 kinase phosphorylation increased over time,despite activation of AMPK,which is often linked to mTOR inhibition. A rapamycin-induced decrease in protein synthesis,a major energy-consuming process,may account for its ATP-saving effect. We propose that a mild reduction in protein synthesis may have the potential to treat mitochondria-related neurodegeneration. View Publication

Predicting the binding mode of flexible polypeptides to proteins is an important task that falls outside the domain of applicability of most small molecule and protein−protein docking tools. Here,we test the small molecule flexible ligand docking program Glide on a set of 19 non-$$-helical peptides and systematically improve pose prediction accuracy by enhancing Glide sampling for flexible polypeptides. In addition,scoring of the poses was improved by post-processing with physics-based implicit solvent MM- GBSA calculations. Using the best RMSD among the top 10 scoring poses as a metric,the success rate (RMSD ≤ 2.0 Å for the interface backbone atoms) increased from 21% with default Glide SP settings to 58% with the enhanced peptide sampling and scoring protocol in the case of redocking to the native protein structure. This approaches the accuracy of the recently developed Rosetta FlexPepDock method (63% success for these 19 peptides) while being over 100 times faster. Cross-docking was performed for a subset of cases where an unbound receptor structure was available,and in that case,40% of peptides were docked successfully. We analyze the results and find that the optimized polypeptide protocol is most accurate for extended peptides of limited size and number of formal charges,defining a domain of applicability for this approach. View Publication

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

Monge's disease,also known as chronic mountain sickness (CMS),is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%,suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however,CMS subjects experience severe hypoxemia,erythrocytosis and many neurologic manifestations including migraine,headache,mental fatigue,confusion,and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS,except for phlebotomy. In the current study,we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs),then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties,but instead by a significantly lowered Na+ channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide,for the first time,evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects,hoping that such studies can pave the way to a better understanding of the neuropathology in CMS. View Publication

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup),carrying different duplication sizes,to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition,using multi-electrodes arrays,we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level,we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor,NCH-51,was validated as a potential clinical candidate. Interestingly,this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders. View Publication

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts,perceptions,and emotions,usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however,most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells,derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient,presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS),when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia,contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening. View Publication

Autism spectrum disorders (ASD) are common,complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies,brain pathology and imaging,but a major impediment to testing ASD hypotheses is the lack of human cell models. Here,we reprogrammed fibroblasts to generate induced pluripotent stem cells,neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a β-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly,defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1),a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1 View Publication

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently,optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally,hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However,these methods have several major limitations,including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods,we have recently developed a new method,which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here,we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor),phenylbenzodioxane (ROCK II inhibitor),and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover,based on NCM,we have demonstrated efficient transfection or transduction of plasmid DNAs,lentiviral particles,and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture,and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies,stem cell research,and drug discovery. View Publication

Purine biosynthesis and metabolism,conserved in all living organisms,is essential for cellular energy homeostasis and nucleic acid synthesis. The de novo synthesis of purine precursors is under tight negative feedback regulation mediated by adenosine and guanine nucleotides. We describe a distinct early-onset neurodegenerative condition resulting from mutations in the adenosine monophosphate deaminase 2 gene (AMPD2). Patients have characteristic brain imaging features of pontocerebellar hypoplasia (PCH) due to loss of brainstem and cerebellar parenchyma. We found that AMPD2 plays an evolutionary conserved role in the maintenance of cellular guanine nucleotide pools by regulating the feedback inhibition of adenosine derivatives on de novo purine synthesis. AMPD2 deficiency results in defective GTP-dependent initiation of protein translation,which can be rescued by administration of purine precursors. These data suggest AMPD2-related PCH as a potentially treatable early-onset neurodegenerative disease. ?? 2013 Elsevier Inc. View Publication