Publications by authors named "Sheena Lee"

28 Publications

  • Page 1 of 1

The long non-coding RNA promotes KAP1-dependent chromatin changes and regulates olfactory bulb neurogenesis.

EMBO J 2018 05 16;37(10). Epub 2018 Apr 16.

Department of Biology and Biochemistry, University of Bath, Bath, UK

Many long non-coding RNAs (lncRNAs) are expressed during central nervous system (CNS) development, yet their roles and mechanisms of action remain poorly understood. , a CNS-expressed lncRNA, controls neuroblastoma cell growth by binding and modulating the activity of transcriptional regulatory elements in a genome-wide manner. We show here that the lncRNA directly binds KAP1, an essential epigenetic regulatory protein, and thereby regulates the expression of shared target genes important for proliferation and neuronal differentiation. promotes KAP1 chromatin occupancy and H3K9me3 deposition at a subset of distal targets, through the formation of a ribonucleoprotein complex containing , KAP1 and the PAX6 transcription factor. -KAP1 genome-wide co-occupancy reveals a fourfold enrichment of overlap between and KAP1 bound sequences, the majority of which also appear to associate with PAX6. Furthermore, both and loss-of-function disrupt olfactory bulb neurogenesis. These observations provide important conceptual insights into the -acting modes of lncRNA-mediated epigenetic regulation and the mechanisms of KAP1 genomic recruitment, and identify and as regulators of neurogenesis .
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http://dx.doi.org/10.15252/embj.201798219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978383PMC
May 2018

Hyperglycaemia induces metabolic dysfunction and glycogen accumulation in pancreatic β-cells.

Nat Commun 2016 11 24;7:13496. Epub 2016 Nov 24.

Department of Physiology, Anatomy and Genetics and OXION, University of Oxford, Parks Road, Oxford OX1 3PT, UK.

Insulin secretion from pancreatic β-cells is impaired in all forms of diabetes. The resultant hyperglycaemia has deleterious effects on many tissues, including β-cells. Here we show that chronic hyperglycaemia impairs glucose metabolism and alters expression of metabolic genes in pancreatic islets. In a mouse model of human neonatal diabetes, hyperglycaemia results in marked glycogen accumulation, and increased apoptosis in β-cells. Sulphonylurea therapy rapidly normalizes blood glucose levels, dissipates glycogen stores, increases autophagy and restores β-cell metabolism. Insulin therapy has the same effect but with slower kinetics. Similar changes are observed in mice expressing an activating glucokinase mutation, in in vitro models of hyperglycaemia, and in islets from type-2 diabetic patients. Altered β-cell metabolism may underlie both the progressive impairment of insulin secretion and reduced β-cell mass in diabetes.
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http://dx.doi.org/10.1038/ncomms13496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123088PMC
November 2016

Genotype-Dependent Effects of COMT Inhibition on Cognitive Function in a Highly Specific, Novel Mouse Model of Altered COMT Activity.

Neuropsychopharmacology 2016 12 8;41(13):3060-3069. Epub 2016 Jul 8.

Department of Psychiatry, University of Oxford, Oxford, UK.

Catechol-O-methyltransferase (COMT) modulates dopamine levels in the prefrontal cortex. The human gene contains a polymorphism (ValMet) that alters enzyme activity and influences PFC function. It has also been linked with cognition and anxiety, but the findings are mixed. We therefore developed a novel mouse model of altered COMT activity. The human Met allele was introduced into the native mouse COMT gene to produce COMT-Met mice, which were compared with their wild-type littermates. The model proved highly specific: COMT-Met mice had reductions in COMT abundance and activity, compared with wild-type mice, explicitly in the absence of off-target changes in the expression of other genes. Despite robust alterations in dopamine metabolism, we found only subtle changes on certain cognitive tasks under baseline conditions (eg, increased spatial novelty preference in COMT-Met mice vs wild-type mice). However, genotype differences emerged after administration of the COMT inhibitor tolcapone: performance of wild-type mice, but not COMT-Met mice, was improved on the 5-choice serial reaction time task after tolcapone administration. There were no changes in anxiety-related behaviors in the tests that we used. Our findings are convergent with human studies of the ValMet polymorphism, and suggest that COMT's effects are most prominent when the dopamine system is challenged. Finally, they demonstrate the importance of considering COMT genotype when examining the therapeutic potential of COMT inhibitors.
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http://dx.doi.org/10.1038/npp.2016.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5101554PMC
December 2016

Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion.

Diabetes 2016 07 18;65(7):1952-61. Epub 2016 Mar 18.

Oxford Centre for Diabetes, Endocrinology & Metabolism, Radcliffe Department of Medicine, Oxford, U.K. Department of Neuroscience and Physiology, University of Göteborg, Göteborg, Sweden Oxford National Institute of Health Research, Biomedical Research Centre, Churchill Hospital, Oxford, U.K.

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.
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http://dx.doi.org/10.2337/db15-1489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996324PMC
July 2016

The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum.

Hum Mol Genet 2015 Jul 23;24(14):4114-25. Epub 2015 Apr 23.

Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK and

The Moonwalker (Mwk) mouse is a model of dominantly inherited cerebellar ataxia caused by a gain-of-function mutation in the transient receptor potential (TRP) channel TRPC3. Here, we report impairments in dendritic growth and synapse formation early on during Purkinje cell development in the Mwk cerebellum that are accompanied by alterations in calcium signaling. To elucidate the molecular effector pathways that regulate Purkinje cell dendritic arborization downstream of mutant TRPC3, we employed transcriptomic analysis of developing Purkinje cells isolated by laser-capture microdissection. We identified significant gene and protein expression changes in molecules involved in lipid metabolism. Consistently, lipid homeostasis in the Mwk cerebellum was found to be disturbed, and treatment of organotypic cerebellar slices with ceramide significantly improved dendritic outgrowth of Mwk Purkinje cells. These findings provide the first mechanistic insights into the TRPC3-dependent mechanisms, by which activated calcium signaling is coupled to lipid metabolism and the regulation of Purkinje cell development in the Mwk cerebellum.
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http://dx.doi.org/10.1093/hmg/ddv150DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4476454PMC
July 2015

Neuron-specific antioxidant OXR1 extends survival of a mouse model of amyotrophic lateral sclerosis.

Brain 2015 May 9;138(Pt 5):1167-81. Epub 2015 Mar 9.

1 Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3QX, UK

Amyotrophic lateral sclerosis is a devastating neurodegenerative disorder characterized by the progressive loss of spinal motor neurons. While the aetiological mechanisms underlying the disease remain poorly understood, oxidative stress is a central component of amyotrophic lateral sclerosis and contributes to motor neuron injury. Recently, oxidation resistance 1 (OXR1) has emerged as a critical regulator of neuronal survival in response to oxidative stress, and is upregulated in the spinal cord of patients with amyotrophic lateral sclerosis. Here, we tested the hypothesis that OXR1 is a key neuroprotective factor during amyotrophic lateral sclerosis pathogenesis by crossing a new transgenic mouse line that overexpresses OXR1 in neurons with the SOD1(G93A) mouse model of amyotrophic lateral sclerosis. Interestingly, we report that overexpression of OXR1 significantly extends survival, improves motor deficits, and delays pathology in the spinal cord and in muscles of SOD1(G93A) mice. Furthermore, we find that overexpression of OXR1 in neurons significantly delays non-cell-autonomous neuroinflammatory response, classic complement system activation, and STAT3 activation through transcriptomic analysis of spinal cords of SOD1(G93A) mice. Taken together, these data identify OXR1 as the first neuron-specific antioxidant modulator of pathogenesis and disease progression in SOD1-mediated amyotrophic lateral sclerosis, and suggest that OXR1 may serve as a novel target for future therapeutic strategies.
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http://dx.doi.org/10.1093/brain/awv039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407188PMC
May 2015

The long non-coding RNA Dali is an epigenetic regulator of neural differentiation.

Elife 2014 Nov 21;3:e04530. Epub 2014 Nov 21.

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

Many intergenic long noncoding RNA (lncRNA) loci regulate the expression of adjacent protein coding genes. Less clear is whether intergenic lncRNAs commonly regulate transcription by modulating chromatin at genomically distant loci. Here, we report both genomically local and distal RNA-dependent roles of Dali, a conserved central nervous system expressed intergenic lncRNA. Dali is transcribed downstream of the Pou3f3 transcription factor gene and its depletion disrupts the differentiation of neuroblastoma cells. Locally, Dali transcript regulates transcription of the Pou3f3 locus. Distally, it preferentially targets active promoters and regulates expression of neural differentiation genes, in part through physical association with the POU3F3 protein. Dali interacts with the DNMT1 DNA methyltransferase in mouse and human and regulates DNA methylation status of CpG island-associated promoters in trans. These results demonstrate, for the first time, that a single intergenic lncRNA controls the activity and methylation of genomically distal regulatory elements to modulate large-scale transcriptional programmes.
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http://dx.doi.org/10.7554/eLife.04530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383022PMC
November 2014

Laf4/Aff3, a gene involved in intellectual disability, is required for cellular migration in the mouse cerebral cortex.

PLoS One 2014 27;9(8):e105933. Epub 2014 Aug 27.

MRC Functional Genomics Unit, University of Oxford, Oxford, United Kingdom.

Members of the AFF (AF4/FMR2) family of putative transcription factors are involved in infant acute leukaemia and intellectual disability (ID), although very little is known about their transcriptional targets. For example, deletion of human lymphoid nuclear protein related to AF4/AFF member 3 (LAF4/AFF3) is known to cause severe neurodevelopmental defects, and silencing of the gene is also associated with ID at the folate-sensitive fragile site (FSFS) FRA2A; yet the normal function of this gene in the nervous system is unclear. The aim of this study was to further investigate the function of Laf4 in the brain by focusing on its role in the cortex. By manipulating expression levels in organotypic slices, we demonstrate here that Laf4 is required for normal cellular migration in the developing cortex and have subsequently identified Mdga2, an important structural protein in neurodevelopment, as a target of Laf4 transcriptional activity. Furthermore, we show that the migration deficit caused by loss of Laf4 can be partially rescued by Mdga2 over-expression, revealing an important functional relationship between these genes. Our study demonstrates the key transcriptional role of Laf4 during early brain development and reveals a novel function for the gene in the process of cortical cell migration relevant to the haploinsufficiency and silencing observed in human neurodevelopmental disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105933PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146563PMC
November 2015

Changes in gene expression associated with FTO overexpression in mice.

PLoS One 2014 19;9(5):e97162. Epub 2014 May 19.

Henry Wellcome Centre for Gene Function, Department of Physiology, Anatomy; and Genetics, University of Oxford, Parks Road, Oxford, United Kingdom.

Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0097162PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4026227PMC
December 2014

The long non-coding RNA Paupar regulates the expression of both local and distal genes.

EMBO J 2014 Feb 1;33(4):296-311. Epub 2014 Feb 1.

MRC Functional Genomics Unit, University of Oxford, Oxford UK.

Although some long noncoding RNAs (lncRNAs) have been shown to regulate gene expression in cis, it remains unclear whether lncRNAs can directly regulate transcription in trans by interacting with chromatin genome-wide independently of their sites of synthesis. Here, we describe the genomically local and more distal functions of Paupar, a vertebrate-conserved and central nervous system-expressed lncRNA transcribed from a locus upstream of the gene encoding the PAX6 transcription factor. Knockdown of Paupar disrupts the normal cell cycle profile of neuroblastoma cells and induces neural differentiation. Paupar acts in a transcript-dependent manner both locally, to regulate Pax6, as well as distally by binding and regulating genes on multiple chromosomes, in part through physical association with PAX6 protein. Paupar binding sites are enriched near promoters and can function as transcriptional regulatory elements whose activity is modulated by Paupar transcript levels. Our findings demonstrate that a lncRNA can function in trans at transcriptional regulatory elements distinct from its site of synthesis to control large-scale transcriptional programmes.
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http://dx.doi.org/10.1002/embj.201386225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983687PMC
February 2014

Expression profiling of mouse subplate reveals a dynamic gene network and disease association with autism and schizophrenia.

Proc Natl Acad Sci U S A 2013 Feb 11;110(9):3555-60. Epub 2013 Feb 11.

Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom.

The subplate zone is a highly dynamic transient sector of the developing cerebral cortex that contains some of the earliest generated neurons and the first functional synapses of the cerebral cortex. Subplate cells have important functions in early establishment and maturation of thalamocortical connections, as well as in the development of inhibitory cortical circuits in sensory areas. So far no role has been identified for cells in the subplate in the mature brain and disease association of the subplate-specific genes has not been analyzed systematically. Here we present gene expression evidence for distinct roles of the mouse subplate across development as well as unique molecular markers to extend the repertoire of subplate labels. Performing systematic comparisons between different ages (embryonic days 15 and 18, postnatal day 8, and adult), we reveal the dynamic and constant features of the markers labeling subplate cells during embryonic and early postnatal development and in the adult. This can be visualized using the online database of subplate gene expression at https://molnar.dpag.ox.ac.uk/subplate/. We also identify embryonic similarities in gene expression between the ventricular zones, intermediate zone, and subplate, and distinct postnatal similarities between subplate, layer 5, and layers 2/3. The genes expressed in a subplate-specific manner at some point during development show a statistically significant enrichment for association with autism spectrum disorders and schizophrenia. Our report emphasizes the importance of the study of transient features of the developing brain to better understand neurodevelopmental disorders.
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http://dx.doi.org/10.1073/pnas.1218510110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587197PMC
February 2013

KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands.

Elife 2012 Dec 18;1:e00205. Epub 2012 Dec 18.

Department of Biochemistry , University of Oxford , Oxford , UK.

CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs.DOI:http://dx.doi.org/10.7554/eLife.00205.001.
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http://dx.doi.org/10.7554/eLife.00205DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3524939PMC
December 2012

Evidence for conserved post-transcriptional roles of unitary pseudogenes and for frequent bifunctionality of mRNAs.

Genome Biol 2012 Nov 15;13(11):R102. Epub 2012 Nov 15.

Background: Recent reports have highlighted instances of mRNAs that, in addition to coding for protein, regulate the abundance of related transcripts by altering microRNA availability. These two mRNA roles - one mediated by RNA and the other by protein - are inter-dependent and hence cannot easily be separated. Whether the RNA-mediated role of transcripts is important, per se, or whether it is a relatively innocuous consequence of competition by different transcripts for microRNA binding remains unknown.

Results: Here we took advantage of 48 loci that encoded proteins in the earliest eutherian ancestor, but whose protein-coding capability has since been lost specifically during rodent evolution. Sixty-five percent of such loci, which we term 'unitary pseudogenes', have retained their expression in mouse and their transcripts exhibit conserved tissue expression profiles. The maintenance of these unitary pseudogenes' spatial expression profiles is associated with conservation of their microRNA response elements and these appear to preserve the post-transcriptional roles of their protein-coding ancestor. We used mouse Pbcas4, an exemplar of these transcribed unitary pseudogenes, to experimentally test our genome-wide predictions. We demonstrate that the role of Pbcas4 as a competitive endogenous RNA has been conserved and has outlived its ancestral gene's loss of protein-coding potential.

Conclusions: These results show that post-transcriptional regulation by bifunctional mRNAs can persist over long evolutionary time periods even after their protein coding ability has been lost.
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http://dx.doi.org/10.1186/gb-2012-13-11-r102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580494PMC
November 2012

Disrupted circadian rhythms in a mouse model of schizophrenia.

Curr Biol 2012 Feb 19;22(4):314-9. Epub 2012 Jan 19.

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3PT, UK.

Sleep and circadian rhythm disruption has been widely observed in neuropsychiatric disorders including schizophrenia [1] and often precedes related symptoms [2]. However, mechanistic basis for this association remains unknown. Therefore, we investigated the circadian phenotype of blind-drunk (Bdr), a mouse model of synaptosomal-associated protein (Snap)-25 exocytotic disruption that displays schizophrenic endophenotypes modulated by prenatal factors and reversible by antipsychotic treatment [3, 4]. Notably, SNAP-25 has been implicated in schizophrenia from genetic [5-8], pathological [9-13], and functional studies [14-16]. We show here that the rest and activity rhythms of Bdr mice are phase advanced and fragmented under a light/dark cycle, reminiscent of the disturbed sleep patterns observed in schizophrenia. Retinal inputs appear normal in mutants, and clock gene rhythms within the suprachiasmatic nucleus (SCN) are normally phased both in vitro and in vivo. However, the 24 hr rhythms of arginine vasopressin within the SCN and plasma corticosterone are both markedly phase advanced in Bdr mice. We suggest that the Bdr circadian phenotype arises from a disruption of synaptic connectivity within the SCN that alters critical output signals. Collectively, our data provide a link between disruption of circadian activity cycles and synaptic dysfunction in a model of neuropsychiatric disease.
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http://dx.doi.org/10.1016/j.cub.2011.12.051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356578PMC
February 2012

FTO is expressed in neurones throughout the brain and its expression is unaltered by fasting.

PLoS One 2011 30;6(11):e27968. Epub 2011 Nov 30.

Department of Physiology, Anatomy and Genetics, OXION Centre for Ion Channel Studies, Henry Wellcome Centre for Gene Function, Oxford, United Kingdom.

Single-nucleotide polymorphisms in the first intron of the ubiquitously expressed FTO gene are associated with obesity. Although the physiological functions of FTO remain unclear, food intake is often altered when Fto expression levels are manipulated. Furthermore, deletion of FTO from neurones alone has a similar effect on food intake to deletion of FTO in all tissues. These results indicate that FTO expression in the brain is particularly important. Considerable focus has been placed on the dynamic regulation of Fto mRNA expression in the hypothalamus after short-term (16-48 hour) fasting, but results have been controversial. There are no studies that quantify FTO protein levels across the brain, and assess its alteration following short-term fasting. Using immunohistochemistry, we found that FTO protein is widely expressed in mouse brain, and present in the majority of neurones. Using quantitative Western blotting and RT-qPCR we show that FTO protein and mRNA levels in the hypothalamus, cerebellum and rostral brain are relatively uniform, and levels in the brain are higher than in skeletal muscles of the lower limbs. Fasting for 18 hours does not alter the expression pattern, or levels, of FTO protein and mRNA. We further show that the majority of POMC neurones, which are critically involved in food intake regulation, also express FTO, but that the percentage of FTO-positive POMC neurones is not altered by fasting. In summary, we find no evidence that Fto/FTO expression is regulated by short-term (18-hour) fasting. Thus, it is unlikely that the hunger and increased post-fasting food intake caused by such food deprivation is driven by alterations in Fto/FTO expression. The widespread expression of FTO in neurones also suggests that physiological studies of this protein should not be limited to the hypothalamus.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027968PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227617PMC
April 2012

Gene expression analysis of the embryonic subplate.

Cereb Cortex 2012 Jun 22;22(6):1343-59. Epub 2011 Aug 22.

Department of Physiology, Anatomy and Genetics, Oxford University, Oxford OX1 3QX, UK.

The subplate layer of the cerebral cortex is comprised of a heterogeneous population of cells and contains some of the earliest-generated neurons. In the embryonic brain, subplate cells contribute to the guidance and areal targeting of thalamocortical axons. At later developmental stages, they are predominantly involved in the maturation and plasticity of the cortical circuitry and the establishment of functional modules. We aimed to further characterize the embryonic murine subplate population by establishing a gene expression profile at embryonic day (E) 15.5 using laser capture microdissection and microarrays. The microarray identified over 300 transcripts with higher expression in the subplate compared with the cortical plate at this stage. Using quantitative reverse transcription-polymerase chain reaction, in situ hybridization (ISH), and immunohistochemistry (IHC), we have confirmed specific expression in the E15.5 subplate for 13 selected genes, which have not been previously associated with this compartment (Abca8a, Cdh10, Cdh18, Csmd3, Gabra5, Kcnt2, Ogfrl1, Pls3, Rcan2, Sv2b, Slc8a2, Unc5c, and Zdhhc2). In the reeler mutant, the expression of the majority of these genes (9 of 13) was shifted in accordance with the altered position of subplate. These genes belong to several functional groups and likely contribute to synapse formation and axonal growth and guidance in subplate cells.
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http://dx.doi.org/10.1093/cercor/bhr197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4972418PMC
June 2012

Transcriptome sequencing, microarray, and proteomic analyses reveal cellular and metabolic impact of hepatitis C virus infection in vitro.

Hepatology 2010 Aug;52(2):443-53

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK.

Unlabelled: Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels.

Conclusion: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus-host interaction which may be highly relevant to pathogenesis in vivo.
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http://dx.doi.org/10.1002/hep.23733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427885PMC
August 2010

Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy.

PLoS Genet 2009 Dec 18;5(12):e1000773. Epub 2009 Dec 18.

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT-PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival.
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http://dx.doi.org/10.1371/journal.pgen.1000773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787017PMC
December 2009

AF4 is a critical regulator of the IGF-1 signaling pathway during Purkinje cell development.

J Neurosci 2009 Dec;29(49):15366-74

Medical Research Council Functional Genomics Unit, and Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, United Kingdom.

Deregulation of the insulin-like growth factor 1 (IGF-1) signaling pathway is a recurrent finding in mouse models and human patients with cerebellar ataxia and thus represents a common pathological cascade in neuronal cell death that may be targeted for therapy. We have previously identified a point mutation in AF4, a transcription cofactor of RNA polymerase II elongation and chromatin remodeling, that causes progressive and highly specific Purkinje cell (PC) death in the ataxic mouse mutant robotic, leading to the accumulation of AF4 in PCs. Here we confirm that the spatiotemporal pattern of PC degeneration in the robotic cerebellum correlates with the specific profile of AF4 upregulation. To identify the underlying molecular pathways, we performed microarray gene expression analysis of PCs obtained by laser capture microdissection (LCM) at the onset of degeneration. Igf-1 was significantly downregulated in robotic PCs compared with wild-type controls before and throughout the degenerative process. Consistently, we observed a decrease in the activation of downstream signaling molecules including type 1 IGF receptor (IGF-1R) and the extracellular signal-regulated kinase (ERK) 1 and ERK2. Chromatin immunoprecipitation confirmed that Igf-1 is a direct and the first validated target of the AF4 transcriptional regulatory complex, and treatment of presymptomatic robotic mice with IGF-1 indeed markedly delayed the progression of PC death. This study demonstrates that small changes in the levels of a single transcriptional cofactor can deleteriously affect normal cerebellum function and opens new avenues of research for the manipulation of the IGF-1 pathway in the treatment of cerebellar ataxia in humans.
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http://dx.doi.org/10.1523/JNEUROSCI.5188-09.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6666107PMC
December 2009

Pre-symptomatic development of lower motor neuron connectivity in a mouse model of severe spinal muscular atrophy.

Hum Mol Genet 2010 Feb 2;19(3):420-33. Epub 2009 Nov 2.

Centre for Integrative Physiology and Euan MacDonald Centre for Motor Neuron Disease Research, University of Edinburgh Medical School, Edinburgh, UK.

The childhood motor neuron disease spinal muscular atrophy (SMA) results from reduced expression of the survival motor neuron (SMN) gene. Previous studies using in vitro model systems and lower organisms have suggested that low levels of Smn protein disrupt prenatal developmental processes in lower motor neurons, influencing neuronal outgrowth, axon branching and neuromuscular connectivity. The extent to which these developmental pathways contribute to selective vulnerability and pathology in the mammalian neuromuscular system in vivo remains unclear. Here, we have investigated the pre-symptomatic development of neuromuscular connectivity in differentially vulnerable motor neuron populations in Smn(-/-);SMN2 mice, a model of severe SMA. We show that reduced Smn levels have no detectable effect on morphological correlates of pre-symptomatic development in either vulnerable or stable motor units, indicating that abnormal pre-symptomatic developmental processes are unlikely to be a prerequisite for subsequent pathological changes to occur in vivo. Microarray analyses of spinal cord from two different severe SMA mouse models demonstrated that only minimal changes in gene expression were present in pre-symptomatic mice. In stark contrast, microarray analysis of late-symptomatic spinal cord revealed widespread changes in gene expression, implicating extracellular matrix integrity, growth factor signalling and myelination pathways in SMA pathogenesis. Taken together, these data suggest that reduced Smn levels induce SMA pathology by instigating rapidly progressive neurodegenerative pathways in lower motor neurons around the time of disease onset rather than by modulating pre-symptomatic neurodevelopmental pathways.
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http://dx.doi.org/10.1093/hmg/ddp506DOI Listing
February 2010

A mouse model for the metabolic effects of the human fat mass and obesity associated FTO gene.

PLoS Genet 2009 Aug 14;5(8):e1000599. Epub 2009 Aug 14.

MRC Harwell, Metabolism and Inflammation, Harwell Science and Innovation Campus, Harwell, UK.

Human FTO gene variants are associated with body mass index and type 2 diabetes. Because the obesity-associated SNPs are intronic, it is unclear whether changes in FTO expression or splicing are the cause of obesity or if regulatory elements within intron 1 influence upstream or downstream genes. We tested the idea that FTO itself is involved in obesity. We show that a dominant point mutation in the mouse Fto gene results in reduced fat mass, increased energy expenditure, and unchanged physical activity. Exposure to a high-fat diet enhances lean mass and lowers fat mass relative to control mice. Biochemical studies suggest the mutation occurs in a structurally novel domain and modifies FTO function, possibly by altering its dimerisation state. Gene expression profiling revealed increased expression of some fat and carbohydrate metabolism genes and an improved inflammatory profile in white adipose tissue of mutant mice. These data provide direct functional evidence that FTO is a causal gene underlying obesity. Compared to the reported mouse FTO knockout, our model more accurately reflects the effect of human FTO variants; we observe a heterozygous as well as homozygous phenotype, a smaller difference in weight and adiposity, and our mice do not show perinatal lethality or an age-related reduction in size and length. Our model suggests that a search for human coding mutations in FTO may be informative and that inhibition of FTO activity is a possible target for the treatment of morbid obesity.
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http://dx.doi.org/10.1371/journal.pgen.1000599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2719869PMC
August 2009

High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection.

BMC Mol Biol 2009 Jul 6;10:69. Epub 2009 Jul 6.

Department of Physiology, Anatomy and Genetics, University of Oxford, Le Gros Clark Building, Oxford, UK.

Background: Laser capture microdissection enables the isolation of single cells or small cell groups from histological sections under direct microscopic observation. Combined with quantitative PCR or microarray, it is a very powerful approach for studying gene expression profiles in discrete cell populations. The major challenge for such studies is to obtain good quality RNA from small amounts of starting material.

Results: We have developed a simple, flexible, and low-cost method for simultaneously producing RNA from discrete cell groups in embryonic day 15 mouse brain. In particular, we have optimized the following key steps in the procedure: staining, cryosectioning, storage of sections and harvesting of microdissected cells. We obtained the best results when staining 20 mum-thick sections with 1% cresyl violet in 70% ethanol and harvesting the microdissected tissue in RNA stabilization solution. In addition, we introduced three stop-points in the protocol which makes the tedious process of laser capture microdissection more flexible, without compromising RNA quality.

Conclusion: Using this optimized method, we have consistently obtained RNA of high quality from all four simultaneously microdissected cell groups. RNA integrity numbers were all above 8, and long cDNA fragments (> 1.2 kb) were successfully amplified by reverse transcription PCR from all four samples. We conclude that RNAs isolated by this method are well suited for downstream quantitative PCR or microarray studies.
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http://dx.doi.org/10.1186/1471-2199-10-69DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2713242PMC
July 2009

Novel markers reveal subpopulations of subplate neurons in the murine cerebral cortex.

Cereb Cortex 2009 Aug 13;19(8):1738-50. Epub 2008 Nov 13.

Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.

The subplate lays the foundation of the developing cerebral cortex, and abnormalities have been suggested to contribute to various brain developmental disorders. The causal relationship between cellular pathologies and cognitive disorders remains unclear, and therefore, a better understanding of the role of subplate cells in cortical development is essential. Only by determining the molecular taxonomy of this diverse class of neurons can we identify the subpopulations that may contribute differentially to cortical development. We identified novel markers for murine subplate cells by comparing gene expression of subplate and layer 6 of primary visual and somatosensory cortical areas of postnatal day (P)8 old mice using a microarray-based approach. We examined the utility of these markers in well-characterized mutants (reeler, scrambler, and p35-KO) where the subplate is displaced in relation to the cortical plate. In situ hybridization or immunohistochemistry confirmed subplate-selective expression of complexin 3, connective tissue growth factor, nuclear receptor-related 1/Nr4a2, and monooxygenase Dbh-like 1 while transmembrane protein 163 also had additional expression in layer 5, and DOPA decarboxylase was also present in the white matter. Localization of marker-positive cells in the reeler and p35-KO cortices suggests different subpopulations of subplate cells. These new markers open up possibilities for further identification of subplate subpopulations in research and in neuropathological diagnosis.
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http://dx.doi.org/10.1093/cercor/bhn195DOI Listing
August 2009

Genes involved in the formation of the earliest cortical circuits.

Novartis Found Symp 2007 ;288:212-24; discussion 224-9, 276-81

Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.

Building the brain is like erecting a house of cards. The early connections provide the foundation of the adult structure, and disruption of these may be the source of many developmental flaws. Cerebral cortical developmental disorders (including schizophrenia and autism) and perinatal injuries involve cortical neurons with early connectivity. The major hindrance of progress in understanding the early neural circuits during cortical development and disease has been the lack of reliable markers for specific cell populations. Due to the advance of powerful approaches in gene expression analysis and the utility of models with reporter gene expressions in specific cortical cell types, our knowledge of the early cortical circuits is rapidly increasing. With focus on the sub-plate, layer VI and layer V projection neurons, we shall illustrate the progress made in the understanding of their neurochemical properties, physiological characteristics and their integration into the early intracortical and extracortical circuitry. This field benefited from recent developments in mouse genetics in generating models with subtype specific gene expression patterns, powerful cell dissection and separation methods combined with microarray analysis. The emergence of cortical cell type specific biomarkers will not only help neuropathological diagnosis, but will also eventually reveal the causal relations in the pathogenesis of various cortical developmental disorders.
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July 2008

Genome-wide expression analysis of a spinal muscular atrophy model: towards discovery of new drug targets.

PLoS One 2008 Jan 2;3(1):e1404. Epub 2008 Jan 2.

OXION Initiative, Oxford Centre for Gene Function, Department of Human Anatomy and Genetics, University of Oxford, Oxford, United Kingdom.

Spinal Muscular Atrophy is a recessive genetic disease and affects lower motor neurones and muscle tissue. A single gene is disrupted in SMA: SMN1 activity is abolished but a second copy of the gene (SMN2) provides limited activity. While the SMN protein has been shown to function in the assembly of RNA-protein complexes, it is unclear how the overall reduction in SMN activity specifically results in the neuromuscular phenotypes. Similar to humans, reduced smn activity in the fly causes earliest phenotypes in neuromuscular tissues. To uncover the effects of reduced SMN activity, we have studied gene expression in control and diseased fly tissues using whole genome micro-arrays. A number of gene expression changes are recovered and independently validated. Identified genes show trends in their predicted function: several are consistent with the function of SMN, in addition some uncover novel pathways. This and subsequent genetic analysis in the fly indicates some of the identified genes could be taken for further studies as potential drug targets for SMA and other neuromuscular disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0001404PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2151137PMC
January 2008

The integration profile of EIAV-based vectors.

Mol Ther 2006 Oct;14(4):536-45

Oxford BioMedica UK Ltd., Medawar Centre, The Oxford Science Park, Oxford OX4 4GA, UK.

Lentiviral vectors based on equine infectious anemia virus (EIAV) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. The integration profile of an EIAV vector was analyzed in dividing HEK293T cells, alongside an HIV-1 vector as a control, and compared to a random dataset generated in silico. A multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a) within/not within a gene, (b) GC content within 20 kb, (c) within 10 kb of a CpG island, (d) gene density within a 2-Mb window, and (e) chromosome number. The majority of the EIAV integration sites (68%; n = 458) and HIV-1 integration sites (72%; n = 162) were within a gene, and both vectors favored AT-rich regions. Sites within genes were examined using a second model to determine the influence of the gene-specific parameters, gene region, and transcriptional activity. Both EIAV and HIV-1 vectors preferentially integrated within active genes. Unlike the gammaretrovirus MLV, EIAV and HIV-1 vectors do not integrate preferentially into the promoter region or the 5' end of the transcription unit.
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http://dx.doi.org/10.1016/j.ymthe.2006.06.006DOI Listing
October 2006

Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis.

Genomics 2004 Jan;83(1):1-8

Biological Systems Group, Oxford BioMedica Ltd., OX4 4GA, Oxford, United Kingdom.

The cellular response to hypoxia involves the promotion of angiogenesis, leading to increased blood flow and oxygenation. The macrophage has been identified as an orchestrator of this response in several pathologies, through the release of angiogenic factors in response to hypoxia. We have produced the first comprehensive transcriptome analysis of hypoxic primary human macrophages with respect to the regulation of angiogenesis. There is a marked induction of genes encoding factors known to stimulate angiogenesis, rather than factors that inhibit this process. We show that overexpression of the transcription factor EPAS1 using a recombinant adenoviral vector amplifies the induction of genes encoding angiogenic proteins in response to hypoxia. This defines a new strategy for enhancing transcriptome and proteome analyses by overexpressing disease-implicated genes using viral gene transfer methodologies.
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http://dx.doi.org/10.1016/s0888-7543(03)00215-5DOI Listing
January 2004

Kinetic Studies of the Reactions of Pentacyanoferrate(II) Complexes with Peroxydisulfate.

Inorg Chem 1996 Apr;35(9):2627-2629

Department of Chemistry, Tunghai Christian University, Taichung, Taiwan, Republic of China.

Reactions of Fe(CN)(5)L(3-) (L = 4-aminopyridine (4-ampy), pyridine (py), 4,4'-bipyridine (4,4'-bpy), and pyrazine (pz)) with peroxydisulfate, Fe(CN)(5)L(3-) + S(2)O(8)(2-) right harpoon over left harpoon Fe(CN)(5)L(2-) + SO(4)(-) + SO(4)(2-), have been found to follow an outer-sphere electron transfer mechanism. The specific rate constants of oxidation are 1.45 +/- 0.01, (9.00 +/- 0.02) x 10(-2), (5.60 +/- 0.01) x 10(-2), and (2.89 +/- 0.01) x 10(-2) M(-1) s(-1), for L = 4-ampy, py, 4,4'-bpy, and pz, respectively, at &mgr; = 0.50 M LiClO(4), T = 25 degrees C, pH = 4.4-8.8. The rate constants of oxidation for the corresponding Ru(NH(3))(5)L(2+) complexes were also measured and were found to be faster than those of Fe(CN)(5)L(3-) complexes by a factor of approximately 10(2) even after the corrections for the differences in reduction potentials and in the charges of the complexes. The difference in reactivity may arise from the hydrogen bonding between peroxydisulfate and the ammonia ligands of Ru(NH(3))(5)L(2+) and nonadiabaticity observed in the Fe(CN)(5)L(3-) complexes.
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http://dx.doi.org/10.1021/ic9505920DOI Listing
April 1996