Publications by authors named "Olivia Sheppard"

15 Publications

  • Page 1 of 1

Beta secretase 1-dependent amyloid precursor protein processing promotes excessive vascular sprouting through NOTCH3 signalling.

Cell Death Dis 2020 02 6;11(2):98. Epub 2020 Feb 6.

John van Geest Centre for Brain Repair, Forvie Site, Robinson Way, Cambridge, CB2 0PY, UK.

Amyloid beta peptides (Aβ) proteins play a key role in vascular pathology in Alzheimer's Disease (AD) including impairment of the blood-brain barrier and aberrant angiogenesis. Although previous work has demonstrated a pro-angiogenic role of Aβ, the exact mechanisms by which amyloid precursor protein (APP) processing and endothelial angiogenic signalling cascades interact in AD remain a largely unsolved problem. Here, we report that increased endothelial sprouting in human-APP transgenic mouse (TgCRND8) tissue is dependent on β-secretase (BACE1) processing of APP. Higher levels of Aβ processing in TgCRND8 tissue coincides with decreased NOTCH3/JAG1 signalling, overproduction of endothelial filopodia and increased numbers of vascular pericytes. Using a novel in vitro approach to study sprouting angiogenesis in TgCRND8 organotypic brain slice cultures (OBSCs), we find that BACE1 inhibition normalises excessive endothelial filopodia formation and restores NOTCH3 signalling. These data present the first evidence for the potential of BACE1 inhibition as an effective therapeutic target for aberrant angiogenesis in AD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-020-2288-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005019PMC
February 2020

Optically Robust and Biocompatible Mechanosensitive Upconverting Nanoparticles.

ACS Cent Sci 2019 Jul 9;5(7):1211-1222. Epub 2019 Jul 9.

Department of Materials Science and Engineering, Stanford University, Stanford, California 94305, United States.

Upconverting nanoparticles (UCNPs) are promising tools for background-free imaging and sensing. However, their usefulness for applications depends on their biocompatibility, which we define by their optical performance in biological environments and their toxicity in living organisms. For UCNPs with a ratiometric color response to mechanical stress, consistent emission intensity and color are desired for the particles under nonmechanical stimuli. Here, we test the biocompatibility and mechanosensitivity of α-NaYF:Yb,Er@NaLuF nanoparticles. First, we ligand-strip these particles to render them dispersible in aqueous media. Then, we characterize their mechanosensitivity (∼30% in the red-to-green spectral ratio per GPa), which is nearly 3-fold greater than those coated in oleic acid. We next design a suite of and tests to investigate their structural and optical properties under several biorelevant conditions: over time in various buffers types, as a function of pH, and along the digestive tract of worms. Finally, to ensure that the particles do not perturb biological function in , we assess the chronic toxicity of nanoparticle ingestion using a reproductive brood assay. In these ways, we determine that mechanosensitive UCNPs are biocompatible, i.e., optically robust and nontoxic, for use as sensors to study animal digestion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscentsci.9b00300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6661856PMC
July 2019

Lipopolysaccharide-induced neuroinflammation induces presynaptic disruption through a direct action on brain tissue involving microglia-derived interleukin 1 beta.

J Neuroinflammation 2019 May 18;16(1):106. Epub 2019 May 18.

John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, E.D Adrian Building, Forvie Site, Robinson Way, Cambridge, CB2 0PY, UK.

Background: Systemic inflammation has been linked to synapse loss and cognitive decline in human patients and animal models. A role for microglial release of pro-inflammatory cytokines has been proposed based on in vivo and primary culture studies. However, mechanisms are hard to study in vivo as specific microglial ablation is challenging and the extracellular fluid cannot be sampled without invasive methods. Primary cultures have different limitations as the intricate multicellular architecture in the brain is not fully reproduced. It is essential to confirm proposed brain-specific mechanisms of inflammatory synapse loss directly in brain tissue. Organotypic hippocampal slice cultures (OHSCs) retain much of the in vivo neuronal architecture, synaptic connections and diversity of cell types whilst providing convenient access to manipulate and sample the culture medium and observe cellular reactions.

Methods: OHSCs were generated from P6-P9 C57BL/6 mice. Inflammation was induced via addition of lipopolysaccharide (LPS), and cultures were analysed for changes in synaptic proteins, gene expression and protein secretion. Microglia were selectively depleted using clodronate, and the effect of IL1β was assessed using a specific neutralising monoclonal antibody.

Results: LPS treatment induced loss of the presynaptic protein synaptophysin without altering PSD95 or Aβ protein levels. Depletion of microglia prior to LPS application prevented the loss of synaptophysin, whilst microglia depletion after the inflammatory insult was partially effective, although less so than pre-emptive treatment, indicating a time-critical window in which microglia can induce synaptic damage. IL1β protein and mRNA were increased after LPS addition, with these effects also prevented by microglia depletion. Direct application of IL1β to OHSCs resulted in synaptophysin loss whilst pre-treatment with IL1β neutralising antibody prior to LPS addition prevented a significant loss of synaptophysin but may also impact basal synaptic levels.

Conclusions: The loss of synaptophysin in this system confirms LPS can act directly within brain tissue to disrupt synapses, and we show that microglia are the relevant cellular target when all major CNS cell types are present. By overcoming limitations of primary culture and in vivo work, our study strengthens the evidence for a key role of microglia-derived IL1β in synaptic dysfunction after inflammatory insult.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12974-019-1490-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6525970PMC
May 2019

Contrasting requirements during disease evolution identify EZH2 as a therapeutic target in AML.

J Exp Med 2019 04 19;216(4):966-981. Epub 2019 Mar 19.

Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute, Cambridge, UK

Epigenetic regulators, such as EZH2, are frequently mutated in cancer, and loss-of-function mutations are common in myeloid malignancies. We have examined the importance of cellular context for Ezh2 loss during the evolution of acute myeloid leukemia (AML), where we observed stage-specific and diametrically opposite functions for Ezh2 at the early and late stages of disease. During disease maintenance, WT Ezh2 exerts an oncogenic function that may be therapeutically targeted. In contrast, Ezh2 acts as a tumor suppressor during AML induction. Transcriptional analysis explains this apparent paradox, demonstrating that loss of derepresses different expression programs during disease induction and maintenance. During disease induction, loss derepresses a subset of bivalent promoters that resolve toward gene activation, inducing a feto-oncogenic program that includes genes such as , whose overexpression phenocopies loss to accelerate AML induction in mouse models. Our data highlight the importance of cellular context and disease phase for the function of Ezh2 and its potential therapeutic implications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20181276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446874PMC
April 2019

Trisomy of human chromosome 21 enhances amyloid-β deposition independently of an extra copy of APP.

Brain 2018 08;141(8):2457-2474

Department of Neurodegenerative Disease, UCL Institute of Neurology, London, WC1N 3BG UK.

Down syndrome, caused by trisomy of chromosome 21, is the single most common risk factor for early-onset Alzheimer's disease. Worldwide approximately 6 million people have Down syndrome, and all these individuals will develop the hallmark amyloid plaques and neurofibrillary tangles of Alzheimer's disease by the age of 40 and the vast majority will go on to develop dementia. Triplication of APP, a gene on chromosome 21, is sufficient to cause early-onset Alzheimer's disease in the absence of Down syndrome. However, whether triplication of other chromosome 21 genes influences disease pathogenesis in the context of Down syndrome is unclear. Here we show, in a mouse model, that triplication of chromosome 21 genes other than APP increases amyloid-β aggregation, deposition of amyloid-β plaques and worsens associated cognitive deficits. This indicates that triplication of chromosome 21 genes other than APP is likely to have an important role to play in Alzheimer's disease pathogenesis in individuals who have Down syndrome. We go on to show that the effect of trisomy of chromosome 21 on amyloid-β aggregation correlates with an unexpected shift in soluble amyloid-β 40/42 ratio. This alteration in amyloid-β isoform ratio occurs independently of a change in the carboxypeptidase activity of the γ-secretase complex, which cleaves the peptide from APP, or the rate of extracellular clearance of amyloid-β. These new mechanistic insights into the role of triplication of genes on chromosome 21, other than APP, in the development of Alzheimer's disease in individuals who have Down syndrome may have implications for the treatment of this common cause of neurodegeneration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/brain/awy159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6061702PMC
August 2018

Early loss of Crebbp confers malignant stem cell properties on lymphoid progenitors.

Nat Cell Biol 2017 Sep 21;19(9):1093-1104. Epub 2017 Aug 21.

Wellcome Trust-MRC Cambridge Stem Cell Institute, Cambridge, UK.

Loss-of-function mutations of cyclic-AMP response element binding protein, binding protein (CREBBP) are prevalent in lymphoid malignancies. However, the tumour suppressor functions of CREBBP remain unclear. We demonstrate that loss of Crebbp in murine haematopoietic stem and progenitor cells (HSPCs) leads to increased development of B-cell lymphomas. This is preceded by accumulation of hyperproliferative lymphoid progenitors with a defective DNA damage response (DDR) due to a failure to acetylate p53. We identify a premalignant lymphoma stem cell population with decreased H3K27ac, which undergoes transcriptional and genetic evolution due to the altered DDR, resulting in lymphomagenesis. Importantly, when Crebbp is lost later in lymphopoiesis, cellular abnormalities are lost and tumour generation is attenuated. We also document that CREBBP mutations may occur in HSPCs from patients with CREBBP-mutated lymphoma. These data suggest that earlier loss of Crebbp is advantageous for lymphoid transformation and inform the cellular origins and subsequent evolution of lymphoid malignancies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ncb3597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633079PMC
September 2017

Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons.

Elife 2016 10 1;5. Epub 2016 Oct 1.

School of Biomedical Sciences, University of Edinburgh, Edinburgh, United Kingdom.

Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.20337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5092045PMC
October 2016

Massively parallel sequencing reveals the complex structure of an irradiated human chromosome on a mouse background in the Tc1 model of Down syndrome.

PLoS One 2013 15;8(4):e60482. Epub 2013 Apr 15.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom.

Down syndrome (DS) is caused by trisomy of chromosome 21 (Hsa21) and presents a complex phenotype that arises from abnormal dosage of genes on this chromosome. However, the individual dosage-sensitive genes underlying each phenotype remain largely unknown. To help dissect genotype--phenotype correlations in this complex syndrome, the first fully transchromosomic mouse model, the Tc1 mouse, which carries a copy of human chromosome 21 was produced in 2005. The Tc1 strain is trisomic for the majority of genes that cause phenotypes associated with DS, and this freely available mouse strain has become used widely to study DS, the effects of gene dosage abnormalities, and the effect on the basic biology of cells when a mouse carries a freely segregating human chromosome. Tc1 mice were created by a process that included irradiation microcell-mediated chromosome transfer of Hsa21 into recipient mouse embryonic stem cells. Here, the combination of next generation sequencing, array-CGH and fluorescence in situ hybridization technologies has enabled us to identify unsuspected rearrangements of Hsa21 in this mouse model; revealing one deletion, six duplications and more than 25 de novo structural rearrangements. Our study is not only essential for informing functional studies of the Tc1 mouse but also (1) presents for the first time a detailed sequence analysis of the effects of gamma radiation on an entire human chromosome, which gives some mechanistic insight into the effects of radiation damage on DNA, and (2) overcomes specific technical difficulties of assaying a human chromosome on a mouse background where highly conserved sequences may confound the analysis. Sequence data generated in this study is deposited in the ENA database, Study Accession number: ERP000439.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060482PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3626651PMC
October 2013

Mouse models of aneuploidy.

ScientificWorldJournal 2012 3;2012:214078. Epub 2012 Jan 3.

Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

Abnormalities of chromosome copy number are called aneuploidies and make up a large health load on the human population. Many aneuploidies are lethal because the resulting abnormal gene dosage is highly deleterious. Nevertheless, some whole chromosome aneuploidies can lead to live births. Alterations in the copy number of sections of chromosomes, which are also known as segmental aneuploidies, are also associated with deleterious effects. Here we examine how aneuploidy of whole chromosomes and segmental aneuploidy of chromosomal regions are modeled in the mouse. These models provide a whole animal system in which we aim to investigate the complex phenotype-genotype interactions that arise from alteration in the copy number of genes. Although our understanding of this subject is still in its infancy, already research in mouse models is highlighting possible therapies that might help alleviate the cognitive effects associated with changes in gene number. Thus, creating and studying mouse models of aneuploidy and copy number variation is important for understanding what it is to be human, in both the normal and genomically altered states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1100/2012/214078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3259538PMC
June 2012

Altered regulation of tau phosphorylation in a mouse model of down syndrome aging.

Neurobiol Aging 2012 Apr 16;33(4):828.e31-44. Epub 2011 Aug 16.

University College London Institute of Neurology, London, UK.

Down syndrome (DS) results from trisomy of human chromosome 21 (Hsa21) and is associated with an increased risk of Alzheimer's disease (AD). Here, using the unique transchromosomic Tc1 mouse model of DS we investigate the influence of trisomy of Hsa21 on the protein tau, which is hyperphosphorylated in Alzheimer's disease. We show that in old, but not young, Tc1 mice increased phosphorylation of tau occurs at a site suggested to be targeted by the Hsa21 encoded kinase, dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A). We show that DYRK1A is upregulated in young and old Tc1 mice, but that young trisomic mice may be protected from accumulating aberrantly phosphorylated tau. We observe that the key tau kinase, glycogen synthase kinase3-β (GSK-3β) is aberrantly phosphorylated at an inhibitory site in the aged Tc1 brain which may reduce total glycogen synthase kinase3-β activity. It is possible that a similar mechanism may also occur in people with DS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neurobiolaging.2011.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314962PMC
April 2012

Generation of a panel of antibodies against proteins encoded on human chromosome 21.

J Negat Results Biomed 2010 Aug 20;9. Epub 2010 Aug 20.

Department of Neurodegenerative Disease, UCL Institute of Neurology, London, UK.

Background: Down syndrome (DS) is caused by trisomy of all or part of chromosome 21. To further understanding of DS we are working with a mouse model, the Tc1 mouse, which carries most of human chromosome 21 in addition to the normal mouse chromosome complement. This mouse is a model for human DS and recapitulates many of the features of the human syndrome such as specific heart defects, and cerebellar neuronal loss. The Tc1 mouse is mosaic for the human chromosome such that not all cells in the model carry it. Thus to help our investigations we aimed to develop a method to identify cells that carry human chromosome 21 in the Tc1 mouse. To this end, we have generated a panel of antibodies raised against proteins encoded by genes on human chromosome 21 that are known to be expressed in the adult brain of Tc1 mice

Results: We attempted to generate human specific antibodies against proteins encoded by human chromosome 21. We selected proteins that are expressed in the adult brain of Tc1 mice and contain regions of moderate/low homology with the mouse ortholog. We produced antibodies to seven human chromosome 21 encoded proteins. Of these, we successfully generated three antibodies that preferentially recognise human compared with mouse SOD1 and RRP1 proteins on western blots. However, these antibodies did not specifically label cells which carry a freely segregating copy of Hsa21 in the brains of our Tc1 mouse model of DS.

Conclusions: Although we have successfully isolated new antibodies to SOD1 and RRP1 for use on western blots, in our hands these antibodies have not been successfully used for immunohistochemistry studies. These antibodies are freely available to other researchers. Our data high-light the technical difficulty of producing species-specific antibodies for both western blotting and immunohistochemistry.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/1477-5751-9-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2936279PMC
August 2010

Down syndrome and the molecular pathogenesis resulting from trisomy of human chromosome 21.

J Biomed Res 2010 Mar;24(2):87-99

Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK.

Chromosome copy number aberrations, anueploidies, are common in the human population but generally lethal. However, trisomy of human chromosome 21 is compatible with life and people born with this form of aneuploidy manifest the features of Down syndrome, named after Langdon Down who was a 19(th) century British physician who first described a group of people with this disorder. Down syndrome includes learning and memory deficits in all cases, as well as many other features which vary in penetrance and expressivity in different people. While Down syndrome clearly has a genetic cause - the extra dose of genes on chromosome 21 - we do not know which genes are important for which aspects of the syndrome, which biochemical pathways are disrupted, or, generally how design therapies to ameliorate the effects of these disruptions. Recently, with new insights gained from studying mouse models of Down syndrome, specific genes and pathways are being shown to be involved in the pathogenesis of the disorder. This is opening the way for exciting new studies of potential therapeutics for aspects of Down syndrome, particularly the learning and memory deficits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/S1674-8301(10)60016-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3596542PMC
March 2010

Positive regulation of the NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F.

Free Radic Biol Med 2008 Sep 29;45(5):679-85. Epub 2008 May 29.

Cardiovascular Division, King's College London British Heart Foundation Centre, James Black Centre, 125 Coldharbour Lane, London SE5 9NU, UK.

The generation of reactive oxygen species (ROS) by the NOX family of NADPH oxidases is known to be involved in the regulation of many physiological cellular functions. Unlike other members of this family, NOX4 generates ROS constitutively without the need for a stimulus. The activity of NOX4 is known to be regulated, at least in part, at the level of mRNA expression. However, nothing is known of the molecular mechanisms which underlie its transcriptional regulation. We have therefore determined the transcriptional initiation site of NOX4 in vascular smooth muscle cells (VSMC) and identified NOX4 genomic sequences necessary to effect high levels of expression of a linked luciferase reporter gene in both rat and mouse VSMCs. A potential binding site for members of the E2F family of transcription factors was identified, and electrophoretic mobility-shift assays (EMSA) and chromatin immunoprecipitation (ChIP) assays confirmed that this site binds E2F1 both in vitro and in vivo. siRNA against E2F1 decreased NOX4 promoter activity, while site-specific mutation of the core-binding site both downregulated the NOX4 promoter and abolished transregulation by E2F1. These data therefore demonstrate that E2F factor(s) are positive regulators of NOX4 transcription in VSMCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.freeradbiomed.2008.05.019DOI Listing
September 2008

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons.

J Neurochem 2007 Jun 7;101(6):1596-606. Epub 2007 Feb 7.

Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, CA, USA.

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mumol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3'-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3'-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1471-4159.2006.04434.xDOI Listing
June 2007