Targeted ablation of oligodendrocytes induces axonal pathology independent of overt demyelination

The critical role of oligodendrocytes in producing and maintaining myelin that supports rapid axonal conduction in CNS neurons is well established. More recently, additional roles for oligodendrocytes have been posited, including provision of trophic factors and metabolic support for neurons. To investigate the functional consequences of oligodendrocyte loss, we have generated a transgenic mouse model of conditional oligodendrocyte ablation. In this model, oligodendrocytes are rendered selectively sensitive to exogenously administered diphtheria toxin (DT) by targeted expression of the diphtheria toxin receptor in oligodendrocytes. Administration of DT resulted in severe clinical dysfunction with an ascending spastic paralysis ultimately resulting in fatal respiratory impairment within 22 d of DT challenge. Pathologically, at this time point, mice exhibited a loss of ∼26% of oligodendrocyte cell bodies throughout the CNS. Oligodendrocyte cell-body loss was associated with moderate microglial activation, but no widespread myelin degradation. These changes were accompanied with acute axonal injury as characterized by structural and biochemical alterations at nodes of Ranvier and reduced somatosensory-evoked potentials. In summary, we have shown that a death signal initiated within oligodendrocytes results in subcellular changes and loss of key symbiotic interactions between the oligodendrocyte and the axons it ensheaths. This produces profound functional consequences that occur before the removal of the myelin membrane, i.e., in the absence of demyelination. These findings have clear implications for the understanding of the pathogenesis of diseases of the CNS such as multiple sclerosis in which the oligodendrocyte is potentially targeted

The Early Postnatal Nonhuman Primate Neocortex Contains Self-Renewing Multipotent Neural Progenitor Cells

The postnatal neocortex has traditionally been considered a non-neurogenic region, under non-pathological conditions. A few studies suggest, however, that a small subpopulation of neural cells born during postnatal life can differentiate into neurons that take up residence within the neocortex, implying that postnatal neurogenesis could occur in this region, albeit at a low level. Evidence to support this hypothesis remains controversial while the source of putative neural progenitors responsible for generating new neurons in the postnatal neocortex is unknown. Here we report the identification of self-renewing multipotent neural progenitor cells (NPCs) derived from the postnatal day 14 (PD14) marmoset monkey primary visual cortex (V1, striate cortex). While neuronal maturation within V1 is well advanced by PD14, we observed cells throughout this region that co-expressed Sox2 and Ki67, defining a population of resident proliferating progenitor cells. When cultured at low density in the presence of epidermal growth factor (EGF) and/or fibroblast growth factor 2 (FGF-2), dissociated V1 tissue gave rise to multipotent neurospheres that exhibited the ability to differentiate into neurons, oligodendrocytes and astrocytes. While the capacity to generate neurones and oligodendrocytes was not observed beyond the third passage, astrocyte-restricted neurospheres could be maintained for up to 6 passages. This study provides the first direct evidence for the existence of multipotent NPCs within the postnatal neocortex of the nonhuman primate. The potential contribution of neocortical NPCs to neural repair following injury raises exciting new possibilities for the field of regenerative medicine

Gene Network Disruptions and Neurogenesis Defects in the Adult Ts1Cje Mouse Model of Down Syndrome

Background: Down syndrome (DS) individuals suffer mental retardation with further cognitive decline and early onset Alzheimer's disease. Methodology/Principal Findings: To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased. Conclusions/Significance: We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals.Chelsee A. Hewitt, King-Hwa Ling, Tobias D. Merson, Ken M. Simpson, Matthew E. Ritchie, Sarah L. King, Melanie A. Pritchard, Gordon K. Smyth, Tim Thomas, Hamish S. Scott and Anne K. Vos

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

V1 derived neurospheres were maintained for at least 6 passages and remained multipotent from passage 0 to 3.

<p>Plot of cell yield at each passage based on a seeding density of 4.8×10⁴ cells per dish (n = 10 independent isolates per condition) (<b><i>A</i></b>). Clonogenicity assays of FGF-2+EGF-generated neurospheres at passages 1–4 (1,000 cell/cm² seeding density) (<b><i>B</i></b>). At passage 3, neurospheres gave rise to MAP2⁺ neurons (<b><i>C</i></b>), O4⁺ oligodendrocytes (<b><i>D</i></b>) and GFAP⁺ astrocytes (<b><i>E</i></b>) after 15 DIV. Data represent mean ± SEM. Scale bar = 50 µm.</p

EGF and/or FGF-2 stimulate the formation of multipotent neurospheres from dissociated V1 tissue.

<p>Plot of primary neurospheres counted in each well of a 96 well plate after 15 DIV. Acutely isolated cells seeded at 500 cells/well (n = 11 independent isolates per condition) (<b><i>A</i></b>). Plot of mean primary neurosphere diameter (n = 6 independent isolates per condition) (<b><i>B</i></b>). After 15 DIV, neurospheres were pulsed with BrdU for 8 hours prior to fixation. BrdU⁺ cells found within the neurospheres (<b><i>C</i></b>, <b><i>D</i></b>, red) were double-labelled with Sox2 (<b><i>C</i></b>, arrowheads), and expressed Nestin (<b><i>D</i></b>). Differentiation of dissociated primary neurospheres for 15 DIV generated MAP2⁺ neurons (<b><i>E</i></b>), O4⁺ oligodendrocytes (<b><i>F</i></b>) and GFAP⁺ astrocytes (<b><i>G</i></b>). Percentages of MAP2⁺ neurons and O4⁺ oligodendrocytes compared to total differentiated cells (<b><i>H</i></b>). Data represent mean ± SEM (*** <i>P</i>&lt;0.0001; **<i>P</i>&lt;0.009, *<i>P</i>&lt;0.03; Mann-Wittney <i>U</i> test <b><i>A,B</i></b>; Kruskal-Wallis test, <b><i>H</i></b>).</p

The neuronal maturation marker NNF is expressed by neurons in marmoset monkey V1 at PD14.

<p><b><i>A</i></b>, Parasagittal section reveals intense expression in layers 3 and 6 of V1, and the absence of labeling in adjacent area V2. <b><i>B</i></b>, Schematic representation of the marmoset neocortex illustrating posterior visual areas. The vertical dashed line illustrates the plane of surgical excision used to isolate V1 tissue. The lateral ventricle is delineated by the gray shaded region based upon our own observations and the analyses of other groups <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034383#pone.0034383-Palazzi1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034383#pone.0034383-Sawamoto1" target="_blank">[39]</a>. <b><i>C</i></b>, Representative piece of resected V1 tissue subsequently processed for cell culture. Calcarine (Ca), calcarine fissure (CaF), hippocampus (Hip), lateral (L), lateral ventricle (LV), medial (M), operculum (Op), primary visual cortex (V1), secondary visual area (V2), white matter (WM). Scale bar = 2000 µm (<b><i>A</i></b>), 1000 µm (<b><i>C</i></b>).</p

DCX, Sox2, Ki67, NG2 and GFAP are expressed in V1 at PD14.

<p>Hoechst staining demarcates cortical layers (<b><i>A</i></b>). Tbr2 was not expressed in V1 but expressed in the SVZ (<b><i>C</i></b>, LV:lateral ventricle). DCX⁺ cell bodies (<b><i>D</i></b>, *blood vessels) were located at the limit between layers 1 and 2 (<b><i>E</i></b>, arrow), and in white matter (<b><i>F</i></b>, arrow). DCX⁺ cells in the white matter extended long radial processes towards the outer layers (<b><i>F</i></b>; arrowheads). Sox2⁺ cells (<b><i>G</i></b>) were distributed across all cortical layers and white matter, of which a subset co-expressed Ki67 (<b><i>H</i></b>, <i>z</i> = z stack). NG2⁺ cells distributed in the white matter and the cortical layers co-expressed Sox2 (<b><i>I</i></b>, <b><i>J</i></b>) and the proliferation marker PCNA (<b><i>K</i></b>). The majority of Sox2+ cells co-expressed GFAP (<b><i>L</i></b>, <b><i>M</i></b>), a subset of astrocytes expressed Ki67 (<b><i>N</i></b>, *blood cell) however most of the Ki67⁺ cells were GFAP⁻ (<b><i>L</i></b>, arrowhead). Scale bar = 100 µm (<b><i>A, B, C, D, G, I, L</i></b>), 50 µm (<b><i>E</i></b>, <b><i>F</i></b>), 20 µm (<b><i>H, J, K, M, N</i></b>).</p

Evidence for Cooperative Selection of Axons for Myelination by Adjacent Oligodendrocytes in the Optic Nerve

<div><p>The cellular mechanisms that regulate the topographic arrangement of myelin internodes along axons remain largely uncharacterized. Recent clonal analysis of oligodendrocyte morphologies in the mouse optic nerve revealed that adjacent oligodendrocytes frequently formed adjacent internodes on one or more axons in common, whereas oligodendrocytes in the optic nerve were never observed to myelinate the same axon more than once. By modelling the process of axonal selection at the single cell level, we demonstrate that internode length and primary process length constrain the capacity of oligodendrocytes to myelinate the same axon more than once. On the other hand, probabilistic analysis reveals that the observed juxtaposition of myelin internodes among common sets of axons by adjacent oligodendrocytes is highly unlikely to occur by chance. Our analysis may reveal a hitherto unknown level of communication between adjacent oligodendrocytes in the selection of axons for myelination. Together, our analyses provide novel insights into the mechanisms that define the spatial organization of myelin internodes within white matter at the single cell level.</p></div