Independent component analysis based gene co-expression network inference (ICAnet) to decipher functional modules for better single-cell clustering and batch integration.

With the tremendous increase of publicly available single-cell RNA-sequencing (scRNA-seq) datasets, bioinformatics methods based on gene co-expression network are becoming efficient tools for analyzing scRNA-seq data, improving cell type prediction accuracy and in turn facilitating biological discovery. However, the current methods are mainly based on overall co-expression correlation and overlook co-expression that exists in only a subset of cells, thus fail to discover certain rare cell types and sensitive to batch effect. Here, we developed independent component analysis-based gene co-expression network inference (ICAnet) that decomposed scRNA-seq data into a series of independent gene expression components and inferred co-expression modules, which improved cell clustering and rare cell-type discovery. ICAnet showed efficient performance for cell clustering and batch integration using scRNA-seq datasets spanning multiple cells/tissues/donors/library types. It works stably on datasets produced by different library construction strategies and with different sequencing depths and cell numbers. We demonstrated the capability of ICAnet to discover rare cell types in multiple independent scRNA-seq datasets from different sources. Importantly, the identified modules activated in acute myeloid leukemia scRNA-seq datasets have the potential to serve as new diagnostic markers. Thus, ICAnet is a competitive tool for cell clustering and biological interpretations of single-cell RNA-seq data analysis.

Review: Challenges in the histopathological classification of ganglioglioma and DNT: microscopic agreement studies and a preliminary genotype-phenotype analysis.

Low-grade epilepsy-associated brain tumours (LEAT) are the second most common cause for drug-resistant, focal epilepsy, that is ganglioglioma (GG) and dysembryoplastic neuroepithelial tumours (DNT). However, molecular pathogenesis, risk factors for malignant progression and their frequent association with drug-resistant focal seizures remain poorly understood. This contrasts recent progress in understanding the molecular-genetic basis and targeted treatment options in diffuse gliomas. The Neuropathology Task Force of the International League Against Epilepsy examined available literature to identify common obstacles in diagnosis and research of LEAT. Analysis of 10 published tumour series from epilepsy surgery pointed to poor inter-rater agreement for the histopathology diagnosis. The Task Force tested this hypothesis using a web-based microscopy agreement study. In a series of 30 LEAT, 25 raters from 18 countries agreed in only 40% of cases. Highest discordance in microscopic diagnosis occurred between GG and DNT variants, when oligodendroglial-like cell patterns prevail, or ganglion cells were difficult to discriminate from pre-existing neurons. Suggesting new terminology or major histopathological criteria did not satisfactorily increase the yield of histopathology agreement in four consecutive trials. To this end, the Task Force applied the WHO 2016 strategy of integrating phenotype analysis with molecular-genetic data obtained from panel sequencing and 450k methylation arrays. This strategy was helpful to distinguish DNT from GG variants in all cases. The Task Force recommends, therefore, to further develop diagnostic panels for the integration of phenotype-genotype analysis in order to reliably classify the spectrum of LEAT, carefully characterize clinically meaningful entities and make better use of published literature.

Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.

The mammalian cerebral cortex supports cognitive functions such as sensorimotor integration, memory, and social behaviors. Normal brain function relies on a diverse set of differentiated cell types, including neurons, glia, and vasculature. Here, we have used large-scale single-cell RNA sequencing (RNA-seq) to classify cells in the mouse somatosensory cortex and hippocampal CA1 region. We found 47 molecularly distinct subclasses, comprising all known major cell types in the cortex. We identified numerous marker genes, which allowed alignment with known cell types, morphology, and location. We found a layer I interneuron expressing Pax6 and a distinct postmitotic oligodendrocyte subclass marked by Itpr2. Across the diversity of cortical cell types, transcription factors formed a complex, layered regulatory code, suggesting a mechanism for the maintenance of adult cell type identity.

Quantification of oligodendrocyte progenitor cells in human and cat optic nerve: implications for endogenous repair in multiple sclerosis.

In multiple sclerosis (MS), one strategy to reduce disability is enhancement of endogenous repair by remyelinating oligodendrocytes derived from oligodendrocyte progenitor cells (OP). An important prerequisite is determining the abundance of OP relative to oligodendrocytes in normal human central nervous system (CNS), which, in turn, requires reliable OP identification. To achieve this, cat and human optic nerves (ON) were subjected to varied preparation protocols, and the resultant neuroglial staining profiles correlated to generate an antigenic phenotype for OP applicable to human autopsy specimens. OP, interchangeably called NG2cells due to universal NG2 expression, were shown to comprise a separate class of neuroglial cells, related to oligodendrocytes by expression of the oligodendrocyte lineage transcription factors, Olig1 and Olig2. Despite their morphological complexity, including contact with axons and other neuroglia, NG2cells all appear capable of responding as OP to counter local oligodendrocyte loss. However, quantification revealed that NG2cells comprised less than 5% of the neuroglia and had a ratio to oligodendrocytes of about 1:10, not only in human and cat ON but also in white and gray-matter regions of cat spinal cord. The finding that NG2cells are not abundant, particularly relative to oligodendrocytes, may have implications for efforts to enhance endogenous repair in MS.

A common progenitor for retinal astrocytes and oligodendrocytes.

Developing neural tissue undergoes a period of neurogenesis followed by a period of gliogenesis. The lineage relationships among glial cell types have not been defined for most areas of the nervous system. Here we use retroviruses to label clones of glial cells in the chick retina. We found that almost every clone had both astrocytes and oligodendrocytes. In addition, we discovered a novel glial cell type, with features intermediate between those of astrocytes and oligodendrocytes, which we have named the diacyte. Diacytes also share a progenitor cell with both astrocytes and oligodendrocytes.

Subclasses of oligodendrocytes populate the mouse hippocampus.

Oligodendrocytes are the myelin-forming cells of the central nervous system that facilitate transmission of axonal electrical impulses. Using transgenic mice expressing 2',3' cyclic nucleotide 3' phosphodiesterase (CNPase)-enhanced green fluorescent protein, a three-dimensional reconstruction tool and analysis, we illustrate that three morphologically different oligodendrocyte types exist in the hippocampus. Those of the ramified type have the most numerous processes, the largest cell body, occupy the largest area and form beaded-like structures, due to mitochondria aggregates, along the processes. Stellar-shaped oligodendrocytes have smaller cell bodies and their processes cover a significantly smaller area. Those of the smooth subtype have a small cell body with at most two processes. In addition to these types, a large number of oligodendrocytes were found that faintly express CNPase-enhanced green fluorescent protein. More than 50% of the faint type colocalized with NG2 and 91% with oligodendrocyte transcription factor-2, whereas 94% of NG2-immunoreactive and 45% of oligodendrocyte transcription factor-2-immunoreactive cells were faintly CNPase-enhanced green fluorescent protein positive. Based on the complexity of the overall structure, the three types probably represent stages of a maturation process such that one subtype can morph into another. Thus, the least complex 'smooth' cell would represent the youngest oligodendrocyte that matures into the stellar type and eventually progresses to become the most complex ramified oligodendrocyte. Investigation of the distribution pattern revealed that the highest density of oligodendrocytes was found in the stratum lacunosum-moleculare and the hilar region. The distribution analysis of oligodendrocyte subclasses revealed a tendency for different cell types to segregate in large non-overlapping areas. This observation suggests that morphologically, and possible functionally, different oligodendrocytes are topographically segregated.

Characteristics and functions of NG2 cells in normal brain and neuropathology.

OBJECTIVE: This review is focused on the current understanding of the roles of the fifth class of non-neuronal cells, NG2 cells, in the central nervous system (CNS). METHODS: We have reviewed some literature on properties of NG2 cells, including cell morphology, expression of receptors and possible functions. RESULTS: Chondroitin sulfate proteoglycan (NG2) is expressed in a high proportion in non-neuronal cells of the CNS. During development, NG2 cells can differentiate into oligodendrocytes, astrocytes and neurons. In the adult, the NG2 cells have a common morphology: multibranched processes and small cell bodies, and are ubiquitously distributed throughout brain parenchyma. They possess some functional receptors and contact neurons at nodes of Ranvier or via synaptic terminals. Some NG2 cells can even fire action potential. Various brain injury models have demonstrated that NG2 cells adjacent to the damage site could increase in number and become hypertrophic. However, there is no clear evidence indicating the function of NG2 cells in the adult brain. DISCUSSION: The function of NG2 cells in the adult brain is still uncertain. The NG2 expressing cells may be progenitor cells in the developing brain. In the adult, the discovery of functional receptors, interactions with neurons and ability to respond to different harmful stimulations have implied roles of NG2 cells in facilitating neuronal network function, which may be important in brain inflammation, neurodegeneration and neuroregeneration.

Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.

A defining feature of glial cells has been their inability to generate action potentials. We show here that there are two distinct types of morphologically identical oligodendrocyte precursor glial cells (OPCs) in situ in rat CNS white matter. One type expresses voltage-gated sodium and potassium channels, generates action potentials when depolarized and senses its environment by receiving excitatory and inhibitory synaptic input from axons. The other type lacks action potentials and synaptic input. We found that when OPCs suffered glutamate-mediated damage, as occurs in cerebral palsy, stroke and spinal cord injury, the action potential-generating OPCs were preferentially damaged, as they expressed more glutamate receptors, and received increased spontaneous glutamatergic synaptic input in ischemia. These data challenge the idea that only neurons generate action potentials in the CNS and imply that the development of therapies for demyelinating disorders will require defining which OPC type can carry out remyelination.

Intracellular translocation of glutathione S-transferase pi during oligodendrocyte differentiation in adult rat cerebral cortex in vivo.

Glutathione S-transferase (GST)-pi is a cytosolic isoenzyme used as a marker for mature oligodendrocytes in the mammalian brain. However, the cellular properties of GST-pi-immunoreactive [GST-pi (+)] cells in adult brain are not completely understood. We immunohistochemically demonstrated the existence of two subtypes of GST-pi (+) cells in the cerebral cortex of adult rats: one subtype exhibited GST-pi in the cytoplasm (C-type cells), while the other did mainly in the nucleus (N-type cells). The GST-pi (+) C-type cells were also immunopositive for 2',3'-cyclic nucleotide 3'-phosphodiesterase and RIP, indicating that they were mature oligodendrocytes, while the GST-pi (+) N-type cells expressed NG2, indicating that they were oligodendrocyte progenitor cells. Furthermore, observation of the fate of newly-generated cells by 5-bromodeoxyuridine-labeling revealed that the GST-pi (+) N-type cells differentiated into C-type cells. These findings indicate translocation of GST-pi from the nucleus to the cytoplasm during oligodendrocyte maturation.

The proneural gene Mash1 specifies an early population of telencephalic oligodendrocytes.

The bHLH (basic helix-loop-helix) transcription factor Mash1 is best known for its role in the regulation of neurogenesis. However, Mash1 is also expressed in oligodendrocyte precursors and has recently been shown to promote the generation of oligodendrocytes in cell culture, suggesting that it may regulate oligodendrogenesis as well. Here, we show that in the developing ventral forebrain, Mash1 is expressed by a subset of oligodendrocyte precursors (OPCs) as soon as they are generated in the ventricular zone. Using reporter mice, we demonstrate that a subset of OPCs in both the embryonic and postnatal forebrain originate from Mash1-positive progenitors, including a large fraction of adult NG2-positive OPCs. Using Mash1 null mutant mice, we show that Mash1 is required for the generation of an early population of OPCs in the ventral forebrain between embryonic day 11.5 (E11.5) and E13.5, whereas OPCs generated later in embryonic development are not affected. Overexpression of Mash1 in the dorsal telencephalon induces expression of PDGFRalpha (platelet-derived growth factor receptor alpha) but not other OPC markers, suggesting that Mash1 specifies oligodendrogenesis in cooperation with other factors. Analysis of double-mutant mice suggests that Olig2 is one of the factors that cooperate with Mash1 for generation of OPCs. Together, our results show for the first time that Mash1 cooperates in vivo with Olig2 in oligodendrocyte specification, demonstrating an essential role for Mash1 in the generation of a subset of oligodendrocytes and revealing a genetic heterogeneity of oligodendrocyte lineages in the mouse forebrain.

Identification and two-photon imaging of oligodendrocyte in CA1 region of hippocampal slices.

Oligodendrocyte (OL) plays a critical role in myelination and axon maintenance in central nervous system. Recent studies show that OL can also express NMDA receptors in development and pathological situations in white matter. There is still lack of studies about OL properties and function in gray matter of brain. Here we reported that some glial cells in CA1 region of rat hippocampal slices (P15-23) had distinct electrophysiological characteristics from the other glia cells in this region, while they displayed uniform properties with OL from white matter in previous report; therefore, they were considered as OL in hippocampus. By loading dye in recording pipette and imaging with two-photon laser scanning microscopy, we acquired the high spatial resolution, three-dimension images of these special cells in live slices. The OL in hippocampus shows a complex process-bearing shape and the distribution of several processes is parallel to Schaffer fiber in CA1 region. When stimulating Schaffer fiber, OL displays a long duration depolarization mediated by inward rectifier potassium channel. This suggested that the OL in CA1 region could sense the neuronal activity and contribute to potassium clearance.

Ehi neonatal: Lem, o el premio a la superviviencia / Ehi newborn: Lem, or the prize to the survival

La hipoxia- isquemica es la principal causa de injuria del sistema nerviosos central en el feto y en el recién nacido, situación agravada, en algunas circunstancias, por infección intrauterina y respuesta inflamatoria fetal. La reducción en las cifras de mortalidad ha producido un incremento de morbilidad, la posibilidad de asistir a las secuelas de aquellas lesiones. En el niño pretérmino que sobrevive, el sustrato morfológico más frecuente de parálisis cerebral es la lucoencefalomalacia pereventricular, la cual forma parte de un expectro más amplio. Las secuelas son déficit en la mielinización, la pérdida de volúmen de sustancia blanca con consecuente ventriculomegalia, el compromiso de vías largas relacionado con el déficit motor del cuadro clínico de páralisis cerebral. En este artículo se sitúa a la leucoencefalomalacia periventricular en el expectro de lesiones hipóxico, isquémica del recién nacido, se detallan sus características clínicas y macra-microscópicas, y se comentan los recientes aportes de la fisiopatología celular. El rol principal del glutamato, la teoría de la excototoxicidad, las citoquinas, y la cascada inflamatoria operan a través de ventanas de vulnerabilidad, que exponen a la oligodendroglia como target esencial, y a la apoptosis como un mecanismo importante de lesión cerebral

Baseline 11C-methionine PET reflects the natural course of grade 2 oligodendrogliomas.

OBJECTIVES: The aim of the present study was to assess the usefulness of positron emission tomography (PET) with the amino acid tracer 11C-methionine (MET) as a predictor of time to progression (TTP) in patients with supratentorial grade 2 gliomas. METHODS: Twenty-seven patients with glioma grade 2 subjected to a baseline PET scan received no anti-tumour treatment except for a diagnostic operation, and were followed until tumour progression. The prognostic impact of the MET uptake and of other prognostic factors was studied. RESULTS: Twenty-five of the patients (93%) experienced tumour progression after a median of 103 weeks. Low uptake of MET was a predictor for long TTP in patients with oligodendrogliomas (p = 0.04) but not in astrocytomas/oligoastrocytomas. Other predictors for long TTP were oligodendroglioma histology (p = 0.009) and seizures as presenting symptom (p = 0.03). Favourable prognostic factors for overall survival were oligodendroglioma histology (p = 0.002) and good performance status (p = 0.03). CONCLUSIONS: PET MET has a definite role in the therapeutic management of grade 2 gliomas. However, for the optimal use of PET MET in the clinical management of these patients, histological subclassification of the tumour is required.

Membrane-associated estrogen receptor and caveolin-1 are present in central nervous system myelin and oligodendrocyte plasma membranes.

The estrogen receptor (ER) is a member of a superfamily of ligand-regulated transcription factors that were thought to localize primarily to the nucleus; however, a membrane-associated ER that can initiate rapid non-genomic cell-signaling events has been identified recently in various cells. The presence of the ER in myelin has not been reported although the nuclear form has been detected in oligodendrocytes. We have shown that an ER with similarities to ERbeta is present in isolated central nervous system (CNS) myelin, the myelin sheath in spinal cord and brain sections, and the oligodendrocyte plasma membrane using two-dimensional (2D) PAGE, mass spectrometry, peptide mass fingerprinting, Western blotting of 1D and 2D gels, and confocal microscopy. Caveolin-1 was also shown to be present in isolated CNS myelin and oligodendrocyte plasma membranes, where it was partially colocalized with ER. After Triton X-100 extraction of myelin, the ER was present in an insoluble low-density glycosphingolipid-enriched fraction and even more in a higher density fraction also containing caveolin and cytoskeletal elements, suggesting that the membrane form of ER may be associated with caveolin or the radial component of myelin. The discovery of the ER in the oligodendrocyte plasma membrane and within the myelin sheath indicates a potential role for estrogen in myelin maintenance or functions.

Sustained hypomyelination and high serum thyroid hormone in aged black tremor hamster.

Oligodendrocytes and myelin in the corpus callosum of black tremor and normal hamsters aged over 1.5 years were ultrastructurally examined to determine the myelination index (ratio of myelin thickness/diameter of axon), percentage of naked axons, and proportions of oligodendroglial subtypes (light, medium and dark). The mutant hamsters were remarkably hypomyelinated, with a low myelination index and a high proportion of naked axons, and high proportions of the dark subtypes. Serum concentrations of thyroid hormones (T3 and T4) in 6-week-old mutant hamsters were 2-fold (T3) to 3-fold (T4) higher than those of age-matched normal animals. However, in the aged animals (over 1.5 years old) only T4 levels of the mutant hamsters were higher in the mutant than normal hamsters. The black tremor hamsters were hypomyelinated throughout their life and high serum level of thyroid hormones might have played a role in the hypomyelination.

Bilirubin is cytotoxic to rat oligodendrocytes in vitro.

High levels of unconjugated bilirubin can be neurotoxic and gliotoxic. However, the effect of bilirubin on oligodendrocyte viability has never been investigated. In the present study, we searched the possible toxic effect of bilirubin on differentiated rat oligodendrocytes. Bilirubin was added to oligodendrocyte cultures at different concentrations varied between 10 and 100 microM, and cultures were incubated for different times (24, 48 and 72 h). Cell viability was evaluated by trypan blue exclusion. The results showed that bilirubin decreased oligodendroglial cell viability in a concentration and time-dependent manner. Bilirubin induced apoptotic cell death as revealed by TUNEL staining and poly(ADP-ribose) polymerase cleavage. We found that bilirubin induced inducible nitric oxide synthase (NOS) mRNA expression in rat oligodendrocytes. Bilirubin also increased oligodendroglial nitrite production in a concentration-dependent manner and NOS inhibitor partly blocked bilirubin-induced cytotoxicity. These results suggest that bilirubin induces cytotoxicity, at least partly, via the induction of nitric oxide production in oligodendrocytes.

Developing chicken oligodendrocytes express the type IV oligodendrocyte marker T4-O in situ, but not in vitro.

Accumulating data suggest that the oligodendrocyte population includes morphological and biochemical subtypes. We recently reported that a polyclonal antiserum against an unknown antigen, the T4-O molecule, labels a subpopulation of chicken oligodendrocytes, obviously representing the type IV variety of Del Rio Hortega. The present study examines the developmental expression of the T4-O molecule in situ and in vitro. The results show that T4-O immunoreactive cells first appear at E15 in the ventral funiculus. But, oligodendrocytes cultured in vitro with or without neurones do not develop a T4-O immunoreactivity. We conclude that oligodendrocytes in the spinal cord of chicken embryos first express the T4-O molecule some time after onset of myelination, and that the T4-O immunoreactive phenotype does not develop in vitro.

Identification of the glial cell types containing carnosine-related peptides in the rat brain.

The cellular localization of carnosine-like immunoreactivity was investigated in the adult rat forebrain and in glial cell cultures obtained from newborn rat brain. Using double staining methods, we showed that in vivo carnosine-like immunoreactivity was occurring in a large number of both glial fibrillary acidic protein (GFAP)-positive astrocytes and 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNP)-positive oligodendrocytes. In vitro, the carnosine-immunoreactive staining was restricted to a subpopulation of completely differentiated oligodendrocytes, whereas no reaction was detected in immature oligodendrocytes and in astrocytes. These observations could have profound physiopathological implications considering the role suggested for carnosine and related peptides as endogenous antioxidants, free radical scavengers and anti-glycating agents of the central nervous system (CNS).

Characterization of a putative novel type of oligodendrocyte in cultures from rat spinal cord.

Oligodendrocytes originate in different neural tube domains, within boundaries of expression of a series of patterning genes which condition the diverse morphogenetic programme of each area. Although neuronal and astrocyte heterogeneity are widely accepted, and despite accumulating evidence for oligodendrocyte heterogeneity in vivo, oligodendrocytes in vitro are currently considered as a homogeneous cell population. The present investigation demonstrates that oligodendrocyte diversity can be detected in vitro and characterizes a novel morphological class of O4-positive oligodendrocyte which is consistently identifiable in rat central nervous system cultures. These cells have a very characteristic epithelioid, unbranched and often lobulated morphology which enables their identification within 2 h of plating. Immunostaining shows that this morphological type is sometimes positive for GD3, A2B5 and vimentin, and most of the time positive for Ranscht antibody, O1 and Rip but negative for glial fibrillary acidic protein, OX-42, neuron-specific enolase, nestin and erbB2. The apparent levels and/or distributions of (i) microtubules, (ii) surface glycolipids recognized by O4, O1 and Ranscht antibody, and (iii) the less specific marker carbonic anhydrase II, typically differ from those of nearby classical, branched oligodendrocytes. Cells with this epithelioid morphology also express myelin basic protein and O10 (a proteolipid protein epitope), both of which are markers for mature oligodendrocytes. Conversely, O4+/O1- cells with this membranous appearance were also seen. Although these atypical oligodendrocytes were most abundant in spinal cord cultures (representing >10% of the O4+ population), they were not exclusive to this region and occurred at a low frequency in neonatal optic nerve cultures.

Discrimination between different types of neuroglial cells in rat central nervous system using combined immuno- and enzyme-histochemical methods.

The central nervous system (CNS) contains several types of neuroglial cells. In the present study, we characterized different types of glial cells in rat CNS by using single and combined immuno- and enzyme-histochemical methods, and immunofluorescence techniques. Two recently developed monoclonal antibodies (mAbs) against rat macrophages-associated antigens appeared to recognize a subpopulation of glial cells in the CNS of normal adult rats. These ED4- and ED8-positive glial cells were predominantly located in the white matter of adult rat CNS and shared morphological features with microglia. ED4 and ED8 were applied in a double staining combined with mAbs and an antiserum raised against galactocerebroside (GalC) to identify oligodendrocytes, or with anti-glial fibrillary acidic protein antiserum (GFA) to identify astrocytes. We also used a mAb against myelin basic protein (MBP) to identify oligodendrocytes. It appeared that ED4 and ED8 recognized a subpopulation of oligodendrocytes. MAbs against GalC and MBP recognized cells in an immunoperoxidase staining with a morphology identical to that of the ED8-positive cells and part of the ED4-positive cells. Frozen sections of Lewis rats CNS with acute experimental allergic encephalomyelitis (EAE) were investigated, where infiltrating brain macrophages could be found which stained positively with ED4 and ED8 as well as with the monocyte/macrophage mAbs ED1 and ED2. These brain macrophages did not stain when GalC, MBP and GFA markers were applied. Furthermore, ED4+GalC+ and ED8+GalC+ oligodendrocytes were present in the CNS white matter of EAE animals with similar appearance as in normal adult rats. With the currently used markers, we could not detect a third type of neuroglial cell, besides the astrocytes and oligodendrocytes. Thus, none of our anti-macrophage monoclonals recognized the presumptive microglia. Only under pathological conditions, e.g., in inflammatory infiltrates in the course of EAE, could brain macrophages be detected in the CNS parenchyma and only in the direct vicinity of blood vessels, indicating their hematogenous origin.