Map5 distribution in neuronal and glial cells of the rabbit and mouse CNS.

<p>A, Map5 is abundant in most populations of mature neurons (top), neuroblasts occurring in germinal layer-derived neurogenic sites or transitory germinative zones (e.g., rabbit subpial layer, SPL; middle), and in neural progenitors of the brain parenchyma (e.g., rabbit striatum, St; bottom, left). White arrows: clusters of newly generated neuroblasts; white arrowheads: chains of neuroblasts. Newly generated neurons in the cerebellum (Crb) and immature neurons of the piriform cortex (Pir) are generally Map5-negative (green arrowheads; bottom, right), apart from some immature neurons which show low level of Map5 staining (yellow arrowheads). Micrograph in panel A are from peripuberal and adult rabbit tissue, except those marked for mouse. For the Map5 staining on the SVZ ependymal wall, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063258#pone.0063258.s004" target="_blank">Fig. S4B</a>. B, In addition to its neuronal localization, Map5 decorates a population of multipolar cells with the morphology of synantocytes (see also red arrows in panel A). These cells are better visualized in rabbit than in mouse by immunocytochemistry, since staining in mice reveals to a lesser extent the ramifications of cell processes (schematically showed by drawings on the right; total length of cell processes quantifications in the two mammalian species is showed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063258#pone.0063258.s002" target="_blank">Fig. S2A</a>). Crb, cerebellum; ML, molecular layer; Cx, cerebral cortex; Cc, corpus callosum; SVZ, subventricular zone; LV, lateral ventricle; Sc, spinal cord; Dg, dentate gyrus of the hippocampus. Scale bars: A, 30 µm; B, 10 µm.</p

Behavior of mMap5 cells under neurodegenerative and traumatic injury conditions in mice.

<p>A, The number of mMap5 is significantly increased in the cerebral cortex (Cx) and corpus callosum (Cc) of APPPS1 trangenic mice. Asterisks, amyloid plaques. B, A slight increase in the amount of mMap5 is detectable after stab wound lesion in the mouse cerebral cortex and corpus callosum. Differences between A and B in the number of mMap5 in WT animals is related to the different ages at which the two lesion models were analysed (12 months for Alzheimer and 3 months for stab wound). Scale bars: Low magnifications, 50 µm; high magnifications, 10 µm.</p

Regional distribution of mMap5 in the mouse CNS.

<p>A, Representative images of mMap5 cells in the cerebral cortex (Cx), corpus callosum (Cc), striatum (St), cerebellum (Crb). g, grey matter; w, white matter. B, Quantitative analysis of mMap5 cell density in different regions, at two different ages. Squares indicate the areas in which cell counts have been carried out. Asterisks/dots indicate significant statistical differences in mMap5 cell densities among different regions/ages (two way ANOVA, p<0,001). Scale bars: Low magnifications, 50 µm; high magnifications, 10 µm.</p

Cellular and Molecular Characterization of Multipolar Map5-Expressing Cells: A Subset of Newly Generated, Stage-Specific Parenchymal Cells in the Mammalian Central Nervous System

<div><p>Although extremely interesting in adult neuro-glio-genesis and promising as an endogenous source for repair, parenchymal progenitors remain largely obscure in their identity and physiology, due to a scarce availability of stage-specific markers. What appears difficult is the distinction between real cell populations and various differentiation stages of the same population. Here we focused on a subset of multipolar, polydendrocyte-like cells (mMap5 cells) expressing the microtubule associated protein 5 (Map5), which is known to be present in most neurons. We characterized the morphology, phenotype, regional distribution, proliferative dynamics, and stage-specific marker expression of these cells in the rabbit and mouse CNS, also assessing their existence in other mammalian species. mMap5 cells were never found to co-express the Ng2 antigen. They appear to be a population of glial cells sharing features but also differences with Ng2+progenitor cells. We show that mMap5 cells are newly generated, postmitotic parenchymal elements of the oligodendroglial lineage, thus being a stage-specific population of polydendrocytes. Finally, we report that the number of mMap5 cells, although reduced within the brain of adult/old animals, can increase in neurodegenerative and traumatic conditions.</p> </div

Primary antibodies used in this study.

<p>Primary antibodies used in this study.</p

Amount of Map5 molecule and mMap5 cells in the rabbit and mouse CNS at different ages.

<p>A, Immunoblot analysis of Map5 (Map1b) expression in different animals at different ages. Autoradiography of the western blot of protein lysate from total brain of mouse 40 days old (a) and 3 months old (b), rabbit 3,5 months old (c) and 1 year old (d), probed with anti-Map5 and anti-vinculin antibodies (3 animals for each condition). On the right, quantitation of the image after normalization with vinculin. The values expressed are a media of the value of the 3 animals for each condition. Values of older animals were expressed relatively to those of young animals of the same species. Level of Map5 in the brain of the older animals does not change significantly with respect to level in the young animals of the same species. B, Trend in the amount of mMap5 cells in the rabbit and mouse CNS at different ages. C, Schematic representation of the data reported in B, and reactivity of mMap5 after injury.</p

Distribution of Map5 in the mammalian CNS as described in literature.

<p>Distribution of Map5 in the mammalian CNS as described in literature.</p

Genesis and differentiation of mMap5 cells in mice.

<p>Analyses carried out with endogenous and exogenous cell proliferation markers in double staining with Map5 and other markers of the oligodendroglial lineage. A, No proliferation of mMap5 cells is detectable by using the endogenous cell proliferation marker Ki67, whereas most Ng2+cells are clearly cycling. Rare Ki67+/GPR17+cells are detectable (A, right), their staining being restricted to the Golgi in the cell body (arrowhead). B–D, Analyses carried out with BrdU i.p. administration followed by different survival times (from 10 to 30 days). BrdU/Map5 double staining is detectable starting from 5–10 days in mice. E (top), quantification of newly generated cells expressing Map5, Ng2, GPR17, GST-π, at different BrdU post-injection survival times, in grey (GM, cortex) and white matter (WM, corpus callosum). Data indicate the percentage of BrdU+/marker+ cells out of total BrdU+ nuclei (raw data are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063258#pone.0063258.s003" target="_blank">Fig. S3</a>). E (bottom), quantification of newly generated mMap5 cells out of the mMap5 cell whole population (analysed in animals with 5+5 days BrdU treatment). Scale bars: 10 µm.</p

Occurrence of mMap5 in different mammals.

<p>Multipolar, Map5+cells showing the same morphology and set of markers found in mouse and rabbit are detectable in various CNS regions of different mammals, including humans. Scale bars: 10 µm.</p

Schematic summary of the spatial and temporal distribution of several markers linked to the mMap5 cell population.

<p>A, The mMap5 cell population can be revealed by Map5/Olig2 double staining, and the subset of newly generated mMap5 cells by Map5/Olig2/BrdU triple staining (left). Double and triple staining with different markers (see colors in the legend on the right) can provide information on the spatial (B) and temporal (C) distribution of different subpopulations. B, The relative amount of each cell population revealed by different markers is indicated by colored squares. There is no overlapping between mMap5 and Ng2+cell population, and only a very small overlapping with the GST-π+ oligodendrocytes. C, Hypothesis on the time course for newly generated mMap5 cells by using endogenous and exogenous cell proliferation markers.</p