10 research outputs found
Data_Sheet_1_Nicotinamide enhances myelin production after demyelination through reduction of astrogliosis and microgliosis.docx
Caloric restriction is the chronic reduction of total caloric intake without malnutrition and has attracted a lot of attention as, among multiple other effects, it attenuates demyelination and stimulates remyelination. In this study we have evaluated the effect of nicotinamide (NAM), a well-known caloric restriction mimetic, on myelin production upon demyelinating conditions. NAM is the derivative of nicotinic acid (vitamin B3) and a precursor of nicotinamide adenine dinucleotide (NAD+), a ubiquitous metabolic cofactor. Here, we use cortical slices ex vivo subjected to demyelination or cultured upon normal conditions, a lysolecithin (LPC)-induced focal demyelination mouse model as well as primary glial cultures. Our data show that NAM enhances both myelination and remyelination ex vivo, while it also induces myelin production after LPC-induced focal demyelination ex vivo and in vivo. The increased myelin production is accompanied by reduction in both astrogliosis and microgliosis in vivo. There is no direct effect of NAM on the oligodendrocyte lineage, as no differences are observed in oligodendrocyte precursor cell proliferation or differentiation or in the number of mature oligodendrocytes. On the other hand, NAM affects both microglia and astrocytes as it decreases the population of M1-activated microglia, while reducing the pro-inflammatory phenotype of astrocytes as assayed by the reduction of TNF-α. Overall, we show that the increased myelin production that follows NAM treatment in vivo is accompanied by a decrease in both astrocyte and microglia accumulation at the lesion site. Our data indicate that NAM influences astrocytes and microglia directly, in favor of the remyelination process by promoting a less inflammatory environment.</p
Normal Precursor Proliferation and Neurogenesis but Loss of OT Neuronal Differentiation in the Absence of SOX1
<p>Coronal brain sections from the ventral telencephalon of wild-type (+/+) and <i>Sox1</i>-null (−/−) embryos. TuJ1 immunolabeling (A and B) at E13 shows no difference in early neuronal differentiation embryos; in situ hybridization at E16 for <i>Brn4</i> (C and D) and <i>Robo</i> (E and F) shows absence of differentiation in the mutant at the prospective OT area. Red arrow in wild-type brain sections indicates OT. Telencephalic sections of wild-type (G, I, and K) and <i>Sox1</i>-null mutant (H, J, and L) embryonic brains were harvested 1 h after BrdU injection at E13 (G and H), E14 (I and J), and E15 (K and L) to detect actively dividing cells of the VZ/SVZ. Positive cells were visualized with anti-BrdU immunofluorescence (G–J) or with DAB staining (K and L). (K and L) show dorsal LGE area at high magnification. No differences were detected in the proliferation precursors at all stages examined, and no ectopic proliferation was observed in the mutant brains<i>.</i> Measurements and statistical analysis of BrdU-positive cells were performed on the DAB-stained sections, showing no significant differences (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030186#st001" target="_blank">Table S1</a>). Scale bar = 300 μm (A and B), 300 μm (C–F), 500 μm (G–J), 500 μm (K and L).</p
<i>Sox1</i> Expression in Precursors Is Not Sufficient for the Emergence of OT/VS Neurons
<div><p>(A–C) X-gal staining of coronal sections from the ventral telencephalon showing <i>Sox1-</i>β<i>geo</i>-expressing OT-prospective neurons at E16-stage embryos with one wild-type <i>Sox1</i> allele, <i>Sox1</i><sup>βgeo/+</sup>, in (A), and none, <i>Sox1</i><sup>βgeo/M1</sup>, in (B), and HoHe (<i>Sox1</i><sup>βgeo/M1</sup>, <i>Sox2</i><sup>R/+</sup>) in (C). Note the absence of X-gal-stained neurons in the area of the VS (red arrowheads), indicating failure of the <i>Sox2</i><sup>R</sup> allele to rescue OT neuron development in the HoHe embryos. Expression of βgeo from the <i>Sox2</i><sup>R</sup> allele in the HoHe (C) may account for the slight increase of X-gal staining compared to (B).</p>
<p>(D and E) SOX1 immunostaining at E16 embryos performed on the other halves of the brains of (A and C), respectively. Note that the level of SOX1 expression in the precursors (yellow arrows) is the same, whether it is expressed from the <i>Sox2</i><sup>R</sup> allele in the HoHe (E) or from one of the <i>Sox1</i> wild-type alleles in the <i>Sox1</i> single heterozygotes (D). Note in the HoHe (E), this expression is not sufficient for the development of OT neurons.</p>
<p>(F and G) DARPP32 immunostaining of coronal brain sections from <i>Sox1</i><sup>+/+</sup><i>Sox2</i><sup>R/+</sup> (F) and <i>Sox1</i><sup>M1/M1</sup><i>Sox2</i><sup>R/+</sup> (G) P10 mice indicating loss of VS neurons.</p>
<p>Red arrowheads indicate OT; red arrows indicate anterior commissure. OC, olfactory cortex. Scale bar = 500 μm (A–E), 1 mm (F and G).</p></div
Normal Generation and Patterning of LGE Precursors in the Absence of SOX1
<div><p>(A and B) Immunocytochemistry and on coronal brain sections of dorsal and ventral telencephalic markers in wild-type (+/+) and <i>Sox1</i>-null (−/−) embryos. PAX6 and GSH2 immunocytochemistry in the dorsal LGE at E12 shows no difference at the expression boundary in the absence of SOX1; the arrows point at the stream of PAX6-positive cells emanating from the boundary.</p>
<p>Double immunostaining for SOX1/PAX6 in wild-type brain (C), and for β-galactosidase/PAX6 (D) in the <i>Sox1</i><sup>βgeo/−</sup> brain, at the VS area at E15. Note the presence of the PAX6-positive neurons in the area of the VS in the <i>Sox1</i><sup>βgeo/−</sup> brain.</p>
<p>(E and F) MASH1 immunocytochemistry in the LGE of wild-type (E) and <i>Sox1</i>-null brain (F), at E13. No changes are detected.</p>
<p>(G and H) The distribution of <i>Dlx1</i>-expressing cells, as detected by in situ hybridization, is similar in both wild-type and mutant brains.</p>
<p>Scale bar = 300 μm (A and B), 200 μm (C–F), 150 μm (G and H).</p></div
The <i>Sox2</i><sup>R</sup> Allele Delivers SOX1 in <i>Sox2</i>-Specific Expression Sites
<div><p>(A) Strategy for targeted replacement of the SOX2 coding region with that of SOX1 and IRES-βgeo. Restriction enzymes: S, SalI; E, EcoRI; Sm, SmaI; X, Xho. Green boxes indicate <i>Sox1</i>; black arrowheads indicate <i>LoxP</i> sites; yellow boxes indicate IRES βgeo; blue lines indicate fragments appearing in Southern blots of EcoR1-digested genomic DNA, hybridized with the external probe, which is shown with red lines. Black arrows show the locus after recombination, homologous and Cre-mediated where is indicated.</p>
<p>(B–E) SOX1 immunostaining of frontal sections from E10 embryos. (B and D) Sox2<sup>+/+</sup> and (C and E) <i>Sox2</i><sup>R/+</sup> showing the ectopic expression of SOX1 in the diencephalon (arrowheads) and the nasal pit (np) at E13.</p></div
SOX2 and SOX3 Down-Regulation in LGE Neurons and SOX1/SOX2 Co-Expression in LGE Precursors
<p>Immunofluorescence of coronal sections at LGE levels in (A–C) E15- and (D–L) E13-stage wild-type embryos visualized on a confocal microscope: antibody staining for (A, D, G, and J) SOX1 (red), (B, E, H, and K) SOX2 (green), (C) SOX3 (green), (D–L) double SOX1 (red) and SOX2 (green), and (F, I, and L) merged. In the OT area and the LGE mantle, there are more neurons expressing SOX1 (A and J) than SOX2 (B and K) and SOX3 (C). Note the extensive co-expression of the SOX1 and SOX2 in precursors (D–I). (G–I) are higher magnifications of the areas within the rectangles. Scale bar = 300 μm.</p
Ectopic Distribution of <i>Sox1</i>-Null Neurons
<p>X-gal staining of mouse forebrains at P16. (A and B) show intact forebrain viewed from the ventral surface, and (C–F) show 150-μm coronal Vibratome sections for <i>Sox1</i><sup>βgeo/+</sup> mice (A, C, and E) and <i>Sox1</i><sup>βgeo/M1</sup> mice (B, D, and E). Red arrows indicate the width of the OT. Red arrowheads indicate increased X-gal staining at more medial and posterior areas of the brain in (B), and in the striatum and septum in (D) and (F). White arrowheads indicate islands other than the medial islands of Calleja. an, accumbens nucleus; I, II, III, cell layers of the OT; ICjM, medial islands of Calleja; lot, lateral olfactory tract; lsn, lateral septal nucleus; ob, olfactory bulb; PC, olfactory (piriform) cortex; S, striatum; sb, striatal bridge Scale bar = 500 μm.</p
Failure of Neurons to Migrate to the VS in the Absence of SOX1
<div><p>This figure shows BrdU labeling of proliferating cells in the developing forebrain. Immunohistochemistry was performed on 5-μm coronal sections, cut at the level of the OT.</p>
<p>(A and B) Sections at E17, after BrdU injection at E14. White arrowheads in (A and B) indicate streams of migrating cells.</p>
<p>(C–H) Sections at P16, after BrdU injection at E13 (C and D), E14 (E and F), or E16 (G and H). The DAB reaction product (C–H) was viewed under dark-field illumination. “II” is layer II of the OT, and the red bracket indicates the olfactory cortex. Note E13-born neurons contribute laterally to the olfactory (piriform) cortex, and medially to the layer II of the OT and the striatal bridges (red arrow). E14-born neurons contribute to more medial VS structures than E15- and E16-born cells, which contribute almost exclusively to the medial islands of Calleja (red arrowheads).</p>
<p>Scale bar = 300 μm (A and B), 1 mm (C–H).</p></div
The Mouse <i>Sox1</i><sup>βgeo</sup> Allele Reveals the Requirement of SOX1 in the Development of VS Neurons
<div><p>(A) Strategy for targeting of the <i>Sox1</i> locus by insertion of β<i>geo.</i> Restriction enzymes: RV, EcoRV; K, KpnI; E, EcoRI; S, SpeI; B, BamHI. Yellow boxes indicate β<i>geo</i>, green, SOX1 exon, and blue lines indicate fragments appearing in Southern blots of EcoR1-digested genomic DNA, hybridized with the external probe, which is shown with red lines.</p>
<p>(B–E) X-gal and SOX1 antibody staining of <i>Sox1</i><sup>βgeo/+</sup>. Comparison of <i>Sox1</i><sup>β<i>geo</i></sup> expression visualized by X-gal staining (B and D) and the endogenous wild-type <i>Sox1</i> gene visualized by whole-mount in situ (C) and SOX1 antibody staining (E). (B and C) show E9-stage embryos and (D and E) show coronal sections of newborn ventral telencephalon.</p>
<p>(F–M) 100-μm coronal sections (Vibratome) were stained with X-gal to identify cells with <i>Sox1</i> promoter activity<i>.</i> (F–I) show <i>Sox1</i><sup>βgeo/+</sup> forebrain sections from E13 to birth (P0) showing normal migration of <i>Sox1</i>-expressing cells from the VZ to the site of the OT, including striatal bridges. (J–M) show sections of <i>Sox1</i><sup>βgeo/M1</sup> forebrain, showing absence of X-gal staining in the OT and the striatal bridges. Red arrowheads show the anterior commissure.</p>
<p>Scale bar = 500 μm for (B) and (C) and 300 μm for (D–M).</p></div
The Distribution of VS Neurons Is Unaffected in Mice Over-Expressing SOX1 from the <i>Sox2</i><sup>R</sup> Allele
<div><p>(A–D), X-gal staining of coronal sections from the ventral telencephalon of P0 mice. Note that there is no difference in the distribution of <i>Sox2</i>-expressing OT neurons with SOX1 (A) or without SOX1 (C), and in <i>Sox2</i><sup>R/<i>+</i></sup> mice with two wild-type <i>Sox1</i> alleles (A) or one (B). Comparison of the number and distribution of neurons expressing <i>Sox2</i><sup>βgeo</sup> in (C) and <i>Sox1</i><sup>βgeo</sup> in (D) shows overlapping expression. (A–C) show 150-μm sections, and (D) shows a 80-μm section.</p>
<p>(E and F) DARPP-32 immunostaining of coronal sections from the ventral telencephalon of P10 <i>Sox2</i><sup>R/<i>+</i></sup> and <i>Sox1</i><sup>βgeo/+</sup> single heterozygous mice, showing no difference in the generation and migration of OT neurons. AC, anterior commissure. Scale bar = 100 μm.</p></div