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. 2007 Oct 26;149(2):328-37.
doi: 10.1016/j.neuroscience.2007.07.044. Epub 2007 Aug 8.

Down-regulation of the axonal polysialic acid-neural cell adhesion molecule expression coincides with the onset of myelination in the human fetal forebrain

I Jakovcevski  1 , Z Mo, N Zecevic
Affiliations

Down-regulation of the axonal polysialic acid-neural cell adhesion molecule expression coincides with the onset of myelination in the human fetal forebrain

I Jakovcevski et al. Neuroscience. .

Abstract

The polysialic acid (PSA) modification of neural cell adhesion molecule, which reduces neural cell adhesion molecule (NCAM) - mediated cell adhesion, is involved in several developmental processes, such as cell migration, axonal growth, path finding, and synaptic plasticity. It has been suggested that PSA-NCAM expression may inhibit myelination. To clarify the relationship between myelination and the expression of PSA-NCAM we systematically investigated its expression in the human forebrain from embryonic stage to midgestation (19-24 gestation weeks, gw). Immunofluorescence on cryosections showed that PSA-NCAM is expressed at the earliest stage studied (5.5 gw) in the primordial plexiform layer of the telencephalon, which mainly consists of neuronal processes. At midgestation, cortical axonal tracts in the emerging white matter were PSA-NCAM+, but they were not yet myelinated, based on the lack of myelin basic protein (MBP) immunoreaction. To follow the progression of myelination we developed organotypic slice cultures that included the subventricular and intermediate zones of the fetal forebrain. In freshly prepared slices, similar to cryosections, axonal tracts were PSA-NCAM+ but did not express MBP. After 5 days in culture there was a dramatic increase in MBP expression around the axons of the intermediate zone, which suggested the onset of myelination. Simultaneously with MBP up-regulation PSA-NCAM expression in axons was completely lost, as demonstrated both with immunofluorescence and Western blot analysis. These results support the idea that in the human fetal forebrain axonal PSA-NCAM expression is inversely related to primary myelination.

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Figures

Figure 1
Figure 1
PSA-NCAM expression in early developmental stages. (A– E) In the embryonic (5.5 gw) forebrain the PSA-NCAM is expressed in the primordial plexiform layer (PPL), above the ventricular zone (VZ). (A, B) Low magnification of the frontal section through the cerebral vesicle immunostained with (A) PSA-NCAM (red) or (B) β-III-tubulin (green). (C–E) Higher magnification of the cerebral vesicle (boxed area in A). Double-immunofluorescence demonstrates PSA-NCAM (red) in neuronal processes labeled with β-III-tubulin (green) (D) Arrow points to a double-labeled cell in the VZ. (E) Overlay. (F–H) At 10 gw, (F) strong PSA-NCAM (red) expression in the cerebral cortex is bilaminar, in layer I and in the pre-subplate (SP), whereas it is faint in the cortical plate (CP). (G) At the same age, PSA-NCAM is expressed in the lateral ganglionic eminence (LGE) and in (H) a “stream” of cells connecting LGE and the lateral cortex (asterisk). LV - lateral ventricle; Cx – cerebral cortex. Scale bars: (F) 20μm (A–E, G) 100μm, (H) 50μm.
Figure 2
Figure 2
PSA-NCAM expression at midgestation (19–24 gw). (A, B) Immunofluorescence for PSA-NCAM (red) in the ventricular zone (VZ). PSA-NCAM is expressed by the neural progenitor cells (arrows). Some of the subventricular zone (SVZ) progenitors are also PSA-NCAM+. On panel A, nuclei are counter-stained with bis-benzamide (blue). (C) Double-immunofluorescence for PDGFRα (green), marker for early oligodendrocyte precursor cells, and PSA-NCAM (red). (D) Double immunofluorescence for Olig2 (green) and PSA-NCAM (red) in the VZ/SVZ. Inset-higher magnification of a cell co-labeled with both markers. (E) Double immunofluorescence for GFAP (green) and PSA-NCAM (red) in the SVZ of the medial cortex of the frontal lobe. Axons in the IZ, the emerging white matter, are strongly PSA-NCAM+ (red). (F) Enlarged detail from the box in E showing VZ/SVZ expression of GFAP (green) and PSA-NCAM (red). LV - the lateral ventricle. Scale bars: 25 μm (A–C), 100 μm (D, E), 20 μm (F).
Figure 3
Figure 3
PSA-NCAM is expressed in neuronal progenitors in vivo and vitro. (A, B) Neuronal progenitors in the 19 gw old cortical subventricular zone (SVZ) labeled with (A) MAP2 (green) and (B) Dll (pan Dlx antibody, green) are co-labeled with PSA-NCAM (red). Nuclei are counter-stained with bis-benzamide (blue). (C, D) In dissociated mixed cell culture at 16 gw (C) GFAP (green) and PSA-NCAM (red) are not co-localized in astroglia, whereas (D) β-III-tubulin+ cells (green, arrows) are co-labeled with PSA-NCAM (red), co-localization seen in yellow. Scale bar (A, B) 10 μm, (C, D) 25 μm.
Figure 4
Figure 4
Axonal expression of PSA-NCAM in the thalamus and forebrain at midgestation. (A–F) Double-immunofluorescence for MBP (A, B, green) and PSA-NCAM (C, D, red) of the thalamus (Th), (A, C, E) and the internal capsule (IC), (B, D, F) at 22 gw Panels E, F show overlapped images. Scale bar: 25 μm.
Figure 5
Figure 5
PSA-NCAM and MBP immunoreactivity in slice cultures. (A, B) Immunoflourescence for PSA-NCAM (red) on slices from human midgestational forebrains fixed immediately (0 DIC). Blue on panels B, D is nuclear counterstaining with bis-benzamide (bb). (C, D) On 5 DIC slice cultures there is virtually no PSA-NCAM expression (a lack of red staining). (E) On the same slices (5 DIC) MBP+ oligodendrocytes (arrows, red) and fibers are frequently demonstrated. Shown are representative images from 4 independent experiments. Scale bars: 25 μm.
Figure 6
Figure 6
Western blot analysis of PSA-NCAM and MBP expression in the slice cultures of the human fetal brain at 19 gw. Panel A shows representative blots at 0 DIC and 5DIC; panel B shows mean values + standard error of mean (SEM). Actin is used as a loading control. In panel B, initial (0 DIC) values for PSA-NCAM and MBP are normalized to 100%. An asterisk indicates significant difference from 0 day (p < 0.05). Protein loading: 20 μg/well.
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