Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 23 (18), 7207-17

Aberrant Growth and Differentiation of Oligodendrocyte Progenitors in Neurofibromatosis Type 1 Mutants

Affiliations

Aberrant Growth and Differentiation of Oligodendrocyte Progenitors in Neurofibromatosis Type 1 Mutants

Michael R Bennett et al. J Neurosci.

Abstract

Neurofibromatosis type 1 (NF1) patients are predisposed to learning disabilities, macrocephaly, and brain tumors as well as abnormalities on magnetic resonance imaging that are postulated to result from abnormal myelination. Here we show that Nf1+/- spinal cords in adult mice have more than twofold-increased numbers of NG2+ progenitor cells. Nf1-/- embryonic spinal cords have increased numbers of Olig2+ progenitors. Also, cultures from Nf1 mutant embryos with hemizygous and biallelic Nf1 mutations have dramatically increased numbers of CNS oligodendrocyte progenitor cells. In medium that allows growth of neuroepithelial cells and glial progenitors, mutant cells hyper-respond to FGF2, have increased basal and FGF-stimulated Ras-GTP, and fail to accumulate when treated with a farnesyltransferase inhibitor. Cell accumulation results in part from increased proliferation and decreased cell death. In contrast to wild-type cells, Nf1-/- progenitors express the glial differentiation marker O4 while retaining expression of the progenitor marker nestin. Nf1 mutant progenitors also abnormally coexpress the glial differentiation markers O4 and GFAP. Importantly, Nf1-/- spinal cord-derived oligodendrocyte progenitors, which are amplified 12-fold, retain the ability to form oligodendrocytes after in vivo transplantation. The data reveal a key role for neurofibromin and Ras signaling in the maintenance of CNS progenitor cell pools and also suggest a potential role for progenitor cell defects in the CNS abnormalities of NF1 patients.

Figures

Figure 1.
Figure 1.
NG2 labeling of adult mouse spinal cords and Olig2 labeling of E12.5 spinal cords. A, B, Photomicrographs of transverse sections of adult wild-type (A) and Nf1+/- (B) spinal cords showing immunostained NG2+ cells (arrows). Scale bar, 20 μm. C, Significantly more NG2+ cells were found in the Nf1+/- (n = 3) versus wild-type (n = 3) spinal cords in both gray matter (*p = 0.01; paired Student's t test) and white matter (*p = 0.001; paired Student's t test). D--F, Photomicrographs of transverse sections of E12.5 wild-type (D), Nf1+/- (E), and Nf1-/- (F) spinal cords showing Olig2+ cells (arrows). Scale bar, 20 μm. G, Significantly more Olig2+ cells were found in the Nf1-/- (n = 3) versus wild-type (n = 3) spinal cords (*p = 0.01; paired Student's t test). Nf1+/- (n = 3) spinal cords were not significantly different from either wild-type or Nf1-/- spinal cords.
Figure 2.
Figure 2.
Growth factor requirements of Nf1 mutant glial progenitors. A--D, Colony number and size from wild-type (n = 9), Nf1+/- (n = 6), and Nf1-/- (n = 2) E12.5 spinal cord cultures at day 5 in the presence of FGF2 plus PDGF (A), FGF2 (B), PDGF (C), and no growth factors (D).
Figure 3.
Figure 3.
FGF2 dose-response curves. A, Number of large (<150 cells) colonies observed at day 5 of wild-type (n = 3), Nf1+/- (n = 3), and Nf1-/- (n = 3) E12.5 spinal cord cultures grown in the presence of 1, 2.5, 5, 10, and 25 ng/ml FGF2. B, Day 5 photographs of wild-type, Nf1+/-, and Nf1-/- E12.5 spinal cord cultures grown in the presence of 10 ng/ml of FGF2.
Figure 4.
Figure 4.
Inhibition of growth by FTI and Ras activation assay. A, B, Colony number and size from wild-type (n = 9), Nf1+/- (n = 6), and Nf1-/- (n = 2) E12.5 spinal cord cultures at day 5 in the presence of FGF2 plus PDGF (A) and FGF2, PDGF, and FTI (B). C, Immunoblot of a Ras-GTP pull-down assay showing increased basal and FGF2-stimulated Ras-GTP in Nf1+/- cells compared with wild-type cells from E12.5 mouse spinal cord cultures.
Figure 5.
Figure 5.
Nf1-/- mixed glial cells, DNA fragmentation, and cell proliferation assays. A, Triple labeling of Nf1-/- spinal cord cultures showing O4+/GFAP+ cells (arrows). Expanded cultures of E12.5 mouse spinal cord cells were assayed for DNA fragmentation and BrdU incorporation. B, Significantly fewer (*p = 0.03; paired Student's t test) Nf1+/- (n = 3) cells were undergoing cell death in the unstimulated condition versus wild-type cells (n = 3). A significant difference was not found when stimulated with 25 ng/ml FGF2. Fewer Nf1-/- (n = 1) cells were dying in each condition. C, A 4 hr pulse revealed a significant increase (*p = 0.02; paired Student's ttest) in BrdU incorporation in the Nf1-/- (n = 3) cultures versus both Nf1+/- (n = 3) and wild-type (n = 3) cultures after 24 hr of FGF2 stimulation (10 ng/ml). There was a slight increase in BrdU incorporation in the Nf1+/- cultures versus wild-type cultures, but it failed to reach significance.
Figure 6.
Figure 6.
E12.5 spinal cord cultures and grafting of dye-labeled Nf1-/- OPCs (O2A cells) into the forebrain of MBP-/- mice.A, OPC cultures from wild-type, Nf1 +/-, and Nf1-/-E12.5 spinal cords stained with cresyl violet. B, Cells expanded from Nf1-/- cultures were identified by double-label immunocytochemistry using A2B5 (left) and anti-GFAP antibodies (right). Left and right images represent the same field of cells in each pair. Top, Colony containing both type 1 astrocytes (A2B5-/GFAP+) and oligodendrocyte progenitor cells (A2B5+/GFAP-). Center, Colony with type 2 (A2B5+/GFAP+) astrocytes. Bottom, Colony with only A2B5+/GFAP- oligodendrocyte progenitor cells. C, A low-magnification micrograph shows the relationship to the third ventricle (*) of dye-labeled grafted OPCs in MBP- mouse brain. A group of dye-labeled cells is shown at higher magnification in D with associated MBP+ myelin sheaths in E. Arrows point to a small group of longitudinally cut myelin sheaths.

Similar articles

See all similar articles

Cited by 26 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback