BDNF+/- mice exhibit deficits in oligodendrocyte lineage cells of the basal forebrain

Abstract

Previous work indicated that brain-derived neurotrophic factor (BDNF), through the trkB receptor, increases DNA synthesis in oligodendrocyte (OLG) progenitor cells (OPCs) and differentiation of postmitotic OLGs of the basal forebrain (BF). In the present studies, BDNF knockout animals were used to investigate BDNF's effects on OLG lineage cells (OLCs) in vivo. OLCs of the BF were found to express the trkB receptor, suggesting they are responsive to BDNF. Immunohistochemistry using NG2 and CC1 antibodies was utilized to examine the numbers of NG2+ OPCs and CC1+ postmitotic BF OLGs. At embryonic day 17 (E17), BDNF-/- animals display reduced NG2+ cells. This reduction was also observed in BDNF+/- mice at E17 and at postnatal day 1 (P1), P14, and adult stage, suggesting that BDNF plays a role in OPC development. BDNF+/- mice do not exhibit deficits in numbers of CC1+ OLGs. However, myelin basic protein, myelin associated glycoprotein, and proteolipid protein are reduced in BDNF+/- mice, suggesting that BDNF plays a role in differentiation. These data indicate that progenitor cells and myelin proteins may be affected in vivo by a decrease in BDNF.

Figure 1

OLCs of the BF express full-length trkB. (A) Western blot of cultured BF OLGs shows the molecular weight of the antibody at 145 kD. The antibody is specific for the full-length form of the receptor, as it does not recognize the truncated form (90 kD). (B) trkB is present on OPCs in the BF. Immunohistochemical analysis indicates that PDGFRa+ cells (green) colocalize with trkB (red) in the P14 BF. Arrows show two co-localized cells. (C) trkB is present on mature OLGs in the mouse BF. Immunohistochemistry shows that CC1+ cells (red) colocalize with trkB (green) in the adult BF of +/+ BDNF mice. The arrow shows a co-localized cell and the arrowhead indicates a cell positive for only one antigen. Scale bar represents 50um.

Figure 2

BDNF +/− mice exhibit reduced BDNF levels in the BF. (A) Western blot shows BDNF protein in the BF of adult BDNF +/+ and +/− mice. (B) Graph is a densitometric analysis of Western blot in (A). Anti-GAPDH was used to normalize the protein. N=5. * Significantly different from control at p < .01. Data were analyzed by Matched-pair t-test.

Figure 3

NG2+ cells are reduced in BDNF −/− and +/− BF. Representative images of BDNF +/+ and +/− mice are shown for E17 (A, B), P1 (C, D), P14 (E, F), and adult (G, H) BF. Scale bar represents 50um. Arrows represent NG2+ cells. Arrowheads represent blood vessels stained in E17 samples. The NG2 antibody stained blood vessels in tissues not perfused, but these were easily distinguishable from NG2+ progenitor cells. E17 BDNF −/− mice (I), as well as E17, P1, P14, and adult BDNF +/− mice (J) exhibit a reduction in numbers of NG2+ cells in the BF. Serial coronal sections of BF were captured every 56 um and every cell was counted. E17 - N=3; P1 - N=6; P14 - N=3; adult - N=3. *Significantly different from control at p < 0.05. Data in (I) were analyzed by Matched-pair t-test and data in (J) were analyzed by ANOVA followed by Fisher’s protected least significant difference posthoc test.

Figure 4

P1, P14, and adult BDNF +/+ and +/− mice exhibit similar numbers of CC1+ cells in the BF. Serial coronal sections of BF were captured every 56 um and every cell was counted. P1 - N=3; P14 - N=3; adult - N=4. Data were analyzed by ANOVA followed by Fisher’s protected least significant difference posthoc test.

Figure 5

Myelin protein levels are reduced in BDNF +/− BF. Western blot was used to examine the expression of MBP (A), MAG (B), and PLP (C) protein levels in P14 and adult BF. Anti-GAPDH was used to normalize to total protein. The graphs represent densitometric analysis of the blots. MBP: N=5 at P14, N=6 at adult. MAG: N=4 at P14 and N=7 at adult. PLP: N=4 at P14 and N=5 at adult. *Significantly different from control +/+ at p < 0.05. **Significantly different from adult +/− at p< 0.05. Data were analyzed using ANOVA followed by Fisher’s protected least significant difference posthoc test.

Figure 6

MBP immunoreactivity is reduced in BDNF +/− BF. Coronal sections of the BF of P14 BDNF +/+ (A) and +/− (C) mice and adult BDNF +/+ (E) and +/− (G) mice were stained with anti-MBP. The myelinated tracts in the BF were compared. Panels (B, D, F, H) are magnified images taken from the boxes in panels (A, C, E, G), respectively. Scale bar represents 50 um. * outlines the area of the fimbria-fornix.

Figure 7

NF-L protein levels are similar in BDNF +/+ and +/− BF. Western blots were used to examine the expression of NF-L protein in P14 and adult BF (A). Anti-GAPDH was used to normalize to total protein. (B) The graph represents densitometric analysis of blots. N=3 at P14 and N=3 at adult stages. Data were analyzed using ANOVA followed by Fisher’s protected least significant difference posthoc test.