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. 2014 Jun 11;34(24):8186-96.
doi: 10.1523/JNEUROSCI.4267-13.2014.

Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination

Clifton G Fulmer  1 , Melissa W VonDran  1 , Althea A Stillman  2 , Yangyang Huang  2 , Barbara L Hempstead  3 , Cheryl F Dreyfus  4
Affiliations

Astrocyte-derived BDNF supports myelin protein synthesis after cuprizone-induced demyelination

Clifton G Fulmer et al. J Neurosci. .

Abstract

It is well established that BDNF may enhance oligodendrocyte differentiation following a demyelinating lesion, however, the endogenous sources of BDNF that may be harnessed to reverse deficits associated with such lesions are poorly defined. Here, we investigate roles of astrocytes in synthesizing and releasing BDNF. These cells are known to express BDNF following injury in vivo. In culture, they increase BDNF synthesis and release in response to glutamate metabotropic stimulation. Following cuprizone-elicited demyelination in mice, astrocytes contain BDNF and increase levels of metabotropic receptors. The metabotropic agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), was therefore injected into the demyelinating lesion. Increases in BDNF, as well as myelin proteins, were observed. Effects of ACPD were eliminated by coinjection of trkB-Fc to locally deplete BDNF and by deletion of astrocyte-derived BDNF. The data indicate that astrocyte-derived BDNF may be a source of trophic support that can be used to reverse deficits elicited following demyelination.

Keywords: ACPD; BDNF; astrocytes; cuprizone; metabotropic receptors; oligodendrocytes.

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Figures

Figure 1.
Figure 1.
Cuprizone-lesioned mice exhibit an increase in BDNF and myelin protein levels 6 h after a single stereotaxic injection of ACPD. a, Western blots demonstrate BDNF, MBP, and MAG protein levels in the corpus callosum of wild-type mice subjected to a 4 week cuprizone lesion and injected with ACPD or 0.9% saline vehicle. GAPDH is shown as a loading control. b, Graphs represent a densitometric analysis of Western blots normalized to GAPDH and are presented as percentage saline-injected control. Levels of mature BDNF are indicated in this analysis. c, Immunohistochemical analysis of MBP, MAG and PLP reactivity in the corpus callosum reveals strong staining intensity in the intact corpus callosum that is decreased following exposure to cuprizone. This MBP, MAG, and PLP deficit is reversed in cuprizone-lesioned animals 6 h after the administration of ACPD. Western blot data analyzed by ANOVA; ***significantly different from saline-injected control at p < 0.0005; *significantly different at p < 0.02. Each Western blot lane is from the corpus callosum of a single mouse within one experiment. Each experiment was repeated eight times. Scale bar, 100 μm. d, Quantification of CC1+ oligodendrocytes in the lesioned corpus callosum revealed that ACPD injection does not affect CC1+ cell numbers; N = 3.
Figure 2.
Figure 2.
The ACPD effect on the expression of myelin proteins in the demyelinated corpus callosum is inhibited by TrkB-Fc. Wild-type mice were fed control or cuprizone-laden food for 4 weeks, then stereotaxically injected with either 0.9% saline or ACPD, along with 1 μg TrkB-Fc or vehicle. a, Western blots demonstrate MBP and MAG protein levels in the corpus callosum of wild-type mice subjected to a 4 week cuprizone lesion. GAPDH is shown as a loading control. b, Graphs represent a densitometric analysis of Western blots normalized to GAPDH and are presented as percentage saline-injected control. Data analyzed by ANOVA; *significantly different from saline-injected control at p < 0.0002; ***significantly different at p < 0.0005. Each Western blot lane is from the corpus callosum of a single mouse within one experiment. Each experiment was repeated four times.
Figure 3.
Figure 3.
Group I and Group II metabotropic glutamate receptor expression is increased in the cuprizone-lesioned corpus callosum. Wild-type mice were fed control or cuprizone-laden food for 4 weeks, then stereotaxically injected with either saline vehicle or ACPD. a, Western blots demonstrate mGluR1, mGluR5, and mGluR2/3 protein levels in the control and cuprizone-lesioned corpus callsoum. Beta-tubulin is shown as a loading control. b, Graphs represent a densitometric analysis of Western blots normalized to GAPDH and are presented as percentage saline-injected control. c, Immunohistochemical visualization of mGluRs and CC1+ oligodendrocytes. Western blot data analyzed by ANOVA; *significantly different from saline-injected control at p < 0.05. Each Western blot lane is from the corpus callosum of a single mouse within one experiment. Each experiment was repeated four times. Scale bar, 20 μm.
Figure 4.
Figure 4.
NF-L+ axonal tracts colocalize with the Group II metabotropic glutamate receptors mGluR2/3 following a demyelinating cuprizone lesion. Wild-type mice were fed control or cuprizone-laden food for 4 weeks. NF-L+ axons fail to colocalize with (a) mGluR1, or (b) mGluR5. However, there is extensive colocalization of NF-L+ axonal tracts with mGluR2/3 immunoreactivity after cuprizone (c). These are representative images seen in four independent experiments. Scale bar, 20 μm.
Figure 5.
Figure 5.
GFAP+ astrocytes express the Group I metabotropic glutamate receptors mGluR1 and mGluR5 in the cuprizone-lesioned corpus callosum. Wild-type mice were fed control or cuprizone-laden food for 4 weeks. There is an increase in the number of GFAP+ astrocytes at this stage of demyelination. These GFAP+ cells colocalize extensively with (a) mGluR1, and (b) mGluR5, but fail to express mGluR2/3 (c). These are representative images seen in four independent experiments. Scale bar, 20 μm.
Figure 6.
Figure 6.
GFAP+ astrocytes express BDNF in the cuprizone-lesioned corpus callosum. Numbers of GFAP+ cells increase after ACPD. a, GFAP+ astrocytes in the cuprizone-lesioned corpus callosum colocalize with BDNF immunoreactivity. NF-L+ axonal tracts are not reactive for BDNF. b, HA-BDNF mice fed control or cuprizone feed for 4 weeks exhibit GFAP and HA immunoreactivity in only a small subset of the GFAP+ cells (arrows) in control mice or those fed cuprizone, whereas HA is predominantly found in non-GFAP+ cells (arrowhead) in controls. After cuprizone, the HA+ non-GFAP+ cells are greatly reduced. When cuprizone-treated mice are injected with ACPD, there is an increase in HA immunoreactivity associated with GFAP+ cells. These are representative images seen in three independent experiments. Scale bar, 20 μm.
Figure 7.
Figure 7.
ACPD-elicited increases in BDNF protein are reduced in cuprizone-treated animals after cre recombination. Cre recombinase + GFAPcreERT2-floxBDNF-ROSA26 mice and Cre recombinase − floxBDNF-ROSA26 controls were injected with tamoxifen and fed cuprizone-laden food for 4 weeks before they received a single stereotaxic injection of either ACPD or saline vehicle. a, Western blots demonstrate BDNF protein in the corpus callosum of these animals. GAPDH is shown as a loading control. b, Graph represents a densitometric analysis of a Western blot of mature BDNF normalized to GAPDH and presented as percentage Cre− saline-injected control. c, Cre recombination results in a decrease in elevations of ACPD-elicited BDNF. Immunohistochemical analysis demonstrates that BDNF is eliminated from ACPD-treated GFAP+ astrocytes in the corpus callosum of cuprizone-treated Cre+ GFAPcreERT2-floxBDNF-ROSA26 mice. Western blot data were analyzed by ANOVA; *significantly different from saline-injected, cre− controls at p < 0.01; ***significantly different at p < 0.01. Each Western blot lane is from the corpus callosum of a single mouse within one experiment. Each experiment was repeated four times. Scale bar, 20 μm.
Figure 8.
Figure 8.
The ACPD effect on myelin protein expression is blocked when astrocyte-derived BDNF is reduced. Cre recombinase + GFAPcreERT2-floxBDNF-ROSA26 mice and Cre recombinase − floxBDNF-ROSA26 controls were injected with tamoxifen and fed cuprizone-laden food for 4 weeks before they received a single stereotaxic injection of either ACPD or saline vehicle. a, Western blots demonstrate MBP and MAG protein in the corpus callosum of these mice. GAPDH is shown as a loading control. b, Graph represents a densitometric analysis of Western blots normalized to GAPDH and are presented as percentage cre- saline-injected control. Western blot data analyzed by ANOVA; *significantly different from saline-injected, cre- controls at p < 0.01; ***significantly different at p < 0.01. Each Western blot lane is from the corpus callosum of a single mouse within one experiment. Each experiment was repeated four times.
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