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BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV - PubMed

  • ️Thu Jan 01 2009

BDNF-mediated cerebellar granule cell development is impaired in mice null for CaMKK2 or CaMKIV

Manabu Kokubo et al. J Neurosci. 2009.

Abstract

The Ca(2+)/calmodulin-activated kinases CaMKK2 and CaMKIV are highly expressed in the brain where they play important roles in activating intracellular responses to elevated Ca(2+). To address the biological functions of Ca(2+) signaling via these kinases during brain development, we have examined cerebellar development in mice null for CaMKK2 or CaMKIV. Here, we demonstrate that CaMKK2/CaMKIV-dependent phosphorylation of cAMP response element-binding protein (CREB) correlates with Bdnf transcription, which is required for normal development of cerebellar granule cell neurons. We show in vivo and in vitro that the absence of either CaMKK2 or CaMKIV disrupts the ability of developing cerebellar granule cells in the external granule cell layer to cease proliferation and begin migration to the internal granule cell layer. Furthermore, loss of CaMKK2 or CaMKIV results in decreased CREB phosphorylation (pCREB), Bdnf exon I and IV-containing mRNAs, and brain-derived neurotrophic factor (BDNF) protein in cerebellar granule cell neurons. Reexpression of CaMKK2 or CaMKIV in granule cells that lack CaMKK2 or CaMKIV, respectively, restores pCREB and BDNF to wild-type levels and addition of BDNF rescues granule cell migration in vitro. These results reveal a previously undefined role for a CaMKK2/CaMKIV cascade involved in cerebellar granule cell development and show specifically that Ca(2+)-dependent regulation of BDNF through CaMKK2/CaMKIV is required for this process.

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Figures

Figure 1.
Figure 1.

Planimetric analysis of the cerebellum from adult WT, Camk4−/−, and Camkk2−/− mice. A, Mid-sagittal cerebellar sections from 3-month-old WT, Camk4−/−, and Camkk2−/− mice were stained with cresyl violet and photographed at 1× magnification. Shown are representative cerebellar sections (scale bars, 1 mm). B, The percentage of the IGL cross-sectional area compared with the whole cerebellum was quantified as described in Materials and Methods. The percentage of the cerebellar cross-sectional area relegated to the IGL was decreased from 44.3% in the wild-type to 38.4% in the Camk4−/− and 37.5% in the Camkk2−/− mice. Values shown are mean ± SEM (*p < 0.01 for difference against WT; n = 5 for each genotype). C, Three-month-old Camk4−/− and Camkk2−/− mice show no difference in IGL cell density compared with WT (values shown are mean ± SEM; n = 5 for each genotype).

Figure 2.
Figure 2.

Structural analysis of the cerebellum from postnatal 7-d-old WT, Camk4−/−, and Camkk2−/− mice. A, Mid-sagittal sections from P7 WT, Camk4−/−, and Camkk2−/− mice were stained with cresyl violet and photographed at 1× magnification. Shown are representative cerebellar sections (scale bars, 1 mm). B, Representative sections of cerebella derived from P7 WT, Camk4−/−, and Camkk2−/− mice. Shown at 20× magnification is the mid-portion of the fifth folia. Note the abnormally thicker EGL in both Camk4−/− and Camkk2−/− mice (scale bars, 100 μm). C, The percentage of the EGL cross-sectional area compared with the whole cerebellum was quantified as described in Materials and Methods. The percentage of the cerebellar cross-sectional area relegated to the EGL was increased from 13.4% in the WT to 18.4% in the Camk4−/− and 18.6% in the Camkk2−/− mice. Values shown are mean ± SEM (*p < 0.01 for difference against WT; n = 5 for each genotype). D, Planimetric measurement of the EGL width shows a significant increase in the Camk4−/− and Camkk2−/− mice compared with WT. Values shown are mean ± SEM (*p < 0.05 for difference against WT; n = 5 for each genotype). E, Seven-day-old Camk4−/− and Camkk2−/− mice show no difference in EGL cell density compared with WT (values shown are mean ± SEM; n = 5 for each genotype).

Figure 3.
Figure 3.

Camk4−/− and Camkk2−/− mice have increased cell proliferation and apoptosis in the EGL at postnatal day 7. A, Representative photograph showing the identification of proliferating cells after immunolabeling against the Ki-67 nuclear antigen. Note the relative increase in cells staining positive in the sections from both the Camk4−/− and Camkk2−/− mice (scale bars, 100 μm). B, The left panel shows the quantification of the total percentage of Ki-67-positive cells in the EGL area. The right panel shows that there was no difference in the average staining intensity of the cells, thus confirming the increase in proliferating cells from both null mice. Values shown are mean ± SEM (*p < 0.05 for difference against WT; p = 0.3 for difference in staining intensity; n = 4 for each genotype). C, The top is a representative photograph of apoptotic GCPs in wild-type, Camk4−/−, and Camkk2−/− mice identified by Tunnel staining (scale bars, 50 μm). The bottom is a higher magnification to demonstrate that the nuclei are indeed positively stained. D, Quantification showing an increase in the number of TUNEL-positive cells per square millimeter in the EGL of Camk4−/− and Camkk2−/− mice compared with WT. Values shown are mean ± SEM (*p < 0.01 for difference against WT; n = 4 for each genotype).

Figure 4.
Figure 4.

In vivo analysis of migrating granule cells. A, Proliferating GCPs in P7 WT, Camk4−/−, and Camkk2−/− mice were labeled by systemic injection of 50 mg/kg BrdU. Cerebella were then collected and processed for BrdU immunohistochemistry at 24 and 48 h after injection. DAPI stain (data not shown) was used to aid in identification of each cellular layer (scale bar, 50 μm). B, Quantification of BrdU-labeled GCPs at 48 h within the three layers of the developing cerebellum. The number of labeled cells in each layer of the cerebellum (EGL, ML, and IGL) was counted in nonadjacent mid-sagittal sections and averaged. Both Camk4−/− and Camkk2−/− mice have a significant increase in the number of BrdU-labeled cells which have not migrated out of the EGL and a significant decrease in the number of cells which have migrated to the ML and IGL. Values shown are mean ± SEM (*p < 0.05 for difference against WT; n = 4 mice from each genotype).

Figure 5.
Figure 5.

In vitro analysis of wild-type, Camk4−/−, and Camkk2−/− cerebella using micro-explants. A, Cerebellar micro-explants derived from P3 mice were cultured in vitro. The top panel shows representative micro-explants from the three genotypes and reveal a reduction in the ability of GCPs from the Camk4−/− and Camkk2−/− micro-explants to migrate as far as those from wild type (zone1 = 0–100 μm; zone 2 = 100–200 μm; zone 3 = 200 μm and beyond). The bottom panel shows a representative set of micro-explants where conditioned medium removed from WT micro-explants was used instead of fresh media in the cultures of Camk4−/− and Camkk2−/− micro-explants. Use of the conditioned medium partially rescues the migration defect (scale bars, 200 μm). B, Quantification of the number of GCPs which have migrated to specified distances (zone1 = 0–100 μm; zone 2 = 100–200 μm; zone 3 = 200 μm and beyond). There is a significant reduction in the number of GCPs from both null micro-explants that migrate further than 100 μm in fresh medium. Addition of the conditioned medium to both null micro-explants significantly improves cellular migration compared with the null micro-explants that received fresh medium. This conditioned medium, however, only partially restores the ability of cells from either null micro-explant to migrate as far as does the WT cells. C, Representative set of micro-explants where conditioned medium removed from Camk4−/− and Camkk2−/− micro-explants and placed on cultures of Camkk2−/− and Camk4−/− micro-explants, respectively. D, Quantification showing that conditioned medium from Camk4−/− micro-explants does not rescue Camkk2−/− micro-explants and vice versa. Values shown are mean ± SEM (*p < 0.01 for nonconditioned and conditioned medium difference against WT; **p < 0.05 for Camk4−/− and Camkk2−/− conditioned medium difference against Camk4−/− and Camkk2−/− nonconditioned medium; n = 4 for each genotype).

Figure 6.
Figure 6.

Addition of exogenous BDNF to cerebellar micro-explants restores GCP migration. A, The top panel shows representative micro-explants from the three genotypes again revealing a reduction in the ability of GCPs from the Camk4−/− and Camkk2−/− micro-explants to migrate as far as those from WT (zone1 = 0–100 μm; zone 2 = 100–200 μm; zone 3 = 200 μm and beyond). The addition of exogenous BDNF (middle panel) rescues both null explants by restoring migration of GCPs to levels seen in WT, which did not received exogenous BDNF. Addition of TrkB-Fc blocks the migration of WT explants and mirrors migration of that seen in both the Camk4−/− and Camkk2−/− micro-explants (scale bars, 200 μm). B, Quantification of the number of GCPs which have migrated to specified distances (zone1 = 0–100 μm; zone 2 = 100–200 μm; zone 3 = 200 μm and beyond). As demonstrated in the previous figure, there is a significant reduction in the number of GCPs from both null micro-explants that migrate further than 100 μm in basal media. Addition of the media containing BDNF (100 ng/ml) to both null micro-explants significantly improves migration to normal levels seen in WT micro-explants, which did not receive exogenous BDNF. The addition of exogenous BDNF also results in an increase in migrating cells in the WT micro-explant. Addition of TrkB-Fc inhibits BDNF mediated migration in WT micro-explants. Values shown are mean ± SEM (*p < 0.01 for difference against WT; **p < 0.01 for difference of Camk4−/− and Camkk2−/− + BDNF vs Camk4−/− and Camkk2−/−, #p < 0.05, ##p < 0.01 for difference of WT + BDNF against WT and +p < 0.01 for difference of WT + TrkB-Fc against WT; n = 4 for each experimental genotype).

Figure 7.
Figure 7.

BDNF protein and mRNA are reduced in both Camk4−/− and Camkk2−/− mice. A, ELISA quantification of BDNF in freshly isolated GCP extracts. Values shown are mean ± SEM (*p < 0.01 for difference against WT; n = 6 for each genotype). B, Real-time PCR analysis of total Bdnf mRNA in freshly isolated GCPs. Values shown are mean ± SEM (*p < 0.05 for difference against WT; n = 7 for each genotype). C, Real-time PCR analysis of Bdnf mRNA in freshly isolated GCPs using specific primers to detect transcripts derived from either exon I or exon IV. Values shown are mean ± SEM (*p < 0.01 for difference against WT; n = 7 for each genotype).

Figure 8.
Figure 8.

Lentiviral-mediated reexpression of CaMKIV or CaMKK2 restores pCREB and BDNF. A, Representative immunoblot of protein extracts derived from cultured WT and Camk4−/− GCPs or Camk4−/− GCPs which have been left uninfected, infected with either a lentiviral-CaMKIV-WT (active) or lentiviral-CaMKIV-K71M (inactive) construct. Note lower level of BDNF and pCREB in Camk4−/− GCPs. The reexpression of a catalytically active form of CaMKIV restored both BDNF and pCREB levels, whereas expression of the catalytically inactive CaMKIV-K71M did not restore BDNF or pCREB levels. B, Quantification of immunoblots after normalizing to total CREB (values shown are mean ± SEM; *p < 0.01 for difference against WT; n = 4 independent experiments). C, Representative Western blot analysis of protein extracts derived from cultured WT and Camkk2−/− GCPs or Camkk2−/− GCPs, which have been left uninfected or infected with either a lentiviral-CaMKK2-WT (active) or lentiviral-CaMKK2-K193E (inactive) construct. The lower molecular weight band seen so prominently in the Camkk2−/− lane is CaMKK1. Note that there is also reduced BDNF and pCREB protein in Camkk2−/− GCPs. The reexpression of a catalytically active form of CaMKK2 also restored both BDNF and pCREB levels, whereas expression of the catalytically inactive CaMKK2-K193E did not restore BDNF or pCREB levels. B, Quantification of immunoblots after normalizing to total CREB (values shown are the mean ± SEM; *p < 0.05 for difference against WT; n = 3 independent experiments).

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