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. 2016 Apr 27:10:104.
doi: 10.3389/fncel.2016.00104. eCollection 2016.

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation

Ana P B Araujo  1 , Luan P Diniz  1 , Cristiane M Eller  1 , Beatriz G de Matos  1 , Rodrigo Martinez  2 , Flávia C A Gomes  1
Affiliations

Effects of Transforming Growth Factor Beta 1 in Cerebellar Development: Role in Synapse Formation

Ana P B Araujo et al. Front Cell Neurosci. .

Abstract

Granule cells (GC) are the most numerous glutamatergic neurons in the cerebellar cortex and represent almost half of the neurons of the central nervous system. Despite recent advances, the mechanisms of how the glutamatergic synapses are formed in the cerebellum remain unclear. Among the TGF-β family, TGF-beta 1 (TGF-β1) has been described as a synaptogenic molecule in invertebrates and in the vertebrate peripheral nervous system. A recent paper from our group demonstrated that TGF-β1 increases the excitatory synapse formation in cortical neurons. Here, we investigated the role of TGF-β1 in glutamatergic cerebellar neurons. We showed that the expression profile of TGF-β1 and its receptor, TβRII, in the cerebellum is consistent with a role in synapse formation in vitro and in vivo. It is low in the early postnatal days (P1-P9), increases after postnatal day 12 (P12), and remains high until adulthood (P30). We also found that granule neurons express the TGF-β receptor mRNA and protein, suggesting that they may be responsive to the synaptogenic effect of TGF-β1. Treatment of granular cell cultures with TGF-β1 increased the number of glutamatergic excitatory synapses by 100%, as shown by immunocytochemistry assays for presynaptic (synaptophysin) and post-synaptic (PSD-95) proteins. This effect was dependent on TβRI activation because addition of a pharmacological inhibitor of TGF-β, SB-431542, impaired the formation of synapses between granular neurons. Together, these findings suggest that TGF-β1 has a specific key function in the cerebellum through regulation of excitatory synapse formation between granule neurons.

Keywords: TGF-β1; cerebellum; development; excitatory synapse.

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Figures

FIGURE 1
FIGURE 1
The expression of TGF-β and TGF-β receptor during early postnatal cerebellar development. mRNA levels of TGF-β1, TGF-β2, TGF-β3, (A) and TβRII (B) were analyzed by qPCR in the cerebellum of mice at different postnatal ages (1, 3, 6, 9, 12, 15, and 30 days). Data are presented as the mean ± error relative to β-actin gene expression (n = 3). TβRII distribution was analyzed in sagittal sections of the postnatal day 6 (P6) mouse cerebellum stained with anti-calbindin (C), anti-β-tubulin III (G) and anti-TβRII (D,H). Note that TβRII colocalizes with calbindin (E,F) in the PCL and with the β-tubulin III neuron marker in the ML (I,J). Scale bar: 40 μm (C,G) and 20 μm (F,J).
FIGURE 2
FIGURE 2
Granule cells are responsive to TGF-β1 in vitro. Granule cells obtained from P6 mice were cultured for 24 h, and the distribution of TβRII and levels of TβRII and TGF-β1 were evaluated by immunocytochemistry (A–C) and PCR (D). Confocal microscopy identified the presence of TβRII throughout the neuronal membrane (A–C). Scale bar: 20 μm. PCR assays demonstrated that GCs express TβRII and TGF-β1 in vitro (D). Treatment of GCs with TGF-β1 (10 ng/mL) for 15 min increased the levels of p-SMAD, a hallmark of TGF-β1 pathway activation (E,F). ∗P < 0.05.
FIGURE 3
FIGURE 3
TGF-β1 does not induce neuronal migration. Cerebellar neuronal migration was assessed with 2 approaches: cerebellar explants (A–C) and cerebellar slices (D–G). (A–C) Cerebellar explants derived from P7 rats were cultured for 48 h in Neurobasal medium supplemented with 2% B27 alone (A) or in the presence of 10 ng/mL of TGF-β1 (B). After that, neuronal migration distance was measured at 6, 12, 24, and 48 h (C). (D–G) Cerebellar slices derived from P7 rats were cultured in Neurobasal medium supplemented with 2% B27 alone (D) and in the presence of 10 ng/mL of TGF-β1 (E) or 10 ng/mL of EGF (F). Neuronal migration was evaluated after 24 h. The results represent the mean of 3 independent experiments. TGF-β1 did not interfere with neuronal migration in either methodological approach. Scale bars: 100 μm. ∗P < 0.05.
FIGURE 4
FIGURE 4
TGF-β1 induces synapse formation between cerebellar neurons in vitro. GCs derived from mice cerebella of P6 were cultured for 5 days in the presence of DMEM-F12 medium (A) and supplemented with 10 ng/mL of TGF-β1 (B) or the pharmacological inhibitor of TβRII, SB-431542, (C) for an additional 24 h. Synapse formation was evaluated by counting the number of synaptophysin/PSD-95 colocalization puncta (D). Levels of synaptic proteins were evaluated by qPCR assays (E,F) and Western blotting (G,H). Scale bars, 10 μm. ∗P < 0.05. TGF-β1 increased the number of excitatory cerebellar synapses and the levels of the pre- and post-synapse proteins, synaptophysin and PSD-95, respectively. ∗∗∗P < 0.001.
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