Robert F. Bulleit

The expression of MEF2 genes is implicated in CNS neuronal differentiation

The myocyte enhancer factor-2 (MEF2) proteins are transcription factors required for muscle differentiation. In the present study we examined MEF2 expression in developing cerebellar granule neurons. In the developing postnatal cerebellum, RNA blot analysis revealed that MEF2A and MEF2D RNA levels increase after birth. The majority of this increase occurs around postnatal day 9 reaching a peak at postnatal day 15-18 which is maintained in adults. This time course of expression coincides with the expression of GABA(A) receptor alpha6 subunit RNA, a marker for the differentiation of the mature cerebellar granule neurons. We further observed, using the polyclonal antibody generated against an MEF2A peptide, that MEF2 protein expression occurs primarily in the internal granule cell layer of the developing cerebellum. Thus, MEF2 expression increases as granule neurons differentiate and mature. Experiments also indicated that MEF2 expression not only occurs in the cerebellum but also in other regions of the CNS. In adult mice, expression of RNA for the MEF2 isoforms A, C and D occurs throughout the CNS. MEF2A and D expression occurs at highest levels in the olfactory bulb, hippocampus and cerebellum. The expression of MEF2C differs with low levels of expression in the cerebellum and hindbrain. Using the MEF2A polyclonal antibody, we observed a similar adult pattern of expression for the MEF2 protein with high level of expression in the olfactory bulb, cortex, hippocampus, thalamus and cerebellum. These observations suggest that MEF2 molecules may be an important factor involved in CNS neuron differentiation similar to their role in muscle differentiation.

Insulin-like growth factor I (IGF-I) is a critical trophic factor for developing cerebellar granule cells

In this study we showed that insulin-like growth factor I (IGF-I) directly increased cell survival in pure cerebellar granule cell cultures established from postnatal day 7 (P7) mice. The maximal survival-promoting effect could be obtained at low IGF-I concentrations (3-5 ng/ml). Withdrawal of IGF-I from differentiated granule neurons resulted in neuronal death which suggests that IGF-I has a survival-promoting effect on differentiated granule neurons. Furthermore, the survival-promoting effect of IGF-I was not attenuated by the addition of K252a, a selective blocker of Trk signaling, indicating that the survival-promoting effect of IGF-I did not require or was not mediated by endogenously produced neurotrophins, such as BDNF and NT3. Further experiments also suggest that IGF-I stimulates proliferation of granule cell precursors and allows terminal granule neuron differentiation to occur, as indicated by the expression of terminal differentiation markers MEF2A and GABA(A) alpha6. Thus, IGF-I could potentially function as both a mitogen and a trophic factor for developing granule cells. This dual action of IGF-I may be important in regulating granule neuron number.

Inhibition of glycogen synthase kinase 3β activity regulates proliferation of cultured cerebellar granule cells

Insulin-like growth factor I (IGF-I) is mitogenic for several types of neuronal progenitors including cerebellar granule neuron progenitors. The present study confirms that IGF-I can function as a mitogen in purified cultures of cerebellar granule cells and identifies intracellular signal transduction molecules that mediate this mitogenesis. In cultured granule cells, IGF-I inhibits GSK-3 activity and leads to phosphorylation of serine9 an inhibitory site on GSK-3beta. Phosphoinositide 3-kinase (PI3-K) activation by IGF-I can lead to phosphorylation and inactivation of GSK-3. A PI3-K inhibitor, LY294002, completely inhibited IGF-I-induced proliferation with half-maximal inhibition occurring at a concentration (1.5 micrograms) close to its reported IC50 value for inhibition of PI3-K. Lithium chloride (LiCl), a direct inhibitor of GSK-3beta, can alone stimulate granule cell proliferation and enhance proliferation induced by IGF-I. LiCl can reverse the inhibitory effect of LY294002 on granule cell proliferation suggesting that GSK-3 inhibition may be downstream of PI3-K activation in IGF-Is mitogenic pathway. Experiments further show that the expression of a dominant active form of GSK-3beta antagonizes IGF-I-induced mitogenesis. These studies support a role for inhibition of GSK-3beta activity in the signal transduction pathway by which IGF-I regulates granule neuron progenitor proliferation.

MEK inhibitors block BDNF-dependent and -independent expression of GABAA receptor subunit mRNAs in cultured mouse cerebellar granule neurons

Brain-derived neurotrophic factor (BDNF) can regulate the maturation of developing cerebellar granule neurons. Within 1-2 days of culture, BDNF induces the expression of granule neuron terminal differentiation markers, particularly GABA(A) receptor alpha6 subunit (GABA(A)alpha6) mRNA. Other trophic factors including insulin-like growth factor, the neurotrophin NT-3, pituitary adenylate cyclase-activating polypeptide (PACAP), and fetal bovine serum failed to induce this early expression. The expression of other GABA(A) receptor subunits, including alpha1 and gamma2, was also enhanced by exposure of developing granule neurons to BDNF. This BDNF-dependent expression of GABA(A) receptor subunit mRNAs could be effectively blocked by treatment with the mitogen-activated protein kinase kinase (MEK) inhibitors, PD98059 or U0126. In the absence of BDNF, GABA(A)alpha6 expression occurs but not until 3-4 days of culture. This BDNF-independent expression of GABA(A)alpha6 was also inhibited by PD98059. Further studies showed that the BDNF-dependent expression GABA(A)alpha6 could also be reduced by LY294002, an inhibitor of the phosphatidylinositol 3-kinase, or depolarizing concentrations of KCl. These results thus suggest that both BDNF-dependent and -independent expressions of GABA(A) receptor subunits require the activation of MEK and the mitogen-activated protein kinase (MAPK) pathway. However, it is also likely that other signaling pathways modulate this maturation process.

POTASSIUM CHLORIDE INHIBITS PROLIFERATION OF CEREBELLAR GRANULE NEURON PROGENITORS

CNS neurogenesis involves a critical transition where neuronal progenitors exit the cell cycle and initiate terminal differentiation. Recent experiments have suggested that depolarization inhibits DNA synthesis in cortical progenitors. Depolarization of proliferating neuronal progenitors may thus activate mechanisms that prevent proliferation and allow the initiation of terminal differentiation. We present evidence that depolarizing concentrations of KCl (25-50 mM) reduce proliferation of developing postnatal cerebellar granule cells in culture. These studies show that KCl antagonizes the mitogenic response of granule cells to insulin-like growth factor-I (IGF-I) and that this reduction in proliferating cells is not the result of a selective cell death. We also examined the differentiation of granule cell cultures using Brn-5 expression as an early differentiation marker. In vivo Brn-5 expression occurs soon after developing granule cells exit the cell cycle and begin their final differentiation. In control cultures and cultures treated with high concentrations of KCl Brn-5 expression increased over 24-48 h of culture. Our results suggest depolarizing concentrations of KCl antagonize proliferation of cerebellar granule neuron progenitors however allow their continued differentiation.

Cell intrinsic mechanisms regulate mouse cerebellar granule neuron differentiation

Cerebellar granule cells isolated from postnatal day 7 mice, and cultured in minimal medium containing only insulin-like growth factor-I (IGF-I), both survive and differentiate. This differentiation is marked by neurite growth and expression of genes associated with terminal differentiation, the myocyte-specific enhancer factor 2A (MEF2A) and the alpha 6 subunit of the gamma-aminobutyric acidA receptor (GABAA alpha 6). Percoll gradient purified granule cells maintained without IGF-I, in minimal medium alone or in medium containing the antioxidant N-acetylcysteine (NAC), also express MEF2A and GABAA alpha 6. Thus, cultured granule neurons can differentiate to some extent cell-autonomously and IGF-I may not be a critical factor for this process.

BDNF ACCELERATES GENE EXPRESSION IN CULTURED CEREBELLAR GRANULE NEURONS

This study reports that in purified cultures of postnatal cerebellar granule cells, BDNF significantly accelerated GABAA receptor alpha 6 subunit (GABAA alpha 6) mRNA expression, a marker for terminally differentiated cerebellar granule neurons, and also accelerated p21cip1 expression. p21cip1 is a general cyclin-dependent kinase (Cdk) inhibitor that can inhibit progression through the cell cycle. Alternatively, the expression of p27kip1, another Cdk inhibitor closely related to p21cip1, is not modified by BDNF. In cultured granule cells, the increase in p21cip1 expression induced by BDNF occurred after dividing granule cells had left the cell cycle and thus was not required to direct granule neuron precursors out of the cell cycle. p21cip1 may have an alterative function during granule neuron terminal differentiation, separate from its ability to regulate cell cycle exit. This report shows that, in vitro, BDNF accelerates granule cell gene expression and may thus modulate cerebellar granule cell differentiation.

Expression of the POU transcription factor Brn-5 is an early event in the terminal differentiation of CNS neurons.

The POU domain transcription factors are a subgroup of homeodomain proteins which have two independent domains that function cooperatively in sequence‐specific DNA binding. Recent experiments suggest that POU proteins may be critical regulators of central nervous system (CNS) neuronal differentiation. The present study evaluated the temporal and spatial expression pattern of the POU protein Brn‐5 in the developing and mature mouse CNS. Immunocytochemistry using a Brn‐5‐specific antiserum revealed that Brn‐5 expression occurs in many CNS neuron populations. In the developing CNS its expression occurs in postmitotic neurons but not in proliferating neuronal progenitors. Experiments in the developing retina suggest that expression of Brn‐5 occurs 24‐48 hours after progenitors exit the cell cycle. Thus, the expression of Brn‐5 appears to be an early event in the process of terminal neuronal differentiation. Brn‐5 may function as a transcriptional regulator involved in specifying the mature phenotype of CNS neurons. J. Neurosci. Res. 52:625–632, 1998.

A novel gene selectively expressed in the cerebellum

A cDNA clone encoding a novel protein (Cer-1) was isolated from a mouse cerebellar cDNA library. The deduced amino acid sequence of Cer-1 is not homologous to other sequences in the protein sequence data base. RNA blot analysis suggests that expression of Cer-1 RNA occurs selectively in the adult cerebellum and in purified cultures of cerebellar granule neurons. An increase in its expression correlates with the timing of granule neuron differentiation in the cerebellum. Thus, the expression of Cer-1 may provide a selective marker for terminal differentiation of cerebellar granule neurons.