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Overexpression of calbindin-D_28K in hippocampal progenitor cells increases neuronal differentiation and neurite outgrowth

Ju Hee Kim^*, Jin-A Lee, Young Mok Song, Chang-Hwan Park, Se-Jin Hwang^||, Yong-Seok Kim^*, Bong-Kiun Kaang and Hyeon Son^*,1

^* Department of Biochemistry and Molecular Biology,
Microbiology,
^|| Anatomy and Cell Biology, Hanyang University College of Medicine, Seoul, Korea;
National Research Laboratory of Neurobiology, Institute of Molecular Biology and Genetics, School of Biological Sciences, College of Natural Sciences, Seoul National University, Seoul, Korea; and
Department of Neurology, Seoul National University Hospital & College of Medicine, Seoul, Korea

¹Correspondence: Department of Biochemistry and Molecular Biology, Hanyang University College of Medicine, 17 Haengdang-dong, Sungdong-gu, Seoul 133-791, Korea. E-mail: hyeonson{at}hanyang.ac.kr

SPECIFIC AIMS

It has been postulated that calcium itself may control the development of its own regulating proteins to modulate Ca²⁺ transients which in turn influence neuronal differentiation. Calbindin-D_28K, a calcium binding protein (CaBP), buffers intracellular Ca²⁺ and modifies synaptic functions in neurons. Therefore, calbindin-D_28K overexpression may alter neural differentiation during development, but its effects on the regulation of activity-induced neuronal differentiation and related biochemical modifications remain unsolved. In the present study, we aimed to investigate whether changes in protein level of calbindin-D_28K influence hippocampal neuronal differentiation and, if so, what could be the biochemical mechanism underlying it.

PRINCIPAL FINDINGS

1. Calbindin-D_28K overexpression induces neuronal differentiation
We performed the gain-of-function experiments using a plasmid containing full-length calbindin cDNA (EF1-calbindin-EGFP), a replication-incompetent retroviral vector containing full-length calbindin cDNA (rt-calbindin-EGFP) and the marker gene EGFP (Fig. 1 ). Calbindin-D_28K overexpression elicited higher expressions of calbindin mRNA (Fig. 1B ) and protein (Fig. 1A, C ) than controls. The calbindin-transfected cells had longer processes (860.3±89.4 µm) than with EF1-EGFP-transfected cells (513.5±26.4 µm).

View larger version (66K): [in this window] [in a new window]   Figure 1. Calbindin-D28K overexpression induces neurite outgrowth and neuronal differentiation. A) Left panel: Retroviruses transduced nearly 100% of proliferating progenitor cells as indicated by GFP staining (green). Total cells are indicated by DAPI (blue). Right panel: arrows, colabeling of GFP and calbindin. Levels of calbindin mRNA by quantitative rtPCR (B) and protein (C) in control vector- and calbindin-transduced cells (n=5). D) Calbindin-transfected GFP(+) cells show extended neurite outgrowth compared with control cells. d1) Length of primary neurites. *Cell bodies; arrows, path of neurites. E, F) Calbindin-transduced neural progenitors selectively differentiate toward a neuronal rather than an astrocytic phenotype in high-density cultures. GFP (green), Tuj1 or GFAP (red). Arrows: colabeling of GFP and Tuj1 or GFAP. Arrowheads: single labeled cells. G) Quantitation of the doubly labeled cells for GFP and Tuj1 or GFAP (n=4, total 1500 cells in each condition). H) Quantitative mRNA analysis of ß-tubulin type III by rtPCR (n=5). I) MAP2 and p-CaMKII immunoreactivities were increased in calbindin-transduced cells. B, d1, G,H) **P < 0.01, ***P < 0.001 (Student’s t test). Error bars ± SE. CTL, control.

Calbindin-D_28K overexpression significantly increased the fraction of cells colabeled with a neuron-specific class III ß-tubulin protein (Tuj1) among GFP (+) cells compared with that of control cells transduced with rt-EGFP [Fig. 1E, G : rt-EGFP, 11.6±1.7%; rt-calbindin-EGFP, 37.6±2.3%; P<0.001]. Calbindin overexpression, however, significantly decreased the fraction of cells colabeled with GFAP, an astrocytic marker, among GFP(+) cells [Fig. 1F, G : rt-EGFP, 26.2±3.9%; rt-calbindin-EGFP, 6.3±0.9%; P<0.001], indicating that calbindin-D_28K facilitated neurogenesis over gliogenesis. Calbindin-D_28K overexpression significantly increased the level of ß-tubulin type III transcript (Fig. 1H ) and the expression of MAP2 (Fig. 1I ). Calbindin overexpression induced phosphorylation of CaMKII but not ERK (Fig. 1I ).

2. Calbindin d-siRNA blocks both endogeneous and overexpressed calbindin-D_28K
To knock down endogeneous calbindin-D_28K expression with a novel gene silencing strategy, recombinant dicer protein was used to create a set of double-stranded 21-mers from a 500 base double-stranded RNA template that encodes part of calbindin-D_28K mRNA. The cotransfection of EF1-calbindin-EGFP and calbindin d-siRNA caused almost 100% knockdown, indicating that calbindin d-siRNA blocked the expression of both endogeneous and exogeneous calbindin-D_28K. Compared with control neurons transfected with EF1-EGFP alone, significant decreases in neurite outgrowth and the number of Tuj1(+) cells among GFP(+) cells were observed in neurons cotransfected with d-siRNAs targeting calbindin, suggesting that blocking the expression of endogeneous calbindin by calbindin d-siRNA efficiently decreased neurite outgrowth and neuronal differentiation. Cotransfection of calbindin-D_28K plasmid with calbindin-D_28K d-siRNA profoundly decreased the number of Tuj1(+) cells among GFP(+) cells and neurite outgrowth. Taken together, these results suggest that calbindin-D_28K might be critically involved in promoting neuronal differentiation and neurite outgrowth.

3. Calbindin-D_28K promotes neuronal differentiation in a CaMKII-dependent manner
To investigate the involvement of CaMKII in calbindin-D_28K-induced neurite elongation, the pharmacological agent KN-62 was used. In the absence of KN-62, calbindin-transduction increased the number of Tuj1(+) cells among GFP(+) cells, consistent with the results shown in Fig. 1E . However, in the presence of KN-62, calbindin-transduced GFP(+) cells were not able to differentiate into neurons. The mRNA expressions of Mash1, NeuroD, and Pax6, which positively regulate hippocampal neuronal development, were significantly increased in the calbindin-D_28K overexpressing cells compared with KN-62-treated calbindin-D_28K overexpressing cells, indicating that calbindin-D_28K facilitates neuronal differentiation via up-regulation of genes such as NeuroD, Pax6, and Mash1.

To confirm the differentiating potential of calbindin-expressing progenitors, we used clonal analysis with fetal hippocampal progenitors transduced with retrovirus before passage. The fraction of clones having > 50% Tuj1(+) cells among total EGFP(+) cells of each clone was increased by fourfold in calbindin-overexpressing clones, compared with control vector clones or KN-62-treated, calbindin-transduced clones. These results confirm that calbindin increases the differentiation of hippocampal neural progenitor cells toward a neuronal phenotype.

The increase of expression of Tuj1 in calbindin-D_28K-transduced cells was inhibited by KN-62, and the phosphorylation of CaMKII was greatly diminished by KN-62 to the level of control cells. Calbindin-transduction was accompanied by increases in the pSer³³⁶NeuroD in a primarily CaMK-dependent manner. An analysis of the calbindin-transduced cells showed a marked correlation between the sites where p-CaMKII and p-NeuroD were enriched in nucleus. Thus, p-CaMKII appears to be in the best possible location to locally regulate phosphorylation of NeuroD.

4. Vitamin D₃ induces calbindin-D_28K, neurite outgrowth, and neuronal differentiation
Induction of calbindin-D_28K in neurons is known to be 1, 25-dihydroxy-vitamin-D₃ (vitD₃)-dependent. The addition of vitD₃ at 200 nM increased both the extent of neurite outgrowth in Tuj1(+) cells and the percentage of Tuj1(+) cells in total cells. The effects of calbindin d-siRNA on neurite outgrowth and neuronal differentiation were inhibited by vitD₃. Vitamin D₃ induced calbindin-D_28K protein in a concentration-dependent manner with maximal expression at 200 nM. Furthermore, vitD₃ induced phosphorylation of NeuroD in a concentration-dependent manner, and phosphorylation of CaMKII was also greatly enhanced by vitD₃, thus indicating that vitD₃ induces calbindin-D_28K and subsequently its function. Treatment of differentiated progenitors with KCl elicited higher expression of calbindin mRNA and protein than control 72 h after single treatment, indicating that calcium itself might control the expression of calbindin-D_28K.

To further confirm the contribution of calbindin-expressing cells to the neurogenic potential of adult hippocampal progenitor cells, we performed similar experiments in vitro in progenitor cells derived from adult hippocampus. Calbindin-D_28K overexpression significantly increased the fraction of cells colabeled with Tuj1 among GFP (+) cells, compared with that of cells transduced with rt-EGFP, whereas it significantly decreased the fraction of cells colabeled with GFAP among GFP(+) cells, indicating that calbindin-D_28K facilitated neurogenesis over gliogenesis. Similarly to the results observed in fetal progenitors, calbindin-transduced cells showed a marked correlation between the sites where p-CaMKII and p-NeuroD were enriched in nucleus compared with cells transduced by control vector.

CONCLUSIONS AND SIGNIFICANCE

In the present study, we demonstrated that calbindin-D_28K promoted the neuronal differentiation of fetal and adult hippocampal progenitor cells in primary cultures. We also demonstrated that adjusting the level of a single CaBP in neural progenitor cell population could alter cell fate and that calbindin expressing cells in vitro might be present in multiple lineages or else a single lineage undergoes a phenotypic change from Tuj1 nonexpressing to Tuj1 expressing. Moreover, a series of experiment using d-siRNA demonstrated that calbindin is effective at physiologically relevant levels. The increased expression of calbindin in response to KCl and vitD₃ further supports that protein level of calbindin can be regulated by neuronal activity and circulating vitD₃ at normal physiological conditions in brain.

A possible mechanism for the effect of calbindin-D_28K on neuronal differentiation is that calbindin-D_28K overexpression increased baseline synaptic transmission. Free intracellular calcium can inactivate high-voltage calcium currents, and such feedback inhibition increases as calbindin levels decrease. Therefore, the overexpression of Ca²⁺ binding protein calbindin-D_28K should decrease such inhibition, resulting in an increase of Ca²⁺ currents (Fig. 2 ). If this is the case, then the next question concerns how an increase of [Ca²⁺]_i could possibly facilitate neuronal differentiation. A recent report indicates that excitatory stimuli can potently induce neuronal production in the adult hippocampal progenitor cells, and that NeuroD expression is increased while the expression of the glial fate genes such as Hes1 and Id2 is repressed. In support of this possibility, we observed in the present study that positive bHLH transcriptional factors, NeuroD and Mash1, were up-regulated, while negative bHLH genes, Hes1 and Hes5, were down-regulated by calbindin-D_28K overexpression.

View larger version (26K): [in this window] [in a new window]   Figure 2. Schematic diagram showing possible roles of calbindin-D28K in neuronal differentiation and neurite outgrowth. Activity-induced increase in free Ca2+ induces the expression of endogeneous calbindin-D28K. Further increase of free calcium might occur as a result of increased Ca2+ current following the increased calbindin expression [1]. An increase in [Ca2+]i activates Ca2+-dependent processes such as an activation of CaMKs. Activation of CaMKs might be involved in the expression of bHLH transcriptional factors via yet unknown mechanisms [2]. Activation of CaMKs, possibly CaMKII in the majority, induces phosphorylation of NeuroD, followed by neurite outgrowth [3]. Astrocytic differentiation might be inhibited in calbindin-overexpressing cells via yet unknown mechanisms [4].

Calcium-dependent phosphorylation of CaMKII and CaMKII-mediated phosphorylation of NeuroD at Ser³³⁶ might be important for neurite outgrowth. In cerebellar granule cells, neuronal activity induces CaMKII-mediated phosphorylation of NeuroD, and pSer³³⁶-NeuroD is necessary for subsequent dendritogenesis. This function is activity-dependent, but separate from the transcriptional activity of NeuroD. The role p-Ser³³⁶NeuroD for the differentiation/production of neurons needs to be clarified in the future.

Although calbindin-overexpressing neural progenitor cells are uniform in vitro with regard to Tuj1 expression, there might be some differences during development between in vitro and in vivo differentiation of embryonic and adult progenitors. If in vitro differentiation and maturation of hippocamapal neurons, including granule cells, resemble in vivo maturation of neurons, our results indicate that an endogeneous expression of calbindin-D_28K may be of functional importance during neuronal differentiation both in vivo embryonic and possibly adult neurogenesis. However, the degree of how much in vitro neurogenic potential reflects its participation in in vivo neurogenesis is not certain, and additional studies are required to determine whether calbindin-expressing cells contribute to adult hippocampal neurogenesis in vivo.

In conclusion, our results constitute one of the first reports on the role of calbindin in neuronal differentiation. As evidenced by its association with synaptic plasticity, calbindin-D_28K might be an important molecule in hippocampal neuronal differentiation and neural plasticity.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4826fje;

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