Abstract
Human Ntera2/cl.D1 (NT2) cells treated with retinoic acid (RA) differentiate towards a well characterized neuronal phenotype sharing many features with human fetal neurons. In view of the emerging role of caspases in murine stem cell/neural precursor differentiation, caspases activity was evaluated during RA differentiation. Caspase-2, -3 and -9 activity was transiently and selectively increased in differentiating and non-apoptotic NT2-cells. SiRNA-mediated selective silencing of either caspase-2 (si-Casp2) or -9 (si-Casp9) was implemented in order to dissect the role of distinct caspases. The RA-induced expression of neuronal markers, i.e. neural cell adhesion molecule (NCAM), microtubule associated protein-2 (MAP2) and tyrosine hydroxylase (TH) mRNAs and proteins, was decreased in si-Casp9, but markedly increased in si-Casp2 cells. During RA-induced NT2 differentiation, the class III histone deacetylase Sirt1, a putative caspase substrate implicated in the regulation of the proneural bHLH MASH1 gene expression, was cleaved to a ∼100 kDa fragment. Sirt1 cleavage was markedly reduced in si-Casp9 cells, even though caspase-3 was normally activated, but was not affected (still cleaved) in si-Casp2 cells, despite a marked reduction of caspase-3 activity. The expression of MASH1 mRNA was higher and occurred earlier in si-Casp2 cells, while was reduced at early time points during differentiation in si-Casp9 cells. Thus, caspase-2 and -9 may perform opposite functions during RA-induced NT2 neuronal differentiation. While caspase-9 activation is relevant for proper neuronal differentiation, likely through the fine tuning of Sirt1 function, caspase-2 activation appears to hinder the RA-induced neuronal differentiation of NT2 cells.
Highlights
The human teratocarcinoma cell line Ntera2/cl.D1 (NT2 cells) represents a well-established model to study the retinoic acid (RA)induced terminal differentiation of human neural progenitors into post-mitotic neurons (NT2-N) [1,2,3]
The many features that NT2-N share with human fetal neurons has generated great interest for their potential use as graft source for cell therapy in neurodegenerative diseases [4], a perspective that warrants a deep understanding of the molecular mechanisms underlying NT2 cell differentiation
The activity of caspase-1,5,6,8 was not significantly affected throughout the differentiation process (Figure 1 D). These results indicate that caspase activation in differentiating NT2 cells is independent of apoptosis, that was found only in a small percentage of cells in the cultures (, 20%) and is known to occur during the first 3–4 days after RA induction [28]
Summary
The human teratocarcinoma cell line Ntera2/cl.D1 (NT2 cells) represents a well-established model to study the retinoic acid (RA)induced terminal differentiation of human neural progenitors into post-mitotic neurons (NT2-N) [1,2,3]. Following the seminal observation by Ishizaki et al [8], the implication of caspases in the differentiation of diverse cell types, and neurons, as well as in various aspects of neuronal plasticity, is becoming more accepted [9,10,11]. Across species, both ‘‘initiator’’ and ‘‘executioner’’ caspases appear involved in neuronal differentiation/maturation, and the evidence gathered far in the mammalian brain appears to suggest the ultimate involvement of caspase-3 [11,12,13,14,15,16]. Whether the latter is a necessary requirement or an epiphenomenon consequent to the hierarchical activation of caspases, as shown to occur following appropriate stimuli leading to apoptosis [5], is so far unclear
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