Knockdown of tissue nonspecific alkaline phosphatase impairs neural stem cell proliferation and differentiation.

In the adult mammalian brain the subependymal layer of the lateral ventricles houses neural stem cells giving rise to young neurons migrating towards the olfactory bulb. The molecular cues controlling essential functions within the neurogenesis pathway such as proliferation, short and long distance migration, differentiation and functional integration are poorly understood. Neural progenitors in situ express the tissue nonspecific form of alkaline phosphatase (TNAP), a cell surface-located nonspecific phosphomonoesterase capable of hydrolyzing extracellular nucleotides. To gain insight into the functional role of TNAP in cultured multipotent neural stem cells we applied a knockdown protocol using RNA interference with shRNA and retroviral infection. We show that TNAP knockdown reduces cell proliferation and differentiation into neurons or oligodendrocytes. This effect is abrogated by addition of alkaline phosphatase to the culture medium. Our results suggest that TNAP is essential for NSC proliferation and differentiation in vitro and possibly also in vivo.

Coordinate pathways for nucleotide and EGF signaling in cultured adult neural progenitor cells.

The adult subventricular zone (SVZ) contains astrocyte-like stem cells capable of generating new neurons for the olfactory bulb. Adult neurogenesis is driven by a variety of signal systems that can induce synergistic or opposing cellular responses. It is therefore important to gain insight into the underlying downstream signaling pathways. We have previously shown that the nucleotides ADPbetaS and UTP induce rapid Ca2+ transients in cultured SVZ-derived adult neural progenitors and augment growth-factor-mediated progenitor cell proliferation. Here, we investigated signaling pathways elicited by ADPbetaS, UTP and epidermal growth factor (EGF). All three agonists elicit ERK1/2 and CREB phosphorylation but the temporal characteristics differ between the nucleotides and EGF. Differentiation of the progenitors alters the receptor profile. Oligodendrocytes and young neurons, but not astrocytes, lose responsiveness to the agonists. Inhibition experiments are indicative of an ADPbetaS-elicited EGF receptor transactivation. Whereas UTP acts via the P2Y2 receptor, ADPbetaS exerts its function via the P2Y1 receptor and the P2Y13 receptor. Our data demonstrate that nucleotides and EGF induce converging, but also differential, intracellular signaling pathways and suggest that they carry the potential to act synergistically in the control of cell proliferation and cell survival in adult neurogenesis.

The bHLH transcription factor Hand2 is essential for the maintenance of noradrenergic properties in differentiated sympathetic neurons.

The basic helix-loop-helix transcription factor Hand2 is essential for the proliferation and noradrenergic differentiation of sympathetic neuron precursors during development. Here we address the function of Hand2 in postmitotic, differentiated sympathetic neurons. Knockdown of endogenous Hand2 in cultured E12 chick sympathetic neurons by siRNA results in a significant (about 60%) decrease in the expression of the noradrenergic marker genes dopamine-beta-hydroxylase (DBH) and tyrosine hydroxylase (TH). In contrast, expression of the pan-neuronal genes TuJ1, HuC and SCG10 was not affected. To analyze the in vivo role of Hand2 in differentiated sympathetic neurons we used mice harboring a conditional Hand2-null allele and excised the gene by expression of Cre recombinase under control of the DBH promotor. Mouse embryos homozygous for Hand2 gene deletion showed decreased sympathetic neuron number and TH expression was strongly reduced in the residual neuron population. The in vitro Hand2 knockdown also enhances the CNTF-induced expression of the cholinergic marker genes vesicular acetylcholine transporter (VAChT) and choline acetyltransferase (ChAT). Taken together, these findings demonstrate that the Hand2 transcription factor plays a key role in maintaining noradrenergic properties in differentiated neurons.

Target-dependent specification of the neurotransmitter phenotype: cholinergic differentiation of sympathetic neurons is mediated in vivo by gp 130 signaling.

Sympathetic neurons are generated through a succession of differentiation steps that initially lead to noradrenergic neurons innervating different peripheral target tissues. Specific targets, like sweat glands in rodent footpads, induce a change from noradrenergic to cholinergic transmitter phenotype. Here, we show that cytokines acting through the gp 130 receptor are present in sweat glands. Selective elimination of the gp 130 receptor in sympathetic neurons prevents the acquisition of cholinergic and peptidergic features (VAChT, ChT1, VIP) without affecting other properties of sweat gland innervation. The vast majority of cholinergic neurons in the stellate ganglion, generated postnatally, are absent in gp 130-deficient mice. These results demonstrate an essential role of gp 130-signaling in the target-dependent specification of the cholinergic neurotransmitter phenotype.

Interaction of Mash1 and Phox2b in sympathetic neuron development.

The transcription factors Mash1 and Phox2b are both essential for sympathetic neuron development. To understand in more detail their function and interaction, Phox2b and Mash1 were ectopically expressed in vivo, in peripheral nerve precursors. Here, we demonstrate that the Phox2b-induced generation of ectopic noradrenergic neurons in chick peripheral nerve involves the induction of Cash1, the chick homolog of Mash1. All Phox2-induced neurons coexpress the noradrenergic marker genes TH and DBH. Conversely, Mash1 induces neuronal differentiation characterized by the expression of generic neuronal genes SCG10, Hu and NF160; however, only a subpopulation of these neurons also displays an autonomic, noradrenergic phenotype. This context-dependent action of Mash1 implicates autonomic codeterminants, required for noradrenergic differentiation in response to Mash1. In contrast, Phox2b coordinates generic and noradrenergic gene expression, recruiting Mash1/Cash1, which may have a major function in the control of pan-neuronal gene expression during noradrenergic neuron development.