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.

Research highlights▶ Identification of the cell surface-associated enzyme tissue nonspecific alkaline phosphatase (TNAP) at cultured adult neural stem cells (NCSs), an enzyme that is also associated with the neurogenic subependymal zone in situ. Analysis of a potential functional involvement of this enzyme in neurogenesis. ▶ Knockdown of TNAP specifically in glial fibrillary acidic protein (GFAP) expressing NSCs. This is achieved by induction of RNA interference through insertion of shRNA cassettes into viral vectors and infection of TVA receptor-expressing NSCs derived from a mutant mouse line expressing the cell surface receptor (TVA) for subgroup A avian leukosis viruses (AlV-A) under the human GFAP-promoter. ▶ Demonstration that TNAP knockdown compromises cell proliferation and differentiation of NSCs into neurons or oligodendrocytes. ▶ This is the first demonstration of a functional role of alkaline phosphatase in NSCs and implies that the enzyme play a significant role in NSC proliferation and differentiation. Since the enzyme is not only expressed by NSCs but also by mouse or human embryonic stem cells this observation is of general interest for stem cell research. TNAP may no longer simply be regarded as a “stem cell marker”.