The bone morphogenetic protein type Ib receptor is a major mediator of glial differentiation and cell survival in adult hippocampal progenitor cell culture.

Bone morphogenetic proteins (BMPs) act as growth regulators and inducers of differentiation. They transduce their signal via three different type I receptors, termed activin receptor-like kinase 2 (Alk2), Alk3, or bone morphogenetic protein receptor Ia (BMPRIa) and Alk6 or BMPRIb. Little is known about functional differences between the three type I receptors. Here, we have investigated consequences of constitutively active (ca) and dominant negative (dn) type I receptor overexpression in adult-derived hippocampal progenitor cells (AHPs). The dn receptors have a nonfunctional intracellular but functional extracellular domain. They thus trap BMPs that are endogenously produced by AHPs. We found that effects obtained by overexpression of dnAlk2 and dnAlk6 were similar, suggesting similar ligand binding patterns for these receptors. Thus, cell survival was decreased, glial fibrillary acidic protein (GFAP) expression was reduced, whereas the number of oligodendrocytes increased. No effect on neuronal differentiation was seen. Whereas the expression of Alk2 and Alk3 mRNA remained unchanged, the Alk6 mRNA was induced after impaired BMP signaling. After dnAlk3 overexpression, cell survival and astroglial differentiation increased in parallel to augmented Alk6 receptor signaling. We conclude that endogenous BMPs mediate cell survival, astroglial differentiation and the suppression of oligodendrocytic cell fate mainly via the Alk6 receptor in AHP culture.

Bone morphogenetic proteins but not growth differentiation factors induce dopaminergic differentiation in mesencephalic precursors.

Bone morphogenetic proteins (BMPs) and growth differentiation factors (GDFs) are potential therapeutic molecules for the treatment of Parkinson's disease (PO). Here we compare the effects of BMP3, 5, 6, and 7 and GDF5 and 6 in a rat mesencephalic cell culture system that reflects the developmental stage of neurons around birth. High concentrations of BMP5, 6, and 7 and GDF5 and 6 induced astroglial cell fate and a depletion of oligodendrocytes. Only BMP5, 6, and 7, however, significantly increased the number of tyrosine hydroxylase (TH)-positive neurons and induced nuclear translocation of the phosphorylated BMP-restricted Smad in a substantial number of TH- and microtubule-associated protein 2(MAP2ab)-positive cells. None of the proteins protected TH-positive cells against 6-hydroxydopamine-induced oxidative stress. BMP3 was without any effect throughout the studies. We conclude that BMP5, 6, and 7 act directly and independently on precursors of the dopaminergic and astroglial lineage and induce their differentiation. In contrast, GDF5 and 6 primarily affect nonneuronal cells in mesencephalic cultures of this stage.

Electroporation of single cells and tissues with an electrolyte-filled capillary.

We show how an electrolyte-filled capillary (EFC) coupled to a high-voltage power supply can be used as a versatile electroporation tool for the delivery of dyes, drugs, and biomolecules to the cytoplasm of single cells and cells in tissues. A large-voltage pulse applied across the EFC (fused silica, 30 cm long, 375-microm o.d., 30-microm i.d.) gives rise to a small electric field outside the terminus of the EFC, which causes pore formation in cell membranes and induces an electroosmotic flow of electrolyte. When the EFC contains cell-loading agents, then the electroosmotic flow delivers the agents at the site of pore formation. The combination of pore formation and delivery enables loading of materials into the cytoplasm. By patch-clamp and fluorescence microscopy, formation of pores was observed at estimated transmembrane voltages of <85 mV with half-maximum values around 206 mV. The electroporation protocol was demonstrated by introduction of fluorogenic dyes into single NG108-15 cells, cellular processes, and small populations of cells in organotypic hippocampal cultures. Preliminary results are shown in which this protocol was employed for in vivo electroporation of ventral mesencephalon in rat brains. The technique was also used to access organelle-based detection systems inside cells. As a demonstration, 1,4,5-inositoltriphosphate was added to the electrolyte and detected by intracellular organelles in electroporated cells.