Protein kinase A activation inhibits oncogenic Sonic hedgehog signalling and suppresses basal cell carcinoma of the skin.

Basal cell carcinoma of the skin (BCC) is caused by constitutive activation of the Sonic hedgehog (Shh) pathway, mainly through mutations either in the Shh receptor Patched (PTCH) or in its co-receptor Smoothened (Smo). Inhibitors of this pathway that are currently in clinical trials inhibit Smo. However, mutations in Smo can result in resistance to these inhibitors. To target most BCCs and avoid acquired resistance because of Smo mutations, inhibiting the Shh-pathway downstream of Smo is critical. Attractive downstream targets would be at the level of Gli proteins, the transcriptional activators of this pathway in BCCs. Previously it has been shown that Gli1 and Gli2, when phosphorylated by protein kinase A (PKA), are targeted for proteosomal degradation. Here we show that PKA activation via the cAMP agonist forskolin is sufficient to completely abolish oncogenic Smo activity in vitro. In an inducible BCC mouse model due to a Smo mutation that confers resistance to current Smo inhibitors, topical forskolin treatment significantly reduced Gli1 mRNA levels and resulted in strongly suppressed BCC tumor growth. Our data show that forskolin inhibits the growth of even those BCCs that are resistant to Smo inhibitors and provide a proof-of-principle framework for the development of topically applied human skin-permeable novel pharmacologic inhibitors of oncogenic Shh-signaling through PKA activation.

Genetic fate mapping of Olig2 progenitors in the injured adult cerebral cortex reveals preferential differentiation into astrocytes.

Olig2 is a basic helix-loop-helix (bHLH) transcription factor essential for development of motoneurons and oligodendrocytes. It is known that Olig2(+) cells persist in the central nervous system (CNS) from embryonic to adult stages and that the number of Olig2(+) progenitors increases in the injured adult CNS. Recent studies have demonstrated an inhibitory action of Olig2 on neurogenesis in adult CNS, but the fate of Olig2(+) cells in the injured state remains largely unknown. To trace directly the fate of Olig2 cells in the adult cerebral cortex after injury, we employed the CreER/loxP system to target the olig2 locus. In this genetic tracing study, green fluorescent protein (GFP) reporter-positive cells labeled after cryoinjury coexpressed glial fibrillary acidic protein (GFAP), an astrocytic marker. Electron microscopy also showed that GFP(+) cells have the ultrastructural characteristics of astrocytes. Furthermore, GFP(+) cells labeled before injury, most of which had been NG2 cells, also produced bushy astrocytes. Here we show direct evidence that Olig2(+) cells preferentially differentiate into astrocytes, which strongly express GFAP, in response to injury in the adult cerebral cortex. These results suggest that reactive astrocytes, known to be the main contributors to glial scars, originate, at least in part, from Olig2(+) cells.

Maternal immune activation in mice delays myelination and axonal development in the hippocampus of the offspring.

Epidemiological data suggest a relationship between maternal infection and a high incidence of schizophrenia in offspring. An animal model based on this hypothesis was made by injecting double-stranded RNA, polyinosinic-polycytidylic acid (poly-I:C), into early pregnant mice, and their offspring were examined for biochemical and histological abnormalities. Mouse brains were examined with special reference to oligodendrocytes, which have been implicated in several neurodevelopmental disorders. We detected a significant decrease of myelin basic protein (MBP) mRNA and protein at early postnatal periods in poly-I:C mice. MBP immunocytochemistry and electron microscopy revealed that the hippocampus of juvenile poly-I:C mice was less myelinated than in PBS mice, with no significant loss of oligodendrocytes. In addition, axonal diameters were significantly smaller in juvenile poly-I:C mice than in control mice. These abnormalities reverted to normal levels when the animals reached the adult stage. These findings suggest that retarded myelination and axonal abnormalities in early postnatal stages caused by maternal immune activation could be related to schizophrenia-related behaviors in adulthood.

Knockdown of the L3/Lhx8 gene suppresses cholinergic differentiation of murine embryonic stem cell-derived spheres.

L3/Lhx8, a member of the Lim-homeobox gene family, is selectively and specifically expressed in the murine embryonic medial ganglionic eminence (MGE). Our previous study demonstrated that L3/Lhx8-deficient mice specifically lack cholinergic neurons in the basal forebrain. In this manuscript, we report the in vitro effects of reduced L3/Lhx8 gene expression on cholinergic differentiation in murine embryonic stem (ES) cell-derived spheres without dissociation. The knockdown of L3/Lhx8 gene expression dramatically decreased the cholinergic phenotype of spheres without altering other known phenotypes (TuJ1, GABA and GFAP). These results strongly suggest that L3/Lhx8 is a key factor for cholinergic differentiation of murine ES cell-derived spheres and is involved in basal forebrain development.

Transient activation of Notch signaling in the injured adult brain.

Brain injury induces various kinds of cellular responses that lead to tissue regeneration and repair. Recent studies have demonstrated that resident progenitors proliferate and then differentiate into mature neuronal cells. We show here that proliferating cells in the cryo-injured cerebral cortex transiently expressed Notch1 immunoreactivity in their cytoplasm. Since activated Notch signaling regulates cellular fate in the developing nervous system, similar regulation may exist in the injured adult brain. To monitor the Notch signaling pathway, we examined whether components of the signaling pathway were co-expressed in Notch1-positive cells. Presenilin-1, a membrane-spanning protease that is required for the release of the Notch intracellular domain, was detected in the Notch1-positive cells and Hes1, a target of the Notch intracellular domain, also co-localized with Notch1 three days after cryo-injury. These results suggest that transient activity of the Notch signaling pathway is involved in the regulation of proliferation and differentiation of progenitors in the injured brain.