Growing evidence indicates that cell cycle arrest and neurogenesis are highly coordinated and interactive processes, governed by cell cycle genes and neural transcription factors. The gene PC3 (Tis21/BTG2) is expressed in the neuroblast throughout the neural tube and inhibits cell cycle progression at the G 1 checkpoint by repressing cyclin D1 transcription. We generated inducible mouse models in which the expression of PC3 was upregulated in neuronal precursors of the neural tube and of the cerebellum. These mice exhibited a marked increase in the production of postmitotic neurons and impairment of cerebellar development. Cerebellar granule precursors of PC3 transgenic mice displayed inhibition of cyclin D1 expression and a strong increase in the expression of Math1, a transcription factor required for their differentiation. Furthermore, PC3, encoded by a recombinant adenovirus, also induced Math1 in postmitotic granule cells in vitro and stimulated the Math1 promoter activity. In contrast, PC3 expression was unaffected in the cerebellar primordium of Math1 null mice, suggesting that PC3 acts upstream to Math1. As a whole, our data suggest that cell cycle exit of cerebellar granule cell precursors and the onset of cerebellar neurogenesis are coordinated by PC3 through transcriptional control of cyclin D1 and Math1, respectively.

Abstract.-Clonal lines of neurons were obtained in culture from a mouse neuroblastoma. The neuroblastoma cells were adapted to culture growth by the animal-culture alternate passage technique and cloned after single-cell plating. The clonal lines retained the ability to form tumors when injected back into mice. A striking morphological change was observed in the cells adapted to culture growth; they appeared as mature neurons, while the cells of the tumor appeared as immature neuroblasts.Acetyleholinesterase and the enzymes for the synthesis of neurotransmitters, cholineacetylase and tyrosine hydroxylase were assayed in the tumor and compared with brain levels; tyrosine hydroxylase was found to be particularly high, as described previously in human neuroblastomas. The three enzymes were found in the clonal cultures at levels comparable to those found in the tumors. Similarly, there were no remarkable differences between the three clones examined.The difficulty in separating glial cells and neurons has proved to be a major obstacle in the biochemical characterization of the components of the nervous system. 1\Jethods of separation, which yield homogeneous populations of cells, are limited by the low amount of cells obtainable.1' 2 On the other hand, the methods described for large scale preparation'-give highly heterogeneous fractions and produce a large amount of cell damage.' Clonal cell lines of the components of nervous tissue would, therefore, provide a useful tool for the study of neurobiology. Recently, a glial cell line which retains in culture the ability to synthesize the brain specific protein S-100 has been developed from a rat brain tumor. Results.-Morphology: Neuroblastomas have been described as highly undifferentiated tumors. The C 1300 Jackson tumor, described as a spontaneous tumor of the region of spinal cord, showed the usual morphology of neuroblastomas. Histological section of the tumor revealed the presence of only round cells. Fibers were absent.When placed into culture, the cells undergo a striking change in morphology. The most striking characteristic of these cells is the large number of elongated processes which emanate from the cell bo4y. These processes begin development soon after subculture or the initiation of primary culture, and within a few days form a complex network. A typical colony of a clonal line is shown in Figure 1.In each colony the cells remain rather sparse. After a few fays in culture, round cells appear on the colonies (Fig. 2). They pile up on the colonies and form clumps, which tend to float away, while the cells with long processes remain attached to the plates. clonal tumor NB41A and NB41B were assayed for acetylcholinesterase, choline acetylase, and tyrosine hydroxylase activities. These results are reported in Table 1. The three enzymes were present in the original tumor. As compared with the brain, the tumor showed a higher content of tyrosine hydroxylase and a lower content of choline acetylase and acetylcholinesterase. The three clones studied, both as tum...

Cultures of Schwann cells from neonatal rat sciatic nerves were treated with acetylcholine agonists and the effects on cell proliferation evaluated. (3)[H]-thymidine incorporation shows that acetylcholine (ACh) receptor agonists inhibit cell proliferation, and FACS analysis demonstrates cell-cycle arrest and accumulation of cells in the G1 phase. The use of arecaidine, a selective agonist of muscarinic M2 receptors reveals that this effect depends mainly on M2 receptor activation. The arecaidine dependent-block in G1 is reversible because removal of arecaidine from the culture medium induces progression to the S phase. The block of the G1-S transition is also characterized by modulation of the expression of several cell-cycle markers. Moreover, treatment with ACh receptor agonist causes both a decrease in the PCNA protein levels in Schwann cell nuclei and an increase in p27 and p53 proteins. Finally, immuno-electron microscopy demonstrates that M2 receptors are expressed by Schwann cells in vivo. These results indicate that ACh, by modulating Schwann cell proliferation through M2 receptor activation, might contribute to their progression to a more differentiated phenotype.

N18TG2 neuroblastoma clone is defective for biosynthetic neurotransmitter enzymes; its inability to establish functional synapses is overcome in the neuroblastoma X glioma 108CC15, where acetylcholine synthesis is also activated. These observations suggest a possible relation between the ability to produce acetylcholine and the capability to advance in the differentiation program and achieve a fully differentiated state. Here, we report the characterization of several clones after transfection of N18TG2 cells with a construct containing a cDNA for rat choline acetyltransferase (ChAT). The ability of these clones to synthesize acetylcholine is demonstrated by HPLC determination on cellular extracts. In the transfected clones, northern blot analysis shows increased expression of mRNAs for a specific neuronal protein associated with synaptic vesicles, synapsin I. Fiber outgrowth of transfected clones is also evaluated to establish whether there is any relation between ChAT levels and morphological differentiation. This analysis shows that the transfected clone 1/2, not expressing ChAT activity, displays a very immature morphology, and its ability to extend fibers also remains rather poor in the presence of "differentiation" agents such as retinoic acid. In contrast, clones 2/4, 3/1, and 3/2, exhibiting high ChAT levels, display higher fiber outgrowth compared with clone 1/2 in both the absence and the presence of differentiating agents.

Acetylcholinesterase (AChE) exists in various molecular forms, depending on alternative splicing of its transcripts and association with structural proteins. Tetramers of the 'tailed' variant (AChE T ), which are anchored in the cell membrane of neurons by the PRiMA (Proline Rich Membrane Anchor) protein, constitute the main form of AChE in the mammalian brain. In the mouse brain, stress and anticholinesterase inhibitors have been reported to induce expression of the unspliced 'readthrough' variant (AChE R ) mRNA which produces a monomeric form. To generalize this observation, we attempted to quantify AChE R and AChE T after organophosphate intoxication in the mouse brain and compared the observed effects with those of stress induced by swimming or immobilization; we also analyzed the effects of heat shock and AChE inhibition on neuroblastoma cells. Active AChE molecular forms were characterized by sedimentation and non-denaturing electrophoresis, and AChE transcripts were quantified by real-time PCR. We observed a moderate increase of the AChE R transcript in some cases, both in the mouse brain and in neuroblastoma cultures, but we did not detect any increase of the corresponding active enzyme.