Gabriella Augusti-Tocco

Dual control of neurogenesis by PC3 through cell cycle inhibition and induction of Math1.

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 G1 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.

The readthrough variant of acetylcholinesterase remains very minor after heat shock, organophosphate inhibition and stress, in cell culture and in vivo

Acetylcholinesterase (AChE) exists in various molecular forms, depending on alternative splicing of its transcripts and association with structural proteins. Tetramers of the ‘tailed’ variant (AChET), 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 (AChER) mRNA which produces a monomeric form. To generalize this observation, we attempted to quantify AChER and AChET 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 AChER 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.

Autocrine activation of nicotinic acetylcholine receptors contributes to Ca2+ spikes in mouse myotubes during myogenesis

It is widely accepted that nicotinic acetylcholine receptor (nAChR) channel activity controls myoblast fusion into myotubes during myogenesis. In this study we explored the possible role of nAChR channels after cell fusion in a murine cell model. Using videoimaging techniques we showed that embryonic muscle nAChR channel openings contribute to the spontaneous transients of intracellular concentration of Ca2+ ([Ca2+]i) and to twitches characteristic of developing myotubes before innervation. Moreover, we observed a choline acetyltransferase immunoreactivity in the myotubes and we detected an acetylcholine‐like compound in the extracellular solution. Therefore, we suggest that the autocrine activation of nAChR channels gives rise to [Ca2+]i spikes and contractions. Spontaneous openings of the nAChR channels may be an alternative, although less efficient, mechanism. We report also that blocking the nAChRs causes a significant reduction in cell survival, detectable as a decreased number of myotubes in culture. This led us to hypothesize a possible functional role for the autocrine activation of the nAChRs. By triggering mechanical activity, such activation could represent a strategy to ensure the trophism of myotubes in the absence of nerves.

Cholinergic modulation of neurofilament expression and neurite outgrowth in chick sensory neurons

The morphogenetic role of the neurotransmitter acetylcholine was studied in cultures of dorsal root ganglia (DRG) neurons obtained from E12 and E18 chick embryos. With this model we have evaluated neurofilament expression and neurite outgrowth following cholinergic agonist and antagonist treatment. Morphometric analysis undertaken to evaluate fiber outgrowth has indicated that E12 DRG cultures treated with cholinergic agonists, such as muscarine and carbachol, when compared with untreated cultures, have longer fibers and a higher number of fibers per neuron. Concomitant treatment with agonists and the antagonists atropine or mecamylamine counteracts the increase in fiber outgrowth, suggesting that the cholinergic agonist effects were mediated by both muscarinic and nicotinic receptors. The expression of the three neurofilament proteins was also evaluated. Western blot analysis showed that, in E12 DRG cultures, both muscarine and carbachol induce a significant increase in neurofilament protein expression and that this effect is inhibited by cholinergic antagonist treatment. Moreover, Northern blot analysis has demonstrated that the increased expression of 68‐ and 145‐kDa neurofilament proteins is dependent on cholinergic modulation of the neurofilament transcripts. Modulated expression of neurofilament proteins by cholinergic agonists was not evident in E18 DRG cultures, suggesting that, when sensory neurons have completed their differentiation, the cholinergic system might be involved in other functions. In conclusion, our data demonstrate that, during sensory neuron development, acetylcholine modulates neurite outgrowth controlling neurospecific marker expression.

Aflatoxin B1 is an inhibitor of cyclic nucleotide phosphodiesterase activity

Aflatoxin B1 (AFB1) action on cyclic nucleotide phosphodiesterase (PDE) activity has been tested on tissue extracts of various organs. In the presence of 100 microM AFB1 a significant inhibition of cAMP and cGMP hydrolytic activity is observed in all tested tissue extracts. However, cGMP hydrolytic activity appears more sensitive to AFB1 inhibition than cAMP hydrolytic activity and a considerably higher inhibition is observed in lung and spleen, than in liver, brain, kidney, and heart. When cGMP is used as substrate, the inhibitory response reaches 72% in lung and spleen extracts. We have also tested AFB1 effects on lung and liver PDE activity peaks separated by DEAE-cellulose chromatography. These data confirm the poor sensitivity to the toxin of all PDE activities present in liver, while the lung peak (where PDE V in present) shows a higher sensitivity to AFB1. In order to establish whether PDE V is in fact more sensitive to AFB1, we have used mouse neuroblastoma cells, in which cGMP hydrolytic activity has been shown to be due to PDE V only. In this case, the calculated IC50 is 24 microM and Dixon plot analysis shows a competitive inhibitory effect with a Ki of 16.7 microM. We have also used aflatoxin B2 and M2, and they proved to be much less effective than AFB1: AFB2 inhibits PDE V with an IC50 of 117 microM, while AFM2 does not show any effect. These results provide the first evidence of a competitive inhibition of AFB1 on an enzymatic activity and suggest that an alteration of cellular cyclic nucleotide levels may play a role in the mechanism of aflatoxin action.

Neuronal and non-neuronal cell populations of the avian dorsal root ganglia express muscarinic acetylcholine receptors

The distribution of muscarinic acetylcholine receptors was investigated by immuno‐light and electron microscopy in the chick dorsal root ganglion during embryonic development (E12 and E18) and after hatching. The monoclonal antibody we used recognizes the acetylcholine binding site shared by all five muscarinic acetylcholine receptor subtypes. At E12, light microscopy reveals several immunopositive neurons with variable degrees of immunolabeling, heterogeneously distributed throughout the ganglion. Later in development and after hatching, the intensity of immunolabeling seems to decrease and the immunopositive neurons, of the small‐medium‐sized type, are located mostly in the medio‐dorsal region of the ganglion. Under the electron microscope, the immunoreaction is associated with the Nissl bodies, budding Golgi cisterns and, especially at E12, with discrete loci along the neuronal plasma membrane. Unmyelinated nerve fibers, in both central and peripheral branches, are also immunopositive, suggesting that muscarinic acetylcholine receptors are transported towards the spinal cord and the periphery, respectively. A large number of perineuronal satellite cells and both myelinating and unmyelinating Schwann cells are intensely labeled. These observations, combined with previous data on the pharmacological and functional characterization of muscarinic acetylcholine receptors in the avian dorsal root ganglion, suggest that both sensory neurons and non‐neuronal cells are able to respond to acetylcholine stimuli. Since muscarinic acetylcholine receptor‐immunoreactivity is restricted to the small‐medium‐sized neurons and their unmyelinated fibers, of the nociceptive type, we suggest that these receptors are involved in modulating the transduction of noxious stimuli from the periphery.

Detection of basal and potassium-evoked acetylcholine release from embryonic DRG explants

Spontaneous and potassium‐induced acetylcholine release from embryonic (E12 and E18) chick dorsal root ganglia explants at 3 and 7 days in culture was investigated using a chemiluminescent procedure. A basal release ranging from 2.4 to 13.8 pm/ganglion/5 min was detected. Potassium application always induced a significant increase over the basal release. The acetylcholine levels measured in E12 explants were 6.3 and 38.4 pm/ganglion/5 min at 3 and 7 days in culture, respectively, while in E18 explant cultures they were 10.7 and 15.5 pm/ganglion/5 min. In experiments performed in the absence of extracellular Ca2+ ions, acetylcholine release, both basal and potassium‐induced, was abolished and it was reduced by cholinergic antagonists. A morphometric analysis of explant fibre length suggested that acetylcholine release was directly correlated to neurite extension. Moreover, treatment of E12 dorsal root ganglion‐dissociated cell cultures with carbachol as cholinergic receptor agonist was shown to induce a higher neurite outgrowth compared with untreated cultures. The concomitant treatment with carbachol and the antagonists at muscarinic receptors atropine and at nicotinic receptors mecamylamine counteracted the increase in fibre outgrowth. Although the present data have not established whether acetylcholine is released by neurones or glial cells, these observations provide the first evidence of a regulated release of acetylcholine in dorsal root ganglia.

Acetylcholinesterase in the development of chick dorsal root ganglia

Acetylcholinesterase is expressed in chick dorsal root ganglia neurons very early in development. Since the physiological role of the enzyme in these cells is still obscure, it appeared of interest to investigate its modifications in the course of development.

Hepatocyte Growth Factor Stimulates Cell Motility in Cultures of the Striatal Progenitor Cells ST14A

Hepatocyte growth factor/scatter factor (HGF/SF) is a growth factor with pleiotropic effects on different cell types. It acts as a mitogen and motility factor for many epithelial cells. HGF/SF and its receptor Met are present in the developing and adult mammalian brain and control neuritogenesis of sympathetic and sensory neurons. We report that the striatal progenitor ST14A cells express the Met receptor, which is activated after binding with HGF/SF. The interaction between Met and HGF/SF triggers a signaling cascade that leads to increased levels of c‐Jun, c‐Fos, and Egr‐1 proteins, in agreement with data reported on the signaling events evoked by HGF in other cellular types. We also studied the effects of the exposure of ST14A cells to HGF/SF. By time‐lapse photography, we observed that a 24‐hr treatment with 50 ng/ml HGF/SF induced modification in cell morphology, with a decrease in cell‐cell interactions and increase of cell motility. In contrast, no effect on cell proliferation was observed. To investigate which intracellular pathway is primarily involved we used PD98059 and LY294002, two specific inhibitors of mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAP‐kinase/ERK‐kinase) and phosphoinositide 3‐OH kinase (PI3‐K), respectively. Cell motility in HGF/SF treated cultures was inhibited by LY294002 but not by PD98059, suggesting that PI3‐K plays a key role in mediating the HGF/SF‐induced dissociation of ST14A cells. Previous evidence of HGF stimulation of motility in nervous system has been obtained on postmitotic neurons, which have already acquired their specificity. Data reported here of a motogenic response of ST14A cell line, which displays properties of neuronal progenitors, seem of interest because they suggest that HGF could play a role in very early steps of neurogenesis.

Expression of muscarinic m2 receptor mRNA in dorsal root ganglia of neonatal rat.

The expression of mRNA coding for m2 subtype of muscarinic cholinergic receptors was assessed in dorsal root ganglia (DRG) of 15-day post-natal rats. Northern blot analysis on total RNA using a mixture of two different oligonucleotide probes, indicated the presence of a single prominent band of approximately 6.5 kb in rat DRG; a band of the same size was observed both in brainstem and cortex taken as positive controls. Analysis by RT-PCR of the mRNA coding for a region of the third cytoplasmic loop of m2 receptor showed a single signal both in rat DRG and hippocampus. In situ hybridization was then used to identify the neuronal subpopulations expressing the mRNA for M2. The transcripts were preferentially localized in medium-small neurons of the ganglion as well as in satellite cells surrounding the neuron cell body. Large neurons were usually negative. Finally, competition binding experiments, performed in the presence of [3H]-quinuclidinyl benzilate (QNB) and methoctramine (a selective competitor for M2 receptors), demonstrated the presence of M2 receptor protein (Ki=100 nM), as previously observed in chick DRG. The preferential localization of M2 in medium-small neurons of the ganglion suggests the involvement of this receptor subtype in the transduction of nociceptive stimuli.