Giancarlo Poiana

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.

Membrane acetylcholinesterase in murine muscular dystrophy In vivo and in cultured myotubes

Murine muscular dystrophy is characterized by a reduction of the 10S molecular form of acetylcholinesterase (AChE); this reduction occurs in both strains of dystrophic mice and at the time of the phenotypic appearance of the disease.

Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.

Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase‐transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1–M4 subtypes and the activation of both IP3 and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor‐1 (EGR‐1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR‐1 induction. The role of EGR‐1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR‐1. EGR‐1 clones show increased neurite extension and a decrease in Repressor Element‐1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc‐finger domain, a dominant negative inhibitor of EGR‐1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR‐1 and REST transcription factors.

5-Hydroxytryptamine 1A and 2B Serotonin Receptors in Neurite Outgrowth: Involvement of Early Growth Response Protein 1

Neurotransmitters play important roles in neurogenesis; in particular, acetylcholine and serotonin may regulate neurite elongation. Acetylcholine may also activate transcription factors such as early growth response protein 1 (EGR-1), which plays a role in neurite extension. N18TG2 neuroblastoma cells (which do not produce neurotransmitters and constitutively express muscarinic acetylcholine receptors) were transfected with constructs containing the cDNA for choline acetyltransferase, 5-hydroxytryptamine 1A (5-HT1A) and 5-HT2B serotonin receptors to study acetylcholine and serotonin interplay in neurite outgrowth. 5-HT1A receptor stimulation causes a decrease in EGR-1 levels and inhibition of neurite outgrowth; 5-HT2B stimulation, however, has no effect. Muscarinic cholinergic stimulation, on the other end, increases EGR-1 levels and fiber outgrowth. Inhibition of EGR-1 binding reduces fiber outgrowth activity. When both cholinergic and 5-HT1A receptors are stimulated, fiber outgrowth is restored; therefore, acetylcholine counterbalances the inhibitory effect of serotonin on neurite outgrowth. These results suggest that EGR-1 plays a role in the interplay of acetylcholine and serotonin in the regulation of neurite extension during development.

Dystrophin localization and gene expression in the developing nervous system of the chick.

The presence and distribution of dystrophin was studied in selected areas of the chick embryo nervous system and in primary cultures. Dystrophin was examined at the protein level by immunocytochemistry and at the transcriptional level by a semiquantitative reverse transcriptase‐polymerase chain reaction analysis.

Muscle Acetylcholinesterase in a Familial Myopathic Disease

Three sisters with myopathy characterized by different degrees of weakness, hypotonia, cramps and a significant hypertrophy of the calves underwent clinical tests. Laboratory examinations (nerve conduction velocity, electromyography and serum enzymes), serial histochemical analyses of muscle specimens and tests for muscular acetylcholinesterase (AChE) activity and its molecular forms were performed. AChE activities did not differ significantly from those of controls, while sedimentation patterns evidenced the disappearance of 16 S, 13 S and 10 S molecular forms in the elder sisters. The genealogical tree of the patients is described and their cases compared to those of others with calf hypertrophy reported in the literature.

Egr-1 Maintains NSC Proliferation and Its Overexpression Counteracts Cell Cycle Exit Triggered by the Withdrawal of Epidermal Growth Factor

In adult mammals, neural stem cells (NSCs) reside in specialized niches at the level of selected CNS regions, such as the subventricular zone (SVZ). The signaling pathways that regulate NSC proliferation and differentiation remain poorly understood. Early growth response protein 1 (Egr-1) is an important transcription factor, widely studied in the adult mammalian brain, mediating the activation of target genes by a variety of extracellular stimuli. In our study, we aimed at testing how Egr-1 regulates adult NSCs derived from mouse SVZ and, in particular, the interplay between Egr-1 and the proliferative factor EGF. We demonstrate that Egr-1 expression in NSCs is induced by growth factor stimulation, and its level decreases after EGF deprivation or by using AG1478, an inhibitor of the EGF/EGFR signaling pathway. We also show that Egr-1 overexpression rescues the cell proliferation decrease observed either after EGF removal or upon treatment with AG1478, suggesting that Egr-1 works downstream of the EGF pathway. To better understand this mechanism, we investigated targets downstream of both the EGF pathway and Egr-1, and found that they regulate genes involved in NSC proliferation, such as cell cycle regulators, cyclins, and cyclin-dependent kinase inhibitors.