Ada Maria Tata

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 Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw’s nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients’ quality of life.

Muscarinic receptor subtypes expression in rat and chick dorsal root ganglia

In the present work we have analyzed by Northern blot, RT-PCR and in situ hybridization the expression of muscarinic receptor subtype mRNAs in rat and chick dorsal root ganglia. Northern blot analysis performed on rat total RNA revealed a strong signal for M(2) while a faint band was observed for M(3) and M(4) subtypes; no signal was evident for M(1) and M(5), while in chick total RNA no signal was detected for any of the analyzed subtypes (M(2), M(3), M(4)). On the other hand, RT-PCR revealed that all muscarinic subtype mRNAs were present both in rat and chick DRG, although the level of their expression may be different. In chick DRG, the presence of various muscarinic subtypes was confirmed by competition binding experiments. In situ hybridization in rat DRG showed that M(3) and M(4) transcripts, similarly to what has been previously described for M(2) mRNA, were preferentially localized in medium-small neurons. Large neurons were usually negative or faintly labelled. No hybridization signal was detected in rat DRG with probes for M(1) and M(5) muscarinic subtypes. The presence of various muscarinic receptors in DRG and their preferential expression in the medium-small sensory neurons suggest their possible involvement in the modulation of nociceptive stimuli transduction.

Muscarinic receptor subtypes as potential targets to modulate oligodendrocyte progenitor survival, proliferation, and differentiation

Acetylcholine (ACh) is a major neurotransmitter but also an important signaling molecule in neuron‐glia interactions. Expression of ACh receptors has been reported in several glial cell populations, including oligodendrocytes (OLs). Nonetheless, the characterization of muscarinic receptors in these cells, as well as the description of the cholinergic effects at different stages of OL development, is still incomplete. In this study, we characterized the pattern of expression of muscarinic receptor subtypes in primary cultures of rat oligodendrocyte progenitor cells (OPC) and mature OLs, at both mRNA and protein levels. We found that muscarinic receptor expression is developmentally regulated. M1, M3, and M4 receptors were the main subtypes expressed in OPC, whereas all receptor subtypes were expressed at low levels in mature OLs. Exposure of OPC to muscarine enhanced cell proliferation, an effect mainly due to M1, M3, and M4 receptor subtypes as demonstrated by pharmacological competition with selective antagonists. Conversely, M2 receptor activation impaired OPC survival. In line with the mitogenic activity, muscarinic receptor activation increased the expression of platelet derived growth factor receptor α. Muscarine stimulation increased CX32 and myelin basic protein expression, left unaffected that of myelin proteolipid protein (PLP), and decreased member of the family of epidermal growth factor receptor (EGFR) ErbB3/ErbB4 receptor expression indicating a predominant role of muscarinic receptors in OPC. These findings suggest that ACh may contribute to the maintenance of an immature proliferating progenitor pool and impair the progression toward mature stage. This hypothesis is further supported by increased expression of Notch‐1 in OL on muscarinic activation.

Serum-activated K and Cl currents underlay U87-MG glioblastoma cell migration

Glioblastoma cells in vivo are exposed to a variety of promigratory signals, including undefined serum components that infiltrate into high grade gliomas as result of blood–brain barrier breakdown. Glioblastoma cell migration has been further shown to depend heavily on ion channels activity. We have then investigated the modulatory effects of fetal calf serum (FCS) on ion channels, and their involvement in U87‐MG cells migration. Using the perforated patch‐clamp technique we have found that, in a subpopulation of cells (42%), FCS induced: (1) an oscillatory activity of TRAM‐34 sensitive, intermediate‐conductance calcium‐activated K (IKCa) channels, mediated by calcium oscillations previously shown to be induced by FCS in this cell line; (2) a stable activation of a DIDS‐ and NPPB‐sensitive Cl current displaying an outward rectifying instantaneous current‐voltage relationship and a slow, voltage‐dependent inactivation. By contrast, in another subpopulation of cells (32%) FCS induced a single, transient IKCa current activation, always accompanied by a stable activation of the Cl current. The remaining cells did not respond to FCS. In order to understand whether the FCS‐induced ion channel activities are instrumental to promoting cell migration, we tested the effects of TRAM‐34 and DIDS on the FCS‐induced U87‐MG cell migration using transwell migration assays. We found that these inhibitors were able to markedly reduce U87‐MG cell migration in the presence of FCS, and that their co‐application resulted in an almost complete arrest of migration. It is concluded that the modulation of K and Cl ion fluxes is essential for the FCS‐induced glioblastoma cell migration. J. Cell. Physiol. 226: 1926–1933, 2011.

Relation between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and neurodegenerative disorder. Since acetylcholine (ACh) is known to participate in the inflammatory response, we investigated the possible relationship between pro-inflammatory cytokines and acetylcholine levels in relapsing-remitting multiple sclerosis (RR-MS) patients. Levels of ACh and pro-inflammatory cytokines IL1-β and IL-17 were measured both in cerebrospinal fluid (CSF) and sera of 22 RR-MS patients in the relapsing phase and in 17 control subjects affected by other non-neurological diseases (OND). We observed higher levels of pro-inflammatory cytokines such as IL-1β and IL-17 in both CSF and serum of RR-MS patients compared to control subjects. Moreover, ACh levels were lower in CSF and serum of RR-MS patients compared to levels of control subjects. Although the relationship between high inflammatory cytokine levels and low ACh levels need to be further investigated in the future, our data suggest that IL-1β, and cytokines induced by it, such as IL-17 and ACh, may be involved in the pathogenesis of MS.

Cholinergic system dysfunction and neurodegenerative diseases: cause or effect?

Acetylcholine (ACh) has been the first molecule to be identified as neurotransmitter. The cholinergic and cholinoceptive areas, both in central and peripheral nervous system, have been well documented. Acetylcholine has been described to control, during embryogenesis, cell proliferation as well as neuron and glial cell survival and differentiation. In the adult, acetylcholine and its receptors are distributed in many tissues other than in the nervous system. More recently, new physiological roles in neuronal and non-neuronal tissues have been proposed for ACh as well as its possible involvement in different pathologies. Altered levels of ACh or modified receptors expression and function, in selected areas of the nervous system, have been described in several neurodegenerative diseases such as Alzheimers, Parkinsons and Huntington as well as in psychiatric disorders such as schizophrenia. Frequently own cognitive, behavioral and motor disabilities that characterize these pathologies are correlated to cholinergic circuit dysfunction. Moreover the involvement of ACh as modulator of the inflammation, in and out of the nervous system, has suggested that its altered functions might represent an additional pathogenetic mechanism negatively influencing the disease outcome as recently suggested in multiple sclerosis. The present review will focus on identifying the cause/effect relationship that may explain the cholinergic dysfunction in several nervous system disorders. Moreover the possible therapeutic novelties including cholinesterase inhibitors, muscarinic agonists and antagonists, and genetic therapy will be discussed.

Subpopulations of rat dorsal root ganglion neurons express active vesicular acetylcholine transporter

The vesicular acetylcholine transporter (VAChT) is a transmembrane protein required, in cholinergic neurons, for selective storage of acetylcholine into synaptic vesicles. Although dorsal root ganglion (DRG) neurons utilize neuropeptides and amino acids for neurotransmission, we have previously demonstrated the presence of a cholinergic system. To investigate whether, in sensory neurons, the vesicular accumulation of acetylcholine relies on the same mechanisms active in classical cholinergic neurons, we investigated VAChT presence, subcellular distribution, and activity. RT‐PCR and Western blot analysis demonstrated the presence of VAChT mRNA and protein product in DRG neurons and in the striatum and cortex, used as positive controls. Moreover, in situ hybridization and immunocytochemistry showed VAChT staining located mainly in the medium/large‐sized subpopulation of the sensory neurons. A few small neurons were also faintly labeled by immunocytochemistry. In the electron microscope, immunolabeling was associated with vesicle‐like elements distributed in the neuronal cytoplasm and in both myelinated and unmyelinated intraganglionic nerve fibers. Finally, [3H]acetylcholine active transport, evaluated either in the presence or in the absence of ATP, also demonstrated that, as previously reported, the uptake of acetylcholine by VAChT is ATP dependent. This study suggests that DRG neurons not only are able to synthesize and degrade ACh and to convey cholinergic stimuli but also are capable of accumulating and, possibly, releasing acetylcholine by the same mechanism used by the better known cholinergic neurons.

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.

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.