<em>Ex Vivo</em> Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Inserm, IRIB, University of Rouen

Although urotensin II (UII) and somatostatin 1 (SS1) exhibit some structural similarities, their precursors do not show any appreciable sequence identity and, thus, it is widely accepted that the UII and SS1 genes do not derive from a common ancestral gene. The recent characterization of novel isoforms of these two peptides, namely urotensin II-related peptide (URP) and somatostatin 2 (SS2)/cortistatin (CST), provides new opportunity to revisit the phylogenetic relationships of UII and SS1 using a comparative genomics approach. In the present study, by radiation hybrid mapping and in silico sequence analysis, we have determined the chromosomal localization of the genes encoding UII- and somatostatin-related peptides in several vertebrate species, including human, chicken, and zebrafish. In most of the species investigated, the UII and URP genes are closely linked to the SS2/CST and SS1 genes, respectively. We also found that the UII-SS2/CST locus and the URP/SS1 locus are paralogous. Taken together, these data indicate that the UII and URP genes, on the one hand, and the SS1 and SS2/CST genes, on the other hand, arose through a segmental duplication of two ancestral genes that were already physically linked to each other. Our results also suggest that these two genes arose themselves through a tandem duplication of a single ancestral gene. It thus appears that the genes encoding UII- and somatostatin-related peptides belong to the same superfamily.

Comparative genomics provides evidence for close evolutionary relationships between the urotensin II and somatostatin gene families.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) are closely related members of the secretin superfamily of neuropeptides expressed in both the brain and peripheral nervous system, and they exhibit neurotrophic and neurodevelopmental effects in vivo. Like the index member of the Trk receptor ligand family, nerve growth factor (NGF), PACAP promotes the differentiation of PC12 cells, a well-established cell culture model, to investigate neuronal differentiation, survival and function. Stimulation of catecholamine secretion and enhanced neuropeptide biosynthesis are effects exerted by PACAP at the adrenomedullary synapse in vivo and on PC12 cells in vitro through stimulation of the specific PAC1 receptor. Induction of neuritogenesis, growth arrest, and promotion of cell survival are effects of PACAP that occur in developing cerebellar, hippocampal and cortical neurons, as well as in the more tractable PC12 cell model. Study of the mechanisms through which PACAP exerts its various effects on cell growth, morphology, gene expression and survival, i.e. its actions as a neurotrophin, in PC12 cells is the subject of this review. The study of neurotrophic signalling by PACAP in PC12 cells reveals that multiple independent pathways are coordinated in the PACAP response, some activated by classical and some by novel or combinatorial signalling mechanisms.

The neurotrophic effects of PACAP in PC12 cells: control by multiple transduction pathways.

The rat pheochromocytoma PC12 cell line has been widely used as a model to study neuronal differentiation. In particular, after serum depletion, PC12 cells stop to proliferate and undergo apoptosis. Under such conditions, treatment with pituitary adenylate cyclase-activating polypeptide (PACAP) promotes cell survival and induces neurite outgrowth. The identification of the proteins regulated by PACAP in PC12 cells under apoptotic conditions should provide valuable information concerning the mechanisms controlling neuronal cell survival and differentiation. To this aim, PC12 cells cultured in serum-free medium were treated with PACAP (10(-7) M), proteins were extracted, separated by two-dimensional gel electrophoresis (2-DE), and identified by MALDI-ToF mass spectrometry. The comparison between 16 2-DE maps led to the characterization of 110 proteins regulated by PACAP among which 22 have been identified by automatic query of the Mascot, Aldente, and Profound servers with the ProGeR-CDD database. Seventy-six percent of these proteins, including the p17 subunit of caspase-3, the heat shock protein hsp60, and the GTPase ran were found to be repressed whereas the others notably hsp27, tubulin beta-5, and calmodulin were overexpressed. Investigation of the putative functions indicated that some of the proteins regulated by PACAP and identified in the present article could control cell survival or differentiation.

Identification of proteins regulated by PACAP in PC12 cells by 2D gel electrophoresis coupled to mass spectrometry.

During early post-natal development of the cerebellum, granule neurons (GN) execute a centripetal migration toward the internal granular layer, whereas basket and stellate cells (B/SC) migrate centrifugally to reach their final position in the molecular layer (ML). We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates in vitro the expression and release of the serine protease tissue-type plasminogen activator (tPA) from GN, but the coordinated role of PACAP and tPA during interneuron migration has not yet been investigated. Here, we show that endogenous PACAP is responsible for the transient arrest phase of GN at the level of the Purkinje cell layer (PCL) but has no effect on B/SC. tPA is devoid of direct effect on GN motility in vitro, although it is widely distributed along interneuron migratory routes in the ML, PCL, and internal granular layer. Interestingly, plasminogen activator inhibitor 1 reduces the migration speed of GN in the ML and PCL, and that of B/SC in the ML. Taken together, these results reveal for the first time that tPA facilitates the migration of both GN and fast B/SC at the level of their intersection in the ML through degradation of the extracellular matrix. Crucial role of tissue plasminogen activator (tPA) in interneuron migration. Interneuron migration is a critical step for normal establishment of neuronal network. This study indicates that, in the post-natal cerebellum, tPA facilitates the opposite migration of immature excitatory granule neurons (GN) and immature inhibitory basket/stellate cells (B/SC) along the same migratory route. These data show that tPA exerts a pivotal role in neurodevelopment.

Cortical-layer-specific effects of PACAP and tPA on interneuron migration during post-natal development of the cerebellum.

In the developing brain, immature neurons migrate from their sites of origin to their final destination, where they reside for the rest of their lives. This active movement of immature neurons is essential for the formation of normal neuronal cytoarchitecture and proper differentiation. Deficits in migration result in the abnormal development of the brain, leading to a variety of neurological disorders. A myriad of extracellular guidance molecules and intracellular effector molecules is involved in controlling the migration of immature neurons in a cell type, cortical layer and birth-date-specific manner. To date, little is known about how extracellular guidance molecules transfer their information to the intracellular effector molecules, which regulate the migration of immature neurons. In this article, to fill the gap between extracellular guidance molecules and intracellular effector molecules, using the migration of cerebellar granule cells as a model system of neuronal cell migration, we explore the role of second messenger signaling (specifically Ca(2+) and cyclic nucleotide signaling) in the regulation of neuronal cell migration. We will, first, describe the cortical layer-specific changes in granule cell migration. Second, we will discuss the roles of Ca(2+) and cyclic nucleotide signaling in controlling granule cell migration. Third, we will present recent studies showing the roles of Ca(2+) and cyclic nucleotide signaling in the deficits in granule cell migration in mouse models of fetal alcohol spectrum disorders and fetal Minamata disease.

Alexis Lebon

PRIMACEN,
Cell Imaging Platform of Normandy,
PRIMACEN, Cell Imaging Platform of Normandy

Inserm, IRIB, University of Rouen

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Alexis Lebon

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Inserm, IRIB, University of Rouen

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

PRIMACEN,
Cell Imaging Platform of Normandy,
PRIMACEN, Cell Imaging Platform of Normandy