Localization and targeting of SCG10 to the trans-Golgi apparatus and growth cone vesicles.

SCG10 is a membrane-associated, microtubule-destabilizing protein of neuronal growth cones. Using immunoelectron microscopy, we show that in the developing cortex of mice, SCG10 is specifically localized to the trans face Golgi complex and apparently associated with vesicular structures in putative growth cones. Consistent with this, subcellular fractionation of rat forebrain extracts demonstrates that the protein is enriched in the fractions containing the Golgi apparatus and growth cone particles. In isolated growth cone particles, SCG10 was found to be particularly concentrated in the growth cone vesicle fraction. To evaluate the molecular determinants of the specific targeting of SCG10 to growth cones, we have transfected PC12 cells and primary neurons in culture with mutant and fusion cDNA constructs. Deletion of the amino-terminal domain or mutations within this domain that prevented palmitoylation at cysteines 22 and 24 abolished Golgi localization as well as growth cone targeting, suggesting that palmitoylation of the amino-terminal domain is a necessary signal for Golgi sorting and possibly transport of SCG10 to growth cones. Fusion proteins consisting of the amino-terminal domain of SCG10 and the cytosolic proteins stathmin or glutathione-S-transferase colocalized with a Golgi marker, alpha-mannosidase II, and accumulated in growth cones of both axons and dendrites. These results reveal a novel axonal/dendritic growth cone targeting sequence that involves palmitoylation.

Targeting of SCG10 to the area of the Golgi complex is mediated by its NH2-terminal region.

SCG10 is a neuronal growth-associated protein that is concentrated in the growth cones of developing neurons. SCG10 shows a high degree of sequence homology to the ubiquitous phosphoprotein stathmin, which has been recently identified as a factor that destabilizes microtubules by increasing their catastrophe rate. Whereas stathmin is a soluble cytosolic protein, SCG10 is membrane-associated, indicating that the protein acts in a distinct subcellular compartment. Identifying the precise intracellular distribution of SCG10 as well as the mechanisms responsible for its specific targeting will contribute to elucidating its function. The main structural feature distinguishing the two proteins is that SCG10 contains an NH2-terminal extension of 34 amino acids. In this study, we have examined the intracellular distribution of SCG10 in PC12 cells and in transfected COS-7 cells and the role of the NH2-terminal domain in membrane-binding and intracellular targeting. SCG10 was found to be localized to the Golgi complex region. We show that the NH2-terminal region (residues 1-34) was necessary for membrane targeting and Golgi localization. Fusion proteins consisting of the NH2-terminal 34 amino acids of SCG10 and the related protein stathmin or the unrelated protein, beta-galactosidase, accumulated in the Golgi, demonstrating that this sequence was sufficient for Golgi localization. Biosynthetic labeling of transfected COS-7 cells with [3H]palmitic acid revealed that two cysteine residues contained within the NH2-terminal domain were sites of palmitoylation.

Regulation of microtubule dynamics by the neuronal growth-associated protein SCG10.

Dynamic assembly and disassembly of microtubules is essential for cell division, cell movements, and intracellular transport. In the developing nervous system, microtubule dynamics play a fundamental role during neurite outgrowth, elongation, and branching, but the molecular mechanisms involved are unknown. SCG10 is a neuron-specific protein that is membrane-associated and highly enriched in growth cones. Here we show that SCG10 binds to microtubules, inhibits their assembly, and can induce microtubule disassembly. We also show that SCG10 overexpression enhances neurite outgrowth in a stably transfected neuronal cell line. These data identify SCG10 as a key regulator of neurite extension through regulation of microtubule instability.

Ultrastructural and cytochemical identification of apoptotic cell death accompanying development of the fetal rat olfactory nerve layer.

It has been previously shown that the embryonic olfactory nerve contains, in addition to glial ensheathing cells, a large population of differentiated neurons that migrate from the developing olfactory epithelium, in close association with the olfactory axon fascicles. The purpose of our study was to verify the hypothesis according to which a process of physiological cell death might be involved in the progressive disappearance of these migrating neurons that has been reported during late embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in rat embryos by using light and electron microscopy, with special reference to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visualization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labeling was performed by combining the DNA-specific bisbenzimide with two neuronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of programmed cell death accompanying the morphogenesis of the olfactory nerve layer. A cell death process was observed within the olfactory nerve layer from its onset at embryonic day 13 (E13). While only few pycnotic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cells were also observed along the olfactory axon fascicles when they penetrated the olfactory nerve layer. Degenerating cells appeared strongly TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apoptotic cells were neurons. These observations indicate that apoptosis may account for the progressive decrease in the number of migrating neurons present within the embryonic olfactory nerve layer. Otherwise, a zone of massive cell death by apoptosis was observed at E14 within the nasal mesenchyme located ventrally and caudally to the olfactory nerve layer. Double-labeling experiments showed that apoptotic cells present within this zone were not neurons. Our findings strongly suggest that apoptotic cell death of migrating neurons may allow the elimination of non-functional cells whereas that of mesenchymal cells may facilitate outgrowth of the newly formed olfactory axon fascicles by pathway formation.

The phosphoprotein stathmin is essential for nerve growth factor-stimulated differentiation.

Stathmin is a ubiquitous cytosolic protein which undergoes extensive phosphorylation in response to a variety of external signals. It is highly abundant in developing neurons. The use of antisense oligonucleotides which selectively block stathmin expression has allowed us to study directly its role in rat PC12 cells. We show that stathmin depletion prevents nerve growth factor (NGF)-stimulated differentiation of PC12 cells into sympathetic-like neurons although the expression of several NGF-inducible genes was not affected. Furthermore, we found that stathmin phosphorylation in PC12 cells which is induced by NGF depends on mitogen-activated protein kinase (MAPK) activity. We conclude that stathmin is an essential component of the NGF-induced MAPK signaling pathway and performs a key role during differentiation of developing neurons.

Immunocytochemical study of the differentiation process of the septal organ of Masera in developing rats.

The septal organ of Masera is a small patch of olfactory epithelium located near the base of the nasal septum. Using the growth-associated protein B-50/GAP-43 as neuronal marker, we have studied the differentiation process of this organ from the olfactory sheet in embryonic and newborn rats. Results show that the septal organ first appeared at embryonic day 16. Even though it was included in the olfactory sheet, the presumptive septal organ could be distinguished by a higher density of B-50/GAP-43-positive neurons. Concomitantly to its morphological development, the septal organ progressively isolated from the main olfactory epithelium. This isolation resulted from the extension of a transitional area which progressively lost its typical features of olfactory epithelium to become a putative respiratory epithelium in late embryonic stages. Results strongly suggest that the septal organ should be a proper chemosensory system with its own time-course of development.

Detection of apoptosis by electron microscopy and in situ labelling in the rat olfactory pit.

The purpose of this study was to provide further information upon the cell death process by apoptosis occurring in the olfactory pit during the primary palate formation and the vomeronasal organ detachment. Apoptotic cells were detected by coupling ultrastructural observations and in situ end-labelling of DNA breaks (TUNEL labelling) in E12-E15 rat embryos. During the primary palate formation and the vomeronasal organ closure, a strong apoptotic cell death process was observed along the midline epithelial seam after the epithelial fusion. The topographical distribution of labelled nuclei was in agreement with the morphological distribution of dying cells. One day before the nasal swellings fused, numerous degenerating cells were also detected in the regions of prospective contact which thus appeared as regions of programmed cell death.

B-50/GAP-43 expression by the olfactory receptor cells and the neurons migrating from the olfactory placode in embryonic rats.

B-50/GAP-43 is a growth-associated phosphoprotein that is commonly expressed in all developing neuronal systems. Using an immunocytochemistry approach, we have investigated the expression of this protein in the rat olfactory system during embryogenesis and neonatal development with a particular emphasis on the early developmental stages of the olfactory placode. Data show that already at embryonic day 12 (E12), a strong B-50/GAP-43 immunoreactivity was detected in few olfactory receptor cells well-recognizable by their positive short neuritic processes. The B-50/GAP-43 expression in the placodal epithelium thus appeared to coincide with the onset of neurite outgrowth. From E13 onwards, there was a rapid increase in the number of B-50/GAP-43-positive olfactory neurons and from E18, the protein was strongly expressed by nearly all neurons. In addition, results clearly demonstrate that as early as E13, B-50/GAP-43 was strongly expressed by many migrating cells which were seen leaving the pit epithelium in association with the first olfactory axons that penetrated the nasal mesenchyme. Many immunoreactive cells were also observed in the presumptive olfactory nerve layer. Experiments of double-labeling showed that B-50/GAP-43-immunostained migrating cells were also stained with anti-neuron-specific enolase (NSE). This confirms the neuronal nature of these early labeled migrating cells. The progressive disappearance of migrating neurons noted during the late stages of embryonic development is discussed in relation with their possible function in the early stages of development of the peripheral olfactory system.

Cell death in the developing olfactory epithelium of rat embryos.

Cell death process in the developing olfactory epithelium was studied by light and electron microscopy in rat embryos from embryonic days 12-18. A massive wave of cell death was observed at embryonic days 12 and 13 and two types of dying cells were seen coexisting. The first type of dying cells exhibited morphological features of apoptosis while the second showed characteristics of a cell death by non-lysosomal disintegration with cytoplasmic swelling and absence of phagocytosis. From embryonic day 14 onward, only apoptotic figures could be still occasionally observed. The significance of such a massive wave of cell death occurring during the earliest developmental stages of the rat olfactory epithelium is discussed in relation with the morphogenesis of the olfactory system.

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode.

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.