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Axons rely on guidance cues to reach remote targets during nervous system development. A well-studied model system for axon guidance is the retinotectal projection. The retina can be divided into halves; the nasal half, next to the nose, and the temporal half. A subset of retinal axons, those from the temporal half, is guided by repulsive cues expressed in a graded fashion in the optic tectum, part of the midbrain. Here we report the cloning and functional characterization of a membrane-associated glycoprotein, which we call RGM (repulsive guidance molecule). This molecule shares no sequence homology with known guidance cues, and its messenger RNA is distributed in a gradient with increasing concentration from the anterior to posterior pole of the embryonic tectum. Recombinant RGM at low nanomolar concentration induces collapse of temporal but not of nasal growth cones and guides temporal retinal axons in vitro, demonstrating its repulsive and axon-specific guiding activity.

Repulsive guidance molecule (RGM) is an axon guidance protein that repels retinal axons upon activation of the neogenin receptor. To understand the functions of RGM-neogenin complexes in vivo, we used gene transfer technology to perturb their expression in the developing neural tube of chick embryos. Surprisingly, neogenin over-expression or RGM down-expression in the neural tube induces apoptosis. Neogenin pro-apoptotic activity in immortalized neuronal cells and in the neural tube is associated with the cleavage of its cytoplasmic domain by caspases. Thus neogenin is a dependence receptor inducing cell death in the absence of RGM, whereas the presence of RGM inhibits this effect.

Although Munc18-1 was originally identified as a syntaxin1-interacting protein, the physiological significance of this interaction remains unclear. In fact, recent studies of Munc18-1 mutants have suggested that Munc18-1 plays a critical role for docking of secretory vesicles, independent of syntaxin1 regulation. Here we investigated the role of Munc18-1 in syntaxin1 localization by generating stable neuroendocrine cell lines in which Munc18-1 was strongly down-regulated. In these cells, the secretion capability, as well as the docking of dense-core vesicles, was significantly reduced. More importantly, not only was the expression level of syntaxin1 reduced, but the localization of syntaxin1 at the plasma membrane was also severely perturbed. The mislocalized syntaxin1 resided primarily in the perinuclear region of the cells, in which it was highly colocalized with Secretogranin II, a marker protein for dense-core vesicles. In contrast, the expression level and the plasma membrane localization of SNAP-25 were not affected. Furthermore, the syntaxin1 localization and the secretion capability were restored upon transfection-mediated reintroduction of Munc18-1. Our results indicate that endogenous Munc18-1 plays a critical role for the plasma membrane localization of syntaxin1 in neuroendocrine cells and therefore necessitates the interpretation of Munc18-1 mutant phenotypes to be in terms of mislocalized syntaxin1.

To promote functional recovery after CNS injuries, it is crucial to develop strategies that enhance both neuronal survival and regeneration. Here, we report that caspase-6 is upregulated in injured retinal ganglion cells and that its inhibition promotes both survival and regeneration in these adult CNS neurons. Treatment of rat retinal whole mounts with Z-VEID-FMK, a selective inhibitor of caspase-6, enhanced ganglion cell survival. Moreover, retinal explants treated with this drug extended neurites on myelin. We also show that caspase-6 inhibition resulted in improved ganglion cell survival and robust axonal regeneration following optic nerve injury in adult rats. The effects of Z-VEID-FMK were similar to other caspase inhibitory peptides including Z-LEHD-FMK and Z-VAD-FMK. In searching for downstream effectors for caspase-6, we identified caspase-8, whose expression pattern resembled that of caspase-6 in the injured eye. We then showed that caspase-8 is activated downstream of caspase-6 in the injured adult retina. Furthermore, we investigated the role of caspase-8 in RGC apoptosis and regenerative failure both in vitro and in vivo. We observed that caspase-8 inhibition by Z-IETD-FMK promoted survival and regeneration to an extent similar to that obtained with caspase-6 inhibition. Our results indicate that caspase-6 and caspase-8 are components of a cellular pathway that prevents neuronal survival and regeneration in the adult mammalian CNS.

Although originally discovered as neuronal growth cone-collapsing factors, repulsive guidance molecules (RGMs) are now known as key players in many fundamental processes, such as cell migration, differentiation, iron homeostasis, and apoptosis, during the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. Furthermore, three RGMs (RGMa, RGMb/DRAGON, and RGMc/hemojuvelin) have been linked to the pathogenesis of various disorders ranging from multiple sclerosis (MS) to cancer and juvenile hemochromatosis (JHH). While the molecular details of these (patho) biological effects and signaling modes have long remained unknown, recent studies unveil several exciting and novel aspects of RGM processing, ligand-receptor interactions, and downstream signaling. In this review, we highlight recent advances in the mechanisms-of-action and function of RGM proteins. RGMs: A Small Gene Family with Widespread EffectsGuidance molecules, initially observed to direct growing axons during embryogenesis [1], also have crucial roles in the morphogenesis and homeostasis of non-neuronal tissues by controlling a plethora of cellular processes, ranging from cell division and migration to differentiation and death. Figure 1A). Each RGM displays tissuespecific expression, is subjected to distinct biosynthetic and processing steps, and has not only unique, but also shared biological functions [2]. RGMs bind the type 1 transmembrane protein Neogenin ( Figure 1A) and many of the reported biological effects of RGMs rely on Neogenin receptor functions, such as axon guidance or neuronal survival [3,4]. RGMs also serve as co-receptors for bone morphogenetic proteins (BMPs) ( Figure 1A) to regulate iron metabolism, skeletal development [5-10] and axon regeneration [11]. In addition to these physiological roles, and as discussed below, RGMs have been implicated in various diseases and are considered to be promising targets in the treatment of MS, spinal cord injury, stroke, anemia, and inflammation [5,[11][12][13][14][15].Although the molecular mechanisms underlying the biological effects and signaling modes of RGMs have long remained unknown, recent work has unveiled several exciting and novel aspects of RGM processing, ligand-receptor interactions, and downstream signaling. These insights include high-resolution structural data of binary or tertiary protein complexes, the unique processing of RGMs into protein fragments with distinct functions, and the identification of a novel molecular mechanism to control ligand-induced ectodomain shedding of Neogenin. In this review, we discuss recent highlights in RGM research, from novel structural data to previously unexplored signaling mechanisms and cellular functions. Structural Insight into Ligand-Receptor InteractionsFor many years, RGMs posed a molecular puzzle because of a general lack of structural homologies to any known protein fold. Recent studies have shed light on their 3D structure and identified two ordered and disulfide-stabiliz...

Single chain variable domain (Fv) fragments (scFv) are powerful tools in research and clinical settings, owing to better pharmacokinetic properties compared to the parent monoclonal antibodies and the relative ease of producing them in large quantities, at low cost. Though they offer several advantages, they suffer from lower binding affinity and rapid clearance from circulation, which limits their therapeutic potential. However, these fragments can be genetically modified to enhance desirable properties, such as multivalency, high target retention and slower blood clearance, and as such, a variety of scFv formats have been generated. ScFvs can be administered by systemic injection for diagnostic and therapeutic purposes. They can be expressed in vivo through viral vectors in instances where large infection rates and sustenance of high levels of the antibody is required. ScFvs have found applications as tools for in vivo loss-of-function studies and inactivation of specific protein domains, diagnostic imaging, tumor therapy and treatment for neurodegenerative and infectious diseases. This review will focus on their in vivo applications

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