Neuropilin and class 3 semaphorins in nervous system regeneration

Injury to the mature mammalian central nervous system (CNS) is often accompanied by permanent loss of function of the damaged neural circuits. The failure of injured CNS axons to regenerate is thought to be caused, in part, by neurite outgrowth inhibitory factors expressed in and around the lesion. These include several myelin associated inhibitors, proteoglycans, and tenascin-R. Recent studies have documented the presence of class 3 semaphorins in fibroblast-like meningeal cells present in the core of the neural scar formed following CNS injury. Class 3 semaphorins display neurite growth-inhibitory effects on growing axons during embryonic development. The induction of the expression of class 3 semaphorins in the neural scar and the persistent expression of their receptors, the neuropilins and plexins, by injured CNS neurons suggest that they contribute to the regenerative failure of CNS neurons. Neuropilins are also expressed in the neural scar in a subpopulation of meningeal fibroblast and in neurons in the vicinity of the scar. Semaphorin/neuropilin signaling might therefore also be important for cell migration, angiogenis and neuronal cell death in or around neural scars. In contrast to neurons in the CNS, neuropilin/plexin positive neurons in the PNS do display long distance regeneration following injury. Injured PNS neurons do not encounter a semaphorin positive neural scar. Furthermore, Semaphorin 3A is downregulated in the regenerating spinal motor neurons themselves. This was accompanied by a transient upregulation of Semaphorin 3A in the target muscle. These observations suggest that the injury induced regulation of Semaphorin 3A in the PNS contributes to successful regeneration and target reinnervation. Future studies in genetically modified mice should provide more insight into the mechanisms by which neuropilins and semaphorins influence nervous system regeneration and degeneration

Manipulation of gene expression in the mammalian nervous system: application in the study of neurite outgrowth and neuroregeneration related proteins

A fundamental issue in neurobiology entails the study of the formation of neuronal connections and their potential to regenerate following injury. In recent years, an expanding number of gene families has been identified involved in different aspects of neurite outgrowth and regeneration. These include neurotrophic factors, cell-adhesion molecules, growth-associated proteins, cytoskeletal proteins and chemorepulsive proteins. Genetic manipulation technology (transgenic mice, knockout mice, viral vectors and antisense oligonucleotides) has been instrumental in defining the function of these neurite outgrowth-related proteins. The aim of this paper is to provide an overview of the above-mentioned four approaches to manipulate gene expression in vivo and to discuss the progress that has been made using this technology in helping to understand the molecular mechanisms that regulate neurite outgrowth. We will show that work with transgenic mice and knockout mice has contributed significantly to the dissection of the function of several proteins with a key role in neurite outgrowth and neuronal survival. Recently developed viral vectors for gene transfer in postmitotic neurons have opened up new avenues to analyze the function of a protein following local expression in naive adult rodents. The initial results with viral vector-based gene transfer provide a conceptual framework for further studies on genetic therapy of neuroregeneration and neurodegenerative diseases

Transgenic expression of B-50/GAP-43 expression in immature olfactory neurons

The adult mammalian olfactory neuroepithelium is an unusual neural tissue, since it maintains its capacity to form new neurons throughout life. Newly formed neurons differentiate in the basal layers of the olfactory neuroepithelium and express B-50/GAP-43, a protein implicated in neurite outgrowth. During maturation these neurons migrate into the upper portion of the epithelium, upregulate expression of olfactory marker protein (OMP) and concomitantly downregulate the expression of B-50/GAP-43. Transgenic mice that exhibit OMP-promoter directed expression of B-50/GAP-43 in mature olfactory neurons display an unexpected decrease in the complement of B-50/GAP-43-positive cells in the lower region of the olfactory epithelium [A.J.G.D. Holtmaat, P.A. Dijkhuizen, A.B. Oestreicher, H.J. Romijn, N.M.T. Van der Lugt, A. Berns, F.L. Margolis, W.H. Gispen, J. Verhaagen, Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular growth, J. Neurosci. 15 (1995) 79537965]. We have investigated whether the decrement in B-50/GAP-43-positive cells in this region was due to a dislocation of the immature neurons to other regions of the olfactory epithelium or to a downregulation of B-50/GAP-43 synthesis in these immature neurons. In eight of nine independent transgenic mouse lines that express the transgene in different numbers of olfactory neurons, a decline in the number of B-50/GAP-43-expressing neurons in the basal portion of the olfactory neuroepithelium was observed, both at the protein level and the mRNA level. An alternative marker for immature cells, a juvenile form of tubulin, was normally expressed in this location, indicating that the olfactory epithelium of OMP-B-50/GAP-43 transgenic mice contains a normal complement of immature olfactory neurons and that most of these neurons display a downregulation of B-50/GAP-43 expression

Efficient adenoviral vector directed expression of a foreign gene to neurons and sustentacular cells in the mouse olfactory neuroepithelium

Replication deficient recombinant adenoviral vectors are efficient gene transfer agents for postmitotic cells, including neurons and glial cells. In this paper we have examined the effectiveness of adenoviral vector-mediated gene transfer to the olfactory epithelium of adult mice. We show that Ad-LacZ, a prototype first generation adenoviral vector containing an expression cassette for the reporter gene LacZ, directs transgene expression to mature and immature olfactory neurons and to sustentacular cells. The technique to apply the vector to the nasal cavity and the amount of viral vector per mouse are important variables that determine the success of viral vector-mediated gene transfer to the mouse olfactory neuroepithelium. Slow infusion of the viral vector solution in fully anaesthetized mice yields the best result in terms of the number of epithelial cells transduced. Infection of the olfactory neuroepithelium with a moderate amount of viral vector (109 plaque-forming units (PFU)) results in transgene expression in many cells throughout the epithelium for 812 days, followed by a decline in transduced cells at 25 days postinstillation of the virus. This decrement in transgene expression is consistent with the natural turnover process that occurs in the epithelium throughout adulthood. At high viral loads (1.3 × 1010 PFU) extinction of transgene expression occurs as early as 8 days postinjection and is accompanied by epithelial degeneration indicating that the vector dose used should be carefully chosen. Taken together, the current observations demonstrate that adenoviral vectors are effective tools to genetically modify the adult mouse olfactory neuroepithelium in vivo