Expression of the gene encoding the chemorepellent semaphorin III is induced in the fibroblast component of neural scar tissue formed following injuries of adult but not neonatal CNS.

This study evaluates the expression of the chemorepellent semaphorin III (D)/collapsin-1 (sema III) following lesions to the rat CNS. Scar tissue, formed after penetrating injuries to the lateral olfactory tract (LOT), cortex, perforant pathway, and spinal cord, contained numerous spindle-shaped cells expressing high levels of sema III mRNA. The properties of these cells were investigated in detail in the lesioned LOT. Most sema III mRNA-positive cells were located in the core of the scar and expressed proteins characteristic for fibroblast-like cells. Neuropilin-1, a sema III receptor, was expressed in injured neurons with projections to the lesion site, in a subpopulation of scar-associated cells and in blood vessels around the scar. In contrast to lesions made in the mature CNS, LOT transection in neonates did not induce sema III mRNA expression within cells in the lesion and was followed by vigorous axonal regeneration. The concomitant expression of sema III and its receptor neuropilin-1 in the scar suggests that sema III/neuropilin-1-mediated mechanisms are involved in CNS scar formation. The expression of the secreted chemorepellent sema III following CNS injury provides the first evidence that chemorepulsive semaphorins may contribute to the inhibitory effects exerted by scars on the outgrowth of injured CNS neurites. The vigorous regrowth of injured axons in the absence of sema III following early neonatal lesions is consistent with this notion. The inactivation of sema III in scar tissue by either antibody perturbation or by genetic or pharmacological intervention could be a powerful means to promote long-distance regeneration in the adult CNS.

Transgenic expression of B-50/GAP-43 in mature olfactory neurons triggers downregulation of native 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) 7953-7965]. 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.

Evidence for a role of the chemorepellent semaphorin III and its receptor neuropilin-1 in the regeneration of primary olfactory axons.

To explore a role for chemorepulsive axon guidance mechanisms in the regeneration of primary olfactory axons, we examined the expression of the chemorepellent semaphorin III (sema III), its receptor neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) during regeneration of the olfactory system. In the intact olfactory system, neuropilin-1 and CRMP-2 mRNA expression define a distinct population of olfactory receptor neurons, corresponding to immature (B-50/GAP-43-positive) and a subset of mature (olfactory marker protein-positive) neurons located in the lower half of the olfactory epithelium. Sema III mRNA is expressed in pial sheet cells and in second-order olfactory neurons that are the target cells of neuropilin-1-positive primary olfactory axons. These data suggest that in the intact olfactory bulb sema III creates a molecular barrier, which helps restrict ingrowing olfactory axons to the nerve and glomerular layers of the bulb. Both axotomy of the primary olfactory nerve and bulbectomy induce the formation of new olfactory receptor neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema III mRNA is transiently induced in cells at the site of the lesion. These cells align regenerating bundles of olfactory axons. In contrast to the transient appearance of sema III-positive cells at the lesion site after axotomy, sema III-positive cells increase progressively after bulbectomy, apparently preventing regenerating neuropilin-1-positive nerve bundles from growing deeper into the lesion area. The presence of sema III in scar tissue and the concomitant expression of its receptor neuropilin-1 on regenerating olfactory axons suggests that semaphorin-mediated chemorepulsive signal transduction may contribute to the regenerative failure of these axons after bulbectomy.

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.

Anatomical distribution of the chemorepellent semaphorin III/collapsin-1 in the adult rat and human brain: predominant expression in structures of the olfactory-hippocampal pathway and the motor system.

Alterations in neuronal connectivity of the mature central nervous system (CNS) appear to depend on a delicate balance between growth-promoting and growth-inhibiting molecules. To begin to address a potential role of the secreted chemorepulsive protein semaphorin(D)III/collapsin-1 (semaIII/coll-1) in structural plasticity during adulthood, we used high-resolution nonradioactive in situ hybridization to identify neural structures that express semaIII/coll-1 mRNA in the mature rat and human brain. SemaIII/coll-1 was expressed in distinct but anatomically and functionally linked structures of the adult nervous system. The olfactory-hippocampal pathway displayed semaIII/coll-1 expression in a continuum of neuronal structures, including mitral and tufted cells of the olfactory bulb, olfactory tubercle, and piriform cortex; and distinct nuclei of the amygdaloid complex, the superficial layers of the entorhinal cortex, and the subiculum of the hippocampal formation. In addition, prominent labeling was found in neuronal components of the motor system, particularly in cerebellar Purkinje cells and in subpopulations of cranial and spinal motoneurons. Retrograde tracing combined with in situ hybridization also revealed that the staining of semaIII/coll-1 within the entorhinal cortex was present in the stellate neurons that project via the perforant path to the molecular layer of the dentate gyrus. Like in the rat, the human brain displayed discrete expression of semaIII/coll-1. Among the structures examined, the most prominent staining was observed in the cellular islands of the superficial layers of the human entorhinal cortex. The constitutive expression of the chemorepellent semaIII/coll-1 in discrete populations of neurons in the mature rat and human CNS raises the possibility that, in addition to its function as repulsive axon guidance cue during development, semaIII/coll-1 might be involved in restricting structural changes that occur in the wiring of the intact CNS.

Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants.

B-50/GAP-43 is a nervous tissue-specific protein, the expression of which is associated with axon growth and regeneration. Its overexpression in transgenic mice produces spontaneous axonal sprouting and enhances induced remodeling in several neuron populations (; ). We examined the capacity of this protein to increase the regenerative potential of injured adult central axons, by inducing targeted B-50/GAP-43 overexpression in Purkinje cells, which normally show poor regenerative capabilities. Thus, transgenic mice were produced in which B-50/GAP-43 overexpression was driven by the Purkinje cell-specific L7 promoter. Uninjured transgenic Purkinje cells displayed normal morphology, indicating that transgene expression does not modify the normal phenotype of these neurons. By contrast, after axotomy numerous transgenic Purkinje cells exhibited profuse sprouting along the axon and at its severed end. Nevertheless, despite these growth phenomena, which never occurred in wild-type mice, the severed transgenic axons were not able to regenerate, either spontaneously or into embryonic neural or Schwann cell grafts placed into the lesion site. Finally, although only a moderate Purkinje cell loss occurred in wild-type cerebella after axotomy, a considerable number of injured transgenic neurons degenerated, but they could be partially rescued by the different transplants placed into the lesion site. Thus, B-50/GAP-43 overexpression substantially modifies Purkinje cell response to axotomy, by inducing growth processes and decreasing their resistance to injury. However, the presence of this protein is not sufficient to enable these neurons to accomplish a full program of axon regeneration.

Adenoviral vector-mediated expression of B-50/GAP-43 induces alterations in the membrane organization of olfactory axon terminals in vivo.

B-50/GAP-43 is an intraneuronal membrane-associated growth cone protein with an important role in axonal growth and regeneration. By using adenoviral vector-directed expression of B-50/GAP-43 we studied the morphogenic action of B-50/GAP-43 in mature primary olfactory neurons that have established functional synaptic connections. B-50/GAP-43 induced gradual alterations in the morphology of olfactory synapses. In the first days after overexpression, small protrusions originating from the preterminal axon shaft and from the actual synaptic bouton were formed. With time the progressive formation of multiple ultraterminal branches resulted in axonal labyrinths composed of tightly packed sheaths of neuronal membrane. Thus, B-50/GAP-43 is a protein that can promote neuronal membrane expansion at synaptic boutons. This function of B-50/GAP-43 suggests that this protein may subserve an important role in ongoing structural synaptic plasticity in adult neurons and in neuronal membrane repair after injury to synaptic fields.

Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS.

In this paper a detailed protocol is presented for neuroscientists planning to start work on first generation recombinant adenoviral vectors as gene transfer agents for the nervous system. The performance of a prototype adenoviral vector encoding the bacterial lacZ gene as a reporter was studied, following direct injection in several regions of the central and peripheral nervous system. The distribution of the cells expressing the transgene appears to be determined by natural anatomical boundaries and possibly by the degree of myelinization of a particular brain region. In highly myelinated areas with a compact cellular structure (e.g. the cortex and olfactory bulb) the spread of the viral vector is limited to the region close to the injection needle, while in areas with a laminar structure (e.g. the hippocampus and the eye) more widespread transgene expression is observed. Retrograde transport of the viral vector may serve as an attractive alternative route of transgene delivery. A time course of expression of beta-galactosidase in neural cells in the facial nucleus revealed high expression during the first week after AdLacZ injection. However, a significant decline in transgene expression during the second and third week was observed. This may be caused by an immune response against the transduced cells or by silencing of the cytomegalovirus promoter used to drive transgene expression. Taken together, the data underscore that for each application of adenoviral vectors as gene transfer agents in the nervous system it is important to examine vector spread in and infectability of the neural structure that is subject to genetic modification.

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 (10(9) plaque-forming units (PFU)) results in transgene expression in many cells throughout the epithelium for 8-12 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 x 10(10) 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.

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 fiber growth.

B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP-positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons.

Use of viral vectors to promote neuroregeneration.

Efficient methods to introduce and express therapeutic genes in the central and peripheral nervous system will find applications in the treatment of neurodegenerative diseases caused by single gene mutations or degeneration with a mechanical, metabolic, or immunological origin. The goal of our research is to develop experimental gene therapy, based on in vivo gene delivery with viral vectors, to promote neuroregeneration in the peripheral and central nervous system. This paper provides an overview of work to determine the capacity of herpes simplex and adenoviral vectors, encoding members of the neurotrophin gene family or the intraneuronal growth-associated protein B-50/GAP-43, to stimulate neurite outgrowth. Initial results demonstrate that viral vector-mediated transfer of genes encoding these neurite growth promoting molecules could be an effective strategy to enhance the growth potential of injured neurons. A number of biological and technical hurdles that have to be settled in order to move closer to future clinical applications will be discussed briefly.

Synchronism of pressor response and grooming behavior in freely moving, conscious rats following intracerebroventricular administration of ACTH/MSH-like peptides.

After the i.c.v. administration of 300 pmol ACTH-(1-24) or [Nle4,D-Phe7]alpha-MSH, a long-lasting increase in blood pressure was observed synchronously with the incidence of excessive grooming. Two structurally related peptides with no grooming behavior-inducing potency, ACTH-(7-16)-NH2 and gamma 2-MSH, in doses of 300 and 500 pmol, respectively, caused a slight and short-lasting increase in blood pressure or had no effect, respectively. When the grooming behavior-inducing effect of ACTH-(1-24) was abolished, either by the prior manipulation of central dopaminergic neurotransmission by the i.c.v. administration of the dopamine receptor antagonist, haloperidol, or, due to the occurrence of single-dose tolerance to ACTH-(1-24), the pressor response was abolished as well. These data are in support of the postulate that the incidence of grooming behavior and the elevation of blood pressure are temporally associated and indicate that the two phenomena are causally related.

Peptide-induced grooming behavior and caudate nucleus dopamine release.

We simultaneously measured the display of grooming behavior and, by monitoring the extracellular dopamine concentration via transversal microdialysis, the release of dopamine in the caudate nucleus in freely moving rats after i.c.v. administration of 1 micrograms adrenocorticotropic hormone-(1-24) (ACTH-(1-24)). During a period of 1 h after administration of the peptide, the incidence of excessive grooming behavior was increased. Concomitantly, the concentration of dopamine in the caudate nucleus dialysates was significantly increased (maximal effect 151% of basal release) whereas that of its metabolite DOPAC was unchanged. The potent alpha-melanocyte stimulating hormone (alpha-MSH) receptor agonist, [Nle4,D-Phe7]alpha-MSH, induced grooming behavior and stimulated caudate nucleus dopamine release (maximal effect 148% of basal release) whereas ACTH-(7-16)-NH2 did neither induce grooming behavior nor cause an increase in caudate nucleus dopamine release. Single-dose tolerance was observed for ACTH-induced grooming but not for ACTH-induced dopamine release. These data are in support of the proposed involvement of brain dopamine systems in grooming behavior of the rat but at the same time suggest that the effect of ACTH/MSH-like peptides on dopaminergic transmission in the caudate nucleus is proximal to the final neural pathway involved in ACTH-induced grooming behavior.