Adenoviral vector-mediated expression of neurotrophin-3 increases neuronal survival in suprachiasmatic nucleus grafts.

To improve transplantation results of fetal suprachiasmatic nucleus (SCN) in SCN-lesioned (SCNX) rats, grafts were ex vivo transduced with an adenoviral vector encoding for neurotrophin-3 (AdNT-3) before implantation. Mock- and AdLacZ-transduced grafts were used as controls. First, transplants were evaluated microscopically and by image analysis for the presence of vasopressinergic (VPergic) and vasoactive intestinal polypeptidergic (VIPergic) SCN neurons at 10 weeks or later postgrafting. Ex vivo AdNT-3-transduced transplants displayed increased volume areas of VPergic and VIPergic SCN cells in comparison with those in mock- and AdLacZ-transduced transplants, but significantly improved graft-to-host VPergic and VIPergic SCN fiber growth was not reached (though AdNT-3-transduced transplants tended to grow more VPergic fibers into the brain of VP-deficient SCNX Brattleboro rat recipients, which were chosen as recipients to circumvent the presence of non-SCN VP fiber staining). Second, a small group of arrhythmic Wistar rats received AdNT-3- or control-treated SCN grafts while continuously on-line for the monitoring of overt circadian activities in the pre- and postgrafting periods. The results indicated that ex vivo transduced SCN grafts can still restore arrhythmia, but that the NT-3-mediated anatomical improvements of the grafting results were not sufficient to enhance efficacy of reinstatement of circadian rhythm in SCN-lesioned rats. However, in this group VIP staining volume area, not VP staining volume area, correlated significantly with reinstatement of circadian rhythm.

Intercostal nerve implants transduced with an adenoviral vector encoding neurotrophin-3 promote regrowth of injured rat corticospinal tract fibers and improve hindlimb function.

Following injury to central nervous tissues, damaged neurons are unable to regenerate their axons spontaneously. Implantation of peripheral nerves into the CNS, however, does result in axonal regeneration into these transplants and is one of the most powerful strategies to promote CNS regeneration. In the present study implantation of peripheral nerve bridges following dorsal hemisection is combined with ex vivo gene transfer with adenoviral vectors encoding neurotrophin-3 (Ad-NT-3) to examine whether this would stimulate regeneration of one of the long descending tracts of the spinal cord, the corticospinal tract (CST), into and beyond the peripheral nerve implant. We chose to use an adenoviral vector encoding NT-3 because CST axons are sensitive to this neurotrophin and Schwann cells in peripheral nerve implants do not express this neurotrophin. At 16 weeks postimplantation of Ad-NT-3-transduced intercostal nerves, approximately three- to fourfold more of the anterogradely traced corticospinal tract fibers had regrown their axons through gray matter below the lesion site when compared to control animals. Regrowth of CST fibers occurred over more than 8 mm distal to the lesion site. No regenerating CST fibers were, however, observed into the transduced peripheral implant. Animals with a peripheral nerve transduced with Ad-NT-3 also exhibited improved function of the hindlimbs when compared to control animals treated with an adenoviral vector encoding LacZ. Thus, transient overexpression of NT-3 in peripheral nerve tissue bridges is apparently sufficient to stimulate regrowth of CST fibers and to promote recovery of hindlimb function, but does not result in regeneration of CST fibers into such transplants. Taken together, combining an established neurotransplantation approach with viral vector-gene transfer promotes the regrowth of injured CST fibers through gray matter and improves the recovery of hindlimb function.

The use of adenoviral vectors and ex vivo transduced neurotransplants: towards promotion of neuroregeneration.

Regeneration of injured axons following injury depends on a delicate balance between growth-promoting and growth-inhibiting factors. Overexpression of neurotrophin genes seems a promising strategy to promote regeneration. Trophic genes can be overexpressed at the site of injury at the axonal stumps, or at the perikaryal level of the injured neuron. Transduction of the neural cells can be achieved by applying adenoviral vectors, either directly in vivo or-in the case of neurotransplantation as an ex vivo approach. In both cases it would create a more permissive environment for axonal growth and therefore in functional regeneration. In this article, the feasibility of the use of adenoviral vectors in several neuroregeneration models--in particularly in spinal cord lesion models and the biological clock transplantation model--is illustrated. The results show that the adenoviral vectors can be a powerful tool to study the effects of overexpression of genes in an in vivo paradigm of nerve regeneration or nerve outgrowth. The potential use of adenoviral vectors and ex vivo transduced neurotransplants is discussed.

Purification of recombinant adeno-associated virus by iodixanol gradient ultracentrifugation allows rapid and reproducible preparation of vector stocks for gene transfer in the nervous system.

Recombinant adeno-associated virus (rAAV) vectors have become attractive tools for in vivo gene transfer. The production and purification of high-titer rAAV vector stocks for experimental and therapeutic gene transfer continue to undergo improvement. Standard rAAV vector purification protocols include the purification of the vector by cesium chloride (CsCl)-density gradient centrifugation followed by extensive desalination via dialysis against a physiological buffer for in vivo use. These procedures are extremely time consuming and frequently result in a substantial loss of the infectious vector titer. As an alternative to CsCl we have investigated the use of Iodixanol, an X-ray contrast solution, as the density-gradient medium. Purification of rAAV vectors by Iodixanol shortened the centrifugation period to 3 hr and resulted in reproducible concentration and purification of rAAV-vector stocks. We show that injection of rAAV derived from an Iodixanol gradient can be used for in vivo gene transfer applications in the brain and spinal cord without detectable cytopathic effects and directing stable transgene expression for at least 2 months.

Ontogeny of neurotrophin receptor trkC expression in the rat forebrain and anterior hypothalamus with emphasis on the suprachiasmatic nucleus.

There is little information about neurotrophic regulation in the developing rat hypothalamus. In the present study, we therefore examined the expression of neurotrophin receptor TrkC in the developing forebrain and hypothalamus. In situ hybridization of coronal sections revealed that on the 15th day of gestation, trkC messenger RNA expression is homogeneously distributed over the neocortex, septum, thalamus, hypothalamus, hippocampus, rhinencephalon and the amygdala. Exceptions were the anteroventral nucleus of the hypothalamus and the striatum, which showed higher levels of trkC messenger RNA expression, and the germinal zones which were devoid of trkC messenger RNA. After birth, the homogeneous staining pattern changes into a heterogeneous staining pattern like that found in adulthood. TrkC expression is observed in the area of the suprachiasmatic nucleus as early as E17 and continues until adulthood. The presence of the TrkC receptor in the E17 suprachiasmatic nucleus suggests that neurotrophin-3 plays a role in development of this structure and that application of neurotrophin-3 could stimulate neuronal survival and neuritic outgrowth in a suprachiasmatic nucleus transplantation model.

Adenoviral vector-mediated expression of a foreign gene in peripheral nerve tissue bridges implanted in the injured peripheral and central nervous system.

Axons of the CNS do normally not regenerate after injury, in contrast to axons of the PNS. This is due to a different microenvironment at the site of the lesion as well as a particular intrinsic program of axonal regrowth. Although transplantation of peripheral nerve tissue bridges is perhaps the most successful approach to promoting regeneration in the CNS, ingrowth of CNS nerve fibers with such transplants is limited. Genetic modification of peripheral nerve bridges to overexpress outgrowth-promoting proteins should, in principle, improve the permissive properties of peripheral nerve transplants. The present study shows that pieces of peripheral intercostal nerve, subjected to ex vivo adenoviral vector-mediated gene transfer and implanted as nerve bridges in transected sciatic nerve, avulsed ventral root, hemi-sected spinal cord and intact brain, are capable of expressing a foreign gene. In vitro studies showed expression of the reporter gene LacZ up to 30 days in Schwann cells. After implantation, LacZ expression could be detected at 7 days postimplantation, but had virtually disappeared at 14 days. Schwann cells of the transduced nerve bridges retained the capacity of guiding regenerative peripheral and central nerve fiber ingrowth. Transduction of intercostal nerve pieces prior to implantation should, in principle, enable enhanced local production of neurotrophic factors within the transplant and has the potential to improve the regeneration of injured axons into the graft.

NT-3 delivered by an adenoviral vector induces injured dorsal root axons to regenerate into the spinal cord of adult rats.

Sensory axons interrupted in the dorsal roots of adult mammals are normally unable to regenerate into the spinal cord. We have investigated whether the introduction of a neurotrophin gene into the spinal cord might offer an approach to otherwise intractable spinal root injuries. The dorsal roots of the 4th, 5th, and 6th lumbar spinal nerves of adult rats were severed and reanastomosed. Fourteen to nineteen days later, adenoviral vectors containing either the LacZ or NT-3 genes were injected into the ventral horn of the lumbar spinal cord, resulting in strong expression of the transgenes in glial cells and motor neurons between 4 and 40 days after injection. When dorsal root axons were transganglionically labelled with HRP conjugated to cholera toxin subunit B, 16 to 37 days after dorsal root injury, large numbers of labelled axons could be seen to have regenerated into the cord, but only in those animals injected with vector carrying the NT-3 gene. The regenerated axons were found at the injection site, mainly in the grey matter, and had penetrated as deep as lamina V. Gene therapy with adenoviral vectors encoding a neurotrophin has therefore been shown to be capable of enhancing and directing the regeneration of a subpopulation of dorsal root axons (probably myelinated A fibres), into and through the CNS environment.

Growth of pyramidal, but not non-pyramidal, dendrites in long-term organotypic explants of neonatal rat neocortex chronically exposed to neurotrophin-3.

The present study was undertaken to determine the effects of neurotrophin-3 (NT3) and spontaneous bioelectric activity (SBA) on dendritic elongation and branching in long-term isolated organotypic explants of rat neocortex. Viral vector-directed expression of NT3 was used as an effective means to ensure a continuous, local production of the neurotrophic factor. Quantitative light microscopic measurement of dendritic branching patterns was carried out on Golgi-stained materials. Explants were exposed to an adenoviral vector encoding the genetic sequence for neurotrophin-3 (Ad-NT3), or to exogenous additions of the neuropeptide NT3. In order to test for activity-dependent growth effects under control and experimental conditions, explants were exposed to glutamatergic blockade using a cocktail of APV and DNQX. Both Ad-NT3 and NT3 peptide potently promoted apical and basal dendritic growth (elongation and branching) in pyramidal neurons. This growth was observed to be significant in layers II-IV and V. These growth effects were also not activity dependent, inasmuch as they were elicited from explants in which spontaneous bioelectric activity had been suppressed. Non-pyramidal neurons, throughout the neocortical slice, showed no significant dendritic responses to the prolonged presence of NT3. These findings show that pyramidal dendritic growth in long-term neocortical explants responds to at least one neurotrophic growth factor, NT3, and is independent of intrinsic bioelectric activity. The use of viral vectors in delivering a continuous high level of neurotrophic factor within developing neural tissues demonstrates its potential application to in vivo tissues during development, or in the stimulation of neuritogenesis and neuroregeneration following injuries.

Adenoviral vector-mediated gene delivery to injured rat peripheral nerve.

Although much progress has been made, current treatments of peripheral nerve damage mostly result in only partial recovery. Local production of neurite outgrowth-promoting molecules, such as neurotrophins and/or cell adhesion molecules, at the site of damage may be used as a new means to promote the regeneration process. We have now explored the ability of an adenoviral vector encoding the reporter gene LacZ (Ad-LacZ) to direct the expression of a foreign gene to Schwann cells of intact and crushed rat sciatic nerves. Infusion of 8 x 10(7) PFU Ad-LacZ in the intact sciatic nerve resulted in the transduction of many Schwann cells with high levels of transgene expression lasting at least up to 12 days following viral vector administration. The efficacy of adenoviral vector delivery to a crushed nerve was investigated using three strategies. Injection of the adenoviral vector at the time of, or immediately after, a crush resulted in the transduction of only a few Schwann cells. Administration of the adenoviral vector the day after the crush resulted in the transduction of a similar number of Schwann cells 5 days after administration, as observed in uncrushed nerves. Regenerating nerve fibers were closely associated with beta-galactosidase-positive Schwann cells, indicating that the capacity of transduced Schwann cells to guide regenerating fibers was not altered. These results imply that the expression of growth-promoting proteins through adenoviral vector-mediated gene transfer may be a realistic option to promote peripheral nerve regeneration.

Adenoviral vector-directed expression of neurotrophin-3 in rat dorsal root ganglion explants results in a robust neurite outgrowth response.

The neurotrophins are a family of proteins that promote neuronal survival and neurite outgrowth during development and can also enhance the regeneration of injured adult neurons. The local and continuous delivery of these proteins at the site of injury is problematic, since this requires repeated intraparenchymal injections or the use of invasive canula-micropump devices. In the present study we report the generation and characterization of an adenoviral vector for a member of the neurotrophins, neurotrophin-3 (Ad-NT-3). Using Ad-NT-3, we examined the expression and biological activity of NT-3 in dorsal root ganglia (DRG) explant cultures. Gene transfer with Ad-NT-3 results in the synthesis of genuine NT-3 and in a dosage-dependent neurite outgrowth response in DRG explants. Transduction of DRG explants with a viral vector dosage of 5 x 10(5) to 5 x 10(6) plaque-forming units induced the formation of a dense halo of neurites comparable to outgrowth observed following the addition of 100 ng/mL exogenous NT-3. In addition, a single infection with Ad-NT-3 produced biologically active NT-3 for at least 20 days in culture, as evidenced by continued neurite extension. This indicates that adenoviral vector-mediated expression of NT-3 results in high-level production of biologically active NT-3 and could therefore be used as a strategy to promote the regeneration of injured peripheral and central nerve projections.

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.

Co-localization of high-affinity neurotrophin receptors in nucleus basalis of Meynert neurons and their differential reduction in Alzheimer's disease.

It has been suggested that degeneration of neurons in Alzheimer's disease is the result of diminished trophic support. However, so far no evidence has been forwarded that neuronal degeneration in Alzheimer's disease is causally related to insufficient production of neurotrophins. The present study deals with (i) the expression and co-localization of tyrosine kinase receptors (trks) in the human nucleus basalis of Meynert and (ii) alterations of these receptors in Alzheimer's disease in the nucleus basalis of Meynert, an area severely affected in Alzheimer's disease. The expression of trkA, trkB and trkC in the nucleus basalis of Meynert of control and Alzheimer's disease brains was studied using three polyclonal antibodies specifically recognizing the extracellular domain of trkA, trkB and trkC. Brain material of eight controls and seven Alzheimer's disease patients was obtained at autopsy, embedded in paraffin and stained immunocytochemically. Using an image analysis system, we determined the proportion of trk neurons expressing the different trk receptors in controls and Alzheimer's disease patients. In control brains, trkA, trkB and trkC were differentially expressed in numerous nucleus basalis of Meynert neurons. The highest proportion of neurons was found to express trkB (75%), followed by trkC (58%) and trkA (54%). Furthermore, using consecutive sections, a clear co-localization of trk receptors was observed in the same neurons. The highest degree of co-localization was observed between trkA and trkB. In Alzheimer's disease patients, the number of immunoreactive neurons and the staining intensity of individual neurons was reduced dramatically. Reduction in the proportion of neurons expressing trkA was 69%, in trkB 47% and in trkC 49%, which indicated a differential reduction in the amount of trk receptors in Alzheimer's disease. These observations indicate that nucleus basalis of Meynert neurons can be supported by more than one neurotrophin and that the degeneration of these neurons in Alzheimer's disease is associated with a decreased expression of trk receptors, suggesting a decreased neurotrophin responsiveness of nucleus basalis of Meynert neurons in Alzheimer's disease.

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.