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The molecular mechanisms by which extracellular guidance cues regulate axonal morphology are not fully understood. Recent findings suggest that increased activity of the protein kinase Akt promotes dendritic branching and elongation in hippocampal neurons. We tested whether expression of constitutively active Akt (CA-Akt) in primary sensory neurons would promote axonal branching and whether targeting CA-Akt to lipid rafts, common sites of Akt function, would differentially regulate axonal morphology. Biolistic transduction of sensory neurons induced a rapid expression of CA-Akt, resulting in increased axonal branching, cell hypertrophy, and growth cone expansion. Additionally, we found that targeting of CA-Akt to lipid rafts significantly potentiated growth cone expansion compared with expression of CA-Akt throughout the neuron. Because lipid rafts are concentrated within the growth cone, this finding suggests that signaling of expansion is likely regulated locally. We found that CA-Akt-mediated growth cone expansion, but not axonal branching, was attenuated by coexpression of dominant-negative Rac1. In contrast, blockade of mammalian target of rapamycin (mTOR) prevented axonal branching and hypertrophy in response to CA-Akt, but not growth cone expansion. These data indicate that Akt activity can regulate growth cone expansion via localized Rac1 signaling and regulate axonal branching and soma size via activation of mTOR., ((c) 2009 Wiley-Liss, Inc.)

Neurotrophins promote sprouting and elongation of central nervous system (CNS) axons following injury. Consequently, it has been suggested that neurotrophins could be used to repair the CNS by inducing axonal sprouting from nearby intact axons, thereby compensating for the loss of recently injured axons. We tested whether long-term overexpression of neurotrophins in the rat cortex would induce sprouting of cortical serotonergic axons following a neurotoxic injury. After a single subcutaneous injection of para-chloroamphetamine (PCA; 9 mg/ml) that lesions the majority of serotonergic axons in the rat cortex, we injected adenoviral vectors containing cDNAs for brain-derived neurotrophic factor (Adv.BDNF), neurotrophin-3 (Adv.NT-3), or nerve growth factor (Adv.NGF) into the rat frontal cortex. Nine days later, we measured significant increases in the concentration of the respective neurotrophins surrounding the vector injection sites, as measured by ELISA. Immunohistochemical localization of serotonin revealed a fourfold increase in the density of serotonergic fibers surrounding the injection sites of Adv.BDNF and Adv.NT-3, corresponding to a 50% increase in cortical serotonin concentration, compared with a control vector containing the cDNA for enhanced green fluorescent protein (Adv.EGFP). In contrast, there was no difference in serotonergic fiber density or cortical serotonin concentration surrounding the injection of Adv.NGF compared with Adv.EGFP. These data demonstrate that localized overexpression of BDNF or NT-3, but not NGF, is sufficient to promote sprouting of serotonergic axons in the cortex following an experimental neurotoxic injury., (Copyright (c) 2005 Wiley-Liss, Inc.)

The mammalian CNS lacks the ability to effectively compensate for injury by the regeneration of damaged axons or axonal plasticity of intact axons. However, reports suggest that molecular or cellular manipulations can induce compensatory processes that could support regeneration or plasticity after trauma. We tested whether local, sustained release of the neurotrophic factor neurotrophin-3 (NT-3) would support axonal plasticity in the spinal cord distal to the site of injury in rats. The corticospinal tract (CST) was cut unilaterally at the level of the medulla. This avoided excessive inflammation, secondary cell death, vascular disruption, and the release of inhibitory molecules in the lumbar spinal cord. A replication-defective adenoviral vector (Adv) carrying the NT-3 gene (Adv.EFalpha-NT3) was delivered to the spinal motoneurons by retrograde transport through the sciatic nerve. Retrograde transport of the adenoviral vectors avoided the inflammatory response that would be associated with direct injection into the spinal cord. Transduction of spinal motoneurons with Adv.EFalpha-NT3 resulted in a significant increase in the concentration of NT-3 in the L3-L6 region of the spinal cord for up to 3 weeks. In animals with a CST lesion, this local expression of NT-3 induced growth of axons from the intact CST across the midline to the denervated side. If the CST remained intact, overexpression of NT-3 did not lead to an increase in the number of axons crossing the midline. These data demonstrate that local, sustained expression of NT-3 will support axonal plasticity of intact CST axons after trauma-induced denervation.

Between December 1996 and September 1998, 13 patients with advanced recurrent malignant brain tumors (9 with glioblastoma multiforme, 1 with gliosarcoma, and 3 with anaplastic astrocytoma) were treated with a single intratumoral injection of 2 x 10(9), 2 x 10(10), 2 x 10(11), or 2 x 10(12) vector particles (VP) of a replication-defective adenoviral vector bearing the herpes simplex virus thymidine kinase gene driven by the Rous sarcoma virus promoter (Adv.RSVtk), followed by ganciclovir (GCV) treatment. The VP to infectious unit ratio was 20:1. Our primary objective was to determine the safety of this treatment. Injection of Adv.RSVtk in doses <==2 x 10(11) VP, followed by GCV, was safely tolerated. Patients treated with the highest dose, 2 x 10(12) VP, exhibited central nervous system toxicity with confusion, hyponatremia, and seizures. One patient is living and stable 29.2 months after treatment. Two patients survived >25 months before succumbing to tumor progression. Ten patients died within 10 months of treatment, 9 from tumor progression and 1 with sepsis and endocarditis. Neuropathologic examination of postmortem tissue demonstrated cavitation at the injection site, intratumoral foci of coagulative necrosis, and variable infiltration of the residual tumor with macrophages and lymphocytes.

Application of neurotrophic factors (NFs) to the cut stump of peripheral nerves confers transient (1- to 2-week) neuroprotection of motoneurons from axotomy-induced death in neonates. We tested whether lumbar spinal motoneurons would be protected from axotomy-induced death when they were genetically modified to produce NFs in situ. Adenoviral (Adv) vectors carrying neurotrophic factor genes under control of the Rous sarcoma virus long terminal repeat promoter (Adv.RSV-nf) or a control vector containing the beta-galactosidase (beta-gal) gene (Adv.RSV-betagal) was injected into the hindlimb muscles of neonatal rats. The Adv were taken up by peripheral nerves and transported to lumbar spinal cord motoneurons where the transgenes were expressed. A fraction (18%) of the motoneurons that projected through the sciatic nerve were transduced with Adv.RSV-betagal. Expression of Adv.RSV-betagal was detected in motoneurons after 7 days and 3 weeks, with no evidence of vector- or beta-gal-induced toxicity or inflammation. PCR, immunocytochemistry, and RT-PCR demonstrated transport of the Adv.RSV-nf vectors to motoneurons and their expression. After retrograde transport of an Adv.RSV-nf vector carrying the gene for glial cell line-derived neurotrophic factor, a substantial proportion of the sciatic nerve motoneurons were resistant to axotomy-induced death 7 days and 3 weeks after sciatic nerve transection (56 and 44%, respectively), compared to Adv.RSV-betagal controls (2.5 and 0%, respectively)., (Copyright 1998 Academic Press.)

We evaluated the efficacy of adenoviral-mediated gene therapy of experimental spinal cord tumors and the functional outcome after this treatment. Spinal cord tumors were generated in the thoracic region of the spinal cord in Fischer 344 rats by stereotaxic intramedullary injection of 1 x 10(4) 9L gliosarcoma cells. Seven days after tumor cell injection, a replication-defective adenoviral vector carrying the herpes simplex virus thymidine kinase gene (ADV-tk) or a control adenoviral vector carrying the beta-galactosidase gene (ADV-beta gal) was injected into the tumors. Beginning 12 h later the animals were treated with the antiviral drug ganciclovir (GCV; 50 mg/kg) or saline twice a day for 6 days. The neurological performance of the animals was assessed during and following treatment. Eighteen days after tumor cell injection, all of the control animals had paraplegia and large tumors. In contrast, no tumors were detected in animals treated with ADV-tk and GCV. In long-term studies, two of the 5 animals treated with ADV-tk and GCV remained tumor-free and remained neurologically intact at 6 months whereas all animals in the control groups became paraplegic within 18 days.

The efficacy of adenovirus (ADV)-mediated gene therapy to treat brain tumors was tested in a syngeneic glioma model. Tumor cells were transduced in situ with a replication-defective ADV carrying the herpes simplex virus thymidine kinase (HSV-tk) gene controlled by the Rous sarcoma virus promoter. Expression of the HSV-tk gene enables the transduced cell to convert the drug ganciclovir to a form that is cytotoxic to dividing cells. Tumors were generated in Fischer 344 rats by stereotaxic implantation of 9L gliosarcoma cells into the caudate nucleus. Eight days later, the tumors were injected either with the ADV carrying the HSV-tk (ADV-tk) gene or a control ADV vector containing the beta-galactosidase (ADV-beta gal) gene and the rats were treated with either ganciclovir or saline. Tumor size was measured 20 days after implantation of 9L cells or at death. Rats treated with ADV-beta gal and ganciclovir or with ADV-tk and saline had large tumors. No tumors were detected in animals treated with ADV-tk and with ganciclovir at doses > or = 80 mg/kg. An infiltrate of macrophages and lymphocytes at the injection site in animals treated with ADV-tk and ganciclovir indicated an active local immune reaction. In survival studies, all animals treated with ADV-tk and ganciclovir have remained alive longer than 80 and up to 120 days after tumor induction whereas all untreated animals died by 22 days. These results demonstrate that ADV-mediated transfer of HSV-tk to glioma cells in vivo confers sensitivity to ganciclovir, and represents a potential method of treatment of brain tumors.

The therapeutic efficacy of adenovirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene transduction of rat C6 glioma cells followed by ganciclovir (GCV) administration was studied in tumors generated in the brains of nude mice. C6 glioma cells were efficiently transduced in vitro by a replicative-defective recombinant adenovirus carrying the HSV-tk gene (ADV/RSV-tk) that rendered them sensitive to GCV in a dose-dependent manner. Tumors were generated by stereotaxic intracerebral injection of 1 x 10(4) C6 cells in nude mice. After 8 days of tumor growth, 3 x 10(8) ADV/RSV-tk viral particles were injected into the tumors and the mice subsequently were treated with GCV for 6 days. Tumor size in untreated and treated animals was compared 20 days after tumor implantation. The mean cross-sectional area of the tumors in the treated animals was 23-fold smaller than in control animals and the tumor volume was reduced by > 500-fold. These results demonstrate that the recombinant adenoviral vector can function as an efficient gene delivery vehicle for the treatment of gliomas by in vivo gene therapy.

We investigated the effects of increasing the concentration of intracellular cyclic adenosine monophosphate (cAMP) on genes associated with oligodendrocyte differentiation in an immortalized glial cell line, 6E12, derived from the spinal cord of an MBP-SV40 large T-antigen transgenic mouse. Raising intracellular levels of cAMP induced expression of oligodendrocyte differentiation antigens recognized by O4 and anti-galactocerebroside antibodies, up-regulated expression of the proteolipid protein (PLP) gene, and down-regulated glial fibrillary acidic protein (GFAP) expression. There was no treatment effect on myelin-associated glycoprotein (MAG) expression. These phenotypic changes are consistent with oligodendrocyte differentiation. Treatment of 6E12 cells with dibutyryl cyclic AMP (DBC) down-regulated myelin basic protein (MBP) gene expression, perhaps, because it also up-regulated expression of a putative MBP repressor SCIP/Tst-1. Moreover, the 6E12 cells expressed high levels of MBP mRNA but no MBP translation products were detected in the presence or absence of DBC. This immortalized glial cell line is proposed as a CNS model for cAMP-modulated myelin gene expression and for post-transcriptional regulation of MBP.

Schwann cells in the intact sciatic nerve express high amounts of ciliary neurotrophic factor (CNTF), but 7 days after injury to the nerve expression dramatically decreases. To determine whether this change occurs only in the region of the injury or throughout the whole nerve we examined the spatial and temporal expression of CNTF after a crush injury. One day after injury the amount of CNTF mRNA and protein decreased within the first 4 mm distal to the crush site. This decrease progressed in a centrifugal manner distally until mRNA and protein were scarcely detectable by 7 days. In nerve proximal to the crush site CNTF expression decreased slightly and was still detectable at all sample times. During regeneration CNTF expression remained very low up to 14 days after injury. By 30 days mRNA and protein were detectable and by 60 days CNTF protein was present at normal amounts. Immunohistochemical analysis of normal nerve revealed CNTF localized in outer portion of the cytoplasm of myelin-forming Schwann cells. Three days after injury CNTF coalesced with pockets of cytoplasm in the Schwann cell and by 5 days was barely detectable. Positive staining remained in proximal segments where little or no degeneration occurred. These results demonstrate that CNTF expression in Schwann cells is synchronized with their functional state. CNTF expression decreases with demyelination during Wallerian degeneration and returns to normal following remyelination during regeneration. These findings also suggest that CNTF expression requires intact axon-Schwann cell interactions.

To study the effect of SV40 large T-antigen expression in myelin-forming cells of both the central and peripheral nervous system, a series of transgenic mice were generated expressing the SV40 large T-antigen under control of the myelin basic protein (MBP) promoter. Two neurologic phenotypes, designated A and B, appeared among individual transgenic founders and their progeny. The A mice developed a severe action tremor at about 10 days of age that progressed into periods of convulsions and early death by three to four weeks of age. In contrast, the B mice exhibited a progressive hindlimb ataxia and had a more normal lifespan. The A mice displayed hypomyelinating lesions in the central nervous system (CNS), whereas the B mice had lesions in either the peripheral nervous system (PNS) alone or in both the PNS and CNS. Immunohistochemical staining of spinal cord sections of a type A mouse showed a substantial depletion in MBP. Moreover, T-antigen-positive cells appeared predominantly in white matter tracts as randomly distributed single cells. Double labeling immunocytochemistry demonstrated that some of these T-antigen-positive cells were positive for oligodendrocyte differentiation markers MBP and O4. Thus, T-antigen expression appeared to coincide with a terminal stage of oligodendrocyte differentiation.

Tight junctions may play an important role in maintaining the integrity of the blood-brain barrier. These junctions can be individually visualized using electron microscopy but no current technique is able to provide a more global picture of the presence and density of tight junctions in central nervous system tissue. We used an antibody that recognizes a high molecular weight protein (ZO-1) associated with tight junctions, to identify these specialized junctions within the rat brain and spinal cord. Immunofluorescent labeling showed a network of tight junctions between cells in the brain vasculature, leptomeninges and choroid plexus, and between tanycytes lining the floor of the third ventricle and the central canal of the spinal cord. Anti-ZO-1 labeled the majority of cells associated with the blood-brain barrier and may prove a useful marker, possibly in conjunction with functional dye studies, in evaluating the anatomical and functional integrity of the blood-brain barrier.

The neurological mutant mice shiverer (shi) and myelin deficient (shimld) lack a functional gene for the myelin basic proteins (MBP), have virtually no myelin in their CNS, shiver, seize, and die early. Mutant mice homozygous for an MBP transgene have MBP mRNA and MBP in net amounts approximately 25% of normal, have compact myelin, do not shiver or seize, and live normal life spans. We bred mice with various combinations of the normal, transgenic, shi, and shimld genes to produce mice that expressed MBP mRNA at levels of 0, 5, 12.5, 17.5, 50, 62.5, and 100% of normal. The CNS of these mice were analyzed for MBP content, tissue localization of MBP, degree of myelination, axon size, and myelin thickness. MBP protein content correlated with predicted MBP gene expression. Immunocytochemical staining localized MBP to white matter in normal and transgenic shi mice with an intensity of staining comparable to the degree of MBP gene expression. An increase in the percentage of myelinated axons and the thickness of myelin correlated with increased gene expression up to 50% of normal. The percentage of myelinated axons and myelin thickness remained constant at expression levels greater than 50%. The presence of axons loosely wrapped with oligodendrocytic membrane in mice expressing lower amounts of MBP mRNA and protein suggested that the oligodendroglia produced sufficient MBP to elicit axon wrapping but not enough to form compact myelin. Mean axon circumference of myelinated axons was greater than axon circumference of unmyelinated axons at each level of gene expression, further evidence that oligodendroglial cells preferentially myelinate axons of larger caliber.(ABSTRACT TRUNCATED AT 250 WORDS)

Ciliary neurotrophic factor (CNTF) is a 200-amino acid protein expressed in high concentrations by peripheral nerves and is thought to be important for the survival and regeneration of injured motoneurons (Lin et al., J Biol Chem 265:8942-8947, 1990). To better understand CNTF's role in nerve injury we have characterized the effects of crush injury on the expression of CNTF in adult rat sciatic nerves using specific antibody and RNA probes. Following a crush injury, both the protein and mRNA levels undergo pronounced decreases distal to the crush. These changes in CNTF expression were qualitatively distinct from changes in the expression of the low-affinity NGF receptor (p75NGFR), which increases following crush. Thus, the changes in CNTF levels do not reflect an overall down-regulation of mRNA during degeneration, and are inconsistent with the proposed role of CNTF in neuronal injury, since its levels are decreasing at the same time as the requirement for neurotrophic support is increasing.

A new type of collagen surface for use with cultures of peripheral nervous system cells is described. Collagen is derivatized to plastic culture dishes by a cross-linking reagent, l-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide-metho-p-toluene sulfonate (carbodiimide), to form a uniform and durable surface for cell attachment and growth that allows dry storage, long-term culture, and improved microscopy. Surfaces of collagen derivatized to plastic were compared to surfaces of adsorbed or ammonia-polymerized collagen in terms of collagen binding and detachment, growth by dorsal root ganglion cells, and electron microscopy appearances. Derivatized collagen surfaces retained more collagen and showed much less evidence of degradation and cellular damage over periods of many weeks than did conventional adsorbed surfaces. Long-term survival of cells on derivatized collagen was far superior to that on the other surfaces, with almost 90% of cultures still viable after 10 wk. Transmission electron microscopy showed an organized layer of single fibrils that supported cell growth well, and scanning electron microscopy demonstrated an increased uniformity of derivatized collagen surfaces compared to ammoniated collagen surfaces. Applications for this improved substrate surface are discussed.

Shiverer (shi) is an autosomal recessive mutation in mice that results in hypomyelination in the central nervous system (CNS) but normal myelination in the peripheral nervous system (PNS). Myelin basic proteins (MBPs) are virtually absent in both PNS and CNS. It is not known whether the cellular target in the PNS is the myelin-forming Schwann cell or another cell type which secondarily affects the Schwann cell. To determine the cellular target of the shi gene, we have adapted tissue culture techniques that allow co-culture of pure populations of mouse sensory neurons of one genotype with Schwann cells and fibroblasts of another genotype under conditions that permit myelin formation. These cultures were stained immunocytochemically as whole mounts to determine whether MBPs were expressed under various in vitro conditions. In single-genotype cultures, presence or absence of MBPs was consistent with earlier in vivo results: +/+ cultures were MBP-positive and shi/shi cultures were MBP-negative. In mixed-genotype cultures, visualization of MBPs in myelin accorded with the genotype of the non-neuronal Schwann cells and fibroblasts and not with the neurons--those cultures that contained +/+ non-neuronal cells were MBP-positive and those with shi/shi non-neuronal cells were MBP-negative, independent of the neuronal genotype. These results rule out neurons or circulating substances as mediators of the influence of the shi genetic locus on MBP synthesis and deposition in peripheral myelin.

Mice homozygous for the mutation myelin deficient (mld), an allele of shiverer, exhibit decreased CNS myelination, tremors, and convulsions of progressively increasing severity leading to an early death. In this report we demonstrate in mld mice that the gene encoding myelin basic protein (MBP) is expressed at decreased levels and on an abnormal temporal schedule relative to the wild-type gene. Southern blot analyses, field-inversion gel electrophoresis studies, and analyses of mld MBP cosmid clones indicate that there are multiple linked copies of the MBP gene in mld mice. We have introduced an MBP transgene into mld mice and found that myelination increases and tremors and convulsions decrease. Mld and shiverer mice with zero, one, or two copies of the MBP transgene express distinct levels of MBP mRNA and myelin. The availability of a range of mice expressing graded levels of myelin should facilitate quantitative analysis of the roles of MBP in the myelination process and of myelin in nerve function.

Mice homozygous for the autosomal recessive mutation shiverer (shi) lack myelin basic protein (MBP) and exhibit a distinct behavioral pattern including tremors (shivering), convulsions, and early death. We have previously demonstrated that shiverer mice have a partial deletion in the gene encoding MBP. We now have introduced the wild-type MBP gene into the germ line of shiverer mice by microinjection into fertilized eggs. Transgenic shiverer mice homozygous for the introduced gene have MBP mRNA and protein levels that are approximately 25% of normal, and produce compacted myelin with major dense lines. Correct temporal and spatial expression of the MBP gene is achieved with a genomic MBP cosmid clone containing 4 kb of 5' flanking sequence and 1 kb of 3' flanking sequence. Moreover, the four different forms of MBP produced by alternative patterns of RNA splicing are present. These homozygous transgenic shiverer mice no longer shiver nor die prematurely.

A new fluorometric assay was developed for the measurement of gamma-glutamyl transpeptidase (gamma-GTP). The assay utilizes as a substrate the synthetic compound 7-gamma-glutamylamido-4-methyl coumarin which is cleaved by gamma-GTP to yield the highly fluorescent product 7-amino-4-methyl coumarin. Optimal excitation and emission wavelengths for the assay are 345 nm and 470 nm, respectively, and the sensitivity of the assay is greatly enhanced by the high-pressure liquid chromatographic separation of the product from the substrate. The assay is minimally 25 times more sensitive than the conventional spectrophotometric assay and permits analysis of as little as 5000 cultured cells of neuronal and glial origin. Analysis of a variety of cultured cells of neuronal and glial origin with this assay suggests that gamma-GTP is largely present in glia and to a lesser extent in neurons.

In order to determine the cellular localization of gamma-glutamyl transpeptidase (gamma-GTP) in the nervous system the enzyme was purified to homogeneity and used to prepare an antiserum in rabbits. The anti-gamma-GTP serum cross-reacted with rodent tissues, but not those of human, monkey, calf, sheep or chicken. Monospecificity of the antibody was demonstrated by immunoelectrophoresis. The antibody inactivated the purified enzyme in a dose-dependent manner. The gamma-GTP antibody was used with the indirect fluorescent method to visualize sites containing gamma-GTP in the CNS. Choroid plexus, capillaries and the luminal side of ependymal cells stained well. Additionally, immunoreactive cells were found in both grey and white matter throughout the CNS which did not have the morphological appearance of neurons and were thought to be glia. These immunocytochemical studies suggest that gamma-GTP is largely though not exclusively associated with glia in the CNS.

Axons of a cut peripheral nerve will grow across a gap (less than or equal to 10 mm in adult rodents) formed when the proximal and distal stumps are placed at opposite ends of an impermeable, inert tube, but will not grow to the end of a blind-ended tube in the absence of the distal stump [Williams et al, 1984]. Work reported here demonstrates that cultured peripheral nervous system (PNS) cells suspended in a collagen matrix will provide an effective milieu that directs and supports axonal regeneration from a severed nerve into a blind-ended tube in the absence of a distal stump. Adult mouse sciatic nerves were cut and the proximal stumps were inserted into close-ended tubes that contained either a collagen matrix containing dissociated cells from embryonic mouse dorsal root ganglia (DRG), a collagen matrix saturated with medium conditioned by cultured DRG cells, or a collagen matrix saturated with fresh medium. In all three cases cellular cables formed that ran the full length of the tubes, but myelinated and unmyelinated axons regenerated the length of the tubes only when cultured cells had been added. The critical factor in influencing axonal regeneration through the length of the tubes was the presence of cultured cells, since collagen alone or collagen saturated with conditioned medium did not support axonal regrowth even though cells had migrated into the chambers from the proximal stumps in all cases. Ordered structure was not a requisite for axonal growth, since the cultures consisted of random arrays of dissociated cells.