Lipid raft-targeted Akt promotes axonal branching and growth cone expansion via mTOR and Rac1, respectively.

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.

In situ expression of brain-derived neurotrophic factor or neurotrophin-3 promotes sprouting of cortical serotonergic axons following a neurotoxic lesion.

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.

In vitro dorsal root ganglia and human prostate cell line interaction: redefining perineural invasion in prostate cancer.

BACKGROUND: Little is understood regarding mechanisms of perineural invasion in prostate cancer progression. We present a novel model system and data that indicate perineural invasion is an active, specific, and reciprocal interaction between nerves and prostate cancer cells. METHODS: Mouse dorsal root ganglia (DRG) and human prostate cancer cells (Du-145, LNCaP, PC3) and stromal cells (HTS-40F) were co-cultured in Matrigel matrix. Control cultures consisted of prostate cancer and stromal cells only and DRG only. Neurite outgrowth, cell colony growth, neurite-colony contact, and retrograde extension were quantitated with dark phase microscopy and image analysis (Optimas 6.1). RESULTS: Directional outgrowth of neurites was observed projecting into DU-145 colonies within 24 hr of co-culture. Cultures with the greatest number of DU-145 cells recruited significantly more neurites and established contact earlier, indicating this process was cell-seeding density dependent. Once neurite/DU-145 cell contact was established neurite growth diminished, suggesting an active neurite recruitment by DU-145 cells. Subsequent to neurite contact, DU-145 cells migrated along neurites in a retrograde fashion into the nerve/ganglion of origin (retrograde extension) establishing perineural invasion. In addition to perineural invasion, DU-145 colony growth was elevated in DRG co-cultures relative to DU-145-only control cell cultures. Similarly, the degree of neurite outgrowth was elevated in DRG-cell co-cultures relative to DRG-only control cultures. The same observations were made with LNCaP and PC3 cells, but interactions between stromal cells and nerves were not found. CONCLUSIONS: This study shows the utility of the prostate cancer/DRG in vitro system to study specific mechanism of prostate cancer cell-nerve interaction. Moreover, these data suggest that perineural invasion mechanisms involve active and reciprocal interactions between carcinoma cells and adjacent nerve/ganglions in prostate cancer progression.

Changes in microtubule stability and density in myelin-deficient shiverer mouse CNS axons.

Altered axon-Schwann cell interactions in PNS myelin-deficient Trembler mice result in changed axonal transport rates, neurofilament and microtubule-associated protein phosphorylation, neurofilament density, and microtubule stability. To determine whether PNS and CNS myelination have equivalent effects on axons, neurofilaments, and microtubules in CNS, myelin-deficient shiverer axons were examined. The genetic defect in shiverer is a deletion in the myelin basic protein (MBP) gene, an essential component of CNS myelin. As a result, shiverer mice have little or no compact CNS myelin. Slow axonal transport rates in shiverer CNS axons were significantly increased, in contrast to the slowing in demyelinated PNS nerves. Even more striking were substantial changes in the composition and properties of microtubules in shiverer CNS axons. The density of axonal microtubules is increased, reflecting increased expression of tubulin in shiverer, and the stability of microtubules is drastically reduced in shiverer axons. Shiverer transgenic mice with two copies of a wild-type myelin basic protein transgene have an intermediate level of compact myelin, making it possible to determine whether the actual level of compact myelin is an important regulator of axonal microtubules. Both increased microtubule density and reduced microtubule stability were still observed in transgenic mouse nerves, indicating that signals beyond synaptogenesis and the mere presence of compact myelin are required for normal regulation of the axonal microtubule cytoskeleton.

Phase I study of adenoviral delivery of the HSV-tk gene and ganciclovir administration in patients with current malignant brain tumors.

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.

Permanent rescue of lesioned neonatal motoneurons and enhanced axonal regeneration by adenovirus-mediated expression of glial cell-line-derived neurotrophic factor.

Axotomy of peripheral nerves in neonatal rats induces motoneuron death that can be delayed but not arrested by the application of several neurotrophic factors (NFs) or adenoviral vectors carrying genes for NFs. We tested whether an adenoviral vector carrying the gene for glial cell-line-derived neurotrophic factor (Adv.RSV-GDNF) would prevent neonatal motoneuron death after facial nerve transection or crush. Nerve transection eliminates the pathway for axonal regeneration, while nerve crush preserves the pathway necessary for target reinnervation that may be required for the permanent rescue of motoneurons. Both types of injury cause substantial motoneuron death in neonatal animals. Adv.RSV-GDNF or a control vector carrying the beta-galactosidase gene (Adv.RSV-betagal) was injected into facial muscles 2 days before the nerve was transected, or Adv.RSV-GDNF, Adv.RSV-betagal, Adv.d1312 (a vector lacking a transgene), or vehicle was injected into facial muscles immediately after nerve crush. Four weeks after nerve transection, few motoneurons survived after Adv.RSV-GDNF and Adv.RSV-betagal treatment (6.1% and 2.4%, respectively). Four weeks after nerve crush, 40% of the motoneurons survived after Adv.RSV-GDNF treatment but only 17% survived in control groups. By 20 weeks, 39% of the motoneurons of the Adv.RSV-GDNF treatment groups survived but only 15-19% survived in controls. The numbers of myelinated axons of the buccal nerve branch of Adv.RSV-GDNF treatment groups were also higher than controls at 4 and 20 weeks (24% and 100% compared to 4.4-6.2% and 25-33% for Adv.RSV-GDNF and controls, respectively). By 20 weeks, Adv.RSV-GDNF-treated animals recovered 50% of the contralateral vibrissal function, while in controls only 5-11% of function was restored.

Neuroprotection of spinal motoneurons following targeted transduction with an adenoviral vector carrying the gene for glial cell line-derived neurotrophic factor.

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).

Targeted transduction of CNS neurons with adenoviral vectors carrying neurotrophic factor genes confers neuroprotection that exceeds the transduced population.

Application of neurotrophic factors (NFs) to the cut stump of motor nerves of neonatal rats confers neuroprotection from trauma-induced neuronal death. To test whether motoneurons are capable of responding to endogenously produced NFs, facial motoneurons were genetically modified in vivo to express several NFs and then tested for their response to peripheral nerve damage. Replication-defective adenoviral vectors [Adv. Rous sarcoma virus (RSV)-nf] representing three families of NFs were constructed that carried genes for brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-derived neurotrophic factor (GDNF), and nerve growth factor. Media from cultured cells transduced with Adv. RSV-nf contained NFs that supported the survival of cultured chick sensory neurons in the same manner as recombinant NF standards. When Adv.RSV-nf or an adenoviral vector containing the beta-galactosidase gene (Adv.RSV-beta-gal) were injected into the facial muscles of neonatal rats the vectors were retrogradely transported to the facial nucleus where the NFs or beta-gal were expressed. A fraction (approximately 10%) of the neurons were transduced as demonstrated by reverse transcriptase-PCR, histochemistry, and immunocytochemistry. In the case of Adv.RSV-BDNF, Adv.RSV-CNTF, and Adv.RSV-GDNF, a significant portion of the facial nucleus neurons was protected, 16.5, 18.2, and 53.3%, respectively, from death after axotomy, showing that neurons are capable of transporting the Adv. RSV-nf, expressing the recombinant NF genes, and responding to the NFs. In the case of Adv.RSV-GDNF, a greater number of facial nucleus motoneurons survived than were transduced, indicating that neighboring untransduced neurons were protected by the GDNF expressed by the transduced neurons by a paracrine mechanism.

Neurotoxicity of intracerebral injection of a replication-defective adenoviral vector in a semipermissive species (cotton rat).

The neurotoxicity of an adenoviral vector (Adv.RSVtk) carrying the gene for herpes simplex virus thymidine kinase (HSVtk) was tested in the cotton rat, a semipermissive host. Adv.RSVtk was injected intracerebrally in cotton rats at a dose of 5.0 x 10(6) or 7.5 x 10(7) p.f.u. No signs of illness were observed. Histological inspection at 12 and 28 days after injection showed inflammation of the ependyma and choroid plexus and at the injection site. No demyelination, viral inclusions, cerebral edema, necrosis, cavities or vascular necrosis were seen in the brains. There was no significant difference between animals injected with 5.0 x 10(6) or 7.5 x 10(7) p.f.u., nor was there a difference between animals analyzed at 12 or 28 days after vector injection. This inflammation was similar in animals that had been preimmunized with wild-type virus and in animals that had been treated with ganciclovir. No histopathology, was observed in the lungs of the animals and no replication-competent virus was detected. These experiments indicate that Adv.RSVtk has limited neurotoxicity which would not prohibit its use in a limited phase I clinical trial in humans that have malignant tumors of the central nervous system.

Oligodendroglia regulate the regional expansion of axon caliber and local accumulation of neurofilaments during development independently of myelin formation.

Axon caliber may be influenced by intrinsic neuronal factors and extrinsic factors related to myelination. To understand these extrinsic influences, we studied how axon-caliber expansion is related to changes in neurofilament and microtubule organization as axons of retinal ganglion cells interact with oligodendroglia and become myelinated during normal mouse brain development. Caliber expanded and neurofilaments accumulated only along regions of the axon invested with oligodendroglia. Very proximal portions of axons within a region of the optic nerve from which oligodendrocytes are excluded remained unchanged. More distally, these axons rapidly expanded an average of fourfold as soon as they were recruited to become myelinated between postnatal days 9 and 120. Unmyelinated axons remained unchanged. Axons ensheathed by oligodendroglial processes, but not yet myelinated, were intermediate in caliber and neurofilament number. That oligodendrocytes can trigger regional caliber expansion in the absence of myelin was confirmed using three strains of mice with different mutations that prevent myelin formation but allow wrapping of some axons by oligodendroglial processes. Unmyelinated axons persistently wrapped by oligodendrocytes showed full axon caliber expansion, neurofilament accumulation, and appropriately increased lateral spacing between neurofilaments. Thus, signals from oligodendrocytes, independent of myelin formation, are sufficient to induce full axon radial growth primarily by triggering local accumulation and reorganization of the neurofilament network.

Adenoviral-mediated thymidine kinase gene transfer into the primate brain followed by systemic ganciclovir: pathologic, radiologic, and molecular studies.

Transduction of experimental gliomas with the herpes simplex virus thymidine kinase gene (HSV-tk) using a replication-defective adenoviral vector (ADV/RSV-tk) confers sensitivity to ganciclovir (GCV) leading to tumor destruction and prolonged host survival in rodents. To determine treatment tolerance prior to clinical trials, we conducted toxicity studies in 6 adult baboons (Papio sp.). The animals received intracerebral injections of either a high dose of ADV/RSV-tk [1.5 x 10(9) plaque-forming units (pfu)] with or without GCV, or a low dose of ADV/RSV-tk (7.5 x 10(7) pfu) with GCV. The low dose corresponded to the anticipated therapeutic dose; the high dose was expected to be toxic. Magnetic resonance imaging (MRI) of the brain was obtained before treatment and at 3 and 6 weeks after treatment. Animals receiving the high-dose vector and GCV either died or became moribund and required euthanasia during the first 8 days of treatment. Necropsies revealed cavities of coagulative necrosis at the injection sites. Animals receiving only the high-dose vector were clinically normal; however, lesions were detected with MRI at the injection sites corresponding to cystic cavities at necropsy. Animals receiving the low-dose vector and GCV were clinically normal, exhibited small MRI abnormalities, and, although no gross lesions were present at necropsy, microscopic foci of necrosis were present. The vector sequence was detected by polymerase chain reaction (PCR) at the injection sites and in non-adjacent central nervous system tissue in all animals. Recombinant DNA sequence was detected outside of the nervous system in some animals, and persisted up to 6 weeks. The viral vector injections stimulated the production of neutralizing antibodies in the animals. No shedding of the vector was found in urine, feces, or serum 7 days after intracerebral injection. This study suggests that further investigations including clinical toxicity trials of this form of brain tumor therapy are warranted.

Adenovirus-mediated gene therapy in an experimental model of breast cancer metastatic to the brain.

We investigated the therapeutic efficacy of adenovirus-mediated gene therapy to treat malignant mammary tumors in vitro and in vivo in the brain. A mammary adenocarcinoma cell line derived from Fischer rats (13762 MAT B III; MAT-B) was used. In vitro studies demonstrated that the MAT-B cells could be efficiently transduced with a replication-defective adenovirus (ADV) vector that carried the herpes simplex virus gene for thymidine kinase (ADV-tk), and that ADV-tk transduction rendered the MAT-B cells sensitive to killing, in a dose-dependent manner, with ganciclovir (GCV). An animal model of a mammary tumor metastatic to the brain was produced by injecting MAT-B cells into the caudate nucleus of Fischer rats. Seven days after MAT-B cell injection, when the tumors were approximately 5 mm2 in cross-sectional size, the tumors were injected with ADV-tk or a control adenovirus vector containing the beta-galactosidase (beta-Gal) gene (ADV-beta gal). After vector injection the animals were treated with GCV or with saline for 6 days. Sixteen days after tumor cell injection, the brains were examined histologically. The rats that were injected with ADV-beta gal and treated with GCV or saline, and those that were injected with ADV-tk and treated with saline had large tumors, whereas the rats that were injected with ADV-tk and treated with GCV had no visible tumor tissue at the site of tumor cell injection. In survival studies animals treated with ADV-tk+GCV survived a significantly longer time than animals treated with ADV-beta gal+GCV. Our results demonstrate that the recombinant adenoviral vector containing the tk gene confers GCV cytotoxic sensitivity to mammary tumor cells in vitro and in the brain, and suggest that this treatment strategy may be useful in treating somatic tumors that metastasize to the brain.

Adenovirus-mediated gene therapy for experimental spinal cord tumors: tumoricidal efficacy and functional outcome.

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.

Adenovirus-mediated gene therapy of experimental gliomas.

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.

Gene therapy for brain tumors: regression of experimental gliomas by adenovirus-mediated gene transfer in vivo.

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.

Cyclic AMP has a differentiative effect on an immortalized oligodendrocyte cell line.

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.

Temporal and spatial expression of ciliary neurotrophic factor after peripheral nerve injury.

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.

Distinct hypomyelinated phenotypes in MBP-SV40 large T transgenic mice.

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.

Immunofluorescent labeling of tight junctions in the rat brain and spinal cord.

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.

Peripheral nerve injury down-regulates CNTF expression in adult rat sciatic nerves.

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.