Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene.

To address the regulation of glial cell line-derived neurotrophic factor (GDNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter. GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle. The GDNF gene was isolated from a human genomic library in a P1 bacteriophage vector.

Functional recovery following nerve injury and repair by silicon tubulization: comparison of laminin-fibronectin, dialyzed plasma, collagen gel, and phosphate buffered solution.

Purpose: This study was designed to investigate the potential for enhancement of peripheral nerve regeneration by the manipulation of the neural microenvironment with laminin-fibronectin solution (LF), dialyzed plasma (DP), collagen gel (CG), or phosphate buffered saline (PBS) in a silicon tubulization repair model.

Method: A rat sciatic nerve model of injury and repair was used to study the effects of exogenous matrix precursors (contained in LF or DP), CG or PBS on nerve regeneration. A total of 50 Sprague-Dawley rats underwent left sciatic nerve transection and repair by silicon tubulization.

Differential regulation of glial cell line-derived neurotrophic factor (GDNF) expression in human neuroblastoma and glioblastoma cell lines.

Human SK-N-AS neuroblastoma and U-87MG glioblastoma cell lines were found to secrete relatively high levels of glial cell line-derived neurotrophic factor (GDNF). In response to growth factors, cytokines, and pharmacophores, the two cell lines differentially regulated GDNF release. A 24-hr exposure to tumor necrosis factor-alpha (TNFalpha; 10 ng/ml) or interleukin-1beta (IL-1,; 10 ng/ml) induced GDNF release in U-87MG cells, but repressed GDNF release from SK-N-AS cells.

Phosphatidylcholine-specific phospholipase inhibitor D609 differentially affects MAP kinases and immediate-early genes in PC12 cells.

The effects of tricyclodecan-9-yl xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipases, on PC12 cells were investigated. D609 repressed nerve growth factor (NGF)-mediated induction of c-fos mRNA with an IC50 approximately 50 microg/ml. Interestingly, maximal c-fos-suppressing doses of D609 did not affect activity of extracellular signal-regulated kinases.

Functional recovery of transected nerves treated with systemic BDNF and CNTF.

The purpose of this study was to investigate the effect of systemic co-injections of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) on the functional recovery of transected sciatic nerves repaired by epineurial coaptation (EC) or collagen tubulization (CT). Forty Sprague-Dawley rats underwent transection of their sciatic nerves and repair by either EC or CT. With each repair technique, systemic injections of neurotrophic factors or control injections of lactated Ringer's solution were given.

Duration and magnitude of nerve growth factor signaling depend on the ratio of p75LNTR to TrkA.

The role of the low affinity neurotrophin receptor p75LNTR in neurotrophin signal transduction remains open. Recent reports show that this receptor generates intracellular signals independent of Trk activity, and others imply that it collaborates with Trk(s) to enhance cellular responses to low neurotrophin concentrations. We have used the Cytosensor microphysiometer as a direct marker of intracellular metabolic activity to address the physiologic role of p75LNTR in nerve growth factor (NGF) signal transduction.

Regulation of glial cell line-derived neurotrophic factor release from rat C6 glioblastoma cells.

We have monitored glial cell line-derived neurotrophic factor (GDNF) secretion from rat C6 glioblastoma cells by ELISA. Representative cytokines, neurotrophins, growth factors, neuropeptides, and pharmacological agents were tested for their ability to modulate GDNF release. Whereas most factors tested had minimal effect, a 24-h treatment with fibroblast growth factor-1, -2, or -9 elevated secreted GDNF protein levels five- to 10-fold.

Simultaneous treatment with BDNF and CNTF after peripheral nerve transection and repair enhances rate of functional recovery compared with BDNF treatment alone.

The objective was to investigate the effects of brain-derived neurotropic factor (BDNF) and ciliary neurotropic factor (CNTF) on peripheral nerve regeneration. Thirty Sprague-Dawley rats underwent left sciatic nerve transection and repair according to three experimental groups: epineurial coaptation (EC), EC with BDNF delivered by an osmotic pump (EC-BDNF), and EC with BDNF and CNTF delivered similarly (EC-BDNF/CNTF). Nerve regeneration was assessed using sciatic functional indices, quantitative histomorphology, and molecular analysis for proteins associated with nerve regeneration.

Conditionally Immortalized Neural Cell Lines: Potential Models for the Study of Neural Cell Function.

Studies on primary cell cultures have contributed significantly to our understanding of neural cell function. Nevertheless, for many studies the value of these primary cell cultures has been limited by the time the cultures survive in vitro, the quantity of cellular material available for analysis, and the need to prepare the cells on a regular basis from fresh tissue. Techniques for immortalizing cells have existed for some time, but the repertoire of immortalizing genes has grown significantly.

p75(NGFR) and TrkA receptors collaborate to rapidly activate a p75(NGFR)-associated protein kinase.

The role of the low affinity nerve growth factor receptor (p75(NGFR)) in NGF-mediated signaling is not yet understood. Here we show by co-immunoprecipitation that NGF activates a protein kinase that is directly associated with p75(NGFR) in dorsal root ganglion (DRG) cells and PC12 cells in culture. Two proteins of 120 and 104 kDa constitute the majority of this activity.

Brain-derived neurotrophic factor and collagen tubulization enhance functional recovery after peripheral nerve transection and repair.

Objective: To investigate the effect of brain-derived neurotrophic factor (BDNF) and collagen tubulization (CT) on the regeneration of transected peripheral nerves. METHODS AND DESIGNS: The left sciatic nerve of 40 Sprague-Dawley rats was transected then repaired using one of four techniques; epineurial coaptation, CT, CT with BDNF delivered by an osmotic pump to the repair site, or CT with BDNF covalently cross-linked to the collagen matrix (CT/linked-BDNF). Sciatic functional indices were measured preoperatively at 10-day intervals for 90 days.

Ciliary neurotrophic factors enhances peripheral nerve regeneration.

Background: Adjunctive measures to enhance nerve repair have focused on a variety of trophic factors that alter the physiologic response to nerve injury through Schwann cell-axonal interactions.

Objective: To evaluate the effects of two trophic factors, ciliary neurotrophic factor and nerve growth factor, on axonal response to injury.

Design: A prospective, randomized, blinded animal study with a placebo control using lactated Ringer's solution.

Interleukin-6 production by Schwann cells and induction in sciatic nerve injury.

Interleukin-6 (IL-6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell-derived IL-6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL-6. Cocultured iSC transcribe IL-6 message as confirmed by northern analysis.

Murine oligodendroglial cells express nerve growth factor.

The studies reported here present evidence for the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) by an oligodendroglial cell line and of NGF by oligodendrocytes in mouse primary culture. An immortalized oligodendroglial cell line (N19) expressing markers for immature oligodendrocytes stimulated PC12 cells to elaborate processes. Polymerase chain reaction analysis with degenerate primers indicated that the N19 cells expressed the mRNAs for the neurotrophic factors NGF and BDNF.

Expression of myelin protein genes and other myelin components in an oligodendrocytic cell line conditionally immortalized with a temperature-sensitive retrovirus.

We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase mRNA.

Generation and analysis of normal and shiverer temperature-sensitive immortalized cell lines exhibiting phenotypic characteristics of oligodendrocytes at several stages of differentiation.

Normal glial cells immortalized at specific developmental stages would be useful tools with which to study glial cell differentiation in vitro. Similarly, immortalized glial cell lines derived from known neurological mutants with identified developmental, molecular genetic defects would also be useful for the in vitro examination of the effects of the mutation on glial cell function. In this report we describe the immortalization of 19 separate oligodendroglial cell lines, 10 derived from normal mice and 9 derived from the neurological mutant shiverer, which is missing a large segment of the myelin basic protein gene.

Posttranscriptional regulation of myelin protein gene expression.

Regulation of myelin protein gene expression occurs at many different levels including transcription, mRNA translocation, translation, and posttranslational modification of myelin proteins prior to their assembly into the membrane. Translocation of myelin basic protein (MBP) mRNAs into oligodendrocyte processes was observed in vivo and in primary cultures, but no such translocation was observed for the mRNAs encoding the proteolipid protein (PLP) or myelin-associated glycoprotein. More than 99% of the mRNAs encoding 2'3'-cyclic nucleotide phosphodiesterase (CNP) remained associated with cell bodies.

Posttranscriptional events in the expression of myelin protein genes.

A number of posttranscriptional events may be involved in regulating the expression of the myelin protein genes. One such event in the expression of the myelin basic protein (MBP) gene is the translocation of MBP mRNAs from oligodendrocyte cell bodies to their processes. This translocation can be observed in vivo and in primary mixed glial cell cultures.

Gene expression in the jimpy mutant: evidence for fewer oligodendrocytes expressing myelin protein genes and impaired translocation of myelin basic protein mRNA.

Myelin basic protein (MBP) and proteolipid protein (PLP) gene expression was investigated in the murine dysmyelinating mutant, jimpy and age-matched normal mice. MBP and PLP mRNA expression was examined in several brain regions by in situ hybridization histochemistry between 10 and 20 days postpartum. The results showed a general reduction in both PLP and MBP mRNA expression throughout in jimpy brain due primarily to fewer numbers of jimpy oligodendrocytes expressing these genes.

Regional expression of myelin protein genes in the developing mouse brain: in situ hybridization studies.

Expression of mRNAs for the two major myelin proteins, myelin basic protein (MBP) and proteolipid protein (PLP), was examined in a number of regions of the developing mouse brain using in situ hybridization. In general, MBP and PLP mRNAs were observed to be coexpressed during ontogeny, prior to the histological appearance of myelin. Expression of both mRNAs was detected as early as 6 hours postpartum in the medulla oblongata and, with development, expression of these mRNAs progressed in a caudal to rostral direction.

Covalent binding of formalin fixed paraffin embedded brain tissue sections to glass slides suitable for in situ hybridization.

A novel method for covalently binding formalin fixed paraffin embedded (FFPE) tissue sections to glass microscope slides is validated suitable for in situ hybridization (ISH). Using the organosilane methodology of Maples (1985), 100% tissue adhesion is reported with no nonspecific probe binding, staining, or autoradiographic artefacts. JC viral nucleic acid sequences are successfully detected in FFPE progressive multifocal leukoencephalopathy brain tissue and the Tm of the hybridized product is estimated.