Ultrafast sound production mechanism in one of the smallest vertebrates.

Motion is the basis of nearly all animal behavior. Evolution has led to some extraordinary specializations of propulsion mechanisms among invertebrates, including the mandibles of the dracula ant and the claw of the pistol shrimp. In contrast, vertebrate skeletal movement is considered to be limited by the speed of muscle, saturating around 250 Hz. Here, we describe the unique propulsion mechanism by which Danionella cerebrum, a miniature cyprinid fish of only 12 mm length, produces high amplitude sounds exceeding 140 dB (re. 1 µPa, at a distance of one body length). Using a combination of high-speed video, micro-computed tomography (micro-CT), RNA profiling, and finite difference simulations, we found that D. cerebrum employ a unique sound production mechanism that involves a drumming cartilage, a specialized rib, and a dedicated muscle adapted for low fatigue. This apparatus accelerates the drumming cartilage at over 2,000 g, shooting it at the swim bladder to generate a rapid, loud pulse. These pulses are chained together to make calls with either bilaterally alternating or unilateral muscle contractions. D. cerebrum use this remarkable mechanism for acoustic communication with conspecifics.

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. Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich. Consensus binding sites for a variety of transcription factors have been identified in the promoter. Promoter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene. The promoter/reporter construct and a number of promoter deletions were transiently transfected into two human cell lines known to express GDNF. Multiple enhancer and silencer regions were revealed as well as a minimal promoter sufficient for basal transcription. Finally, a RT-PCR assay, specific for transcripts originating from this GDNF promoter, was developed and used to show that this promoter is active in vivo. The results suggest GDNF is regulated in a complex manner.

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. The silicon tubules were either left empty (E), or filled with solutions of LF, DP, CG, or PBS. Nerve function was assessed preoperatively and then postoperatively, every 10 days for 90 days using sciatic functional indexes (SFI). On postoperative day 90, the sciatic nerves were harvested for histologic analysis and the posterior compartment muscles of each animal were harvested and weighed. Molecular analysis for two proteins associated with neural regeneration was performed on the nerve segments. RESULTS: All five animal groups demonstrated equivalent functional recovery. Comparison of the rate of recovery and mean maximal recovery between each group revealed no statistically significant differences, with P-values ranging from 0.30 to 0.95. Posterior compartment muscle masses were similar in all groups except for LF, whose animals had muscle masses 8-9% lower than CG, PBS, or E (P < 0.05). CONCLUSION: Alteration of the regenerating neural microenvironment with exogenous matrix precursors (LF, DP), CG or PBS failed to improve sciatic functional recovery after nerve transection and silicon tubulization in this model. From this study, we conclude that LF, DP, CG, and PBS do not enhance the rate or degree of recovery of peripheral nerve function across a narrow gap when nerves are repaired 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. Fibroblast growth factors (FGF)-1, -2, and -9 (50 ng/ml), the prostaglandins PGA2, PGE2, and PGI2 (10 microM), phorbol 12,13-didecanoate (PDD; 10 nM), okadaic acid (10 nM), dexamethasone (1 microM), and vitamin D3 (1 microm) also differentially effected GDNF release from U-87MG and SK-N-AS cells. A result shared by both cell lines, was a two- to threefold increase in GDNF release by db-cAMP (1 mM), or forskolin (10 microM). In general, analysis of steady-state GDNF mRNA levels correlated with changes in extracellular GDNF levels in U-87MG cells but remained static in SK-N-AS cells. The data suggest that human GDNF synthesis/release can be regulated by numerous factors, signaling through multiple and diverse secondary messenger systems. Furthermore, we provide evidence of differential regulation of human GDNF synthesis/release in cells of glial (U-87MG) and neuronal (SK-N-AS) origin.

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. Surprisingly, D609 enhanced the extracellular acidification rate of PC12 cells, even in the absence of NGF. D609 alone induced c-jun mRNA with the same potency as it repressed the NGF-induced expression of c-fos. Like NGF, D609 alone induced c-jun even in the presence of dominant-negative Ras. Immediate-early induction of c-jun mRNA by NGF and D609 was abrogated by pretreatment with the kinase inhibitor olomoucine. Jun kinase, which is inhibited by olomoucine, was found to be activated by D609. Thus, D609 might induce c-jun in PC12 cells as a consequence of Jun kinase activation through a Ras-independent pathway. Under the same conditions, D609 repressed NGF-mediated induction of c-fos mRNA.

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. This resulted in four treatment groups: EC, EC + BDNF/CNTF, CT, and CT + BDNF/CNTF. Nerve function was assessed using sciatic functional indices (SFI). Animals whose nerves were repaired by CT (P = 0.01), CT + BDNF/CNTF (P = 0.04), and EC + BDNF/CNTF (P = 0.04) all had better functional recovery than those whose nerves were repaired by EC. There were no significant differences among these three groups, however. Animals in the CT group manifested the most rapid rate of recovery (P = 0.02 compared with EC). Collagen tubulization and systemic co-injections of BDNF/CNTF improve the rate and extent of sciatic functional recovery after nerve repair. The improvement in recovery conferred is not additive.

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. NGF treatment of PC12 or TrkA-transfected Chinese hamster ovary (CHO) cells results in a rapid, transient increase in the extracellular acidification rate as measured by the Cytosensor; in both cell types, p75LNTR enhances this response. p75LNTR affects both the magnitude of and the duration of the extracellular acidification response to NGF. Moreover, it is not merely the presence of p75LNTR, but also the ratio of p75LNTR:TrkA which determines cellular responsiveness to NGF. In transiently transfected CHO cells, a 5:1 ratio of p75LNTR:trkA cDNAs produced the greatest change in NGF-induced acid secretion. Pretreatment of PC12 cells with anti-p75LNTR antibodies decreased the responsiveness to NGF. However, long-term NGF exposure to PC12 cells in which p75LNTR expression was decreased to approximately 10% of wild-type levels showed a longer duration of acid secretion compared to wild-type PC12 cells. Together, these data suggest that p75LNTR may play a dual role in modulating NGF signal transduction by enhancing and extending cellular responses to short-term ligand exposures while attenuating the metabolic response to long-term ligand exposures. With regard to potential Trk-independent p75LNTR signal transduction mechanisms, we detected no change in extracellular acidification response in 75LNTR-transfected CHO cells, PCNA-15 fibroblasts, or Schwann cells, all of which express large amounts of p75LNTR and no Trk. Thus, p75LNTR cannot produce any signal detected by microphysiometry in the absence of TrkA.

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. The proinflammatory cytokines interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and lipopolysaccharide elevated GDNF release 1.5- to twofold. Parallel studies aimed at elucidating intracellular events that may regulate GDNF synthesis/release demonstrated the involvement of multiple signaling pathways. GDNF levels were increased by phorbol 12,13-didecanoate (10 nM) activation of protein kinase C, the Ca2+ ionophore A23187 (1 microM), okadaic acid (10 nM) inhibition of type-2A protein phosphatases, nitric oxide donors (1 mM), and H2O2 (1 mM)-induced oxidative stress. Elevation of cyclic AMP levels by either forskolin (10 microM) or dibutyryl cyclic AMP (1 mM) repressed GDNF secretion, as did treatment with the glucocorticoid dexamethasone (1 microM). Our results demonstrate that diverse biological factors are capable of modulating GDNF protein levels and that multiple signal transduction systems can regulate GDNF synthesis and/or release.

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. Analysis of variance (ANOVA) comparing all groups at each time point demonstrated significant differences between groups on days 20, 30, 40, 50, 60, and 80. A paired, two-tailed Student's t-test with the Bonferroni correction for multiple comparisons demonstrated that at 40 days postoperatively, animals in the EC-BDNF/CNTF group (n = 7) manifested superior functional recovery compared with those in the EC group (n = 9) and those in the EC-BDNF group (n = 9) (P < 0.001 and P < 0.05, respectively). At 80 days, the animals in both the EC-BDNF (P < 0.01) and EC-BDNF/CNTF (P < 0.05) groups demonstrated greater functional recovery compared with those in the EC group, with no significant difference between the two factor groups at the endpoint. Morphometric analysis demonstrated that nerves from animals in the EC-BDNF/CNTF group had the largest mean axon diameters as compared with those from the EC (proximal: P < 0.001, distal: P < 0.05) and EC-BDNF (proximal: P < 0.01) groups. No significant differences were seen in nerve cross-sectional area. In distal nerve segments, Western blot analysis revealed that expression of myelin-associated glycoprotein was higher than control for the EC group and lower than control for both the EC-BDNF and EC-BDNF/CNTF groups. We conclude that BDNF/CNTF combined treatment increases the early rate of functional sciatic nerve regeneration over treatment with BDNF alone, although the degree of maximal recovery was similar at the conclusion of the experiment.

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. In PC12 cells, TrkA activation was necessary to elicit p75(NGFR)-associated kinase activity. Although NGF binding to p75(NGFR) was not necessary for kinase activation, it accelerated the activation of the kinase at low NGF concentrations. Deletion analysis showed that a 43 amino acid region in the cytoplasmic domain of p75(NGFR) was responsible for this effect. These findings show that p75(NGFR) accelerates TrkA-mediated signaling and, in addition, demonstrate that p75NGFR and TrkA collaborate to activate a previously undescribed p75(NGFR)-associated protein kinase.

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. INTERVENTION: Rat sciatic nerves were transected and repaired as a model of injury following which experimental factors were delivered in vivo through an implantable osmotic pump. OUTCOME MEASURES: Functional nerve recovery, muscle mass, and gene expression in the three experimental groups were evaluated. RESULTS: The ciliary neurotrophic factor group (n=6) showed a higher sciatic functional recovery (P=.003) and preservation of affected muscle mass (P=.03) compared with the nerve growth factor (n=8) and control (n=8) groups. Molecular analysis of injured nerves showed no difference in expression of ciliary neurotrophic factor, myelin basic protein, or low-affinity neurotrophin receptor messenger RNA among the three groups. CONCLUSION: These data suggest that ciliary neurotrophic factor may serve as an important neurocytokine for axonal regrowth during peripheral nerve regeneration.

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. Segments of the sciatic nerves proximal and distal to the repair site were harvested at 90 days for histologic and morphometric evaluation. RESULTS: Animals repaired by CT/linked-BDNF (n=10) demonstrated the most favorable functional recovery of all groups, with statistically significant differences seen compared with animals repaired by CT (n=10, P=.05) and epineural coaptation (n=9, P<.001). Animals repaired by CT with BDNF delivered by an osmotic pump (n=8) and CT also showed statistically superior functional recovery compared with those repaired by epineurial coaptation (P=.005 and P=.02, respectively). Nerves repaired by CT/linked-BDNF had the largest mean axon diameters proximal and distal to the repair site. CONCLUSION: Brain-derived neurotrophic factor and CT improve the rate and the degree to which recovery of sciatic function occurs after nerve transection and repair. Animals repaired by CT/BDNF-linked demonstrated the most favorable functional recovery of all groups. Animals whose repair technique included BDNF had the largest mean axon diameters of all groups.

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. Stimuli that induce IL-6 production in the hematopoietic lineage induced transcription and production of IL-6 by iSC cells. Lipopolysaccharide, tumor necrosis factor-alpha, IL-1 alpha, IL-6, and serum withdrawal induced iSC cell IL-6 mRNA. The kinetics of IL-6 production was confirmed in the mouse IL-6-dependent B9 bioassay and that activity could be neutralized with antisera to IL-6. Expression of both the IL-6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL-6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL-6 message is induced. IL-6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL-6 in response to PC12 cell coculture and to stimuli that induce IL-6 production in the hematopoietic lineage. The induction of IL-6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.

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. Northern blot analysis confirmed that the N19 cells expressed the 1.3-kb NGF mRNA and the 1.4- and 4-kb BDNF mRNAs. In situ hybridization histochemistry identified the presence of NGF mRNAs in 9-day primary oligodendroglial cultures. Combined immunocytochemistry and in situ hybridization histochemistry colocalized NGF mRNA within primary cultured cells that immunostained for the oligodendrocyte marker galactocerebroside (GC). Double-immunofluorescence analysis also colocalized NGF protein within GC+ cells and within A2B5+ cells, a marker for oligodendrocyte progenitors. These results show that oligodendroglia and their precursor cells can express the neurotrophic factor NGF. They suggest that cells in the oligodendrocyte lineage may play an active role in neurite extension through fiber tracts in addition to myelination.

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. These cell lines grew continuously at the permissive temperature (34 degrees C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39 degrees C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39 degrees C for > 9 days compared with cells maintained at 34 degrees C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers--galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 18.5-, and 21.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a "mature" oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of "maturation." This cell line has now been passaged > 40 times with fidelity of phenotype and genotype.

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. Enriched oligodendrocyte cultures prepared at 7 days in vitro, a time when the majority of the cells were oligodendrocyte precursors undergoing transition into mature oligodendrocytes, were immortalized with pZIPSVtsA58, which carries a temperature-sensitive immortalizing oncogene. All of the immortalized cell lines grew rapidly at the permissive temperature of 34 degrees C and exhibited a dramatic decrease in growth rate at the nonpermissive temperature of 39 degrees C. The cell lines were characterized by immunocytochemistry and Northern blot analysis for a number of glial cell markers, and the phenotype of all the lines were consistent with their being in the oligodendroglial cell lineage. The phenotypes of the cell lines varied significantly with representatives of oligodendrocyte precursors, and immature and mature oligodendrocytes being present within the population of immortalized lines. All the cell lines appeared to be clonal based upon Southern blot analysis and all have been passaged at least 40 times with retention of stable phenotype.

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. In the jimpy mutant, MBP mRNA translocation appeared to be impaired, but translocation occurred normally in quaking brains in vivo. We have found that steroids, such as glucocorticoids, stimulate the translation of MBP and PLP mRNAs in cell-free systems and inhibit the translation of CNP mRNA. This pattern of regulation is consistent with compositional changes noted in myelin during development. We have localized a nine nucleotide segment within the 5'-untranslated region of the MBP mRNA that is involved in the action of steroids on translation of this mRNA. We have also determined that the protein synthetic step modulated by the steroids is chain initiation, enhancing the rate at which new ribosomal subunits bind to the MBP mRNAs.

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. In jimpy brains the translocation of MBP mRNA appears to be disrupted, so that most of the mRNA remains associated with cell bodies. This apparent failure of translocation may account for the lack of incorporation of newly synthesized MBP into jimpy myelin. In quaking myelin, where MBP assembly is also defective, translocation appears to be normal, suggesting that incorporation of MBP into the membrane also is regulated posttranslationally. We have identified a number of the structural features of MBP mRNAs that influence the efficiencies with which they are translated and may be involved in regulating the levels of individual MBP produced. We also found that glucocorticoids stimulate the translation of MBP and PLP mRNAs and inhibit the translation of CNP mRNA in cell-free systems. Our results suggest that this pattern of translational regulation may be physiologically meaningful, especially during maturation of myelin. The mechanism by which the steroids modulate translation of these messages appears to be novel. Analysis of the effect of steroids on cRNAs produced from engineered MBP cDNA constructs has permitted the identification of a nine nucleotide element involved in this steroid modulation within the 5' untranslated region of the MBP mRNA.

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. A less severe, but significant, reduction in the level of PLP expression per oligodendrocyte was also observed in the mutant brain. Because of the diffuse pattern of MBP mRNA distribution in the controls it was not possible to determine whether MBP expression was reduced on a per cell basis in the jimpy mutant. Silver grains representing MBP mRNAs were diffusely distributed over myelinating regions in normal mice but, in contrast, the grains were primarily clustered over oligodendrocyte cell bodies in the jimpy brains. No significant differences in jimpy oligodendrocyte morphology were evident by galactocerebroside immunohistochemistry. These results suggested that the apparent MBP mRNA clustering was not due to truncated or reduced numbers of oligodendrocyte processes. Rather, they suggest that the inability of jimpy mice to incorporate MBP into myelin may be due, in part, to a disruption in the translocation of MBP mRNAs within the oligodendrocyte.

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. Peak expression occurred at approximately postnatal day 20 in most regions examined, regardless of time of onset of expression. As myelination proceeded, two different labeling patterns were observed with the PLP and MBP 35S-labeled cDNA probes. In the earliest stages of myelinogenesis MBP mRNA labeling was restricted to oligodendrocyte cell bodies, but shortly after the gene began to be expressed the labeling became more diffuse. In contrast, PLP mRNA labeling remained over or surrounding oligodendrocyte cell bodies at all stages of myelinogenesis. These two distinctly different patterns of labeling are consistent with alternative intracellular trafficking of MBP and PLP mRNAs, in which PLP mRNAs remain associated with ribosomes within the cell soma and MBP mRNAs move from the cell soma to the oligodendrocyte processes at a specific stage early in myelinogenesis. However, there appeared to be a clear time lag between the onset of MBP mRNA expression and the movement of ribosomes carrying MBP mRNAs into the oligodendrocyte processes. Additionally, the in situ hybridization studies revealed a population of unidentified cells residing in cortical molecular layers that express PLP mRNA in the absence of MBP mRNA.