Proteolipid protein mRNA stability is regulated by axonal contact in the rodent peripheral nervous system.

Proteolipid protein (PLP) and its alternatively spliced isoform, DM20, are the main intrinsic membrane proteins of compact myelin in the CNS. PLP and DM20 are also expressed by Schwann cells, the myelin-forming cells in the PNS, and are necessary for normal PNS function in humans. We have investigated the expression of PLP in the PNS by examining transgenic mice expressing a LacZ transgene under the control of the PLP promoter. In these animals, myelinating Schwann cells expressed beta-galactosidase more prominently than nonmyelinating Schwann cells. PLP/DM20 mRNA levels, but not those of LacZ mRNA, increased during sciatic nerve development and decreased after axotomy, with resultant Wallerian degeneration. PLP/DM20 transcription rates, in nuclear run off experiments, however, did not increase in developing rat sciatic nerve despite robust increases in PLP/DM20 mRNA levels during the same period. In RNAse protection studies, PLP mRNA levels fell to undetectable levels following nerve transection whereas levels of DM20 were essentially unchanged despite both being transcribed from the same promoter. Finally, cotransfection studies demonstrated that PLP-GFP, but not DM20-GFP mRNA is down-regulated in Schwann cells cultured in the absence of forskolin. Taken together these data demonstrate that steady state levels of PLP mRNA are regulated at a posttranscriptional level in Schwann cells, and that this regulation is mediated by Schwann cell-axonal contact. Since the difference between these two mRNAs is a 105-bp sequence in PLP and not in DM20, this sequence is likely to play a role in the regulation of PLP mRNA.

Disruption of the murine nuclear factor I-A gene (Nfia) results in perinatal lethality, hydrocephalus, and agenesis of the corpus callosum.

The phylogenetically conserved nuclear factor I (NFI) family of transcription/replication proteins is essential both for adenoviral DNA replication and for the transcription of many cellular genes. We showed previously that the four murine NFI genes (Nfia, Nfib, Nfic, and Nfix) are expressed in unique but overlapping patterns during mouse development and in adult tissues. Here we show that disruption of the Nfia gene causes perinatal lethality, with >95% of homozygous Nfia(-/-) animals dying within 2 weeks after birth. Newborn Nfia(-/-) animals lack a corpus callosum and show ventricular dilation indicating early hydrocephalus. Rare surviving homozygous Nfia(-/-) mice lack a corpus callosum, show severe communicating hydrocephalus, a full-axial tremor indicative of neurological defects, male-sterility, low female fertility, but near normal life spans. These findings indicate that while the Nfia gene appears nonessential for cell viability and DNA replication in embryonic stem cells and fibroblasts, loss of Nfia function causes severe developmental defects. This finding of an NFI gene required for a developmental process suggests that the four NFI genes may have distinct roles in vertebrate development.

Expression of molecular chaperones and vesicle transport proteins in differentiating oligodendrocytes.

The major stages of oligodendrocyte differentiation are defined by the presence or absence of certain myelin-specific proteins. Events leading to the successful processing of these proteins, such as the folding, assembly, and trafficking of these proteins through the biosynthetic pathway, are largely undefined. In the present study, we have examined both cultured primary oligodendrocytes and immortalized oligodendrocyte cell lines for the presence of molecular chaperones and/or vesicle transport proteins. We find that a select set of these proteins are expressed relatively early in oligodendrocyte differentiation, whereas a characteristically different set of proteins are expressed at later stages of oligodendrocyte differentiation. In other systems, these proteins participate in the folding and assembly of protein complexes, in the prevention of protein aggregation, as well as the trafficking of proteins via vesicles to specific subcellular destinations including the plasma membrane. Some of the chaperones and/or vesicle transport proteins investigated in this study may play a pivotal role in the certain aspect of myelin biogenesis.

Expression of cell surface markers and myelin proteins in cultured oligodendrocytes from neonatal brain of rat and mouse: a comparative study.

Dissociated brain cell cultures are a useful model for investigating development and differentiation of oligodendrocytes in vitro. The current studies compare the developmental patterns of expression for oligodendrocyte lineage/myelin markers in both primary and secondary oligodendrocyte cultures derived from mouse and rat neonates. The rat and mouse dissociated brain cell cultures express the same myelin-specific antigens, but mouse oligodendrocytes produce a larger and more elaborate sheet-like membrane than rat oligodendrocytes, and some of the myelin markers (O4, GC, and MBP) show more intense membrane staining in mouse cultures. GD3 appears to be a good oligodendrocyte marker for rat cells, but it is nonspecific in mouse cells. There are fewer oligodendrocytes in mouse cultures, and they appear to require a longer differentiation time than rat oligodendrocytes. These same results are also observed in secondary oligodendrocyte cultures, although in general late myelin markers such as MBP and PLP are expressed at a much lower level in mouse cells than rat cells.

A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice.

Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta-galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane.

Control of virus-induced cell fusion by host cell lipid composition.

Virus-induced cell fusion has been examined in a series of stable cell lines which were originally selected for resistance to the fusogenic effects of polyethylene glycol (PEG). For a wide variety of viruses, including murine hepatitis virus (a coronavirus), vesicular stomatitis virus (a rhabdovirus), and two paramyxoviruses (Sendai virus and SV5), susceptibility to virus-induced fusion was found to be inversely correlated with susceptibility to PEG-induced fusion. This phenomenon was observed both for cell fusion occurring in the course of viral infection and for fusion induced "from without" by the addition of high titers of noninfectious or inactivated virus. The fusion-altered cell lines (fusible by virus but not by PEG) are characterized by their unusual lipid composition, including marked elevation of saturated fatty acids and the presence of an unusual ether-linked neutral lipid. To test the association between lipid composition and fusion, acyl chain saturation was manipulated by supplementing the culture medium with exogenous fatty acids. In such experiments, it was possible to control the responses of these cells to both viral and chemical fusogens. Increasing the cellular content of saturated fatty acyl chains increased the susceptibility of cells to viral fusion and decreased susceptibility to PEG-induced fusion, whereas lowering fatty acid saturation had the opposite effect. Thus, parallel cultures of cells can be either driven toward the PEG-fusible/virus-fusion-resistant phenotype of the parental cells or rendered susceptible to viral fusion but resistant to PEG-induced fusion, solely by the alteration of cellular lipids. The ability of cellular lipid composition to regulate virus-induced membrane fusion suggests a possible role for lipids in viral infection and pathogenesis.

Preliminary studies on the biology of Borna disease virus.

Borna disease virus (BDV) is an unclassified agent that causes neurological disease in a wide range of animal species and possibly in humans. The infectious nature of BDV has been long established but, despite extensive progress on the pathogenesis of the infection, the aetiological agent is still uncharacterized. Recent studies have shown that BDV replicates productively in cultures of foetal rabbit glial cells (FRG) which produce a virus-specific protein that is easily detected immunocytochemically. This provides a marker for BDV infectivity. This cell culture system was used to investigate the replication cycle of BDV. The agent required at least 1 h to bind to and penetrate the cells and the antigen was detected 24 h later. Cycloheximide and actinomycin D inhibited production of the antigen in inoculated cells, indicating that both protein synthesis and a DNA-dependent function were required for the production of viral antigen. Cocultivation of BDV-infected FRG cells with Vero cells resulted in a persistent productive infection in the latter. Use of these cells showed that the infectious agent matured exclusively in the cytoplasm and within the plasma membrane of the cell. Antigen-laden nuclei did not have infectivity. These studies showed that BDV has the physical and replicative properties typical of conventional viruses but its mechanism of replication and site of morphogenesis may be unique.

Astrocytes and Schwann cells are virus-host cells in the nervous system of rats with Borna disease.

Borna disease virus (BDV) replicates only in cells in the central (CNS) and peripheral (PNS) nervous system in adult rats. Infection of the nervous system is associated with a transient, intense mononuclear meningoencephalitis and immunemediated loss of BDV-infected neurons. The identification of BDV antigen in neurons and the accompanying immunologically-specific lysis of these cells led to the prediction that the CNS would be virus-free after the animal had recovered from encephalitis. However, BDV infectivity and antigen persist for the lifetime of the animal. It appeared, therefore, that other neural cells might be hosts for viral replication and provide a reservoir for the virus. Morphological criteria were used to identify astrocytes and Schwann cells which expressed BDV antigens in vivo. Borna disease virus (BDV) infected astrocytes were identified by double labeling tissue sections with combined cell-specific and BDV-specific antibodies in an avidin-biotin immunocytochemical assay. Examination of serial I micrometer-thick cryosections of hippocampus and sciatic nerve preparations revealed several cells that expressed both glial and BDV antigens. Infectious virus was recovered from cultures of Schwann cells from infected rats. Borna disease virus-infected glial elements persisted beyond the period of inflammation and massive neuronal destruction, and represented a major class of infected cells during chronic disease.

Neurotransmitter abnormalities in Borna disease.

Borna disease (BD) agent is an infectious pathogen that causes progressive central nervous system (CNS) dysfunction in a wide range of vertebrate hosts. The course of BD in adult rats is biphasic. The acute phase is characterized by aggressive behavior and inflammatory cell infiltrates in brain. With chronic infection animals become listless and inflammation resolves. BD antigens are similarly distributed in neurons in hippocampus, neocortex, cerebellum and brainstem in acutely and chronically infected animals. We have recently examined brain levels of neuronal transcripts in rats with acute and chronic BD. Levels for 3 of these mRNAs, cholecystokinin, glutamic acid decarboxylase and somatostatin, were decreased in acutely infected rats and increased toward control values in chronically infected rats. A fourth transcript, MuBr8, correlated in distribution with BD antigen, was persistently decreased throughout the course of infection. These data may have implications for understanding the pathogenesis of neurologic disturbances in BD and other inflammatory CNS diseases.

Pathogenesis of Borna disease in rats: evidence that intra-axonal spread is the major route for virus dissemination and the determinant for disease incubation.

Borna disease virus is an uncharacterized agent that causes sporadic but fatal neurological disease in horses and sheep in Europe. Studies of the infection in rats have shown that the agent has a strict tropism for neural tissues, in which it persists indefinitely. Inoculated rats developed encephalitis after an incubation period of 17 to 90 days. This report shows that the incubation period is the time required for transport of the agent in dendritic-axonal processes from the site of inoculation to the hippocampus. The immune responses to the agent had no effect on replication or transport of the virus. The neural conduit to the brain was proven by intranasal inoculation of virus that resulted in rapid transport of the agent via olfactory nerves to the hippocampus and in development of disease in 20 days. Virus inoculation into the feet resulted in spread along nerve fibers from neuron to neuron. There was sequential replication in neurons of the dorsal root ganglia adjacent to the lumbar spinal cord, the gracilis nucleus in the medulla, and pyramidal cells in the cerebral cortex, followed by infection of the hippocampal neurons and onset of disease. This progression required 50 to 60 days. The exclusiveness of the neural conduit was proven by failure to cause infection after injection of the virus intravenously or into the feet of neurectomized rats.

Increased levels of myelin basic protein transcripts in virus-induced demyelination.

In multiple sclerosis, a demyelinating disease of young adults, there is a paucity of myelin repair in the central nervous system (CNS) which is necessary for the restoration of fast saltatory conduction in axons. Consequently, this relapsing disease often causes marked disability. In similar diseases of small rodents, however, remyelination can be quite extensive, as in the demyelinating disease caused by the A59 strain of mouse hepatitis virus (MHV-A59), a coronavirus of mice. To investigate when and where oligodendrocytes are first triggered to repair CNS myelin in such disease, we have used a complementary DNA probe specific for one major myelin protein gene, myelin basic protein (MBP), which hybridizes with the four forms of MBP messenger RNA in rodents. Using Northern blot and in situ hybridization techniques, we previously found that MBP mRNA is first detected at about 5 days after birth, peaks at 18 days and progressively decreases to 25% of the peak levels in the adult. We now report that in spinal cord sections of adult animals with active demyelination and inflammatory cells, in situ hybridization reveals a dramatic increase in probe binding to MBP-specific mRNA at 2-3 weeks after virus inoculation and before remyelination can be detected by morphological methods. This increase of MBP-specific mRNA is found at the edge of the demyelinating area and extends into surrounding areas of normal-appearing white matter. Thus, in situ hybridization with myelin-specific probes appears to be a useful method for detecting the timing, intensity and location of myelin protein gene reactivation preceding remyelination. This method could be used to elucidate whether such a reactivation occurs in multiple sclerosis brain tissue. Our results suggest that in mice, glial cells react to a demyelinating process with widespread MBP mRNA synthesis which may be triggered by a diffusible factor released in the demyelinated areas.

Comparison of monoamine concentrations in the brains of adult male and female Japanese quail.

A fluorometric assay measuring brain tissue concentrations of norepinephrine, dopamine, and serotonin has been validated for Japanese quail. Accuracy, precision, specificity, and parallelism were determined. The sensitivity of the assays was 6 ng/tube, which allowed individual assay of 1 to 2 mg hypothalamic tissue. In Experiment 1, relatively large areas of brain from adult, reproductively active males and females were found to differ significantly in norepinephrine content in optic lobes and for dopamine in right telencephalon. A microdissection technique was used in Experiment 2 to sample small portions of hypothalamic tissue. Sex differences were observed for norepinephrine in the sections containing the lobus paraolfactorius and the preoptic, anterior, and medial hypothalamus. Differences in monoamine content were most apparent when smaller areas dissected by microdissection were analyzed. These results give evidence for sex differences in the monoamine content in specific areas of the brain.

The developmental distribution of monoamines in the brain of male Japanese quail (Coturnix coturnix japonica).

Changes in the levels of the brain catecholamines, norepinephrine (NE) and dopamine (DA), were studied in the male Japanese quail during maturation. In addition the neurotransmitter, serotonin (5HT), its precursor, tryptophan ( TRYP ), and its metabolite, 5-hydroxyindoleacetic acid (5HIAA), were measured. Birds were sampled at weekly intervals between the ages of 1 and 63 days of age. Brains of the quail were dissected into cerebral hemispheres, diencephalon, brain stem, cerebellum, and optic lobes. Spectrophotofluorometric analyses of the monoamines in the brain parts were performed following the validation of this technique for use with the avian brain. Norepinephrine increased significantly (P less than .05) with age in all brain regions except the cerebellum, whereas dopamine increased significantly (P less than .05) only in the cerebral hemispheres. Serotonin levels increased significantly (P less than .05) in all brain regions except the cerebellum. The most dramatic change over maturation was observed in the cerebral hemispheres, and relatively smaller increases occurred in the diencephalon. Tryptophan decreased significantly (P less than .05) in concentration in all brain regions throughout the 9 weeks of development. However, 5HIAA did not change significantly in brain regions. Serum testosterone, as measured by testosterone radioimmunoassay, increased in conjunction with sexual maturation. In general, the monoamine levels rose with age. Additionally, the monoamine distribution appeared to increase in caudal to rostral direction. Changes in the monoamines, particularly in the diencephalon, occurred at approximately the time that increased peripheral concentrations of testosterone were also observed. This observation supports the contention that monoamines may be involved in regulating gonadotropin release during maturation.

Age-related reproductive decline in the male Japanese quail.

A series of experiments was conducted to study the decline in reproduction associated with age in the male Japanese quail. In Experiment 1, eggs were collected from pairs that were 28, 56, 107, and 149 weeks of age. Pairs that were 56 weeks of age or older showed a sharp drop in fertility and hatchability. Subsequent experiments were designed to study the endocrine and behavioral basis for this decline in the male. In Experiment 2, males that were between 23 and 70 weeks of age were tested for mating behavior, plasma testosterone was measured, and testes wet weight was determined. There were no significant differences between the age groups. However, samples taken from an additional group of males 168 weeks of age showed significantly (P less than 0.05) lower serum testosterone and testes weight. Therefore, the third experiment was designed to include age groups of males between 4 and 126 weeks of age in order to further examine this decline in males older than 70 weeks of age. There was a significant (P less than 0.05) drop in this number of males showing sexual behavior and of those males showing sexual behavior; the quantity of mating activity also declined. Plasma testosterone was reduced in the older groups; however, the change was not significant. In addition, the size of the cloacal gland, an androgen-dependent organ, declined with age (P less than 0.05). These results argue that the age-related reproductive decline may have behavioral as well as endocrine basis. Possibly, the behavioral changes may result from altered receptor sensitivity at the level of areas of the brain, which control reproductive behavior, and at the level of the testes, which affect hormone production and spermatogenesis.