Intracellular distribution of myelin protein gene products is altered in oligodendrocytes of the taiep rat.

Hypomyelination and subsequent demyelination of the taiep rat CNS are thought to result from the abnormal accumulation of microtubules (MTs) in oligodendrocytes that disrupts intracellular transport of components needed to form and maintain the myelin sheath. In this study, myelin gene expression was evaluated in mutant and age-matched controls to determine if MT abnormalities affect the distribution of myelin proteins and their mRNAs. Immunohistochemical analysis of taiep brains and spinal cords revealed a gradual decrease in levels of several myelin proteins including myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Accompanying early declines in MAG and PLP, accumulations of immunoreactive products were detected within oligodendrocytes, consistent with a defect in protein trafficking. Northern blot analysis indicated that diminishing protein levels could not be attributed to changes in transcriptional activity, except for MBP of which mRNA levels decreased with age. Cellular localization of MBP mRNA by in situ hybridization further revealed that transcripts were concentrated within oligodendrocyte cell bodies instead of uniformly distributed throughout processes. These results demonstrate that changes in expression and intracellular localization of myelin gene products are concurrent with increases in MT mass in taiep oligodendrocytes and support our hypothesis that cytoskeletal defects prevent the normal transport of elements required for the formation and maintenance of the myelin sheath.

Abnormal expression of SNS/PN3 sodium channel in cerebellar Purkinje cells following loss of myelin in the taiep rat.

Using in situ hybridization and immunochemical methods, we have observed an increase in the expression of SNS/PN3 sodium channel mRNA and protein in cerebellar Purkinje cells of the taiep rat. These changes are present in taiep rats at 12 months of age, following loss of myelin, but not at one month, prior to loss of myelin. Increased SNS/PN3 expression is not associated with aging per se, because it was not observed in control rats at 12 months of age. These results suggest that altered sodium channel expression in Purkinje cells may contribute to the ataxia that occurs in taiep rats.

Insertion of a retrotransposon in Mbp disrupts mRNA splicing and myelination in a new mutant rat.

Our understanding of myelination has been greatly enhanced via the study of spontaneous mutants that harbor a defect in a gene encoding one of the major myelin proteins (myelin mutants). In this study, we describe a unique genetic defect in a new myelin mutant called the Long Evans shaker (les) rat that causes severe dysmyelination of the CNS. Myelin deficits result from disruption of the myelin basic protein (Mbp) gene caused by the insertion of an endogenous retrotransposon [early transposons (ETn) element] into a noncoding region (intron 3) of the gene. The ETn element alters the normal splicing dynamics of MBP mRNA, leading to a dramatic reduction in the levels of full-length isoforms (<5% of normal) and the appearance of improperly spliced, chimeric transcripts. Although these aberrant transcripts contain proximal coding regions of the MBP gene (exons 1-3), they are unable to encode functional proteins required to maintain the structural integrity of the myelin sheath. These chimeric transcripts seem capable, however, of producing the necessary signal to initiate and coordinate myelin gene expression because normal numbers of mature oligodendrocytes synthesizing abundant levels of other myelin proteins are present in the mutant CNS. The les rat is thus an excellent model to study alternative functions of MBP beyond its well characterized role in myelin compaction.

Microtubule alterations in cultured taiep rat oligodendrocytes lead to deficits in myelin membrane formation.

The taiep rat is a myelin mutant in which hypomyelination and progressive demyelination of the CNS are accompanied by an accumulation of microtubules within oligodendrocytes. To investigate whether and how the myelin defects were caused by microtubule abnormalities, we have established a taiep oligodendrocyte culture system in which mutant cells produce abnormally high levels of tubulin and microtubule-associated proteins and exhibit myelin defects. The studies show that abnormal microtubule accumulation and tight microtubule bundles developed in the taiep oligodendrocytes, with a higher ratio of minus-end-distal to plus-end-distal microtubules in their processes. Initially, in culture, immature taiep oligodendrocytes which have higher levels of tubulin than controls extend roughly twice as much membrane sheet as controls. The membrane sheets of the mature taiep oligodendrocytes which display the microtubule accumulation, however, grew much less rapidly compared to controls. By the fifth day in culture, a majority of the taiep oligodendrocytes had ceased the expansion of their membrane sheets and in some cases the sheets retracted. The levels of the myelin proteins, proteolipid protein and myelin-associated glycoprotein, were also markedly diminished in the mature taiep oligodendrocytes. Treatment with the microtubule depolymerizing drug nocodazole prevented not only the accumulation of microtubules but also restored the normal distribution of proteolipid proteins within the taiep oligodendrocytes. These data demonstrate that myelin synthesis in the oligodendrocyte cultures relies on the formation of a normal microtubule array, and the microtubule abnormalities are directly responsible for the myelin deficit in the taiep oligodendrocytes.

Morphological and morphometric studies of the dysmyelinating mutant, the Long Evans shaker rat.

The Long Evans shaker (les) rat is a recently identified CNS myelin mutant with an autosomal recessive mode of inheritance. Although scattered myelin sheaths are present in some areas of the CNS, most notably the ventral spinal cord in the young neonatal rat, this myelin is gradually lost, and 8-12 weeks little myelin is present throughout the CNS. Despite this severe myelin deficiency, some mutants may live beyond one year of age. Rare, thin myelin sheaths that are present early in development lack myelin basic protein (MBP) and on ultrastructural examination are poorly compacted and lack a major dense line. Many oligodendrocytes develop an accumulation of vesicles and membranous bodies, but no abnormal cell death is observed. In the optic nerve, cell kinetic studies show an increase in proliferation at early time points in les, while total glial cell counts are also increased in les from 2 months of age. In situ hybridization studies demonstrate that the numbers of mature oligodendrocytes are similar to controls early in life and increase with time compared to controls. There is both a progressive astrocyte hypertrophy and microgliosis. While les has a mutation in the myelin basic protein (mbp) gene, it is dissimilar in both genotype and phenotype to the previously described mbp mouse mutants, shiverer (shi) and shiverer(mld). Unlike shi and its allele, where myelin increases with time and oligodendrocytes become ultrastructurally normal, les oligodendrocytes are permanently disabled, continue to demonstrate cytoplasmic abnormalities, and fail to produce myelin beyond the first weeks of life.

Generation of oligodendroglial progenitors from neural stem cells.

To understand how the differentiation of stem cells to oligodendroglial progenitors is regulated, we established cultures of neural stem cells from neonatal rat striatum in the presence of epidermal growth factor (EGF) as free-floating neurospheres that were then exposed to an increasing amount of B104 cell-conditioned medium (B104CM). The resultant cells proliferated in response to B104CM but no longer to EGF. In vitro analysis and transplantation studies indicated that these cells were committed to the oligodendroglial lineage, and they were thus referred to as oligospheres. Further characterization of their expression of early markers, cell cycle, migration, and self-renewal suggests that they were pre-O2A progenitors. RT-PCR analysis indicated that the oligosphere cells expressed mRNAs of platelet-derived growth factor alpha receptor in addition to fibroblast growth factor receptor but not EGF receptor; the latter two receptor mRNAs were expressed by neurosphere cells. Thus, the progression of stem cells to oligodendroglial progenitors is likely induced by factors in B104CM.

In vivo cystometric evaluation of progressive bladder outlet obstruction in rats.

PURPOSE: Bladder outlet obstruction in man is a common medical disorder that may result from benign prostatic hyperplasia, urethral stricture disease, or congenital anomaly. The functional changes that develop in response to obstruction include detrusor instability, elevated voiding pressures, and the presence of a residual urine. The aim of this study was to document the development of progressive bladder outlet obstruction over time in a rat model using conscious, in vivo urodynamics. MATERIALS AND METHODS: Infravesical bladder outlet obstruction was created in female rats by placing a jeweler's jump ring loosely around the proximal urethra. Gradual development of outlet obstruction was followed urodynamically in awake animals at 3, 7, 14, 21, and 28 days post obstruction using a subcutaneously implanted mediport. For each group n = 5-8 animals. RESULTS: Animals developed large capacity bladders with increased compliance, a high residual urine volume, and spontaneous activity. Bladder capacity increased from 0.20 + 0.02 ml. to 6.30 + 1.59 ml. at 28 days post obstruction (p < 0.05). Residual volume increased from 0.06 + 0.01 ml. to 5.95 + 1.54 ml. (p < 0.05). Percent void decreases from 72 + 3.7% in sham controls to 6.7 + 2.5% at 28 d (p < 0.05). Voiding pressure increased from 12 + 1.6 mm. Hg in sham animals to a maximum of 42 + 6.1 mm. Hg at 21 d (p < 0.05). Compliance was significantly higher at 28 d when compared to all other time points. 89% of obstructed animals developed bladder instability. CONCLUSIONS: This study provides clear evidence of the progressive change in bladder function which occurs following outlet obstruction. Implantation of a subcutaneous mediport allows in vivo recording of both the filling and voiding phases of micturition in awake animals that have intact neural responses. This is a precise and easily reproducible method for producing obstruction in a small animal which can provide a continuum of tissue and urodynamic data that may be used to further study the pathophysiologic changes underlying bladder outlet obstruction or other models of bladder dysfunction.

Selective regulation of agrin mRNA induction and alternative splicing in PC12 cells by Ras-dependent actions of nerve growth factor.

The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction. However, regulation of agrin gene expression and pre-mRNA splicing, important in determining the biological actions of agrin, is not well understood. To begin to identify mechanisms controlling agrin expression, quantitative polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat pheochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacked inserts in the Y and Z sites (agriny0z0), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily non-neuronal tissue distribution. Transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution (agriny4z8, agriny4z11, and agriny4z19) were not detected. Treatment of PC12 cells with nerve growth factor (NGF) caused a significant increase in total agrin mRNA. In contrast, exposure to epidermal growth factor had no effect. Analysis of alternative splicing of agrin mRNA revealed that NGF elicited a specific increase in agriny4 and agrinz8 mRNAs that did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably overexpressing a dominant inhibitory form of p21 Ras indicated that NGF induced changes in levels of agrin mRNA and alternative splicing required Ras activity. The results show that NGF can influence important aspects of neuronal differentiation by regulating alternative splicing. Furthermore, these data provide insight into the mechanisms governing agrin gene expression and suggest that neurotrophic factors may play a role in regulating agrin expression in vivo.

Expression of agrin mRNA is altered following seizures in adult rat brain.

Agrin mRNA is broadly distributed throughout the adult rat brain, consistent with its proposed role in synaptogenesis and the organization of synaptic proteins in the central nervous system. The present study examined the effect of neuronal activity on agrin mRNA expression in adult rat forebrain using the hilus lesion paradigm for seizure induction and in situ hybridization and polymerase chain reaction techniques for quantification and characterization of agrin mRNA content. Seizures induced rapid, prolonged, and region-specific changes in agrin mRNA expression with the most prominent alterations occurring in hippocampal and cortical neurons. However, there were no detectable perturbations in the relative abundance of alternatively spliced agrin transcripts in affected brain regions. Activity-dependent changes in agrin expression suggest a role for this protein in modifications of synaptic structure associated with functional synaptic plasticity.

Developmental expression of alpha 7 neuronal nicotinic receptor messenger RNA in rat sensory cortex and thalamus.

The distribution of alpha 7 messenger RNA expression was characterized in developing rat cortex and thalamus. Northern blot analysis of neonatal and adult cortex revealed a single messenger RNA transcript of 5.7 kb. Using in situ hybridization with both full length and short 35S-labeled alpha 7 riboprobes, a distinct transient expression of messenger RNA within sensory cortex and thalamus, during early postnatal development, was observed. alpha 7 transcripts were expressed in low levels as early as embryonic day 13 in the ventricular zone of the neocortex, and as early as embryonic day 15 in the thalamic neuroepithelium. A marked increase in messenger RNA levels was observed during the late prenatal period in both sensory and non-sensory regions of the cortex and thalamus. Moderate to high levels of messenger RNA were maintained into the first postnatal week, followed by a decline into adulthood. alpha 7 messenger RNA expression was significantly higher in the anterodorsal, lateral dorsal, ventral posterior medial and ventral posterior lateral thalamic nuclei of postnatal day 7 pups than in adult brains. Expression of messenger RNA within dorsal lateral geniculate, ventral lateral geniculate and medial geniculate did not show a significant reduction with age. Within the developing cortex, messenger RNA expression delineated the primary somatosensory, auditory and visual cortices in a unique laminar pattern that was consistently and significantly higher than in the adult in superficial layer VI. Higher levels of expression were also observed in retrosplenial cortex at postnatal day 7 than in the adult. Tangential sections through postnatal day 7 cortex revealed low levels of alpha 7 messenger RNA expression delineating the primary sensory areas in layer IV, corresponding to acetylcholinesterase-labeled thalamocortical afferents. However, these sensory areas exhibited higher levels of alpha 7 messenger RNA expression and were more clearly defined in layer VI, but not by acetylcholinesterase staining. The distribution of alpha 7 messenger RNA within the developing thalamocortical system parallels the distribution of alpha-bungarotoxin binding sites and suggests that the receptor is localized on both thalamic cells and their cortical target neurons. This transient and distinct pattern of distribution of the alpha 7 neuronal nicotinic receptor, which coincides with the major phase of thalamocortical development, suggests that it may play a functional role in the development of cortical circuitry.

Localization and alternative splicing of agrin mRNA in adult rat brain: transcripts encoding isoforms that aggregate acetylcholine receptors are not restricted to cholinergic regions.

Agrin is a protein implicated in the formation and maintenance of the neuromuscular junction. In addition to motor neurons, agrin mRNA has been detected in the brains of embryonic rat and chick and adult marine ray, suggesting that this molecule may also be involved in the formation of synapses between neurons. As a step toward understanding agrin's role in the CNS, we utilized Northern blot and in situ hybridization techniques to analyze the regional distribution and cellular localization of agrin mRNA in the spinal cord and brain of adult rats. The results of these studies indicate that the agrin mRNA is expressed predominantly by neurons broadly distributed throughout the adult CNS. Moreover, expression of agrin mRNA is not restricted to cholinergic structures or regions of the brain receiving cholinergic input. Recently, RNA isolated from rat embryonic spinal cord was shown to contain four alternatively spliced agrin mRNAs, referred to as agrin0, agrin8, agrin11, and agrin19, each of which encodes agrin proteins that are active in acetylcholine receptor aggregating assays (Ferns et al., 1992). Using the polymerase chain reaction we demonstrate that all four of these agrin transcripts are expressed within the adult CNS. Agrin0, agrin8, and agrin19 were present in all regions analyzed. In contrast, agrin11 was detected only in forebrain. Results of these studies indicate that both the level of expression and pattern of alternative splicing of agrin mRNA are differentially regulated in the brain. The broad and predominantly neuronal distribution of agrin mRNA in the adult brain suggests that, in addition to its role at the neuromuscular junction, agrin may play a role in formation and maintenance of synapses between neurons in the CNS.

Characterization of an activity from the strict anaerobe Roseburia cecicola that degrades DNA when exposed to air.

Roseburia cecicola is an obligately anaerobic bacterium that is extremely sensitive to oxygen. Genomic DNA isolated from cells exposed to air for even a brief period (< 5 min) is partially degraded, while DNA extracted from cells maintained in an anaerobic environment remains intact. Cells exposed to air for longer and longer periods yield DNA which is progressively degraded into fragments with decreasing sizes. Oxygen toxicity for this anaerobe appears to result, at least in part, from degradation of its genomic DNA. Cell lysates of the organism exhibited a similar ability to degrade exogenous sources of DNA when assayed in vitro under aerobic conditions. A substance that degrades both DNA and RNA when incubated aerobically was partially purified from such lysates. It has an approximate molecular weight of 2,800 and is unlikely to be a protein. It requires a reducing agent for activity and can be inhibited by catalase and peroxidase but not superoxide dismutase. The rate at which it degrades DNA in vitro can be enhanced by temperatures above 37 degrees C or by oxygen at partial pressures above atmospheric pressure. These results suggest that this substance degrades nucleic acids by a mechanism involving oxygen radicals.