Cytoprotective effect of estrogen on ammonium chloride-treated C6-glioma cells.

The potential cytoprotective effects of estrogen in the brain are of special interest in aging, neurodegenerative diseases, exposure to toxins, and trauma. Estrogen effects on neurons have been widely explored, but less is known about estrogen effects on glia. Glial cells are primary targets of ammonia toxicity, which arises from liver disease or failure (such as from cirrhosis in alcoholics), urea cycle disorders, or inborn errors of metabolism. We examined the ability of estrogen to protect glial cells from ammonium chloride toxicity using an in vitro model system. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well established model system for this work. When C6-glioma cells were exposed to 15 mM ammonium chloride, we observed major cell death (only 32% cell survival relative to control) within 72 h. Pretreatment with 17beta-estradiol (10 microM) significantly protected C6-glioma cells from ammonia toxicity (99% cell survival relative to control). In addition to enhancing the viability of C6-glioma cells against ammonia challenge, estrogen pretreatment was also found to protect mitochondrial function as assayed using the MTT reduction assay. Mitochondrial function was reduced to 39% of control levels in ammonia-challenged cultures and was mostly protected by estrogen (72% of control levels). The findings are potentially relevant for the development of therapeutic strategies to protect glial cells against ammonia toxicity resulting from hepatic failure or other causes.

Estrogen effects on neurite outgrowth and cytoskeletal gene expression in ERalpha-transfected PC12 cell lines.

The potential of gonadal steroids like estrogen (E) to promote neurite sprouting is of interest in development and aging, as well as after neural trauma. The specific roles of the two main estrogen receptors, ERalpha and ERbeta, in neuronal sprouting are not yet well understood. We examined the hypothesis that E can enhance nerve growth factor (NGF)-stimulated neurite sprouting in an ERalpha-dependent manner. PC12 cells that were stably transfected with the full-length rat ERalpha gene (PCER) and a control line of cells transfected with vector DNA alone (PCCON) were compared. Both cell lines vigorously differentiate neurites when treated with NGF. We determined that both lines show basal expression of ERbeta mRNA, but only the PCER cells express ERalpha mRNA. Estrogen treatment markedly enhanced NGF-stimulated neurite outgrowth from PCER but not from PCCON cells. Significantly larger proportions of PCER cells (34 and 53% at 24 and 48 h, respectively) had neurites than did the PCCON cells (17 and 26% at 24 and 48 h) after E plus NGF treatment. We also examined the effects of E and NGF treatment of PCER and PCCON cells on peripherin, alpha-tubulin, and tau mRNA expression. In undifferentiated PCER cells, E treatment increased peripherin, reduced alpha-tubulin, and did not alter tau mRNA levels. No changes in these mRNAs were observed in the controls (undifferentiated PCCON cells) after E treatment. NGF treatment markedly stimulated expression of peripherin, alpha-tubulin, and tau mRNAs in both PCER and PCCON cells. From these observations we conclude that E synergizes with NGF and stimulates neurite sprouting and also modulates expression of several cytoskeletal mRNAs through ERalpha.

Differential regulation of cytoskeletal gene expression in hamster facial motoneurons: effects of axotomy and testosterone treatment.

We have previously demonstrated that systemic administration of testosterone increases the rate of axonal regeneration following facial nerve crush in adult male hamsters. In the present study, the molecular mechanisms by which androgens could enhance axonal regeneration were examined at a cellular level. Specifically, the following question was addressed using quantitative in situ hybridization with cDNA probes complementary to betaII, and alpha1 tubulin mRNAs: Does exogenous testosterone augment axotomy-induced changes in tubulin mRNA expression in hamster facial motoneurons (FMN)? Castrated adult male hamsters were subjected to right facial nerve severance, with the left side serving as internal control. One-half of the animals received testosterone replacement in the form of subcutaneously implanted silastic capsules containing crystalline testosterone propionate, and the other half were implanted with blank capsules immediately following the axotomy. Postoperative survival times from 2-14 days were examined. Axotomy alone resulted in a significant increase in the levels of both betaII and alpha1 tubulin mRNAs in facial motor neurons between 2-14 days after injury. Administration of testosterone selectively augmented the axotomy-induced increases in betaII-tubulin, but not alpha1 tubulin, mRNA, levels at 7 and 14 days post axotomy. These results demonstrating an effect of testosterone in altering the neuronal cytoskeletal response to axotomy suggest that testosterone may enhance the regenerative properties of motor neurons via molecular mechanisms that involve selective alterations of the neuronal cytoskeleton.

Differential expression of estrogen receptor alpha and beta in rat dorsal root ganglion neurons.

Estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) mRNAs are both expressed in rat dorsal root ganglion (DRG) neurons, but the distribution of these two mRNAs differs markedly. Radiolabeled probes highly specific to ERalpha or ERbeta mRNAs were used for in situ hybridization studies; two antibodies specific to ERalpha protein were used for immunocytochemistry and specific primers were used for reverse transcription polymerase chain reaction (RT-PCR) studies. These revealed that ERbeta mRNA is widely expressed in the DRG of both male and female rats, with virtually all neurons showing positive signals. In contrast, ERalpha mRNA, as well as nuclear localized ERalpha protein, is more restricted in its localization and is present in many, but not all, of the small-sized (<600 microm(2)) DRG neurons, but is only rarely present in larger neurons. The L6-S1 DRG levels, which contain sensory neurons that innervate reproductive tissues, are relatively enriched in ERalpha compared to L3-L5 DRG levels, which contain sensory neurons that innervate hind limb regions. Long-term estrogen treatment of ovariectomized rats (21-28 days) dramatically reduces immunocytochemically detectable ERalpha protein in the DRG relative to that in ovariectomized controls. RT-PCR studies also showed that long-term estrogen treatment of ovariectomized rats downregulates the levels of ERalpha mRNA, but upregulates the levels of ERbeta mRNA in the DRG. Interestingly, in intact cycling female rats, ERalpha and ERbeta mRNA levels in the DRG were both higher during proestrus compared to metestrus. These findings suggest that the changes in expression of estrogen receptors which occur dynamically during the estrus cycle differ from those induced by long-term estrogen treatment of ovariectomized animals.

Estrogen and NGF synergistically protect terminally differentiated, ERalpha-transfected PC12 cells from apoptosis.

The potential cytoprotective effects of the gonadal steroid estrogen, acting via its receptor (ER) on target neurons, are of considerable interest in aging, disease, and trauma. In this study, we examined the effects of estrogen on a prototypical neuronal-like cell, namely, nerve growth factor (NGF) differentiated PC12 cells when these cells are placed into an apoptosis-inducing environment. A clonal line of PC12 cells stably transfected to express full-length rat ERalpha mRNA and protein (PCER cells), as well as control cells (vector DNA transfected: PCCON cells), were differentiated into neurite bearing cells by 14 days of NGF treatment. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry for ERalpha revealed that terminally differentiated PCER cells continue to show robust ER mRNA and protein expression after differentiation. PCCON cells do not express ERalpha either before or after NGF-induced differentiation. After differentiation, serum and NGF withdrawal from wild type PC12 cells is well known to induce cell death via apoptotic mechanisms. We challenged differentiated PCER and PCCON cells with serum-free media and assessed the effects of various treatments on cell survival and apoptosis using trypan blue staining and quantitative TUNEL staining. When differentiated PCER cells were treated with 17beta-estradiol (E2) plus NGF during a 2-day serum-free media challenge, cell survival was significantly greater, and the apoptotic index was significantly lower, than when PCER cells were treated with NGF only during the challenge. No additional improvement in cell survival, and no further reduction in apoptosis, was observed in non-ER expressing cells (PCCON) by E2 plus NGF treatment over that obtained by NGF alone. The effects of E2 on the expression of several apoptosis-related genes in this system were also examined. We found that E2 treatment of differentiated PCER cells in serum-free media increased the levels of Bcl-XL mRNA and reduced the levels of BAD mRNA relative to those in vehicle-treated PCER cells, and also relative to those in PCCON cells. Thus, estrogen's protective effects on PCER cells appear to, at least partly, involve a modulation of apoptosis-regulating genes.

Stable transfection of PC12 cells with estrogen receptor (ERalpha): protective effects of estrogen on cell survival after serum deprivation.

Potential protective effects of the gonadal steroid estrogen on neurons are of particular interest in aging, neurodegenerative disease, and other traumatic conditions. In this study, we examined the hypothesis that estrogen, acting through the estrogen receptor (ERalpha), can enhance neuronal cell survival in the face of serious apoptotic challenge. PC12 cells were transfected with full-length rat ERalpha cDNA and a number of stable transfectants that expressed ER mRNA and protein (PCER cells) at levels comparable to those present in uterus or the MCF7 breast cancer cell line were obtained. A control line of cells transfected with vector DNA alone (PCCON cells) was used for comparisons. The apoptotic challenge used in the experiments was serum-free media, as it is well established that undifferentiated PC12 cells rapidly undergo cell death via apoptosis under those conditions. Estrogen treatment of PCER cells markedly increased the viability of these cells relative to PCCON cells in serum-free media, as assessed by trypan blue staining and TUNEL staining. We also examined the mitotic effects of estrogen treatment. While estrogen significantly stimulated bromodeoxy uridine (BrdU) incorporation into PCER cells in low-serum, but otherwise steroid-free media, no BrdU incorporation occurred in serum-free media. Mitotic effects of estrogen in low-serum steroid-free media were completely abolished by treatment with the estrogen receptor antagonist ICI 182,780. From this we conclude that the effects of estrogen on PCER cells in serum-free media can be attributed to increased cell survival, rather than proliferation.

Alterations in glial fibrillary acidic protein (GFAP) mRNA levels in the hamster facial motor nucleus: effects of axotomy and testosterone.

Testosterone propionate (TP) administered at the time of facial nerve injury in the hamster accelerates the rate of regeneration. In this study, we tested the hypothesis that the mechanism by which TP augments peripheral nerve regeneration involves regulation of glial fibrillary acidic protein (GFAP) mRNA in the facial motor nucleus. Castrated male hamsters were subjected to right facial nerve transection, with half the animals implanted subcutaneously with Silastic capsules containing exogenous TP and the remainder sham implanted. Postoperative survival times were 0.25, 1, 2, 4, 7, and 14 d. Qualitative/quantitative analyses of both film and emulsion autoradiograms were accomplished. Axotomy, with or without TP, resulted in a dramatic increase in GFAP mRNA levels by 1 d post-operative on the axotomized side, relative to controls. GFAP mRNA levels remained elevated throughout all postoperative times in both the nonhormone- and TP-treated animals. Qualitative examination of the film autoradiograms indicated a generalized decrease in the amount of GFAP mRNA in the control and axotomized nuclei of TP-treated animals when compared to the control and axotomized nuclei, respectively, of nonhormone-treated animals. Statistical comparison of the values obtained for both the film and emulsion autoradiograms confirmed this impression. Thus, while the injury-induced increases in GFAP mRNA expression were not blocked by TP, the overall extent of the increase was significantly tempered by steroid treatment. These data suggest that hormonal modulation of the astrocytic response to peripheral nerve injury may be a contributing factor in the ability of steroids to enhance the regenerative capacities of injured motor neurons.

Gonadal steroid regulation of growth-associated protein GAP-43 mRNA expression in axotomized hamster facial motor neurons.

Treatment with testosterone propionate (TP) after nerve injury is known to accelerate both the rate of axonal regeneration and functional recovery from facial paralysis in the adult male hamster. Peripheral nerve injury is also known to increase the expression of a 43 kilodalton growth-associated protein (GAP-43). In the intact brain, GAP-43 expression is affected by gonadal steroids. We thus postulated that steroidal modulation of GAP-43 gene expression may be a component of the neurotrophic action of TP in regenerating neurons. This issue was examined in hamster facial motor neurons (FMN) which contain androgen receptors and which have been shown to respond to exogenous steroids in a number of previous studies. Castrated adult male hamsters were subjected to right facial nerve transection and treated with either TP via subcutaneous hormone capsule implants, or left untreated (no hormone replacement). At post-injury/treatment times of 0.25, 2, 4, 7, and 14 d, the brain stem regions were harvested, cryostat sections were collected through the facial motor nucleus, and in situ hybridization was done using a 33P-labeled GAP-43 cDNA probe. Quantitative analysis of the autoradiograms by computer assisted grain counting revealed that axotomy produced a dramatic increase in GAP-43 mRNA levels in FMN by 2 d post-axotomy and that this increase remained through 14 d post-injury in both the TP-treated and the untreated group. In the nonhormone-treated group, there was a statistically significant dip in GAP-43 mRNA levels in FMN at 7 d post-operative, relative to 4 d post-operative levels. TP-treatment prevented this transient decline in GAP-43 mRNA levels in axotomized FMN.

Distribution of growth-associated class I alpha-tubulin and class II beta-tubulin mRNAs in adult rat brain.

A comprehensive survey of class I alpha-tubulin (alpha 1) and class II beta-tubulin (beta II) mRNAs was performed using in situ hybridization in order to determine the extent of continued expression of these immature tubulin isotype mRNAs in the adult rat brain. Qualitatively similar distributions of the two isotype mRNAs were observed, with marked variations in hybridization intensity of both probes apparent across different brain regions. Neurons in a wide variety of structures throughout the brain exhibited intense hybridization signals. While the presence of large numbers of neurons with a moderate hybridization intensity could account for the relatively high level of total binding in some regions such as the cerebellar and dentate granule layers, in most cases higher regional mRNA levels reflected greater hybridization intensity per neuron. Little variability in hybridization intensity was typically seen between individual cells within specific nuclei throughout the brain. The presence of occasional intensely labeled neurons scattered throughout the basal ganglia provided the most striking exception to this pattern. While no qualitative differences between the distributions of alpha 1-tubulin and beta II-tubulin mRNAs were observed, consistent differences in the relative intensity of hybridization for alpha 1-tubulin versus beta II-tubulin mRNA were apparent in a few brain regions. Expression by glia did not appear to contribute significantly to detectable levels of either alpha 1-tubulin or beta II-tubulin mRNA. These findings suggest that continued expression of growth-associated tubulin isotype mRNAs may have functional significance in specific neuronal populations of the adult brain. Partial overlap between the distributions of alpha 1- and beta II-tubulin mRNAs and that of GAP-43 mRNA is discussed, as are potential roles for growth-associated tubulin gene expression in supporting cytoskeletal turnover, reactive axonal growth, and dendritic remodeling in the adult brain.

Coordinated upregulation of alpha 1- and beta II-tubulin mRNAs during collateral axonal sprouting of central peptidergic neurons.

An in situ hybridization study was performed to determine the relationship between levels of mRNAs for the axonal growth-associated alpha 1-tubulin and beta II-tubulin isotypes and the process of collateral axonal sprouting by identified central nervous system (CNS) neurons. A unilateral hypothalamic knife-cut was used to hemisect the hypothalamoneurohypophysial tract, which results in a robust collateral sprouting response by the uninjured neurons of the contralateral supraoptic nucleus (SON) (Watt and Paden: Exp Neurol 111:9-24, 1991). At 10 and 30-35 days after the lesion, cryosections of the SON were obtained and hybridized with 35S-labeled cDNA probes specific to alpha 1- and beta II-tubulin mRNAs. Quantitative evaluation of the resulting autoradiographs revealed that alpha 1-tubulin mRNA levels were significantly increased by 10 days in SON neurons that were undergoing collateral sprouting compared to controls and that this increase was sustained at 30-35 days post-lesion. Less marked increases in hybridization intensity of the beta II-tubulin probe were also apparent in sprouting neurons at both 10 and 30-35 days after the lesion, but were statistically significant only at 10 days. The measured increases in intensity of hybridization of alpha 1- and beta II-tubulin probes are likely to be conservative estimates of the underlying increase in alpha 1- and beta II-tubulin mRNA levels because sprouting SON neurons undergo significant hypertrophy. High levels of both alpha 1- and beta II-tubulin mRNAs were also seen in surviving axotomized SON neurons ipsilateral to the hypothalamic lesion. We conclude that the pattern of regulation of alpha 1- and beta II-tubulin mRNAs in CNS neurons which are capable of supporting new axonal growth includes three elements: maintenance of significant basal alpha 1- and beta II-tubulin mRNA pools in mature neurons, rapid increases in the pool size of the mRNAs following stimulation of collateral sprouting, and sustained elevation of mRNA levels during the period of axonal sprouting.

Transcriptional and post-transcriptional mechanisms regulating neurofilament and tubulin gene expression during normal development of the rat brain.

The transcription of the beta II-, beta IV- and alpha 1-tubulin genes as well as that of the three neurofilament genes, NF-L, NF-M and NF-H, was examined during the course of postnatal brain development. Changes in the transcriptional activity of these genes were studied using run-off transcription assays with nuclei isolated from the rat cerebral cortex at postnatal days P2, P5, P10 and adult stages. Northern blotting of total RNA isolated from the cerebral cortex was used to compare changes in steady-state mRNA levels with transcriptional changes that occurred in the cerebral cortex during the postnatal interval. Nuclear run-off assays showed that beta II- and alpha 1-tubulin gene transcription rates were maximal from P2-P5 and declined at later times. Changes in the steady-state mRNA levels for these two genes followed the same general pattern as transcription, but in the case of beta II-tubulin mRNA, were more dramatic. beta IV-tubulin gene transcription dropped between P2 and P5 and then increased progressively to the adult stage, coordinate with an increase in beta IV-tubulin steady-state mRNA levels. NF-L and NF-H genes showed similar patterns of transcriptional change during the postnatal interval, with maximal rates of transcription at P5 followed by a decline at later times. The steady-state levels of NF-L and NF-H mRNAs changed in a manner opposite to that of transcription and increased progressively during the postnatal interval. This suggests that mRNA stabilization is the main factor regulating the steady-state levels of NF-L and NF-H mRNAs in postnatal brain. For the NF-M gene, the developmental transcription pattern was also dissociated from steady-state mRNA level changes, but differed from the transcription patterns of the NF-L and NF-H genes. This suggests the importance of post-transcriptional mechanisms in regulating NF-M mRNA levels in brain and also indicates that some differences exist in the regulatory mechanisms which control NF-M compared to NF-L and NF-H mRNA levels.

Sensory neurons selectively upregulate synthesis and transport of the beta III-tubulin protein during axonal regeneration.

The effects of peripheral nerve injury on the content, synthesis, and axonal transport of the class III beta-tubulin protein in adult rat dorsal root ganglion (DRG) neurons were examined. Recent reports of selective increases in the steady-state levels of the beta III-tubulin mRNA during axonal regeneration (Moskowitz et al., 1993) led to the hypothesis that upregulated levels of expression of the beta III-tubulin isotype that alter the composition of neuronal microtubules is important for effective axonal regrowth. If this is the case, the increases in mRNA levels must be translated into increased beta III-tubulin protein levels and subsequently modify the axonal cytoskeleton via axonal transport mechanisms. The present study assessed whether or not this occurs by examining beta III-tubulin protein content in adult rat lumbar DRG neurons at different times (1-14 d) after a distal sciatic nerve crush (approximately 55 mm from the DRG) by Western blotting and immunocytochemistry with a beta III-tubulin specific monoclonal antibody. These studies showed substantial increases in beta III-tubulin content in DRG neurons, as well as in proximal regions of peripheral sensory axons (0-6 mm from the DRG), from 1-2 weeks after a distal nerve injury. Pulse labeling of DRG neurons with 35S-methionine and 35S-cysteine and immunoprecipitation of labeled beta III-tubulin indicated that the synthesis of beta III-tubulin was increased in the DRG after axotomy. Studies of axonal transport, wherein L5 DRG proteins were labeled with 35S-methionine and 35S-cysteine by microinjection, revealed that slow component b(SCb) of axonal transport conveyed more labeled tubulin moving at apparently faster rates through the intact regions of sciatic nerve axons in response to crush injury of the distal sciatic nerve. Immunoprecipitation experiments using proximal peripheral nerve segments showed that SCb in distally injured DRG neurons was enriched in the beta III-tubulin isotype. These findings demonstrate that the augmented synthesis of beta III-tubulin after axotomy alters the composition of the axonally transported cytoskeleton that moves with SCb. The increased amounts and rate of delivery of beta III-tubulin in axons of regenerating DRG neurons suggest that the altered pattern of tubulin gene expression that is initiated by axotomy impacts on the composition and organization of the axonal cytoskeleton in a manner that can facilitate axonal regrowth.

Androgenic regulation of tubulin gene expression in axotomized hamster facial motoneurons.

We have previously demonstrated that systemic administration of testosterone increases the rate of axonal regeneration following facial nerve crush in adult male hamsters. In this investigation, the molecular mechanisms by which androgens may enhance axonal regeneration were examined. Specifically, the following question was addressed using Northern blot and in situ hybridization with three cytoskeletal cDNA probes complementary to beta II-, beta III-, and alpha 1-tubulin mRNA: does exogenous testosterone augment axotomy-induced changes in tubulin mRNA expression in hamster facial motoneurons (FMN)? Adult male hamsters were subjected to unilateral facial nerve severance, with the opposite side serving as an internal control. One-half of the animals were subcutaneously implanted with Silastic capsules containing crystalline testosterone propionate and the other half implanted with blank capsules. Postoperative survival times were 2 and 7 d. At 2 d after axotomy alone, no changes in levels of any of the three tubulin mRNAs were observed in the injured FMN. However, by 7 d after axotomy, significant increases in all three tubulin mRNAs were observed. This time course of axotomy-induced changes in tubulin gene expression is consistent with findings in other injured neuronal populations. Administration of testosterone at the time of injury had two major effects on the cytoskeletal response pattern in axotomized FMN. First, testosterone differentially regulated the set of tubulin mRNAs examined, in that beta II-tubulin mRNA levels were selectively altered by the steroid, whereas beta III- or alpha 1-tubulin mRNAs were not.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects of axotomy on beta-tubulin and NF gene expression in rat DRG neurons.

To compare the long-term recovery of gene expression in dorsal root ganglion (DRG) neurons under conditions of regeneration vs. non-regeneration, Northern blotting and in situ hybridization were used to assess steady-state neurofilament (NF) and beta tubulin mRNA levels 12 weeks following axonal injury. Adult male rats sustained either a crush lesion of the mid-sciatic nerve (regeneration occurs), or a cut lesion of the sciatic nerve combined with ligation of the proximal nerve stump and removal of a large segment of the distal nerve (regeneration does not occur). In the latter case, neuroma formation physically prevented axonal regeneration. Results of Northern blotting of total RNA obtained from the DRG indicated that NF-L and NF-M mRNA levels had largely returned to control levels at 12 weeks following crush axotomy but were still substantially depressed following cut/ligation injury of the sciatic nerve at that time. In situ hybridization studies indicated that both crush and cut/ligation axotomy resulted in significantly lower NF-L mRNA levels in large-sized (> 1000 micron2) DRG neurons at 12 weeks post-axotomy. Discrepancies in the conclusions from Northern blotting and in situ hybridization experiments were also noted in the case of tubulin mRNA changes at long intervals after axotomy. In situ hybridization data derived from the large-sized DRG neurons using a coding region beta-tubulin cDNA (which recognizes both beta II and beta III mRNAs) showed complete recovery of beta-tubulin mRNA levels in surviving large-sized DRG neurons after crush axotomy, but significantly elevated tubulin mRNA levels in surviving large DRG cells at 12 weeks after cut/ligation axotomy. In contrast, Northern blotting results indicated that beta II-tubulin mRNA levels in the crush axotomy condition remained elevated relative to control while they were substantially lower than control in cut/ligation axotomy samples. Results from analysis of beta III-tubulin mRNA changes were not conclusive. The lack of complete correspondence in the results from the two different methods of analysis of mRNA changes (blotting vs. in situ) is likely to be due to selective loss of large-sized DRG neurons in the long-standing cut/ligation injury condition. This would influence results from blotting data, where RNA is derived from the DRG as a whole, more so than in situ hybridization experiments which specifically focus on the surviving large-sized neurons. Overall, data from these experiments indicate that altered patterns of gene expression remain in the DRG for long intervals after axonal injury, whether or not axonal regeneration has been successful.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of changes in beta-tubulin and NF gene expression in rat DRG neurons under regeneration-permissive and regeneration-prohibitive conditions.

To examine the question of whether or not prevention of axonal regrowth after injury affects the molecular responses of neurons to axotomy, Northern blotting and in situ hybridization were used to study changes in the mRNA levels of neurofilament (NF) proteins and tubulins in rat dorsal root ganglion (DRG) cells. Adult male rats sustained either a crush lesion of the mid-sciatic nerve (regeneration-permissive condition) or a cut lesion of the sciatic nerve combined with ligation of the proximal nerve stump and removal of a large segment of the distal nerve (regeneration-prohibitive condition). At 14 days post-injury, the relative levels of the low (NF-L) and middle (NF-M) molecular weight NF protein mRNAs, as well as those of beta II- and beta III-tubulin, were examined in the L4 and L5 DRG. The data showed that the levels of NF-L and NF-M mRNAs decreased while beta II- and beta III-tubulin mRNA levels increased in the DRG after either crush axotomy or cut/ligation axotomy of the sciatic nerve, suggesting that the elicitation of these molecular changes by axon disconnection is independent of the ultimate success or failure of the axonal regrowth process. However, cut/ligation axotomy had a more pronounced effect than did crush injury on the mRNA changes. This result suggests that feedback mechanisms from regrowing axons are important in regulating the extent of the cytoskeletal mRNA changes in injured neurons.

Coordinate regulation of tubulin and microtubule associated protein genes during development of hamster brain.

The present study documents the patterns of mRNA expression for 5 different tubulin genes and 4 of the structural microtubule-associated protein (MAP) genes during normal development of hamster forebrain. Northern blotting in conjunction with densitometric analysis was used to study changes in the levels of the mRNAs for alpha 1-tubulin, classes beta I-, beta II-, beta III- and beta IV-tubulin, as well as the mRNAs for tau, MAP1A, MAP1B and MAP2, using total RNA isolated from hamster forebrain at various embryonic (E) and postnatal (P) stages. Densitometric analyses of the autoradiograms from the Northern blots revealed that each of the tubulin genes exhibited distinct developmental patterns of expression, several of which appeared to be temporally correlated with the expression of specific MAP mRNAs. The beta I-, beta II- and beta III-tubulin mRNAs increased rapidly between late embryonic stages to birth, reached peak levels early in the first postnatal week, and declined thereafter. alpha 1-Tubulin mRNA was easily detected during embryonic stages, rose to peak levels at P7-P9 and then gradually declined. A similar pattern was seen for tau mRNA. After the first postnatal week, the size of the tau mRNA also shifted to a slightly larger size, presumably due to differential splicing. beta IV-Tubulin mRNA levels did not become significant until very late in postnatal development (3-4 weeks). MAP2 mRNA expression was unusual in that peak levels were reached at two different stages of development--an initial peak occurred in the first postnatal week, followed by a decline, and then a second rise occurred during the third and fourth postnatal weeks. The expression of the beta IV-tubulin mRNA coincided temporally with the second peak in MAP2 mRNA expression. MAP1B mRNA abruptly reached high levels at birth, remained abundant during the first two postnatal weeks, and then decreased. In contrast, MAP1A mRNA levels were low in the initial postnatal interval and increased only at very late developmental stages. The findings of a temporal correspondence in expression of high levels of tau and MAP1B with beta I-, beta II-, beta III- and alpha 1-tubulin mRNAs suggest that this profile of gene expression is one that endows greater plasticity to the neuronal cytoskeleton. Conversely, the transition to increased expression of beta IV-tubulin, MAP1A, and a larger tau mRNA species defines a portion of the molecular pattern that underpins the increased stability of neuronal form during maturation.

Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury.

We have examined the injury response of astrocytes in the immature hamster brain in this study, focusing on alterations in the expression of glial fibrillary acidic protein (GFAP) and vimentin. In the adult CNS these two type III intermediate filament (IF) proteins have been shown to undergo robust increases in expression in response to axonal injury. Since injury to the immature CNS reportedly elicits less glial scar formation than adult brain injury, we examined the possibility that immature astrocytes respond differently than adult astrocytes to CNS injury with respect to IF gene expression. In situ hybridization using a 35S-labeled cDNA GFAP probe was done on brainstem sections obtained 2, 7 and 14 days after unilateral transection of the corticospinal tract in P8 hamster pups. The results indicated that substantial increases in GFAP mRNA were associated with the degenerating portion of the corticospinal tract by 2 days after axotomy and that the levels remained elevated for at least 14 days. Double-label immunofluorescence studies of this material suggested that GFAP as well as vimentin protein levels were also increased in many astrocytes in and around the degenerating corticospinal tract 2-14 days after axotomy. Most of the reactive astrocytes in the degenerating regions exhibited increases in GFAP and vimentin immunostaining but some vimentin-negative GFAP-positive reactive astrocytes were also observed, particularly in regions surrounding the actual degenerative zones.(ABSTRACT TRUNCATED AT 250 WORDS)

Immature corticospinal neurons respond to axotomy with changes in tubulin gene expression.

We have examined the expression of two different tubulin mRNAs in hamster corticospinal neurons that were axotomized at three different developmental stages; postnatal day 8 (P8), P20, and adult. In situ hybridization of histological sections of the sensorimotor cortex was done with 35S-labeled cDNA probes specific to alpha 1-tubulin and beta III-tubulin mRNAs at 2-14 days following unilateral transection of the corticospinal tract in the caudal medulla. Both film and emulsion autoradiography were used to detect changes in tubulin mRNA levels. Qualitative assessment indicated substantial decreases in both alpha 1-tubulin and beta III-tubulin mRNA levels in layer V neurons of the sensorimotor cortex following axotomy. The changes were apparent as early as 2 days postinjury for P20 and adult operates, but not for P8 operates. However, by 14 days postinjury, decreases in alpha 1-tubulin and beta III-tubulin gene expression were apparent in animals operated at all three developmental stages. These findings indicate that both immature and adult corticospinal neurons respond to axonal injury in a manner that is distinctly different from the peripheral neuron response.

Expression of the class III beta-tubulin gene during axonal regeneration of rat dorsal root ganglion neurons.

The effect of peripheral axotomy on the expression of the class III beta-tubulin gene in adult dorsal root ganglion (DRG) neurons was examined. Of the 5 isotypic classes of beta-tubulin expressed in the mammalian nervous system, only the class III beta-tubulin is neuron specific. While information about the expression of several of the tubulin genes during neuronal development and regeneration has become available recently, very little is known about the expression of beta III-tubulin during axonal regeneration. To explore this issue, we examined axotomy-induced changes in beta III-tubulin mRNA levels in adult rat lumbar dorsal root ganglion (DRG) neurons at different times (1-28 days) after unilateral sciatic nerve crush using northern blotting of total RNA and quantitative in situ hybridization. These studies showed an initial decrease in beta III-tubulin mRNA levels in axotomized DRG neurons as compared to contralateral controls at 1 day after injury followed by robust increases in beta III-tubulin mRNA levels relative to contralateral controls from 1 to 4 weeks after injury. We postulate that beta III-tubulin may play an essential role in axonal growth because of its unique neuron-specific pattern of expression and its substantial increase in neurons that have been stimulated to regrow their axons.

Differential regulation of beta III and other tubulin genes during peripheral and central neuron development.

Mammalian peripheral and central neurons differ considerably in the composition and properties of their axonal cytoskeletons. Recent reports of the selective expression of a high molecular weight (HMW) tau protein in neurons with peripherally projecting axons have furthered the idea that the microtubules in central and peripheral neurons are disparate. In the present study, we examined the possibility that the various tubulin genes are differentially expressed in central versus peripheral neurons. To examine this, we compared the expression of four of the beta-tubulin mRNAs (classes beta I, beta II, beta III, beta IV) and the alpha 1-tubulin mRNA in rat dorsal root ganglion (DRG) neurons with their expression in cerebral cortex during postnatal development (P5-90), using northern blots and in situ hybridization. We document both similarities and differences in tubulin gene expression in these two regions of the neuraxis during postnatal development. In both DRG and cortex, the expression of the class beta I- and beta II-tubulin mRNAs and the alpha 1-tubulin mRNA was higher at earlier stages of postnatal development than in the adult. However, class beta IV-tubulin mRNA levels increased during cortical development but decreased during DRG postnatal development. The opposite pattern was found for the neuron-specific class beta III-tubulin gene, the mRNA levels of which were high in cortex, at birth and then decreased with increasing postnatal development. In DRG, the beta III-tubulin mRNA levels generally increased during postnatal development. Beta III-tubulin protein levels were examined qualitatively at different developmental stages (5-90 days) by immunoblotting and immunocytochemistry.(ABSTRACT TRUNCATED AT 250 WORDS)