Androgen receptor mRNA regulation in adult male and female hamster facial motoneurons: effects of axotomy and exogenous androgens.

Testosterone propionate (TP) administration at the time of facial nerve injury in the adult hamster augments the regenerative properties of the injured facial motoneurons (FMN), with the androgen receptor (AR) playing a key role in mediating the actions of TP on facial nerve regeneration. The purpose of the present study was to determine the effects of axotomy on AR mRNA expression in FMN. This was accomplished using in situ hybridization in conjunction with a (35)S-labeled AR riboprobe. Gonadally intact adult male and gonadectomized (gdx) adult female hamsters were subjected to a right facial nerve axotomy, with the left side serving as internal, unoperated control. Half the animals were subcutaneously implanted with a 10-mm TP Silastic capsule, and the other half were sham-implanted. An additional group of nonaxotomized, gonadally intact males was also included. Postaxotomy survival times were 1, 4, and 7 days. At 1 postoperative day 1, there were no effects of axotomy on AR mRNA levels. By postoperative days 4 and 7, axotomy caused a significant decrease in AR mRNA levels in FMN of gonadally intact males, relative to either the contralateral control FMN of the same animals or FMN from the group of gonadally intact males that were not subjected to facial nerve axotomy. There were no significant differences between AR mRNA levels in contralateral control FMN and FMN from the gonadally intact group of nonaxotomized males. TP administration at the time of axotomy had no effect on AR mRNA levels in either the axotomized or contrala(teral control FMN of gonadally intact males, relative to the nonaxotomized, gonadally intact male group. Corroborating our previous work, AR mRNA levels were reduced in the contralateral control FMN of gdx females, relative to the nonaxotomized, gonadally intact male group, with axotomy having no additional effects. The data are discussed in a mechanistic framework suggesting how TP acts to augment facial nerve regeneration.

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.

Ribosomal RNA transcriptional activation and processing in hamster facial motoneurons: effects of axotomy with or without exposure to testosterone.

A key step in the ability of neurons to survive injury and successfully regenerate involves ribosomal RNA production. Testosterone propionate (TP), augments facial nerve regeneration in the adult hamster. TP modulates the nucleolar reaction in injured facial motoneurons, such that mature ribosome levels increase more rapidly and in greater magnitude than with injury only. In this study, molecular and electron microscopic stereologic approaches were used to determine the effects of axotomy and steroid treatment on ribosomal transcription and processing in facial motoneurons. Castrated adult male hamsters were subjected to right facial nerve transection at the stylomastoid foramen. Half the animals were subcutaneously implanted with one Silastic TP capsule, with the remainder sham implanted. For the in situ hybridization experiments, postoperative survival times were 0.5, 2, or 6 hours. In situ hybridization with a ribosomal DNA probe specific to the external transcribed spacer region located at the 5' end of the ribosomal gene was accomplished. Transcriptional activation of the rRNA gene occurred rapidly, within 2 hours, after injury only. Unexpectedly, TP treatment did not alter the time course or magnitude of rRNA transcriptional activity. For the electron microscope experiments, the postoperative time of 12 hours was selected. Stereologic analysis of 3 nucleolar subcomponents, fibrillar centers (site of rRNA transcription), nucleolonema (site of rRNA processing), and granular material (site of preribosome storage), was accomplished. TP decreased the nucleolonemal strands and the granular material, relative to injury only. These results suggest that, although rRNA transcription is rapidly activated by axotomy, rRNA processing is temporarily stalled. TP does not affect the early, axotomy-induced transcriptional activation of the ribosomal gene, but may, instead, prevent the subsequent disruption in rRNA processing. An hypothesis for the molecular mechanism by which steroids augment the regenerative capabilities of injured facial motoneurons is presented.

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.

Effects of axotomy and testosterone on androgen receptor mRNA expression in hamster facial motoneurons.

We have previously demonstrated that testosterone propionate (TP) treatment accelerates the rate of regeneration following facial nerve crush axotomy in adult male hamsters. These effects are mediated by androgen receptor (AR) activation and are blocked by pretreatment with the AR antagonist, flutamide. In addition to its beneficial effects on regeneration, TP regulates AR mRNA levels in facial motor neurons (FMN). Gonadectomized (gdx) male hamsters have been shown to have approximately 50% of the AR mRNA levels found in gonadally intact males. Administration of TP to gdx males results in an upregulation in AR mRNA levels after 1 day of treatment. Recent reports in the literature suggest that axotomy also may regulate the expression of AR in motor neurons. In this study, we examined the effects of axotomy and exogenous steroid treatment on the regulation of AR mRNA in hamster FMN. Five days after castration, adult male hamsters were subjected to a right facial nerve axotomy. Half the animals received one 10-mm Silastic capsule filled with 100% crystalline TP, and the remainder were sham implanted. Postoperative survival times were 6 h or 1, 2, 4, 7, or 14 days. In situ hybridization in conjunction with an AR riboprobe and computerized image analysis were used to quantify AR mRNA levels. The contralateral FMN served as internal controls for these experiments, and FMN of gonadally intact males served as additional nonaxotomized controls. As predicted, AR mRNA levels were upregulated in contralateral control FMN after TP treatment. However, this TP-induced upregulation of AR mRNA levels did not occur in the axotomized FMN. These results indicate that axonal injury can disrupt the normal regulatory pattern of AR mRNA expression by exogenous steroids in motoneurons. We conclude that the potentiation of regenerative events by TP does not require augmented synthesis of AR, but, instead, enhanced stabilization of existing receptors.

Regulation of androgen receptor mRNA expression in hamster facial motoneurons: differential effects of non-aromatizable and aromatizable androgens.

We have previously shown inherent sex differences in the levels of androgen receptor mRNA (AR mRNA) in hamster facial motor neurons (FMN). FMN of intact females contained approximately 50% less AR mRNA than their male counterparts. Gonadectomy in males down-regulated AR mRNA levels in FMN by approximately 50%, whereas no effects of gonadectomy were observed in females. Sex differences in the regulation of AR mRNA levels by exogenous testosterone propionate (TP) were also observed. In those studies, AR mRNA levels were up-regulated after 1 day of treatment with exogenous TP in FMN of gonadectomized (GDX) males and after 7 days in FMN of intact females, with no effects in GDX females. Since TP is aromatizable to estrogen, and given recent findings of transient expression of estrogen receptors (ER) in rodent FMN, the effects of dihydrotestosterone (DHT), a non-aromatizable form of the steroid, on AR mRNA expression in hamster FMN were examined in the present study. If testosterone (TES) were the active hormone regulating AR mRNA levels in FMN, DHT treatment should render a similar regulatory pattern as TP, but if metabolism of TES to estradiol plays a role in AR mRNA regulation, effects of the two treatments should differ. In situ hybridization and computerized image analysis were used to quantify the regulation of AR mRNA by DHT in individual FMN of hamsters of both sexes. Exogenous DHT was administered to intact and gonadectomized (GDX) male and female hamsters by implantation of one 10-mm Silastic capsule for 1, 2 or 7 days. AR mRNA levels were significantly up-regulated in intact females at all time points of DHT exposure, with no effects in GDX groups. These results differ from previous work using TP, in which a modest up-regulation in AR mRNA levels was observed in FMN of intact females only after 7 days. As with TP, DHT exposure gradually down-regulated AR mRNA levels in FMN of intact males. Thus, DHT only regulated AR mRNA levels in intact animals, with endogenous sources of estrogen available, but not in GDX animals, with endogenous estrogens reduced by gonadectomy. Taken together, these results substantiate our previous findings of sex differences in AR mRNA levels/regulation and suggest a synergism between estrogen and androgen in the regulation of AR mRNA levels in peripheral motor neurons.

Adrenergic agents inhibit rapid increases in cerebellar Purkinje cell glutamic acid decarboxylase (GAD67) mRNA levels after climbing fiber lesions or reserpine treatment.

Loss of the inferior olive-climbing fiber input to the cerebellar cortex after treatment with the neurotoxin 3-acetylpyridine (3-AP) has been reported to double the simple spike activity of the cerebellar Purkinje cell and eliminates complex spike activity. This is quickly followed by a three- to fourfold increase in Purkinje cell mRNA for the 67 kDa form of glutamic acid decarboxylase (GAD), a synthetic enzyme for the neurotransmitter GABA. Treatment with the indirectly acting sympathomimetic amphetamine or the direct acting beta 2 adrenergic agonist clenbuterol inhibited the increase in GAD67 mRNA, and this inhibition was blocked by pretreatment with the beta receptor antagonist propranolol. The activity-enhancing effect of 3-AP treatment on cerebellar neurons was confirmed by extracellular recordings. Clenbuterol treatment prevented the increase in neuronal firing without altering lesion induction or the loss of complex spikes, and propranolol treatment produced a partial reversal of the inhibitory effect of clenbuterol on the neuronal firing rate. These results suggest that beta receptor-mediated effects on cerebellar neuronal activity may prevent the increase in mRNA levels, but that firing rate-independent beta-mediated effects on genomic expression may also play a role. A role for noradrenergic systems in modulating GAD67 mRNA is also supported by the finding that reducing endogenous cerebellar norepinephrine levels by treatment with reserpine increased Purkinje cell GAD67 mRNA levels (250% of control), and this also was inhibited by clenbuterol treatment.

Sex differences in androgen receptor mRNA levels and regulation in hamster facial motoneurons.

We have previously shown that testosterone propionate augments hamster facial nerve regeneration to a greater extent in males than females. Further, sex differences in facial nerve regeneration have been observed. From those studies, we hypothesized that sex differences in nerve regeneration could be due to inherent differences in androgen receptor (AR) mRNA content within facial motor neurons (FMN) of male and female hamsters. In the present study, that hypothesis was tested using in situ hybridization, and computerized image analysis to quantify levels and regulation of AR mRNA in individual FMN of hamsters of both sexes. Intact and gonadectomized (gdx) male and female hamsters were used in the initial experiments in the study. In subsequent experiments, exogenous testosterone propionate (TP) was administered to the aforementioned groups of animals by subcutaneous implantation of one 10-mm Silastic capsule for 1, 2 or 7 days. FMN of intact females contained approximately 50% less AR mRNA than their male counterparts. Gonadectomy in males downregulated AR mRNA levels by approximately 50%, whereas no effects of gonadectomy were observed in females. Thus, in all paradigms where TP levels were low relative to the intact males, AR mRNA levels were approximately half of those in the intact male FMN. TP administration induced AR mRNA levels in gdx males within 1 day. Significant effects of TP were not detected in gdx females, and only after 7 days in the intact females. To our knowledge, the results of this study are the first quantitative demonstration of sex differences in steroid receptor mRNA content in a given neuronal population and substantiate the idea that sex differences in the effects of androgens on peripheral nerve regeneration are based on intrinsic sex differences in the levels and regulation of receptor mRNA in motor neurons.

Rapid increases in cerebellar Purkinje cell glutamic acid decarboxylase (GAD67) mRNA after lesion-induced increases in cell firing.

Loss of the inferior olive-climbing fiber input to the cerebellar cortex doubles the simple spike activity of the cerebellar Purkinje cell. There is a 3- to 4-fold increase in Purkinje cell messenger RNA for the 67 kDa form of glutamic acid decarboxylase (a synthetic enzyme for the neurotransmitter GABA) within 4-5 h of the increase in electrical activity, suggesting a rapid response of mechanisms influencing neurotransmitter synthesis or stability to altered electrophysiological activity.

Interaction of apolipoprotein A-I with dimyristoylphosphatidylcholine particles of various sizes.

Dimyristoylphosphatidylcholine (DMPC) particles of different sizes were prepared by a short sonication of the lipid followed by fractionation on a Sepharose CL-4B column. Column fractions were pooled to give DMPC multilamellar liposomes, and vesicles ranging in average diameters from 300 to 220 A. These particles were used to prepare complexes with bovine apolipoprotein A-I (apo A-I). Reaction mixtures covering molar ratios from 2000:1 to 100:1 DMPC/apo A-I were equilibrated at 25 degrees C for over 15 h and were fractionated by gel filtration in order to separate vesicular and micellar protein.lipid complexes. The results indicate that under identical reaction conditions, larger particles with a smaller total surface area per mol of lipid give proportionately more micellar complexes. In fact, the multilamellar liposomes give only micellar complexes at all the initial molar ratios. For the smaller DMPC vesicles, the vesicular complexes are saturated when 6 or 7 apo A-I molecules are bound per particle, which corresponds to 2,83 X 10(4) A2 of vesicle surface per apo A-I and an approximate 16% coverage of the surface by apo A-I.

Kinetics and mechanism of apolipoprotein A-I interaction with L-alpha-dimyristoylphosphatidylcholine vesicles.

The dynamics of human apolipoprotein A-I (apo A-I) interaction with dimyristoylphosphatidylcholine (DMPC) vesicles were investigated in a 4000:1 DMPC/apo A-I (mol/mol) mixture where all the protein is bound to DMPC in stable vesicular complexes, and in a 100:1 DMPC/apo A-I (mol/mol) mixture which gives micellar complexes at equilibrium. Gel filtration and fluorescence methods (polarization and intensity) were used to follow the reaction kinetics. The binding of apo A-I to DMPC vesicles is a very rapid process which takes only a few minutes, while the formation of micellar complexes takes several hours at 25 degrees C and involves saturated complexes of apo A-I . DMPC and free apo A-I. The rate-limiting step in micellar complex formation is the breakdown of saturated vesicle . apo A-I complexes, a process that exhibits first order kinetics with a rate constant k = 0.22 h-1 and a half-life t 1/2 = 3 h 9 min.

Incorporation of excess cholesterol by high density serum lipoproteins.

Excess cholesterol was added to human HDL3 and to bovine mammalian high density serum lipoprotein (HDL) by incubating aqueous lipoprotein solutions with solid dispersions of [4-(14)C]cholesterol on Celite. Lipoprotein cholesterol complexes were isolated by centrifugation and filtration through a Sepharose 4B column. The pure complexes were analyzed for protein and lipid content and composition and were subsequently investigated by physical methods (analytical ultracentrifugation, circular dichroism, and fluorescence spectroscopy), in order to detect any structural changes induced by added cholesterol. The rates of cholesterol uptake varied as an inverse function of the intrinsic cholesterol present in the native lipoproteins. The maximum cholesterol taken up by human HDL3 increased the free cholesterol content from 3--4% (initial) up to 22% of the total lipoprotein weight. Bovine HDL was observed to increase its free cholesterol content from 2--4% (initial) up to 11--17% of the total lipoprotein weight, before denaturation. At maximum levels of added cholesterol, both lipoproteins had increased molecular weights and sedimentation velocity coefficients corresponding to the increased mass of the particles. No major changes in the hydrodynamic properties were observed. At the molecular level, the protein components only showed a 15--20% decrease in fluorescence intensity, possibly a consequence of a modified environment of the aromatic amino acid residues. In the human HDL3, added cholesterol increased the microviscosity of the lipid domains by 1.2 P at 25 degrees C (from 3.4 to 4.6 P), but did not affect the fluidity of bovine HDL lipids (5.9 P).