With a little help from my friends: androgens tap BDNF signaling pathways to alter neural circuits.

Gonadal androgens are critical for the development and maintenance of sexually dimorphic regions of the male nervous system, which is critical for male-specific behavior and physiological functioning. In rodents, the motoneurons of the spinal nucleus of the bulbocavernosus (SNB) provide a useful example of a neural system dependent on androgen. Unless rescued by perinatal androgens, the SNB motoneurons will undergo apoptotic cell death. In adulthood, SNB motoneurons remain dependent on androgen, as castration leads to somal atrophy and dendritic retraction. In a second vertebrate model, the zebra finch, androgens are critical for the development of several brain nuclei involved in song production in males. Androgen deprivation during a critical period during postnatal development disrupts song acquisition and dimorphic size-associated nuclei. Mechanisms by which androgens exert masculinizing effects in each model system remain elusive. Recent studies suggest that brain-derived neurotrophic factor (BDNF) may play a role in androgen-dependent masculinization and maintenance of both SNB motoneurons and song nuclei of birds. This review aims to summarize studies demonstrating that BDNF signaling via its tyrosine receptor kinase (TrkB) receptor may work cooperatively with androgens to maintain somal and dendritic morphology of SNB motoneurons. We further describe studies that suggest the cellular origin of BDNF is of particular importance in androgen-dependent regulation of SNB motoneurons. We review evidence that androgens and BDNF may synergistically influence song development and plasticity in bird species. Finally, we provide hypothetical models of mechanisms that may underlie androgen- and BDNF-dependent signaling pathways.

Androgen receptor expression in the levator ani muscle of male mice.

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurones that innervates the bulbocavernosus (BC) and levator ani (LA), skeletal muscles that attach to the base of the penis. In many species, including mice, rats and hamsters, the LA and BC have been found to be highly responsive to androgen and, in rats, these muscles mediate several effects of androgen on the SNB system. However, characterising the SNB system in mice is important because of the availability of genetic models in this species. In the present study, we examined AR expression in skeletal muscles of C57/BlJ6 adult male mice using immunoblotting and immunocytochemistry, comparing the BC/LA to the androgen-unresponsive extensor digitorum longus (EDL). We found similar differences in AR expression for these muscles in the mouse as previously reported for rats. In mice, the BC/LA contains more AR protein than does the EDL. At the cellular level, the LA contains a higher percentage of AR positive myonuclei and fibroblasts than does the EDL. Finally, AR expression is enriched at the neuromuscular junction of mouse LA fibres. The increased expression of AR in the LA compared to the EDL in both muscle fibres and fibroblasts indicates that each cell type may critically mediate androgen action on the SNB system in mice.

Seasonal control of penile development of Siberian hamsters (Phodopus sungorus) by daylength and testicular hormones.

Seasonal variation in prepubertal penile growth has not previously been studied. The present study assessed the influence of daylength and androgens on penile development in the Siberian hamster (Phodopus sungorus). Adult penile masses were achieved at 18 and 8 weeks of age in hamsters maintained from birth under short (10 h light:14 h dark) versus long (14 h light:10 h dark) daylengths, respectively. Insulin-like growth factor I concentrations, previously implicated in penile growth, did not differ between hamsters maintained in short versus long daylengths. Gonadectomized juvenile males maintained in short and long daylengths and administered testosterone attained adult penile masses well in advance of untreated gonad-intact males maintained in short daylengths. Hamsters from both photoperiods, castrated as juveniles and first treated with testosterone in adulthood, also achieved adult penile masses. The photoinhibited gonad is insufficient to promote penile growth, and prepubertal gonadal secretions during short daylengths are not necessary for eventual penile development. Among young born near the end of the mating season, onset of neuroendocrine refractoriness to short daylengths at about 100 days of age and subsequent gonadal development induces growth in all reproductive tissues. Timing of puberty and increased androgen secretion controlled by daylength are the primary determinants of postnatal penile growth, which may also be affected by prenatal and early postnatal organizational actions of androgens.

Photoperiod and androgens act independently to induce spinal nucleus of the bulbocavernosus neuromuscular plasticity in the Siberian hamster, Phodopus sungorus.

In the Siberian hamster, Phodopus sungorus, short-day photoperiods induce the winter phenotype, which in males includes a decrease in the production of androgens and changes in physiology to inhibit reproduction. Motoneurones of the spinal nucleus of the bulbocavernosus (SNB) and their target muscles, the bulbocavernosus and the levator ani, a neuromuscular system involved in male copulation, also display seasonal plasticity in P. sungorus. It is not known whether the plasticity seen in the SNB system of gonadally intact hamsters is due to the effects of photoperiod per se, or to the photoperiod-induced changes in androgen production. To answer this question, we castrated adult male hamsters from long days and then implanted them with capsules containing either testosterone or blanks. Half of the hamsters from each hormone condition were moved into short photoperiod (8 : 16 h light/dark cycle) while the rest were maintained under long-day conditions (15 : 9 h light/dark cycle). After 15 weeks, many measures of the SNB system, such as somata size and weight of target muscles, responded only to androgen, not to photoperiod. However, there were effects of photoperiod on the neuromuscular junctions (NMJs) that were independent of androgen status. For example, the number of synaptic zones per NMJ and the area of the NMJs were significantly increased by short days and/or testosterone treatment. The two factors exerted an additive, rather than an interactive, effect on these measures. Another striated muscle, the extensor digitorum longus, which is present in both sexes and plays no specialized role in reproduction, displayed neither an effect of androgen nor of photoperiod on fibre size or NMJ structure. These results suggest that, in addition to androgenic effects on SNB plasticity, there is also an androgen-independent effect of photoperiod on the SNB neuromuscular system.

Androgens regulate the mammalian homologues of invertebrate sex determination genes tra-2 and fox-1.

Androgens, like other steroid hormones, exert profound effects on cell growth and survival by modulating the expression of target genes. In vertebrates, androgens play a critical role downstream of the testis determination pathway, influencing the expression of sexually dimorphic traits. Among cells of the nervous system, motor neurons respond to trophic effects of androgen stimulation, with a subpopulation of spinal motor neurons exhibiting sexually dimorphic survival. To study the mechanisms of androgen action in these cells, we performed a subtractive screen for genes upregulated by androgen in a motor neuron cell line. We show androgen-inducible expression of two RNA-binding proteins that are the mammalian homologues of invertebrate sex determination genes. Androgens upregulate the expression of tra-2alpha, an enhancer of RNA splicing homologous to Drosophila tra-2, and promote redistribution of the protein from a diffuse to a speckled pattern within the nucleus. Similarly, androgens upregulate the expression of a novel gene homologous to Caenorhabditis elegans fox-1. These data indicate that androgens exert their effects, in part, by modulating the expression and function of genes involved in RNA processing, and identify homologues of invertebrate sex determination genes as androgen-responsive genes in mammals.

Testosterone regulates terminal Schwann cell number and junctional size during developmental synapse elimination.

Previous work has shown that exposure to exogenous testosterone during synapse elimination permanently stabilizes synapses that would normally be lost in the androgen-sensitive levator ani (LA) muscle, indicating that testosterone is a potent stabilizing factor for developing LA synapses. Terminal Schwann cells (TSCs), which cap the neuromuscular junction, have also been implicated in the control of synaptic stability and may play a decisive role in the selective stabilization of synapses during synapse elimination. In this study, we begin to investigate the possible role of TSCs in the effect of testosterone on synapse elimination by determining whether testosterone influences their number. As the number of TSCs generally correlates with the size of endplates, we also measured endplate size. Male rats were castrated or sham gonadectomized at postnatal day (P) 7 and given capsules containing either testosterone or nothing. Three weeks later (P27-28), LA neuromuscular junctions (NMJs) were stained using immune and nonimmune markers. As expected, testosterone treatment during synapse elimination maintained synapses that would ordinarily be eliminated. In addition, we find that the size of LA endplates and the number of TSCs per LA junction were also increased by the testosterone treatment. However, testosterone significantly increased the number of TSCs on both singly and multiply innervated fibers, indicating that the effect of testosterone on the number of TSCs is not specific to its effect on synapse maintenance. Rather the testosterone-induced increase in the number of TSCs appears related to the size of LA NMJs, a relationship that has precedence.

The orthodox view of brain sexual differentiation.

The standard view of sexual differentiation of the brain, derived primarily from work with mammals, is that the same steroidal signal which permanently masculinizes the body early in life, androgen, also permanently masculinizes the nervous system. This oversimplified view overlooks the rich diversity of mechanisms produced by natural selection. We review the mechanisms underlying sexual differentiation of what may be the simplest mammalian model, the spinal nucleus of the bulbocavernosus (SNB), a system that is intimately associated with sexual differentiation of the periphery. Indeed, in many instances, early androgen can permanently masculinize the SNB system but, surprisingly, these early influences may depend to some extent on social mediating factors. Furthermore, in adulthood, androgen continues to affect the SNB system in diverse ways, acting on several different loci, indicating a life-long plasticity in even this simple system. Finally, there is evidence that adult androgens interact with social experience in order to affect the SNB system. Thus the SNB system displays a far richer array of interactions than the standard view of sexual differentiation would predict. Examination of other systems and other species, as depicted in the following reports, reveals a far more complicated, and far more interesting perspective on how the brains and behaviors of males and females diverge.

Glia as mediators of steroid hormone action on the nervous system: An overview.

This special issue on steroids and glia represents the intersection of two emerging themes in the neurosciences: (a) Glia actively modulate and participate in brain function throughout life, and (b) glia are sensitive to steroid hormones. This overview begins by reviewing some of the basic principles of steroid hormone action on the brain and introducing the various glia that inhabit the peripheral and central nervous system. A prominent theme among the articles that follow is that glia may be direct targets for steroid hormones since they possess steroid receptors and the promoter region of glial-specific genes such as glutamine synthetase contain hormone-responsive elements. The articles in this special issue discuss evidence that glia may mediate steroid action on the nervous system in the context of (a) steroid metabolism, which may control the hormonal microenvironment of neurons both in the normal and injured brain; (b) brain development including sexual differentiation; (c) synaptic plasticity which may underlie the cyclic release of luteinizing hormone releasing hormone in the female rodent brain; (d) neural repair and aging; and (e) brain immune function. Another theme among these articles is that glia influence neurons via specific secreted and cell-surface molecules, and that steroids affect this mode of communication by altering the level of glial production of these signaling molecules and/or the sensitivity of neurons to such signals.

Ontogeny of androgen receptor immunoreactivity in lumbar motoneurons and in the sexually dimorphic levator ani muscle of male rats.

We documented the ontogeny of androgen receptor (AR) immunoreactivity for rat lumbar motoneurons of the sexually dimorphic motor pools, the spinal nucleus of the bulbocavernosus (SNB) and the dorsolateral nucleus (DLN), and for the sexually monomorphic retrodorsolateral nucleus (RDLN). We also assessed the ontogeny of AR immunoreactivity in the rat sexually dimorphic levator ani (LA), which is a target muscle for SNB motoneurons. Lumbar spinal cords and LA muscles from gonadally intact males at ages postnatal days (P)7, P10, and P14 and as adults were incubated with the rabbit antiserum PG-21. Half of the prepubertal males (P7-P14) received 200 micrograms of testosterone propionate (TP) 2 hours prior to death to enhance immunodetection of ARs. We found that SNB motoneurons developed AR immunoreactivity at first and achieved adult levels by P10. In contrast, the number of RDLN motoneurons with AR-immunopositive nuclei during development remained well below the adult number. Development of AR immunoreactivity in the DLN shared characteristics with both the SNB and the RDLN. AR immunoreactivity developed in some DLN motoneurons by P10, although the percentage of labelled motoneurons remained below that in adulthood. Acute TP treatment significantly increased the number of SNB motoneurons with AR-positive nuclei at P7. The LA showed a robust pattern of AR immunostaining from P7 to adulthood. Immunostaining was present only in nuclei and constituted only a subpopulation of the nuclei present in muscle. The present results confirm and extend previous results based on steroid autoradiography and steroid binding assays regarding regional and developmental differences in the expression of ARs.

Phenethylthiazolylthiourea (PETT) compounds as a new class of HIV-1 reverse transcriptase inhibitors. 2. Synthesis and further structure-activity relationship studies of PETT analogs.

Phenylethylthiazolylthiourea (PETT) derivatives have been identified as a new series of non-nucleoside inhibitors of HIV-1 RT. Structure-activity relationship studies of this class of compounds resulted in the identification of N-[2-(2-pyridyl)ethyl]-N'-[2-(5-bromopyridyl)]-thiourea hydrochloride (trovirdine; LY300046.HCl) as a highly potent anti-HIV-1 agent. Trovirdine is currently in phase one clinical trials for potential use in the treatment of AIDS. Extension of these structure-activity relationship studies to identify additional compounds in this series with improved properties is ongoing. A part of this work is described here. Replacement of the two aromatic moieties of the PETT compounds by various substituted or unsubstituted heteroaromatic rings was investigated. In addition, the effects of multiple substitution in the phenyl ring were also studied. The antiviral activities were determined on wild-type and constructed mutants of HIV-1 RT and on wild-type HIV-1 and mutant viruses derived thereof, Ile100 and Cys181, in cell culture assays. Some selected compounds were determined on double-mutant viruses, HIV-1 (Ile 100/Asn103) and HIV-1 (Ile100/Cys181). A number of highly potent analogs were synthesized. These compounds displayed IC50's against wild-type RT between 0.6 and 5 nM. In cell culture, these agents inhibited wild-type HIV-1 with ED50's between 1 and 5 nM in MT-4 cells. In addition, these derivatives inhibited mutant HIV-1 RT (Ile 100) with IC50's between 20 and 50 nM and mutant HIV-1 RT (Cys 181) with IC50's between 4 and 10 nM, and in cell culture they inhibited mutant HIV-1 (Ile100) with ED50's between 9 and 100 nM and mutant HIV-1 (Cys181) with ED50's between 3 and 20 nM.

Morphological effects of ciliary neurotrophic factor treatment during neuromuscular synapse elimination.

In adult skeletal muscles, exogenous ciliary neurotrophic factor (CNTF) induces axons and their nerve terminals to sprout. CNTF also regulates the amount of multiple innervation in developing skeletal muscles during synapse elimination, maintaining multiple innervation of muscle fibers. While CNTF may maintain multiple innervation by regulating developmental synapse elimination, it is also possible that CNTF induces the formation of new multiple innervation through a sprouting response. In this study I examined morphologically the effects of CNTF during synapse elimination in the extensor digitorum longus (EDL) muscle. Rat pups received injections of CNTF in one leg and vehicle in the other either early [postnatal day 7 (P7)-P13] or late (P14-P20) in development. The early treatment period corresponds to that time when the pattern of innervation in the EDL is converted from predominantly multiple to single innervation. The late treatment period is at the end of synapse elimination for the EDL but corresponds to the major period of synapse elimination in the levator ani (LA), allowing a comparison of effects on these two muscles from the same animals. On the day after the final injection, EDL muscles were dissected and stained with tetranitroblue tetrazolium and the resulting pattern of innervation was assessed. The present findings indicate that only the early CNTF treatment regulates the level of multiple innervation in the EDL. Moreover, the effect o early CNTF treatment was local, affecting multiple innervation only in the EDL from the CNTF-treated leg. CNTF injected during the late treatment period had no apparent effects on the EDL but had a potent effect on the pattern of innervation in the LA, significantly increasing the level of multiple innervation in this muscle. Thus, CNTF affected multiple innervation in these two muscles only if provided during the period when single innervation normally develops. There was no evidence to indicate that CNTF induced axons or their terminals to sprout during either treatment period. In conclusion, CNTF increases the level of multiple innervation, probably by regulating synapse elimination, and skeletal muscles themselves may be an important target site for CNTF action. Presumably, the sprouting response to CNTF found in adult muscle develops sometime after P21.

Ciliary neurotrophic factor may act in target musculature to regulate developmental synapse elimination.

During development of mammalian skeletal muscles, synaptic contacts from different motoneurons are made on to individual muscle fibers but then are progressively lost until the adult pattern of single innervation is established. Although this process of synapse elimination occurs throughout the developing nervous system, little is known about the underlying mechanisms that drive this process. Competition for target-derived trophic substances has been proposed as one mechanism whereby synapses are selectively maintained or eliminated. To directly test whether exogenous trophic substance could alter neuromuscular synapse elimination, levator ani (LA) muscles of male rats were treated during the period of synapse elimination with either human recombinant ciliary neurotrophic factor (CNTF-a putative motoneuronal survival factor), or vehicle. LA muscles were stained with tetranitroblue tetrazolium at the end of treatment and the number of axonal inputs per muscle fiber was quantified. Effects of CNTF on other parameters such as body weight, axonal sprouting, muscle fiber and motoneuronal growth were also assessed. CNTF-treated muscles contained 3 times more multiple innervation than did vehicle-treated muscles, suggesting that CNTF can regulate synapse elimination in the LA. Moreover, CNTF delivered near the LA was more potent in blocking synapse elimination than the same dose of CNTF delivered at a site distant from the LA, suggesting that the target muscle is an important site, either for direct CNTF action on synapse elimination, and/or for directed transport of CNTF to motoneuronal cell bodies.

Phenethylthiazolethiourea (PETT) compounds, a new class of HIV-1 reverse transcriptase inhibitors. 1. Synthesis and basic structure-activity relationship studies of PETT analogs.

A novel series of potent specific HIV-1 inhibitory compounds is described. The lead compound in the series, N-(2-phenethyl)-N'-(2-thiazolyl)thiourea (1), inhibits HIV-1 RT using rCdG as the template with an IC50 of 0.9 microM. In MT-4 cells, compound 1 inhibits HIV-1 with an ED50 of 1.3 microM. The 50% cytotoxic dose in cell culture is > 380 microM. The chemical structure-activity relationship (SAR) was developed by notionally dividing the lead compound in four quadrants. The SAR strategy had two phases. The first phase involved optimization of antiviral activity through independent variation of quadrants 1-4. The second phase involved the preparation of hybrid structures combining the best of these substituents. Further SAR studies and pharmacokinetic considerations led to the identification of N-(2-pyridyl)-N'-(5-bromo-2-pyridyl)-thiourea (62; LY300046.HCl) as a candidate for clinical evaluation. LY300046.HCl inhibits HIV-1 RT with an IC50 of 15 nM and in cell culture has an ED50 of 20 nM.

Comparison of panel-reactive antibody levels in Caucasian and African American renal transplant candidates.

PRA levels from 58 Caucasian and 70 African American ESRD patients were compared against a panel of cryopreserved lymphocytes from 60 donors (40 Caucasian, 15 African American, 5 others) to determine whether there was significant racial influence on PRA outcome. African Americans were found to have significantly higher mean PRA levels than Caucasians (27% vs. 18%, P = 0.02). Restricting this analysis to only 1 degree transplant candidates showed predictably lower mean PRAs: 6% in Caucasians and 15% in African Americans, but the difference between the two groups remained significant (P = 0.015). The percentage of patients with PRA > or = 10% was also greater among African Americans than Caucasians (43% vs. 24%, P = 0.026). For patients not previously transplanted, the difference between these frequencies remained significant: 11% in Caucasians, 30% in African Americans (P = 0.025). Untransplanted African American patients with positive PRAs (> or = 10%) had significantly higher PRA against African American cell donors (mean = 55%) than against Caucasian cell donors (mean = 44%) (mean difference = 10.6%, P = 0.0056). African Americans were more frequently transfused than Caucasians. The percentage of patients not previously transplanted receiving 0, 1-5, and > 5 transfusions were 69%, 22%, and 9% for Caucasians and 43%, 44%, and 13% for African Americans (P = 0.03). This higher transfusion rate is the most likely contributor to the elevated PRA levels observed in African Americans.

Medicinal chemistry of difluoropurines.

A series of over 70 difluoropurine analogs was synthesized by varying the C-2, 6 and 8 substituents about the purine ring system. After initial in vitro and in vivo screening, testing concentrated on the 2,6-diaminopurine analog (dFdAP) and the guanosine analog (dFdG). dFDAP appears to be a prodrug for dFdG. Both compounds significantly inhibited mammary tumor growth in mice, caused a moderate inhibition in ovarian and lymphosarcoma models, and demonstrated no activity in lung and melanoma models. This is a narrower spectrum of activity than that of gemcitabine (dFdC). The antitumor activity of dFdAP in human xenografts that are refractory to standard clinical agents was comparable or superior to that of gemcitabine. However, during the preliminary toxicology testing, dFdG was associated with several deaths caused by cardiac toxicity. Therefore, although dFdG is a potentially useful oncolytic, further investigation is required.

Androgenic, not estrogenic, steroids alter neuromuscular synapse elimination in the rat levator ani.

Developmental synapse elimination in the rat levator ani (LA) muscle is sensitive to gonadal androgen. This process occurs faster in castrated male rats that lack gonadal testosterone and is largely prevented by testosterone treatment. Because testosterone can be irreversibly converted into either androgenic metabolites such as dihydrotestosterone or estrogenic metabolites such as estradiol, the present experiment sought to determine which of these metabolites account for testosterone's effect. Male rat pups at postnatal day 7 (P7) were castrated and given daily subcutaneous injections of one of 5 possible treatments for 3 weeks (P7-P28): (1) testosterone propionate (TP), (2) dihydrotestosterone propionate (DHTP), (3) estradiol benzoate (EB), (4) a combination of DHTP and EB or (5) sesame oil vehicle. At the end of treatment, the LA and extensor digitorum longus (EDL) muscles were dissected and their motor nerve terminals were stained with tetranitroblue tetrazolium. Hormone effects on synapse elimination were evaluated by counting the number of motor axons that contacted individual muscle fibers. The lumbosacral spinal cord was also dissected and processed histologically to examine the motoneurons in the spinal nucleus of the bulbocavernosus (SNB), which innervates the LA. Hormone effects on SNB motoneuron size were assessed by measuring the cross-sectional area of SNB motoneuronal somata and nuclei. We report that DHTP mimics the effects of TP on synapse elimination in the LA muscle, but that EB, acting either alone or together with DHTP, has little or no effect on this process. Synapse elimination in the EDL was unaffected by any hormone treatment. TP or DHTP, but not EB, increase the size of SNB motoneurons. We conclude that testosterone or its androgenic metabolites influence synapse elimination in the LA and probably exert these effects via androgen receptors.

Synthesis and anaerobic activity of novel 1-carba-1-dethiacephalosporins.

The synthesis and biological evaluation of novel 1-carba-1-dethiacephalosporins exhibiting activity against anaerobic pathogens are described. The nitrothiazole substituent was determined to be crucial to maintaining this activity. The pharmacokinetic parameters and initial toxicological profile of the lead compound are discussed.