Breedlove Jordan Lab

Ongoing Research Projects

1. Spinal Nucleus of the Bulbocavernous (SNB)

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In rats, the motoneurons of the spinal nucleus of the bulbocavernosus (SNB) innervate striated muscles (bulbocavernosus and levator ani) attached to the base of the penis. Before birth, male and female rats have the muscles and the SNB motoneurons innervating them, but both die in females in the first week of life. The SNB system survives in males because they secrete androgenic hormones such as testosterone to spare the system from cell death. Thus in adulthood there is a prominent sexual dimorphism in the SNB region of the spinal cord (Figure 1). So if you expose newborn female rats to testosterone, you prevent the death of the motoneurons and their targets for life. We have evidence that the SNB motoneurons are not directly responding to the androgen. By elimination, it seems likely that androgen acts upon the target muscles to keep them alive and that the muscles in turn keep the SNB motoneurons alive. We would like to confirm this idea that the muscles are the primary site of androgen action to spare the SNB system, and ask which particular cell population within the muscles (muscle fibers, fibroblasts, Schwann cells, endothelial cells) are responding to the hormone.

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In adulthood, the SNB system continues to respond to androgen. Castrating adult male rats causes the somata and dendrites of SNB motoneurons to shrink. Treating castrates with androgen can prevent or reverse this effect. Androgen acts directly on the muscle fibers to cause them to enlarge, too. The effect of androgen on SNB motoneuron somata is a direct, cell-autonomous response, as only SNB motoneurons that make functional androgen receptors can expand their somata after androgen treatment. On the other hand, androgen acts indirectly to expand SNB motoneuronal dendrites, because androgen treatment at the muscle can cause the dendrites to expand (Figure 2). We would like to know how the target muscles direct their innervating SNB motoneurons to expand dendrites and make new synapses.

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NMDA-type glutamate receptors play a role in the androgen-responsiveness of adult SNB motoneurons. Pharmacological blockade of NMDA receptors prevents the motoneurons from expanding their somata in response to adult androgen treatment (Figure 3). We would like to identify the neurons that are providing glutamate stimulation to SNB motoneurons and ask how they contribute to androgen-mediated neural plasticity in this system. (E. Ottem, J. Johansen, D. Zuloaga)

2. Spinal Bulbar Muscular Atrophy (SBMA, Kennedy's Disease)

A recent focus of our lab has been studying a new transgenic mouse model of SBMA. Quite unexpectedly, our mice developed a severe disease phenotype, characterized by progressive loss of motor function, rapid weight loss and eventual death. This was a surprising find, since this model was developed to study questions on sexual differentiation and the SNB system.

What makes our mouse model unique? We have inserted a transgene for the wildtype androgen receptor (AR) in the mouse genome that is selectively expressed by muscle fibers and not by any other cell types, most particularly, not by cells that populate the nervous system, such as neurons and glia. Expression of the AR transgene is controlled by a strong skeletal muscle-specific promoter, which causes ARs to be over-expressed in muscle fibers compared to their normal wildtype levels. This over-expression of AR in muscle fibers induces an androgen-dependent disease phenotype characteristic of SBMA (Table 1).

Over-expression of wildtype AR in muscle fibers of transgenic male mice leads to a loss in motor function.

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Figure 1. Left: Paw Print analysis of sick transgenic (Tg) males show a shortened stride compared to wildtype (Wt) males. Right: Sick transgenic males are unable to perform the hang test compared to Wt and healthy Tg males. These behavioral tests suggest a loss in motor function, strength and coordination. * = Significantly different from wt and transgenic, p < .001.

Over-expression of wildtype AR in muscle fibers of transgenic male mice leads to muscle pathology.

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Figure 2. H&E staining of EDL muscles of wildtype (Wt) and transgenic male mice indicate that transgenic muscles contain both abnormally small, angular fibers (black arrow) and hypertrophied fibers (white arrow). Both fiber types contain internal nuclei (stars).

Over-expression of wildtype AR leads to increased oxidative metabolism as seen in NADH stain.

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Figure 3. EDL muscle from wildtype male mice stain moderately for NADH (left). In contrast, the EDL from adult transgenic male mice show increased NADH staining, indicating a switch to a more oxidative metabolism. "Ring" fibers (arrow) are also evident in EDL transgenic muscles stained for NADH.

What is SBMA? SBMA is a heritable X-linked, slowly progressive lower motor neuron disease that usually emerges in adulthood. The known cause of this disease is a mutation in the AR gene. This disease is characterized by a loss of motor function that is associated with muscle weakness and atrophy and a loss of motoneurons. SBMA is typically viewed as a "motoneuron disease"; i.e., muscles become dysfunctional because motoneurons die first, causing muscles to become denervated. However, contrary to prevailing theory, our mouse model suggests that SBMA is in fact a myogenic disease. We think that the disease may be triggered by problems that originate in the muscle, not the motoneurons.

This new model will allow us to directly examine the previously unsuspected role of AR in muscle fibers in triggering SBMA. Our model is also likely to provide insight into the critical mechanisms underlying other so-called "motoneuron diseases" such as Amyotrophic Lateral Sclerosis (ALS) since recent evidence suggests they too have a myogenic origin. We are very excited about this new model and we have many questions yet to answer. (J. Johansen, D. A. Monks)

Summary of SBMA Pathology

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Table 1. Comparison of disease phenotype in SBMA patients, current transgenic mouse models of SBMA and our transgenic mouse model of SBMA. We find that over-expression of wildtype AR in skeletal muscle fibers causes a neuromuscular disease phenotype that matches SBMA.

3. Sexual Dimorphism in the Medial Amygdala

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The medial amygdala in rats and mice is sexually dimorphic: about 150% larger in volume in males as in females (Figure 4). The individual neurons within the medial amygdala are also larger in males than in females. This sexual dimorphism is not 'organized' by perinatal hormones, because castration of adult males results, a month later, in a medial amygdala that has shrunk to the size of that in normal females. Conversely, treating adult female rats with an androgen such as testosterone for a month causes the medial amygdala to grow to the size seen in normal males. Thus this brain region seems to retain its plasticity throughout life so that it can always be 'organized' in a masculine fashion by androgen.

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We have evidence that both androgen receptors and estrogen receptors must be activated in order for testosterone to fully masculinize the medial amygdala (Figure 5). We would like to know more about the cellular mechanisms and the sites of action by which steroid hormones alter the structure and function of the medial amygdala. (A. Durazzo, R. Johnson)

4. Do Fetal Androgens Affect Human Sexual Orientation?

In animal models of sex differences in behavior, early exposure to testosterone and other androgens masculinizes the brain so that, in adulthood, the animal displays male-typical behaviors. There are many sex differences in human behavior, but it is not clear whether these are due to the sex difference in fetal exposure to testosterone (as in animal models), or due to the sex difference in social stimulation provided to children. It is not easy to disentangle these two hypotheses in people who show gender-typical behaviors because males are exposed to both high levels of fetal androgens and male socialization pressures, while the females are exposed to both low levels of fetal androgen and female socialization pressures. But in those minority of humans who do not show all gender-typical behaviors, one can ask whether differences in social rearing and/or fetal testosterone plays a role.

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There is a sex difference in the ratio of the length of the index finger divided by the length of the ring finger (2D:4D). The 2D:4D tends to be smaller in males than in females (Figure 8). This sex difference has been reported to be present in two year old children, which suggests that it is due to the effects of fetal androgen reducing 2D:4D. We have found support for that notion, as the 2D:4D of people with congenital adrenal hyperplasia (CAH) are lower than that of same-sex controls. We would like to learn more about how fetal androgen affects the development of finger lengths.

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We have found that the 2D:4D of homosexual women is, on average, lower than that of heterosexual women (Figure 9), a finding that has been replicated at least three times by other laboratories. We have also found evidence that 2D:4D is lower in males with older brothers than in males without older brothers. We would like to learn how older brothers influence finger development in subsequent brothers. (D. Puts)

5. The Role of Co-Factors in Steroid-Mediated Neural Plasticity

For the first two model systems described above (the SNB and the medial amygdala), you read about how gonadal hormones (androgens and/or estrogens) are capable of changing neuronal morphology. You also read that in some cases we know where hormones act to alter neuronal morphology. For example, we know that androgens induce adult SNB motoneuronal cell bodies to grow by activating androgen receptors in SNB motoneurons, and that without functional androgen receptors, SNB motoneurons do not grow in response to androgens. We also know that normally only some adult SNB motoneurons grow in response to adult androgens (increasing cell body size), while others do not. However, just about all motoneurons have androgen receptors, indicating that whether or not a motoneuron has androgen receptors can not explain why some motoneurons grow in response to androgens, while others do not. Thus, we have become interested in steroid receptor cofactors and whether they will explain these differences in androgen-responsiveness. Steroid receptor cofactors can be viewed as 'helper proteins' that are recruited to the AR after androgens bind and activate the receptor. Such cofactors are thought to play an essential role in the regulation of gene transcription by steroid receptors. We would like to know whether differences in steroid receptor cofactors exist that might account for differences in androgen-responsiveness among motoneurons.

Recall that the medial amygdala in Siberian hamsters exposed to short days loses its responsiveness to hormones, failing to grow in response to hormones as it does in hamsters exposed to long days. We are also interested in whether steroid receptor cofactors have a role in the change in hormone responsiveness that we observe in this model system. (C. Jordan)