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

Abstract

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.

Androgen regulation of axon growth and neurite extension in motoneurons

“…Importantly, tubulin mRNAs are upregulated by androgen treatment selectively. While α Itubulin, β II -tubulin, and β III -tubulin are all upregulated by axotomy, only β II -tubulin has been shown to be under the control of androgen in this model (Jones and Oblinger, 1994;Jones et al, 1999b). Tubulin genes are also regulated by androgen in axotomized sciatic motoneurons ) and rubrospinal motoneurons (Storer et al, 2002;DeLucia et al, 2007), and in the spinal nucleus of the bulbocavernosus (SNB; Matsumoto et al, 1993Matsumoto et al, , 1994, a group of motoneurons whose dendritic extension and retraction is under the control of androgens (Fargo and Sengelaub, 2007b;Kurz et al, 1986), raising the possibility that this may be a general phenomenon in motoneurons.…”

“…First, extrapolation of hamster facial motoneuron axon regeneration data indicates that axon sprouting occurs at about two days post-injury (see Figure 3; Kujawa et al, 1991). Second, androgen treatment enhances axotomy-induced upregulation of β II -tubulin expression between two days and seven days post-injury (Jones and Oblinger, 1994;Jones et al, 1999b). Considered together with the fact that neuritin is an essential mediator of androgen-induced axon outgrowth in NSC34/mAR cells (Marron et al, 2005), these data suggest the hypothesis that neuritin may be involved in coordinating androgen-enhanced axon regeneration in the injured facial nerve.…”

“…Note that the data extrapolate to approximately 2 days post-injury for the zero outgrowth distance. This time point corresponds both to the androgen-induced spike in neuritin mRNA levels (see Section 3.4) and to the earliest time point at which androgen treatment augments tubulin upregulation following axotomy (Jones and Oblinger, 1994;Jones et al, 1999b). (After Kujawa et al, 1991.…”

“…The primary cytoskeletal structures in axons are microtubules, which are composed of tubulin proteins. Tubulin mRNAs are upregulated in facial motoneurons as the axons regenerate following axotomy (Jones and Oblinger, 1994;Jones et al, 1999b;Tetzlaff et al, 1991); and androgen treatment, which enhances axon regeneration rates, causes an additional upregulation of tubulin mRNAs above axotomy alone (Jones and Oblinger, 1994;Jones et al, 1999b). Importantly, tubulin mRNAs are upregulated by androgen treatment selectively.…”

“…Testosterone upregulates α-tubulin, β II -tubulin, and β III -tubulin in cultured SH-SY5Y cells in an AR-dependent manner (Butler et al, 2001). In vivo, β II -tubulin expression is upregulated during androgen-enhanced nerve regeneration in axotomized facial (Jones and Oblinger, 1994;Jones et al, 1999b) and sciatic (Brown et al, 1999) motoneurons, and androgens regulate levels of both β-actin and β-tubulin in SNB motoneurons (Matsumoto et al, 1992(Matsumoto et al, , 1993(Matsumoto et al, , 1994.…”