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. Author manuscript; available in PMC: 2023 Jan 18.
Published in final edited form as: J Neurotrauma. 2008 May;25(5):561–566. doi: 10.1089/neu.2007.0466

Androgen Regulates Neuritin mRNA Levels in an In Vivo Model of Steroid-Enhanced Peripheral Nerve Regeneration

KEITH N FARGO 1,2, THOMAS D ALEXANDER 2, LISA TANZER 1, ANGELO POLETTI 3, KATHRYN J JONES 1,2
PMCID: PMC9848905  NIHMSID: NIHMS1862967  PMID: 18419250

Abstract

Following crush injury to the facial nerve in Syrian hamsters, treatment with androgens enhances axonal regeneration rates and decreases time to recovery. It has been demonstrated in vitro that the ability of androgen to enhance neurite outgrowth in motoneurons is dependent on neuritin–a protein that is involved in the re-establisment of neuronal connectivity following traumatic damage to the central nervous system and that is under the control of several neurotrophic and neuroregenerative factors–and we have hypothesized that neuritin is a mediator of the ability of androgen to increase peripheral nerve regeneration rates in vivo. Testosterone treatment of facial nerve-axotomized hamsters resulted in a ~300% increase in neuritin mRNA levels 2 days post-injury. Simultaneous treatment with flutamide, an androgen receptor blocker that is known to prevent androgen enhancement of nerve regeneration, abolished the ability of testosterone to upregulate neuritin mRNA levels. In a corroborative in vitro experiment, the androgen dihydrotestosterone induced a ~100% increase in neuritin mRNA levels in motoneuron-neuroblastoma cells transfected with androgen receptors, but not in cells without androgen receptors. These data confirm that neuritin is under the control of androgens, and suggest that neuritin is an important effector of androgen in enhancing peripheral nerve regeneration following injury. Given that neuritin has now been shown to be involved in responses to both central and peripheral injuries, and appears to be a common effector molecule for several neurotrophic and neurotherapeutic agents, understanding the neuritin pathway is an important goal for the clinical management of traumatic nervous system injuries.

Keywords: androgens, facial nerve injuries, nerve regeneration, neuronal plasticity, steroids

INTRODUCTION

Neuritin, also referred to as candidate plasticity-related gene 15 or CPG15, is a protein concentrated in neural tissue and related to neuroplasticity (Naeve et al., 1997; Nedivi et al., 1993). The neuritin gene is an activity-dependent immediate-early gene (Fujino et al., 2003) that codes for a glycosylphosphatidylinositol-anchored protein and is located on the surface of both developing and mature neurons (Naeve et al., 1997). Neuritin is present in highest concentrations in neural tissues undergoing rapid changes in connectivity, and experimental conditions and trophic factors that stimulate changes in connectivity upregulate neuritin expression (Corriveau et al., 1999; Lee and Nedivi, 2002; Naeve et al., 1997; Nedivi et al., 1996; Newton et al., 2003; Han et al., 2007; Harwell et al., 2005; Wibrand et al., 2006; Pahnke et al., 2004; Lee et al., 2005; Rickhag et al., 2007).

Neuritin is thought to play a role in the structural plasticity of both axons and dendrites. In cultured cells, recombinant neuritin protein enhances neurite extension and branching (Naeve et al., 1997), and neuritin mRNA is critical in directed neuronal differentiation (Lee et al., 2005; Karamoysoyli et al., 2007; Cappelletti et al., 2007; Marron et al., 2005). In vivo, overexpression of neuritin in Xenopus laevis optic tectal neurons enhances dendritic growth (Nedivi et al., 1998) and induces axonal elaboration of retinal ganglion cells (Cantallops et al., 2000). Neuritin also promotes axonal arbor elaboration and branching when expressed in Xenopus laevis motor axons (Javaherian and Cline, 2005).

Moreover, neuritin may be critically involved in regulating the re-establishment of appropriate neuronal connectivity following injury to the nervous system, and its expression is selectively modulated following both spinal cord injury and cerebral ischemia (Han et al., 2007; Di Giovanni et al., 2005; Rickhag et al., 2007). Our laboratory has been studying nerve regeneration processes, using the axotomized hamster facial nerve as a model, and has found a neurotherapeutic role for androgens (Jones et al., 2001). Treatment of axotomized animals with exogenous androgens leads to accelerated functional recovery (Jones, 1993; Kujawa et al., 1989) by enhancing axonal regeneration rates (Kujawa et al., 1991; Tanzer and Jones, 1997, 2004). Androgen treatment regulates a variety of cellular and molecular events in the facial motor nucleus following injury (Jones et al., 2001), highlighted by the acceleration of tubulin upregulation between 2 and 7 days post-axotomy (Jones and Oblinger, 1994; Jones et al., 1999). Recently, neuritin has been shown to be a downstream effector of androgen in promoting neurite outgrowth in motoneurons in vitro (Marron et al., 2005). Thus, we have hypothesized that the ability of androgens to enhance injury-induced structural plasticity in vivo may involve neuritin.

In the present paper, we report that neuritin mRNA levels are regulated by androgen treatment, in an androgen receptor-dependent manner, in a well-defined, in vivo model of steroid-enhanced nerve regeneration. In addition, we extend the observation that androgens regulate neuritin mRNA levels in cultured motoneuron hybrid cells, using a different cell line than that of Marron et al. (2005). These results support the hypothesis that neuritin may be a significant molecular mediator of the neurotherapeutic actions of gonadal steroids on injured motoneurons.

METHODS

Facial motoneurons were axotomized by severing the right facial nerve at its exit from the stylomastoid foramen in adult, male Syrian hamsters. At the same time, some of the animals were given subcutaneous, interscapular implants of Silastic capsules containing testosterone propionate (3.18 mm outer diameter [OD], 1.57 mm inner diameter [ID], 10 mm long). These capsules reliably deliver a steady, supraphysiological dose of systemic testosterone (Smith et al., 1977). Postoperative survival times were 6 hours, or 1, 2, 4, 6, or 7 days. Based on initial results, another group of animals was axotomized and treated with testosterone as described, but also given daily subcutaneous injections of flutamide (15 mg in 0.2 mL of a 50:50 v/v solution of ethanol and propylene glycol; Schering-Plough, Kenilworth, NJ), a non-steroidal androgen receptor blocker; this group had a postoperative survival time of 2 days. Tissue punches containing the facial nucleus were harvested from both sides of the brainstem, with the left side serving as an internal control for each animal. Group size was three to six animals per group.

A complementary in vitro experiment was performed using a line of mouse motoneuron-neuroblastoma hybrid (MN) cells (clone 2F1.10.14.7) (Salazar-Grueso et al., 1991) transfected with human androgen-receptor-positive (AR+; plasmid construct pCMV-AR) (Brooks et al., 1998). MN cells that did not contain the androgen receptor gene (i.e., androgen receptor-negative [AR−]), but did contain an empty cassette, were used as a control. MN cells were a gift of Drs. B.P. Brooks and K.H. Fischbeck. All MN cells were differentiated to a neuronal phenotype by treating with neuronal differentiation medium for approximately 72 h before harvest (Brooks et al., 1998). During the final 48 h of neuronal differentiation, three groups each of AR+ and AR− MN cells were treated with either 100 nM dihydrotestosterone (DHT; a potent androgenic derivative of testosterone) or vehicle control. Approximately 5 × 105 cells were harvested from each group.

RNA was isolated from both tissue punches and harvested cells by guanidinium-thiocyanate extraction (Chomczynski and Sacchi, 1987), and neuritin mRNA levels were assessed by reverse transcription and real-time polymerase chain reaction (PCR). Because the neuritin sequence in hamsters is unknown, primers were designed to target a region that is conserved between the rat (gi 2062677) and mouse (gi 23272029) neuritin sequences. The selected region corresponds to bases 318–432 of the mouse sequence. The forward and reverse neuritin primers used in this experiment were 5’-GCATGGCCAACTACCC-3’ and 5’-CCTTCCTGGCAATCCGT-3’, respectively. GAPDH served as the reference gene, and amplification was detected with SYBR Green fluorescent dye. PCRs were run in triplicate for each sample, with the average result serving as the sample score. For each sample, percent change in neuritin mRNA levels was calculated using the formula % = (2−ΔΔCt − 1) × 100.

To verify the specificity of the amplified products, we performed melt curve analyses immediately following each PCR. As an additional control, PCR product from several different animals at the 2-day post-axotomy time point was sequenced to ensure that it matched the expected neuritin amplicon. Briefly, cDNA was purified from PCR product and sequenced on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The resultant sequences exhibited an average of 98.44% homology with the mouse neuritin transcript, and no significant homology with any other sequence from the BLAST databases.

Statistical Analysis

Data were analyzed using the analysis of variance (ANOVA) method, followed by post hoc comparisons using Fisher’s least significant difference (LSD).

RESULTS

As shown in Figure 1, a two-way ANOVA for changes in neuritin mRNA levels following axotomy revealed a main effect of time point [F(5, 28) = 7.67, p < 0.001], a main effect of testosterone treatment [F(1, 28) = 8.17, p < 0.01], and an interaction between time point and testosterone treatment [F(5, 28) = 12.64, p < 0.001]. Post hoc analysis indicated that neuritin mRNA levels were significantly higher in testosterone-treated animals 2 days post-axotomy than in any other group (LSD, p < 0.001), increasing by approximately 300% above the uninjured control side. However, concurrent treatment with flutamide completely abolished the ability of testosterone to increase neuritin mRNA levels, suggesting that this effect was androgen receptor-dependent.

FIG. 1.

FIG. 1.

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Neuritin mRNA levels in axotomized animals (open bars) and axotomized animals treated with testosterone (black bars) at 6 h, and 1, 2, 4, 6, and 7 days post-axotomy. Also shown are axotomized animals treated with both testosterone and the androgen receptor blocker flutamide for 2 days following axotomy (gray bar). Neuritin mRNA levels are displayed as the percent change between the axotomized side and the uninjured control side within each animal. At 2 days post-axotomy, testosterone treatment caused a dramatic increase in neuritin mRNA levels. Because these data represent percent change with axotomy relative to the uninjured control side, this increase is above and beyond any potential general effect of androgens in the facial nucleus. This increase was completely prevented by flutamide treatment. Bar heights represent means (±SEM).

In the in vitro experiment, treatment with DHT caused neuritin mRNA levels to double in cultured AR+ MN cells [F(2, 6) = 14.96, p < 0.01; post hoc analysis, LSD, p < 0.01; Fig. 2]. However, DHT had no effect on neuritin mRNA levels in AR− MN cells, again indicating that the effect of DHT on neuritin mRNA levels is androgen receptor-dependent.

FIG. 2.

FIG. 2.

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Neuritin mRNA levels in androgen receptor-negative (AR−) and androgen receptor-positive (AR+) cultured MN cells, treated with either dihydrotestosterone (DHT; filled bars) or vehicle only (open bars). DHT treatment doubled neuritin mRNA levels, but only in AR+ cells. Bar heights represent means (±SEM).

DISCUSSION

The results from the in vivo experiment (Fig. 1) demonstrate that neuritin mRNA levels are upregulated by testosterone in our in vivo model of androgen-enhanced nerve regeneration. Furthermore, these data are consistent with our hypothesis that neuritin is an important effector molecule in androgen’s ability to increase axonal regeneration rates. This idea is strengthened by the fact that flutamide treatment, which completely blocked the ability of androgen to upregulate neuritin in the present experiment, is known to block the ability of androgen to increase axon regeneration rates in this model (Kujawa et al., 1995).

The results from the in vitro experiment (Fig. 2) confirm and extend those of Marron et al. (2005) by using a line of cultured motoneurons that is of different origin, but widely used as a model to study androgen effects in motoneurons (Brooks et al., 1998). In addition to using a different cell line from that used in the Marron et al. (2005) paper, this experiment was conducted at a different facility, by different personnel, and with a different method to evaluate androgen receptor dependence. Thus, these data provide important evidence of the reliability and generalizability of the previous report’s conclusions (Marron et al., 2005).

The possibility that androgen regulation of neuritin mRNA levels is a simple anabolic effect of androgenic steroids must be considered. This is a valid concern with the in vitro experiment. However, it cannot be the case with the in vivo experiment. In the in vivo experiment, only the right side of each animal was axotomized, and neuritin mRNA levels are measured as a change in the axotomized side relative to the untreated control side. Furthermore, androgen treatment was given systemically, so both sides of the animal received the same amount of androgen. Therefore, if the effect of androgen was merely anabolic, neuritin mRNA levels would be expected to rise equally on both sides. Instead, neuritin mRNA on the axotomized side rose to approximately 300% of those on the uninjured side (Fig. 1). Therefore, androgen treatment has an effect on neuritin mRNA levels on the injured side above and beyond any simple anabolic effect that might be expected on the control side as well.

It is notable that the effect of androgen treatment on the neuritin mRNA levels on the axotomized side was transient, only increasing at 2 days post-injury even though the androgen treatment used in this experiment provides a long-term increase in testosterone levels and provides a sustained effect on axon regeneration rates (Kujawa et al., 1989, 1991). Neuritin is an immediate early gene (Fujino et al., 2003), and one of the hallmarks of immediate early genes is that a transient change in their expression can have relatively long lasting effects. We find the 2-day time point to be particularly intriguing for two reasons. First, previous studies of hamster facial motoneuron axon regeneration indicate that axon sprouting occurs at about 2 days post-injury (Kujawa et al., 1991). Second, androgenic enhancement of axotomy-induced upregulation of βII-tubulin occurs 2–7 days post-injury (Jones and Oblinger, 1994; Jones et al., 1999). Taken together with the fact that neuritin has been shown to be both necessary and sufficient to induce neurite outgrowth in at least some cell culture models (Marron et al, 2005; Naeve et al., 1997; Cappelletti et al., 2007), a case can be made for the involvement of neuritin in androgen-enhanced nerve regeneration.

In summary, we have demonstrated that testosterone increases neuritin mRNA levels in the axotomized hamster facial motor nucleus, an in vivo model of steroid-enhanced nerve regeneration. To our knowledge, this is the first demonstration that androgen regulates neuritin mRNA levels in vivo. Immunocytochemistry or in situ hybridization experiments would prove valuable in determining whether testosterone regulation of neuritin mRNA occurs in the injured motoneurons themselves, or in the supportive cells of the facial nucleus. Further, we have shown that the effect of testosterone in this model is dependent on the androgen receptor, and have confirmed that androgen regulates neuritin mRNA levels in cultured motoneurons. These data suggest that neuritin might act as an effector of androgen in enhancing nerve regeneration.

Neuritin has now been shown to be under the control of several molecules that exert powerful neuroprotective and neuroregenerative effects, including NGF, GDNF, BDNF, NT-3, and androgens, in both the central and peripheral nervous systems (Naeve et al., 1997; Marron et al., 2005; Pahnke et al., 2004; Wibrand et al., 2006; Karamoysoyli et al., 2007; Cappelletti et al., 2007). Furthermore, it has been demonstrated with both NGF and androgens that blocking neuritin upregulation through RNA interference abolishes the ability of these molecules to enhance neurite extension (Marron et al., 2005; Cappelletti et al., 2007). These findings suggest that neuritin may be a common downstream effector molecule for neurotrophic and neuroregenerative factors. If this is the case, then understanding how to control the neuritin pathway could contribute greatly to the clinical management of traumatic nervous system injuries.

ACKNOWLEDGMENTS

This research was supported by NIH NINDS (grant NS28238, to K.J.J.; grant NS052997, to K.N.F.) and Telethon, Italy (grant GGP06063, to A.P.).

Footnotes

DISCLOSURE STATEMENT

No conflicting financial interests exist.

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