Androgen‐induced neurite outgrowth is mediated by neuritin in motor neurones

Abstract

In the brain, the spinal cord motor neurones express the highest levels of the androgen receptor (AR). Experimental data have suggested that neurite outgrowth in these neurones may be regulated by testosterone or its derivative 5a-dihydrotestosterone (DHT), formed by the 5a-reductase type 2 enzyme. In this study we have produced and characterized a model of immortalized motor neuronal cells expressing the mouse AR (mAR) [neuroblastoma-spinal cord (NSC) 34/mAR] and analysed the role of androgens in motor neurones. Androgens either activated or repressed several genes; one has been identified as the mouse neuritin, a protein responsible for neurite elongation. Real-time PCR analysis has shown that the neuritin gene is expressed in the basal condition in immortalized motor neurones and is selectively upregulated by androgens in NSC34/mAR cells; the DHT effect is counteracted by the anti-androgen Casodex. Moreover, DHT induced neurite outgrowth in NSC34/mAR, while testosterone was less effective and its action was counteracted by the 5a-reductase type 2 enzyme inhibitor finasteride. Finally, the androgenic effect on neurite outgrowth was abolished by silencing neuritin with siRNA. Therefore, the trophic effects of androgens in motor neurones may be explained by the androgenic regulation of neuritin, a protein linked to neurone development, elongation and regeneration.

Androgen regulation of axon growth and neurite extension in motoneurons

“…As with MN cells, the fact that NSC34 cells only express AR when transfected allows us to use these cells to discriminate between AR-dependent and AR-independent androgen effects. In this model, androgens enhance motoneuron differentiation and neurite outgrowth via activation of the AR (Marron et al, 2005). In particular, while untreated cells are characterized by short cell processes, androgen-treated NSC34/mAR cells have a much higher number of cell processes and display a marked motoneuron-like morphology.…”

“…Androgen-induced neurites are quite long, with a mean length ranging from five to ten times the soma size. Interestingly, these neurites are also immunoreactive for SMI312 (Marron et al, 2005), a cocktail of monoclonal IgG1 antibodies that is specific for phosphorylated medium-and high-molecular weight neurofilaments, selected to provide a marker for axons (Sternberger et al, 1982). Thus, androgen treatment results in the differentiation of axon-like cell processes, which normally do not develop easily in neuron cultures.…”

“…We have used NSC34/mAR cells to examine the differential effects of these two androgens in promoting motoneuron differentiation (Marron et al, 2005). Cells were treated for 48 hours with either testosterone or DHT, and axon outgrowth was measured by counting the number of neurites positive for SMI312 (considered a specific marker for axons; Sternberger et al, 1982;see above).…”

“…We have used differential display PCR (DD-PCR), confirmed with RT-PCR, to identify several genes that are either activated or repressed by androgen treatment (Marron et al, 2005). One of the primary genes upregulated by androgen is neuritin, also known as cpg15 (for candidate plasticity-related gene 15; Naeve et al, 1997;Nedivi et al, 1993).…”

“…Moreover, an in silico analysis performed using MatInspector (www.genomatix.de) identified seven putative androgen response elements (AREs) in the promoter region of the neuritin gene, suggesting that neuritin expression may be under the direct control of steroid-bound AR. We have used NSC34/mAR cells to study the interaction between androgens and neuritin in promoting neurite outgrowth in motoneurons (Marron et al, 2005). NSC34/mAR cells express neuritin at basal levels even in the absence of androgens.…”