Proceedings of The Physiological Society

King's College London (2011) Proc Physiol Soc 22, PC10

Poster Communications

The neuronal actions of anti-Mnllerian hormone appear to be independent of amyloid precursor like protein 2.

F. Imhoff1, I. S. McLennan1

1. Anatomy, University of Otago, Dunedin, New Zealand.

Anti-Müllerian Hormone (AMH) is a regulator of neurons with multiple unrelated functions. In embryos it is a testicular hormone, which contributes to the male bias in the brain and behaviour (1). In mature animals, AMH is produced by neurons of both sexes where it may serve as an anterograde regulator of neural networks (2,3). AMH is a member of the transforming growth factor β superfamily of proteins that are synthesised as large precursor proteins and then cleaved to give an N-terminal peptide and a C-terminal signalling peptide. The C-terminal of AMH signals through an AMH-specific type II receptor and a type I receptor that is shared with the bone morphogenetic proteins. In sperm and transfected CHO cells, AMH has non-classical signalling, involving the N-terminal fragment binding to amyloid precursor like protein 2 (APLP2), leading to activation of the extracellular regulated kinases 1/2 (ERK1/2) via a G0 protein mechanism (4). APLP2 and amyloid precursor protein are redundant regulators, without which synapses degrade (5). Furthermore, the ERK pathways mediate part of the neurotrophin signalling. We have therefore examined the possibility that AMH activates ERK1/2 in neurons through APLP2. Hippocampal neurons from 16-day-old mouse embryos were cultured for 4 days, and were then treated with either an AMH-related peptide (an N-terminal AMH peptide, the C-terminal fragment or the full-length AMH), brain-derived neurotrophic factor (BDNF) as a positive control or vehicle. The dose of the AMH peptides ranged from 0 to 75 nM, with both their acute (0-30 min) and chronic (0-6 days) effects on the phosphorylation of ERK1 and ERK2 being measured. For each condition, 3 independent primary cultures with 2 replicates were used. The phosphorylation of ERK1/2 was detected by Western blot using anti-ERK and anti-phospho-ERK antibodies and IRDye-conjugated secondary antibodies. The presence of APLP2 in the cultures was verified by Western blots and immunohistochemistry. All three isoforms (a-c) of APLP2 were detected in the cultures and the brain, whereas only the b and c isoforms were present in the testes. The positive control, BDNF, increased the ratio of phosphorylated-ERK1/2 to ERK1/2 by over 3 fold (6.00±0.21, vehicle 2.66±0.46, n=2). In contrast, none of the AMH variants altered the phosphorylation of ERK1 or ERK2. For example, the p-ERK1/ERK1 ratio with 75 nM of the N-terminal AMH was 0.23±0.11 compared to a control of 0.20±0.11(n=6, P>0.05, Student T). The cultured neurons secreted AMH but this was not responsible for the low level of basal phosphorylation of ERK, as it persisted when Amh-/- neurons were cultured (0.27±0.05). In conclusion, the reported data is inconsistent with AMH regulating neurons via ERK1/2, suggesting that AMH regulation of the brain is solely through the classical pathway.

Where applicable, experiments conform with Society ethical requirements