The role of RNF38 in reproductive brain ageing

Abstract

Brain ageing deteriorates both cognitive and motor functions. One of the age-related molecular changes is the up-regulation of RING finger protein 38 (RNF38), which is found in the hypothalamus. Despite the function of RNF38 in the brain remains unknown, Rnf38 transcript is highly expressed in rat testis, which speculates its relationship with reproductive system. Male reproductive ageing is characterized by reduced activation of reproductive neuropeptide gonadotropin-releasing hormone (GnRH) neurons in the preoptic area (POA), impairing firing rate and pulsation, leading to abnormal Leydig cell function. Serotonin (5-hydroxytryptamine, 5-HT) is a key neurotransmitter that regulates reproductive system, as 5-HT-immunoreactive boutons are localized in the region where GnRH neurons are located; and 5-HT receptors are found on GnRH neurons. 5-HT is susceptible towards ageing as lack of 5-HT fibre projections have been observed in ageing brains. This study investigates the potential role of RNF38 in brain reproductive ageing and its relationship with GnRH and serotonergic system. Firstly, the mRNA levels of Rnf38, Gnrh, 5-HT receptors were determined in the POA of young (9-12 weeks), middle-aged (52-55 weeks) and aged (79 – 85 weeks) male mice. Age down-regulates Rnf38 mRNA level in the POA despite Gnrh gene expression remains unchanged. This hints the potential of Rnf38 as an ageing marker in the POA. Secondly, age decreases the expression of both 5-HT1A and 5-HT1B receptors. This may be due to low serotonergic neurotransmission as age-reduced 5-HT levels were reported in several brain regions. Preoptic Rnf38 gene expression was up-regulated following 4 weeks of selective serotonin reuptake inhibitor (SSRI), citalopram treatment in young adult mice. This indicates potential interaction between RNF38 and serotonergic system. Immunohistochemical (IHC) studies were conducted to characterize the functionality of RNF38 in the brain. RNF38 peptides were observed in medial septum and POA, the site where GnRH neurons are found. Double-labeled IHC studies showed RNF38 is expressed by preoptic neurons, despite no co-localization between GnRH neurons and RNF38. This is validated through absence of Rnf38 gene expression at single cell GnRH neurons. Therefore, RNF38 is expressed in non-GnRH neurons within the POA. The close proximity of GnRH neurons with RNF38 implies that RNF38 may aid in signaling GnRH pathways indirectly via interneurons. Lastly, biochemical study based on Rnf38 and 5-HT related genes were conducted after the treatment of plant extract from Leucaeuna leucocephala (L. leucocephala) as it has binding affinity of 5-HT2A receptors and carries anxiolytic effect. Single central injection of crude extract of L. leucocephala seems to cause an increase in Rnf38 mRNA in the POA region despite no changes were observed between control and treated animals.