OR08-3 The Role Of Neuronal Plasticity In The Timing Of Puberty Onset: Insights From A Mkrn3 Deficient Mouse Model.

Abstract

Makorin ring finger protein 3 (MKRN3) was identified as a regulator of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty (CPP). MKRN3 is a maternally imprinted gene located on chromosome 15q11.2 in humans. In juvenile mice, Mkrn3 is highly expressed in the preoptic area and the arcuate nucleus of the hypothalamus, with a decrease in expression before puberty initiation. Together, these findings suggest an inhibitory role of MKRN3 on GnRH secretion during the prepubertal quiescent period. MKRN3 contains a RING finger motif and several zinc finger motifs, suggesting E3 ubiquitin ligase and RNA binding activities, respectively. To date, the mechanisms of action of MKRN3 in puberty initiation remain unknown. In this study, we used a Mkrn3 deficient mouse model to identify hypothalamic targets of MKRN3’s inhibitory effects. By Western blot and immunohistochemistry, we confirmed that Mkrn3 protein was undetectable in Mkrn3+/- mice (which inherited the mutant allele from their father), but present in wildtype (Mkrn3+/+) mice. Mkrn3 deletion was associated with accelerated puberty onset in female mice and a trend towards early puberty onset in male mice, as shown by an advanced day of female first estrus and a trend towards earlier age of male preputial separation in Mkrn3 deficient (Mkrn3+/-) compared to Mkrn3+/+animals. Hypothalamic mRNA levels of Gnrh1, Kiss1, Tac2 and Tac1, regulators of pubertal initiation, were measured by RT-qPCR in females at different ages across postnatal development (postnatal day (PND)10, PND15, PND20 and PND25). No significant difference was found in Gnrh1 and Kiss1 expression in the preoptic area, nor in Kiss1, Tac2 and Tac1 expression in the arcuate nucleus, between Mkrn3+/+ and Mkrn3+/- mice, at any of the postnatal day ages tested. Thus, to further investigate possible targets of MKRN3 action using an unbiased approach, we compared the transcriptome (via RNA-sequencing) and proteome (via Tandem Mass Tagging (TMT) method) of the arcuate nucleus of Mkrn3+/+ and Mkrn3+/- female mice at PND15. These analyses revealed differences in gene or protein expression between Mkrn3+/+ and Mkrn3+/- animals in factors involved in extracellular matrix organization, cell adhesion, and axon guidance pathways, which together control neuronal development and synaptic plasticity. These findings suggest that Mkrn3 may be important as a postnatal/prepubertal regulator of hypothalamic neuronal plasticity in mice. In humans, puberty has been shown to be a critical time window of neuronal plasticity. Thus, understanding the involvement of MKRN3 in these processes will provide new insights into pubertal disorders such as CPP and delayed puberty, and may lead to the development of new treatment strategies.