The mechanism of action of MKRN3 and its implication in central precocious puberty
MetadataShow full item record
BACKGROUND: MKRN3 is a gene recently identified to encode the first known inhibitor of puberty initiation. MKRN3 mutations have been identified in children diagnosed with familial central precocious puberty. MKRN3 is a maternally imprinted gene; only the father’s allele is expressed by the child. Family studies and patterns of inheritance affirm that mutant alleles only result in CPP when the mutation was inherited from the father. Although mutations in MKRN3 were found to have implications in development of central precocious puberty, its mechanism of action remains a mystery. Previous studies in our laboratory have focused on MKRN3’s function as an E3 ubiquitin ligase and thus sought to investigate its potential targets in the cell. Because kisspeptin is the most potent known activator of GnRH neurons, it seemed to be the most likely candidate. Despite efforts to identify an association between kisspeptin levels and MKRN3 expression, little headway has been made. OBJECTIVE: Beyond the most common known function of E3 ubiquitin ligases in protein degradation, increasing evidence suggests that E3 ubiquitin ligases contribute to aiding human brain development throughout childhood and into adolescence. This proposed action of E3 ubiquitin ligases led us to propose a role for MKRN3 as a potential regulator of GnRH neuronal plasticity and maturation. I hypothesized that MKRN3 plays a role in delaying GnRH neuronal maturation and neuronal plasticity until puberty onset. Thus, MKRN3 deficiency would result in premature GnRH neuron maturation. METHODS: A mouse model of Mkrn3 knockout mice (Mkrn3 +/p-) was compared to wildtype mice (Mkrn3+/+). Using DAB IHC, GnRH neurons in the rostral pre-optic area (rPOA) area were labeled and morphologically analyzed. Additionally, Golgi staining of neurons in the arcuate nucleus was done to visualize details of neuronal synapses. Neurons were visualized under 40X and 100X magnification. To visualize spines, confocal images of dendrites from Mkrn3+/p- and Mkrn3+/+ mice were captured. Images were visualized and analyzed using both ImageJ and NeuronStudio software (for spine analysis only). RESULTS: Initial morphological analysis of GnRH neurons failed to show any significant effect of the Mkrn3 genotype on dendrite arrangement, varying among complex, bipolar, and unipolar. Similarly, Golgi staining analysis revealed that the Mkrn3 genotype did not have a measurable effect on dendritic spine type percentages. Mkrn3+/p- and Mkrn3+/+ mice showed no apparent difference in the percentage of stubby, thin, and mushroom shaped spines. However, Mkrn3 deficiency did show an effect on spine density, with neurons from the arcuate nucleus of the hypothalamus of Mkrn3+/p- mice having an increased total number of dendritic spines compared to neurons from wildtype mice. CONCLUSIONS: Mkrn3 does not appear to have an effect on dendrite architecture of GnRH neurons nor change in spine type of arcuate nucleus neurons. However, the Mkrn3 genotype does seem to affect neuronal spine density, at least in the population of neurons in the arcuate nucleus. Future research is needed to conclusively determine the mechanism of action of MKRN3 and its specific role in central precocious puberty.