7381 Overexpression of Makorin Ring Finger Protein 3 in the Arcuate Nucleus of Postpubertal Female Mice Impacts Neurokinin B and Kisspeptin Co-Expressing Neurons

Abstract Disclosure: S.A. Roberts: None. A. Fontes: None. S. Blau: None. M. Magnuson: None. K.M. McKnight: None. M. Sena-Esteves: None. R.S. Carroll: None. U.B. Kaiser: None. Background: Makorin ring finger protein 3 (MKRN3), an upstream inhibitor of GnRH release, is an important regulator of the age of menarche. Loss-of-function mutations in MKRN3 are the most common genetic etiology of central precocious puberty. However, its potential role in delayed puberty or hypogonadism is not well defined. In mice, expression of Mkrn3 in the hypothalamic arcuate nucleus (ARC), where kisspeptin, neurokinin B and dynorphin (KNDy) co-expressing neurons are located, is high early in life and declines before pubertal onset. Neonatal hypothalamic overexpression of Mkrn3 leads to delayed puberty in female mice and decreased neurokinin B (NKB) and kisspeptin protein levels in the ARC peripubertally. Conversely, Mkrn3-deficient peripubertal mice exhibit increased NKB protein levels in the ARC. We hypothesized that Mkrn3 overexpression in the ARC of postpubertal female mice would result in suppression of the HPG axis, manifested as hypogonadotropic hypogonadism. Methods and Results: Twelve week old adult wild type female mice were injected stereotaxically into the ARC with a novel adeno-associated virus overexpressing Mkrn3 (AAV-Mkrn3-IRES-EGFP; denoted AAV-Mkrn3) or a control virus (AAV-EGFP). Mean body weight did not differ between groups post-injection. AAV-Mkrn3 injected mice spent significantly more time (62.7%) in diestrus three to six weeks post-injection compared to controls (41.3%; P<0.01). Eight weeks post-injection, gonad-intact females were injected with an NKB receptor (NK3R) agonist, senktide (5 nmol or 10 nmol ip). AAV-Mkrn3 mice had a decreased peak LH response twenty minutes post-injection, compared to controls (P<0.001) at both doses. Peak LH levels were also reduced in response to senktide (5 nmol ip) in ovariectomized and estradiol capsule replaced (OVX+E2) AAV-Mkrn3 injected females, compared to controls (P< 0.001). In contrast, in both gonad-intact and OVX+E2 female mice, kisspeptin-10 (1 nmol ip) resulted in similar peak serum LH levels in AAV-Mkrn3 injected mice compared to controls. Immunohistochemistry in the ARC of intact and OVX+E2 AAV-Mkrn3 injected females showed significantly decreased NKB protein expression in AAV-Mkrn3 mice compared to controls. In response to OVX, serum LH levels increased similarly between AAV-Mkrn3 and control-injected mice. LH pulsatility in OVX females, assessed over 3 hours, showed that mean basal LH levels, number of pulses, pulse amplitude and area under the curve did not differ significantly between groups. Conclusions: These findings indicate that MKRN3 can exert inhibitory effects on the reproductive axis in female mice beyond the juvenile period, which has implications as a potential therapeutic target. The blunted response to senktide, and decreased NKB protein levels in the setting of Mkrn3 overexpression, further supports the impact of Mkrn3 on the neuropeptides and/or receptors of KNDy neurons. Presentation: 6/2/2024