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The Role of Circadian Transcription Factors in Gonadotropin-Releasing Hormone Gene Expression
Circadian regulation is essential for many physiological functions, from sleep/wake patterns to hormonal rhythms. While the brainās biological clock, the suprachiasmatic nucleus (SCN), likely plays a role in reproductive rhythms, it remains unknown if intracellular circadian clocks elsewhere contribute to the neuroendocrine control of reproduction. Gonadotropin-releasing hormone (GnRH) stimulates pituitary gonadotropin secretion, which plays crucial roles in reproduction. My research examined if the clock may regulate GnRH gene expression by determining whether the intrinsic proteins of the clock regulate GnRH transcription in an in vitro model of GnRH neurons, GT1-7 cells. These cells were transfected with full-length and truncated regions of the GnRH reporter combined with luciferase reporters, and co-transfected with clock proteins. Using a luminometer, GnRH-Luc activity was quantified, revealing that overexpression of core clock proteins inhibit transcription of all GnRH constructs used. Since this is contrary to the established function of these clock components, we have concluded that transcriptional repressors activated by the clock machinery bind to the GnRH promoter, likely via multiple repressor sites, as revealed by truncations. Further study of the GnRH promoter, using electromobility shift assays, will likely determine where clock-controlled transcription factors are binding, and the use of stably-cloned secretable luciferase GnRH promoter plasmids will allow measuring of oscillatory promoter activity. This knowledge could aid in a better understanding of the neuroendocrine regulation of reproduction and etiology of various reproductive disorders
Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipid accumulation: possible implications of lysosomal dysfunction in macular degeneration
Age-related macular degeneration (AMD) is the leading cause of vision loss in elderly people over 60. The pathogenesis is still unclear. It has been suggested that lysosomal stress may lead to drusen formation, a biomarker of AMD. In this study, ARPE-19 cells were treated with chloroquine to inhibit lysosomal function. Chloroquine-treated ARPE-19 cells demonstrate a marked increase in vacuolation and dense intracellular debris. These are identified as chloroquine-dilated lysosomes and lipid bodies with LAMP-2 and LipidTOX co-localization, respectively. Dilation is an indicator of lysosomal dysfunction. Chloroquine disrupts uptake of exogenously applied rhodamine-labeled dextran by these cells. This suggests a disruption in the phagocytic pathway. The increase in LAMP protein levels, as assessed by Western blots, suggests the possible involvement in autophagy. Oxidative stress with H2O2 does not induce vacuolation or lipid accumulation. These findings suggest a possible role for lysosomes in AMD. Chloroquine treatment of RPE cells may provide insights into the cellular mechanisms underlying AMD