Investigating the Role of MAGEL2 in Regulating Gene Expression, mRNA Translation, and Neuroendocrine Function in the HPA Axis of a Prader-Willi Syndrome Mouse Model

Prader–Willi Syndrome (PWS) and Schaaf–Yang Syndrome (SYS) are rare neurodevelopmental disorders resulting from deletions of MAGEL2 and other genes within the imprinted region of chromosome 15, or from mutations in the MAGEL2 gene alone, respectively. Both syndromes are characterized by hypothalamic dysfunction, leading to disruptions in the neuroendocrine system, growth and development, circadian rhythm, and metabolism. Despite recognition of MAGEL2’s role in the development of both syndromes, which affects protein trafficking at the cellular level, as well as hormone and neuropeptide secretion in the hypothalamus, the underlying molecular mechanisms governing its function remain unclear. In particular, nothing is known about its role in the translational regulation of proteins and neuropeptides. This dissertation addresses this gap by investigating the effects of Magel2 loss on gene expression and mRNA translation within the hypothalamus and pituitary gland. Using Magel2 knockout (KO) mice as a model for PWS and SYS, we optimized polysome profiling, the quintessential technique for studying protein translation, in the mouse hypothalamus and pituitary, and applied it to analyze mRNA transcription and translation. Since part of our aims involved confirming previously reported results in the same mouse model, which were conducted using male mice, we used male mice for all primary experiments. However, several experiments were also repeated in female mice. With our work, we have, for the first time, optimized polysome profiling in the mouse hypothalamus and pituitary. Analysis of polysome profiles suggested that global translation rates were not significantly altered in the hypothalamus or pituitary of Magel2 KO male mice. However, follow-up gene expression analysis of polysome fractions revealed several genes that were differentially translated or expressed in the Magel2 KO pituitary, indicating an effect on specialized translation. These genes are involved in hormone secretion, circadian rhythm, and translation control, implicating a role for MAGEL2 in regulating these processes at the translational level. A 24-hour fasting protocol was then implemented to evaluate how nutritional stress affects the hypothalamus and pituitary and to investigate the role of Magel2 in this context. Fasting did not alter global translation; however, preliminary data from the pituitary warrant further investigation of the effects of fasting and Magel2 loss on selective mRNA translation under nutrient stress using RNA sequencing analysis. To complement our translatome studies, we performed proteomic analyses of hypothalamus and plasma upon 24-hour fasting and for plasma also under 24-hour refeeding. These analyses revealed significant alterations in hypothalamic and plasma protein composition, including changes in hormones, components of the translation machinery, and PWS-associated proteins. Our findings suggest a broader role for MAGEL2 in coordinating hypothalamic–pituitary function and systemic metabolic regulation. In addition to its recently described roles in protein recycling, intracellular sorting, and regulated secretion of hormones and neuropeptides, MAGEL2 may also influence processes relevant to PWS and SYS at both transcriptional and translational levels, including endocrine and metabolic homeostasis as well as circadian rhythm. Finally, we developed an in vitro model, induced pluripotent stem cell (iPSC)- derived cerebral organoids, which will be instrumental for future mechanistic studies and importantly, for drug discovery. MAGEL2 expression was validated in human iPSC-derived cerebral organoids and induced neurons (iNeurons), establishing the validity of these as translational models for future investigations of PWS, SYS, and related disorders. Overall, our work confirmed that MAGEL2 is essential for maintaining hypothalamic–pituitary function and supporting endocrine, metabolic, and circadian balance. In addition, it highlights potential novel roles of MAGEL2, including its regulation at the levels of translation and gene expression—mechanisms not previously demonstrated. Taken together, our findings provide new molecular insights into how MAGEL2 dysfunction contributes to PWS and SYS, laying a foundation for future mechanistic and therapeutic studies.