PROJECT SUMMARY Magnesium (Mg2+) is the fourth-most abundant ion the human body. Participating in over 300 enzymatic pathways, intracellular Mg2+ is crucial for life, affecting fundamental cellular process such as metabolism, DNA replication, mRNA transcription and protein translation. Consequently, the amount of intracellular Mg2+ in cells and in the body are tightly regulated, within narrow sub-millimolar ranges. Alterations in Mg2+ homeostasis are associated with numerous clinical phenotypes, including cardiovascular disease, diabetes mellitus, hypertension as well as neuropsychiatric and neurodevelopmental disorders like anxiety, intellectual disability and developmental delay. Cyclin M2 (CNNM2) is a transmembrane protein that mediates cellular transport of Mg2+. The protein is highly expressed in the kidney and the brain, where it is principally expressed in neurons. Proteomic studies by the Runnels laboratory have demonstrated that CNNM2 is part of protein complex. Human mutations in CNNM2 give rise to a monogenic disease called Hypomagnesemia, Seizures, and Intellectual Disability (HSMR) Syndrome. HSMR patients excrete more Mg2+ from the kidney into the urine, causing Mg2+ deficiency, are also obese and have seizures, motor skills difficulties, intellectual disability and developmental delay. The HSMR symptoms are consistent with intracellular Mg2+ levels being critical for neuronal development and function. The importance of intracellular Mg2+ in neurobiology is supported by a wealth of literature, but surprisingly despite its significance, Mg2+ homeostasis remains poorly understood in neurons. Moreover, CNNM2 neuronal expression and its protein composition remains undefined. We will address this gap in knowledge and hypothesize that i) mouse and human CNNM2 will have a specific neuronal expression and protein composition and ii) CNNM2 mutations will disrupt Mg2+ flux in both mouse and human neurons, and be associated with developmental, synaptic and proteomic changes that are rescued once intracellular Mg2+ levels are restored. To test this hypothesis, in Aim 1 we will determine i) Cnnm2 expression in the developing and adult mouse brain using complementary approaches (IHC, western, GFP transgenic) and ii) the composition of CNNM2 complex in moue and human induced neurons (iNs). In Aim 2, we will rigorously define the function and impact of CNNM2 isogenic KO and HSMR disease-causing mutations on neuronal development and function. Mg2+ levels, neurite and synaptic development, and Ca2+ signaling will be examined in mouse and human iNs. Proteomics will determine expression changes and rescue experiments will uncover which phenotypes are Mg2+ specific. By using both mouse and human stem cell lines, we will be able to determine whether CNNM2 function is conserved across species. Unbiased, high throughput methodologies (robotics, high content CellInsight imaging platform, proteomics) will be utilized to increase productivity, statistical power and rigor. These studies will provide critical understanding of how intracellular neuronal Mg2+ homeostasis is maintained and how its disruption affects neuronal function and development and contributes to CNS disorders, including epilepsy and HSMR.

PROJECT NARRATIVE Mg2+ plays an essential role in neurobiology but despite its importance, how neuronal Mg2+ homeostasis is regulated and how its dysfunction contributes to clinical symptoms remains unexplored. In this proposal CNNM2, a Mg2+ transporter, is leveraged as a biologically relevant entry point to understand these questions. CNNM2 expression and protein composition is characterized in both mouse and human neurons and the effect of disease-relevant mutations on neuronal development is determined.