Mechanisms of leptin transport across the human blood-brain barrier

Novel strategies are needed to treat obesity and its associated metabolic detriments. This research proposal seeks to clarify the mechanisms by which leptin, an integral hormone that regulates energy utilization and metabolism, is transported through the blood-ˇbrain barrier (BBB) and into the brain where it exerts its primary effects. Paradoxically, obese individuals have increased levels of leptin in the bloodstream, but leptin transport across the BBB is reduced. The mechanisms behind this so-ˇcalled “peripheral leptin resistance” are unknown, mainly because the receptors and transporters that shuttle leptin across the BBB are also unknown. We hypothesize that if this leptin transport machinery is properly characterized, it can be directly manipulated to increase leptin brain uptake in obese individuals, thereby providing a novel route for treating obesity and obesity-ˇrelated behaviors. To identify this leptin transport machinery, we will utilize: (1) robust human brain endothelial cell sources that appropriately mimic BBB behavior, and (2) cutting-ˇedge targeted and high throughput CRISPR techniques. Aim 1 will assess leptin uptake in BBB endothelial cells derived from human induced pluripotent stem cells (iPSCs) after treatment with a library of small molecule agonists. After identification of compounds that increase leptin uptake, RNA sequencing will be conducted to profile upregulated genes that are potentially responsible for this increase. Gene knockouts will then be generated in the iPSC-ˇderived BBB model to validate their role in leptin transport. Aim 2 will assess leptin uptake in an immortalized BBB endothelial cell line after it is transduced with a genome-ˇwide CRISPR activation library, such that every cell overexpresses a single gene. Positive selection and RNA sequencing will be used to determine genes whose upregulation consequently increases leptin uptake. The relevance of each gene for leptin transport will then be confirmed in the iPSC-ˇderived BBB model. The combination of targeted and high throughput assays maximizes the odds of success for identifying novel contributors and regulators of leptin transport into the brain. This work is expected to have an important positive impact on the field of obesity research by providing insight into a central metabolic question that has remained unanswered for over 20 years. Furthermore, outcomes from this research will motivate exciting in vivo studies to validate the identified mechanisms, as well as create immediate opportunities for drug screening campaigns to specifically target the leptin transport machinery.