Developing a toolbox to study proteome plasticity in brain endothelial cells - Neurons lack substantial energy stores and thus their function is critically dependent on the timely delivery of energy substrates in the blood. Precise control of brain blood flow is therefore essential for brain health. However, the exact mechanisms through which cerebral hemodynamics are regulated remain unclear. Furthering our understanding of this control is critical, as it is increasingly appreciated that disruption of brain blood flow is one of the earliest pathological events in Alzheimer’s disease and related disorders and may be a key contributory factor to disease progression and neurodegeneration. Thus, advancing our understanding of the mechanisms of brain blood flow control may reveal novel and much needed targets for therapeutic interventions. Neuronal energy requirements vary greatly across the wide range of tasks that they perform, and this raises the possibility that energy delivery mechanisms have to be flexibly adapted to meet these needs. Our recent work has revealed a novel and surprising form of plasticity in brain blood flow control mechanisms, which we refer to as “Vascular Signaling Plasticity” (VSP). Here, the strength of mechanisms for blood flow control in brain capillaries are reprogrammed according to changes in local neuronal energy needs. Indeed, our data show that when neuronal energy demands are increased in the barrel cortex, the strength of a key Kir2.1 channel- dependent capillary-to-arteriole electrical signaling pathway that controls blood flow is augmented through a mechanism involving epigenetic reprogramming of capillary endothelial cells which ultimately modifies membrane density of Kir2.1. Surprisingly, our further investigations into the molecular mechanism of VSP strongly suggest that this process involves ribogenesis which in turn reprograms the translatome of the cell. Using these exciting findings as a springboard, we propose to apply two technologies to capillary endothelial cells—RiboTag and Puro-PLA—to enable us to elucidate the mechanism of VSP in granular detail. In Aim 1, we will directly quantify ribogenesis in capillary endothelial cells undergoing VSP and we will use RiboTag technology to isolate ribosomes from brain capillaries and perform an unbiased RNA-seq screen to identify changes to the translatome of these cells during VSP. In Aim 2 we will apply puro-PLA to capillaries undergoing VSP, which will enable both the broad visualization of proteins undergoing translation, and the specific tracking of proteins of interest involved in blood flow control. Completion of this work will thus reveal novel aspects of the VSP mechanism and may identify novel therapeutic targets in brain capillaries and could lay the groundwork for much needed treatments aimed at protecting or rescuing blood flow in brain disorders with a vascular component.
