Novel tracers for in vivo studies of waste transport by fluid flows in the brain

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SUMMARY Interstitial fluid motion in the form of convective flow is hypothesized to contribute to amyloid beta (Aβ) and waste clearance by carrying extracellular proteins out of the brain. Failures of this system could contribute to Alzheimer’s disease (AD), but are some aspects of this theory are still speculative. For examples, while the inflow of fluid to the brain parenchyma is well documented, the outflow path is less clear. How exogenous tracers and brain proteins get from the tissue into the lymphatics is still an open question. There are at least two ideas. The glymphatic/lymphatic transport mechanism predicts that the inflow of fluid from the arteriole perivascular spaces would be balanced by an outflow through the venular perivascular spaces. The Intermural Peri-Arterial Drainage (IPAD) camp has identified that extracellular dyes and proteins aggregate near the basement membrane of the arteriole vessels and hypothesizes that this compartment provides a conduit for waste clearance. The IPAD idea predicts that waste exits along the arterioles, in the opposite direction of the glymphatic prediction, although both are thought to feed into the lymphatics. There are experimental challenges in studying this transport because fluid motion is usually tracked by injection of exogenous indicators that alter pressure balances and can only be used for acute measurements. To overcome these barriers, new genetically engineered secreted tracers (GESTs) which mimic endogenous protein production and are not complicated by artifacts from injection have been prototyped. Microinjected adeno-associated virus (AAV) vectors drive the expression of GESTs in neurons within a small volume of brain. The distribution of the secreted fluorescent protein using fluorescence imaging in both tissue sections and intravital imaging can be used to map the transport. Because this tracer is produced by neurons long after the injection of the viral vectors, pressures and fluid flow in the brain are not disturbed. In addition, proteins such as Aβ are also secreted by neurons, so that the GEST distribution will replicate native protein transport rather than the motion of exogenously injected tracers. While this strategy has shown promise, new capabilities are needed to address the specific questions about fluid transport of waste proteins. First, interstitial fluid flow rates in the brain require faster time resolution on the scale of minutes. A photoactivable version of the GEST is proposed so that a volume of tracer can be “highlighted” and tracked as it moves through the brain tissue. The direction and speed of motion could support or refute the different theories of flow-mediated waste clearance. Second, it is not possible to detect GESTs in areas of faster flow or low concentration such as the lymphatics. To detect the paths taken by secreted proteins, a new secreted, cell-permeant Cre is developed. This will be used in floxed-stopped reporter animals to turn on reporter genes in cells that encounter fluids carrying the Cre. Because the gene activation is permanent, this provides a cumulative assay of exposure to Cre with good sensitivity to low concentrations. Finally, these new assays will be used to investigate whether transport is altered in AD mouse models.