Abstract Body: Introduction
Apolipoprotein E (apoE) is a major risk factor for Alzheimer's, Parkinson’s, and cardiovascular disease. ApoE is secreted by hepatocytes in the liver where apoE incorporates into VLDL particles for systemic circulation and by astrocytes in the central nervous system (CNS) on discoidal HDL particles. Understanding the biology of apoE is challenging due to the number of cell types that secrete apoE and the wide variety of receptors that can bind and internalize apoE, including LDLR and LRP1.

Hypothesis
We hypothesized that a photoconvertible tagged apoE would enable precise spatiotemporal tracking of apoE trafficking between organs and cell types. We predicted that: 1) phototagged apoE would maintain normal lipidation and trafficking patterns when expressed in the liver, 2) photoconversion would allow us to track tissue-specific uptake of hepatic apoE, and 3) this system would reveal isoform-specific differences in apoE trafficking and distribution.

Method
We developed a phototagged form of apoE with N-terminal Kikume Green-Red (KikGR), a photoconvertible fluorophore that irreversibly converts from green to red fluorescence after excitation with a short wavelength light (405nm). After validating proper expression, lipidation, and photoconversion in vitro, we achieved robust liver expression using AAV-mediated transduction with successful in vivo photoconversion.

Results
Hepatic expressed KikGR-apoE was uptaken by enteric neurons in the small intestine and the proximal colon; a result of particular interest in the context of Parkinson’s disease as α-synuclein initially aggregates in enteric neurons before spreading in a prion-like fashion to the CNS. We also observed hepatic KikGR-apoE in the glomeruli of the kidney, consistent with the accumulation of apoE in glomerular disorders. We observed isoform-specific patterns of expression between KikGR-apoE2 and KikGR-apoE4 when expressed in apoE KO mice. Fractionation of the plasma showed that KiKGR-apoE was lipidated and present on the VLDL and HDL fractions, as expected. Plasma levels of KiKGR-apoE2 were much higher than KiKGR-apoE4, consistent with the known inability of apoE2 to bind LDLR.

Future Directions
To study apoE in the CNS, we are currently using an AAV9 vector in order to transduce astrocytes. Future work will utilize this model to understand apoE trafficking throughout systemic circulation and lymphatics in the context of both homeostasis and in disease pathogenesis.