A lab-on-a-chip platform for studying the subcellular functional proteome of neuronal axons

Abstract

Axons are long, slender processes extending from the cell bodies of neurons and play diverse and crucial roles in the development and function of nervous systems. Here, we describe the development of a chip device that can be used to produce large quantities of axons for proteomic and RNA analyses. On the chip surface, bundles of axons of rat hippocampal neurons in culture are guided to grow in areas distinct and distant from where their cell bodies reside. Fluorescence immunocytochemical studies have confirmed that the areas where these axons are guided to grow are occupied exclusively by axons and not by neuronal somatodendrites or astroglial cells. These axon-occupied parts are easily separated from the remainder of the chip and collected by breaking the chip along the well-positioned linear grooves made on the backside. One- and two-dimensional gel electrophoresis and Western blotting analyses reveal that the axons and whole cells differ in their protein compositions. RT-PCR analyses also indicate that the axons contain only a subset of neuronal RNAs. Furthermore, the chip device could be easily modified to address other issues concerning neuronal axons, such as the molecular composition of the axon substructure, the growth cone and shaft, the degeneration and regeneration processes associated with injured axons and the effects of extrinsic molecules, such as axon guidance cues and cell adhesion molecules, on the axon. With these diverse applications, the chip device described here will serve as a powerful platform for studying the functional proteome of neuronal axons.