Background

In the vertebrate visual system, retinal ganglion cells (RGCs) in the eye extend axons through the optic nerve to retinorecipient target areas in the brain that process visual information. Proteins perform many essential functions in cells, and the capacity to perform these functions depends on their subcellular distribution. Protein distribution in cells is tightly regulated, particularly in neurons, which are highly polarized with different cellular compartments in axons, dendrites, and cell bodies. A primary means of regulating protein distribution is by controlling their transport into axons and dendrites. To identify proteins that are transported from RGC somata into axons and synaptic targets in vivo, we labeled proteins in the retina in rodents using intravitreal injections of N-hydroxysuccinimidobiotin (NHS-biotin) and analyzed biotinylated proteins in optic nerves and their projections in the visual system using immunohistochemical, biochemical, and mass spectrometry methods. These studies identified thousands of proteins that are transported along RGC axons from the cell bodies in the retina to the presynaptic axon terminals in the target areas, including the lateral geniculate nucleus (LGN) and superior colliculus (SC) [1–3].

In vivo protein biotinylation with NHS-biotin has many advantages for the investigation of protein identification, distribution, and function: NHS-biotin is a membrane-permeable biotin reagent that binds covalently to exposed lysines and N-terminal amino acids of proteins, resulting in extensive protein labeling. Biotin is a naturally occurring molecule that binds to many proteins without altering their function or activities. NHS-biotin cannot be re-incorporated into other proteins after protein degradation because the succinimide group is quenched after reacting with amino groups and because the resulting biotin-tagged amino acids, such as biotinyl-lysine, cannot be charged onto tRNAs [4]. Injecting NHS-biotin in the relatively large intravitreal space in the eye, ~2 mm away from the neural retina, avoids damaging the retina or the optic nerve and allows NHS-biotin to be taken up by retinal cells that line the back of the eye, including RGC cell bodies. The intravitreal chamber serves as a natural reservoir for the injected NHS-biotin, increasing the opportunity for protein labeling. NHS-biotin-labeled proteins distribute throughout RGC cells, including axons and dendrites. The optic nerve exits the back of the eye and extends to the brain, so that proteins that are transported along the optic nerve to the LGN, SC, and other targets in the brain can be captured by isolating the optic nerves. Proteins that are transported to the retinorecipient targets can be identified and characterized by local dissection of the target areas [2]. We call this population of proteins the transported proteome, or the transportome. A subset of proteins that are transported from the RGC cell bodies to the LGN are further transported via geniculocortical axons to the visual cortex [1]. As additional uses, we applied this in vivo intravitreal protein labeling strategy to mice with mutations in motor proteins and demonstrated molecular mechanisms underlying protein transport [3]. Also, we used intravitreal injection of the noncanonical amino acid azidohomoalanine to label newly synthesized RGC proteins in a model of glaucoma-induced optic nerve injury, and identified changes in transport of newly synthesized proteins in response to optic nerve injury [5]. Additionally, we injected NHS-biotin unilaterally into one cortical lobe in mouse pups to label intercortical callosal axons [1]. Together, these studies represent a range of applications of intravitreal protein labeling strategies.

Here, we present a detailed protocol for intravitreal NHS-biotin injections, optic nerve dissection, and methods for minimally invasive and unbiased in vivo labeling of proteins and immunostaining of whole optic nerve samples to investigate protein transport along the entire optic nerve, enabling analysis of protein transport in the optic nerves of animals of different ages and genotypes.