Researchers at the Woods Hole Marine Biological Laboratory have for the first time identified a short peptide which ‘tags’ cellular cargo for transport along a neuron’s axon. The peptide is a short fragment of amyloid precursor protein, which many believe is responsible for the neuropathology of Alzheimer’s disease.
Axonal transport is the mechanism by which materials are moved along the axon. This transport can take place slowly or quickly, in two directions: in anterograde (or ‘forward’) transport, cargo is moved away from the cell body, and in retrograde (‘backward’) transport, it is moved towards the cell body. For example, voltage-gated ion channels and receptors destined for the synapse are synthesized in the cell body and packaged into membrane-bound vesicles, which are then latched onto the transport machinery and moved along the axon to the nerve terminal. Neurotrophic factors, which are essential for maintenance of the neuron, bind to cell surface receptors and transported back towards the nucleus, where they alter gene expression. Axon transport is also required for repairing any damage that a neuron might incur. A hard knock to the elbow, for example, may bruise axons in the ulnar nerve. In order for this damage to be repaired, proteins synthesized in the neuronal cell bodies near the spinal cord are transported along the arm to the damaged site.
The axonal transport system is exploited by herpes viruses during infection of sensory neurons. Herpes simplex virus (HSV) causes cold sores. It enters the neuron at the nerve terminal, and is transported retrogradely to the cell body in the trigeminal ganglion, so that its genome can be integrated into that of its host cell. Another type of herpes virus, Herpes (or varicella) zoster, causes chicken pox and shingles. One of the symptoms of shingles is a band of small blisters on the chest and/or back. These bands are produced by the virus as it it is transported slowly along the axons toward the spinal cord. Inside the nucleus, herpes viruses replicate their genetic material, which is exported to the cell body and packaged to produce new viral particles. These new particles are then transported back to the nerve terminal, from where they escape to infect other cells.
Elaine Bearer and her colleagues have now elucidated some of the molecular mechanisms that make axonal transport possible. They have identified a peptide which tethers cargo that is destined to be moved to the axon transport infrastructure – the motor proteins which actively drag cellular components back and forth along networks of microtubules in the cell. The experiments were carried out in the Atlantic squid, Loligo pealei. Squid have giant axons that can be up to 10 cm in length; this axon has a diameter about 1,000 times that of an average human axon, and runs longitudinally. The giant axons originate in the stellate ganglion and project to muscles in the mantle, enabling the squid to swim quickly.
From earlier studies, Bearer’s team already knew that a fragment of amyloid precursor protein (APP) was associated with the herpes virus; their previous work had shown that individual viral particles have some 3,000 copies of the fragment attached to them. In light of this, they decided to investigate whether or not the fragment could mediate axonal transport by acting as a receptor for the transport machinery.
To test this possibility, they used the DNA sequence of APP to chemically synthesize the peptide. They then conjugated the peptide to herpes virus-sized fluorescent polystyrene microspheres. When these beads were injected into the giant squid axon, they were immediately transported away from the cell body, whereas beads conjugated to other peptides remained stationary at the injection site. When bead injection into the axons was preceded by an injection of a solution of the peptide, transport of the beads was reduced four-fold, most likely because the peptide bound to the transport motors, so that fewer binding sites were available for the peptide-bead conjugate.
Analysis of time-lapse sequences taken with a confocal microscope showed that the transport was sustained for more than an hour, during which the beads were transported about 2mm. The transport velocity was measured to be 0.5-0.9 micrometres per second. During transportation, the beads paused now and then for 4-6 seconds. Imaging also showed that multiple beads were being trans- ported in parallel tracks as well as in chains along the same microtubule. Some of the beads were seen to disappear and reappear, suggesting that some tracks
The peptide consists of the last 15 amino acid residues from one end (the carboxy terminus) of APP, and has been named APP-C. The peptide is highly conserved, and has an almost identical sequence in fruit flies, squid and humans, suggesting that it plays a crucial role in axon transport. This role is to tether cargo to the molecular motors then transport that cargo along microtubules in the nerve fibre. APP-C mediated only anterograde transport of the beads, which is carried out by the kinesin motor (left). It is likely that another tag is required for tethering cargo to dynein, the motor which transports cargo in the other direction.
Cellular processing of APP is complex; a number of enzymes are known to cleave the full-length protein to produce various fragments, which are probably involved in many different cellular functions. One of these fragments, amyloid-β (Aβ) consists of 39-43 amino acid residues; it is generated by the sequential cleavage of APP by two enzymes, β- and γ-secretase, and aggregates to form the plaques which are a characteristic pathology of Alzheimer’s disease.
The identification of the APP-C molecular ‘tag’ will provide a new tool for researchers investigating axonal transport; the ‘tag’ may also be used in anatomical studies to delineate neural pathways. Because disrupted axonal transport has recently been implicated in neurodegenerative diseases, the discovery of the tag may eventually lead to therapies for these conditions. It may also provide a means by which therapeutic proteins can be delivered to synapses damaged in disease or by toxins.