Axonal Protein Synthesis Research Articles

Local protein synthesis in invertebrate axons: from dogma to dilemma.

In the past decade, it has become increasingly apparent that both axonal and dendritic domains of neurons have a natural propensity to synthesize proteins locally, i.e., independently of their cell bodies. A combined cell biological, molecular and neurobiological approach has now provided unequivocal evidence which serves to demonstrate not only the presence of various mRNA transcripts in the extrasomal regions, but also local protein synthesis. The identity and functional significance of various mRNAs and their translated products in axons and dendrites is also being revealed. Moreover, evidence is accumulating to suggest that the expression profiles of both mRNAs and their encoded proteins in the extrasomal regions are highly regulated by a variety of extrinsic stimuli. Because most evidence in support of axonal synthesis of proteins has primarily emanated from molluscan preparations, this review will thus remain mainly focused on invertebrate species such as squid, Aplysia and Lymnaea. Specifically, we will present evidence in support of the idea that both intact and isolated axons from the above molluscan species not only harbor a vast majority of mRNA species and protein synthetic machinery, but that they are also capable of de novo protein synthesis. The functional significance of local protein synthesis will also be discussed in the context of developmental and adult plasticity.KeywordsRough Endoplasmic ReticulumMolluscan SpeciesGiant AxonSquid Giant AxonRadiolabeled Amino AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cryptic Peripheral Ribosomal Domains Distributed Intermittently along Mammalian Myelinated Axons

A growing body of metabolic and molecular evidence of an endogenous protein-synthesizing machinery in the mature axon is a challenge to the prevailing dogma that the latter is dependent exclusively on slow axoplasmic transport to maintain protein mass in a steady state. However, evidence for a systematic occurrence of ribosomes in mature vertebrate axons has been lacking until recently, when restricted ribosomal domains, called "periaxoplasmic plaques," were described in goldfish CNS myelinated axons. Comparable restricted RNA/ribosomal "plaque" domains now have been identified in myelinated axons of lumbar spinal nerve roots in rabbit and rat on the basis of RNase sensitivity of YOYO-1-binding fluorescence, immunofluorescence of ribosome-specific antibodies, and ribosome phosphorus mapping by electron spectroscopic imaging (ESI). The findings were derived from examination of the axoplasm isolated from myelinated fibers as axoplasmic whole mounts and delipidated spinal nerve roots. Ribosomal periaxoplasmic plaque domains in rabbit axons were typically narrow ( approximately 2 microm), elongated ( approximately 10 microm) sites that frequently were marked by a protruding structure. The domain complexity included an apparent ribosome-binding matrix. The small size, random distribution, and variable intermittent axial spacing of plaques around the periphery of axoplasm near the axon-myelin border are likely reasons why their systematic occurrence has remained undetected in ensheathed axons. The periodic but regular incidence of ribosomal domains provides a structural basis for previous metabolic evidence of protein synthesis in myelinated axons.

Protein synthesis in axons and its possible functions.

Proteins synthesized in neuronal cell bodies are transported along axons by fast and slow axonal transport. Cytoskeletal proteins and cytosolic proteins that travel by slow axonal transport could take years to reach the terminals of meter-long axons, and it is difficult to see how proteins could last long enough to make this journey. How then are proteins supplied to the distal regions of long axons? Evidence has accumulated indicating that axons contain specific mRNAs and ribosomes and can synthesize cytoskeletal proteins and some other proteins. This review considers the direct evidence that proteins can be synthesized in axons and considers the possible functional significance of axonal protein synthesis. It remains unclear whether local protein synthesis could supply the cytoskeletal proteins and other slow-transported proteins required for the maintenance, plasticity, and regeneration of long axons.

Axonal protein synthesis and transport.

Recent evidence has challenged our ideas about the nature of axonal protein synthesis and transport. Previous metabolic labeling evidence supported the idea that all axonal proteins were synthesized in the cell body and then transported as formed cytoplasmic structures into the axon. Recent evidence suggests that neither the synthesis nor the transport of axonal proteins is that simple. Though most axonal proteins do appear to be synthesized in the neuronal cell body, a small amount of protein appears to be synthesized intra-axonally in some axons. Though small in amount, intra-axonal protein synthesis may be important functionally in some axons. Recent experiments have also begun to identify the presence of a rich array of transport motors in axons, including many members of the kinesin, dynein and myosin families. Progress is being made in identifying which cargoes are being transported by which of these motors. Finally, recent experiments have addressed an old question about whether axoplasmic proteins are transported as filamentous polymers or as soluble components in axons. The answer is that both mechanism can be used in axons. For example, neurofilament protein can move in its particulate or polymeric state, while tubulin can move in its soluble or unpolymerized state.

Systemic hypothermia following spinal cord compression injury in the rat: axonal changes studied by β-APP, ubiquitin, and PGP 9.5 immunohistochemistry

Systemic hypothermia exerts neuroprotective effects in experimental ischemic CNS models caused by vascular occlusions. Recent experimental and clinical studies have also demonstrated beneficial effects of hypothermic treatment following brain trauma. The present study addresses the question as to whether systemic hypothermia has similar protective qualities following severe spinal cord compression trauma using beta-APP-, ubiquitin-, and PGP-9.5-immunohistochemistry combined with the ABC complex method as markers to identify axonal changes. Fifteen rats were randomized into three equally large groups and sustained to either thoracic laminectomy or to severe spinal cord compression trauma of the Th 8 - 9 segments. The non-trauma group contained laminectomized animals submitted to a hypothermic procedure in which the core temperature was reduced from 38 to 30 degrees C. The two trauma groups were either submitted to the same hypothermic procedure or kept normothermic during the corresponding time. All animals were sacrificed 24 h following the surgical procedure. In the hypothermic non-trauma group no axonal changes were seen. The number of abnormal axons, as indicated by accumulation of immunoreactive material in enlarged axons, was lower in the peri-injury zones of the hypothermic trauma group than in the normothermic trauma group. This difference was most obvious in the cranial peri-injury zones. No differences were seen between the groups in the trauma zones. This study demonstrates reduced axonal swelling in the peri-injury zones of spinal cord injured rats treated with systemic hypothermia. These changes could either indicate neuroprotective effects of the hypothermic treatment, or be results of reduced axonal transport or protein synthesis. To evaluate the clinical importance of our findings, further studies including reliable outcome measures of the animals must be performed.

Synthesis of β-Tubulin, Actin, and Other Proteins in Axons of Sympathetic Neurons in Compartmented Cultures

The proteins needed for growth and maintenance of the axon are generally believed to be synthesized in the cell bodies and delivered to the axons by anterograde transport. However, recent reports suggest that some proteins can also be synthesized within axons. We used [35S]methionine metabolic labeling to investigate axonal protein synthesis in compartmented cultures of sympathetic neurons from newborn rats. Incubation of distal axons for 4 hr with [35S]methionine resulted in a highly specific pattern of labeled axonal proteins on SDS-PAGE, with 4 prominent bands in the 43-55 kDa range. The labeled proteins in axons were not synthesized in the cell bodies, because they were also produced by axons after the cell bodies had been removed. Two of the proteins were identified by immunoprecipitation as actin and beta-tubulin. Axons synthesized <1% of the actin and tubulin synthesized in the cell bodies and transported into the axons, and 75-85% inhibition of axonal protein synthesis by cycloheximide and puromycin failed to inhibit axonal elongation. Nonetheless, the specific production by axons of the major proteins of the axonal cytoskeleton suggests that axonal protein synthesis arises from specific mechanisms and likely has biological significance. One hypothetical scenario involves neurons with long axons in vivo in which losses from turnover during axonal transport may limit the availability of cell body synthesized proteins to the distal axons. In this case, a significant fraction of axonal proteins might be supplied by axonal synthesis, which could, therefore, play important roles in axonal maintenance, regeneration, and sprouting.

Reduced protein synthesis in diabetic retina and secondary reduction of slow axonal transport

Protein synthesis in the retina in diabetic rabbits decreased as the severity of the diabetes increased, but protein degradation and replacement were not affected. The reduction in the amount of orthograde slow axonal transport in diabetic rabbits closely paralleled the reduction in the amount of fast axonal transport and protein synthesis in the retina. The reduction in slow flow is most probably a simple reflection of reduced protein synthesis in the retinal ganglion cells. The relationships among reduction in axon diameter, volume of perikaryon, slow axonal transport and diabetic neuropathy are discussed.

Release of protein from axons during rapid axonal transport: An in vitro preparation

An in vitro system from the frog was used to study fast axonal transport and determine if transported protein is released from the axons. This preparation included the eighth and ninth dorsal root ganglia with their roots, sciatic nerve and gastrocnemius muscle. The preparation was placed in a three-compartment chamber with each compartment separated by a silicone grease barrier. The dorsal root ganglia were incubated in [ 14C]leucine for 5 h in compartment A. The labeled protein was transported down the axon from compartment A to compartment B. The sciatic nerve in compartment B was superfused with frog Ringer. This solution was collected in hourly samples and dialyzed to remove unincorporated leucine before counting. Incubating the ganglia in 100 μg/ml cycloheximide in frog Ringer blocked the release of labeled protein from the axon. Superfusing compartment B with solution containing 100 μg/ml cycloheximide inhibited axonal and Schwann cell protein synthesis, but did not block the release of labeled protein. It was concluded that the labeled protein released into the superfusing solution was synthesized in the ganglia and transported to the axon before release. SDS acrylamide gels were used to separate the labeled proteins. Sectioning the gels in 2 mm slices and determining the radioactivity showed that 80–85% of the counts were contained in two fast moving bands.

The action of cycloheximide on the action potential and protein synthesis in medullated Xenopus axons.

Cycloheximide depresses maximum rate of change in membrane potential observed during the rising phase of the action potential in single medullated axons of Xenopus. To,e course of depression is independent of cycloheximide concentration over a range that almost completely inhibits leucine incorporation into axonal proteins.

Influence of nerve cell body and neurolemma cell on local axonal protein synthesis following neurotomy

The influence of the nerve cell body and neurolemma cell on selected aspects of local axonal protein synthesis as a consequence of neurotomy was studied in the hypoglossal nerve. The onset of the phase of rapid local protein synthesis in axons of the proximal stump, occurring 12–15 hr after transection, was not affected by decentralization at the time of neurotomy. This indicated that the perikaryon was not required to induce the rapid local synthesis response. Moreover, other experiments demonstrated that this response could be induced in intact nerve in regions subjected to gentle blunt dissection of the nerve away from surrounding tissue. Incorporation of [ 3H] leucine into axonal protein in proximal stumps, 18 hr after transection, occurred without apparent delay and proceeded at a linear rate through 2 hr in vitro. In addition, incubation in vitro with colchicine produced no significant effect on local synthesis of axonal protein. These findings were consistent with an intra-axonal site of protein synthesis and a local induction of the phase of rapid axonal protein synthesis.

Axonal protein synthesizing activity during the early outgrowth period following neurotomy

Axonal protein synthesizing activity in the rabbit hypoglossal nerve was assayed in vitro 12–96 hr following unilateral nerve transection. Microscopic samples of myelin-free axons from the central stump region and a more proximal region were analyzed directly for total protein content and corresponding radioactivity using microchemical methods. Fifteen to 21 hr following transection, there was a doubling of protein content in axons from the proximal stump region, and a delayed increase in axons located more proximally. These findings were consistent with the reported damming of cytoplasmic debris within axons following nerve injury. At the same time intervals, there was a greater than 20-fold increase in protein synthesizing activity, normalized to axonal protein content of zero-hr control nerve. With [ 3H] Leucine incorporation into axonal protein, almost complete inhibition by cycloheximide but not by chloramphenicol occurred. The results show that there is a phase of rapid local axonal protein synthesis in the region of nerve injury which is induced after a latent period of 12–15 hr following transection, and which peaks at 18 hr. This phase is almost completed by 24 hr, and is followed by a phase of elevated protein synthesis which is about twice that of control and constant through 96 hr after transection.

The transport of proteins in Mauthner axon in fish as studied by autoradiography and interference microscopy

AbstractUsing 1 C‐Leucine the rate of transport of proteins in single nerve fiber was investigated by quantitative autoradiography using the desheathed, hand‐free isolated Mauthner axons of fish.After different times of incubation of living fish in the radioactive medium a non‐uniform labelling of the isolated axons along their length was observed, which showed a characteristic pattern. The distribution of the label indicates: (a) a continuous supply of labelled proteins from the cell body to the axon, (b) accumulation of labelled proteins near the terminal end of the axon, (c) two different rates of the somato‐axonal transport of proteins (30–40 mm 24hr; 3.5 mm/24 hr).After the transection of the spinal cord no incorporation into the segments of the axon below the lesion was detectable.No changes of the dry mass between the proximal and distal parts of the Mauthner axon were detectable in interference microscope.The alternative explanations for the observed pattern of axonal labelling, i.e. local protein synthesis in axons and somato‐axonal flow of proteins, are discussed.