Edward Koenig

Axonal and presynaptic protein synthesis: new insights into the biology of the neuron.

The presence of a local mRNA translation system in axons and terminals was proposed almost 40 years ago. Over the ensuing period, an impressive body of evidence has grown to support this proposal -- yet the nerve cell body is still considered to be the only source of axonal and presynaptic proteins. To dispel this lingering neglect, we now present the wealth of recent observations bearing on this central idea, and consider their impact on our understanding of the biology of the neuron. We demonstrate that extrasomatic translation sites, which are now well recognized in dendrites, are also present in axonal and presynaptic compartments.

Synthetic mechanisms in the axon. IV. In vitro incorporation of [3H]precursors into axonal protein and RNA.

PREVIOUS studies (KOENIG and KOELLE, 1961; KOENIG, 1965~; KOENIG, 1967) provided evidence of a local, autochthonous synthesis of acetylcholinesterase (AChE) in the axon. In addition, it was observed that actinomycin-D, an inhibitor of DNAdependent synthesis (see REICH, 1964), when applied directly to peripheral nerve, produced an increased AChE activity, limited to the nerve segment treated (KOENIG, 1967). The actinomycin-D-induced increase was antagonized by puromycin and by 5-fluororotic acid, and therefore, indicated de novo enzyme synthesis. This finding suggested that the synthesis of protein in the axon might be regulated by a local DNA mediated mechanism. Since RNA was demonstrated in the mammalian axon (KOENIG, 1965b), the necessary protein synthesizing machinery appeared to be available. Hence, it seemed unlikely that AChE might be just an isolated example of protein synthesis, and likely that a significant protein synthesis could be demonstrated in uitro by measuring the incorporation of [3H]leucine into axonal protein. In addition, the in uitro incorporation of labelled precursors into axonal RNA and its inhibition by actinomycin-D would constitute a presumptive basis for a local DNA-dependent mechanism. This report is concerned, therefore, with: (1) the in vitro incorporation of [3H]leucine into axonal protein and the effects of actinomycin-D and chloramphenicol upon it; and (2) the in vitro incorporation of [3H]orotic acid and [3H]adenine into axonal RNA and the effects of actinomycin-D upon it.

SYNTHETIC MECHANISMS IN THE AXON—IIRNA IN MYELIN-FREE AXONS OF THE CAT*

ALTHOUGH differentiated for conducting excitation over long distances without attenuation, the axon in fulfilling this function appears as an enormous appendage of the cell. A comparison of the ultrastructure of the axon with that of the perikaryon reveals characteristicdifferences that reflect specialization both in terms of biochemistry and function. One of the earliest indications of regional cellular differences was the apparent lack of Nissl substance in the axon in contrast to its presence in the perikaryon and dendrites (SCHAFFER, 1893). This observation was repeatedly confirmed over the years with classical methods of staining with basic dyes. Confirmation of these observations was obtained at the ultrastructural level with the study of PALAY and PALADE (1955). These investigators, and others since, failed to discern ribonucleoprotein granules (i.e., ribosomes) in the axoplasm, in contrast to its rich distribution elsewhere in the neuron. Earlier, NURNBERGER, ENGSTROM and LINSTROM (1952) also reported that RNA was lacking in the axon on the basis of ultraviolet microspectrophotometry. The apparent absence of RNA in the axon seemed compatible with the view that axonal proteins were synthesized in the soma. An indication that all proteins were not exclusively synthesized in the cell body was shown by the studies of KOENIG and KOELLE (1961) and CLOUET and WAELscH( 1961) on the restoration of acetylcholinesterase (AChE) in the axon following irreversible inactivation by organophosphorus compounds. The results pointed to an axonal capacity for synthesizing AChE. More recently, observations were extended to axotomized, cholinergic nerves which provided additional evidence that the axon synthesizes AChE (KOENIG, 1965). I t was postulated that the axonal synthesis of AChE was an example of a local mechanism concerned with synthesis of proteins forming integral substituents of the axolemma (i.e. plasma membrane). A question at the time of the early study was how the apparent absence of RNA in the mammalian axon could be consistent with axonal synthesis of an enzyme. A qualified answer was forthcoming when .I.-E. EDSTROM, EICHNER and A. EDSTROM (1962) demonstrated that the giant axon of the Mauthner cell in young gold fish contained RNA. An extension of the work was carried on by A. EDSTROM (1964). In addition GRAMPP and EDSTROM (1963) reported that the lobster sensory stretch receptor axon contained RNA. In both species the concentration of RNA was less than that found generally in cell bodies.

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 (∼2 μm), elongated (∼10 μm) 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.

SYNTHETIC MECHANISMS IN THE AXON. I. LOCAL AXONAL SYNTHESIS OF ACETYLCHOLINESTERASE.

ON THE basis of a number of morphological and biochemical findings (WEISS and HISCOE, 1948; SAMUELS, BYARSKY, GERARD, LIBET and BRUST, 1951 ; WAELSCH, 1958; KOENIG, 1958; LAJTHA, 1961; MIANI, 1960, 1963; DROZ and LEBLOND, 1963; and others), the concept of a soma1 origin for axoplasmic constituents, particularly proteins, has gained general acceptance in recent years. The lack of demonstrable Nissl substance (SCHAFFER, 1893) and its ultrastructural counterpart ribonucleoprotein (RNP) granules (PALAY and PALADE, 1955)-presumably requisite protein-synthesizing machinery-in the axon further seemed to strengthen the hypothesis of axonal proteins being synthesized in the cell body and convected distally in the axon. However, observations contradicting an exclusive synthesis of axonal proteins in the perikaryon emerged from studies of AChE restoration following irreversible inactivation by organophosphorus inhibitors (KOENIG and KOELLE, 1960, 1961 ; CLOUET and WAELSCH, 1961a, 1961~). As stated earlier (KOENIG and KOELLE, 1961), the rationale for using organophosphorus compounds (e.g., DFP) was based upon the importance of not producing atypical alterations in the regional disposition of AChE which appeared likely to be induced by a state of growth (i.e. regeneration). Histochemical evidence available at the time (SCHWARZACHER, 1958) showed that AChE disappears from the cell body during the period of axon outgrowth following axotomy. Hopefully, what is revealed following irreversible inactivation is a restoration of AChE a t a rate which reflects its normal turnover. If the cell body serves as a continuous source of newly synthesized AChE, then a proximo-distal gradient should be established during the early period of AChE return, and this should diminish in steepness with time. On the other hand, if no proximo-distal gradient becomes evident and the rate of enzyme restoration is uniform along the nerve, a local axonal synthesis would be indicated. The results (KOENIG and KOELLE, 1961) showed a uniform rate of AChE restoration along the nerve. In view of the importance of validating the existence of a local mechanism for protein synthesis in the axon, it was deemed essential to repeat a part of the earlier

Ribosomal RNA in Mauthner axon: Implications for a protein synthesizing machinery in the myelinated axon

RNA was extracted from myelin-free Mauthner axons of the goldfish on a microscale and fractionated by microelectrophoresis. Microextracts showed the presence of nominal 26 SE, 18 SE, 5 SE and 4 SE components, which co-migrated with rRNA from fish brain. In addition, a non-ribosomal 15 SE component was present in axon microextracts, but not in RNA extracts of fish brain or of myelin sheath from Mauthner axon, indicating an unusual enrichment of a putative mRNA class. Evidence was presented to support the contention that axonal rRNA was not due to contamination from the myelin sheath. Possible reasons for the lack of ultrastructural evidence for axoplasmic ribosomes are discussed.

RNA Trafficking in Axons

A substantial number of studies over a period of four decades have indicated that axons contain mRNAs and ribosomes, and are metabolically active in synthesizing proteins locally. For the most part, little attention has been paid to these findings until recently when the concept of targeting of specific mRNAs and translation in subcellular domains in polarized cells emerged to contribute to the likelihood and acceptance of mRNA targeting to axons as well. Trans‐acting factor proteins bind to cis‐acting sequences in the untranslated region of mRNAs integrated in ribonucleoprotein (RNPs) complexes determine its targeting in neurons. In vitro studies in immature axons have shown that molecular motors proteins (kinesins and myosins) associate to RNPs suggesting they would participate in its transport to growth cones. Tau and actin mRNAs are transported as RNPs, and targeted to axons as well as ribosomes. Periaxoplasmic ribosomal plaques (PARPs), which are systematically distributed discrete peripheral ribosome‐containing, actin‐rich formations in myelinated axons, also are enriched with actin and myosin Va mRNAs and additional regulatory proteins. The localization of mRNAs in PARPs probably means that PARPs are local centers of translational activity, and that these domains are the final destination in the axon compartment for targeted macromolecular traffic originating in the cell body. The role of glial cells as a potentially complementary source of axonal mRNAs and ribosomes is discussed in light of early reports and recent ultrastructural observations related to the possibility of glial‐axon trans‐endocytosis.

Evaluation of local synthesis of axonal proteins in the goldfish Mauthner cell axon and axons of dorsal and ventral roots of the rat in vitro

Polypeptides labeled in axons, in the myelin sheath, and in nerve cell bodies with [(35)S]methionine were analyzed separately in cytologically pure microscopic samples after incubation in vitro. Labeled polypeptides were evaluated and compared in (i) the goldfish Mauthner (M) cell axon and its myelin sheath and (ii) dorsal and ventral root axons, corresponding Schwann cell/myelin sheaths, and dorsal root ganglion cells in the rat. In all cases, polypeptides in axonal samples were not labeled in the presence of cycloheximide. Labeling patterns in the M-cell axon and in axons of dorsal and ventral roots were complex, neuronal in character, and distinctive in comparison with the myelin sheath. In all instances, tubulin and actin were labeled. Evidence is adduced that the myelin sheath of the M-cell axon appears also to have a local capacity for endogenous protein synthesis. The results reported here suggest that a number of constituent proteins of slow transport groups in myelinated axons probably turnover during transport and are replaced by local endogenous protein synthesis.

Axonal protein synthesizing activity during the early outgrowth period following neurotomy

Abstract 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 [ 3 H] 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.

Myelinated axons contain β-actin mRNA and ZBP-1 in periaxoplasmic ribosomal plaques and depend on cyclic AMP and F-actin integrity for in vitro translation

Periaxoplasmic ribosomal plaques (PARPs) are periodic structural formations containing ribosomes, which are likely cortical sites of translation along myelinated fibers. β‐actin mRNA, and its trans‐acting binding factor, zipcode‐binding protein‐1, were co‐distributed within PARP domains of axoplasmic whole‐mounts isolated from goldfish Mauthner, rabbit and rat nerve fibers. The distribution of co‐localization signals of fluorophore pixels, however, was asymmetric in PARP domains, possibly indicative of endpoint trafficking of RNPs. β‐actin mRNA in RNA extracted from axoplasm of single Mauthner fibers was confirmed by RT‐PCR. A metabolically active isolated Mauthner fiber system, which required cAMP to activate translation, was developed in order to probe cycloheximide‐sensitivity, and the importance of the actin cytoskeleton. cAMP greatly stimulated protein synthesis in axoplasm after a period of pre‐incubation, while being inhibited strongly by cycloheximide, or by cytochalasin D. Cytochalasin D reduced incorporation only modestly in the associated myelin sheath. We conclude that mechanisms for targeting and localizing β‐actin mRNA to discrete PARP domains are probably similar to those described for dendritic synaptic domains. Moreover, optimal translation in axoplasm depends on the integrity of the actin cytoskeleton, and can be modulated by cAMP as well.