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Biological Research

Print version ISSN 0716-9760

Biol. Res. vol.34 n.2 Santiago  2001

http://dx.doi.org/10.4067/S0716-97602001000200014 

The autonomous axon: a model based on local synthesis
of proteins

JAIME ALVAREZ

Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.

Corresponding Author: Jaime Alvarez. Facultad de Ciencias Biológicas. Pontificia Universidad Católica de Chile. Casilla 114-D. Santiago, Chile. e-mail: jalvarez@genes.bio.puc.cl. Tel.: 686 2609. Fax: 686 2717

Received: May 29, 2001. Accepted: July 10, 2001

ABSTRACT

In the current understanding of axons, axoplasmic synthesis of proteins is negated, and it is asserted that proteins are transported from perikarya to axons. This 'transport model' in which axons are fully dependent of perikarya is seriously flawed. The 'autonomous axon' proposed here negates in turn transport of proteins, and asserts (i) local synthesis of axoplasmic proteins, corner stone of this model, (ii) existence of internal programs in axon and terminals, (iii) external control of programs resulting in local regulation of phenotype of axons and terminals, hence they are autonomous from perikarya; (iv) participation of perikarya through the fast transport in setting up the axonal programs but not in their immediate regulation. The word merotrophism (meros meaning part) denotes post-transcriptional regulation of phenotype of restricted regions of a cell. We surmise that merotrophism is at the base of many plastic phenomena.

Key words: plasticity, regeneration, Schwann cell, slow transport, sprouting, trophism, Wlds

In the fifties, a picture of axons was emerging based on the idea that perikarya supplied axons with proteins and that the axoplasm did not synthesize proteins. The study of protein synthesis in axoplasm began in the sixties. Today, the transport model is accepted, i.e., perikarya are believed to supply intrinsic axoplasmic proteins by a slow transport mechanism. I contend that this model is wrong, and present instead a novel axon model based on local synthesis of proteins. The essentials of the models are shown in Fig. 1.

Slow axoplasmic transport model and its flaws

Weiss and Hiscoe (1948) studied the reversible ballooning of axons central to a constriction and their distalward thinning, and proposed that the axoplasm was streaming from perikaryon to terminals at about 1-2 mm/day, which roughly agreed with the velocity of nerve regeneration. This model predicts that proteins should spend in transit several years in axons of man and about half of a century in those of blue whales, which is sufficient for complete degradation of proteins along the way. This conundrum was overcome by assuming that proteins were degraded only at the terminals. This very assumption, however, called for further assumptions as it predicted an infinite swelling of axons above constrictions, which was not the case. Ribosomes, as studied with the conventional electron microscope, were not observed in the axoplasm (Palay and Palade, 1955). This has been taken ever since to signify that axons cannot synthesize proteins.

Droz and Leblond (1963) observed radioactive waves in nerves after supplying amino acids to perikarya, and Hoffman and Lasek (1975) established that the label was held in axoskeletal proteins. Waves lost radioactivity as they moved in time and space, good evidence for protein degradation, despite this, they reasserted the metabolic stability of proteins in axons (Lasek and Hoffman, 1976). That the axoplasm was unable to synthesize proteins and that perikarya supplied axons with proteins became standard textbooks notions, and so remain to the present day (Kandel et al, 2000). It is currently debated whether proteins are transported as soluble subunits (Hirokawa et al, 1997) or as assembled polymers (Baas and Brown, 1997).

The vulnerable spot of the transport model was, and still is, the assertion of virtual indestructibility of proteins in axons upon which the whole construct rests. On the one hand, this assertion is extreme, and on other, it is contradicted by experimental evidence (vide supra). Nixon (1980) addressed in optic nerves the issue of protein degradation, and established a half life of less than two weeks for the slowly transported proteins, and re-cycling of released amino acids. Hence proteins should disappear in the first few cm of axons, which makes the transport model untenable. Supporters of this model have not discussed the crucial consequences of protein degradation in axoplasm.

Since the transport model is flawed at its root, unsupported assumptions must be added time and again to rescue the model.

As a token, sensory perikarya should maintain axoplasmic masses whose range is 1:5500, which is not reflected in the perikaryonal protein synthesis whose range is barely 1:35 (von Bernhardi and Alvarez, 1989). For an extensive discussion of flaws, see Alvarez et al (2000).

Local synthesis of axoplasmic proteins

The study of protein synthesis in axons began in the sixties. In squid, fish and mammals, axons were shown to incorporate amino acids into proteins by a mechanism sensitive to ordinary inhibitors of protein synthesis (for reviews, see Koenig, 1984; Koenig and Giuditta, 1999; Alvarez et al, 2000). The bulk of protein synthesized in the axoplasm is sizeable. In the Mauthner neuron of the goldfish, incorporation of amino acids into the axoplasm is about 2.5% of that in the perikaryon by unit of volume; however, owing to the enormous axoplasmic volume, total incorporation is 30 times that of the perikaryon (Alvarez and Benech, 1983).

The kinetics of protein synthesis in axons is amenable to experimental manipulation. Inhibitors of protein synthesis should unbalance the steady state (see Fig. 1, Local synth.) resulting in a depletion of cytoskeletal proteins. Cycloheximide and emetine applied locally to sciatic nerves for a week reduced the microtubule content by half in a reversible manner, while adjacent segments remained normal (Bustos et al, 1991). The explanation of this result is trivial within the local synthesis model, while it requires ad hoc assumptions within the transport model.

Biochemical evidence for tRNA, rRNA and mRNA coding for axoplasmic proteins has been produced as well (for reviews, see Koenig, 1984; Koenig and Giuditta, 1999; Tiedge et al, 1999). Recently, Spencer et al (2000) showed that an isolated axon of snail injected with a foreign mRNA coding a G-protein-coupled conopressin receptor expressed the protein and responded to the specific ligands. That is, an axon is able to translate mRNAs and to direct the proteins to their target.

Both slow transport and local synthesis can supply the axoplasm with proteins as they do not exclude each other. Although this compromise is logically consistent, it is not biologically appealing because defined proteins should spend years in transit to reach their destination and the same time they are produced locally. We surmised that the axoplasm had only one source for its intrinsic proteins. Hence we dismissed the transport model because neurons cannot comply with its tenets whereas the local synthesis model can in principle account for axons of any length in steady or dynamic states (Alvarez, 1992). The fact that ribosomes were not seen in the axoplasm was a serious drawback for the model. We argued, though, that not to see belongs to the observer while not to exist belongs to the object. As a research program, the local synthesis model had two major challenges, (i) explaining the radioactive waves moving distalward -hard evidence for the transport of proteins- without further assumptions, and (ii) producing experimental evidence for ribosomes in axons.

Axons can be represented as an array of compartments (Fig. 1). In the transport model, velocity of translocation is the only variable subject to regulation. The local synthesis model involves ordinary processes that are subject to regulation: synthesis and degradation of proteins (which remain stationary), and uptake, leakage, and diffusion of amino acids. In the local synthesis model, transfer of amino acid residues (half arrows in Fig. 1, Local synth.) define a system of differential equations. The system was solved by the method of finite differences with parameters taken or derived from published data. The simulated pulse of radioactivity given to the perikaryon retrieved a set of curves similar to published experimental waves (Alvarez and Torres, 1985). Thus radioactive waves can move distalward through stationary proteins. The first challenge of the model was overcome and made the notion of transport a misinterpretation of data; however the transport model has not been discredited nor the notion of local synthesis has been accredited.

Figure 1. Models of axons. Drawing represents the Aut(onomous) axon. The axon has a machinery for protein synthesis (polyribosomes), a sprouting program (fork), and a destruction program (bomb). The Schwann cell represses the axon (-) which in turn specifies the Schwann cell (); this arm of the regulatory loop is beyond the scope of this paper. Diagrams, kinetics of local synthesis and slow transport models. Each diagram represents one of a linear array of compartments; axoplasm is between parallel lines; P is protein, and AA, amino acids. Local synth.: AA are used for synthesis of P, and released upon degradation of P (upper); P remain stationary; AA diffuse axially (), are taken up, and leak across the membrane (lower ). Slow transp.: P are synthesized in the perikaryon and transported distalward (); synthesis of P in axoplasm is formally negated; degradation of P () is formally negated but supported by data (see text); in this model AA play no role but were included for symmetry (redrawn from Alvarez et al, 2000).

Axoplasmic ribosomes

In recent years, evidence for axoplasmic ribosomes has been produced with electron spectroscopic imaging, YOYO-1 (a specific dye for RNA), and immunohistochemistry (Martin et al, 1989; Koenig and Martin, 1996; Sotelo et al, 1999; Koenig et al, 2000). Thus the other major challenge of the local synthesis model was conquered. Recently, with conventional electron microscopy, we found abundant ribosome-like particles in decentralized axons of Wlds mice (Fig. 2). These particles could be labelled with [H3]uridine administered to decentralzed Wlds nerves and also decorated with RNase-colloidal gold (Fig. 2). The evidence indicated that these particles were ribosomes, and that they contained newly synthesized RNA (Court and Alvarez, unpublished data). The up regulation of axonal ribosomes in decentralized Wlds axons excluded perikarya as their source while their labelling with uridine indicated a local source. Interestingly, Koenig (1970) had already proposed that Schwann cells transfer RNA to axons. The fine anatomy of Wlds fibers also supported the transfer hypothesis in that ribosomes were seen in the outer and the inner layers of the myelin sheath, between Schwann cell and axon, and in the cortical region of axons. Once we were trained to recognize the array of particles, we could find them also in intact axons of mice and rats, albeit they were few and widely scattered, in accordance with the low rate of amino acids incorporation into the axoplasm. Transfer of a ribosome implies transfer of a mRNA since it is required to organize the particle. In this regard, Schwann cells have been shown to contain mRNAs coding for neurofilament proteins, which they barely translate (Roberson et al, 1992; Sotelo-Silveira et al, 2000). A bold conjecture is that glia cells and more generally target cells supply axons and terminals with specific mRNAs to regulate locally their phenotype.

Figure 2. Ribosomes in severed Wlds axons. A, ribosomes in axoplasm appear as 19x23 nm particles. The black structure is the myelin sheath. Insets: enlargements to show ribosomes decorating membranes and a polyribosome. B, severed nerve injected with [3H]uridine and processed for high resolution autoradiography; silver grains overlay a field of ribosomes, a group of which is indicated (arrow). Inset, light autoradiography, the axoplasm of myelinated fibres is overlaid with silver grains. Bar, 0.2 µm.

Schwann cell control of axons and sprouting program

Motor axons span from the spinal cord through the roots to peripheral nerves, as also do sensory axons. Their microtubule density is twice as great in the spinal cord or peripheral nerves as it is in the roots (Fadic et al, 1985, Saitua and Alvarez, 1989). We proposed that local cues regulate axonal microtubules, and we allowed central branches of nodosal sensory neurons -that correspond to dorsal root fibers- to regenerate along the hypoglossal nerve.

Regenerated axons acquired the high microtubule density typical of peripheral axons. (Serra and Alvarez, 1989), and from this we proposed that Schwann cells were regulating axonal microtubules on a local basis. To test this hypothesis, we inhibited transcription in a short span of sciatic nerve with actinomycin D for a week. Schwann cells were destroyed while axons were healthy, presented sprouts, and had twice as many microtubules; in contrast, the adjacent segments were normal (Bustos et al, 1991). Thus we proposed (i) that axons embody a distributed sprouting program ready to go, (ii) that Schwann cells down regulate axonal microtubules and repress the sprouting program, and (iii) owing to the local nature of the effects, that the axonal responses are not directly controlled by perikarya. That is, the axon has intrinsic programs locally regulated whereby it is autonomous from the perikaryon.

In the frame of the local synthesis model, blockade of protein degradation predicts a local accumulation of cytoskeletal components. As a trial, we administered three serpins, leupeptin that permeates membranes, and aprotinin and the amyloid precursor protein with the Kunitz insert that do not. Within a week, inhibitors induced a mitotic burst in Schwann cells and a sprouting response in axons (Alvarez et al, 1992; 1995). Unknowingly, we had triggered the sprouting program. Schwann cells were present albeit proliferating. Thus repression of the sprouting program involved the differentiated Schwann cell and an extracellular protease; in contrast, the undifferentiated or proliferating cell became permissive for axonal growth. In this scenario, nerve regeneration was viewed as follows: interruption of an axon is followed by degeneration of the distal segment, next the corresponding Schwann cells dedifferentiate becoming permissive for growth, and finally sprouts extend at the amputated zone; the delay of nerve regeneration, 1-2 days in rodents, would reflect the fading of the repressive phenotype of distal Schwann cells. This conjecture was tested in the sciatic nerve; a segment was injected with aprotinin to induce Schwann cell proliferation and then the nerve was crushed: the delay of regeneration was obliterated (Tapia et al, 1995). This result supports that regeneration is a case of the ubiquitous sprouting program of axons, and is largely controlled on a local basis, without direct involvement of the perikaryon.

The Wlds strain (Wallerian degeneration slow) is a mutant of the C57BL inbred mouse. Cut Wlds nerves present a long delay of degeneration, 4 weeks rather than 1-2 days, and regeneration is also delayed even though amputated axons develop sprouts at the site of the lesion (Brown et al, 1994). This mutant was appropriate for further exploration of our ideas. Schwann cells of the distal stump remained differentiated after a lesion hence their repressive phenotype should be instrumental to prevent axonal regeneration. To test this conjecture, we crippled Schwann cells of a nerve segment with actinomycin D prior to the crush. Elongation of Wlds axons normalized (Court and Alvarez, 2000), supporting the notion that differentiated Schwann cells impair regeneration. The long survival of severed Wlds axons allowed us also to test whether or not the perikaryon was required for the sprouting response. We cut Wlds sciatic nerves and crushed a short span of the distal stump to destroy Schwann cells (Fig. 3). The resulting acellular nerve segment was invaded by sprouts extended retrogradely from the surviving decentralized axons (Iñiguez and Alvarez, 1999). Therefore, signals in the vicinity of axons are sufficient to trigger the sprouting program. Perikarya may enable the axonal programs through the fast transport but not control them directly.

Hyperactivity increases the size of motor nerve axons (Edds, 1950), and hyperactivity of sympathetic trunk for less than three days increases microtubule content and size axons (Alvarez et al, 1982). Within the frame of the local synthesis model, such increases require a complement of proteins hence their turnover in axons should be regulated by their discharge. We explored the rate of protein synthesis in the Mauthner axon of the goldfish, the spontaneous discharge of which is occasional (startling response). A steady stimulation of the axon at 0.3-0.8 Hz for 18 hours doubled the local incorporation of amino acids into axoplasmic proteins, returning to the baseline in one day, while stimulation for 4 hours produced no effect (Eugenín and Alvarez, 1995).

Figure 3. Sprouting of Wlds axons deprived of perikaron. Diagram of experimental condition. Vertical line is a cut through the sciatic nerve; the distal stump to the right has an extended crush (broken line) that destroyed Schwann cells. The undamaged axons (continuous line to the right) survive and extend retrograde sprouts into the crushed domain (see Iñiguez and Alvarez, 1999).


Destruction program

In normal nerves, a lesion determines the rapid destruction of distal axons. In Wlds nerves, the inordinately delayed degeneration is an intrinsic property of axons (Glass et al, 1993). In general, mutations cripple functions. Therefore, the long survival of Wlds axons is due to the missing function while the normal function determines the quick and active destruction of severed axons. Axonal degeneration has been called cytoplasmic apoptosis (Court and Alvarez, 2000; cf Ribchester et al, 1995) in that the destruction is active but involves only a part of the cell while the nucleus plays no role. Activation of calpains with calcium is instrumetal for axonal degeneration, and when calcium rises in Wlds axons, they are digested (Glass et al, 1994). Therefore, the Wlds mutation should alter the degeneration cascade previous to the rise of calcium. In cultured Wlds neurons, we found that the calcium flow through L-type channels was drastically reduced (Benavides and Alvarez, unpublished data). A lesion arrests the fast transport and induces degeneration of normal axons as also does colchicine, it is then plausible that the fast transport prevents the destruction of axons by controlling directly or indirectly the flow of calcium into the axoplasm. The link between fast transport and axonal degeneration was proposed by Lubinska (1977) when she found that both progressed at the same rate. The destruction program is the inverse of the sprouting program, and both probably operate in harmony for the continuous reshaping of axons and terminals.

Merotrophism

I want to comment on the control of the anatomy of axons in the frame of trophic phenomena. Trophism deals with control of phenotype and the mechanism implies regulation of gene expression; as a consequence the whole cell is involved. We have shown already that the axon varies its size, extends branches, or modifies its microtubule content on a local basis and without involvement of its perikaryon. The regulation of these phenotypic features of axons belongs to trophism but excludes as a mechanism the control of nuclear genes. Two important consequences follow. First, the expression of a given genetic program can give rise to more than one phenotype by controlling translation of existing mRNAs; second, a part of the cell can modify its phenotype while the rest of the cell remains unchanged. The word 'merotrophism' was coined to denote trophic phenomena of restricted region of a cell without involvement of the nucleus (Court and Alvarez, 2000). It is likely that merotrophic phenomena are more apparent in cells that have extended processes, e.g., neurons and oligodendrocytes, than in ordinary cells, although polarization of epithelial cells may be a case at stake.

Concluding remarks

The model proposed here breaks with the accepted notion of transport of axoplasmic proteins. In contrast, axons and terminals are endowed with the ability to synthesize proteins whereby they become autonomous from perikarya although greatly dependent upon local regulators. Axons and terminals are viewed as highly dynamic entities with internal programs to extend sprouts and to destroy axonal segments or sprouts. These programs support the local regulation of the phenotype of axons and terminals in response to their history and local cues. In the autonomous axon model, perikarya would enable the axonal programs through the fast transport but would not control directly the behaviour of axons. Many plastic phenomena are predicted to be macroscopic expressions of this autonomy. The autonomous axon model presented here sets many issues on a new scenario whereby it suggests a novel, and hopefully fruitful, avenue of experimental inquiry.

ACKNOWLEDGEMENTS

I dedicate this paper to all my students as this model was created by us all. I thank C.S. Koenig for critical reading of the manuscript. This work was supported by grants FONDECYT 1980973, 1990151, and FONDAP-Biomedicine 1398001, Chile.

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