Raymond J. Lasek

Afferents to the rat caudoputamen studied with horseradish peroxidase. An evaluation of a retrograde neuroanatomical research method

The potential for using the physiologically occurring retrograde axonal transport of protein as a neuroanatomical tool for identifying the origin of neuronal connections within the adult central nervous system has been evaluated for the case of the rat caudoputamen. Following injections of a marker protein, in this case horseradish peroxidase (HRP), centering on but not limited solely to the caudoputamen, peroxidase labeled cell bodies could be identified in several cell territories known or thought to contain afferents to this structure. These included the pars compacta of the substantia nigra, the intralaminar and parafascicular nuclei of the thalamus, and the dorsal nucleus of the midbrain raphe. In cases where only the caudoputamen and external segment of the globus pallidus were labeled directly at the injection site, no peroxidase- containing cells could be identified in the neocortex or in the thalamus outside the intralaminar and parafascicular nuclei. The evidence presented suggests first, that the degree of localization at the injection site is compatible with approaching some problems in neuroanatomy; second, that anterograde transport of the marker protein, if it occurs, does not appear to confound the interpretation of retrogradely labeled cell bodies; third, that many, though not all, afferent cell populations can be identified; and fourth, that labeling by axons in passage does not appear to be a problem. A detailed description of the method, abilities and limitations of the technique, and sources of misinterpretation are also provided.

Heat shock-like protein is transferred from glia to axon

Glia-axon protein transfer was examined in the squid giant axon. Proteins synthesized by the glial sheath surrounding the axon were labeled with [3H]leucine. Raising the temperature of the incubation medium from 20 degrees C to 30 degrees C increased the synthesis of glial proteins that resembled heat-shock proteins. These proteins were among the group known to be transferred into the axon. Thus, glia provide the axon with proteins that may be involved in the reaction to trauma.

Axonal transport of the cytoskeleton in regenerating motor neurons: constancy and change

We have examined slow axonal transport in regenerating motor neurons of the rat sciatic nerve. Using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) we previously found that the slow component is the vehicle for the axonal cytoskeletal proteins, i.e. the neurofilament triplet proteins, tubulin and actin. When these proteins are pulse-labeled by injecting [3H]- or [35S]-amino acids into the spinal cord, they are transported distally in the nerve as two distinguishable waves of radioactivity, SCa and SCb. In normal motor neurons, the neurofilament triplet proteins and the tubulin are transported in SCa at an average velocity of 1.7 mm/day; the less heavily labeled SCb which moves at 2-5 mm/day is the primary vehicle for actin. We now find that during regeneration the velocity of SCa is unchanged in the region of the axon between the cell body and the lesion, but the amount of labeled neurofilament triplet and associated tubulin transported in the axon is decreased in neurons which had been labeled 20 days post-lesion. In contrast, the labeling of the slowly transported proteins moving ahead of the neurofilament triplet is greater in regenerating nerves than in controls. On the basis of our findings, we propose that in motor axons the normal supply of cytoskeletal protein, which is continuously transported in the slow component, is sufficient to support regeneration. Nevertheless, the neuron cell body can alter the supply of these cytoskeletal proteins so as to enhance its regenerative capacity.

Nerve-specific enolase and creatine phosphokinase in axonal transport: soluble proteins and the axoplasmic matrix

The axonal transport of two soluble enzymes of intermediary metabolism was evaluated: the nerve-specific form of the glycolytic enzyme enolase (NSE) and the brain isozyme of creatine phosphokinase (CPK). Previously, little was known about the intracellular movements of the soluble proteins of the cell. Although the soluble enzymes of glycolysis and other pathways of intermediary metabolism have been thought to be freely diffusing in the cytosol, many are required in the axonal extremities of the neuron and must be transported to the sites of utilization. Comigration of purified enzymes with radioactive polypeptides associated with specific rate components of axonal transport in two-dimensional gel electrophoresis indicates that both NSE and CPK move in the axon solely as part of the group of proteins known as slow component b (SCb) at a rate of 2 mm/day. Peptide mapping following limited proteolysis confirmed identification of NSE and CPK in SCb. Materials associated with SCb have been shown to move coherently along the axon and to behave as a discrete cellular structure, the axoplasmic matrix. Association of two soluble enzymes, NSE and CPK, with the SCb complex of proteins requires a reevaluation of the assumption that these and other soluble proteins of the axon are freely diffusible.

Axonal transport of actin: Slow component b is the principal source of actin for the axon

Axonally transported proteins were studied in guinea pig retinal ganglion cells using the standard radioisotopic labeling procedure. Two slowly moving groups of proteins were identified in guinea pig retinal ganglion cells. The more slowly moving group of proteins, designated slow component a (SCa) was transported at 0.2-0.5 mm/day. Five polypeptides contained greater than 75% of the total radioactivity transported in SCa. Two of these polypeptides correspond to the subunits of tubulin, while the other three correspond to the slow component triplet. The other slowly moving group of proteins, which is designated slow component b (SCb), was transported at approximately 2 mm/day. Twenty labeled polypeptides were identified in SCb. The major labeled polypeptides transported in SCb differ from those transported in SCa. One of the polypeptides transported in SCb co-migrates with skeletal muscle actin in SDS-polyacrylamide slab gels. This polypeptide behaved identically to skeletal muscle actin on DNaseI affinity columns. Since DNaseI is a highly specific affinity ligand for actin, we conclude that the labeled SCb polypeptide which comigrates with actin in SDS-gels is actin. Between 1.4 and 5.7% of the total radioactivity transported in SCb is attributable to action. Detailed comparison of the distribution of total radioactivity in the optic axons with the distribution of radioactive actin in the optic axons at post-injection times between 6 and 77 days showed that actin was transported specifically in SCb, and not in SCa. Furthermore, analyses of the proteins transported in the fast component of guinea pig retinal ganglion cells by DNaseI affinity chromatography failed to reveal an actin-like moiety. Slow component a, SCb and the fast component are the major components of axonal transport in guinea pig retinal ganglion cells. Thus, in these neurons, actin is transported principally and possibly only in SCb. Guinea pig retinal ganglion cell axons project principally to the lateral geniculate nucleus and superior colliculus. The fate of actin axonally transported to the region of the axon terminals was studied by determining the kinetics by which radioactivity associated with actin accumulates and then decays in the superior colliculus. The results of these studies indicate that labeled actin has a half-life in the superior colliculus of approximately 28 days.

Electron microscopic observations of horseradish peroxidase transported from the caudoputamen to the substantia nigra in the rat: possible involvement of the agranular reticulum.

The intracellular distribution of horeseradish peroxidase (HRP) transported intraaxonally from the caudoputaminal complex to the substantia nigra has been examined with the electron microscope. The reciprocal axonal connections between the caudoputamen and the substantia nigra permitted observation not only of HRP transported retrogradely from axons and axon terminals in the caudoputamen to the cell bodies of origin in the pars compacta of the substantia nigra, but also provided information suggesting that HRP may be transported anterogradely by neurons of the caudoputamen to their terminals, which are especially numerous in the pars reticulata of the substantia nigra. Special attention was focused on observations which might elucidate the manner in which exogenous proteins are compartmentalized and transported intracellularly. It is suggested that the agranular reitculum is involved in the retrograde transport of proteins which are pinocytosed near the axon terminal and ultimately reach lysosomes in the perikaryon. A possible anterograde movement of HRP may also involve the agranular reticulum. The implications such findings have on the use of HRP in neuroanatomical tracing techniques are also discussed.

Polymer sliding in axons.

SUMMARY In axons the cytoskeletal polymers are transported by slow axonal transport. Microtubules, microfilaments and neurofilaments move at different rates in the axon. On the basis of their transport rates, two populations of polymers can be distinguished: SCb polymers are transported at 2–4 mm day−1 and SCa polymers are transported at 0.25–1 mm day−1. As they move within the axon, the faster moving SCb polymers must pass the slower moving SCa polymers. This observation and others indicate that polymers slide in the axon. A model of polymer sliding is presented. This model provides a dynamic architectural framework for studies of the mechanisms of slow axonal transport.

Retardation in the slow axonal transport of cytoskeletal elements during maturation and aging

Using the pulse-labeling method, the rate of the slow component (SC) of axonal transport was analyzed during maturation and aging. Ventral motor neurons and retinal ganglion cells of 3-, 6-, and 24-month-old Fischer 344 rats were radiolabeled with 35S-methionine. To measure the rates of SCa and SCb subcomponents, distributions of the total radiolabeled proteins and certain cytoskeletal proteins (actin, clathrin, tubulin, and the neurofilament proteins) were analyzed in the ventral root-sciatic nerve and optic nerve. Our results show that the rate of transport for both SCa and SCb proteins decreases with age in ventral motor axons and optic axons. For example, in ventral motor axons the rates of both SCa and SCb decreased 40% between 6 and 24 months. These results, with those of others, show that the rate of slow transport gradually decreases in the neurons of adult rats (7,11) The factors that may contribute to the slowing are discussed.

Function and evolution of neurofilament proteins.

The intermediate filament (IF) proteins have evolved by repeated duplications of a single ancestral gene (FIGURE 1). At least five subfamilies of IF proteins have been identified in mammals, and each subfamily is expressed preferentially in a particular type of differentiated cell: cytokeratins in epithelia, neurofilament (NF) proteins in neurons, glial fibrillary acidic protein (GFAP) in glia, desmin in myogenic cells, and vimentin in early embryonic cells and in mesenchymal cells.’-’ All of the IF proteins have a conserved 40 kD rod-shaped domain, which has probably been derived from the ancestral IF gene?-” This 40 kD domain is distinguished by its large content of alpha helical structure and by the presence of a highly conserved epitope that is recognized by a mouse monoclonal antibody that reacts specifically with all IF proteins.” It appears that the 40 kD domain has been highly conserved because of its essential role in IF IF proteins also contain two hypervariable regions that flank the conserved 40 kD d ~ m a i n . ~ These regions form the amino-terminal (head) and the carboxy-terminal (tail) portions of the IF proteins. Evolutionary variation in both of these regions has led to substantial differences between the subfamilies of IF proteins? In particular, the tail segment of the NF proteins has varied extensively and now contains a number of features not found in other IF proteins. One of the most evident differences between the tail segment of the NF proteins and other IF proteins is size. The tail segment of many NF proteins is larger than 100 kD, whereas the typical tail segment of other IF proteins is less than 5 kD.5,’ The tail region is heavily phosphorylated in higher molecular weight NF subunits16 and amino acid sequencing of the 68 kD subunit has identified a unique domain, the b domain, at the carboxy-terminal end of the protein.’ The b domain is characterized by an unusually high content of acidic residues (44% glutamic acid), and also contains the binding site for the Bodian

Bidirectional transport of radioactively labelled axoplasmic components.

IT is well established that axoplasm moves somatofugally from the neurone cell body toward the axon terminals1. Somatofugal axoplasmic transport was first demonstrated by Weiss and Hiscoe2, who found that if a peripheral nerve was constricted the axons proximal to the site of constriction became engorged with axoplasm. Recent variations of these experiments have shown that certain axoplasmic components (acetylcholinesterase1, catechol-amine storage granules3 and labelled phospholipids4) accumulate distal as well as proximal to the constriction. This tendency for axoplasmic components to accumulate on both sides of a lesion in a nerve has been interpreted as support for the idea that axoplasmic components move along the axons both from the cell body toward the axon terminals and back in the direction of the cell body.