The slow component of axonal transport. Identification of major structural polypeptides of the axon and their generality among mammalian neurons.

This study of the slow component of axonal transport was aimed at two problems: the specific identification of polypeptides transported into the axon from the cell body, and the identification of structural polypeptides of the axoplasm. The axonal transport paradigm was used to obtain radioactively labeled axonal polypeptides in the rat ventral motor neuron and the cat spinal ganglion sensory neuron. Comparison of the slow component polypeptides from these two sources using sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis revealed that they are identical. In both cases five polypeptides account for more than 75% of the total radioactivity present in the slow component. Two of these polypeptides have been tentatively identified as tubulin, the microtubule protein, on the basis of their molecular weights. The three remaining polypeptides with molecular weights of 212,000, 160,000, and 68,000 daltons are constitutive, and as such appear to be associated with a single structure which has been tentatively identified as the 10-nm neurofilament. The 212,000-dalton polypeptide was found to comigrate in SDS gels with the heavy chain of chick muscle myosin. The demonstration on SDS gels that the slow component is composed of a small number of polypeptides which have identical molecular weights in neurons from different mammalian species suggests that these polypeptides comprise fundamental structures of vertebrate neurons.

Control of axonal caliber by neurofilament transport.

The role of neurofilaments, the intermediate filaments of nerve cells, has been conjectural. Previous morphological studies have suggested a close relationship between neurofilament content and axonal caliber. In this study, the regenerating neuron was used as a model system for testing the hypotheses that neurofilaments are intrinsic determinants of axonal caliber, and that neurofilament content is controlled by the axonal transport of neurofilaments. This system was chosen because previous studies had shown that, after axotomy, axonal caliber was reduced within the proximal stump of the regenerating nerve and, because the relative amount of neurofilament protein undergoing axonal transport in regenerating axons was selectively reduced. The relationship between axonal caliber and neurofilament number was examined in a systematic fashion in both regenerating and control motor axons in rat L5 ventral root. Reconstruction of the spatial and temporal sequences of axonal atrophy in the proximal stump after axotomy showed that reductions in axonal caliber were first detected in the most proximal region of the root and subsequently progressed in a proximal-to-distal direction at a rate of 1.7 mm/day, which is identical to the rate of neurofilament transport in these neurons. Quantitative ultrastructural studies showed that these reductions in caliber correlated with a proportional decrease in the number of axonal neurofilaments but not microtubules. These results support the hypotheses that neurofilament content is a major intrinsic determinant of axonal caliber and that neurofilament content is controlled by the axonal transport of neurofilaments. On this basis, we suggest a role for neurofilaments in the control of axonal volume.

Expression of NF-L in both neuronal and nonneuronal cells of transgenic mice: increased neurofilament density in axons without affecting caliber.

We have generated transgenic mice containing additional copies of the murine NF-L gene in order to examine the consequences of neurofilament-L overexpression on axonal morphology. Founder mice were constructed to carry a transgene in which the presumptive 5' promoter sequences of NF-L were replaced with the strong murine sarcoma virus long terminal repeat promoter. The transgenes were expressed prominently in several tissues, including skeletal muscle and kidney where NF-L accumulated to approximately 2% of cell protein. This was not accompanied by an overt phenotype, except that expression in lens led to cataract formation. In the brains of these animals, transgene RNA levels exceeded the endogenous NF-L RNAs by up to 20-fold, although no additional protein accumulated, indicating posttranscriptional regulation of NF-L expression. However, in peripheral neurons transgene RNA was approximately fourfold higher than endogenous NF-L mRNAs, and a corresponding increase in NF-L subunits was found in axons arising from these neurons. Myelinated nerve fibers of transgenic animals contained increased numbers of NFs, assembled predominantly of NF-L. This was reflected in an increase in the density of axonal NFs; axonal caliber was not affected.

Axonal transport of class II and III beta-tubulin: evidence that the slow component wave represents the movement of only a small fraction of the tubulin in mature motor axons.

Pulse-labeling studies demonstrate that tubulin synthesized in the neuron cell body (soma) moves somatofugally within the axon (at a rate of several millimeters per day) as a well-defined wave corresponding to the slow component of axonal transport. A major goal of the present study was to determine what proportion of the tubulin in mature motor axons is transported in this wave. Lumbar motor neurons in 9-wk-old rats were labeled by injecting [35S]methionine into the spinal cord 2 wk after motor axons were injured (axotomized) by crushing the sciatic nerve. Immunoprecipitation with mAbs which recognize either class II or III beta-tubulin were used to analyze the distributions of radioactivity in these isotypes in intact and axotomized motor fibers 5 d after labeling. We found that both isotypes were associated with the slow component wave, and that the leading edge of this wave was enriched in the class III isotype. Axotomy resulted in significant increases in the labeling and transport rates of both isotypes. Immunohistochemical examination of peripheral nerve fibers demonstrated that nearly all of the class II and III beta-tubulin in nerve fibers is located within axons. Although the amounts of radioactivity per millimeter of nerve in class II and III beta-tubulin were significantly greater in axotomized than in control nerves (with increases of +160% and +58%, respectively), immunoassay revealed no differences in the amounts of these isotypes in axotomized and control motor fibers. We consider several explanations for this paradox; these include the possibility that the total tubulin content is relatively insensitive to changes in the amount of tubulin transported in the slow component wave because this wave represents the movement of only a small fraction of the tubulin in these motor fibers.

Changes in neurofilament transport coincide temporally with alterations in the caliber of axons in regenerating motor fibers.

The delivery of neurofilaments via axonal transport has been proposed as an important mechanism for regulating axonal caliber. If this hypothesis is correct, alterations in axonal caliber should appear coincident with changes in the delivery of neurofilaments to the axon. The purpose of this study was to determine whether alterations in the caliber of axons in the proximal stumps of transected motor fibers precede, coincide with, or occur substantially later than changes in the delivery of neurofilaments via axonal transport. Between 3 d and 12 wk after crushing the sciatic nerves of 7-wk-old rats, lumbar motor neurons were labeled by the intraspinal injection of [35S]methionine. In neurons labeled between 3 d and 6 wk after axotomy, the relative amount of neurofilament protein in the slow component, as reflected by the ratio of the radioactivities of the 145-kD neurofilament protein to tubulin, was reduced to 30-40% of the control value. Moreover, as determined by immunoreactivity on blots, the amounts of neurofilament protein and tubulin in these nerve fibers were reduced fourfold and twofold, respectively. Thus, changes in the ratio of labeled neurofilament protein to tubulin correlated with comparable changes in the quantities of these proteins in nerve fibers. This decrease in the quantity of neurofilament proteins delivered to axons coincided temporally with reductions in axonal caliber. After regeneration occurred, the delivery of neurofilament proteins returned to pre-axotomy levels (i.e., 8 wk after axotomy), and caliber was restored with resumption of normal age-related radial growth of these axons. Thus, changes in axonal caliber coincided temporally with alterations in the delivery of neurofilament proteins. These results suggest that the majority of neurofilaments in these motor fibers continuously move in the anterograde direction as part of the slow component of axonal transport and that the transport of neurofilaments plays an important role in regulating the caliber of these axons.

Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport.

To examine the mechanism through which neurofilaments regulate the caliber of myelinated axons and to test how aberrant accumulations of neurofilaments cause motor neuron disease, mice have been constructed that express wild-type mouse NF-H up to 4.5 times the normal level. Small increases in NF-H expression lead to increased total neurofilament content and larger myelinated axons, whereas larger increases in NF-H decrease total neurofilament content and strongly inhibit radial growth. Increasing NF-H expression selectively slow neurofilament transport into and along axons, resulting in severe perikaryal accumulation of neurofilaments and proximal axonal swellings in motor neurons. Unlike the situation in transgenic mice expressing modest levels of human NF-H (Cote, F., J.F. Collard, and J.P. Julien. 1993. Cell. 73:35-46), even 4.5 times the normal level of wild-type mouse NF-H does not result in any overt phenotype or enhanced motor neuron degeneration or loss. Rather, motor neurons are extraordinarily tolerant of wild-type murine NF-H, whereas wild-type human NF-H, which differs from the mouse homolog at > 160 residue positions, mediates motor neuron disease in mice by acting as an aberrant, mutant subunit.

Slow axonal transport of neurofilament proteins: impairment of beta,beta'-iminodipropionitrile administration.

beta,beta'-Iminodipropionitrile (IDPN) administration prevented normal slow axonal transport of [35S]methionine- or [3H]leucine-labeled proteins in rat sciatic motor axons. Ultrastructural and electrophoretic studies showed that the neurofilament triplet proteins in particular were retained within the initial 5 millimeters of the axons, resulting in neurofilament-filled axonal swellings. Fast anterograde and retrograde axonal transport were not affected. The IDPN thus selectively impaired slow axonal transport. The neurofibrillary pathology in this model is the result of the defective slow transport of neurofilaments.

Guidance for the decontamination of intracavity medical devices: the report of a working group of the Healthcare Infection Society.

BACKGROUND: Intracavity medical devices (ICMDs) are used in a wide variety of healthcare settings. The approach to their decontamination and the resources available also differ widely. Their potential for infection transmission is considerable. AIM: To produce a comprehensive risk assessment-based approach to the decontamination of ICMDs, accompanied by an adaptable audit tool.

Uniform: an evidence review of the microbiological significance of uniforms and uniform policy in the prevention and control of healthcare-associated infections. Report to the Department of Health (England).

A systematic search and quality assessment of published literature was conducted to establish current knowledge on the role of healthcare workers uniforms' as vehicles for the transfer of healthcare-associated infections. This review comprised a systematic search of national and international guidance, published literature and data on recent advances in laundry technology and processes. We found only a small number of relevant studies that provided limited evidence directly related to the decontamination of uniforms. Studies concerning domestic laundry processes are small scale and largely observational. Current practice and guidance for laundering uniforms is extrapolated from studies of industrial hospital linen processing. Healthcare workers' uniforms, including white coats, become progressively contaminated in use with bacteria of low pathogenicity from the wearer and of mixed pathogenicity from the clinical environment and patients. The hypothesis that uniforms/clothing could be a vehicle for the transmission of infections is not supported by existing evidence. All components of the laundering process contribute to the removal or killing of micro-organisms on fabric. There is no robust evidence of a difference in efficacy of decontamination of uniforms/clothing between industrial and domestic laundry processes, or that the home laundering of uniforms provides inadequate decontamination.

Neuronal and glial cytoskeletons.

Long-awaited evidence for in vivo functions of the major neuronal microtubule associated proteins indicates that they are directly involved in neurite extension. Companion evidence reveals an intrinsic role for glial intermediate filaments in glial cell extension along neurites and for neurofilaments in establishing axonal caliber. New fluorescence and photoactivation experiments require a re-thinking of models of slow axonal transport and of the part the cytoskeleton plays in axonal guidance.

Neurofilament antigens in acrylamide neuropathy.

After repeated exposure, acrylamide (AC) produces degeneration of distal axons. Because neurons whose axons have been injured (e.g. by axotomy) show alterations in their structural and chemical properties, the present study was designed to differentiate the direct effects of AC intoxication from neuronal responses secondary to axonal injury caused by AC. Rats were given AC as either a single high dose (75 mg/kg), or as daily intraperitoneal injections (30 mg/kg, six days per week for four weeks). Dorsal root ganglia of the fifth lumbar level, L5, were examined using a variety of monoclonal antibodies directed against nonphosphorylated (2-135) and phosphorylated (03-44, 06-17, 07-05) epitopes of 145 and 200 kilodalton neurofilament proteins. In control rats, antibody 2-135 stained axons and neuronal cell bodies; antibodies against phosphorylated epitopes of neurofilaments stained only axons distal to the glomerulus. Following chronic AC intoxication, all three antibodies directed against phosphorylated epitopes of neurofilaments (particularly 07-05) demonstrated intense immunoreactivity in 20-30% of neuronal cell bodies. In addition, the glomerular region of these axons was stained. Electron microscopy revealed many chromatolytic cells containing few neurofilaments. In contrast, a single high dose of AC produced no abnormal staining of neuronal cell bodies at a time when slow axonal transport was impaired. Our findings are compared to those observed following axotomy and to those occurring in aluminum-intoxicated rabbits, two experimental disorders in which altered distributions of phosphorylated filaments have been documented.

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.

Changes in the isotype composition of beta-tubulin delivered to regenerating sensory axons by slow axonal transport.

beta-Tubulin is encoded by a family of genes that produces at least five distinct polypeptide isotypes in neurons. Two of these isotypes (i.e., classes II and III) preferentially accumulate in axons, and the expression of one of them (i.e., class II) correlates closely with axonal outgrowth during development and regeneration. In dorsal root ganglion (DRG) neurons, expression of the class II isotype declines to relatively low levels during early postnatal development, and increases dramatically in mature neurons during axon regeneration (i.e., to a level comparable to that in developing neurons). In contrast, expression of the class III isotype, which rises slightly during postnatal development, increases much less than the class II isotype during regeneration. We now document that these changes in gene expression are associated with an increase in the relative amount of class II as compared to class III beta-tubulin delivered to regenerating sensory axons of rat sciatic nerve by slow axonal transport. In this study, the tubulin transported in sensory axons was labeled by injecting [35S]methionine into the L5 DRG either 7 or 14 days after crushing the sciatic nerve; pulse-labeled class II and class III beta-tubulin were identified using immunoprecipitation. This change in the isotype composition of beta-tubulin transported in regenerating axons may influence outgrowth by altering the assembly and dynamic properties of axonal microtubules.

The axonal transport of beta III-tubulin is altered in both branches of sensory axons after injury of the rat sciatic nerve.

We have analyzed the axonal transport of beta III-tubulin in the central (dorsal root) and peripheral (sciatic nerve) branches of sensory axons after injury of the sciatic nerve. Our finding that the relative amount of beta III-tubulin transported in slow component b (SCb) is increased in both axonal branches does not support the generally accepted hypothesis that the transport of cytoskeletal proteins is altered in the peripheral, but not the central branch after injury of the sciatic nerve.

Rapid transport of proteins in the sonic motor system of the toadfish.

A pure cholinergic, motor system of a marine fish has been utilized to study the kinetics and characteristics of proteins rapidly transported from the sonic motor nucleus to the musculature enveloping the swim bladder. Following microinjection of [3H]leucine, [3H]lysine, [35S]methionine, or [3H]fucose into the nucleus a wave of radioactivity was observed moving along the sonic motor nerves with an apparent rate of 96-120 mm/day. Analysis of the rapidly transported methionine-labeled protein using SDS gel electrophoresis revealed at least 9 major peaks of activity. Eight of these proteins were found to incorporate fucose, suggesting that most of the rapidly transported material consists of glycoproteins. These results are consistent with the previously suggested hypothesis relating the function of rapid transport to synaptic vesicles and the maintenance of pre-synaptic terminal membranes.

Levels of neurotransmitter and cytoskeletal protein mRNAs during nerve regeneration in sympathetic ganglia.

The present study examines levels of neurotransmitter messenger RNA (mRNA) at various stages after crush of postganglionic nerves in the superior cervical ganglia. Using complementary DNA (cDNA) probes, we demonstrated a reduction in ganglionic mRNA levels for tyrosine hydroxylase (TH) after axotomy. Concomitantly, actin and tubulin mRNA levels in ganglia were increased. Thus, in neurons of sympathetic ganglia, axotomy appears to be associated with a selective reduction in levels of TH mRNA, and, in turn, alters levels of protein and enzyme activities.

Selective interruption of axonal transport of neurofilament proteins in the visual system by beta,beta'-iminodipropionitrile (IDPN) intoxication.

Beta,beta'-iminodipropionitrile (IDPN) is an agent that produces a marked impairment in the transport of neurofilaments. Its effect on other slowly transported cytoskeletal components such as tubulin and actin is variable. Previous studies have evaluated transport of neurofilaments after IDPN intoxication in a neurofilament-rich system (sciatic motor nerves) and in a system devoid of neurofilaments (axons of the dorsal motor nucleus of the vagus). In the former, IDPN impairs the transport of tubulin and actin but to a lesser degree than it does neurofilament proteins. In the latter, tubulin and actin transport were not impaired, and neurofilament proteins were not present. In this study we evaluated the transport of the cytoskeletal components in a system with an intermediate amount of neurofilaments (the visual system). In the visual system, there is a selective and marked (50%) impairment in the transport of neurofilaments with no impairment in transport of tubulin or microtubule-associated proteins (tau group). We conclude that these different patterns of impairment in transport reflect the differences in pre-intoxication neurofilament content of the nerves examined, the effect of IDPN on the transport of the other components of slow transport being secondary to the presence of stagnated neurofilaments. This model also suggests that transport of neurofilaments can be selectively impaired without producing an effect on other major slow transport components.

Immunocytochemical studies of neurofilament antigens in the neurofibrillary pathology induced by aluminum.

Intrathecal administration of aluminum salts induces accumulation of neurofilaments in axons and perikarya of motor neurons and is associated with impaired axonal transport of neurofilament proteins. Because phosphorylation of the 200-kilodalton (kd) neurofilament protein, thought to be a major component of the sidearms, seems to be important in interactions of neurofilaments with other cytoskeletal elements, we have postulated that aluminum may produce neurofibrillary pathology by altering patterns of neurofilament phosphorylation. To test this hypothesis, antibodies against phosphorylated and non-phosphorylated neurofilament epitopes were used for immunocytochemical analysis of spinal cord sections from aluminum-injected rabbits. In control animals, phosphorylated 200-kd neurofilament proteins were not demonstrable in perikarya of motor neurons. In experimental rabbits, perikarya and proximal axons of affected motor neurons showed striking accumulations of immunoreactivity of one phosphorylated epitope. The presence of phosphorylated 200-kd neurofilament proteins in these regions may have important consequences for the organization of the cytoskeleton and for the transport of neurofilaments. A similar, but not identical, pattern of accumulation of phosphorylated neurofilament immunoreactivity has recently been observed in neurofibrillary tangles in Alzheimer's disease.

Aluminum intoxication: a disorder of neurofilament transport in motor neurons.

In the rabbit, intrathecal administration of aluminum salts (AlCl3) induces accumulation of neurofilaments in nerve cells of the central nervous system. In motor neurons, the spatial pattern of neurofilamentous accumulation following aluminum intoxication suggests a defect in the axonal transport of neurofilament proteins. To test this hypothesis, we examined the distribution of radioactive cytoskeletal proteins in sciatic nerves of intoxicated and control animals. In the nerves of aluminum-injected animals, there was a 40% reduction in the relative amount of radioactive neurofilament proteins compared to tubulin. These results suggest that an abnormality in neurofilament transport may be important in the pathogenesis of the neurofibrillary pathology induced by aluminum intoxication.