Disruption of the NF-H Gene Increases Axonal Microtubule Content and Velocity of Neurofilament Transport: Relief of Axonopathy Resulting from the Toxin beta ,beta '-Iminodipropionitrile (original) (raw)
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Brain Research, 2000
Neurofilament proteins (NFs) are made by co-polymerization of three intermediate filament proteins, NF-L, NF-M and NF-H and constitute the most abundant cytoskeletal element in large myelinated axons. NFs have a well-established role as intrinsic determinants of axon caliber with all the functional implications, but the role of each individual NF subunit is much less clear. The aim of our study was to examine functional properties of large myelinated axons with altered morphology from mice bearing a targeted disruption of each NF genes (NF-L 2 / 2, NF-M2 / 2 and NF-H 2 / 2 mice). Membrane properties, action potentials and single axon refractory period were measured in isolated sciatic nerves in vitro, using intra-axonal microelectrode recording in conjunction with current-clamp technique. Some results were obtained from whole nerves by sucrose-gap recording. The NF-knockout mice showed several deficits in physiological properties of low-threshold fibers. In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L 2 / 2 and NF-M 2 / 2 transgenic animals (control, 39.961.8 m / s, NF-M 2 / 2; 23.561.4 m / s, and NF-L2 / 2; 12.060.7 m / s, mean6S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22-268C). However, in spite of their preserved caliber, large myelinated axons in NF-H 2 / 2 mice also showed a significant decrease in conduction velocity (22.861.0 m / s, mean6S.E.M.). Although action potential amplitudes, duration and shape did not differ between control axons and transgenic animals, the refractory period was prolonged in NF-H 2 / 2 and NF-M 2 / 2 animals. Intracellular injections of 200 ms depolarizing and hyperpolarizing currents revealed outward and inward rectification in all animal groups. In comparison to control animals, NF-H 2 / 2 mice expressed a significant decrease in outward rectification. Potassium channel blockers (4AP and TEA) and cesium ions were able to block outward and inward rectification in all myelinated axons in qualitatively the same manner. These results suggest that NF-H may have a specific role in modulating ion channel functions in large myelinated fibers.
The Journal of Neuroscience, 2007
We generated mice with doxycycline control of a human neurofilament light (NF-L) transgene in the context of the absence (tTA;hNF-L;NF-L−/−) or presence (tTA;hNF-L;NF-L+/−) of endogenous mouse NF-L proteins. Doxycycline treatment caused the rapid disappearance of human NF-L (hNF-L) mRNA in tTA;hNF-L mice, but the hNF-L proteins remained with a half-life of 3 weeks in the brain. In the sciatic nerve, the disappearance of hNF-L proteins after doxycycline treatment occurred in synchrony along the sciatic nerve, suggesting a proteolysis of NF proteins along the entire axon. The presence of permanent NF network in tTA;hNF-L;NF-L+/−mice further stabilized and extended longevity of hNF-L proteins by several months. Surprisingly, after cessation of doxycycline treatment, there was no evidence of leading front of newly synthesized hNF-L proteins migrating into sciatic nerve axons devoid of NF structures. The hNF-L proteins detected at weekly intervals reappeared and accumulated in synchrony ...
Brain Research, 1989
The neurotoxic compound 2,5-hexanedione (2,5-HD) causes an axonopathy characterized by the presence of neurofilament (NF)-containing enlargements in the preterminal segments of central and peripheral axons. The 2,5-HD axonopathy is a good model for human acquired and inherited giant axonal neuropathies. Recently, we reported that following 2,5-HD administration, axonal transport of NF is markedly and selectively accelerated in the primary visual system. We have now studied slow axonal transport in the sciatic system of rats intoxicated with 0.5% 2,5-HD in drinking water. Following radiolabeling, transported proteins were examined after polyacrylamide gel electrophoresis and fluorography. The bulk of radiolabeled NF subunits was located 30-50 mm from the spinal cord in 2,5-HD treated animals and 10-25 mm in controls. The rate of transport of the three NF subunits was 0.7 mm/day in controls and 1.2 mm/day in 2,5-HD treated animals. The rate of transport of tubulin was not significantly changed. Electrophysiological studies of soleus nerve and muscle showed no evidence of denervation after 6 weeks of intoxication. It is concluded that, following 2,5-HD administration, transport of NF is preferentially accelerated in both central and peripheral axons. A pathogenetic mechanism based on the acceleration of NF transport is proposed, which may explain the formation and the distal or proximal location of NF-containing axonal enlargements in giant axonopathies.
Journal of Neurochemistry, 2001
To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two-to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-MϪ/Ϫ mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about twofold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and ␣-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-MϪ/Ϫ;NF-HϪ/Ϫ mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [ 35 S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-MϪ/Ϫ;NF-HϪ/Ϫ mice. The combined results demonstrate a requirement of the high-molecularweight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.
Transport of neurofilaments in growing axons requires microtubules but not actin filaments
Journal of Neuroscience Research, 2005
Neurofilament (NF) polymers are conveyed from cell body to axon tip by slow axonal transport, and disruption of this process is implicated in several neuronal pathologies. This movement occurs in both anterograde and retrograde directions and is characterized by relatively rapid but brief movements of neurofilaments, interrupted by prolonged pauses. The present studies combine pharmacologic treatments that target actin filaments or microtubules with imaging of NF polymer transport in living axons to examine the dependence of neurofilament transport on these cytoskeletal systems. The heavy NF subunit tagged with green fluorescent protein was expressed in cultured sympathetic neurons to visualize NF transport. Depletion of axonal actin filaments by treatment with 5 μM latrunculin for 6 hr had no detectable effect on directionality or transport rate of NFs, but frequency of movement events was reduced from 1/3.1 min of imaging time to 1/4.9 min. Depolymerization of axonal microtubules using either 5 μM vinblastine for 3 hr or 5 μg/ml nocodazole for 4–6 hr profoundly suppressed neurofilament transport. In 92% of treated neurons, NF transport was undetected. These observations indicate that actin filaments are not required for neurofilament transport, although they may have subtle effects on neurofilament movements. In contrast, axonal transport of NFs requires microtubules, suggesting that anterograde and retrograde NF transport is powered by microtubule-based motors. © 2005 Wiley-Liss, Inc.
Dissociation of Axonal Neurofilament Content from Its Transport Rate
PloS one, 2015
The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with pr...
Science, 1978
We have investigated the axonal transport of neurofilament protein in cultured neurons by constricting single axons with fine glass fibers. We observed a rapid accumulation of anterogradely and retrogradely transported membranous organelles on both sides of the constrictions and a more gradual accumulation of neurofilament protein proximal to the constrictions. Neurofilament protein accumulation was dependent on the presence of metabolic substrates and was blocked by iodoacetate, which is an inhibitor of glycolysis. These data indicate that neurofilament protein moves anterogradely in these axons by a mechanism that is directly or indirectly dependent on nucleoside triphosphates. The average transport rate was estimated to be at least 130 m/h (3.1 mm/d), and ف 90% of the accu-mulated neurofilament protein remained in the axon after detergent extraction, suggesting that it was present in a polymerized form. Electron microscopy demonstrated that there were an abnormally large number of neurofilament polymers proximal to the constrictions. These data suggest that the neurofilament proteins were transported either as assembled polymers or in a nonpolymeric form that assembled locally at the site of accumulation. This study represents the first demonstration of the axonal transport of neurofilament protein in cultured neurons.
3,4-Dimethyl-2,5-hexanedione impairs the axonal transport of neurofilament proteins
The Journal of Neuroscience, 1984
Accumulations of neurofilaments are observed in a variety of neurological disorders, and their pathogenesis is a fundamental problem of neuropathology. 2,5-Hexanedione (HD) neurotoxicity provides an extensively studied model of axonal neurofibrillary changes in which the pathogenetic mechanisms have been conjectural. Chronic exposure to HD results in neurofilament-filled swellings in the distal regions of large axons of exposed humans and experimental animals. In this report we describe the changes produced by a potent analogue of HD, 3,4- dimethyl-2,5-hexanedione ( DMHD ), in slow axonal transport in the rat sciatic motor axons. Young rats received 0.6 mmol/kg of DMHD for 5 days before [35S]methionine was injected into the lumbar ventral horns. Slow axonal transport of the neurofilament proteins, tubulin, and selected slow component b (SCb) proteins in DMHD -treated animals was compared to the profiles found in age-matched control animals. DMHD administration reduced the rate of tr...
1999
Changes in the synthesis and axonal transport of neurofila- ment (NF) proteins and tubulin were examined after various selective axotomies of adult rat DRG cells. For axonal trans- port studies, DRGs were labeled by microinjection of 35S-methionine 14 d after axonal injuries, and nerves were retrieved 7 or 14 d after labeling. Slowly transported proteins were examined by quantitative PAGE/fluorography. After distal peripheral nerve crush (SO-55 mm from the DRG), the cytoskeleton that entered undamaged regions of peripheral branch DRG axons by slow axonal transport differed from normal, while the cytoskeleton that entered dorsal root ax- ons did not. Specifically, smaller-than-normal ratios of la- beled NF protein/tubulin were transported in peripheral DRG axons after distal peripheral nerve crush. This change was almost entirely due to a selective decrease in the output of labeled NF proteins rather than to an increase in the amount of tubulin transported with NF proteins. Since the...