CALCIUM-DEPENDENT ALTERATIONS OF NEUROFILAMENT PROTEINS OF RAT PERIPHERAL NERVE

Segments of rat peripheral nerve were excised and incubated in vitro under conditions which hasten or retard the penetration of calcium into tissues. The nerve segments were then homogenized in 1%SDS, 8 M-urea and 50mM-dithioerythritol and the extracted proteins were analyzed by SDS polyacrylamide disc gel electrophoresis. Representative samples were also fixed and processed for electron microscopic examinations. Incubational conditions which favored the penetration of calcium into the tissues caused widespread granular disruption of neurofilaments as well as the selective loss of 200,000, 150,000 and 69,000 MW proteins from the nerve segments. These structural and chemical changes occurred in the 24-48 h interval during incubation at 37^oC under nutrient conditions, but were accelerated under energy-deprived conditions and with high-calcium media. The same changes occurred in nerve segments incubated for 2h in media containing calcium with Triton X-100 detergent or A23187 ionophore and in nerve segments freeze-thawed in 0.5 mM-calcium. These changes did not occur after parallel incubations in calcium-free media. These findings support the view that 200,000, 150,000 and 69,000 MW proteins are components of neurofilaments and that they are susceptible to a calcium-induced disruption.     

Chemical and structural changes of neurofilaments in transected rat sciatic nerve

The sequence of changes occurring in transected rat sciatic nerve was examined by electron microscopy and by sodium dodecyl sulfate (SDS) polyacrylamide disc gel electrophoresis. Representative segments of transected nerves were processed for ultrastructural examinations between 0 and 34 days after the transection of sciatic nerves immediately below the sacro-sciatic notch. The remainder of the transected nerves and the intact portions of sciatic nerves were desheathed and immediately homogenized in 1 percent SDS containing 8 M urea and 50 mM dithioerythritol. Solubilized proteins were analyzed on 12 percent gels at pH 8.3 in a discontinuous electrophoretic system. Initial changes were limited to the axons of transected nerve fibers and were characterized by the loss of microtubules and neurofilaments and their replacement by an amorphous floccular material. These changes became widespread between 24 and 48 h after transection. The disruption of neurofilaments during this interval occurred in parallel with a selective loss of 69,000, 150,000 and 200,000 mol wt proteins from nerve homogenates, thus corroborating the view that these proteins represent component subunits of mammalian neurofilaments. Furthermore, the selective changes of neurofilament proteins in transected nerves indicate their inherent lability and suggest their susceptibility to calcium-mediated alterations. Electrophoretic profiles of nerve proteins during the 4-34-day interval after nerve transection reflected the breakdown and removal of myelin, the proliferation of Schwann cells and the deposition of endoneurial collagen. A marked increase of intermediate-sized filaments within proliferating Schwann cell processes was not accompanied by the appearance of neurofilamentlike proteins in gels of nerve homogenates.     

Neurofilament proteins of rat peripheral nerve and spinal cord

Intact neurofilaments were isolated in parallel from rat peripheral nerve and spinal cord by osmotic shock into hypotonic media containing divalent cation chelators. Isolated neurofilaments were washed and separated by multiple centrifugations in 0.1 M NaCl. Abundant intact neurofilaments were identified in the washed pellets by negative staining techniques. Their origin from neurofilaments was confirmed by immune electron microscopy. Washed neurofilaments were extracted from lipid and membranous components with 8 M urea. Analyses of neurofilament isolates on sodium dodecyl sulfate gels showed that proteins of 200,000, 150,000, and 69,000 mol wt were the major components of intact neurofilaments derived from rat peripheral and central nervous systems. These same proteins were identified in whole tissue homogenates of both sources and became enriched during the isolation of intact neurofilaments. A minor component of 64,000 mol wt arose during isolation. Other proteins were identified as contaminants. Small amounts of proteins with electrophoretic migration of tubulin and actin remain in neurofilament isolates.     

Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross- bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick- freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.     

[32P]orthophosphate and [35S]methionine label separate pools of neurofilaments with markedly different axonal transport kinetics in mouse retinal ganglion cells in vivo

Newly synthesized neurofilament proteins become highly phosphorylated within axons. Within 2 days after intravitreously injecting normal adult mice with [³²P]orthophosphate, we observed that neurofilaments along the entire length of optic axons were radiolabeled by a soluble³²P-carrier that was axonally transported faster than neurofilaments.³²P-incorporation into neurofilament proteins synthesized at the time of injection was comparatively low and minimally influenced the labeling pattern along axons.³²P-incorporation into axonal neurofilaments was considerably higher in the middle region of the optic axons. This characteristic non-uniform distribution of radiolabel remained nearly unchanged for at least 22 days. During this interval, less than 10% of the total³²P-labeled neurofilaments redistributed from the optic nerve to the optic tract. By contrast, newly synthesized neurofilaments were selectively pulse-labeled in ganglion cell bodies by intravitreous injection of [³⁵S]methionine and about 60% of this pool translocated by slow axoplasmic transport to the optic tract during the same time interval. These findings indicate that the steady-state or resident pool of neurofilaments in axons is not identical to the newly synthesized neurofilament pool, the major portion of which moves at the slowest rate of axoplasmic transport. Taken together with earlier studies, these results support the idea that, depending in part on their phosphorylation state, transported neurofilaments can interact for short or very long periods with a stationary but dynamic neurofilament lattice in axons.Special issue dedicated to Dr. Sidney Ochs.     

The differential appearance of neurofilament triplet polypeptides in the developing rat optic nerve

The ontogenetic appearance of the individual triplet polypeptides that comprise mammalian neurofilaments was studied in the developing rat optic nerve. Triton-insoluble cytoskeletal preparations from the optic nerves of rats of postnatal ages 1 Day (P1), 6 days (P6), 10 days (P10), 20 days (P20), and 3 months (adult) were analyzed for protein composition by one and two-dimensional gel electrophoresis. Results indicate that at P1, both the 150- and 68-kDa neurofilament subunit proteins are present. The 200-kDa subunit first becomes discernible at P20, but, at this age, it is still present in considerably less quantity than in the adult. Immunocytochemical verification of the presence of neurofilament protein was accomplished by staining tissue sections with specific antibodies against the 150- and the 68-kDa neurofilament subunits using the peroxidase-antiperoxidase technique. Results of the morphological analyses have shown that neurofilaments are not present in quantity until P10, which coincides with the time when the 68-kDa subunit increases in quantity by one dimensional gel analysis. Thus, the 150- and 68-kDa subunits can be detected prior to the appearance of neurofilaments, and the 200-kDa protein is not observed until sometime later. The potential physiological significance of the differential subunit transport is discussed with respect to neuronal differentiation in the developing mammalian CNS.     

Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport

The COOH-terminal tail of mammalian neurofilament heavy subunit (NF-H), the largest neurofilament subunit, contains 44-51 lysine-serine-proline repeats that are nearly stoichiometrically phosphorylated after assembly into neurofilaments in axons. Phosphorylation of these repeats has been implicated in promotion of radial growth of axons, control of nearest neighbor distances between neurofilaments or from neurofilaments to other structural components in axons, and as a determinant of slow axonal transport. These roles have now been tested through analysis of mice in which the NF-H gene was replaced by one deleted in the NF-H tail. Loss of the NF-H tail and all of its phosphorylation sites does not affect the number of neurofilaments, alter the ratios of the three neurofilament subunits, or affect the number of microtubules in axons. Additionally, it does not reduce interfilament spacing of most neurofilaments, the speed of action potential propagation, or mature cross-sectional areas of large motor or sensory axons, although its absence slows the speed of acquisition of normal diameters. Most surprisingly, at least in optic nerve axons, loss of the NF-H tail does not affect the rate of transport of neurofilament subunits.     

GTP regeneration influences interactions of microtubules, neurofilaments, and microtubule-associated proteins in vitro

Interactions of microtubules, neurofilaments, and microtubule-associated proteins were investigated by turbidity and falling-ball viscometry measurements. We found evidence of endogenous GTPase activity in neurofilaments and microtubule-associated proteins (MAPs) in preparations that do not include urea or heat treatment, respectively. The absence or presence of either adenyl-5'-yl imidodiphosphonic acid or a GTP-regenerating system markedly influenced observed polymerization and gelation characteristics. Most significantly, the apparent viscosity of neurofilament and microtubule samples did not display a biphasic optimal MAP concentration profile when a GTP-regenerating system was operant. Likewise, GTP regeneration promoted the recovery of gelation following mechanical disruption of neurofilament/MAP/microtubule mixtures. These and other observations require some reassessment of proposed roles for microtubule-associated proteins in modulating neurofilament-microtubule interactions in vitro.     

Phosphorylated epitopes of neurofilaments have been conserved during chordate evolution

We have characterized some rabbit polyclonal responses as strictly specific for phosphorylated epitopes located in the carboxyterminal (tail) domain of the H or the M subunits of mammalian neurofilaments. These antibodies have been used to confirm the occurrence in lizard neurofilaments of a single heavy subunit cross-reacting with both H and M from mammals. A heavy subunit with similar cross-reactivity has been detected in neurofilaments preparations from fishes, whereas more primitive Chordata possess a HMW polypeptide cross-reacting with only the M subunit. We could also demonstrate in frog spinal cord two distinct heavy subunits cross-reacting with either the M or the H subunit from mammals, a fact which suggests a convergent evolution for phosphorylated epitopes of neurofilaments.     

Axonal cytoskeletal changes after non-disruptive axonal injury

In animal models of human diffuse axonal injury, axonal swellings leading to secondary axotomy occur between 2 and 6 h after injury. But, analysis of cytoskeletal changes associated with secondary axotomy has not been undertaken. We have carried out a quantitative analysis of cytoskeletal changes in a model of diffuse axonal injury 4 h after stretch-injury to adult guinea-pig optic nerves. The major site of axonal damage was the middle portion of the nerve. There was a statistically significant increase in the proportion of small axons with a diameter of 0.5 μm and smaller in which there was compaction of neurofilaments. Axons with a diameter greater than 2.0 μm demonstrated an increased spacing between cytoskeletal elements throughout the length of the nerve. However, in the middle segment of the nerve these larger axons demonstrated two different types of response. Either, where periaxonal spaces occurred, there was a reduction in axonal calibre, compaction of neurofilaments but no change in their number, and a loss of microtubules. Or, where intramyelinic spaces occurred there was an increased spacing between neurofilaments and microtubules with a significant loss in the number of both. Longitudinal sections showed foci of compaction of neurofilaments interspersed between regions where axonal structure was apparently normal. Neurofilament compaction was correlated with disruption of the axolemma at these foci present some hours after injury. We suggest that the time course of these axonal cytoskeletal changes after stretch-injury to central axons is shorter than those changes documented to occur during Wallerian degeneration.     

Influence of pH of the medium on free fatty acid utilization by isolated mammalian cells

Studies with Ehrlich ascites tumor cells showed that small decreases in the pH of the incubation medium from 7.4 increase the magnitude of incorporation of free fatty acid (FFA) into the cells from an albumin solution. A similar effect occurred when rabbit erythrocytes, rat heart slices, or rat liver slices were incubated with FFA-bovine albumin solutions and when tumor cells were incubated with FFA in media containing human albumin, -lactoglobulin, or rat plasma. The effect was not seen when the medium contained no protein. When the pH of the albumin-containing medium was lowered from 7.4 to 6.6, oxidation of FFA to CO(2) by the tumor cells increased, esterification of the FFA (mostly into phospholipids and triglycerides) increased, and less esterified radio-active fatty acid was depleted from the cells. Hence, more fatty acid accumulated in the cells in more acid media. These findings suggest that small changes in extracellular pH might regulate FFA utilization and lipid accumulation in mammalian tissues.     

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70–79) than in younger (aged 40–59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule. The level of these anticholinergic NF-H IgG are significantly higher in AD sera than in those of both normal controls and patients with multi-infarct dementia.     

Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation

Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC) disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) and murine double minute (Mdm2) E3 ligase. Growth cone collapse induced by genetic (npc1−/−) or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK)-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1−/− mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.     

In vitro culture ofDrosophila ovarian follicles: The influence of different media on development,RNA synthesis,protein synthesis and potassium uptake

Summary Contradictory electrophysiological results were recently obtained inDrosophila ovarian follicles kept in different salines or complete media during measurements. Therefore, I checked follicles maintained in various solutions using morphological, physiological and biochemical criteria. Defined complete media were the best for supporting development from stage 10 to stage 14 (end of oogenesis). Supplementation of the solutions with insect pupal haemolymph had negative effects. For the maintenance of RNA synthesis, complete media were again superior to simple salines. Total protein synthesis was not very sensitive to the culture conditions during short incubation periods, but electrophoretic protein patterns were slightly less complex in the salines than in complete media. Furthermore, some major proteins (e.g. chorion proteins) synthesized during longterm culture failed to appear in the salines. In view of extrafollicular electrical currents and intracellular electrical potentials, potassium uptake experiments were conducted with several inhibitors, using rubidium-86 as a probe. Both potassium concentration and osmolarity were found to exert strong influences on total potassium uptake of the follicles. In the tested media and salines differing amounts of potassium were taken up via (Na⁺, K⁺)-ATPase, via other K⁺-pump(s) or passively. The possible influences of several parameters on the outcome of earlier in vitro experiments withDrosophila follicles are discussed. For further in vitro studies the chemically defined complete R-14 medium seems to be the most suitable.     

Characterization of two proteolytically derived soluble polypeptides from the neurofilament triplet components NFM and NFH.

We have purified to homogeneity the regions derived by chymotryptic digestion of the ox neurofilament polypeptides NFH and NFM; the regions, called M1 and M2, are thought to form part of the projecting sidearms of mammalian neurofilaments [Chin, Eagles & Maggs (1983) Biochem. J. 215, 239-252]. They were isolated and purified under non-denaturing conditions and showed no tendency to interact with each other in solution. The Mr values obtained by sedimentation are approx. 61,000 for M1 and 42,000 for M2, considerably lower than the values obtained by SDS/polyacrylamide-gel electrophoresis. These Mr values were unchanged in the presence of 6 M-guanidine hydrochloride, suggesting that the regions exist as monomers in solution. Both M1 and M2 are highly phosphorylated, and there is only a slight change in the sedimentation value upon dephosphorylation. Dephosphorylation of M1 with alkaline phosphatase was more than 90% efficient but was never absolute. Dephosphorylation of M2 was complete. Both M1 and M2 bind Ca2+; in the case of M1, this binding is phosphorylation-dependent. M1 also binds cytochrome c, and dephosphorylation affects binding. In similar conditions, neurofilaments bind at least twice their own mass of cytochrome c, owing to their opposite net charges. No interactions were observed between native or dephosphorylated M1 and M2, and intact neurofilaments under a wide variety of conditions. These results are discussed in terms of the possible roles that neurofilament sidearms might play and throw doubt upon their supposed function of rigidly cross-linking neurofilaments together within the axoplasm of neurons.     

Promotion of Microtubule Assembly by Neurofilament-Associated Microtubule-Associated Proteins

Abstract: Intact neurofilaments (NF) purified from mammalian brain and spinal cord promote the assembly of microtubules in solutions of pure phosphocellulose (PC)-purified tubulin. This assembly is temperature-dependent and is inhibited by mitotic spindle inhibitors. The ability of NF to induce microtubule formation is 20% of that of purified microtubule-associated proteins (MAPs), whereas MAPs comprise less than 5% of the protein in the NF preparations. The inducing activity of NF is rapidly lost on boiling. When intact NF are incubated with PC-tubulin and then centrifuged, tubulin is sedimented together with the filaments. This association is inhibited by colchicine and podophyllotoxin and is cold-sensitive. NF purified to homogeneity under denaturing conditions and then reassembled completely lack the ability to promote the assembly of PC-tubulin or to bind tubulin on a centrifugation assay. No MAPs are present in these preparations, though these filaments have the ability to bind exogenous MAPs. While these experiments do not rule out an intrinsic microtubule-assembly-promoting activity, they suggest that this activity is due to nontriplet proteins in the preparation, most likely filament-associated MAPs.     

Neurofilament homeostasis and motoneurone degeneration

Neurofilament disorganisation is a hallmark of various neurodegenerative diseases. We review here current knowledge of neurofilament structure, gene expression and function. Neurofilament involvement in motoneurone neurological diseases is discussed in view of recent data from transgenic and spontaneous mouse mutants. In the mammalian neurone, the three neurofilament subunits are assembled into intermediate filaments as obligate heteropolymers. The subunits are expressed differentially during development and adult life according to the cell type and its physiological state. In addition to the well-established role of neurofilaments in the control of axonal calibre, there is increasing evidence that neurofilaments can interact with other cytoskeletal components and can modulate the axoplasmic flow. Although the extent to which neurofilament abnormalities contribute to the pathogenesis in human diseases remains unknown, emerging evidence suggests that disorganised neurofilaments can provoke degeneration and death of neurones. BioEssays 23:24-33, 2001.     

Normal role of the low-molecular-weight neurofilament protein in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease

Intermediate filaments serve important structural roles, but other cellular functions are increasingly recognized. This study demonstrated normal function of the low-molecular-weight neurofilament protein (NFL) in mitochondrial dynamics and disruption in Charcot-Marie-Tooth disease (CMT) due to mutations in the Nefl gene. In motor neurons of spinal cord cultured from Nefl-knockout mice, mitochondrial length and the rate of fusion were decreased concomitant with increased motility. These parameters were normalized after expression of NFL(wt) on the Nefl(-/-) background, but not by overexpression of the profusion protein, mitofusin 2 (MFN2). The effects of CMT-causing NFL mutants bore similarities to and differences from Nefl knockout. In the early phase of toxicity before disruption of the neurofilament network, NFL(Q333P) and NFL(P8R) integrated into neurofilaments and had effects on mitochondria similar to those with Nefl knockout. The reduction of fusion rate by NFL(Q333P) was partly due to interference with the function of the profusion protein MFN2, which is mutated in CMT2A, functionally linking these forms of CMT. In the later phase of toxicity, mitochondria essentially stopped moving in neurons expressing NFL mutants, probably a consequence of cytoskeletal disruption. Overall, the data point to important functions of neurofilaments in mitochondrial dynamics as well as primary involvement in CMT2E/1F.