Subunit composition of neurofilaments specifies axonal diameter

Neurofilaments (NFs), which are composed of NF-L, NF-M, and NF-H, are required for the development of normal axonal caliber, a property that in turn is a critical determinant of axonal conduction velocity. To investigate how each subunit contributes to the radial growth of axons, we used transgenic mice to alter the subunit composition of NFs. Increasing each NF subunit individually inhibits radial axonal growth, while increasing both NF-M and NF-H reduces growth even more severely. An increase in NF-L results in an increased filament number but reduced interfilament distance. Conversely, increasing NF-M, NF-H, or both reduces filament number, but does not alter nearest neighbor interfilament distance. Only a combined increase of NF-L with either NF- M or NF-H promotes radial axonal growth. These results demonstrate that both NF-M and NF-H play complementary roles with NF-L in determining normal axonal calibers.     

Requirement of Heavy Neurofilament Subunit in the Development of Axons with Large Calibers

Neurofilaments (NFs) are prominent components of large myelinated axons. Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber. We created NF-H knockout mice to assess the contribution of NF-H to the development of axon size as well as its effect on the amounts of low and mid-sized NF subunits (NF-L and NF-M respectively). Surprisingly, we found that NF-L levels were reduced only slightly whereas NF-M and tubulin proteins were unchanged in NF-H–null mice. However, the calibers of both large and small diameter myelinated axons were diminished in NF-H–null mice despite the fact that these mice showed only a slight decrease in NF density and that filaments in the mutant were most frequently spaced at the same interfilament distance found in control. Significantly, large diameter axons failed to develop in both the central and peripheral nervous systems. These results demonstrate directly that unlike losing the NF-L or NF-M subunits, loss of NF-H has only a slight effect on NF number in axons. Yet NF-H plays a major role in the development of large diameter axons.     

Phosphorylation-mediated conformational changes in the mouse neurofilament architecture: insight from a neurofilament brush model

Neurofilaments (NFs) are important cytoskeletal filaments that consist of long flexible C-terminal tails that are abundant with charges. The tails attain additional negative charges through serine phosphorylation of Lys-Ser-Pro (KSP) repeat motifs that are particularly found in neurofilament heavy (NF-H) and neurofilament medium (NF-M) proteins. These side-arm protrusions mediate the interaction between neighboring filaments and maintain axonal diameter. However, the precise role of NF proteins and their phosphorylation in regulating interfilament distances and axonal diameter still remains unclear. In this regard, a recent gene replacement study revealed that the phosphorylation of mouse NF-M KSP repeats does not affect axonal cytoarchitecture, challenging the conventional viewpoint on the role of NF phosphorylation. To better understand the effect of phosphorylation, particularly NF-M phosphorylation, we applied a computational method to reveal phosphorylation-mediated conformational changes in mouse NF architecture. We employed a three-dimensional sequence-based coarse-grained NF brush model to perform Monte Carlo simulations of mouse NF by using the sequence and stoichiometry of mouse NF proteins. Our result shows that the phosphorylation of mouse NF-M does not change the radial extension of NF-M side arms under a salt-free condition and in ionic solution, highlighting a structural factor that supports the notion that NF-M KSP phosphorylation has no effect on the axonal diameter of mouse. On the other hand, significant phosphorylation-mediated conformational changes were found in NF-H side arms under the salt-free condition, while the changes in ionic solution are not significant. However, NF-H side arms are found at the periphery of mouse NF architecture, implying a role in linking neighboring filaments.     

Relating interactions between neurofilaments to the structure of axonal neurofilament distributions through polymer brush models

Neurofilaments (NFs) have been proposed to interact with one another through mutual steric exclusion of their unstructured C-terminal "sidearm" domains, producing order in axonal NF distributions and conferring mechanical strength to the axon. Here we apply theory developed for polymer brushes to examine the relationship between the brush properties of the sidearms and NF organization in axons. We first measure NF-NF radial distribution functions and occupancy probability distributions for adult mice. Interpreting the probability distributions using information theory, we show that the NF distributions may be represented by a single pair potential of mean force. Then, to explore the relationship between model parameters and NF architecture, we conduct two-dimensional Monte Carlo simulations of NF cross-sectional distributions. We impose purely repulsive interaction potentials in which the sidearms are represented as neutral and polyelectrolyte chains. By treating the NFs as telechelic polymer brushes, we also incorporate cross-bridging interactions. Both repulsive potentials are capable of reproducing NF cross-sectional densities and their pair correlations. We find that NF structure is sensitive to changes in brush thickness mediated by chain charge, consistent with the experimental observation that sidearm phosphorylation regulates interfilament spacing. The presence of attractive cross-bridging interactions contributes only modestly to structure for moderate degrees of cross-bridging and leads to NF aggregation for extensive cross-bridging.     

Absence of the Mid-sized Neurofilament Subunit Decreases Axonal Calibers,Levels of Light Neurofilament (NF-L), and Neurofilament Content

Neurofilaments (NFs) are prominent components of large myelinated axons and probably the most abundant of neuronal intermediate filament proteins. Here we show that mice with a null mutation in the mid-sized NF (NF-M) subunit have dramatically decreased levels of light NF (NF-L) and increased levels of heavy NF (NF-H). The calibers of both large and small diameter axons in the central and peripheral nervous systems are diminished. Axons of mutant animals contain fewer neurofilaments and increased numbers of microtubules. Yet the mice lack any overt behavioral phenotype or gross structural defects in the nervous system. These studies suggest that the NF-M subunit is a major regulator of the level of NF-L and that its presence is required to achieve maximal axonal diameter in all size classes of myelinated axons.Neurofilaments (NFs)¹ are the most prominent cytoskeletal components in large myelinated axons and probably the most abundant and widely expressed of neuronal intermediate filament (IF) proteins. In mammals, NFs are composed of three proteins termed light (NF-L), mid-sized (NF-M), and heavy (NF-H) NFs. These proteins are encoded by separate genes (17, 21, 27) and have apparent molecular weights of ∼68,000, 150,000, and 200,000, respectively, when separated on SDS-PAGE gels.Like all IFs, NF proteins contain a relatively well-conserved α helical rod domain of ∼310 amino acids with variable NH₂-terminal and COOH-terminal regions (33). In NFs, the COOH-terminal domains are greatly extended relative to other IFs and contain a glutamic acid–rich region of unknown significance and in NF-M and NF-H a series of lysine-serine-proline-valine (KSPV) repeats (21, 27) which are major sites of phosphorylation in both proteins. In axons, NFs form bundles of 10-nm diameter “core filaments” with sidearms consisting of phosphorylated COOH-terminal tail sequences of NF-M and NF-H (12, 13, 26, 29) that have been thought to extend and maintain the spacing between filaments (4). Similar sidearm extensions are not found in IFs composed of other IF proteins such as desmin, glial fibrillary acidic protein, or vimentin. In NFs assembled in vitro, all three subunits appear to be incorporated into core filaments (12, 26). Thus, current models of NF assembly suggest that NF-M and NF-H are the major components of sidearm extensions and are anchored to a core of NF-L via their central rod domains.Although much is known about NF structure and assembly, questions remain concerning NF function. A primarily structural role for NFs is suggested by their prominence in large axons (41). Small unmyelinated axons contain few NFs (9) and some small neurons lack morphologically identifiable NFs (3, 32, 38). Most dendrites contain few NFs and only in dendrites of large neurons such as motor neurons are NFs numerous (41).A role for NFs as a major determinant of axonal diameter has long been suspected from the correlation between NF content in axonal cross sections and axonal caliber (16). This correlation persists during axonal degeneration and regeneration (14) and changes in NF transport correlate temporally with alterations in the caliber of axons in regenerating nerves (15). Additionally, fewer NFs occur at nodes of Ranvier where axonal diameter is reduced (1), and certain NF epitopes are found only in regions where maximal axonal caliber has developed (6).Several animal models have supported a role for NFs in establishing axonal diameter. One is a Japanese quail (Quiverer) with a spontaneous mutation in NF-L that generates a truncated protein incapable of forming NFs (31). Homozygous mutants contain no axonal NFs and exhibit a mild generalized quivering. In these animals, radial growth of myelinated axons is severely attenuated (44) with a consequent reduction in axonal conduction velocity (37). In transgenic mice, Eyer and Petersen (8) expressed an NF-H/β-galactosidase fusion protein in which the COOH terminus of NF-H was replaced by β-galactosidase. NF inclusions were found in the perikarya of neurons and the resulting NF aggregates blocked all NF transport into axons resulting in axons with reduced calibers. More recently, Zhu et al. (45) have shown that mice lacking NFs due to a targeted disruption of the NF-L gene have diminished axonal calibers and delayed maturation of regenerating myelinated axons.Although these models clearly suggest a role for NFs in establishing axonal diameter, they contribute only limited information concerning the roles of the individual NF subunits. During development, NF-L and NF-M are coexpressed initially whereas NF-H appears later (4). Studies in transgenic mice have found that overexpressing mouse NF-L leads to an increased density of NFs, but no increase in axonal caliber (25). More recently, Xu et al. (43) overexpressed each of the mouse NF subunits either individually or in various combinations. They found that only when NF-L was overexpressed in combination with either NF-M or NF-H was axonal growth significantly increased. Interestingly, when NF-M and NF-H were overexpressed alone or in combination with one another, radial axonal growth was inhibited.It also remains incompletely understood how NF stoichiometries are regulated and the degree to which any one NF subunit is dominant in this regulation. Recently, conflicting data has appeared concerning the role of NF-M in regulating NF stoichiometries. We found that overexpression of human NF-M in transgenic mice increases the levels of endogenous mouse NF-L protein and decreases the extent of phosphorylation of NF-H (39). These results imply that NF-M may play a dominant role in regulating the levels of NF-L protein, the relative stoichiometry of NF subunits, and the phosphorylation status of NF-H. However different results were obtained by Wong et al. (40) who found that overexpression of mouse NF-M in transgenic mice did not effect the levels of axonal NF-L, and although it reduced NF-H, it did not effect its phosphorylation status.To further address these issues we generated mice bearing a null mutation in the mouse NF-M gene. Here we describe the effects of this mutation on nervous system development with particular reference to the role of the NF-M subunit in specifying axonal diameter and its effect on levels of the remaining NF subunits.     

Conformational properties of interacting neurofilaments: Monte Carlo simulations of cylindrically grafted apposing neurofilament brushes

Neurofilaments are essential cytoskeletal filaments that impart mechanical stability to axons. They are mostly assembled from three neurofilament proteins that form the core of the filament and its sidearms. Adjacent neurofilaments interact with each other through their apposing sidearms and attain unique conformations depending on the ionic condition, phosphorylation state, and interfilament separations. To understand the conformational properties of apposing sidearms under various conditions and gain insight into interfilament interactions, we performed Monte Carlo simulations of neurofilament pairs. We employed a sequence-based coarse-grained model of apposing NF sidearms that are end-tethered to cylindrical geometries according to the stoichiometry of the three neurofilament subunits. Monte Carlo simulations were conducted under different conditions such as phosphorylation state, ionic condition, and interfilament separations. Under salt-free conditions, apposing sidearms are found to adopt mutually excluding stretched but bent away conformations that are reminiscent of a repulsive type of interaction. Under physiological conditions, apposing sidearms are found to be in a coiled conformation, suggesting a short-range steric repulsive type of interaction. Increased sidearm mutual interpenetration and a simultaneous decrease in the individual brush heights were observed as the interfilament separation was reduced from 60 to 40 nm. The observed conformations suggest entropic interaction as a likely mechanism for sidearm-mediated interfilament interactions under physiological conditions.     

Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms.

We carried out immunolabeling studies of purified bovine spinal cord neurofilaments (NFs) and filaments reconstituted from several combinations of the NF triplet polypeptides, NF-H, NF-M, and NF-L. Six antibodies with known epitopes in either the rod domains or the tailpiece extensions of the NF triplet were used in these studies, and the immune complexes were visualized directly by the glycerol-spray, rotary shadowing technique, which permitted unambiguous identification of the NF sidearms. Antibodies directed against the tailpiece extensions of NF-H and NF-M labeled the sidearms of native NFs and reconstituted filaments containing those two polypeptides, but not the backbone of the filaments. Combining these two antibodies in the same labeling experiment resulted in more intense labeling than either of the antibodies alone, indicating that both NF-H and NF-M are capable of forming sidearms. The anti-NF-L tailpiece antibody recognized only a limited number of sites along native NFs, but labeled reconstituted NF-L homopolymers uniformly and heavily. This suggests that the NF-L tailpiece extension is relatively inaccessible in native filaments, but is accessible in reconstituted homopolymers. One possible explanation is that, in native NFs, the NF-H- and NF-M-containing sidearms curtailed antibody access to NF-L. A second possibility that is not mutually exclusive with the first is that, when both NF-L and another triplet polypeptide are present, they preferentially form heterodimers such that the NF-L tailpiece epitope becomes hidden. Taken collectively, and in combination with published structural information, our data are consistent with a subunit packing scheme in which an NF-L-containing dimer serves as the fundamental building block of most mammalian NFs, such that their sidearms consist of pairs of NF-H/NF-L, NF-M/NF-L, or NF-L/NF-L tailpiece extensions.     

Modulation of repulsive forces between neurofilaments by sidearm phosphorylation

Recent studies have advanced the notion that the axonal organization of neurofilaments (NFs) is based on mutual steric repulsion between the unstructured "sidearm" domains of adjacent NFs. Here, we present experimental evidence that these repulsive forces are modulated by the degree of sidearm phosphorylation. When NFs are sedimented into a gelatinous pellet, pellet volume falls with increasing ionic strength and enzymatic dephosphorylation; sedimentation of phosphorylated NFs in the presence of divalent cations also dramatically reduces pellet volume. Further, atomic force microscopy imaging of isolated mammalian NFs reveals robust exclusion of colloidal particles from the NF backbone that is reduced at high ionic strength and attenuated when the filaments are enzymatically dephosphorylated. Phosphate-phosphate repulsion on the NF sidearm appears to modulate NF excluded volume in a graded fashion, thereby controlling axonal NF organization through interfilament forces.     

Interplay between liquid crystalline and isotropic gels in self-assembled neurofilament networks

Neurofilaments (NFs) are a major constituent of nerve cell axons that assemble from three subunit proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight into a 10 nm diameter rod with radiating sidearms to form a bottle-brush-like structure. Here, we reassemble NFs in vitro from varying weight ratios of the subunit proteins, purified from bovine spinal cord, to form homopolymers of NF-L or filaments composed of NF-L and NF-M (NF-LM), NF-L and NF-H (NF-LH), or all three subunits (NF-LMH). At high protein concentrations, NFs align to form a nematic liquid crystalline gel with a well-defined spacing determined with synchrotron small angle x-ray scattering. Near physiological conditions (86 mM monovalent salt and pH 6.8), NF-LM networks with a high NF-M grafting density favor nematic ordering whereas filaments composed of NF-LH transition to an isotropic gel at low protein concentrations as a function of increasing mole fraction of NF-H subunits. The interfilament distance decreases with NF-M grafting density, opposite the trend seen with NF-LH networks. This suggests a competition between the more attractive NF-M sidearms, forming a compact aligned nematic gel, and the repulsive NF-H sidearms, favoring a more expansive isotropic gel, at 86 mM monovalent salt. These interactions are highly salt dependent and the nematic gel phase is stabilized with increasing monovalent salt.     

Role of phosphorylation on the structural dynamics and function of types III and IV intermediate filaments

Phosphorylation of types III and IV intermediate filaments (IFs) is known to regulate their organization and function. Phosphorylation of the amino-terminal head domain sites on types III and IV IF proteins plays a key role in the assembly/disassembly of IF subunits into 10 nm filaments, and influences the phosphorylation of sites on the carboxyl-terminal tail domain. These phosphorylation events are largely under the control of second messenger-dependent protein kinases and provide the cells a mechanism to reorganize the IFs in response to the changes in second messenger levels. In mitotic cells, Cdk1, Rho kinase, PAK1 and Aurora-B kinase are believed to regulate vimentin and glial fibrillary acidic protein phosphorylation in a spatio-temporal manner. In neurons, the carboxyl-terminal tail domains of the NF-M and NF-H subunits of heteropolymeric neurofilaments (NFs) are highly phosphorylated by proline-directed protein kinases. The phosphorylation of carboxyl-terminal tail domains of NFs has been suspected to play roles in forming cross-bridges between NFs and microtubules, slowing axonal transport and promoting their integration into cytoskeleton lattice and, in doing so, to control axonal caliber and stabilize the axon. The role of IF phosphorylation in disease pathobiology is discussed.     

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Neurofilament networks: Salt-responsive hydrogels with sidearm-dependent phase behavior

BackgroundNeurofilaments (NFs) — the neuron-specific intermediate filament proteins — are assembled into 10 nm wide filaments in a tightly controlled ratio of three different monomer types: NF-Low (NF-L), NF-Medium (NF-M), and NF-High (NF-H). Previous work on reconstituted bovine NF hydrogels has shown the dependence of network properties, including filament alignment and spacing, on the subunit composition.MethodsWe use polarized optical microscopy and SAXS to explore the full salt-dependent phase behavior of reconstituted bovine NF networks as a function of various binary and ternary subunit ratios.ResultsWe observe three salt-induced liquid crystalline phases: the liquid-ordered B_G and N_G phases, and the disordered I_G phase. We note the emergent sidearm roles, particularly that of NF-H in driving the parallel to cross-filament transition, and the counter-role of NF-M in suppressing the I_G phase.ConclusionsIn copolymers of NF-LH, NF-H shifts the I_G to N_G transition to nearer physiological salt concentrations, as compared to NF-M in copolymers of NF-LM. For ternary mixtures, the role of NF-H is modulated by the ratio of NF-M, where beneath 10 wt.% NF-M, NF-H drives the transition to the disordered phase, and above which NF-H increases interfilament spacing.General significanceUnderstanding the role of individual subunits in regulating the network structure will enable us to understand the mechanisms that drive the dysfunction of these networks, as observed in diseased conditions.     

Increased Solubility of High-Molecular-Mass Neurofilament Subunit by Suppression of Dephosphorylation: Its Relation to Axonal Transport

Abstract: To investigate the role of phosphorylation in the turnover and transport of neurofilament (NF) proteins in vivo, we studied their solubility properties and axonal transport in the rat sciatic nerve using phosphatase inhibitors to minimize dephosphorylation during preparation. About 20% of the 200-kDa subunit (NF-H) in the axon was soluble in the 1% Triton-containing buffer under the present conditions, whereas this amount was less and more variable in the absence of phosphatase inhibitors. The 68-kDa subunit (NF-L) was exclusively insoluble and not affected by the inhibitors. Such selective solubilization of NF-H by phosphorylation differed significantly from the in vitro phosphorylation with cyclic AMP-dependent protein kinase, which resulted in NF disassembly. The carboxy-terminal phosphorylation state of NF-H probed with the phosphorylation-sensitive antibodies was also not directly related to solubility. The solubility of NF-H did not differ along the nerve. In contrast, the solubility of l -[³⁵S]methionine-labeled, transported NF-H was lowest at the peak of radioactivity. Higher solubility at the leading edge, regardless of its location along the nerve, indicates that NF-H solubility is positively correlated with the rate of NF transport.     

Assembly and structure of neurofilaments isolated from bovine spinal cord

Neurofilaments (NFs) are neuron-specific intermediate filaments. The NFs were isolated from bovine spinal cord by differential centrifugation. The NFs were detected with electron microscopy and scanning tunneling microscopy (STM). Under STM, two kinds of sidearm of NFs were revealed: one was short, the other was long. They were arrayed along the 10-nm width core filaments one by one. The intervals between two adjacent long sidearms or two short sidearms were 20–22 nm, while those between two adjacent long and short sidearms were 10–11 nm. It was proposed that the rod domain of NF triplet proteins was 3/4-staggered. The assembly properties of NF triplet proteins were also studied. Immuno-colloidal-gold labeling assay showed that NF-M and NF-H are able to co-assemble into long filaments with NF-L. NF-M and NF-H can also co-constitute into winding filaments.     

The single neurofilament subunit of the lamprey forms filaments and regulates axonal caliber and neuronal size in vivo

Neurofilaments (NFs) are composed of a heteropolymer of three related subunits in mammalian neurons, where they are a major component of the cytoskeleton in large neurons and are thought to regulate axonal diameter. NFs in the lamprey, while ultrastructurally and functionally indistinguishable from mammalian NFs, are polymers of a single subunit protein, NF180. In this study, we use the simplicity of lamprey NFs and the accessibility of the lamprey central nervous system (CNS) to examine the effects of overproducing NFs in an identified giant neuron in vivo, and thus to elucidate the role of NFs in regulating neuronal size and axonal caliber in the vertebrate CNS. We show that overexpression of NF180 tagged with a variant of Green Fluorescent Protein (EYFP) in identified lamprey neurons (ABCs) and in human neuroblastoma (NB2a) cells results in the assembly of exogenous NF180 into ultrastructurally normal NFs that are tightly packed and unphosphorylated. These accumulate in the somata of NB2a cells and produce somatic swelling by 3 days post-transfection. NF180 overexpression in lamprey ABCs in vivo causes exogenous NFs to accumulate in ABC axons, somata, and dendrites, and induces a significant increase in axonal diameter without increasing axonal NF packing density. Overexpression of EYFP alone has none of these effects. We conclude that NF180 normally plays a critical role in determining axonal caliber in ABCs and may influence neuronal size in situations where NFs accumulate in the soma, such as after axonal injury.     

Hypophosphorylated neurofilament subunits undergo axonal transport more rapidly than more extensively phosphorylated subunits in situ

Axonal transport of neurofilaments (NFs) has long been considered to be regulated by phosphorylation. We present evidence that in optic axons of normal mice, the rate of NF axonal transport is inversely correlated with the NF phosphorylation state. In addition to 200 kDa NF-H and 145 kDa NF-M, axonal cytoskeletons from CNS contained a range of phospho-variants of NF-H migrating between 160-200 kDa, and of NF-M migrating at 97-145 kDa. While 160 kDa phospho-variants of NF-H have been well characterized, we confirmed the identity of the previously-described 97 kDa species as a hypophospho-variant of NF-M since (1) pulse-chase metabolic labeling confirmed the 97 kDa species to be a new synthesis product that was converted by phosphorylation over time into a form migrating at 145 kDa, (2) the 97 kDa protein reacted with multiple NF-M antibodies, including one specific for hypophosphorylated NF-M, and (3) dephosphorylation converted NF-M isoforms to 97 kDa. Autoradiographic analyses following metabolic radiolabeling demonstrated that hypophosphorylated NF-H and NF-M isoforms underwent substantially more rapid transport in situ than did extensively phosphorylated isoforms, while NF-H subunits bearing a developmentally delayed C-terminal phospho-epitope transported at a rate slower than that of total 200 kDa NF-H. Differential transport of phospho-variants also highlights that these variants are not homogeneously distributed among NFs, but are segregated to some extent among distinct, although probably overlapping, NF populations, indicating that axonal NFs are not homogeneous with respect to phosphorylation state.     

Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits

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-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, 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 alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- 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 [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.     

Regulation of neurofilament axonal transport by phosphorylation in optic axons in situ

Axonal transport of neurofilament (NFs) is considered to be regulated by phosphorylation. While existing evidence for this hypothesis is compelling, supportive studies have been largely restricted to correlative evidence and/or experimental systems involving mutants. We tested this hypothesis in retinal ganglion cells of normal mice in situ by comparing subunit transport with regional phosphorylation state coupled with inhibition of phosphatases. NF subunits were radiolabeled by intravitreal injection of 35S-methionine. NF axonal transport was monitored by following the location of the peak of radiolabeled subunits immunoprecipitated from 9x1.1 mm segments of optic axons. An abrupt decline transport rate was observed between days 1 and 6, which corresponded to translocation of the peak of radiolabeled subunits from axonal segment 2 into segment 3. Notably, this is far downstream from the only caliber increase of optic axons at 150 mu from the retina. Immunoblot analysis demonstrated a unique threefold increase between segments 2 and 3 in levels of a "late-appearing" C-terminal NF-H phospho-epitope (RT97). Intravitreal injection of the phosphatase inhibitor okadaic acid increased RT97 immunoreactivity within retinas and proximal axons, and markedly decreased NF transport rate out of retinas and proximal axons. These findings provide in situ experimental evidence for regulation of NF transport by site-specific phosphorylation.     

Assembly and structure of neurofllaments isolated from bovine spinal cord

Neurofilaments (NFs) are neuron-specific intermediate filaments. The NFs were isolated from bovine spinal cord by differential centrifugation. The NFs were detected with electron microscopy and scanning tunneling microscopy (STM). Under STM, two kinds of sidearm of NFs were revealed: one was short, the other was long. They were arrayed along the 10-nm width core filaments one by one. The intervals between two adjacent long sidearms or two short sidearms were 20—22 nm, while those between two adjacent long and short sidearms were 10—11 nm. It was proposed that the rod domain of NF triplet prnteins was 3/4-staggered. The assembly properties of NF triplet proteins were also studied. Immuno-colloidal-gold labeling assay showed that NF-M and NF-H are able to co-assemble into long filaments with NF-L. NF-M and NF-H can also co-constitute into winding filaments.     

CDK-5-Mediated Neurofilament Phosphorylation in SHSY5Y Human Neuroblastoma Cells

Cyclin-dependent kinase-5 (CDK-5) has been shown to play important roles in neuronal development and neurogenesis. In vitro studies indicate a role of CDK-5 in phosphorylation of neurofilaments (NFs). In this study, we have chosen the human neuroblastoma cell line SHSY5Y as a model system to study the in vivo phosphorylation of NF proteins by CDK-5. Upon differentiation of SHSY5Y cells with retinoic acid, we found that the phosphorylation of high molecular mass (NF-H) and medium molecular mass (NF-M) NFs increased, whereas the CDK-5 protein level and kinase activity were unaffected. The role of CDK-5 in the phosphorylation of cytoskeletal proteins was studied by using antisense oligonucleotides (ONs) to inhibit the expression of the CDK-5 gene. We found that inhibition of CDK-5 levels by antisense ON treatment resulted in a decrease in phosphorylation of NF-H that correlated with a decline in neurite outgrowth. These results demonstrate that CDK-5 is a major proline-directed kinase phosphorylating the human NF-H tail domain.