Multiple interactions of aluminum with neurofilament subunits: Regulation by phosphate-dependent interactions between C-terminal extensions of the high and middle molecular weight subunits

Exposure of individual purified neurofilament (NF) proteins to AlCl₃ alters their electrophoretic properties in a time- and concentration-dependent manner, as visualized by their failure to migrate into SDS gels. Co-incubation of purified high (NF-H) and middle (NF-M) but not low (NF-L) molecular weight NF subunits prevents this AlCl₃-induced alteration in electrophoretic migration. This latter finding suggested that specific interactions between NF-H and NF-M other than filament formation influenced their interaction with AlCl₃. Co-incubation of the 160 kDa α-chymotryptic cleavage product of NF-H (corresponding to the highly phosphorylated C-terminal sidearm domain) with native NF-M prevented alteration in subunit electrophoretic migration by AlCl₃. By contrast, intact, dephosphorylated NF-H subunits were unable to prevent AlCl₃-induced alteration of native NF-M electrophoretic migration. Taken together, these findings suggest that phosphate-dependent interactions between the sidearm extensions of NF-H and NF-M diminish the ability of AlCl₃ to associate with either subunit in a manner that alters their electrophoretic migration. This interaction of NF-H and NF-M sidearms is SDS-sensitive, while AlCl₃-induced alteration in electrophoretic migration of individual subunits is SDS-resistant. Addition of SDS to mixtures of NF-H and NF-M subunits disrupted the protective effect, and promoted AlCl₃-induced alterations in subunit electrophoretic migration. These findings support and extend the current hypothesis that the ability of aluminum to interact with NF subunits is a function of subunit phosphorylation, assembly, and extent of neurofilament-neurofilament cross-linking. © 1994 Wiley-Liss, Inc.     

A molecular dissection of the carboxyterminal tails of the major neurofilament subunits NF-M and NF-H.

We have initiated a multidisciplinary project that aims to dissect and ultimately define the functions of the long and unusual C-terminal "tail" sequences of the two high molecular weight neurofilament subunits, NF-M and NF-H. A series of recombinant fusion proteins containing selected NF-M and NF-H tail sequences were constructed using appropriate cDNAs. These fusion proteins were used to further define the epitopes for a variety of widely used neurofilament antibodies, including NN18 and N52, which are now available commercially from several companies. We also measured the SDS-PAGE mobility of the fusion proteins and found that, like the native neurofilament tails, the fusion proteins ran considerably slower than predicted from their molecular weight. Since all fusion proteins produced so far exhibit this characteristic we conclude that all segments of the NF-M and NF-H tail share this unusual property. Finally we were able to produce novel and potentially useful polyclonal and monoclonal antibodies to selected segments of NF-M and NF-H sequence. These antibody studies showed that the extreme C-termini of NF-M and NF-H are immunologically absolutely distinct from one another and also indicate that the extreme C-terminus of NF-M is immunologically much more conserved than the analogous region of NF-H. These findings are in complete agreement with our conclusions derived from amino acid sequence analysis, and further underline the possible functional importance of the extreme C-terminus of NF-M. We also show that the unusual immunological properties of the bovine NF-M tail we have previously observed do not extend to the extreme C-terminal region, which appears immunologically no different from the analogous region of other NF-M molecules. The peculiarities of bovine NF-M could be explained by the presence of a KSP motif that resembles the NF-H KSP prototype.     

Sequence of the rabbit neurofilament protein L.

In the course of screening a rabbit brain cDNA library with a probe for the H neurofilament protein, we identified a neurofilament L-cDNA. Its nucleotide sequence is 88% identical to that of human, indicating that L is highly conserved among species. The similarities between the sequences of L from rabbit and mouse suggest that the species-specific accumulation of neurofilaments that occurs in rabbit during aluminum intoxication is not a consequence of the primary structure of L.     

Assembly of glial intermediate filament protein is initiated in the centriolar region

Assembly of glial intermediate filament protein (GFP) into intermediate filaments (IF) was first detected by immunofluorescence in the perinuclear region of astrocytes differentiating in colony cultures before the rest of the cytoplasm was labeled. Double labeling with antisera specific for centrioles indicated that this site corresponds to the centriolar region. These studies suggest that the centriolar region plays an important role in the assembly of some types of IF as well as microtubules.     

The structure of the largest murine neurofilament protein (NF-H) as revealed by cDNA and genomic sequences

The complete primary structure of the largest mammalian neurofilament component, NF-H, is predicted from mouse cDNA and genomic clones, revealing a protein of molecular weight ca. 115,000. A central filament-forming domain structurally typical of all intermediate filament proteins is present, but anomalies are noted which may place constraints on the mechanism of NF-H assembly into filaments. The COOH-terminal portion of the protein is extremely long (661 amino acids) by comparison to non-neuronal intermediate filament components and has a remarkably monotonous, highly charged composition (Glu and Lys at 20% each). Its most remarkable feature is a tandem repeat of a 6 amino acid sequence containing the motif Lys-Ser-Pro that extends for more than half the length of the COOH-terminus. The Lys-Ser-Pro motif appears 48 times and since it is now known that the serine therein is a target for in vivo kinases, the massive axonal phosphorylation of NF-H is explained. Comparison of mouse and human NF-H reveals that otherwise conserved proteins have been subjected to evolutionary mutation within their multiphosphorylation repeat domains, although the Lys-Ser-Pro motif has been conserved.     

C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity

We probed the relationship of NF axonal transport of neurofilaments (NFs) to their phosphorylation state by comparing these parameters in two closely-aged groups of young adult mice - 2 and 5 months of age. This particular time interval was selected since prior studies demonstrate that optic axons have already completed axonal caliber expansion and attained adult NF levels by 2 months but, as shown herein, continue to increase NF-H C-terminal phosphorylation. NF axonal transport was monitored by autoradiographic analysis of the distribution of radiolabeled subunits immunoprecipitated from optic axon segments at intervals following intravitreal injection of 35S-methionine. Both the peak and front of radiolabeled NFs translocated faster in 2- vs. 5-month-old mice. This developmental decline in NF transport rate was not due to reduced incorporation of NFs into the cytoskeleton, nor to an overall decline in slow axonal transport. By excluding or minimizing other factors, these findings support previous conclusions that C-terminal NF phosphorylation regulates NF axonal transport.     

The high and middle molecular weight neurofilament subunits regulate the association of neurofilaments with kinesin: inhibition by phosphorylation of the high molecular weight subunit

Kinesin participates in axonal transport of neurofilaments (NFs), but the mode by which they attach to kinesin is unclear. We compared the association of NFs with kinesin in mice expressing or lacking NF-H or NF-M. In normal and M-/- mice, the leading edge of metabolically labeled NF subunits was selectively co-precipitated with kinesin. By contrast, the entire wave of radiolabeled subunits co-precipitated with kinesin in H-/- mice. Similar bulk levels of NFs co-precipitated with kinesin from normal and H-/- mice, but reduced levels co-precipitated from M-/- mice. These data suggest that both NF-H and NF-M regulate the association of NFs with kinesin. They further indicate that phosphorylation of NF-H dissociates NFs from kinesin and provides a mechanism by which NF-H phosphorylation can contribute to the slowing of NF axonal transport.     

Formation of 10-nanometer filaments from the 150K-dalton neurofilament protein in vitro

In the present study we report self-assembly of individual neurofilament (NF) triplet proteins (70K, 150K, and 200K daltons) isolated by anion exchange chromatography from bovine spinal cord. Formation of smooth 10-nm filaments by both NF 150K and NF 70K is shown. Optimal conditions for NK 150K filament formation were incubation in 100 mM MES, 0.2 M NaCl, 1 mM DTT, 0.5 mM EGTA, pH 6.5, at 37 degrees C for 24 hr. Under the same assembly conditions, NF 200K formed 7-nm coiled structures. These thin filaments were similar to those formed by NF 70K and 150K under less than optimal conditions. Our results indicate that NF 150K is an integral part of the filament (self-assembly of NF 70K was previously demonstrated by others). We suggest that the optimal conditions resulting in the formation of a 10-nm 200K homopolymer remain to be determined and that the thin coiled structures formed by all three NF proteins are protofilaments that coalesce to form a double helical 10-nm filament.     

Differential appearance of extensively phosphorylated forms of the high molecular weight neurofilament protein in regions of mouse brain during postnatal development

The appearance and accumulation of extensively phosphorylated forms of the high molecular weight neurofilament protein (H-phos) was studied in six regions of mouse brain during postnatal development by quantitative immunoblot analyses. H-phos (migrating at 200 kDa) was detected in brainstem, cerebellum, cortex and hippocampus as early as postnatal day 1. While NF-H levels increased dramatically during subsequent postnatal development in these regions, and reached levels similar to those observed in adult brain by postnatal day 14, quantitative differences were observed in both the rate and the extent of increase among individual regions. The most rapid accumulation of H-phos was observed in brainstem and cortex, where H-phos increased within the first postnatal week to levels comparable to those of adult brain. However, H-phos exhibited a slower developmental change in cerebellum, where the levels increased uniformly over the first two postnatal weeks. In hippocampus, the major increase in H-phos levels was delayed until the second postnatal week. In contrast to its early detection in the above regions, H-phos was not detected in immunoblot analyses of olfactory bulb or hypothalamus cytoskeletons at postnatal day 1, indicating that in these regions the accumulated levels of posttranslationally modified forms of this protein appeared relatively late. Furthermore, H-phos levels in hippocampus did not level off at postnatal day 14 and continued to increase until at least postnatal day 21. Immunoblot analyses of whole embryonic brain revealed the presence of H-phos as early as embryonic day 17, demonstrating that some mouse brain regions carry out extensive phosphorylation of NF-H during embryonic development.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identities, antigenic determinants, and topographic distributions of neurofilament proteins in the nervous systems of adult frogs and tadpoles of Xenopus laevis

Three proteins with nominal molecular weights of 73 kDa (XNF-L), 175 kDa (XNF-M), and 205 kDa (XNF-H) were identified as putative neurofilament proteins in the nervous system of the frog, Xenopus laevis. These conclusions were based on four criteria: (1) these proteins were enriched in cytoskeletal preparations; (2) they reacted with a monoclonal antibody (anti-IFA) that cross-reacts with an epitope found in all intermediate filament proteins; (3) they cross-reacted with monoclonal antibodies directed against specific mammalian neurofilaments; and (4) antibodies that reacted with these proteins on Western blots specifically stained neurons in immunohistochemical analyses. The neurofilament proteins in Xenopus were antigenically similar, but not identical to mammalian neurofilament proteins. The principal difference was that four antibodies that reacted on Western blots with rat NF-H reacted with XNF-M in Xenopus. However, similarly to mammals, antibodies against phosphorylated XNF-M specifically labeled axons, whereas an antibody that reacted only with dephosphorylated epitopes on XNF-M specifically labeled neuronal cell bodies in immunohistochemistry. Three other antibodies that reacted equally well with untreated or alkaline-phosphatase-treated XNF-M or XNF-H proteins also showed axonally restricted staining in the adult Xenopus nervous system. An XNF-L (XC5D10) antibody was produced which stained axons and cell bodies equivalently throughout the adult Xenopus nervous system. By 3 days of development (stage 42; Xenopus tadpoles), antibodies to all three molecular weight forms of the frog neurofilament proteins detected specific neurons in the brainstem and spinal cord; and antibodies to phosphorylated and dephosphorylated epitopes on XNF-M could discriminate between axons and cell bodies in the rhombencephalon. The immunoreactivities of four antibodies directed at XNF-L, -M, or -H, which were unaffected by alkaline phosphatase treatment, differed significantly in their immunohistochemical staining patterns in adult vs. premetamorphic frogs.     

Site-specific phosphorylation of the middle molecular weight human neurofilament protein in transfected non-neuronal cells.

We expressed the human midsized neurofilament subunit (NF-M) using genomic DNA in mouse L cells and showed that it is transcribed and translated into a protein capable of assembly into the cytoskeleton and of forming a filamentous network that colocalizes with the endogenous vimentin filaments. Moreover, human NF-M expressed in L cells is phosphorylated at sites within the multiphosphorylation repeat (MPR), i.e., the major sites of phosphorylation of NF-M in vivo. We also expressed a genomic construct lacking the MPR domain in the native molecule and showed that this MPR(-) protein also was expressed and formed a filamentous network despite diminished incorporation of radiolabeled phosphate. Two major conclusions emerged from the work described in this paper: human NF-M is translated, assembled, and phosphorylated at physiological sites without the need of any other specific neuronal proteins; phosphorylation sites other than the MPR are present within NF-M which may play a role in synthesis, assembly, and degradation of NF protein in humans.     

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.     

Phosphorylation of human high molecular weight neurofilament protein (hNF-H) by neuronal cyclin-dependent kinase 5 (cdk5)

Neurofilaments (NFs), the neuron-specific intermediate (i.e. ∼10-nm diameter) filaments are major cytoskeletal components of most neurons. In a mature mammalian neuron, NFs are co-assembled from three subunits, NF-L (low), NF-M (medium), and NF-H (high), with molecular masses of 68, 95, and 115 kDa, respectively. Neurofilament proteins (NF-Ps), particularly, NF-H, are most extensively phosphorylated in large myelinated axons under normal conditions. This phosphorylation occurs on the serine residues of the lysine (Lys)–serine (Ser)–proline (Pro) (KSP) multiple amino acid repeats of the carboxy-terminal tail domain. Phosphorylation of KSP motifs affects physical, biochemical, and immunological properties of NF-H. For example, phosphorylation is thought to play a pivotal role in the maintenance of the neuronal cytoskeletal structure which influences the conduction velocity of the nerve fiber. The key components responsible for phosphorylation are not known. In this study, an identified cyclin-dependent kinase 5 (cdk5), isolated from nervous tissue, has been shown to phosphorylate the human NF-H (hNF-H) and affects its electrophoretic mobility. On the basis of the following observations, it is suggested that neuronal cdk5 (cdk5) phosphorylates KSPXK motifs in the human high molecular weight neurofilament (hNF-H) and affects its electrophoretic mobility. (1) A 14-mer synthetic peptide (KSPEKAKSPVKEEA) derived from hNF-H; (2) a bacterially expressed protein containing 14 KSPXK multiple repeats of hNF-H in C-terminal tail domain; and (3) a dephosphorylated hNF-H in neurofilament preparation are phosphorylated by cdk5. The decrease in molecular mass of hNF-H caused by dephosphorylation was completely recovered upon cdk5 phosphorylation. It is proposed that neuronal cdk5 regulates phosphorylation of the KSPXK motif in hNF-H and other cytoskeletal proteins with similar motifs in the nervous system.     

Similarity of neurofilament proteins from different parts of the rabbit nervous system

In the nervous system, the various populations of neurons perform a large spectrum of functions. Although neurofilaments are a major constituent of the different neurons, the neurofilament protein composition and the expression the genes specifying these proteins may not be the same throughout the entire nervous system. To investigate these two aspects of the biology of neurofilaments, we have prepared neurofilament-rich fractions from different regions of the nervous system of strains of rabbits known to present a genetically determined polymorphism involving one of the neurofilament polypeptides (P200). Filaments were isolated from brain, spinal cord, sciatic, optic and trigeminal nerves, and lumbar ventral and dorsal roots by a procedure not involving axonal flotation and yielding material suitable for comparative analysis within a single animal. The filaments were compared for their variability as a function of the region from which they were prepared. For any given animal, the neurofilament peptides migrate to identical positions on SDS-gel electropherograms. Whatever allele of P200 is expressed in filaments from one region, the same allele is also expressed in all of the other filament preparations from that animal. On two-dimensional analysis isomorphs of the P68 neurofilament protein are not present in the same amounts in different regions of the nervous system. These results indicate that, although it seems that the gene for the P200 neurofilament protein is expressed uniformly throughout the nervous system, there may be some topographic specificity in the distribution of the other constituent proteins of neurofilaments.     

Appearance and localization of phosphorylated variants of the high molecular weight neurofilament protein in NB2a/d1 cytoskeletons during differentiation

We used immunoblot and immunocytochemical methodologies to characterize the appearance and intracellular localization of the high molecular weight neurofilament subunit (NF-H) within the Triton-insoluble cytoskeleton during the first 5 days of differentiation of mouse NB2a/d1 neuroblastoma cells. Hypophosphorylated and partially phosphorylated forms of NF-H were detected in cells before and throughout differentiation. By contrast, some extensively phosphorylated forms of NF-H were first detected on the third day of differentiation and at least one additional 200 kDa isoform was visualized in cytoskeletons only after five days of differentiation. Extensively phosphorylated forms of NF-H were restricted to axonal neurites; by contrast, hypophosphorylated and partially phosphorylated forms of NF-H were present throughout undifferentiated and differentiated cells.     

Triton-soluble phosphovariants of the heavy neurofilament subunit in developing and mature mouse central nervous system

The low abundance of soluble neurofilament (NF) subunits in mature axons has suggested that newly synthesized NF proteins rapidly assemble into highly stable polymers and associate with the Triton X-100–insoluble cytoskeleton. The dynamic nature of these subunit associations in vivo remains unresolved, and the applicability of this assembly model to NFs in other neuronal compartments or to developing neurons is unknown. Here, we report that a unique pool of Triton X-100–soluble, extensively phosphorylated, high molecular weight NF subunits (NF-H, or H-200) are abundantly expressed in the mouse CNS during early postnatal development and persist in the perikaryal compartment of some mature neurons. Triton-soluble H-200 subunits appeared at postnatal day 14 (P14) and remained high through P60, beyond which the percentage declined to marginal levels by P120. Medium and low molecular weight NF (NF-M and NF-L, respectively) were at all times only detectable within the cytoskeleton. Comparison of soluble and cytoskeleton-associated H-200 immunoreactivity indicated that certain phosphorylation-dependent epitopes were confined to the cytoskeleton. Pulse-chase radiolabeling analyses in optic pathway demonstrated that some Triton-soluble NF-H subunits are extensively phosphorylated within retinal perikarya before they are incorporated into Triton-insoluble structures. These findings indicate that the assembly behaviors of NF-H differ substantially from those of NF-M and NF-L, and that the interaction of NF-H with NFs may be more dynamic than is generally recognized, especially during brain development and within specific compartments of mature neurons. J. Neurosci. Res. 48:515–523, 1997. © 1997 Wiley-Liss Inc.