Monoclonal antibodies to the heavy neurofilament subunit (NF-H) of Torpedo cholinergic neurons.

Previous studies from our laboratory suggest that Alzheimer's disease sera contain a repertoire of antibodies to the heavy neurofilament subunit (NF-H) and that a subpopulation of these antibodies bind specifically to epitopes highly enriched in NF-H isolated from the purely cholinergic electromotor neurons of Torpedo. In the present study, we prepared and characterized monoclonal antibodies (MAbs) that bind to epitopes specifically enriched in Torpedo cholinergic neurons. This was performed by a differential enzyme-linked immunosorbent assay (ELISA) in which MAbs were selected that bind to epitopes much more abundant in the NF-H protein of Torpedo cholinergic neurons than in NF-H from the chemically heterogeneous Torpedo spinal cord. This yielded four MAbs, three of which (TC4, TC8, and TC21) were found to be specific to NF-H and one (TC15) that reacts with both NF-H and the medium-size neurofilament subunit NF-M. Dephosphorylation abolishes the binding of MAbs TC4 and TC15 to Torpedo cholinergic NF-H, partially reduces that of MAb TC21 and has no effect on the binding of MAb TC8. This suggests that the antigenic sites specific to Torpedo cholinergic NF-H contain phosphorylated as well as non phosphorylated epitopes. All the MAbs cross-react with rat brain NF-H.     

Varying degrees of phosphorylation determine microheterogeneity of the heavy neurofilament polypeptide (Nf-H)

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that possess phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.     

Transfected rat high-molecular-weight neurofilament (NF-H) coassembles with vimentin in a predominantly nonphosphorylated form

A fully encoding cDNA for the high-molecular-weight rat neurofilament protein (NF-H) has been isolated from a lambda gt11 library, sequenced and subcloned into eukaryotic expression vectors. Sequence analysis shows that rat NF-H has an overall homology of 72 and 88% with human and mouse NF-H, respectively. The head and rod domains are almost entirely identical, and the divergences are due to differences in the long C-terminal extensions of the molecule. The consensus phosphorylation sequence for neurofilaments Lys-Ser-Pro (KSP) is present 52 times. The predicted molecular mass of the protein is 115 kDa, 42% lower than that observed by SDS-PAGE. Upon transfection into vimentin-containing fibroblasts, such as L tk-, L929, and 3T6 cells, NF-H is seen distributed with vimentin by light and electron microscopic examinations indicating that copolymers of NF-H and vimentin are formed in these cells. Only a negligible proportion of the cells is positive when stained with a number of antibodies directed against phosphorylated NF-H epitopes. This is in contrast with the middle molecular weight NF protein (NF-M) transfected into L tk- and L929 cells, which can readily be detected by antibodies against phosphorylated neurofilament epitopes. The mobilities of the transfected protein on 1- and 2-dimensional gels confirm that NF-H is predominantly in a nonphosphorylated form. These results indicate that phosphorylation of NF-H, but not NF-M, on the KSP sequence is due to protein kinases, which are not present in fibroblasts and are presumably NF-H specific. The stable NF-H-expressing cell lines can therefore be used to study these putative neurofilament kinases in vitro and in vivo.     

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.     

Cyclin dependent kinase-5 (CDK-5) phosphorylates neurofilament heavy (NF-H) chain to generate epitopes for antibodies that label neurofilament accumulations in amyotrophic lateral sclerosis (ALS) and is present in affected motor neurones in ALS.

1. Accumulations of phosphorylated neurofilaments are seen in affected motor neurones in amyotrophic lateral sclerosis (ALS). 2. The authors demonstrate that cyclin dependent kinase-5 (cdk-5) will induce cellular phosphorylation of neurofilament heavy chain side-arms to generate epitopes for several antibodies that label these neurofilament accumulations. 3. By creating recombinant neurofilament fragments of NF-H side-arm domains, the authors also map the epitopes for several of these antibodies. 4. Finally, the authors demonstrate that cdk-5 is also present in affected motor neurones in ALS. These studies implicate cdk-5 in the pathogenesis of ALS.     

In situ localization of NF-H neurofilament subunit mRNAs in rat brain

The expression of NF-H neurofilament subunit mRNAs was investigated in the rat brain at different ontogenic stages. The levels of NF-H mRNAs vary 15-fold among brain regions with the highest level in the brainstem. In situ localization studies revealed that the NF-H mRNAs are mainly concentrated in the brainstem motoneuron nuclei. By increasing the sensitivity of the hybridization method, NF-H mRNAs could also be localized in neurons present in the cortex, thalamus and hippocampus areas. Minor amounts of NF-H mRNAs were already detected at 17-day embryonic stages.     

Selective changes in the neurofilament and microtubule cytoskeleton of NF-H/LacZ mice

This study focused mainly on changes in the microtubule cytoskeleton in a transgenic mouse where beta-galactosidase fused to a truncated neurofilament subunit led to a decrease in neurofilament triplet protein expression and a loss in neurofilament assembly and abolished transport into neuronal processes in spinal cord and brain. Although all neurofilament subunits accumulated in neuronal cell bodies, our data suggest an increased solubility of all three subunits, rather than increased precipitation, and point to a perturbed filament assembly. In addition, reduced neurofilament phosphorylation may favor an increased filament degradation. The function of microtubules seemed largely unaffected, in that tubulin and microtubule-associated proteins (MAP) expression and their distribution were largely unchanged in transgenic animals. MAP1A was the only MAP with a reduced signal in spinal cord tissue, and differences in immunostaining in various brain regions corroborate a relationship between MAP1A and neurofilaments.     

Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells

Adenosine 3',5'-cyclic monophosphate (cAMP) content of neurons is determined not only by the rate of synthesis but also by the rate of hydrolysis by cyclic nucleotide phosphodiesterases. Multiple forms of cyclic nucleotide phosphodiesterase exist in brain and other tissues, and these may be regulated by various hormones and neuromodulators. The present study examines this regulation in a cloned line of neuroblastoma cells (N18TG2). A biphasic Lineweaver-Burk plot of cAMP hydrolysis revealed two Kms approximating 5 and 25 microM. Lineweaver-Burk plots of cGMP hydrolysis were linear over a range of 1 microM to 1 mM and exhibited a Km of 37 microM. Neither cAMP nor cGMP competed for hydrolysis of the alternative cyclic nucleotide. No evidence for an allosteric activation of cAMP phosphodiesterase by cGMP was found. Calcium regulation of phosphodiesterase was not found in spite of preparation of the cell extract with several protease inhibitors, and addition of exogenous calmodulin. No effect of calmodulin antagonists (calmidazolium, W7, or trifluoperazine) was observed in vitro or in situ. Growth of the cells in the presence of 200 nM 3,5,3'-triiodothyronine (T3) resulted in an increased hydrolysis of cAMP but of cGMP. This increase was attributed to an increase in Vmax with no change in either high or low Km. This response was blocked by cycloheximide, suggesting that the thyroid hormone effect requires protein synthesis. The thyroid hormone response in neuroblastoma cells is compared with the results of other studies of thyroid hormone effects on phosphodiesterase in other tissues in vivo.     

Tpc1808 increases expression of NF-H in PC12 cells

Tpc1808 is a candidate chemotropic factor induced by nerve injury, and we report here the role of rat Tpc1808 in the expression of high-molecular-weight neurofilament (NF-H) in PC12 cells. A full-length Tpc1808 cDNA encoding the 275-amino-acid protein was constructed into pcDNA-HA and transfected into PC12 cells. The increased expression of NF-H was detectable in PC12-Tpc1808 clones by semi-qRT-PCR, real-time PCR, Western blot, and immunofluorescence. Such an increase could also be observed in PC12 cells subjected to recombinant Tpc1808 protein by real-time PCR and immunohistochemistry. At a concentration of 0.1 μg/mL, Tpc1808 protein, similar to NGF, could promote the expression of NF-H in a time-dependent manner. Our findings suggest that Tpc1808 is the gene related to promotion of nerve growth, and both the Tpc1808 gene and the Tpc1808 recombinant protein up-regulate the expression of NF-H in PC12 cells.     

Compartments of tubulin and tubulin-like proteins in differentiating neuroblastoma cells

Cytoplasmic, tubular and particulate fractions of differentiating neuroblastoma cells were prepared and the tubulin together with tubulin-like proteins was measured in each cell fraction during different stages of cell differentiation. In undifferentiated cells, 73%, 5% and 22% of the tubulin and tubulin-like proteins were contained in the cytoplasmic, tubular and particulate fractions, respectively. After 5 days of differentiation, the overall content of tubulin and tubulin-like proteins had increased by 73%. This corresponded to increases of 45%, 145% and 100% in the cytoplasmic, microtubular and particulate fractions, respectively. The increase in membrane-bound (particulate) tubulin and tubulin-like proteins was significantly greater that the total increase of proteins in the particulate fraction.Polyacrylamide gel electrophoresis of the proteins in each subcellular fraction revealed the presence of protein bands corresponding to the α and β subunits of tubulin. Whereas these bands indicated equal amounts of protein in the α and β positions for the tubular and particulate cell fractions, an analysis of the cytoplasmic fraction revealed much more protein migrating to the α-tubulin position than to the β-tubulin position, especially during cell differentiation. Furthermore, two overlapping but distinct protein bands were demonstrable in the position of the α-tubulin from the cytoplasmic fraction. These bands were designatedα₁ andα₂. The particulate fraction contained only theα₁ and the tubular fraction only theα₂ protein band.The addition of 1 mM dibutyryl cyclic AMP to the neuroblastoma cells, at the time when the serum was withdrawn, enhanced the rate of differentiation and the redistribution of tubulin and tubulin-like proteins within the 3 cellular compartments. These results are discussed as they relate to the regulation, biosynthesis, turnover and compartmentation of tubulin and tubulin-like proteins in differentiating neuroblastoma cells.     

Cyclic AMP-mediated potentiation of muscarinic hyperpolarization in neuroblastoma cells

Adenosine, 2-chloroadenosine and prostaglandin E₁ which are known to increase cyclic AMP in neuroblastoma cells potentiated the acetylcholine-induced muscarinic hyperpolarization of the cells without changing the resting membrane potential. The potentiation caused by 2-chloroadenosine was further augmented by Ro 20-1724, a phosphodiesterase inhibitor. A direct intracellular pressure application of cyclic AMP potentiated the muscarinic hyperpolarization without changing the resting membrane potential. Morphine which inhibits adenylate cyclase antagonized 2-chloroadenosine-induced potentiation of the muscarinic hyperpolarization. These results suggest that changes in cyclic AMP level modulate the muscarinic response of neuroblastoma cells.     

Spectrin expression in neuroblastoma cells

Mouse neuroblastoma cells express a spectrin-related molecule containing 240 kDal (kiloDalton) and 235 kDal subunits in a 1:1 ratio. The 240 kDal and 235 kDal subunits are nearly identical to the alpha and beta subunits respectively of brain spectrin by two dimensional chymotryptic peptide mapping analysis. The neuroblastoma cells do not express a measureable quantity of a red blood cell (rbc)-type spectrin molecule. Neuroblastoma spectrin has been localized throughout the cell body, and neurites of these cells by indirect immunofluorescence studies. As neuroblastoma cells are homogeneous, neuron-like, available in large quantity, and synthesize a single variant of spectrin which is closely related to brain spectrin(240/235), it is the best available model system for the study of the synthesis, assembly and turnover of a neuronal spectrin subtype.     

cDNA coding for the tail region of the high molecular weight rabbit neurofilament protein NF-H

A cDNA library was prepared from rabbit brain mRNA, in the expression vector, lambda gt11. The library was screened with polyclonal antibodies raised against the neurofilament protein NF-H, and a cloned cDNA (KMRH-1) was selected and characterized. The fusion protein coded for by KMRH-1 includes epitopes for two monoclonal antibodies which react with nonphosphorylated sites in the tail region of NF-H. The selected cDNA includes 891 base pairs. It hybridizes to human genomic DNA, as well as to rabbit genomic DNA, and to a rabbit brain mRNA with a size of 4.7 kilobases (kb). The sequence of KMRH-1 includes extensive repeating regions, including one duplicated 60-base segment. Within the first 196 bases, one 13-base segment is repeated 9 times. The cDNA codes for the carboxy-terminal 184 amino acid residues of NF-H, including a series of 9 serines, each surrounded by a similar group of amino acids: ..Ala.Lys.Ser.Pro.(Glu./Val.).Lys.. Comparison of the derived amino acid sequence for KMRH-1 indicates considerable divergence from the sequence information available for rodent NF-H (Robinson et al.: FEBS Lett 209:203-205, 1986). This diversity in amino acid sequence may account for the failure to induce tangles of neurofilaments in animals, such as rats, following treatment with doses of aluminum which are sufficient to induce such tangles in rabbits and to bring on seizures and behavioral pathology in both species.     

The neurofilament heavy chain (NfH) in the cerebrospinal fluid diagnosis of Alzheimer's disease

BACKGROUND/AIMS: Attempting to improve the cerebrospinal fluid (CSF) diagnosis of Alzheimer's disease (AD), the neurofilament heavy chain isoform, NfH(SMI35) was compared to other CSF markers [total tau, phospho-tau, amyloid beta 1-42 (Abeta42), the ratio of amyloid beta fragments Abeta42/Abeta40 (Abeta ratio)]. METHODS: CSF levels were determined in patients with AD (n = 109), mild cognitive impairment (MCI, n = 25), frontotemporal dementia (n = 15), vascular dementia (VD, n = 41), and controls (n = 58). RESULTS: CSF NfH(SMI35) was elevated in AD and VD as compared to controls (p < 0.05). Total tau was higher in AD as compared to controls (p < 0.05). CSF phospho-tau was elevated in AD as compared to controls and VD (p < 0.05 each). CSF Abeta42 and Abeta ratios in AD were lower than in MCI and controls (p < 0.05 each). CONCLUSION: The diagnostic potential of NfH(SMI35) is not superior to that of other CSF markers.     

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.     

Cyclic AMP-dependent protein phosphorylation in the rat anterior pituitary

The activation of cyclic adenosine 3'5'-monophosphate (cAMP)-dependent protein kinases has been implicated as an integral mechanism in stimulus-secretion coupling in the anterior pituitary. Therefore, we have investigated phosphorylation of endogenous protein substrates both in the presence and absence of cAMP in cell-free extracts of the rodent anterior pituitary. Specific phosphoprotein substrates in the rat anterior pituitary, which are phosphorylated by a cAMP-dependent protein kinase in vitro, were identified. Cyclic AMP potentiated the phosphorylation of proteins with apparent molecular weights of 85,000, 77,000, 63,000, 53,000, 39,000, and 33,000 as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins with apparent molecular weights of 124,000, 93,000, 48,000, and 43,000 were phosphorylated only in the presence of cAMP and not in the basal condition. The results highlight endogenous protein substrates that may potentially be involved in cAMP-dependent stimulus-secretion coupling in the anterior pituitary.