G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
neurofilament, medium polypeptide
G00000567 (Mus musculus)

Databases (6)

ENSG00000104722 (Ensembl human gene)
4741 (Entrez Gene)
119 (G2Cdb plasticity & disease)
162250 (OMIM)
Marker Symbol
HGNC:7734 (HGNC)
Protein Sequence
P07197 (UniProt)

Synonyms (2)

  • NF-M
  • NFM

Literature (26)

Pubmed - other

  • Alterations in oligodendrocyte proteins, calcium homeostasis and new potential markers in schizophrenia anterior temporal lobe are revealed by shotgun proteome analysis.

    Martins-de-Souza D, Gattaz WF, Schmitt A, Rewerts C, Marangoni S, Novello JC, Maccarrone G, Turck CW and Dias-Neto E

    Laboratório de Neurociências, Faculdade de Medicina da USP, Instituto de Psiquiatria, Universidade de São Paulo, Rua Dr. Ovídio Pires de Campos, No 785, s/n Consolação, São Paulo, SP, CEP 05403-010, Brazil. danms90@gmail.com

    Global proteomic analysis of post-mortem anterior temporal lobe samples from schizophrenia patients and non-schizophrenia individuals was performed using stable isotope labeling and shotgun proteomics. Our analysis resulted in the identification of 479 proteins, 37 of which showed statistically significant differential expression. Pathways affected by differential protein expression include transport, signal transduction, energy pathways, cell growth and maintenance and protein metabolism. The collection of protein alterations identified here reinforces the importance of myelin/oligodendrocyte and calcium homeostasis in schizophrenia, and reveals a number of new potential markers that may contribute to the understanding of the pathogenesis of this complex disease.

    Journal of neural transmission (Vienna, Austria : 1996) 2009;116;3;275-89

  • Association of the dopamine receptor interacting protein gene, NEF3, with early response to antipsychotic medication.

    Strous RD, Greenbaum L, Kanyas K, Merbl Y, Horowitz A, Karni O, Viglin D, Olender T, Deshpande SN, Lancet D, Ben-Asher E and Lerer B

    Beer Yaakov Mental Health Center, Beer Yaakov and Sackler Faculty of Medicine, Tel Aviv, Israel.

    Genetic variation in antipsychotic drug targets could underlie variability among patients in the time required for antipsychotic effects to be elicited. In a clinical, pharmacogenetic study we focused on the dopamine receptor interacting protein (DRIP) gene family. DRIPs are pivotally involved in regulating dopamine receptor signal transduction. Consecutively hospitalized, acutely psychotic patients with DSM-IV schizophrenia (n=121) were included in the study if they received treatment with typical antipsychotic medication (TYP, n=72) or TYP plus risperidone (TYP-R, n=49) for at least 2 wk. Clinical state and adverse effects were rated at baseline and after 2 wk. Patients improved significantly on both TYP and TYP-R with no significant difference between them. Early responders were defined as patients whose PANSS change scores were greater than the median. Twenty-two single nucleotide polymorphisms (SNPs) were analysed in five DRIP-encoding genes. Two SNPs in NEF3, which encodes the DRIP, neurofilament-medium (NF-M), were associated with early response (rs1457266, p=0.01; rs1379357, p=0.006). A 5 SNP haplotype spanning NEF3 was over-represented in early responders (p=0.015), in the combined patient group and in the TYP group alone. These findings suggest that variation in NEF3, most likely functional variants that are in linkage disequilibrium with the SNPs that we studied, influences rate of response to TYP. Since NEF3 is primarily associated with dopamine D1 receptor function, the evidence for a complementary role of dopamine D1 receptors in antipsychotic effects is considered. The findings reported here open an interesting research avenue in the pharmacogenetics of antipsychotic effects but require replication in larger samples treated in a controlled context.

    The international journal of neuropsychopharmacology 2007;10;3;321-33

  • Mutations in neurofilament genes are not a significant primary cause of non-SOD1-mediated amyotrophic lateral sclerosis.

    Garcia ML, Singleton AB, Hernandez D, Ward CM, Evey C, Sapp PA, Hardy J, Brown RH and Cleveland DW

    Ludwig Institute for Cancer Research and Department of Neurosciences, University of California at San Diego, 9500 Gilman Drive, CMM-E/Room 3072, La Jolla, CA 92093-0670, USA.

    While 1 to 2% of amyotrophic lateral sclerosis (ALS) is caused by mutations in the SOD1 gene, the basis of the remaining instances of inherited disease is unknown. Neuropathology, mouse modeling, and human genetics have implicated neurofilaments in the pathogenesis of motor neuron diseases such as ALS and Charcot-Marie-Tooth disease (CMT). A systematic analysis of the coding region and intron-exon boundaries of all three neurofilament genes is now reported from DNA samples derived from more than 200 non-SOD1 linked familial and sporadic ALS patients, along with >400 non-disease control individuals. Rare variants within each of the three neurofilament subunits that are predicted to affect neurofilament assembly properties were identified at higher frequency in non-SOD1 mutant ALS samples. However, none could be unambiguously linked to dominantly inherited disease. Thus, mutations in neurofilaments are possible risk factors that may contribute to pathogenesis in ALS in conjunction with one or more additional genetic or environmental factors, but are not significant primary causes of ALS.

    Funded by: NINDS NIH HHS: R37 NS027036

    Neurobiology of disease 2006;21;1;102-9

  • O-glycosylation of the tail domain of neurofilament protein M in human neurons and in spinal cord tissue of a rat model of amyotrophic lateral sclerosis (ALS).

    Lüdemann N, Clement A, Hans VH, Leschik J, Behl C and Brandt R

    Department of Neurobiology, University of Osnabrück, Barbarastrasse 11, D-49076 Osnabrück.

    Mammalian neurofilaments (NFs) are modified by post-translational modifications that are thought to regulate NF assembly and organization. Whereas phosphorylation has been intensely studied, the role of another common modification, the attachment of O-linked N-acetylglucosamine (GlcNAc) to individual serine and threonine residues, is hardly understood. We generated a novel monoclonal antibody that specifically recognizes an O-glycosylated epitope in the tail domain of NF-M and allows determination of the glycosylation state at this residue. The antibody displays strong species preference for human NF-M, shows some reactivity with rat but not with mouse or bovine NF-M. By immunohistochemistry and Western blot analysis of biopsy-derived human temporal lobe tissue we show that immunoreactivity is highly enriched in axons parallel to hyperphosphorylated NFs. Treatment of cultured neurons with the GlcNAcase inhibitor PUGNAc causes a 40% increase in immunoreactivity within 1 h, which is completely reversible and parallels the total increase in cellular O-GlcNAc modification. Treatment with the mitogen-activated protein kinase kinase inhibitor PD-98059 leads to a similar increase in immunoreactivity. In spinal cord tissue of a transgenic rat model for amyotrophic lateral sclerosis, immunoreactivity is strongly decreased compared with wild-type animals while phosphorylation is increased. The data suggest that hyperphosphorylation and tail domain O-glycosylation of NFs are synchronously regulated in axons of human neurons in situ and that O-glycosylation of NF-M is highly dynamic and closely interweaved with phosphorylation cascades and may have a pathophysiological role.

    The Journal of biological chemistry 2005;280;36;31648-58

  • Phosphoproteomic analysis of synaptosomes from human cerebral cortex.

    DeGiorgis JA, Jaffe H, Moreira JE, Carlotti CG, Leite JP, Pant HC and Dosemeci A

    Laboratory of Neurobiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.

    Journal of proteome research 2005;4;2;306-15

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • Sequence comparison of human and mouse genes reveals a homologous block structure in the promoter regions.

    Suzuki Y, Yamashita R, Shirota M, Sakakibara Y, Chiba J, Mizushima-Sugano J, Nakai K and Sugano S

    Human Genome Center, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, 108-8639, Japan. ysuzuki@ims.u-tokyo.ac.jp

    Comparative sequence analysis was carried out for the regions adjacent to experimentally validated transcriptional start sites (TSSs), using 3324 pairs of human and mouse genes. We aligned the upstream putative promoter sequences over the 1-kb proximal regions and found that the sequence conservation could not be further extended at, on average, 510 bp upstream positions of the TSSs. This discontinuous manner of the sequence conservation revealed a "block" structure in about one-third of the putative promoter regions. Consistently, we also observed that G+C content and CpG frequency were significantly different inside and outside the blocks. Within the blocks, the sequence identity was uniformly 65% regardless of their length. About 90% of the previously characterized transcription factor binding sites were located within those blocks. In 46% of the blocks, the 5' ends were bounded by interspersed repetitive elements, some of which may have nucleated the genomic rearrangements. The length of the blocks was shortest in the promoters of genes encoding transcription factors and of genes whose expression patterns are brain specific, which suggests that the evolutional diversifications in the transcriptional modulations should be the most marked in these populations of genes.

    Genome research 2004;14;9;1711-8

  • The G336S variant in the human neurofilament-M gene does not affect its assembly or distribution: importance of the functional analysis of neurofilament variants.

    Perez-Olle R, Lopez-Toledano MA and Liem RK

    Department of Pathology, Columbia University Medical Center, New York, New York 10032, USA.

    The human neurofilament medium (hNFM) subunit is one of the 3 neurofilament (NF) polypeptides, which are the most abundant intermediate filament (IF) proteins in post-mitotic neurons. The formation of neurofilamentous aggregates is a pathological hallmark of many neurodegenerative diseases, including the Lewy bodies found in Parkinson disease (PD). A Gly336Ser (G336S) variant in the rod domain of hNFM has recently been described in a patient with early-onset autosomal-dominant PD. In this study, we have generated a mammalian expression vector encoding the variant hNFM cDNA and characterized its effects on the formation of heteropolymeric IFs in heterologous cell lines. We have also investigated the distribution of the (G336S) hNFM variant protein in neuronal CAD cells, as well as the effects of the variant on the distribution of other cellular organelles and proteins. Our results demonstrate that the G336S variant does not affect the formation of IF networks nor the distribution of the variant hNFM protein. Our data suggest that if the G336S variant is involved in the development of PD, it does not appear to be due to defects in the assembly and distribution of NFs.

    Funded by: NINDS NIH HHS: NS15182

    Journal of neuropathology and experimental neurology 2004;63;7;759-74

  • Computational and experimental studies on human misshapen/NIK-related kinase MINK-1.

    Qu K, Lu Y, Lin N, Singh R, Xu X, Payan DG and Xu D

    Rigel, Inc., 1180 Veterans Boulevard, South San Francisco, CA 94080, USA. kqu@rigel.com

    We have studied the structure and function of Human Misshapen/NIK-related kinase (MINK-1) through a combination of computational methods and experimental approaches, including (1) fold recognition and sequence-structure alignment for each structural domain using the threading program PROSPECT, (2) gene expression and protein-protein interaction analysis of yeast homologs of human MINK-1 domains, and (3) yeast two-hybrid screening for proteins that interact with human MINK-1. Our structure prediction dissects MINK-1 into four domains: a conserved N-terminal kinase domain, followed by a coiled-coil region and a proline-rich region, and a C-terminal GCK domain. Gene expression and yeast two-hybrid analysis of yeast homologs of the MINK-1 domains suggest that MINK-1 may be involved in cell-cycle progression and cytoskeletal control. Consistent with these predicted functions, our in-house yeast two-hybrid screen for proteins that interact with human MINK-1 provides strong evidence that the coiled-coil and proline-rich domains of MINK-1 participate in the regulation of cytoskeletal organization, cell-cycle control and apoptosis. A homology model of the MINK-1 kinase domain was used to screen the NCI open compound database in DOCK, and chemical compounds with pharmaceutically acceptable properties were identified. Further medicinal chemistry compound structure optimization and kinase assays are underway.

    Current medicinal chemistry 2004;11;5;569-82

  • Co-purification and localization of Munc18-1 (p67) and Cdk5 with neuronal cytoskeletal proteins.

    Bhaskar K, Shareef MM, Sharma VM, Shetty AP, Ramamohan Y, Pant HC, Raju TR and Shetty KT

    Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore 560029, India.

    Munc18-1 (p67, nSec1, rbSec1), a neuron-specific 67kDa protein was independently identified as a syntaxin-binding protein, and as a component that co-purifies with, and regulates the kinase activity of cyclin dependent kinase (Cdk5). Gene knockout studies have demonstrated a role for Munc18-1 in synaptic vesicle docking and neurotransmitter release. Mice lacking Munc18-1 gene were synaptically silent, but the gene deletion did not prevent normal brain assembly, including the formation of layered structures, fiber pathways and morphologically defined synapses. Previous study has shown that Munc18-1 facilitates Cdk5 mediated phosphorylation of KSPXK domains of the neuronal cytoskeletal elements, suggesting that Munc18-1 may function in the regulation of cytoskeletal dynamics. Present study demonstrates the co-purification and co-localization of Munc18 with cytoskeletal elements and forms first step towards understanding the role for Munc18-1 in cytoskeletal dynamics. Conversely, the cytoskeletal proteins and Cdk5 co-purifies with Munc18-1 in a Munc18-1 immuno-affinity chromatography, suggesting a strong protein-protein interaction. Findings from immunofluorescence studies in PC12 cells have shown co-localization of Munc18-1 and Cdk5 with neurofilaments and microtubules. Further, immunohistochemical and immuno-electron microscopic studies of rat olfactory bulb also demonstrated co-localization of Munc18-1 and Cdk5 with cytoskeletal elements. Thus, the biochemical evidence of strong interaction between Munc18-1 with cytoskeletal proteins and morphological evidence of their (Munc18 and cytoskeletal elements) identical sub-cellular localization is suggestive of the possible role for Munc18-1 in cytoskeletal dynamics.

    Neurochemistry international 2004;44;1;35-44

  • NF-M is an essential target for the myelin-directed "outside-in" signaling cascade that mediates radial axonal growth.

    Garcia ML, Lobsiger CS, Shah SB, Deerinck TJ, Crum J, Young D, Ward CM, Crawford TO, Gotow T, Uchiyama Y, Ellisman MH, Calcutt NA and Cleveland DW

    Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA.

    Neurofilaments are essential for acquisition of normal axonal calibers. Several lines of evidence have suggested that neurofilament-dependent structuring of axoplasm arises through an "outside-in" signaling cascade originating from myelinating cells. Implicated as targets in this cascade are the highly phosphorylated KSP domains of neurofilament subunits NF-H and NF-M. These are nearly stoichiometrically phosphorylated in myelinated internodes where radial axonal growth takes place, but not in the smaller, unmyelinated nodes. Gene replacement has now been used to produce mice expressing normal levels of the three neurofilament subunits, but which are deleted in the known phosphorylation sites within either NF-M or within both NF-M and NF-H. This has revealed that the tail domain of NF-M, with seven KSP motifs, is an essential target for the myelination-dependent outside-in signaling cascade that determines axonal caliber and conduction velocity of motor axons.

    Funded by: NCRR NIH HHS: P41 RR004050, P41 RR04050; NINDS NIH HHS: NS 27036, NS 38855, R01 NS027036, R37 NS027036

    The Journal of cell biology 2003;163;5;1011-20

  • Mutation analysis of the neurofilament M gene in Parkinson's disease.

    Krüger R, Fischer C, Schulte T, Strauss KM, Müller T, Woitalla D, Berg D, Hungs M, Gobbele R, Berger K, Epplen JT, Riess O and Schöls L

    Department of Neurology, Neurodegeneration Laboratory, University of Tübingen, Hoppe-Seyler-Strasse 3, D-72076, Tübingen, Germany. reijko.krueger@uni-tuebingen.de

    Neurofilament M, a major component of Lewy bodies, represents an interesting candidate in the pathogenesis of Parkinson's disease (PD). We performed detailed mutation analyses of the NF-M gene in 322 familial and sporadic PD patients. Two polymorphisms (Ala475Thr and Gly697Arg) occurred at similar frequencies in PD patients and controls. A Pro725Gln substitution and a deletion of valine in position 829 were identified in two PD patients. These substitutions affect residues of the NF-M protein that are highly conserved among different species. None of our patients carried the Gly336Ser substitution, which has been described in familial PD. Our results argue against a major role of NF-M in PD. However, rare variants of the NF-M gene may act as susceptibility factors for PD and functional analyses of the identified variations are warranted to decipher possible mechanisms in neurodegeneration.

    Neuroscience letters 2003;351;2;125-9

  • [Detection of level and mutation of neurofilament mRNA in Alzheimer's disease].

    Wang Y, Wang Q and Wang J

    Department of Pathophysiology, Research Institute of Neuroscience, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

    Objective: To study if the increase of levels of neurofilament (NF)-M and NF-L in brain of Alzheimer's diseases (AD) is caused by increase of NF-M and NF-L.

    Methods: Semi-quantitative reverse transcription PCR was used to measure the NF-M and NF-L mRNA levels in the brain gray matter obtained by autopsy from 8 patients with AD and 7 age-matched patients with Huntington's disease (HD) used as controls. The mutation of NF-M and NF-L was detected by single strand DNA conformation polymorphism technique.

    Results: The levels of NF-M mRNA and NF-L mRNA were significantly lower in the brains of AD patients (0.3 +/- 0.03 * OD(NF-M)/OD(GAPDH) or OD(NF-L)/OD(GAPDH) and 0.44 +/- 0.16 * OD(NF-M)/OD(GAPDH) or OD(NF-L)/OD(GAPDH) respectively) than in the brains of HD patients (0.42 +/- 0.07 OD(NF-M)/OD(GAPDH) or OD(NF-L)/OD(GAPDH) and 0.79 +/- 0.09 OD(NF-M)/OD(GAPDH) or OD(NF-L)/OD(GAPDH) respectively) (P < 0.005 and P < 0.007 respectively). NF-M mutation was detected in one AD patient. No NF-L mutation was found in the samples examined.

    Conclusion: The increase of neurofilament protein observed in the brains of AD patients is related neither to increased gene transcription nor to mutation of NF mRNA.

    Zhonghua yi xue za zhi 2002;82;8;519-22

  • Transcription of intermediate filament genes is enhanced in focal cortical dysplasia.

    Taylor JP, Sater R, French J, Baltuch G and Crino PB

    Department of Neurology, University of Pennsylvania Medical Center, Philadelphia 19104, USA.

    Focal cortical dysplasia (FCD) is characterized by disorganized cerebral cortical cytoarchitecture. Increased expression of several intermediate filament (IF) proteins such as neurofilament, vimentin, alpha-internexin, and nestin observed in dysplastic "balloon" neurons (DN) may contribute to disrupted cortical lamination. We hypothesized that increased IF protein expression results from enhanced IF gene transcription within dysplastic neurons. We used a novel strategy to evaluate IF mRNA expression in three FCD specimens from medically intractable epilepsy patients. Poly(A) mRNA was amplified (aRNA) from single microdissected DN, morphologically normal neurons at the margin of the FCD resection, morphologically normal neurons in non-FCD cortex from epilepsy patients, and normal control neurons. Radiolabeled aRNA from single neurons was used to probe cDNA arrays containing the low (NFL), medium (NFM) and high (NFH) molecular weight neurofilament isoform, alpha-internexin, desmin, vimentin, peripherin (PRPH), nestin, and glial fibrillary acidic protein (GFAP) cDNAs. Hybridization intensity of aRNA-cDNA hybrids was used to quantify relative IF abundance. Increased expression of nestin, alpha-internexin, PRPH, vimentin, NFL, NFM, and NFH mRNAs was found in DN when compared with the three control neuronal subtypes. Desmin and GFAP mRNAs were not detected in any cell types. Expression of PRPH mRNA and protein in select DN was confirmed by reverse transcription-polymerase chain reaction and immunohistochemistry. We conclude that aberrant expression of IF proteins in FCD likely results from enhanced transcription of IF genes in dysplastic neurons and propose that future analysis of transcriptional elements that regulate IF expression be evaluated in FCD.

    Funded by: NIMH NIH HHS: K08 MH 01658

    Acta neuropathologica 2001;102;2;141-8

  • Cdk5 and MAPK are associated with complexes of cytoskeletal proteins in rat brain.

    Veeranna, Shetty KT, Takahashi M, Grant P, Pant HC and Veeranna GJ

    Laboratory of Neurochemistry, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bldg. 36, Rm. 4D20, Bethesda, MD 20892-4130, USA.

    Neurofilament proteins, the major cytoskeletal components of large myelinated axons, are highly phosphorylated by second messenger-dependent and -independent kinases. These kinases, together with tubulins and other cytoskeletal proteins, have been shown to bind to neurofilament preparations. Cdk5 and Erk2, proline-directed kinases in neuronal tissues, phosphorylate the Lys-Ser-Pro (KSP) repeats in tail domains of NF-H, NF-M, and other axonal proteins such as tau and synapsin. In neurofilament and microtubule preparations from rat brain, we demonstrated by Western blot analysis that cdk5, a neuronal cyclin dependent kinase and Erk1/2 were associated with complexes of NF proteins, tubulins and tau. Using P13(suc1) affinity chromatography, a procedure known to bind cdc2-like kinases in proliferating cells with high affinity, we obtained a P13 complex from a rat brain extract exhibiting the same profiles of cdk5 and Erk2 bound to cytoskeletal proteins. The phosphorylation activities of these preparations and the effect of the cdk5 inhibitor, butyrolactone, were consistent with the presence of active kinases. Finally, during a column fractionation and purification of Erk kinases from rat brain extracts, fractions enriched in Erk kinase activity also exhibited co-elution of phosphorylated NF-H, tubulin, tau and cdk5. We suggest that in mammalian brain, different kinases, their regulators and phosphatases form multimeric complexes with cytoskeletal proteins and regulate multisite phosphorylation from synthesis in the cell body to transport and assembly in the axon.

    Brain research. Molecular brain research 2000;76;2;229-36

  • Cytoplasmic O-GlcNAc modification of the head domain and the KSP repeat motif of the neurofilament protein neurofilament-H.

    Dong DL, Xu ZS, Hart GW and Cleveland DW

    Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    Neurofilaments, the major intermediate filaments in large myelinated neurons, are essential for specifying proper axonal caliber. Mammalian neurofilaments are obligate heteropolymers assembled from three polypeptides, neurofilament (NF)-H, NF-M, and NF-L, each of which undergoes phosphorylation at multiple sites. NF-M and NF-L are known to be modified by O-linked N-acetylglucosamine (O-GlcNAc) (Dong, D. L.-Y., Xu, Z.-S., Chevrier, M. R., Cotter, R. J., Cleveland, D. W., and Hart, G. W. (1993) J. Biol. Chem. 268, 16679-16687). Here we further report that NF-H is extensively modified by O-GlcNAc at Thr53, Ser54, and Ser56 in the head domain and, somewhat surprisingly, at multiple sites within the Lys-Ser-Pro repeat motif in the tail domain, a region in assembled neurofilaments known to be nearly stoichiometrically phosphorylated on each of the approximately 50 KSP repeats. Beyond the earlier identified sites on NF-M and NF-L, O-GlcNAc sites on Thr19 and Ser34 of NF-M and Ser34 and Ser48 of NF-L are also determined here, all of which are localized in head domain sequences critical for filament assembly. The proximity of O-GlcNAc and phosphorylation sites in both head and tail domains of each subunit indicates that these modifications may influence one another and play a role in filament assembly and network formation.

    Funded by: NICHD NIH HHS: HD13563; NINDS NIH HHS: NS27036

    The Journal of biological chemistry 1996;271;34;20845-52

  • Differential binding of apolipoprotein E isoforms to tau and other cytoskeletal proteins.

    Fleming LM, Weisgraber KH, Strittmatter WJ, Troncoso JC and Johnson GV

    Department of Psychiatry, University of Alabama, Birmingham, 35294-0017, USA.

    The apolipoprotein E4 (apoE4) gene dose is a major risk factor for late-onset and sporadic Alzheimer's disease with 50% of homozygous patients developing the disease by age 70. Previous studies have shown localization of apoE to the cytoplasm of certain neurons within the brain. In addition, apoE3, but not apoE4, forms SDS-stable complexes with the microtubule-associated proteins tau and MAP-2. To extend these studies and quantitate the association of apoE with other proteins, the association of apoE3 and apoE4 with several cytoskeletal proteins was examined using both gel shift and overlay assays. In the gel shift assay, apoE3 formed SDS-stable complexes with the longest isoform of human recombinant tau (T4L), the shortest isoform of human recombinant tau (T3), and the 160-kDa neurofilament protein (NFM). ApoE4 did not bind T3, T4L, or NFM in this assay. The association of apoE3 and apoE4 with T4L, actin, or tubulin was further examined in an overlay assay with known amounts of the cytoskeletal proteins slot-blotted onto nitrocellulose and incubated in 0.15 microM (5 microg/ml) apoE3 or apoE4. In this assay, apoE3 and apoE4 bound T4L and tubulin equally well. In contrast, apoE3 bound actin with a significantly greater affinity than did apoE4. These results indicate that apoE isoforms interact with cytoskeletal proteins with at least two different binding affinities. The more avid interaction results in the formation of complexes which are SDS stable and occurs almost exclusively with apoE3, while the other interactions between apoE and cytoskeletal proteins are specific for apoE3.

    Funded by: NHLBI NIH HHS: HL41633; NIA NIH HHS: AG06569; NINDS NIH HHS: NS27538

    Experimental neurology 1996;138;2;252-60

  • Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits.

    Tu PH, Elder G, Lazzarini RA, Nelson D, Trojanowski JQ and Lee VM

    Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-4283, USA.

    Neurofilaments (NFs), the major intermediate filaments of central nervous system (CNS) and peripheral nervous system (PNS) neurons, are heteropolymers formed from the high (NFH), middle (NFM), and low (NFL) molecular weight NF subunits. To gain insights into how the expression of NF subunit proteins is regulated in vivo, two transgenes harboring coding sequences for human NFM (hNFM) with or without the hNFM multiphosphorylation repeat domain were introduced into mice. Expression of both hNFM constructs was driven by the hNFM promoter and resulted in increased levels of hNFM subunits concomitant with an elevation in the levels of mouse NFL (mNFL) proteins in the CNS of both lines of transgenic mice. The increased levels of mNFL appear specific to NFM because previous studies of transgenic mice overexpressing either NFL or NFH did not result in increased expression of either of the other two NF subunits. Further, levels of the most heavily phosphorylated isoforms of mouse NFH (mNFH) were reduced in the brains of these transgenic mice, and electron microscopic studies showed a higher packing density of NFs in large-diameter CNS axons of transgenic versus wild-type mice. Thus, reduced phosphorylation of the mNFH carboxy terminal domain may be a compensatory response of CNS neurons to the increase in NFs, and reduced negative charges on mNFH sidearms may allow axons to accommodate more NFs by increasing their packing density. Taken together, these studies imply that NFM may play a dominant role in the in vivo regulation of the levels of NFL protein, the stoichiometry of NF subunits, and the phosphorylation state of NFH. NFM and NFH proteins may assume similar functions in regulation of NF packing density in vivo.

    The Journal of cell biology 1995;129;6;1629-40

  • Isolation of novel and known genes from a human fetal cochlear cDNA library using subtractive hybridization and differential screening.

    Robertson NG, Khetarpal U, Gutiérrez-Espeleta GA, Bieber FR and Morton CC

    Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115.

    We used a combination of subtractive hybridization and differential screening strategies to identify genes that may function normally in hearing and, when mutated, result in deafness. A human fetal cochlear (membranous labyrinth) cDNA library was subtracted against total human fetal brain RNAs by an avidin-biotin-based procedure to enrich for cochlear transcripts. Subtracted cochlear clones were differentially screened with 32P-labeled total cochlear and total brain cDNA probes. Sequence analysis of clones that hybridized more intensely with cochlear than with brain cDNA probes revealed some previously characterized genes, including mitochondrial sequences, collagen type I alpha-2 (COL1A2), collagen type II alpha-1 (COL2A1), collagen type III alpha-1 (COL3A1), spermidine/spermine N1-acetyltransferase (SAT), osteonectin (SPARC), and peripheral myelin protein 22 (PMP22). Also identified were clones that are potential novel cochlear genes. Northern blots of cochlear and brain RNAs probed with COL1A2, COL2A1, COL3A1, SAT, SPARC, PMP22, and a novel sequence, designated Coch-5B2, confirm results of the subtractive procedure by showing preferential cochlear expression. A number of these genes serve structural or regulatory functions in extracellular matrix or neural conduction; defects in some of these genes are associated with disorders involving hearing loss. Partial sequence analysis of Coch-5B2 reveals a von Willebrand factor type A-like domain in this cDNA. To assess the cochlear specificity of Coch-5B2, a Northern blot panel of 14 human fetal tissue RNAs was probed with Coch-5B2, showing differential expression of this novel gene in the cochlea.

    Funded by: NHLBI NIH HHS: T32 HL07627; NIDCD NIH HHS: DC00871

    Genomics 1994;23;1;42-50

  • Glycosylation of mammalian neurofilaments. Localization of multiple O-linked N-acetylglucosamine moieties on neurofilament polypeptides L and M.

    Dong DL, Xu ZS, Chevrier MR, Cotter RJ, Cleveland DW and Hart GW

    Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.

    Neurofilaments are neuronal intermediate filaments that play an important role in the growth and maintenance of large myelinated axons. Mammalian neurofilaments are composed of three polypeptide subunits, designed as NF-L, NF-M, and NF-H, all of which are phosphorylated. Here, we demonstrate by several criteria that neurofilament polypeptides are also modified by an abundant type of intracellular protein glycosylation in which single N-acetylglucosamine monosaccharides are O-glycosidically (O-GlcNAc) linked to serine or threonine residues. In purified neurofilament proteins, the O-GlcNAc modifications occur at a stoichiometry of approximately 0.1 and 0.15 mol of GlcNAc/mol of NF-L and NF-M, respectively. The predominant sites of O-GlcNAc attachment on NF-L and NF-M are identified using proteolysis, purification of the glycopeptides, and subsequent analysis by automated gas-phase sequencing, manual Edman degradation, and laser desorption mass spectrometry. For NF-L, both major sites of glycosylation (Thr21 and Ser27) are located at the NH2-terminal head domain. For NF-M, one major site (Thr48) lies within the NH2-terminal head domain, whereas the other (Thr431) is located at the tail domain. Deletions encompassing these sites have been shown previously to have a dominant detrimental effect upon neurofilament assembly, raising questions about the specific function(s) of the saccharide moieties at these sites. Specific identification of these O-GlcNAc attachment sites has set the stage for more detailed mutagenic analysis of O-GlcNAc functions on neurofilaments.

    Funded by: NICHD NIH HHS: HD13563; NINDS NIH HHS: NS27036; PHS HHS: 1P30A128748

    The Journal of biological chemistry 1993;268;22;16679-87

  • RFLP for BgI II at the human neurofilament medium chain (NEF3) gene locus.

    Ding Y, Reed DR, Baltazar MC and Price RA

    Department of Psychiatry, University of Pennsylvania, Philadelphia 19104-6141.

    Funded by: NIDDK NIH HHS: DK44073; NIMH NIH HHS: MH43880, MH44210

    Nucleic acids research 1992;20;6;1429

  • Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M.

    Xu ZS, Liu WS and Willard MB

    Department of Anatomy and Neurobiology and Biochemistry, Washington University School of Medicine, St. Louis, Missouri 63110.

    The COOH-terminal tail domain of the neurofilament polypeptide M from rat nervous tissue contains approximately six molecules of phosphate. We report here that protein kinases in a crude cytoskeleton preparation of rat nervous tissue phosphorylated a set of tryptic peptides of M similar (but not identical) to those phosphorylated by living dorsal root ganglion cells in culture. Using these phosphopeptides as markers, we purified these same peptides from rat spinal cord and identified six specific phosphorylation sites in M by enzymatic and chemical criteria. These sites, serines 502, 506, 536, 606, 608, and 666, are all located in the COOH-terminal tail domain. Four are embedded in the repeated motif KSP whereas two are within variants of this motif, KSD and ESP. All of the sites that were preceded by lysine were resistant to alkaline phosphatase prior to modification of the lysine with citraconic anhydride. The identification of these sites should aid in investigations of the function of the phosphorylation of this protein and provides criteria for identifying the relevant kinases.

    Funded by: NEI NIH HHS: EYO 2682

    The Journal of biological chemistry 1992;267;7;4467-71

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

    Chin TK, Harding SE and Eagles PA

    Department of Biophysics, King's College London, U.K.

    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.

    Funded by: Wellcome Trust

    The Biochemical journal 1989;264;1;53-60

  • Identification of the major multiphosphorylation site in mammalian neurofilaments.

    Lee VM, Otvos L, Carden MJ, Hollosi M, Dietzschold B and Lazzarini RA

    Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia.

    The sequence Lys-Ser-Pro-Val-Pro-Lys-Ser-Pro-Val-Glu-Glu-Lys-Gly repeats six times serially in the human midsized neurofilament (NF) protein (NF-M). To establish whether Lys-Ser-Pro-Val(Ala) is the major site for in vivo NF phosphorylation, peptides based on the human NF-M repeat were synthesized and chemically phosphorylated. These synthetic peptides were probed with 515 monoclonal antibodies (mAbs) that were raised to, and distinguished, several differentially phosphorylated forms of NF proteins. Studies with 95 of those mAbs that recognized the peptides before and after chemical phosphorylation demonstrated that a highly immunogenic epitope shared by the peptides is present in NFs from all species tested, including invertebrates. This suggests the phylogenetic conservation of a major NF phosphorylation site. Lastly, a cross-reactive antigenic determinant shared by the peptides and the major NF phosphorylation site was shown to exist in neurofibrillary tangles of patients with Alzheimer disease as well as in two neuron-specific microtubule-associated proteins (MAPs)--i.e., MAP2 and tau.

    Funded by: NIA NIH HHS: AG-06107; NINDS NIH HHS: NS-15722, NS-18616

    Proceedings of the National Academy of Sciences of the United States of America 1988;85;6;1998-2002

  • The human mid-size neurofilament subunit: a repeated protein sequence and the relationship of its gene to the intermediate filament gene family.

    Myers MW, Lazzarini RA, Lee VM, Schlaepfer WW and Nelson DL

    We report the isolation and sequence of cDNA and genomic clones for one of the two large subunits of human neurofilament, NF-M. Analysis of the sequence has allowed us to investigate the structure of the carboxy-terminal tail of this protein, and to compare it to that of the small neurofilament as well as to other intermediate filaments. The carboxy-terminal region of the protein contains a 13 amino acid proline- and serine-rich sequence repeated six times in succession. Within each repeat unit are two smaller repeats of the sequence Lys-Ser-Pro-Val. The four amino acid repeat may represent a kinase recognition site in a region of the protein that is known to be highly phosphorylated. We also note the presence of an additional heptad repeat at the extreme carboxy terminus of the protein. This region of 60 amino acids may be involved in coiled-coil interactions similar to those that facilitate the filament formation in the rod region. The human gene contains only two introns. Their positions correspond to two of the three introns found in the small neurofilament of the mouse. Thus, two of the three neurofilament genes of mammals have similar structures which are quite different from those of the other intermediate filaments. This finding suggests a common origin of the neurofilament subunits, whose evolutionary relationship to other intermediate filament genes is uncertain.

    The EMBO journal 1987;6;6;1617-26

Gene lists (8)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000037 G2C Homo sapiens Pocklington H6 Human orthologues of cluster 6 (mouse) from Pocklington et al (2006) 5
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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