Disease relevance of Nefm

• The pattern of increased NFM mRNA during development, prior to the appearance of the wobbler phenotype, among littermates (from heterozygous carriers) conforms to a mendelian inheritance of a single gene defect 1:2:1 (wr/wr:wr/+:+/+) [1].

Psychiatry related information on Nefm

• In addition, spatial learning was impaired in transgenic mice expressing transgenes for NFH and NFM, similar to the memory deficits reported in patients with ALS [2].

High impact information on Nefm

• The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking [3].
• 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 [4].
• By contrast, the preserved dorsal root axons of NF-M-null mutant animals do not show a similar depletion of neurofilaments [5].
• 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) [6].
• Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites [7].

Biological context of Nefm

• The 5' regions of all three NF genes are identified as CpG islands that remain unmethylated in expressing and non-expressing tissues, although partial methylation occurs at -795 in NF-H and at -525 in NF-M [8].
• Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal alpha-helical region and the carboxy-terminal domains of the protein [9].
• Progressive deletion of 5' flanking sequence to -385 in NF-H, to -325 in NF-L and to -505 in NF-M caused a several-fold increase of transcription from the transfected plasmids [10].
• These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene [9].
• The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L [11].

Anatomical context of Nefm

• At this age, NF-L and NF-M are detected primarily in the processes of horizontal cells and retinal ganglion cells, but are rarely found in amacrine cell processes [12].
• They also show that NF-M can partner with intermediate filament proteins other than the NF-H and NF-L subunits in neurons to support slow transport and possibly other functions of neuronal intermediate filaments [13].
• In vivo pulse radiolabeling analyses in retinal ganglion cell neurons revealed that NF-L alone is incapable of efficient transport, whereas nearly one-half of the normal level of NF-M is transported along optic axons in the absence of the other triplet subunits [13].
• We have generated a set of amino- and carboxy-terminal deletions of the neurofilament NF-M gene and determined the molecular consequences of forced expression of these mutant constructs in mouse fibroblasts [14].
• Both adult and embryonic neurites were highly immunoreactive for NF-L and NF-M [15].

Associations of Nefm with chemical compounds

• The sole amino acid difference, E567K in the glutamate rich region of Nfm, between BALB/c and CXSD was found to be a simple genetic polymorphism because the same substitution existed in STS, a non-cataract mouse strain [16].
• Immunoblots of axonal proteins showed that HNE adducts are only detected in neurofilament heavy subunit (NFH) and, to a lesser extent, neurofilament medium subunit (NFM), both lysine-rich proteins, consistent with the adducts being limited to lysine residues [17].
• In this study, we have demonstrated that integrin-matrix interactions promote KSP tail-domain phosphorylation of neurofilament medium molecular weight subunits (NF-M) in cultured rat spinal cord motoneurons and NF-M transfected NIH 3T3 cells [18].
• The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M [19].
• In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L -/- and NF-M -/- transgenic animals (control, 39.9+/-1.8 m/s, NF-M -/-; 23.5+/-1. 4 m/s, and NF-L-/-; 12.0+/-0.7 m/s, mean+/-S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22-26 degrees C) [20].

Physical interactions of Nefm

• Affinity purification of NF-M 3'-UTR-binding proteins from rat brain followed by mass spectrometry and immunoprecipitation assays identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP E1 as the proteins forming the 70- and 47-kDa complexes, respectively [21].

Enzymatic interactions of Nefm

• Under these conditions, however, NF-M transport is completely abolished by deleting alpha-internexin [13].

Regulatory relationships of Nefm

• We found that laminin and fibronectin induce NF-M tail-domain phosphorylation in motoneurons and NIH 3T3 cells transfected with NF-M, respectively [18].

Other interactions of Nefm

• 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 [6].
• 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 [22].
• Therefore we excluded Nefl and Nef3 from the candidate genes for lr2 based on expression and mutation analyses [16].
• Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits [23].
• Both clonal lines can serve as host cells for expressible cDNAs introduced by transfection, such as the neurofilament protein, NF-M, and the small myelin basic protein [24].

Analytical, diagnostic and therapeutic context of Nefm

• On the other hand, laterally migrating neurons in the basal part of the mesencephalon were observed by electron microscopy to contact with tangentially arranged nerve fibers which were immunopositive for the 160 kDa neurofilament polypeptide at the light microscopic level from E10 [25].
• Increased expression of medium neurofilament (NFM) is an early molecular marker of wobbler mouse, an animal model of motoneuron disease [26].
• Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology [27].
• Additionally, transfected and control cells were proliferative (Ki67+) and expressed several neural markers (nestin, MAP-2, and NF160) as determined by immunofluorescence [28].
• Western blot analysis demonstrated that P19 EC cells contain significant levels of NF-L protein in the insoluble fraction but undetectable levels of NF-M and NF-H protein in either the insoluble or total cell fractions [29].

References