Disease relevance of Nefh

• NF-H is not encountered in either the horizontal cell processes or the ganglion nerve fibers until P5 [1].
• By 3-4 months of age, these NF-H transgenics progressively develop neurological defects and abnormal neurofilamentous swellings that are highly reminiscent of those found in amyotrophic lateral sclerosis (ALS) [2].
• We propose that a modest up-regulation of NF-H cross-linkers can result in an impairment of neurofilament transport, causing neuronal swellings with ensuing axonopathy and muscle atrophy, a mechanism of pathogenesis pertinent to the possible etiology of ALS [2].
• Microscopic examination corroborated the protective effect of NF-H protein against SOD1 toxicity [3].
• We have found that constructs containing 5' region from -1314 to -115 exhibit a 3-5-fold higher level of NF-H promoter activity, relative to the adenovirus major late promoter (pML), in brain versus liver extracts [4].

Psychiatry related information on Nefh

• 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 [5].
• By immunohistochemical techniques, the accumulation of 200 kDa neurofilament protein was demonstrated in affected neurites in murine CJD [6].

High impact information on Nefh

• First, transgenic mice that overexpress neurofilament proteins show motor neuron degeneration and, second, variant alleles of the neurofilament heavy-subunit gene (NF-H) have been found in some human ALS patients [7].
• To investigate how disorganized neurofilaments might cause neurodegeneration, we examined axonal transport of newly synthesized proteins in mice that overexpress the human NF-H gene [7].
• The transgene, regulated by NFH sequences, was expressed in projection neurons [8].
• 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 [9].
• In young mutant animals, fast axonal transport appeared to be intact, but NF-H, as well as NF-M and NF-L, accumulated in the cell bodies of peripheral sensory neurons accompanied by a reduction in sensory axon caliber [10].

Chemical compound and disease context of Nefh

• The differentiation states of neurons in primary cultures were determined by the sensitivity of neurons to glutamate toxicity and the expression of specific proteins such as the phosphorylated form of a 200-kDa neurofilament, HPC-1/syntaxin 1A, and cell adhesion molecule L1 [11].
• Cyclic AMP-dependent expression of the heavy neurofilament (NF-H) polypeptide in differentiating neuroblastoma cells [12].
• 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 [13].

Biological context of Nefh

• Previous studies have suggested that NF number as well as the phosphorylation state of the COOH-terminal tail of the heavy neurofilament (NF-H) subunit are major determinants of axonal caliber [14].
• Axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed an increased transport velocity of newly synthesized NF-L and NF-M proteins in motor axons of NF-H knockout mice [15].
• 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 [16].
• Unlike the situation in transgenic mice expressing modest levels of human NF-H (Cote, F., J.F. Collard, and J.P. Julien. 1993. Cell. 73:35-46), even 4.5 times the normal level of wild-type mouse NF-H does not result in any overt phenotype or enhanced motor neuron degeneration or loss [17].
• Studies on synthetic peptide analogs of KSP repeats with substitution of specific residues, known to be present in the C-terminal regions of NF-H, revealed a consensus sequence of X(S/T)PXK, characteristic of the p34cdc2 kinase substrate [18].

Anatomical context of Nefh

• When treated with beta,beta'-iminodipropionitrile (IDPN), a neurotoxin that segregates microtubules and retards neurofilament transport, mice heterozygous or homozygous for the NF-H null mutation did not develop neurofilamentous swellings in motor neurons, unlike normal mouse littermates [15].
• Disruption of the NF-H gene increases axonal microtubule content and velocity of neurofilament transport: relief of axonopathy resulting from the toxin beta,beta'-iminodipropionitrile [15].
• However, our analysis of NF-H knockout mice revealed an approximately 2.4-fold increase of microtubule density in their large ventral root axons [15].
• The light (NF-L), mid-sized (NF-M) and heavy (NF-H) neurofilament (NF) genes were probed with methylation-sensitive restriction enzymes and patterns of methylation and expression of the NF genes were compared in tissues and cell lines of the mouse [16].
• Neurofilaments (NFs), a major component of the neuronal cytoskeleton, are composed of three polypeptide subunits, NF-H, NF-M, and NF-L, and are abundant in large dendritic trees [19].

Associations of Nefh with chemical compounds

• Immunoelectron microscopy investigations of the incorporation sites of NF-H labeled with biotin compounds also revealed the lateral insertion of NF-H subunits into the preexisting NF array, taking after the pattern seen in the case of NF-L [20].
• 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 [21].
• Although the expression levels of phosphorylated NF-H and synaptophysin were reduced in the cerebral cortex and the hippocampus of Abeta(25-35)-injected mice, their levels in ginsenoside Rb1- and M1-treated mice were almost completely recovered up to control levels [22].
• We examined by cell-free analyses whether or not this Triton-soluble NF-H pool was assembly-competent in cell-free analyses [23].
• The location of this serine-rich repeat in the phosphorylated domain of NF-H indicates that it represents the major protein kinase recognition site [24].

Physical interactions of Nefh

• Using a complementary approach, we confirmed an association of synapsin with NFs by demonstrating that immunoprecipitated synapsin I complexes contained NF-H and NF medium (160-kDa) subunits [25].

Regulatory relationships of Nefh

• By cotransfecting these fragments with the neurofilament kinases cyclin-dependent kinase-5 (cdk-5)/p35 and glycogen synthase kinase-3alpha (GSK-3alpha), we show that cdk-5 induces cellular phosphorylation of the KSPXK-containing fragment of NF-H [26].
• Additionally, these latter deletions appeared to act as dominant negative mutants which induced the collapse of the endogenous vimentin cytoskeleton as well as the constitutively expressed NF-H and NF-M cytoskeletons in stably transfected cell lines [27].

Other interactions of Nefh

• 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 [28].
• Using the transfected fragments, we also map the epitopes for several commonly utilised NF-H monoclonal antibodies and describe the effects that phosphorylation by cdk-5 and GSK-3alpha have on their reactivities [26].
• There was also increased expression of phosphorylated high molecular weight neurofilament subunit (NF-H), NF-M, and MAP1B [29].
• MAP2 (dendritic) immunostaining in the post-ischemic hippocampus showed little difference but NF200 (axonal) immunoreactivity was reduced in GFAP(-/-) [30].
• The second conserved linkage involves human chromosome 22 and mouse chromosome 11 and contains two genetically and physically linked loci, Lif and Nfh [31].

Analytical, diagnostic and therapeutic context of Nefh

• Surprisingly, the lack of NF-H subunits had little effect on axonal calibers and electron microscopy revealed no significant changes in the number and packing density of neurofilaments made up of only the neurofilament light (NF-L) and neurofilament medium (NF-M) subunits [15].
• To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching [20].
• TGFbeta treatment further increased the synthesis of NF200 and GAP43 in the transfected clones as revealed by Western blot analysis [32].
• An 80-kDa doublet that coimmunoprecipitated with NF-H complexes, recognized by MAb 223, was shown by immunocytochemistry and immunoblotting to be synapsin Ia and Ib [25].
• Following overnight dialysis to remove urea, low-speed centrifugation to sediment Triton-insoluble cytoskeletons resulted in the co-sedimentation of radiolabeled NF-H, indicating that Triton-soluble NF-H was capable of association with Triton-insoluble structures [23].

References