The reduction of NADH ubiquinone oxidoreductase 24- and 75-kDa subunits in brains of patients with Down syndrome and Alzheimer's disease

NADH: ubiquinone oxidoreductase (complex I), one of the most complicated multi-protein enzyme complexes, is important for energy metabolism because it is the initial enzyme of the mitochondrial respiratory chain. Deficiency of complex I is frequently found in various tissues of patients with neurodegenerative disease. Here we studied the protein levels of complex I 24- and 75-kDa subunits in several brain regions from patients with Down syndrome (DS) and Alzheimer's disease (AD). We determined protein levels of complex I 24-, 75-kDa subunits and mitochondrial marker proteins mitochondrial matrix protein P1 (hsp60) and aconitate hydratase from seven brain regions of patients with DS, AD and controls. Proteins were separated by two-dimensional (2-D) gel electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Complex I 24-kDa subunit was significantly reduced in occipital cortex and thalamus in patients with DS and temporal and occipital cortices in patients with AD. Complex I 75-kDa subunit was significantly reduced in brain regions from patients with DS (temporal, occipital and caudate nucleus) and AD (parietal cortex). Reductions of two subunits of complex I may lead to the impairment of energy metabolism and result in neuronal cell death (apoptosis), a hallmark of both neurodegenerative disorders.     

Antagonistic Roles of Neurofilament Subunits NF-H and NF-M Against NF-L in Shaping Dendritic Arborization in Spinal Motor Neurons

Dendrites play important roles in neuronal function. However, the cellular mechanism for the growth and maintenance of dendritic arborization is unclear. 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. By overexpressing each of the three NF subunits in transgenic mice, we altered subunit composition and found that increasing NF-H and/or NF-M inhibited dendritic arborization, whereas increasing NF-L alleviated this inhibition. Examination of cytoskeletal organization revealed that increasing NF-H and/or NF-M caused NF aggregation and dissociation of the NF network from the microtubule (MT) network. Increasing NF-H or NF-H together with NF-M further reduced NFs from dendrites. However, these changes were reversed by elevating the level of NF-L with either NF-H or NF-M. Thus, NF-L antagonizes NF-H and NF-M in organizing the NF network and maintaining a lower ratio of NF-H and NF-M to NF-L is critical for the growth of complex dendritic trees in motor neurons.     

Changes in Neurofilament Protein NF-L and NF-H Immunoreactivity Following Kainic Acid-Induced Seizures

Abstract: Postlesion plasticity of neuronal processes might contribute to secondary spontaneous seizures after kainic acid administration. In this study, neurofilament (NF) proteins were examined following intraperitoneal injection of kainic acid, and special reference was given to temporal changes in quantity and quality of the NF light (NF-L) and heavy (NF-H) subunits. A pronounced decrease in phosphorylation-related immunoreactivity of NF-H occurred as early as 1 day after the injection in the amygdala/pyriform cortex, hippocampus, striatum, and dorsal cerebral cortex. A shift of NF-H from the phosphorylated to nonphosphorylated form was evident in immunoblots, suggesting dephosphorylation contributed to the decrease. Decreases in NF-L and phosphorylated NF-H contents in the limbic structure at 3 days were correlated with the increasing kainic acid doses from 2.5 to 10 mg/kg. The degradation pattern in immunoblots with antibodies against NF-L indicated that the decrease in NF-L was probably due to calcium-activated proteolysis. NF-L and phosphorylated NF-H contents secondarily increased from 9 days onward, with ∼20% above the control level of phosphorylated NF-H immunoreactivity at 27 days in the amygdala/pyriform cortex and ventral hippocampus. Immunohistochemical examination of the hippocampus revealed that an increase of NF staining in the mossy fiber system may contribute to the NF recovery in this region. Furthermore, the temporal changes of NF-L and phosphorylated NF-H contents were positively correlated with those of the neuronal cell adhesion molecule, a neuritic growth cone marker, substantiating postlesion regenerative reactions of NF proteins. Functional consequences of the NF plasticity remain to be identified.     

Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome

In brain, nucleoside diphosphate kinase (NDPK) and its coding gene, nm23, have been implicated to modulate neuronal cell proliferation, differentiation, and neurite outgrowth. However, a role of NDPK in neurodegenerative diseases has not been reported yet. Using proteomics techniques, we evaluated the protein levels of NDPK-A in seven brain regions from patients with Alzheimer's disease (AD) and Down syndrome (DS) showing AD-like neuropathology. NDPK-A was significantly decreased in brain regions (frontal, occipital, and parietal cortices) of both disorders. Due to the limitation of brain samples, the activity of NDPK was measured in three brain regions (frontal cortex, temporal cortex, and cerebellum). The specific activity of NDPK was significantly decreased in AD (frontal cortex) and DS (frontal and temporal cortices). Since NDPK-B could also drive the activity of NDPK, protein expression levels of both NDPK-A and NDPK-B were studied in frontal cortex by Western blot analysis. NDPK-A was significantly decreased in AD, which was consistent with the results of proteomics. However, NDPK-A was slightly decreased in DS and protein expression levels of NDPK-B in both DS and AD were moderately decreased, without reaching statistical significance. We propose that oxidative modification of NDPK could lead to the decreased activity of NDPK and, subsequently, influence several neuronal functions in neurodegenerative diseases as multifunctional enzyme through several mechanisms.     

Neuroendocrine-specific protein C, a marker of neuronal differentiation, is reduced in brain of patients with Down syndrome and Alzheimer's disease

Neuroendocrine-specific protein C (NSP-C) is found in neural and neuroendocrine cells and associated with the endoplasmic reticulum. Its expression was found to correlate with the degree of neuronal differentiation. As the neuropathological findings in Down syndrome (DS) includes deficits of differentiation, and we detected a downregulated sequence with 100% homology with NSP-C homolog mRNA in temporal cortex of patients with DS as well as Alzheimer's disease (AD) using differential display-polymerase chain reaction (DD-PCR), we decided to examine the protein levels of NSP-C in temporal, frontal cortex and cerebellum of DS and AD. To normalize NSP-C versus neuronal density, we also determined neuron-specific enolase (NSE) levels and calculated the ratios. NSP-C was significantly reduced in DS (temporal and frontal cortex) and AD (frontal cortex) compared to controls. The significant decrease of NSP-C in DS was even more pronounced when related to NSE levels. Impaired differentiation in DS brain may well be due to absolutely and relatively decreased NSP-C levels in temporal and frontal cortex. As NSP-C was also reduced in AD frontal cortex, NSP-C deficits in these disorders may be reflecting neurodegenerative changes rather than a primary and specific finding of DS or AD pathogenesis.     

Antibody labeling of bovine neurofilaments: implications on the structure of neurofilament sidearms.

We carried out immunolabeling studies of purified bovine spinal cord neurofilaments (NFs) and filaments reconstituted from several combinations of the NF triplet polypeptides, NF-H, NF-M, and NF-L. Six antibodies with known epitopes in either the rod domains or the tailpiece extensions of the NF triplet were used in these studies, and the immune complexes were visualized directly by the glycerol-spray, rotary shadowing technique, which permitted unambiguous identification of the NF sidearms. Antibodies directed against the tailpiece extensions of NF-H and NF-M labeled the sidearms of native NFs and reconstituted filaments containing those two polypeptides, but not the backbone of the filaments. Combining these two antibodies in the same labeling experiment resulted in more intense labeling than either of the antibodies alone, indicating that both NF-H and NF-M are capable of forming sidearms. The anti-NF-L tailpiece antibody recognized only a limited number of sites along native NFs, but labeled reconstituted NF-L homopolymers uniformly and heavily. This suggests that the NF-L tailpiece extension is relatively inaccessible in native filaments, but is accessible in reconstituted homopolymers. One possible explanation is that, in native NFs, the NF-H- and NF-M-containing sidearms curtailed antibody access to NF-L. A second possibility that is not mutually exclusive with the first is that, when both NF-L and another triplet polypeptide are present, they preferentially form heterodimers such that the NF-L tailpiece epitope becomes hidden. Taken collectively, and in combination with published structural information, our data are consistent with a subunit packing scheme in which an NF-L-containing dimer serves as the fundamental building block of most mammalian NFs, such that their sidearms consist of pairs of NF-H/NF-L, NF-M/NF-L, or NF-L/NF-L tailpiece extensions.     

Macromolecular structure of reassembled neurofilaments as revealed by the quick-freeze deep-etch mica method: difference between NF-M and NF-H subunits in their ability to form cross-bridges.

Neurofilament (NF) structure and ability to form cross-bridges were examined by quick-freeze deep-etch mica and low-angle rotary-shadow electron microscopy in NFs purified from bovine spinal cord and reassembled in various combinations of NF subunits. When NFs were reassembled from triplet proteins, NF-L, NF-M and NF-H, they were oriented randomly and often fragmented, but their elongated filaments (12-15 nm wide) and the cross-bridges (4-5 nm wide) connecting them were similar in appearance to those of isolated bovine NFs or in vivo rat NFs. Projections extended from the wall of the core filament in almost the same pattern as the cross-bridges and were the same in width and interval (minimum interval, 20-25 nm) as the cross-bridges. Projections were more conspicuous when core filaments were separated by 60 to 80 nm or more, while cross-bridges were more conspicuous when core filaments were close to each other. Projections or cross-bridges extended bilaterally at intervals of 20 to 25 nm where core filaments expanded and formed a network between filaments which were far from one another. When NFs were reconstructed from NF-L alone, only core filaments appeared, the same width as the filaments of triplet NFs. The core filaments were occasionally in almost direct contact with each other, with no projection or cross-bridge. When NFs were reassembled from NF-M alone or NF-L + NF-M, although NF-M core filaments were shorter and slightly thinner than NF-L + NF-M core filaments, both had projections, and both had cross-bridges, but cross-bridges were less evident. Cross-bridges were almost the same in width as those of triplet NFs, but significantly shorter and much less frequent although the minimum interval was the same, and core filaments were not attached to each other. In contrast, when NFs were reconstituted from NF-H alone or NF-L + NF-H, both had conspicuous projections and cross-bridges, similar to those of triplet NFs. Thus, when NFs contained NF-H, they formed frequent cross-bridges and long projections with extensive peripheral branching. When NFs contained NF-M but no NF-H, they tended to form cross-bridges, and to form projections that were shorter and straighter and without peripheral branching. That is, there appears to be a significant difference between NF-M and NF-H in ability to form cross-bridges and thus in interaction with adjacent NFs.     

Selective accumulation of the high molecular weight neurofilament subunit within the distal region of growing axonal neurites.

Axonal maturation in situ is accompanied by the transition of neurofilaments (NFs) comprised of only NF-M and NF-L to those also containing NF-H. Since NF-H participates in interactions of NFs with each other and with other cytoskeletal constituents, its appearance represents a critical event in the stabilization of axons that accompanies their maturation. Whether this transition is effected by replacement of "doublet" NFs with "triplet" NFs, or by incorporation of NF-H into existing doublet NFs is unclear. To address this issue, we examined the distribution of NF subunit immunoreactivity within axonal cytoskeletons of differentiated NB2a/d1 cell and DRG neurons between days 3-7 of outgrowth. Endogenous immunoreactivity either declined in a proximal-distal gradient or was relatively uniform along axons. This distribution was paralleled by microinjected biotinylated NF-L. By contrast, biotinylated NF-H displayed a bipolar distribution, with immunoreactivity concentrated within the proximal- and distal-most axonal regions. Proximal biotinylated NF-H accumulation paralleled that of endogenous NF immunoreactivity; however, distal-most biotinylated NF-H accumulation dramatically exceeded that of endogenous NFs and microinjected NF-L. This phenomenon was not due to co-polymerization of biotin-H with vimentin or alpha-internexin. This phenomenon declined with continued time in culture. These data suggest that NF-H can incorporate into existing cytoskeletal structures, and therefore suggest that this mechanism accounts for at least a portion of the accumulation of triplet NFs during axonal maturation. Selective NF-H accumulation into existing cytoskeletal structures within the distal-most region may provide de novo cytoskeletal stability for continued axon extension and/or stabilization.     

Subunit composition of neurofilaments specifies axonal diameter

Neurofilaments (NFs), which are composed of NF-L, NF-M, and NF-H, are required for the development of normal axonal caliber, a property that in turn is a critical determinant of axonal conduction velocity. To investigate how each subunit contributes to the radial growth of axons, we used transgenic mice to alter the subunit composition of NFs. Increasing each NF subunit individually inhibits radial axonal growth, while increasing both NF-M and NF-H reduces growth even more severely. An increase in NF-L results in an increased filament number but reduced interfilament distance. Conversely, increasing NF-M, NF-H, or both reduces filament number, but does not alter nearest neighbor interfilament distance. Only a combined increase of NF-L with either NF- M or NF-H promotes radial axonal growth. These results demonstrate that both NF-M and NF-H play complementary roles with NF-L in determining normal axonal calibers.     

Interplay between liquid crystalline and isotropic gels in self-assembled neurofilament networks

Neurofilaments (NFs) are a major constituent of nerve cell axons that assemble from three subunit proteins of low (NF-L), medium (NF-M), and high (NF-H) molecular weight into a 10 nm diameter rod with radiating sidearms to form a bottle-brush-like structure. Here, we reassemble NFs in vitro from varying weight ratios of the subunit proteins, purified from bovine spinal cord, to form homopolymers of NF-L or filaments composed of NF-L and NF-M (NF-LM), NF-L and NF-H (NF-LH), or all three subunits (NF-LMH). At high protein concentrations, NFs align to form a nematic liquid crystalline gel with a well-defined spacing determined with synchrotron small angle x-ray scattering. Near physiological conditions (86 mM monovalent salt and pH 6.8), NF-LM networks with a high NF-M grafting density favor nematic ordering whereas filaments composed of NF-LH transition to an isotropic gel at low protein concentrations as a function of increasing mole fraction of NF-H subunits. The interfilament distance decreases with NF-M grafting density, opposite the trend seen with NF-LH networks. This suggests a competition between the more attractive NF-M sidearms, forming a compact aligned nematic gel, and the repulsive NF-H sidearms, favoring a more expansive isotropic gel, at 86 mM monovalent salt. These interactions are highly salt dependent and the nematic gel phase is stabilized with increasing monovalent salt.     

Thyroid stimulating hormone-receptor overexpression in brain of patients with Down syndrome and Alzheimer's disease

Thyroid hormone abnormalities are strongly associated with Down Syndrome (DS) with elevated thyroid stimulating hormone (TSH) levels as the most consistent finding. Using subtractive hybridization for gene hunting we found significant overexpression of mRNA levels for the TSH-receptor (TSH-R) in brain of a fetus with DS. Based upon this observation we determined TSH-R protein levels in five brain regions of patients with DS (n=8), Alzheimer disease (AD, n=8) and controls (C, n=8). Western blots revealed significantly elevated immunoreactive TSH-R protein(s) 40 kD and 61 kD in temporal and frontal cortex of patients with DS and, unexpectedly, in AD. Levels for the 40 kD protein in temporal cortex were 1.00+/-0.036 (arbitrary units+/-SD) in C, 1.35+/-0.143 in DS, 1.52+/-0.128 in AD; in frontal cortex: 1.00+/-0.046 in C, 1.10+/-0.03 in DS, 1.10+/-0.038 in AD. Levels for the 61 kD protein in temporal cortex were 1.01+/-0.015 in C, 1.47+/-0.013 in DS, 1.623+/-0.026 in AD; in frontal cortex: 1.02+/-0.020 in C, 1.18 +/-0.123 in DS, 1.48+/-0.020 in AD. These results show that elevated brain immunoreactive TSH-R is not specific for DS and maybe reflecting apoptosis, a hallmark of both neurodegenerative disorders, as it is well-documented that the thyroid hormone system is involved in the control of programmed cell death.     

Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons

Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. 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. In the case of NF-L protein, the bleached zone gradually restored its fluorescence intensity with a recovery half time of approximately 35 min. On the other hand, recovery of the bleached zone of NF-H was considerably faster, taking place in approximately 19 min. However, in both cases the bleached zone was stationary. Thus, it was suggested that NF-H is the dynamic component of the NF array and is interchangeable, but that it assembles with the other neurofilament triplet proteins in a more exchangeable way, implying that the location of NF-H is in the periphery of the core NF array mainly composed of NF- L subunits. 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. In summary, our results demonstrate that the dynamics of the L and H subunit proteins in situ are quite different from each other, suggesting different and separated mechanisms or structural specialization underlying the behavior of the two proteins.     

Dephosphorylation of Neurofilaments by Exogenous Phosphatases Has No Effect on Reassembly of Subunits

Exhaustive in vitro dephosphorylation of porcine neurofilaments (NFs) by alkaline or acid phosphatase did not cause a dissociation of the 210-kD (NF-H), 160-kD (NF-M), or 70-kD (NF-L) subunits and had no effect on the reassembly of NFs from urea or guanidine solution. Electron microscopy revealed that the NFs reassembled from isolated or dephosphorylated subunits had similar morphologies. Phosphatase treatment caused significant increases in the mobilities of NF-M and NF-H on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the subunits underwent marked conformational changes after dephosphorylation. Chemical phosphate analysis showed that as isolated NF-H, NF-M, and NF-L contained about 22, 11, and 3 mol phosphate/mol polypeptide, respectively. The corresponding values for the three subunits from alkaline phosphatase-treated NFs were about 8, 6, and 2 mol phosphate/mol polypeptide, respectively. These results indicate the occurrence of a class of phosphate moieties that is not accessible to exogenous phosphatases.     

Assembly and structure of neurofilaments isolated from bovine spinal cord

Neurofilaments (NFs) are neuron-specific intermediate filaments. The NFs were isolated from bovine spinal cord by differential centrifugation. The NFs were detected with electron microscopy and scanning tunneling microscopy (STM). Under STM, two kinds of sidearm of NFs were revealed: one was short, the other was long. They were arrayed along the 10-nm width core filaments one by one. The intervals between two adjacent long sidearms or two short sidearms were 20–22 nm, while those between two adjacent long and short sidearms were 10–11 nm. It was proposed that the rod domain of NF triplet proteins was 3/4-staggered. The assembly properties of NF triplet proteins were also studied. Immuno-colloidal-gold labeling assay showed that NF-M and NF-H are able to co-assemble into long filaments with NF-L. NF-M and NF-H can also co-constitute into winding filaments.     

Structure of the peripheral domains of neurofilaments revealed by low angle rotary shadowing

The structure of the peripheral domains of neurofilaments (NFs) was revealed by rotary shadowing electron microscopy. NFs were isolated from bovine spinal cords by Sepharose CL-4B gel filtration and examined by low angle rotary shadowing. The peripheral domains appeared as thin, flexible, filamentous structures projecting from the intermediate filament core, with a constant density along their entire length. The average length of the projections was approximately 85 nm and the width about 4 nm. These projections appeared from regularly distributed sites, at 22 nm spacing, which seemed to correspond to the typical repeat of the alpha-helix-rich rod domain of the core filament. The density of the projections was found to be 4.1 (+/- 0.6) per 22 nm. We performed reconstitution experiments using purified NF polypeptides to confirm that the projection was indeed the NF peripheral domain. Individual components of the NF triplet, i.e. NF-L, NF-M and NF-H, were purified by DE-52 and Mono-Q anion exchange chromatographies in the presence of 6 M-urea and were assembled in various combinations into filaments. Reassembled filaments were somewhat more slender than the isolated NFs and exhibited a distinct 22 nm axial periodicity. While prominent projections were not observed in the filaments assembled from NF-L alone, reconstructed filaments containing NF-L plus either NF-M or NF-H revealed many projections. The average length of the projections in the filaments reconstructed from NF-L and NF-H was about 63 nm. The projections of reconstructed filaments from NF-L and NF-M were about 55 nm in length. The difference in the lengths of the projections might reflect the difference in the length of the carboxy-terminal tail domain between NF-M and NF-H. The results are interpreted to show that the carboxy-terminal tail domains of NFs project in a regular pattern from the core filament, which is consistent with a half-staggered organization of the tetrameric subunits.     

Neurofilaments are obligate heteropolymers in vivo

Neurofilaments (NFs), composed of three distinct subunits NF-L, NF-M, and NF-H, are neuron-specific intermediate filaments present in most mature neurons. Using DNA transfection and mice expressing NF transgenes, we find that despite the ability of NF-L alone to assemble into short filaments in vitro NF-L cannot form filament arrays in vivo after expression either in cultured cells or in transgenic oligodendrocytes that otherwise do not contain a cytoplasmic intermediate filament (IF) array. Instead, NF-L aggregates into punctate or sheet like structures. Similar nonfilamentous structures are also formed when NF-M or NF-H is expressed alone. The competence of NF-L to assemble into filaments is fully restored by coexpression of NF- M or NF-H to a level approximately 10% of that of NF-L. Deletion of the head or tail domain of NF-M or substitution of the NF-H tail onto an NF- L subunit reveals that restoration of in vivo NF-L assembly competence requires an interaction provided by the NF-M or NF-H head domains. We conclude that, contrary to the expectation drawn from earlier in vitro assembly studies, NF-L is not sufficient to assemble an extended filament network in an in vivo context and that neurofilaments are obligate heteropolymers requiring NF-L and NF-M or NF-H.     

The Cerebrocortical Areas in Normal Brain Aging and in Alzheimer's Disease: Noticeable Differences in the Lipid Peroxidation Level and in Antioxidant Defense

The markers of oxidative stress were measured in four cerebrocortical regions of Alzheimer's disease (AD) and age-matched control brains. In controls the levels of diene conjugates (DC) and lipid peroxides (LOOH) were significantly higher in the sensory postcentral and occipital primary cortex than in the temporal inferior or frontal inferior cortex. The antioxidant capacity (AOC) was highest in the temporal, and GSH in the frontal inferior cortex. The highest activity of superoxide dismutase (SOD) and catalase (CAT) was found in the occipital primary cortex. Compared with controls, significantly higher level of DC and LOOH and attenuated AOC were evident in AD temporal inferior cortex. In AD frontal inferior cortex moderate increase in LOOH was associated with positive correlation between SOD activity and counts of senile plaques. Our data suggest that in AD cerebral cortex, the oxidative stress is expressed in the reducing sequence: temporal inferior cortex > frontal inferior cortex > sensory postcentral cortex occipital primary cortex, corresponding to the histopathological spreading of AD from the associative to primary cortical areas.     

Assembly and structure of neurofllaments isolated from bovine spinal cord

Neurofilaments (NFs) are neuron-specific intermediate filaments. The NFs were isolated from bovine spinal cord by differential centrifugation. The NFs were detected with electron microscopy and scanning tunneling microscopy (STM). Under STM, two kinds of sidearm of NFs were revealed: one was short, the other was long. They were arrayed along the 10-nm width core filaments one by one. The intervals between two adjacent long sidearms or two short sidearms were 20—22 nm, while those between two adjacent long and short sidearms were 10—11 nm. It was proposed that the rod domain of NF triplet prnteins was 3/4-staggered. The assembly properties of NF triplet proteins were also studied. Immuno-colloidal-gold labeling assay showed that NF-M and NF-H are able to co-assemble into long filaments with NF-L. NF-M and NF-H can also co-constitute into winding filaments.     

Assembly Properties of Amino- and Carboxyl-Terminally Truncated Neurofilament NF-H Proteins with NF-L and NF-M in the Presence and Absence of Vimentin

Abstract: To understand the assembly characteristics of the high-molecular-weight neurofilament protein (NF-H), carboxyl- and amino-terminally deleted NF-H proteins were examined by transiently cotransfecting mutant NF-H constructs with the other neurofilament triplet proteins, low- and middle-molecular-weight neurofilament protein (NF-L and NF-M, respectively), in the presence or absence of cytoplasmic vimentin. The results confirm that NF-H can coassemble with vimentin and NF-L but not with NF-M into filamentous networks. Deletions from the amino-terminus show that the N-terminal head is necessary for the coassembly of NF-H with vimentin, NF-L, or NF-M/vimentin. However, headless NF-H or NF-H from which the head and a part of the rod is removed can still incorporate into an NF-L/vimentin network. Deletion of the carboxyl-terminal tail of NF-H shows that this region is not essential for coassembly with vimentin but is important for coassembly with NF-L into an extensive filamentous network. Carboxyl-terminal deletion into the α-helical rod results in a dominant-negative mutant, which disrupts all the intermediate filament networks. These results indicate that NF-L is the preferred partner of NF-H over vimentin and NF-M, the head region of NF-H is important for the formation of NF-L/NF-H filaments, and the tail region of NF-H is important to form an extensive network of NF-L/NF-H filaments.