Compartmentation of alpha-internexin and neurofilament triplet proteins in cultured hippocampal neurons

Summary Intermediate filaments comprise an integral part of the neuronal cytoskeleton. However, little is known about their function, and there remains some uncertainty about their precise subcellular localization. We examined the timingof expression and distribution of -internexin, neurofilament triplet proteins and peripherin using immunocytochemistry in cultured hippocampal neurons. -Internexin immunostaining was present in all neurons at all developmental stages. Immunostaining appeared as long filaments in axons and short fragments in dendrites which extended into dendritic spines. The presence of -internexin in dendritic spines was confirmedin situ by electron microscopy of rat hippocampal tissue sections and suggests that this intermediate filament may serve as a link between cytoskeletal elements in dendritic shafts and spines. In culture, immunostaining using antibodies against individual triplet protein subunits indicated that light (NF-L) and middle (NF-M) subunits were first expressed in cells shortly after the initiation of axonal outgrowth. Expression of the heavy (NF-H) subunit occurred a few days later. Although timing and localization of expression did not correlate with the initiation of axonal or dendritic processes, it was coincident with periods of rapid outgrowth. Triplet proteins were more abundant in axons and appeared to be incorporated into lengthier filaments than in dendrites. Highly phosphorylated NFH/M immunoreactivity was polarized to axons after 6 days in culture. The distribution of one NF-H epitope was restricted to GABAergic neurons in mature cultures, suggesting a cell-type specific modification. Peripherin was not detectable at any time in hippocampal cultures. Our results show that intermediate filaments are integral components of the neuronal cytoskeleton of cultured hippocampal neurons throughout development. Furthermore, the localization of -internexin suggests that it may be involved in the formation or maintenance of dendritic spines.     

Phosphate-dependent and independent neurofilament protein epitopes are expressed throughout the cell cycle in human medulloblastoma (D283 MED) cells.

The low (NF-L) and middle (NF-M) molecular weight (Mr) neurofilament (NF) subunits are expressed before the high (NF-H) Mr NF subunit in embryonic neurons. Thereafter, NF-M attains its mature state of phosphorylation more rapidly than does NF-H. However, little is known about NF subunit expression during cell division. A rapidly dividing medulloblastoma cell line (D283 MED), therefore, was examined using flow cytometry, immunochemistry, and a large panel of NF subunit-specific polyclonal and monoclonal antibodies. Many of the monoclonal antibodies (MAbs) distinguished NF-H and NF-M in different states of phosphorylation. By flow cytometry, more than 90% of the D283 cells expressed NF-H and NF-M in different states of phosphorylation, and an antiserum specific for the carboxy terminus of NF-L labeled more than 60% of these cells. Furthermore, the fluorescence intensity produced by MAbs that detected phosphorylated versus nonphosphorylated NF-H and/or NF-M epitopes, appropriately decreased or increased, respectively, by preincubating the D283 cells with alkaline phosphatase. In contrast, cell staining with antibodies specific for phosphate-independent NF protein epitopes did not change substantially as a result of enzymatic dephosphorylation. These results agreed closely with those obtained from studies of normal human spinal cord NF extracts. However, NF-H, NF-M, and NF-L were expressed throughout the cell cycle in dual parameter studies of D283 cells labeled with an antibody and propidium iodide. Nevertheless, reductions in the fluorescence intensity produced with most of these antibodies late in the cell cycle suggested that NF proteins may be subject to modifications in their structure or accessibility to antibody probes during different phases of the cell cycle. These data led to the conclusion that NF subunits are expressed throughout the cell cycle in cultured human medulloblastoma cells, but that subtle changes in the immunoreactivity of these proteins occur during cell division.     

Expression and phosphorylation of neurofilament protein in different neuronal tissues

Expression and phosphorylation of neurofilament protein in different neuronal tissues     

Expression of transient receptor potential (TRP) channel mRNAs in the mouse olfactory bulb

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET.     

Co-expression of low molecular weight neurofilament protein and glial fibrillary acidic protein in established human glioma cell lines.

This report describes the expression of glial and neuronal cytoskeletal proteins and their messenger RNAs (mRNAs) in established cell lines derived from human primitive neuroectodermal tumors (PNETs) and malignant gliomas. Northern blot analyses revealed neurofilament (NF) protein mRNAs in 6 of 7 PNET cell lines but no glial fibrillary acidic protein (GFAP) mRNA. Six of these cell lines contained mRNA for the microtubule-associated protein (MAP) known as MAP1b, whereas MAP2 mRNAs were detected only in 1 of the PNET cell lines. These findings closely paralleled previously published data on the expression of these cytoskeletal proteins in the same group of PNET cell lines. Although GFAP mRNA was detected in only 2 of 5 glioma cell lines, 4 of these cell lines contained mRNAs for the low-molecular-weight (M(r)) NF protein (NF-L). Western blot analysis confirmed the expression of both GFAP and NF-L protein in 2 of the glioma cell lines (U251 MG and U373 MG) that contained GFAP and NF-L mRNAs. Further, double immunofluorescence studies showed that GFAP and NF-L co-localized in the same glioma cells. In contrast, neither the middle- (NF-M) or high- (NF-H) M(r) NF proteins or their mRNAs were detected in any of these glioma cell lines. Finally, MAP1b mRNA was expressed in all 5 glioma cell lines, whereas MAP2 mRNAs were detected in only 3 of the cell lines. This is the first documentation of the expression of both glial-specific and neuron-specific cytoskeletal proteins in human malignant glioma-derived cell lines. These data may reflect the aberrant induction of neuron-specific gene products in some neoplastic glial cell lines. Alternatively, our findings may indicate that some glioma cell lines correspond to transformed bipotential human central nervous system precursors of cells restricted to a neuronal or glial lineage.     

Expression of medium and heavy chain neurofilaments in the developing human auditory cortex

Neurofilament medium (NF-M) and heavy (NF-H) chain proteins have been used as markers for maturity in the developing brain since their accumulation in axons leads to an increase in conduction velocity. Earlier studies have demonstrated immunoreactivity of neurofilaments in Layer I of the human auditory cortex at 22 gestation weeks (GW), whereas that in other layers developed between 1 and 12 postnatal years, suggesting a gradual increase in the processing of sounds. However, third trimester fetuses and infants are fairly sophisticated in their ability to discern different aspects of complex sounds. Given these contradictory findings, we decided to study the expression of neurofilaments in human auditory cortex between 15 GW and adulthood. We found that mRNA and protein for both NF-M and NF-H were present in the presumptive human auditory cortex in the second trimester and during the postnatal period (1 year—adulthood). Axons in all layers of the auditory cortex were immunoreactive for neurofilaments by 25 GW and the density of the neurofilament-rich plexus in the cortical wall became adult-like during the first postnatal year in humans (9 postnatal months). Our results suggest that in terms of neurofilament expression, axons within the preterm human auditory cortex may be more mature than previously thought.     

The effects of quisqualate and nocodazole on the organization of MAP2 and neurofilaments in spinal cord neurons in vitro.

The relationship between microtubules, neurofilaments and microtubule-associated protein (MAP)2 was investigated in spinal cord neurons grown for up to 14 days in vitro. Neurons were labelled using antibodies against MAP2, neurofilaments and tubulin, and immunofluorescence analyzed by confocal microscopy. A well-structured network of neurofilaments and microtubules was observed in unstimulated cultures. MAP2 staining was poorly structured but became more filamentous following depolymerization of microtubules with nocodazole. Double-staining experiments suggested that MAP2 was now closely associated with neurofilaments in cell bodies and dendrites. Stimulation of cultures with excitatory amino acids increased the resistance of the microtubular cytoskeleton to depolymerization by nocodazole. Again double-labelling experiments demonstrated an increased association between neurofilaments and MAP2 immunofluorescence. Previous results suggested that the stability of the neuronal cytoskeleton could be modulated by glutamate receptors acting through an increased binding of MAP2 to microtubules. From the results presented here, we further suggest that cross-linking of neurofilaments to microtubules may also play a role in this process.     

Neurofilament proteins form an annular superstructure in guinea-pig type I vestibular hair cells

Summary Neurofilaments, the neuron-specific intermediate filaments, are composed of three immunochemically distinct subunits: NF-L, NF-M and NF-H that can be either phosphorylated or unphosphorylated. In mammals, the distribution of these subunits has been described in vestibular ganglion neurons, but there are no reports on the presence of neurofilaments in vestibular hair cells. We investigated, by immunocytochemistry, neurofilaments in vestibular hair cells from rat and guinea-pig using antibodies against the three subunits and to dephosphorylated NF-H (clone SMI 32, recognizes also NF-M on immunoblots), on Vibratome sections of the vestibular end-organs and on isolated hair cells. Various immunostaining protocols were used, as appropriate for the method of observation: laser scanning confocal microscopy (immunofluorescence) and transmission electron microscopy (immunoperoxidase, pre-embedding technique). In rat and guinea-pig cristae and utricles, neurofilament immunoreactivity was observed in axons inside and below the sensory epithelia. In guinea-pig, in addition to this staining, intensely immunoreactive annular structures were found in the basal regions of hair cells. These rings were detected with anti-NF-L, -NF-M and -dephosphorylated NF-H/M antibodies, but not with anti-phosphorylation-independent NF-H. Ring-containing hair cells were present in all regions of the sensory epithelia but were more abundant in the peripheral areas. All levels of observation (Vibratome and thin sections, and isolated hair cells) showed that only the guinea-pig type I hair cells contained a neurofilament ring. High-resolution observations showed that the ring was located below the nucleus, often close to smooth endoplasmic reticulum and the cell membrane.     

Transgenic Mice in the Study of ALS: The Role of Neurofilaments

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurological disorder of multiple etiologies that affects primarily motor neurons in the brain and spinal cord. Abnormal accumulations of neurofilaments (NFs) in motor neurons and a down-regulation of mRNA for the NF light subunit (NF-L) are associated with ALS, but it remains unclear to what extent these NF perturbations contribute to human disease. Transgenic mouse studies demonstrated that overexpression of normal and mutant NF proteins can sometimes provoke a motor neuronopathy characterized by the presence of abnormal NF accumulations resembling those found in ALS. Remarkably, the motor neuronopathy in transgenic mice overexpressing human NF heavy (NF-H) sub-units was rescued by the co- expression of a human NF-L transgene at levels that restored a correct stoichiometry of NF-L to NF-H subunits. Transgenic approaches have also been used to investigate the role of NFs in disease caused by Cu/Zn superoxide dismutase (SOD1) mutations, which is responsible for ˜2% cases of ALS. Studies with transgenic mice expressing low levels of a fusion NF-H/lacZ protein, in which NFs are withheld from the axonal compartment, suggested that axonal NFs are not toxic intermediates required for SOD1 -mediated disease. On the contrary, overexpression of human NF-H proteins was found to confer an effective protection against mutant SOD1 toxicity in transgenic mice, a phenomenon that may be due to the ability of NF proteins to chelate calcium. In conclusion, transgenic studies showed that disorganized NFs can sometimes have noxious effects resulting in neuronopathy. However, in the context of motor neuron disease caused by mutant SOD1, there is emerging evidence that NF proteins rather play a protective role.     

Elevated levels of phosphorylated neurofilament proteins in cerebrospinal fluid of Alzheimer disease patients

Neurofilament (NF) subunits NF-H, NF-M and NF-L are hyperphosphorylated and elevated in Alzheimer disease (AD) brain. We investigated the level and phosphorylation states of NF subunits in lumbar cerebrospinal fluid (CSF) from living patients by bienzyme substrate-recycle enzyme-linked immunosorbent assay. We found: (i), that the levels of phosphorylated NF-H/M (pNF-H/M), non-phosphorylated NF-H/M (npNF-H/M) and NF-L were significantly higher (pNF-H/M, 12–24-fold; npNF-H/M, 3–4-fold) in neurologically healthy aged people than young control individuals; (ii), that in AD, the levels of npNF-H/M, and NF-L were similar to vascular dementia (VaD), and higher than in age-matched controls; and (iii), that the levels of pNF-H/M were significantly higher than in aged controls, non-AD neurological disorders and VaD. Based on these findings, it is suggested that the increased level of total NF proteins in CSF could be used as a marker for brain aging and neurodegenerative disorders in general, and the levels of pNF-H/M as a marker to discriminate AD from normal brain aging and as well as neurological conditions including VaD.     

Neurofilaments in health and disease

With dendritic neurofilaments (NFs) and NF reassembly experiments, the phosphorylation of NF-H was found related to development of crossbridges, resulting in alignment of core filaments. When treated with aluminum chloride, rabbits died acutely with tetanic spasm in which NFs were accumulated in neuronal perikarya and proximal axons. Compared with axonal NFs, the NFs accumulated in the perikarya were composed of less-developed cross-bridges and more irregularly aligned core filaments, and their NF-H, although it became phosphorylated, was less phosphorylated. Transgenic mice expressing NF-H-beta-galactosidase protein also showed NF accumulation in the perikarya, which was similar in organization and NF-H phosphorylation to that in aluminum-treated rabbits, but NFs were almost absent from the axonal compartment in these mice that did not show any overt phenotype. Jimpy mutant mice, with dysmyelinated axons and a short lifespan, showed a significant increase in NF density in the axonal compartment. NF-H and its mRNA were drastically enhanced in expression in these mice, whereas enhancement in expression of NF-L and its mRNA was slight. Most increased NF-H, and probably NF-M also, in the axons was of the nonphosphrylated form. NFs that increased in the axons were also constructed of irregularly organized core filaments linked with fewer crossbridges. Another dysmyelinating mutant type of mice, shiverer mice, also showed similar morphological, immunocytochemical, and behavioral characteristics. Taken together, axonal NF accumulation rather than that in the perikarya must be toxic for neurons to provoke axonal degeneration, possibly resulting in reduction of lifespan. In other transgenic mice, however, the elimination of NFs from the axonal compartment seems to make the neuron vulnerable. Nevertheless, because overexpression of NF-H displayed severe neurological disorder while elimination of this protein appeared to be more resistant to some neurotoxic agent, NF-H appears to function as an exacerbation factor when it exists in the neurologically disordered condition. However, as NF-H is provided with a unique carboxy-terminal tail domain that is highly phosphorylated in the axon and because disruption of its gene affected the survival of axons, which did not develop normal axonal caliber, NF-H should play an important role in healthy neurons.     

Effects of the ecdysoneless mutant on synaptic efficacy and structure at the neuromuscular junction in Drosophila larvae during normal and prolonged development

Neuronal axons are cellular extensions that can reach more than a meter in length. To maintain such a structure, macromolecules synthesized in cell bodies must be transported to the distal axons. Proteins associated with membranous organelles are generally transported in several fast transported groups, while cytoplasmic proteins, mostly composed of cytoskeletal proteins, are transported in slowly transported groups. Neurofilaments are a main component in the slowly transported group. Composed of three polypeptide subunits (NF-H, NF-M and NF-L), they are the most abundant cytoskeletal element in large myelinated axons. In various neurological or neurotoxic disorders, selective accumulation of neurofilaments was observed in different compartments of a neuron (cell bodies, proximal or distal axons). The underlying mechanism for this regional selectivity has been unclear. Using the classical pulse labeling method, we examined the changes in neurofilament transport velocity in transgenic mice that overexpress different neurofilament subunits. We present evidence that at least three velocities of neurofilament transport exist along peripheral motor axons. Each of these velocities was altered differently depending on which neurofilament subunit was overexpressed. We suggest that neurofilament transport in motor axons consists of multiple successive stages and that each of these stages is carried out by different transport mechanisms. These differences provide a basis for the regional deficiencies in axonal transport associated with several neurological disorders.     

Co-expression of four intermediate filament subclasses in childhood glial neoplasms.

Immunohistochemical analysis of intermediate filament (IF) proteins was performed on frozen sections of 16 childhood glial tumors using a library of 10 antigen-specific IF protein directed monoclonal antibodies (MoABs) and a four-step biotin-streptavidin-alkaline phosphatase conjugated antigen detection immunocytochemical technique. Human glial fibrillary acidic protein (GFAP) and vimentin were expressed in all brain tumors. High molecular weight (200 kDa) neurofilament (NF-H) protein was expressed in 15 out of 16 tumors; medium molecular weight (160 kDa) neurofilament (NF-M) in seven out of 16 tumors; and low molecular weight (68 kDa) neurofilament (NF-L) in five out of 16. Positive acidic keratin reactivity was found in five out of 16 tumors using MoAB AE1. Expression of a keratin pair was detected with MoAB AE2 in five out of 16 tumors. A second keratin pair in 14 out of 16 glial tumors was demonstrated with MoAB AE3. Immunostaining with AE5 defined the expression of another basic keratin (64 kDa) in nine out of 16 glial tumors. Finally, in 14 out of 16 astrocytomas an individual 51 kDa acidic keratin (detected with MoAB AE8) was expressed. Glial tumor cells contain cell lineage specific and nonspecific IF proteins in the following IF pattern: AE3+, AE8+, GFAP+, vimentin+, and NF-H+. The heterogenous composition of these cytoskeletal IF proteins in childhood glial tumors may reflect a direct stage dependent correlation with their neoplastic transformation.     

Immunofluorescence demonstration of neurofilament polypeptide expression in fetal human neurons in culture

Sensory neurons from 10-week human fetal dorsal root ganglia were dissociated and grown in vitro and immunostained with monoclonal antibodies specific for neurofilament triplet polypeptides (non-phosphorylated 70 kDa peptide, NF-L; phosphorylated 150 kDa peptide, NF-M; and phosphorylated 200 kDa peptide, NF-H). All 3 neurofilament subunits appeared early in neuronal perikarya, but they were demonstrated in neurites at different times of culture. The phosphorylated 150 kDa subunit was the first to be expressed in neurites and was followed first by the phosphorylated 200 kDa subunit, and then by the 70 kDa subunit.     

Deleting the phosphorylated tail domain of the neurofilament heavy subunit does not alter neurofilament transport rate in vivo

Phosphorylation of the carboxyl tail domains of the neurofilament heavy (NF-H) and middle molecular weight (NF-M) subunits has been proposed to regulate the axonal transport of neurofilaments. To test this hypothesis, we recently constructed mice lacking the extensively phosphorylated NF-H tail domain (NF-HtailDelta) and showed that the transport rate of neurofilaments in optic axons is unaltered in the absence of this domain [M.V. Rao, M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.A. Calcutt, Y. Uchiyama, R.A. Nixon, D.W. Cleveland, Gene replacement in mice reveals that the heavily phosphorylated tail of neurofilament heavy subunit does not affect axonal caliber or the transit of cargoes in slow axonal transport, J. Cell Biol. 158 (2002) 681-693]. However, Shea et al. proposed that deletion of NF-H carboxyl-terminal region accelerates the transport of a subpopulation of neurofilaments based on minor differences between tail-deleted and control mice in our axonal transport analysis. Here, we present additional evidence that neurofilament transport rate is unchanged after deleting the phosphorylated NF-H tail domain, establishing unequivocally that the NF-H tail domain alone does not regulate the rate of neurofilament transport in optic axons in vivo. Possible roles for tail domains as cross-bridges between a neurofilament and its neighbors or other cytoskeletal elements is discussed.     

Morphometric analysis of Mauthner axon cytoskeletal components in adult and subadult fish.

A previous cytoskeletal analysis on trout MA during developmental stages demonstrated, during the subadult stages, neurofilaments (NF) as main components as expressed by the high values of neurofilament to microtubules (MT) ratio which was found to be of the order of 300:1. Since the MA cytoskeletal composition is not known in the adult fish, the MA cytoskeletal composition has been compared to other axons of much smaller diameter of the fasciculus longitudinalis medialis (flm) among which the MA run in the ventral spinal cord. The following parameters were measured on conventional electron microscopy in MA and flm axons cross sections micrographs by means of a computer linked graphic tablet (Apple II): axonal caliber, number of microtubules (MT), microtubular (MT/microns2) and neurofilament (NF/microns2) densities. The analysis of these parameters demonstrated that neurofilaments are the main architectural components in the adult and subadult fish MA and flm axons. However, MA cytoskeletal composition differs from the other flm axons because of its particular very high ratio of neurofilaments to microtubules. The inverse relationship of axonal caliber to microtubular density, previously found in the trout during developmental stages and suggested also for many other vertebrate species, was further confirmed for flm axons which, with calibers 10 times smaller than MA, exhibit a microtubular density 10 times larger.     

Overexpression of neurofilament subunit NF-L and NF-H extends survival of a mouse model for amyotrophic lateral sclerosis

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS) in a subset of patients. Neurofilaments (NFs), the most abundant protein in motoneurons, may play a role in motoneuron degeneration. To investigate this role, we crossed transgenic mice expressing SOD1 mutant G93A (G93A mice) with mice overexpressing mouse neurofilament subunit H (H mice) or L (L mice). G93A mice overexpressing either NF-L or NF-H developed ALS later and survived longer than the G93A mice on a wild type background. These results illustrate a beneficial role of neurofilaments in ALS and call into question of several hypotheses regarding the role of neurofilaments in the development of ALS.     

BHA通过ERK信号转导途径促进小鼠胎肝细胞神经组织细胞特异基因表达的研究

目的：了解丁羟回香醚（BHA）对小鼠胎肝（FL）细胞神经组织特异基因表达的影响及其信号途径。 方法： 采用MACS试剂盒分离小鼠胚胎肝Sca-1+细胞，以DMEM+10%胎牛血清培养液培养；第4 d后，加入或者不加入细胞外信号调节蛋白激酶特异抑制剂PD98059 (25 μmol/L)处理24 h，再加入BHA处理24 h（0.2 mmol/L）。然后在无血清培养基中培养5 d。用Western blotting和半定量RT-PCR方法分析BHA处理前后基因表达。 结果： 经BHA诱导处理后，细胞表达神经组织细胞特异蛋白显著增加如神经丝轻链（NF-L）、神经丝重链（NF-H）、脑因子1（BF-1）和酪氨酸羟化酶（TH）。NF-L、NF-H、BF-1和TH分别增加6.32倍、2.73倍、3.37倍、2.68倍。而PD98059能明显抑制BHA诱导的神经组织细胞特异蛋白NF-L、NF-H、BF-1和TH的表达。 结论： BHA通过细胞外信号调节蛋白激酶途径促进小鼠FL Sca-1+细胞表达神经组织细胞特异抗原、结构和功能基因。     

Epitope map of neurofilament protein domains in cortical and peripheral nervous system Lewy bodies.

A subset of demented elderly patients exhibit large numbers of cortical intraneuronal inclusions similar to the neurofilament (NF)-rich Lewy bodies (LB) found in pigmented subcortical neurons of patients with Parkinson's disease (PD). Because these cortical inclusions may contribute to the emergence of cognitive impairments in afflicted individuals, the authors mapped the distribution of NF epitopes in these so-called cortical LBs. This was done using ethanol-fixed tissues and a large library of monoclonal antibodies (MAbs) with well-characterized binding specificities to various regions of each NF triplet protein. Cortical LBs were examined by light, confocal, and electron microscopy, and they were compared with the subcortical LBs of PD and LBs in the peripheral nervous system (PNS). Monoclonal antibodies specific for the rod regions of each of the three NF subunits, or for phosphate-dependent and independent antigenic sites in the tail region of the high- (NF-H) and middle- (NF-M) molecular weight (Mr) NF subunits as well as other MAbs to the extreme COOH terminus of NF-L and NF-M or the head region of NF-M labeled a variable number of cortical LBs. Remarkably one of these anti-NF MAbs, RMO32, which recognized a phosphorylated epitope in the tail region of NF-M, immunolabeled nearly all cortical LBs, whereas each of the other anti-NF MAbs never labeled more than 10% of ubiquitin- or RMO32-positive cortical LBs. Further LBs in the PNS resembled those in the central nervous system (CNS) in their immunologic properties, and LBs in both sites were dominated by filamentous aggregates at the ultrastructural level. These findings suggest that NF proteins are profoundly altered during their incorporation into cortical and PNS LBs. Further the authors here identified immunologic and ultrastructural properties common to cortical LBs, PNS LBs, and classic substantia nigra LBs in PD. The accumulation of filamentous, perikaryal inclusions rich in NF proteins at diverse sites in the CNS and PNS of patients with a variety of neurodegenerative disorders suggests a widespread disruption of NF metabolism or transport.