Peripherin Is Tyrosine-Phosphorylated at Its Carboxyl-Terminal Tyrosine

Abstract: Peripherin is a type III intermediate filament present in peripheral and certain CNS neurons. We report here that peripherin contains a phosphotyrosine residue and, as such, is the only identified intermediate filament protein known to be modified in this manner. Antiserum specific for phosphotyrosine recognizes peripherin present in PC12 cells (with or without nerve growth factor treatment) and in rat sciatic nerve as well as that expressed in Sf-9 cells and SW-13 cl. 2 vim⁻ cells. The identity of peripherin as a tyrosine-phosphorylated protein in PC12 cells was confirmed by immunoprecipitation, two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, and phosphoamino acid analysis. Unlike serine/threonine phosphorylation, tyrosine phosphorylation of peripherin is not regulated by depolarization or nerve growth factor treatment. To identify the site of tyrosine phosphorylation, rat peripherin was mutated at several tyrosine residues and expressed in SW-13 cl. 2 vim⁻ cells. Tyrosine phosphorylation was selectively lost only for peripherin mutants in which the carboxy-terminal tyrosine (Y474) was mutated. Indirect immunofluorescence staining indicated that both wild-type peripherin and peripherin Y474F form a filamentous network in SW-13 cl. 2 vim⁻ cells. This indicates that tyrosine phosphorylation of the peripherin C-terminal residue is not required for assembly and leaves open the possibility that this modification serves other functions.     

Tyrosine Nitration is a Novel Post-translational Modification Occurring on the Neural Intermediate Filament Protein Peripherin

The biological implication of protein tyrosine nitration in signaling pathways triggered by nitric oxide is recently emerging. Here we report for the first time that nitrotyrosination occurs in the neural intermediate filament protein peripherin. In neuron-like PC12 cells, nitrated peripherin is associated with the cytoskeleton fraction, its level increases during the progression of NGF-induced differentiation and the nitrated protein remains closely associated with stable microtubules. Tyr 17 and Tyr 376 were identified by MALDI-TOF analyses as two specific residues endogenously nitrated. Finally, peripherin nitration is not restricted to PC12 cells but it is also present in vivo in rat brain. Gabriella Tedeschi and Graziella Cappelletti contributed equally to this work.     

Analysis and Comparison of In Vitro Synthesized Glial Fibrillary Acidic Protein with Rat CNS Intermediate Filament Proteins

Intermediate filament (IF) proteins from rat spinal cord were analyzed by two-dimensional gel electrophoresis and compared with the in vitro translation products of a messenger RNA-dependent reticulocyte lysate system stimulated with 16-day-old rat brain polysomes. In two dimensions, the molecular weight 49,000 to 50,000 band of the IF preparation resolved to seven spots, whereas antiserum to glial fibrillary acidic (GFA) protein precipitated only two immediately adjacent radiolabeled in vitro synthesized products, with molecular weights of 49,000 to 50,000. Autoradiographs of two-dimensional gels of extracted IF proteins incubated with iodinated IgG fraction of GFA protein antiserum showed that all seven spots were recognized by the antiserum. These observations suggest that the primary gene product of GFA protein is modified either by post-translational processing or experimental artifact.     

Relationship Between the Nerve Growth Factor-Regulated Clone 73 Gene Product and the 58-Kilodalton Neuronal Intermediate Filament Protein (Peripherin)

Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an mRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF-treated PC12 cell cytoskeletons derived by extraction with 1% Triton X-100 is of apparent Mr = 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6-5.8, and is elevated relative to non-NGF-treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14-residue sequence encoded by the NGF-regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19-residue synthetic peptide corresponding to the deduced C-terminus of the protein encoded by the NGF-regulated clone 73 mRNA specifically recognizes the 58,000-Mr protein. Properties of the 58-kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF-induced message should facilitate studies of its regulation and function.     

58,000 Dalton Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in the Goldfish Visual Pathway

A group of proteins in the goldfish optic nerve with a molecular weight of 58K daltons was analyzed by two-dimensional gel electrophoresis. Results show that the proteins are differentially phosphorylated and found exclusively in a cytoskeletal-enriched fraction. The proteins from this fraction can be reconstituted into typical intermediate filament structures, as shown by electron microscopy. Two components which are of neuronal origin are transported within the slow phase of transport. The 58K proteins are the most abundant proteins in the optic nerve, and they are distinct from actin and tubulin. It was concluded that they are intermediate filament proteins. Cytoskeletal preparations of rat spinal cord, rat optic nerve, and goldfish optic nerve were compared by one-dimensional gel electrophoresis. The rat spinal cord contains glial fibrillary acidic protein (GFAP), and the rat optic nerve contains vimentin and GFAP, in addition to the neurofilament triplet. A typical mammalian neurofilament triplet is not detected in the goldfish optic nerve, while the major cytoskeletal constituent is a 58K band which coelectrophoreses with vimentin in the rat optic nerve by one-dimensional gel electrophoresis.     

Differential Regulation of Two Classes of Neuronal Intermediate Filament Proteins During Optic Nerve Regeneration

Abstract: The regulation of expression of two different types of neuronal intermediate filament proteins, ON₁/ON₂ and plasticin, was studied during optic nerve regeneration in the goldfish. During regenerative growth of optic axons, there is a rapid and dramatically increased expression of plasticin, a recently cloned, novel type III intermediate filament protein, in the retinal ganglion cells. At the time when the growing axons reinnervate the optic tectum, expression of plasticin declines and there is an increased expression of ON₁ and ON₂. This time course suggests that the target tissue participates in the regulation of these proteins. The aim of this study was to characterize the regulatory role played by the optic tectum. To address this issue, a repeated-crush paradigm was used whereby growing axons were hindered from reaching their target. It was found that in absence of tectal contact, the increased expression of ON₁ and ON₂ normally seen during regeneration was not induced. In contrast, expression of plasticin increased both in the presence and in the absence of tectal contact.     

Studies on the Biosynthesis of Intermediate Filament Proteins in the Rat CNS

Abstract: The biosynthesis of brain intermediate filament proteins [neurofilament proteins and glial fibrillary acidic protein (GFA)] was studied with cell-free systems containing either rat spinal cord polysomes (free polysomes or rough microsomes) and rabbit reticulocyte factors or wheat germ homogenate containing spinal cord messenger RNA. The products of translation were isoated by immunoaffinity chromatography and then analyzed by two-dimensional gel electrophoresis (2DGE) followed by fluorography. The free polysome population was found to synthesize two neurofilament proteins (MW 145K, p15.4, and MW 70K, pl 5.3) and three isomers of GFA (α, β, and γ) that differ in isoelectric point. Wheat germ homogenate containing messenger RNA extracted from free cord polysomes synthesized two proteins that comigrated with neurofilament protein standards at 145K 5.4 and 70K 5.3; these proteins were partially purified by neurofilament affinity chromatography. The wheat germ system also synthesized the α, β, and γ isomers of GFA as characterized by immunoaffinity chromatographic purification and comigration with standards in 2DGE analysis. Our data are consistent with the conclusion that synthesis of neurofilament proteins requires multiple messenger RNAs. Also, synthesis of intermediate filament proteins occurs in the free polysome population; detectable amounts of these proteins were not synthcsized by the rough microsomes.     

The Intermediate Filament Protein Peripherin Is the Specific Interaction Partner of Mouse BPAG1-n (Dystonin) in Neurons

The dystonia musculorum (dt) mouse suffers from severe degeneration of primary sensory neurons. The mutated gene product is named dystonin and is identical to the neuronal isoform of bullous pemphigoid antigen 1 (BPAG1-n). BPAG1-n contains an actin-binding domain at its NH₂ terminus and a putative intermediate filament-binding domain at its COOH terminus. Because the degenerating sensory neurons of dt mice display abnormal accumulations of intermediate filaments in the axons, BPAG1-n has been postulated to organize the neuronal cytoskeleton by interacting with both the neurofilament triplet proteins (NFTPs) and microfilaments. In this paper we show by a variety of methods that the COOH-terminal tail domain of mouse BPAG1 interacts specifically with peripherin, but in contrast to a previous study (Yang, Y., J. Dowling, Q.C. Yu, P. Kouklis, D.W. Cleveland, and E. Fuchs. 1996. Cell. 86:655–665), mouse BPAG1 fails to associate with full-length NFTPs. The tail domains interfered with the association of the NFTPs with BPAG1. In dt mice, peripherin is present in axonal swellings of degenerating sensory neurons in the dorsal root ganglia and is downregulated even in other neural regions, which have no obvious signs of pathology. Since peripherin and BPAG1-n also display similar expression patterns in the nervous system, we suggest that peripherin is the specific interaction partner of BPAG1-n in vivo.     

Identification of a Peripherin Dimer: Changes During Axonal Development and Regeneration of the Rat Sciatic Nerve

Abstract: Western blotting of rat dorsal root ganglion (DRG) and sciatic nerve under nonreducing conditions revealed that a peripherin-specific antibody recognized a protein species of 116/130 kDa, pi 5.6, in addition to peripherin (56 kDa, pl 5.6). We showed that this 116/130 kDa protein is a disulfide dimer of peripherin, because it gave rise to a single protein band comigrating with peripherin under reducing conditions and yielded the same proteolytic pattern as peripherin upon N-chlorosuccinimide digestion. In addition, the immunological characteristics of the resulting peptides were identical to those of peripherin. We investigated the changes in peripherin monomer and dimer protein levels during axonal development and regeneration. During postnatal development, quantitative analysis of western blots of DRG proteins showed a significant increase in peripherin monomer (+52%) and dimer (+33%) levels from the day of birth [postnatal day 0 (PO)] to P7. The monomer levels remained high until P14 and then decreased so that at P21 and later ages, the monomer levels were similar to those observed at birth. In contrast, the dimer levels decreased continuously after P7, and in the adult, its level represented only 30% of the level at birth. Changes in [³⁵S]methionine incorporation into adult DRG proteins were studied during regeneration of axotomized sciatic axons. Quantitative analysis of proteins showed a strong increase in labeling of both peripherin monomer (+56%) and dimer (+88%) 7 days after the crush. These levels, which remained high until 28 days after the axotomy, had returned to normal 70 days post axotomy. Our results show that peripherin monomer and dimer greatly increase during DRG fiber development and regeneration, suggesting that the two forms are involved in the growth of axons.     

Intermediate Filament Disassembly in Cultured Dorsal Root Ganglion Neurons Is Associated with Amino-Terminal Head Domain Phosphorylation of Specific Subunits

Abstract: We previously reported that activation of protein kinase A in cultured rat dorsal root ganglion neurons, treated concomitantly with low concentrations of okadaic acid that selectively inhibit protein phosphatase-2A, enhanced the Triton X-100 solubility of neurofilament triplet proteins. We now show that peripherin and α-internexin follow the same fragmentation profile as the neurofilament subunits, consistent with the notion that all five cytoplasmic intermediate filament proteins in these neurons form an integrated filamentous network whose assembly can be modulated by protein kinase A. Similar to the situation previously observed for the light neurofilament subunit, there was a strong correlation between phosphorylation of the amino-terminal head domain of peripherin and filament fragmentation. In contrast, insignificant levels of ³²P were incorporated into α-internexin under conditions promoting disassembly, indicating that phosphorylation of this protein is not involved directly in filament fragmentation. The situation for the mid-sized neurofilament subunit (NFM) was not as clear-cut. Phosphopeptide mapping of NFM revealed many head and tail domain phosphorylation sites. However, changes in NFM head domain phosphorylation under conditions promoting filament disassembly were not as pronounced as for peripherin.     

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.     

拟南芥细胞中存在中间纤维的研究

利用整装电镜制样与选择性抽提技术,在拟南芥（Ａｒａｂｉｄｏｐｓｉｓｔｈａｌｉａｎａ （Ｌ．） Ｈｅｙｎｈ） 愈伤组织细胞质中观察到直径１０ ｎｍ 左右的纤维网络结构。免疫印迹分析表明纤维的主要成分是６ 种多肽,它们分别与动物角蛋白单克隆抗体ＡＥ１ 、ＡＥ３ 有免疫交叉反应。利用间接免疫荧光技术,与ＡＥ１ 和ＡＥ３ 反应的抗原呈弥散状定位于整个细胞质中,而且１０ ｎｍ 纤维可以在体外重新组装。以上结果表明,在拟南芥细胞质中存在类角蛋白的中间纤维。以动物中间纤维基因的保守序列为引物,采用ＲＴ＿ＰＣＲ技术,进一步从这一模式植物中克隆到一个ｃＤＮＡ片段,这可能为从分子水平上证明植物中间纤维的存在提供了一个线索     

The Existence of Intermediate Filament in Arabidopsis thaliana Cell

The network structure of cytoplasmic filaments of 10 nm in diameter was detected from callus cells of Arabidopsis thaliana (L.) Heynh by selective extraction combined with whole mount electron microscopy. Western blot analysis showed that the major filament components were 6 polypeptides, which reacted with keratin monoclonal antibody of AE1 or AE3 respectively. By indirect immunofluorescence technique, the AE1 and AE3-reactive antigens were localized throughout the cytoplasm in a diffused pattern. The 10 nm-plant filaments could be reassembled in vitro. These results demonstrated that keratin-like intermediate filaments exist in the cytoplasm of A. thaliana. Using conservative sequence of animal IF genes as primer, a cDNA fragment was further cloned from this model material by RT-PCR, which might shed more light on molecular characterization of IF existence in higher plant.     

The intermediate filament protein, synemin, is an AKAP in the heart

Targeting of protein kinase A (PKA) by A-kinase anchoring proteins (AKAPs) contributes to high specificity of PKA signaling pathways. PKA phosphorylation of myofilament and cytoskeletal proteins may regulate myofibrillogenesis and myocyte remodeling during heart disease; however, known cardiac AKAPs do not localize to these regions. To identify novel AKAPs which target PKA to the cytoskeleton or myofilaments, a human heart cDNA library was screened and the intermediate filament (IF) protein, synemin, was identified as a putative RII (PKA regulatory subunit type II) binding protein. A predicted RII binding region was mutated and resulted in loss of RII binding. Furthermore, synemin co-localized with RII in SW13/cl.1-vim+ cells and co-immunoprecipitated with RII from adult rat cardiomyocytes. Synemin was localized at the level of Z-lines with RII and desmin in adult hearts, however, neonatal cardiomyocytes showed differential synemin and desmin localization. Quantitative Western blots also showed significantly more synemin was present in failing human hearts. We propose that synemin provides temporal and spatial targeting of PKA in adult and neonatal cardiac myocytes.     

Analysis of Cyclic AMP-Dependent Changes in Intermediate Filament Protein Phosphorylation and Cell Morphology in Cultured Astroglia

Receptor agonists that increase cyclic AMP levels in cultured astroglia have been shown to increase 32P-labeling of the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin in these cells. Experiments were designed to determine if the increase in 32P-labeling resulted from either an increase in the turnover or net number of phosphates associated with the intermediate filament proteins and if the phosphorylation of these proteins causally affected astroglial morphology. Time course experiments indicated that 6-8 h were required to reach steady-state 32P-labeling of both GFAP and vimentin. Treatment with forskolin (10 microM) after steady-state 32P-labeling increased GFAP and vimentin phosphorylation fourfold and twofold, respectively, and also induced a morphological change from polygonal to process-bearing cells within 20-30 min of drug addition. Cells incubated in media containing brain extract (30%) for 24 h at 37 degrees C and then 3 h at 23 degrees C underwent changes from polygonal to process-bearing cells with no apparent increase in the phosphorylation of either GFAP or vimentin. Treatment of process-bearing cells (induced by brain extract) or polygonal cells with 10 microM forskolin at 23 degrees C resulted in a three- to fourfold increase in GFAP phosphorylation without significant morphological changes. These results suggest that forskolin stimulation of GFAP and vimentin increases net number of phosphates associated with these intermediate filament proteins and that the resulting increase in phosphorylation can be dissociated from morphological changes.     

Receptor-Mediated Phosphorylation of Astroglial Intermediate Filament Proteins in Cultured Astroglia

Primary cultures of purified astroglia have been shown to exhibit a variety of membrane receptors that regulate intracellular cyclic AMP levels. The experiments described in this paper were completed to examine the effect of such receptor agonists on protein phosphorylation in intact astroglia. An analysis of 32P-labelled proteins derived from whole cell extracts and separated via two-dimensional gel electrophoresis indicated that increasing cyclic AMP levels in astroglia stimulated the phosphorylation of two distinct proteins that had apparent molecular weights/isoelectric points (pI) of 51K/6.0 and 57K/5.7. Similar experiments with cultured meningeal cells indicated that only the 57K/5.7 protein was phosphorylated in response to elevated levels of cyclic AMP. The 51K/6.0 protein was never observed in gels derived from meningeal cells. Immunoblot experiments indicated that the 51K/6.0 protein stained with antiserum to glial fibrillary acidic protein (GFAP) and the 57K/5.7 protein stained with antibodies to vimentin. Concentration-effect studies indicate that these proteins are maximally phosphorylated at concentrations of receptor agonists that only slightly elevate cyclic AMP levels. All receptor agonists that have been shown to increase cyclic AMP levels appear similarly efficacious with respect to increasing the phosphorylation of the two proteins. These experiments suggest that the membrane receptors present on astroglia function, in part, to regulate phosphorylation of the intermediate filament proteins GFAP and vimentin.     

Homology and Diversity Between Intermediate Filament Proteins of Neuronal and Nonneuronal Origin in Goldfish Optic Nerve

The predominant intermediate filament proteins of the goldfish optic nerve have molecular weights of 58K. They can be separated into a series of four major isoelectric variants of neuronal (ON1 and ON2) and nonneuronal (ON3 and ON4) origin. The extent of homology between the goldfish 58K intermediate filament proteins themselves and to rat optic nerve vimentin and glial fibrillary acidic protein (GFAP) was investigated. Unlabeled and [32P]orthophosphate-labeled proteins were subjected to partial hydrolysis by V8 protease, chymotrypsin, and CNBr. The results show that the goldfish intermediate filament proteins share with vimentin and GFAP a 40K chymotrypsin-resistant core fragment. Phosphorylated moieties appear to be located outside the core region since they are preferentially cleaved off by chymotrypsin and not found associated with the 40K core. In addition, the goldfish ON proteins contain the antigenic site within the core that is common to most intermediate filaments. V8 or CNBr digestion indicates that many fragments that are common to ON1 and ON2 are clearly distinct from fragments that are common to ON3 and ON4. In addition, structural variability is observed between the goldfish intermediate filament proteins and vimentin and GFAP. The results are discussed in terms of intermediate filament structure and their possible role in nerve growth.     

Interleukin-6 Induces Expression of Peripherin and Cooperates with Trk Receptor Signaling to Promote Neuronal Differentiation in PC12 Cells

Abstract: In contrast to the intensively studied nerve growth factor (NGF)-related family of cytokines, relatively little is known about the mechanisms of neurotrophic activity elicited by the cytokine interleukin-6 (IL-6). We have examined the mechanisms of IL-6-induced neuronal differentiation of the pheochromocytoma cell line PC12. IL-6 independently induced the expression of peripherin , identifying this gene as the first neuronal-specific target of IL-6. However, IL-6 alone failed to elicit neurite outgrowth in PC12 cells and instead required low levels of Trk/NGF receptor tyrosine kinase activity to induce neuronal differentiation. The cooperating Trk signal could be provided by either overexpression of Trk or exposure to low concentrations of NGF. IL-6 also functioned cooperatively with basic fibroblast growth factor to promote PC12 differentiation. IL-6 and Trk/NGF synergized in enhancing tyrosine phosphorylation of the Erk-1 mitogen-activated protein kinase and in activating expression of certain NGF target genes. NGF also induced expression of the gp80 subunit of the IL-6 receptor, providing another potential mechanism of cooperativity between NGF and IL-6 signaling. We propose that IL-6 functions as an enhancer of NGF signaling rather than as an autonomous neuronal differentiation signal. Moreover, our results demonstrate that a Trk receptor-specific cellular response can be achieved in the absence of NGF through amplification of its basal signaling activity by the IL-6 receptor system.     

Intracellular assembly and sorting of intermediate filament proteins: role of the 42 amino acid lamin insert

Nuclear and cytoplasmic intermediate filament (IF) proteins segregate into two independent cellular networks by mechanisms that are poorly understood. We examined the role of a 42 amino acid (aa) insert unique to vertebrate lamin rod domains in the coassembly of nuclear and cytoplasmic IF proteins by overexpressing chimeric IF proteins in human SW13+ and SW13- cells, which contain and lack endogenous cytoplasmic IF proteins, respectively. The chimeric IF proteins consisted of the rod domain of human nuclear lamin A/C protein fused to the amino and carboxyl-terminal domains of the mouse neurofilament light subunit (NF-L), which contained or lacked the 42 aa insert. Immunofluorescence microscopy was used to follow assembly and targeting of the proteins in cells. Chimeric proteins that lacked the 42 aa insert colocalized with vimentin, whereas those that contained the 42 aa insert did not. When overexpressed in SW13- cells, chimeric proteins containing the 42 aa formed very short or broken cytoplasmic filaments, whereas chimeric proteins that lacked the insert assembled efficiently into long, stable cytoplasmic filaments. To examine the roles of other structural motifs in intracellular targeting, we added two additional sequences to the chimera, a nuclear localization signal (NLS) and a CAAX motif, which are found in nuclear IF proteins. Addition of an NLS alone or an NLS in combination with the CAAX motif to the chimera with the 42 aa insert resulted in cagelike filament that assembled close to the nuclear envelope and nuclear lamina-like targeting, respectively. Our results suggest that the rod domains of eukaryotic nuclear and cytoplasmic IF proteins, which are related to each other, are still compatible upon deletion of the 42 aa insert of coassembly. In addition, NF-L end domains can substitute for the corresponding lamin domains in nuclear lamina targeting.     

Quantitative and Qualitative Alterations of Neuronal and Glial Intermediate Filaments in Rat Nervous System After Exposure to 2, 5-Hexanedione

The precise mechanism for the neurotoxicity of 2,5-hexanedione is not known, but cross-linking of neurofilament proteins has been suggested as one possibility. In this study the effects of long-term exposure to 2,5-hexanedione were studied in the rat nervous system with special reference to regional changes in the quantities of neuronal and glial intermediate filaments. Using enzyme-linked immunosorbent assays the concentrations of 68- and 200-kDa neurofilament polypeptides were shown to be reduced in all brain regions studied. Similar results were obtained in the sciatic nerve. The concentration of glial fibrillary acidic protein was decreased in the cerebellar vermis and the dorsal cerebral cortex, whereas it was increased in the spinal cord, a result suggesting a regional variation in glial sensitivity. The intermediate filaments of the exposed animals were also immunoblotted using polyclonal antisera against the various neurofilament polypeptides and glial fibrillary acidic protein. In all tissues studied, several aggregates with molecular weights higher than those of the monomeric polypeptides were demonstrated. Contrary to clinical observations, these data indicate pronounced effects in both CNS and PNS and call for further studies on CNS effects in humans.