Tubulin Synthesis in Rat Forebrain: Studies with Free and Membrane-Bound Polysomes

Abstract: Free and membrane-bound polysomes were prepared from rat forebrain and added to a cell-free system containing rabbit reticulocyte factors and L-[³⁵S]methionine. The translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The free polysomes synthesized actin and at least four major tubulin subunits (α¹, α², β¹, and α²) that are found in rat forebrain cytoplasm. The membrane-bound polysomes synthesized predominantly one protein (MB) in the tubulin region of the two-dimensional gel. MB has a molecular weight and isoelectric point similar to α-tubulin. Only trace amounts of α- and β-tubulin and actin were synthesized by the membrane-bound polysomes. MB co-purified with cytoplasmic tubulin after two cycles of aggregation and disaggregation. MB synthesized in vitro (from membrane-bound polysomes) and α- and β-tubulin and actin subunits (synthesized from free polysomes) were digested with Staphylococcus aureus V8 protease, and the resulting peptides were separated by slab gel electrophoresis followed by autoradiography. The peptide pattern of MB was similar but not identical to the peptide patterns of α- and β-tubulin; MB yielded peptides not found in tubulin. We conclude that membrane-bound polysomes from rat forebrain do not synthesize significant amounts of the predominant tubulin subunits synthesized by free polysomes. A major protein (MB) is synthesized by membrane-bound polysomes and is similar, but not identical, to α-tubulin synthesized by free polysomes on the basis of molecular weight, isoelectric point, and peptide analysis.     

Principal neurofilament-associated protein kinase in squid axoplasm is related to casein kinase I

A cytoskeletal extract of pure axoplasm, highly enriched with neurofilaments (ANF), was prepared from the giant axon of the squid. This ANF preparation also contained potent kinase activities which phosphorylated the Mr greater than 400,000 (high molecular weight) and Mr 220,000 squid neurofilament protein subunits. High salt (1 M) extraction of this ANF preparation solubilized most of the neurofilament proteins and kinase activities and gel filtration on an AcA 44 column separated these two components. The neurofilaments eluted in the void volume of the column while the kinase activities eluted in the 17-44-kDa range of the column. Two major kinase activities were measured in this peak of activity. One of these strongly phosphorylated the phosphate acceptor peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and was completely inhibited by the selective inhibitor of cAMP-dependent kinase Thr-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-Ile- NH2 (Wiptide). Since addition of cAMP did not stimulate activity, this suggested that this kinase was a free catalytic subunit of cAMP-dependent kinase associated with the neurofilaments. The second kinase activity most effectively phosphorylated alpha-casein, and this activity was not affected by Wiptide. The alpha-casein phosphorylating activity (ANF kinase) was the principal activity responsible for neurofilament protein phosphorylation, and was not inhibited by various inhibitors against second messenger regulated kinases, suggesting it was related to the casein kinase family. Four lines of evidence indicate ANF kinase was similar to casein kinase I. These were: 1) the apparent molecular weight determined by gel filtration and the chromatographic elution profile on phosphocellulose column corresponded to casein kinase I; 2) heparin, an inhibitor of casein kinase II at 2-5 micrograms/ml, stimulated both ANF kinase and purified casein kinase I at these concentrations, while CKI-7, a relatively selective inhibitor of casein kinase I, inhibited ANF kinase in a comparable dose-response fashion; 3) purified casein kinase I strongly phosphorylated both ANF protein subunits (like ANF kinase) whereas casein kinase II was relatively ineffective; and 4) tryptic peptide maps of the HMW and Mr 220,000 neurofilament proteins after phosphorylation by ANF kinase or purified casein kinase I showed similar 32P-peptide patterns.     

Purification, Properties, and Immunohistochemical Localisation of Human Brain 14-3-3 Protein

Abstract: A protein has been purified from human brain that appears to be the human equivalent of bovine 14-3-3 protein. On polyacrylamide gel electrophoresis the protein migrates as a faster major component, termed 14-3-3-2 protein, and a slower minor component, termed 14-3-3-1 protein, which consists of approximately 12% of the total protein. Both 14-3-3-1 and 14-3-3-2 have a native molecular weight of approximately 67,000. 14-3-3-2 appears to have the subunit composition (αβ; 14-3-3-1 has the composition ββ. Peptide mapping with Stuphvlococcus aureus V8 proteinase shows that α and β subunits are unrelated but the β and β' subunits show some common peptides. Immunoperoxidase labelling shows that 14-3-3 is localised in neurones in the human cerebral cortex. 14-3-3 shows no enolase, creatine kinase, triose phosphate isomerase, ATPase, cyclic nucleotide-dependent protein kinase, or purine nucleoside phosphorylase activity. 14-3-3 does not bind calcium and does not appear to be related to calmodulin, calcineurin, tubulin, neurofilament proteins, clathrin-associated proteins, or tropomyosin. The functional significance of this neuronal protein remains obscure.     

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.     

Synthesis of Cytoskeletal Proteins in Bulk-Isolated Neuronal Perikarya Takashi Nakayama

Abstract: Neuronal perikarya were isolated from young rat brain by sucrose density gradient centrifugation of the tissue, dissociated with a low concentration of trypsin. The isolated cells retained their endogenous proteins, and were capable of active protein synthesis. After incubation with L-[³⁵S]methionine, perikarya were homogenised and separated into soluble and particulate fractions by centrifugation at 70,000 g. Newly synthesised polypeptides in each fraction were resolved by SDS-gel and two-dimensional gel electrophoresis coupled with fluorography. Neuronal perikarya synthesised predominantly actin, and α¹-, α² and β-tubulin. In addition, polypeptides with molecular weights of 35,000, 68,000 and 85,000 were heavily labelled. On two-dimensional electrophoresis, microheterogeneities were seen in soluble actin as well as in soluble tubulins, indicating that heterogeneities reported for brain actin and tubulins are inherent in neuronal actin and tubulins, but not owing to the heterogeneity of cells in the brain tissue. Structural differences between soluble tubulins and those associated with the particulate fraction were indicated by two-dimensional gel electrophoresis and also by one-dimensional peptide maps. The 68,000 molecular weight polypeptide synthesised in neuronal perikarya in vitro yielded a peptide map virtually identical with that generated from the major component of the neurofilament triplet polypeptides that were synthesised in situ. The 160,000 and 200,000 components of the neurofilament triplet were also synthesised in perikarya in vitro , but to disproportionately weaker extents compared with the 68,000 component.     

Comparative studies of the neurofilament triplet protein peptide mapping by chemical cleavage

Peptide mapping of the three neurofilament protein subunits with apparent mol. weights of 210 kDa, 160 kDa and 70 kDa was performed with two different reagents: CNBr, BNPS-Skatole leading to the cleavage of methionyl and tryptophanyl bonds respectively. With BrCN we obtained two large fragments resistant to the cleavage, with mol. wts of 85 kDa for the 160 kDa and 135 kDa for the 210 kDa neurofilament proteins respectively. These fragments were located on the C-terminal part of the proteins (the tails) and correspond to specific regions responsible for their physiological identity. On the other hand, the cleavage with BNPS-Skatole at the tryptophanyl bonds gave similar patterns. The 210 kDa and 160 kDa neurofilament proteins gave a doublet of high mol. wt resistant to the cleavage, corresponding very likely to the C-terminal part and 4 fragments of mol. wt between 30 and 40 kDa corresponding to the N-terminal part. The neurofilament triplet share a common 30.5 kDa fragment located on the N-terminal part. From these peptide mapping studies, we conclude that the two neurofilament subunit proteins with mol. wts of 160 kDa and 210 kDa are different but related structures and that the CNBr characterized cleavage fragments of mol. wt 85,000 and 135 kDa are suitable polypeptides for sequence and immunological studies of the C-terminal part of these proteins.     

Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis

Protein carbonylation, the non-enzymatic addition of aldehydes or ketones to specific amino acid residues, has been implicated in the pathophysiology of multiple sclerosis. In this study, we investigated whether protein carbonyls also accumulate in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE). Western blots analysis after derivatization with dinitrophenyl hydrazine (oxyblot) showed elevated protein carbonylation at the time of maximal clinical disability. During the same period glutathione levels were substantially reduced, suggesting a causal relationship between these two markers. In contrast, lipid peroxidation products accumulated in EAE spinal cord well before the appearance of neurological symptoms. Carbonyl staining was not restricted to inflammatory lesions but present throughout the spinal cord particularly in neuronal cell bodies and axons. By 2-dimensional-oxyblot, we identified several cytoskeletal proteins, including β-actin, glial acidic fibrillary protein, and the neurofilament proteins as the major targets of carbonylation. These findings were confirmed by pull-down experiments, which also showed an increase in the number of carbonylated β-actin molecules and a decrease in that of oxidized neurofilament proteins in EAE. These data suggest the possibility that oxidation targets neurofilament proteins for degradation, which may contribute to axonal pathology observed in multiple sclerosis and EAE.     

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.     

The regulatory role of calmodulin in the proteolysis of individual neurofilament proteins by calpain

The in vitro degradation of individual neurofilament proteins by calpain and the effects of calmodulin on this proteolysis were studied. Two major results are reported. First, in the presence of calcium, calmodulin binds to the 200-kD neurofilament protein, but only weakly associates with the 150-kD neurofilament protein. The 70-kD neurofilament protein shows no specific calmodulin-binding. Second, calmodulin inhibits the calpain-mediated degradation of the 200-kD neurofilament protein, but does not alter the hydrolysis of the 150-kD and 70-kD neurofilament proteins. In addition, calmodulin is able to bind to the 200-kD neurofilament protein in the presence of other neurofilament subunits, indicating that calmodulin may play a role in the regulation of the metabolism of the 200-kD neurofilament protein in vivo.     

Brush-border alpha-actinin? Comparison of two proteins of the microvillus core with alpha-actinin by two-dimensional peptide mapping

The bundle of filaments within the intestinal microvillus contains four major polypeptides in addition to actin calmodulin, a 70-kdalton subunit and two polypeptides with molecular masses similar to that of the Z-line component alpha-actinin (95 and 105 kdaltons). Two- dimensional mapping of tryptic peptides indicates that (a) alpha- actinins from chicken skeletal, cardiac, and smooth muscle are similar but not identical proteins and that skeletal alpha-actinin in more similar to the cardiac subunit than to the alpha-actinin from gizzard; (b) the brush-border 95- and 105-kdalton subunits are closely related to each other, but the smaller subunit is not a proteolytic fragment of the 105-kdalton subunit; and (c) although there is considerable peptide overlap between the brush-border subunits and the three alpha-actinins, the peptide maps of the 95- and 105-kdalton proteins are substantially distinct from the various alpha-actinin maps, suggesting that neither brush-border subunit is a bona fide alpha-actinin. Nevertheless, on the basis of peptide mapping criteria alone, one cannot exclude the possibility that the brush-border subunits are "alpha-actinin-like." However, there is no immunological cross-reactivity between the brush- border subunits and alpha-actinins, using antibodies prepared against gizzard alpha actinin.     

Effect of Melatonin and Melatonylvalpromide on β-amyloid and Neurofilaments in N2a Cells

In the present study, we have studied the effect of melatonin (Mt) and melatonin derivative, i.e., melatonylvalpromide (Mtv), on cell viability, β-amyloid (Aβ) production, cell morphology, and expression and phosphorylation of neurofilament proteins in wild-type murine neuroblastoma N2a (N2a/wt) and N2a stably transfected with amyloid precursor protein (N2a/APP) cell lines. The study used MTT assay, Sandwich ELISA, immunocytochemistry and Western blots techniques. The results showed that both Mt and Mtv could increase cell viability, but Mtv did so more effectively. The N2a/APP showed shorter and less amount of cell processes than N2a/wt, and Mtv but not Mt slightly improved the morphological changes in N2A/APP. Both Mt and Mtv suppressed the Aβ level in cell lysates, but the effect of Mtv was stronger than Mt. The immunoreaction to the non-phosphorylated neurofilament proteins probed by SMI32 and SMI33 were remarkably weaker in N2a/APP than N2a/wt, while the immunoreaction to the phosphorylated neurofilament proteins at SMI34 epitopes was slightly stronger in N2a/APP than N2a/wt, suggesting higher phosphorylation level of neurofilament proteins in N2a/APP. Treatment of the cells with Mt and Mtv increased the immunoreaction at SMI32 and SMI33 epitopes, while only Mtv but not Mt decreased the staining at SMI34 epitope, suggesting both Mt and Mtv promote dephosphorylation of neurofilament at SMI32 and SMI33 epitopes, while Mtv stimulates dephosphorylation of neurofilament at SMI34 epitope. These results suggest that Mtv may be a better candidate in arresting the intracellular accumulation of Aβ and protecting the cells from Aβ-related toxicity. Xiao-Chuan Wang and Yin-Chun Zhang equally contributed to the work.     

Sequences in the Amino Termini of GABA ρ and GABAA Subunits Specify Their Selective Interaction In Vitro

Abstract: Molecular cloning has revealed that there are six classes of subunits capable of forming GABA-gated chloride channel receptors. GABA_A receptors are composed of α, β, γ, δ, and ε/χ subunits, whereas GABA_C receptors appear to contain ρ subunits. However, retinal cells exhibiting GABA_C responses express α, β, and ρ subunits, raising the possibility that GABA_C receptors may be a mixture of subunit classes. Using in vitro translated protein, we determined that human GABA_A receptor subunits α1, α5, and β1 did not coimmunoprecipitate with full-length ρ1, ρ2, or the N-terminal domain of ρ1 that contains signals for ρ-subunit interaction. To explore the molecular mechanism underlying these apparently exclusive combinations, chimeric subunits were created and tested for interaction with the wild-type subunits. Transfer of the N terminus of β1 to ρ1 created a β1ρ1 chimera that coimmunoprecipitated with the α1 subunit but not with the ρ2 subunit. Furthermore, exchanging the N terminus of the ρ1 subunit with the corresponding region of β1 produced a ρ1β1 chimera that interfered with ρ1 receptor expression in Xenopus oocytes, whereas the full-length β1 subunit had no effect. Together, these results indicate that sequences in the N termini direct assembly of ρ subunits and GABA_A subunits into GABA_C and GABA_A receptors, respectively.     

The Postsynaptic Density: Constituent and Associated Proteins Characterized by Electrophoresis, Immunoblotting, and Peptide Sequencing

The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two-dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N-terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tublin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1 alpha, and receptor protein. The three neurofilament proteins are detected but a 58-kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66-kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68-kDa protein is identified as a member of the hsp70/BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N-ethylmaleimide-sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115-kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.     

Selective Dephosphorylation of the Subunits of Skeletal Muscle Calcium Channels by Purified Phosphoprotein Phosphatases

Abstract: Multiple sites on the α1 and β subunits of purified skeletal muscle calcium channels are phosphorylated by cyclic AMP-dependent protein kinase, resulting in three different tryptic phosphopeptides derived from each subunit. Phosphoprotein phosphatases dephosphorylated these sites selectively. Phosphoprotein phosphatase 1 (PP1) and phosphoprotein phosphatase 2A (PP2A) dephosphorylated both α1 and β subunits at similar rates, whereas calcineurin dephosphorylated β subunits preferentially. PP1 dephosphorylated phosphopeptides 1 and 2 of the α1 subunit more rapidly than phosphopeptide 3. In contrast, PP2A dephosphorylated phosphopeptide 3 of the α1 subunit preferentially. All three phosphoprotein phosphatases preferentially dephosphorylated phosphopeptide 1 of the β subunit and dephosphorylated phosphopeptides 2 and 3 more slowly. Mn²⁺ increased the rate and extent of dephosphorylation of all sites by calcineurin so that >80% dephosphorylation of both α1 and β sub-units was obtained. The results demonstrate selective dephosphorylation of different phosphorylation sites on the α1 and β subunits of skeletal muscle calcium channels by the three principal serine/threonine phosphoprotein phosphatases.     

A Comparison of In Vitro- and In Vivo Phosphorylated Neurofilament Polypeptides

Abstract: Neurofilament polypeptides phosphorylated in vitro by incubation of neurofilament-enriched preparations from rat CNS with [γ-³²P]ATP were compared with the corresponding polypeptides labeled in vivo by injection of ³²P_i into the lateral ventricles of rats. Autoradiography of sodium dodecyl sulfate (SDS)-polyacrylamide gels revealed that the major phosphorylated species in both preparations were the three neurofilament subunits, which have molecular weights of 200K, 145K, and 68K. However, the relative levels of ³²P detected in the three in vitro -labeled subunits differed from the relative in vivo levels. The two larger neurofilament polypeptides displayed similar ³²P isoprotein distribution patterns on two-dimensional gels, whereas additional isoproteins were seen in the in vitro -labeled 68K species. Limited proteolysis in SDS-polyacrylamide gels revealed the presence of common phosphopeptides in the corresponding pairs of in vitro- and in vivo-labeled subunits, but the in vivo -labeled 145K and in vitro -labeled 200K polypeptides contained additional digestion products. Two-dimensional peptide mapping of the 68K polypeptide digested with a mixture of trypsin and chymotrypsin indicated that this component was phosphorylated at a single, identical site, both in vivo and in vitro. These results indicate that the protein kinase that copurifies with neurofilament preparations may be involved in their in vivo phosphorylation.     

Acrylamide alters neurofilament protein gene expression in rat brain

Acrylamide, a prototype neurotoxin, alters neurofilament protein (NF) gene expression in rat brain. Levels of mRNA coding for neurofilament protein subunits NF-L, NF-M, and NF-H have been determined by Northern blot analysis using³²P-labeled cDNA probes. Acrylamide given acutely (100 mg/kg, single intraperitoneal injection) causes a selective increase in NF-M mRNA (approximately 50%) compared to controls. The expression of NF-L or NF-H mRNA is not affected by acrylamide. In contrast, chronic treatment with acrylamide [0.03% (w/v) in drinking water for 4 weeks] induces a modest but significant increase (approximately 22%) in NF-L mRNA compared to controls. Levels of NF-M, and NF-H mRNA are not altered by acrylamide treatment. The expression of -actin mRNA, an ubiquitous protein, is not affected by either treatment regimen of acrylamide. The results of this study show that acrylamide increases the expression of mRNA for NF protein subunits in rat brain. The increase of specific mRNA for NF subunits depends on the dose, duration and route of acrylamide administration.     

CHARACTERIZATION OF MULTIPLE FORMS OF BRAIN TUBULIN SUBUNITS

Abstract— Microtubular protein was isolated from rat forebrain by biochemical purification (ammonium sulfate precipitation followed by DEAE cellulose chromatography) or by two cycles of aggregation-disaggregation. The protein subunit structure was examined on two-dimensional electrophoretograms: first dimension, urea isoelectric focusing gel; second dimension, sodium dodecyl sulfate exponential acrylamide slab gel. Two forms of α tubulin were separated in the second dimension on the basis of different rates of migration (α and α₂). Each of these species was further separated into at least three forms with different isoelectric points. β Tubulin was separated into a minor species (BI) and a major species β₂). Multiple subunits were observed using protein from either purification method and in a two-dimensional electrophoretogram of total supernatant proteins from rat brain. Separation and visualization of multiple forms of α and β tubulin is consistent with reports that provide evidence for post-translational modification of these proteins.     

Carbon Disulfide-Induced Changes in Cytoskeleton Protein Content of Rat Cerebral Cortex

To investigate the mechanism of carbon disulfide-induced neuropathy, male wistar rats were administrated by gavage at dosage of 300 or 500 mg/kg carbon disulfide, five times per week for 12 weeks. By the end of the exposure, the animals produced a slight or moderate level of neurological deficits, respectively. Cerebrums of carbon disulfide-intoxicated rats and their age-matched controls were Triton-extracted and centrifuged at a high speed (100,000 × g) to yield a pellet fraction of NF polymer and a corresponding supernatant fraction, which presumably contained mobile monomer. Then, the contents of six cytoskeletal protein (NF-L, NF-M, NF-H, α-tubulin, β-tubulin, and β-actin) in both fractions were determined by immunoblotting. Results showed that the contents of the three neurofilament subunits in the pellet and the supernatant fraction decreased significantly regardless of dose levels (P < 0.01). As for microtubule proteins, in the pellet fraction of cerebrum, the levels of α-tubulin and β-tubulin demonstrated some inconsistent changes. However, in the supernatant fractions, the content of α-tubulin and β-tubulin increased significantly in both two dose groups (P < 0.01). In comparison to neurofilament and tubulin proteins, the content of β-actin changed less markedly, only the supernatant fraction of the high dose group displayed significant increase (P < 0.01), but the others remained unaffected. These findings suggested that the changes of cytoskeleton protein contents in rat cerebrum were associated with the intoxication of carbon disulfide, which might be involved in the development of carbon disulfide neurotoxicity.