[Lewy bodies in Parkinson's disease: histological, immunohistochemical, and interferometric examinations]. / Tel'tsa Levi pri bolezni Parkinsona (gistologicheskoe, immunogistokhimicheskoe i interferometricheskoe issledovanie).

OBJECTIVE: To quantify the morphochemical characteristics of Lewy bodies detected in the substantia nigra in patients with Parkinson's disease (PD). MATERIAL AND METHODS: The investigators studied the localization of alpha-synuclein (α-Syn) and the distribution of neurofilament protein and synaptophysin by immunohistochemical assas and compared with the results of interferometry and computer-assisted morphometry of Lewy bodies in the autopsy specimens of the substantia nigra from PD patients. RESULTS: Three groups of synuclein-positive aggregates differing in shape were identified. Mature Lewy bodies had a rounded shape, a concentric structure, a poorly stained core, and, as compared with neuropil, a high phase difference value. Comparison of the localization of α-Syn, neurofilaments, and synaptophysin showed that immunostaining of neurofilaments in the peripheral layer of Lewy bodies was shifted closer to the nucleus and the localization of synaptophysin and α-Syn coincided. CONCLUSION: Synuclein-positive protein aggregates showed heterogeneity in structure, shape, and protein composition in PD. The localization of neurofilament protein and synaptophysin in Lewy bodies attests that the cytoskeleton and neuronal synaptic vesicle trafficking in the substantia nigra are impaired in BP.

Immunohistochemical evaluation of neuroreceptors in healthy and pathological temporo-mandibular joint.

AIM: A study was performed on the articular disk and periarticular tissues of the temporo-mandibular joint (TMJ) with immunohistochemical techniques to give evidence to the presence of neuroreceptors (NRec) in these sites. METHODS: The study was carried out on tissue samples obtained from 10 subjects without TMJ disease and from 7 patients with severe TMJ arthritis and arthrosis. We use antibodies directed against following antigens: Gliofibrillary Acidic Protein (GFAP), Leu-7, Myelin Basic Protein (MBP), Neurofilaments 68 kD (NF), Neuron Specific Enolase (NSE), S-100 protein (S-100) and Synaptophysin (SYN). RESULTS: This study revealed that Ruffini's-like, Pacini's-like and Golgi's-like receptors can be demonstrated in TMJ periarticular tissues and that free nervous endings are present in the subsynovial tissues but not within the articular disk. We observed elongated cytoplamic processes of chondrocytes that demonstrated strong S-100 immunoreactivity but they were unreactive with all other antibodies. These cytoplamic processes were more abundant and thicker in the samples obtained from patients with disease TMJ. CONCLUSION: The results of this study confirm that different Nrec are detectable in TMJ periarticular tissues but they are absent within the articular disk. In the latter site, only condrocytic processes are evident, especially in diseased TMJ, and they might have been confused with nervous endings in previous morphological studies. Nevertheless the absence of immunoreactivity for NF, NSE and SYN proves that they are not of neural origin.

Oxidative modification of neurofilament-L and neuronal cell death induced by the catechol neurotoxin, tetrahydropapaveroline.

Tetrahydropapaveroline (THP), which is an endogenous neurotoxin, has been suspected to be associated with dopaminergic neurotoxicity of l-DOPA. In this study, we examined oxidative modification of neurofilament-L (NF-L) and neuronal cell death induced by THP. When disassembled NF-L was incubated with THP, protein aggregation was increased in a time- and THP dose-dependent manner. The formation of carbonyl compounds and dityrosine were observed in the THP-mediated NF-L aggregates. Radical scavengers reduced THP-mediated NF-L modification. These results suggest that the modification of NF-L by THP may be due to oxidative damage resulting from the generation of reactive oxygen species (ROS). When THP exposed NF-L was subjected to amino acid analysis, glutamate, proline and lysine residues were found to be particularly sensitive. We also investigated the effects of copper ions on THP-mediated NF-L modification. At a low concentration of THP, copper ions enhanced the modification of NF-L. Treatment of C6 astrocyte cells with THP led to a concentration-dependent reduction in cell viability. When these cells were treated with 100µM THP, the levels of ROS increased 3.5-fold compared with control cells. Furthermore, treatment of cells with THP increased NF-L aggregate formation, suggesting the involvement of NF-L modification in THP-induced cell damage.

Multiple neurofilament subunits are present in lamprey CNS.

In mammals, there are three neurofilament (NF) subunits (NF-L, NF-M, and NF-H), but it was thought that only a single NF, NF180, exists in lamprey. However, NF180 lacked the ability to self-assemble, suggesting that like mammalian NFs, lamprey NFs are heteropolymers, and that additional NF subunits may exist. The present study provides evidence for the existence of a lamprey NF-L homolog (L-NFL). Genes encoding two new NF-M isoforms (NF132 and NF95) also have been isolated and characterized. With NF180, this makes three NF-M-like isoforms. In situ hybridization showed that all three newly cloned NFs are expressed in spinal cord neurons and in spinal-projecting neurons of the brainstem. Like NF180, there were no KSP multiphosphorylation repeat motifs in the tail regions of NF132 or NF95. NF95 was highly identical to homologous parts of NF180, sharing 2 common pieces of DNA with it. Northern blots suggested that NF95 may be expressed at very low levels in older larvae. The presence of L-NFL in lamprey CNS may support the hypothesis that as in mammals, NFs in lamprey are obligate heteropolymers, in which NF-L is a required subunit.

Isolation of intermediate filaments.

Intermediate filaments (IFs) are found in most eukaryotic cells and are made up of various IF proteins. IFs are highly insoluble in conventional extraction buffers and are therefore commonly purified under denaturing condition. Purified IF proteins can be reassembled into filaments by dialysis. At least 65 IF proteins are found in humans, and the procedures for the purification of each subunit vary somewhat, although many basic steps are similar. To illustrate the isolation of IFs, a detailed protocol is described for purifying neurofilament proteins (NFL, NFM, and NFH subunits) from bovine spinal cord. These three proteins form the predominant IF network in mature neurons. An alternative method for the purification of NFL from a prokaryotic expression system is also included. The isolation of recombinant proteins from bacteria is quite straightforward and may therefore be the method of choice for producing and purifying IFs. Finally, there is a discussion of the purification methods of other IF proteins.

Identification of nitrated proteins in the normal rat brain using a proteomics approach.

BACKGROUND: The nitration of tyrosine has been suggested to play a role in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). METHODS: In the present study, we identified four targets of protein nitration, T-complex polypeptide 1 alpha subunit (TCP-1), neurofilament L (NFL), glial fibrillary acidic protein (GFAP) and clathrin heavy chain (CHC), in the normal rat cortex using a proteomics approach. CONCLUSIONS: There have been no reports on these proteins being identified by proteomics as nitrated forms in the brain. For further study, we have to investigate alterations in these nitrated proteins during aging and in neurodegenerative disorders.

Transdiferenciación neuronal de células madre mesenquimales de médula ósea humana / Neuronal transdifferentiation of human bone marrow mesenchymal stem cells

En el presente estudio se muestra la posibilidad de lograruna transdiferenciación de células madre mesenquimales humanas,obtenidas del estroma de médula ósea, hacia neuronasadultas. Los resultados obtenidos confirman experiencias previasacerca de que las células madre del estroma de la médulaósea experimentan cambios fenotípicos y expresan marcadoresde diferenciación neuronal cuando se cultivan in vitrocon determinadas sustancias químicas. Por otra parte, el presenteestudio demuestra que la transdiferenciación neuronalde estas células puede ser obtenida también por medio de unco-cultivo con células de Schwann. La transdiferenciación enco-cultivo comienza más tardíamente, y aunque la expresiónde nestina, como primer marcador neural, se observa ya a las4 horas del co-cultivo, esta expresión disminuye entre las 24 y72 horas, momento en que comienzan a observarse células conexpresión de marcadores de diferenciación neuronal, aumentandoprogresivamente el número de células que expresanEnolasa específica neuronal, Proteína de neurofilamentos yBeta-tubulina

This study describes the possibility to obtain neuronaltransdifferentiation of human mesenchymal stem cells, obtainedfrom the bone marrow stroma. Our present resultsconfirm previous experiences showing that these cells expressmarkers of neuronal differentiation when they arecultured with certain chemical factors. On the other hand,the present study demonstrates that a neuronal transdifferentiationof these cells can also be obtained by means of aco-culture with Schwann cells. In co-culture, transdifferentiationbegins later, although nestin expression is first observed4 hours after beginning the co-culture, it decreasedbetween 24 and 72 hours, and at this time, a clear increasein the number of NSE, NF and beta-tubulin-positive cells,can be seen

Lamprey neurofilaments contain a previously unreported 50-kDa protein.

We have previously hypothesized that regeneration of axons after spinal cord injury in the lamprey may involve assembly and transport of neurofilaments (NFs) into the growing tip. A single NF, NF-180, has been cloned in this laboratory and until now was thought to be the only NF subunit in lamprey nervous system. However, homopolymerization of NF-180 has not been observed either in experiments on transfected cells or in self-assembly tests in vitro. Forty-three monoclonal antibodies designated as LCM series were generated previously against cytoskeletal proteins of the lamprey nervous system. Seven LCMs were NF specific, and five were keratin specific, as demonstrated by immunohistochemistry. In the present study, one antibody, LCM40, selectively labeled axons in immunohistochemical sections and recognized a single 50-kDa protein in Western blots. Other neuron-specific LCMs and anti-NF antibodies, e.g., LCM39, recognized a known NF subunit, NF-180. Two-dimensional (2-D) gel electrophoresis was employed to separate otherwise indistinguishable individual cytoskeletal proteins. Western blot analysis with an antibody (IFA) that selectively labels all known intermediate filaments indicated that this 50-kDa protein is an intermediate filament (IF). The new protein was incorporated into IF polymers in vitro. Immunoelectron microscopy confirmed that neuronal IFs contain this novel protein. These results suggest that the 50-kDa protein is a previously unrecognized neuronal IF subunit in the lamprey.

Identification of a neurofilament-like protein in the protocerebral tract of the crab Ucides cordatus.

Neurofilaments (NFs) have not been observed in crustaceans using conventional electron microscopy, and intermediate filaments have never been described in crustaceans and other arthropods by immunocytochemistry. Since polypeptides, labeled by the NN18-clone antibody, were revealed on microtubule side-arms of crayfish, we have tested, in this study, whether proteins similar to mammalian NFs are present in the protocerebral tract (PCT) of the crab Ucides cordatus. We used immunohistochemistry for light microscopy with monoclonal antibodies against three different NF subunits, high (NF-H), medium (NF-M), and light (NF-L). Labeling was observed with the NN18-clone, which recognizes NF-M. In order to confirm the results obtained with the immunohistochemical reactions, Western blotting, using the three primary antibodies, was performed and the presence of NF-M was confirmed. The NN18-clone monoclonal antibody recognized a protein of approximately 160 kDa, similar to the mammalian NF-M protein, but NF-L and NF-H were not recognized. Conventional transmission electron microscopy was used to observe the ultrastructural components of the axons and immunoelectron microscopy was used to show the distribution of the NF-M-like polypeptides along cytoskeletal elements of the PCT. Our results agree with previous studies on crustacean NF proteins that have reported negative immunoreactions against NF-H and NF-L subunits and positive immunoreactions against the mammalian NF-M subunit. However, the protein previously referred to as P600 and recognized by the NN18-clone, has a very high molecular weight, thus, being different from mammalian NF-M subunit and from the protein revealed now in our study.

Identification of endogenous phosphorylation sites of bovine medium and low molecular weight neurofilament proteins by tandem mass spectrometry.

Neurofilament proteins (NFP) are intermediate filaments found in the neuronal cytoskeleton. They are highly phosphorylated, a condition that is believed to be responsible for the assembly and stability of the filaments. Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) shows molecular masses for bovine NFP subunits of 63, 105, and 125 kDa for NFL, NFM, and NFH. Mass spectrometric de novo sequencing was used to determine the N-terminal sequence of bovine NFM (115 amino acids), which was previously unknown. Molecular mass information shows that there is one-half equivalent phosphate group on NFL and 24 on NFM. For the first time, it is shown that bovine NFL has three phosphorylation sites (Ser(55), Ser(66), and Ser(472)) and NFM has 22 (Ser(512), Ser(546), Ser(554), Ser(560), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), Ser(750), Ser(756), Ser(770), and Ser(846)) and two tentative sites (Ser(659)/Thr(661) and Thr(840)). Ser(66) was previously not known to be phosphorylated in NFL of other species, while two sites (Ser(55) and Ser(472)) are consistent with the phosphorylations observed in other mammalian NFLs. The three sites, Ser(55), Ser(66), Ser(472), are heterogeneously phosphorylated. Phosphorylation in bovine NFM occurs mainly in the Lys-Ser-Pro (KSP) region, but the Val-Ser-Pro and Ser-Glu-Lys motifs are also phosphorylated. Most of the phosphorylation sites are in accordance with those previously identified in other mammalian NFMs. In bovine NFM, 16 out of the 22 sites are always phosphorylated (Ser(512), Thr(627), Ser(629), Ser(634), Ser(639), Thr(646), Ser(649), Ser(654), Ser(664), Ser(669), Thr(676), Ser(679), Ser(684), Ser(694), Ser(726), and Ser(750)), all of which are contained in the KSP region, and six are sometimes phosphorylated (Ser(546), Ser(554), Ser(560), Ser(756), Ser(770), and Ser(846)). The NFPs have other modifications, including deamidation, oxidation, and N-terminal acetylation. Pyroglutamic acid formation also occurs.

Cdk5 regulates axonal transport and phosphorylation of neurofilaments in cultured neurons.

Phosphorylation has long been considered to regulate neurofilament (NF) interaction and axonal transport, and, in turn, to influence axonal stability and their maturation to large-caliber axons. Cdk5, a serine/threonine kinase homologous to the mitotic cyclin-dependent kinases, phosphorylates NF subunits in intact cells. In this study, we used two different haptenized NF subunits and manipulated cdk5 activity by microinjection, transfection and pharmacological inhibition to monitor the effect of Cdk5-p35 on NF dynamics and transport. We demonstrate that overexpression of cdk5 increases NF phosphorylation and inhibits NF axonal transport, whereas inhibition both reduces NF phosphorylation and enhances NF axonal transport in cultured chicken dorsal-root-ganglion neurons. Large phosphorylated-NF 'bundles' were prominent in perikarya following cdk5 overexpression. These findings suggest that Cdk5-p35 activity regulates normal NF distribution and that overexpression of Cdk5-p35 induces perikaryal accumulation of phosphorylated-NFs similar to those observed under pathological conditions.

In vitro phosphorylation of cytoskeletal proteins from cerebral cortex of rats.

Procedures for the preparation of high- and low-salt Triton insoluble cytoskeletal fractions from rat brain suitable for studying in vitro phosphorylation by endogenous kinases and phosphatases are described. The high-salt Triton insoluble cytoskeletal fraction is enriched in neurofilament subunits (NF-H, NF-M and NF-L), vimentin and glial fibrillary acidic protein (GFAP), while the low-salt Triton insoluble cytoskeletal fraction contains detergent insoluble cytoskeletal elements such as intermediate filament subunits and tubulins. One of our approaches is to incubate cerebral cortex slices with [32P]orthophosphate before the cytoskeletal fraction extraction, which allows the in vitro phosphorylation of cytoskeletal constituents in an intact intracellular environment. On the other hand, we also incubate low- or high-salt cytoskeletal fractions previously prepared with [gamma(32)P]ATP. By doing so, we are able to study the direct effects of substances on the kinase and phosphatase activities associated with the cytoskeletal fraction. Moreover by using specific activators or inhibitors of protein kinases and phosphatases we can obtain more detailed information on the alterations provoked by these substances. These approaches are useful for the investigation of the neurotoxic effects of various drugs and metabolites affecting the cytoskeletal-associated phosphorylation system in the brain.

Identification and characterization of Scp15, a protein from Streptomyces coelicolor A3(2) inducing neurites in PC12 cells.

We previously showed that a fungal protein, p15, induces neurite outgrowth and differentiation of rat pheochromocytoma PC12 cells. We report here the identification and characterization of a protein similar to p15, found in Streptomyces coelicolor A3(2). This hypothetical protein, tentatively named Scp15, has significant similarity with p15, including conserved positions of four cysteine residues involved in the formation of essential disulfide bonds in p15. Hexahistidine-tagged recombinant Scp15 proteins were produced in Escherichia coli, purified, and analyzed for their neurite-inducing activity. Although they were less active than p15, they dose-dependently induced neurites and the expression of neurofilament M. Neurite outgrowth by Scp15 was inhibited by nicardipine, suggesting that Scp15 induces neurites via activation of a calcium signaling pathway.

Phosphorylation state of the native high-molecular-weight neurofilament subunit protein from cervical spinal cord in sporadic amyotrophic lateral sclerosis.

The intraneuronal aggregation of phosphorylated high-molecular-weight neurofilament protein (NFH) in spinal cord motor neurons is considered to be a key pathological marker of amyotrophic lateral sclerosis (ALS). In order to determine whether this observation is due to the aberrant or hyper-phosphorylation of NFH, we have purified and characterized NFH from the cervical spinal cords of ALS patients and controls. We observed no differences between ALS and normal controls in the physicochemical properties of NFH in Triton X-100 insoluble protein fractions, with respect to migration patterns on 2D-iso electrofocusing (IEF) gels, the rate of Escherichia coli alkaline phosphatase mediated dephosphorylation, or the rate of calpain-mediated proteolysis. The rate of calpain-mediated proteolysis was unaffected by either exhaustive NFH dephosphorylation or by the addition of calmodulin to the reaction. Phosphopeptides and the phosphorylated motifs characterized by liquid chromatography tandem mass spectroscopy (LC/MS/MS) analysis demonstrated that all the phosphorylated residues found in ALS NFH were also found to be phosphorylated in normal human NFH samples. Hence, we have observed no difference in the physicochemical properties of normal and ALS NFH extracted from cervical spinal cords, suggesting that the perikaryal aggregation of highly phosphorylated NF in ALS neurons reflects the aberrant somatotopic localization of normally phosphorylated NFH.

Characterization of the phosphorylation sites of the squid (Loligo pealei) high-molecular-weight neurofilament protein from giant axon axoplasm.

Axonal caliber in vertebrates is attributed, in part, to the extensive phosphorylation of NFM and NFH C-terminal tail domain KSP repeats by proline-directed kinases. The squid giant axon, primarily involved in rapid impulse conduction during jet propulsion motility, is enriched in squid-specific neurofilaments, particularly the highly phosphorylated NF-220. Of the 228 serine-threonine candidate phosphate acceptor sites in the NF-220 tail domain (residues 401-1220), 82 are found in numerous repeats of three different motifs SAR/K, SEK/R, K/RSP, with 62 of these tightly clustered in the C-terminal repeat segment (residues 840-1160). Characterization of the in vivo NF-220 phosphorylated sites should provide clues as to the relevant kinases. To characterize these sites, proteolytic digests of NF-220 were analyzed by a combination of HPLC, electrospray tandem mass spectrometry and database searching. A total of 53 phosphorylation sites were characterized, with 47 clustered in the C-terminal repeat segment (residues 840-1160), representing 76% (47/62) of the total acceptor sites in the region. As in mammalian NFH, approximately 64% of the K/RSP sites (14/22) in this region were found to be phosphorylated implicating proline-directed kinases. Significantly, 78% of serines (31/40) in the KAES*EK and EKS*ARSP motifs were also phosphorylated suggesting that non proline-directed kinases such as CKI may also be involved. This is consistent with previous studies showing that CKI is the principal kinase associated with axoplasmic NF preparations. It also suggests that phosphorylation of large macromolecules with multiple phospho-sites requires sequential phosphorylation by several kinases.

gamma-diketone peripheral neuropathy. II. Neurofilament subunit content.

Quantitative morphometric analyses have demonstrated that axon atrophy is the primary neuropathic alteration in peripheral nerve of 2,5-hexanedione (HD)-intoxicated rats (Lehning et al., Toxicol. Appl. Pharmacol. 165, 127-140, 2000). Research suggests that axon caliber is regulated by neurofilament (NF) content and density. Therefore, as a possible mechanism of atrophy, NF subunit (NF-L, -M, and -H) proteins were quantitated in moderately affected rats intoxicated with HD at three daily dosing rates (175, 250, and 400 mg/kg/day). Analyses of subunit protein contents in proximal sciatic nerves indicated uniformly small decreases, which corresponded to minimal changes in axon area occurring in this region. In distal tibial nerve, subunit proteins were decreased substantially (40-70%) when rats were exposed to the 175 and 250 mg/kg/day doses. These reductions in NFs corresponded to significant decreases (approximately 50%) in tibial axon area induced by lower dosing rates. In contrast, 400 mg/kg/day produced similar changes in caliber but smaller reductions (18-25%) in NF-L, -M, and -H levels. This suggests that a decrement in axonal NF content is unlikely to be solely responsible for gamma-diketone-induced axon atrophy and that the corresponding mechanism probably involves additional changes in factors regulating NF density. Analysis of NF content in peripheral nerve also identified the presence of anomolous higher molecular weight NF-H proteins. However, the neurotoxicological significance of these abnormal subunits is uncertain based on their limited occurrence and inconsistent spatiotemporal expression.

Neurofilament-L is a protein phosphatase-1-binding protein associated with neuronal plasma membrane and post-synaptic density.

Far Westerns with digoxigenin-conjugated protein phosphatase-1 (PP1) catalytic subunit identified PP1-binding proteins in extracts from bovine, rat, and human brain. A major 70-kDa PP1-binding protein was purified from bovine brain cortex plasma membranes, using affinity chromatography on the immobilized phosphatase inhibitor, microcystin-LR. Mixed peptide sequencing following cyanogen bromide digestion identified the 70-kDa membrane-bound PP1-binding protein as bovine neurofilament-L (NF-L). NF-L was the major PP1-binding protein in purified preparations of bovine spinal cord neurofilaments and the cytoskeletal compartment known as post-synaptic density, purified from rat brain cortex. Bovine neurofilaments, at nanomolar concentrations, inhibited the phosphorylase phosphatase activity of rabbit skeletal muscle PP1 catalytic subunit but not the activity of PP2A, another major serine/threonine phosphatase. PP1 binding to bovine NF-L was mapped to the head region. This was confirmed by both binding and inhibition of PP1 by recombinant human NF-L fragments. Together, these studies indicate that NF-L fulfills many of the biochemical criteria established for a PP1-targeting subunit and suggest that NF-L may target the functions of PP1 in membranes and cytoskeleton of mammalian neurons.

Topographic regulation of cytoskeletal protein phosphorylation by multimeric complexes in the squid giant fiber system.

In mammalian and squid nervous systems, the phosphorylation of neurofilament proteins (NFs) seems to be topographically regulated. Although NFs and relevant kinases are synthesized in cell bodies, phosphorylation of NFs, particularly in the lys-ser-pro (KSP) repeats in NF-M and NF-H tail domains, seem to be restricted to axons. To explore the factors regulating the cellular compartmentalization of NF phosphorylation, we separated cell bodies (GFL) from axons in the squid stellate ganglion and compared the kinase activity in the respective lysates. Although total kinase activity was similar in each lysate, the profile of endogenous phosphorylated substrates was strikingly different. Neurofilament protein 220 (NF220), high-molecular-weight NF protein (HMW), and tubulin were the principal phosphorylated substrates in axoplasm, while tubulin was the principal GFL phosphorylated substrate, in addition to highly phosphorylated low-molecular-weight proteins. Western blot analysis showed that whereas both lysates contained similar kinases and cytoskeletal proteins, phosphorylated NF220 and HMW were completely absent from the GFL lysate. These differences were highlighted by P13(suc1) affinity chromatography, which revealed in axoplasm an active multimeric phosphorylation complex(es), enriched in cytoskeletal proteins and kinases; the equivalent P13 GFL complex exhibited six to 20 times less endogenous and exogenous phosphorylation activity, respectively, contained fewer cytoskeletal proteins and kinases, and expressed a qualitatively different cdc2-like kinase epitope, 34 kDa rather than 49 kDa. Cell bodies and axons share a similar repertoire of molecular consitutents; however, the data suggest that the cytoskeletal/kinase phosphorylation complexes extracted from each cellular compartment by P13 are fundamentally different.

Identification of novel in vitro PKA phosphorylation sites on the low and middle molecular mass neurofilament subunits by mass spectrometry.

Phosphorylation of the head domains of intermediate filament proteins by second messenger-dependent kinases is important in regulating filament assembly. In the case of neurofilaments, head domain phosphorylation is known to be important in assembly, but few sites have been identified. Using matrix-assisted laser desorption-ionization (MALDI) and nano-electrospray mass spectrometry, we report the identification of several novel in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites in the low (NF-L) and middle (NF-M) molecular mass neurofilament subunits. Neurofilament polypeptides were purified from adult rat brain, and fractions containing a mixture of NF-L and NF-M were nonradioisotopically phosphorylated with PKA prior to proteolytic digestion of the polypeptides in situ in polyacrylamide excised from SDS gels. Sites of phosphorylation were determined by mass spectrometric analysis of mixtures enriched in tryptic phosphopeptides. In NF-L, four novel sites were identified: serines 12, 41, and 49 in the head domain and serine 435 in the carboxyl-terminal tail domain, and data consistent with phosphorylation of serine 2 were obtained. Recombinant rat NF-L protein was also phosphorylated with PKA, and the same serines were identified as phosphorylation sites, with two additional sites, serine 43 and probable phosphorylation of serine 55. In NF-M, one novel site, serine 1 in the amino-terminal head domain, was found to be phosphorylated, and serine 46, also in the amino-terminal head domain, was confirmed as a PKA phosphorylation site.

[The assembly of low molecular weight neurofilament protein (NF-L) in vitro].

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.