Neurofibrillar differentiation during embryonary growth

The spinal cord and the acoustic primordium of the chick embryo (3 to 7-day-old) were investigated by electron microscopy. Special attention was devoted to the structure of neurofilaments and tubules in the neuroblasts and cells of the crista acustica. The neurofilaments were observed to be related at early stages of development to the RNA particles of the cytoplasm (Palade's particles). This relationship is observed in particular fields which are clearly distinguished because they show fewer particles than the surrounding cytoplasm. The first stage is characterized by the presence in these fields of short branched filaments, which frequently are in contact with the ribosomes. The filaments grow in length and become ordered during a later period. At the same time, the fields become free of the basophilic particles. Tubules about 300 Å thick were found in neuroblasts and their prolongations (central nervous system); in the acoustic nerve, fibers of the “tubule type” or of the “neurofilament type” are described. Filaments and tubules showing similar morphology were also observed in the acoustic epithelium (crista acustica). The filaments seem to originate in the same way as in the neuroblasts. A close relationship between tubules and centrioles was found. The origin of the neurofilaments is discussed in relation to the filaments of proteinic nature described in other cell types.     

The effects of dephosphorylation on the structure of the projections of neurofilament

Carboxy-terminal tail domains of larger molecular mass subunits (NF-M and NF-H) of neurofilaments (NFs), which are the highly phosphorylated moieties, were observed as thin flexible filaments projecting from NF core filaments by rotary shadowing (Hisanaga and Hirokawa, 1988). Dephosphorylation of NFs has been suspected to affect the structures and the functions of the carboxy-terminal tail projections. We report here the effects of the dephosphorylation on the structure of NFs studied by electron microscopy. (1) The structures of carboxy-terminal tail projections after dephosphorylation were compared with those of the control NFs by low-angle rotary shadowing. This was examined with 2 samples; the isolated neurofilaments and the short filaments assembled from NF-H. Both the dephosphorylated NFs and the short filaments showed many projections laterally extending from core filaments similar to those observed in the control samples. (2) With respect to the structure of NF in physiological solution, the density of NFs in the precipitates was examined by thin-section electron microscopy. No difference in the density was noted between control and dephosphorylated NFs. (3) The ability to form cross-bridges in vitro was examined by quick-freeze, deep-etch electron microscopy. The structure and frequency of cross-bridges appeared to be similar in both control and dephosphorylated NFs. (4) Phosphate determination revealed that about 90% of the phosphate groups of NF-H subunit were removed by treatment with E. coli alkaline phosphatase. These results indicated that the dephosphorylation of NF did not affect the structure and the ability to form cross-bridges of the carboxy-terminal tail projections in vitro.     

Aluminum treatment of intact neuroblastoma cells alters neurofilament subunit phosphorylation,solubility, and proteolysis

Addition of 400 μM AlCl₃ to the culture medium for 72 h has been previously shown to induce perikaryal whorls of intermediatesized filaments in intact mouse NB2a/d1 neuroblastoma cells. Immunoblot analysis demonstrated that in vivo treatment of cells with aluminum induced thede novo appearance of extensively phosphorylated NF-H isoforms in cytoskeletons of undifferentiated cells and increased levels of these isoforms in differentiated cells. Neurofilament subunits isolated from intact cells treated with aluminum were resistant to dephosphorylation in vitro by alkaline phosphatase and to in vitro degradation by endogenous calcium-dependent protease(s). These alterations were accompanied by a greater tendency of neurofilaments to form insoluble aggregates after isolation. These findings demonstrate direct effects of aluminum on neurofilament subunits within intact neuronal cells similar to those previously demonstrated following in vitro exposure of isolated neurofilaments to aluminum.     

An immunocytochemical comparison of cytoskeletal proteins in aluminum-induced and Alzheimer-type neurofibrillary tangles

Summary Exposure of the central nervous system (CNS) of rabbits to aluminum salts produces a progressive encephalopathy. Examination of CNS strucres discloses widespread perikaryal neurofibrillary tangle (NFTs) formation. The aluminum-induced NFTs consist of collections of normal neurofilaments, and differ ultrastructurally and in their solubility characteristics from Alzheimer-type NFTs, the latter being composed of largely insoluble paired helical filaments. The present study compares NFTs found in the rabbit to those of Alzheimer's disease, using monoclonal antibodies (SMI 31, SMI 32) that recognize phosphorylated and non-phosphorylated determinants respectively in normal neurofilaments, and an antiserum raised against purified microtubules. Paraffin-embedded sections were stained by the avidin-biotin immunocytochemical method. Intense staining of aluminum-induced NFTs was found after processing with SMI 31 and SMI 32, while no staining of non-tangled perikarya of control rabbits or of Alzheimer-type NFTs was seen. Antimicrotubule antiserum gave weak, nonfocal staining in the aluminum-treated and control rabbits, while Alzheimer-type NFTs were stained intensely. These results show that phosphorylated and non-phosphorylated neurofilaments accumulate in aluminum-induced NFTs, thus complementing the previously demonstrated specific slowing of the axonal transport of neurofilaments in aluminum intoxication. Further, they suggest that the presence of microtubular proteins may be necessary for altered neurofilaments to take on a paired helical configuration.Supported by NIH grant No. 1 KO8 AG00258     

In vitro assembly and isolation of neurofilaments and microtubules from mammalian CNS

Neurofilaments were assembled in vitro from the high speed supernatant of mammalian CNS homogenate in a disassembly buffer containing 4-morpholine-ethane sulfonic acid, MgCl2 and EGTA at 4 degrees C in the presence of 4 M glycerol. The assembled neurofilaments were depolymerized by dialysis against the disassembly buffer and repolymerized by the addition of glycerol to the clarified supernatant obtained afer disassembly. The filament assembly reaction was complete in less than 30 s as measured by turbidimetric changes at 415 nm and did not require any added nucleotide. No assembly of filaments was detected when using frozen tissue. The assembled filaments corresponded to the enrichment of neurofilament triplet, the 210,000, 160,000 and 70,000 dalton polypeptides on SDS-polyacrylamide gels and appeared morphologically and immunochemically identical to neurofilaments isolated by axonal flotation methods. These studies demonstrate in vitro assembly of neurofilaments under native conditions which raises the possibility that like microtubules, neurofilaments or a subpopulation of neurofilaments might be in a dynamic state of assembly--disassembly in situ.     

Mice with disrupted midsized and heavy neurofilament genes lack axonal neurofilaments but have unaltered numbers of axonal microtubules.

Mammalian neurofilaments are assembled from the light (NF-L), midsized (NF-M), and heavy (NF-H) neurofilament proteins. While NF-M and NF-H cannot self-assemble into homopolymers, the data concerning NF-L has been more contradictory. In vitro bovine, porcine, and murine NF-L can homopolymerize in the absence of other subunits. However, in vivo studies suggest that neither rat nor mouse NF-L can form filaments when transfected alone into cells lacking endogenous intermediate filaments. By contrast, human NF-L forms homopolymers in similar cell lines. Recently we generated mice with null mutations in the NF-M and NF-H genes. To determine if mouse NF-L can homopolymerize in mouse axons, NF-M and NF-H null mutants were bred to create a line of double mutant animals. Here we show that axons in NF-M/H double mutant animals are largely devoid of 10-nm filaments. Instead, the axoplasm is transformed to a microtubule-based cytoskeleton-although the lack of any increase in tubulin levels per unit length of nerve or of increases in microtubule numbers relative to myelin sheath thickness argues that microtubules are not increased in response to the loss of neurofilaments. Thus in vivo rodent neurofilaments are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form a filamentous network.     

Stable tubule only polypeptides (STOP) proteins co-aggregate with spheroid neurofilaments in amyotrophic lateral sclerosis

A major cytopathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of axonal spheroids containing abnormally accumulated neurofilaments. The mechanism of their formation, their contribution to the disease, and the possibility of other co-aggregated components are still enigmatic. Here we analyze the composition of such lesions with special reference to stable tubule only polypeptide (STOP), a protein responsible for microtubule cold stabilization. In normal human brain and spinal cord, the distribution of STOP proteins is uniform between the cytoplasm and neurites of neurons. However, all the neurofilament-rich spheroids present in the tissues of affected patients are intensely labeled with 3 different anti-STOP antibodies. Moreover, when neurofilaments and microtubules are isolated from spinal cord and brain, STOP proteins are systematically co-purified with neurofilaments. By SDS-PAGE analysis, no alteration of the migration profile of STOP proteins is observed in pathological samples. Other microtubular proteins, like tubulin or kinesin, are inconstantly present in spheroids, suggesting that a microtubule destabilizing process may be involved in the pathogenesis of ALS. These results indicate that the selective co-aggregation of neurofilament and STOP proteins represent a new cytopathological marker for spheroids.     

Deformation of isolated neurofilaments and the pathogenesis of neurofibrillary pathology

Experimental structural alterations were studied in neurofilaments isolated from rat peripheral nerve. Structural alterations were induced in vitro by prolonged incubations of neurofilament-rich fractions in 0.1 M KCl prior to fixation and examination of neurofilaments by negative staining techniques. Neurofilaments appeared to retain their structural integrity during the first week of incubation. Subsequently, a progressive reduction in the numbers of intact neurofilaments coincided with the increasing appearance of neurofilamentous breakdown products. During the period of neurofilament breakdown, an increasing number of altered neurofilaments were observed. These abnormal neurofilaments were characterized primarily by axial enlargements with diameters frequently occurring in the 150-200 A range but measuring up to 250 A. Enlarged neurofilaments also showed a greater irregularity of their lateral margins and less ridigity in their linear course. A striking feature of enlarged neurofilaments was their tendency to display twisting deformations along their longitudinal axes. Twists occurred most frequently along neurofilaments of greatest diameter. The largest abnormal neurofilaments reveal multiple periodic twists, sometimes occurring at 800-1000 A intervals. These enlarged and twisted neurofilaments resembled the abnormal filaments described in neurofibrillary tangles and plaques. The demonstration of neurofilament plasticity as well as the resemblance of neurofilaments deformed in vitro to the abnormal filamentous structures of neurofibrillary tangles and plaques has led to the suggestion that the latter structures may be derived from alterations of normally-formed neurofilaments. Furthermore, it is proposed that the enlarged and twisted filaments of neurofibrillary pathology may arise from alterations in the turnover of neurofilaments or, more specifically, from deficiencies in the degradative processes by which these organelles are broken down.     

Induction of neurofibrillary degeneration following treatment with maytansine in vivo

The effects of maytansine (MYT), a naturally occurring ansa macrolide and potent antimitotic drug that binds to tubulin, were studied by light and electron microscopy in the central nervous system of rabbits. Respectively, 17 and 5 animals were sacrificed at various time intervals following a single intrathecal or intraocular injection of the agent. The rabbits responded to the intrathecal injection with progressively severe weakness. By 19 h following injection the neurons of the cervical spinal cord, medulla and pons showed, by light microscopy, a marked clumping of the Nissl substance, while on the third day and later the nerve cell perikarya and dendrites displayed severe neurofibrillary changes.By electron microscopy several cytological alterations were observed as early as 19 h; among them were the clumping of the rough endoplasmic reticulum, the reduction in number of microtubules, and the presence of a fine, floccular and amorphous material. The perikaryonal and dendritic neurofibrillary changes appeared as tangles and/or bundles of 10 nm neurofilaments. In the intraocularly injected rabbits the earliest changes observed in retinal ganglion cells were the severe reduction in microtubule number and the presence of an amorphous material. The neurofibrillary changes seen at later times were comparable with those observed in the cervical cord and brain stem.Although the molecular events occurring between the formation of the intracytoplasmic MYT-tubulin complexes and the accumulation of filaments are not known, the present results indicate that the proliferation of neurofilaments is chronologically preceded by the reduction in number of the microtubules and by the appearance of an amorphous floccular material in the cytoplasm. It is emphasized that despite differences in binding characteristics and physico-chemical properties, maytansine, colchicine and the Vinca alkaloids have, as a common denominator, the property of interfering with the process of utilization of tubulin and in that way they seem to differ from other chemical agents known to induce neurofibrillary degeneration.     

MAP2 is a component of crossbridges between microtubules and neurofilaments in the neuronal cytoskeleton: quick-freeze, deep-etch immunoelectron microscopy and reconstitution studies

Microtubules (MT) and neurofilaments (NF) are linked by frequent crossbridges in situ. In order to answer the question of what makes these crossbridges, we performed the immunogold procedure on rat spinal cord motor neurons using an affinity-purified polyclonal antibody against rat brain MAP2 and gold-labeled anti-rabbit IgG goat IgG. A quick-freeze, deep-etch technique (QF-DE) in conjunction with decoration with anti-MAP2 antibody and ferritin-labeled second antibody was also used. In motor neuron dendrites crossbridges were clearly displayed between MTs and NFs by QF-DE. These crossbridges were revealed in thin sections as fuzzy filamentous structures between MT and NF. Gold particles studded the fuzzy structures associated with MT. Many such structures connected MTs to NFs. Furthermore, antibody complexes containing ferritin were localized on the crossbridges between MTs and NFs by the QF-DE study. In addition, we performed reconstitution experiments. We isolated 70 kDa (L) protein of neurofilaments from calf spinal cords and assembled L to form neurofilaments in vitro. MAP2 bound these neurofilaments according to both SDS-PAGE and QF-DE electron microscopy of the pellets of suspensions containing L proteins and MAP2. When we added tubulin to this suspension and polymerized it in the presence of taxol, neurofilaments were crosslinked with microtubules by MAP2 crossbridges. Hence, from these 2 approaches we concluded that MAP2 is a component of crossbridges between MTs and NFs in the neuronal cytoskeleton in vivo and in vitro.     

Accumulation of neurofilaments and SOD1-immunoreactive products in a patient with familial amyotrophic lateral sclerosis with I113T SOD1 mutation

OBJECTIVE: To report neuropathologic features of argyrophilic inclusions in the anterior horn cells, motor cortex Betz cells, and neurons of the medullary reticular formation, spinal posterior horn, and Clarke column in a Japanese case of familial amyotrophic lateral sclerosis with I113T substitution in exon 4 of the copper-zinc superoxide dismutase (SOD1) gene. METHODS AND RESULTS: These inclusions were stained pale pink on the hematoxylin-eosin stain and dark on the Bielschowsky stain. They were positive for antibodies to phosphorylated neurofilaments, ubiquitin, and SOD1. On electron microscopy, they consisted of abundant intermediate filaments of 10 to 20 nm in diameter with disordered array indicating neurofilaments. CONCLUSION: These findings suggest that the I113T mutation induces accumulation of neurofilaments and SOD1 in the central nervous system neurons.     

Identification of a tektin-like protein associated with neurofilaments in the developing chick nervous system.

A 160-kD polypeptide, which is recognized by an affinity-purified polyclonal antibody to the 55-kD tektin-A polypeptide from sea urchin sperm flagellar microtubules, is associated with neurofilaments in embryonic chick nerve cells. Antibodies to tektin-A and monoclonal antibodies to the neurofilament triplet proteins colocalize to filaments in cultured nerve cells and to filaments in extracts of chick spinal cord, using indirect immunofluorescence microscopy and immunogold electron microscopy. The antigen reacting with anti-tektin-A in chick brain and spinal cord extracts has been identified as a 160-kD polypeptide by SDS-PAGE and has been shown to be distinct from the known neurofilament-triplet proteins by two-dimensional immunoblot analysis. These data suggest that a unique protein with limited sequence homology to tektin-A is a component of the neuronal cytoskeleton and is incorporated into or associated with neurofilaments.     

Neuropil threads of Alzheimer's disease show a marked alteration of the normal cytoskeleton

Abnormal neurites, neuropil threads, are a widespread and characteristic lesion of Alzheimer's disease likely to play a major role in the cognitive impairment of this disease. Contrary to normal neurites, neuropil threads contain straight and paired helical filaments that contain the microtubule-associated protein tau and ubiquitin. It is not known whether these abnormal filaments are added to or replace the normal cytoskeleton. In this study, we examined the fine structure of neuropil threads and carried out a morphometric analysis of the neurofilaments and abnormal filaments contained in the neuropil threads by using an antiserum to tau and colloidal gold immuno-electron microscopy. Almost 70% of the neuropil threads contained straight or paired helical filaments with no neurofilaments. The total number of filaments in each neuropil thread remained essentially unchanged either when straight or paired helical filaments were present alone or when they coexisted either together or with neurofilaments. When the three types of filaments were expressed as a proportion of the total, a linear inverse correlation was found between neurofilaments and straight filaments as well as between straight and paired helical filaments. Approximately 10% of the neuropil threads were found to be myelinated axons. It is concluded that straight filaments are likely to replace neurofilaments, that they in turn might be replaced by paired helical filaments, and that this process occurs in axons as well as dendrites.     

小鼠神经干细胞的生物学特性观察

目的探讨小鼠神经干细胞体外原代培养的生长特性.方法用无血清与单细胞克隆技术对小鼠胚胎脑组织进行分离、培养,用光镜、免疫组织化学进行鉴定,并对其不同生长时期的生物学特性进行透射电镜观察.结果鼠胚胎分离细胞具有连续分化及克隆能力,克隆球和早期的原始细胞神经上皮干细胞蛋白(nestin)抗原呈阳性.成熟分化后胶质纤维酸性蛋白(GFAP)及神经丝-200(NF-200)抗原呈阳性.不同期电镜结果:培养2周的神经干细胞较原始,核大,细胞质少,细胞器不发达.将其继续培养到4周,部分细胞内可见到发达的细胞器,并在分化细胞中观察到神经微管、微丝、胶样丝和细胞间连接样结构.结论胚胎脑组织在体外培养并克隆成神经球,具有很强的增殖能力,是多分化潜能干细胞.     

Fulvic acid inhibits aggregation and promotes disassembly of tau fibrils associated with Alzheimer's disease

Alzheimer's disease is a neurodegenerative disorder involving extracellular plaques (amyloid-β) and intracellular tangles of tau protein. Recently, tangle formation has been identified as a major event involved in the neurodegenerative process, due to the conversion of either soluble peptides or oligomers into insoluble filaments. At present, the current therapeutic strategies are aimed at natural phytocomplexes and polyphenolics compounds able to either inhibit the formation of tau filaments or disaggregate them. However, only a few polyphenolic molecules have emerged to prevent tau aggregation, and natural drugs targeting tau have not been approved yet. Fulvic acid, a humic substance, has several nutraceutical properties with potential activity to protect cognitive impairment. In this work we provide evidence to show that the aggregation process of tau protein, forming paired helical filaments (PHFs) in vitro, is inhibited by fulvic acid affecting the length of fibrils and their morphology. In addition, we investigated whether fulvic acid is capable of disassembling preformed PHFs. We show that the fulvic acid is an active compound against preformed fibrils affecting the whole structure by diminishing length of PHFs and probably acting at the hydrophobic level, as we observed by atomic force techniques. Thus, fulvic acid is likely to provide new insights in the development of potential treatments for Alzheimer's disease using natural products.     

Identification of serine 473 as a major phosphorylation site in the neurofilament polypeptide NF-L

Neurofilaments are composed of 3 polypeptides designated NF-H, NF-M, and NF-L, all of which are subject to posttranslational phosphorylation. It has been suggested that phosphorylation of the NF-L polypeptide can influence the assembly of NF-L into filaments, but the sites at which NF-L is phosphorylated are unknown. To locate these phosphorylation sites, we have identified phosphopeptides of NF-L by labeling them with 32P both in vitro and in cultured neurons and also by observing their change in chromatographic behavior after they have been treated with phosphatase. We report here that serine 473, in the carboxy-terminal tail domain of NF-L, is a major substrate in vitro for protein kinases endogenous to a crude cytoskeleton-containing fraction. Moreover, serine 473 is a major phosphorylation site in vivo; in neurofilaments isolated from rat spinal cord, approximately 73% of serine 473 was phosphorylated, and accounted for at least one-third of the total phosphate associated with NF-L. The identification of this phosphorylation site in NF-L provides a criterion for identifying the protein kinase that phosphorylates NF-L and raises the question of its function.     

A case of giant axonal neuropathy showing focal aggregation and hypophosphorylation of intermediate filaments

We report here the clinicopathological features of a typical case of giant axonal neuropathy (GAN). Scanning electron microscopy of the hair of this case revealed an extraordinarily irregular cuticle. Focal accumulation of intermediate filaments in axons, Schwann cells, muscle fibers and skin fibroblasts were also found under an electron microscopy. When examined immunocytochemically, muscle fibers exhibited local disruption of the filamentous network in the subsarcolemmal space and in the central cytoplasm accompanied by focal accumulation of desmin. The intracellular network of vimentin was also disrupted, exhibiting global accumulation in some of the cultured skin fibroblasts. Decreased interneurofilament spacing was found in enlarged axons, suggesting the presence of hypophosphorylation of neurofilaments in this patient. These findings suggest general disorganization, abnormal distribution and possible defective phosphorylation of intermediate filaments in GAN.     

Assembled tau filaments differ from native paired helical filaments as determined by scanning transmission electron microscopy (STEM)

Paired helical filaments (PHF) are abnormal, approximately 20–25-nm wide periodically twisted filaments, which accumulate in Alzheimer's disease (AD) brain and other neurodegenerative disorders, including corticobasal degeneration (CBD). PHF are primarily composed of highly phosphorylated tau protein. However, both phosphorylated and non-phosphorylated forms of tau are able to assemble in vitro into filaments similar in the ultrastructural appearance to PHF. In the present study, filaments were assembled in vitro from unmodified recombinant human tau and the physical mass per unit length of filaments and the mass density were determined using scanning transmission electron microscopy (STEM). Two general types of filaments were observed. One type was composed of 11.4 nm-wide, 10–75 nm long, frequently twisted and PHF-like filaments, with a mass per unit length (44 kDa/nm) approximately one third of that observed in isolated AD filaments. The other were straight filaments, approximately 6.8-nm wide and 0.2–2 μm long, which often formed parallel clusters of two or more filaments. Triple clusters were 19.2-nm wide and had a mass per unit length (70 kDa/nm) approximately two thirds of that seen in isolated AD filaments. Despite different morphology, both twisted and straight filaments had mass densities between 0.48–0.55 kDa/nm³. These values are significantly higher than those reported for PHF found either in AD (0.40 kDa/nm³) or CBD (0.33 kDa/nm³). These results suggest that the packing of tau differs in vivo from that observed in vitro and that specific tau isoform content, elongation of tau molecules by phosphorylation or other factors may be required to reproduce pathological assembly. Therefore mass density determinations appear to be an important criterion in comparing various filaments.     

Astrocyte cell lineage. II. Mouse fibrous astrocytes and reactive astrocytes in cultures have vimentin- and GFP-containing intermediate filaments

When cells from mouse neopallium are grown in colony cultures for 10-12 days, small cells with many processes, resembling normal fibrous astrocytes, form on top of the astrocyte precursor cells independently of the presence of dBcAMP in the culture medium. These cells are distinctly different from the much larger, previously described reactive astrocytes which also form in colony cultures and whose maturation is greatly enhanced by the presence of dBcAMP in the culture medium. Immunofluorescence studies showed that both vimentin-containing and glial filament protein (GFP)-containing intermediate filaments (IF) are present in the small normal fibrous astrocytes as well as in the larger reactive astrocytes. The vimentin-containing IF are assembled first in astrocyte precursor cells, whereas GFP-containing IF are assembled later toward the final stages of astrocyte differentiation both in vivo and in vitro. Thus in respect to the expression of the two types of IF, astrocyte differentiation in vitro closely resembles that in vivo. Parallel studies by electron microscopy showed that the vimentin-positive but GFP-negative astrocyte precursor cells contain single IF or small groups of IF, whereas in the more differentiated normal fibrous astrocytes and reactive astrocytes which are also GFP-positive, additional IF arranged in large bundles are present.     

Axonal neurofilamentous accumulations: a comparison between human and canine giant axonal neuropathy and 2,5-HD neuropathy

The neuropathy produced by the hexacarbon 2,5-hexanedione (2,5-HD) resembles human and canine inherited giant axonal neuropathy (GAN) in the presence of giant axonal swellings that contain accumulations of neurofilaments. The accumulations are both paranodal and internodal in GAN and 2,5-HD induced neuropathy. Detailed morphometry on the neurofilaments reveals that the changes in human and canine GAN are closely similar and differ from those of 2,5-HD neuropathy, suggesting that the mechanisms underlying the formation of the axonal neurofilamentous accumulations differ between the two conditions. In both human and canine GAN, the neurofilaments are more closely spaced and are of greater diameter than in 2,5-HD neuropathy. The changes in the NF in GAN may be the consequence of flattening of the side-arms of the neurofilaments against the axis of the filaments