Journal of Nrunrchumrsrr) V a l 33. pp 5 In 13 Pergamon Press Ltd 1979. Printed in Great Hritain

SHORT REVIEW

NEUROFILAMENTS MICHAEL L. SHELANSKI and RONALDK. H. LIEM Department of Pharmacology, New York University School of Medicine, New York, NY 10016, U.S.A

THE PRESENCE of linear fibrillar structures within case with tubulin and actin, or are they highly indinerve cells was first appreciated in the early part of vidualized? In this review, two decades after the first the 19th century. The introduction of silver staining biochemical studies on neurofilaments, there is adetechniques made these structures readily apparent quate evidence to provide answers to these questions and many early theories of neuronal connectivity and and, in spite of a sizable number of loose ends, pose conductivity involved them. From a medical point of a set of new and more functionally oriented questions. view, the fibrils were seen to undergo an apparent thickening and increase in number in a variety of conCOMPOSITION OF NEUROFILAMENTS ditions including the human presenile dementia referred to as Alzheimer’s disease (TERRY, 1979). Attempts to isolate and identify the chemical subWhen ultrastructural methods became available it structures of neurofilaments began in the laboratory was discovered that the silver staining in normal of Professor F. 0. SCHMITTat M.I.T. in the 1950s neurons and in the toxic neuropathies was due to using the axoplasm of the squid giant axon (DAVISON the presence of 8-9 nm dia. neurofilaments. The stain- &TAYLOR, 1960). More recent studies have used both ing in Alzheimer’s disease was related to the presence invertebrate and vertebrate materials. of twisted fibrillar structures which have a maximum diameter of 19 nm. In this article we will concentrate Inuertebrutes on the normally-occurring neurofilament and return The giant axon of the marine worm Myxicolu is only briefly to the more specialized structures in Alz- packed with a tight helically wound bundle of neuroheimer’s disease and other human dementias. filaments which can be removed intact with a pair The use of histological and ultrastructural methods of forceps (GILBERT,1975). This unique ‘package’ is also indicated that astroglial cells in the vertebrate ideally suited to both structural and biochemical brain contain filaments which require silver tech- studies. GILBERT and his colleagues have demonniques different from those of neurons for observa- strated that these filaments have two major proteins tion. These glial filaments are similar in appearance with molecular weights of 152,000 and 160,000. The to, but thinner than, the neurofilaments (WUERKER, axoplasm contains a calcium activated protease which 1970). Other filaments of the ‘intermediate’ class will cleave the filaments to one or more fragments (7-11 nm) have also been observed in a wide variety with molecular weights in the 50,000 range (GILBERT of non-neural cells and tissues (GASKIN & SHELANSKI,et a[., 1975). These filaments can also be purified as 1976). ring-shaped aggregates by coprecipitation with A pattern of replacement in the numbers of micro- cytochrome-c (GILBERT,1977). tubules with neurofilaments has been observed with The Myxicola filaments have a high a-helix content & VAUGHN,1967). and the subunits sediment is a hypersharp peak with age in the rat optic nerve (PETERS In addition to this observation there was also a an S,,,, of 6.5 suggesting considerable subunit asymsuggestion of a reciprocal relationship between the metry in a protein of mol. wt. 15Ck160,OOO (BELL& number of neurofilaments and microtubules in en- GILBERT,1977). cephalopathy induced by mitotic spindle inhibitors The Myxicola axoplasm is lacking in microtubules and the characteristic cross linking of filaments and et at., 1968). (WISNIEWSKI These observations led to the framing of three tubules seen in other invertebrates and mammalian major questions: (a) What is the composition of the axons. neurofilaments? (b) Does the reciprocal relationship Studies on squid axoplasm which possess the more in number of filaments and tubules indicate that they usual structure of filaments and tubules have led to are polymorphic assembly forms of the same protein the purification of filaments in which two major comor that they are under reciprocal assembly control? ponents are detected: a major protein with a molecu(c) Are the proteins of intermediate filaments similar lar weight of 200,000 and a minor protein with a molin different cell types and across species, as is the ecular weight of 60,000. In spite of the differences 5

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MICHAEL L. SHELANSKI and RONALD K. H. LIEM

between molecular weights in squid and Myxicola, they have been reported to be immunologically crossreactive (LASEK,1977). A much larger band with an apparent molecular weight in excess of 4 x lo5 is also seen at the top of SDS gels and may be a neurofilament protein (LASEKet al., in press). All squid neurofilament components except the mol. wt. 60,000 band are phosphorylated (PANTet al., 1978) and both Myxicola subunits are phosphorylated (LASEK,personal communication).

Mammaliun neurojilaments The direct isolation of neurofilaments from brain in which larger numbers of astroglial filaments of similar, but not identical morphology are present poses a problem. In an attempt to circumvent this problem a procedure was developed in which the myelin around the axon was used as a flotation device to float the axon and its contents away from capillaries, cells and other components. Electron microscopic observation confirmed that well preserved myelinated axons are obtained in this manner. In the initial studies, myelin was removed by homogenization and brief exposure to low ionic strength media yielding unmyelinated axons and filaments with a rather limited number of proteins (Fig. 1). Bands with molecular weights of 160,000, 68,000 and 51,000 are especially prominent in these preparations. The purified filaments showed a combination of ‘loose bundles’ in which the individual filaments are clearly defined and ‘tight bundles’ in which the fibrillar elements are in close apposition (SHELANSKI et al., 1971). The former resembled the axoplasmic filaments while the latter appeared similar to the filaments of fibrous astrocytes. Subsequent modification of the purification in which overnight exposure at low ionic strength was used to remove myelin led to diminution of the higher molecular weight components to the point that the mol. wt. 51,000 material comprised over 90% of the protein in the preparation (YENet al., 1976). Biochemical studies showed these ‘brain filament’ preparations to differ completely from tubulin (LIEMet al., 1977) and to be both immunologically and biochemically similar to the glial fibrillary acidic protein, the putative subunit of astroglial filaments (YEN et al., 1976). Antisera to this P51 band were found to stain astroglia and Bergmann glial fibers very strongly, and peripheral and central neurons only et al., 1977). A protein which weakly (SHELANSKI cross-reacts immunologically with the P51 band, and has a similar molecular weight, increases in parallel with the development of gliosis in the mutant staggerer mouse (LEEer al., 1977). More recently, purified intermediate filaments of definite glial origin have been isolated and were found to be biochemically and immunologically similar to the P51 band obtained by the axonal flotation method (GOLDMANet al., 1978). These results strongly suggest that the major protein component of the brain filament preparations (P51)is of astroglial rather than of neuronal origin.

Thus it would appear that in spite of enrichment in myelinated axons in early stages of the preparation, the subsequent steps led to amplification of glial filaments. A possible reason for this is suggested by the (1971, 1978) that the flaobservation of SCHLAEPFER ments of peripheral axons were disrupted by exposure to low ionic strength medium--conditions used in the removal of myelin in the CNS preparation and conditions resulting in the amplification of P51 at the expense of the higher molecular weight components in the axon preparation. Return to 1 h low ionic strength extraction or use of detergent to remove the myelin gives a preparation with considerable amounts of the 210,000, 150,000 and 68,000 dalton ‘triplet’ as well as P51. The molecular weights of these ‘triplet’ polypeptides correspond to the molecular weights of the ‘triplet’ of radioactively labeled peptides transported along with tubulin in the slowest portion of the slow component of axoplasmic flow and postulated to be components of the neurofilaments (HOFFMAN & LASEK,1975). Recent work has shown decreases in these components in Wallerian degeneration and calcium-induced axonal degeneration (SCHLAEPFER & MICKO, 1978, 1979). Tubulin does not seem to be significantly altered in quantity in any of these conditions. To avoid the problem of astroglial Contamination it was advantageous to use peripheral nervous tissue. Extraction of peripheral nerve in low ionic strength buffer led to a preparation of some intact filaments which quickly broke down and the appearance in solution of a protein with a molecular weight of 69,000 (SCHLAEPFER, 1977). An antiserum raised against this material and absorbed in an albumen & column gave clear neuronal staining (SCHLAEPFER LYNCH,1977). Therefore it appeared that in spite of the fact that the major protein migrating at mol. wt. 69,000 was probably derived from serum proteins, as was suggested by biochemical analysis and extractions of rat tail under the same conditions (SHELANSKI et al., 1977), there was also a protein with this molecular weight derived from neurons and corresponding to one component of the axoplasmic transport ‘triplet’. Direct purification of intact filaments from rabbit intradural spinal nerve roots produced a preparation of high purity with molecular weights of 200,000, 150,000 and 68,000 (LIEMet ul., 1978). No P51 was present in these preparations reflecting the fact that the few astroglial filaments which enter the root terminate in the most proximal portion. Nor was there any significant amount of collagen present. There was a band with a molecular weight of 60,000 which is not present in CNS and which could well be derived from Schwann cell intermediate filaments. A quite different purification method which does not use axonal flotation but rather direct isolation from homogenates also leads to enriched filament preparations with enhancement of the ‘triplet’ proteins. The proteins from the PNS consist of the ‘triplet’ plus a mol. wt. 64,000 protein while the CNS

FIG.1. SDS gel electrophoresis pattern of filaments obtained by the method of SHELANSKI rt trl. (1971). Intact unmyelinated axons on the left; filaments centrifuged through 2 M-sucrose on the right.

FIG.2. Peptide maps of the filament polypeptides obtained by limited proteolysis by the S. rrureus protease in the presence of SDS (CLEVELAND et ul., 1977). From left: P51; P200; P160; P68.

8

Neurofilaments

material is reported to consist of the ‘triplet’ plus tubulin (SCHLAEPFER & FREEMAN, 1978). However, since this work was done on rat where the ‘brain filament’ or ‘P51’ material migrates as a doublet close to tubulin (GOLDMAN et a/., 1977), this identification is not conclusive. Indeed, all the intermediate filament proteins of brain show variability in their molecular weights from species to species (LIEMet al., 1978). Antisera raised against P68 and P160 from bovine central nervous system show strong staining of peripheral nerve axons and of neurons. Aluminuminduced neurofibrillary bundles in spinal neurons are also stained intensely by these antisera. Isolated neurons with aluminum induced neurofibrillary bundles showed an enrichment in polypeptides of mol. wt. 210,000, 160,000 and 68,000 when compared to control neurons (LIEMet al., 1979; SELKOEet al., 1979). No enrichment in P51 was seen. In spite of the weight of this evidence, there exists some data suggesting that the mol. wt. 51,000 protein may, at least in some part, be derived from the neurofilament either directly or as a breakdown product of one of the triplet proteins. BIGNAMI has shown that central nervous system and peripheral nerve extracts enriched in a mol. wt. 54,000 component show strong staining of peripheral nerve and aluminum induced neurofibrillary bundles (BIGNAMIet al., 1977). SCHACHNER has reported that mouse antisera raised against P51 or total filaments isolated from bovine brain by the axonal flotation method (SHELANSKI et ul., 1971) also give neuronal staining while anti-GFA does not (SCHACHNER et a/., 1978). Our own data shows immunochemical cross reaction between P51 and the triplet proteins (LIEMet al., 1978). These apparently conflicting results could be explained if P51 contained a component derived from breakdown of the neurofilament. One possible mechanism would be through the action of a calcium-activated protease such as has been reported in squid and Myxicola axoplasm. (SCHLAEPFER & MICKO,1978). RELATIONSHIP BETWEEN THE TRIPLET COMPONENTS

Microtubules and F-actin are composed of only one major subunit species in each case and therefore give no guidance in how to approach the neurofilament in which three distinct polypeptides are involved. This dilemma could be resolved if each of the triplet polypeptides was derived by a specific cellular processing mechanism from a larger parent molecule-perhaps the mol. wt. 210,000 species and perhaps an even larger ‘profilament protein’. Some support for this possibility is obtained from the fact that antibodies raised against any one of the triplet proteins recognize, to some extent at least, the other two components (LIEMet al., 1978). This similarity, however, is not strongly supported by peptide mapping studies done in our laboratory using the limited proteolytic hydrolysis method (CLEVELAND et al.,

9

1977).These studies show some limited similarity, especially when any two of the components are compared (Fig. 2) but there is no clear indication of relatedness and no indication that these molecules are cleaved from a parent unless there is little or no overlap between cleavage products. Such a parent molecule would, therefore, have a molecular weight in the range of the sum of the triplet molecular weights, or approx 410,00~450,000. This precursor could be even larger if chains were duplicated or not all of the molecule went into the filament. N o evidence of such a protein has been seen in mammalian neurons, but it has not been systematically sought. It is clear that this question of the origin and relationship between the triplet polypeptides is a major one and must be systematically investigated. These data will be of importance in understanding filament reassembly and the subunit packing in the filament. These reassembly studies will require the purification-in a soluble state-of each of the triplet proteins and their combination in various manners to see if each of the subunits can make a filament or whether mixtures are needed. Structural studies using circular dichroism and X-ray diffraction might also indicate whether each of these subunits has a similar structural ‘core’ and a differing ‘variable’ region. Some indication of secondary structure similarities between biochemically dissimilar intermediate filament proteins et has already appeared in the literature (STEINERT a/., 1978). NEUROFILAMENTS AND MICROTUBULES

While there are examples in which neurofilaments exist in the presence of few microtubules and where microtubules are present in considerable exces., compared to neurofilaments there are also abundant morphological observations which show small ‘arms’ linking neurofilaments to microtubules and reporting rather fixed relationships between the number of neurofilaments and microtubules in the mature axon (FRIEDE,1970). Similarly, the physiological data on axonal transport of radioactively labeled proteins shows that tubulin and the filament triplet are transported together in relatively fixed ratios and presumably as intact organelles in the slowest component of axonal transport. There is considerable evidence that the ‘arms’ on microtubules are composed of the high molecular weight microtubule-associated proteins though other molecules have not been excluded. Recently, the co-purification of intermediate filaments and ‘triplet’ proteins in a standard reassembly purification of brain tubulin has been reported (BERKOWITZ et al., 1977). Numerous other groups using similar purification methods for tubulin have failed to note ‘triplet’ proteins even though accessory proteins were actively sought. The reasons for these differences have been explored systematically in our laboratory (LETERRIER, LIEM& SHELANSKI, unpublished observations) with the conclusion that vigorous homogeniza-

10

MIVHAEI. L. SHELANSKI and RONALDK. H. LIEM

tion in a blade-type homogenizer followed by a reas- et ul., 1968), n-hexane and methyl butyl ketone et ul., 1975). It also occurs in axotomy (BARsembly procedure using relatively low (less than (SPENCER 60,000 g-h) centrifugal forces favors co-purification of RON et al., 1975) and in sporadic motorneuron disease. triplet proteins with tubulin, while gentle homogeni- This group can be further divided into the mitotic zation in a Potter homogenizer followed by centrifu- spindle inhibitors which induce such changes in a gation at greater than 100,000g gives preparations wide variety of cell types including neurons and the free of triplet, as previously described (SHELANSKI et ‘specific’ neurotoxins which induce neurofibrillary ul., 1973). The filaments in the former preparation change only in neurons such as n-hexane, methyl et al., 1978) and aluminum (SHEseem to remain in an assembled state under both butyl ketone (SELKOE unpublished observations). Unfortubulin assembly and disassembly conditions and can LANSKI & ROBBINS, be removed from the preparation by centrifugation tunately, most other ‘neurotoxins’ have not been at 250,OOOg for 1 h at any point with no adverse effect assayed on non-neuronal cells. Since the mitotic on the ability of tubulin to assemble. However, at spindle inhibitors have a direct effect on microtubules low forces there are indications that tubulin and the and their effects on intermediate filaments could triplet protein specifically and reversibly associate simply be due to a ‘collapse’ of the cytoskeleton we under tubulin assembly conditions. When brain shall eliminate them from consideration here. Howhomogenates are prepared so as to contain both fila- ever neither aluminum nor methyl butyl ketone have ments and tubulin and tubulin is then assembled at direct effects on microtubules. In aluminum-induced 37°C in the presence of GTP, both filaments and neurofibrillary proliferation, as stated above, there is tubules may be pelleted by centrifugation at 50,000 g. an increase in total neuronal protein and this increase When the tubules are depolymerized at 4°C and the is limited almost exclusively to the neurofilament trippreparation is again centrifuged at the same force the let. Similarly, axoplasmic transport studies done in triplet proteins as well as tubulin are found in the IDPN intoxication show an accumulation of radiosupernates. This is in spite of the fact that the vis- actively labeled proteins corresponding to the triplet et al.; 1978). Therefore, it appears that the cosity of the solutions during the first centrifugation is (GRIFFEN higher due to the presence of 4M-glycerol which is filamentous accumulation in each of these cases is absent from the second centrifugation. This reversible due to a build-up of normal neurofilaments. The second ultrastructural substratum for neurofico-sedimentation occurs through many cycles of assembly and disassembly and suggests that the fila- brillary proliferation is the ‘twisted tubule’ or ‘paired ment fragments are reversibly attaching to the assem- helicial filament’ (TERRY,1963) of human senile and bled microtubules which then ‘pull’ the filaments into presenile dementia and certain other human neuroet a/., 1971). Preliminary the pellet. Disassembled filaments obtained in this logical diseases (WISNIEWSKI way seem to be tightly bound to a tubulin molecule data based on comparisons of gel electrophoretic patand show interactions with both tau and high mol- terns of neurons isolated from patients with Alzecular weight microtubule-associated proteins. The heimer’s disease and from age-matched controls sugnature of these interactions and their specificity are gested that a protein with a molecular weight of a major focus of investigation at this time. It is inter- 50,000 was increased in the diseased neurons (IQBAL esting that triplet preparations obtained by co-purifi- et al., 1974). This band was reported to be immunolocation with tubulin are completely free of the P51 gically and biochemically similar to the brain filament protein, while filaments obtained by axonal flotation protein and to tubulin (IQBALet al., 1976). However, are heavily contaminated by P51 but almost free of the more recent identification of the brain filament tubulin. The tubulin-filament preparation also shows protein as a glial filament protein has raised a quesa magnesium-activated ATPase activity which is tion of whether this increase was due to the extensive absent from pure tubulin prepared by phosphocellu- gliosis in the diseased brain rather than to an intrinsic lose chromatography (SHELANSKI, unpublished obser- neuronal change. Other studies (YEN,personal communication) have vation). The nature and specificity of this activity are unexplored. failed to show staining of these tangles in tissue section or in separated cells having neurofibrillary tangles with specific antibodies against bovine tubuNEUROFILAMENTS, NEUROFIBRILLARY lin, bovine glial filament protein or the mol. wt. PROLIFERATIONS AND ALZHEIMER’S 200,000 or 160,000 component of the bovine neuroDISEASE filament triplet. The wide variety of neuronal diseases which are characterized as ‘neurofibrillary degenerations’ on the NON-NEURONAL INTERMEDIATE basis of silver staining at the light microscopic level FILAMENTS can be divided into two major classes with the electron microscope. The first of these have great increases Within the nervous system intermediate filaments in the apparent number of neurofilaments. This pic- are found in astrocytes where they are composed of ture is caused by a multitude of agents including col- the glial fibrillary acidic protein (GFA). This protein chicine, vinca alkaloids, podophyllotoxin (WISNIEWSKIwhich exists in both soluble and particulate forms

Neurofilaments

11

has a molecular weight of 51,000 in bovine brain (ENG composed of two proteins with molecular weights of e t al., 1971) and this varies slightly in different species. 150,000 and 160,000. The relationship between the inImmunological evidence suggests that in the mature vertebrate and vertebrate filaments is still not clear. brain this protein is limited to astroglia (BIGNAMIet The similarity of the molecular weights of at least ul., 1973). The factors controlling the assembly and one polypeptide in each of the squid, worm and mamdisassembly of the filaments have not been identified. malian neurofilament components as well as some Reactive Schwann cells in the peripheral nervous sys- degree of immunological cross-reactivity between tem show accumulations of intermediate filaments them suggests that a limited amount of evolutionary which have not yet been biochemically identified. conservation might exist. However, even within differHowever, the loss of neurofilament peptides under ent mammalian species there are differences in the conditions where Schwann cell filaments proliferate molecular weights of the three polypeptide subunits would suggest that they differ from neurofilaments, suggesting that they are not as highly conserved as while the lack of peripheral nerve staining with anti- microtubules and microfilaments. Certainly the bulk GFA suggests differences from astroglial filaments. of the available evidence suggests that intermediate Thus it would appear that the nervous system con- filaments are highly tissue or cell-specific. The physiotains at least three biochemically distinct types of logical implications of this lack of conservation are still obscure. These data also argue against the presintermediate filaments. ence of neurofilament proteins as a component of the Intermediate filaments have also been isolated in varying degrees of purity from a wide variety of cells. post-synaptic density (COHENe t al., 1977; Y E N e t d., 1977). Similarly, it is clear that the filaments-neuroSmooth muscle intermediate filaments have been isofilaments and microtubules-are not alternate assemlated (COOKE,1976; SMALL& SOBIESZEK, 1977) and shown to have a molecular weight of 55,000. The pro- bly forms of each other and the depolymerization of tein, later named Desmin, was studied by LAZARIDES both by calcium suggests that assembly control is not strictly reciprocal. & HUBBARD (1976) who determined that it localized The data presented here on microtubule-neurofilato Z-bands in striated muscle and to filament systems in non-muscle cells. Intermediate filaments have also ment interactions raise the possibility of direct interactions between these organelles, perhaps mediated et ul., been isolated from fibroblasts (SCHOLLMEYER 1976). Highly purified intermediate filaments capable by their accessory proteins. These interactions and of assembly at high ionic strength and disassembly other possible interactions with actin, myosin and the at low ionic strength with a molecular weight of membrane must be systematically explored to under58,000 have been isolated from BHK cells (STARGER stand the role of these cytoskeletal elements in axonal transport and neuronal function. & GOLDMAN,1977). Tonofilaments from epidermis also fall in this mol. wt. 50,000-60,000 class (STEINERT, 1975; BRYSKe t al., 1977) and show no biochemical REFERENCES or immunological relationship to either neurofilament BARRONK. D., DENTINGER M. P., NELSON L. R. & MINCY or glial filament proteins (YEN, LEM, JENQ& SHEJ . E. (1975) Ultrastructure of axonal reaction in red nucLANSKI, in preparation). lues of cat. J. Neuropath. exp. Neurol. 34, 222-248. Thus, except for the neurofilament and the possible BELLC. & GILBERT D. S. (1977) Neurofilament structure. occurrence of polypeptides with molecule weights Proc. Int. Soc. Neurochem. 6, 102b. similar to the ‘triplet’ in the cytoskeleton of human BENNETTG. S., FELLIN] S. A,, CROOPJ. M., OTTOJ. J., fibroblasts (LEHTOe t al., 1978) the ‘intermediate filaBRYAN J. & HOLTZER H. (1978) Differences among 100 A filaments from different cell types. Proc. natn. Acud. ment’ subunits tend to cluster in a narrow molecular Sci., U.S.A. 75, 43664368. weight range. They show, however, significant bioS. A., KATAGIRI J., BINDERH. K. & WILLIAMS chemical and immunological differences. The few BERKOWITZ R. C. (1977) Separation and characterization of micropapers reporting cross-reactivity of anti-P51 with entubule proteins from brain. Biochemistry 16, 561b-5617. 1977) and neuroblastoma dothelial cells (BLOSEet d., BIGNAMIA,, ENGL. F., DAHLD. & UYEDAC. T. (1973) (JORGENSEN et a/., 1976) must be interpreted with cauLocalization of the glial fibrillary acidic protein in astrotion in light of recent findings on autoantibodies cytes by immunofluorescence. Brain Res. 43, 429-435. against intermediate filaments (BENNETT et al., 1978). BIGNAMIA,, DAHLD. & RUEGERD. C. (1977) Isolation of neurofilament and glial filament proteins from water and urea extracts of nerve tissue, in Mechanisms, ReguluCONCLUSION From the data presented above, it is possible to return to the questions asked at the start of this review. It is now agreed that the mammalian neurofilaments are composed of a triplet of proteins with molecular weights of approx 200,000, 160,000 and 68,000. The squid neurofilament has two components with molecular weights of 200,000 and 60,000, while the worm Myxicola has neurofilaments which are also

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MICHAELL. SHELANSKI and RONALDK. H. LIEM

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