Similarity of neurofilament proteins from different parts of the rabbit nervous system

Brain Research, 216 (1981) 387-398 © Elsevier/North-Holland Biomedical Press

387

SIMILARITY OF N E U R O F I L A M E N T PROTEINS F R O M D I F F E R E N T PARTS OF T H E RABBIT NERVOUS SYSTEM

HENRYK CZOSNEK, DAVID WISNIEWSKI

SOIFER*,

KATHRYN MACK

and HENRYK M.

Department of Pathological Neurobiology, Institute for Basic Research in Mental Retardation, 1050 Forest Hill Rd., Staten Island, N.Y. 10314 (U.S.A.)

(Accepted November 20th, 1980) Key words: neurofilaments -- peripheral nerve - - central nerve - - two-dimensional electrophoresis -

-

intermediate filaments - - neurofilament genes

SUMMARY In the nervous system, the various populations of neurons perform a large spectrum of functions. Although neurofilaments are a major constituent of the different neurons, the neurofilament protein composition and the expression of the genes specifying these proteins may not be the same throughout the entire nervous system. To investigate these two aspects of the biology of neurofilaments, we have prepared neurofilament-rich fractions from different regions of the nervous system of strains of rabbits known to present a genetically determined polymorphism involving one of the neurofilament polypeptides (P200). Filaments were isolated from brain, spinal cord, sciatic, optic and trigeminal nerves, and lumbar ventral and dorsal roots by a procedure not involving axonal flotation and yielding material suitable for comparative analysis within a single animal. The filaments were compared for their variability as a function of the region from which they were prepared. For any given animal, the neurofilament peptides migrate to identical positions on SDS-gel electropherograms. Whatever allele of P200 is expressed in filaments from one region, the same allele is also expressed in all of the other filament preparations from that animal. On two-dimensional analysis isomorphs of the P68 neurofilament protein are not present in the same amounts in different regions of the nervous system. These results indicate that, although it seems that the gene for the P200 neurofilament protein is expressed uniformly throughout the nervous system, there may be some topographic specificity in the distribution of the other constituent proteins of neurofilaments.

* To whom correspondence should be addressed.

388 INTRODUCTION Neurofilaments are part of the structural network of most neuronal cells and comprise a major component of the axoplasm of central and peripheral neural tissues 36. In sectioned material, they appear to be 10 nm thick. Preparations of 10 nm filaments from brainl,8,8, 23,37,43, spinal cord6, 42, spinal nerve roots 23, cranial nerves 10 and sciatic nervesa,7,30, a2 all include 3 polypeptides ('triplet') of molecular weights in the range of 200,000 (P200), 150,000 (P150) and 68,000 (P68) daltons. These polypeptides appear to be the constitutive proteins of neurofilaments. The neurofilament polypeptides are synthesized in the neuronal cell bodies as individual proteins by free polyribosomes 5 and are transported down the axon in the slow component of axonal flow20,24,31. The neurofilament polypeptides appear to be less conserved than other flamentous proteins of the nervous system, such as tubulin or actin. Their exact molecular weights vary from one mammalian species to another3,10. Major aspects of the biology of neurofilaments remain unclear. Although the neurofilaments are a conspicuous element of neurons throughout the entire nervous system, sharing similar ultrastructural features, we do not know whether their chemical composition is uniform in all types of neurons. We also do not know whether the genes which specify the neurofilament prote:ns are expressed invariably in all neurons, regardless of their localization and function. The questions of the generality of neurofilament gene expression and of neurofilament protein composition in all the different types of neurons throughout the nervous system are of particular interest when one considers the very narrow regional susceptibility of fibrous proteins to neuropathies 47. To examine some of the questions raised above - - composition of the neurofilament in different regions of the nervous system, expression of neurofilament genes - - we have isolated filaments from different regions of the rabbit nervous system. To compare the chemical composition of isolated filaments from various parts of the nervous system within the same individual, we have developed a method which yields filaments from samples of nervous tissue weighing as little as 20 mg. To study the expression of the neurofilament genes, we have taken advantage of a particular feature of one of the 'triplet' proteins. It has been shown that the P200 component of neurofilaments may exist in a genetically altered form in the central nervous system of certain rabbits 45. The two forms of the protein, termed H I and H 2, may be discriminated by electrophoresis on SDS-gels. We used the dimorphism of P200 as a probe to investigate whether neurofilament genes are expressed similarly or differently in various groups of neurons. Using rabbits which were homozygous for either H a o r H 2, as well as animals carrying both forms of P200, we have isolated l0 nm filaments from optic nerves, trigeminal nerves, lumbar ventral roots, lumbar dorsal roots, sciatic nerves, spinal cord and brain. The regions were sampled to provide filaments from peripheral neurons (dorsal roots), from central neurons (ventral roots, optic nerves), and from mixtures of both. The results presented in this communication demonstrate that the neuro-

389 filaments are formed of similar proteins regardless of their source. The type of P200 is constant for all regions sampled in a given animal. Differences may appear in the distribution of isomorphs of P68 in different parts of the nervous system. MATERIALSAND METHODS

Animals Three- to four-month-old male rabbits (obtained from The Jackson Laboratory) from the partially inbred strains IIIVO/J (98 ~ inbred) and X/J (91 ~ inbred) 14 were used as the source of neurofilaments with the P200 component present in the form ofH 1 and H 2, respectively4L New Zealand White rabbits (Penn-Dutch Farms, Denver, Pa.) were used as a source of H I alone or for hybrid rabbits (H 1 + Ha).

Preparation of filaments Immediately after the rabbits were killed, brain, spinal cord (excluding the lumbar region), optic nerves, trigeminal nerves (mostly Gasserian ganglia) and sciatic nerves were dissected, immersed in 0.25 M sucrose/TKM and frozen on dry-ice (TKM is 50 mM Tris, pH 7.6, 25 mM KC1, I0 mM MgC12). The spinal nerve roots from the lumbar region were exposed by cutting the dura. The dorsal and ventral roots weIe separated under a dissecting microscope. Samples of each of the roots were taken from the region between the dorsal root ganglia and the spinal cord, immersed in 0.25 M sucrose/TKM and frozen on dry-ice. The samples remained frozen at --80 °C for 1-2 weeks before the isolation of filaments was performed. The tissues were then thawed, cleaned of meninges and blood vessels, weighed and homogenized with TKM containing 5 mM EGTA and 3 mM DTT (about 10 ml/g tissue wet weight) using a conical ground glass homogenizer (Kontes Duall tissue grinder). The homogenate was made 1 ~o~ in Triton X-100 using a 20 % solution. After stirring for 30 min at 2 °C, the homogenates were layered on a 2 ml cushion of 1 M sucrose/TKM and were centrifuged for 2 h at 35,000 rpm in a Beckman 75 Ti rotor (78,000 × gay). The pellet of filamentous material was suspended in water, frozen in dry-ice and stoled at 1 8 0 °C.

Eleetrophoresis of proteins on polyaerylamide gels Filament proteins were analyzed on one- and two-dimensional polyacrylamide gels. For analysis in one dimension, samples were made 2 % (w/v) SDS, 1% (w/v)/3mercaptoethanol, 10 % (v/v) glycerol, 0.001% (w/v) bromphenol blue and 20 mM Trisphosphate, pH 7.6, and heated for 5 rain at 95 °C. The electrophoresis was carried out on 0.75 mm thick 7 % acrylamide slab gels employing the discontinuous buffer system of Laemmli~L For two-dimensional analysis, samples were prepared and run in the first dimension according to O'Farrell 3a, using ampholytes from LKB. In the second dimension, the isoelectric focusing tube gels were applied on 0.75-mm thick 5-20 acrylamide gradient slab gels and samples were run using the buffer system of Laemmli21. The slab gels used in the electrophoresis in both one and two dimensions were stained with Coomassie blue R-250 and destained with 10 ~o ~ acetic acid/20 methanol.

390

8

b wt~

id.

200

m200

150 B

~130 ~94

~6B

--f ~43

123456 I I 2 I3 4 5 6ll 2 3456Jl 2 3 45 6 Fig. 1. Electrophoretic comparison of filaments isolated from different regions of the rabbit nervous system. Filaments from spinal cord (1), sciatic nerves (2), optic nerves (3), trigeminal nerves (4), lumbar ventral roots (5) and lumbar dorsal roots (6) were isolated from the same animal and compared by electrophoresis on SDS-polyacrylamide gels. The samples were from inbred animals of strains IIIVO/J (a) and X/J (b) carrying respectively the H 1 (a) and H ~ (b) form of the P200 neurofilament protein, and from outbred animals (c) and (d). The apparent molecular weights (in kdaltons) of filament proteins are indicated at left. The apparent molecular weights of standards (in kdaltons) are indicated at right (200: myosin; 130 :fl-galactosidase; 94: phosphorylase b; 68 : bovine serum albumin; 43 : ovalbumin), f, c and t indicate the position of polypeptides comigrating with the actin-binding protein, collagen and tubulin, respectively.

RESULTS Neurofilament-rich fractions were prepared by a simple one-step procedure which does not involve axonal flotation 11. The tissues were disrupted in a buffer containing 25 m M KC1, 10 m M MgClz, a calcium ion chelator and a disulphide reducing agent. The filaments were freed from myelin with detergent. After centrifugation through a sucrose cushion, the filaments were found as a pellet while the solubilized material (myelin, membranes) remained at the interface or above the cushion. With this procedure, we were able to process tissue samples weighing as little as 20 mg wet weight, and to isolate filaments from them in such amounts that it was feasible to examine this material by electron microscopy and to analyze its protein composition by electrophoresis on gels in one and two dimensions. The advantages o f the preparation are obvious when one wants to compare filaments isolated from different regions o f tile nervous system o f a given individual. In the gels presented in Fig. 1, we have c o m p a r e d the polypeptide composition o f filaments isolated from spinal cord, sciatic nerves, optic nerves, trigeminal nerves and lumbar ventral and dorsal roots; all samples were dissected from the same animal. The electropherograms show that all samples include 3 major polypeptides with

391 apparent molecular weights of 68,000 (P68), 150,000 (P150) and 200,000 (P200) daltons. These are considered to be the constituent proteins of neurofilaments 1,3,5-s, lO,20,23,24,a0-32,37,43,46. In addition to this 'triplet', two other proteins are prominent components on the electropherograms: a polypeptide of molecular weight of 60,000 (P60) and another of molecular weight of 50,000 daltons (P50). P50 is present in the spinal cord, optic and cranial nerves. This protein seems to be of glial origin12,26,23,39. This is confirmed here; P50 is present only in samples prepared from tissues rich in astroglial cells. The origin of P60 is more mysterious. This is probably the same as the 60,000 dalton protein described by Liem et al. z3 and is not to be confused with the neuron-specific non-filamentous 65,000 dalton protein, P65, which we have described 41. Although present in all filament preparations isolated from the peripheral nervous system, it is also a major component of filaments isolated from optic nerve. The protein composition of the filaments isolated from sciatic nerve and from dorsal and ventral roots is identical; that of the optic and cranial nerves is a mixture of polypeptides present in the spinal cord and polypeptides present in sciatic nerve and roots. Other minor protein bands are present in these preparations as contaminants. A doublet of molecular weight of about 230,000 daltons, which co-migrates with the actin-binding protein 44, was found in all samples; collagen polypeptides were detected by their metachromatic characteristics when stained with Coomassie blue R25029 and were observed mainly in samples prepared from sciatic nerve and ventral roots; tubulin was present in small amounts in all samples. The P200 component of the rabbit neurofilament 'triplet' from spinal cord and optic nerve has been reported to exist in two forms, H 1 and H z. This dimorphism was found to be genetically determined 4~. Three polypeptide phenotypes, H 1, H z and H 1 + H 2 corresponding to all possible genotypes - - homozygotes H1H 1 and H2H 2 and heterozygote H I H 2 - - are present in outbred rabbit populations, although H2H z is very rare. The inbred rabbit strains show either the H 1 or the H 2 phenotype 45. To determine if the gene coding for the P200 was expressed similarly or differently throughout the different groups of neurons, we have studied the distribution of this polymorphic protein in filament preparations isolated from different regions of the nervous system within the same animal. Partially inbred rabbits from the strains IIIVO/J and X/J carrying the genotype H 1 and H z, respectively, and outbred animals were used for this purpose. The two forms of P200 were differentiated by gel electrophoresis. When electrophoretically compared, the filaments isolated from different regions of the same animal exhibited a uniform composition regarding the molecular weight of the P200. All filaments isolated from IIIVO/J animals carry the H 1 form of P200 (Fig. 1a); all filaments isolated from X/J animals carry the H 2 form of P200 (Fig. lb). The outbred animals carry either H 1 (Fig. lc) or the mixture of H 1 -}H 2 (Fig. ld). The form of P200 is constant for all regions sampled within a given individual. We have not been able to find outbred animals with H z alone; this is not surprising in view of the extreme rarity (0.5 ~o) of the H 2 phenotype in outbred populations 45. Although we have sampled only a limited number of regions throughout the rabbit nervous system, the results indicate that the P200 gene expresses itself in a constant fashion, regardless of the localization and function of the neurons involved.

392 From the analysis presented in Fig. 1, it is apparent that the neurofilament moiety of the filaments isolated from the different regions of the rabbit nervous system is identical for each individual as far as the molecular weight of the 'triplet' proteins is concerned. This uniformity in apparent molecular weight does not exclude the possibility of some fine differences that are not detectable by SDS-gel electrophoresis. For example, multiple forms of cytoskeletal proteins from the nervous system have been described, using two-dimensional gel analysis; brain a- and fl-tubulins9,13,1s,25,26 as well as brain actin 4,27appear as several isoproteins of the same molecular weight but different isoelectric points. Neurofilament polypeptides from rabbit spinal cord also display a certain degree of heterogeneity6. Moreover, neurofilaments present in cells which perform different functions may have different patterns of znicroheterogeneity. To test this hypothesis, the filament samples which were analyzed by electrophoresis on SDS ,ief

200 J

130 94

68 ,~ll

,3~b

6,5

4.5

'

393

Fig. 2. Two-dimensional analysis of filaments isolated from different regions of the rabbit nervous system. Electropherograms of filaments from brain (a), spinal cord (b), sciatic nerves (c), optic nerves (d), trigeminal nerves (e), lumbar ventral roots (f), lumbar dorsal roots (g). All filaments were isolated from rabbit (c) of Fig. 1. Molecular weights of main filament proteins are indicated in kdaltons. Arrows mark the additional spot of P68, when present. The limits of the pH gradient for each isoelectric focusing gel (ief) are indicated at the bottom of gel (a). The molecular weights (in kdaltons) of the proteins used as standards in the second dimension (sds) are indicated at the left of gel (a).

gels (Fig. 1) were c o m p a r e d by gel electrophoresis in two dimensions. The twodimensional electropherograms (Fig. 2) include the characteristic pattern of the neurofilament 'triplet' proteins6, 7. The isoelectric pHs of P68 and P150 are very similar to one another, although P68 is slightly more acidic. P200 focuses at a more alkaline p H range. The distribution o f the spots on the gels, reflecting the microheterogeneity o f these proteins, is not identical for all the filaments sampled. Filaments from brain (Fig. 2a) and spinal cord (Fig. 2b) include a doublet in the P68 protein. The isomorphic forms o f P68 are either present in about equal mounts 6, or the more acidic o f the doublet spots comprises the bulk o f the P687. Filaments from sciatic (Fig. 2c), optic (Fig. 2d) and trigeminal nerves (Fig. 2e), and filaments from the lumbar roots (Fig. 2f and g), all have a P68 which resolves either into a single spot or has a small

394 secondary spot as a tail on the two-dimensional gels. In trigeminal nerves (Fig. 2e) and dorsal roots (Fig. 2g) a small amount of the more basic form of P68 is just barely detectable. In addition to the 'triplet' protein of neurofilaments, the additional proteins detected in Fig. 1 are distributed at characteristic topographical localizations on the 2-dimensional electropherograms. P60, when present, appears as two adjacent spots. P50, when present, appears as a heterogeneous collection of spots of different intensities with pls more alkaline than the other proteins. The isomorphs which focus at various pH values of the P200 component of the neurofilament 'triplet' do not seem to vary whether they are present as a doublet or as a singlet 6. DISCUSSION Although neurofilaments were among the first cytoskeletal filamentous structures described in neurons, understanding their properties and function has been a slow process. Attempts to elucidate the chemical composition of the mammalian neurofilaments have been impaired by the complexity of the central nervous system tissues and by the intrinsic properties of the organelle. That neurofilaments depolymerize at low ionic strength aa and are very susceptible to proteolysis40 has long been the source of misleading results. By now, it is agreed that the mammalian neurofilaments are composed of at least 3 proteins, as demonstrated by electrophoretic analysis of isolated filaments on SDS-polyacrylamide gels. Although the 'triplet' polypeptide is a common component of filaments isolated from any mammalian nervous tissue, the identity of the chemical composition of neurofilaments from different neurons throughout the entire nervous system is questionable in regard to the variety of localizations and functions played by each of those neurons. No systematic investigation has been carried out along these lines. Only a very limited number of communications present comparative data. Isolated filaments from rat brain and sciatic nerve have been compared 1, as have filaments from rabbit intradural spinal nerve roots and brain 23 and filaments from rabbit spinal cord and sciatic nerve 7. In all cases, the 'triplet' proteins were shown to be common to all neurofilament preparations. In this communication, we present an electrophoretic analysis of filaments isolated from different regions of the rabbit nervous system. Since various factors, which influence metabolic processes (such as age, sex, degree of inbreeding, etc.) may be reflected in the protein composition of different organelles, we have compared the chemical composition of neurofilaments isolated from the same animal. As pooling samples was out of the question, we developed a new method to isolate filaments from tissue samples weighing as little as 20 mg. The procedure, which is derived from our previous methodsS, 6, but which does not require the usual axonal flotation step, permits the isolation of filaments from all types of axons, not only those which are extensively myelinated. In effect, the Triton extraction of the homogenate is analogous to the procedure applied in the preparation of intact 'cytoskeleton' from culture cells2,19,22,a4. Instead of intact cytoskeleton, the extraction of disrupted cells and subsequent sedimentation of the residue through a sucrose density gradient yields a mixture of protein which is effectively a highly enriched fraction of intermediate

395 filament protein. The amount of filaments recovered is adequate to provide extensive chemical and morphological analysis. The electrophoretic analysis shows that the filaments isolated from all the nerve tissue samples always contain the 'triplet' proteins of neurofilaments as a prominent component, although in variable percentages. Two other proteins present in large amounts are found among the filament polypeptides. One, with a molecular weight of 50,000 daltons (P50), is found in the samples isolated from astroglial-rich tissues (spinal cord and optic nerve). The other, which has a molecular weight of about 60,000 daltons (P60), is of unknown origin. The P60 is present in all nerve samples which include large numbers of fibroblasts; it is not detected only in the spinal cord and brain preparations. The possibility that it might be a unique constituent of Schwann cells23, is unlikely because P60 is found in optic nerve, which lacks Schwann cells. Of the 5 major proteins found in the filament fractions, only P200, P150 and P68 are neuron-specific. This is best demonstrated in studies involving experimental Wallerian degeneration of optic nerve and sciatic nerve. While the triplet ascribed to neurofilament disappears within two weeks in both tissues following section of the nerves, P50 and P60 in the optic nerve and P60 in the sciatic nerve are not reduced41,42. The fact that the SDS-gel electrophoretic analysis of filament proteins does not reveal any differences in the molecular weights or in the distribution of the neurofilament polypeptides in samples of nervous system tissues does not demonstrate an obligatory identity of these proteins throughout the entire nervous system. A general phenomenon characteristic of mammalian cytoskeletal proteins is their high degree of microheterogeneity. Multiple forms of tubulin9,13,1s,25,26, actin 4,27 and neurofilaments have been described. Although each family of protein has about the same molecular weight, each individual form can be segregated by its different isoelectric point. While some of the isoproteins described are the translation products of different mRNAslV, 2s and seem to be specified at the genome level, others are the products of post-translational modifications~5, 35. It is not unreasonable to suspect that a protein with multiple forms may be used to perform distinct functions in different cell types. Alternatively, different forms of the same protein may co-exist in different cells or in different cellular substructures, as was shown for actin 27. In a similar fashion, one can postulate that the neurofilaments which are present in different regions of the nervous system are represented by different isoproteins. The results presented in this communication point toward the possibility that at least some of the P68 protein of the spinal cord and brain differs in its isoelectric pH from most of the P68 of optic, trigeminal and sciatic nerves and lumbar roots. The biological significance of this observation is not yet known. The only information concerning the genetics of neurofilament proteins is provided by an electrophoretic analysis of the constitutive proteins of particulate fractions isolated from nervous tissue of rabbits of different strains45. One of these proteins, which was later identified as the P200 component of isolated filaments6, 46, may occur in a genetically altered form in certain animals. Two forms of P200, termed H 1 and H ~, have been described; they differ from each other by 10,000 daltons, H 1 having the higher molecular weight45. It is not known whether H 1 and H e are two

396 different proteins with two different sequences or whether H 2 has the a m i n o acid sequence o f H 1 minus 50-80 a m i n o acids. The availability o f animals h o m o z y g o u s for the two forms o f P200 was exploited to see if the P200 gene is expressed uniformly in all groups o f neurons. By following the type o f P200 present in filaments isolated from different regions o f the nervous system o f rabbits with H 1, H 2 or H 1 6- H 2 phenotype, we expected to be able to o b t a i n some i n f o r m a t i o n on the expression o f the P200 gene. O u r observations d o n o t p o i n t to a differential genetic expression o f P200 in different regions o f the nervous system. The P200 protein o f the neurofilament 'triplet' is u n i f o r m l y H 1 or H 2 in inbred r a b b i t strains, H I or H 1 -]- H 2 in outbred populations*. The results are best explained if we p o s t u l a t e that the P200 gene is present as a single copy. The m u t a t i o n which p r o d u c e d H 2 affected this unique gene, a n d H 2 is an allelic form o f H 1. W h e n H 1 a n d H 2 genes are present in the same animals, they are expressed simultaneously a n d equally in all neurons. The absence o f a p r o p e r m a r k e r precludes the application, to the o t h e r two neurofilament proteins, o f the sort o f analysis that has been used for P200. The P200 o f h u m a n a n d calf brain filaments m a y also a p p e a r as a singlet or as a d o u b l e t suggesting the possibility o f allelic forms o f this protein in other m a m m a l i a n species than the r a b b i t (unpublished observation). The significance o f this polym o r p h i s m is n o t known. It does n o t seem that the a l t e r a t i o n in P200 brings a b o u t physiological changes involving the biology o f neurofilaments. F o r example, rabbits h o m o z y g o u s for H 1 or H 2 exhibit a similar degree o f susceptibility to a l u m i n u m induced e n c e p h a l o m y e l o p a t h y 47, as do heterozygous animals where the P200 is present as H 1 -t- H 2 (unpublished observation). ACKNOWLEDGEMENTS This research was s u p p o r t e d by the Office o f M e n t a l R e t a r d a t i o n a n d Developmental Disabilities o f the State o f New York., W e t h a n k R i c h a r d Weed a n d K e n W o l f s o n for their excellent assistance with p h o t o g r a p h i c reproduction.

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* In the course of preliminary experiments one-dimensional gels of sciatic nerve filaments of a single New Zealand White rabbit showed the presence of H 1 + H 2 while gels of spinal cord filaments had only a single H 1 band. We have never been able to find other evidence for a difference between the P200 proteins of one part of the nervous system and another in any of the more than 50 animals in which such comparisons could be made. The fact that it can occur is reported here even though our data cannot explain this single event.

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