Brain Research, 163 (1979) 235-252 © Elsevier/North-Holland BiomedicalPress

235

BIOCHEMICAL AND IMMUNOLOGICAL CHARACTERIZATION OF NEUROFILAMENTS IN EXPERIMENTAL NEUROFIBRILLARY DEGENERATION INDUCED BY A L U M I N U M

DENNIS J. SELKOE, RONALD K. H. LIEM, SHU-HUI YEN and MICHAEL L. SHELANSKI Department of Neuroscience, Harvard Medical School, Children's Hospital Medical Cehter, Boston, Mass. 02115 (U.S.A.)

(Accepted July 5th, 1978)

SUMMARY In order to identify the protein composition of 10 nm neuronal filaments, we prepared enriched fractions of rabbit spinal neurons undergoing experimental neurofilamentous degeneration induced by aluminum. Electron microscopy of the isolated perikarya showed well-preserved, large perinuclear masses of neurofilaments, which were not found in similarly isolated control perikarya. Comparison of these glial-free fractions by SDS-polyacrylamide gel electrophoresis revealed several-fold augmentation in the filament-enriched neurons of proteins migrating at 68,000 and 160,000 daltons, with an additional component at 200,000 daltons. Otherwise, the protein patterns were identical; no band was found at 51,000 daltons, the molecular weight assigned to the major proteins both of glial filaments and of a previously reported bovine brain filament preparation. An antiserum raised against the 160,000 dalton component of a modified bovine brain filament fraction produced specific and intense fluorescent staining of the aluminum-induced neurofilament bundles. Antibodies to the 51,000 dalton protein of brain filaments and to tubulin failed to stain the induced filaments. The results strongly support the hypothesis that both normal and aluminuminduced neuronal filaments are composed of 68,000, 160,000 and 200,000 dalton polypeptides and do not contain significant amounts of the 51,000 dalton filament protein. The likelihood of biochemical heterogeneity among organelles with similar morphology, namely the glial and neuronal filaments, is raised.

INTRODUCTION Despite the abundance of 10 nm cytoplasmic filaments in the cell bodies and processes of all neurons, efforts to elucidate the biochemistry of these organelles have

236 begun only recently and have led to many contradictions. Isolation and identification of the proteins comprising the neuronal filament have been particularly complicated by the fact that morphologically similar filaments are found in great numbers in glial cells a2. In addition to impeding an understanding of the function of the filament in the normal neuron, this imperfect biochemical knowledge has retarded our understanding of the many neurological diseases characterized by accumulation of normal or abnormal neurofilaments. By far the most common examples of such disorders in man are the late-life degenerative dementias, i.e., Alzheimer's disease and senile dementia of the Alzheimer type. These disorders are characterized ultrastructurally by the presence of abnormal fibrillar organelles in perikarya and in neurites which are made up of paired helically wound 10 nm filaments with a periodicity of 80 nm 17,29. Several experimental models of neurofilamentous proliferation have been described which rely on the filament-inducing action of neurotoxins such as colchicine, vinblastine or podophyllotoxing,31. A model with particular application to the biochemical study of the neurofilament is the fibrillary degeneration induced in certain mammals by aluminum ions, since, in contrast to the other agents, its cytopathological effects are limited to the neuronlS, 28. Interest in this model has been heightened by the discovery of elevated cortical levels of aluminum in topographical proximity to the filamentous lesions in Alzheimer brain 5& In the present study, we have isolated spinal neurons undergoing fibrillary degeneration from aluminum in order to identify the proteins of an enriched filament preparation of indisputably neuronal origin. The results are compared to available data on the composition of mammalian brain filaments and strongly suggest that the neurofilament is composed of three proteins with molecular weights of 68,000, 160,000 and 200,000 daltons respectively. MATERIALS AND METHODS

Isolation of neurons undergoing neurofibrillary degeneration Albino rabbits (2-3 kg) were anesthetized intravenously with thiopental and placed in a stereotactic apparatus. Sterile 1 ~ A1C13 (0.22-0.28 ml) in artificial cerebrospinal fluid was injected slowly (30-45 sec) into the cisterna magna via cisternal puncture. Following an asymptomatic interval of 9-16 days, the animals developed an encephalomyelopathy which progressed over 3-5 days to a paralytic convulsive state. The rabbits were sacrificed by pentothal overdose in the terminal portion of the syndrome. In animals to be used for neuronal isolation and biochemical studies, the entire spinal cord and the basal portions of the medulla and pons were rapidly removed at 4 °C via a total laminectomy. All further operations were carried out at 4 °C.

To isolate neuronal perikarya from rabbit spinal cord, we modified portions of several available methods for rat brain and bovine cord ~,15,22. Freshly removed cord was kept moist with cold hexose-phosphate buffer (HP) consisting of 10 m M KH2PO4, 5 ~ fructose, 5 ~o glucose, 1 ~ Ficoll, pH 6.0 (modified from Norton and Poduslo22). The meninges were carefully trimmed. In initial trial separations, the dorsal funiculi were removed, but this step did not improve cell yield and whole cord

237 was used thereafter. The cord was weighed and then minced 4 times at perpendicular planes on a Mcllwain tissue chopper set at 0.45 mm. The minced tissue was taken up in 10 ml HP and 2 cords (total wet weight 5-6 g) were dissociated together. Incubation at 37 °C under oxygen for 15, 30 or 60 min did not appreciably alter cell yield and was abandoned. The combined cords were mechanically dissociated by sieving once through 335 #m nylon mesh (Ernst Tobler, N.Y.), a fresh sieve being used for each one-third aliquot. Sieves were attached to the end of a cylinder made by cutting the end off the barrel of a 20 ml polyethylene syringe. Sieving was accomplished using gentle positive pressure from the syringe plunger, each sieve being rinsed with 20 ml of 0.9 M sucrose in HP. The tissue was then passed twice through 153/~m nylon mesh, a fresh sieve being used for each one-third aliquot during the first passage. Each passage was again followed by repeated rinsing with 2 ml aliquots of 0.9 M sucrose-HP. The resultant suspension (total volume 125 ml) was layered over discontinuous sucrose gradients composed of 6 ml of 1.8 M sucrose, 7 ml of 1.3 M sucrose, 4 ml of 0.9 M sucrose, all prepared in HP. The gradients were spun at 4500 x g (max) for 15 min at 4 °C in a SW27 swinging bucket rotor. Fractions were collected from each sucrose interface with a pasteur pipette. The 0.9 M sucrose supernatant was rich in myelinated fragments but contained very few recognizable neuronal perikarya. The 0.9-1.3 M interface contained many capillary fragments, myelin and scattered small cells that resembled fibrillary astrocytes, plus a few neuronal perikarya. The 1.3-1.8 M interface held most of the identifiable neuronal perikarya recovered in the procedure but was contaminated with small fragments of cytoplasm surrounding nuclei and with free nuclei per se. A very small pellet consisted primarily of free nuclei and a few neurons. The neuronal fraction was slowly diluted 6-fold with cold HP and pelleted at 700 x g for 12 min. Perikarya were examined with a Leitz inverted phase microscope and cell counts performed with a hemacytometer.

Polyacrylamide gel electrophoresis Pellets of control and experimental neuronal perikarya were stored in HP buffer at --70 °C until used. Two to three cell pellets (4-6 cords) were combined for electrophoretic studies. The pellets were washed 3 times in cold phosphate buffered saline (PBS), pH 6.0, to remove sugars and were then solubilized in a sample buffer (1% sodium dodecyl sulfate (SDS), 8 M urea, 0.03 M Tris, pH 8.0) by repetitive stirring followed by boiling for 5 min. The SDS-solubilized protein was assayed by the method of Lowry et al. 21. Samples were then made 2 % in fl-mercaptoethanol, boiled for 1 min, made 0.001% in bromophenol blue (tracking dye) and loaded on 0.1% SDS, 5-15 % polyacrylamide gradient slab gels. Identical amounts of protein (10-20/~g) from control and experimental cells were loaded. Preparation of gels and electrophoresis were performed in general accordance with the method of Laemmli19. Gels were electrophoresed at 30 mA (constant current) for about 4 h. They were fixed in 10 % acetic acid-50 % methanol overnight, stained in 0.1% Coomassie blue for 1 h and destained in 10% acetic acid. Molecular weight markers were: myosin (200,000

238 daltons), fl-galactosidase (130,000), bovine serum albumin (68,000), bovine brain filament major band 33 (51,000), ovalbumin (43,000) and cytochrome ( < 12,000). The gels were scanned at 565 nm with a Schoeffel densitometer.

Light and electron microscopy For morphological correlation, coronal sections of the cervical enlargement were taken from each control and experimental cord undergoing neuronal separation and fixed by immersion in 10~ formalin. For histopathological studies, affected or control rabbits were anesthetized and sacrificed by intracardiac perfusion with 10 ~/,, buffered formalin or Bouin's solution (1 ~ picric acid in 10~ formalin). Ten /~m sections from various brain regions and spinal cord were stained with toluidine blue or cresyl violet (Nissl method) and with the Bodian argyrophilic stain. For electron microscopy (EM), rabbits were perfused through the heart with warm one-quarter strength Karnovsky solution (1.0~ paraformaldehyde, 1.25% glutaraldehyde (Fisher biol. grade) in 0.1 M sodium phosphate buffer, pH 7.5) followed by the half strength fixative (2.0 ~ paraformaldehyde, 2.5 ~ glutaraldehyde in phosphate buffer). Tissue blocks were left in cold half strength fixative for 24 h, then washed in cold 0.18 M sucrose in phosphate buffer overnight. The blocks were postfixed in cold 2 ~ OsO4 in 0.I M phosphate buffer containing 0.05 M sucrose, dehydrated in ascending grades of acetone with the 7 0 ~ acetone containing 2°/ /o uranyl acetate and embedded in Epon-Araldite 11. Thin sections were stained with 2 o/ /O aqueous uranyl acetate and lead citrate, or with lead citrate alone. One micron thick sections stained with toluidine blue were used to monitor the areas sectioned with the light microscope. EM of the isolated perikaryal fractions was performed by slowly diluting the fractions obtained from the sucrose gradients with an equal volume of cold 4 glutaraldehyde in HP, pH 6.0. After 1 h fixation, the cells were diluted 3-fold with cold HP, pelleted (700 × g for 12 min), washed once in HP and stored overnight (4 °C) in 0.18 M sucrose-HP, pH 6.0. The pellets were postfixed and dehydrated as above but beginning with a 30 ~ rather than a 70 ~ acetone solution. The cells were embedded in Epon-Araldite in a conical Beem capsule prior to thin sectioning and staining as above.

Immunofluorescent studies Sections of fresh cervical and lumbar cord from experimental and control rabbits were quick frozen on dry ice immediately upon removal. Ten /~m frozen coronal sections were placed on albuminized glass slides. Smears of control and experimental isolated neuronal perikarya were also used for immunofluorescent studies. The slides were washed with PBS, pH 7.2, and incubated for 1 h with appropriate dilutions of preimmune or immune sera from rabbits or guinea pigs immunized with various gel-purified components of a bovine brain filament preparation 2°. An antiserum prepared against purified bovine brain tubulin 27 was also employed. Following repetitive washing with PBS for 1 h, the slides were incubated with fluorescein-conjugated goat anti-rabbit or anti-guinea pig serum for 1 h. After

239 washing again with PBS for 1 h, the sections were covered with glass slips mounted with a few drops of PBS-glycerol (1:1) and viewed under a Zeiss microscope outfitted with epifluorescence. RESULTS

Aluminum-induced encephalomyelopathy The initial signs and evolution of the aluminum encephalomyelopathy were highly consistent from animal to animal, as were the neuropathological lesions produced. The onset of the neurological illness, however, varied between 9 and 16 days following intracisternal aluminum administration. This interval did not correlate with the weight-adjusted dose of aluminum ions injected. The earliest clinical sign was an increased irritability to external stimuli which progressed in 24-48 h to repetitive myoclonic jerks elicited by sudden tactile or auditory stimuli. Gait impairment, due to progressive weakness and incoordination of limbs and beginning in the hind limbs, was prominent throughout. As the animal lost the ability to hop, the righting reflex became weak and it disappeared by days 3-5 of the syndrome. Abnormal tonic posturing of limbs could be induced by placing the animal on its side and often led to frank ophisthotonus. Terminally, the animal ceased eating and developed repetitive clonic convulsions. Death from starvation or continuous convulsions occurred between 3 and 6 days following onset of the disorder. All experimental animals were sacrificed at a time estimated to be apploximately 12-24 h prior to death, since it is known that masses of neuronal filaments accumulate progressively throughout the asymptomatic and symptomatic stages of the illness a0.

In situ light and electron microscopy Cresyl violet stained coronal sections of formalin perfused cord revealed the widespread presence at all levels of spinal neurons containing large pale-blue cytoplasmic 'clearings', as shown in Fig. 1A and B. These lesions were usually multiple and were grouped around the nucleus, which maintained its normal position and staining characteristics. The affected cells appeared swollen compared to control neurons. The greatest density of abnormal perikarya occurred around the central canal and in the adjacent gray matter within one millimeter on either side. In these areas, cytoplasmic lesions were seen in over three-fourths of the identifiable neurons in a 10/~m thick cross-section. These cells were primarily intermediate sized neurons (diameter 25-50 #m). As one approached the depth of the anterior horn, the proportion of abnormal cells declined, so that the very largest anterior horn cells (diameter 65-90 #m) which presumably represent alpha motoneurons, were infrequently affected. Neurons of the dorsal horns likewise showed few changes. Bodian preparations, seen in Fig. 1C and D, demonstrated a homogeneous magenta-colored argyrophillic staining of the clear zones, indicating their content of masses of neurofibrils. Topographically, the greatest abundance of pathological nerve cells occurred throughout the central gray matter of the cord from cervical to sacral levels. The gray matter of brain stem tegmentum was uninvolved, but prominent

240

Fig. 1. Light microscopy of perfused rabbit spinal cord. A : toluidine blue stained section of anterior horn in experimental aluminum myelopathy. The great majority of neurons contain single or multiple light-staining cytoplasmic 'clearings'. B: higher magnification shows distension of the neurons by these clear zones, which contain masses of 10 nm filaments. C: control anterior horn shows normal toluidine blue stained neurons. D: Bodian stain of basis pontis in experimental animal reveals dark, argyrophilic neurons containing neurofilament tangles (Bouin's fixative). E: affected anterior horn cells near central canal show prominent filament bundles staining darkly with Bodian stain (Karnovsky fixative). F: control anterior horn cells display normal light cytoplasm with Bodian stain (Karnovsky fixative). All bars represent 50 Ibm.

241

Fig. 2. Electron microscopy of in situ filament-containing neurons. A: large perinuclear swirl of neurofilaments (nf) displaces other organelles in an anterior horn cell. Bar represents 2 ~m. B and C : higher magnifications. Bar in B represents 1/~m; bar in C represents 100 rim.

intracellular lesions were seen in the nuclei of the basis pontis. The hippocampus and parahippocampal gyrus showed only a few scattered abnormal cells. There were essentially no neuronal changes in the cerebral hemispheres, including the deep gray nuclei and cerebral cortex. This distribution is similar to that reported by Klatzo et al. la, although the greater involvement of central thalamus and certain brain stem nuclei as well as the more widespread neuronal change throughout the anterior horn in the latter study may be explained by their use of a second intracisternal injection on the 8th day following the initial dose. The predominance of affected neurons in the pericentral and anteromedial gray matter of cord, which was invariably seen in the large number of rabbits we examined, might be expected from the fact that the subarachnoid space lies closest to the central gray isthmus due to the deep indentation of the cord by the anterior median fissure. Others have also found the earliest and most severe changes in medial areas of cord 1°,3°. The site of injection of aluminum ions (intracerebral, intraspinal or subarachnoid) appears not to influence the topography or number of altered nerve cells 3°. Electron microscopy (Fig. 2) of the perfused cord showed that the perinuclear cytoplasmic clearings consisted of large swirls of linear rod-like filaments, which had a diameter of approximately 10 nm, and conformed to the appearance of normal neuronal filaments. High power electron micrographs revealed that some of the induced filaments bore small 'side arms', as can be found in normal 10 nm neuronal filaments. The filament bundles appeared to lie very close to the nuclear membrane. An occasional thin section demonstrated bundles on either side of the nucleus,

242

Fig. 3. Phase microscopy of isolated spinal neurons. A : several isolated control anterior horn cells, showing multiple processes and nuclei with dark nucleoli. B: higher magnification of experimental neuron shows good preservation of cell form despite disruption of plasma membrane found by EM. Bars represent 50/~m.

indicating their multiplicity or, alternatively, their continuous concentric configuration around the nucleus. The masses of filaments pushed normal cytoplasmic organelles aside; occasional mitochondria, microtubules or lysosomes could be seen within a bundle. No limiting membrane was noted. The neuron's remaining cytoplasmic organelles were unremarkable. Occasional sections revealed degenerating neuronal processes.

Isolation of enriched neuronal fractions Initial cell separation of trimmed cord using methods devised for rat and human brain15, z2 gave very low neuronal yields: 3000-12,000 large and medium sized perikarya, with a mean count of 6000 cells per cord. It was clear that these apparently tiny yields were a function not only of our initial techniques but also of the method of cell enumeration and the neuronal content of rabbit cord. We chose to count only those perikarya which were clearly identifiable as neurons; that is, cells that had a single centrally placed nucleus with one or two distinct nucleoli, abundant cytoplasm completely surrounding the nucleus and two or more cytoplasmic extensions, the length of which was at least half the diameter of the perikaryon (see for example Fig. 3). In the vast majority of enumerated cells, the processes were at least 1-2 times the somal diameter; in some particularly well preserved cells, up to 6-8 processes extended from the soma. Most of the perikarya had a large cytoplasm to nucleus ratio. The filament bundles were not visible in the unstained experimental neurons viewed by phase microscopy. Our criteria excluded counting as 'cells' the small perikarya shorn of all processes that were present in our preparations. Although such smaller 'cells' were not counted, they were included in our final 'neuronal' fractions taken for analytical protein separation. We were unable to find reports of manual microscopic counts of anterior horn cells in rabbit spinal cord. However, some estimates are available for several other mammals. Interpolating from rough estimates (derived from Blinkov and Glezer 2) of

243 < 104 anterior horn cells for whole mouse cord, approximately 5 × 104 for the macaque monkey, 2 × 105 for the human, and several times the latter figure in the cow3, we approximated the number of neurons in both anterior horns of the entire rabbit cord as being between 4 and 8 × 104. Modification of the cell separation to the final procedure outlined in the Methods section led to cell counts in control animals ranging from 11,000 to 40,000 cells per cord with an average yield of 21,000 perikarya per cord. Therefore, our average yield of large and medium spinal neurons represents approximately 25 % of the total anterior horn cells we have estimated to exist in rabbit cord. This figure compares favorably with published yields of 6 % bovine cord 8 and 15-20 ~ from rat brain 23, using similar cell separation techniques. Modifications that increased cell yield included: elimination of trypsin and initial low-speed centrifugation; the use of 153 #m nylon mesh for the final sieving rather than the 74 #m stainless steel screen employed previously15,21, and a large buffer to-tissue ratio during repetitive sieving. Yields were routinely somewhat lower in experimental animals, with a mean of 13,000 cells per cord and a range of 6000 to 28,000 cells. Isolated perikarya were preserved by freezing at --70 °C. The cells were well maintained and showed no morphological changes when examined by phase microscopy after up to 6 weel~s of storage. Pooled neuronal fractions were taken up in 1 ~ SDS, 8 M urea sample buffer. Estimated total SDS-soluble protein in control animals was approximately 95-115 #g per neuronal fraction from one cord. The figure for experimental animals was approximately 65-85/~g of protein per neuronal fraction from one cord. An estimate of protein per isolated neuron has been derived by others 3,1a by dividing the total protein per fraction by the total number of neuronal perikarya in the fraction, Applying this method to our fractions, the protein per perikaryon averaged about 5.3 ng in isolated control neurons and about 6.4 ng in experimental neurons. These values are in rough agreement with similarly calculated estimates of 8.2 ng/cel113 and 9.7 ng/cell 3 for isolated large bovine anterior horn cells. The lower protein content in our perikarya may partially be accounted for by their smaller size compared to bovine spinal neurons.

Electron microscopy of isolated neurons The experimental and control neuronal fractions were compared by EM prior to electrophoresis. The isolated perikarya retained their outlines despite an almost total lack of plasma membrane. The cytoplasmic matrix was looser in its packing density in the isolated cells. Prominent bundles of 10 nm filaments were found in the characteristic perinuclear location in electron micrographs of isolated experimental cells, as seen in Fig. 4. Large cell-free swirls of 10 nm filaments were also scattered throughout the experimental sections. No perinuclear or free filament bundles were to be found in the control sections; only small numbers of isolated free 10 nm filaments were noted. Otherwise, there were no significant differences between the electron micrographs of the control and experimental fractions. Gel electrophoresis Polyacrylamide slab gels of control and experimental neuronal fractions re-

244

Fig. 4. Electron microscopy of isolated experimental spinal neurons. A: isolated perikaryon from aluminum-treated rabbit demonstrates preservation of large perinuclear neurofilament bundle, n, nucleus. B : higher magnification of neurofilaments. Bars represent 1/~m.

vealed very similar protein patterns which were highly reproducible from preparation to preparation (Fig. 5). Visual inspection revealed two distinct protein bands that were significantly augmented in the aluminum-treated neurons compared to controls. The first augmented band had an estimated molecular weight of approximately 68,000 daltons and co-migrated with bovine serum albumin. The second accentuated band migrated at approximately 160,000 daltons, somewhat behind the fl-galactosidase standard. In addition, the experimental fractions contained a small doublet of approximately 200,000 daltons, migrating with myosin, which was not visible at all in the control gels. Densitometric scanning confirmed these differences and is shown in Fig. 6. The 68,000 dalton peak in the experimental cells was approximately 4 times greater than the same peak in the controls. A similar 4-fold augmentation was found at 160,000 daltons. The small 200,000 doublet was well demonstrated in the experimental but not the control scans. Of particular interest was the observation that no enhanced band

245

Fig. 5. SDS-polyacrylamide gel electrophoresis of isolated neuronal fractions. Experimental perikarya (E) have augmented protein bands compared to control cells (C) at approximately 68,000 and 160,000 daltons, plus an additional faint doublet at 200,000 daltons. Otherwise, the electrophoretic patterns were visibly indistinguishable, as confirmed by densitometry (see Fig. 6). Of interest is the absence of a polypeptide at 50-51,000 daltons in either preparation. BF, purified bovine brain filaments prepared by the method of Yen et al. 83, with the major protein at 51,000 daltons and several minor components, including bands at approximately 68,000 and 160,000 daltons. S, molecular weight standards (in descending order): myosin (200,000), fl-galactosidase (130,000), bovine serum albumin (68,000), BF major band (51,000), ovalbumin (45,000), cytochrome (< 12,000).

was found at 51,000 daltons in the experimental cells. Indeed, no band was noted at the 50-51,000 dalton position in either control or experimental neurons. Otherwise, the scans of experimental and control fractions were qualitatively and quantitatively indistinguishable.

Immunofluorescent studies In order to identify further the proteins comprising the aluminum-induced filaments, unfixed frozen sections of affected and control cervical and lumbar cord were reacted with antisera to brain tubulin and to various components of bovine brain filament preparations. An indirect immunofluorescent technique was employed. Antisera to bovine tubulin 27 failed to show any fluorescent staining of the filamentous neuronal masses. Likewise, antisera raised against the 51,000 dalton major band of a bovine brain filament preparation prepared by the method of Yen et al. 88 did not react with the aluminum-induced filaments. Instead, the experimental sections showed

246 EXPERIMENTAL

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Fig. 6. Densitometric scans of electrophoretic gels of isolated neuronal fractions. A: comparison of control and experimental fractions reveals specific enrichment of 68,000 and 160,000 dalton proteins in the neurons containing neurofilament bundles, as well as additional small peak at 200,000 daltons. No 50,000 dalton peak is noted. B: superimposition of experimental (dotted line) and control (solid line) profiles demonstrates absence of differences in protein patterns except at the triplet proteins.

247

Fig. 7. Indirect fluorescent immunohistology of control and experimental neurons. A: section of experimental cord treated with antiserum raised against the gel-purified 160,000 dalton polypeptide of bovine brain filament preparation shows intense fluorescence of cytoplasmic filament bundles in several anterior horn cells. B : control cord treated with same antiserum as in A shows faint staining of neuronal processes and no significant fluorescence of cell bodies. C: experimental cord treated with preimmune serum shows no fluorescence. D: isolated neuron from experimental rabbit confirms preservation of brightly fluorescent cytoplasmic filaments after cell separation (anti-P160 antiserum). E: isolated neuron from control rabbit shows no fluorescence (anti-P160 antiserum). All bars represent 50/~m. intensely black 'holes' without even the usual pale green background fluorescence in the cytoplasmic areas containing the filament bundles. In contrast, an antiserum raised against the gel-purified 160,000 dalton polypeptide of a modified bovine brain filament preparation 2° gave intense staining of the perikaryal filament bundles in cord sections of the aluminum-treated animals, as seen in Fig. 7A. The same serum gave faint staining of neuronal processes and only very slight staining of perikarya in control cord (Fig. 7B), This procedure was then carried out on the isolated neuronal perikaryal fractions and again showed very bright fluorescence of many perikarya in the experimental isolates but not in the control fractions (Fig. 7D and E). Treatment with preimmune serum from the guinea pig in which the anti-160,000 antiserum was raised produced no staining in either experimental or control cord sections (Fig. 7C). An additional control experiment was the preabsorption of the anti-160,000 antiserum with the brain filament preparation of Liem et al. 20, resulting in abolition of the fluorescence of the filament bundles in the experimental sections.

248 DISCUSSION Two fundamental questions regarding the biochemistry of neuronal filaments have been the source of controversy: (1) what are the molecular weights of the major protein or proteins comprising the mammalian neurofilament; (2) to what extent are the filament proteins from neurons and astroglia biochemically homologous? The initial isolation and characterization of brain intermediate filaments employed the axonal flotation method, in which neurofilament-rich myelinated axons of bovine cerebral white matter were exposed briefly to hypotonic medium followed by homogenization to strip their myelin, and the axoplasm was then purified by gradient centrifugation26. A modification of this procedure using long exposures to low ionic strength media resulted in a morphologically pure filament preparation with a major protein species migrating at 51,000 daltons on electrophoretic gels33. Fluorescent immunohistology using antibodies raised against this major protein showed strong staining of glial cells and processes in the brain, but not of central neurons and axons. This reaction pattern was very similar to that reported for an antibody raised against the glial fibrillary acidic protein 1, a soluble protein isolated from the gliotic plaques of multiple sclerosis brain and therefore presumed to originate from predominantly astroglial filaments. These results are compatible with co-purification of neuronal and astroglial filaments by the axonal flotation procedure a3 and subsequent raising of a mixed antiserum. Furthermore, the recent work of Schlaepfer z5 demonstrating the solubilization of peripheral nerve filaments by low ionic strength media raises the likelihood that neurofilaments exposed to such media during the axonal purification procedure of Yen et al. 33 are solubilized and degraded, leaving the preparation enriched in glial filaments. In addition to the 51,000 dalton protein component, such filament-rich fractions have consistenly demonstrated minor bands of approximately 68,000 (P68), 160,000 (P160) and 200,000 (P200) daltons. We have recently modified the procedure for purifying intermediate filaments from central and peripheral nervous system'~°. Filaments isolated in this manner have as major protein components a polypeptide triplet of P68, P160 and P200. In some mammalian species such as the cow, the 51,000 dalton band remains more prominent than each of the three triplet proteins. However, we have recently examined these proteins in similar axonal preparations from human cerebral white matter as well as in isolated human cortical neurons. In such preparations, P68, P160 and P200 are each more prominent than the 51,000 dalton moiety. The same appears to be true in isolated axonal filaments from the guinea pig. Immunoprecipitation assays and immunofluorescent histology of peripheral and central nervous tissue using antibodies raised to these gel-purified protein species strongly support the hypothesis that the three peptides are major components of the mammalian neurofilament and that the 51,000 dalton brain filament protein is the subunit of the astroglial filament20. In the present study, we have shown that isolated mammalian neurons markedly enriched in 10 nm neuronal filaments induced by aluminum do not show any protein band at 50-51,000 daltons nor do they react immunologically with antisera prepared

249 against the original 51,000 dalton major protein (P51) of bovine brain filaments isolated by the procedure of Yen et al. aS. Rather, these filament-enriched neurons have specifically agumented protein species at approximate molecular weights of 68,000 and 160,000 daltons, plus an additional component at about 200,000 daltons. No other differences in the electrophoretic patterns of control and experimental neurons were detected. Furthermore, an antibody raised against the 160,000 dalton band of the modified bovine brain filament preparation ~0 shows intense immunofluorescent staining of the aluminum-treated neurons in precisely the cytoplasmic areas which stain with silver stains in histological procedures. When this antiserum is reacted against normal rabbit spinal cord, only staining of neuronal processes and slight, wispy fluorescence in the cell body is found. No staining occurs when preimmune guinea pig serum is used. Preabsorption of the immune serum with the triplet proteins purified from brain markedly reduces the neurofilament staining. These biochemical and immunological results may be correlated with electron microscopy of the isolated neuronal fractions, which reveals no differences between control and experimental isolates other than the presence of the large neurofilament bundles in the latter fractions. Taken together, the results strongly support the idea that the 68,000, 160,000 and 200,000 dalton species represent forms of the subunit protein of the aluminuminduced neuronal filament. A major advantage of the present approach, compared to previous biochemical studies of brain filaments, is the fact that the neuronal origin of the filaments under study is virtually indisputable. The induction of filamentous proliferation by aluminum was chosen for these studies because, in contrast to other agents such as colchicine and the vinca alkaloids, its effects are limited to the neuron. No reactive fibrillary gliosis occurs in this model. Therefore, any increase in the number of filaments will be in those of neuronal origin. Very few astroglial cells were present in the perikaryal fractions obtained. There was no difference in the number of these glial contaminants between experimental and control fractions, since our neuronal/glial separation procedure was carried out identically in both sets of animals. The aluminum-induced filaments are not likely to be derived from a simple rearrangement of soluble subunits, since the proliferation is paralleled by an increase of mass in the cell. In these studies, the increase in protein per cell was approximately 20 ~ and similar changes have been reported by others 1°. Light microscopy of the affected anterior horn cells in situ also demonstrated visible swelling of the perikarya compared to similar cells in control sections. The attribution of this increase in protein entirely to the increase in neurofilaments is not possible. However, the electrophoretic patterns of the isolated neurons show intensification only in polypeptides corresponding to the triplet proteins we have identified in human and bovine brain filament preparations. This group of three proteins also corresponds closely to three non-tubulin polypeptides present in the slow component of axoplasmic transport and tentatively identified as subunits of the neurofilament by Hoffman and Lasek 14. Moreover, the relative proportion of protein in the three bands was similar in our preparations and those of Hoffman and Lasek. In both studies there was more protein or label in the 68,000 dalton moiety than in the 160,000

250 and 200,000 dalton positions. These similarities could be spurious, however, since it has been shown that the relative proportions of the polypeptides derived from brain filaments can vary with the conditions of isolation s . The studies on axoplasmic transport, these studies, and an analysis of peripheral nerve neurofilaments 20,25, all show a lack of P51, which is the major component of the brain filament preparation described by Yen et al. 33. Of specific interest in the present study was the absence of any polypeptide migrating at 50,000 daltons in both the control and experimental neurons. An electrophoretic comparison of filament preparations isolated from rabbit and bovine brains indicates that in the rabbit the band corresponding to the 51,000 dalton bovine component actually migrates slightly further to a 50,000 dalton position. Thus, the absence of any protein at the latter position in our isolated rabbit neuronal fractions may be correlated with the lack of glia in these fractions, making it likely that no significant astroglial filament component was present. The recent report that the P51 is the subunit of the glial filaments ~2 and the immunohistological localization of antibody to glial filament subunits exclusively to astroglial filaments 24 also argue that this component is of glial origin. Staining of control and aluminum treated cords with anti-P51 revealed only astroglial staining with absolutely no reaction over the neurofilament bundles. Other reports have claimed staining of aluminum-induced filament bundles with antibody against a 54,000 dalton protein extracted by denaturing methods from peripheral nerve 7. It is possible that this antibody is actually against a degradation product derived from the triplet rather than against one of the native components. The present results and other recent work on the neurofilament raise the rather unexpected problem of considerable biochemical heterogeneity among organelles with similar morphology. Such a conclusion is directly contrary to experience with microtubules and with actin filaments (microfilaments) but is strongly supported by current studies of neurofilaments and glial filaments. The use of the aluminum model raises the question of whether the neurons undergoing proliferation of filaments are representative of all neurons or whether other neurons may contain filaments having the 51,000 dalton peptide as the major subunit and thus remain unaffected by aluminum while perhaps responding to other filament-inducing agents. The effect of aluminum, while showing a distinct topography, involves a large variety of neuronal groups and does not occur exclusively in certain specific functional or morphological classes of nerve cells 3°. Moreover, a very recent study 4 of rat motor neurons undergoing filamentous hyperplasia induced by the toxin, fl,fl-iminodipropionitrile revealed that slow axoplasmic transport of a peptide triplet similar to that described here was impaired, causing these proteins to accumulate in the proximal axon where massive filamentous swellings developed. Since the characterization of the aluminum-induced filament reported here is closely similar to the apparent protein composition we have found in normal human neurofilaments, it may be speculated that the paired helical filaments accumulating in neurons in Alzheimer's disease (presenile and senile dementia) will be shown to have the same subunits. However, despite its definite neuronal toxicity in susceptible lower mammals like the rabbit, aluminum cannot yet be etiologically related to the

251 pathogenesis of neurofibrillary degeneration in man. Its apparent accumulation in Alzheimer brain may represent a secondary phenomenon which follows the filamentous degeneration of neurons. A single electrophoretic study 16 of neuronal fractions from Alzheimer brain said to be enriched in paired helical filaments revealed a unique polypeptide band migrating at 50,000 daltons which the authors postulated might represent the protein subunit of these abnormal filamentous organelles. However, our results in both normal bovine and rabbit brain, as well as in the aluminum model, indicate that a protein of this molecular weight is not a component of the neurofilament but is probably a glial filament subunit, It is possible that the 50,000 dalton band noted by Iqbal et al. 16 represented a glial filament component in the fractions, since they were taken from the highly gliotic hippocampal cortex of Alzheimer brain. W o r k on the aluminum model has confirmed that pathological neurons can be successfully separated from other brain constituents with morphological preservations of the filamentous intraneuronal lesions. Techniques such as those utilized in the present study are now being applied by us to an analysis of neurons undergoing spontaneous neurofilamentous degeneration in Alzheimer's disease. ACKNOWLEDGEMENTS We wish to thank Ms. Carol Van Horn for her expert assistance in electron microscopy and Ms. Alice Magnet for valuable assistance in all aspects of this study. The work was supported by N.I.H. Grants AG00704, NS11504 and a grant from the McKnight Foundation. Dr. Selkoe is the recipient of the Whittier Fellowship of the Committee to C o m b a t Huntington's Disease.

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