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Aluminum-Induced Dendritic Pathology Revisited: Cytochemical and Electron Microscopic Studies of Rabbit Cortical Pyramidal Neurons

Address correspondence to John Savory, Ph.D., Department of Pathology, University of Virginia School of Medicine, P.O. Box 800214,Charlottesville, VA 22908, USA; tel 434 924 5682; fax 434 924 2574; e-mail js2r{at}virginia.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Intracisternal administration of aluminum maltolate induces biochemical and histological changes in the rabbit brain. The primary histological response to this aluminum intoxication is the appearance within many neuronal somata and dendrites of intensely argyrophilic masses of fibrillar material. Ultrastructural examination of these bodies in both conventionally-prepared and silver-stained sections shows them to be composed of neurofilaments. For this reason, we have elected to call these argyrophilic masses "neurofilamentous arrays (NFAs)." At their zenith, NFAs in cortical pyramidal neurons comprise thousands of filaments interconnected by periodic crossbridges. NFAs begin to be formed within both somata and dendrites as isolated groups of neurofilaments, which apparently go on to assemble en masse within the cytoplasm. In symptomatic animals, many cortical neurons are rich in NFAs, yet lack classic cytological signs of degeneration, such as nuclear pyknosis. Though silver staining reveals extensive NFAs only in aluminum-exposed brains, there is a strong degree of immunostaining for phosphorylated neurofilamentous epitopes in both untreated and Al-injected animals. This suggests that protein subunits that are already present in the neurons under normal circumstances are recruited, in the presence of aluminum, to form NFAs through the directed assembly of masses of oriented filaments.

(received 30 August 2001; accepted 12 October 2001)

Keywords: Alzheimer’s disease, aluminum neurotoxicity, neurofilamentous arrays, aluminum maltolate

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The neurotoxicity of the aluminum ion has long been established. A seminal finding in aluminum neurointoxication has been the presence in certain neurons of readily-visible cytoplasmic inclusions that upon examination by various histological staining regimens and electron microscopy prove to be masses of filaments. This observation has lent credence to the use of this system for the study of neurofibrillary pathology and related human neurodegenerative disorders, including Alzheimer’s disease [1]. Although there are distinct differences between the aluminum-induced inclusions and the Alzheimer "tangles," there nevertheless remains between the two pathologies a similarity that involves considerable alteration to the cytoskeletal components of neurons. Apoptosis is another feature that is associated with severe aluminum neurotoxicity in rabbit brain [2], and this same cell-death pathway may play a role in the neuronal loss observed in Alzheimer’s disease [3,4]. It remains to be clarified, however, just how the various abnormalities seen in Alzheimer’s disease brain come eventually to result in neuronal loss.

Although a considerable body of earlier work has been devoted to the presence, regional distribution, and general structural and staining characteristics of aluminum-induced neuronal inclusions [5–9], we have initiated a closer examination of their microscopic architecture and the steps by which their development might proceed. We have chosen to term these bodies "neurofilamentous arrays" (NFAs) to distinguish them from superficially similar aggregates found in other neuropathologies.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Animals.  Young female white rabbits of the New Zealand strain were used for this study. The animals ranged from 8–12 mo in age and 2.4–3.6 kg in weight. Experimental animals were administered a single dose of 50 mM aluminum maltolate in 100 ml of sterile saline, injected directly into the cisterna magna with a sterile 25-gauge needle. Control animals were left untreated in order to provide an appreciation of the baseline "normal" ultrastructure of rabbit brain. Both in this and our previous biochemical studies it was determined that one week of survival is sufficient for histological alterations to become visible in spinal cord, brainstem, and cerebral cortex. By that time, aluminum-treated animals usually evince a distinct symptomatology that consists of loss to some degree of motor function, particularly in the hind limbs, with accompanying lethargy and loss of appetite.

Tissue processing.  Brains from control and experimental animals were prepared for microscopic examination by vascular perfusion with aldehyde fixative. Following administration via the ear vein of a euthanizing dose of pentobarbital, each animal was rapidly thoracotomized, its descending aorta clamped, and a 16-gauge needle inserted into the apex of the left ventricle. The right atrium was then cut and a gravity-feed perfusion begun, consisting in sequence of 300 ml of glucose/sucrose/saline solution (0.4%/0.8%/0.8%, respectively), followed immediately by 500 ml of a solution containing 0.05% glutaraldehyde, 4% paraformaldehyde, 15% (v/v) saturated aqueous picric acid, and 0.1 M sodium phosphate buffer, pH 7.4. Total perfusion time was approximately 10 min, after which time the brain was quickly exposed, removed from the skull, and immersed for 3 hr in fresh fixative solution. The brain was then sliced into four separate coronal segments (forebrain/midbrain/cerebellum + brainstem/spinal cord), which were reimmersed in fixative at 4°C overnight. The segments were washed in several changes of 0.1 M sodium phosphate and stored in phosphate buffer with 0.01% sodium azide to avoid microbial contamination.

For sectioning, the individual segments were encapsulated in 15% gelatin, and this stabilizing coating was further hardened by immersion for 24–48 hr in aldehyde fixative. Selected segments were cut into 50-µm coronal sections with injector-type razorblades mounted in a vibrating microtome (DSK Microslicer DTK-3000, Ted Pella, Inc., Redding, CA), and returned to azide-phosphate solution until further processing was undertaken. All sections were processed in free-floating form, and not until the conclusion of each procedure were those sections intended for light microscopic documentation mounted on slides (see details, below).

Bielschowsky’s silver staining of neurofilamentous arrays: Selected 50-µm sections were stained in free-floating form by a protocol used in this laboratory to reveal argyrophilic components in neurons. In summary, sections were immersed in darkness in 20% aqueous silver nitrate at 37°C for 30 min, followed by 15 min incubation in "ammoniacal silver" (prepared by adding concentrated ammonium hydroxide to the silver nitrate, while stirring, until a clear solution appears). The silver reaction was developed by adding ~10 drops/50 ml solution of a "developer" consisting of 2% formalin and 0.4% citric acid in distilled water acidified with concentrated nitric acid (2 drops/250 ml).

Whereas in our experience 14-µm-thick, formaldehyde-fixed sections mounted on slides require only about 15–20 min to show a suitable degree of staining in this solution, the considerably thicker sections used in this study required as much as 2 hr to display thorough argyrophilia of neuronal cytoskeletal elements (as monitored, during the development step, by light-microscopic examination of the wet sections). The reaction was stopped by transfer of sections into 1% aqueous ammonium hydroxide, with a subsequent "fix" in 5% sodium thiosulfate. Some sections were washed in distilled water, affixed to gelatin-coated slides, allowed to dry, and then cleared in xylenes and coverslipped for light microscopic examination, while others were directed to a plastic-embedment regimen detailed below.

Immunocytochemical staining for phosphorylated neurofilaments.  Pre-embedding immunocytochemistry on brain sections was carried out as previously described [10]. To summarize, sections were infiltrated 30 min with a cryoprotectant solution (2% DMSO and 20% glycerol in 0.1 M sodium cacodylate) and collected on segments of glass microscope slides. These were placed into aluminum weighing dishes, which were set on a liquid-nitrogen surface for 2 cycles of freezing and thawing; this procedure renders sections more permeable to antibody penetration.

The sections were washed thoroughly in phosphate buffer, blocked with 10% normal horse serum and 1% BSA (Sigma #7030) in 0.1 M sodium phosphate, pH 7.4, and then incubated in vials of primary antibody solution (see primary antibody concentrations below; carrier solution consisted of 1% BSA in 0.1 M sodium phosphate buffer, pH 7.4). Incubation continued overnight at 4°C with constant agitation on a Nutator (model 1105, Clay-Adams, Parsippany, NJ).

The next morning, sections were washed in buffer and then exposed to secondary antibody in carrier (1:200 biotinylated horse anti-mouse IgG, Vector BA-2000) at RT for 2 hr, followed by rinses and incubation in avidin-solution (Vector Vectastain ABC Elite Kit) for 2 hr at room temperature. Sections were then rinsed and the reaction developed for 30 min by the glucose oxidase-diaminobenzidine method of Itoh et al [11].

As with the silver-stained sections that were intended for light microscopic examination alone, immunostained sections were affixed to gelatin-coated slides, allowed to dry, cleared in a series of xylene solutions, and coverslipped.

A monoclonal antibody that reacts with phosphorylated neurofilament epitopes (SMI-31, Sternberger Monoclonals, Inc., Lutherville, MD) was tested on sections at dilutions of 1:1000 and 1:5000.

Electron microscopy.  For conventional TEM examination, the 50-µm sections were washed in 0.1 M Na cacodylate solution and postfixed gradually (to minimize section curling) in ascending concentrations of osmium tetroxide (final concentration 1%) in cacodylate buffer (pH 7.4) for a total postfixation period of 30 min. En bloc immersion in saturated aqueous uranyl acetate was carried out for 30 min. Some of the silver-stained sections were prepared without osmium postfixation and uranium block-staining.

All sections were dehydrated in an ascending series of ethanols, passed through propylene oxide, and infiltrated under vacuum in Poly/Bed 812 resin (Polysciences, Warrington, PA). The free-floating sections were transferred to microscope slides coated with liquid-release agent (Electron Microscopy Sciences, Fort Washington, PA) and topped with similarly-coated coverslips, which were weighted with coins to flatten the sections. The embedments were then cured at 60°C for two days. Sections were examined on the slides with a light microscope and areas of interest selected. The coverslip portions over these regions were removed with a scribing tool and dissecting pin, and a trapezoidal portion of the brain section was cut out with a razorblade and glued with cyanoacrylate to an epoxy capsule.

Semi-thin (0.25–1.0 µm) sections were prepared with glass knifes on a Sorvall MT-2B ultramicrotome and stained with 1% toluidine blue in 1% sodium borate for orientation and detailed light microscopic documentation; for transmission electron microscopy (TEM), 7–10 nm "ultrathin" sections were cut with a DuPont diamond knife.

Sections were collected on copper-mesh grids and stained with saturated uranyl acetate in 50% acetone (1–2 min) and 0.5% alkaline lead citrate (1–2 min). All sections were examined in a Zeiss EM-10CA transmission electron microscope at an accelerating voltage of 60 keV. This instrument was calibrated for crucial magnifications against a replica of an optical grating, and measurements of cell structures were made directly from high-magnification photographs with a vernier caliper.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Light microscopic observations:  The cytoskeletal elements of neurons are clearly delineated with the Bielschowsky’s method of silver staining; their three-dimensional interrelationships are particularly well demonstrated in 50-µm-thick sections stained with this procedure (Figs. 1–6). In the frontal lobe cortex of untreated animals, the cytoskeleton is revealed as dark threadlike profiles located primarily in axons (Figs. 1, 3), whereas equivalent sections of Al-treated brain offer a striking contrast, with numerous pyramidal cell bodies standing out in silhouette because of prominent silver-stained profiles within them (Figs. 2, 4–6); cells affected in this manner are found in varying numbers throughout the frontal lobe, but are particularly numerous in the more caudal frontal lobe, over the medial convexity and in the cingulate gyri, tending to be most concentrated on either side of the dorsal midline.

View larger version (197K): [in this window] [in a new window]   Fig. 1–6. Silver-stained (Bielschowsky’s method) coronal 50-µm sections through frontal cortex showing pyramidal neuron cytoskeletal components in untreated young female rabbit (Figs. 1,3) and aluminum-treated female (50 mM aluminum maltolate, 7 days exposure) (Figs. 2,4–6). Fig. 1. Silver staining confers a delicate, threadlike pattern running vertically throughout much of the cortex Although the neuronal nuclei stain lightly with silver (cf. Fig. 3), outlines of the perinuclear cytoplasm are not evident in control tissue. 40x. Fig. 2. An equivalent section from an animal exposed to aluminum for 7 days. A layer of cortical pyramidal neurons stands out in silhouette because of the argyrophilic staining of their cytoplasm. 40x. Fig. 3. Detail of control cortex. The lightly opacified dots scattered throughout this field are cell nuclei (Nu). The pattern of silver staining here is limited to extensive thin profiles of cytoskeletal material, the majority associated with axons. 110x. Figs. 4–7. Frontal cerebral cortex from aluminum-treated animal (treatment as described in Fig. 1). Fig. 4. Aluminum-treated animal. Although some nuclei can be discerned, the picture is dominated by densely-staining cortical cell cytoplasm, made evident by its prominent apical (AD) and basal (BD) dendrites, the latter fanning out from the base of the neuronal cell bodies. 110x. Fig. 5. Aluminum-treated. The elongate silver-positive inclusions within apical dendrites in some instances display a twisted or "corkscrew" morphology; in places along their lengths, parallel separate strands of stained material can be discerned. Equivalent masses in the basal dendrites are heavily stained, but are less extensive. 280x. Fig. 6. In this pyramidal cortical neuron, the silver-stained cytoskeletal material is continuous from the apical dendrite, extending as several thin strands through the cell body that pass around the nuclear zone (Nu) to merge with the material in each basal dendrite. 565x. Fig. 7. "Semi-thin" (~0.25 µm) section from plastic-embedded cortex, stained with toluidine blue and viewed in an orientation similar to that seen in Fig. 6. Here the nucleus (Nu) is viewed in section, partly surrounded by a contiguous mass of proliferated cytoskeletal material, which in this preparation appears lucent against the basophilia of the other cytoplasmic contents, and extends from the apical dendrite into the cell soma. 1140x.

In the "whole-mount" sort of display afforded by the thick-section, Bielschowsky’s-stained preparations, some cells (Fig. 6) demonstrate continuity between the apical and basal argyrophilic masses. This can be confirmed in toluidine-blue-stained, "semi-thin" sections of plastic-embedded material (Fig. 7), where because of their lack of osmiophilia the neuronal inclusions appear unstained against the darker background of contrasted nucleus and cytoplasm. Although nuclei are difficult to detect in Bielschowsky’s-stained cortex of aluminum-treated rabbits, being largely obscured by the intensely silver-stained masses superimposed on them, they are readily identified in semi-thin and ultrathin sections, and are normal in appearance (Figs. 7–9).

View larger version (322K): [in this window] [in a new window]   Fig. 8–16. Transmission electron micrographs of cerebral cortex from aluminum-treated rabbit brain, documenting the ultrastructure of neurofilamentous arrays (NFAs) that characterize the aluminum-induced changes. Fig. 8. Survey of pyramidal neuron (approximately same orientation as previous illustrations); within the apical dendrite and the perinuclear soma (Nu, nucleus) appear lucent zones (*) that represent sections through masses of filaments. Toward the bottom of the field, similar masses are directed away from the lower portion of the cell into the cytoplasmic extensions that form basal dendrites (BD). No substantial alterations in ultrastructure appear in either the nuclear profile nor do any of the other cytoplasmic contents appear altered. 1800x. Fig. 9. Cortical neuron in orientation similar to that in Fig. 8, but from a Bielschowsky’s silver-stained section for comparison. Dominating the field is an opacified neurofilamentous array (NFA) that extends from the cell body up into the apical dendrite. The fibrillar component of the neuron’s nucleolus (Nuc) is also stained. 3000x. Fig. 10. In this somatic NFA, the majority of filaments are cut in transverse section. The NFA is almost entirely composed of 14-nm-diameter neurofilaments (detail shown in inset) with only an occasional profile of a mitochondrion (Mi) or ER tubule. 10,400x; inset 37,000x. Fig. 11. Longitudinal section through NFA in apical dendrite. The closely-packed neurofilaments observe a strict parallel arrangement. Periodic crossbridges extend between adjacent filaments, merging with an amorphous substance that coats the filament shafts. 80,000x. Fig. 12. Transversely-cut NFA, showing distribution of neurofilaments, many of which are connected laterally by crossbridges that hold the adjacent filaments in register with a center-to-center spacing of ~33 nm. Amorphous material adheres to the profiles of the individual filaments as well. 134,000x. Fig. 13. One of two small perinuclear concentrations of neurofilaments in a nerve cell found adjacent to other neurons that contained large NFAs. The component neurofilaments are flanked by Golgi saccules (GA), and, as in the larger NFAs, are oriented mostly parallel to one another. 24,500x. Fig. 14. A skein of parallel neurofilaments is suspended in the long axis of an apical dendrite. This sort of assembly, along with somatic accumulations like those seen in Fig. 13, appears to represent the beginnings of NFA formation. Although this collection of filaments is less extensive than the NFAs in fully-involved neurons, its components possess similar spacing, coatings and crossbridges (see inset). 24,500x; inset 87,500x.

Inspection at high magnification shows the filaments to be closely packed and uniform in appearance (Figs. 11, 12). On the basis of their 14-nm diameter (14.1±1.7 nm, n =15), similar to that of neurofilaments that form sparse populations in dendrites of untreated animals (14.9±0.9 nm, n = 15) and to axonal filaments (13.2±0.9 nm, n = 15), they can be classified as belonging to the cytoskeletal fibril subcategory commonly known as "neurofilaments." Because of this, we designate these particular argyrophilic masses as "neurofilamentous arrays" (NFAs).

While neurofilaments and glial filaments alike are considered to belong to the general category known as "10-nm" or "intermediate" filaments, it is clear that the neurofilaments, whether found in unaffected dendrites, axons, or NFAs, are typically larger in overall diameter, consistently displaying an amorphous coating that is lacking from glial filaments. In contrast, the glial filaments—as measured in the same sections—average about 10 nm in diameter (10.2±1.0 nm, n = 15). (All measurements of diameters of different filament categories were made on thin sections from conventionally prepared material convention: ie, aldehyde-fixed and osmium-postfixed, with uranyl acetate block-staining and on-section staining with both uranium and lead.)

Closely-packed neurofilaments that compose NFAs are joined by numerous cross-bridging structures (Figs. 11, 12) that hook the filaments together in a lattice-like array, in which adjacent filaments commonly observe parallel orientation and a center-to-center spacing of ~33 nm (33.4±2.6 nm, n = 17) (Fig. 12). The degree of NFA development is not the same in all cortical neurons, even at the same coronal level; fully-formed populations of NFAs may be evident throughout the dendrites and cell body of a neuron, while only small filament groups appear in nearby cells (Figs. 13, 14). Regardless of the extent of NFAs, there is no overt ultra-structural sign of degeneration in any of the cells that contain them. That is, NFA-containing cells, except for their masses of filaments, are qualitatively similar to their unaffected neighbors, displaying no sign of nuclear pyknosis, Golgi swelling, or obvious pathology in the ER and mitochondria.

Immunocytochemical observations.  Despite the profound histological differences between normal and aluminum-treated cortical neurons, the immunochemical pattern for the phosphorylated neurofilament epitope is similar, with abundant staining throughout the dendrites and somata in both untreated (Fig. 15) and aluminum-treated cortical pyramidal neurons (Fig. 16). The overall staining appears more intense in aluminum-treated animals, and close examination shows this to result from the density of immunoreactive material within the apical and basal dendrites (Fig. 17).

View larger version (123K): [in this window] [in a new window]   Fig. 15–18. Immunostaining for phosphorylated neurofilament epitope SMI-31. Fig. 15. Untreated cortex. Staining is evident throughout the cell bodies and dendrites of pyramidal neurons. 270x. Figs. 16,17. Aluminum-exposed cortex. Although casual inspection of Fig. 16 shows these neurons to be similar in size and appearance to those in Fig. 15, the regions of immunostaining are notable in the degree of opacification of their contents. This is particularly visible in the apical (AD) and basal dendrites (BD), which at higher magnification (Fig. 17) appear darker and thicker than those in untreated cells. Fig. 16, 270x; Fig. 17, 700x.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

The resulting biochemical changes in proteins such as cytochrome c, Bax, and Bcl-2 are virtually identical in both cortex and neighboring hippocampus (12), despite the paucity of NFAs in hippocampal neurons. The time-course of development of cortical NFAs is consistent, even when partial neuroprotection is bestowed by glial cell-derived nerve growth factor (GDNF), a protein which when injected simultaneously with aluminum is associated with the animals’ ability to survive well beyond a week of aluminum exposure [13]. After this time, many of the NFAs eventually wane (Forbes, unpublished observations) suggesting that NFA formation may be a largely transitory event, embodying a defensive, "filtering" response by certain cells to the presence of the aluminum ion.

NFAs in aluminum-treated rabbit brain are composed of normal-appearing neurofilaments. The same three polypeptide species (68, 150–160, and 200 kDa) are found in "normal" and NFA-component neurofilaments [14,15], and all such neurofilaments are immunologically distinct from glial cell filaments [16], which are composed primarily of glial fibrillary acidic protein (GFAP).

Commercially available monoclonal antibodies for the phosphorylated and non-phosphorylated epitopes of neurofilaments have been utilized in several studies of NFAs in the aluminum-rabbit model [5,17–19]. In these studies, SMI-31 (which detects phosphorylated NF antigens) intensely stained NFAs of aluminum-exposed brain, while in equivalent neurons of control animals, SMI-31 immunoreactivity was restricted to the axons, with no reactivity at all appearing in either dendrites or perikarya. Our present experience with this same antibody, however, agrees with the findings of Katsetos et al [20], since it demonstrates a considerable presence of phosphorylated NF epitope throughout cortical pyramidal neurons in control and aluminum-treated rabbit brain alike.

Interestingly, it has been found that the topical application of SMI-31 antibody to acetone-fixed cultured spinal neurons gave results similar to the other studies–ie, axons were strongly labeled, but cell bodies were not [21]. However, actual injection of antibody into the living cells or immunoreaction of Triton-extracted cells prior to fixation both resulted in positive staining of perikaryal and dendritic filament complements [21]. In that report it was proposed that the question of SMI-31 immunoreactivity is not dictated by the actual degree of neurofilament phosphorylation, but rather by the relative accessibility of the epitopes [21]. These findings are consistent with previous findings from our laboratory (22) that detected no significant changes between control and aluminum-exposed rabbit brain, either in the degree of phosphorylation or gene expression of any of the neurofilament isoforms. The immunohistochemical method used in this and other laboratories for "permeabilization" of sections, then, assumes special significance. As noted in our Methods section, we utilize a process of repeated freeze/thaw cycles, in the presence of cryoprotectant solution. It is thought that this procedure produces submicroscopic fissures in cell membranes, which enhance penetration of the subsequent reagent solutions, in particular the antibodies. This regimen, then, may optimize the immunological reactivity of phosphorylated neurofilament epitopes, whether they are incorporated into actual filaments or not.

By and large, neuronal dendrites, despite their newly-appreciated functionalities [23–25], are not under ordinary circumstances striking in terms of their architecture. However, upon exposure of the CNS to aluminum there rapidly appear, within both apical and basal dendrites of cortical pyramidal neurons, populations of oriented neurofilaments tightly bound together in massive rods or bundles that may nearly fill the dendritic processes, and occupy substantial somatic cytoplasm besides. The groups of filaments appear to begin forming in situ from nucleating points in both soma and dendrites to create tightly-organized skeins of parallel neurofilaments.

These aluminum-induced cytoskeletal changes may result largely from utilization of the protein-aceous material—already present in non-filamentous form—which is induced to adopt fibrillar configuration and create large three-dimensional aggregates. Overall, this indicates that phosphorylated neurofilament epitopes are already in place, and abundantly so, in the normal brain. The fundamental difference between control neurons and aluminum-exposed, NFA-containing cells is the structural form taken by the majority of neurofilament proteins; ie, they may exist largely in a non-fibrillar form in control animals, while in aluminum-exposed rabbits they are displayed mainly as large, extensive masses of long filaments. There appears, then, to occur a rapid and extensive reconfiguration of existing cytoskeletal subunits into fullyformed cytoskeletal fibrils, interconnected by numerous crossbridge structures. In vitro treatment of neuroblastoma cells [26] and fetal rabbit midbrain neurons [27] with aluminum salts has been shown to produce Bielschowsky-silver positive whorls consisting of neurofilaments; these whorled accumulations were not seen in the somata of untreated cells, however. This fits in well with our observations on treated brain, where it also appears that aluminum elicits the formation of great numbers of "assembled" (polymerized) neurofilaments. Our finding in untreated neurons of an abundance of phosphorylated epitopes thus suggests that a neurofilament’s architecture, as much as or more than its degree of phosphorylation, is the basis for Bielschowsky’s silver staining in rabbit brain.

Although, in general, neurofibrillary inclusions have been linked to one or another disease process, we here make the case that NFAs in rabbit brain are transitory and/or reversible in nature, given the right circumstances; under those circumstances, furthermore, their rise and fall can occur quickly. Given the far longer time-frame characteristic of Alzheimer’s disease, however, it has recently been proposed that in addition to the lack of a universal occurrence of neurofibrillary tangles (NFTs), the cells which do develop them may continue to survive in spite of their presence for long periods of time (10–20 yr on average) [25]. Even though there exist substantial differences between NFTs and NFAs, not the least of which is the relative lifespan of their host, it appears that the conclusions of Morsch et al [28] regarding Alzheimer’s tangles are equally applicable to aluminum-induced neurofilamentous arrays.

What, then, is the reason for NFA occurrence? Even though there was evidence of hind-limb malfunction in rabbits 7–8 days after intraventricular or intracisternal aluminum injection, Simpson et al. (29) concluded that no "gross effect on neuronal metabolism" had been wrought either by aluminum or the presence of NFAs. In the absence of overt mobilization of protein synthetic machinery, such as might be indicated at the fine-structural level by enlarged Golgi saccules, increased ribosomal rosettes and proliferated endoplasmic reticulum, and considering that a great deal of neurofilament protein is already present (even in neurons not exposed to aluminum), we have suggested that the filaments which make up NFAs are assembled in large part from proteins already on hand.

It has been proposed that, in Alzheimer’s disease, paired helical filaments are able to form and accumulate because their composition is so similar to "normal" cytoskeletal proteins as to escape the degradative and exocytotic mechanisms that ordinarily would be triggered by the presence of foreign materials [30]. The situation for aluminum-induced NFAs might be even more subtle.

Aluminum’s effects are multifocal; some studies have indicated that nuclear chromatin is especially sensitive to this ion [31], and histochemical stains specific for aluminum furthermore localize on chromatin [9,32], implying that aluminum and chromatin have in fact a certain, perhaps deadly, affinity for one another. While aluminum may bind directly to genetic material, it appears also to be at work in the cytoplasm. In an early study with the Solochrome-Azurine method for aluminum localization, Klatzo et al [6] found staining of both nucleoli and NFAs. It appears that the presence of aluminum ion promotes the aggregation of phosphorylated cytoskeletal proteins [26,33].

It seems likely that there exists some balance between buildup and breakdown of various structures, including neurofilaments. Goldstein et al [34] found that phosphorylated neurofilaments are resistant to proteinase action, while dephosphorylated ones are more susceptible. In their comparison of aluminum intoxication with Alzheimer’s disease, Yokel et al [35] proposed that sufficient inhibition of proteinase activity would interrupt the normal cycle of filament breakdown in the neurons, thus leading to the production of large filamentous aggregations. One may speculate therefore that aluminum may subvert the production of proteinase at the nuclear transcription/translation level, while in the cytoplasm it simultaneously promotes both the assembly and architectural stability of neurofilament arrays.

We conclude, therefore, that the formation of NFAs in aluminum-treated animals can help our understanding of the role of NFTs in the Alzheimer’s disease brain, and that both—although classical neuropathological hallmarks of their respective disorders— might not cause neuronal death. Rather, these fibrillar accumulations might represent a protective response by neurons, buffering the effect of neurotoxins in an attempt to minimize apoptotic (and perhaps necrotic) cell death.

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Supported by a grant (DAMD 1799-1-9552) from the US Department of the Army to John Savory. The transmission electron microscope was provided and maintained by the Central Electron Microscope Facility of the University of Virginia School of Medicine. The authors thank Carlo Bruni, Professor Emeritus at the University of Virginia, for helpful discussions.

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

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