Neurobiology of Aging, Vol. 10, pp. 125-132. © Pergamon Press plc, 1989. Printed in the U.S.A. 0197-4580/89 $3.00 + .00

Immunoreactivity of Neuronal Lipofuscin With Monoclonal Antibodies to the

Amyloid [3-Protein

C. B A N C H E R , I. G R U N D K E - I Q B A L , K. I Q B A L K. S. K I M A N D H. M. W I S N I E W S K I

New York State Institute f o r Basic Research in Developmental Disabilities 1050 Forest Hill Road, Staten Island, N Y 10314

Rece ived 19 July 1988; Accep ted 14 December 1988

BANCHER, C., I. GRUNDKE-IQBAL, K. IQBAL, K. S. KIM AND H. M. WISNIEWSKI. Immunoreactivity of neuronal lipofuscin with monoclonal antibodies to the amyloid ~3-protein. NEUROBIOL AGING 10(2) 125-132, 1989.--Monoclonal antibodies generated against a synthetic peptide corresponding to amino acids 1 to 24 of cerebrovascular amyloid [3-protein do not only stain amyloidotic blood vessels and the amyloid deposits of the (senile) neuritic plaques, but also the neuronal pigment lipofuscin. Staining of lipofuscin is observed in both cerebral and cerebellar cortices, subcortical nuclei as well as the brain stem, and is identical in Alzheimer and normal control brain. Western blots of a lipofuscin enriched fraction show an anti-13-protein reactive polypeptide migrating at approximately 31 kDa position on SDS-polyacrylamide gel electrophoresis. These results suggest that this polypeptide is associated with lipofuscin and is most likely derived from the predicted amyloid precursor protein. This implicates that, unlike in Alzheimer's disease where this protein is also processed extraneuronally in a manner to release an amyloid fiber forming fragment, the end point of its processing in the nerve cell seems to accumulate on a lipopigment characteristic for normal aging.

Aging Alzheimer's disease Amyloid precursor protein 13-protein Lipofuscin Monoclonal antibodies

THE deposition of amyloid in the form of (senile) neuritic plaques (NP) and in the walls of blood vessels is one of the major events in the pathogenesis of the brain lesions typical for Alzheimer's disease (AD). Ultrastructurally these deposits consist of 4-8 nm helical filaments (19,20) which are found extracellularly as bun- dles in the cortical neuropil, and which also aggregate into large compact stellate masses forming the cores of many NP. The same material is also found in parenchymal and meningeal blood vessels and represents the correlate of congophilic angiopathy.

The major protein component of these fibrillar deposits consists of a self-aggregating 4-5 kDa polypeptide (3,4) termed [3-protein (6) or A4 (17) due to its 13-pleated sheet structure or its molecular weight, respectively. Part of this protein has been sequenced by Glenner and Wong in 1984 (6). Little is known about the mechanism leading to the formation and the deposition of this fibrillar polypeptide, however, recent studies using synthetic oligonucleotide probes based on the amino acid sequence of the 13-protein predict that it originates from a 695 amino acid precursor with the characteristics of a membrane-spanning glycoprotein resembling a cell surface receptor (5,11). More recently two additional mRNAs containing domains homologous to the Kunitz type of protease inhibitors coding for proteins of 751 and 770 amino acids, respectively, have been shown to contain a sequence coding for the [3-protein (13, 22, 28). In the central nervous system the mRNA encoding these proteins has been localized to neurons (1, 7, 16, 26) and various nonneuronal cell types (Brown et al., unpublished observation), but it has also been found in

many peripheral tissues (27). Immunocytochemical studies indi- cate that the amyloid precursor protein may be localized to some nuclear envelopes of adult human muscle (33) and to the plasma membrane of nerve cells (26). Immunoreactivity with monoclonal antibodies (mAbs) to the 13-protein has been observed in neuronal cytoplasm (8,9).

This study uses a battery of mAbs raised to a synthetic cerebrovascular amyloid [3-protein and shows by immunocy- tochemistry that the epitopes recognized by these antibodies are not only present on amyloid deposits in NP and amyloidotic blood vessels, but also in the neuronal aging pigment lipofuscin. Western blots of lipofuscin enriched fractions reveal that these epitopes are localized on a polypeptide with an apparent molecular weight of 31 kDa.

METHOD

Tissue Investigated

Immunostaining was performed on 4% paraformaldehyde fixed paraffin embedded human autopsy material. The data presented were obtained from the brains of 9 AD patients (age: 58-93 years) and 16 control cases (age: 20 months to 85 years). Criteria for the diagnosis of AD included a clinical history of dementia and, on histopathological observation, the presence of numerous NP and neurofibrillary tangles in the neocortex. Young control cases (n=9 , age: 20 months-43 years) were devoid of plaques and tangles. Older controls (n=7 , age: 65-85 years) had varying numbers of NP and neurofibrillary tangles in the hippocampus and

125

126 BANCHER ET AL.

adjacent temporal cortex and, for one case aged 75 years, also in the frontal lobe. Sections through the following brain regions were investigated: hippocampus and adjacent temporal cortex, frontal cortex, precentral gyrus, cerebellum including the dentate nucleus, tail of the caudate nucleus, midbrain including the substantia nigra, pons and medulla oblongata at various levels. Not every brain area was available for a given case, and the number of blocks studied varied from 2 to 4 paraffin blocks per case.

All paraffin blocks were cut into 5 txm thick gapless serial sections. Of each block, sections were mounted unstained for autofluorescence and stained with periodic acid Schiff (PAS) and Sudan Black B for optimum visualization of lipofuscin. Adjacent sections were stained with thioflavin S and Bielschowsky's silver method to assess the presence of NP and neurofibrillary tangles.

Monoclonal Antibodies and Immunocytochemisto'

Mouse mAbs were generated against a synthetic polypeptide corresponding to the first 24 amino acids of cerebrovascular amyloid [3-protein. By ELISA these mAbs react with residues 17-24 of the 13-protein. Details of antibody production have been described elsewhere (12). Immunoglobulins were purified from ascites fluid on a Protein A column and immunoglobulin concen- tration was adjusted to 0.4 mg/ml for all antibodies.

Antibodies, their immunoglobulin classes and dilutions of purified mAbs for immunocytochemistry were the following: 2B8, IgG1, 1:500; 2F9, IgG1, 1:1,000; 4G5, lgG1, 1:20 (tissue culture supernatant); 4El 1, IgG2a, 1:1,000; 4G8, IgG2b, 1:2,000. For Western blots, antibodies were used at double concentration.

Controls included 2 mAbs to Cytomegalovirus (4DI1, IgG1 and 48E10, IgG2a) produced in the same laboratory. These antibodies were equally adjusted to an immunoglobulin concen- tration of 0.4 mg/ml and were used at a dilution of 1:100. Additional controls consisted of a mAb to tau [Tau-1 (2), 1:50,000] and a mAb raised to paired helical filaments recognizing a determinant of ubiquitin [3-39 (29), 1:10,000].

Secondary antisera and immunohistochemical reagents in- cluded biotinylated species specific anti-mouse immunoglobins (Amersham, 1:200) and horseradish peroxidase labelled avidin (Sigma, 14 p~g/ml).

Sections were deparaffinized in xylene/graded alcohols and endogenous peroxidase was inactivated by 0.2% hydrogen perox- ide in methanol for 30 minutes. The sections were then incubated in 10% fetal calf serum in PBS pH 7.4 for 30 minutes followed by the primary antibodies, then by the secondary biotinylated antise- rum and the avidin/peroxidase complex. Primary antibody incu- bations were performed at 4°C overnight, all other incubations were done for one hour at room temperature. Peroxidase was visualized with diaminobenzidine according to Reese and Kar- novsky (23). All sections were counterstained with hematoxilin.

To enhance the immunoreactivity of amyloid on formaldehyde fixed tissue, sections were treated with concentrated formic acid for 1 hour at room temperature prior to immunostaining (14).

Immune Electron Microscopy

For electron microscopic investigation, appropriate areas with high density of lipofuscin containing cells were dissected from 3 paraffin blocks originating from 3 different cases (temporal cortex, subiculum, inferior olivary nucleus), cut into 100 Ixm sections, deparaffinized and reembedded without osmication in the hydro- philic resin LR White (London Resin Co.). Ultrathin sections were prepared and mounted on gold grids.

Unspecific binding sites were blocked by incubating the grids of 0.5% bovine serum albumin, in which all immunological

reagents were diluted. Primary antibody incubations were per- formed overnight as described for paraffin sections, however, at double antibody concentration. Bound immunoglobulin was de- tected with 10 nm gold labelled Protein A (Sigma, 1:50). To enhance the intensity of immunolabetling rabbit anti-mouse im- munoglobulin (DAKO, 1:50) was used as an intermediate step. After immunolabelling the grids were routinely stained with uranyl acetate and lead citrate.

Isolation of Neurons and Neuronal LipoJuscin

Neuronal cell bodies and lipofuscin were isolated from normal cerebral cortex of a 67-year-old subject obtained 11 hours post- mortem, and lipofuscin and membrane fractions from an 81- year-old normal case obtained 4 hours post-mortem. A neuronal cell body enriched fraction obtained from the first sucrose gradient of the tangle preparation according to Iqbal et al, (10) was used as such, treated with 2% SDS for 5 minutes at room temperature or digested overnight at 37°C with 2 mg/ml collagenase in 50 mM Tris-HC1, pH 7.4. The fraction was brought to 1 M sucrose concentration, 10 ml layered over 15 ml of 1.15 M sucrose and centrifuged at 4,800 × g for 60 minutes. The top 1 M sucrose layer containing lipofuscin and membranes was diluted with 2 volumes of water and centrifuged at 4,800 x g for 30 minutes. The pellet was resuspended in water and centrifuged at 200,000×g for 10 minutes in a Beckman TL-100 ultracentrifuge. In the case of the collagenase treated preparation, a yellowish brown hard pellet with a fluffy white layer at the top was obtained. The top white layer, which is rich in membranes, was carefully washed off from the lower brown pellet by squirting with water and decanting until almost all the white layer was separated from the yellowish brown pellet. The first two milky washes containing membranes were designated as membrane enriched fraction, the next two slightly brownish washings containing a mixture of membranes and lipofuscin were discarded. The final yellowish brown pellet free from white layer was enriched in lipofuscin. The lipofuscin enriched pellet was frozen in dry ice, embedded in 2% agarose, fixed, dehydrated and embedded in resin. Electron microscopic observation of the pellet revealed that the fraction contained a low amount of membranous debris and rarely small bundles of amyloid fibres as contaminants.

SDS-Polyacrylamide Gel Electrophoresis and Western Blots

Samples for gel electrophoresis were heated in a boiling water bath for 3 minutes in 50 mM Tris-HC1, pH 7.4 in the presence of 1% sodium dodecyl sulfate (SDS) and 1% 13-mercaptoethanol (ME). In addition, 8 M urea or 0.5% Triton X-100 were added to the sample buffer prior to heating. Fractions were electrophoresed on a 0.75 mm SDS-polyacrylamide gel (5-15% polyacrylamide gradient) using Laemmli's (15) buffer system and electrophoreti- cally transferred to a polyvinylidene difluoride transfer membrane (Immobilon, Millipore). Immunostaining was performed using alkaline phosphatase labeled goat anti-mouse immunoglobulins (Bio Rad, 1:2,000) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt and p-nitro blue tetrazolium chloride as a substrate. Alternatively a peroxidase antiperoxidase technique (Sternberger and Meyer Inc.) was employed.

RESULTS

lmmunocytochernistry

Immunostaining of tissue sections of AD brain containing NP and amyloidotic blood vessels led to strong reactivity of amyloid deposits with all five mAbs to the synthetic [3-protein. Classical

L I P O F U S C I N A N D THE [3-PROTEIN 127

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FIG. 1. Immunostaining of neuritic plaques and lipofuscin containing nerve cells with a monoclonal antibody to a synthetic cerebrovascular amyloid [3-protein. a: Staining of a neuritic plaque and lipofuscin containing nerve cells. Temporal cortex of Alzheimer patient ( × 450). b: Numerous lipofuscin containing nerve cells in subiculum of the same patient. Neuritic plaque in upper left ( × 100). c: Immunostaining of nerve cells of the inferior olivary nucleus. Normal control case aged 77 years ( × 66). d,e: Higher magnification of neurons of inferior olivary nucleus shows immunostaining of lipofuscin. Same case as c. (d: x 200, e: × 600).

128 BANCHER UI" AL.

NP presented as a dense star-shaped core surrounded by a halo of dot and wisp like profiles (Fig. la). Pretreatment of the sections with formic acid led to strong enhancement of the immunostaining (14). Amyloid deposits are then not only found in the form of NP, but also as diffuse infiltrates occupying large areas of the cerebral cortex. These infiltrates were often located in a ribbon-like manner on the surface of the brain (32).

Staining of neurofibrillary tangles or plaque neurites was never observed with any of these antibodies.

However, a varying number of neurons presented with granular immunomactive material in their cytoplasm (Fig. l a, b). The material was often arranged in a cap-like manner adjacent to the nucleus. Sometimes, in the subiculum, immunoreactive granules were arranged in chain-like fashion in the neuropil, suggesting their presence in apical dendrites of pyramidal cells. The immu- nostained material was especially abundant in large neurons of the medulla oblongata and the lateral geniculate body. In the brain stem, the inferior olivary nucleus was constantly the most heavily involved nerve cell group (Fig. lc, d, e). The immunostained granular material was also found in many cortical neurons, the hippocampus, the basal ganglia, the midbrain and the pons. In the cerebellt, m, the dentate nucleus was heavily involved, whereas the Purkinje cells were often devoid of this material or contained only a few scattered immunostained grains.

Adjacent serial sections of all tissue blocks were stained with PAS and Sudan Black B and viewed unstained in a fluorescence microscope. The intraneuronal granular material was found to be positive in both histological stains and appeared strongly autoflu- orescent. Similar results were obtained in every brain area studied, autofluomscent, PAS- and Sudan Black B-positive granules al- ways matching the distribution of the immunostained material, and vice versa. From its morphological appearance, its regional distribution and its histochemical properties we conclude that the immunostained material is lipofuscin and that its reactivity with [3-protein antibodies is identical in different nerve cell populations. Reactivity of lipofuscin was independent from the presence of NP and anayloidotic blood vessels in the same brain. No difference was found in the immunoreactivity of this material in AD and normal control cases. Young adult controls and children presented with a lower number of lipofuscin containing nerve cells, often restricted to specific predilection sites, and, compared to aged individuals, the amount of pigment per neuron was also reduced. In the brain stem of children, the lipofuscin granules appeared smaller and less well demarcated than in the adult brain. Neu- romelanin remained unstained with all antibodies.

Pretreatment of tissue sections with formic acid led to enhance- ment of the immunostaining of lipofuscin which was, however, less dramatic than the effect observed on the reactivity of NP and amyloidotic blood vessels.

Control qf Specificiu

In order to ensure the specificity of the immunostaining the following control experiments were undertaken: 1) No staining was observed if the primary antibody incubation was omitted, for both the avidin/biotin system and the Protein A system. 2) Immunostaining with two mAbs to Cytomegalovirus belonging to

two different lgG subclasses at higher concentration than the [3-protein antibodies did neither stain lipofuscin nor amyloid deposits. A mAb to tau [Tau-I (2)] and a mAb to paired helical filaments [3-39 (29)], both staining neurofibrillary tangles and plaque neurites, did not stain lipofuscin or amyloid. 31 To rule out any unspecific antibody binding to lipofuscin due to electrostatic interactions, primary antibody incubation was performed at pH 5, pH 7.4 and pH 9. In all three cases specific staining of the lipopigment was maintained. 4) Absorption of the primary anti- body with 5 ~tg of synthetic [3-protein per ml diluted antibody abolished the staining of both, anwloid deposits and lipofuscin.

Immune Electron Microscopy

Electron microscopy confirmed the immunoreactivity to be localized to lipofuscin inside the cytoplasm of nerve cells (Fig. 2a). The reactivity was localized to the electron dense proteina- ceous matrix part of the lipofuscin granules (Fig. 2b, c). No fibers were detected in the lipofuscin matrix (Fig. 2d).

In view of the different ultrastructural morphology of lipofus- cin from amyloid deposits, the question arises whether the epitopes recognized by the mAbs to the [3-protein are localized on the same molecule in these two structures or whether the same reactive amino acid sequence is part of two different proteins. In order to further characterize the immunoreactive constituent of tipofuscin we assessed its electrophoretic mobility on a SDS- polyacrylamide gel.

Western Blots

The electrophoretic pattern of the lipofuscin enriched fraction stained with Coomassie Brilliant Blue is shown in Fig. 3, lane 2. lmmunoblotting of the same fraction with mAbs to synthetic 13-protein revealed a single reactive polypeptide migrating at approximately 31 kDa (Fig. 3, lanes 3, 5 and 7). Heating the samples in the presence of 8 M urea or 0.5% Triton X-100 did not lead to the release of any subunit polypeptides. A membrane fraction obtained from the same pellet did not yield any positive reaction. No positive reaction of the lipofuscin fraction was found when blots were reacted with a mAb to Cytomegalovirus (figure not shown).

A preparation of isolated neuronal cell bodies immunoblotted with a mAb to synthetic [3-protein showed staining of a band of identical molecular weight as that observed in the lipofuscin fraction (Fig. 3, lane 8).

DISCUSSION

The immunoreactivity of amyloid [3-protein antibodies with neurons has been shown previously at the light microscopic level (8,9). In the present study, we confirm this observation and we identify the immunolabelled neuronal component as lipofuscin. At both, the light and electron microscopic levels, we demonstrate reactivity of this neuronal pigment with 5 mAbs to a synthetic peptide corresponding to the first 24 amino acids of the amyloid [3-protein. On Western blots, these mAbs have been shown to label a polypeptide doublet of approximately 125 kDa, most likely

FACING PAGE

FIG. 2. Immune electron microscopy of neuronal lipofuscin with a monoclonal antibody to a synthetic cerebrovascular amyloid 13-protein. Subiculum of a 65-year-old control case. a: Subicular neuron with high quantity of lipofuscin. High density of 10 nm gold grains on lipofuscin granules ( x 10,000). b: Labelling of the proteinaceous matrix component of lipofuscin granule. Most of the lipid component has been washed out by the embedding procedure ( x 30,000). c: Dense immunolabelling of lipofuscin granules ( x 30,000). d: High magnification of the lipofuscin matrix shows the absence of filamentous profiles ( x 250,000).

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130 BANCHER ET AL.

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FIG. 3. Protein profiles and Western blots reacted with a monoclonal antibody to a synthetic cerebrovascular amyloid 13-protein. Lane 1: Molecular weight markers: Ovalbumin (43 kDa), c~-Chymotrypsinogen (25.7 kDa), 13-Lactoglobulin (18.4 kDa), Lysozyme (14.3 kDa), Cytochrome C ( 12.3 kDa), Bovine trypsin inhibitor (6.2 kDa). Lane 2: Electrophoretic pattern of l ipofuscin enriched fraction. The lane was loaded with 40 ixg of protein and stained with Coomassie Brilliant Blue. Lanes 3, 5, 7: The same fraction electroblotted to transfer membrane and reacted with anti-[3-protein. The sample was heated in 50 mM Tris-HCl, pH 7.4 in the presence of 1% SDS and 1% ME (lane 3); 1% SDS, 1% ME and 0.5% Triton X-100 (lane 5); I% SDS, 1% ME and 8 M urea (lane 7). Lanes 4 and 6: Synthetic amyloid 13-protein (amino acids 1-24) heated in the presence of 1% SDS, 1% ME and 8 M urea (lane 4) and 1% SDS and 1% ME (lane 6) and reacted with anti-13-protein. The synthetic amyloid [3-protein is a highly aggregating polypeptide and migrates more slowly than expected from its subunit Mr. of about 2.7 kDa, Lane 8: Fraction of isolated neuronal cell bodies reacted with a mAb to synthetic [?,-protein.

corresponding to two different molecular species of the predicted amyloid precursor protein(s) (9).

Specificity of the immune reaction was confirmed by using five different mAbs to synthetic 13-protein belonging to three different tgG subclasses, by using four control antibodies, by ruling out any electrostatic interactions between antibodies and the reactive part of lipofuscin and by absorbing the antibodies with their specific antigen. These findings, therefore, suggest the presence of the amino acid sequence recognized by our mAbs (amino acids 17-24 of the 13-protein) to be present on lipofuscin.

Immune electron microscopy showed that the reactive epitopes were localized on the proteinaceous matrix part of lipofuscin which does not contain any fibrous elements. This finding indi- cates that the domain of the 13-peptide present in both, lipofuscin and amyloid deposits is part of either two distinct or same but differently processed proteins in these two structures.

Western blots of lipofuscin enriched fraction revealed a 13- protein immunoreactive polypeptide with an apparent molecular weight of approximately 31 kDa. The molecular weight of the polypeptide forming amyloid fibers is approximately 4.5 kDa. However, this protein has a strong tendency to aggregate and

therefore can display a lower migrating velocity on polyacryla- mide gels and elute earlier from HPLC columns. In the literature apparent molecular weights of amyloid plaque core proteins and their polymers range from 4 to 64 kDa, according to preparation (4, 17, 18, 24). However, a band around Mr 31,000 has not been reported and polymers of the 13-protein do not focus in a sharp solitary band as observed in the present study. These results together with the fact that preparation of the lipofuscin sample in the presence of denaturing agents like 8 M urea or 0.5 % Triton X-100 does not release any lower molecular weight peptides suggest that the epitopes of lipofuscin recognized by our mAbs are unlikely to be localized on an octamer of the [3-protein. This indicates that two different size polypeptides react with these mAbs in brain amyloid and lipofuscin.

Several studies have shown the amyloid 13-protein to be derived from large precursor proteins with predicted molecular weights above 78 kDa (11, 13, 22, 28). On Western blots, an in vitro translation product corresponding to an amyloid precursor protein has been shown to migrate at 91.5 kDa (5). In brain homogenate and in extracts of muscle biopsies, polypeptides of 125 kDa (25) and 93 kDa (33), respectively, have been shown to react with

LIPOFUSCIN AND THE 13-PROTEIN 131

antibodies to the cytoplasmic domain of the amyloid precursor. Little is know yet about the biochemical events which lead to the cleavage of these proteins in a manner to release an amyloidogenic fragment. However, microglial cells are suspected to be involved in this process (30,31). In the central nervous system in situ hybridization studies suggest that the mRNA for the amyloid precursor protein is present in neurons (1, 7, 16, 26). However, neurons do not form amyloid fibers of the type found in NP. If one excludes a fortuitous amino acid homology between amino acid residues 17-24 of the [3-protein and the 31 kDa polypeptide associated with neuronal lipofuscin, our results suggest that 1) the latter protein may, as the B-protein itself, originate from the same precursor and 2) processing of the precursor in the neuron leads to different fragments than its breakdown in the microglial cell or in the extracellular space.

Lipofuscin is a lipopigment which physiologically accumulates in certain cell types during aging (21). Its gradual intracellular accumulation is probably the most characteristic cytological change associated with the process of aging of the brain. Cytochemically, it is considered to be an inert end product of lysosomal breakdown.

Immunoreactivity of lipofuscin was identical in AD patients and in aged controls. Moreover, areas of the brain rich in lipofuscin containing cells like the inferior olivary nucleus do not

match the predilection sites for the histopathology characteristic for AD. These findings support the opinion that the amyloid precursor protein is a normal constituent of certain nerve cells (1, 7, 16, 26).

A recent study reports that the amount of lipofuscin correlates with amyloid precursor protein mRNA levels in neurons of the rat brain (26), further supporting the connection between the amyloid precursor protein and the neuronal aging pigment. More studies are needed to further characterize this relationship.

ACKNOWLEDGEMENTS

The authors wish to express their thanks to Dr. J. Shek, Mrs. M. Lee and Mr. J. Holshek for expert technical assistance, to Mr. J. Chen and Mr. V. J. Sapienza for help in monoclonal antibody production and to Mrs. T. Zaidi for preparing lipofuscin fractions. Human autopsy material was kindly provided by Dr. H. Budka, Neurological Institute, University of Vienna, Austria. Dr. H. Lassmann provided helpful discussion. Mono- clonal antibody Tau-1 was generously supplied by Dr. L. I. Binder. We also express appreciation to Mrs. J. Kay for skillful secretarial assistance and to Mr. R. Weed for photographic supervision. This study was supported in part by grants NS 18105, AG 05892 and AG 04220 from the National Institutes of Health.

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