Journal of Neuroscience Research 33:355-369 (1992)

Human Serum Stimulates Alzheimer Markers in Cultured Hippocampal Neurons G.J. Brewer and J.W. Ashford Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield (G.J.B.); Department of Psychiatry, University of California, Davis, VA Medical Center, Martinez, California (J.W.A.) The mechanism for promoting the distinct types of lesions in the Alzheimer disease (AD) brain and other changes outside the brain is unknown. We examined neurons in culture, unprotected by glia or a bloodbrain barrier, to determine if exposure to serum from Alzheimer patients would affect markers for Alzheimer brain lesions. Rat hippocampal neurons were first grown for 4 days in a new serum-free culture medium, then exposed for 24 hr to human sera. Sera from 12 AD patients or their spouses increased four molecular markers characteristic of Alzheimer senile plaques and neurofibrillary tangles: Alz-50, P-amyloid (p/A4), MAP2, and ubiquitin, each with their expected cytologic distributions. Sera from ten young adults produced significantly less stimulation. By quantitative immunofluorescence, neuronal exposure to the elderly human sera produced 1.8- to 2.5-fold increases in mean fluorescent aredcell for each of these four markers relative to no serum exposure. As controls, an unrelated neuronal marker, enolase, was unaffected and fetal bovine serum did not stimulate immunoreactivity. Neuron viability and soma1 area were unaffected at 24 hours. The MAP2 increases were dose dependent with negligible effect at 2% serum and maximum effect at 10% serum after 24 hr. The MAP2 increase was greater after 48 hr of exposure than 24 hr and negligible at 2 hr. This stimulation of AD markers by human serum suggests that the genesis of both neuronal plaques and tangles may arise from access of toxic serum factors to susceptible neurons and/or failure to detoxify these factors. 0 1992 Wiley-Liss, Inc.

Key words: Ah-50, P-amyloid, MAP2, ubiquitin, immunocytochemistry, immunofluorescence INTRODUCTION Alzheimer’s disease (AD) is characterized by an age-related progressive loss of memory and other higher cognitive functions (Ashford et al., 1989; McKhann et al., 1984) in association with a critical concentration of 0 1992 Wiley-Liss, Inc.

senile plaques (SP) and neurofibrillary tangles (NFT) (Khachaturian, 1985). These brain lesions are not usually present in young individuals, but commonly occur in the elderly (Hauw et al., 1986; Ulrich, 1985). There is no evidence for a common mechanism of inducing these distinct pathological entities despite considerable progress toward identifying the molecular components of SP (Joachim and Selkoe, 1989) and NFT (Kosik, 1989) brain lesions. Additional findings of AD-related abnormalities outside the brain (Aubenko et al., 1987; Blass et al., 1989; Chakravarti et al., 1989; Foley et al., 1988; Giometto et al., 1988; Ingram et al., 1974; Jarvik et al., 1982, Jarvik and Matsuyama, 1984; Joachim el al., 1989; Kumar et al., 1988; Matsuyama and Fu, 1983; Peterson et al., 1986) suggest that a systemically acting factor or environmental influence may be responsible for inducing the brain lesions as well. However, only certain regions of the brain are particularly susceptible to the AD lesions. The susceptible regions, such as the hippocampus (Brun, 1983; Hirano and Zimmerman, 1962; Hyman et al., 1984), function heavily in memory processing (Coburn et al., 1990; Mishkin, 1978). The synaptic plasticity required for memory may be the basis for vulnerability to the Alzheimer pathology (Ashford and Jarvik, 1985; Butcher and Woolf, 1988; Represa et al., 1988). Therefore, we have developed a hippocampal neuron culture system to test for responses to systemic factors by expression of molecular markers that have been associated with SP and NFT. We show that human serum from either probable AD patients or their nondemented spouses produces increased immunoreactivity for four such markers, Alz-50, P-amyloid (P/A4), MAP2, and ubiquitin, in cultured hippocampal neurons. Neurons in this preparation may respond because they have virtually no protection by glia to detoxify a systemically borne

Received January 22, 1992; revised May 1, 1992; accepted June 1, 1992. Address reprint requests to G.J. Brewer, Department of Medical Microbiology and Immunology, Southern Illinois University School of Medicine, Springfield, IL 62704-9230.

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factor (Crols et al., 1986; Nandy and Reisberg, 1983; Panter et al., 1985; Yamaguchi et al., 1987), no layer of endothelial cells (the blood-brain barrier) for isolation from such a factor (Elovaara et al., 1987; Ishii and Haga, 1976; Pappolla and Andorn, 1987; Wisniewski and Kozlowski, 1982), and no immunoprotection (Farrar et al., 1987; Rogers et al., 1988). Since immunocompetence and other defenses deteriorate with age, stimulation of AD markers in culture is consistent with brain access of a systemic substance(s) or ineffective detoxification of that substance contributing to Alzheimer pathogenesis.

MATERIALS AND METHODS Cell Culture and Serum Treatment A novel serum-free medium was used to grow embryonic rat hippocampal neurons in culture (Brewer and Cotman, 1989). To prepare neurons for the immunofluorescence assay, four hippocampi were dissected from the brains of two 18-19 day gestation Sprague/Dawley rat embryos (Banker and Cowan, 1977). Neurons were isolated and plated at 40,000 cells/cm2 substrate, which results in >80% pyramidal neurons (Brewer and Cotman, 1989). Cells were plated in 0.4 ml of defined medium onto 13-mm-diameter coverslips precoated with polylysine in a 24-well dish. Cultures were incubated face up at 37°C in an atmosphere of 5% CO,, 9% oxygen in an automatic O2/CO, incubator (Forma, Marietta, Ohio). These target neurons develop an extensive network of neuronal processes during 4 days in vitro, yet individual neurons are clearly visualized. At this time, the hippocampal neurons were exposed to 10% serum from AD patients [Table I, six males, six females, mean age -+ S.D. = 73 -t 10, diagnoses (McKhann et al., 1984): 11 probable, one possible AD], or their unaffected spouses or siblings (four males, seven females, mean age = 68 -+ 7), or young adults (six females, four males, mean age = 25 5 3). The AD patients had complete dementia evaluations. None had diabetes, unstable hypertension, active infection, or other neurological disease that would have precluded a diagnosis of probable AD. All subjects were free of any significant illness and had taken no centrally active medications within the last 24 hr. Sera were collected by centrifugation of blood samples allowed to clot overnight at 4°C and stored in aliquots at -70°C. Viability was assessed by counting cells excluding 0.2% trypan blue or by metabolic labeling with fluorescein diacetate (Jones and Senft, 1985). Cell size was obtained from phase contrast images on 50 X magnification negatives. These images were digitized by video input into a PC/AT computer and the phase bright boundaries including apical dendrites down

to one-fourth the soma1 diameter were traced with a digitizing tablet and analyzed with Bioquant software.

Quantitative Immunofluorescence Molecular markers characteristic of AD were evaluated by immunofluorescence techniques. Since some molecules known to be abnormally present in AD pathological lesions are naturally present in fetal neurons (Selkoe et al., 1988; Wolozin et al., 1988), it was necessary to rigorously quantitate the amount of fluorescence produced by serum in comparison to various control and background levels. At 24 hr after addition of serum, neurons were rinsed with PBS, fixed in salinephosphate buffered 3.7% formaldehyde, and AD-marker antibodies were applied. An appropriate fluorescejn-labeled secondary antibody was used to visualize the distribution of the primary antibody-antigen complex. Two measures of fluorescence were obtained from each photograph: mean cellular brightness (intensity) and total fluorescent area per cell (see below). By these techniques, the distribution of cellular brightness could range from localized to relatively uniform, and the mean fluorescent area/cell could vary with how much of each neuron is fluorescent and the fraction of fluorescent neurons over nonfluorescent neurons. Quantitative immunofluorescence was achieved with a Nikon Diaphot microscope by minimum constant excitation through a 0.5 neutral density filter, 60 X /1.4 n.a. plan-apo phase contrast oil immersion objective, B 1A filter set and photography on Ektachrome P800 slide film, 8-sec constant exposure in a darkened room. After parallel E6 development of multiple rolls with push processing one stop, illuminated slides were digitized with a computer and video camera set to constant gain and contrast (Bioquant MegIV, R & M Biometrics). The photographic step was included because a direct video link from the fluorescent microscope to the digitizing computer was not available. Initial variations in excitation intensity and film exposure were conducted to insure that exposures did not saturate the film. The neutral density filter minimized fluorescent bleaching to 9% over the course of exposure. Linearity of the digitization process was confirmed by varying the film exposure, measuring the transilluminated light flux with a power meter, calibration with neutral density filters, and comparing to digitized brightness values. Under phase contrast optics, photographic fields were selected to contain a minimum of four cells, avoiding fields with debris or overlapping cells bodies, i.e., fields with clusters containing more than four cells in a cluster. Since this precluded knowledge of fluorescence and the sample identity was coded, experimental bias in quantitation was precluded (i.e., “blind” analysis). Four types of controls were a) no serum exposure of the neurons, b) no primary antibody, c) no cells, and d) an

Alzheimer Markers Induced by Serum

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TABLE I. Patient and Control Subject Sera Tested for Effects on Cultured Neurons Immunofluorescence probabilities" Subjects

1. la. Wife Ib. Father (single stroke) 2. 2a. Wife

3. 3a. Wife 4. Mother of 3 5. 5a. Sister 6. 6a. Husband 7. 7a. Wife

8. 8a. Husband

9.

9a. Husband 10. 10a. Wife 11. Brother of 10 1 la. Wife 12. 12a. Husband 13. Multiple strokes Young adults 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Acre/Sex

Duration

DX

MMS

EVA4

Alz-SO

MAP2

65 M 65 F 90 M

13

1 4

0

,001 ,000

n.s. n.s.

5

30

.019

n.s. ,024 ,000

64 M 62 F 68 M 67 F 89 F 84 F 75 F 65 F 66 M 58 M 56 F 78 F 83 M 71 F 69 M 83 M 63 F 83 M 72 F 70 F 72 M 81 M

4

1

22 12

n.s. n.s. ,008

0 21

,011 ,000

4

7 9 5 4

1 4 1

I 4 1 4

22

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,000 ,002 ,000 .035

.001

,005 n.s. n.s. n.s.

,013 ,004

,010 ,001

,002 ,002

,017 .018 ,001

20

1

4

4 2 4

11

4

1

7

3

4 1 4

5

n.s. ns. ,002

5

1

3

,036

.ooo

.014

n.s. ,002 ns. ,007

ns. ns. n.s. n.s. .026 n.s. n.s. n.s. n.s. n.s.

n.s. n.s. n.s. n.s. n.s. n.s. .042 n.s. n.s. ns.

13

26 F 26 M 25 M 22 M 26 F 28 F 25 F 29 M 23 F 21 F

4 1 4 5

19

,000

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,000

I 0

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,005

,039 ,006

,046

,001

.ooo .ooo ,000 ,001 ,012

.03 ns. n.s. ns. n.s. n.s. ns. n.s. n.s. n.s.

Abbreviations: DX (diagnosis, Khachaturian, 1985): 1 = probable AD; 2 = possible AD; 3 = not AD; 4 = normal; 5 = neurological control (stroke). Duration = date of evaluation - reported date of onset in years. MMS = Mini-mental State scale (Folstein et al., 1975): 30 = no impairment; 0 = maximum impairment. Spouses were all estimated to have no impairment. n.s., not significant. aProbability that immunofluorescence is significantly greater than neurons not treated with serum, Hotelling's TZstatistic, multivariate analysis of variance (MANOVA). Anti-P/A4, 2 X 20 degrees of freedom; Alz-50 (2 X 25 degrees of freedom), and anti-MAP2 (2 X 13 degrees of freedom).

irrelevant antibody of the same immunoglobulin type: an IgG monoclonal against a mouse mammary tumor antigen (courtesy of Walter Myers) and an IgM monoclonal against a mouse red cell antigen (courtesy of Edward Moticka). Approximately 100 cells were analyzed at 150 x film magnification from at least four color slides for each serum treatment.

Calibration of Immunofluorescence Calibration with purified antigens was conducted with the P/A4 peptide, residues 12-28 (Peninsula Lab-

oratories, Belmont, CA). Peptide dissolved in 99% formic acid was spotted onto glass coverslips at the indicated concentrations, air dried, and processed as for immunocytochemistry . Testing other antigens would be desirable, but they were not available.

Calculations Camera brightness and contrast settings were adjusted once for each AD-markeriantibody to count pixels over a range of brightness bins, with bins 1 to 4 contain-

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ing all the background (the no cell, full antibody condition, not shown in the figures). Settings were further adjusted so that bins 5 to 21 contained pixels of cellular fluorescence (10 grey scale units per bin) taking care not to allow pixels in bin 21, which would fail to accurately represent the brightest areas. A magnification of seven pixels per km2 was determined. The average fluorescent arealcell was obtained from each slide by determining the total fluorescent area divided by the number of cells in that slide. Weighted mean brightness was calculated from digitized slides as the average brightness of each slide. The weighted brightness was the product of the fluorescent area at each brightness bin times that brightness bin number divided by the total fluorescent area on the slide. Statistical analysis between groups was performed by multivariate analysis of variance (MANOVA for brightness and area). Probabilities were calculated from Hotelling’s ? test with the SPSS-X program on an IBM main frame with the assistance of Dr. Paul Kolm, Division of Statistics and Research Consulting. Student’s two-tailed r test was used for univariate analyses. The null hypothesis was rejected at P < 0.05.

normal neurons, was kindly provided by Kenneth Kosik (Harvard, Boston, MA). Washed and fixed neurons were blocked with 1% BSA, 1% normal sheep serum, 0.05% Triton X-100, followed by 5F9 at a dilution of 1:20. The secondary antibody was a 1:lOO dilution of sheep antimouse IgG (Fab’,) coupled to fluorescein (Sigma).

Neuron-Specific Enolase (NSE) Immunofluorescence NSE antibody E8F9 (Boss et al., 1987; Haan et a]. , 1982) was kindly provided by Barbara Boss (Hana Biologics, Alameda, CA). Washed neurons were fixed 5 min with 2% paraformaldehyde, 0.1 M sodium acetate, pH 6, rinsed and fixed with 2% paraformaldehyde, 0.1 M sodium borate, pH l l . After rinsing, neurons were treated with the same block as in the P/A4 tests, followed by E8F9 at a dilution of 1:6,000 in blocking solution. The secondary antibody was a 150 dilution of goat anti-mouse IgG (Fab’ ,human serum adsorbed) coupled to fluorescein (Tago).

RESULTS Effect of Serum on Cell Viability, Morphology, and Size Alz-50 Immunofluorescence To examine effects of serum on cell viability after Alz-50 monoclonal antibody (Wolozin et al., 1986) 4 days in serum-free medium, fetal rat hippocampal neuculture supernatant was kindly provided by Peter Davies rons in culture were exposed for 24 hr to 10% serum (Albert Einstein College of Medicine, Bronx, NY). For from two AD patients, two spouses of AD patients, two washed and fixed neurons, nonspecific sites were young subjects, or no serum. Viable cell counts by flublocked with 1% BSA, 1% normal goat serum, 0.05% orescence of hydrolysed fluorescein diacetate (Favaron Triton X-100 in PBS for 5 min. AIz-50 hybridoma culet al., 1988) were similar for all sera, averaging 105 2 ture supernatant was applied at 1:25 dilution in blocking 6% (mean i S.E.) of the no serum values and 98 t 5% solution for 1 hr at room temperature. After rinsing, goat of the day-before counts (three 0.3 mm2 fields on each of anti-mouse IgM conjugated to fluorescein (Sigma, St. three coverslips); Student’s two-tailed t test indicated no Louis) was used for detection at a 1 5 0 dilution in blocksignificant differences. Figure 1B shows a phase contrast ing solution. Coverslips rinsed in PBS were mounted on image in which a rich neuropil of healthy cells is evident slides with Aquamount and photographed. in cultures not exposed to serum. Figure 1A shows a P-Amyloid (PlA4) Immunofluorescence reduction in neuropil in a culture exposed to serum from To expose the @/A4epitope, washed and fixed cul- the spouse of an AD patient. In cultures exposed to eltures were treated for 30 min with 70% formic acid (Ki- derly serum, reductions in neuritic- processes were obtamoto et al., 1987) and rinsed with PBS. Nonspecific served consistently. Quantitation of this effect will be the sites were blocked as above with the addition of 1% subject of a future communication. Since immunofluorescence is dependent on the size normal human serum. Hybridoma culture supernatant 4G8 (Kim et al., 1988), kindly provided by K.S. Kim and of the fluorescent cells, the effect of serum on the area of Henry Wisniewski (New York Institute for Developmen- individual neurons was assessed from phase contrast imtal Disabilities, Staten Island, NY), was diluted 1:lOO in ages. Computer digital analysis of the outlines of 66 blocking solution and incubated 30 min at room temper- neurons from four coverslips (soma + apical dendrite) ature. Bound antibody was detected with a 1:50 dilution treated with one spousal serum (subject 10a) yielded a of goat anti-mouse IgG (adsorbed with rat and human mean area of 176 i 9 pm2 (S.E.). The mean area of 81 IgG) conjugated to fluorescein (Tago, Burlingame, CA). neurons treated with young serum (subject 14) was 163 f 8 pm2. This 7% difference is not significant. MAP2 Immunofluorescence MAP2 monoclonal antibody 5F9, which reacts Induction of a Plaque and Tangle Marker strongly with dystrophic neurons and some NFT (Kosik The first marker studied was a 68-Kd tau protein et a]. , 1984; McKee et al., 1989) and less with MAP2 in recognized by the Alz-50 monoclonal antibody that was

Fig. 1. Phase contrast (A,B) and corresponding A h 5 0 immunofluorescence (C,D) for (A,C) neurons treated with spousal serum (subject 3a) compared to (B,D) untreated neurons reveals brighter fluorescence, especially in axon-like processes, and a greater cellular fluorescent area above the background. Fluorescence images are printed as negatives for better contrast, magnification = 5 17 x .

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Fig. 2 . A: Digitization confirms the increase in brightness and area for Alz-50 immunoreactivity, especially for the brightest processes in the brightest bin. B: Quantitative Alz-50 immunofluorescenceof neurons exposed to 12 AD, 11 spousal, two stroke sera, and three AD or spousal sera reacted with an irrelevant primary antibody compared to untreated neurons and neurons treated with nine of 10 young adult sera (mean S.E.).

*

obtained by immunization of a mouse with an AD brain was fetal bovine serum (data not shown). Nineteen of 25 homogenate (Wolozin et al., 1988). Alz-50 immunore- elderly sera tested (76%, including two elderly stroke activity is greatest in NFT but also reacts with SP and sera) produced significantly greater immunof luorescence neurons possibly in early stages of tangle formation than no serum ( P = 0.01, Fig. 2b, Table I). The in(Hyman et al., 1988). The antibody recognizes aphosphor- creases in mean fluorescent area per cell for elderly seylated epitope on the axonal microtubule associated pro- rum above young serum treatments could be due to an tein, tau (Ksiezak-Reding et al., 1988; Ueda et al., actual increase in fluorescent area of each cell or an 1990). In the fields corresponding to the phase contrast increase in the number of fluorescent cells within the images in Figures lA,B, Alz-50 immunofluorescence is sampled population. Comparisons with cell numbers shown in Figures lC,D for neurons treated with serum from phase contrast images indicate more fluorescent from spousal subject 3a (Table I) in comparison to un- neurons and fewer nonfluorescent neurons, not an intreated neurons. The fluorescence images show that the crease in the fluorescent area of each neuron. serum caused large increases in perikaryon fluorescence, Further possible insight into the relationship of elas seen in neurons from AD brains. Digital analysis of derly sera to stimulation of AD-like immunoreactivity the effects of this serum indicates both an increase in was investigated in correlations within the data. A dosebrightness and a higher mean fluorescent areaineuron dependent environmental factor might produce evidence (Fig. 2A). However, treatments with ten young sera in- of high immunoreactivity from spouse sera whose AD dicated that only the mean areaheuron increase was spe- cohort was high and vice-versa. This correlation was not cific for the elderly sera (Fig. 2B). evident (R2 = .17, F = 0.24, P = .63, data not shown). When compared to young serum treatment, there The strong correlation of AD with age might be reflected was significantly greater Alz-50 immunofluorescence for in the potency of the sera to stimulate Alz-50 immunoAD, spouse, and elderly stroke serum treatments (Table reactivity. Figure 3 shows a significant correlation of 11). The difference between treatments with AD and Alz-50 immunoreactivity with age of the individual spousal sera were insignificant. Compared to young sera, providing the serum (R2 = .455, F = 7.8, P = .009). the AD sera treatments averaged 2.1 times higher in However, insignificant correlations were found for AD fluorescent areaineuron. Specificity is indicated by weak sera alone (R2 = .278, F = .75, P = .4), spouse sera fluorescence obtained when an irrelevant primary IgM alone (R2 = .172, F = .24, P = .6), or combined was substituted for the Alz-50 (Fig. 2B). Nine of ten sera (R2 = .188, F = .77, P = .4). Therefore, the major from young adults were not significantly different from effect is due to the low Alz-50 immunoreactivity of the untreated neurons (Fig. 2B, Tables I and II), and neither young sera rather than any relation among the elderly sera.

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Brightness and Area Measures of Immunofluorescence* Serum [n] mean area (S.D.) (w2) mean brightness (S.D.) Alz-50 AD [53] 97 (84) 9.2 (1.2) Spouse [47] 68 (51) 9.4 (1.5) Young (601 46 (45) 9.7 (2.1) Stroke [4] 160 (37) 11.1 (0.2) None [42] 43 (33) 8.9 (1.6) plA4 AD [50] 69 (49) 6.4 (1.2) Spouse [52] 80 (63) 6.4 (1.1) Young [60] 55 (44) 6.8 (1.7) Stroke [8] 81 (38) 6.2 (0.6) None [49] 41 (44) 6.1 (1.1) Fetal calf [8] 8 (6) 6.4 (1.3)

MAP2 AD [25] 74 (46) 7.5 (1.7) Spouse [48] 94 (80) 8.2 (2.5) Young [49] 59 (34) 6.8 (1.1) None [31] 45 (45) 6.9 (1.7)

0 N

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subjezt age (years) F(2,99) = .9 F(2,107) = 5.5 n.s P = ,005

F(2,96) = 4.4 P = .O1

F(2,109) = 11.4 F(2,98) = 7.3 P < .0005 P = ,001 F(2,106) = 3.3 P = .04 F(2,57) = . 3 n.s.b

F(2,65) = 5.8 P = .W5

F(2,55) = 4.3 F(2,63) = 3.4 P = .02 P = .04

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F(2,87) = 6.3 P = .003 F(2,88) = 2.6 n.s.

*Hotelling's T2 statistic, multivariate analysis of variance (MANOVA), P > .05 = n.s., not significant. "Univariate F test for brightness. Multivariate test was n.s. bThe difference between stroke sera and AD sera was similar. For Alz-50, stroke sera were even greater than spouse or AD sera treatments.

Fig. 3. Correlation of age of subject with Alz-50 immunofluorescence of neurons treated with sera.

Induction of Ubiquitin Immunoreactivity Another antigen associated with both SP and NFT in Alzheimer's disease is ubiquitin (Perry et al., 1987). Ubiquitin is a 76 residue protein that becomes covalently linked to other proteins to target them for degradation in response to stress (Munro and Pelhan, 1985). The addition of ubiquitin to tau correlates with the loss of Alz-50 immunoreactivity in AD brain sections (Bancher et al., 1989a; Vincent and Davies, 1990). Figure 4 shows a histogram of the ubiquitin immunofluorescent brightness and area measures of neurons treated with either young serum (Fig. 4A) or AD serum (Fig. 4B). In addition to the weak fluorescent population of neurons seen for either treatment, the AD serum produced a new population of neurons with both greater fluorescent brightness and areahemon. These data suggest that a subpopulation of neurons in our hippocampal cultures is selectively more susceptible to stimulation by elderly serum. Additional experiments can determine whether these are the CAI pyramidal neurons that also show increased sensitivity to glutamate toxicity in culture (Mattson and Kater, 1989; Mattson, 1990). Induction of a Plaque Marker Another AD-associated antigen examined was pamyloid (P/A4), the primary peptide constituent of SP (Joachim et al., 1989), although possibly present in NFT (Hyman et al., 1989) and lipofuscin (Bancher et al.,

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Brewer and Ashford

A

B

Fig. 4. Anti-ubiquitin immunofluorescenceis mostly brighter for a subpopulation of neurons treated with AD serum (B) in comparison to young serum (A). 1989b). The cDNA sequence of amyloid precursor protein (APP) predicts that the 4-kD P/A4 fragment is derived by posttranslational processing from either a 770, 75 1 , or 695 residue, membrane-associated precursor (Dyrks et al., 1988; Kang et al., 1987; Tanzi et al., 1988). Levels of certain APP fragments increase severalfold in the brain with age (Nordstedt et al., 1991). To detect the P/A4 sequence in the hippocampal neurons, we used monoclonal antibody 4G8 against a synthetic peptide fragment of PIA4, residues 17-24, whose exposure is greatly increased in AD brain (Kim et al., 1988). Quantification of the fluorescence immunoreactivity of the isolated peptide indicates extraordinary linearity of response over three orders of magnitude (Fig. 5 ) . These data permit estimation of P/A4 or p-APP concentration in individual neurons. Figure 6A shows anti-P/A4 immunofluorescence from neurons treated with AD serum (subject 4), in comparison to neurons not exposed to serum, as shown in Figure 6B. The immunofluorescence of many treated cells covers a larger area per neuron than untreated neurons. This was not due to larger cells, as noted above. Deposition from the serum seems unlikely since immunofluorescence was not increased by serum exposure for only 2 hr. Therefore, the pericellular distribution of the marker, similar to the distribution of P/A4 in SP, suggests secretion and is consistent with a cell adhesion or extracellular matrix molecule (Dyrks et al., 1988; Glenner and Wong, 1984; Kang et al., 1987; Klier et al., 1990; Selkoe et al., 1988). Computer digitized fluorescence analysis of this serum (Fig. 7A) and ten other sera (Fig. 7B) indicates that elderly human serum produced a mean 1 .%fold greater fluorescent area per cell than untreated neurons. Compared to young serum, the spousal

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Fig. 5. Calibration of anti-P/A4 immunofluorescence digitization with synthetic P/A4 peptide (residues 12-28). Peptide was dissolved in formic acid, applied to glass coverslips at the indicated concentrations, dried, and processed for immunoreactivity and digitization. sera as a group and the AD sera as a group produced significantly more immunofluorescence (Table 11). Figure 7B also shows the specificity of the immunoreactivity by leaving out the primary antibody or reacting with an irrelevant IgG antibody. As with Alz-50, there was no

Alzheimer Markers Induced by Serum

363

Fig. 6. Anti-PlA4 immunofluorescence is both brighter and originates from a larger areal neuron for (A) neurons treated with AD serum (subject 4)compared to (B) untreated neurons. Magnification = 5 17 X significant difference between sera from AD patients and their spouses (Table 11) and nine of ten sera from young adults were not significantly different from untreated neurons (Fig. 7B, Tables I and II), nor was fetal calf serum (data not shown). There was insignificant correlation between the magnitude of fluorescence produced by AD serum vs. spouse serum (R2 = .43). Age of the serum donor was not found to be significantly correlated with P/A4 immunoreactivity of neurons.

Induction of a Cytoskeletal Marker The last AD-related antigen whose expression is reported is a form of the brain dendrite-specific microtubule associated protein 2 (MAP2). The epitope of MAP2 recognized by monoclonal antibody 5F9 was originally reported to stain N I T in the brain (Kosik et al., 1984); however, a more recent report with a different fixative failed to demonstrate reactivity in classical N I T , but found staining of CAI neurons with severely altered architecture (McKee et al., 1989), perhaps precursors of N I T . The 5F9 immunofluorescence of cultured neurons

treated with AD serum (subject 7) is both brighter and originates from a larger fraction of fluorescent neurons than untreated cells (Fig. 8A,B). The distribution of fluorescence in the perikarya and apical dendrites resembles that seen in dystrophic neurons in AD brains. From this pair of serum-treated and untreated neurons (Fig. 9A) and other such digitized fluorescence analyses (Fig. 9B), eight of the ten elderly sera tested showed statistically significant increases in MAP2 immunoreactivity, compared to untreated neurons (Table I). Compared to young sera, sera from AD patients, as a group, and their spouses, as a group, produced greater MAP2 immunofluorescence (Table 11). This immunofluorescence for AD sera was only significant for the brightness measure. For the spouse sera, the increased brightness was more significant than the increased fluorescent area. AD and spousal sera produced a mean 40% increase in brightness measure per unit area and a mean 1.9-fold increase in the number of fluorescent neurons (to yield a higher area/ neuron average). Again, the difference between AD and spousal serum treatments was not significant ( P = 0.377); there was insignificant correlation between the

364

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weighted meon brightness

Fig. 7. A: Digitized anti-P/A4 immunofluorescence reveals a greater fluorescent areakell and more fluorescent cells for neurons treated with the AD serum shown in Figure 6A compared to 6B. B: Quantitative immunofluorescence of neurons exposed to 22 AD and spousal sera compared to untreated neurons or neurons exposed to serum but reacted with an irrelevant antibody or neurons exposed to nine of ten young adult sera.

Fig. 8. MAP2 irnmunofluorescence reveals brighter perikarya fluorescence and a greater number of fluorescent neurons above the background for neurons treated with (A) AD serum (subject 7) compared to (B) no serum. Magnification = 517 X .

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1' spouse irreievont serum Ab : i

EiEB no serum

160 15

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4

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serum

young

I

100

= 10 V

-2 Y

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Fig. 9. A: Digitization confirms the increases in MAP2 immunoreactivity. B: Quantitative immunofluorescence of neurons exposed to ten AD and spousal sera compared to untreated neurons or neurons exposed to nine of ten young adult sera. 250H

200 -

MI

AD serum young serum

B T

150100-

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hours of s s r u m exposure

Z serum

Fig. 10. Increase in MAP2 immunofluorescent cell area with duration of exposure to 10% serum (A) or with serum concentration after 24-hr exposure (B) (mean S.E.).

*

magnitude of fluorescence produced by AD serum vs. spouse serum (R2 = .69); and nine of ten sera from young adults produced immunofluorescence similar to no serum treatment (Fig. 9B, Table I), as did fetal calf serum (data not shown). For neurons treated with spousal serum, the mean MAP2 immunofluorescent area of individual neurons above background was 170 pm2, not significantly different from the phase area. However, for neurons treated with young serum, the diminished brightness resulted in significantly less mean fluorescent area above background, 24 km2/cell. These numbers were obtained by analysis of individual neurons and are not comparable to those presented in Figures 2, 7, and 8, which are obtained more rapidly as averages of four to 30 cells per photographic slide. Also, the lack of localization of Alz50 and @/A4immunofluorescence to the soma often pre-

cludes assignment of immunoreactivity to particular cells. The kinetics of 5F9 MAP2 immunoreactivity was examined for one spouse serum (subject 9a) and one young serum (subject 18). Figure 10A shows that 2-hr serum exposure is insufficient to produce an increased immunoreactive cell area. This argues against simple adsorption of immunoreactive serum components to the neurons. The process documented above at 24 hr is up to sevenfold stronger than young serum after 48 hr of serum exposure. Similar results were obtained for mean cell brightness. Figure 1OB indicates the dose dependence of 24 hr serum exposure on 5F9 immunoreactive cell area. Differences between AD and young serum were not significant at 2% or 5% serum. The major effect at 10% serum is not further increased by treatment with 20%

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serum. Similar results were obtained for mean cell brightness.

Induction of Any Marker? As a negative control for the pan-induction of neuronal markers, we investigated the glycolytic enzyme enolase whose isozyine is specific to neurons (Boss et al., 1987; Haan et al., 1982). One AD serum (subject 5 , Table I), a “spouse” serum (subject 5a), a young serum (subject 14), and no serum treatments were compared in triplicate for differences in immunofluorescence. Strong immunoreactivity characteristic of neuronal staining was evident in all samples. Immunofluorescence differences between AD or “spouse” sera and young or no serum were not significant by MANOVA (maximum F(2,20) = 2.1, minimum P = 0.15).

et al., 1988, 1991) and P/A4 fragments (Whitson et al., 1989) increase survival and neuritogenesis of cultured cortical and hippocampal neurons. Other APP fragments can be trophic or toxic to cultured neurons depending on the age of the neurons and the concentration of the fragments (Yankner et al., 1990). Blass et al. (1991) have also observed increased expression of Alz-50 and PHF immunoreactivities in fibroblasts cultured from AD patients compared to cultures from control subjects. In support of the rapidity of the response, increased expression of APP mRNA occurs 24 hr after treating cultured rat neurons with heparin (Octave et al., 1989) and glutamate increases Alz-50 immunoreactivity in cultured hippocampal neurons within 4 hr (Mattson, 1990). All of these observations could be due to induction of proteins such as P/A4 that have a stress-related heat shock promoter (Salbaum et al., 1989; Abe et al., 1991; Papasozomenos and Su, 1991).

DISCUSSION Molecular Basis Context These results demonstrate that human sera from If the marker changes observed in culture are reprobable AD patients or their elderly spouses can produce lated to those observed in AD, the culture system could major increases in both plaque and tangle markers in aid investigation of the molecular basis for these cultured rat hippocampal neurons within 24 hr, while changes. Plausible mechanisms include glutamate toxicnone of the effects were observed with only 2 hr of ity (Deboni and Crapper-McLachlan, 1985; Greenamyre exposure. An increase in mean fluorescent areakell could and Young, 1989), aberrant stimulation by a growth facbe stimulated by an increased fraction of fluorescent cells, tor (Butcher, and Woolf, 1988; Mattson et al., 1989; an increase in fluorescent area of individual cells, or a Mobley et al., 1988), protease or protease inhibitor decrease in cell area. Examination of phase contrast im- (Wagner et al., 1989), or perturbation by antibodies ages showed no effect of any serum on size of the soma (Chapman et al., 1989; Foley et al., 1988) and/or comand apical dendrite. Therefore, the observed increases in plement (Eikelenboom et al., 1989; McGeer et al., immunof luorescence due to elderly human sera represent 1991). Mattson (1990) reported that glutamate excitotoxincreases in antibody binding sites due to either an in- icity or calcium influx increases immunoreactivity to creased number of cells producing antigen, as in the case Alz-50 with an axonal distribution similar to our results. of Alz-50 and MAP2, or an increase in antigens in a fixed He also reported elevated tau and ubiquitin immunoreportion of cells (ubiquitin, P/A4, and MAP2). This in- activities caused by the same agents, but failed to obcrease was intracellular for Alz-50, ubiquitin, and MAP2, serve changes in reactivity with a different MAP2 antilocalized to the soma and apical dendrite, and both in- body (AP9) whose immunoreactivity is not affected in tracellular and extracellular for P/A4 immunogenicity . AD. It seems unlikely that glutamate levels are suffiThe background levels of Alz-50 and MAP2 immunore- ciently different in the sera we tested to account for our active neurons treated with young serum is concordant results by this mechanism. Initial characterization of the with the staining of postnatal hippocampal neurons in the serum factor indicates some heat lability and partial rerat brain slices with Alz-50 and with a different MAP2 tention of activity after dialysis, properties inconsistent antibody (Hamre et al., 1989). Since there are many with glutamate. More likely, other serum factors activate cytoplasmic components, colocalization of these markers a common pathway that leads to a pleiotropic response, in neonatal brain does not imply cross-reactive epitopes. possibly through elevated intracellular calcium. Since these markers are greatly reduced in normal adult brain, the increases seen in AD and in our cultures treated Significance with elderly serum may indicate reinduction of developThe increased expression of multiple AD markers mentally controlled expression. in culture supports a conjunctive mechanism of induction These changes in culture could be related to the of SP and NFT in AD, as opposed to two independent abnormal phosphorylation of a number of proteins in AD mechanisms. However, these data do not resolve the lesions (Zhang et al., 1989). Other effects of factors in issue of whether one factor induces only one type of AD brain extracts (Uchida and Tomonaga, 1989; Uchida abnormality that leads to other types (Joachim et

Alzheimer Markers Induced by Serum

al., 1989) (a linear model of causation) or whether an exogenous factor or group of factors can stimulate all of the molecular changes (divergent or pleiotropic model). In contrast to the years required for P/A4 induction in humans (Rumble et al., 1989), the rapidity of the increases in AD markers in culture may be related to the increased access of the serum to the neurons in vitro or the absence of detoxification. The finding that spousal sera are just as effective as AD sera may relate to a common environmental agent and a preclinical state of spouses of AD patients. Aged controls with no history of AD in the family are being recruited to test this possibility. Alternatively, a factor that promotes the development of AD may develop in the sera of all elderly individuals, with the development of AD due to a loss of defense, such as a breakdown of the blood-brain barrier, immunodeficiency, or altered lysosomal processing (Cataldo et al., 1991). The observation of SP and/or NFT in nearly all elderly brains (Hamre et al., 1989; Ulrich, 1985j , the presence of brain-reactive antibodies in the sera of elderly humans (Ingram et al., 1974), and discordance of AD among monozygotic twins (Rapoport et al., 1991) suggests a role for differential susceptibility to Alzheimer dementia among all the elderly. The smaller increases in immunoreactivity of neurons exposed to young subject serum further suggest that an active serum factor may be present but in lower concentrations or abrogated by an inhibitor. The net result would be a quantitative difference in production of SP and NFT.

Future The simplicity of this culture model provides a useful means to simulate the induction of AD-like pathologic markers. This will facilitate direct tests of specific pathogenic mechanisms such as glutamate toxicity, antibody binding, or induction by specific growth factors or proteases. Finally, this model will facilitate assays for isolating a factor from fractionated serum that appears to stimulate AD-like markers. ACKNOWLEDGMENTS We thank Drs. Kim and Wisniewski for provision of the anti-P/A4 antibody, Dr. Peter Davies for the Alz50 antibody, and Dr. Kenneth Kosik for the anti-MAP2 and anti-ubiquitin antibodies. Early discussions with Dr. Lawrence Domino and the excellent technical assistance of Danielle Richer, Melanie Bedolli, and John Torricelli are gratefully acknowledged. We also thank Drs. Paul Kolm and Jerry Colliver for assistance in statistical evaluations. This work was supported in part by the Illinois Department of Public Health, Alzheimer Research Program, the Pearson Family Foundation, NIH BRSG 2 SO7 RR05843-0 and SIU School of Medicine.

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REFERENCES Abe K, St. George-Hyslop PH, Tanzi RE, Kogure K (1991): Induction of amyloid precursor protein mRNA after heat shock in cultured human lymphoblastoid cells. Neurosci Lett 125: 169-171. Ashford JW, Jarvik L (1985): Alzheimer’s disease: Does neuron plasticity predispose to axonal neurofibrillary degeneration? N Engl J Med 313:388-389. Ashford JW, Kolm P, Colliver JA, Bekian C, Hsu LN (1989): Alzheimer patient evaluation and the mini-mental state: Item characteristic curve analysis. J Gerontol 44:139-146. Aubenko GS, Wusylko M, Cohen BM, Boller F, Teply I (1987): Family study of platelet membrane fluidity in Alzheimer’s disease. Science 238539-542. Bancher C, Brunner C, Lassmann H, Budka H, Jellinger K, Seitelberger F, Grundke-Iqbal I, Iqbal K, Wisniewski HM (1989a): Tau and ubiquitin immunoreactivity at different stages of formation of Alzheimer neurofibrillary tangles. Prog Clin Biol Res 317:837-848. Bancher C, Grundke-Iqbal I, Iqbal K, Kim KS, Wisniewski HM (1989b): Immunoreactivity of neuronal lipofuscin with monoclonal antibodies to the amyloid @-protein. Neurobiol Aging 10:125-132. Banker GA, Cowan WM (1977): Rat hippocampal neurons in dispersed cell culture. Brain Res 126:397-425. Blass JP, Baker AC, Sheu K-FR,KO L, Black RS, Smith A (1989): Use of cultured skin fibroblasts in studies of Alzheimer’s disease. In Boller S, Katzman R, Rascol A (eds.): “Biological Markers of Alzheimer Disease.” New York: Springer Verlag, pp 153-162. Blass JP, Baker AC, Sheu K-FR, KO L, Sheu R, Black RS (1991): Expression of ‘Alzheimer antigens’ in cultured skin fibroblasts. Arch Neurol 48:709-717. Boss B, Gozes I, Cowan W (1987): The survival of dentate gyms neurons in dissociated culture. Dev Brain Res 36: 199-218. Brewer G, Cotman C (1989): Survival and growth of hippocampal neurons in defined medium at low density: Advantages of a sandwich culture technique or low oxygen. Brain Res 494: 65-74, Brun A (1983): An overview of light and electron microscopic changes. In Reisberg B (ed): “Alzheimer’s Disease, the Standard Reference.” New York: The Free Press Publisher, pp 37-47. Butcher LL, Woolf NJ (1988): Neurotrophic agents may exacerbate the pathologic cascade of Alzheimer’s disease. Neurobiol Aging 10557-570. Cataldo AM, Paskevich PA, Kominami E, Nixon RA (1991): Lysosoma1 hydrolases of different classes are abnormally distributed in brains of patients with Alzheimer disease. Proc Natl Acad Sci USA 88:10998-11002. Chakravarti A, Slaugenhaupt S, Zubenko G (1989): Inheritance pattern of platelet membrane fluidity in Alzheimer disease. Am J Human Genet 44:799-805. Chapman J, Bachar 0, Korczyn A, Wertman E, Michaelson D (1989): Alzheimer’s disease antibodies bind specifically to a neurofilament protein in torpedo cholinergic neurons. J Neurosci 9: 27 10-27 17. Coburn KL, Ashford JW, Fuster JM (1990): Visual response latencies in temporal lobe structures as a function of stimulus information load. Behav Neurosci 104:183-194. Crols R, Saerens J, Noppe M, Lowenthal A (1986): Increased GFAP levels in CSF as a marker of organicity in patients with Alzheimer’s disease and other types of irreversible chronic organic brain syndrome. J Neurol 233:157-160.

368

Brewer and Ashford

DeBoni U, Crapper-McLachlan D (1985): Controlled induction of paired helical filaments of the Alzheimer type in cultured human neurons by glutamate and aspartate. J Neurol Sci 68:10.5118. Dyrks T, Weidemann A , Multhaup G , Salbaum J, Lemaire H, Kang J, Muller-Hill B, Masters C, Beyreuther K (1988): Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer’s disease. EMBO J 7:949-957. Eikelenboom P, Hack C, Rozemuller J , Stam F (1989): Complement activation in amyloid plaques in Alzheimer’s dementia. Virchows Arch [B] 56:259-262. Elovaara I, Palo J, Erkinjuntti T, Sulkava R (1987): Serum and cerebrospinal fluid proteins and the blood-brain barrier in Alzheimer’s disease and multi-infarct dementia. Eur Neurol 26:229234. Farrar W, Hill J , Harel-Bellan A, Vinocour M (1987): The immune logical brain. Immunol Rev 100:372-377. Favaron M, Manev H, Alho H, Bertolino M, Ferret B, Guidotti A, Costa E (1988): Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal culture of neonatal rat cerebellum and cortex. Proc Natl Acad Sci USA 8.5:7351-735.5. Foley P, Bradford H, Docherty M, Fillit H, Luine V, McEwen B, Bucht G, Winblad B, Hardy J (1988): Evidence for the presence of antibodies to cholinergic neurons in the serum of patients with Alzheimer’s disease. J Neurol 235:466-471. Folstein MF, Folstein SE, McHugh PR (1975): “Mini-mental state”-a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res 12:189-198. Giometto B, Argentiero V, Sanson F, Ongaro G, Tavolato B (1988): Acute-phase proteins in Alzheimer’s disease. Eur Neurol 28: 30-33. Glenner GG, Wong CW (1984): Alzheimer’s disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885890. Greenamyre J , Young A (1989): Excitatory amino acids and Alzheimer’s disease. Neurobiol Aging 10593-602. Haan E, Boss B, Cowan W (1982): Production and characterization of monoclonal antibodies against the ‘neuron-specific’ proteins 14-3-2 and S-100. Proc Natl Acad Sci USA 79:7585-7589. Hamre K, Hyman B, Goodlett C, West J, Van Hoesen G (1989): Alz-50 immunoreactivity in the neonatal rat: Changes in development and co-distribution with MAP2 immunoreactivity. Neurosci Lett 98:264-271. Hauw JJ, Vignolo P, Duyckaerts C, Beck H, Forette F, Henry JF, Laurent M, Piette F, Sachet A, Berthaux P (1986): Etude neuropathologique de 12 centenaires. Rev Neurol 142: 107-1 15. Hirano A, Zimmerman HM (1962): Alzheimer’s neurofibrillary changes. Arch Neurol 7:227-242. Hyman BT, Van Hoesen GW, Damasio A, Barnes C (1984): Alzheimer’s disease: Cell-specific pathology isolates the hippocampal formation. Science 2.55:1168-1170. Hyrnan BT, Van Hoesen GW, Beyreuther K, Masters CL (1989): A4 amyloid protein immunoreactivity is present in Alzheimer’s disease neurofibrillary tangles. Neurosci Lett 101:352-355. Hyman BT, Van Hoesen GW, Wolozin BL, Davies P, Kromer LJ, Damasio AR (1988): Alz-50 antibody recognizes Alzheimerrelated neuronal changes. Ann Neurol 23:371-379. Ingram CR, Phegan KJ, Blumenthal HT (1974): Significance of an aging-linked neuron binding gamma globulin fraction of human sera. J Gerontol 29:20-27. Ishii T, Haga S (1976): Immuno-electron microscopic localization of immunoglobulins in amyloid fibrils of senile plaques. Acta Neuropathol 36:243-249.

Jarvik L, Matsuyama S (1984): The philothermal response and the diagnosis of dementia of the Alzheimer’s type. In Solomon CA (ed): “Biology and Treatment of Dementia in the Elderly.” Washington, DC: American Psychiatric Association, pp 5058. Jarvik LF, Matsuyama SS, Kessler JO, Fu T-K, Tsai SY, Clark EO (1982): Philothermal response of polymorphonuclear leukocytes in dementia of the Alzheimer type. Neurobiol Aging 3: 93-99. Joachim C, Selkoe D (1989): Minireview: Amyloid protein in Alzheimer’s disease. J Gerontol 44:B77-B82. Joachim C, Mori H, Selkoe D (1989): Amyloid beta-protein deposition in tissues other than brain in Alzheimer’s disease. Nature 341 1226-230. Jones KH, Senft JA (1985): An improved method to determine cell viability by simultaneous staining with fluorescein diacetatepropidium iodide. J Histochem Cytochem 33:77-79. Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzexchik K-H, Multhaup G , Beyreuther K, Muller-Hill B (1987): The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature 325:733-737. Khachaturian ZS (1985): Diagnosis of Alzheimer’s disease. Arch Neurol 42: 1097-1 104. Kim KS, Miller DL, Chen C-M, Sapienza VJ, Bai C, Grundke-Iqbal I, Currie JR, Wisniewski HM (1988): Production and characterization of monoclonal antibodies reactive to synthetic cerebrovascular amyloid peptide. Neurosci Res Commun 2: 121130. Kitamoto T, Ogomori K , Tateishi J , Prusiner S (1987): Methods in laboratory investigation: Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 571230-236. Klier F, Cole C, Stallcup W, Schubert D (1990): Amyloid P-protein precursor is associated with extracellular matrix. Brain Res .515:336-342. Kosik K (1989): Minireview: The molecular and cellular pathology of Alzheimer neurofibrillary lesions. J Gerontol Biol Sci 44:B55 B.58. Kosik KS, Duffy LK, Dowling MM, Abraham C, McCluskey A, Selkoe D (1984): Microtubule-associated protein 2: Monoclonal antibodies demonstrate the selective incorporation of certain epitopes into Alzheimer neurofibrillary tangles. Proc Natl Acad Sci USA 81:7941-7945. Ksiezak-Reding H, Davies P, Yen S (1988): Alz 50, a monoclonal antibody to Alzheimer’s disease antigen, cross-reacts with tau proteins from bovine and normal human brain. J Biol Chem 263:7943 -7947. Kumar M, Cohen D, Eisdorfer C (1988): Serum IgG brain reactive antibodies in Alzheimer’s disease and Down syndrome. Alzheimer’s Dis Assoc Disord 250-55. Matsuyama SS, Fu T-K (1983): Inhibition of normal polymorphonuclear leukocyte philothermal response by serum from dementia of the Alzheimer-type patients. Age 6:72-75. Mattson MP (1990): Antigenic changes similar to those seen in neurofibrillary tangles are elicited by glutamate and Ca2+ influx in cultured hippocampal neurons. Neuron 4:105-117. Mattson MP, Kater SB (1989): Development and selective neurodegeneration in cultures from different hippocampal regions. Brain Res 49O:llO-125. Mattson MP, Murrain M, Guthrie PB, Kater SB (1989): Fibroblast growth factor and glutamate: Opposing roles in the generation and degeneration of hippocampal neuroarchitecture. J Neurosci 9:3728-3740. McGeer P, Walker D, Akiyama H, Kawamata T , Guan A, Parker C,

Alzheimer Markers Induced by Serum Okada N, McGeer E (1991): Detection of the membrane inhibitor of reactive Iysis (CD59) in diseased neurons of Alzheimer brain. Brain Res 544:315-319. McKee A, Kowall N, Kosik K (1989): Microtubular reorganization and dendritic growth response in Alzheimer’s disease. Ann Neurol 26:652-659. McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan E (1984): Clinical diagnosis of Alzheimer’s disease: Report of the NINCDS-ADRDA work group under the auspices of department of health and human services task force on Alzheimer’s disease. Neurol 34:939-944. Mishkin M (1978): Memory in monkeys severely impaired by combined but not by separate removal of amygdala and hippocampus. Nature 273:297-298. Mobley W, Neve R, Prusiner S, McKinley M (1988): Nerve growth factor increases mRNA levels for the prion protein and the B-amyloid protein precursor in developing hamster brain. Proc Natl Acad Sci USA 85:9811-9815. Munro S, Pelhan H (1985): What turns on heat shock genes? Nature 3 17:477-478. Nandy K, Reisberg B (1983): Immunologic factors. Alzheimer’s Dis 18:135-138. Nordstedt C, Gandy SE, Alafuzoff I, Caporaso GL, Iverfeldt K, Grebb JA, Winblad B, Greengard P (1991): Alzheimer P/A4 amyloid precursor protein in human brain: Aging-associated increases in holoprotein and in a proteolytic fragment. Proc Natl Acad Sci USA 88:8910-8914. Octave J, de Sauvage F, Maloteaux J (1989): Modification of neuronal cell adhesion affects the genetic expression of the A4 amyloid peptide precursor. Brain Res 486:369-37 1. Panter S S , McSwigan JD, Sheppard JR, Emory CR, Frey WH (1985): Glial fibrillary acidic protein and Alzheimer’s disease. Neurochem Res 10:1567-1576. Papasozomenos SC, Su Y (1991): Altered phosphorylation of tau protein in heat-shocked rats and patients with Alzheimer disease. Proc Natl Acad Sci USA 88:4543-4547. Pappolla M, Andorn A (1987): Serum protein leakage in aged human brain and inhibition of ligand binding at alpha2-adrenergic and cholinergic binding sites. Synapse 1:82-89. Perry G , Friedman R, Shaw G, Chau V (1987): Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites of Alzheimer disease brains. Proc Natl Acad Sci USA 84:3033-3036. Peterson C, Ratan RR, Shelanski ML, Goldman JE (1986): Cytosolic free calcium and cell spreading decrease in fibroblasts from aged and Alzheimer donors. Proc Natl Acad Sci USA 83: 7999 - 800 1. Rapoport SI, Pettigrew KD, Schapiro MB (1991): Discordance of dementia of the Alzheimer type (DAT) in monozygotic twins indicate heritable and sporadic forms of Alzheimer’s disease. Neurology 41: 1549-1553. Represa A, Duyckaerts C, Tremblay E, Hauw J, Ben-Ari Y (1988): Is senile dementia of the Alzheimer type associated with hippocampal plasticity? Brain Res 457:355-359. Rogers J, Luber-Narod J, Styren S , Civin W (1988): Expression of immune system-associated antigens by cells of the human central nervous system: Relationship to the pathology of Alzheimer’s disease. Neurobiol Aging 9:339-349. Rumble B, Retallack R, Hilbich C, Simms G, Multhaup G, Martins R, Hockey A, Montgomery P, Beyreuther K, Masters CL (1989): Amyloid A4 protein and its precursor in Down’s syndrome and Alzheimer’s disease. N Engl J Med 320: 1446-1 452.

369

Salbaum J, Weidemann A, Masters C, Beyreuther K (1989): The promoter of Alzheimer’s disease amyloid A4 precursor gene. In Igbal K, Wisniewski HM, Winblad B (eds): “Alzheimer’s Disease and Related Disorders.” New York: Alan R. Liss, pp. 277-283. Selkoe D, Podlisny M, Joachim C, Vickers E, Lee G, Fritz L, Oltersdorf T (1988): P-Amyloid precursor protein of Alzheimer disease occurs as 100- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues. Proc Natl Acad Sci USA 85:7341-7345. Tanzi R, McClatchey A, Lamperti E, Villa-Komaroff L, Gusella J, Neve R (1988): Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer’s disease. Nature 31 1528-530. Uchida Y, Tomonaga M (1989): Neurotrophic action of Alzheimer’s disease brain extract is due to the loss of inhibitory factors for survival and neurite formation of cerebral cortical neurons. Brain Res 481:190-193. Uchida Y, Ihara Y, Tomonaga M (1988): Alzheimer’s disease brain extract stimulates the survival of cerebral cortical neurons from neonatal rats. Biochem Biophys Res Commun 150:1263-1267. Uchida Y, Takio K, Titani K, Ihara Y, Tomanaga M (1991): The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein. Neuron 7:337-347. Ueda K, Masliah E, Saitoh T, Bakalis S , Scoble H, Kosik K (1990): Alz-50 recognizes a phosphorylated epitope of tau-protein. J Neurosci 10:3295-3304, Ulrich J (1985): Alzheimer changes in nondemented patients younger than sixty-five: Possible early stages of Alzheimer’s disease and senile dementia of Alzheimer type. Ann Neurol 17:273277. Vincent IJ, Davies P (1990): ATP-induced loss of Alz-50 immunoreactivity with the A68 proteins from Alzheimer brain is mediated by ubiquitin. Proc Natl Acad Sci USA 87:4840-4844. Wagner S , Geddes J, Cotman C, Lau A, Gurwitz D, Isackson P, Cunningham D (1989): Protease Nexin-I, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer disease. Proc Natl Acad Sci USA 86:8284-8288. Whitson J, Selkoe D, Cotman C (1989): Amyloid p protein enhances the survival of hippocampal neurons in vitro. Science 243: 1488-1489. Wisniewski H, Kozlowski P (1982): Evidence for blood-brain barrier changes in senile dementia of the Alzheimer type (SDAT). Ann NY Acad Sci 396:119-129. Wolozin BL, Pruchnicki A, Dickson DW, Davies PA (1986): Neuronal antigen in the brains of Alzheimer patients. Science 232: 648-650. Wolozin B, Scicutella A, Davies P (1988): Reexpression of a developmentally regulated antigen in Down syndrome and Alzheimer disease. Proc Natl Acad Sci USA 85:6202-6206. Yamaguchi H, Morimatsu M, Hirai S, Takahashi K (1987): Alzheimer’s neurofibrillary tangles are penetrated by astroglial processes and appear eosinophilic in their final stages. Acta Neuropathol 72:2 14-217. Yankner B, Duffy LK, Kirschner DA (1990): Neurotrophic and neurotoxic effects of amyloid P protein: Reversal by tachykinin neuropeptides. Science 250:279-282. Zhang H, Sternberger NH, Rubinstein LJ, Herman MM, Binder LI, Sternberger LA (1989): Abnormal processing of multiple proteins in Alzheimer disease. Proc Natl Acad Sci USA 86:80458049.