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Resting [Ca2+]i and [Ca2+]i transients are similar in fibroblasts from normal and Alzheimer's donors
Neurobiologyof Aging, Vol. 13, pp. 33-38. ©Pergamon Press plc, 1991. Printed in the U.S.A.
0197-4580/92 $5.00 + .00
Resting [Ca2+]i and [Ca2+ ]i Transients Are Similar in Fibroblasts From Normal and Alzheimer' s Donors LAURENCE
A. B O R D E N , F R E D E R I C K R. M A X F I E L D ,
J A M E S E. G O L D M A N
A N D M I C H A E L L. S H E L A N S K I 1
Department of Pathology, Columbia University, College of Physicians and Surgeons 630 W. 168th Street, New York, NY 11203 R e c e i v e d 9 A u g u s t 1990; A c c e p t e d 27 A u g u s t 1991 BORDEN, L. A., F. R. MAXFIELD, J. E. GOLDMAN AND M. L. SHELANSKI. Resting [Ca2+]i and [Ca2+]i transients are similar in fibroblasts from normal and Alzheimer's donors. NEUROBIOL AGING 13(1) 33-38, 1992.--Previous studies have reported that resting concentrations of intracellular calcium ion were markedly reduced in cultured dermal fibroblasts from Alzheimer's disease patients and that the ability of these cells to respond ro serum stimulation was also decreased as compared to both young and age-matched control cells. In this study we have carefully reexamined these parameters in several of the same lines of fibroblasts and fail to find major differences in resting cytosolic calcium [Ca 2÷ ]~, in the response of [Ca 2+ ]i to serum stimulation or in cell spreading in the AD cells as compared to young controls. The present findings suggest that cytoplasmic ionic calcium levels are neither pathognomonic for Alzheimer's cells nor of diagnostic value. Alzheimer's disease
Fibroblasts
Calcium
Bradykinin
A L Z H E I M E R ' S disease is a progressive neurological disease of unknown etiology, characterized by severe behavioral changes and memory deficits. The brains of Alzheimer's patients display characteristic senile plaques and neurofibrillary tangles, as well as alterations in neurotransmitters and their receptors. However, the relationship between the morphological and biochemical changes on the one hand, and the behavioral deficits on the other, remains to be determined. It is also not clear how Alzheimer's disease relates to the normal process of aging. A major impediment to our understanding of the pathophysiology of Alzheimer's disease is the lack of an animal model and the obvious difficulty in obtaining biopsy material from the brains of affected individuals. Recent data, however, suggest that biochemical abnormalities are present in nonneural cells from aged and Alzheimer's patients, raising the possibility that the metabolic changes are not limited to the central nervous system. For example, protein synthesis, oxidative metabolism (7), and DNA repair mechanisms (5) are deficient in skin fibroblasts from Alzheimer's patients. Additionally, Jarvik et al. (3) demonstrated that the tendency for neutrophils to migrate along a temperature gradient (philothermal response) was diminished in cells from patients with Alzheimer's disease. A number of studies have suggested alterations in calcium homeostasis associated with aging and Alzheimer's disease. For example, Miller (6) showed that in response to mitogenic stimuli the proliferation of T-lymphocytes from old mice was diminished compared to cells from young animals, and that this defect could be overcome with a combination of phorbol ester and cal-
cium ionophore. Diminished mitogen-induced calcium uptake has been observed in lymphocytes from Alzheimer's patients (1), and the calcium-dependence of the Ca 2 ÷-Mg 2÷ -ATPase present in Alzheimer's fibroblasts differs from that in their normal counterparts (10). In previous studies it was reported that cell spreading, resting levels of cytosolic calcium ([Ca 2÷ ]i), and responses to calcium-elevating drugs were decreased in skin fibroblasts from aged donors as compared to normal controls, and that these parameters were further diminished in Alzheimer's donors (8,9). While reinvestigating these phenomena as part of a larger study to understand the molecular mechanisms underlying Alzheimer's disease, we found unexpectedly that we were unable to reproduce the earlier observations. Utilizing the same cell lines as previously used, we now find that cell spreading and cytosolic calcium are similar in fibroblasts from young and Alzheimer's donors. These findings suggest that the previously observed defects in calcium homeostasis in Alzheimer's fibroblasts are not of diagnostic value. We cannot rule out the possibility that the previously observed differences may be dependent on as-yet unidentified variations in cell culture conditions. METHOD Cultured skin fibroblast cell lines were obtained from the National Institute on Aging Cell Repository (Camden, N J). The lines used in the present study were from normal young donors (GM3652A, AG3440, GM03651 C, AG7720, G M 1891, GM04390,
~Requests for reprints should be addressed to Michael L. Shelanski.
33
34
and GM03523; mean age, 23 years) and from Alzheimer's patients (AG04400, AG0364D, AG08245, AG04402A, AG6264A, AG5809A, and AG5810C; mean age, 63 years). Cells were used between passages 6 and 19, and no differences were observed as a result of passage number. lntracellular calcium levels were determined essentially as described by Kruskal et al. (4). Dishes were prepared by punching a hole (12 mm) into the bottom of a plastic 35-mm tissues culture dish and attaching a glass coverslip to the bottom. In some cases, the coverslips were coated with poly-D-lysine, whereas other dishes were left uncoated; the results for coated and uncoated dishes are presented separately. Cells were cultured as described previously (9). On day 0, 1,000 cells were added to each 12-mm well in 0.2 ml DMEM (Gibco) containing penicillin-streptomycin and 16.7% heat-inactivated (56 ° . 30 minutes) fetal calf serum. Three to four hours later, by which time the cells had adhered to the glass coverslip, 2 ml of the same medium was added to each dish. On day 3, the medium was replaced with DMEM lacking both serum and antibiotics. On day 4, cells were loaded with the calcium-sensitive dye fura-2 and [ C a 2 ~ ]i was measured by microspectrofluorometry. To load cells with fura-2, they were washed 3 × with medium 1 (in mM: NaC1, 150; KCI, 5; CaC12, 1; MgC12; 1; glucose, 10; HEPES, 20; pH 7.4), and incubated for 1 hour at room temperature with medium 1 containing 5 IxM fura-2AM and 5 mg/ml fatty acid-free bovine serum albumin. Cells were then washed 3 x with medium 1 and further incubated for 1 hour (room temperature) to allow dye cleavage. Cells were then washed again, 2 ml of med 1 (37 °) was added, and dishes were placed on the microscope for determination of [Ca 2 * ],. [Ca2 ~ L was measured using a Leitz-Diavert microscope equipped for UV-transmission epifluorescence (4), using a 40 x Nikon objective. Fluorescence illumination was provided by a 100-W Hg arc lamp. Fura-2 fluorescence was alternately excited at 340 and 380 nm using a motorized filter wheel which contained the appropriate band pass filters (10 nm full width at half maximum, Oriel, Stamford, CT). The duration of excitation was 0.25 seconds, and a pair of reading, was taken every 2 seconds. Fluorescence emission (500 nm long pass filter) was detected with a Kinetek photometer (Yonkers, NY). The photometry system was controlled by a personal computer interfaced to a data acquisition and control unit from Kinetek. For each dish examined, resting [Ca 2. ]i was measured in 5 individual fields, where each field typically contained 1-5 cells. A new field of cells was then chosen, [Ca 2÷ ]~ was measured for about 30 seconds to determine baseline, and then 1 ml of bradykinin (Sigma; final concentration=25 nM) or fetal calf serum (final= 1% v/v) was added and measurements continued for about 5 minutes. During measurements, the cells were maintained at 35-37°C with an air curtain. To determine [Ca 2+ ]i from the experimental data, background fluorescence from an acellular area of the dish was subtracted, and the corrected ratios were converted to [Ca2+]~ by comparison with buffers containing saturating and low free calcium, assuming a K D of 224 nM (2). To measure cell spreading, cells were plated onto glass coverslip dishes in DMEM with antibiotics and 16.7% heat-inactivated fetal calf serum (700-800 cells/well). Coverslips cleaned with Nochromix (Godax Laboratories, NY) were employed for spreading experiments, as preliminary experiments demonstrated that spreading on uncleaned coverslip dishes was not uniform. After incubation for 2 or 3 hours (37°C), cells were rinsed 3 x with PBS (in mM: NaC1, 136.9; KCI, 2.7; NazHPO4, 8.1; KH2PO 4, 1.5), and fixed with 10% formaldehyde in PBS. Following fixation, cells were rinsed 3 x with PBS, permeabilized with 1% Nonidet P-40 (Sigma) (30 min, room temperature), and filamentous actin was stained (30-45 rain, 37°C) with rhodamine
B{)RI)EN E'! A i .
'FABLE 1 RESTING [Ca 2+ ]i IN HUMAN FIBROBLASTS (.'ell Line
I('a':
" ]i, nM:i
Young
AG3440 GM3651C GM3652 AG7720 GM1891 GM03523
46 +_ 5 (25) 81-_ 7 i4) 39 _= 7 (1% 51 ~ 4 (3~ 34 ± 2 (4) 41 ~- 11 __~3t 45 2: 3 (58~
Alzheimer's
AG08245 AGO4402A AG6264A AG4400 AG5809A AG5810C
48 32 54 34 53 28 40
~ = -" ~_+ :~:
6 (17) 4 (22) 8 !5 2 (3i 12 (3) .3 I3)_ 3 (53)$
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated glass coverslip dishes. On day 3 the medium was switched to serum-flee DMEM, and on day 4 resting [Ca:+]i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values are mean - sem of the number of dishes indicated in parentheses; the value for each dish was determined from 5 microscopic fields, each consisting of 1-5 cells. -tPooted data for all fields of young cell lines. ~:Pooled data for all fields of Alzheimer's cell lines.
phalloidin (Molecular Probes). The cells were examined with a Leitz Diavert inverted microscope and an RCA SIT video camera, and the fluorescent images were recorded on optical disk using a Panasonic optical disk recorder. The images were digitized and cell size was quantified using a Vicom-Gould IP8500 image processor, interfaced with a Microvax II computer. Two dishes of each cell line were examined at each time point, and at least 20 cells per dish were analyzed. RESULTS
Resting [Ca 2 +]i Resting levels of cytosolic calcium were measured in serum-deprived young normal and Alzheimer's flbroblasts maintained on both uncoated and polylysine-coated glass coverslip dishes. For cells grown on uncoated glass, the values were 45 nM and 40 nM (see Table for 1 values for individual cell lines). As a control for effects of the substrate, several of these lines (Table 2) were grown on poly-D-lysine-coated glass. In this case, resting [Ca2+]i was 27 nM and 29 nM for young and Alzheimer's cells, respectively. Importantly, on either substrate, r e s t i n g [Ca2+]i was similar for young and Alzheimer's fibroblasts. This is in contrast to the results of Peterson et al. (8), who found that resting calcium in Alzheimer's fibroblasts (16 nM) was about 4-fold lower than resting calcium in fibroblasts from young donors (60 nM).
Response to Bradykinin and Fetal Calf Serum We next examined the ability of serum-deprived fibroblasts to respond to bradykinin. Typical responses to 25 nM bradykinin for young and Alzheimer's fibroblasts plated on uncoated glass are shown in Fig. 1A and B, respectively. In both cell
C A L C I U M T R A N S I E N T S IN A L Z H E I M E R ' S F I B R O B L A S T S
35
TABLE 2
TABLE 3
RESTING [Ca2+]x IN HUMAN FIBROBLASTS
CHANGES IN [Ca:+]i IN HUMAN FIBROBLASTS IN RESPONSE TO BRADYKININ
Cell Line
[Ca2+]i, nM* Cell Line
Young
AG3440 GM3652
22 ± 3 (16) 34 ± 4 (15)
Young
GM3652 AG3440 GM03651C AG7720 GM03523 GM1891 AG4390
27 ± 3 (31)t AGO4400 AG08245 AG00364D AG04402A
Alzheimer' s
29 34 18 31
± ± ± ---
3 4 6 3
D[Ca2+]i, riM*
(7) (9) (8) (9)
257 320 441 566 287 105 660
29 --- 2 (33):~ Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on polyD-lysine-coated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 resting [Ca2+]i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values are mean ± sem of the number of dishes indicated in parentheses; the value for each dish was determined from 5 microscopic fields, each consisting of 1-5 cells. tPooled data for all fields of young cell lines. :~Pooled data for all fields of Alzheimer's cell lines.
types, [Ca 2+ ]i b e g a n to increase within 1 0 - 2 0 s e c o n d s o f bradykinin application, quickly reached a peak, and then declined. In general, [Ca2+L returned to a plateau value w h i c h w a s h i g h e r than the resting level prior to drug application. A l t h o u g h res p o n s e s to bradykinin were variable, the r e s p o n s e s o f Alzheim e r ' s fibroblasts were similar in shape and amplitude to those o f y o u n g fibroblasts. R e s p o n s e s to 250 n M bradykinin were also similar in y o u n g fibroblasts and A l z h e i m e r ' s fibroblasts (not s h o w n ) . To c o m p a r e the r e s p o n s e s o f the cells in a quanti-
1000
I
I
I
I
58 52 53 42 81 73 81
353 ± Alzheimer' s
AG08245 AG04402A AG6264A AG4400 AG5809A AG5810C
566 488 445 327 395 367
(ll) (17) (4) (3) (3) (4) (6)
34 (48)1
± 141 ___ 62 ± 68 ± 74 ± 183 ± 99
471 ±
(9) (14) (5) (3) (3) (3)
45 (37):~
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca 2÷ ]i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values show the maximal increase in [Ca 2+ ], in response to 25 nM bradykinin (peak response minus preapplication value). Each value is the mean ± sem of the number of determinations indicated in parentheses. CPooled data for all fields of young cell lines. :~Pooled data for all fields of Alzheimer's cell lines.
tative m a n n e r , we d e t e r m i n e d the m a x i m u m c h a n g e in [Ca2+]i (D[Ca2+]i) for each treatment. T h i s value w a s calcu-
1000
I
1
1
1
1
1
Alzheimer's
Young 800
c
± ± ± ± ± ± ±
B
800
\
600
~_~c 600
+ t~
8 400 ,--,
o
o
200
400
2O0
0 0
I
I
I
I
I
60
120
180
240
.300
seconds
0 .360
0
60
120
180
240
300
360
seconds
FIG. 1. Fibroblasts from young (A) and Alzheimer's (B) donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca2+]i was measured as described in the Method section. Bradykinin (25 nM) was added at the arrow. The data in (A) shows 3 traces each from GM3440 and AG7720, and the data in (B) show 3 traces each from AG04402A and AG5810C. Each trace is from a single dish, and represents a field of 1-5 cells.
36
!~ORI)EN
1000
I
I
I
[
- -
1000
'1
r-
i
-1. . . . .
i
T. . . . . . . . . .
.....
800
.,\~
T. . . . B
Alzheilner's
Young
[i~l "
800
I =1
600
-
600
+
,~. 400
4oo
-
200 -
2OO
0 0
60
I
I
I
I
1 20
180
240
300
I
360
0
60
120
180
240
300
36C
seconds
seconds
FIG. 2. Fibroblasts from young (A) and Alzheimer's (B) donors were plated as described in Fig. 1. One percent fetal calf serum was added at the arrow. The data in (A) show 2 traces each from GM3652 and GM3440, and the data in (B) show 2 traces each from AG08245 and AG04402A. Each trace is from a single dish, and represents a field of 1-5 cells.
lated as the peak h e i g h t in r e s p o n s e to bradykinin, m i n u s the c o r r e s p o n d i n g predrug resting level. T h e values for the individual cell lines e x a m i n e d are s h o w n in Table 3 (uncoated glass) and in Table 4 for several o f the lines on polylysine-coated glass. W h e n the data for the various cell lines e x a m i n e d is pooled, the increase in [Ca 2+ ]i in A l z h e i m e r ' s fibroblasts plated on u n c o a t e d glass (471 n M ) w a s f o u n d to be s o m e w h a t greater than the increase in y o u n g fibroblasts (353 nM). Similarly, alt h o u g h r e s p o n s e s o f cells plated on p o l y - D - l y s i n e were smaller
than those o f cells plated on uncoated glass, the m a x i m a l increase in [Ca 2÷ ]i in A l z h e i m e r ' s fibroblasts (298 n M ) w a s again s o m e w h a t greater than the increase o f y o u n g fibroblasts (193 nM). Typical r e s p o n s e s o f y o u n g and A l z h e i m e r ' s fibroblasts on uncoated glass in r e s p o n s e to 1% fetal calf s e r u m are s h o w n in Fig. 2 A a n d B, respectively. T h e r e s p o n s e s are similar in shape to those elicited with bradykinin, consisting o f an inital peak followed by a plateau. Interestingly, quantitation o f the data d e m o n s t r a t e s that the m a x i m a l increase in [Ca2+]i is nearly
TABLE 4 CHANGES IN [cae+]i IN HUMAN FIBROBLASTS IN RESPONSE TO BRADYKININ Cell Line Young
Alzheimer' s
AG3440 AG3652
AG04400 AG0364D AG04402A AG08245
TABLE 5
D[Ca2+]i, nM* 163 + 17 (13) 222 +-- 43 (13) 193 + 23 (26)? 356 -+ 322267 -+ 281 +298 +
109 41 24 46 30
(8) (3) (12) (11) (34)$
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on polyD-lysine-coated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca2+] i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values show the maximal increase in [Ca2÷]i in response to 25 nM bradykinin (peak response minus preapplication value). Each value is the mean +_ sem of the number of determinations indicated in parentheses. ?Pooled data for all fields of young ceil lines. :~Pooled data for all fields of Alzheimer's cell lines
CHANGES IN [Ca2+] i IN HUMAN FIBROBLASTS IN RESPONSE TO FETAL CALF SERUM Cell Line
D[Ca 2 ÷ ]i, nM*
Young
GM3652 AG3440
466 _+ 128 (5) _424 _+ 50 (6) 443 ___ 61 ( l l ) t
Alzheimer's
AG04402A AG08245
423 +- 35 (6) 464 -+ 80 (6) 444 -+ 42 (12):I:
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca2+] i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values show the maximal increase in [Ca2+]i in response to 1% fetal calf serum (peak response minus preapplication value). Each value is the mean _+ sere of the number of determinations indicated in parentheses. ?Pooled data for all fields of young cell lines. ,Pooled data for all fields of Alzheirner's cell lines.
CALCIUM TRANSIENTS IN ALZHEIMER'S FIBROBLASTS
identical for young and Alzheimer's fibroblasts (Table 5). While only two control lines and two AD lines were tested here, these data are clearly in disagreement with the results of Peterson et al. (9) in which all AD lines were unresponsive and all control lines were responsive. Similar results were obtained with 10% fetal calf serum (not shown), although this was examined in only a few experiments. The responses to bradykinin and fetal calf serum in young and Alzheimer's cells that were serum-starved for about 80 hours were similar to those obtained in cells serum-starved for 24 hours (data not shown). In summary, the present findings differ from those of Peterson et al. (9), who found that the response of Alzheimer's fibroblasts to bradykinin or fetal calf serum was greatly diminished when compared to the response of fibroblasts from young donors. In the previous study (9), cells were maintained in antibioticfree medium, whereas in the present study, cells were switched to antibiotic-free medium the day before [Ca 2÷ ]i was measured. To determine if this accounted for the discrepant results, young (cell lines AG7720 and AG3440) and Alzheimer's (AG6264 and AG364D) cells were plated on uncoated glass on day 0 in medium with serum but lacking antibiotics, on day 3 they were switched to serum-free medium (also lacking antibiotics), and on day 4 [Ca2+] i was measured. Resting [Ca2+] i was 36---2 nM and 4 2 - 5 n M , and D[Ca2+]i was 3 8 9 ± 4 9 nM and 315---32 nM for young and Alzheimer's cells, respectively, where each value is the mean ± SEM of 2 cell lines, 3 dishes/line. These results are similar to those obtained under control conditions, indicating that the discrepancy with the earlier reports is not due to an effect of antibiotics.
Spreading It was previously reported (8) that during a 2-hour period, 40% of Alzheimer's cells spread as compared with 80% for fibroblasts from young donors. In these previous studies, cells were considered spread when they had attained a polygonal shape. To examine spreading in a more quantitative manner, cells were fixed at various times (generally 2 or 3 hours) after plating and stained with rhodamine phalloidin. Cell size was determined by digital image analysis of the fluorescent images. In the first set of experiments, we compared the spreading of the Alzheimer's line AG4402A (passages 11 and 12) with that of the young cell line GM3440 (passages 12-14). In qualitative agreement with Peterson et al. (8), the Alzheimer's line spread more slowly: at 2 and 3 hours after plating, the Alzheimer's cells were 70--5% and 6 0 _ 6% the size of the young cells, respectively, where each value represents the mean -- SEM of three independent experiments. To determine whether these results reflected a general property of young vs. Alzheimer's cells, we examined the spreading of other cell lines. In contrast to the previous results, the Alzheimer's cell line AG04400 (passages 9 and 10) spread more rapidly than the young cell line GM3440 (passages 10 and 11): at 2 and 3 hours, the Alzheimer's cells were 108% and 126%, respectively, the size of the young cells (mean of 2 independent experiments). Similarly, the Alzheimer's line AG6264A (passage 10) spread more rapidly than the young line GM03651C (passage 10) (131% and 114% of young at 3 and 4 hours, respectively). Thus spreading is variable among cell lines, and there is no consistent deficit in the ability of Alzheimer's fibroblasts to undergo spreading. DISCUSSION
In the present study we found that cell spreading, resting cytosolic calcium, and the response to serum were similar in flbroblasts derived from normal young donors and from older patients
37
with Alzheimer's disease, while the response to bradykinin was, if anything, slightly greater in Alzheimer's cells. These results disagree with previous studies (8,9) in which these parameters were found to be decreased in cells from aged donors, and further diminished in Alzheimer's disease. Further experiments employing additional Alzheimer's cell lines and age-matched controls would be necessary to determine if the heightened response to bradykinin is significant, and if so, whether it reflects differences in the age of the donors (young vs. old) or is a direct consequence of Alzheimer's disease. As shown by the representative traces in Figs. 1 and 2, the responses of both young and Alzheimer's fibroblasts to agents that elevate cytosolic calcium are variable, even within a cell line, The variability was observed within an experiment, and thus was not due to day-to-day variability. Although the responses of Alzheimer's cells to bradykinin were slightly greater than those of young cells (Tables 3 and 4), it is apparent from Fig. 1 that there is considerable overlap in the responses; a similar overlap is seen in the responses to fetal calf serum (Fig. 2). While occasional cells responded poorly to bradykinin or fetal calf serum, this was observed both in young and Alzheimer's cells. It should be stressed that no cell line examined consistently gave poor responses, nor were poor responses consistently observed within any particular experiment. This is in marked contrast to the previous study (9), in which Alzheimer's cells were consistently found to be poor responders. It is presently not clear why our results differ from those of the earlier study. Although we attempted to perform the experiments under similar conditions to those used previously, a number of factors were different. First, although six of seven Alzheimer's cell lines employed in the present study were the same as in the previous study, and were obtained from the same source, they were derived from different frozen stocks. Thus it is possible that the various frozen stocks differ in their properties. Second, the cells were used at later passage number (7-19) than in the previous study (average, 7.5). This does not seem to account for the discrepancy because the responses to bradykinin and fetal calf serum did not appear to differ with passage number. Last, different batches of serum were used in the growth medium. Although the cells were serum-starved for one day before [Ca 2+ ]i was measured, long-lasting changes may have taken place during their growth in serum-containing medium. Thus it is possible that certain batches of serum have differential effects on fibroblasts from young, old, or aged donors, while other batches do not. Control experiments revealed that maintaining the cells with or without antibiotics did not account for the discrepancy with the previous findings. Recently, Sisken et al. (11) presented an abstract in which they reported that the responses to 1% fetal calf calf serum are similar in Alzheimer's and control fibroblasts, whereas the responses to 10% serum are greater in Alzheimer's cells. These findings are similar to our current results and indicate that, under certain conditions, the responses of Alzheimer's fibroblasts are not diminished compared to cells from normal donors. However, it can not be ruled out that defects in calcium homeostasis may exist in certain subsets of Alzheimer's patients. In summary, we now find that cell spreading, resting cytosolic calcium, and response to serum in fibroblasts from patients with Alzheimer's disease do not differ greatly from those of cells from young, normal donors. The discrepancy from the earlier findings of diminished responses in Alzheimer's fibroblasts is not understood, but may relate to poorly understood variations in cell culture conditions. In any event, the present findings suggest that the previously observed deficits in fibroblasts are neither pathognomonic for Alzheimer's cells nor of diagnostic value.
~8
fgORDEN [ ! t A l
ACKNOWLEDGEMENTS We would like to thank Ms. Bernetta Abramson for expert assistance with cell culture, and Dr. Jesse Sisken for helpful discussions. This work was supported by grants from the NIH (NS27680) and the American Health Assistance Foundation. L . A B was a trainee of the National Institute of Aging (AG00189).
REFERENCES 1. Gibson, G. E.; Nielsen, P.; Sherman, K. A.; Blass, J. P. Diminished mitogen-induced calcium uptake by lymphocytes from Alzheimer patients. Biol. Psychiatry 22:1079-1086; 1987. 2. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260:3440-3450; 1985. 3. Jarvik, L. F.; Matsuyama, S. S.; Kessler, J. O.; Fu, T.-K.; Tsai, S. Y.; Clark, E. O. Philothermal response of polymorphonuclear leukocytes in dementia of the Alzheimer type. Neurobiol. Aging 3:93-99; 1982. 4. Kruskal, B. A.; Shak, S.; Maxfield, F. R. Spreading of human neutrophils is immediately preceeded by a large increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. USA 83:2919-2923; 1986. 5. Li, J. C.; Kaminiskas, K. Deficient repair of DNA lesions in Alzheimer's disease fibroblasts. Biochem. Biophys. Res. Commun. 129:733-738; 1985. 6. Miller, R. Immunodeficiency of aging: Restorative effects of phorbol ester combined with calcium ionophore. J. Immunol. 137:805-
808; 1986. 7. Peterson, C.; Goldman, J. E. Alterations in calcium content and biochemical processes during aging and Alzheimer's disease. Proc. Natl. Acad. Sci. USA 83:2758-2761; 1986. 8. Peterson, C.; Ratan, R. R.; Shelanski, M. L.; Goldman, J. E. Cytosolic free calcium and cell spreading decrease in fibrobtasts from aged and Alzheimer donors. Proc. Natl. Acad. Sci. USA 83:79998001; 1986. 9. Peterson, C.; Ratan, R. R.; Shelanski, M. L.; Goldman, J. E. Altered response of fibroblasts from aged and Alzheimer donors to drugs that elevate cytosolic free calcium. Neurobiol. Aging 9:261266; 1988. 10. Rizopoulos, E.; Chambers, J. P.; Wayner, M. J.; Martinez, A. O.; Armstrong, L. S. Effects of free Ca 2+ on the [Ca 2+ + Mg2*] dependent adenosinetriphosphatase (ATPase) of Alzheimer and normal fibroblasts. Neurobiol. Aging 10:717-720; 1989. I1. Sisken, J. E.; McCoy, K. R.; Newcomb, T. G.; You, J. Seruminduced calcium transients in fibroblasts from individuals with familial Alzheimer's disease. J. Cell Biol. 109:301a; 1989.
0197-4580/92 $5.00 + .00
Resting [Ca2+]i and [Ca2+ ]i Transients Are Similar in Fibroblasts From Normal and Alzheimer' s Donors LAURENCE
A. B O R D E N , F R E D E R I C K R. M A X F I E L D ,
J A M E S E. G O L D M A N
A N D M I C H A E L L. S H E L A N S K I 1
Department of Pathology, Columbia University, College of Physicians and Surgeons 630 W. 168th Street, New York, NY 11203 R e c e i v e d 9 A u g u s t 1990; A c c e p t e d 27 A u g u s t 1991 BORDEN, L. A., F. R. MAXFIELD, J. E. GOLDMAN AND M. L. SHELANSKI. Resting [Ca2+]i and [Ca2+]i transients are similar in fibroblasts from normal and Alzheimer's donors. NEUROBIOL AGING 13(1) 33-38, 1992.--Previous studies have reported that resting concentrations of intracellular calcium ion were markedly reduced in cultured dermal fibroblasts from Alzheimer's disease patients and that the ability of these cells to respond ro serum stimulation was also decreased as compared to both young and age-matched control cells. In this study we have carefully reexamined these parameters in several of the same lines of fibroblasts and fail to find major differences in resting cytosolic calcium [Ca 2÷ ]~, in the response of [Ca 2+ ]i to serum stimulation or in cell spreading in the AD cells as compared to young controls. The present findings suggest that cytoplasmic ionic calcium levels are neither pathognomonic for Alzheimer's cells nor of diagnostic value. Alzheimer's disease
Fibroblasts
Calcium
Bradykinin
A L Z H E I M E R ' S disease is a progressive neurological disease of unknown etiology, characterized by severe behavioral changes and memory deficits. The brains of Alzheimer's patients display characteristic senile plaques and neurofibrillary tangles, as well as alterations in neurotransmitters and their receptors. However, the relationship between the morphological and biochemical changes on the one hand, and the behavioral deficits on the other, remains to be determined. It is also not clear how Alzheimer's disease relates to the normal process of aging. A major impediment to our understanding of the pathophysiology of Alzheimer's disease is the lack of an animal model and the obvious difficulty in obtaining biopsy material from the brains of affected individuals. Recent data, however, suggest that biochemical abnormalities are present in nonneural cells from aged and Alzheimer's patients, raising the possibility that the metabolic changes are not limited to the central nervous system. For example, protein synthesis, oxidative metabolism (7), and DNA repair mechanisms (5) are deficient in skin fibroblasts from Alzheimer's patients. Additionally, Jarvik et al. (3) demonstrated that the tendency for neutrophils to migrate along a temperature gradient (philothermal response) was diminished in cells from patients with Alzheimer's disease. A number of studies have suggested alterations in calcium homeostasis associated with aging and Alzheimer's disease. For example, Miller (6) showed that in response to mitogenic stimuli the proliferation of T-lymphocytes from old mice was diminished compared to cells from young animals, and that this defect could be overcome with a combination of phorbol ester and cal-
cium ionophore. Diminished mitogen-induced calcium uptake has been observed in lymphocytes from Alzheimer's patients (1), and the calcium-dependence of the Ca 2 ÷-Mg 2÷ -ATPase present in Alzheimer's fibroblasts differs from that in their normal counterparts (10). In previous studies it was reported that cell spreading, resting levels of cytosolic calcium ([Ca 2÷ ]i), and responses to calcium-elevating drugs were decreased in skin fibroblasts from aged donors as compared to normal controls, and that these parameters were further diminished in Alzheimer's donors (8,9). While reinvestigating these phenomena as part of a larger study to understand the molecular mechanisms underlying Alzheimer's disease, we found unexpectedly that we were unable to reproduce the earlier observations. Utilizing the same cell lines as previously used, we now find that cell spreading and cytosolic calcium are similar in fibroblasts from young and Alzheimer's donors. These findings suggest that the previously observed defects in calcium homeostasis in Alzheimer's fibroblasts are not of diagnostic value. We cannot rule out the possibility that the previously observed differences may be dependent on as-yet unidentified variations in cell culture conditions. METHOD Cultured skin fibroblast cell lines were obtained from the National Institute on Aging Cell Repository (Camden, N J). The lines used in the present study were from normal young donors (GM3652A, AG3440, GM03651 C, AG7720, G M 1891, GM04390,
~Requests for reprints should be addressed to Michael L. Shelanski.
33
34
and GM03523; mean age, 23 years) and from Alzheimer's patients (AG04400, AG0364D, AG08245, AG04402A, AG6264A, AG5809A, and AG5810C; mean age, 63 years). Cells were used between passages 6 and 19, and no differences were observed as a result of passage number. lntracellular calcium levels were determined essentially as described by Kruskal et al. (4). Dishes were prepared by punching a hole (12 mm) into the bottom of a plastic 35-mm tissues culture dish and attaching a glass coverslip to the bottom. In some cases, the coverslips were coated with poly-D-lysine, whereas other dishes were left uncoated; the results for coated and uncoated dishes are presented separately. Cells were cultured as described previously (9). On day 0, 1,000 cells were added to each 12-mm well in 0.2 ml DMEM (Gibco) containing penicillin-streptomycin and 16.7% heat-inactivated (56 ° . 30 minutes) fetal calf serum. Three to four hours later, by which time the cells had adhered to the glass coverslip, 2 ml of the same medium was added to each dish. On day 3, the medium was replaced with DMEM lacking both serum and antibiotics. On day 4, cells were loaded with the calcium-sensitive dye fura-2 and [ C a 2 ~ ]i was measured by microspectrofluorometry. To load cells with fura-2, they were washed 3 × with medium 1 (in mM: NaC1, 150; KCI, 5; CaC12, 1; MgC12; 1; glucose, 10; HEPES, 20; pH 7.4), and incubated for 1 hour at room temperature with medium 1 containing 5 IxM fura-2AM and 5 mg/ml fatty acid-free bovine serum albumin. Cells were then washed 3 x with medium 1 and further incubated for 1 hour (room temperature) to allow dye cleavage. Cells were then washed again, 2 ml of med 1 (37 °) was added, and dishes were placed on the microscope for determination of [Ca 2 * ],. [Ca2 ~ L was measured using a Leitz-Diavert microscope equipped for UV-transmission epifluorescence (4), using a 40 x Nikon objective. Fluorescence illumination was provided by a 100-W Hg arc lamp. Fura-2 fluorescence was alternately excited at 340 and 380 nm using a motorized filter wheel which contained the appropriate band pass filters (10 nm full width at half maximum, Oriel, Stamford, CT). The duration of excitation was 0.25 seconds, and a pair of reading, was taken every 2 seconds. Fluorescence emission (500 nm long pass filter) was detected with a Kinetek photometer (Yonkers, NY). The photometry system was controlled by a personal computer interfaced to a data acquisition and control unit from Kinetek. For each dish examined, resting [Ca 2. ]i was measured in 5 individual fields, where each field typically contained 1-5 cells. A new field of cells was then chosen, [Ca 2÷ ]~ was measured for about 30 seconds to determine baseline, and then 1 ml of bradykinin (Sigma; final concentration=25 nM) or fetal calf serum (final= 1% v/v) was added and measurements continued for about 5 minutes. During measurements, the cells were maintained at 35-37°C with an air curtain. To determine [Ca 2+ ]i from the experimental data, background fluorescence from an acellular area of the dish was subtracted, and the corrected ratios were converted to [Ca2+]~ by comparison with buffers containing saturating and low free calcium, assuming a K D of 224 nM (2). To measure cell spreading, cells were plated onto glass coverslip dishes in DMEM with antibiotics and 16.7% heat-inactivated fetal calf serum (700-800 cells/well). Coverslips cleaned with Nochromix (Godax Laboratories, NY) were employed for spreading experiments, as preliminary experiments demonstrated that spreading on uncleaned coverslip dishes was not uniform. After incubation for 2 or 3 hours (37°C), cells were rinsed 3 x with PBS (in mM: NaC1, 136.9; KCI, 2.7; NazHPO4, 8.1; KH2PO 4, 1.5), and fixed with 10% formaldehyde in PBS. Following fixation, cells were rinsed 3 x with PBS, permeabilized with 1% Nonidet P-40 (Sigma) (30 min, room temperature), and filamentous actin was stained (30-45 rain, 37°C) with rhodamine
B{)RI)EN E'! A i .
'FABLE 1 RESTING [Ca 2+ ]i IN HUMAN FIBROBLASTS (.'ell Line
I('a':
" ]i, nM:i
Young
AG3440 GM3651C GM3652 AG7720 GM1891 GM03523
46 +_ 5 (25) 81-_ 7 i4) 39 _= 7 (1% 51 ~ 4 (3~ 34 ± 2 (4) 41 ~- 11 __~3t 45 2: 3 (58~
Alzheimer's
AG08245 AGO4402A AG6264A AG4400 AG5809A AG5810C
48 32 54 34 53 28 40
~ = -" ~_+ :~:
6 (17) 4 (22) 8 !5 2 (3i 12 (3) .3 I3)_ 3 (53)$
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated glass coverslip dishes. On day 3 the medium was switched to serum-flee DMEM, and on day 4 resting [Ca:+]i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values are mean - sem of the number of dishes indicated in parentheses; the value for each dish was determined from 5 microscopic fields, each consisting of 1-5 cells. -tPooted data for all fields of young cell lines. ~:Pooled data for all fields of Alzheimer's cell lines.
phalloidin (Molecular Probes). The cells were examined with a Leitz Diavert inverted microscope and an RCA SIT video camera, and the fluorescent images were recorded on optical disk using a Panasonic optical disk recorder. The images were digitized and cell size was quantified using a Vicom-Gould IP8500 image processor, interfaced with a Microvax II computer. Two dishes of each cell line were examined at each time point, and at least 20 cells per dish were analyzed. RESULTS
Resting [Ca 2 +]i Resting levels of cytosolic calcium were measured in serum-deprived young normal and Alzheimer's flbroblasts maintained on both uncoated and polylysine-coated glass coverslip dishes. For cells grown on uncoated glass, the values were 45 nM and 40 nM (see Table for 1 values for individual cell lines). As a control for effects of the substrate, several of these lines (Table 2) were grown on poly-D-lysine-coated glass. In this case, resting [Ca2+]i was 27 nM and 29 nM for young and Alzheimer's cells, respectively. Importantly, on either substrate, r e s t i n g [Ca2+]i was similar for young and Alzheimer's fibroblasts. This is in contrast to the results of Peterson et al. (8), who found that resting calcium in Alzheimer's fibroblasts (16 nM) was about 4-fold lower than resting calcium in fibroblasts from young donors (60 nM).
Response to Bradykinin and Fetal Calf Serum We next examined the ability of serum-deprived fibroblasts to respond to bradykinin. Typical responses to 25 nM bradykinin for young and Alzheimer's fibroblasts plated on uncoated glass are shown in Fig. 1A and B, respectively. In both cell
C A L C I U M T R A N S I E N T S IN A L Z H E I M E R ' S F I B R O B L A S T S
35
TABLE 2
TABLE 3
RESTING [Ca2+]x IN HUMAN FIBROBLASTS
CHANGES IN [Ca:+]i IN HUMAN FIBROBLASTS IN RESPONSE TO BRADYKININ
Cell Line
[Ca2+]i, nM* Cell Line
Young
AG3440 GM3652
22 ± 3 (16) 34 ± 4 (15)
Young
GM3652 AG3440 GM03651C AG7720 GM03523 GM1891 AG4390
27 ± 3 (31)t AGO4400 AG08245 AG00364D AG04402A
Alzheimer' s
29 34 18 31
± ± ± ---
3 4 6 3
D[Ca2+]i, riM*
(7) (9) (8) (9)
257 320 441 566 287 105 660
29 --- 2 (33):~ Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on polyD-lysine-coated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 resting [Ca2+]i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values are mean ± sem of the number of dishes indicated in parentheses; the value for each dish was determined from 5 microscopic fields, each consisting of 1-5 cells. tPooled data for all fields of young cell lines. :~Pooled data for all fields of Alzheimer's cell lines.
types, [Ca 2+ ]i b e g a n to increase within 1 0 - 2 0 s e c o n d s o f bradykinin application, quickly reached a peak, and then declined. In general, [Ca2+L returned to a plateau value w h i c h w a s h i g h e r than the resting level prior to drug application. A l t h o u g h res p o n s e s to bradykinin were variable, the r e s p o n s e s o f Alzheim e r ' s fibroblasts were similar in shape and amplitude to those o f y o u n g fibroblasts. R e s p o n s e s to 250 n M bradykinin were also similar in y o u n g fibroblasts and A l z h e i m e r ' s fibroblasts (not s h o w n ) . To c o m p a r e the r e s p o n s e s o f the cells in a quanti-
1000
I
I
I
I
58 52 53 42 81 73 81
353 ± Alzheimer' s
AG08245 AG04402A AG6264A AG4400 AG5809A AG5810C
566 488 445 327 395 367
(ll) (17) (4) (3) (3) (4) (6)
34 (48)1
± 141 ___ 62 ± 68 ± 74 ± 183 ± 99
471 ±
(9) (14) (5) (3) (3) (3)
45 (37):~
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca 2÷ ]i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values show the maximal increase in [Ca 2+ ], in response to 25 nM bradykinin (peak response minus preapplication value). Each value is the mean ± sem of the number of determinations indicated in parentheses. CPooled data for all fields of young cell lines. :~Pooled data for all fields of Alzheimer's cell lines.
tative m a n n e r , we d e t e r m i n e d the m a x i m u m c h a n g e in [Ca2+]i (D[Ca2+]i) for each treatment. T h i s value w a s calcu-
1000
I
1
1
1
1
1
Alzheimer's
Young 800
c
± ± ± ± ± ± ±
B
800
\
600
~_~c 600
+ t~
8 400 ,--,
o
o
200
400
2O0
0 0
I
I
I
I
I
60
120
180
240
.300
seconds
0 .360
0
60
120
180
240
300
360
seconds
FIG. 1. Fibroblasts from young (A) and Alzheimer's (B) donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca2+]i was measured as described in the Method section. Bradykinin (25 nM) was added at the arrow. The data in (A) shows 3 traces each from GM3440 and AG7720, and the data in (B) show 3 traces each from AG04402A and AG5810C. Each trace is from a single dish, and represents a field of 1-5 cells.
36
!~ORI)EN
1000
I
I
I
[
- -
1000
'1
r-
i
-1. . . . .
i
T. . . . . . . . . .
.....
800
.,\~
T. . . . B
Alzheilner's
Young
[i~l "
800
I =1
600
-
600
+
,~. 400
4oo
-
200 -
2OO
0 0
60
I
I
I
I
1 20
180
240
300
I
360
0
60
120
180
240
300
36C
seconds
seconds
FIG. 2. Fibroblasts from young (A) and Alzheimer's (B) donors were plated as described in Fig. 1. One percent fetal calf serum was added at the arrow. The data in (A) show 2 traces each from GM3652 and GM3440, and the data in (B) show 2 traces each from AG08245 and AG04402A. Each trace is from a single dish, and represents a field of 1-5 cells.
lated as the peak h e i g h t in r e s p o n s e to bradykinin, m i n u s the c o r r e s p o n d i n g predrug resting level. T h e values for the individual cell lines e x a m i n e d are s h o w n in Table 3 (uncoated glass) and in Table 4 for several o f the lines on polylysine-coated glass. W h e n the data for the various cell lines e x a m i n e d is pooled, the increase in [Ca 2+ ]i in A l z h e i m e r ' s fibroblasts plated on u n c o a t e d glass (471 n M ) w a s f o u n d to be s o m e w h a t greater than the increase in y o u n g fibroblasts (353 nM). Similarly, alt h o u g h r e s p o n s e s o f cells plated on p o l y - D - l y s i n e were smaller
than those o f cells plated on uncoated glass, the m a x i m a l increase in [Ca 2÷ ]i in A l z h e i m e r ' s fibroblasts (298 n M ) w a s again s o m e w h a t greater than the increase o f y o u n g fibroblasts (193 nM). Typical r e s p o n s e s o f y o u n g and A l z h e i m e r ' s fibroblasts on uncoated glass in r e s p o n s e to 1% fetal calf s e r u m are s h o w n in Fig. 2 A a n d B, respectively. T h e r e s p o n s e s are similar in shape to those elicited with bradykinin, consisting o f an inital peak followed by a plateau. Interestingly, quantitation o f the data d e m o n s t r a t e s that the m a x i m a l increase in [Ca2+]i is nearly
TABLE 4 CHANGES IN [cae+]i IN HUMAN FIBROBLASTS IN RESPONSE TO BRADYKININ Cell Line Young
Alzheimer' s
AG3440 AG3652
AG04400 AG0364D AG04402A AG08245
TABLE 5
D[Ca2+]i, nM* 163 + 17 (13) 222 +-- 43 (13) 193 + 23 (26)? 356 -+ 322267 -+ 281 +298 +
109 41 24 46 30
(8) (3) (12) (11) (34)$
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on polyD-lysine-coated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca2+] i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values show the maximal increase in [Ca2÷]i in response to 25 nM bradykinin (peak response minus preapplication value). Each value is the mean +_ sem of the number of determinations indicated in parentheses. ?Pooled data for all fields of young ceil lines. :~Pooled data for all fields of Alzheimer's cell lines
CHANGES IN [Ca2+] i IN HUMAN FIBROBLASTS IN RESPONSE TO FETAL CALF SERUM Cell Line
D[Ca 2 ÷ ]i, nM*
Young
GM3652 AG3440
466 _+ 128 (5) _424 _+ 50 (6) 443 ___ 61 ( l l ) t
Alzheimer's
AG04402A AG08245
423 +- 35 (6) 464 -+ 80 (6) 444 -+ 42 (12):I:
Fibroblasts from young normal or Alzheimer's donors were plated on day 0 in DMEM with 16.7% heat-inactivated fetal calf serum, on uncoated glass coverslip dishes. On day 3 the medium was switched to serum-free DMEM, and on day 4 [Ca2+] i was measured with fura-2 and microspectrofluorometry as described in the Method section. *Values show the maximal increase in [Ca2+]i in response to 1% fetal calf serum (peak response minus preapplication value). Each value is the mean _+ sere of the number of determinations indicated in parentheses. ?Pooled data for all fields of young cell lines. ,Pooled data for all fields of Alzheirner's cell lines.
CALCIUM TRANSIENTS IN ALZHEIMER'S FIBROBLASTS
identical for young and Alzheimer's fibroblasts (Table 5). While only two control lines and two AD lines were tested here, these data are clearly in disagreement with the results of Peterson et al. (9) in which all AD lines were unresponsive and all control lines were responsive. Similar results were obtained with 10% fetal calf serum (not shown), although this was examined in only a few experiments. The responses to bradykinin and fetal calf serum in young and Alzheimer's cells that were serum-starved for about 80 hours were similar to those obtained in cells serum-starved for 24 hours (data not shown). In summary, the present findings differ from those of Peterson et al. (9), who found that the response of Alzheimer's fibroblasts to bradykinin or fetal calf serum was greatly diminished when compared to the response of fibroblasts from young donors. In the previous study (9), cells were maintained in antibioticfree medium, whereas in the present study, cells were switched to antibiotic-free medium the day before [Ca 2÷ ]i was measured. To determine if this accounted for the discrepant results, young (cell lines AG7720 and AG3440) and Alzheimer's (AG6264 and AG364D) cells were plated on uncoated glass on day 0 in medium with serum but lacking antibiotics, on day 3 they were switched to serum-free medium (also lacking antibiotics), and on day 4 [Ca2+] i was measured. Resting [Ca2+] i was 36---2 nM and 4 2 - 5 n M , and D[Ca2+]i was 3 8 9 ± 4 9 nM and 315---32 nM for young and Alzheimer's cells, respectively, where each value is the mean ± SEM of 2 cell lines, 3 dishes/line. These results are similar to those obtained under control conditions, indicating that the discrepancy with the earlier reports is not due to an effect of antibiotics.
Spreading It was previously reported (8) that during a 2-hour period, 40% of Alzheimer's cells spread as compared with 80% for fibroblasts from young donors. In these previous studies, cells were considered spread when they had attained a polygonal shape. To examine spreading in a more quantitative manner, cells were fixed at various times (generally 2 or 3 hours) after plating and stained with rhodamine phalloidin. Cell size was determined by digital image analysis of the fluorescent images. In the first set of experiments, we compared the spreading of the Alzheimer's line AG4402A (passages 11 and 12) with that of the young cell line GM3440 (passages 12-14). In qualitative agreement with Peterson et al. (8), the Alzheimer's line spread more slowly: at 2 and 3 hours after plating, the Alzheimer's cells were 70--5% and 6 0 _ 6% the size of the young cells, respectively, where each value represents the mean -- SEM of three independent experiments. To determine whether these results reflected a general property of young vs. Alzheimer's cells, we examined the spreading of other cell lines. In contrast to the previous results, the Alzheimer's cell line AG04400 (passages 9 and 10) spread more rapidly than the young cell line GM3440 (passages 10 and 11): at 2 and 3 hours, the Alzheimer's cells were 108% and 126%, respectively, the size of the young cells (mean of 2 independent experiments). Similarly, the Alzheimer's line AG6264A (passage 10) spread more rapidly than the young line GM03651C (passage 10) (131% and 114% of young at 3 and 4 hours, respectively). Thus spreading is variable among cell lines, and there is no consistent deficit in the ability of Alzheimer's fibroblasts to undergo spreading. DISCUSSION
In the present study we found that cell spreading, resting cytosolic calcium, and the response to serum were similar in flbroblasts derived from normal young donors and from older patients
37
with Alzheimer's disease, while the response to bradykinin was, if anything, slightly greater in Alzheimer's cells. These results disagree with previous studies (8,9) in which these parameters were found to be decreased in cells from aged donors, and further diminished in Alzheimer's disease. Further experiments employing additional Alzheimer's cell lines and age-matched controls would be necessary to determine if the heightened response to bradykinin is significant, and if so, whether it reflects differences in the age of the donors (young vs. old) or is a direct consequence of Alzheimer's disease. As shown by the representative traces in Figs. 1 and 2, the responses of both young and Alzheimer's fibroblasts to agents that elevate cytosolic calcium are variable, even within a cell line, The variability was observed within an experiment, and thus was not due to day-to-day variability. Although the responses of Alzheimer's cells to bradykinin were slightly greater than those of young cells (Tables 3 and 4), it is apparent from Fig. 1 that there is considerable overlap in the responses; a similar overlap is seen in the responses to fetal calf serum (Fig. 2). While occasional cells responded poorly to bradykinin or fetal calf serum, this was observed both in young and Alzheimer's cells. It should be stressed that no cell line examined consistently gave poor responses, nor were poor responses consistently observed within any particular experiment. This is in marked contrast to the previous study (9), in which Alzheimer's cells were consistently found to be poor responders. It is presently not clear why our results differ from those of the earlier study. Although we attempted to perform the experiments under similar conditions to those used previously, a number of factors were different. First, although six of seven Alzheimer's cell lines employed in the present study were the same as in the previous study, and were obtained from the same source, they were derived from different frozen stocks. Thus it is possible that the various frozen stocks differ in their properties. Second, the cells were used at later passage number (7-19) than in the previous study (average, 7.5). This does not seem to account for the discrepancy because the responses to bradykinin and fetal calf serum did not appear to differ with passage number. Last, different batches of serum were used in the growth medium. Although the cells were serum-starved for one day before [Ca 2+ ]i was measured, long-lasting changes may have taken place during their growth in serum-containing medium. Thus it is possible that certain batches of serum have differential effects on fibroblasts from young, old, or aged donors, while other batches do not. Control experiments revealed that maintaining the cells with or without antibiotics did not account for the discrepancy with the previous findings. Recently, Sisken et al. (11) presented an abstract in which they reported that the responses to 1% fetal calf calf serum are similar in Alzheimer's and control fibroblasts, whereas the responses to 10% serum are greater in Alzheimer's cells. These findings are similar to our current results and indicate that, under certain conditions, the responses of Alzheimer's fibroblasts are not diminished compared to cells from normal donors. However, it can not be ruled out that defects in calcium homeostasis may exist in certain subsets of Alzheimer's patients. In summary, we now find that cell spreading, resting cytosolic calcium, and response to serum in fibroblasts from patients with Alzheimer's disease do not differ greatly from those of cells from young, normal donors. The discrepancy from the earlier findings of diminished responses in Alzheimer's fibroblasts is not understood, but may relate to poorly understood variations in cell culture conditions. In any event, the present findings suggest that the previously observed deficits in fibroblasts are neither pathognomonic for Alzheimer's cells nor of diagnostic value.
~8
fgORDEN [ ! t A l
ACKNOWLEDGEMENTS We would like to thank Ms. Bernetta Abramson for expert assistance with cell culture, and Dr. Jesse Sisken for helpful discussions. This work was supported by grants from the NIH (NS27680) and the American Health Assistance Foundation. L . A B was a trainee of the National Institute of Aging (AG00189).
REFERENCES 1. Gibson, G. E.; Nielsen, P.; Sherman, K. A.; Blass, J. P. Diminished mitogen-induced calcium uptake by lymphocytes from Alzheimer patients. Biol. Psychiatry 22:1079-1086; 1987. 2. Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260:3440-3450; 1985. 3. Jarvik, L. F.; Matsuyama, S. S.; Kessler, J. O.; Fu, T.-K.; Tsai, S. Y.; Clark, E. O. Philothermal response of polymorphonuclear leukocytes in dementia of the Alzheimer type. Neurobiol. Aging 3:93-99; 1982. 4. Kruskal, B. A.; Shak, S.; Maxfield, F. R. Spreading of human neutrophils is immediately preceeded by a large increase in cytoplasmic free calcium. Proc. Natl. Acad. Sci. USA 83:2919-2923; 1986. 5. Li, J. C.; Kaminiskas, K. Deficient repair of DNA lesions in Alzheimer's disease fibroblasts. Biochem. Biophys. Res. Commun. 129:733-738; 1985. 6. Miller, R. Immunodeficiency of aging: Restorative effects of phorbol ester combined with calcium ionophore. J. Immunol. 137:805-
808; 1986. 7. Peterson, C.; Goldman, J. E. Alterations in calcium content and biochemical processes during aging and Alzheimer's disease. Proc. Natl. Acad. Sci. USA 83:2758-2761; 1986. 8. Peterson, C.; Ratan, R. R.; Shelanski, M. L.; Goldman, J. E. Cytosolic free calcium and cell spreading decrease in fibrobtasts from aged and Alzheimer donors. Proc. Natl. Acad. Sci. USA 83:79998001; 1986. 9. Peterson, C.; Ratan, R. R.; Shelanski, M. L.; Goldman, J. E. Altered response of fibroblasts from aged and Alzheimer donors to drugs that elevate cytosolic free calcium. Neurobiol. Aging 9:261266; 1988. 10. Rizopoulos, E.; Chambers, J. P.; Wayner, M. J.; Martinez, A. O.; Armstrong, L. S. Effects of free Ca 2+ on the [Ca 2+ + Mg2*] dependent adenosinetriphosphatase (ATPase) of Alzheimer and normal fibroblasts. Neurobiol. Aging 10:717-720; 1989. I1. Sisken, J. E.; McCoy, K. R.; Newcomb, T. G.; You, J. Seruminduced calcium transients in fibroblasts from individuals with familial Alzheimer's disease. J. Cell Biol. 109:301a; 1989.