Decreased survival of hippocampal neurons in medium conditioned by fibroblasts from aged and Alzheimer donors

Brain Research, 515 (1990) 39-44

39

Elsevier BRES 15392

Decreased survival of hippocampal neurons in medium conditioned by fibroblasts from aged and Alzheimer donors Christine Peterson and Carl W. Cotman Department of Psychobiology, University of California, Irvtne, CA 92717 (U.S.A ) (Accepted 26 September 1989)

Key words. Alzheimer's disease; Aging; Tissue culture; Fibroblast; Conditioned medium; Neuron; Hippocampus; Neurite, Survival

Medium conditioned by fibroblasts promotes survival and neunte outgrowth of cultured hippocampal neurons. Sub-confluent cultures of cells from normal young, aged and Alzhelmer donors conditioned serum-free medium for 2 days. Hippocampal neurons from 18- to 19-day-old embryos were maintained in medium conditioned by skin fibroblasts from young, aged or Alzhelmer donors. One, 4 and 7 days after plating the cells were examined for morphological differences. Neuron survival four days after plating was found to be the highest in medium conditioned by young > aged > Alzheimer cells. These findings suggest that both aged and Alzheimer donors secrete less of the substances that are necessary for survival and neurite outgrowth of hlppocampal neurons. This provides further evidence that non-neuronal cells demonstrate abnormalities during aging and Alzheimer's disease INTRODUCTION A l z h e i m e r ' s disease is a neurodegenerative disease characterized by severe n e u r o n a l loss in specific cortical and subcortical areas 3A3,a4. The reported n e u r o n a l degeneration in the entorhinal cortex, subiculum and the hippocampus may serve to functionally disconnect the cortex from the hippocampus 14. Golgi impregnation reveals a decrease in hippocampal pyramidal cells n u m ber and size in the brains of Alzheimer patients 3°. The precise mechanism for this loss is currently u n k n o w n . A m o n g the molecules critical to the growth of n e u r o n s are neurotrophic factors. It has been suggested that n e u r o n a l loss in Alzheimer's disease may be related to either a lack of neurotrophic factors ~'26 or decreased responsiveness of n e u r o n s to these factors 11. N o n - n e u ronal cells such as fibroblasts secrete substances that support n e u r o n a l growth. Cholinergic function of sympathetic n e u r o n s can be stimulated by soluble factors that are released by both normal h u m a n ~5 and rat ~7 fibroblasts. R e d u c e d neurotrophic activity is also observed in sensory ganglion cells treated with conditioned media from individuals with d y s a u t o n o m i a29, a disease in which a b n o r m a l nerve growth factor ( N G F ) production has been postulated 18'2s. This suggests that peripheral tissues produce factors similar to those that normally act on cells of the central nervous system (for review see refs. 5, 22). The present study examined whether h u m a n skin fibroblasts release soluble substances that have neurotrophic

activity on hippocampal n e u r o n s in vitro a n d whether these compounds are altered by aging or Alzheimer's disease.

MATERIALS AND METHODS Timed pregnant Sprague-Dawley rats that were obtained from Charles Rivers Breeding Labs (Wilmington, MA) were housed individually. Heat-inactivated fetal bovine serum, Trypan blue and Dulbecco's modified Eagles' medium (DMEM) were obtained from Gibco (Long Island City, NY). Defined medium components and all other chemicals were from Sigma. Cultured skin fibroblasts were obtained from the NIA Aging Cell Repository (Camden, NjI9). Cells from normal young (AG6310, AG7720, AG7804, AG7306, GM1891, GM3440, GM3523, AG4390), aged (AG6241, AG6010, AG4560, AG4149, AG7139, GM1681, AG7803) and Alzhelmer patients with neuropathologically confirmed Alzheimer's disease or first degree relatives with confirmed Alzheimer's disease (AG00364A, AG4400, AG4401B, AG6262, AG5770B, AG6262, AG6264, AG6848) were used in these studies.

Skin fibroblast cultures Cultured skin fibroblasts from young, aged and Alzheimer donors were seeded into 75 cm2 flasks at a density of 10'*cells per cm2. The cells were maintained in 10 ml of 16 6% heat-inactivated fetal bovine serum diluted in DMEM as described prevaously19 Two days after plating, the serum-containing medium was removed and the cells were nnsed 5 times with 10 ml of Hank's balanced salt solution without calcium or magnesium. Three ml of new serum-free DMEM that also contained 25 mM N-2-hydroxyethylplperazme-N'-2-ethanesulfonic acid (HEPES) and bovine serum albumin (BSA; 3 mg/ml) was added to each of the flasks. Forty-eight hours later the medium was removed and centrifuged for 3 rain at 400 g and an ahquot (1.5 ml) was placed Into 35 mm2 dishes. None of the cultures were confluent during the experiment. Condmoned medium was

Correspondence C. Peterson, Department of Psychobiology, University of Cahfornla, Irvine, CA 92717, U S A 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B V. (Biomedical Division)

40 prepared from each cell line at three different passages, all the media were tested separately (n = 9 determinations per cell line)

Htppocampal cultures Hippocampl from rat embryos (18-19 days old) were dissected an Hank's balanced salts solutaon without calcmm or magnesium but with 4.2 mM sodium bicarbonate, 1 mM pyruvate, 20 mM HEPES and 3 mg/ml BSA 2°. The tissue was mechanically dissociated in 2 ml of Hank's dissecting buffer by triturataon through a fire-polished sihcomzed glass papette tip (0.5-1 mm dmmeter) An equal volume of DMEM with 10% heat-inactavated fetal bovine serum (final concentration), 20 mM HEPES, 26.2 mM sodium bicarbonate and 1 mM pyruvate was added and the cell suspensaon was mixed and non-triturated debris was allowed to settle for 3 min The supernatant was transferred to a new tube and centrifuged at room temperature for 3 min at 400 g The supernatant was discarded and the pellet resuspended in 4 ml of DMEM that contained 2.4 mg/ml of bovine serum albumin, 26.2 mM sodium bicarbonate, 1 mM sodium pyruvate and 20 mM HEPES (DMEM-BSA). The suspensaon was recentnfuged and the pellet resuspended in DMEM-BSA. An aliquot was removed for counting with a hemocytometer; vmble neurons excluded Trypan blue. Cells were plated (day 0) onto 12 mm poly-D-lyslne (50 ag/ml) coated covershps at a densaty of 75 viable cells/ram2. One hour after plating, the excess medium, that contained non-attached cells and debris, was aspirated. Four coverslips were placed with the cell sade down into 35 mm 2 plastac tissue culture dishes that contained 1 5 ml of the medium condi-

tloned by the subconfluent fibroblast cultures There is no neuron survwal in uncondmoned D M E M - B S A As a means to monitor the hlppocampal preparation cultures under controlled conditions, neurons were maintained m 1.5 ml of antibiotic-free D M E M - B S A medium that was supplemented with 0.41 a M biotin, 0.1 mg/ml bovine serum albumin, 10 a M carnitine-HCl, 10 a M ethanolamineHCI, 85 a M galactose, 5 ag/ml insulin, 100 a M putresclne-HCl, 0.03 a M selenite, 0.003 a M tmodo-L-thyronlne, 5 ag/ml transferrin, 0.25 a M vitamin B12, 0.06 a M cortlcosterone, 3.6 a M hnolelc acid, 3.6 a M hnolemc acid, 0.02 a M progesterone, 0 3 a M retinol-all trans, 0 35 a M retlnyl acetate, 2 3 a M tocopherol, 2.1 a M tocopheroi acetate 21 Neurons plated m this manner were generally viable for up to 3 weeks 2°. The hippocampal neurons were examined at 1, 4 and 7 days after plating. Four days after plating neuron survival was determined by Trypan blue exclusion The 4 coverslips per dish were removed and placed on top of 0.003 ml of Trypan blue Five fields of neurons were counted at 20x magmfication The total number of neurons per field was simdar m all of the conditions tested. Different cell types were adentified immunocytochemlcally Briefly, the cells were fixed wath 3 7% buffered formalin (30 min), rinsed with phosphate-buffered saline, incubated with 1.25% ovalbumin; 1% non-fat dry milk (15 min) to block non-specific binding, primary antibody (45 mm), phosphate-buffered saline (3 min), secondary antibody conjugated to peroxidase antl-peroxldase (45 mln), phosphate-buffered saline (3 man), 3,3"-diaminobenzidine (8 min), phosphate-buffered saline (3 min), series of upgraded alcohols

Fig. 1. Hippocampal neurons 1 day after plating. Hippocampt from embryonic rat were dissociated and plated (75 cells/mm2) on 13 mm covershps that had been coated with poly-lysine One hour after plating, excess medium was aspirated and the coverslips were inverted and placed into 35 mm dishes that contamed either (A) defined media or medium conditioned by cultured skin fibroblasts from either (B) young (AG7720), (C) aged (AG7139) or (D) Aizhelmer (AG4401B) donors. (x 320 )

41 %

5

f

Fig. 2 Hippoeampal neurons 4 days after plating. Hippocampi from embryonic rat were dissociated and plated (75 cells/mm 2) on 13 mm coverslips that had been coated with poly-lysme. One hour after plating, excess medium was aspirated and the coverslips were inverted and placed into 35 mm dishes that contained either (A) defined media or medium conditioned by cultured skin fibroblasts from either (B) young (GM3523), (C) aged (AG6241) or (D) Alzheimer (AG6264) donors. (x 320.). (3 min), xylene (3 rain) and mounted. Rabbit antlsera to glial fibrillary acidic protein and mouse monoclonal to phosphorylated and non-phosphorylated neurofilament antibodies were used2°. Astrocytes were very flat cells and dearly distinct from neurons. The percentage of glial fibnllary acidic protein stained cells (2.3 + 0.1%; n = 4 preparations) was much lower than the neurons stained with neurofilament antibody (98.5 + 1.2%; n = 4 preparations) There were no unidentified cells types in the preparation used. Only those cells that resembled neurofilament stained cells were counted in the remainder of the expenments. RESULTS H i p p o c a m p a l n e u r o n s that are m a i n t a i n e d in D M E M with defined c o m p o n e n t s sprouted neurites within two hours after plating. The n u m b e r of Trypan blue excluding n e u r o n s was 82.7 + 1.0% of the total n u m b e r of neurons. N e u r o n survival in fibroblast conditioned m e d i u m from young cells was slightly less (78.5 + 1.3% of the total n u m b e r of neurons counted) than that of neurons in defined m e d i u m . Survival of h i p p o c a m p a l neurons at 24 h was similar w h e t h e r the m e d i u m was conditioned by young, aged or A l z h e i m e r donors.

F o u r days after plating in defined m e d i u m , n e u r o n survival was still very high (81.0 + 0.6% of the total n u m b e r of neurons). F i b r o b l a s t - c o n d i t i o n e d m e d i u m was still not as effective as the defined m e d i u m c o m p o n e n t s in p r o m o t i n g n e u r o n survival. W h e n the m e d i u m conditioned by the 3 cell groups is c o m p a r e d , neuronal survival is highest with young donors, less with aged donors and even further r e d u c e d with A l z h e i m e r donors. This observation was not due to a m e a s u r a b l e alteration in the protein content of the m e d i u m conditioned by the different cell lines (3.96 + 0.07 mg/ml (young; n = 24), 3.98 + 0.06 mg/ml (aged; n = 24) and 3.95 + 0.06 mg/ml ( A l z h e i m e r ; n = 24). By 24 h the neurites were generally longer than the d i a m e t e r of 4 cell bodies in the cultures m a i n t a i n e d in defined m e d i u m (Fig. 2A). T h e dendrites h a d m a n y branches and p r o n o u n c e d growth cones. W h e n neurons were m a i n t a i n e d in D M E M - B S A that was conditioned by cells from either young (Fig. 1B), aged (Fig. 1C) or A l z h e i m e r (Fig. 1D) donors neurite outgrowth was slower and less extensive than those cells grown in

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Fig. 3. Hippocampal neurons 7 days after plating. Hippocampl from embryomc rat were dissociated and plated (75 cells/mm2) on 13 mm coverslips that had been coated with poly-lysme. One hour after plating, excess medmm was aspirated and the coverslips were inverted and placed into 35 mm dishes that contained either (A) defined media or medium conditioned by cultured skin fibroblasts from either (B) young (GM4390), (C) aged (AG6010) or (D) Alzheimer (AG4400) donors (x 320 )

defined media (Fig. 1A). The neurites were not as long nor as branched but growth cones could be easily observed. At 24 h there appeared to be no differences qualitatively in neuron morphology between cells maintained in medium conditioned by cells from young, aged or Alzheimer donors. Four days after plating, the dendritic arbors were very extensive and elaborate in the cells maintained in D M E M - B S A with defined medium components (Figs. 2A and 3A). Neurons grown in media conditioned by cells from young donors also had many dendrites but they did not appear to be as many when compared to neurons maintained in defined medium (Figs. 2B and 3B). The hippocampal neurons in medium conditioned by aged donors had dendrites that appeared to be finer and not as extensive as those in either defined medium or medium conditioned by young cells (Figs. 2C and 3C). Furthermore there were areas that showed degeneration. In medium conditioned by fibroblasts from Alzheimer donors the cell bodies appeared shrunken and the

dendrites were thin. There were swellings at the branch points and along other areas of the dendrites as well (Figs. 2D and 3D). Seven days after plating, the hippocampal neurons in defined medium still looked healthy and had extensive dendritic arbors. In defined media there was more dendritic cabling which appeared as very thick processes. Those neurons in medium, conditioned by aged and Alzheimer donors showed marked degeneration of both cell bodies and dendrites, when compared to medium conditioned by young donors. DISCUSSION These data demonstrate that medium conditioned by fibroblasts promotes the survival and growth of hippocampal neurons m vitro. Protein synthesis in cultured skm fibroblasts, as measured radioisotopically is depressed by aging and Alzheimer's disease despite the similarities in extruded protein (present study) and cell

43 n u m b e r 19. It is not yet clear whether the reduced activity of the fibroblast conditioned medium represents diminished synthesis, deficient secretory mechanisms or release of a modified molecule with less or inhibitory activity. N G F production by fibroblasts in vitro was reported several years ago but N G F synthesis by fibroblasts in vivo has yet to be demonstrated. Unfortunately the synthesis of N G F by fibroblasts does not fit with the target tissue hypothesis since fibroblasts are not a target for sensory or sympathetic nerves. Fibroblasts, however, may provide N G F under special conditions, such as nerve regeneration 12. Fibroblasts also secrete extracellular matrix proteins (for review see ref. 22). Three extracellular matrix proteins that interact with neurons are tenascin, fibronectin, heparan sulfate proteoglycan as well as the membrane-associated adhesion molecule N C A M . All of these compounds accumulate in the interstitial areas surrounding denervated synaptic sites 24 and appear to be synthesized by fibroblasts that proliferate in perisynaptic areas following denervation 4'7's. When regenerating axons approach synaptic sites the secretion of these neuroactive extracellular matrix molecules by fibroblasts may guide neuronal regrowth. W h e t h e r decreased production of trophic factors contributes to Alzheimer's disease is controversial. Alz-

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