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Canine leptomeningeal organ culture: a new experimental model for cerebrovascular β-amyloidosis
Journal of Neuroscience
Methods
68 (1996)
143- 148
Canine leptomeningeal organ culture: a new experimental model for cerebrovascular l3-amyloidosis Reinhard Prior a.* , Donatella D’Urso a, Rainer Frank ‘, Ingrid Prikulis ‘, Giinther Wihl a, Goran Pavlakovic a a Department
of Neurology. Heinrich-Heine-University. h Centerfor Molecular Biology Uniuersi& Received
4 October
1995; revised
Moorenstr. of Heidelberg.
29 January
5. D-40225 Diisseldorj~ Heidelberg, Grrmun?
1996: accepted 30 January
Germun\,
1996
Abstract Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzheimer’s disease and a common cause of cerebral hemorrhage in the elderly. The pathogenetic mechanisms leading to the deposition of Alzheimer amyloid p-protein (A@) in cortical and leptomeningeal vessel walls are unknown. There are no experimental models which reproduce the pathological changes of CAA. In this study, leptomeninges from young and old dogs with pre-existing CAA were cultured in cell culture medium or cerebrospinal fluid and their viability, histological appearance and metabolic activity were analyzed during the culture. In addition, living leptomeninges of old and young dogs were incubated with fluorescein-conjugated AP and the uptake of AP was studied by fluorescence microscopy. Leptomeninges from young and old dogs were viable up to 8 weeks in culture. They contain many small- and medium-sized arterioles, the main vessel type affected by CAA. Histology and immunohistochemistry showed excellent preservation of the vessel wall microarchitecture up to 4 weeks in culture. The cultures were metabolically active as shown by the de novo production of P-amyloid precursor protein. Exogenously added A(3 was focally deposited in the vessel walls of old, but not young dogs. In conclusion, the organ culture of canine leptomeninges is easy to perform and appears suitable to investigate the pathogenesis and the progression of CAA. Kryvords:
Cerebral
amyloid
angiopathy;
Aizheimer’s
disease;
Amyloid
P-protein:
Organ
culture -____
1. Introduction Intracerebral (senile plaques) and cerebrovascular amyloid deposits (cerebral amyloid angiopathy/CAA) of Alzheimer amyloid P-protein CAP; Glenner and Wong, 1984) are a neuropathological feature of Alzheimer’s disease (AD) and are central to its pathogenesis (for review see Maury, 1995; Selkoe, 1994). CAA may also develop in the absence of AD and is a common cause of cerebral hemorrhage in the elderly (Vonsattel et al., 1991; Itoh et al., 1993). AP is derived from the transmembrane amyloid precursor protein, APP (Kang et al., 1987). Proteolytic cleavage of APP generates AP species of heterogeneous length; whereas in senile plaques AP(I--42) is the most prevalent form (Masters et al., 1985; Iwatsubo et al., 1994), AP(l-
-.--
-.---
-- -.--
..---
391, A@l-40) and A@(1 -42) have been isolated from cerebrovascular amyloid (Prelli et al., 1988: Joachim et al., 1988; Roher et al., 1993). Increased brain concentrations of Aj3(1-40) have been linked to the development of CAA (Suzuki et al., 1994). The experimental reproduction of AP deposits is important to understand the pathogenesisof AD and CAA and
different approaches have been employed to reproduce AQ deposits. Synthetic A@ was shown to form amyioid fibrils in cell-free in vitro systems (Castano et al., 1986; Kirschner et al., 1987; Hilbich et al., 1991). Intracellular A@ aggregates were observed in neuronal cell cultures overexpressing the C-terminal part of APP (Maruyama et al., 1990). Recently, the first transgenic mouse model with substantial intracerebral A/3 deposits has been described (Games et al., 1995). However, cerebrovascular deposits were not reported.
Corresponding 17803.
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Various proteins give rise to amyloid formation in different tissues causing a group of heterogeneous diseases collectively termed amyloidoses. Any type of amyloidosis
Q 1996 Elsevier
Science
B.V. All rights reserved
R. Prior et al. /Journal of Neuroscience Methods 68 (1996) 143-148
144
(i.e. the pathological extracellular deposition of insoluble and fibrillar proteins which stain with Congo Red and show’green-red birefringence with polarized light) shows a highly specific tissue distribution. Whereas APP is ubiquitously expressed (Golde et al., 1990) and soluble AP is present in blood serum and cerebrospinal fluid (CSF) (Seubert et al., 1992; Vigo-Pelfrey et al., 1993), AP amyloidosis occurs exclusively within the brain cortex or within the walls of cortical and leptomeningeal arterioles and capillaries. It is not known what tissue-specific molecular mechanisms underlie cerebrovascular AP deposition and what is the origin of cerebrovascular AP. Since soluble AP is produced by cultured cells during normal metabolism (Haass et al., 1992) and since A@ is detected at low concentrations in cerebrospinal fluid and blood serum under physiological conditions (Seubert et al., 1992; Vigo-Pelfrey et al., 1993), the cerebrovascular A@ deposits may be derived from blood serum or cerebrospinal fluid. Alternatively, A@ may originate from APP produced locally by vascular smooth muscle cells (Tagliavini et al., 1990; Frackowiak et al., 1995). Dogs are one of the few species which develop amyloid deposits with age (Uchida et al., 1993; Cummings et al., 1993). In addition, the amino acid sequences of human and canine AP are identical (Johnstone et al., 1991). Therefore, living canine tissue may be a suitable model for studying the pathogenesis of AP amyloidosis. The purpose of the present study was to examine survival, metabolic activity and histological changes in cultured leptomeninges obtained from young and old dogs and to determine, whether living canine leptomeninges might be useful for identifying the origin of cerebrovascular A p and to characterize conditions that induce or promote the pathogenesis of CAA.
2. Materials
2.2. Viability testing The leptomeningeal viability was analyzed immediately after dissection, after 2, 5 and 7 days in culture and then at weekly intervals. Tissue samples were incubated with fluorescein diacetate (FDA, 2 p,g/ml, Molecular Probes) for 5 min. After washing, the leptomeningeal membranes were distended on a microscopical slide, overlayed with phosphate-buffered saline (PBS), coverslipped and immediately analyzed by fluorescence microscopy. 2.3. Histology and immunohistochemistry For histological studies, leptomeningeal tissue was sampled immediately after the removal of the leptomeninges and after each week of organ culture. The tissue was fixed in 4% buffered formalin and embedded in paraffin. 8 p,rn sections were cut and stained with hematoxylin-eosin, Masson-trichrome and thioflavin S. For immunohistochemistry, deparaffinized sections were treated with 1% hydrogen peroxide in methanol to block endogenous peroxidase, washed in PBS and incubated overnight with the monoclonal primary antibodies against smooth muscle actin (Dako, clone lA4, 2 mg/ml), amyloid precursor protein (Boehringer, clone 22Cl1, 4 p.g/ml) and with a polyclonal antiserum raised against synthetic AP(2-43) (1:200 dilution, gently provided by K. Beyreuther). Following wash-out, sections were incubated with biotinylated antimouse or anti-rabbit antibodies for 1 h, washed in PBS and incubated with avidin-biotin peroxidase complex (Vector) for 45 min. Sections were developed in 0.05% 3,3-diaminobenzidine tetrahydrochloride (Sigma) plus O,Ol% hydrogen peroxide in PBS before counterstaining with hematoxylin. Negative controls were obtained by omitting the primary antibody or by using normal rabbit serum instead of the primary antibody.
and methods 2.4. Biosynthetic labeling and immunoprecipitation
2.1. Preparation
of APP
and organ culture of leptomeninges
Craniotomy was performed under sterile conditions immediately after the dogs (5 old dogs with mean age 14.8 + 1.3 years; 5 young dogs less than 1 year old) had been euthanized by a veterinarian for incurable non-neurological disease or had been killed for unrelated orthopedic studies. The whole brain was removed and transported on ice in physiologic saline supplemented with 5% glucose and a mixture of antibiotics. After dissection, leptomeninges were cultured in cell culture medium (DMEM containing 20% fetal calf serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 p,g/ml streptomycin) or in pooled human cerebrospinal fluid (CSF) supplemented with 0.45% glucose. The leptomeningeal membrane was kept freely floating in 20 ml of culture medium per 85 mm culture dish. The medium was completely exchanged after the first day and every other day of culture thereafter.
Samples (1 cm* of membranes) which had been kept for 1 week in culture were incubated for 3 h in methionine-free MEM (Gibco) supplemented with 150 $i of [35S]methionine. After removal of the medium, the tissue was washed twice in PBS and homogenized in 200 ~1 of STEN buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA), supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF), 2% Nonidet P-40 and 2% Triton X-100. After 10 min on ice, the samples were centrifuged at 10 000 X g for 5 min and the supematant stored at - 20°C until further analysis. For immunoprecipitation, the sample was diluted 1:5 with STEN and incubated with 7 ~1 of a polyclonal antiserum against recombinant full-length APP (provided by K. Beyreuther, Heidelberg) for 2 h at room temperature with constant shaking. Next, IO ~1 (1.5 mg) of protein A-Sepharose (Pharmacia) was added for 1 h. The insoluble complexes were washed three times, resus-
per&d in 50 ~1 of 2 X Laemmb sample buffer, boiled for 5 min and centrifuged at 10000 X g for 5 min. 20 p,g of total pro&& per sample was loaded onto an 8% SDS-PAGE gel. As a positive control, neuroblastoma cells (SYSY, 3 X 10’ cells), which c~~~vely express high levels of APP, were processed us&g the same protocol and loaded on the same gel. Following electrophoresis, the gel was fixed, dried and after overnight exposure analyzed with a Fuji BAS loo0 pkosphoimaging system. 2.5. Incubation conjugated A p
of the cultures with synthetic fluorescein-
A B(l-40) and an unrelated control peptide (GVEAYVDLKPASLEDIERLLFEDRRLMAYYC) were synthesized on polyoxyethylene-polysterene graft resin in a continuous fiow instrument constructed and operated as described (Frank and Gausepohl, 1988). Peptide chain assembly was performed using Fmoc chemistry and in situ activation of amino acid building blocks by PyBOP. Fluorescent was coupled to the N-terminal residue via its succinimidyl ester (5(and-6)carboxyfluorescein succinimidyl ester, Molecular Probes). The peptides were purified by reversed-phase HPLC and characterized by laser desorption mass spectrometry. From a 100 p,M stock solution in water. the peptides were added to the culture medium to a final concentration of 1 p,M. Leptomeninges from old and young dogs were incubated for 15 h with fluorescein-coupled ABC1 -40) and control peptide. The leptomeninges were frozen and 8 p,m cryosections were cut and embedded for analysis with a fluorescence microscope.
Fig. 1. Fluorescence microscopy of FDA tomeninges after 7 days of culture. Note the majority of the vessels indicating their viability.
3.1. Vessel iiahili~ Up to 4 weeks of culture, viability was shown by bright green light emission after FDA staining observed in the majority of vessels which were typically labeled over their full length (Fig. 1). Only occasionally were short segments of the vessels negative for FDA, indicating necrotic areas. After 4 weeks. the vessel walls showed a progressively discontinuous pattern of FDA staining as expression of vascular cell death. However, continuous segments of viable vessel walls were observed even after 8 weeks in culture. When leptomeninges were cultured in CSF, the majority of vessels were viable up to 3 weeks. There was no significant difference in the FDA staining pattern of leptomeninges obtained from young and old dogs. 3.2. Histology
anti immunohistochemist~
Paraffin sections and cryosections were analyzed after hematoxylin-eosin and Masson-trichrome staining. Small-
of
canine
Magnification.
lepof the
20X
and medium-sized arteries, which are the vessels most affected by CAA, represented the main vessel type in the cultured leptomeninges (Fig. 2a). The vessel wall architecture was well preserved during the culture. Sight thickening of the leptomeningeal membranes was noticed after 2 weeks of culture (Fig. 2b) and progressive connective tissue proliferation was observed after 4 weeks of culture (Fig. 2~). All of the old dogs showed thioflavin S-positive amyloid in the vessel walls (Fig. 3a) and the presence of AB in the vessels was confirmed immunohistochemically (Fig. 3b). Smooth muscle actin (Fig. 2c) and APP (not shown) were detectable in arteriolar vessel walls of young and old dogs even after several weeks of culture, indicating conserved structural integrity of the vessel walls. 3.3. Immzmc~precipitation
3. Results and discussion
staining
continuous staining
of biosynthetically lubeled APP
The immunoprecipitation studies following metabolic labeling of APP with [35S]methionine revealed that the canine leptomeningeal organ cultures had preserved the ability to synthesize APP (Fig. 4). Three protein bands between 95 and 115 kDa were observed after immunoprecipitation of the tissue homogenates (Fig. 4, lane 2). These bands are of the same molecular weight as the bands obtained by APP immunoprecipitation from cell lysates of neuroblastoma cells (Fig. 4. lane 3) which served as a positive control. The lower band (Fig. 4. lane 2) is identical to the APP band immunoprecipitated from the supernatant of neuroblastoma cells (Fig. 4, lane 4), suggesting that it represents the secreted form of APP (Weidemann et al.. 1989). 3.4. Orgun culture o~cnnine leptomcninges us u model,fku cerebrr~-l,asc~lllNr A/3 depositkm The organ culture of canine leptomeninges appears to be a useful model for studying the development of CAA, since the cultured leptomeninges of young and old dogs are viable for several weeks with only minor morphologi-
146
R. Prior
et al. /Journal
of Neuroscience
Methods
68 f 19961 143-148
Fig. 3. Cerebrovascular AP deposits in leptomeninges of old dogs after 1 week in culture: (a) stained with thioflavin S, and (b) immunostained for AP (counterstained with hematoxylin). Magnification, 40 X
Fig. 2. Paraffin sections of canine leptomeninges stained with hematoxylin-eosin immediately after dissection (a) and after 2 weeks (b). Panel c shows leptomeninges after 4 weeks of culture immunostained for smooth muscle actin and counterstained with hematoxylin. Cytoplasmic staining for smooth muscle actin can be observed in the vessel walls. The canine leptomeninges are abundant with small and medium sized arteries and the vessel wall architecture is well preserved. Magnification, 40 X
cal changes and good preservation of the vessel wall architecture. In addition, the cultured leptomeninges are metabolically active as demonstrated by the de novo production of APP. Our experimental model uses living tissue of a species in which CAA develops with age and preserves both the extracellular matrix and the microarchitecture of those vessel walls where Al3 is deposited in vivo. This might be important, particularly in view of the finding that extracerebral matrix molecules such as heparan sulfate
proteoglycans play an important role in AP-amyloid formation (Snow et al., 1994). Therefore, this experimental model may be useful to study the process of Al3 deposition and the effect of pharmacological manipulation on Al3 deposition. The leptomeninges can also be cultured in CSF for a prolonged period of time. As CSF may contain specific, brain-derived factors which play a role in amyloid deposition, it may be important to study the pathogenesis of CAA under these experimental conditions, which closely resemble the in vivo situation, where leptomeningeal vessels in the subarachnoid space are bathed by CSF. 1
2
3
4
5
115 kD b
79.5 kD + ”:
,
Fig. 4. Biosynthetic labeling and immunoprecipitation of APP from old dog leptomeningeal tissue lysate after 1 week in culture (lanes l-21, neuroblastoma SYSY cell lysate (lanes 3,5) and culture medium (lane 4). Samples in lanes 2-4 were immunoprecipitated with me polyclonal APP antiserum, while samples in lanes 1 and 5 are negative controls incubated with normal rabbit serum.
R. Prior
et al. / Journal
of Neuroscience
Methods
6X ( IY%)6i 14%
i-i8
147
experimentally induced Al3 deposits strongly resembles the pattern of focal amyloid deposits in CAA, it is possible that exogenous AB deposited to the sites of pre-existing amyloid. This is in accordance with the findings of Maggie et al. (19921, who have previously reported that radiolabeled Al3 binds to senile plaques and blood vessel walls in post-mortem brain tissue. Our method, however, enables the study of CAA progression in living vessels with the possibility of microscopical analysis and colocalization of potential AP binding molecules. A detailed confocal laser scanning microscopy analysis of the AP binding in living leptomeningeal vessels of old dogs is reported elsewhere (Prior et al., 19951. In conclusion, the organ culture of canine leptomeninges is technically easy to perform and the cultures are viable for prolonged time. The model appears suitable to study the pathogenesis of CAA. Various issues, such as the origin of cerebrovascular A& the influence of the CNS microenvironment, the role of apolipoprotein E and other amyloid-associated proteins and the differential role of AP(l-40) and AB(l -42) can be explored using this experimental system.
Acknowledgements Fig. 5. Fluorescence dog incubated with tissue was frozen jugated AB binds arterial wall (a) or
photomicrographs of the leptomeninges from an old 1 PM fluorescein-conjugated AB(l-40) for 15 h. The and 8 Frn cryosections were cut. Fluorescein-confocally to either only outer and middle layers of the throughout the vessel wall (b). Magnification. 60 X
Finally, our model may be useful to investigate the source of the Al3 deposited in CAA. Al3 may be derived from the APP produced by vessel wall myocytes or from soluble AB contained in the CSF or blood serum. These hypotheses can be studied in organ culture. 3.5. Incubation exogeneous A /3
qf
liuing
canine
leptomeninges
with
In order to assess whether exogenous Al3 deposits in the leptomeningeal vessel walls, the cultured leptomeninges were incubated with 1 FM fluorescein-conjguated AP( l-40). We used a fluorescein-conjugation procedure that labeled only the N-terminal residue of the A@ probes. Since the carboxy terminus of AB is critical for the formation of A&amyloid (Jarrett et al., 1993), the N-terminal modification is unlikely to change the aggregation properties of fluorescein-conjugated AB. We observed focal deposition of exogenous AB mainly in the outer and middle layers of leptomeningeal arterioles of old dogs (Fig. 5a), whereas in some vessel walls AB was present throughout the vessel wall (Fig. 5b). The fluorescein-conjugated control peptide (unrelated to Al31 showed a weak and diffuse binding pattern while young dogs showed no A@ deposits at all (not shown). As the focal nature of the
This study was supported by grants to R.P. from DFG (Pr 299/3- 1; Forschergruppe Molekularbiologie neurodegenerativer Erkrankungen, Mu 630/5-l 1. D.D. is an Alexander von Humboldt fellow on leave from Istituto Superiore di Sanid, Rome, Italy. We are grateful to the Institute of Neuropathology/University of Dusseldorf (Prof. Wechsler) for providing laboratory facilities. The authors thank Prof. Konrad Beyreuther (Heidelberg) for providing the antisera against AB and APP
References Castano, E.. Ghiso, J., Prelli, F.P., Gorevic. P.D., Migheli. A. and Frangione, B. (1986) In vitro formation of amyioid fibrils from two synthetic peptides of different lengths homologous to Alzheimer’s disease B-protein. Biochem. Biophys. Res. Commun.. 141: 782-789. Cummings, B.J.. Su. J.H.. Cotman, C.W.. White, R. and Russell, M.J. (1993) B-amyloid accumulation in aged canine brain: a model of early plaque formation in Alzheimer’s disease. Neurobio!. Aging, 14: 547560. Frackowiak, J., Mazur-Kolecka, B.. Wisniewski, H.M., Potempska. A.. Carroll. R.T., Emmerling. M.R. and Soo Kim, K (1995) Secretion and accumulation of Alzheimer’s B-protein by cultured vascular smooth muscle cells from old and young dogs, Brain. Res.. 676: 225-230. Frank, R. and Gausepohl, H. (1988) Continuous flow peptide synthesis. In H. Tschesche (Ed.). Modern Methods in Protein Chemistry, Vol. 3. De Gruyter. Berlin, pp. 42-60. Games, D.. Adams, D.. Alessandrini. R., Barbour. R., Berthelene. P.. Blackwell. C., CUT. T.. Clemens. J.. Donaldson, T., Gillespie. F.. Guido, T.. Hagopian. S.. Johnson-Wood. K., Khan, K.. Lee. M., Leibowitz. P.. Lieherburg. I.. Litrle, S.. Masliah. E Mctoniogue. L...
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Montoya-Zavala, M., Mucke, L., Paganini, L., Penninman, E., Power, M., Schenk, D., Seubert, P., Snyder, B., Soriano, F., Tan, H., Vitale, J., Wadsworth, S., Wolozin, B. and Zhao, J (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F B-amyloid precursor protein, Nature, 373: 523-527. Glenner, G.G. and Wang, C.W. (1984) Alzheimer’s disease and Down’s syndrome: Sharing of a unique cerebrovascular amyloid fibril protein, Biochem. Biophys. Res. Commun., 122: 1131-l 135. Golde, T.E., Estus, S., Usiak, M., Younkin, L.H. and Younkin, S.G. (1990) Expression of B amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimer’s disease using PCR, Neuron, 4: 253-267. Haass, C., Schlossmacher, M.G., Hung, A.Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B.L., Lieberburg, I., Koo, E.H., Schenk, D., Teplow, D.B. and Selkoe, D.J. (1992) Amyloid B-peptide is produced by cultured cells during normal metabolism, Nature, 359: 322-325. Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. and Beyreuther, K. (1991) Aggregation and secondary structure of synthetic amyloid PA4 peptides of Alzheimer’s disease, J. Mol. Biol., 218: 149-163. Itoh, Y., Yamada, M., Hayakawa, M., Otomo, E. and Miyatake, T. (1993) Cerebral amyloid angiopathy: a significant cause of cerebellar as well as lobar cerebral hemorrhage in the elderly, J. Neurol. Sci., 116: 135-141. Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N. and Ihara, Y. (1994) Visualization of AB beta 42(43) and Al340 in senile plaques with end-specific A@ monoclonals: evidence that an initially deposited species is AB42(43), Neuron, 13: 45-53. Jarrett, J.T., Berger, P.B. and Lansbury, P.T. Jr (1993) The carboxy terminus of the B amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer’s disease, Biochemistry, 32: 4693-4697. Joacbim, C.L., Duffy, L.K., Morris, J.H and Selkoe, D.J. (1988) Protein, chemical and immunocytochemical studies of meningovascular betaamyloid protein in Alzheimer’s disease and normal aging, Brain Res., 474: 100-111. Johnstone, E.M., Chaney, M.O., Norris, F.H., Pascual, R. and Little, S.P. (1991) Conservation of the sequence of the Alzheimer’s disease amyloid peptide in dog, polar bear and five other mammals by cross-species polymerase chain reaction analysis, Brain Res. Mol. Brain Res., 10: 299-305. Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K. and Miller-Hill B. (1987) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor, Nature, 325: 733-736. Kirschner, D.A., Inoue, H., Duffy, N.L.K., Sinclar, A., Lind, M. and Selkoe, D.J. (1987) Synthetic peptide homologous to B-protein from Alzheimer disease forms amyloid like fibrils in vitro, Proc. Natl. Acad. Sci. USA, 84: 6953-6957. Maggio, J.E., Stimson, E.R., Ghilardi, J.R., Allen, C.J., Dahl, C.E., Whitcomb, D.C., Vigna, S.R., Vinters, H.V., Labenski, M.E. and Mantyh, P.W. (1992) Reversible in vitro growth of Alzheimer disease beta-amyloid plaques by deposition of labeled amyloid peptide, Proc. Natl. Acad. Sci. USA, .89: 5462-5466. Maruyama, K., Ten&ado, K., Usami, M. and Yoshikawa, K. (1990) Formation of amyloid-like fibrils in COS cells overexpressing part of the Alzheimer amyloid protein precursor, Nature, 347: 566-569.
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Masters, CL., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L. and Beyreuther, K. (1985) Amyloid plaque core protein in Alzheimer disease and Down syndrome, Proc. Natl. Acad. Sci. USA, 82: 4245-4249. Maury, C.P.J. (1995) Molecular pathogenesis of B-amyloidosis in Alzheimer’s disease and other cerebral amyloidoses, Lab. Invest., 72: 4-16. Prelli, F., Castano, E., Glenner, G.G. and Frangione, B. (1988) Differences between vascular and plaque core amyloid in Alzheimer’s disease, J. Neurochem., 51: 648-651. Prior, R., D’Urso, D., Frank, R., Prikulis, I. and Pavlakovic, G. (1995) Experimental deposition of Alzheimer amyloid B-protein in canine ieptomeningeal vessels, Nemoreport, 6: 1747-175 1. Roher, A.E., Lowenson, J.D., Clarke, S., Woods, A.S., Cotter, R.J., Gowing, E. and Ball, M.J. (1993) B-Amyloid-(l-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer’s disease, Proc. Nat. Acad. Sci. USA, 90: 10836-10840. Selkoe, D.J. (1994) Cell biology of the amyloid beta-protein precursor and the mechanism of Alzheimer’s disease, Annu. Rev. Cell. Biol., 10: 373-403. Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., Swindlehurst, C., McCormack, R., Wolfert, R., Selkoe, D., Lieberburg, I. and Schenk, D (1992) Isolation and quantification of soluble Alzheimer’s B-peptide from biological fluids, Nature, 359: 325-327. Snow, A.D., Sekiguchi, R., Nochlin, D., Fraser, P., Kimata, K., Mizutani, A., Arai, M., Schreier, W.A. and Morgan, D.G. (1994) An important role of heparan sulfate proteoglycan (Perlecan) in a model system for the deposition and persistence of flbrillar A B-amyloid in rat brain, Neuron, 12: 219-234. Suzuki, N., Iwatsubo, T. and Odaka, A. (19941 High tissue content of soluble Bl-40 is linked to cerebral amyloid angiopathy, Am. J. Pathol., 145: 452-460. Tagliavini, F., Ghiso, J., Timmers, W.F., Giaccone, G., Bugiani, 0. and Frangione, B. (1990) Coexistence of Alzheimer’s amyloid precursor protein and amyloid protein in cerebral vessel walls, Lab. Invest., 62: 761-767. Uchida, K., Okuda, R., Yamaguchi, R., Tateyama, S., Nakayama, H. and Goto, N. (1993) Double-labeling immunohistochemical studies on canine senile plaques and cerebral amyloid angiopathy, J. Vet. Med. Sci., 55: 637-642. Vigo-Pelfrey, C., Lee, D., Keim, P., Lieberburg, I. and Schenk, D.B. (1993) Characterization of B-amyloid peptide from human cerebrospinal fluid, J. Neurochem., 61: 1965-1968. Vonsattel, J.P., Myers, R.H., Hedley-Whyte, E.T., Ropper, A.H., Bird, E.D. and Richardson, E.P. (1991) Cerebral amyloid angiopathy without and with cerebral hemorrhages: a comparative histological study, Ann. Neurol., 30: 637-649. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L. and Beyreuther, K. (1989) Identification, biogenesis and localization of precursors of Alzheimer’s disease A4 amyloid protein, Cell, 57: 115-126.
Methods
68 (1996)
143- 148
Canine leptomeningeal organ culture: a new experimental model for cerebrovascular l3-amyloidosis Reinhard Prior a.* , Donatella D’Urso a, Rainer Frank ‘, Ingrid Prikulis ‘, Giinther Wihl a, Goran Pavlakovic a a Department
of Neurology. Heinrich-Heine-University. h Centerfor Molecular Biology Uniuersi& Received
4 October
1995; revised
Moorenstr. of Heidelberg.
29 January
5. D-40225 Diisseldorj~ Heidelberg, Grrmun?
1996: accepted 30 January
Germun\,
1996
Abstract Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzheimer’s disease and a common cause of cerebral hemorrhage in the elderly. The pathogenetic mechanisms leading to the deposition of Alzheimer amyloid p-protein (A@) in cortical and leptomeningeal vessel walls are unknown. There are no experimental models which reproduce the pathological changes of CAA. In this study, leptomeninges from young and old dogs with pre-existing CAA were cultured in cell culture medium or cerebrospinal fluid and their viability, histological appearance and metabolic activity were analyzed during the culture. In addition, living leptomeninges of old and young dogs were incubated with fluorescein-conjugated AP and the uptake of AP was studied by fluorescence microscopy. Leptomeninges from young and old dogs were viable up to 8 weeks in culture. They contain many small- and medium-sized arterioles, the main vessel type affected by CAA. Histology and immunohistochemistry showed excellent preservation of the vessel wall microarchitecture up to 4 weeks in culture. The cultures were metabolically active as shown by the de novo production of P-amyloid precursor protein. Exogenously added A(3 was focally deposited in the vessel walls of old, but not young dogs. In conclusion, the organ culture of canine leptomeninges is easy to perform and appears suitable to investigate the pathogenesis and the progression of CAA. Kryvords:
Cerebral
amyloid
angiopathy;
Aizheimer’s
disease;
Amyloid
P-protein:
Organ
culture -____
1. Introduction Intracerebral (senile plaques) and cerebrovascular amyloid deposits (cerebral amyloid angiopathy/CAA) of Alzheimer amyloid P-protein CAP; Glenner and Wong, 1984) are a neuropathological feature of Alzheimer’s disease (AD) and are central to its pathogenesis (for review see Maury, 1995; Selkoe, 1994). CAA may also develop in the absence of AD and is a common cause of cerebral hemorrhage in the elderly (Vonsattel et al., 1991; Itoh et al., 1993). AP is derived from the transmembrane amyloid precursor protein, APP (Kang et al., 1987). Proteolytic cleavage of APP generates AP species of heterogeneous length; whereas in senile plaques AP(I--42) is the most prevalent form (Masters et al., 1985; Iwatsubo et al., 1994), AP(l-
-.--
-.---
-- -.--
..---
391, A@l-40) and A@(1 -42) have been isolated from cerebrovascular amyloid (Prelli et al., 1988: Joachim et al., 1988; Roher et al., 1993). Increased brain concentrations of Aj3(1-40) have been linked to the development of CAA (Suzuki et al., 1994). The experimental reproduction of AP deposits is important to understand the pathogenesisof AD and CAA and
different approaches have been employed to reproduce AQ deposits. Synthetic A@ was shown to form amyioid fibrils in cell-free in vitro systems (Castano et al., 1986; Kirschner et al., 1987; Hilbich et al., 1991). Intracellular A@ aggregates were observed in neuronal cell cultures overexpressing the C-terminal part of APP (Maruyama et al., 1990). Recently, the first transgenic mouse model with substantial intracerebral A/3 deposits has been described (Games et al., 1995). However, cerebrovascular deposits were not reported.
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Various proteins give rise to amyloid formation in different tissues causing a group of heterogeneous diseases collectively termed amyloidoses. Any type of amyloidosis
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R. Prior et al. /Journal of Neuroscience Methods 68 (1996) 143-148
144
(i.e. the pathological extracellular deposition of insoluble and fibrillar proteins which stain with Congo Red and show’green-red birefringence with polarized light) shows a highly specific tissue distribution. Whereas APP is ubiquitously expressed (Golde et al., 1990) and soluble AP is present in blood serum and cerebrospinal fluid (CSF) (Seubert et al., 1992; Vigo-Pelfrey et al., 1993), AP amyloidosis occurs exclusively within the brain cortex or within the walls of cortical and leptomeningeal arterioles and capillaries. It is not known what tissue-specific molecular mechanisms underlie cerebrovascular AP deposition and what is the origin of cerebrovascular AP. Since soluble AP is produced by cultured cells during normal metabolism (Haass et al., 1992) and since A@ is detected at low concentrations in cerebrospinal fluid and blood serum under physiological conditions (Seubert et al., 1992; Vigo-Pelfrey et al., 1993), the cerebrovascular A@ deposits may be derived from blood serum or cerebrospinal fluid. Alternatively, A@ may originate from APP produced locally by vascular smooth muscle cells (Tagliavini et al., 1990; Frackowiak et al., 1995). Dogs are one of the few species which develop amyloid deposits with age (Uchida et al., 1993; Cummings et al., 1993). In addition, the amino acid sequences of human and canine AP are identical (Johnstone et al., 1991). Therefore, living canine tissue may be a suitable model for studying the pathogenesis of AP amyloidosis. The purpose of the present study was to examine survival, metabolic activity and histological changes in cultured leptomeninges obtained from young and old dogs and to determine, whether living canine leptomeninges might be useful for identifying the origin of cerebrovascular A p and to characterize conditions that induce or promote the pathogenesis of CAA.
2. Materials
2.2. Viability testing The leptomeningeal viability was analyzed immediately after dissection, after 2, 5 and 7 days in culture and then at weekly intervals. Tissue samples were incubated with fluorescein diacetate (FDA, 2 p,g/ml, Molecular Probes) for 5 min. After washing, the leptomeningeal membranes were distended on a microscopical slide, overlayed with phosphate-buffered saline (PBS), coverslipped and immediately analyzed by fluorescence microscopy. 2.3. Histology and immunohistochemistry For histological studies, leptomeningeal tissue was sampled immediately after the removal of the leptomeninges and after each week of organ culture. The tissue was fixed in 4% buffered formalin and embedded in paraffin. 8 p,rn sections were cut and stained with hematoxylin-eosin, Masson-trichrome and thioflavin S. For immunohistochemistry, deparaffinized sections were treated with 1% hydrogen peroxide in methanol to block endogenous peroxidase, washed in PBS and incubated overnight with the monoclonal primary antibodies against smooth muscle actin (Dako, clone lA4, 2 mg/ml), amyloid precursor protein (Boehringer, clone 22Cl1, 4 p.g/ml) and with a polyclonal antiserum raised against synthetic AP(2-43) (1:200 dilution, gently provided by K. Beyreuther). Following wash-out, sections were incubated with biotinylated antimouse or anti-rabbit antibodies for 1 h, washed in PBS and incubated with avidin-biotin peroxidase complex (Vector) for 45 min. Sections were developed in 0.05% 3,3-diaminobenzidine tetrahydrochloride (Sigma) plus O,Ol% hydrogen peroxide in PBS before counterstaining with hematoxylin. Negative controls were obtained by omitting the primary antibody or by using normal rabbit serum instead of the primary antibody.
and methods 2.4. Biosynthetic labeling and immunoprecipitation
2.1. Preparation
of APP
and organ culture of leptomeninges
Craniotomy was performed under sterile conditions immediately after the dogs (5 old dogs with mean age 14.8 + 1.3 years; 5 young dogs less than 1 year old) had been euthanized by a veterinarian for incurable non-neurological disease or had been killed for unrelated orthopedic studies. The whole brain was removed and transported on ice in physiologic saline supplemented with 5% glucose and a mixture of antibiotics. After dissection, leptomeninges were cultured in cell culture medium (DMEM containing 20% fetal calf serum, 4 mM L-glutamine, 100 U/ml penicillin and 100 p,g/ml streptomycin) or in pooled human cerebrospinal fluid (CSF) supplemented with 0.45% glucose. The leptomeningeal membrane was kept freely floating in 20 ml of culture medium per 85 mm culture dish. The medium was completely exchanged after the first day and every other day of culture thereafter.
Samples (1 cm* of membranes) which had been kept for 1 week in culture were incubated for 3 h in methionine-free MEM (Gibco) supplemented with 150 $i of [35S]methionine. After removal of the medium, the tissue was washed twice in PBS and homogenized in 200 ~1 of STEN buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA), supplemented with 2 mM phenylmethylsulfonyl fluoride (PMSF), 2% Nonidet P-40 and 2% Triton X-100. After 10 min on ice, the samples were centrifuged at 10 000 X g for 5 min and the supematant stored at - 20°C until further analysis. For immunoprecipitation, the sample was diluted 1:5 with STEN and incubated with 7 ~1 of a polyclonal antiserum against recombinant full-length APP (provided by K. Beyreuther, Heidelberg) for 2 h at room temperature with constant shaking. Next, IO ~1 (1.5 mg) of protein A-Sepharose (Pharmacia) was added for 1 h. The insoluble complexes were washed three times, resus-
per&d in 50 ~1 of 2 X Laemmb sample buffer, boiled for 5 min and centrifuged at 10000 X g for 5 min. 20 p,g of total pro&& per sample was loaded onto an 8% SDS-PAGE gel. As a positive control, neuroblastoma cells (SYSY, 3 X 10’ cells), which c~~~vely express high levels of APP, were processed us&g the same protocol and loaded on the same gel. Following electrophoresis, the gel was fixed, dried and after overnight exposure analyzed with a Fuji BAS loo0 pkosphoimaging system. 2.5. Incubation conjugated A p
of the cultures with synthetic fluorescein-
A B(l-40) and an unrelated control peptide (GVEAYVDLKPASLEDIERLLFEDRRLMAYYC) were synthesized on polyoxyethylene-polysterene graft resin in a continuous fiow instrument constructed and operated as described (Frank and Gausepohl, 1988). Peptide chain assembly was performed using Fmoc chemistry and in situ activation of amino acid building blocks by PyBOP. Fluorescent was coupled to the N-terminal residue via its succinimidyl ester (5(and-6)carboxyfluorescein succinimidyl ester, Molecular Probes). The peptides were purified by reversed-phase HPLC and characterized by laser desorption mass spectrometry. From a 100 p,M stock solution in water. the peptides were added to the culture medium to a final concentration of 1 p,M. Leptomeninges from old and young dogs were incubated for 15 h with fluorescein-coupled ABC1 -40) and control peptide. The leptomeninges were frozen and 8 p,m cryosections were cut and embedded for analysis with a fluorescence microscope.
Fig. 1. Fluorescence microscopy of FDA tomeninges after 7 days of culture. Note the majority of the vessels indicating their viability.
3.1. Vessel iiahili~ Up to 4 weeks of culture, viability was shown by bright green light emission after FDA staining observed in the majority of vessels which were typically labeled over their full length (Fig. 1). Only occasionally were short segments of the vessels negative for FDA, indicating necrotic areas. After 4 weeks. the vessel walls showed a progressively discontinuous pattern of FDA staining as expression of vascular cell death. However, continuous segments of viable vessel walls were observed even after 8 weeks in culture. When leptomeninges were cultured in CSF, the majority of vessels were viable up to 3 weeks. There was no significant difference in the FDA staining pattern of leptomeninges obtained from young and old dogs. 3.2. Histology
anti immunohistochemist~
Paraffin sections and cryosections were analyzed after hematoxylin-eosin and Masson-trichrome staining. Small-
of
canine
Magnification.
lepof the
20X
and medium-sized arteries, which are the vessels most affected by CAA, represented the main vessel type in the cultured leptomeninges (Fig. 2a). The vessel wall architecture was well preserved during the culture. Sight thickening of the leptomeningeal membranes was noticed after 2 weeks of culture (Fig. 2b) and progressive connective tissue proliferation was observed after 4 weeks of culture (Fig. 2~). All of the old dogs showed thioflavin S-positive amyloid in the vessel walls (Fig. 3a) and the presence of AB in the vessels was confirmed immunohistochemically (Fig. 3b). Smooth muscle actin (Fig. 2c) and APP (not shown) were detectable in arteriolar vessel walls of young and old dogs even after several weeks of culture, indicating conserved structural integrity of the vessel walls. 3.3. Immzmc~precipitation
3. Results and discussion
staining
continuous staining
of biosynthetically lubeled APP
The immunoprecipitation studies following metabolic labeling of APP with [35S]methionine revealed that the canine leptomeningeal organ cultures had preserved the ability to synthesize APP (Fig. 4). Three protein bands between 95 and 115 kDa were observed after immunoprecipitation of the tissue homogenates (Fig. 4, lane 2). These bands are of the same molecular weight as the bands obtained by APP immunoprecipitation from cell lysates of neuroblastoma cells (Fig. 4. lane 3) which served as a positive control. The lower band (Fig. 4. lane 2) is identical to the APP band immunoprecipitated from the supernatant of neuroblastoma cells (Fig. 4, lane 4), suggesting that it represents the secreted form of APP (Weidemann et al.. 1989). 3.4. Orgun culture o~cnnine leptomcninges us u model,fku cerebrr~-l,asc~lllNr A/3 depositkm The organ culture of canine leptomeninges appears to be a useful model for studying the development of CAA, since the cultured leptomeninges of young and old dogs are viable for several weeks with only minor morphologi-
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68 f 19961 143-148
Fig. 3. Cerebrovascular AP deposits in leptomeninges of old dogs after 1 week in culture: (a) stained with thioflavin S, and (b) immunostained for AP (counterstained with hematoxylin). Magnification, 40 X
Fig. 2. Paraffin sections of canine leptomeninges stained with hematoxylin-eosin immediately after dissection (a) and after 2 weeks (b). Panel c shows leptomeninges after 4 weeks of culture immunostained for smooth muscle actin and counterstained with hematoxylin. Cytoplasmic staining for smooth muscle actin can be observed in the vessel walls. The canine leptomeninges are abundant with small and medium sized arteries and the vessel wall architecture is well preserved. Magnification, 40 X
cal changes and good preservation of the vessel wall architecture. In addition, the cultured leptomeninges are metabolically active as demonstrated by the de novo production of APP. Our experimental model uses living tissue of a species in which CAA develops with age and preserves both the extracellular matrix and the microarchitecture of those vessel walls where Al3 is deposited in vivo. This might be important, particularly in view of the finding that extracerebral matrix molecules such as heparan sulfate
proteoglycans play an important role in AP-amyloid formation (Snow et al., 1994). Therefore, this experimental model may be useful to study the process of Al3 deposition and the effect of pharmacological manipulation on Al3 deposition. The leptomeninges can also be cultured in CSF for a prolonged period of time. As CSF may contain specific, brain-derived factors which play a role in amyloid deposition, it may be important to study the pathogenesis of CAA under these experimental conditions, which closely resemble the in vivo situation, where leptomeningeal vessels in the subarachnoid space are bathed by CSF. 1
2
3
4
5
115 kD b
79.5 kD + ”:
,
Fig. 4. Biosynthetic labeling and immunoprecipitation of APP from old dog leptomeningeal tissue lysate after 1 week in culture (lanes l-21, neuroblastoma SYSY cell lysate (lanes 3,5) and culture medium (lane 4). Samples in lanes 2-4 were immunoprecipitated with me polyclonal APP antiserum, while samples in lanes 1 and 5 are negative controls incubated with normal rabbit serum.
R. Prior
et al. / Journal
of Neuroscience
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6X ( IY%)6i 14%
i-i8
147
experimentally induced Al3 deposits strongly resembles the pattern of focal amyloid deposits in CAA, it is possible that exogenous AB deposited to the sites of pre-existing amyloid. This is in accordance with the findings of Maggie et al. (19921, who have previously reported that radiolabeled Al3 binds to senile plaques and blood vessel walls in post-mortem brain tissue. Our method, however, enables the study of CAA progression in living vessels with the possibility of microscopical analysis and colocalization of potential AP binding molecules. A detailed confocal laser scanning microscopy analysis of the AP binding in living leptomeningeal vessels of old dogs is reported elsewhere (Prior et al., 19951. In conclusion, the organ culture of canine leptomeninges is technically easy to perform and the cultures are viable for prolonged time. The model appears suitable to study the pathogenesis of CAA. Various issues, such as the origin of cerebrovascular A& the influence of the CNS microenvironment, the role of apolipoprotein E and other amyloid-associated proteins and the differential role of AP(l-40) and AB(l -42) can be explored using this experimental system.
Acknowledgements Fig. 5. Fluorescence dog incubated with tissue was frozen jugated AB binds arterial wall (a) or
photomicrographs of the leptomeninges from an old 1 PM fluorescein-conjugated AB(l-40) for 15 h. The and 8 Frn cryosections were cut. Fluorescein-confocally to either only outer and middle layers of the throughout the vessel wall (b). Magnification. 60 X
Finally, our model may be useful to investigate the source of the Al3 deposited in CAA. Al3 may be derived from the APP produced by vessel wall myocytes or from soluble AB contained in the CSF or blood serum. These hypotheses can be studied in organ culture. 3.5. Incubation exogeneous A /3
qf
liuing
canine
leptomeninges
with
In order to assess whether exogenous Al3 deposits in the leptomeningeal vessel walls, the cultured leptomeninges were incubated with 1 FM fluorescein-conjguated AP( l-40). We used a fluorescein-conjugation procedure that labeled only the N-terminal residue of the A@ probes. Since the carboxy terminus of AB is critical for the formation of A&amyloid (Jarrett et al., 1993), the N-terminal modification is unlikely to change the aggregation properties of fluorescein-conjugated AB. We observed focal deposition of exogenous AB mainly in the outer and middle layers of leptomeningeal arterioles of old dogs (Fig. 5a), whereas in some vessel walls AB was present throughout the vessel wall (Fig. 5b). The fluorescein-conjugated control peptide (unrelated to Al31 showed a weak and diffuse binding pattern while young dogs showed no A@ deposits at all (not shown). As the focal nature of the
This study was supported by grants to R.P. from DFG (Pr 299/3- 1; Forschergruppe Molekularbiologie neurodegenerativer Erkrankungen, Mu 630/5-l 1. D.D. is an Alexander von Humboldt fellow on leave from Istituto Superiore di Sanid, Rome, Italy. We are grateful to the Institute of Neuropathology/University of Dusseldorf (Prof. Wechsler) for providing laboratory facilities. The authors thank Prof. Konrad Beyreuther (Heidelberg) for providing the antisera against AB and APP
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