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synaptophysin is a common feature of brains in Alzheimer and Pick disease
V o l u m e 263, n u m b e r 2, 3 3 7 - 3 3 9
F E B S 08364
A p r i l 1990
A high ratio of chromogranin A to synaptin/synaptophysin is a common feature of brains in Alzheimer and Pick disease R o s a Weiler, H a n s L a s s m a n n +, Peter Fischer +, K u r t Jellinger ° a n d H a n s W i n k l e r Department of Pharmacology, University of lnnsbruck, Peter Mayr-Strafle la, A-6020 Innsbruck, +Institute of Brain Research, Austrian Academy of Sciences and Neurological Institute, University of Vienna a n d °Ludwig Boltzmann Institute for Clinical Neurobiology, Lainz Hospital, A-1090 Vienna, Austria R e c e i v e d 7 M a r c h 1990 Chromogranin A and synaptin/synaptophysin were characterized by immunological methods in human autopsy brain tissue from patients with Alzheimer's and Pick's disease. In immunoblots there was no qualitative difference between the antigens in control and diseased brain, but significant quantitative differences were found. In all Alzheimer cases there was a significantly lower level of synaptin/synaptophysin, whereas chromogranin A was higher in 4 out of 5 cases and in all cases relative to synaptin/synaptophysin. An analogous finding was obtained for Pick's disease. Immunohistologically a consistent staining of neuritic plaques for chromogranin A, but not for secretogranin I! was found in Alzheimer cases. In Pick's disease the characteristic Pick bodies showed an analogous specific immunostaining. Alzheimer's disease; Pick's disease; Chromogranin A; Secretogranin II; Synaptin/synaptophysin
1. I N T R O D U C T I O N Chromogranin A and secretogranin 1I (chromogranin C) are proteins present in endocrine [1,2] and in large dense core vesicles of brain [3]. Synaptin/synaptophysin [4] is found in high concentrations in synaptic vesicles [5-7], but also in large dense core vesicles and endocrine vesicles [5,8,9]. Recently it has been reported that a monoclonal antibody against chromogranin A recognizes the characteristic plaques of Alzheimer's disease [10] indicating the presence of chromogranin immunoreactive material in these lesions. We report now for Alzheimer's and Pick's disease that a high chromogranin A/synaptin(synaptophysin) ratio is a c o m m o n characteristic for these two brain diseases leading to dementia. 2. MATERIALS AND M E T H O D S The study was performed on human autopsy brain tissue from the Vienna Prospective Longitudinal Study on Dementia [1 II. It included 21 cases of Alzheimer's disease (age: 59-88), 3 cases of Pick's disease (age: 66-75), 4 cases of mixed dementia (multiple cerebral infarcts together with lesions of Alzheimer's type, age: 77-92), and 5 nondemented control cases (age 57-91). lmmunostaining for chromogranin A and secretogranin 1I was performed on paraffin sections. The sections were deparaffinized, unspecific binding was blocked with 10070 fetal calf serum. Polyclonal rabbit primary antisera were used in dilutions of 1:300. Bound antibody was visualized with an alkaline-phosphatase/anti-alkaline phosphatase technique [12]. As controls the primary antiserum was
Correspondenceaddress: H. Winkler, Department of Pharmacology, Peter Mayr-Strasse la, A-6020 Innsbruck, Austria
omitted or replaced by normal rabbit serum in identical dilutions. The techniques of Bielschowsky silver impregnation and immunocytochemistry for tau were described in detail earlier [13]. For immunoblotting with a polyclonal antiserum [14] against human chromogranin (dilution 1:200) heat stable extracts (corresponding to 1 g wet tissue) of lyophilized brain tissue [15] were used. For synaptin/synaptopbysin (polyclonal antiserum against rat synaptin/synaptophysin kindly provided by R. Jahn, Munich, FRG) lyophilized tissue samples (corresponding to 25 mg wet tissue) were dissolved directly in electrophoresis sample buffer.
3. RESULTS In the normal human neocortex and hippocampus the distribution of chromogranin A-like immunoreactivity was identical to that described previously in sheep brain [3]. Within neurons, chromogranin A was found in a granular distribution in the cytoplasm of nerve cells (results not shown). No chromogranin A-like immunoreactivity was found in the white matter. Secretogranin II reactivity was identical to that of chromogranin A, although the overall intensity of the immune reaction was lower. In Alzheimer's disease, in addition to the pattern found in normal brains numerous senile plaques contained chromogranin Areactive, dystrophic neurites. A detailed quantitative analysis of these plaques in comparison with those stained by Bielschowsky's silver technique revealed that, dependent upon the case, 2-100°70 (50 +_ 5, n = 24) of plaques in the tissue visualized by the modified Bielschowsky technique contained chromogranin A like immunoreactivity. This highly divergent incidence was due to the fact, that chromogranin A was exclusively present in plaques with a neuritic component. Diffuse
Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies
337
Volume 263, number 2
I
FEBS LETTERS
anti- A
I I
anti-Syn
]
PG-A t
94
68-
I
! AM
C
P
A1 A2
CG C
P
A1 A2
Fig. 1. Immunoblots of tissue samples of control and Alzheimer brain, lmmunostaining (anti-A: anti chromogranin A; anti-Syn: anti synaptin/synaptophysin) of samples from temporal cortex of brains from a control patient (C: 57 M), from two Alzheimer cases (AI: 75 M; A2:79 F) and from a case with morbus Pick (P) are shown. A quantitative comparison of the various samples is not possible from this figure since the film obtained from the labelled blot was overexposed to show as many bands as possible in a way suitable for printing. PG-A: proteoglycan-chromogranin A; AM: heat stable extract from human adrenal medulla.
( ' e a r l y ' ) plaques d i d n o t reveal c h r o m o g r a n i n A imm u n o r e a c t i v i t y . In P i c k ' s disease all P i c k bodies (as d e t e r m i n e d b y staining a d j a c e n t sections with either B i e l s c h o w s k y silver i m p r e g n a t i o n or i m m u n o c y t o c h e m i s t r y for tau) were intensively stained with antib o d i e s a g a i n s t c h r o m o g r a n i n A . In b o t h A l z h e i m e r ' s a n d P i c k ' s disease s e c r e t o g r a n i n II like i m m u n o r e a c -
Table I Levels of chromogranin A and synaptin/synaptophysin Subject Controls
Mean +_ SE Alzheimer
Mean + SE Pick
Age Sex (years) 57 91 54 67
M M M M
77 73 72 75 79
M F F M F
75
F
Ch-A
Syn
Ch-A/Syn
145 48 79.4 127.5
104.4 94.1 93 108.4
1.4 0.5 0.9 1.2
99.9_+22.2 301 272 200 281 78 226.4_+40.8* 396.7
99.9_+3.8 1.0___0.2 69 4.4 87.2 3.1 74.5 2.7 65.1 4.3 19.1 4.1 63-+ 11,6" 3.7-+0.3** 36.8 _+8.7 10.8
Heat stable extracts of brains from non-demented controls and from cases of Alzheimer's and Pick's disease were subjected to quantitative immunoblotting [15]. Four controls and 5 Alzheimer cases were available, For Pick's disease only one brain was analyzed; the results of several measurements (n = 2 for Ch-A; n = 4 for syn) for different brain samples are presented. Statistical differences: * P < 0.05; • * P <0.001. 338
April 1990
tivity was exclusively present in g r a n u l a r d i s t r i b u t i o n in the c y t o p l a s m o f nerve cells. P i c k b o d i e s a n d p l a q u e neurites were negative for s e c r e t o g r a n i n II. These imm u n o h i s t o c h e m i c a l results d e m o n s t r a t e that c h r o m o g r a n i n A i m m u n o r e a c t i v i t y is consistently f o u n d b o t h in A l z h e i m e r plaques a n d P i c k bodies; however, this m e t h o d does not allow a n y statements on the p r o p e r t i e s a n d relative q u a n t i t y o f the i m m u n o r e a c t i v e m a t e r i a l . In o r d e r to define this i m m u n o r e a c t i v e m a t e r i a l in m o l e c u l a r terms i m m u n o b l o t s were p e r f o r m e d . P r e vious studies (see [1]) have e s t a b l i s h e d t h a t in extracts o f i m m e d i a t e l y processed e n d o c r i n e a n d b r a i n tissues a n t i s e r a against c h r o m o g r a n i n A react with the p r o p r o tein c h r o m o g r a n i n A a n d several smaller p r o t e i n s w h i c h are f o r m e d within the s t o r a g e vesicles b y e n d o g e n o u s processing. F o r tissues n o t i m m e d i a t e l y p r o cessed a f u r t h e r b r e a k d o w n leading to smaller p r o t e i n s b e c o m e s a p p a r e n t [16]. F o r h u m a n b r a i n , as s h o w n in Fig. 1, the p r o p r o t e i n c h r o m o g r a n i n A can still be d e m o n s t r a t e d (A in Fig. 1). In a d d i t i o n a faster m o v i n g b r e a k d o w n p r o d u c t (A1 in Fig. 1) is present in v a r y i n g relative a m o u n t s . A slower m o v i n g b a n d ( P G - A in Fig. 1) has been p r e v i o u s l y [17] identified as the p r o t e o g l y c a n f o r m o f c h r o m o g r a n i n A , which can be f o u n d consistently in b r a i n tissue, e.g. o f rat [1,18]. As s h o w n in Fig. 1 there is no significant qualitative difference b e t w e e n the c h r o m o g r a n i n A - r e a c t i v e b a n d s in c o n t r o l or diseased brains. T h e a p p a r e n t p r o m i n e n c e o f the p r o t e o g l y c a n - c h r o m o g r a n i n A in P i c k ' s b r a i n s is d u e to the fact t h a t higher levels o f t o t a l c h r o m o g r a n i n were present in these samples. S y n a p t i n / s y n a p t o p h y s i n (see Fig. 1) c o u l d also be d e m o n s t r a t e d in h u m a n brain. F o r s e c r e t o g r a n i n II o u r m e t h o d was n o t sensitive enough. The quantitative evaluation of chromogranin A and s y n a p t i n / s y n a p t o p h y s i n levels is s h o w n in T a b l e I, A c o n s i s t e n t finding for all A l z h e i m e r cases was the lower level o f s y n a p t i n / s y n a p t o p h y s i n ; o n the o t h e r h a n d in 4 o u t o f 5 A l z h e i m e r cases the c h r o m o g r a n i n A level was r e m a r k a b l y elevated. In a fifth case the c h r o m o g r a n i n A level was below c o n t r o l , but the c h r o m o g r a n i n A / s y n a p t i n ( s y n a p t o p h y s i n ) ratio was still high. It seems likely t h a t in this case a c o n s i d e r a b l e cell loss has reduced a previously elevated c h r o m o g r a n i n A level b e l o w c o n t r o l . O n l y one b r a i n o f P i c k ' s disease was available for i m m u n o b l o t t i n g ; nevertheless, several s a m p l e s were a n a l y z e d a n d a very high level o f c h r o m o g r a n i n A a n d o f the c h r o m o g r a n i n A / s y n a p t i n ( s y n a p t o p h y s i n ) ratio was detected.
4. D I S C U S S I O N S y n a p t i n / s y n a p t o p h y s i n is a p r o t e i n f o u n d in high c o n c e n t r a t i o n s in s y n a p t i c vesicles [ 5 - 7 ] , a n d in lower c o n c e n t r a t i o n in large dense core vesicles a n d in end o c r i n e vesicles like c h r o m a f f i n granules [5,8,9]. Its
Volume 263, number 2
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consistent low levels in Alzheimer's (which is in agreement with immunohistochemical data [19,20]) and Pick's brains can simply be an expression of neuronal and synaptic loss occurring in these diseases, but a specific loss o f synaptic vesicles should also be considered. On the other hand, chromogranin A levels are increased. This protein is found in endocrine vesicles and large dense core vesicles o f brain and is secreted together with other constituents o f these vesicles by exocytosis. This property is shared with neuropeptides [1]. For some neuropeptides, an immunohistochemical staining o f Alzheimer plaques has been observed [21], but we are unaware o f a report describing high levels o f any o f these peptides in brain. In fact reduced levels have been reported (see also [22]) for neuropeptide Y [23] and somatostatin [24]. In addition we have not found immunostaining o f Alzheimer plaques or of Pick bodies with an antiserum against secretogranin II which at least in chromaffin granules is colocalized with chromogranin A [25]. Therefore our results on chromogranin A levels seem quite specific for this secretory protein. Unfortunately the function o f this protein is still not very well defined, although peptides derived from it (e.g. pancreastatin) have been shown to inhibit the secretion o f hormones [26,27]. Elevated levels o f chromogranin A in diseased brain might be a consequence o f an increased synthesis or decreased secretion or breakdown. Our present study does not yet allow us to state whether chromogranin A is only accumulating in the typical lesions or is elevated in all neurons. This can only be elucidated in further studies including mRNA-measurements. In this context it is interesting to note that one report has claimed that in adrenal medulla of Alzheimer patients large vacuoles with electron dense content (protein?) have been found [28]. The adrenal medulla contains a high concentration of chromogranin A [1]. Does this suggest that peripheral organs also contain more chromogranin as it has recently been shown for the amyloid fl protein precursor [29]? In any case we consider our results important for the following reasons: we have found chromogranin A immunostaining both in Alzheimer plaques and Pick bodies. The immunoreactive material was identified as chromogranin A. In both diseases the brain contains increased levels of this secretory protein found in large dense core vesicles whereas synaptin/synaptophysin levels are low. These data for chromogranin A appear specific, since other secretory constituents o f large dense core vesicles (secretogranin II, neuropeptides) behave differently. Thus for these two diseases leading to dementia but with significantly differing pathohistology a high chromogranin A/synaptin(synaptophysin) ratio is a c o m m o n and specific denominator. These data might also become relevant for diagnostic purposes: does the cerebrospinal fluid of Alzheimer and Pick patients contain higher levels o f chromogranin A or o f peptides derived from it?
April 1990
Acknowledgements: The study was partly funded by a grant of the Austrian Ministry for Science and Research, by the Ludwig Boltzmann Institute of Aging Research (W. Danielczyk), by the Fonds zur F6rderung der Wissenschaftlichen Forschung and by the Dr Legerlotz-Stiftung. We are indebted to Mrs H. Breitschopf, Ms M. Leiszer and Ms S. Katzensteiner for expert technical assistance.
REFERENCES [l] Winkler, H., Apps, D.K. and Fischer-Colbrie, R. (1986) Neuroscience 18, 261-290. [2] Simon, J.-P. and Aunis, D. (1989) Biochem. J. 262, 1-13. [3] Somogyi, P., Hodgson, A.J., De Potter, R.W., FischerColbrie, R., Schober, M., Winkler, H. and Chubb, I.W. (1984) Brain Res. Rev. 8, 193-230. [4] Gaardsvoll, H., Obendorf, D., Winkler, H. and Bock, E. (1988) FEBS Lett. 242, 117-120. [5] Bock, E. and Helle, K.B. (1977) FEBS Lett. 82, 175-178. [6] Navone, F., Jahn, R., Gioia, G.D., Stukenbrok, H., Greengard, P. and De Camilli, P. (1986) J. Cell Biol. 103, 2511-2527. [7] De Camilli, P. and Jahn, R. (1990) Annu. Rev. Physiol. 52, in press.
[8] Lowe, A.W., Maddedu, L. and Kelly, R.B. (1988) J. Cell Biol. 106, 51-59. [9] Obendorf, D., Schwarzenbrunner, U., Fischer-Colbrie, R., Laslop, A. and Winkler, H. (1988) J. Neurochem. 51, 1573-1580. [10] Munoz, D.G. (1989) Neurology 39, $396. [11] Fischer, P., Gatterer, G., Marterer, A. and Danielczyk, W. (1988) Arch. Neurol. 45, 1341-1343. [12] Vass, K., Berger, M.L., Nowak, T.S., Welch, W.J. and Lassmann, H. (1989) Neurosci. Lett. 100, 259-264. [13] Bancher, C., Brunner, C., Lassmann, H., Budka, H., Jellinger, K., Wiche, G., Seitelberger, F., Grundke Iqbal, I., lqbal, K. and Wisniewski, H.M. (1989) Brain Res. 477, 90-99. [14] Weiler, R., Fischer-Colbrie, R., Schmid, K.W., Feichtinger, H., Bussolati, G., Grimelius, L., Krisch, K., Kerl, H., O'Connor, D. and Winkler, H. (1988) Am. J. Surg. Pathol. 12, 877-884. [15] Schober, M., Howe, P.R.C., Sperk, G., Fischer-Colbrie, R. and Winkler, H. (1989) Hypertension 13, 469-474. [16] Schober, M., Fischer-Colbrie, R., Schmid, K.W., Bussolati, G., O'Connor, D.T. and Winkler, H. (1987) Lab. Invest. 57, 385-391. [171 Falkensammer, G., Fischer-Colbrie, R. and Winkler, H. (1985) J. Neurochem. 45, 1475-1480. [18] Weiler, R., Wohlfarter, T., Marksteiner, J., Schmid, K.W., Sperk, G. and Winkler, H. (1989) J. Neurochem. 52, $84A. [19] Masliah, E., Terry, R.D., DeTheresa, R., Alford, M. and Hansen, L.A. (1989) J. Neuropathol. Exp. Neurol. 48, 333. [20] Hamos, J.E., De Gennaro, L.J. and Drachman, D.A. (1989) Neurology 39, 355-361. [21] Armstrong, D.M., Benzing, W.C., Evans, J., Terry, R.D., Shields, D. and Hansen, A. 0989) Neuroscience 31,663-671. [22] Mann, D.M.A. and Yates, P.O. (1986) Human Neurobiol. 5, 147-158. [23] Beal, M.F., Mazurek, M.F., Chartha, G.K., Svendsen, C.N., Bird, E.D. and Martin, J.B. (1986) Ann. Neurol. 20, 282-288. [24] Candy, J.M., Gascoigne, A.D., Biggins, J.A., Smith, A.I., Perry, R.H., McDermott, J.R. and Edwardson, J.A. (1985) J. Neurol. Sci. 71, 315-323. [25] Steiner, H.-J., Schmid, K.W., Fischer-Colbrie, R., Sperk, G. and Winkler, H. (1989) Histochemistry 91, 473-477. [26] Tatemoto, K., Efendic, S., Mutt, V., Makk, G., Feistner, G.J. and Barchas, J.D. (1986) Nature 324, 476-478. [27] Simon, J.-P., Bader, M.-F. and Aunis, D. (1988) Proc. Natl. Acad. Sci. USA 85, 1712-1716. [281 Averback, P. (1983) Lancet ii, 1203. [29] Joachim, C.L., Mori, H. and Selkoe, D.J. (1989) Nature 341, 226-230.
339
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A high ratio of chromogranin A to synaptin/synaptophysin is a common feature of brains in Alzheimer and Pick disease R o s a Weiler, H a n s L a s s m a n n +, Peter Fischer +, K u r t Jellinger ° a n d H a n s W i n k l e r Department of Pharmacology, University of lnnsbruck, Peter Mayr-Strafle la, A-6020 Innsbruck, +Institute of Brain Research, Austrian Academy of Sciences and Neurological Institute, University of Vienna a n d °Ludwig Boltzmann Institute for Clinical Neurobiology, Lainz Hospital, A-1090 Vienna, Austria R e c e i v e d 7 M a r c h 1990 Chromogranin A and synaptin/synaptophysin were characterized by immunological methods in human autopsy brain tissue from patients with Alzheimer's and Pick's disease. In immunoblots there was no qualitative difference between the antigens in control and diseased brain, but significant quantitative differences were found. In all Alzheimer cases there was a significantly lower level of synaptin/synaptophysin, whereas chromogranin A was higher in 4 out of 5 cases and in all cases relative to synaptin/synaptophysin. An analogous finding was obtained for Pick's disease. Immunohistologically a consistent staining of neuritic plaques for chromogranin A, but not for secretogranin I! was found in Alzheimer cases. In Pick's disease the characteristic Pick bodies showed an analogous specific immunostaining. Alzheimer's disease; Pick's disease; Chromogranin A; Secretogranin II; Synaptin/synaptophysin
1. I N T R O D U C T I O N Chromogranin A and secretogranin 1I (chromogranin C) are proteins present in endocrine [1,2] and in large dense core vesicles of brain [3]. Synaptin/synaptophysin [4] is found in high concentrations in synaptic vesicles [5-7], but also in large dense core vesicles and endocrine vesicles [5,8,9]. Recently it has been reported that a monoclonal antibody against chromogranin A recognizes the characteristic plaques of Alzheimer's disease [10] indicating the presence of chromogranin immunoreactive material in these lesions. We report now for Alzheimer's and Pick's disease that a high chromogranin A/synaptin(synaptophysin) ratio is a c o m m o n characteristic for these two brain diseases leading to dementia. 2. MATERIALS AND M E T H O D S The study was performed on human autopsy brain tissue from the Vienna Prospective Longitudinal Study on Dementia [1 II. It included 21 cases of Alzheimer's disease (age: 59-88), 3 cases of Pick's disease (age: 66-75), 4 cases of mixed dementia (multiple cerebral infarcts together with lesions of Alzheimer's type, age: 77-92), and 5 nondemented control cases (age 57-91). lmmunostaining for chromogranin A and secretogranin 1I was performed on paraffin sections. The sections were deparaffinized, unspecific binding was blocked with 10070 fetal calf serum. Polyclonal rabbit primary antisera were used in dilutions of 1:300. Bound antibody was visualized with an alkaline-phosphatase/anti-alkaline phosphatase technique [12]. As controls the primary antiserum was
Correspondenceaddress: H. Winkler, Department of Pharmacology, Peter Mayr-Strasse la, A-6020 Innsbruck, Austria
omitted or replaced by normal rabbit serum in identical dilutions. The techniques of Bielschowsky silver impregnation and immunocytochemistry for tau were described in detail earlier [13]. For immunoblotting with a polyclonal antiserum [14] against human chromogranin (dilution 1:200) heat stable extracts (corresponding to 1 g wet tissue) of lyophilized brain tissue [15] were used. For synaptin/synaptopbysin (polyclonal antiserum against rat synaptin/synaptophysin kindly provided by R. Jahn, Munich, FRG) lyophilized tissue samples (corresponding to 25 mg wet tissue) were dissolved directly in electrophoresis sample buffer.
3. RESULTS In the normal human neocortex and hippocampus the distribution of chromogranin A-like immunoreactivity was identical to that described previously in sheep brain [3]. Within neurons, chromogranin A was found in a granular distribution in the cytoplasm of nerve cells (results not shown). No chromogranin A-like immunoreactivity was found in the white matter. Secretogranin II reactivity was identical to that of chromogranin A, although the overall intensity of the immune reaction was lower. In Alzheimer's disease, in addition to the pattern found in normal brains numerous senile plaques contained chromogranin Areactive, dystrophic neurites. A detailed quantitative analysis of these plaques in comparison with those stained by Bielschowsky's silver technique revealed that, dependent upon the case, 2-100°70 (50 +_ 5, n = 24) of plaques in the tissue visualized by the modified Bielschowsky technique contained chromogranin A like immunoreactivity. This highly divergent incidence was due to the fact, that chromogranin A was exclusively present in plaques with a neuritic component. Diffuse
Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/90/$3.50 © 1990 Federation of European Biochemical Societies
337
Volume 263, number 2
I
FEBS LETTERS
anti- A
I I
anti-Syn
]
PG-A t
94
68-
I
! AM
C
P
A1 A2
CG C
P
A1 A2
Fig. 1. Immunoblots of tissue samples of control and Alzheimer brain, lmmunostaining (anti-A: anti chromogranin A; anti-Syn: anti synaptin/synaptophysin) of samples from temporal cortex of brains from a control patient (C: 57 M), from two Alzheimer cases (AI: 75 M; A2:79 F) and from a case with morbus Pick (P) are shown. A quantitative comparison of the various samples is not possible from this figure since the film obtained from the labelled blot was overexposed to show as many bands as possible in a way suitable for printing. PG-A: proteoglycan-chromogranin A; AM: heat stable extract from human adrenal medulla.
( ' e a r l y ' ) plaques d i d n o t reveal c h r o m o g r a n i n A imm u n o r e a c t i v i t y . In P i c k ' s disease all P i c k bodies (as d e t e r m i n e d b y staining a d j a c e n t sections with either B i e l s c h o w s k y silver i m p r e g n a t i o n or i m m u n o c y t o c h e m i s t r y for tau) were intensively stained with antib o d i e s a g a i n s t c h r o m o g r a n i n A . In b o t h A l z h e i m e r ' s a n d P i c k ' s disease s e c r e t o g r a n i n II like i m m u n o r e a c -
Table I Levels of chromogranin A and synaptin/synaptophysin Subject Controls
Mean +_ SE Alzheimer
Mean + SE Pick
Age Sex (years) 57 91 54 67
M M M M
77 73 72 75 79
M F F M F
75
F
Ch-A
Syn
Ch-A/Syn
145 48 79.4 127.5
104.4 94.1 93 108.4
1.4 0.5 0.9 1.2
99.9_+22.2 301 272 200 281 78 226.4_+40.8* 396.7
99.9_+3.8 1.0___0.2 69 4.4 87.2 3.1 74.5 2.7 65.1 4.3 19.1 4.1 63-+ 11,6" 3.7-+0.3** 36.8 _+8.7 10.8
Heat stable extracts of brains from non-demented controls and from cases of Alzheimer's and Pick's disease were subjected to quantitative immunoblotting [15]. Four controls and 5 Alzheimer cases were available, For Pick's disease only one brain was analyzed; the results of several measurements (n = 2 for Ch-A; n = 4 for syn) for different brain samples are presented. Statistical differences: * P < 0.05; • * P <0.001. 338
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tivity was exclusively present in g r a n u l a r d i s t r i b u t i o n in the c y t o p l a s m o f nerve cells. P i c k b o d i e s a n d p l a q u e neurites were negative for s e c r e t o g r a n i n II. These imm u n o h i s t o c h e m i c a l results d e m o n s t r a t e that c h r o m o g r a n i n A i m m u n o r e a c t i v i t y is consistently f o u n d b o t h in A l z h e i m e r plaques a n d P i c k bodies; however, this m e t h o d does not allow a n y statements on the p r o p e r t i e s a n d relative q u a n t i t y o f the i m m u n o r e a c t i v e m a t e r i a l . In o r d e r to define this i m m u n o r e a c t i v e m a t e r i a l in m o l e c u l a r terms i m m u n o b l o t s were p e r f o r m e d . P r e vious studies (see [1]) have e s t a b l i s h e d t h a t in extracts o f i m m e d i a t e l y processed e n d o c r i n e a n d b r a i n tissues a n t i s e r a against c h r o m o g r a n i n A react with the p r o p r o tein c h r o m o g r a n i n A a n d several smaller p r o t e i n s w h i c h are f o r m e d within the s t o r a g e vesicles b y e n d o g e n o u s processing. F o r tissues n o t i m m e d i a t e l y p r o cessed a f u r t h e r b r e a k d o w n leading to smaller p r o t e i n s b e c o m e s a p p a r e n t [16]. F o r h u m a n b r a i n , as s h o w n in Fig. 1, the p r o p r o t e i n c h r o m o g r a n i n A can still be d e m o n s t r a t e d (A in Fig. 1). In a d d i t i o n a faster m o v i n g b r e a k d o w n p r o d u c t (A1 in Fig. 1) is present in v a r y i n g relative a m o u n t s . A slower m o v i n g b a n d ( P G - A in Fig. 1) has been p r e v i o u s l y [17] identified as the p r o t e o g l y c a n f o r m o f c h r o m o g r a n i n A , which can be f o u n d consistently in b r a i n tissue, e.g. o f rat [1,18]. As s h o w n in Fig. 1 there is no significant qualitative difference b e t w e e n the c h r o m o g r a n i n A - r e a c t i v e b a n d s in c o n t r o l or diseased brains. T h e a p p a r e n t p r o m i n e n c e o f the p r o t e o g l y c a n - c h r o m o g r a n i n A in P i c k ' s b r a i n s is d u e to the fact t h a t higher levels o f t o t a l c h r o m o g r a n i n were present in these samples. S y n a p t i n / s y n a p t o p h y s i n (see Fig. 1) c o u l d also be d e m o n s t r a t e d in h u m a n brain. F o r s e c r e t o g r a n i n II o u r m e t h o d was n o t sensitive enough. The quantitative evaluation of chromogranin A and s y n a p t i n / s y n a p t o p h y s i n levels is s h o w n in T a b l e I, A c o n s i s t e n t finding for all A l z h e i m e r cases was the lower level o f s y n a p t i n / s y n a p t o p h y s i n ; o n the o t h e r h a n d in 4 o u t o f 5 A l z h e i m e r cases the c h r o m o g r a n i n A level was r e m a r k a b l y elevated. In a fifth case the c h r o m o g r a n i n A level was below c o n t r o l , but the c h r o m o g r a n i n A / s y n a p t i n ( s y n a p t o p h y s i n ) ratio was still high. It seems likely t h a t in this case a c o n s i d e r a b l e cell loss has reduced a previously elevated c h r o m o g r a n i n A level b e l o w c o n t r o l . O n l y one b r a i n o f P i c k ' s disease was available for i m m u n o b l o t t i n g ; nevertheless, several s a m p l e s were a n a l y z e d a n d a very high level o f c h r o m o g r a n i n A a n d o f the c h r o m o g r a n i n A / s y n a p t i n ( s y n a p t o p h y s i n ) ratio was detected.
4. D I S C U S S I O N S y n a p t i n / s y n a p t o p h y s i n is a p r o t e i n f o u n d in high c o n c e n t r a t i o n s in s y n a p t i c vesicles [ 5 - 7 ] , a n d in lower c o n c e n t r a t i o n in large dense core vesicles a n d in end o c r i n e vesicles like c h r o m a f f i n granules [5,8,9]. Its
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consistent low levels in Alzheimer's (which is in agreement with immunohistochemical data [19,20]) and Pick's brains can simply be an expression of neuronal and synaptic loss occurring in these diseases, but a specific loss o f synaptic vesicles should also be considered. On the other hand, chromogranin A levels are increased. This protein is found in endocrine vesicles and large dense core vesicles o f brain and is secreted together with other constituents o f these vesicles by exocytosis. This property is shared with neuropeptides [1]. For some neuropeptides, an immunohistochemical staining o f Alzheimer plaques has been observed [21], but we are unaware o f a report describing high levels o f any o f these peptides in brain. In fact reduced levels have been reported (see also [22]) for neuropeptide Y [23] and somatostatin [24]. In addition we have not found immunostaining o f Alzheimer plaques or of Pick bodies with an antiserum against secretogranin II which at least in chromaffin granules is colocalized with chromogranin A [25]. Therefore our results on chromogranin A levels seem quite specific for this secretory protein. Unfortunately the function o f this protein is still not very well defined, although peptides derived from it (e.g. pancreastatin) have been shown to inhibit the secretion o f hormones [26,27]. Elevated levels o f chromogranin A in diseased brain might be a consequence o f an increased synthesis or decreased secretion or breakdown. Our present study does not yet allow us to state whether chromogranin A is only accumulating in the typical lesions or is elevated in all neurons. This can only be elucidated in further studies including mRNA-measurements. In this context it is interesting to note that one report has claimed that in adrenal medulla of Alzheimer patients large vacuoles with electron dense content (protein?) have been found [28]. The adrenal medulla contains a high concentration of chromogranin A [1]. Does this suggest that peripheral organs also contain more chromogranin as it has recently been shown for the amyloid fl protein precursor [29]? In any case we consider our results important for the following reasons: we have found chromogranin A immunostaining both in Alzheimer plaques and Pick bodies. The immunoreactive material was identified as chromogranin A. In both diseases the brain contains increased levels of this secretory protein found in large dense core vesicles whereas synaptin/synaptophysin levels are low. These data for chromogranin A appear specific, since other secretory constituents o f large dense core vesicles (secretogranin II, neuropeptides) behave differently. Thus for these two diseases leading to dementia but with significantly differing pathohistology a high chromogranin A/synaptin(synaptophysin) ratio is a c o m m o n and specific denominator. These data might also become relevant for diagnostic purposes: does the cerebrospinal fluid of Alzheimer and Pick patients contain higher levels o f chromogranin A or o f peptides derived from it?
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Acknowledgements: The study was partly funded by a grant of the Austrian Ministry for Science and Research, by the Ludwig Boltzmann Institute of Aging Research (W. Danielczyk), by the Fonds zur F6rderung der Wissenschaftlichen Forschung and by the Dr Legerlotz-Stiftung. We are indebted to Mrs H. Breitschopf, Ms M. Leiszer and Ms S. Katzensteiner for expert technical assistance.
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