- Email: [email protected]
Cystatin C containing neurons in human postmortem hypothalamus
Neuroscienee Letters, 88 (1988) 131 134 Elsevier Scientific Publishers Ireland Ltd.
13I
NSL 05308
Cystatin C containing neurons in human postmortem hypothalamus* H a n s - G e r t B e r n s t e i n I , M i k k o J f i r v i n e n 2, R a i m o P611finen 2, H e n d r i k S c h i r p k e I, B r i g i t t e K n 6 f e l 1 a n d R i i t t a R i n n e 3 Zlnstitute qf Anatomy, Medical Academy, Magdeburg (G.D.R.), 2Institute of Pathology, University cq Oulu, Oulu (Finland) andSDepartment ~f Neurology, University c¢ Tromso, Tromso (Norway
(Received 27 October 1987: Revised version received 1 February 1988: Accepted 3 February 1988) Key words. Cystatin C: Human brain: Hypothalamus; Immunocytochemistry
The regional distribution and cellular localization of cystatin C in neurons of human postmortem hypothalamus was studied by use of peroxidase--anti-peroxidase technique. Cystatin C (earlier named ~,trace) was found to be present in multiple nerve cells belonging to nuclei supraopticus, paraventricularis and arcuatus. We speculate that the occurrence of cystatin C in human cerebrospinal fluid is the result of a release of the protein from these neurons into the ventricular system.
C y s t a t i n C (7-trace) is a basic m i c r o p r o t e i n , which was originally identified in hum a n c e r e b r o s p i n a l fluid ( C S F ) a n d subsequently, also in m o s t o t h e r b o d y fluids [3, 4, 8, 9, 12]. Its m o l e c u l a r weight was established to be a b o u t 13,000. C y s t a t i n C is a m e m b e r o f the so-called cystatin s u p e r f a m i l y [2]. It c o n t a i n s two disulfide l o o p s t o w a r d s the c a r b o x y l terminus o f the p e p t i d e chain [2]. F r o m the p i o n e e r w o r k o f the G r u b b a n d L6fberg g r o u p [6, 7, 10] it is now clear that cystatin C is also present in different n e u r o e n d o c r i n e cells o f the g a s t r o e n t e r o p a n c r e a t i c system. This, a n d the recently established role o f the p r o t e i n as a p o t e n t inhibitor o f cathepsins B a n d L [1], m a k e it r e a s o n a b l e to s u p p o s e a function o f c y s t a tin C in the subtle r e g u l a t i o n o f p r o t e i n a n d n e u r o p e p t i d e turnover. Cystatin C was f o u n d in certain n e u r o n s o f h u m a n neocortical biopsies [1 I]. Recently, a slightly modified m o l e c u l a r v a r i a n t o f cystatin C was shown to be responsible for the expression o f a rare form o f cerebral h e m o r r h a g e (Iceland h e r e d i t a r y h e m o r r h a g e with a m y l o i d o s i s ) [5]. The c i r c u m s t a n c e that (a) cystatin C is o b v i o u s l y associated with n e u r o e n d o c r i n e ( p e p t i d e - p r o d u c i n g ) cells and (b) that the p r o t e i n occurs in the C S F at c o n c e n t r a t i o n s several fold higher t h a n that o f the b l o o d led us to h y p o t h e s i z e that there must be o t h e r sources o f cystatin C in the h u m a n brain *Presented in part at the second World Congress of Neuroscience, Budapest, 1987. Correspondence." H.G, Bernstein, Institute of Anatomy, Medical Academy, Leipziger Str. 44, DDR-3090 Magdeburg, G.D.R. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
132 apart from neocortex. We herein show that hypothalamic neurons are rich m cystatin C. Six human postmortem hypothalami were investigated. The brain material was obtained from persons aged between 67 and 83 years. The preparation of the hypo.thalami was performed between 4 and 11 h after the death of the persons. Brain material was fixed in 4% ice-cold formalin, embedded in paraffin, and cut at 6/tm. For purposes of a positive reference tissue, 4 pituitary glands of human cadavers were processed the same way. Dewaxed sections were either stained for morphological orientation (Nissl) or immunostained. The antigen was purified to apparent homogeneity from urine of patients with renal failure as recommended [7]. The antiserum was raised in rabbits and its speciticity was thoroughly checked including immuno-electrophoresis and Western blot analysis. The antiserum did non cross-react with cystatin A. Furthermore, our human anti-cystatin C shows a very weak cross-reactivity with rat cystatin C as shown by immunostaining of rat adenohypophysis. Previously the antiserum was successfully applied to quantitative studies on the concentration ofcystatin C in human CSF by use of fluorescence immunoassay [8]. The immunohistochemical procedure involved the following steps: equilibration of the sections with phosphate-buffered saline (PBS, pH 7.2), blocking of endogenous peroxidase activity by treatment with H202 and methanol, incubation with the primary antiserum at a dilution of 1:t00 and 1:200 for 15 h at 4°C, incubation with antirabbit immunoglobulin (swine; Dako) at a dilution of 1:100 for I h at room temperature, and incubation with peroxidaseanti-peroxidase complex according to Sternberger [13] for i h at room temperature. The immunoreactive material was visualized by use of H202 and 3,3'-diaminobenzidine as a chromogen. The sections were washed 3 times in PBS after each step. Control sections with the first antiserum replaced by either non-immune serum or antiserum preabsorbed with pure cystatin C never revealed staining (Fig. 4). Anti-cystatin C preabsorbed with either pancreatic type glucagon or cholecystokinin did not change its immunological properties, i.e. after its application there was still a well-pronounced immunostaining. Incubation of the sections with cystatin C antiserum revealed multiple large and medium sized neurons of human postmortem hypothalamus to contain the protein. These nerve cells belonged to nuclei supraopticus, paraventricularis (Fig. 1) and arcuatus (Fig. 2). It is of interest to note that we were able to demonstrate cystatin C in postmortem material, since the experience of L6fberg and Grubb speakes against a postmortem stability of the protein [10]. Obviously, the concentration of cystatin C in hypothalamic neurons is high enough to guarantee the survival of some antigenity in postmortem material. In 2 of 6 brains (those with a delay in material acquisition of 8 or more hours after death) no cystatin C immunoreaction was demonstrable. Intraneuronally, the reaction product formed juxtanuclearly located clusters. At higher magnification, the granular nature of the immunoreactive material was evident (Fig. 1). The human adenohypophysis, which was investigated for purposes of a positive reference organ. showed multiple cystatin C-positive cells (Fig. 3) [6] as described by others.
133
1
2
3
4
Fig. 1. Two magnocellular neurons belonging to nucleus paravcntricularis displaying cystatin C immunoreactivity. Note the granular nature of the immunoreactive material. Magnification ca. x 600. |Zig. 2. Neurons belonging to nucleus arcuatus with juxtranuclear clusters of cystatin C immunorcaclive material, Magnitication ca. x 480. Fig. 3. Adenohypophyseal cells containing cystatin C (positive reference). Magnilication ca. × 150. Fig. 4. t t u m a n adenohypophysis (parallel section to that shown in Fig. 3). Cystatin C antiserum was preabsorbed with pure cystatin C. An immunoreaction is nol demonstrable. Magnification ca. × 15~).
The presence of cystatin C in hypothalamic neurons gives a simple and plausible explanation for the elevated levels of cystatin C in human CSF in comparison with blood [4, 7, 12], since it is known that (at least in several non-human mammals) some of the hypothalamic neurons project directly to the third ventricle, thereby being able to release cell products into the CSF [14, 15]. Another possible route by which CSF might receive 'hypothalamic' cystatin C would be via the median eminence. So, prob-
134 ably, C S F levels o f c y s t a t i n C reflect p h y s i o l o g i c a l (or p a t h o p h y s i o l o g i c a l ) p r o c e s s e s g o i n g o n in b r a i n p a r e n c h y m a ( a n d e s p e c i a l l y h y p o t h a l a m u s ) . T h e r e f o r e , it is n e c e s s a r y to p a y m o r e a t t e n t i o n to this b r a i n area, w h e n c y s t a t i n C is used as a p a r a m e t e r o f n e u r o l o g i c a l d i s o r d e r s [5, 9, 10, 12]. I Barrett, A.J., Davies, M.E. and Grubb, A., The place of y-trace (cystatin C) amongst the cysteine proteinase inhibitors, Biochem. Biophys. Res. Commun., 120 (1984)631-636. 2 Barrett, A.J., The cystatins: a new class of peptidase inhibitors, Trends Biochem. Sci., 12 (1987) 193 196. 3 Cejka, J. and Fleischmann, L.E., Post ?,-globulin: isolation and physiochemical characterization, Arch. Biochem. Biophys., 157 (1973) 168 -176. 4 Clausen, J., Proteins in normal cerebrospinal fluid not found in serum, Proc. Soc. Exp. Biol. Med., 107 (1961) 170~172. 5 Cohen, D.H., Feiner, H., Jensson, O. and Pragnione, B., Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein. gamma trace, J. Exp. Med., 158 (1983) 623 628. 6 Grubb, A. and L6fberg, H., Human 7-trace, a basic microprotein: its amino acid sequence and presence in the adenohypophysis, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3024-3027. 7 Grubb, A. and L6fberg, H., Human y-trace. Structure, function and clinical use of concentration measurements, Scand. J. Clin. Lab. Invest., 45 Suppl. 177 (1985) 7-13. 8 Grubb, A.O., Weiber, H. and L6fberg, H., The ),-trace concentration of normal human seminal plasma is thirty-six times higher that of normal human blood plasma, Scand. J. Clin. Lab. Invest., 43 (1983) 421 425. 9 Joronen, I., Hopsu-Havu, V.K., Manninen, M., Rinne, A., J/irvinen, M. and Halonen, P., Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoro-immunoassay, J. lmmunol. Methods, 86 (1986) 243 247. 10 L6fberg, H. and Grubb, A.O., Quantitation of y-trace in human biological fluids: indications for production in the central nervous system, Scand. J. Clin. Lab. Invest., 39 (1979) 619-626. 11 L6fberg, H., Grubb, A.O. and Brun, A., Human brain cortical neurons contain ),-trace. Rapid isolation, immunohistochemical and physiochemical characterization of human ),-trace, Biomed. Res., 2 (1981) 298 306. 12 L6fberg, H., Grubb, A.O., Sveger, T. and Olsson, J.E., The cerebrospinal fluid and plasma concentration of )'-grace and E2-microglobulin at various stages and in neurological disorders, J. Neurol., 223 (1980) 159 170. 13 Sternberger, L.A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979. 14 Vigh-Teichmann, 1. and Vigh, B., Correlation of CSF contacting neuronal elements to neurosecretory and ependymsecretory structure. In A. Mitro (Ed.), Ependyma and Neurohormonal regulation, VEDA, Bratislava, 1974, pp. 281-295. 15 Weindl, A., Sofroniew, M.V. and Schinko, I., Psychotrope Wirkungen hypothalamischer Neurone: Immunohistochemische Identifikation extrahypophys/irer Verbindungen neuroendokriner Neurone, Arzneim.-Forsch., 26 (1976) 1191 1194.
13I
NSL 05308
Cystatin C containing neurons in human postmortem hypothalamus* H a n s - G e r t B e r n s t e i n I , M i k k o J f i r v i n e n 2, R a i m o P611finen 2, H e n d r i k S c h i r p k e I, B r i g i t t e K n 6 f e l 1 a n d R i i t t a R i n n e 3 Zlnstitute qf Anatomy, Medical Academy, Magdeburg (G.D.R.), 2Institute of Pathology, University cq Oulu, Oulu (Finland) andSDepartment ~f Neurology, University c¢ Tromso, Tromso (Norway
(Received 27 October 1987: Revised version received 1 February 1988: Accepted 3 February 1988) Key words. Cystatin C: Human brain: Hypothalamus; Immunocytochemistry
The regional distribution and cellular localization of cystatin C in neurons of human postmortem hypothalamus was studied by use of peroxidase--anti-peroxidase technique. Cystatin C (earlier named ~,trace) was found to be present in multiple nerve cells belonging to nuclei supraopticus, paraventricularis and arcuatus. We speculate that the occurrence of cystatin C in human cerebrospinal fluid is the result of a release of the protein from these neurons into the ventricular system.
C y s t a t i n C (7-trace) is a basic m i c r o p r o t e i n , which was originally identified in hum a n c e r e b r o s p i n a l fluid ( C S F ) a n d subsequently, also in m o s t o t h e r b o d y fluids [3, 4, 8, 9, 12]. Its m o l e c u l a r weight was established to be a b o u t 13,000. C y s t a t i n C is a m e m b e r o f the so-called cystatin s u p e r f a m i l y [2]. It c o n t a i n s two disulfide l o o p s t o w a r d s the c a r b o x y l terminus o f the p e p t i d e chain [2]. F r o m the p i o n e e r w o r k o f the G r u b b a n d L6fberg g r o u p [6, 7, 10] it is now clear that cystatin C is also present in different n e u r o e n d o c r i n e cells o f the g a s t r o e n t e r o p a n c r e a t i c system. This, a n d the recently established role o f the p r o t e i n as a p o t e n t inhibitor o f cathepsins B a n d L [1], m a k e it r e a s o n a b l e to s u p p o s e a function o f c y s t a tin C in the subtle r e g u l a t i o n o f p r o t e i n a n d n e u r o p e p t i d e turnover. Cystatin C was f o u n d in certain n e u r o n s o f h u m a n neocortical biopsies [1 I]. Recently, a slightly modified m o l e c u l a r v a r i a n t o f cystatin C was shown to be responsible for the expression o f a rare form o f cerebral h e m o r r h a g e (Iceland h e r e d i t a r y h e m o r r h a g e with a m y l o i d o s i s ) [5]. The c i r c u m s t a n c e that (a) cystatin C is o b v i o u s l y associated with n e u r o e n d o c r i n e ( p e p t i d e - p r o d u c i n g ) cells and (b) that the p r o t e i n occurs in the C S F at c o n c e n t r a t i o n s several fold higher t h a n that o f the b l o o d led us to h y p o t h e s i z e that there must be o t h e r sources o f cystatin C in the h u m a n brain *Presented in part at the second World Congress of Neuroscience, Budapest, 1987. Correspondence." H.G, Bernstein, Institute of Anatomy, Medical Academy, Leipziger Str. 44, DDR-3090 Magdeburg, G.D.R. 0304-3940/88/$ 03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd.
132 apart from neocortex. We herein show that hypothalamic neurons are rich m cystatin C. Six human postmortem hypothalami were investigated. The brain material was obtained from persons aged between 67 and 83 years. The preparation of the hypo.thalami was performed between 4 and 11 h after the death of the persons. Brain material was fixed in 4% ice-cold formalin, embedded in paraffin, and cut at 6/tm. For purposes of a positive reference tissue, 4 pituitary glands of human cadavers were processed the same way. Dewaxed sections were either stained for morphological orientation (Nissl) or immunostained. The antigen was purified to apparent homogeneity from urine of patients with renal failure as recommended [7]. The antiserum was raised in rabbits and its speciticity was thoroughly checked including immuno-electrophoresis and Western blot analysis. The antiserum did non cross-react with cystatin A. Furthermore, our human anti-cystatin C shows a very weak cross-reactivity with rat cystatin C as shown by immunostaining of rat adenohypophysis. Previously the antiserum was successfully applied to quantitative studies on the concentration ofcystatin C in human CSF by use of fluorescence immunoassay [8]. The immunohistochemical procedure involved the following steps: equilibration of the sections with phosphate-buffered saline (PBS, pH 7.2), blocking of endogenous peroxidase activity by treatment with H202 and methanol, incubation with the primary antiserum at a dilution of 1:t00 and 1:200 for 15 h at 4°C, incubation with antirabbit immunoglobulin (swine; Dako) at a dilution of 1:100 for I h at room temperature, and incubation with peroxidaseanti-peroxidase complex according to Sternberger [13] for i h at room temperature. The immunoreactive material was visualized by use of H202 and 3,3'-diaminobenzidine as a chromogen. The sections were washed 3 times in PBS after each step. Control sections with the first antiserum replaced by either non-immune serum or antiserum preabsorbed with pure cystatin C never revealed staining (Fig. 4). Anti-cystatin C preabsorbed with either pancreatic type glucagon or cholecystokinin did not change its immunological properties, i.e. after its application there was still a well-pronounced immunostaining. Incubation of the sections with cystatin C antiserum revealed multiple large and medium sized neurons of human postmortem hypothalamus to contain the protein. These nerve cells belonged to nuclei supraopticus, paraventricularis (Fig. 1) and arcuatus (Fig. 2). It is of interest to note that we were able to demonstrate cystatin C in postmortem material, since the experience of L6fberg and Grubb speakes against a postmortem stability of the protein [10]. Obviously, the concentration of cystatin C in hypothalamic neurons is high enough to guarantee the survival of some antigenity in postmortem material. In 2 of 6 brains (those with a delay in material acquisition of 8 or more hours after death) no cystatin C immunoreaction was demonstrable. Intraneuronally, the reaction product formed juxtanuclearly located clusters. At higher magnification, the granular nature of the immunoreactive material was evident (Fig. 1). The human adenohypophysis, which was investigated for purposes of a positive reference organ. showed multiple cystatin C-positive cells (Fig. 3) [6] as described by others.
133
1
2
3
4
Fig. 1. Two magnocellular neurons belonging to nucleus paravcntricularis displaying cystatin C immunoreactivity. Note the granular nature of the immunoreactive material. Magnification ca. x 600. |Zig. 2. Neurons belonging to nucleus arcuatus with juxtranuclear clusters of cystatin C immunorcaclive material, Magnitication ca. x 480. Fig. 3. Adenohypophyseal cells containing cystatin C (positive reference). Magnilication ca. × 150. Fig. 4. t t u m a n adenohypophysis (parallel section to that shown in Fig. 3). Cystatin C antiserum was preabsorbed with pure cystatin C. An immunoreaction is nol demonstrable. Magnification ca. × 15~).
The presence of cystatin C in hypothalamic neurons gives a simple and plausible explanation for the elevated levels of cystatin C in human CSF in comparison with blood [4, 7, 12], since it is known that (at least in several non-human mammals) some of the hypothalamic neurons project directly to the third ventricle, thereby being able to release cell products into the CSF [14, 15]. Another possible route by which CSF might receive 'hypothalamic' cystatin C would be via the median eminence. So, prob-
134 ably, C S F levels o f c y s t a t i n C reflect p h y s i o l o g i c a l (or p a t h o p h y s i o l o g i c a l ) p r o c e s s e s g o i n g o n in b r a i n p a r e n c h y m a ( a n d e s p e c i a l l y h y p o t h a l a m u s ) . T h e r e f o r e , it is n e c e s s a r y to p a y m o r e a t t e n t i o n to this b r a i n area, w h e n c y s t a t i n C is used as a p a r a m e t e r o f n e u r o l o g i c a l d i s o r d e r s [5, 9, 10, 12]. I Barrett, A.J., Davies, M.E. and Grubb, A., The place of y-trace (cystatin C) amongst the cysteine proteinase inhibitors, Biochem. Biophys. Res. Commun., 120 (1984)631-636. 2 Barrett, A.J., The cystatins: a new class of peptidase inhibitors, Trends Biochem. Sci., 12 (1987) 193 196. 3 Cejka, J. and Fleischmann, L.E., Post ?,-globulin: isolation and physiochemical characterization, Arch. Biochem. Biophys., 157 (1973) 168 -176. 4 Clausen, J., Proteins in normal cerebrospinal fluid not found in serum, Proc. Soc. Exp. Biol. Med., 107 (1961) 170~172. 5 Cohen, D.H., Feiner, H., Jensson, O. and Pragnione, B., Amyloid fibril in hereditary cerebral hemorrhage with amyloidosis (HCHWA) is related to the gastroentero-pancreatic neuroendocrine protein. gamma trace, J. Exp. Med., 158 (1983) 623 628. 6 Grubb, A. and L6fberg, H., Human 7-trace, a basic microprotein: its amino acid sequence and presence in the adenohypophysis, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3024-3027. 7 Grubb, A. and L6fberg, H., Human y-trace. Structure, function and clinical use of concentration measurements, Scand. J. Clin. Lab. Invest., 45 Suppl. 177 (1985) 7-13. 8 Grubb, A.O., Weiber, H. and L6fberg, H., The ),-trace concentration of normal human seminal plasma is thirty-six times higher that of normal human blood plasma, Scand. J. Clin. Lab. Invest., 43 (1983) 421 425. 9 Joronen, I., Hopsu-Havu, V.K., Manninen, M., Rinne, A., J/irvinen, M. and Halonen, P., Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoro-immunoassay, J. lmmunol. Methods, 86 (1986) 243 247. 10 L6fberg, H. and Grubb, A.O., Quantitation of y-trace in human biological fluids: indications for production in the central nervous system, Scand. J. Clin. Lab. Invest., 39 (1979) 619-626. 11 L6fberg, H., Grubb, A.O. and Brun, A., Human brain cortical neurons contain ),-trace. Rapid isolation, immunohistochemical and physiochemical characterization of human ),-trace, Biomed. Res., 2 (1981) 298 306. 12 L6fberg, H., Grubb, A.O., Sveger, T. and Olsson, J.E., The cerebrospinal fluid and plasma concentration of )'-grace and E2-microglobulin at various stages and in neurological disorders, J. Neurol., 223 (1980) 159 170. 13 Sternberger, L.A., Immunocytochemistry, 2nd edn., Wiley, New York, 1979. 14 Vigh-Teichmann, 1. and Vigh, B., Correlation of CSF contacting neuronal elements to neurosecretory and ependymsecretory structure. In A. Mitro (Ed.), Ependyma and Neurohormonal regulation, VEDA, Bratislava, 1974, pp. 281-295. 15 Weindl, A., Sofroniew, M.V. and Schinko, I., Psychotrope Wirkungen hypothalamischer Neurone: Immunohistochemische Identifikation extrahypophys/irer Verbindungen neuroendokriner Neurone, Arzneim.-Forsch., 26 (1976) 1191 1194.