Abstract

Recently we identified a novel EF-hand Ca-binding protein termed secretagogin, which is expressed in neuroendocrine cells. Immunohistochemical investigations, using a murine monoclonal and an affinity purified rabbit polyclonal anti-secretagogin antibody as well as Northern-blot and Western-blot analysis revealed a neuron-specific cerebral expression pattern. Secretagogin was detected in high quantity in basket and stellate cells of the cerebellar cortex, in secretory neurons of the anterior part of the pituitary gland and in singular neurons of the frontal and parietal neocortex. Remarkable staining intensity was observed in hypothalamic and in hippocampal neurons. Using a newly developed sandwich capture ELISA we show presence of secretagogin in serum of patients suffering from hypoxic neuronal damage. In sera obtained from 32 patients with different forms of neurological symptoms due to focal cerebral ischemia, secretagogin levels ranged from 3 to 236 pg/ml, with highest levels observed on days 2 and 3 after infarction. Three patients exhibiting minor, reversible neurological deficits had nondetectable serum secretagogin levels at time points of testing. In 50 control sera, secretagogin was below the detection limit of our ELISA. Parallel analysis of secretagogin and the established neurobiochemical marker S-100B in 14 representative patients revealed comparable results. However, S-100B levels were higher and exhibited different kinetics than secretagogin. Our data present the cerebral expression pattern of secretagogin and give evidence that this protein might represent a clinically relevant serum marker indicative for neuronal damage.

Introduction

The EF-hand is a highly conserved Ca-binding motif found in a large number of intracellular proteins — EF-hand proteins (Celio et al., 1996). Some members of the EF-hand protein family are expressed ubiquitously in human tissues. However, most EF-hand proteins are characterized by a specific tissue distribution with predominant expression in the CNS (Heizmann and Hunziker, 1991; Yamaguchi et al., 1999). Intracerebrally, the Ca-binding proteins calretinin, calbindin D28k and parvalbumin are present in essentially different subsets of neurons (Celio, 1990; DeFelipe, 1997; Fortin et al., 1998; Munkle et al., 1999). Although the function of Ca-binding proteins in the CNS still remains to be fully elucidated, it has become clear that EF-hand proteins are involved in several Ca-dependent processes (Lledo et al., 1992; Ghosh and Greenberg, 1995; Niki et al., 1996). Besides the function as intracellular Ca-buffers, recent findings indicate that EF-hand proteins play a role in cell-cycle regulation, developmental processes and in apoptosis, as well as in transcription and secretion control (Sheng et al., 1991; Chard et al., 1993; Fierro and Llano, 1996; Asai et al., 1999; Carrion et al., 1999; Lo et al., 1999; Li et al., 2000; Maki, 2000; Peracchia et al., 2000; Rodney et al., 2000). Furthermore, Ca-binding proteins seem to be involved in several pathological conditions in the CNS, such as epilepsy or neurodegenerative disorders, for example Alzheimer's disease (Heizmann and Braun, 1992; Fonseca and Soriano, 1995; Maguire-Zeiss et al., 1995; Vito et al., 1996; Magloczky et al., 1997; Buxbaum et al., 1998). Moreover, S-100B, also a member of the EF-hand family with specific expression in astroglial cells, can be detected in the cerebrospinal fluid and in the peripheral blood under specific pathological conditions associated with neuronal death and impairment of the blood–brain barrier (Hardemark et al., 1989; Fano et al., 1995; Buttner et al., 1997). Elevated S-100B serum concentration after ischemic brain damage [TIA (transient ischemic attack), prolonged ischemic neurological deficit, stroke], intracerebral hemorrhage and traumatic brain injury has been the subject of investigations in recent years and it has become well-known that S-100B is a sensitive marker of neuronal damage (Kim et al., 1996; Abraha et al., 1997; Rosen et al., 1998; Wunderlich et al., 1999). However, the determination of a panel of proteins released into the serum after brain damage, as well as the identification of new proteins indicating neuronal death, could increase the sensitivity of neurobiochemical markers (Hill et al., 2000).

We recently identified a novel Ca-binding protein named secretagogin. This protein consists of six EF-hand Ca-binding domains and is characterized by a mol. wt of 32 kDa. First data on its tissue distribution indicated that secretagogin expression was restricted to neuroendocrine cells (Wagner et al., 2000). Herewith, we present Northern-blot data, as well as Western-blot and immunohistochemical analyses, which demonstrate expression of secretagogin in the CNS, particularly in the cerebellum, pituitary gland and hypothalamus, but also in thalamic tissue, neocortex, hippocampus and basal ganglia. Moreover, we show that secretagogin is detectable in human serum after ischemic brain damage, employing a recently developed sandwich capture ELISA and compare these data with S-100B values. Our findings might represent the basis for the establishment of an additional neurobiochemical marker indicative of neuronal death.

Patients and Methods

Patients

In the present study, we included 32 patients presenting with neurological symptoms due to cerebral ischemia. The diagnosis of stroke was confirmed by the clinical syndrome and by brain imaging (CCT or MRI). Criteria for clinically definite TIAs were: Sudden onset of focal neurological symptoms such as unilateral weakness or sensory symptoms, aphasia (dysphasia) or homonymous hemianopsia with symptoms being maximal at onset. Events which are clinically classified as probable TIAs were not included in the present study.

Thirty-two patients (14 female) aged between 43 and 93 years participated in the study after having given informed consent. Thirty patients suffered from focal brain ischemia, two patients from primary (hypertensive) intracerebral hemorrhage (small hemorrhage in the internal capsule; hemorrhage in the basal ganglia), five patients were classified to have a clinically definite TIA, 25 patients suffered from different types of ischemic stroke [TACS, total anterior circulation syndrome; PACS, partial anterior circulation syndrome; POCS, posterior circulation syndrome; LACS, lacunar syndrome; (Bamford et al., 1991)] caused by different etiologies (Table 1).

Control sera were obtained from age matched healthy blood donors (n = 50). Excluded were patients with cardiological as well as those with neuroendocrine diseases. None of the control subjects had any neurological symptoms or history of a neurological disease.

Blood Sample Collection

Serial venous blood samples were obtained from the patients by puncture of the cubital vein at admission (day 1, n = 32), on the four following days (day 2, n = 29; day 3, n = 31; day 4, n = 28; day 5, n = 21) and on the day of discharge from the department of neurology (n = 2). The time from stroke onset to initial phlebotomy was 8 ± 2 h. Blood was allowed to clot at 4°C and after centrifugation within 2 h (2500 U/min, 5 min), serum was stored for later analysis at –20°C. Hemolytic sera were excluded from analysis. The blood collected for protein assay was obtained in the course of routine blood sampling. This study had been approved by the local Research ethics committee.

Brain Tissue Processing

Aliquots of post-mortem tissue derived from various brain regions were frozen immediately after autopsy in liquid nitrogen until processing. Brain tissue (300–350 mg) was homogenized in 200 μl ice-cold homogenization buffer (TBS containing 1% Triton X-100, 10 μg/ml aprotinin, 1 μg/ml pepstatin, 10 μg/ml leupeptin, 50 mM PMSF and 0.8 mM Pefabloc). A Polytron® homogenizer (Kinematica, Kriens, Switzerland) set on medium speed for 60 s was used for homogenization. Homogenates were then centrifuged at 10000 g for 10 min at 4°C and aliquots of the supernatants were immediately analysed or frozen at –80°C. Total protein content of precleared brain lysate was determined using a Micro BCA Protein assay kit (Pierce, Rockford, IL).

SDS–Polyacrylamide Electrophoresis and Immunoblotting

Secretagogin content in brain homogenates was analysed under reducing conditions by loading aliquots (15 μg of total protein per lane) onto a 12.5% SDS-polyacrylamide gel. After electrophoretically transfering the protein onto nitrocellulose (Amersham, Little Chalfont, UK), blotted membranes were blocked with blocking reagent (Roche Molecular Biochemicals, Vienna, Austria) diluted 1:5 in phosphate-buffered saline (PBS) for 1 h and then incubated at 4°C overnight with affinity purified polyclonal rabbit anti-secretagogin antibody diluted 1:2000 in PBS containing 0.1% Tween 20 and 3% FCS, followed by peroxidase conjugated goat anti-rabbit immunoglobulin (Kirkegaard & Perry, Gaithersburg, MD) diluted 1:10 000 in PBS containing 0.1% Tween 20 for 1 h. Finally, blots were developed by BM chemiluminescent reagent (Roche Molecular Biochemicals, Vienna, Austria). Each incubation step was followed by two washes with PBS containing 0.1% Tween 20 for 10 min.

Secretagogin Sandwich Capture ELISA

Aliquots (100 μl) of the tissue culture supernatant containing the murine monoclonal anti-secretagogin antibody mAb D24 were incubated for 16 h at 4°C in flat-bottomed, 96-well Reacti-Bind goat anti-mouse coated plates (Pierce, Rockford, IL). After a brief wash with PBS, 100 μl of biological fluid was added and incubated for 2 h at room temperature under constant shaking. Serial dilutions of human recombinant secretagogin in human serum were included as standard in all experiments. The plate was washed three times with PBS containing 0.1% Tween 20 in an ELISA wash station (ELX-50 Auto Strip Washer, Biotek Instruments, Vinooski, VT). Rabbit anti-human secretagogin antiserum, diluted 1:1000 in PBS containing 10% BSA (Kirkegaard & Perry, Gaithersburg, MD), was then incubated for 1.5 h under constant shaking with a Behring shaker (Behring AG, Marburg, Germany) at room temperature. After three washes, the plate was incubated with polyclonal horseradish peroxidase conjugated goat anti-rabbit immunoglobulin diluted 1:10000 (Kirkegaard & Perry, Gaithersburg, MD) in PBS containing 3% goat serum and 3% human serum for 1.5 h at room temperature under constant shaking. Wells were washed four times with PBS containing 0.1% Tween 20 and developed using the TMB two component peroxidase substrate solution (Kirkegaard & Perry). The reaction was stopped by adding 1 M H3PO4 and quantitated by absorbance at 450 nm using a Powerwave ELISA reader (Biotek Instruments, Vinooski, VT).

S-100B Luminometric Immunoassay

Serum concentration of S-100B protein was determined using the LIA-mat® Sangtec®100 immunoluminometric assay (AB Sangtec Medical, Bromma, Sweden) according to the manufacturer's instructions. Briefly, 100 μl of standards (0–20 μg/l) or serum samples were diluted in 100 μl BSA buffer and added to S-100B antibody coated tubes. After 1 h incubation under constant shaking with a Behring shaker (Behring AG, Marburg, Germany) at room temperature, tubes were washed three times with 2 ml of wash solution and 200 μl of luminescence labelled monoclonal anti-S-100B antibody was applied into each tube. Following a second incubation step (2 h) under constant shaking at room temperature and three further washes with wash fluid, chemiluminescence reaction was started by sequentially adding alkaline peroxide solution and catalyst solution (LIA-mat® Starter Kit, Byk-Sangtec Diagnostica, Dietzenbach, Germany). The luminescence signal was measured in a Berthold LB 95003 luminometer (Berthold, Bad Wildbad, Germany).

Northern-blot Analysis

The multiple human tissue Northern blot (brain and MTN™ Human) was obtained from Clontech (Palo Alto, CA). Hybridization was performed according to the manufacturers's instructions, using the 32P-Klonow-labeled cDNA insert encoding secretagogin. Blots were washed and exposed to X-ray films at –70°C for 12 h. In order to control for RNA loading the blot was rehybridized using 32P-Klonow-labeled cDNA encoding GAPDH.

Affinity Purification of Polyclonal Rabbit Anti-secretagogin Antibody

Recombinant protein was prepared as indicated previously (Wagner et al. 2000). In brief, secretagogin GST (glutathione S-transferase) fusion protein was purified using Glutathion Sepharose. Secretagogin was liberated by thrombin cutting at the cleavage site located in between GST and secretagogin. Recombinant secretagogin was dialysed in coupling buffer and coupled onto freeze-dried cyanogen bromide activated Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer's instructions. The secretagogin–Sepharose column was prewashed twice with Tris buffer containing 0.5 M NaCl, 0.1% Triton X-100 and 50 mM Tris–HCl, pH 8 and pH 9 respectively. The column was mock-eluted with 50 mM TEA buffer (triethanolamine; pH 11.3), 0.1% Triton X-100 and 0.15 M NaCl. After equilibration of the column with 50 mM Tris–HCl buffer (pH 8), 5 ml of rabbit anti-secretagogin polyclonal antiserum was loaded on the column and recirculated twice. The column was then washed with wash buffer (0.01 M Tris–HCl, pH 8.0 at 4°C, 0.15 M NaCl, 0.025% NaN3). Finally, the antibody was eluted with TEA (50 mM), 0.15 M NaCl elution buffer, pH 11 into 1 M Tris–HCl, pH 6.7. Antibody-containing fractions were pooled and dialysed against PBS and stored at 4°C for further use.

Immunohistochemistry

Cryopreserved tissue

Cryostat sections of human brain tissue were air dried, fixed in acetone and stained within 6 h or stored at –20°C wrapped in aluminium foil. For secretagogin immunostaining, slides were brought to room temperature before unwrapping. PBS-wetted tissue sections were incubated with the monoclonal murine antibody mAb D24 (tissue culture supernatant) or with the polyclonal, affinity purified rabbit anti-secretagogin antibody (diluted 1:300) for 1 h. The secretagogin bound monoclonal murine antibody was detected employing the APAAP and the ABC methods, respectively. For the APAAP method, slides were incubated sequentially with rabbit anti-mouse immunoglobulin diluted 1:20 for 1 h, the APAAP complex diluted 1:20 for 1 h (both from DAKO, Glostrup, Denmark) and Fast Red/naphthol ASBI phosphate (Sigma, Vienna, Austria) as chromogenic substrate. Endogenous alkaline phosphatase was blocked by levamisole (1 mM). The ABC method was performed using biotinylated anti-mouse immunoglobulin and avidin-conjugated peroxidase (both Vector Laboratories, Burlingame, CA), followed by development with diaminobenzidine (DAKO, Glostrup, Denmark).

Tissue sections incubated with polyclonal anitbody were stained sequentially with swine anti-rabbit antibody (DAKO, Glostrup, Denmark) followed by rabbit PAP complex (DAKO, Glostrup, Denmark), both diluted 1:100 in PBS containing BSA (10 ng/μl), or with peroxidase labeled affinity purified goat anti-rabbit antibody (diluted 1:300) (Kirkegaard & Perry, Gaithersburg, MD) as second anitbody and then developed with diaminobenzidine (DAKO, Glostrup, Denmark) as chromogenic substrate. Slides were counterstained with hematoxylin and mounted with Aquamount, improved (BDH, UK). All incubations were performed in a moist chamber at room temperature. Between each incubation step, the slides were washed twice for 5 min with PBS.

In each experiment, two different negative controls were included. These consisted of incubation with tissue culture medium without the primary antibody and incubation of primary antibody pre-absorbed with recombinant secretagogin.

Paraffin-embedded Tissue

Paraffin-embedded tissue sections were deparaffinized and rehydrated by sequential incubations in xylene and a graded alcohol series. Prior to immunohistochemical analysis, tissue sections were regenerated in citrate buffer (DAKO, Glostrup, Denemark) at 80°C overnight. Antigen detection was performed using the affinity purified polyclonal rabbit anti-secretagogin antibody as described above.

Results

Detection of Secretagogin at Protein and mRNA Level in Various Regions of Human Brain

Northern-blot Analysis

In order to investigate secretagogin tissue distribution, we analysed human multiple tissue Northern blots with a 32P-labeled cDNA probe specific for secretagogin. Besides the previously described expression of secretagogin in various neuroendocrine organs (Wagner et al., 2000), substantial expression of secretagogin encoding mRNA was observed in human brain when whole brain mRNA was subject for testing. In comparison with the level of secretagogin expression observed in human pancreatic tissue, the quantity of secretagogin encoding mRNA originating from whole brain extracts was much lower (Fig. 1A) and only a single band at ~1500 bp was detectable. Analysis of various regions of the CNS revealed highest expression in the cerebellum. Moderate quantities of secretagogin encoding mRNA were detectable in most regions of the neocortex (frontal and temporal lobe, occipital lobe) as well as in the medulla oblongata (Fig. 1B). All secretagogin encoding mRNA transcripts are characterized by the same length of ~1500 bp. The multiple tissue Northern-blot was rehybridized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control for RNA loading.

Western-blot Analysis

To confirm and to expand our Northern-blot data, we analysed the secretagogin protein content of tissue homogenates derived from various brain regions (cerebellum, pituitary gland, hypothalamus, basal ganglia, cortical motoric region, temporal cortex, subcortical white matter, substantia nigra) by immuno-blotting. Western-blot analysis (Fig. 2), performed by loading 15 μg protein aliquots of homogenate derived from different brain regions, revealed highest amounts of secretagogin in samples originating from the pituitary gland. In concordance with our Northern-blot data, secretagogin content was also high in cerebellar homogenates. Minor quantities of secretagogin were detected in tissue samples derived from hypothalamus, cortical motoric region, basal ganglia and temporal cortex. Furthermore, a scant quantity of secretagogin was found in tissue homogenates of substantia nigra. Secretagogin was detected as a 32 kDa protein band. Recombinant secretagogin was included in all experiments as control.

Quantification of Secretagogin Protein Expression in Various Brain Regions

In order to investigate brain tissue derived secretagogin detectability and solubility in human serum, tissue homogenates were diluted in human serum and presence of secretagogin was quantitated using the newly developed sandwich capture ELISA (Fig. 3). Highest amounts of secretagogin were found in pituitary and cerebellar tissue (143.2 and 39.3 ng of 100 μg total protein, respectively). Tissue homogenates derived from other cerebral regions contained remarkably less secretagogin, ranging from 185 pg of 100 μg total protein in the basal ganglia to 2.9 ng of 100 μg total protein in thalamic tissue.

Immunohistochemistry

Immunohistochemistry was employed to identify secretagogin expressing cells. Immunohistochemical data obtained in cryopreserved tissue as well as on paraffin embedded tissue sections were in line with results obtained by Northern- and Western-blot analysis. Strong secretagogin immunostaining was observed in cerebellar basket and stellate cells located at the molecular layer with both mAb D24 (APAAP and ABC methods; data not shown) and affinity purified polyclonal Ab (PAP method) (Fig. 4A,D,F). Interestingly, throughout the molecular layer, dendrites (→) of basket and stellate cells were visualized (Fig. 4A,F). Within the granular layer, only infrequent positively stained cells were observed and staining intensity was lower than in cells of the molecular layer (Fig. 4D,E).

Secretagogin protein expression was further found in singular neurons of the temporal cortex and, to a higher extent, in neurons of the motoric region in the frontal neocortex. In a minor subpopulation (<1%), high expression was detected (Fig. 4C). This type of cell had one or two prominent neurites which also stained for the protein. Furthermore, positive staining was seen in pyramidal neurons but at very low intensity (data not shown). In the hippocampal region (paraffin embedded tissue), a high percentage (~70%) of neurons exhibited remarkable immunoreactivity for secretagogin (data not shown). These neurons were located in a distinct compartment of the hippo-campus, situated beyond a superficial layer of immunonegative hippocampal neurons. Like neocortical neurons, hippocampal cells also presented with immunoreactive neurites. Moreover, in neurons of the basal ganglia secretagogin staining was detected to a slightly higher extent when compared with neurons of the neocortex (data not shown). Hypothalamic neurons exhibited considerable secretagogin immunoreactivity (Fig. 4B) and the density of secretagogin expressing cells was higher than in neocortical regions. Finally, the density of immunoreactive cells was highest in the pituitary gland and staining of these cells was as strong as in cerebellar basket and stellate cells (Fig. 4HJ). Staining in the posterior part of the pituitary gland was less intensive and the number of positively stained cells was much lower (data not shown).

There was broad variability of staining intensity between different cells and differences in protein detection at subcellular level. In immunopositive neurons staining was observed in the cytoplasm, but in most cells immunoreactivity was also seen to a varying extent within the nucleus (Fig. 4J). Furthermore, a considerable number of neurons of the cerebral and cerebellar cortex exhibited immunostaining within dendrites and axons (Fig. 4A,F,G).

Secretagogin and S-100B Detection in Human Serum

Determination of Serum Secretagogin Concentration following Cerebral Ischemia

As evaluated above, secretagogin, a highly soluble cytoplasmic protein, is detectable in tissue homogenates of human brain diluted in serum by using the newly developed sandwich capture ELISA. In order to investigate if secretagogin is released into circulation following neuronal damage, we analysed sera derived from 32 patients suffering from an episode of neurological deficit due to ischemia using the newly developed ELISA. For demographic data and description of different stroke subtypes see Tables 1 and 2. As controls, sera from 50 neurologically healthy donors were tested.

Under the experimental conditions used, the detection limit was 1.7 pg/ml. The standard curve using recombinant secretagogin as standard was linear from 1.7 to 400 pg/ml. None of the tested samples contained >400 pg/ml secretagogin. In sera obtained from the control group, secretagogin concentrations were below the detection limit of our ELISA. In contrast, the majority (n = 29, 90.6%) of the patients admitted to the hospital due to neurological symptoms presented with elevated serum secretagogin levels at least once during the observation period. Patients suffering from stroke with severe neurological deficits (TACS and POCS) tested positive at three to five time points of measurement. This was in contrast to patients presenting with transient ischemic attacks. Patients suffering from episodes of TIA had only short periods of detectable secretagogin levels in peripheral blood and values were lower. Positive measurement was seen in these patients as soon as 6 h after onset of neurological symptoms, with absence of immunoreactivity after 12–24 h. Peak secretagogin concentrations ranged from 3 to 238 pg/ml. In three patients, secretagogin was undetectable in the serum after the onset of only minor reversible neurological symptoms. In patients suffering from stroke or TIA, mean serum secretagogin concentrations exhibited a characteristic time course (Fig. 5A). Highest values were measured on days 2 and 3 (mean secretagogin level day 2, 20.4 pg/ml; mean secretagogin level day 3, 19.2 pg/ml), with a consecutive decrease to levels measured at admission (mean secretagogin level day 1, 5.7 pg/ml; day 4, 6.9 pg/ml; day 5, 6.4 pg/ml). Characteristic secretagogin releasing patterns of three individual patients with persistent severe neurological deficit due to cerebral infarction are shown in Figure 6. Two patients presented with undulating serum secretagogin concentrations, showing two phases of increase during the observation period (Fig. 6A,B). Clinically, these patients exhibited a progression of neurological symptoms (progressive stroke). At discharge from the neurological department, serum secretagogin levels were under the detection limit of our ELISA (Fig. 6A). Focusing on stroke subtype, highest secretagogin values were found in sera obtained from patients presenting with TACS and from patients suffering from POCS. Lower levels of secretagogin were observed in sera derived from patients suffering from PACS, LACS, TIA and hemorrhage (Table 2).

Comparison between S-100B and Secretagogin Serum Concentration following Cerebral Ischemia

In order to compare the releasing patterns of secretagogin and S-100B following an ischemic episode, we determined the serum concentration of this well-established neurobiochemical marker in 14 representative patients. The course and level of mean S-100B serum concentration in these patients was similar to releasing patterns of S-100B described by other investigators (Buttner et al., 1997; Missler et al., 1997; Wunderlich et al., 1999). When compared with secretagogin serum concentration following cerebral infarction, we observed three differences: first, mean S-100B serum levels exceeded secretagogin values by a magnitude of ~100×. Secondly, in contrast to secretagogin, which reached peak levels on days 2 and 3 after admission, S-100B serum concentration was highest on days 4 and 5 (Fig. 5B). Finally, most patients exhibited elevated S-100B values over a period of several days, whereas serum secretagogin concentration declined more rapidly after the onset of clinical symptoms (Fig. 6A,B).

Discussion

In this study, we describe the cerebral distribution pattern of secretagogin, a recently identified hexa-EF-hand domain Ca-binding protein (Wagner et al., 2000). In addition, we show that secretagogin is detectable in human serum after ischemic neuronal damage and compare the releasing patterns of secretagogin and S-100B following cerebral ischemia.

Cerebral Expression of Secretagogin

In the brain, secretagogin expression is restricted to distinct subtypes of neurons, with highest expression in the molecular layer of the cerebellum, in the anterior part of the pituitary gland, in the thalamus, in the hypothalamus and in a subgroup of neocortical neurons. Secretagogin was also detectable in tissue homogenates of various other brain regions, but only to a very minor extent. The strong expression of secretagogin protein in neurons of the molecular layer of the cerebellum is in concordance with the results of Northern-blot analysis, which revealed highest amounts of secretagogin encoding mRNA in the cerebellum. The main cell types present in the molecular layer of the cerebellum are stellate and basket cells. Both cell types express secretagogin. This fact is not surprising, as stellate and basket cells seem to belong to a single class of neurons which are known to exert inhibitory action upon Purkinje cells. Secretagogin is also expressed in varying quantity in some neurons of the neocortex. Distinct, singular cells of the neocortex, characterized by one or two prominent neurites, exhibit a high degree of secretagogin expression. The specific intracerebral restriction of Ca-binding protein expression to cell subtypes is a well-known fact (Seto-Ohshima, 1994). Especially, the localization of calbindin D28k, calretinin and parvalbumin has been extensively investigated in recent years. These proteins are used as markers of specific neuronal subpopulations (Celio, 1990; DeFelipe, 1997; Munkle et al., 1999). Recent immunohistochemical single and double antigen localization procedures performed on tissue sections originating from primate cerebellum revealed strong expression of parvalbumin and calbindin in stellate as well as basket cells of the cerebellar cortex (Fortin et al., 1998). Although there is evidence of varying expression of Ca-binding proteins among the different mammalian species (DeFelipe, 1997), colocalization of secretagogin with parvalbumin and calbindin in basket and stellate cells is likely. In cerebellar neurons, secretagogin is mainly located in the cell bodies, in varying amounts in the nucleus but also in axons and dendrites, whereas calbindin is mainly located in dendrites (Fortin et al., 1998). Although secretagogin and calbindin exhibit some sequence homology (Wagner et al., 2000), the differences in intracellular localization indicate functional differences between these proteins. In addition to secretagogin expression in cortical cells of the human brain, we found remarkable quantities of secretagogin in the thalamus. This is in line with previous reports of other investigators, showing thalamic expression of parvalbumin, calbindin and calretinin (Munkle et al., 1999).

Furthermore, secretagogin expression reaches a high extent in the hypothalamus and especially in the pituitary gland. Due to the cell morphology, the anatomic location of these cells and the fact that neuroendocrine cells are characterized by secretagogin immunoreactivity (Wagner et al., 2000), it is most likely that the cells staining positively for secretagogin are neuroendocrine active cells involved in the hypothalamic–pituitary endocrine axis. Nearly all immunoreactive pituitary cells exhibited nuclear staining to a varying extent. Although intranuclear localization of secretagogin was also observed in neurons of other brain regions and in neuroendocrine cells (Wagner et al., 2000), the high staining intensity as well as the high percentage of immunoreactive nuclei of pituitary neurons is surprising and indicates intranuclear functions of secretagogin. The fact that secretagogin was detectable in almost all tested brain regions at protein or mRNA level indicates that secretagogin containing neurons are distributed throughout the whole cerebrum. However, the degree of secretagogin expression seems to be specific and tightly regulated.

This study does not provide insight into the function of secretagogin. However, the characteristic distribution pattern, the abundant expression at cerebellar level and recently gained insights in the function of other homologous EF-hand proteins suggest that secretagogin plays distinct roles in the function of specific neurons or neuronal circuits. For example, findings in mice carrying a null mutation of the calbindin gene show that this member of the EF-hand family plays a crucial role in the control of dendritic calcium signaling, particularly at the level of Purkinje cells. Alteration of Ca homeostasis, resulting from the calbindin deficiency in these cells of the cerebellar cortex, appears to contribute to the severe impairment in motor coordination of these mice (Airaksinen et al., 1997).

Serum Detection of Secretagogin after Ischemic Brain Damage

Various proteins expressed by cells of the CNS are released into the circulation due to disturbance of cellular integrity, cell death or impairment of blood brain barrier (Hardemark et al., 1989; Buttner et al., 1997; Horn et al. 1995; Hill et al., 2000). Consequently, we screened blood samples derived from patients suffering from stroke or TIA for elevated secretagogin levels with the newly developed sandwich capture ELISA. In nearly all of these patients, secretagogin was detectable in the serum. In contrast, in blood samples obtained from age-matched healthy blood donors, secretagogin concentration was under the detection limit of our ELISA. Secretagogin levels seem to be higher in patients suffering from TACS and POCS when compared with concentrations in sera derived from patients presenting with LACS and PACS. This finding suggests that the serum level of secretagogin after neuronal death depends on two facts: firstly, the number of affected secretagogin expressing cells and, secondly, it mirrors the location of neuronal damage as a minor cerebellar lesion might cause an inadequate high serum level. Secretagogin is detectable in sera originating from patients with TIA, except with different kinetics when compared with stroke. This shows that secretagogin might be a very sensitive neurobiochemical marker, even under conditions without neuroradiological correlation.

The release of secretagogin into the extracellular fluid is most probably a consequence of hypoxic neuronal damage and impairment of the blood–brain barrier (Lee et al., 1999). Recent findings indicate that necrotic as well as apoptotic mechanisms account for neuronal death following cerebral hypoxia (Tarkowski et al., 1999; Mattson et al., 2000). Whereas necrosis occurs within hours after the ischemic neuronal injury, apotosis leads to delayed neuronal cell death over a period of several days, which is following excitotoxic effects of neurotransmitter release in the marginal portion of the ischemic brain lesion (Nitatori et al., 1995; Chen et al., 1998; Lee et al., 1999). The finding of maximal serum levels on days 2 and 3 after infarction may be due to release of secretagogin in the course of the apoptotic cascade. During apoptosis, alteration of intracellular Ca-homeostasis plays a pivotal role and transcription induction of EF-hand Ca-binding proteins, i.e. calbindin D28k, has been described (Cheng et al., 1994; Choi, 1995; Mattson et al., 1995, 2000; Krebs, 1998; Kristian and Siesjo, 1998; Asai et al., 1999; Christakos et al., 2000). The data obtained by other investigators suggest that secretagogin transcription might also be induced in neurons in the course of apoptosis.

Comparison of Secretagogin and S-100B Releasing Pattern following Cerebral Ischemia

In this study, we compared serum concentrations of S-100B, a well-established neurobiochemical marker, and of secretagogin following cerebral ischemia. Our findings concerning the S-100B serum concentrations are in line with previously described results (Buttner et al., 1997; Missler et al., 1997; Rosen et al., 1998; Wunderlich et al., 1999). S-100B serum values are much higher than secretagogin levels. This finding may be due to two facts. Firstly, the extent of secretagogin expression in human brain tissue is lower than that of S-100B. Secondly, S-100B is an astroglial marker, whereas secretagogin exhibits a characteristic neuronal expression pattern. Consequently, the amount of secretagogin released into circulation depends on the region affected by the ischemic event. In contrast to secretagogin levels, which rapidly decline after 48–72 h, S-100B serum concentration remains elevated for several days. The delayed increase and prolonged elevation of S-100B in comparison to NSE, a neuron specific neurobiochemical marker, has already been described and may reflect later responses in the pathophysiological cascade and microglial reaction to the ischemic damage (Wunderlich et al., 1999).

Conclusion

We describe the specific cerebral expression pattern of secretagogin, a recently identified member of the EF-hand family of Ca-binding proteins. Our data encourage the extension of investigations of the role of Ca-binding proteins in physiological and pathological processes of neurons. We demonstrate that secretagogin is released into the serum after clinical manifestation of neurological deficit in cerebral ischemic disease. This contributes to the development of a new neurobiochemical serum marker indicative for neuronal death.

Notes

W.G. was a recipient of a research fellowship provided by the University of Vienna and the Facultas Verlagsund Buchhandels AG. We want to thank Professor Budka and Dr Hainfellner for support. We also want to thank Ernst Reinberger for photographic work.

Table 1

Demographic and clinical data of the patients included in the present study

 Cerebral infarctions TIA Cerebral hemorrhage 
No. of patients (%) 25 (78%)  5 (15%)  2 (6%) 
Age (mean ± SD) 69 ± 12 78 ± 10 51 ± 12 
Sex, male/female 15/10  2/3  1/1 
Lesion location, left/right 13/12  2/3  1/1 
Etiology,n (%) 
    Embolism  9 (36%)  2 (40%)  
    Small artery occlusion  5 (20%)   
    Large art. atherosclerosis  7 (28%)  2 (40%)  
    Undeterminable  4 (16%)  1 (20%)  
 Cerebral infarctions TIA Cerebral hemorrhage 
No. of patients (%) 25 (78%)  5 (15%)  2 (6%) 
Age (mean ± SD) 69 ± 12 78 ± 10 51 ± 12 
Sex, male/female 15/10  2/3  1/1 
Lesion location, left/right 13/12  2/3  1/1 
Etiology,n (%) 
    Embolism  9 (36%)  2 (40%)  
    Small artery occlusion  5 (20%)   
    Large art. atherosclerosis  7 (28%)  2 (40%)  
    Undeterminable  4 (16%)  1 (20%)  
Table 2

Mean peak secretagogin level in stroke patients grouped by clinical subtype

 Stroke subtype 
 TACS POCS PACS LACS TIA Hem. 
TACS, total anterior cerebral syndrome; POCS, posterior cerebral syndrome; PACS, partial anterior cerebral syndrome; LACS, lacunar syndrome; TIA, transient ischemic attack; Hem, hemorrhage; SE, standard error. 
No. of pat. 
Max. sec. level ± SE (pg/ml) 70.7 ± 22.9 46.4 ± 38.5 10.2 ± 7.1 10.4 ± 3.3 13.0 ± 3.5 5.0 ± 2.2 
 Stroke subtype 
 TACS POCS PACS LACS TIA Hem. 
TACS, total anterior cerebral syndrome; POCS, posterior cerebral syndrome; PACS, partial anterior cerebral syndrome; LACS, lacunar syndrome; TIA, transient ischemic attack; Hem, hemorrhage; SE, standard error. 
No. of pat. 
Max. sec. level ± SE (pg/ml) 70.7 ± 22.9 46.4 ± 38.5 10.2 ± 7.1 10.4 ± 3.3 13.0 ± 3.5 5.0 ± 2.2 
Figure 1.

Detection of secretagogin mRNA in human brain and pancreas. 2 μg of poly-A mRNA originating from pancreatic and whole brain tissue (A) and from different human brain regions as indicated (B) was hybridized with 32P-labeled secretagogin cDNA. The stripped blot was rehybridized with GAPDH cDNA for comparison of mRNA loading. Molecular size markers are presented at the left.

Figure 1.

Detection of secretagogin mRNA in human brain and pancreas. 2 μg of poly-A mRNA originating from pancreatic and whole brain tissue (A) and from different human brain regions as indicated (B) was hybridized with 32P-labeled secretagogin cDNA. The stripped blot was rehybridized with GAPDH cDNA for comparison of mRNA loading. Molecular size markers are presented at the left.

Figure 2.

Western-blot analysis of secretagogin expression in various brain regions. Brain tissue homogenates containing 15 μg of total protein were loaded onto a 12.5% PAGE gel under reducing conditions. Secretagogin was identified using polyclonal affinity purified rabbit anti-secretagogin antibody. Highest quantities of secretagogin were found in the pituitary gland and in the cerebellum. To a minor degree, secretagogin is also expressed in various other regions of the human brain. Recombinant secretagogin was included in each experiment as control.

Figure 2.

Western-blot analysis of secretagogin expression in various brain regions. Brain tissue homogenates containing 15 μg of total protein were loaded onto a 12.5% PAGE gel under reducing conditions. Secretagogin was identified using polyclonal affinity purified rabbit anti-secretagogin antibody. Highest quantities of secretagogin were found in the pituitary gland and in the cerebellum. To a minor degree, secretagogin is also expressed in various other regions of the human brain. Recombinant secretagogin was included in each experiment as control.

Figure 3.

Quantification of secretagogin in human brain tissue using a sandwich capture ELISA. To quantitate secretagogin expression in various brain regions, tissue homogenates were diluted in human control serum and secretagogin concentration was measured using the newly developed sandwich capture ELISA. The measured secretagogin content of the tested samples was normalized to 100 μg of total brain protein. Shown is a representative experiment out of two; data represent mean ± SD of two individual measurements.

Figure 3.

Quantification of secretagogin in human brain tissue using a sandwich capture ELISA. To quantitate secretagogin expression in various brain regions, tissue homogenates were diluted in human control serum and secretagogin concentration was measured using the newly developed sandwich capture ELISA. The measured secretagogin content of the tested samples was normalized to 100 μg of total brain protein. Shown is a representative experiment out of two; data represent mean ± SD of two individual measurements.

Figure 4.

Immunohistochemical localization of secretagogin. (A) Cryosections (4 μm) of cerebellar tissue were stained using affinity purified polyclonal rabbit anti-secretagogin antibody and the PAP method. Basket and stellate cells, both located at the molecular layer of the cerebellar cortex, exhibit strong immunoreactivity. The positively stained cell is surrounded by a dense network of faintly stained neurites. Original magnification, ×1100. (B) Cryosections (4 μm) of hypothalamic tissue were stained using affinity purified rabbit polyclonal anti-secretagogin Ab and the PAP method. The antibody labels a high number of hypothalamic neurons. Immunoreactive cells are characterized by a mainly cytoplasmic staining pattern. Original magnification, ×1100. (C) Cryosections of the cortical motoric region were immunostained using polyclonal rabbit anti-secretagogin antibody and the PAP method. Only singular cortical neurons exhibited substantial immunoreactivity. These neurons were characterized by one or two positively stained neurites. Original magnification, ×1100. (D) Frozen cerebellar tissue sections (4 μm) were stained with polyclonal affinity purified-anti secretagogin Ab and the PAP method. Whereas the density of immunoreactive cells within the molecular layer was very high (~90%), only a minority of cells of the granular layer exhibited positive staining. Original magnification, ×200. (E) Positively stained neurons within the stratum granulosum, characterized by lower staining intensity than neurons of the molecular layer. Original magnification, ×1100. (F) Neurons of the molecular layer, stained by polyclonal rabbit anti-secretagogin antibody and the PAP method, exhibited not only cytoplasmic, but also axonal staining. Original magnification, ×1100. (G) At the border between molecular and granular layer of the cerebellar cortex, a network of immunopositive neurites is detectable using polyclonal rabbit anti-secretagogin antibody and the PAP method (cryostat section). Original magnification, ×1100. (H) Cryostat sections of the pituitary gland were fixed in acetone and stained using mAb D24 and the APAAP method. Nearly all cells of the anterior part of the pituitary gland exhibited considerable immunostaining. Original magnification, ×200. Similar results were obtained by staining cryosections with affinity purified rabbit polyclonal antibody and using the PAP method (I,J). (I) Pituitary cells are characterized by a reticular cytoplasmatic staining pattern. Original magnification, ×800. (J) A high number of cells exhibited nuclear staining with varying intensity, often reaching a very high level of immunoreactivity. Original magnification, ×1100.

Figure 4.

Immunohistochemical localization of secretagogin. (A) Cryosections (4 μm) of cerebellar tissue were stained using affinity purified polyclonal rabbit anti-secretagogin antibody and the PAP method. Basket and stellate cells, both located at the molecular layer of the cerebellar cortex, exhibit strong immunoreactivity. The positively stained cell is surrounded by a dense network of faintly stained neurites. Original magnification, ×1100. (B) Cryosections (4 μm) of hypothalamic tissue were stained using affinity purified rabbit polyclonal anti-secretagogin Ab and the PAP method. The antibody labels a high number of hypothalamic neurons. Immunoreactive cells are characterized by a mainly cytoplasmic staining pattern. Original magnification, ×1100. (C) Cryosections of the cortical motoric region were immunostained using polyclonal rabbit anti-secretagogin antibody and the PAP method. Only singular cortical neurons exhibited substantial immunoreactivity. These neurons were characterized by one or two positively stained neurites. Original magnification, ×1100. (D) Frozen cerebellar tissue sections (4 μm) were stained with polyclonal affinity purified-anti secretagogin Ab and the PAP method. Whereas the density of immunoreactive cells within the molecular layer was very high (~90%), only a minority of cells of the granular layer exhibited positive staining. Original magnification, ×200. (E) Positively stained neurons within the stratum granulosum, characterized by lower staining intensity than neurons of the molecular layer. Original magnification, ×1100. (F) Neurons of the molecular layer, stained by polyclonal rabbit anti-secretagogin antibody and the PAP method, exhibited not only cytoplasmic, but also axonal staining. Original magnification, ×1100. (G) At the border between molecular and granular layer of the cerebellar cortex, a network of immunopositive neurites is detectable using polyclonal rabbit anti-secretagogin antibody and the PAP method (cryostat section). Original magnification, ×1100. (H) Cryostat sections of the pituitary gland were fixed in acetone and stained using mAb D24 and the APAAP method. Nearly all cells of the anterior part of the pituitary gland exhibited considerable immunostaining. Original magnification, ×200. Similar results were obtained by staining cryosections with affinity purified rabbit polyclonal antibody and using the PAP method (I,J). (I) Pituitary cells are characterized by a reticular cytoplasmatic staining pattern. Original magnification, ×800. (J) A high number of cells exhibited nuclear staining with varying intensity, often reaching a very high level of immunoreactivity. Original magnification, ×1100.

Figure 5.

Releasing pattern of secretagogin and S-100B. (A) Mean secretagogin concentration ± SE (pg/ml) in sera collected from patients after cerebral infarction or TIA over a period of 5 days. Peak levels are found on days 2 and 3, with decreasing concentrations on days 4 and 5 [mean secretagogin concentration: day 1 (32 patients), 5.7 pg/ml; day 2 (29 patients), 20.4 pg/ml; day 3 (31 patients), 19.2 pg/ml; day 4 (28 patients), 6.9 pg/ml; day 5 (21 patients), 6.4 pg/ml]. (B) Mean S-100B serum concentration ± SE (ng/ml) following an ischemic cerebral damage was higher by a magnitude of 102 than secretagogin values and increased until day 4 and 5 after admission to the hospital [mean S-100B concentration: day 1 (14 patients), 1.13 ng/ml; day 2 (13 patients), 1.61 ng/ml; day 3 (12 patients), 1.96 ng/ml); day 4 (12 patients), 2.47 ng/ml; day 5 (11 patients), 2.48 ng/ml]

Figure 5.

Releasing pattern of secretagogin and S-100B. (A) Mean secretagogin concentration ± SE (pg/ml) in sera collected from patients after cerebral infarction or TIA over a period of 5 days. Peak levels are found on days 2 and 3, with decreasing concentrations on days 4 and 5 [mean secretagogin concentration: day 1 (32 patients), 5.7 pg/ml; day 2 (29 patients), 20.4 pg/ml; day 3 (31 patients), 19.2 pg/ml; day 4 (28 patients), 6.9 pg/ml; day 5 (21 patients), 6.4 pg/ml]. (B) Mean S-100B serum concentration ± SE (ng/ml) following an ischemic cerebral damage was higher by a magnitude of 102 than secretagogin values and increased until day 4 and 5 after admission to the hospital [mean S-100B concentration: day 1 (14 patients), 1.13 ng/ml; day 2 (13 patients), 1.61 ng/ml; day 3 (12 patients), 1.96 ng/ml); day 4 (12 patients), 2.47 ng/ml; day 5 (11 patients), 2.48 ng/ml]

Figure 6.

Course of secretagogin and S-100B level in peripheral blood of individual patients suffering from severe and prolonged symptoms of neurological deficit. (A) This patient suffered from TACS and had severe neurological symptoms. He experienced a second episode of worsening neurological deficit followed by a second but lower secretagogin serum peak. S-100B level peaked on day 3 and remained high for several days. Finally, after >4 weeks of neurological rehabilitation, the patient was discharged with undetectable secretagogin values and an S-100B concentration of 0.30 ng/ml in peripheral blood. (B) This patient suffered from TACS and presented worsening clinical symptoms at day 4 of hospitalization. The secretagogin level was already elevated in the first blood sample and the peak concentration was measured at day 2 of hospitalization. The second episode of cerebral ischemia was followed by an increase of secretagogin serum concentration. S-100B serum levels were high during the whole observation period, reaching a peak level 72 h after admission. (C) In this patient suffering from TACS, S-100B and secretagogin serum concentrations exhibit a similar releasing pattern, reaching the peak level 96 h after admission.

Figure 6.

Course of secretagogin and S-100B level in peripheral blood of individual patients suffering from severe and prolonged symptoms of neurological deficit. (A) This patient suffered from TACS and had severe neurological symptoms. He experienced a second episode of worsening neurological deficit followed by a second but lower secretagogin serum peak. S-100B level peaked on day 3 and remained high for several days. Finally, after >4 weeks of neurological rehabilitation, the patient was discharged with undetectable secretagogin values and an S-100B concentration of 0.30 ng/ml in peripheral blood. (B) This patient suffered from TACS and presented worsening clinical symptoms at day 4 of hospitalization. The secretagogin level was already elevated in the first blood sample and the peak concentration was measured at day 2 of hospitalization. The second episode of cerebral ischemia was followed by an increase of secretagogin serum concentration. S-100B serum levels were high during the whole observation period, reaching a peak level 72 h after admission. (C) In this patient suffering from TACS, S-100B and secretagogin serum concentrations exhibit a similar releasing pattern, reaching the peak level 96 h after admission.

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