Distribution of Secretory Pathway Ca²⁺ ATPase (SPCA1) in Neuronal and Glial Cell Cultures

Abstract

1. Secretory pathway Ca²⁺ ATPase type 1 (SPCA1) is a newly recognized Ca²⁺/Mn²⁺-transporting pump localized in membranes of the Golgi apparatus.

2. The expression level of SPCA1 in brain tissue is relatively high in comparison with other tissues.

3. With the aim to determine the expression of SPCA1 within the different types of neural cells, we investigated the distribution of SPCA1 in neuronal, astroglial, oligodendroglial, ependymal, and microglial cell cultures derived from rat brains.

4. Western Blot analysis with rabbit anti-SPCA1 antibodies revealed the presence of SPCA1 in homogenates derived from neuronal, astroglial, ependymal, and oligodendroglial, but not from microglial cells.

5. Cell cultures that gave rise to positive signal in the immunoblot analysis were also examined immunocytochemically.

6. Immunocytochemical double-labeling experiments with anti-SPCA1 serum in combination with antibodies against cell-type specific proteins showed a localization of the SPCA1signal within cells stained positively also for GFAP, α-tubulin or MBP.

7. These results definitely established the expression of SPCA1 in astroglial, ependymal, and oligodendroglial cells.

8. In addition, the evaluation of neuronal cultures for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal in cells identified as neurons.

INTRODUCTION

Besides the endoplasmic reticulum, all other intracellular organelles can serve as mobilizable calcium stores (for review, see Michelangeli et al., 2005). Among them, the Golgi apparatus has a relatively high capacity for sequestering (Chandra et al., 1991) and storing of Ca²⁺ (Chandra et al., 1994; Pezzati et al., 1997) and furthermore is capable of responding to inositol-1,4,5-triphosphate stimulation by Ca²⁺ release (Pinton et al., 1998; Surroca and Wolff, 2000). This capability of the Golgi allows it to contribute to the complexity of spatial and temporal cytosolic Ca²⁺ signaling. The presence of Ca²⁺ in the Golgi lumen is also fundamental for maintaining other essential functions of the Golgi apparatus, including luminal and membrane protein trafficking (Carnell and Moore, 1994; Canaff et al., 1996). Recent studies have shown that calcium is transported into the lumen of the Golgi by two subfamilies of P-type Ca²⁺-ATPases. Besides the thapsigargin (Tg)-sensitive sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPases (SERCA), a newly recognized group of Tg-insensitive Ca²⁺ and Mn²⁺ pumps—the secretory pathway Ca²⁺-ATPases (SPCA)—is involved in the translocation of cytoplasmic Ca²⁺ into the Golgi apparatus (for a review, see Wuytack et al., 2002). Two isoforms sharing 64% of sequence identity, namely SPCA1 (Van Baelen et al., 2003) and SPCA2 (Vanoevelen et al., 2005), are expressed in mammalian cells (Wootton et al., 2004; Xiang et al., 2005).

SPCA1 has been shown to participate in various Ca²⁺ signaling events, for instance by modulating Ca²⁺ spiking in response to histamine in HeLa cells or by facilitating baseline oscillations in response to ATP in COS-1 cells (Van Baelen et al., 2004). In human beings, haploinsufficiency of hSPCA1, caused by mutations in one allele of the ATP2C1 gene, leads to Hailey-Hailey disease, a blistering disease of the skin characterized by disruption of desmosomal contacts in keratinocytes (Hu et al., 2000; Sudbrak et al., 2000).

Among the different tissues of the rat, SPCA1 mRNA and protein are predominantly present in brain, testis, and epididymal fat pads (Wootton et al., 2004). The higher expression levels of SPCA1 in these rat tissues coincide with a relatively high ratio of SPCA activity to the total activity of Ca²⁺-dependent ATPases, implying a significant role of SPCA-facilitated transport of Ca²⁺ for calcium storage within these tissues (Wootton et al., 2004).

The high ratio of Tg-insensitive to total Ca²⁺-ATPase activity together with the high expression level of SPCA1 in brain evokes the question about the neural cell type-specific localization of SPCA1. Therefore, cell cultures derived from rat brains and highly enriched with neurons, astrocytes, oligodendrocytes, ependymocytes, and microglia were probed for the expression of SPCA1 by Western blotting and immunocytochemistry. The results indicate, that SPCA1 is expressed in neuronal, astroglial, oligodendroglial, and ependymal cells in vitro.

MATERIALS AND METHODS

Materials

Dulbecco's modified Eagle's medium (DMEM), horse serum, Minimal Essential Medium (MEM), and transferrin were from Gibco-Invitrogen Corporation (Karlsruhe, Germany). Fetal calf serum (FCS) was from Biochrom AG (Berlin, Germany). Aqueous-based mounting medium “Immu-mount” was from Thermo Shandon (Pittsburgh, USA). Penicillin G and streptomycin sulfate were from Serva (Heidelberg, Germany). Protein molecular weight markers solution “Full Range Rainbow” was from Amersham Life Science (Braunschweig, Germany). Skimmed milk powder was from Töpfer GmbH (Dietmansried, Germany). Sodium selenite and sodium dodecyl sulfate (SDS) were from Fluka (Deisenhofen, Germany). Human thrombin was a gift from Aventis Behring (Marburg, Germany). All other chemicals were obtained at analytical grade from E. Merck (Darmstadt, Germany). Sterile plastic material, culture dishes, and flasks were from Nunc (Wiesbaden, Germany) and Greiner (Frickenhausen, Germany).

Antibodies

The preparation and characterization of a rabbit anti-serum against the large cytosolic loop between transmembrane segments 4 and 5 of SPCA1 has been described elsewhere (Van Baelen et al., 2003). The mouse monoclonal anti-glial fibrillary acidic protein (GFAP) and anti-α-tubulin antibodies were purchased from Sigma-Aldrich (Deisenhofen, Germany). Alexa-Fluor-conjugated antigoat, antirabbit, and antimouse IgG antibodies were from Molecular Probes Europe (Leiden, The Netherlands). Goat polyclonal antiserum against myelin basic protein (MBP) and anti-rabbit IgG conjugated with alkaline phosphatase were from Santa Cruz Biotechnology (Heidelberg, Germany).

Cell Cultures

Neuron-rich primary cultures were prepared from the brains of rats at embryonic day and maintained as described (Löffler et al., 1986). Astroglia-rich or ependymal-rich primary cultures were prepared from brains of neonatal rats and maintained according to methods described by Hamprecht and Löffler (1985) and Prothmann et al., (2001), respectively. Oligodendroglia-rich secondary cultures derived from astroglia-rich primary cultures were prepared as described elsewhere (Hirrlinger et al., 2002). A modification (Hirrlinger et al., 2000) of the method described by Giulian and Baker (1986) was used to prepare microglia-rich secondary cultures from astroglia-rich primary cultures.

Immunocytochemistry

For immunocytochemistry, cells were grown on square (18 mm×18 mm) coverslips that had been attached to the surface of a culture dish with sterile silicon grease. After removal of culture media, the cells on coverslips were fixed by immersion in 3.5% (w/v) paraformaldehyde/phosphate-buffered saline (PBS) at room temperature for 10 min. Fixed cells were washed twice in PBS for 5 min and once in PBS containing 0.1% (w/v) glycine for 10 min. To enhance antibody penetration, cells were permeabilized in PBS containing 0.3% (w/v) Triton X-100 for 10 min. To minimize unspecific signals, the cells were incubated with PBS/0.1% (w/v) Triton X-100/10 mg/mL BSA solution at room temperature for 30 min. For immunofluorescence double labeling experiments, the coverslips were covered with a mixture of two appropriately diluted primary antibodies in PBS/0.1% Triton X-100/1 mg/mL BSA (anti-SPCA, 1:1000; anti-GFAP, 1:400; anti-tubulin, 1:500) and incubated (4°C, overnight) in a humidified chamber. After washing three times with PBS/0.1% (w/v) Triton X-100, the cells were incubated with a mixture of diluted (1:1000) Alexa Fluor-conjugated secondary antibodies in a dark humidified chamber at room temperature for 2 h. Subsequently, the cells were washed three times in PBS/0.1% (w/v) Triton X-100 at room temperature for 15 min.

The protocol for double-labeling experiments with anti-MBP serum (diluted 1:100) in combination with anti-SPCA serum was similar but modified as follows: Primary as well as secondary antibodies were not mixed but they were applied separately in four successive cycles in the order (i) goat anti-MBP serum; (ii) chicken anti-goat IgG; (iii) rabbit anti-SPCA serum; (iv) goat anti-rabbit IgG.

Thereafter, coverslips were mounted cells down on slides in “Immu-mount” mounting medium. The preparations were viewed by glycerol immersion optics (Zeiss fluorescence microscope IM35 with a Plan-Neofluar 253 objective).

Immunoblot Analysis

The proteins of cell-culture homogenates were separated by SDS–PAGE in a 7% gel. Proteins were transferred onto a nitrocellulose membrane by electroblotting (100 V, 1 h), and unspecific binding sites on the blot were saturated with 5% (w/v) skimmed milk powder solution in PBS/0.05% (w/v) Tween 20 at room temperature for 1 h. After three washing steps with PBS/0.05% (w/v) Tween 20, membranes were incubated with SPCA antiserum (diluted 1:500 in washing buffer) at room temperature for 2 h, washed again in PBS/0.05% (w/v) Tween 20 and subsequently incubated with goat anti-rabbit IgG-alkaline phosphatase conjugate [diluted 1:10000 in PBS/0.05% (w/v) Tween 20] at room temperature for 1 h. The chromogenic signal was developed by immersing the membrane in 0.2 M Tris/HCl pH 9.5, 10 mM MgCl₂, 0.39 mM 4-nitroblue tetrazolium chloride and 0.37 mM 5-bromo-4-chloro-3-indolylphosphate. The reaction was stopped by rinsing the membrane with water.

RESULTS

Wootton et al. (2004) have already demonstrated the presence of SPCA1 in rat brain by the method of immunoblotting. Therefore, in the present study we used homogenate derived from rat brain as a positive control for the detection of SPCA1 by immunoblot in homogenates of cultured neuronal, astroglial, ependymal, oligodendroglial, and microglial cells. The protein band corresponding to SPCA1 (approximate molecular mass of 100 kDa) appeared in the lanes loaded with samples from neuronal, astroglial, ependymal, and oligodendroglial cultures (Fig. 1), but not in the lanes loaded with either 10 or 30 μg of the microglial protein sample (Fig. 1, lane M1 and M2, respectively).

Fig. 1.

Western blotting analysis of rat brain homogenate (B) and homogenates from cultured astroglial (A), ependymal (E), oligodendroglial (O), microglial (M1, M2), and neuronal (N) cells for the expression of SPCA1. The proteins from respective homogenates were separated by SDS-PAGE, electroblotted onto a nitrocellulose membrane and Western Blot analysis with rabbit anti-SPCA1 serum was performed as described in Materials and Methods. The amount of applied protein in each lane was 10 μg, except for lane M2, where 30 μg of protein was used.

The possible presence of other contaminating SPCA1-containing cells in the cell cultures analyzed limits the significance of the results of the Western Blot analyses. Therefore, in order to firmly assign the presence of SPCA1 to particular neural cell type(s), the cell cultures which gave rise to a positive immunoblotting signal were subjected to further immunocytochemical analysis. Thus, in double labeling experiments anti-SPCA1 antibodies were used in combination with antibodies against marker protein specific for astroglial, ependymal, or oligodendroglial cells.

The cells of astroglia-rich primary culture were fixed and subsequently treated with a mixture of rabbit anti-SPCA1 antiserum (Fig. 2(A)) and mouse monoclonal antibodies against GFAP (Fig. 2(B)). The SPCA1-specific immunofluorescence, was present in the cytosol of the cells positively stained for GFAP, with predominantly juxtanuclear localization and punctuated and/or rod-like appearance.

Fig. 2.

Photomicrograph of immunocytochemical double labeling of SPCA1 and GFAP in astroglial culture. The fixed cells were treated with a mixture of rabbit serum against SPCA1 (A) and mouse monoclonal antibodies against GFAP (B) and subsequently with a mixture of Alexa Fluor 486-conjugated anti-rabbit IgG and Alexa Fluor 566-conjugated anti-mouse IgG. The phase contrast view corresponding to the region depicted in A and B is shown in (C). The negative control (D) was performed by omitting the SPCA1 antiserum. The scale bar on C represents 50 μm.

Immunocytochemical examination of ependyma-rich primary cultures revealed that the SPCA1 immunofluorescence signal was observable in the cytosol of the majority of cells (Fig. 3(A)). Since no ependymocyte-specific immunological markers have been identified to date, the presence of cilia was used as identifier for ependymal cells. Although hard to discern in phase contrast view (Fig. 3(C)), the cilia can be clearly distinguished after staining with antibodies directed against their major constituent, α-tubulin (Prothmann et al., 2001). Stained cilia appeared as fine hair-like structures on the surface of the cells (Fig. 3(B)), which also displayed a strong immunofluorescence signal due to SPCA1.

Fig. 3.

Immunocytochemical double-staining for SPCA1 (A) and ciliated cells/α-tubulin (B) in ependyma-rich primary culture. The fixed cells were first treated with a mixture of rabbit SPCA1-antiserum and mouse anti-α-tubulin monoclonal antibodies, and subsequently with a mixture of Alexa Fluor 486-conjugated anti-rabbit IgG and Alexa Fluor 566-conjugated anti-mouse IgG. The phase contrast view corresponding to A and B is shown in C. A negative control (D) was performed by omitting anti-SPCA serum. The scale bar represents 50 μm.

Cells in oligodendroglia-rich secondary culture were also positively stained with SPCA antisera (Fig. 4(A)). The SPCA1 signal localized to small rounded somata of cells that exhibited a surrounding halo when viewed by phase-contrast (Fig. 4(C)). These cells also contained branched processes stained positively with antibodies against MBP (Fig. 4(B)). Thus, they were identified as oligodendrocytes.

Fig. 4.

Double immunocytochemical labeling of SPCA1 and MBP in oligodendroglial culture. The fixed cells were treated with the rabbit serum against SPCA1 (A) followed by goat anti-rabbit IgG Alexa Fluor 566 conjugate and with goat serum against MBP (B) and subsequently with Alexa Fluor 486-conjugated chicken anti-goat IgG as described in Materials and Methods. The phase contrast view (C) depicts the same area as A and B. The negative control (D) was performed by omitting the SPCA1-antiserum. The scale bar on C represents 50 μm.

In addition, the evaluation of neuronal culture for the presence of SPCA1 revealed an SPCA1-specific immunofluorescence signal (Fig. 5(A)) in cells with small rounded somata and thin long processes, which formed characteristic cell-clusters known as “small brains” (Fig. 5(B)). Based on this morphology these cells have to be considered as neurons.

Fig. 5.

Photomicrograph of immunocytochemical labeling of a neuronal primary culture for SPCA1 (A). The cells were fixed and treated with rabbit serum against SPCA1 (A), followed by affinity-purified goat serum against rabbit IgG conjugated with Alexa Fluor 486. The phase contrast view (B) corresponds to the view in A. The scale bar on C represents 50 μm.

The specificity of the immunocytochemical staining of astroglial, ependymal, and oligodendroglial cells with anti-SPCA1 antiserum was evaluated in control experiments, in which antiSPCA antiserum was omitted. The omission of antiserum completely prevented the specific signal observed in its presence and only the background signal was visible in astroglial, ependymal, and oligodendroglial cultures, as shown in Figs. 2(D), 3(D), and 4(D), respectively.

DISCUSSION

In this study, we have confirmed the earlier observation of Wootton et al., (2004) that SPCA1 is present in rat brain. In addition, we have definitely established the expression of SPCA1 in cultured neuronal, astroglial, ependymal, and oligodendroglial cells. Furthermore, we have not detected the SPCA1-specific immunoblotting signal in homogenate protein derived from microglial cells, indicating that either this cell type does not express SPCA1 or the amount of the SPCA1 protein is below the detection capability of the immunoblotting method applied.

SPCA1 is a member of a recently recognized subfamily of SPCA pumps (Wuytack et al., 2002) localized in the membranes of the Golgi apparatus (Ton et al., 2002). It is well known that the Golgi lumen is rich in Ca²⁺ (Chandra et al., 1994; Pezzati et al., 1997), an ion with important functions in the regulation of protein sorting, glycosylation, and secretion. The Golgi is involved in the regulation of cytosolic calcium signaling by sequestering or releasing Ca²⁺ ions at appropriate times (Michelangeli et al., 2005). Because some neural functions depend on the release of specific compounds into the extracellular milieu and on the delivery of required receptors and glycoproteins to the plasma membrane, the expression of SPCA1 may be essential for neurons and macroglial cells in maintaining their physiological properties.

Furthermore, neurons are specialized cells for reception, conduction and transmission of signals. The transmission of signal is facilitated by synapses, those formation in the CNS is a highly complex process that needs to be orchestrated with high temporal and spatial precision. Synapse formation is accompanied by accumulation of synaptic organelles and proteins at “contact” sites between axons and dendrites. These synaptic precursors are derived from the Golgi apparatus (for review see Sytnyk et al., 2004). Besides the neurons, astroglial cells have been shown to release different regulatory molecules, neurotrophic factors, and neuropeptides by calcium-triggered exocytosis. Gliotransmitters (Calegari et al., 1999) released by astrocytes are involved in the stimulation and/or modulation of neuronal synaptic transmission (Evanko et al., 2004) and intercellular communication (Volterra and Meldolesi, 2005). All these functions of neuronal cells are also dependent of the functional Golgi apparatus, and therefore this may be in the agreement with proposed importance of the SPCA1 in the signal transmission maintaining process.

One of the SPCA1 functions is also the modulation of calcium signaling (Van Baelen et al., 2004). While Ca²⁺ signaling has a profound impact on cellular functions, especially in neurons, concise data about the relevance of SPCA1 for and its influence on calcium signaling in neuronal and glial cells are still missing. The differences among the neural cells in their physiological functions and behavior may also determine the function and composition of their respective cellular secretory machineries and their expression of particular SPCA isoforms. Taken together, further studies on the distribution and function of SPCA1 in neurons and glial cells are required. Such experiments will have to be performed not only with cell cultures, but also under in vivo conditions.

In addition to the expression of SPCA1, also SPCA2 is present in mammalian cells. Its level of expression in brain tissue remains controversial in literature (Vanoevelen et al., 2005; Xiang et al., 2005). Nevertheless, SPCA2 is expressed in brain to some extent, and it might be present in microglial cells to compensate the lack of SPCA1 in this cell type.

CONCLUSION

The results presented here suggest that in spite of morphological and functional differences between neurons and the different types of macroglial cells, they all express the SPCA1 protein. Immunocytochemical experiments confirmed the localization of SPCA1 to the juxtanuclear region of the cytoplasm.