Purkinje neurons express the SERCA3 isoform of the organellar type Ca2+-transport ATPase

doi:10.1016/0169-328X(96)00088-5

Molecular Brain Research

Volume 41, Issues 1–2, 5 September 1996, Pages 169-174

https://doi.org/10.1016/0169-328X(96)00088-5 Get rights and content

Abstract

We report the distribution of the sarco(endo)plasmic reticulum Ca²⁺ ATPase 3 (SERCA3) isoform in the rat brain. Compared to SERCA2 isoform, which is found in all brain regions, SERCA3 is specifically expressed in the Purkinje neurons. This conclusion is based on immunochemical observations using SERCA3- and SERCA2b-specific antibodies, in-situ hybridization using SERCA3-specific oligonucleotide probes and single-cell reverse transcription-polymerase chain reaction (RT-PCR). Immunocytochemistry clearly revealed the expression of SERCA3 in the cell body and in the dentritic processes of the Purkinje neurons. Single-cell ratio RT-PCR showed that Purkinje neurons expressed 3-fold lower levels of SERCA3 mRNA compared to SERCA2 mRNA. SERCA3 expression is very low or absent in the rat cerebrum and brainstem. It is known that the SERCA3 Ca²⁺ pump has an approximately 5-fold lower affinity for Ca²⁺ when expressed in COS cells as compared to other SERCA members [15]. If this property is also valid in a neuronal context, the expression of the SERCA3 Ca²⁺-pump isoform could have important functional implications for the regulation of the cytosolic Ca²⁺ concentration in Purkinje neurons.

Introduction

Precisely controlled variations in the cytosolic Ca²⁺ concentration play a key role in neuronal cell function. Both presynaptic transmitter release and postsynaptic effects such as modulation of responses to receptor activation and gene expression are regulated by the level of free intracellular Ca²⁺ [1],[12],[16]. One of the major components of the complex Ca²⁺ buffering system in neurons is the intracellular Ca²⁺ store constituted by the endoplasmic reticulum and related compartments 2, 11. The membranes of these stores contain Ca²⁺-transport ATPases and Ca²⁺ channels that mediate, respectively, the uptake of Ca²⁺ into the store and its release. The Ca²⁺-transport ATPases belong to the SERCA (Sarco(Endo)plasmic Reticulum Calcium ATPase) gene family. The SERCA family consists of three genes: SERCA1, SERCA2, and SERCA3 4, 5. The SERCA2 gene is ubiquitously expressed, whereas the expression of SERCA1 has been detected only in fast skeletal muscle. The SERCA2 gene gives rise to two protein isoforms by alternative splicing of the primary transcript: SERCA2a and SERCA2b 10, 14. The SERCA2a isoform is present in cardiac muscle, slow skeletal muscle and to some extent also in smooth muscle [9]. SERCA2b is the ubiquitously expressed house-keeping isoform. It differs from SERCA2a in that the last four amino acids of the latter are replaced by a stretch of 49 amino acids. SERCA2b presents a 2-fold higher affinity for Ca²⁺ but a lower catalytic turnover rate than SERCA2a 18, 26. SERCA3 is thought to be expressed only in cells of the hemopoietic lineage and in some endothelial cells 3, 26. When expressed in COS cells, SERCA3 shows an approximately 5-fold lower Ca²⁺ affinity compared to the SERCA1 and SERCA2 pumps [22].

Up until now, only the SERCA2 isoform has been demonstrated in the brain. SERCA2 mRNA is expressed in all regions of the brain, as determined by in-situ hybridization [17]. At the protein level, unusually high levels of SERCA2b [19]have been detected in the Purkinje neurons of the cerebellum. These neurons are also particularly rich in other proteins involved in Ca²⁺ homeostasis, such as inositol 1,4,5-trisphosphate (IP₃) receptors [20], calbindin and parvalbumin [6]. We now report that SERCA3 is expressed along with SERCA2b in cerebellar Purkinje neurons. The results were corroborated by using immunocytochemistry, Western blotting, in-situ hybridization and single-cell RT-PCR. The expression of SERCA3 may have important functional consequences because of the much lower affinity of this Ca²⁺-pump isoform as compared to the housekeeping SERCA2b.

Section snippets

Animal and tissue processing

Adult rats were anaesthetized with Ketalar and killed by an overdose of Nembutal. The brains were perfused in situ via the aorta with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 100 mM sodium phosphate buffer (PB) (pH 7.4). The cerebrum and cerebellum were removed from the skull, post-fixed for 4 h at 4°C and rinsed overnight in PB. Sections (50 μm thick) were cut using a vibratome and collected in PB. Alternatively the cerebellum was soaked overnight in 30% sucrose in

Western blot analysis

On Western blots, the SERCA3-specific antiserum N89 labeled a single band at the expected position for the SERCA3 protein in the membranes prepared from the cerebellum. No reaction was seen in the membrane fractions obtained from the brainstem or the cerebrum (Fig. 1A). In contrast, the SERCA2b antiserum stained a single band in the cerebellum, in the cerebrum and in the brainstem (Fig. 1B).

Immunocytochemistry

In accordance with the results obtained by Western blot analysis, immunocytochemical analysis showed that

Discussion

Purkinje cells constitute the sole neuronal output of the cerebellum. For this reason and because of their unique electrophysiological and biochemical properties, they have been a focus of interest. Purkinje neurons express a great variety of proteins involved in the regulation of cytosolic Ca²⁺, often at high levels. The Ca²⁺-binding proteins calbindin D28k and parvalbumin [6], the inositol trisphosphate receptors (IP₃) and ryanodine receptors [23], the plasma membrane Ca²⁺-transport ATPase

Acknowledgements

We thank Prof. A. Konnerth, Dr. T. Plant, Dr. C. Schirra for help with the single-cell PCR. We thank Dr. L. Arkens of the Laboratory of Neuroendocrinology and Immunological Biotechnology, Katholieke Universiteit Leuven, Dr. F. Viana, Dr. B. Himpens and Dr. P. Stalmans (Laboratorium voor Fysiologie) for advice. This work was supported by a grant from the IUAP, Belgium.

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The identification of mRNAs that have restricted expression patterns in the brain represents powerful tools with which to characterize and manipulate the nervous system. Here, we describe a strategy using microarray technology (Affymetrix Mouse Genome 430 2.0 Arrays) to identify mRNA transcripts that are candidate markers of cerebellar Purkinje neurons. Initially, gene expression profiles were compared between cerebella of 4-month-old Purkinje cell degeneration (pcd^3J) mice, in which most Purkinje cells had already degenerated and wild-type littermates with a normal complement of Purkinje neurons. Of 14,563 probe sets expressed in wild-type cerebellum, 797 showed a significant (p<0.0001) reduction in pcd^3J mice. These probes could represent transcripts with varying levels of specificity for Purkinje cells as well as transcripts in other cell types that decline as a secondary consequence of Purkinje cell loss. Ranking of the probe signals revealed that well-known Purkinje cell-specific transcripts such as calbindin and L7/pcp2 clustered in a group that was <33% of wild-type levels. Therefore, to identify potentially new Purkinje cell-specific transcripts that cluster with the known markers, more stringent selection criteria were applied (<33% of wild-type signal and p<0.0001). With these criteria, 55 independent transcripts were identified of which 33 were annotated genes and 22 were ESTs and RIKEN cDNAs. A literature search revealed that 25 of the 33 annotated genes were expressed in Purkinje cells, with no data being available on the other 8. Thus, the additional 8 annotated and 22 un-annotated genes are clustered with many genes expressed in Purkinje cells making them candidate markers. To confirm the microarray data, eight representative annotated genes were selected including five reported to be in Purkinje neurons and three for which no data was available. Semi-quantitative RT-PCR demonstrated reduced expression of all eight transcripts in cerebella from pcd^3J mice. The promoters of genes expressed selectively in subsets of neurons can be used to direct heterologous gene expression in transgenic mice and the more restricted the expression pattern the greater their utility. Therefore, microarray analysis was used to assess expression levels of all 55 transcripts in cerebral cortex, striatum, substantia nigra and ventral tegmental area. This permitted the identification of a set of genes whose promoters might have utility for selectively targeting gene expression to cerebellar Purkinje cells.
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Research reportPurkinje neurons express the SERCA3 isoform of the organellar type Ca2+-transport ATPase

Abstract

Introduction

Section snippets

Animal and tissue processing

Western blot analysis

Immunocytochemistry

Discussion

Acknowledgements

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Research report
Purkinje neurons express the SERCA3 isoform of the organellar type Ca²⁺-transport ATPase