Research ArticleNeuroscience

STIM2 Regulates Capacitive Ca2+ Entry in Neurons and Plays a Key Role in Hypoxic Neuronal Cell Death

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Science Signaling  20 Oct 2009:
Vol. 2, Issue 93, pp. ra67
DOI: 10.1126/scisignal.2000522
  • Fig. 1

    STIM2 is the main STIM isoform in neurons. (A) Western blot analysis of STIM1 and STIM2 abundance in various organs of 2-month-old mice; α-tubulin expression was used as loading control. LN, lymph node; Plt, platelet. (B) Immunofluorescence staining of STIM1 (top) and STIM2 (middle) in cultured hippocampal neurons [major microtubule-associated protein 2a/b (MAP2a/b), green] and CD4+ T cells from wild-type mice. Immunofluorescence staining of STIM2 in cultured hippocampal neurons (MAP2a/b, green) from Stim2−/− (bottom) mice. Cell nuclei are counterstained with DAPI (blue). Scale bars, 10 μm. (C) RT-PCR of neuronal and heart complementary DNA with Stim and Orai primers. (D) Targeting strategy for the generation of Stim2−/− mice (see Materials and Methods for details); Neo-LacZ, neomycin resistance and LacZ cassettes. (E) Southern blot analysis of Bam HI–digested genomic DNA of wild-type (+/+), heterozygous (+/), or Stim2 knockout (−/−) mice labeled with the external probe. (F) Western blot analysis of STIM2 and STIM1 expression in brain and lymph node (LN) cells of adult wild-type and Stim2−/− mice.

  • Fig. 2

    Stim2−/− and wild-type (WT) littermates in the Morris Water Maze task. (A) Total duration and distance mice swam before finding the hidden platform; the arrows indicate the start of the reversal trials, where the platform was moved to a different position. Values are shown as mean ± SEM; F1,12 = 19.73, P < 0.001 [comparison of Stim2−/− and wild-type mice in the acquisition phase (trials 1 to 12)], analysis of variance (ANOVA) for repeated measures. (B) Analysis of Stim2−/− mice and wild-type littermates in the elevated plus maze to assess anxiety-like behavior. Values are shown as mean ± SEM, Student’s t test. not significant. The top and middle panels represent the percent of total time mice spent in either the open or the closed arm of the maze, respectively. The last panel displays the number of total visits to the closed arms.

  • Fig. 3

    STIM2 regulates Ca2+ homeostasis in cortical neurons. (A) Neuronal cultures [5 to 9 days in vitro (DIV 5 to 9)] were loaded with fura-2 and averaged [Ca2+]i responses in Stim2−/− neurons were compared to those in wild-type (WT) cells, in Stim1−/− compared to Stim1+/+ cells, and in Orai1−/− compared to Orai1+/+ cells (n = 5 to 7 experiments per group, each representing the average signal from 20 to 35 cells). Cells were stimulated with CPA (20 μM) followed by replacement of 1 mM EGTA with 2 mM Ca2+. Basal and peak [Ca2+]i were determined during the time intervals indicated. Store-operated [Ca2+]i increases (Δ[Ca2+]i) were calculated by subtracting basal [Ca2+]i from peak [Ca2+]i. (B) Ca2+ release from intracellular stores was elicited by the addition of 5 μM ionomycin in the presence of EGTA. This Ca2+ release was normalized to the maximum response observed after replacement of 1 mM EGTA with 2 mM Ca2+. (C) Effect of combined OGD on [Ca2+]i. Cultured neurons (DIV 12 to 14) were exposed for 1 hour (WT) or 2 hours (Stim2−/−) to a glucose-free bath solution continuously bubbled with N2. The slight [Ca2+]i increase after about 70 min in the Stim2−/− neurons occurred only in this particular experiment. (D) Chemical anoxia was induced in Stim2−/− or WT cells by CCCP (2 μM, 30 min). Recovery from increased [Ca2+]i was determined after washout of CCCP for 60 min. All bars represent means of five to seven experiments. Error bars indicate SEM. *P < 0.05, two-tailed Mann-Whitney U test.

  • Fig. 4

    Lack of STIM2 is neuroprotective under ischemic conditions in vitro and ex vivo. (A) Representative images of apoptotic (caspase-3 positive, Casp3, red) cultured hippocampal neurons (MAP2a/b, green) from wild-type and Stim2−/− mice under control (0 hours, O2) conditions and after in vitro ischemia (6 hours, N2). Scale bar represents 50 μm. (B) Bar graph representation of dead neurons (%) under the different experimental conditions. (C) Bar graph representation of dead WT and Stim2−/− neurons (%) under the different experimental conditions. Staurosporine (Stauro.; 300 nM) was used to induce an alternative Ca2+-dependent cell death pathway. (D) TPEN (2 μM) was added to neuronal cell cultures for 10 hours to induce mainly Ca2+-independent cell death. (E) Representative images of caspase-3 (Casp3, red)–positive hippocampal neurons (neuron-specific antigen-positive cells, NeuN, green) in brain slices under ischemic and control conditions. DAPI counterstaining (DAPI, blue). White arrows indicate Casp3-positive neurons. Scale bars represent 10 (left) or 100 μm (right). (F) Bar graph representation of neuronal cell death (dead neurons per square millimeter) under control (6 hours, O2 black columns) and ischemic conditions (6 hours, N2, gray columns) in Stim2−/− mice and control littermates. The results are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, modified Student’s t test.

  • Fig. 5

    Stim2−/− mice are protected from neuronal damage after cerebral ischemia. (A to D) Wild-type and Stim2−/− mice were subjected to tMCAO and analyzed after 24 hours. Experiments were also performed with wild-type mice transplanted with Stim2−/− bone marrow (Stim2+/+BM−/−) and Stim2−/− mice transplanted with wild-type bone marrow (Stim2−/−BM+/+). (A) Representative TTC stains of three corresponding coronal brain sections of the groups. Infarcted areas are indicated by arrows. (B) Brain infarct volumes as measured by planimetry at day 1 after tMCAO (n = 8 to 10 per group). (C) Neurological Bederson score and (D) grip test as assessed at day 1 after tMCAO. Graphs plot mean ± SD (n = 8 to 10 mice per group). (B to D) *P < 0.05, **P < 0.01, Bonferroni one-way ANOVA tested against wild-type mice. (E) Hematoxylin and eosin–stained sections in the ischemic hemispheres of wild-type (WT) and Stim2−/− mice. Infarcts (white area) are restricted to the basal ganglia in Stim2−/− mice, but consistently include the neocortex in the wild-type mice (10 of 10). Scale bars, 300 μm.

Additional Files

  • Supplementary Materials for:

    STIM2 Regulates Capacitive Ca2+ Entry in Neurons and Plays a Key Role in Hypoxic Neuronal Cell Death

    Alejandro Berna-Erro, Attila Braun, Robert Kraft, Christoph Kleinschnitz, Michael K. Schuhmann, David Stegner, Thomas Wultsch, Jens Eilers, Sven G. Meuth, Guido Stoll, Bernhard Nieswandt*

    *To whom correspondence should be addressed. E-mail: bernhard.nieswandt{at}virchow.uni-wuerzburg.de

    This PDF file includes:

    • Fig. S1. Characterization of Stim2–/– mice.
    • Fig. S2. Brain structure of Stim2–/– mice.
    • Fig. S3. Stim2–/– neurons are protected from hypoxia-induced apoptosis.
    • Fig. S4. Sustained neuroprotection after tMCAO in Stim2–/– mice.

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    Format: Adobe Acrobat PDF

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    Citation: A. Berna-Erro, A. Braun, R. Kraft, C. Kleinschnitz, M. K. Schuhmann, D. Stegner, T. Wultsch, J. Eilers, S. G. Meuth, G. Stoll, B. Nieswandt, STIM2 regulates capacitive Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death. Sci. Signal. 2, ra67 (2009).

    © 2009 American Association for the Advancement of Science

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