1. Mechanism of ER Stress-Induced Brain Damage by IP3 Receptor
Takayasu Higo, Kozo Hamada, Chihiro Hisatsune, Nobuyuki Nukina, Tsutomu Hashikawa, Mitsuharu Hattori, Takeshi Nakamura, Katsuhiko Mikoshiba 
Neuron 
Volume 68, Issue 5, Pages (December 2010)
DOI: /j.neuron
Copyright © 2010 Elsevier Inc. Terms and Conditions

2. Figure 1
The Loss of IP3R1 Function Enhances ER Stress-Induced Neuronal Death
(A) Expression levels of IP3R1 and an ER lumenal protein, GRP78, in crude microsome prepared from cerebella of IP3R1 (+/+), IP3R1 (+/−), and IP3R1 (−/−) mice.
(B and C) In vivo effect of ER stress on Purkinje neurons in wild-type (WT) and heterozygous IP3R1 knockout mice (IP3R1+/−). Representative hematoxylin and eosin (HE)-stained (B) and α-calbindin-stained (C) cerebellum sections from WT and IP3R1+/− mice sacrificed 2 days after injection of Tun (1 μg/g) or control phosphate-buffered saline. Note marked increase of shrunken neurons (B) and decrease of calbindin signals (C) in the cerebellum lobule VI of IP3R1+/−. Arrowheads indicate Purkinje cell layer in WT (black) and IP3R1+/− (red). We confirmed reproducibility using five and two littermate pairs (WT and IP3R1+/−) for HE staining and for calbindin immunostaining, respectively.
(D and E) Enhancements of apoptosis by IP3R1 knockdown. HeLa cells transfected with IP3R1 siRNA were treated with Tun (5 μg/ml) or Tg (2 μM) or DTT (2 mM) for 12, 24, or 48 hr. The cells were stained with Annexin-V FITC/PI (D) or MitoCapture (E). (D) The percentage of apoptosis (Annexin-positive/PI-negative) in at least 10,000 cells was determined by flow cytometer analysis, in which the cells treated with 1 μM staurosporine for 4 hr were used as a positive-control. (E) A cationic MitoCapture dye selectively enters into mitochondria to fluoresce red in healthy cells, and changes reversely its color from red to green (FL1) as membrane potential decreases in apoptotic cells. The percentage of cells showing a green fluorescence (FL1) in at least 10,000 cells was determined by flow cytometer analysis. Data in (D) and (E) are presented as mean ± SEM from at least three independent experiments. ∗p < 0.05; ∗∗p <
See also Figure S1.
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3. Figure 2 ER Stress Impairs IP3R1-Mediated Ca2+ Release
(A and B) Representative Ca2+ responses in N1E-115 cells treated with control DMSO (black), DTT (red), or Tun (blue) for 16 hr are shown in (a, left traces). N1E-115 (A) and HeLa (B) cells were stimulated with 1 μM bradykinin (BK) and 3 μM histamine in the absence of extracellular Ca2+, respectively. Bar charts summarize average peak amplitude of Ca2+ release in N1E-115 (A) and HeLa (B) cells treated with control DMSO (gray), DTT (red), or Tun (blue).
(C) ER stress attenuates IP3-induced Ca2+ release. After ATP-dependent uptake of Ca2+ into crude microsomes from HeLa cells pretreated with DMSO (black), DTT (red), or Tun (blue), for 48 hr, the indicated concentrations of IP3 (μM) were sequentially added.
(D) ER stress impairs IP3R1-mediated Ca2+ release in primary neurons. Primary striatal neurons were treated with Tun (5 μg/ml) for 12 hr and then stimulated with 100 μM 3,5-dihydroxyphenylglycine (DHPG), a specific agonist for group 1 and 5 metabotropic glutamate receptors, after depolarization with 30 mM KCl to fill the Ca2+ store. Data are presented from at least three independent experiments. Data in (A) to (D) are presented as mean ± SEM from at least three independent experiments. ∗p < 0.05.
See also Figure S2.
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4. Figure 3 Identification of GRP78 as an IP3R1-Associated Protein
(A) Schematic representation of IP3R. IP3R contains six transmembrane domains (TMs) and three lumenal loops, L1, L2, and L3. L3 is divided into L3V (red) with two glycosylations (blue) and L3C. IP3 binds to the N-terminal cytosolic region.
(B) GRP78 specifically interacts with the L3V of IP3R1, but not that of IP3R2 or IP3R3. Purified GST, GST-1L3V, GST-2L3V, or GST-3L3V was incubated with maltose-binding protein (MBP)-GRP78. Protein complexes were isolated using amylose resin and analyzed by western blotting with α-GST (top and middle) or α-MBP (bottom).
(C) GRP78 colocalizes with IP3R1 in ER. COS-7 cells expressing Myc-GRP78 and green fluorescent protein-tagged IP3R1 (GFP-IP3R1) were stained with α-Myc (panels 1–3). The boxed regions in panels 1–3 are shown at increased magnification in panels 4–6, respectively. Scale bar, 5 μm.
(D) In vivo interaction between GRP78 and IP3R1. HeLa cells were treated with a membrane-permeable crosslinker, DSP, and the cell lysates were subjected to immunoprecipitation (IP) with control rabbit IgG or α-GRP78. The lysate (Input) and IP samples were analyzed as in (B).
(E) GRP78 inhibits binding of ERp44 to IP3R1. COS-7 cells were transfected with expression vectors for GFP-IP3R1 and HA-ERp44 or GFP-IP3R1, HA-ERp44, and Myc-GRP78. Forty-eight hours after transfection, cells were treated with DSP for 30 min. After washing with PBS, the cells were solubilized and then subjected to IP with α-HA (3F10). The lysate (Input) and IP samples were analyzed as in (D).
(F) GRP78 colocalizes with IP3R1 in the brain. Mouse cortical and striatal sections were immunostained with α-IP3R1 and α-GRP78. Scale bar, 10 μm.
(G and H) Interaction of IP3R1 with GRP78 in the brain. (G) Crude microsome obtained from P14 mouse brain was solubilized and the cleared fraction was immunoprecipitated with α-IP3R1 (10A6). The lysate (Input) and IP samples were analyzed as described above. (H) Primary cortical neurons were treated with DSP and the cell lysates were subjected to IP and then analyzed as in (G).
(I and J) ER stress inhibits the interaction of IP3R1 with GRP78. (I) HeLa cells were treated with DMSO, Tun (2 μg/ml), Tg (2 μM), and DTT (2 mM) for 24 hr. After cross-linking with DSP, the cell lysates were subjected to IP with α-IP3R1. (J) Forty-eight hours after Tun injection to P14 mice, the cerebra were dissected and homogenized. The crude microsome was solubilized and the cleared fractions were immunoprecipitated with α-IP3R1. The lysate (Input) and IP samples were analyzed as in (G).
See also Figure S3.
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5. Figure 4
IP3R1-GRP78 Interaction Is Inhibited in the Model Mice of the Huntington's Disease
(A) IP3R1 and GRP78 interaction in model mice of Huntington's disease (HD). Crude microsome fraction was obtained from cerebrum of WT or R6/2 mice (8 weeks) that have been widely used as a model mouse of HD. The detergent-solubilized fractions (Input) were immunoprecipitated with α-IP3R1 (10A6) and the IP samples were analyzed by western blotting.
(B) Decreased interaction of IP3R1 with GRP78 in the HD model mice. The homogenates of the R6/2 mice striatum were solubilized and the cleared fraction was immunoprecipitated with α-IP3R1. The lysate (Input) and IP samples were analyzed as described above.
(C) Impaired Ca2+ release activity of IP3R1 in the striatum of HD model mice. Primary striatal neurons were stimulated with 100 μM DHPG after depolarization with 30 mM KCl.
(D) Decreased interaction of IP3R1 with GRP78 in the HD model mice. Crude microsome fraction was obtained from cerebellum of WT or R6/2 mice. The detergent-solubilized fractions (Input) were immunoprecipitated with α-IP3R1 (10A6) and the IP samples were analyzed as in (B). The ratio of the densities of the IP samples (GRP78/IP3R1) in WT and R6/2 are 0.35 ± 0.03 (mean ± standard error of mean [SEM], n = 3) and 0.21 ± 0.05 (mean ± SEM, n = 3) respectively, and these results show the significant reduction in interaction of the two proteins in the cerebellum of R6/2 mice compared with that in WT (p < 0.05).
(E) Impaired Ca2+ release activity of IP3R1 in the cerebellum of HD model mice. Ca2+ uptake into the microsomes obtained from cerebellum of WT or R6/2 mice was initiated by addition of ATP and then 0.1 μM IP3 was added to monitor IP3R1 activity. Data in (C) and (E) are presented as mean ± SEM from at least three independent experiments. ∗p < 0.05.
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6. Figure 5 GRP78 Knockdown Inhibits IP3R1-Mediated Ca2+ Release
(A) Knockdown of GRP78 in HeLa and COS-7 cells. The detergent-solubilized fractions of HeLa and COS-7 cells transfected with siRNAs were analyzed by western blotting with the antibodies for IP3R1, IP3R2, IP3R3, GRP78, protein disulfide isomerase (PDI), calreticulin (CRT), and actin.
(B and C) Knockdown of GRP78 inhibits Ca2+ release in HeLa cells. (B) Seventy-two to ninety-six hours after siRNA transfection, cells were stimulated with 1, 3, and 10 μM ATP in the presence of extracellular Ca2+. (C) The average peak amplitude was significantly lower in GRP78 siRNA-transfected cells (red) (0.004 ± 0.001 [1 μM], 0.36 ± 0.06 [3 μM], 0.62 ± 0.1 [10 μM]) than in the control cells (gray) (0.15 ± 0.05 [1 μM], 0.88 ± 0.13 [3 μM], 1.71 ± 0.12 [10 μM]).
(D) Seventy-two hours after siRNA transfection, COS-7 cells were stimulated with 0.1, 0.3, and 1 μM ATP in the presence of extracellular Ca2+.
(E) Average peak amplitude of the Ca2+ response in COS-7 cells transfected with control siRNA (gray) (0.74 ± 0.15 [0.3 μM], 1.35 ± 0.12 [1 μM]) or GRP78 siRNA (red) (0.79 ± 0.07 [0.3 μM], 1.38 ± 0.04 [1 μM]).
(F) Knockdown of GRP78 in N1E-115 cells. The soluble fractions of N1E-115 cells transfected with indicated siRNA were analyzed as in (A).
(G) Seventy-two hours after siRNA transfection, N1E-115 cells were stimulated with 0.01, 0.1, and 1 μM bradykinin (BK) in the presence of extracellular Ca2+.
(H) The average peak amplitude was significantly lower in GRP78 siRNA-transfected cells (red) (0.02 ± 0.02 [0.01 μM], 0.46 ± 0.08 [0.1 μM], 0.74 ± 0.16 [1 μM]) than in the control cells (gray) (0.79 ± 0.19 [0.01 μM], 0.81 ± 0.13 [0.1 μM], 1.03 ± 0.22 [1 μM]).
(I) Knockdown of GRP78 did not decrease ER Ca2+ content in N1E-115 cells. (Left) Cells were stimulated with 1 μM Tg in the absence of extracellular Ca2+. (Right) The average peak amplitudes in control siRNA- and GRP78 siRNA-transfected cells were 0.97 ± 0.05 and 0.98 ± 0.13, respectively. Representative Ca2+ responses in control siRNA-transfected (black) or GRP78 siRNA-transfected cells (red) are shown in (B), (D), (G), and (I).
(J) GRP78 re-expression restored attenuated IP3R1-mediated Ca2+ release. HeLa cells were first transfected with siRNA against the GRP78 3′UTR. Forty-eight hours after transfection, the cells were transfected with expression vectors for monomeric RFP (red) and full-length (blue) GRP78. Similarly, HeLa cells were transfected with control siRNA and control vectors (black). Forty-eight hours after the second transfection, the cells were stimulated with 10 μM ATP in nominally Ca2+-free medium. Representative Ca2+ responses (left) and the average peak amplitudes (right) are shown. Data in (C), (E), (H), (I), and (J) are presented as mean ± SEM from at least three independent experiments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p <
See also Figure S4.
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Copyright © 2010 Elsevier Inc. Terms and Conditions

7. Figure 6 GRP78 Knockdown Impairs IP3R1 Assembly
(A) HeLa cells transfected with control siRNA or GRP78 siRNA for 96 hr were crosslinked with DSP and the soluble fractions were subjected to gel filtration. Resulting fractions were analyzed by western blotting with indicated antibodies. Exclusion volume of molecular size markers is shown at the top of the panel (A).
(B) Calibration of elution profiles of gel filtration. Vo is the void volume of the column estimated with blue dextran 2000 and Ve is the elution volumes of the proteins.
(C) Quantified levels of IP3R1 in gel filtration fractions from three independent experiments using western blotting analysis.
(D and E) Gel filtration was performed using the soluble fractions of N1E-115 (D) and COS-7 cells (E) as in (A). Resulting fractions were analyzed as in (A).
(F) HeLa cells were treated with control (DMSO), Tun (4 μg/ml), or Tg (2 μM) for 16 hr. After crosslinking with DSP, the detergent-solubled cell lysates were subject to FPLC. The resultant fractions were analyzed by western blotting.
(G) GRP78 overexpression ameliorates ER stress-dependent impairment of IP3R1 assembly. Thirty-six hours after transfection with a vector for control or Myc-GRP78 in HeLa cells, the cells were treated with control DMSO or Tun and then were analyzed as described above.
See also Figure S5.
Neuron  , DOI: ( /j.neuron )
Copyright © 2010 Elsevier Inc. Terms and Conditions

8. Figure 7
GRP78 Knockdown Inhibits the Tetrameric Assembly of Newly Synthesized IP3R1
(A and B) HeLa, N1E-115, and COS-7 cells transfected with indicated siRNA, GFP-IP3R1, or GFP-IP3R1-deltaL3V were solubilized and then subjected to sedimentation in a linear 5%–20% sucrose gradient. Fractions were collected from top to bottom and analyzed by western blotting with indicated antibodies.
(C) HeLa cells were transfected with control siRNA (left panel) or GRP78 siRNA (right panel), and then crosslinked with a noncleavage crosslinker, disuccinimidylsuberate (DSS). The soluble fractions were subjected to sedimentation in a linear 5%–20% sucrose gradient and resulting fractions were separated on native-PAGE in the absence of both SDS and a reducing agent. Migration of molecular size markers is shown at right panel.
(D) Experimental procedure of pulse labeling.
(E) The IP samples were separated in SDS-PAGE and detected by autoradiography. The representative results of IP with α-IP3R1 and α-IP3R3 are shown from three and two independent experiments, respectively.
Neuron  , DOI: ( /j.neuron )
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9. Figure 8 GRP78 Regulates the IP3R1 Assembly Depending on Its ATPase
(A) ATP-dependent binding behavior of GRP78 to 1L3V. 1,500 resonance unit (RU) of GST-1L3V were immobilized. At time 0, a solution containing the indicated concentration of GRP78 was introduced and passed over the chip for 120 s. Association and dissociation were followed in the presence of 5 mM ATP or ADP. The association kinetics between GRP78 and GST-1L3V (kon = 8.6 × 103 [M−1s−1]) and the dissociation kinetics of GRP78 from 1L3V (koff = 3.6 × 10−4 [s−1] and Kd = 4.49 × 10−8 [M]) in the presence of ATP are obtained from three independent experiments.
(B) HeLa cells were transfected with vectors for WT or an ATPase mutant of GRP78, T37G. Using the lysates, IP was performed with α-Myc. The lysates (Input) and IP samples were analyzed by western blotting with α-IP3R1 (top and middle) or α-Myc (bottom).
(C) HeLa cells were transfected with vectors for WT or an ATPase mutant of GRP78, T37G. After crosslinking with DSP, the detergent-solubled cell lysates were subject to FPLC. The resultant fractions were analyzed by western blotting.
(D) HeLa cells were cotransfected with vectors for RFP-KDEL and GRP78 ATPase mutants. Forty-eight hours after transfection, the cells were stimulated with 3 μM histamine in the absence of extracellular Ca2+. Representative Ca2+ responses in RFP-positive cells are shown.
(E) The average peak amplitudes were 1.56 ± 0.10 (control), 2.26 ± 0.20, (GRP78 WT), 1.62 ± 0.17 (T37G), 1.80 ± 0.23 (G226D), and 1.60 ± 0.11 (Delta A). Results are mean ± SEM from at least three independent experiments. ∗p < 0.05.
(F) The Ca2+ storage capacity is unchanged in HeLa cells expressing GRP78. Quantification of the peak amplitude in cells stimulated with 1 μM Tg.
(G and H) Primary cortical neurons were cotransfected with expression vectors for monomeric RFP and GRP78 WT. Forty-eight hours after transfection, the cells were stimulated with 100 μM DHPG, followed by depolarization with 30 mM KCl to fill the Ca2+ store. (G) Representative Ca2+ responses in RFP-positive cells are shown. (H) The average peak amplitudes were ± 0.03 (control) and ± (GRP78 WT). Data are presented mean ± SEM from six independent experiments. ∗p < 0.05.
See also Figure S6.
Neuron  , DOI: ( /j.neuron )
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