Annexin 7 Mobilizes Calcium from Endoplasmic Reticulum Stores in Brainb

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Biochimica et Biophysica Acta 1742 (2004) 151–160 http://www.elsevier.com/locate/bba Review Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brainB

W.D. Watsona,1, M. Srivastavab,1, X. Leightonb, M. Glasmanb, M. Faradayc, L.H. Fossamd, H.B. Pollardb,*, A. Vermae

aNeurology Department, National Naval Medical Center, Bethesda, MD 20814, United States bDepartment of Anatomy, Physiology, and Genetics, 4301 Jones Bridge Road, Bethesda, MD 20814, United States cDepartment of Medical Psychology, Bethesda, MD 20814, United States dDivision of Neuropharmacological Drug Products, CDER, FDA, Rockville, MD 20852, United States eDepartment of Neurology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, United States

Received 18 September 2004; received in revised form 5 October 2004; accepted 12 October 2004 Available online 29 October 2004

Abstract

Mobilization of intracellular calcium from inositol-1,4,5-triphosphate (IP3)-sensitive endoplasmic reticulum (ER) stores plays a prominent role in brain function. Mice heterozygous for the annexin A7 (Anx7) gene have a profound reduction in IP3 receptor function in pancreatic islets along with defective insulin secretion. We examined IP3-sensitive calcium pools in the brains of Anx7 (+/À) mice by utilizing ATP/Mg2+-dependent 45Ca2+ uptake into brain membrane preparations and tissue sections. Although the Anx7 (+/À) mouse brain displayed similar levels of IP3 binding sites and thapsigargin-sensitive 45Ca2+ uptake as that seen in wild-type mouse brain, the Anx7 (+/À) mouse brain Ca2+ pools showed markedly reduced sensitivity to IP3. A potent and saturable Ca2+-releasing effect of recombinant ANX7 protein was demonstrated in mouse and rat brain membrane preparations, which was additive with that of IP3. We propose that ANX7 mobilizes Ca2+ from an endoplasmic reticulum-like pool, which can be recruited to enhance IP3-mediated Ca2+ release. D 2004 Elsevier B.V. All rights reserved.

Keywords: Annexin 7; Knockout mouse; Endoplasmic reticulum; Calcium; IP3 receptor; Signaling; Brain

2+ 1. Introduction second messenger-induced [Ca ]cyt changes. Inositol-1,4,5- triphosphate (IP3), an intracellular second messenger gen- Precise regulation of cytoplasmic calcium levels erated in response to neurotransmitter receptor activation, 2+ ([Ca ]cyt) is required to control the electrical, secretory, mediates the release of calcium from a subset of intracellular and metabolic activities of brain cells. Cytoplasmic calcium ER stores by binding to its receptors (IP3R; 2). The IP3R1 is levels are controlled by a diverse set of mechanisms, the predominant subtype of IP3 receptor in the central including calcium binding proteins; plasma membrane nervous system and allows variable control of local intra- channels, pumps, and exchangers; mitochondria; and endo- cellular calcium concentrations, which can modulate the plasmic reticulum (ER) uptake and release mechanisms [1]. efficacy of both excitatory and inhibitory inputs [2]. The ER calcium machinery has a major impact on neuronal Homozygote mutants for IP3R1 die in utero while heterozygotes manifest epilepsy with polyspike electroencephalo- grams, opisthotonus, motor discoordination, ataxia, and B The opinions and assertions contained herein are the private views of altered synaptic function and plasticity [3]. These observa- the authors and are not to be construed as official or reflecting the views of tions indicate the importance of IP3-regulated Ca2+ release the Department of Defense. * Corresponding author. Tel.: +1 301 295 3661; fax: +1 301 295 2822. mechanisms in normal brain function. Proteins that interact E-mail address: [email protected] (H.B. Pollard). with the ER and with the IP3R directly can also influence 1 Dr. Watson and Dr. Srivastava are to be considered as co-first authors. IP3-mediated Ca2+ release. For example, the homer family of

0167-4889/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamcr.2004.10.008 152 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 proteins enhances IP3-mediated calcium release by bringing was maintained at 23 8C at 50% relative humidity on a 12-h plasma membrane proteins such as metabotropic receptors reverse light/dark cycle (lights on at 1900 h). Behaviors into close proximity with IP3R [4]. Coupling of IP3-sensitive were measured during the dark portion of the circadian and IP3-insensitive compartments by putative GTP-binding cycle for response stability and for face validity (i.e., proteins has also been proposed as a mechanism for animals were in the active portion of their daily cycle). increasing agonist mobilization of calcium pools [5]. Calm- Open-field apparatus: Open-field activity was measured odulin and Transient Receptor Potential channels (TRPs) also using an Omnitech Electronics Digiscan infrared photocell appear to regulate the function of IP3 receptors [6]. system [Test box model RXYZCM (16 TAO); Omnitech Members of the annexin gene family are a diverse group Electronics, Columbus, OH], located in a dedicated room of highly conserved Ca2+-dependent phospholipid binding constructed so that sound is kept to a minimum. Animals proteins found ubiquitously in higher organisms [7]. were placed singly in a 40Â40Â30-cm clear Plexiglas Although most annexins have poorly understood functions, arena. A Plexiglas lid with multiple 3.5-cm diameter they contain a conserved C-terminal region, which in many ventilation holes was placed on top of the arena. A photocell members of the annexin gene family possesses calcium array measured horizontal locomotor activity using 16 pairs channel activity [8]. Annexin A7 (ANXA7; ANX7; of infrared photocells located every 2.5 cm from side-to-side synexin) is a Ca2+-dependent membrane fusion protein with and 16 pairs of infrared photocells located front-to-back in a intrinsic GTPase activity. Ca2+, GTP, and PKC are proposed plane 2 cm above the floor of the arena. Data were to converge on ANXA7 to drive membrane fusion activity automatically gathered and transmitted to a computer via required during exocytosis [9]. Besides this activity, an Omnitech Model DCM-I-BBU analyzer. The apparatus ANXA7 has increasingly been implicated in the regulation monitored animal activity continuously with data recorded of intracellular calcium homeostasis in several cell types and as cumulative activity every 5 min for a total testing period species [11–13]. We have shown that Anx7 heterozygous of 2 h. Once animals were placed in the test arenas, the knockout Anx7 (+/À) mice display an insulin secretion experimenter turned off the lights and left animals undis- deficit from pancreatic islets of Langerhans due to defective turbed during the testing period. For data analysis purposes, intracellular pancreatic Ca2+ signaling processes with a activity was summed over the total 2-h testing period. Total marked reduction in IP3-sensitive Ca2+ [13]. Given the activity over the 2-h testing period was used to determine importance of ER Ca2+ in brain function and the reduced whether or not Anx7 (+/À) knockout male and female mice expression of Anx7 in all tissues of Anx7 (+/À) mice, we and wild-type littermates exhibit differences in activity. explored the possible impact of this deficiency on brain ER During the Baseline Phase (10 days), animals were calcium pools. acclimated to the facility and were handled every day to minimize any stress that might occur as a result of routine handling for body weight measurement, open-field testing, 2. Methods and startle testing. Animals underwent two acclimation exposures to the open-field apparatus to minimize effects of 2.1. Animals stress or novelty. Acclimation sessions consisted of placing the animal in the apparatus for 1 h on two separate days. Adult male Sprague–Dawley rats (300–350 g) were Baseline open-field measurements were then obtained. All obtained from Charles River. The Anx7 targeting vector, the statistical analysis tests were two-tailed. Results are sig- transfection and selection of embryonic stem cells, the nificant at Pb0.05, unless otherwise noted. preparation of chimeras, and the breeding protocols were as described by Srivastava et al. [13]. All animals described in 2.3. Tissue preparation this article were the progeny of at least six successive backcrosses into the wild-type C57BL6/J strain. Adult wild-type mice, Anx7 (+/À) mice or Sprague– Dawley rats were sacrificed using CO2 narcosis followed by 2.2. Behavioral studies decapitation. Brains were rapidly removed, minced with a razor, and placed in ice-cold homogenization buffer (10 ml/ To determine whether knockout of the Anx7 gene has any g) containing 25 mM N-2hydroxyethylpiperazine-NV-2 general effects on behavior, we tested open-field behavior in ethanesulfonic acid (HEPES, pH 7.3 with potassium 28 7-month-old mice: 7 male Anx7 (+/À) mice with 7 wild- hydroxide), 0.25 M sucrose, 100 mM EDTA, and protease type male littermates and 7 female Anx7 (+/À) mice with 7 inhibitors (100 mg/ml phenylmethanesulfonylfluoride, 10 wild-type female littermates. Open-field testing was per- Ag/ml leupeptin, and 10 Ag/ml aprotinin). A glass-teflon formed essentially as described previously [14]. Animals dounce homogenizer was used to homogenize tissue using were individually housed in standard polycarbonate shoe- five up-and-down strokes of a T-Line laboratory stirrer box cages (42Â20.5Â20 cm) on hardwood chip bedding (Talboys Engineering, Montrose, PA) at 70% RPM, (Pine-Dri). Throughout the study, the animals had contin- followed by ultracentrifugation at 100,000Âg with a Sorvall uous access to rodent chow and water. The housing room TH-641 swinging bucket rotor in a Sorvall Ultra Pro 80 at 4 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 153

8C for 60 min. The pellet, containing whole cell membranes, digitonin at 25 8C for 15 min. Slides were incubated in assay was rinsed and resuspended using ice-cold homogenization buffer (same as used for membranes) at 37 8C for 60 min. buffer lacking EDTA and enzyme inhibitors. Protein content Slides were rinsed twice for 5 min in an ice-cold wash was determined by the Bradford method using protein dye buffer containing (10 mM HEPES pH 7.3 with KOH, 10 concentrate (Bio-Rad, Hercules, CA) and adjusted to a mM dipotassium oxalate, 3% PEG, 5 mM sodium azide, 5 concentration of 1.25–5.0 mg/ml prior to storage at À80 8C. mM MgCl2, 2 mM EGTA) thoroughly dried by vacuum-air, Microsomes were prepared as outlined for homogenates and exposed to film for 48 h. For quantification, tissue except the glass-teflon dounced homogenate was subjected sections were completely wiped off slides using filter paper to 10,000Âg centrifugation at 4 8C for 15 min. Supernatant and tissue radioactivity was measured in 4-ml scintillation was then ultracentrifuged at 4 8C for 60 min, with resultant cocktail in a Wallac beta counter. pellet rinsed, suspended in homogenization buffer lacking EDTA and protease inhibitors, and stored after protein 2.5. Measurement of Ca2+-ATPase activity concentration was determined. Fresh frozen sections were obtained by rapidly freezing Ca2+-ATPase activity was determined in brain micro- brain in Tissue Freezing Medium (Triangle Biomedical somal membranes by the measurement of liberated inor- Sciences, Durham, NC). Cryostat sections (18 Am) were ganic phosphate. The assay was performed in a 20 mM thaw mounted at À20 8C onto Fisherbrand Superfrost/ HEPES–KOH (pH 7.3) buffer containing 80 mM KCl, 10 Plus slides (Fisher Scientific, Pittsburgh, PA) and stored at mM potassium oxalate, 5 mM sodium azide, 3% PEG and À80 8C. the concentration of free Ca2+ was adjusted to 1 AM with EGTA titration using a calcium-sensitive electrode. Follow- 2+ 2.4. Ca uptake assay ing fresh additions of 5 mM DTT, 1 mM MgCl2, and brain microsomes (100 Ag/ml final protein), reactions were started Radiolabeled calcium uptake into whole brain mem- by the addition of 2 mM ATP in a final volume of 250 Al, branes was performed as described previously [16]. Briefly, and continued at 37 8C for 30 min. Vacuum filtration of the studies were performed in 20 mM HEPES (pH 7.35 with 96-well MAFB microplate into a polystyrene 96-well KOH), with 50 mM dipotassium oxalate, 3% polyethylene culture microplate halted the assay. Ca2+-dependent ATPase glycol (PEG, avg mol wt 10,000), 5 mM sodium azide, 5 activity was subsequently determined by measuring liber- mM dithiothreotol (DTT), 5 mM phosphocreatine, 20 U/ml ated phosphate in the filtrate as reported previously [15]. creatine phosphokinase, 2 mM ATP, 1 mM MgCl2,50AM 45 2+ total CaCl2,1ACi/ml Ca , and indicated additions. Free 2.6. Ins(1,4,5)P3 autoradiography Ca2+ concentrations were adjusted with EGTA titration 2+ using a Ca -sensitive electrode (Orion, Charlestown, MA) IP3 binding sites were visualized as previously described calibrated with Ca2+ standards (World Precision Instru- [16] with minor modifications. Fresh frozen 18-Am-thick rat ments, Sarasota, FL). Digitonin (10 AM) was added to the brain sections were incubated for 15 min at 4 8Cin0.1M membrane preparations prior to experimentation to elimi- KCl, 20 mM NaCl, 20 mM Tris–HEPES, pH 8.0, 1 mM nate contribution from plasma membrane vesicles. To EDTA, 5 mM digitonin, and 2 mM h-mercaptoethanol was 45 2+ 3 initiate Ca uptake assays, brain membranes were added freshly added with 50 nM [ H] Ins [1,4,5] P3 (Perkin to a final protein concentration of 50 Ag/ml in a total Elmer). Sections were then washed twice for 1 min each in reaction volume of 0.5 ml at 37 8C. 45Ca2+ accumulation in ice-cold buffer, air-dried under a cool stream, and apposed the presence of the calcium ionophore A23187 (10 AM) was to hyperfilm. Nonspecific binding (blank) was determined considered nonspecific uptake and was subtracted from all in the presence of 25 AMIP3, and was less than 5% under values to yield specific uptake. All reported 45Ca2+ these conditions. accumulation is expressed as specific uptake unless otherwise specified. Assays routinely proceeded for 45 min and 2.7. IP3 receptor reverse transcription and polymerase were terminated by rapid filtration through Whatman GF/C chain reaction (RT-PCR) filters pre-soaked in 0.3% polyethyleneimine (PEI) using a Brandel cell harvester (Gaithersburg, MD). The filters were Total cellular RNA was extracted from wild-type mice washed twice with 3 ml of ice-cold wash buffer containing and Anx7 (+/À) mice by a one-step acid guanidine 20 mM HEPES (pH 7.35 with KOH), 5 mM MgCl2,5mM isothiocyanate–phenol method using Trizol reagent (GIBCO sodium azide, 2 mM EGTA, and 3% PEG. Radioactivity BRL, Gaithersburg, MD), precipitated with ethanol and was measured in 4 ml of CytoScint liquid scintillation quantitated by spectrophotometry. Total RNA (1 Ag) was cocktail (ICN Pharmaceuticals, Costa Mesa, CA) using a used for each RT-PCR. Two hundred units of Moloney Wallac model 1409 beta counter (Gaithersburg, MD). murine leukemia virus reverse transcriptase (GIBCO BRL) Histochemical 45Ca2+ uptake assays were performed in was added in a typical 50-Al reaction (1 Ag RNA, 1X first- thaw-mounted fresh frozen brain sections. Slides were pre- strand buffer, 1 mM DTT, 0.125 mM dNTPs, random incubated in a permeabilization buffer containing 10 AM primers and RNAse H) and incubated for 1 h at 37 8C. The 154 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 purified cDNA template was amplified using different sets 65 8C for 1 min and extension at 72 8C for 1 min. RT-PCR of primers. products were then separated on a 1.2% agarose gel and IP3 receptors come in three subtypes, in which only a visualized by staining with ethidium bromide. The inten- discrete domain varies among the three. The following sities of the bands corresponding to the RT-PCR products primers were prepared and tested in these experiments. were quantified using a phosphorimager (Bio-Rad) and normalized with respect to the h-actin product. Type1=[forward primer: 5V-CACCGCGGCAGAGATT- GACAC-3V; reverse primer: 5V-CCAGCTGCCCGGA- GATTTC-3V]; 3. Results Type2=[forward primer: 5V-CTGGGGCCAACGCTAA- TACTACTT-3V; reverse primer: 5V-GAACCCCGTGAT- 3.1. Wild-type and Anx7 heterozygous mice differ in TACCTGTGACTG-3V]; behavioral responses to novelty Type3=[forward primer: 5V-GCGGGCCTGTGACAC- TCTACTTAT-3V; reverse primer: 5V-CGCCGCTCAC- The Anx7 (+/À) mice have a 50% reduction in the Anx7 CAGGGACAT-3V]. protein [13]. These animals display a sexually dimorphic increase in overall body and organ mass (Fig. 1a–c). h-Actin was used as an internal control. PCRs were However, a statistical difference in brain mass between performed in a 25-Al volume in which 1Â PCR buffer, 0.2 wild-type and heterozygotes was not observed. Multivariate mM dNTPs, 1.5 mM MgCl2, 2.5 U of Taq polymerase and analyses of open-field behavioral testing showed some sex- 100 ng of the specific primers were added. A brief initial specific differences (females tended to be more active than denaturation at 95 8C for 2 min was followed by 30 cycles males). However, no significant differences were observed with the following steps: 94 8C for 1 min, annealing at 55– between Anx7 (+/+) and Anx7 (+/À) mice in measures of

Fig. 1. Anx7 knockout mice (+/À) are physically larger and have a more rapid growth rate than wild-type (+/+) mice. (a) Photograph of adult Anx7 (+/+) and Anx7 (+/À) littermates. The Anx7 (+/+) mice are white-furred, and (+/À) mice are brown. (b) Representative growth curve of 30 Anx7 (+/+) and (+/À) littermates as a function of age. Size by weight of mice is a function of Anx7 gene copy number. Anx7 (+/+) is represented by a dotted line and (+/À) by a solid line. (C) Male Anx7 (+/À) mice have increased organ weight compared to wild-type (+/+). Mean and 95% confidence interval of organ weights of 30 control and anx7 (+/À) mice are shown. The percent increase or decrease of organ weight in knockout mice compared with control mice is plotted for males and females and, thus, Anx7 (+/À) mice exhibit sexual dimorphism compared to their wild-type littermates. *Pb0.05, **Pb0.01. W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 155

Fig. 2. Open-field behavioral trials show that Anx7 (+/À) mice display anxiety to novel stimuli as compared to wild-type mice. Wild-type and knockout littermates were tested in an open-field apparatus in two 3-h sessions run 3 days apart. Nine behaviors were recorded and analyzed, including measuresof general locomotion (e.g., total distance traveled, Panel a), rearing (vertical activity, Panel b), stereotypies (Panel c), and emotionality (e.g., time spent in center of the field vs. the margins, Panel d). Multivariate analysis showed some sex-specific differences (females tended to be more active than males), and the only significant differences detected between Anx7 (+/À) and wild-type mice of the same sex were in the emotionality test. *Pb0.05.

general locomotion, rearing, and stereotypy (Fig. 2a–c). uptake activities in Anx7 (+/+) and Anx7 (+/À) brain When tested with the emotionality paradigm, the Anx7 (+/À) membranes, which were comparable to those seen in rats mice spent far less time in the center of the cage than the (Table 1). wild-type littermates during the initial testing trial, a pattern We also incorporate the Ca2+ precipitating anion oxalate of behavior reflecting anxiety to novel stimuli (Fig. 2d). in our assays. At the low free Ca2+ levels in the uptake buffer Ca2+ oxalate precipitation is prevented. However, the 2+ 3.2. Anx7 (+/À) mice brain calcium pools have reduced IP3 high level of sequestered Ca in ER luminal compartments sensitivity allows the co-transported oxalate to form precipitates, thus markedly enhancing ER uptake [15,16]. Inclusion of ER 45Ca2+ uptake was monitored using whole brain mem- Ca2+ channel openers such as IP3 in the uptake assay branes from Anx7 (+/+) and Anx7 (+/À) mice as described produces a leak, which over time results in diminished net in Methods. Although we employed whole brain mem- 45Ca2+ oxalate accumulation [15,16]. Approximately 35– branes for higher yield, our assay is specific for brain ER 40% of the total brain ER calcium pool is known to be IP3- compartments and does not reflect uptake into mitochondria sensitive and this pool is a subset of the thapsigargin- or plasma membrane vesicles [15,16]. This selectivity was sensitive ER compartment [16,17]. In the brain membrane provided by the incorporation of digitonin to eliminate sequestration by plasma membrane vesicles and the use of a 2+ Table 1 low free [Ca ] and azide to inhibit mitochondrial accumu- 45 2+ 45 2+ Oxalate supported Ca uptake in rat, wild-type mouse and Anx7 (+/À) lation. Ca uptake in this assay was also insensitive to mouse brain membranes + + + H ,Na, and K ionophores but was completely releasable 45 2+ 2+ Ca uptake by mouse and rat brain membrane preparations by the calcium ionophore A23187 [15,16].ERCa is (nmol/mg protein) driven by a family of Mg2+-dependent Ca2+ ATPases Total uptake Thapsigargin-resistant referred to as SERCAs (sarco-endoplamic reticulum Wild-type mice Anx7 (+/+) 6.8F0.6 1.56F0.5 ATPases), which are all selectively inhibited by submicro- Heterozygote mice Anx7 (+/À) 7.1F0.4 1.6F0.3 molar doses of thapsigargin. In the brain, between 15% and Rats 5.8F0.4 1.33F0.2 20% of the ER calcium is also driven by a thapsigargin- 45Ca2+ uptake was performed as described in Methods. insensitive ATPase [15]. We observed similar levels of total, Values represent the meanFS.E. of three experiments performed in thapsigargin-sensitive, and thapsigargin-resistant 45Ca2+ triplicates. 156 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 preparations from Anx7 (+/+) mice, we observed a dose- the incubation. The right half of each panel shows the ER dependent reduction of net 45Ca2+ accumulation by IP3 with 45Ca2+ accumulated in a serial section in the presence of a maximal effect at 10 AM IP3 (Fig. 3a). This action of IP3 added IP3. The image of one brain hemisphere from each reflects its binding to, and opening of, IP3 receptor channels condition has been cropped together to allow anatomical and is consistent with previously reported potencies of IP3 comparison. Thus, at 10 AM IP3, a clear reduction of net in rat brain [18,19]. Although a dose-dependent reduction of 45Ca2+ accumulation is seen in the cerebral cortex, striatum, net 45Ca2+ calcium accumulation by IP3 was also observed hippocampus and cerebellar cortex of wild-type mouse in Anx7 (+/À) membranes, the potency of IP3 was horizontal brain sections (Fig. 3b, upper left panel). As significantly diminished as compared with that in Anx7 previously reported for rat brain, the septum and thalamus, (+/+) membranes (Fig. 3a). which are known to have Ca2+ pools with minimal IP3 The reduced effectiveness of IP3 in releasing Ca2+ from sensitivity [16], show a minimal effect of IP3. At this same Anx7 (+/À) IP3-sensitive Ca2+ pools was also observed in dose of IP3, a much smaller reduction of net 45Ca2+ mouse brain sections analyzed via 45Ca2+ autoradiography accumulation was seen in brain sections from Anx7 (+/À) (Fig. 3b). In this approach, fresh frozen tissue sections are mice (Fig. 3b, upper right panel). At a higher dose (30 AM) substituted for brain homogenate preparations in the 45Ca2+ of IP3, a clear effect on net 45Ca2+ uptake was seen in brain uptake assay, thus allowing anatomical localization of membranes (Fig. 3a) and sections (Fig. 3b, lower panels) 45Ca2+ sequestering pools [16]. For the analysis in Fig. 3, from both Anx7 (+/+) and Anx7 (+/À) mice. This finding brain cryostat sections were assayed for 45Ca2+ accumu- suggested that the reduced effectiveness of IP3 in the Anx7 lation in the absence of presence of IP3 at the indicated (+/À) mice was not due to a marked loss of IP3 receptor concentrations. For the autoradiographic images shown in expression. Indeed, RT-PCR measurement of mRNA for the Fig. 3b, the left half of each panel shows an image of the ER IP3 receptor subtypes 1, 2, and 3 (IP3R1, IP3R2, IP3R3) in 45Ca2+ accumulated in sections where IP3 was not added to the cerebral cortex and striatum revealed similar expression

Fig. 3. Anx7 (+/À) mice have reduced IP3 sensitivity of brain ER calcium pools. (a) IP3 dose–response curves for reducing net Ca2+ accumulation in wild-type and Anx7 (+/À) mouse brain membranes. Differences at 1, 3, and 10 AM IP3 are statistically significant Pb0.05. (b) Localization of IP3-sensitive Ca2+ pools in wild-type and Anx7 (+/À) brain sections. See text for detail. (c) Preserved expression of IP3R1 mRNA in wild-type and Anx7 (+/À) mice. (d) Preserved IP3 binding in wild-type and Anx7 (+/À) mouse brain sections. W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 157 levels in Anx7 (+/+) and Anx7 (+/À) mice (Fig. 3c). Direct broad dose range of IP3 (Fig. 4d) and did not display any autoradiographic analysis of [3H] IP3 binding also revealed inhibitory effect on brain homogenate Ca2+-stimulated similar IP3 receptor expression in the Anx7 (+/+) and Anx7 ATPase activity (Fig. 4e), suggesting that ANX7 was (+/À) mice (Fig. 3d). reducing the signal via calcium release rather than by Anx7 is a Ca2+-dependent phospholipid binding protein, inhibition of uptake. Using rat brain sections, we also which when mutated produces abnormalities of intracellular explored whether the Ca2+-releasing effect of ANX7 was calcium homeostasis in cells from species as diverse as reversible. To investigate this, we pre-incubated sections for dictyostelium and mouse [10–13]. Anx7 (+/À) mice have a 30 min with 1 ng/ml ANX7 and then used two 5-min defect in agonist-mediated mobilization of pancreatic islet washes to remove the protein. As shown in Fig. 4f, ANX7 IP3-sensitive calcium pools [13]. These previous findings only elicited Ca2+ release when present during the uptake and the data shown above led us to speculate that the ANX7 assay and the effect was reversible upon removing the protein might play a role in the mobilization of ER calcium protein. pools. To investigate this possibility, we explored the effect of exogenously added human recombinant ANX7 protein 3.3. ANX7 and IP3 release calcium from overlapping brain on ER calcium pools in mouse and rat brain homogenates. ER stores Using our ER specific 45Ca2+ uptake assay in rat brain membranes, we found that recombinant ANX7 protein Having demonstrated a potent and reversible Ca2+- potently reduced the net accumulation of 45Ca2+ (Fig. 4a). releasing effect of ANX7 which was additive with that of Although high amounts of ANX7 could conceivably IP3, we next explored the anatomical localization of the produce a nonspecific Ca2+ leak through the formation of ANX7-sensitive Ca2+ pool. Examination of ER-specific channels [8], we observed a plateau effect of ANX7 over the 45Ca2+ uptake in rat brain sections by autoradiography (Fig. range of 1 pg/ml to 1 ng/ml. Moreover, although this effect 5a) revealed a heterogeneous distribution as seen previously was specific to the thapsigargin-sensitive component of ER [16]. IP3 selectively reduced net 45Ca2+ accumulation in the 45Ca2+ uptake (Fig. 4b), it was additive with the effects of cerebral cortex, striatum, hippocampus, and substantia nigra IP3 in brain membrane preparations from rat, wild-type among other regions. However, several areas such as the mice, and Anx7 (+/À) mice (Fig. 4c). ANX7 exerted a septum, thalamus, corpus callosum and midbrain were similar additive effect on IP3-mediated Ca2+ release across a insensitive to IP3 (Fig. 5b). By contrast, ANX7 appeared

Fig. 4. Anx7 protein release of brain ER calcium. (a) Dose–response curve for recombinant human ANX7 protein-mediated reduction of net calcium accumulation in rat brain membranes. (b) ANX7 effect is selective for the Thapsigargin-sensitive calcium pool. (c) ANX7 is active in rat and mouse brain and is additive with IP3. (d) Additivity of ANX7 over a range of IP3 doses in rat brain membranes. (e) Lack of effect of ANX7 on rat brain Ca2+ ATPase activity. (f) Reversibility of ANX7 Ca2+-releasing effect. Rat brain sections were exposed to 1 nM ANX7 either during a 30-min pre-incubation or during the uptake assay as indicated. Results are shown as the means (N=5) of three independent calcium uptake experiments performed in triplicate; +S.E. 158 W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160

Fig. 5. Anatomical overlap of IP3 and ANX7-sensitive Ca2+ pools in rat forebrain. (a) Heterogeneous 45Ca2+ uptake in control rat brain sections. (b) Net 45Ca2+ accumulation in the presence of 10 AM IP3. (c) Net 45Ca2+ accumulation in the presence of 1 nM ANX7. (d) Net 45Ca2+ accumulation in the presence of 10 AM IP3 and 1 nM ANX7. Cerb. Ctx., cerebral cortex; Hipp., hippocampus; Sep., septum; Str., striatum; Thal., thalamus; S.N., substantia nigra; *Midbrain region.

to be more active in releasing 45Ca2+ in these same areas higher uptake in the CA3 and dentate gyrus regions than in than IP3 (Fig. 5c). In several areas where both IP3 and the CA1 region (Fig. 6a). IP3 released Ca2+ more effectively ANX7 were effective, they exhibited overlapping patterns from the CA1 region than from the CA3 or dentate gyrus of release. For example, IP3 released ER 45Ca2+ from both (Fig. 6b) while ANX7 displayed the opposite pattern (Fig. the superficial and the deeper layers of the cerebral cortex 6c) and showed an additive effect with IP3 (Fig. 6d). while ANX7 was more effective in the deeper cortex than the superficial layer (Fig. 5c). IP3 and ANX7 together displayed an additive effect in releasing 45Ca2+ from the 4. Discussion cortex and other regions (Fig. 5d). Regional differences in the sensitivity of hippocampal ER Ca2+ to IP3 and ANX7 The annexin protein family has been shown to play were also appreciated at higher magnification (Fig. 6). diverse roles in such critical cell processes as secretion, Hippocampal ER 45Ca2+ uptake showed heterogeneity with inflammation, and blood clotting [7]. Transgenic mouse models with reduced expression of annexin proteins are only now beginning to reveal the impact of these proteins in normal physiology and disease processes. Anx7 (+/À) mice have been shown to develop a glucose intolerant diabetic state due to an insulin secretory deficit [13]. Anx7 (+/À) mice also develop neoplasms in several organs including breast, pancreas, and prostate, which are characterized by a reduced rate of cellular apoptosis [18]. This observation has been clinically translated into the identification of ANX7 as tumor suppressor gene in human cancers [19,20]. Our current report provides the first evidence for a possible role of ANX7 in normal function and diseases of the central nervous system. Anx7 (+/À) mice, despite having similar brain size and morphology as their wild-type littermates, display anxious behavior when presented with novel stimuli (Fig. 2). This phenotype is likely to arise from a perturbation of brain cell signaling mechanisms. Our observation that the Anx7 (+/À) mice have a reduced IP3 sensitivity of their ER Ca2+ stores provides significant support for this notion (Fig. 3). The Fig. 6. Anatomical overlap of IP3 and ANX7-sensitive Ca2+ pools in rat 45 2+ reduction of IP3 sensitivity in the Anx7 (+/À) brains is not hippocampus. (a) Heterogeneous net Ca uptake in rat hippocampus. (b) 2+ Net 45Ca2+ accumulation in the presence of 10 AM IP3. (c) Net 45Ca2+ due to a deficiency in overall ER Ca uptake (Table 1) nor accumulation in the presence of 1 nM ANX7. (d) Net 45Ca2+ accumulation to the lack of IP3 receptor expression (Fig. 3c and d). This in the presence of 10 AM IP3 and 1 nM ANX7. finding is consistent with our prior report of defective IP3- W.D. Watson et al. / Biochimica et Biophysica Acta 1742 (2004) 151–160 159 sensitive Ca2+ release in pancreatic islet of Langerhans cells manifestations in Anx7 (+/À) mice ranging from neoplasia from Anx7 (+/À) mice but is unique in that the islet cells to anxiety. displayed a markedly reduced expression of IP3R [13]. Loss of IP3R1 expression leads to severe neurological dysfunc- tion [3] and it is therefore remarkable that the reduced IP3 Acknowledgements sensitivity seen in the Anx7 (+/À) brain ER pools is associated only with an anxious phenotype. Given the This work was supported by DOD grants F292AH, important role of IP3-mediated signaling in brain function F192AN, Department of Clinical Investigations WU no. however, it is likely that the Anx7 (+/À) mice will show 0371020, Walter Reed Army Medical Center, and USUHS additional abnormalities in cognition, learning, or sensitivity grant C070IV. to traumatic or ischemic insults. In an attempt to explore a direct effect of the ANX7 2+ protein on ER Ca handling, we were surprised to discover References a novel ER Ca2+-releasing action of the recombinant ANX7 protein. This effect was potent, reversible and selective for [1] M.J. Berridge, Neuronal calcium signaling, Neuron 21 (1) (1998 Jul) ER pools loaded by SERCAs, but not mediated by 13–26. inhibition of SERCA activity. The ANX7 effect was also [2] K. Mikoshiba, Inositol 1,4,5 trisphosphate (IP3) receptor, Nippon additive with the effect of IP3 and unique to specific brain Yakurigaku Zasshi 121 (4) (2003 Apr) 241–253. [3] M. 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