Differential neuronal localizations and dynamics of phosphorylated and unphosphorylated type 1 inositol 1,4,5-trisphosphate receptors

Inositol 1,4,5-trisphosphate receptors

Pergamon

PII: S0306-4522(00)00470-X

Neuroscience Vol. 102, No. 2, pp. 433±444, 2001 433 q 2001 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/01 $20.00+0.00

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DIFFERENTIAL NEURONAL LOCALIZATIONS AND DYNAMICS OF PHOSPHORYLATED AND UNPHOSPHORYLATED TYPE 1 INOSITOL 1,4,5-TRISPHOSPHATE RECEPTORS A. A. PIEPER, a D. J. BRAT, b E. O'HEARN, a,c D. K. KRUG, a A. I. KAPLIN, d K. TAKAHASHI, e J. H. GREENBERG, e D. GINTY, a M. E. MOLLIVER a,c and S. H. SNYDER a,d,f* a

The Johns Hopkins University, School of Medicine, Department of Neuroscience, 725 N. Wolfe Street, Baltimore, MD 21205, USA b

Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 1364 Clifton Rd. NE, Atlanta, GA 30322, USA

c

The Johns Hopkins University, School of Medicine, Department of Neurology, 725 N. Wolfe Street, Baltimore, MD 21205, USA

d

The Johns Hopkins University, School of Medicine, Department of Psychiatry, 725 N. Wolfe Street, Baltimore, MD 21205, USA e

Cerebrovascular Research Center, Department of Neurology, University of Pennsylvania School of Medicine, 415 Stemmler, 3450 Hamilton Walk, Philadelphia, PA 19104-6063, USA

f

The Johns Hopkins University, School of Medicine, Department of Pharmacology and Molecular Sciences, 725 N. Wolfe Street, Baltimore, MD 21205, USA

AbstractÐType 1 inositol 1,4,5-trisphosphate receptors are phosphorylated by cyclic-AMP-dependent protein kinase A at serines 1589 and 1755, with serine 1755 phosphorylation greatly predominating in the brain. Inositol 1,4,5-trisphosphate receptor protein kinase A phosphorylation augments Ca 21 release. To assess type 1 protein kinase A phosphorylation dynamics in the intact organism, we developed antibodies selective for either serine 1755 phosphorylated or unphosphorylated species. Immunohistochemical studies reveal marked variation in localization. For example, in the hippocampus the phosphorylated type 1 inositol 1,4,5-trisphosphate receptor is restricted to CA1, while the unphosphorylated receptor occurs ubiquitously in CA1±CA3 and dentate gyrus granule cells. Throughout the brain the phosphorylated type 1 inositol 1,4,5trisphosphate receptor is selectively enriched in dendrites, while the unphosphorylated receptor predominates in cell bodies. Focal cerebral ischemia in rats and humans is associated with dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors, and glutamatergic excitation of cerebellar Purkinje cells mediated by ibogaine elicits dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors that precedes evidence of excitotoxic neuronal degeneration. We have demonstrated striking variations in regional and subcellular distribution of inositol 1,4,5-trisphosphate receptor phosphorylation that may in¯uence normal physiological intracellular Ca 21 signaling in rat and human brain. We have further shown that the subcellular distribution of inositol 1,4,5-trisphosphate receptor phosphorylation in neurons is regulated by excitatory neurotransmission, as well as excitotoxic insult and neuronal ischemia±reperfusion. Phosphorylation dynamics of type 1 inositol 1,4,5-trisphosphate receptors may modulate intracellular Ca 21 release and in¯uence the cellular response to neurotoxic insults. q 2001 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: receptor phosphorylation, inositol 1,4,5-trisphosphate receptor, cyclic-AMP-dependent protein kinase A, ibogaine, Purkinje cells, stroke.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) couple release of endoplasmic reticulum (ER) Ca 21 stores to activation of a wide variety of cell surface receptors. Modi®cation of IP3Rs may contribute to differences in frequency, amplitude and spatial distribution of Ca 21

currents that facilitate exquisite signal±response coupling speci®city with inositol 1,4,5-trisphosphate (IP3). Cross-talk between IP3R signaling and other intracellular signaling systems, such as cyclic-AMP (cAMP)/ protein kinase A (PKA) signaling, may impact physiological events mediated by IP3. These events include, but are not limited to, cellular proliferation, development, transcription, secretion, muscular contraction, cell death, neuronal excitability, neurotransmitter release, synaptic plasticity and memory. Puri®ed IP3Rs can be phosphorylated by PKA, 6,8,28 protein kinase C, 9,27 calcium/calmodulin-dependent protein kinase type II 9 and nitric oxide (NO)/cyclic-GMPdependent protein kinase. 21,22 Puri®ed IP3R can also autophosphorylate, 10 and IP3R tyrosine phosphorylation augments calcium release by IP3. 13,16 PKA phosphorylates type 1 IP3Rs at two sites, serines 1589 and 1755 (S-1589

*Corresponding author. Tel.: 11-410-955-3024; fax: 11-410-9553623. E-mail address: [email protected] (S. H. Snyder). Abbreviations: BSA, bovine serum albumin; cAMP, cyclic-AMP; EDTA, ethylenediaminetetra-acetate; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; LTD, long-term depression; mGluR, metabotropic glutamate receptor; NO, nitric oxide; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PIPES, piperazineN,N 0 -bis(2-ethanesulfonic acid); PKA, protein kinase A; S-1589, serine 1589; S-1755, serine 1755; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline. 433

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and S-1755). 8 The importance of these phosphorylation sites is underscored by their conservation in both mouse and human type 1 IP3Rs. 12,52 Type 1 IP3R is also alternatively spliced, such that a form lacking 40 amino acids between the two PKA phosphorylation sites is enriched in peripheral tissues, while IP3Rs without this deletion predominate in the brain. 6,29,37 Peripheral type 1 IP3Rs are more heavily PKA phosphorylated at S-1589, while type 1 IP3Rs in the brain are more heavily PKA phosphorylated at S-1755. 6 Both sites, however, may be phosphorylated in vivo to some extent throughout the body. In rat cerebellar slices, for example, S-1589 is predominantly phosphorylated by cyclic-GMP-dependent protein kinase, while S-1755 is largely phosphorylated by PKA. 14 Type 1 IP3R predominates in most tissues and is particularly abundant in the cerebellum, type 2 IP3R is minimally expressed in peripheral tissues, and type 3 IP3R is enriched in the olfactory bulb of the brain. 40 S-1589 and S-1755 phosphorylation appears to be restricted to type 1 IP3R, as type 2 and 3 IP3Rs lack consensus sites for PKA phosphorylation surrounding these serine residues. However, other serines in PKA consensus sequences are present elsewhere within type 2 and 3 IP3Rs, 2,26,43 and in vivo PKA phosphorylation of these IP3Rs has been observed with different stoichiometry than type 1 IP3R. 50 Thus, PKA phosphorylation of S-1755 in type 1 IP3R is likely to modulate intracellular Ca 21 signaling in the nervous system. Initial studies in our laboratory 45 and others 34 suggested that PKA phosphorylation decreases Ca 21 release. However, it was dif®cult to discriminate the enhancement of Ca 21 pump activity by PKA phosphorylation in microsomal preparations from its in¯uences on Ca 21 release. PKA phosphorylation in intact megakaryocytes inhibits IP3-induced Ca 21 release, 48 but phosphorylation of proteins other than IP3Rs could be involved and cells of hematopoietic lineage simultaneously express multiple IP3R subtypes. 44 More de®nitive studies 28 have now established that PKA phosphorylation of type 1 IP3R increases sensitivity of Ca 21 release to IP3 by about twofold. While the role of cAMP/PKA signaling in modulating IP3-mediated intracellular Ca 21 signaling is not fully understood, it has been observed that ER Ca 21 stores are released in a wave-like regenerative manner 17 within a restricted range of cellular IP3 concentration that is widened by cAMP. 5 IP3R function may vary in different parts of neurons, with associated Ca 21 release in¯uencing local dendritic responses to neurotransmitter input and Ca 21 release in cell bodies and terminals having different roles. To monitor IP3R phosphorylation in vivo, we developed antibodies that differentiate type 1 IP3R phosphorylated at S-1755 from the unphosphorylated form. We now show differential IP3R phosphorylation in neuronal cell bodies and processes and alterations in phosphorylation dynamics associated with neuronal insults. EXPERIMENTAL PROCEDURES

PC12 cell culture, metabolic labeling and forskolin treatment PC12 rat pheochromocytoma cells were plated at three million

cells per 65-mm plate and maintained at 378C and 5% CO2 in incubation medium (Dulbecco's modi®ed Eagle's medium supplemented with 5% heat-inactivated horse serum and 10% heat-inactivated fetal bovine serum). Horse serum was dialysed overnight against 2 £ 4 l of Tris±HCl buffer (pH 7.4). At approximately 75% con¯uence, cells were washed in phosphate-free medium (1% dialysed horse serum with glutamine in phosphatefree Dulbecco's modi®ed Eagle's medium, 3 £ 5 ml) and incubated in phosphate-free medium for 30 min at 378C and 5% CO2. [ 32P]Orthophosphate (0.5 mCi) was added to each 65-mm plate and cells were incubated at 378C and 5% CO2 for 2 h. Metabolically labeled PC12 cells were exposed to 10 mM forskolin (brought up in phosphate-free incubation medium from 10 mM stock forskolin in 100% ethanol). Controls were exposed for 10 min to an equivalent volume of 0.1% ethanol in phosphatefree incubation medium. Inositol 1,4,5-trisphosphate receptor immunoprecipitation from PC12 cells Following treatment, PC12 cells were washed with 10 ml of ice-cold phosphate-buffered saline (PBS; pH 7.4) and incubated in 2 ml of NP-40 lysis buffer (50 mM b-glycerophosphate, pH 7.4, 10% glycerol, 1% NP-40, 50 mM sodium ¯uoride) with 40 mM phenylmethylsulfonyl ¯uoride, 0.2 mg/ml leupeptin, 0.2 mg/ml aprotinin and 0.2 mM EDTA for 15 min on ice with gentle rocking. Cells were transferred to 2-ml screwtop Eppendorf tubes and centrifuged at maximum speed for 15 min at 48C in an Eppendorf Centrifuge 5415c. Supernatant was incubated in new Eppendorf tubes with 10 ml of polyclonal goat antiIP3R antibody (1 mg/ml). After 2 h of tumbling at 48C, 50 ml of protein-G±Sepharose were added and tubes were tumbled for 1 h at 48C. Controls were performed by incubating samples with protein-G±Sepharose alone without IP3R antibody. Samples were centrifuged at maximum speed for 30 s at 48C in an Eppendorf Centrifuge 5415c and pellets were washed three times in 1 ml NP-40 lysis buffer, followed by one wash in 1 ml NP-40 lysis buffer containing 0.5 M NaCl. Pellet and supernatant were analysed by sodium dodecyl sulfate±polyacrylamide gel electrophoresis (SDS±PAGE) to assess purity. Sodium dodecyl sulfate±polyacrylamide gel electrophoresis and autoradiographic visualization of immunoprecipitated inositol 1,4,5-trisphosphate receptor Protein content of immunoprecipitated samples was determined with the bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Samples were boiled in 2% SDS and 50 mg of protein from each sample was electrophoretically resolved in a 7% polyacrylamide gel. The gel was dried and exposed to X-ray ®lm overnight for autoradiographic visualization of radioactively labeled IP3R. Production and af®nity puri®cation of phospho-speci®c inositol 1,4,5-trisphosphate receptor antibodies Peptides corresponding to a short amino acid sequence surrounding type I IP3R S-1755 in the rat (KPSGRRESLTSFGCONH2) were synthesized (Johns Hopkins University Peptide Synthesis Facility) and some of this peptide was chemically phosphorylated. Antibodies against either phosphorylated or unphosphorylated peptide were raised in New Zealand White rabbits injected with appropriate peptide according to established protocols (Covance, Denver, PA, USA). Later production bleeds were highly speci®c for phosphorylated/unphosphorylated peptide and phosphorylated/unphosphorylated IP3R, as assessed through dotblot procedures with progressively increasing dilutions of antibodies exposed to nitrocellulose coated with 10 mg of phosphorylated peptide, unphosphorylated peptide and a non-sense peptide control composed of the same amino acid sequence synthesized in reverse order (data not shown). Earlier production bleeds were af®nity puri®ed. Crude serum was passed over a Protein-A± Sepharose column for 1 h with repeated re-addition of the eluate. After washing the column (2 £ 5 ml of 100 mM Tris±HCl, pH

Inositol 1,4,5-trisphosphate receptors

8.0, and 2 £ 5 ml of 10 mM Tris±HCl, pH 8.0), immunoglobulin G was eluted with 10 ml of 100 mM glycine (pH 3.0). Aliquots were collected in tubes containing 0.1 ml of 1 M Tris±HCl (pH 8.0). Fractions containing peptide were combined and diluted to 10 ml with 100 mM Tris±HCl (pH 8.0) and passed at a slow ¯ow rate for 1 h through Af®gel-10 columns (Bio-Rad, Hercules, CA, USA) containing either phosphorylated or unphosphorylated peptide. Puri®ed antibodies were obtained by combining acid (4.5 ml of 100 mM glycine, pH 3.0, eluate collected 1:1 into 1 M Tris±HCl, pH 8.0) and base (4.5 ml of 100 mM tetraethylammonium, pH 11.5, eluate collected 1:1 into 1 M Tris±HCl, pH 8.0) fractions. Antibody eluate was dialysed overnight against 2 £ 2 l PBS (pH 7.4) and stored in 0.02% sodium azide at 2708C until use. Both af®nity-puri®ed and highly speci®c crude serum antibodies for phosphorylated and unphosphorylated peptides were used to immunoprecipitate IP3R from PC12 cells with 10 ml (1 mg/ml) of antibody, according to a protocol otherwise identical to that described above. SDS±PAGE of equal aliquots (20 mg/lane) of immunoprecipitated proteins and subsequent western blotting with whole IP3R antibody, as described below, further established that our phospho-speci®c antibodies recognize IP3R (data not shown). Puri®cation of inositol 1,4,5-trisphosphate receptor IP3R was puri®ed from various rat tissues as described previously from rat cerebellum, 38 with the inclusion of 0.2 mM phenylmethylsulfonyl ¯uoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin and 0.5 mM sodium orthovanadate. Protein kinase A phosphorylation of puri®ed inositol 1,4,5trisphosphate receptor Puri®ed IP3R (0.5 mg) from male Sprague±Dawley rat cerebella was incubated with PIPES/MgCl2 buffer (50 mM PIPES, pH 7.4, 10 mM MgCl2), 5 mM ATP, 5 mCi [ 32P]a-ATP (NEN Life Sciences) and 6 ml PKA catalytic subunit (Sigma, St Louis, MO, USA) in 50 ml for 20 min at 308C. IP3R was then either dissolved in 0.2 ml of 2 £ SDS solubilization buffer and aliquoted into 50ng samples or treated with alkaline phosphatase for 10 min at room temperature and then solubilized and aliquoted. Western blot with whole inositol 1,4,5-trisphosphate receptor antibody and phospho-speci®c inositol 1,4,5-trisphosphate receptor antibodies SDS±PAGE-resolved proteins (20 mg/lane) were transferred to nitrocellulose membranes, which were blocked using 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 45 min at room temperature with gentle shaking, and subsequently incubated for 2 h at room temperature with either a 1:5000 dilution of goat polyclonal antibody to whole IP3R or a 1:2000 dilution of rabbit polyclonal antibodies against either phosphorylated or unphosphorylated IP3R. Nitrocellulose membranes were then washed in 25 ml of 0.5% BSA in TBS (3 £ 5 min) and incubated for 30 min at room temperature with either a 1:15,000 dilution of horseradish peroxidase-conjugated anti-goat secondary antibody or a 1:15,000 dilution of horseradish peroxidase-conjugated anti-rabbit secondary antibody (Amersham, Arlington Heights, IL, USA). After washing in 25 ml of 0.5% BSA in TBS (3 £ 5 min), nitrocellulose blots were developed by chemiluminescence using the Renaissance kit (NEN Life Sciences, Boston, MA, USA) according to the manufacturer's protocol. Rat brain ischemia±reperfusion Focal cerebral ischemia was produced in adult male Sprague± Dawley rats (Charles River Laboratory, MA, USA) as described previously. 46 Bilateral temporary common carotid artery occlusion with cauterization of the distal portion of the right middle cerebral artery was performed, and the middle cerebral artery distal to its crossing with the inferior cerebral vein was lifted by a ®ne stainless steel hook attached to a micromanipulator. After 90 min of occlusion, carotid loops were released and animals were allowed 10 min of reperfusion before being

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transcardially perfused and prepared for immunohistochemistry as described below. Infarct area was determined with a computerbased image analyser (NIH Image version 1.59). All experiments conformed to NIH guidelines on the ethical use of animals, and all efforts were made to minimize the number of animals used and their suffering. Ibogaine treatment Ibogaine hydrochloride (NIDA) was administered intraperitoneally (i.p.) at varying doses (stock ibogaine hydrochloride, 10 mg/ml distilled water) to adult male Sprague±Dawley rats as described previously. 31 Control rats received one injection of normal saline in a volume equal to that of ibogaine administered to treated rats. Rats were allowed to survive for the times indicated after injection and then transcardially perfused and prepared for immunohistochemistry as described below. Immunohistochemistry of phosphorylated and unphosphorylated inositol 1,4,5-trisphosphate receptor in rat tissue A total of seven rats was subjected to ischemia±reperfusion as described above, and then anesthetized with sodium pentobarbital (80 mg/kg i.p.) and perfused through the left ventricle with ice-cold 4% paraformaldehyde in 0.15 M phosphate buffer (pH 7.4). Brains were post®xed in perfusion solution for 4±6 h at 48C and cryoprotected in 10% dimethylsulfoxide in PBS. Brain sections were cut at 40 mm on a freezing-sliding microtome. Adjacent sections were collected for Nissl stain to assess cytology. Immunohistochemical staining was performed on free-¯oating sections. Primary antibodies included calbindin (1:8000; Swant, Bellinzona, Switzerland), anti-OX-42 (1:1000; Serotec, Oxford, UK), anti-phosphorylated IP3R (1:3000) and anti-unphosphorylated IP3R (1:3500). Tissues were blocked for 1 h at room temperature with shaking in 0.2% Triton, 2% normal horse serum and Blotto, and then incubated in primary antibody suspended in this same solution for 24±48 h at 48C with shaking. Primary antibodies were visualized using Vectastain ABC-Elite reagents (Vector Labs, Burlingame, CA, USA) and the chromogen 3,3 0 -diaminobenzidine (Sigma, St Louis, MO, USA). Sections from control sham surgical rat brains exhibited staining for poly(ADP-ribose) identical to that seen on the non-surgical side of brains of rats subjected to unilateral ischemia±reperfusion as described above (data not shown). Human stroke tissue Formalin-®xed, paraf®n-embedded archival tissue was obtained from surgical pathology (Johns Hopkins University School of Medicine) for four cases in which CNS tissue was resected due to mass effect following an acute ischemic event. Patients included three females and one male, ages 30, 40, 60 and 69 years, who presented with symptoms of an acute ischemic event 20±40 h prior to operation. Archival tissue from temporal lobe resections for seizure disorders from two females and one male, ages 31, 34 and 41 years, provided controls. Temporal lobe specimens contained both the seizure focus, characterized by neuronal loss and extensive gliosis, and adjacent temporal neocortex that was uninvolved clinically and appeared normal by histological examination. For both ischemic and seizure cases, surgically removed tissue from the operating room was ®xed in 10% buffered formalin between 20 and 45 min after resection. In addition to surgical specimens, formalin-®xed, paraf®n-embedded post mortem brain tissue (temporal lobe including the hippocampus, cerebellum and frontal lobe) was retrieved from the Johns Hopkins Brain Research Center for three patients who died of non-neurological causes (one male and two females, ages 46, 52 and 56 years). Post mortem intervals for these cases ranged from 12 to 22 h. Surgically resected and post mortem tissues were sectioned at 4 mm, and stained by the hematoxylin and eosin method. For immunohistochemical studies, sections were deparaf®nized and subjected to antigen retrieval by either limited protein digestion or steaming (20 min at 808C). Slides were then incubated at room temperature with a 1:2000 dilution of polyclonal antibodies against either phosphorylated or unphosphorylated IP3R. Sections incubated without primary antibody served as

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Fig. 1. Detection of in vitro and in vivo IP3R phosphorylation. (A) IP3R immunoprecipitated from PC12 cells incubated with [ 32P]orthophosphate is prominently radiolabeled following forskolin (FSK) treatment to stimulate PKA. Faint radiolabeling in non-FSK-treated PC12 cells may indicate IP3R autophosphorylation or the action of endogenous protein kinases. (B) Antibodies generated against a 13-amino acid sequence containing unphosphorylated S-1755 [anti-(unP)S-1755] speci®cally recognize with western blot IP3R isolated from rat cerebellum. Antibody binding is abolished when puri®ed IP3R is phosphorylated by PKA, and is enhanced when puri®ed IP3R that has been phosphorylated by PKA is subsequently dephosphorylated by alkaline phosphatase. (C) Antibodies generated against the same 13-amino acid sequence containing phosphorylated S-1755 [anti-(P)S-1755] also speci®cally recognize with western blot IP3R isolated from rat cerebellum. Antibody binding is enhanced when puri®ed IP3R is phosphorylated by PKA, and is abolished when puri®ed IP3R that has been phosphorylated by PKA is subsequently dephosphorylated by alkaline phosphatase. The less prominently labeled band at 230,000 mol. wt (230 kDa) presumably represents an IP3R breakdown product. 49

negative controls. The higher antibody concentration required for optimal staining of human brain tissue relative to rat brain tissue may re¯ect species-speci®c differences in antigenic presentation, as well as the different techniques of antigen ®xation that were employed (transcardial perfusion with paraformaldehyde in the rat vs post mortem formaldehyde ®xation of human brain sections). Primary antibodies were detected using the avidin±biotin complex method, and 3,3 0 -diaminobenzidine served as the chromogen. RESULTS

In vivo inositol 1,4,5-trisphosphate phosphorylation at serine-1755 To ascertain whether IP3R PKA phosphorylation occurs in intact cells, we incubated PC12 cells with

[ 32P]orthophosphate and stimulated adenyl cyclase with forskolin. IP3R was subsequently immunoprecipitated and resolved through SDS±PAGE (Fig. 1A). Forskolintreated cells display a prominent radiolabeled band at about 260,000 mol. wt, where IP3R typically migrates in SDS±PAGE, as well as a less prominently labeled band at 230,000 mol. wt, thought to represent an IP3R breakdown product. We observe faint IP3R radiolabeling in cells not treated with forskolin, which may re¯ect IP3R autophosphorylation 10 or endogenous protein kinase activity. Recently, Haug et al. 14 have also detected IP3R PKA phosphorylation in cerebellar slices labeled with [ 32P]orthophosphate. We synthesized a short amino acid peptide incorporating

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Fig. 2. Western blot of rat tissue extract with phospho-speci®c antibodies. Lanes 1 and 2 serve as controls for antibody speci®city. Lane 1 (IP3R 1 PKA) is puri®ed IP3R in vitro phosphorylated by PKA. Lane 2 (IP3R 1 ALK PHOS) is puri®ed IP3R in vitro phosphorylated by PKA and subsequently dephosphorylated by alkaline phosphatase. Cerebellar (CB) IP3R is heavily phosphorylated (P) at S-1755, although a substantial amount of IP3R unphosphorylated (unP) at S-1755 exists as well. In the cerebral cortex (CORTEX), there is much less IP3R overall, and it is predominantly phosphorylated at S-1755. Cerebral cortex IP3R migrates somewhat more slowly than IP3R from other tissues. In the vas deferens (VAS DEF), aorta, thymus and lung, IP3R is predominantly unphosphorylated at S-1755. Among the peripheral tissues examined, the vas deferens contains the most IP3R, as determined by western blot with antibody to whole IP3R.

S-1755 in order to develop antisera that differentiate phosphorylated and unphosphorylated type 1 IP3Rs. Some of this peptide was chemically phosphorylated. We selected S-1755 because it is the principal if not sole site of type 1 IP3R PKA phosphorylation in the brain. 6 Antibody to phosphorylated peptide recognizes IP3R in control brain tissue, but provides markedly augmented detection of puri®ed IP3R that has been in vitro PKA phosphorylated. Antibody binding is abolished by in vitro alkaline phosphatase treatment of puri®ed IP3R (Fig. 1C). In contrast, antibody to unphosphorylated receptor recognizes IP3R most strongly following treatment with alkaline phosphatase and displays negligible recognition of PKA phosphorylated IP3R (Fig. 1B). Antisera speci®city is equivalent with crude diluted serum or af®nity-puri®ed antibody.

Untreated, puri®ed cerebellar IP3R reacts with antibodies against both phosphorylated and unphosphorylated IP3Rs, indicating substantial basal in vivo type 1 IP3R phosphorylation at S-1755 in rat cerebellum. Speci®c differentiation of phosphorylated and unphosphorylated type 1 IP3Rs with these antibodies permitted evaluation of the extent to which type 1 IP3R is PKA phosphorylated in various tissues (Fig. 2). Cerebellar type 1 IP3R is heavily phosphorylated. In the cerebral cortex, type 1 IP3R is also predominantly phosphorylated, although much less than in the cerebellum where there is a very high density of type 1 IP3Rs. By contrast, in peripheral tissues, including the vas deferens, aorta, thymus and lung, the majority of IP3Rs are not phosphorylated at S-1755. Staining with antibody against whole IP3R protein con®rms that the greatest IP3R

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Fig. 3. Type 1 IP3R phosphorylation at S-1755 in rat and human hippocampus. (A) Type 1 IP3R phosphorylated at S-1755 is enriched in cell bodies and processes of pyramidal cell neurons of rat CA1 hippocampus. CA2, CA3 and dentate gyrus granule cells display much less staining with anti-(P)S-1755 IP3R antibodies. Staining with anti-(unP)S-1755 IP3R antibodies is seen ubiquitously in pyramidal cell bodies of CA1, CA2 and CA3 rat hippocampus, as well as dentate gyrus granule cell bodies. (B) Staining with phospho-speci®c IP3R antibodies in human dentate gyrus granule cells shows a similar distribution as in the rat. Robust immunostaining is seen with anti-(unP)S-1755 IP3R antibodies, while almost no staining is seen with anti-(P)S-1755 IP3R antibodies.

density occurs in the cerebellum. This antibody may also recognize other isoforms of IP3Rs in accordance with their relative concentrations in tissue. Among peripheral tissues, IP3R levels are highest in the vas deferens. Owing to potential differences in antibody af®nity, however, the apparent relative differences in phosphorylation must be interpreted with caution. IP3R molecular weight is similar in all tissues examined, except for the cerebral cortex where the band migrates somewhat more slowly than in other tissues for reasons that are not evident.

CA1. 40 Thus, dendritic IP3R in the hippocampus is con®ned to CA1 neurons. Similar to rats, human dentate gyrus granule cells also display robust immunostaining for unphosphorylated receptor and negligible staining for phosphorylated receptor (Fig. 3). Preferential distribution of dendritic phosphorylated type 1 IP3R is not restricted to the hippocampus. In rat cerebral cortex, phosphorylated type 1 IP3R is enriched in the dendrites and cell bodies of pyramidal neurons, while unphosphorylated type 1 IP3R is largely restricted to cell bodies (Fig. 4).

Selective dendritic localizations of phosphorylated type 1 inositol 1,4,5-trisphosphate receptors

Dephosphorylation of type 1 inositol 1,4,5-trisphosphate receptors following cerebral ischemia

Immunohistochemistry with phospho-speci®c antibodies reveals marked differences in distribution of phosphorylated and unphosphorylated type 1 IP3Rs in both rat and human brain (Fig. 3). Phosphorylated receptor is greatly enriched in pyramidal bodies and dendrites throughout rat hippocampus CA1. This enrichment terminates abruptly at the CA1/CA2 border, with much less immunoreactivity in CA2, CA3 and the dentate gyrus. Under high magni®cation, we observe some phosphorylated type 1 IP3R in pyramidal cells and their processes in CA2 and CA3, but no phosphorylated receptor is seen in dentate gyrus granule cells. By contrast, the unphosphorylated type 1 IP3R occurs ubiquitously in pyramidal cell bodies but not in dendrites throughout CA1, CA2 and CA3, with a slight decrease in CA2/ CA3. Low levels are detected in dentate gyrus granule cell bodies. IP3R protein immunohistochemical mapping reveals somatic staining in hippocampal layers and the dentate gyrus, but dendritic staining occurs only in

We examined the cerebral cortex to ascertain whether ischemic insult in¯uences type 1 IP3R phosphorylation. Rats were subjected to 90 min of middle cerebral artery occlusion followed by 10 min of reperfusion. In ischemic areas of the cortex, phosphorylated type 1 IP3R density is markedly diminished, while unphosphorylated type 1 IP3R staining is substantially augmented in both cell bodies and processes (Fig. 4). We evaluated type 1 IP3R phosphorylation in the cerebral cortex of three human subjects that developed vascular stroke and compared them with histopathologically unaffected human temporal lobe cortical tissue resected from three patients with seizure disorders (Fig. 5). Control human cortex reveals a greater density of phosphorylated than unphosphorylated type 1 IP3R. As in the rat, staining for unphosphorylated type 1 IP3R is augmented in ischemic areas. The most pronounced increase in unphosphorylated compared to phosphorylated receptor is in penumbra of ischemic tissue, where cells are

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Fig. 4. Type 1 IP3R is dephosphorylated at S-1755 in rat cerebral cortical (CX) neurons after ischemia. Rat cerebral cortical neurons reveal that staining with anti-(P)S-1755 IP3R antibodies is markedly decreased in both cell bodies and dendrites, while staining with anti-(unP)S-1755 IP3R antibodies is reciprocally increased after 90 min of middle cerebral artery occlusion and 10 min of reperfusion (I/R).

hypoxic, but there is minimal cell death. Differences between ischemic and control cortex are consistent in all human specimens evaluated. Dephosphorylation of cerebellar type 1 inositol 1,4,5trisphosphate receptors following ibogaine neurotoxicity Cerebellar Purkinje cells display differential intracellular distribution between phosphorylated and unphosphorylated type 1 IP3Rs similar to that observed in the hippocampus and cerebral cortex (Figs 6, 7). Phosphorylated type 1 IP3R is abundant throughout Purkinje cell dendrites, with substantial staining in cell bodies as well. By contrast, unphosphorylated type 1 IP3R is abundant in Purkinje cell bodies but barely detectable in dendrites (Figs 6, 7). To evaluate the dynamics of Purkinje cell type 1 IP3R phosphorylation at S-1755, we employed ibogaine, a psychotomimetic stimulant drug that produces selective Purkinje cell degeneration within discrete parasagittal bands primarily in the vermis of the cerebellum. This neurotoxicity is presumably excitotoxic in origin, since ibogaine excitation of inferior olive neurons produces prolonged glutamate release from climbing ®bers and prior ablation of inferior olive neurons prevents ibogaine-induced Purkinje cell degeneration. 31 Selective Purkinje cell vulnerability to ibogaine toxicity probably re¯ects simultaneous release of glutamate at hundreds of synapses due to extremely dense termination of climbing ®bers on Purkinje cells. We treated rats with a dose of ibogaine (100 mg/kg, i.p.; Fig. 7) known to be reliable in

production of Purkinje cell degeneration. 31 At 5 h after ibogaine, phosphorylated type 1 IP3R is markedly decreased within Purkinje cells in discrete parasagittal bands and unphosphorylated type 1 IP3R is reciprocally increased, especially in dendrites (Fig. 7). Twelve hours after ibogaine, type 1 IP3R dephosphorylation is observed within the same bands in which staining is lost for calbindin, a Purkinje cell marker that is decreased after injury (Fig. 7). Microglial activation, evident by immunoreactivity with the OX-42 antibody against complement receptor 3, also increases at 12 h in areas with diminished staining for calbindin and phosphorylated type 1 IP3R (data not shown). Five hours after ibogaine, there is no change in calbindin (Fig. 7) or OX-42 (data not shown) staining, despite a pronounced decline in phosphorylated type 1 IP3R. This shift in type 1 IP3R phosphorylation at 5 h is the earliest reported sign of Purkinje cell change induced by ibogaine and may predict ensuing neuronal injury. In some areas of the cerebellum 12 h after ibogaine, we see loss of staining for both phosphorylated and unphosphorylated type 1 IP3Rs, consistent with presumed damage to these cells (data not shown). Whether loss of type 1 IP3R precedes major cellular damage and death is not clear. In patients with olivopontocerebellar atrophy, Mikoshiba and associates 18 have observed decreased IP3R in Purkinje cells that have not degenerated. We questioned whether type 1 IP3R dephosphorylation might result from increased climbing ®ber activity without Purkinje cell neurotoxicity. Doses of 10 mg/kg of ibogaine, which do not kill Purkinje cells, produce

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Fig. 5. Type 1 IP3R is dephosphorylated at S-1755 in human cerebral cortical neurons within ischemic brain and penumbra. Staining with phospho-speci®c IP3R antibodies in human cerebral cortical neurons reveals similar phosphorylation dynamics at S-1755 after ischemia as in the rat. Human temporal lobe control (CTRL) cortical tissue, from temporal lobe resections for seizure disorders in which temporal neocortex was not involved, shows greater density of phosphorylated [A; IP3R (P)S-1755] than non-phosphorylated [B; (IP3R (unP)S-1755] IP3R at S-1755. Human temporal lobe cortical tissue which was resected due to mass effect following an acute ischemic insult (STROKE), however, shows augmented staining for unphosphorylated IP3R in cell bodies and proximal dendrites [D; IP3R (unP)S-1755] without a dramatic decrease in staining for phosphorylated IP3R [C; IP3R (P)S-1755]. This increase in unphosphorylated compared to phosphorylated IP3R is most pronounced in the penumbra of ischemic tissue (E, F).

readily detectable Purkinje cell type 1 IP3R dephosphorylation (data not shown). Thus, type 1 IP3R dephosphorylation at S-1755 in rat cerebellar Purkinje cells re¯ects increased excitatory synaptic activity as well as excitotoxic insult. DISCUSSION

Our discrimination of PKA phosphorylated and unphosphorylated type 1 IP3Rs at S-1755 reveals notable

distinctions between brain and periphery, with speci®c variations in IP3R phosphorylation in different parts of neurons. We have also discovered alterations in phosphorylation associated with increased neuronal activity ranging from augmented excitatory synaptic activity to excitotoxic insult. Type 1 IP3R phosphorylated at S-1755 is greatly enriched in the brain, while unphosphorylated receptor predominates in several peripheral tissues. These ®ndings in intact tissues con®rm our earlier ®ndings with puri®ed IP3R revealing preferential

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Fig. 6. Type 1 IP3R phosphorylation at S-1755 in rat cerebellar Purkinje neurons. Type 1 IP3R phosphorylated at S-1755 [IP3R (P)S1755] exhibits abundant immunostaining throughout dendrites and cell bodies of rat cerebellar Purkinje neurons. Unphosphorylated type 1 IP3R [IP3R (unP)S-1755] is enriched in Purkinje cell bodies and minimally present in processes.

phosphorylation on S-1755 of brain IP3R and S-1589 of peripheral IP3R. 6 Peripheral IP3R is alternatively spliced such that 40 amino acids are deleted between S-1755 and S-1589. Presumably, this deletion alters IP3R conformation and changes the phosphorylation dynamics at these two sites. Phosphorylation of S-1755 augments type 1 IP3R-mediated Ca 21 release. 28 Therefore, differences in phosphorylation status of central and peripheral receptors may impact IP3-induced intracellular Ca 21 release with tissue speci®city. Phosphorylated type 1 IP3R predominates in dendritic processes in several brain regions. Since IP3Rs regulate intracellular Ca 21 release following synaptic activity, phosphorylation of dendritic type 1 IP3R ought to in¯uence synaptic signaling. Our ®nding of preferential distribution of phosphorylated type 1 IP3Rs in dendrites and unphosphorylated receptors in neuronal cell bodies and proximal dendrites suggests that cAMP-mediated potentiation of IP3R-mediated Ca 21 release may be an important regulator of synaptic neurotransmission. Variations in type 1 IP3R phosphorylation between dendrites and cell bodies may be relevant to a large body of work showing intracellular variations in Ca 21 signaling. Although we do not know the precise role of IP3R phosphorylation dynamics in synaptic signaling, our observation that increased climbing ®ber activity

leads to Purkinje cell type 1 IP3R dephosphorylation implies that excitatory synaptic input modulates intracellular dendritic Ca 21 signaling. It has been shown previously that functional postsynaptic compartments in cerebellar Purkinje cell spines and dendrites exist through attachment of neuronal IP3Rs to metabotropic glutamate receptors (mGluRs). 49 Long-term depression (LTD), a form of spatially restricted cerebellar synaptic plasticity at the glutamatergic parallel ®ber±Purkinje cell synapse, requires simultaneous mGluR and IP3R activation. 24 Cells from mutant mice lacking IP3Rs exhibit marked impairment of LTD. 15 In normal animals, each parallel ®ber stimulus produces subthreshold amounts of IP3, so that ER Ca 21 release suf®cient to establish LTD requires repetitive synaptic activity mediated by localized mGluRs. 11 Activation of postsynaptic IP3Rs ranges from individual spines to small spinodendritic compartments depending on stimulation frequency. 11,47 Thus, local postsynaptic IP3 signaling in¯uences spatial encoding of information in neuronal dendrites and phosphorylation dynamics of type 1 IP3R could in¯uence neuronal plasticity. IP3R signaling may also be relevant to neuronal cell death. The distribution of neurons most vulnerable to CNS hypoxia, such as cerebellar Purkinje cells, CA1 hippocampus pyramidal neurons, neurons in cortical

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Fig. 7. Type 1 IP3R is dephosphorylated at S-1755 in cerebellar Purkinje dendrites following ibogaine stimulation. At 12 h after excitotoxic injury to Purkinje neurons with 100 mg/kg ibogaine (i.p.), a decrease in S-1755 phosphorylated type 1 IP3R immunostaining [IP3R (P)S-1755] is seen in dendrites and cell bodies. The unphosphorylated form is increased in the same locations, as shown in adjacent sections. This shift occurs in parasagittal bands within the vermis of rat cerebellum at both 5 h (arrows) and 12 h (arrowheads) after ibogaine treatment. This shift in phosphorylation at 12 h is observed within the same parasagittal zones in which staining for the Purkinje cell marker calbindin is lost (arrowheads), a sign of Purkinje cell injury. At 5 h after ibogaine, before loss of calbindin staining is seen, immunostaining for S-1755 phosphorylated IP3R [IP3R (P)S-1755] is markedly reduced in parasagittal zones and immunostaining for S-1755 unphosphorylated IP3R is reciprocally increased (arrows).

layers III, V and VI, and small- and medium-sized striatal neurons, 23,41 corresponds to brain regions of greatest IP3R density. 30,39,42,51 We observe dephosphorylation at S1755 of cerebral cortical type 1 IP3R in pyramidal cells in the early phase of reperfusion following in vivo focal hypoxia elicited by middle cerebral artery occlusion. Middle cerebral artery occlusion produces massive increases in extracellular glutamate, whose excitotoxic actions may lead to IP3R dephosphorylation. While we do not know the precise in vivo mechanisms or consequences of type 1 IP3R dephosphorylation at S-1755 following neuronal excitation, we speculate that dephosphorylation affords neuroprotection by diminishing intracellular Ca 21 release by type 1 IP3R. While dendritic IP3R may be basally phosphorylated at S-1755 in order to potentiate intracellular Ca 21 release in normal synaptic signaling, dephosphorylation in response to excitotoxic signaling might delay excessive Ca 21 release and subsequent activation of Ca 21-sensitive proteases and DNAases that contribute to cell death. We also observe type 1 IP3R dephosphorylation at S-1755 in human stroke brain. This dephosphorylation is most pronounced in the penumbra of human stroke tissue, supporting the notion that receptor dephosphorylation related to synaptic activation depends on neuronal activity with intact cellular metabolism. Toxic doses of ibogaine elicit dephosphorylation of dendritic Purkinje cell type 1 IP3R long before cells exhibit signs of injury, and dephosphorylation is also observed at lower doses of ibogaine that stimulate the

inferior olive without killing Purkinje neurons. The large range of synaptic activity over which type 1 IP3R dephosphorylation occurs suggests a broad spectrum of synaptic modulation of IP3R function, ranging from normal physiological signaling to glutamatergic excitotoxicity. As a precedent for a continuum of glutamatergic synaptic activity ranging from basal synaptic transmission to excitotoxicity, we have recently shown that basal N-methyl-d-aspartate/NO-mediated glutamatergic neurotransmission in rat brain elicits substantial basal DNA damage that is exacerbated with augmentation and decreased with inhibition of N-methyl-d-aspartate/NOmediated synaptic activity. 33 This same signaling system mediates excitotoxicity following cerebral cortical hypoxia. 32 IP3R dephosphorylation may be an early indication of transition of excitatory synaptic signaling from normal physiology to excitotoxicity. Dephosphorylation in response to neurotoxic insult may in¯uence cellular survival strategies by limiting elevation of intracellular Ca 21 levels. Since type 1 IP3R S-1755 is predominantly phosphorylated by PKA in cerebellar brain tissue, 14 the phosphorylation dynamics that we describe here further substantiate a physiological link between cAMP- and IP3R-mediated Ca 21 release mechanisms. Associations between cAMP and Ca 21 second messenger systems have been recognized for over 25 years, 1,35,36 and there are numerous physiological instances in which selective phosphorylation in¯uences IP3 signaling speci®city. For example, activation of platelet a-2A adrenergic receptors

Inositol 1,4,5-trisphosphate receptors

relieves basal cAMP/PKA-mediated suppression of IP3dependent Ca 21 release, 20 and frog esophageal mucociliary epithelial ciliary beating is maintained through oscillatory Ca 21 signaling controlled by cAMP/PKA and IP3. 3 Speci®c targeting of PKA to subcellular locations, 7 including the ER, probably contributes to IP3/ Ca 21 ±cAMP/PKA signaling speci®city, so that agonist response may be speci®ed by subcellular spatial distribution of IP3Rs and PKA. A variety of receptor systems activates phospholipase C leading to IP3 production, while simultaneously modulating adenylyl cyclase

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activity leading to changes in cAMP concentration. 4 Receptor-mediated effects on these signaling systems vary between receptors, cell types and species, and cAMP may potentiate or inhibit agonist-induced intracellular Ca 21 elevation in different tissues. 19,25 AcknowledgementsÐThis work was supported by USPHS grants MH18501 (to S.H.S.), Research Scientist Award DA00074 (to S.H.S.), DA08692 (to M.E.M.), DA00225 (to E.O'H.), National Institutes of Health Grant NS32017 (to J.H.G.) and National Institute of Mental Health Training Grant MH418 (to A.A.P.).

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