Rolf Stricker

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor.

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.

Protease-activated receptor-1 protects rat astrocytes from apoptotic cell death via JNK-mediated release of the chemokine GRO/CINC-1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin‐induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up‐regulated secretion of the chemokine growth‐regulated oncogene/cytokine‐induced neutrophil chemoattractant‐1 (GRO/CINC‐1) in primary rat astrocytes in a concentration‐dependent manner. However, we found no increase of interleukin (IL)‐6, IL‐1β and tumour necrosis factor‐α secretion. Protease‐activated receptor 1 (PAR‐1)‐induced GRO/CINC‐1 release was mainly mediated by c‐Jun N‐terminal kinase (JNK) activation. Extracellular signal‐regulated kinase 1/2 might be partially involved, but not p38 mitogen‐activated protein kinase. Further studies demonstrated that PAR‐1 activation, as well as application of recombinant GRO/CINC‐1, protected astrocytes from C2‐ceramide‐induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB‐332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR‐1. These results demonstrate for the first time that PAR‐1 plays an important role in anti‐apoptosis in the brain by regulating the release of chemokine GRO/CINC‐1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.

Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.

Jab1, a Novel Protease-activated Receptor-2 (PAR-2)-interacting Protein, Is Involved in PAR-2-induced Activation of Activator Protein-1

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor for trypsin and tryptase, exerts important physiological and pathological functions in multiple systems. However, unlike PAR-1, the PAR-2-mediated intracellular signal transductions are hardly known. Here, using yeast two-hybrid screening with a human brain cDNA library, we identified an interacting partner of human PAR-2, the Jun activation domain-binding protein 1 (Jab1). The interaction was confirmed by glutathione S-transferase pull-down assays in vitro, and by co-immunoprecipitation assays in vivo. Jab1 was also shown to be colocalized with PAR-2 in both transfected HEK293 cells and in normal primary human astrocytes by double immunofluorescence staining. Further experiments demonstrated that multiple intracellular domains of PAR-2 are required for the interaction with Jab1. We then showed that agonist stimulation of PAR-2 disrupted the interaction, which could be prevented by the inhibitor of receptor endocytosis phenylarsine oxide, but not by the lysosomal protease inhibitor ZPAD. Importantly, we found that activation of PAR-2 induced the redistribution of Jab1 from the plasma membrane to the cytosol, but did not influence expression of Jab1. Furthermore, Jab1 mediated PAR-2-induced c-Jun activation, which was followed by increased activation of activator protein-1. Loss-of-function studies, using Jab1 small interfering RNA, demonstrated that Jab1 knockdown blocked PAR-2-induced activator protein-1 activation. Taken together, our data demonstrate that Jab1 is an important effector that mediates a novel signal transduction pathway for PAR-2-dependent gene expression.

Cellular expression and subcellular localization of the human Ins(1,3,4,5)P4-binding protein, p42IP4, in human brain and in neuronal cells

In this study we describe for the human inositol-(1,3,4,5)-tetrakisphosphate (InsP4)-binding protein, p42IP4, the cellular distribution and subcellular localization in human brain and in transfected neuronal cells. The cDNA sequence of the human p42IP4 containing a single open reading frame yields a peptide of 374 amino acids with a calculated molecular mass of 43.4 kDa with a zinc-finger motif at the N-terminus, followed by two pleckstrin homology (PH) domains. Using a peptide-specific antiserum, p42IP4 protein was localized in a majority of neuronal cells of human brain sections. In the hypothalamus a subpopulation of paraventricular and infundibular nucleus neurons were strongly immunoreactive for p42IP4. In cortical areas the protein was predominantly found in large pyramidal cells. Some immunoreactivity for p42IP4 was also observed in the pyramidal cells of the hippocampal formation. Functional expression of p42IP4 protein in neuronal (NG108-15) and non-neuronal (CHO-K1) cells stably transfected with GFP-p42IP4 was shown in all cell fractions (homogenate, cytosol and membranes) by specific [3H]Ins(1,3,4,5)P4 binding activity, which correlated with p42IP4 protein detection by Western blot analysis. Using confocal laser scanning microscopy we showed that in NG108-15 and CHO-K1 cells stably transfected with GFP-p42IP4 the full length p42IP4 protein was localized in the cytoplasm, at the plasma membrane and in the nucleus. A deletion mutant of p42IP4 lacking the zinc finger domain resulted in solely a cytosolic and membrane localization but was not found in the nucleus. Thus we can conclude that human p42IP4 shows a region-specific localization in the human brain and the zinc finger motif within the protein is responsible for the localization of the protein in the cell nucleus.

IDENTIFICATION OF RAT BRAIN P42IP4, A HIGH-AFFINITY INOSITOL(1,3,4,5)TETRAKISPHOSPHATE/ PHOSPHATIDYLINOSITOL(3,4,5)TRISPHOSPHATE BINDING PROTEIN

Inositol(1,3,4,5)tetrakisphosphate (InsP4) and phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) are two potential second messengers with a still largely unknown mode of action. We recently cloned the 42 kDa protein p42IP4 previously purified from pig cerebellum, which binds InsP4 (Kd approximately 2 nM) and PtdInsP3 with comparable affinities (Stricker et al., FEBS Lett. 405 (1997) 229). The protein p42IP4 (pig) is highly homologous to centaurin-alpha, a larger protein of 46 kDa, derived from a rat brain cDNA library clone (Hammonds-Odie et al., J. Biol. Chem. 271 (1996) 18859). Here we investigated whether also p42IP4 is expressed in rat brain and how it might be related to centaurin-alpha. When we carried out RT-PCR using mRNA from brain of rats of different ages we obtained several clones corresponding to p42IP4, but not to centaurin-alpha. The existence of p42IP4 in rat brain is supported by the following findings: (1) biochemical analysis of the purified rat brain protein shows inositol phosphate ligand affinities identical to those of the protein from other species; (2) Western blot analysis of rat brain membrane fractions using a peptide-specific antiserum revealed only the 42 kDa protein (p42IP4), but did not give evidence for the occurrence of a larger 46 kDa centaurin-alpha-like protein in rat brain; and (3) the amino acid sequences deduced from p42IP4 cDNA are highly homologous in several species and are confirmed by protein fragment microsequences. Thus, p42IP4 from rat brain which has two pleckstrin homology domains is a protein largely conserved between different species and most likely has an important function in inositol phosphate or inositol lipid signal transduction.

Reduced neuronal co-localisation of nardilysin and the putative α-secretases ADAM10 and ADAM17 in Alzheimer’s disease and Down syndrome brains

The peptidase nardilysin is involved in degradation of neuropeptides and limited intracellular proteolysis. Recent reports point to an involvement of nardilysin in the pathophysiology of Alzheimer’s disease. Nardilysin enhances the α-secretase activity of the disintegrin and metalloproteases (ADAMs) 10 and 17, thereby possibly contributing to reduced generation of amyloidogenic fragments from the amyloid precursor protein. A prerequisite for the α-secretase-stimulating effect of nardilysin on the activity of ADAMs in vivo is cellular co-expression of nardilysin with ADAM10 and/or ADAM17. We immunolocalised nardilysin, ADAM10, and ADAM17 in cortical regions of normal aged brain, in Alzheimer’s disease, and in Down syndrome brains and counted the number of protease-expressing neurons. A considerable portion of neurons co-express nardilysin together with either ADAM10 or ADAM17. Compared to controls, in Alzheimer’s disease and in Down syndrome brains there is a decreased cellular expression of all three antigens, and a reduction in the number of those neurons that co-express nardilysin with ADAM10 or with ADAM17. Our data are consistent with the notion that the proposed α-secretase-enhancing activity of nardilysin might play a role in human brain pathology.

Interaction of the brain-specific protein p42IP4/centaurin-α1 with the peptidase nardilysin is regulated by the cognate ligands of p42IP4, PtdIns(3,4,5)P3 and Ins(1,3,4,5)P4, with stereospecificity

The brain‐specific protein p42IP4, also called centaurin‐α1, specifically binds phosphatidylinositol 3,4,5‐trisphosphate [PtdIns(3,4,5)P3] and inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4]. Here, we investigate the interaction of p42IP4/centaurin‐α1 with nardilysin (NRDc), a member of the M16 family of zinc metalloendopeptidases. Members of this peptidase family exhibit enzymatic activity and also act as receptors for other proteins. We found that p42IP4/centaurin‐α1 binds specifically to NRDc from rat brain. We further detected that centaurin‐α2, a protein that is highly homologuous to p42IP4/centaurin‐α1 and expressed ubiquitously, also binds to NRDc. In vivo interaction was demonstrated by co‐immunoprecipitation of p42IP4/centaurin‐α1 with NRDc from rat brain. The acidic domain of NRDc (NRDc‐AD), which does not participate in catalysis, is sufficient for the protein interaction with p42IP4. Interestingly, preincubation of p42IP4 with its cognate ligands d‐Ins(1,3,4,5)P4 and the lipid diC8PtdIns(3,4,5)P3 negatively modulates the interaction between the two proteins. d‐Ins(1,3,4,5)P4 and diC8PtdIns(3,4,5)P3 suppress the interaction with virtually identical concentration dependencies. This inhibition is highly ligand specific. The enantiomer l‐Ins(1,3,4,5)P4 is not effective. Similarly, the phosphoinositides diC8PtdIns(3,4)P2, diC8PtdIns(3,5)P2 and diC8PtdIns(4,5)P2 all have no influence on the interaction. Further experiments revealed that endogenous p42IP4 from rat brain binds to glutathione‐S‐transferase (GST)‐NRDc‐AD. The proteins dissociate from each other when incubated with d‐Ins(1,3,4,5)P4, but not with inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3]. In summary, we demonstrate that p42IP4 binds to NRDc via the NRDc‐AD, and that this interaction is controlled by the cognate cellular ligands of p42IP4/centaurin‐α1. Thus, specific ligands of p42IP4 can modulate the recruitment of proteins, which are docked to p42IP4, to specific cellular compartments.

The brain‐specific protein, p42IP4 (ADAP 1) is localized in mitochondria and involved in regulation of mitochondrial Ca2+

In brain, p42IP4 (centaurin‐α1; recently named ADAP 1, which signifies ADP ribosylation factor GTPase activating protein with dual PH domains 1, within the large family of Arf‐GTPase activating proteins) is mainly expressed in neurons. p42IP4 operates as a dual receptor recognising two second messengers, the soluble inositol(1,3,4,5)tetrakisphosphate and the lipid phosphatidylinositol(3,4,5)trisphosphate. We show here for the first time that p42IP4 is localized in mitochondria, isolated from rat brain and from cells transfected with p42IP4. In rat brain mitochondria we additionally found interaction of p42IP4 with 2′, 3′‐cyclic nucleotide 3′‐phosphodiesterase and α‐tubulin by pull‐down binding assay and by immunoprecipitation. In mitochondria from Chinese hamster ovary cells, p42IP4 is predominantly associated with the intermembrane space and the inner membrane. This localization of p42IP4 indicates that p42IP4 might have a still unknown mitochondrial function. We studied whether p42IP4 is involved in Ca2+‐induced permeability transition pore opening, which is important in mitochondrial events leading to programmed cell death. We used mouse neuroblastoma cells as a model for the functional studies of p42IP4 in mitochondria. In mitochondria isolated from p42IP4‐transfected mouse neuroblastoma cells, over‐expression of p42IP4 significantly decreased Ca2+ capacity and lag time for Ca2+ retention. Thus, we suggest that p42IP4 is involved in the regulation of Ca2+ transport in mitochondria. We propose that p42IP4 promotes Ca2+‐induced permeability transition pore opening and thus destabilizes mitochondria.

Extracellular α-crystallin protects astrocytes from cell death through activation of MAPK, PI3K/Akt signaling pathway and blockade of ROS release from mitochondria

α-Crystallin with two isoforms, αA-crystallin (HSPB4) and αB-crystallin (HSPB5), is found in eye lens, spleen, lung, kidney, cornea, skin, but also in brain. Several studies revealed roles of αA/αB-crystallin in regulating cell viability and protection in the central nervous system. We previously demonstrated that α-crystallin serves as an intracellular protectant in astrocytes. Compared to well-studied intracellular functions of α-crystallin, there is limited proof for the role of α-crystallin as extracellular protectant. In order to clarify protective effects of extracellular αA/αB-crystallin, we exposed astrocytes to the toxic agents, staurosporine or C2-ceramide, or serum-starvation in the presence of αA/αB-crystallin. Extracellular αA/αB-crystallin protected astrocytes from staurosporine- and C2-ceramide-induced cell death. In addition, extracellular αB-crystallin/HSPB5 effectively promoted astrocytes viability through phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinases (JNK) signaling pathways under serum-deprivation. Furthermore, αB-crystallin/HSPB5 decreases the staurosporine-mediated cleavage of caspase 3 through PI3K/Akt signaling preventing apoptosis of astrocytes. Thus, the current study indicates that extracellular αA/αB-crystallin protects astrocytes exposed to various harmful stimuli. Furthermore, application of αB-crystallin/HSPB5 to isolated rat brain mitochondria inhibits ROS generation induced by complex III inhibition with Antimycin A.