Parkin deficiency disrupts calcium homeostasis by modulating phospholipase C signalling Anna Sandebring 1,2, *, Nodi Dehvari 1, *, Monica Perez-Manso 1 , Kelly Jean Thomas 2 , Elena Karpilovski 1 , Mark R. Cookson 2 , Richard F. Cowburn 1,3 and Angel Cedazo-Mı ´ nguez 1 1 Karolinska Institutet, Department of NVS, KI-Alzheimer’s Disease Research Center, Stockholm, Sweden 2 Laboratory of Neurogenetics, National Institute on Aging ⁄ NIH, Bethesda, MD, USA 3 AstraZeneca R&D, Local Discovery RA CNS & Pain Control, Disease Biology, So ¨ derta ¨ lje, Sweden Introduction Parkinson’s disease (PD) is a neurodegenerative disor- der involving cell loss in various brain regions, espe- cially dopamine neurons in the substantia nigra pars compacta (snpc) [1]. In recent years, mechanism of action studies on gene mutations causing rare familial forms of disease have provided important new insights into PD pathogenesis. Of the familial PD genes, mutations in parkin are the most common cause of autosomal-recessive juvenile parkinsonism (ARJP). Parkin is an E3 ubiquitin ligase in which mutations have been shown to alter the level, activity, aggregation or localization of its substrates. Some studies have proposed parkin deficiency- related consequences for intracellular signalling, Keywords autosomal recessive juvenile Parkinsonism; calcium; parkin; Parkinson’s disease; phospholipase C Correspondence A. Cedazo-Minguez, Karolinska Institutet, Department of NVS, KI-Alzheimer’s Disease Research Center, NOVUM Floor 5, 141 57 Huddinge, Sweden Fax: +46 8 585 83880 Tel: +46 8 585 83751 E-mail: Angel.Cedazo-Minguez@ki.se *These authors contributed equally to this work (Received 9 April 2009, revised 24 June 2009, accepted 7 July 2009) doi:10.1111/j.1742-4658.2009.07201.x Mutations in the E3 ubiquitin ligase parkin cause early-onset, autosomal- recessive juvenile parkinsonism (AJRP), presumably as a result of a lack of function that alters the level, activity, aggregation or localization of its sub- strates. Recently, we have reported that phospholipase Cc1 is a substrate for parkin. In this article, we show that parkin mutants and siRNA parkin knockdown cells possess enhanced levels of phospholipase Cc1 phosphory- lation, basal phosphoinositide hydrolysis and intracellular Ca 2+ concentra- tion. The protein levels of Ca 2+ -regulated protein kinase Ca were decreased in AJRP parkin mutant cells. Neomycin and dantrolene both decreased the intracellular Ca 2+ levels in parkin mutants in comparison with those seen in wild-type parkin cells, suggesting that the differences were a consequence of altered phospholipase C activity. The protection of wild-type parkin against 6-hydroxydopamine (6OHDA) toxicity was also established in ARJP mutants on pretreatment with dantrolene, implying that a balancing Ca 2+ release from ryanodine-sensitive stores decreases the toxic effects of 6OHDA. Our findings suggest that parkin is an important factor for maintaining Ca 2+ homeostasis and that parkin deficiency leads to a phospholipase C-dependent increase in intracellular Ca 2+ levels, which make cells more vulnerable to neurotoxins, such as 6OHDA. Abbreviations 6OHDA, 6-hydroxydopamine; ARJP, autosomal-recessive juvenile parkinsonism; [Ca 2+ ] i, intracellular Ca 2+ concentration; DAG, diacylglycerol; EGF, epidermal growth factor; ER, endoplasmic reticulum; Fluo-3AM, Fluo-3-acetoxymethyl; IP 3 , inositol 1,4,5-trisphosphate; KHB ⁄ Li, Krebs– Henseleit bicarbonate buffer ⁄ LiCl; KO, knockout; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide; NT, nontransfected; Pael- R, parkin-associated endothelial-like receptor; PD, Parkinson’s disease; PI, , phosphoinositide; PINK1, PTEN-induced kinase-1; PKC, protein kinase C; PLCc1, phospholipase Cc1; PLSD, protected least-significant difference; PS1, presenilin 1; RyR, ryanodine receptors; snpc, substantia nigra pars compacta; TG, thapsigargin. FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS 5041 including altered apoptotic stress-activated protein kinase signalling [2]. Parkin has also been suggested to promote Akt signalling by preventing the endocytosis and trafficking of the epidermal growth factor (EGF) receptor via proteasome-independent ubiquitination of the parkin substrate Eps15 [3]. In the same signalling pathway, we have found that parkin interacts with and ubiquitinates phospholipase Cc1 (PLCc1). Although mutant parkin interacts with PLCc1, it shows less potency to ubiquitinate this substrate than does wild- type (WT) parkin, and PLCc1 levels are enhanced in brain homogenates from parkin knockout (KO) mice [4]. Parkin has also been shown to protect cells against damage induced by several agents, including dopamine [5], ceramide [6] and a mutant of a-synuclein [7], the major component of Lewy body inclusions, a patho- logical hallmark of PD. In parallel, a number of differ- ent pathways have been proposed by which parkin mutations and deficiency can induce cellular toxicity [8]. Several studies have demonstrated that parkin is protective against oxidative stress and is important for the maintenance of mitochondrial morphology and function (reviewed in [9]). Whether the protective effects of parkin are a result of its E3 ligase activity or of additional functions of the protein remains unknown at present. In support of the first idea, parkin has been shown to protect against the neuro- toxicity induced by unfolded protein stress, suggesting that its function in the ubiquitination pathway may be to target for degradation of misfolded proteins derived from the endoplasmic reticulum (ER) [10,11]. The ER plays a pivotal role in the processing and folding of proteins, as well as in the regulation of calcium (Ca 2+ ) homeostasis. The ER stress response interferes with the role of the ER as both a protein factory and a Ca 2+ storage organelle, and excessively high intracellu- lar Ca 2+ concentrations ([Ca 2+ ] i ) can initiate apopto- sis [12]. In contrast, low Ca 2+ levels induce the ER stress response by promoting the accumulation of ER chaperones and Ca 2+ transporting proteins [13]. One important phenotypic trait that distinguishes snpc dopaminergic neurons is that they are autonomously active and require a constant clearance of Ca 2+ , compared with other neurons that are activated by synaptic input. In addition, dopaminergic neurons rely on L-type Ca 2+ channels [14], whereas the activity of other neuron types mainly depends on Na + channels. Thus, snpc dopaminergic neurons have unique features that may make them more vulnerable to disrupted calcium homeostasis [15]. Indeed, Ca 2+ toxicity has been a subject of interest in neurodegenerative patho- genesis, including PD, for many years [16–18], and there is some evidence that the use of Ca 2+ channel blockers may even reduce the risk of disease [19]. Two of the parkin identified substrates, parkin-associated endothelial-like receptor (Pael-R) [11] and PLCc1 [4], are known to be involved in the regulation of [Ca 2+ ] i [20]. It is therefore possible that an impairment of the parkin substrate-dependent regulation of [Ca 2+ ] i could be part of the mechanism by which parkin mutations lead to ARJP. In the present study, we show that PLC signalling is altered in parkin-deficient human neuroblastoma cell lines, resulting in a disrupted [Ca 2+ ] homeostasis and increased vulnerability to 6-hydroxydopamine (6OHDA). We also show that blocking of either PLC activity or ryanodine receptors (RyR) can reverse these effects. Results Effects of parkin deficiency on PLCc1 activation, phosphoinositide (PI) hydrolysis and [Ca 2+ ] i In order to address the functional role of the parkin ubiquitination of PLCc1, we investigated PLC activity in human neuroblastoma SH-SY5Y cell lines stably transfected with either WT or mutant R42P or G328E parkin. We chose to utilize human neuroblastoma cell lines as parkin KO in rodents does not result in the key pathological events seen in humans, such as dopa- minergic cell death in snpc and substantial motor impairment [21]. Moreover, some of the experiments required the stable expression of exogenous parkin. The protein levels of parkin and PLCc1 were the same as those published previously [4]. The treatment of cells with EGF leads to the direct phosphorylation and activation of PLCc1 via the EGF receptor [22]. In our hands, treatment of SH-SY5Y cells with EGF gave a significantly higher phosphorylation of PLCc1 in R42P and G328E parkin mutants when compared with both nontransfected (NT) and WT parkin cells (Fig. 1). PLC activation leads to PI hydrolysis, resulting in diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP 3 ), the latter being an important intracellular second messenger for the control of ER Ca 2+ levels [23]. We therefore determined whether the between cell type dif- ferences in PLCc1 phosphorylation were reflected at the level of PI hydrolysis. Our results showed that basal PI hydrolysis was also significantly higher in both R42P and G328E parkin mutants when com- pared with NT and WT parkin cells (Fig. 2A). In addi- tion, siRNA knockdown of endogenous parkin in NT SH-SY5Y cells gave a similar increase in basal PI hydrolysis as that seen in parkin mutant cells Parkin deficiency disrupts calcium homeostasis A. Sandebring et al. 5042 FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS (Fig. 2B). Moreover, we tested for the consequences of knocking down c-Cbl, another known E3 ligase for PLCc1 [24]. Similar to the effects seen with parkin knockdown, siRNA knockdown of c-Cbl gave signifi- cantly increased basal PI hydrolysis (Fig. 2B). Parkin and c-Cbl siRNA knockdowns were verified by immu- noblotting (Fig. 2B). We next investigated the effects of parkin and parkin R42P and G328E mutations on [Ca 2+ ] i .As shown in Fig. 3A, both R42P and G328E mutant cells possessed significantly higher basal [Ca 2+ ] i when com- pared with NT and WT parkin transfected cells. Simi- larly, knockdown of parkin or c-Cbl expression by siRNA also resulted in significantly higher basal [Ca 2+ ] i when compared with control cells (Fig. 3B). These between cell differences were found when mea- surements were performed in either Ca 2+ -free NaCl ⁄ P i or MEM buffer (Fig. 3A,B). Increased cytosolic Ca 2+ levels in ARJP parkin mutants are a result of altered PLC activity We next investigated whether the primary cause of the increased basal [Ca 2+ ] i levels seen in ARJP parkin mutant cells resulted from increased PLC activity or from altered Ca 2+ influx from extracellular sources. Treatment with the PLC inhibitor neomycin (500 lm) reduced basal [Ca 2+ ] i in R42P and G328E cells to the estimated levels in WT parkin cells (Fig. 4A). In addi- tion, the RyR antagonist dantrolene (10 lm) gave a similar reversal of the R42P and G328E mutant Ca 2+ levels, indicating that altered levels were a result of a subsequent increased Ca 2+ -induced Ca 2+ release response in these cells (Fig. 4A). Blocking of either L-type or N-type Ca 2+ channels with nimodipine (1 lm) and x-conotoxin (1 lm) did not change [Ca 2+ ] i in any of the cell types (Fig. 4B,C), confirming that the observed differences were a result of altered intracellular Ca 2+ handling. Treatment of cells with thapsigargin (TG) (50 nm), an inhibitor of the ER Ca 2+ pump (SERCA) that depletes intracellular Ca 2+ stores [25], gave a rapid Ratio of + EGF NT PLCγ1 pTyr783 EGF NT WT R42P G328E –+ –+ –+ –+ * # # * 0 1 2 3 4 5 6 7 PLCγ1 pTyr783 β-Actin Fig. 1. EGF-mediated PLCc1 phosphorylation is increased in ARJP parkin cell lines. Immunoblotting of phosphoTyr783-PLCc1inNT and in stably transfected human SH-SY5Y neuroblastoma cells with WT parkin and the ARJP parkin mutations R42P and G328E treated or untreated with EGF for 2 min. Histogram shows the quantifica- tion (mean ± SEM) of phosphoTyr783-PLCc1 normalized to actin from five independent experiments. *P < 0.05 ANOVA, Fisher’s post-hoc test for the comparison of treated versus basal. #P < 0.05 ANOVA, Fisher’s post-hoc test for the comparison of treated condi- tion versus treated NT cells. AB Fig. 2. PI hydrolysis is enhanced in parkin-deficient cell lines. Histograms show means ± SEM of PI hydrolysis measured in basal conditions of NT, parkin WT, R42P and G328E transfected cells (n = 5) (A) and in the different siRNA-transfected cells (n = 3) (B). Parkin and c-Cbl protein levels were detected by western blot analysis in SH-SY5Y neuroblastoma cells after siRNA knockdown of parkin and c-Cbl. A. Sandebring et al. Parkin deficiency disrupts calcium homeostasis FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS 5043 increase in [Ca 2+ ] i in all cells, followed by a slow increase that reached a plateau during the time of exposure. The shapes of the TG-induced Ca 2+ curves were parallel in all cell types, indicating that the between cell type differences were attributable to differences in basal [Ca 2+ ] i (Fig. 4D). We have demon- strated previously that PLCb levels are not altered by the overexpression of WT parkin or R42P and G328E parkin mutants [4]. In this study, we investigated whether changes in PLCb activity could contribute to the effects of these parkin mutants on [Ca 2+ ] i . For this, we treated cells with carbachol (100 lm), an agonist of muscarinic acetylcholine receptors that specifically activates PLCb but not PLCc. Treatment of cells with carbachol gave a rapid increase in [Ca 2+ ] i . Peak increases were found 30 s after the addition of carbachol and were significantly higher in both R42P and G328E mutants when com- pared with NT and WT parkin cells (Fig. 4E). How- ever, no significant differences were found when the data were expressed as ratios (peak ⁄ basal), suggesting that peak differences were a result of basal [Ca 2+ ] i dif- ferences among cell types. In both R42P and G328E cells, the effects of carbachol on [Ca 2+ ] i lasted for a longer time, and a long tail-off effect was seen that was more pronounced in G328E mutants when compared with R42P cells (Fig. 4E). Protein kinase Ca (PKCa) levels are lower in ARJP parkin mutant cells PLC signalling regulates not only the release of ER Ca 2+ , but also the formation of DAG, which results in the activation of PKC. We next explored whether the increased PLCc activity and [Ca 2+ ] i seen in ARJP parkin mutants have consequences for PKC. From the multiple PKC isoforms [26], we chose to investigate effects on the Ca 2+ -dependent PKCa and the Ca 2+ - independent PKCe. ARJP parkin mutant cells showed reduced protein levels of PKCa, whereas PKCe levels were unchanged (Fig. 5A,B). PKC activity was deter- mined by measuring the translocation from soluble to particulate fractions, as described previously [27]. There were no significant differences in the transloca- tion of either PKC isoform (Fig. 5C,D). Dantrolene reverses the higher sensitivity to 6OHDA neurotoxicity seen in ARJP parkin mutants to the levels of WT parkin overexpressing cells Both dopamine and its analogue 6OHDA have been shown to be toxic to SH-SY5Y cells, with such toxicity being attenuated by the overexpression of WT but not ARJP mutant forms of parkin [5]. We first confirmed these findings by measuring the effect of 6OHDA in our NT, WT parkin and parkin mutant (R42P, G328E) transfected cells. Treatment with 40 lm 6OHDA gave an 15% decrease in cell viability with no significant differences among cell lines (Fig. 6A). However, as shown in Fig. 6A, and in agreement with the data of Jiang et al. [5], treatment with 120 lm 6OHDA was significantly less toxic in cells over- expressing WT parkin compared with NT and parkin mutant cells. As we have shown that the RyR anta- gonist dantrolene reverses the increase in [Ca 2+ ] i seen in R42P and G328E parkin mutants, we next studied the effect of dantrolene on the toxicity caused by 6OHDA (120 lm). Cells were pretreated with dantro- lene (10 lm) for 30 min and prior co-incubation with A B Fig. 3. Parkin-deficient cells possess higher levels of [Ca 2+ ]. Mea- surements were performed in Ca 2+ -free NaCl ⁄ P i ()) and in MEM (+) containing normal (1 m M)Ca 2+ . Experiments were performed in parkin-transfected cells (n = 12) (A) and in siRNA-transfected cells (n = 3) (B). For the siRNA experiments, two groups were used as controls (NT cells treated with Darmafect and control siRNA). Statistical analyses of the results were carried out using ANOVA followed by Fisher’s PLSD post-hoc test. *P < 0.05; **P < 0.01 against the respective value in both NT and WT. # P < 0.05 against R42P. Parkin deficiency disrupts calcium homeostasis A. Sandebring et al. 5044 FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS A B C D E Fig. 4. Altered Ca 2+ homeostasis in ARJP parkin cell lines is dependent on PLC signalling. (A) Both the PLC inhibitor neomycin (500 lM) and the RyR antagonist dantrolene (10 l M) reduced basal [Ca 2+ ] i in R42P and G328E mutants to that seen in WT parkin cells (n = 3). [Ca 2+ ] i mea- surements were performed after the addition of nimodipine (1 l M) (B) or x-conotoxin (1 lM) (C) in NaCl ⁄ P i ()[Ca 2+ ] e ) and MEM (1 mM [Ca 2+ ] e ), respectively, as described in Materials and methods. (D) Basal and TG (50 nM)-stimulated Ca 2+ measurements were made in MEM. Basal [Ca 2+ ] i was higher in parkin mutants and TG induced similar responses in all cell types. (E) Basal and carbachol (100 lM)-stimulated measurements were made in NaCl ⁄ P i . In (B)–(E), lines show the average value of three independent experiments, in each of which between 9 and 12 wells were analysed per group. Statistical analysis was carried out using ANOVA followed by Fisher’s PLSD post-hoc test. *P < 0.05. **P < 0.01. A. Sandebring et al. Parkin deficiency disrupts calcium homeostasis FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS 5045 6OHDA and dantrolene for 6 h. Blocking of RyR significantly reduced the amount of cell death in par- kin mutant cells when exposed to 6OHDA, resulting in an equal cell viability level to that in WT parkin cells with or without dantrolene (Fig. 6B). We also explored whether compromised mitochondrial Ca 2+ buffering could participate in 6OHDA-mediated toxicity in ARJP parkin mutant cells by blocking the mitochon- drial permeability transition pore with cyclosporine A prior to and during 6OHDA treatment. This treatment A B C D Fig. 5. ARJP parkin mutant cells possess lower protein levels of PKCa. (A) Immunoblotting of PKCa and PKCe levels in NT and in stably transfected human SH-SY5Y neuroblastoma cells with WT parkin and the ARJP parkin mutations R42P and G328E. (B) Repre- sentative immunoblots of PKCa and PKCe levels in soluble and par- ticulate fractions. (C) Histogram shows total PKCa levels (mean ± SEM, n = 4) normalized to actin. (D) Histogram showing the ratio of particulate and total fractions representing the relative activity of PKCa (mean ± SEM, n = 4). Statistical analyses of the results were carried out using ANOVA followed by Fisher’s PLSD post-hoc test. *P < 0.05; **P < 0.01 against the respective value in both NT* and WT#. A B Fig. 6. ARJP parkin mutations confer a higher sensitivity to 6OHDA neurotoxicity, which is reversed by dantrolene. (A) Effects of 6OHDA on MTT reduction in NT, WT parkin and in ARJP parkin mutant R42P and G328E cells. Cells were treated with 40 or 120 l M 6OHDA for 6 h (n = 6). (B) Dantrolene reverses 6OHDA toxicity in ARJP mutants to the levels seen in WT parkin. Cells were treated with 120 l M 6OHDA with or without 10 lM dantro- lene for 6 h. In dantrolene-treated cells, an additional pretreatment for 30 min was also performed. Untreated cells were used as a control. Cell viability was analysed by the MTT assay (n = 3). Data (mean ± SEM) are expressed as the percentage of values in untreated NT cells (*P < 0.05; ANOVA followed by Fisher’s PLSD post-hoc test). Parkin deficiency disrupts calcium homeostasis A. Sandebring et al. 5046 FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS failed to rescue ARJP parkin mutant cells from 6OHDA-induced cell death (data not shown), a finding in accordance with previous studies [28,29]. Discussion The identification of parkin as an E3 ligase suggested that a deficient protein ubiquitination and ⁄ or degrada- tion of substrates was behind the pathological mecha- nisms linking parkin mutations with ARJP. We have demonstrated previously that PLCc1 is ubiquitinated by parkin and that R42P and G328E parkin mutations generate significantly lower levels of ubiquitinated PLCc1 than does WT parkin in vitro. WT parkin expression significantly reduced the levels of PLCc1in human neuroblastoma cells. We further showed that PLCc1 levels were increased in parkin KO mice brain homogenates [4]. PLCc1 has been implicated in multi- ple signalling pathways that control cell division, differentiation, motility and apoptosis, and is activated on stimulation of receptors for growth factors, includ- ing EGFR [30]. The activation of EGFR results in PLCc1 phosphorylation at several sites, Tyr783 being the most crucial [31]. In addition, PLCc1, together with other PLC isoforms and phosphoinositide 3-kinase, hydrolyse PIs, resulting in the formation of two major second messengers, IP 3 and DAG. IP 3 releases Ca 2+ from the ER, through the activation of IP 3 receptors and, subsequently, also RyR [30]. In the present study, we have demonstrated that ARJP parkin mutations (R42P and G328E) and partial knockdown of parkin by siRNA result in increased phosphorylation of PLCc1 after EGF treatment, as well as enhanced basal PI hydrolysis and [Ca 2+ ] i (summarized in Fig. 7). PLC isoforms are known to control, independently of lipase activity, the size and duration of PLCb-medi- ated Ca 2+ signals by regulating a secondary Ca 2+ entry via ionotropic channels [32,33]. Both WT and ARJP parkin mutants showed similar responses to car- bachol (which activates PI hydrolysis via G-protein- coupled acetylcholine muscarinic receptors and PLCb), suggesting no differences in PLCb activity among these cell types. However, compared with WT parkin cells, ARJP parkin mutants showed longer lasting responses to carbachol, that are seen as long tail-off effects after Ca 2+ peaks. These long tail-off effects were also blocked by neomycin and dantrolene, which is consis- tent with the effects being mediated by PLC iso- enzymes and by RyR, respectively. A secondary Ca 2+ entry after the depletion of Ca 2+ stores by TG or the Ca 2+ ionophore ionomycin, which is independent of PLCc, has been described previously [33]. We investi- gated the contribution of this mechanism to the differ- ences seen in [Ca 2+ ] i . Both WT and ARJP parkin mutants responded similarly to TG and to ionomycin (data not shown), indicating no differences in second- ary Ca 2+ entry among these cells. To better define the mechanism responsible for the enhanced [Ca 2+ ] i seen in ARJP parkin mutants, we used specific blockers of PLC, RyR and different Ca 2+ channels. Both the PLC inhibitor neomycin and the RyR antagonist dantrolene reversed the high basal [Ca 2+ ] i levels seen in R42P and G328E parkin cells to those seen in WT parkin cells. In contrast, blocking plasma membrane L-type and N-type Ca 2+ channels with nimodipine and x-conotoxin, respectively, had no effect. Together, these results indicate that the increased basal [Ca 2+ ] i levels seen in R42P and G328E parkin cells are a result of enhanced PLC activity, and are mediated via RyR. The fact that siRNA knockdown of both parkin and c-Cbl also resulted in higher PI hydrolysis and [Ca 2+ ] i levels confirms that these effects are a consequence of a loss of parkin function leading to deregulated PLCc1 ubiquitination. Rare mutations in c-Cbl have been associated with myeloid leukaemia [34,35]; however, to date, there is no correlation between c-Cbl mutations and any neuronal diseases. An appropriate regulation of Ca 2+ homeostasis is crucial for maintaining balanced concentrations in the cell; thus, during normal conditions, changes are tran- sient and do not cause adverse effects. However, when Fig. 7. Summary of proposed mechanism for PLCc1-induced calcium toxicity in ARJP parkin cells. Parkin has been shown previ- ously to ubiquitinate PLCc1. In this study, we have shown that ARJP parkin mutant cells and parkin siRNA lead to enhanced PI hydrolysis and increased release of Ca 2+ from intracellular stores, increasing sensitivity to cell death induced by 6OHDA. Disrupted Ca 2+ homeostasis and ⁄ or other parkin-related functions ultimately alter PKCa protein levels. A. Sandebring et al. Parkin deficiency disrupts calcium homeostasis FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS 5047 components that influence Ca 2+ homeostasis are altered, transient increases from normal activity can lead to toxicity, which has been suggested to be part of the pathogenesis in several neurodegenerative diseases [17,18,36,37]. Moreover, the pathological cell death in snpc of PD patients has been proposed to be caused by the increased vulnerability of these cells because of their higher metabolic activity and Ca 2+ load [18]. In a recent report, transfections with mutated PTEN- induced kinase-1 (PINK1), another ARJP causative gene, have been shown to increase cytosolic Ca 2+ , which is associated with mitochondrial impairment [38]. Ca 2+ is a powerful secondary messenger which, when present in excess, activates degrading caspases and cal- pains which disrupt cytoskeletal proteins, membrane receptors and metabolic enzymes [39,40]. Furthermore, disrupted Ca 2+ homeostasis causes oxidative stress [41,42] and induces apoptosis by mechanisms that per- turb mitochondrial function [43], leading to energetic deficiency and the release of pro-apoptotic proteins [12] and reactive oxygen species [44,45]. In agreement with others [2,5], we have shown that the overexpression of WT parkin is partially protective when challenging the cells with the dopamine metabolite 6OHDA, and that this protective effect is attenuated in the ARJP parkin mutants. We have also shown that this lack of protection in parkin mutants is reversed by the RyR antagonist dantrolene, suggesting that higher sen- sitivity to 6OHDA seen in ARJP parkin mutants is a result of altered IP 3 ⁄ Ca 2+ signalling. PLC activation has two major outcomes: the release of calcium from ER and the activation of PKC. The conventional subclass of PKC isoforms is regulated by both DAG and Ca 2+ . Our results show that parkin deficiency leads to enhanced [Ca 2+ ] i levels which could have an impact on PKC activity. Therefore, we investi- gated the protein levels and activity of the Ca 2+ - dependent and Ca 2+ -independent PKCa and PKCe, respectively. We detected reduced protein levels of PKCa in ARJP parkin mutant cells; however, the rela- tive activity was similar to that in WT parkin and NT cells. It has been consistently reported that an overacti- vation of PKC results in a downregulation of enzyme levels [46]. Another possibility is that parkin regulates the gene transcription of PKCa. This has been reported previously for other genes [47–49], but further experiments need to be performed to determine the link between PKCa and parkin. In view of the vast number of identified parkin sub- strates [50], the vulnerability to toxic insults in parkin ARJP must be a combination of an imbalance in many systems, some of which might overlap. One other possible parkin substrate that may coincide with the same toxic pathway as that described here is the G- protein-coupled receptor Pael-R1 which has been shown to regulate PLC activity [51] and to subse- quently mobilize [Ca 2+ ] i [20]. Our results suggest that the accumulation of Pael-R1 in ARJP parkin mutants and knockdown by parkin siRNA may also contribute to an unbalanced Ca 2+ homeostasis and thus a higher sensitivity to toxic agents. Therefore, we suggest that PLCc1 may not act alone to change Ca 2+ responses, but in concert with additional substrates of parkin. In summary, we have demonstrated that ARJP par- kin mutants show enhanced PLCc1 activity and conse- quently increased basal levels of PI hydrolysis and disturbances in PLC-mediated Ca 2+ homeostasis. We have also demonstrated that the increased [Ca 2+ ] i seen in ARJP parkin mutants confers a higher sensitivity to the toxicity of 6OHDA, which can be reversed by blocking RyR. Our findings suggest that the disruption of PLCc1 signalling ⁄ Ca 2+ homeostasis could be one of the mechanisms by which ARJP parkin mutations mediate neuronal death. Materials and methods Materials EGF, Dowex 1X8-200 (chloride form), 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), pro- benecid, neomycin, dantrolene and 6OHDA were purchased from Sigma-Aldrich (Munich, Germany). Fluo-3-acetoxy- methyl (Fluo-3AM) ester and PluronicF-127 were pur- chased from Molecular Probes (Leiden, The Netherlands). Myo-[2- 3 H]inositol (10 CiÆmmol )1 ) was obtained from Perkin-Elmer Life Science (Boston, MA, USA). Nimodipine and x-conotoxin were purchased from Alomon Laborato- ries (Jerusalem, Israel). All other chemicals were standard laboratory reagents. DNA constructs, transfections and cell culture Human dopaminergic SH-SY5Y neuroblastoma cells were stably transfected with WT, R42P and G328E parkin constructs, as described previously [4]. Cells were cul- tured at 37 °C, 5% CO 2 , in Eagle’s MEM with Gluta- max containing 10% fetal bovine serum. Transfected cells were supplemented with 200 lgÆmL )1 geneticin. All cell culture supplies were purchased from Invitrogen (Ta ¨ by, Sweden). Parkin, c-Cbl and control siRNA knockdown were performed in SH-SY5Y cells by trans- fecting 30 nm siRNA with DarmaFECT (Dharmacon, Chicago, IL, USA) following the manufacturer’s instruc- tions. In all cases, transfections were performed for 72 h. The knockdown of the different proteins was confirmed by western blotting. Parkin deficiency disrupts calcium homeostasis A. Sandebring et al. 5048 FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS Immunoblot analysis EGF treatment of SH-SY5Y cells was performed at a concentration of 100 ngÆmL )1 for 2 min at 37 °C. Prior to EGF treatment, cells were maintained for 2 h in serum-free conditions. Cells were lysed in lysis buffer (20 mm Tris ⁄ HCl, 137 mm NaCl, 2 mm EDTA, 2% Non- idet P-40, 2% Triton X-100 and protease and phospha- tase inhibitor cocktails) (Sigma-Aldrich). Cell extract protein amounts were quantified using the BCA protein assay kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were separated using 10% acrylamide gels. Proteins were transferred to a nitrocellulose mem- brane (Schleicher & Schuell, Dassel, Germany). Western immunoblotting was performed using anti-phospho-PLCc1 (Tyr783) rabbit polyclonal IgG (Upstate, Lake Placid, NY, USA), anti-parkin (Cell Signaling, Danvers, MA, USA), anti-PLCc1 or anti-c-Cbl (BD Transduction Labo- ratories, Heidelberg, Germany), with overnight incuba- tions at 1 : 1000 dilution. The secondary antibodies were anti-rabbit or anti-mouse horseradish peroxidase-linked (Amersham, Little chalfont, UK), and were used at 1 : 2000 dilution for 1 h at room temperature. Detection was made by the ECL method (Amersham) and exposure to Hyper film MP (Amersham). PI hydrolysis assay Cells were cultured to 75–80% confluence in 10 cm Petri dishes. One day prior to the experiment, cells were changed to serum-free medium containing 5 lCiÆ mL )1 myo- [2- 3 H]inositol and incubated for 24 h. Basal PI hydrolysis was measured as described previously [52,53]. Cells were harvested by scraping with a rubber policeman in 4 mL of NaCl ⁄ P i . The contents were centrifuged at 500 g for 15 min. The pellets were washed twice at 37 °C with NaCl ⁄ P i and resuspended in 3 mL of Krebs–Henseleit bicarbonate buffer containing 10 mm LiCl (KHB ⁄ Li) at 37 °C, gassed with 5% CO 2 , 95% O 2 and centrifuged again (4300 g, 15 min). Cell pellets were resuspended in 210 lLof KHB ⁄ Li, regassed and 50 lL was added to glass centrifuge tubes containing 250 lL of KHB ⁄ Li buffer. The tubes were incubated at 37 °C under an atmosphere of 5% CO 2 , 95% O 2 with gentle agitation for 25 min. Incubations were stopped by the addition of 940 lL of chloroform–methanol (1 : 2). Tubes were incubated on ice for 30 min and the phases were separated by the addition of 310 lL of chloro- form and 310 lL of water, followed by vortexing and centrifugation; 750 lL of the aqueous phase were removed and labelled IPs were separated from myo-[2- 3 H]inositol by Dowex chromatography. The chloroform phase was removed, placed into scintillation vials and allowed to eva- porate before determination of ‘lipid d.p.m.’ by scintillation spectroscopy. The results were expressed as d.p.m. IPs ⁄ (d.p.m. IPs + d.p.m. lipid). PKC translocation PKC translocation was determined as described previously [27]. Approximately 5 · 10 6 cells were washed with ice-cold NaCl ⁄ P i and harvested by scraping in lysis buffer contain- ing 20 mm Tris ⁄ HCl, pH 7.4, 0.32 mm sucrose, 2 mm EDTA, 50 mm b-mercaptoethanol (Sigma-Aldrich) and protease inhibitor cocktail, and sonicated (12 s, 22 microns) on ice. A sample from this fraction was saved for total lysate analysis. The lysates were then ultracentrifuged at 100 000 g for 30 min at +4 °C. The supernatants were des- ignated as soluble fractions. The samples were analysed by western blotting, and the ratio of particulate to total frac- tions is referred to as the translocation between cytosol and membrane compartments. [Ca 2+ ] i measurements [Ca 2+ ] i measurements were essentially determined as described previously [52,53]. In brief, cells cultured in 96-well plates were loaded with MEM without phenol red containing 5 lm Fluo-3AM ester, 0.5% (v ⁄ v) Pluronic F-127 and 0.1 mm probenecid (90 min in the dark at room temperature). After loading, the cells were incubated for 120 min in MEM without phenol red with 1 mm probene- cid in the dark at room temperature to allow intracellular esterases to decompose the Fluo-3AM ester. Basal [Ca 2+ ] i was measured in both Ca 2+ -free NaCl ⁄ P i and phenol red- free MEM (containing 1 mm Ca 2+ ). In the experiments including nimodipine (1 lm)orx-conotoxin (1 lm), [Ca 2+ ] i was measured first in NaCl ⁄ P i . The NaCl ⁄ P i was removed and MEM was added to the cells, with the respective blocker being present during all the measurements. TG was used at 50 nm and [Ca 2+ ] i was measured in phenol red-free MEM. Measurements with carbachol were performed after measuring basal apparent [Ca 2+ ] i ; NaCl ⁄ P i was then removed, and 100 lm carbachol in NaCl⁄ P i solution at 37 °C was added. Carbachol was present for all subsequent apparent [Ca 2+ ] i measurements. For the experiments with neomycin or dantrolene, the agents were included in MEM without phenol red and used during a 120 min incubation period, and also in NaCl ⁄ P i for a 10 min incubation period when basal [Ca 2+ ] i levels were measured MTT assay Cell viability was determined by the MTT assay. MTT powder was dissolved in MEM without phenol red at 0.3 mgÆmL )1 and then added to the cells. After 1 h at 37 °C, the medium was removed and formazan crystals were dissolved in isopropanol. Aliquots were moved to a 96-well plate and optical densities were read at 540 nm in a Molecular Devices Spectra MAX 250 plate reader (Ramsey, MN, USA). For the experiments with dantrolene, cells were A. Sandebring et al. Parkin deficiency disrupts calcium homeostasis FEBS Journal 276 (2009) 5041–5052 ª 2009 The Authors Journal compilation ª 2009 FEBS 5049 pretreated with 10 lm dantrolene for 30 min, followed by treatment with 120 lm 6OHDA for 6 h. Control cells received an equivalent amount of vehicle. The results were expressed as a percentage of the values obtained for non- treated cells. Statistical analyses Analyses of differences were carried out by analysis of vari- ance (ANOVA), followed by Fisher’s protected least-signifi- cant difference (PLSD) post-hoc test. P < 0.05 was considered to be statistically significant. Acknowledgements This work was supported by grants from the following Swedish foundations: Swedish Brain Power, Parkin- sonsfonden, Riskbankens Jubileum Fond, Karolinska Institutets Foundation for Geriatric Research, Loo and Hans Ostermans Foundation, Gun and Bertil Stohnes Foundation, K.A. Wallenberg, Stiftelsen fo ¨ r Gamla Tja ¨ narinnor and A ˚ ke Wibergs Foundation. This research was also supported (in part) by the Intramural Research Program of the National Institute on Aging, National Institutes of Health, Ayudas Postdoctorales, Gobierno de Navarra and LIONS Foundation for Research of Age Related Disorders. References 1 Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN & Braak E (2003) Staging of brain pathology related to sporadic Parkinson’s disease. 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Parkin deficiency disrupts calcium homeostasis by modulating phospholipase C signalling Anna Sandebring 1,2, *, Nodi Dehvari 1, *, Monica Perez-Manso 1 ,. show that blocking of either PLC activity or ryanodine receptors (RyR) can reverse these effects. Results Effects of parkin deficiency on PLCc1 activation, phosphoinositide