Apoptosis DOI 10.1007/s10495-014-1027-7

ORIGINAL PAPER

The serine protease inhibitor TLCK attenuates intrinsic death pathways in neurons upstream of mitochondrial demise C. Reuther • G. K. Ganjam • A. M. Dolga C. Culmsee

•

Ó Springer Science+Business Media New York 2014

Abstract It is well-established that activation of proteases, such as caspases, calpains and cathepsins are essential components in signaling pathways of programmed cell death (PCD). Although these proteases have also been linked to mechanisms of neuronal cell death, they are dispensable in paradigms of intrinsic death pathways, e.g. induced by oxidative stress. However, emerging evidence implicated a particular role for serine proteases in mechanisms of PCD in neurons. Here, we investigated the role of trypsin-like serine proteases in a model of glutamate toxicity in HT-22 cells. In these cells glutamate induces oxytosis, a form of caspase-independent cell death that involves activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid), leading to mitochondrial demise and ensuing cell death. In this model system, the trypsin-like serine protease inhibitor Na-tosylL-lysine chloromethyl ketone hydrochloride (TLCK) inhibited mitochondrial damage and cell death. Mitochondrial morphology alterations, the impairment of the mitochondrial membrane potential and ATP depletion were prevented and, moreover, lipid peroxidation induced by glutamate was completely abolished. Strikingly, truncated

C. Reuther
G. K. Ganjam
A. M. Dolga
C. Culmsee (&) Institut fu¨r Pharmakologie und Klinische Pharmazie, Fachbereich Pharmazie, Philipps-Universita¨t Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg, Germany e-mail: [email protected] C. Reuther e-mail: [email protected] Present Address: C. Reuther Institut fu¨r Schlaganfall- und Demenzforschung (ISD), Klinikum der Universita¨t Mu¨nchen, Max-Lebsche-Platz 30, 81377 Munich, Germany

Bid-induced cell death was not affected by TLCK, suggesting a detrimental activity of serine proteases upstream of Bid activation and mitochondrial demise. In summary, this study demonstrates the protective effect of serine protease inhibition by TLCK against oxytosis-induced mitochondrial damage and cell death. These findings indicate that TLCK-sensitive serine proteases play a crucial role in cell death mechanisms upstream of mitochondrial demise and thus, may serve as therapeutic targets in diseases, where oxidative stress and intrinsic pathways of PCD mediate neuronal cell death. Keywords Trypsin-like serine proteases
Oxidative stress
Mitochondria
Bid
TLCK
Oxytosis Abbreviations AD Alzheimer’s disease AIF Apoptosis-inducing-factor Bax Bcl-2-associated X protein Bcl-2 B-cell lymphoma 2 Bid BH3 interacting-domain death agonist BODIPY 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4bora-3a,4a-diaza-sindacene-3-undecanoic acid CCCP Carbonylcyanide-3-chlorophenylhydrazone HtrA2 High temperature requirement factor A2 MMP Mitochondrial membrane potential MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide NF-jB Nuclear factor kappa B OCR Oxygen consumption rate PCD Programmed cell death PD Parkinson’s disease ROS Reactive oxygen species TLCK Na-tosyl-L-lysine chloromethyl ketone hydrochloride

123

Apoptosis

TPCK TMRE

N-p-tosyl-L-phenylalanine chloromethyl ketone Tetramethylrhodamine ethyl ester

Introduction Programmed cell death (PCD) is a highly conserved cell death mechanism essential for various physiological processes such as maintenance of embryonic development, tissue homeostasis and removal of injured cells. Many studies detected paradigms of PCD in neurodegenerative diseases such as Alzheimer’s disease (AD) or Parkinson’s disease (PD) and after acute ischemic brain damage [1–3]. Mitochondrial damage is a well-established hallmark in the so called intrinsic pathway of PCD in neurons, where mitochondrial membrane permeabilization designates a pivotal decision point for survival or death of cells [4]. In particular, the activation of the pro-apoptotic protein BH3 interacting-domain death agonist (Bid) and its translocation to the mitochondria, the highly detrimental generation of ROS and the release of pro-apoptotic proteins such as cytochrome c (Cytc), apoptosis-inducing-factor (AIF) or endonucleaseG (endoG) from the mitochondrial intermembrane space are associated with this intrinsic apoptotic pathway [4–7]. Major downstream targets of the mitochondrial death signaling are proteases, e.g. caspases, which are essential mediators accelerating PCD mechanisms [4, 7–9]. Serine proteases, such as chymotrypsin and trypsin, were also related to PCD pathways. Introducing these proteases in tumor cells led to cell death with characteristics of apoptosis [10]. Other lysosomal proteases, such as cathepsins, granzyme B or chymotrypsin-like serine proteases were later on associated with apoptotic mechanisms [11–14]. Upon lysosomal membrane permeabilization the distribution of lysosomal proteases into the cytosol mediates Bid cleavage and Cytc release from the mitochondria by cathepsin B, H, L and S and cathepsin D, respectively [11, 15–17]. Recent studies showed that a lysosomal serine protease, chymotrypsin B, is also able to cleave Bid and thereby potentiates apoptosis in rat hepatoma cells [18]. Generally, it is reported that serine proteases are not only involved in development, plasticity and pathology of the nervous system but also in apoptosis in neuronal cells as well as in non-neuronal cell types [19–22]. Inhibiting serine proteases with the irreversible inhibitors for serine proteases Na-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK) and N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK) mediated protective effects in different model systems of neuronal cell death in previous

123

studies [23, 24]. Downstream mechanisms of serine protease-mediated cell death may involve, for instance, the degradation of the anti-apoptotic protein Bcl-2, the upregulation/activation of pro-apoptotic proteins such as Bax and Bid, and the activation of caspases [25–28]. Further, recent evidence suggests that serine proteases, such as neutrophil elastase and trypsin directly increase intracellular ROS levels arising from the mitochondria which contribute to cytotoxicity. A direct influence of serine proteases on the generation of intracellular ROS levels was concluded from studies showing that proteaseinduced ROS increase and subsequent cell death were attenuated by catalase treatment, and accelerated by aminotriazole, a catalase inhibitor [29]. In addition, oxidative stress induced apoptosis through the enhanced expression of the mitochondrial serine protease Omi/HtrA2 [30]. Since oxidative stress is a trigger of neuronal cell death associated with different neurodegenerative diseases including AD, PD or ischemic stroke [31], the present study focused on the involvement of trypsin-like serine proteases in a model of oxidative stress-induced cell death. For this purpose, we applied the model of glutamate toxicity in immortalized murine hippocampal cell line (HT-22 cells). These cells lack the expression of ionotropic glutamate receptors and are widely used as a model system to investigate mechanisms of oxytosis, a form of cell death that occurs in a caspase-independent manner. Oxytosis involves the activation of the pro-apoptotic protein Bid, the impairment of mitochondrial function, the release of AIF from the mitochondrial intermembrane space and the generation of highly detrimental ROS levels [5, 32]. TLCK and TPCK were used for serine protease inhibition after glutamate challenge and the impact on cell viability and mitochondrial integrity were subsequently analyzed. Since TLCK, but not TPCK, showed protective effects on cell viability in HT-22 cells and primary cortical neurons, and protective effects on mitochondrial function in HT-22 cells, our data suggests a pivotal role of trypsin-like serine proteases in glutamate-induced neurotoxicity upstream of mitochondrial demise.

Results The pharmacological trypsin-like serine protease inhibitor TLCK provides neuroprotection in HT-22 cells and primary cortical neurons. To test the protective potential of serine protease inhibitors against oxytosis in immortalized hippocampal mouse neurons, we applied TLCK and TPCK to HT-22 cells. Upon induction of oxytosis with 2–5 mM glutamate, HT-22 cells change their morphology from spindle-shaped morphology in control conditions to round cells which

Apoptosis

detach from the bottom of the cell culture dish (Fig. 1a, upper panels). These morphological alterations after glutamate treatment were prevented by the serine protease inhibitor TLCK (Fig. 1a, lower right panel). The protective effect of TLCK was verified by cell viability measurements using the MTT assay after 17 h of glutamate treatment, demonstrating that serine protease inhibition increased cell viability (Fig. 1b). To further confirm these neuroprotective effects of TLCK we quantified cell death by AnnexinV/ propidium iodide (AVPI) staining. Cells were treated with 3 mM glutamate with or without 50 lM TLCK (co-treatment) for 11.5 h and as shown in Fig. 1c, d the number of double positive stained cells was increased after glutamate exposure, which could be reduced to control levels by TLCK. In this model of oxytosis, TPCK had no protective effects against glutamate toxicity. Interestingly, TPCK decreased cell viability in a concentration-dependent manner (Fig. 1e). These results suggest that TLCK, but not TPCK, attenuates cell death in HT-22 neurons upon glutamate challenge. After characterizing the protective effect of serine protease inhibition against glutamate-induced toxicity in a neuronal cell line, we next analyzed the impact of TLCK on primary cortical neurons. Cortical neurons were treated after 8–10 days in culture with 25 lM glutamate to induce cell death. As shown in Fig. 1f, TLCK is also able to attenuate glutamate-induced excitotoxicity in primary cortical neurons in a concentration-dependent manner. The protective effect of TLCK in HT-22 cells was also confirmed by real-time impedance measurements using the xCELLigence system. Proliferating cells induce an increase in cell impedance, while cell death is linked to decreased cell index values due to cell detachment from the cell culture dish. This decrease of cell impedance upon glutamate toxicity could be completely prevented by TLCK treatment (Fig. 2a). To examine whether serine protease activation occurs early or late in response to oxidative stress, TLCK was applied 2, 4, and 6 h after the onset of glutamate and cell viability was analyzed by the xCELLigence system over time. A post-treatment with TLCK up to 2 h prevented cell death, while 4 and 6 h of post-treatment did not increase cell viability, which indicates an activation of serine proteases in the initiating phase of the cell death cascade initiated by glutamate treatment (Fig. 2b). Taken together, these results show that TLCK is able to significantly reduce glutamate-induced cell death in HT-22 neurons and in primary cortical neurons. Serine proteases act upstream of Bid The pro-apoptotic protein Bid is an important mediator of caspase-independent pathways of neuronal cell death in HT-22 cells and an important regulator of mitochondrial

demise [5, 6]. Since serine proteases were linked to Bid activation thereby inducing cell death, the next issue of interest was to investigate if TLCK-sensitive serine proteases act upstream or downstream of Bid activation in HT22 cells. For this purpose, cells were pre-treated with 50 lM TLCK for 1 h followed by transfection with the truncated Bid (tBid) plasmid (2 lg/well, 18 h) to induce apoptosis as previously described by Grohm et al. [6]. After 17 h of tBid treatment cell death was examined by AVPI staining and subsequently FACS-analysis. TLCK was not able to prevent cell death induced by tBid, indicating that activation of trypsin-like serine proteases occurred upstream of Bid activation (Fig. 3a, b). This finding was substantiated by the fact that TLCK was able to prevent glutamate-induced translocation of Bid from the cytosol to the mitochondria (Fig. 3c). Cells were transfected with the two plasmids DsRed2-Bid and mitoEGFP (green). In control cells the red-fluorescening Bid is localized in the cytosol and cannot be found at the level of mitochondria. However, after glutamate exposure Bid translocated to the mitochondria, which was illustrated by the yellow color in the confocal pictures, indicating a redistribution of Bid from the cytoplasm to the mitochondria. This translocation could be inhibited by TLCK as after serine-protease inhibition Bid resided in the cytosol and the relocation to the mitochondria was abolished (Fig. 3c). Serine protease inhibition abolishes lipid peroxidation In HT-22 cells glutamate exposure results in increased detrimental ROS accumulation and lipid peroxide production. As the work of Tobaben et al. showed, a first increase in lipid peroxidation within the first hours after glutamate exposure was followed by a second much more pronounced increase resulting in cell death. The first ROS increase led to the activation of Bid, its translocation to the mitochondria and ensuing irreversible mitochondrial damage which was followed by the second ROS boost representing a major feature of oxidative stress in HT-22 cells [32]. To investigate the influence of serine protease activation on lipid peroxide production in conditions of oxidative stress, HT-22 cells were treated in the presence or absence of TLCK with 3 mM glutamate for 10 h. Lipid peroxidation was examined by FACS-analysis of BODIPY stained cells. Interestingly, TLCK completely blocked glutamate-induced lipid peroxide production, even to control levels (Fig. 4a, b). This strong decrease of lipid peroxidation after glutamate toxicity indicates that TLCK acts even before the first wave of ROS, which provides additional evidence for the conclusion that trypsin-like serine proteases are activated upstream of mitochondrial demise.

123

Apoptosis

123

Apoptosis b Fig. 1 Neuroprotective effects of the trypsin-like serine protease

inhibitor TLCK in cultured neurons and primary cortical neurons.a Light microscopy images show protective effects of TLCK against glutamate toxicity in HT-22 cells. The upper panels show control cells, the lower panels cells treated with TLCK (50 lM) in the presence (left panel) or absence (right panel) of glutamate (3 mM, 11.5 h). b The MTT assay confirmed the concentration-dependent protective effect of TLCK against glutamate-induced neurotoxicity (3 mM, 17 h; n = 8). c Cell death of HT-22 cells after glutamate treatment was analyzed by AVPI staining and subsequently FACSanalysis (3 mM, 13.5). At least 10,000 cells were counted for each sample (representative dot plots). d Quantification of dead cells (AV and PI positive cells) of the plots from Fig. 1c (n = 4). e In contrast to TLCK, the chymotrypsin-like serine protease inhibitor TPCK had no protective effect against glutamate toxicity (3 mM, 17 h) in HT-22 cells determined by MTT assay (n = 8). f The MTT assay revealed a protective effect of TLCK against glutamate-induced neurotoxicity (25 lM, 21 h) in a concentration-dependent manner in primary cortical neurons (n = 6). All experiments were repeated at least three times (***p \ 0.001, ANOVA, Scheffe´’s test)

Recently it was reported that TLCK exhibited antioxidative properties and due to this feature TLCK rescued H9C2 cells from H2O2-induced cell death [33]. In this study, glutamate-induced lipid peroxidation was completely abolished by TLCK (Fig. 4a, b), imposing the hypothesis that TLCK may act as an antioxidant in glutamate toxicity and thereby rescues HT-22 cells from cell death. To examine the protective potential of TLCK in H2O2-induced cell death, cell viability was determined by MTT assay after 15.5 h of treatment. H2O2 decreased cell viability in a concentration-dependent manner, however, TLCK was not able to prevent it, leading to the conclusion that TLCK is not acting as an antioxidant in this cell death model system (Fig. 4c). TLCK preserves mitochondrial integrity and mitochondrial respiration and prevents the loss of MMP Oxidative stress causes alterations in mitochondrial morphology and mitochondrial function [34]. Mitochondrial fragmentation and the loss of mitochondrial membrane potential (MMP) are also associated with glutamateinduced cell death in HT-22 neurons. In order to investigate the role of TLCK at the level of mitochondria, we examined mitochondrial integrity and function. First, we detected changes in mitochondrial morphology after glutamate exposure compared to control cells (Fig. 5a, upper right panel). In un-treated control cells mitochondria exhibit an elongated and tubular-like shape, while in glutamate-treated neurons increased mitochondrial fission and mitochondrial fragmentation were observed. To quantify these mitochondrial changes three categories of mitochondrial morphology were defined (see [6] and ‘‘Materials and methods’’ section). The observed mitochondrial

fragmentation after glutamate toxicity was significantly reduced by treating cells with TLCK. In the presence of TLCK, the number of category I mitochondria in glutamate-treated cells were maintained similar to vehicletreated control cells. The increase in category III mitochondria after induction of cell death was attenuated by cotreating cells with TLCK (Fig. 5b). Furthermore, glutamate toxicity in HT-22 cells was associated with the loss of MMP as measured in glutamatetreated control cells. TLCK significantly restored the impairment of the MMP after glutamate exposure (3 mM, 12.5 h) as detected by tetramethylrhodamine ethyl ester (TMRE) staining and subsequent FACS-analysis. The uncoupler of the MMP carbonylcyanide-3-chlorophenylhydrazone (CCCP) was used as a positive control (Fig. 6a). The quantification of the peak shift is shown in Fig. 6b, confirming that TLCK is able to prevent mitochondrial depolarization. Another hallmark of neuronal cell death is the impaired energy metabolism as detected by reduced ATP levels. As mitochondrial respiration is one of the major metabolic reactions to produce ATP, we directly analyzed the effect of TLCK after glutamate exposure on the mitochondrial respiratory capacity by measuring the oxygen consumption rate (OCR) with the Seahorse XF96 system and the impact of TLCK on ATP levels after glutamate toxicity. Compared to un-treated controls, cells treated with glutamate exhibit a decrease in basal respiration, decreased ATP generation and a reduction in the maximal respiration. TLCK was able to abolish all these detrimental effects caused by glutamate nearly to control levels (Fig. 6c, d). Taken together, our data suggests that trypsin-like serine proteases are involved in oxidative stress-induced neuronal cell death pathways in HT-22 cells and primary cortical neurons, as inhibition with TLCK increases cell viability and prevents cell death. Furthermore, mitoprotective effects obtained with TLCK indicate an activation of trypsin-like serine proteases upstream of mitochondrial demise after induction of oxidative stress in HT-22 neurons. Moreover, these data exhibit an initial activation of trypsin-like serine proteases even before the activation of Bid in glutamate toxicity in HT-22 cells.

Discussion Oxidative stress-induced cell death and the contribution of ROS are linked to many neurodegenerative diseases such as AD, PD and to cell death occurring after stroke or ischemia [35–37]. The intrinsic apoptotic pathway is triggered by enhanced levels of ROS and is associated with mitochondrial demise and enhanced fission, amongst others [38]. Another mediator of the oxidative stress-induced

123

Apoptosis Fig. 2 Protective effects of the serine protease inhibitor TLCK in conditions of post-treatment. a HT-22 cells were plated in 96-well E-plates and cell impedance was measured continuously by the xCELLigence system. TLCK (50 lM) mediated persistent protective effects against glutamate toxicity compared to control cells exposed to glutamate alone (n = 6). b Impedance was measured also in post-treatment conditions when TLCK (50 lM) was applied 2, 4 and 6 h after the onset of glutamate. Posttreatment with TLCK (50 lM) attenuated cell death compared to glutamate-treated controls when applied up to 2 h after the onset of glutamate, while a posttreatment with TLCK after 4 and 6 h after glutamate exposure did not exert protective effects (n = 7). c Bar graph evaluation at the 12 h time point of the xCELLigence recordings (right black arrow) from Fig. 2b. TLCK significantly blocked glutamateinduced cell death in conditions of co-treatment and up to 2 h of post-treatment. In contrary, cells exposed only to glutamate and in conditions of post-treatment after 4 and 6 h TLCK exhibited no protective effects. All experiments were repeated at least three times (***p \ 0.001, ANOVA, Scheffe´’s test)

mitochondrial intrinsic cell death pathways is the proapoptotic protein Bid, which is involved in the regulation of the mitochondrial membrane permeabilization and the subsequent lethal AIF release from the mitochondria

123

[5, 32]. However, lysosomes are also an upcoming contributor to apoptotic mechanisms induced by oxidative stress, as the release of lysosomal proteases into the cytosol is a crucial step in apoptosis in different cell types [39, 40].

Apoptosis

Fig. 3 TLCK does not prevent tBid induced cell death. a ptBidinduced cell death in HT-22 cells was analyzed by AVPI staining and subsequently FACS-analysis (2 lg plasmid/24-well, 18 h). Pre-incubation for 1 h with 50 lM TLCK could not prevent tBid-induced cell death (representative dot plots). The empty vector pcDNA3.1 was used as a control plasmid. At least 10,000 cells were counted for each sample. b Quantification of dot plots shown in Fig. 3a (n = 4).

c Confocal pictures illustrated the translocation of Bid (red) to the mitochondria (green) after glutamate exposure (yellow). The nuclei were counterstained with DAPI (blue). As depicted in the very right row TLCK was able to block the translocation of Bid in the presence of glutamate. All experiments were repeated at least three times (***p \ 0.001, ANOVA, Scheffe´’s test) (Color figure online)

123

Apoptosis

Fig. 4 TLCK blocks lipid peroxide production. a Cells were stained with BODIPY 581/591 and lipid peroxides were measured by FACSanalysis after 10.5 h of glutamate treatment (3 mM). TLCK significantly reduced the lipid peroxide production to control levels (representative dot plots). b Quantification of the plots of Fig. 4a (n = 4). c HT-22 cells were treated with different concentrations of H2O2 and 50 lM TLCK. Cell viability was determined by MTT assay after 15.5 h of treatment. The serine protease inhibitor TLCK did not prevent the decrease of cell viability induced by H2O2 (n = 8). All experiments were repeated at least three times (***p \ 0.001, ANOVA, Scheffe´’s test)

In the present study, we investigated the role of trypsinlike serine proteases in a model of oxidative stress caused by high extracellular glutamate concentrations and the impact on cell viability, mitochondrial function, the generation of

123

Fig. 5 TLCK preserves mitochondrial fragmentation. a Confocal pictures were made after staining of HT-22 cells with MitoTracker DeepRed. Glutamate treatment caused mitochondrial fragmentation (upper right panel) which was prevented by TLCK treatment (lower right panel). b Quantification of mitochondrial morphology: mitochondrial structures were divided into three categories: Category I: elongated, fused mitochondria, Category II: intermediate mitochondria and Category III: fragmented mitochondria. TLCK preserved mitochondrial tubular, elongated morphology after exposure to glutamate (2 mM, 13.5 h). At least 500 cells per condition were counted without knowledge of treatment history. All experiments were repeated at least three times (**p \ 0.01; ***p \ 0.001, ANOVA, Scheffe´’s test)

lipid peroxides and the role of the pro-apoptotic protein Bid. Since caspases are very well-studied contributors in PCD pathways, little is known about the involvement of noncaspase death regulators, such as calpains, lysosomal cathepsins and serine proteases, however, in the last years they have been associated with apoptosis [10, 13, 18, 41, 42]. Depending on the cell type and the apoptosis inducing agent, different serine proteases are involved in cell death mechanisms, e.g. TLCK-sensitive serine proteases are involved in colchicine-induced cell death of sympathetic neurons and chymotrypsin B is involved in apoptosis in rat

Apoptosis

Fig. 6 TLCK preserves the impairment of MMP, prevents ATP depletion and maintains mitochondrial respiration a MMP was analyzed by TMRE staining and following FACS-analysis. After 12.5 h of glutamate (3 mM) treatment impaired MMP was detected by a loss of red fluorescence, as indicated by a peak shift to the left. Serine protease inhibition by TLCK prevented the impairment of the MMP (representative dot plots). CCCP was used as a positive control. b Quantification of peak percentages of red fluorescence depicted in

Fig. 6a (n = 4). c After 15 h of glutamate exposure (3 mM) ATP levels were measured. TLCK prevented ATP depletion compared to glutamate-treated control cells (n = 8). d Measurement of the oxygen consumption rate (OCR) after glutamate exposure (3 mM, 15 h). TLCK treatment prohibited the decrease of the basal respiration and the loss of ATP and the maximal respiration could be restored after glutamate toxicity. All experiments were repeated at least three times (***p \ 0.001, ANOVA, Scheffe´’s test) (Color figure online)

hepatoma cells through a mitochondrial pathway [13, 24]. Due to oxidative stress stimuli the mitochondrial serine protease Omi/HtrA2 is also activated and involved in cell death pathways [30] and Ucf-101, a specific Omi/HtrA2 inhibitor, protected neurons against cerebral oxidative injury [43]. Further, an inhibition of Omi/HtrA2 attenuated oxytosis in HT-22 cells. The activation and release of Omi/ HtrA2, however, occur mostly during the course of mitochondrial loss of function and integrity and then initiates the execution phase of PCD. In the present study, we showed that TLCK prevented glutamate toxicity upstream of mitochondrial damage, suggesting that trypsin-like serine proteases are activated very early in pathways of oxytosis, in the initiation phase of this caspase-independent form of PCD (Figs. 1, 2). Recently, it was shown that mitochondrial fission and the loss of MMP are associated with oxidative stress-

induced cell death in HT-22 cells in a caspase-independent cell death pathway mediated by the translocation of Bid to the mitochondria and the subsequent crucial release of AIF [5, 6]. Assuming that serine proteases are activated at an early time point we addressed if serine protease activity was linked to mitochondrial dysfunction and if Bid was an upstream or downstream target of serine proteases. The translocation of Bid to the mitochondria linked key regulating steps of mitochondrial fragmentation and cell death in the model of glutamate toxicity in HT-22 cells [5]. As previously reported Bid can be activated by different lysosomal proteases and thereby increases cell death. We revealed in our study that tBid-induced cell death could not be abolished by TLCK treatment and moreover, that TLCK prevented the translocation of Bid to the mitochondria, indicating Bid being a downstream target of trypsin-like serine proteases in our model system of oxidative stress

123

Apoptosis

[18]. In answer to the second question, we show in our work that the crucial ROS increase, mitochondrial fission and the impairment of the MMP after glutamate exposure could be completely blocked by co-treating cells with TLCK. The detrimental loss of ATP after glutamate-induced cell death was significantly abolished by inhibiting trypsin-like serine proteases. TLCK prevented the decrease of the mitochondrial basal respiration and restored mitochondrial respiratory capacity nearly to control levels, while glutamate exposure led to impaired mitochondrial respiration. This leads to the conclusion that trypsin-like serine proteases act upstream of mitochondrial levels and that an inhibition does not only lead to a decrease in cell death but also to mitoprotection. Our results are in line with findings from studies in models of DNA-damage-induced neuronal death, where TLCK-sensitive serine proteases were involved in apoptotic mechanisms upstream of mitochondrial Cytc release in PC12 cells and embryonic cortical neurons exposed to camptothecin. We revealed that this protective effect upstream of mitochondrial demise is also linked to PCD induced by oxidative stress in HT-22 cells [44]. Inhibition of trypsin-like serine proteases with TLCK significantly prevented cell death also in primary cortical neurons, where glutamate-induced excitotoxicity led to cell death. This underlines that TLCK-sensitive trypsin-like serine proteases become relevant executioners in oxidative stressinduced neuronal cell death. In the present study, we revealed an important role of trypsin-like serine proteases in glutamate-induced cell death in HT-22 cells. There are a plenty of trypsin-like serine proteases which are expressed in neurons and play important roles in neuronal plasticity and neurodegeneration, e.g. trypsin IV, P22 or neuropsin to name just a few [45]. In principal, all these proteases may contribute to the effects observed after oxytosis and could be possible targets to prevent cell death. However, neither the one protease nor the exact mechanism of protease activation and the following signaling cascade of glutamate-induced cell death in HT-22 cells is known to date. The most pronounced effect of TLCK is indeed the inhibition of trypsinlike serine proteases, but TLCK has also been shown to have a variety of actions besides the serine protease inhibitory effect, e.g. inactivating protein kinase C [46] or blockage of cyclic AMP-dependent protein kinase [47]. It is reported by different groups that another property of TLCK is the inhibition of nuclear factor ‘kappa-lightchain-enhancer’ of activated B-cells (NF-jB) activation, which occurs due to different stimuli, e.g. interleukin 1b treatment, and subsequently leads to cell death [48, 49]. Although TLCK can interfere with NF-jB activation the current protective effect is unlikely attributed to this mechanism, because NF-jB activation was not detected after the onset of glutamate in HT-22 cells.

123

Serine proteases have been implicated to act both upstream and downstream of caspases and, for instance, serine proteases such as Omi/HtrA2 or granzyme B were identified as caspase activators [26, 27, 50]. However, as shown in our earlier work, caspases are not involved in PCD mechanisms in HT-22 cells and inhibitors of caspase-8, caspase-2 and the general caspase inhibitor z-VAD-fmk did not exert protection against glutamate toxicity. Therefore, the protective effect of serine protease inhibition in HT-22 cells is likely not mediated through the inhibition of the caspase–cascade in contrast to findings in a model of hypoxia-reoxygenation in rat kidney proximal tubule cells [5, 27]. Despite recent observations indicate a possible inhibitory effect of activated caspase-3, caspase-6 and caspase-7 of TLCK, these findings play a minor role in our model system of PCD, as it is caspase-independent and nevertheless TLCK exhibited protective effects in conditions of glutamate toxicity in HT-22 neurons [51]. The antioxidative effect of TLCK is discussed to contribute to cell survival. A recent study demonstrated an antioxidative action of TLCK, which prevented H2O2induced cell death accompanied by the inhibitory effect on p53 function [33]. However, in glutamate-induced cell death in HT-22 cells an antioxidative action of TLCK does not account for the observed protective effect, since cell death induced by H2O2 was not prevented by this serine protease inhibitor (Fig. 4c). Accordingly, the target of action of TLCK remains unknown, however, based on our current findings it is likely to be a trypsin-like serine protease which is activated prior to mitochondrial demise and at an early point in this cell death cascade. In-depth studies would further reveal the underlying specific proteases contributing to this cell death model of oxytosis in HT-22 cells. A schematic illustration summarizes our findings of glutamate-induced cell death in HT-22 cells based on the data presented in the current study (Fig. 7). Taken together, our findings reveal a strong protective effect of TLCK after glutamate-induced cell death in neuronal cells by inhibiting trypsin-like serine proteases and furthermore, mitochondrial damage could be significantly prevented, identifying serine proteases as a promising target for neuroprotection upstream of mitochondrial demise.

Materials and methods Cell culture and materials Immortalized hippocampal mouse neurons (HT-22 cells) were cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented

Apoptosis

Fig. 7 Schematic illustration of glutamate-induced cell death in HT22 cells high extracellular glutamate concentrations block the cystine/ glutamate antiporter which leads to increased cystine levels within the cell, resulting in glutathione depletion. This leads to an increase in ROS which is accompanied by elevated lipid peroxidation and the translocation of Bid from the cytoplasm to the mitochondria.

Following, mitochondrial fission and a second increase in ROS formation lead to cell death. TLCK prevented both waves of ROS formation, the translocation of Bid to the mitochondria, the impairment of MMP and ensuing cell death, which places the activation of trypsin-like serine proteases in a very initial phase in this model of glutamate-induced cell death

with 10 % heat-inactivated fetal calf serum (PAA Laboratories GmbH, Co¨lbe, Germany), 100 U/ml penicillin, 100 lg/ml streptomycin and 2 mM glutamine (all Sigma Aldrich, Taufkirchen, Germany). Thirty hours after seeding the cells, oxytosis was induced by adding 2–5 mM glutamate for 10–17 h. Depending on the cell density, the passage number of the cells and the method of analysis the time frame and glutamate concentrations ranging from 3 to 5 mM were adjusted. TLCK (Sigma Aldrich) was applied with or without glutamate at a final concentration of 50 lM, if not otherwise declared. DMSO (Sigma Aldrich, Taufkirchen, Germany) was used as solvent in all experiments. For hydrogen peroxide-induced cell death (H2O2, Sigma-Aldrich, Taufkirchen, Germany), a stock solution of H2O2 with a concentration of 30 % was diluted in HT-22 medium to final concentrations of 800–900 lM prior to use.

(CAF) was added at a final concentration of 0.25 lM. Since cultures of primary neurons develop functional glutamate receptors after 6–8 days in culture, treatment was performed within day 8–10. Excitotoxicity was induced by glutamate (25 lM) in neurobasal medium supplemented with B27 and pen/strep and cell viability was analyzed after 18–21 h of treatment by MTT assay.

Primary cortical neurons For culturing of primary embryonic mouse cortical neurons, cell culture dishes were coated 1 day before the preparation with 5 % polyethylenimine. Primary neurons were cultured in neurobasal medium (Invitrogen, San Diego, CA, USA) supplemented with 2 % B27 (v/v) (Gibco, Life Technologies, Darmstadt, Germany), 2 mM glutamine and 100 U/ml penicillin/streptomycin (Invitrogen, San Diego, USA). Cells were plated in 96-well plates at a density of 60,000–80,000 cells/well. On day 2 medium was changed completely and cytosine arabinofuranoside

Viability assays In 96-well plates 10,000 cells/well were seeded and after 24 h of growing cell death was induced by glutamate (3 mM). TLCK was added with or without glutamate and after indicated time points cell viability was measured by reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) at a concentration of 0.5 mg/ ml for 1 h. After 1 h at -80 °C and after dissolving the reduced MTT dye for 1 h at 37 °C in DMSO the absorbance was determined at 570 versus 630 nm (FluoStar, BMG Labtech, Offenburg, Germany). In addition, cellular viability was detected over time in a real-time impedance measurement system (xCELLigence system, Roche, Penzberg, Germany) as previously described [52]. Cell death analysis Cells were seeded in 24-well plates (60,000 cells/well) and after 24 h of growing glutamate (3 mM) and TLCK (50 lM) were added and cell death was quantified after 13.5 h by AVPI staining and subsequent FACS-analysis.

123

Apoptosis

Cells were harvested by using 19 TE (Trypsin/EDTA, Sigma-Aldrich, Taufkirchen, Germany), washed once in 19 PBS and stained according to the manufacturer’s protocol (Annexin-V-FITC Detection Kit, PromoKine, Promocell, Heidelberg, Germany). Afterwards stained cells were analyzed by FACS-analysis (Guava easyCyte System, Millipore, USA). Annexin-V-FITC was excited at 488 nm and emission was detected through a 530 ± 40 nm band pass filter. Propidium iodide was excited at 488 nm and fluorescence emission was detected using a 680 ± 30 nm band pass filter. At least 10,000 gated events per sample were analyzed. Data are representative of at least three independent experiments. Plasmid transfection The tBid plasmid was generated as described previously [53]. The plasmid pcDNA 3.1 was used as a control vector and was obtained from Invitrogen (Karlsruhe, Germany). For plasmid transfection 40,000 cells/well were seeded in 24-well plates and were allowed to grow overnight. The next day cells were pre-treated for 1 h with 50 lM TLCK in 500 ll of culture medium and afterwards transfected with 2 lg tBid plasmid or pcDNA 3.1 (empty vector) dissolved in OptiMEM I (Karlsruhe, Invitrogen, Germany). Attractene (4.5 ll/well; Qiagen, Hilden, Germany) was used as transfection reagent. After 20 min of incubation at RT, the transfection mix (OptiMEM I, attractene and plasmid) was applied to each well and after 17–18 h of treatment cell death was analyzed by AVPI staining and subsequent FACS-analysis. Confocal laser scanning microscopy For mitochondrial translocation of Bid analysis, HT-22 cells cultured in ibidi l-slide eight-well plates at a densitiy of 12,000 cells/well and were co-transfected with pDsRed2-Bid and pAAV2-CMV-mitoEGFP vectors. Where, pDsRed2-Bid encodes a fusion of Discosoma sp. Red fluorescent protein with human Bid and pAAV2-CMVmitoEGFP encodes mitochondrial targeted EGFP to visualize the mitochondria. Plasmid co-transfection was performed with attractene as mentioned above. Following 24 h of transfection, cells were cotreated with glutamate (5 mM) and TLCK (50 lM) for 16 h. After fixation of cells with 4 % paraformaldehyde, nuclei were stained with Dapi (1 lg/ml) in 1XPBS for 15 min at room temperature and washed with PBS. Bid translocation images were acquired through a 63X1.4 NA, oil immersion objective of cofocal laser microscope (Leica SP5; Leica, Wetzler, Germany). Where mitoEGFP flurosecence was excited at 488 nm, and emissions were detected between 500 and

123

535 nm bandwidth. DsRed2-Bid fluorescence was detected by excitation at 633 nm and emission between 640 and 750 nm window. Digital images were obtained by LSM image browser 4.2.0 (Carl Zeiss). Lipid peroxidation Cells were seeded in 24-well plates (60,000 cells/well) and after growing for 24 h glutamate and TLCK were added and cellular lipid peroxides were detected by loading the cells after indicated time points with 2 mM BODIPY (boron-dipyrromethene) 581/591 C11 (Invitrogen, Karlsruhe, Germany) in HT-22 medium. After 1 h of staining cells were collected, washed once in 19 PBS and re-suspended in 19 PBS for FACS-analysis. FACS-analysis was performed using 488 nm UV line argon laser for excitation and BODIPY emission was recorded at 530 nm (reduced dye) and 585 nm (oxidized dye). At least 10,000 gated events per sample were analyzed. Data are representative of at least three independent experiments. Mitochondrial membrane potential measurements For detection of changes in the MMP in whole cells the TMRE assay (MitoPTTM TMRE kit (Immunochemistry Technologies, Hamburg, Germany)) was used. In 24-well plates 60,000 cells/well were seeded and after growing for 24 h glutamate and TLCK were added. After induction of oxytosis cells were collected and incubated for 20 min at 37 °C with TMRE (Tetramethylrhodamine ethyl ester). Afterwards, cells were washed in 19 PBS and re-suspended in 19 assay buffer. CCCP (50 lM) protonophore was used as a positive control to induce a complete loss of MMP. It was applied to cells with intact mitochondria for at least 30 min before TMRE staining. FACS-analysis was performed using 488 nm UV line argon laser for excitation and the emission was performed at 680 nm. At least 10,000 gated events per sample were analyzed. Data are representative of at least three independent experiments. Mitochondrial morphology Cells were stained with MitoTracker DeepRed (Invitrogen, Karlsruhe, Germany) to analyze mitochondrial morphology alterations. In eight-well ibidi slides 17,000 cells/well were seeded and staining was performed before glutamate exposure. After the indicated time point cells were fixed with 4 % paraformaldehyde for 20 min at RT. Blinded counting of at least 500 cells per condition was examined for at least three independent times. Images were acquired using a confocal microscope (Carl Zeiss, LSM 150, Jena,

Apoptosis

Germany). MitoTracker DeepRed fluorescence was excited at a wavelength of 620 nm band pass filter and emissions were detected using 670 nm long pass filter (red). ATP measurements For luminescence measurements cells were seeded in white 96-well plates (10,000 cells/well, Greiner Bio one, Frickenhausen, Germany). At indicated time points after glutamate treatment ATP levels were analyzed by luminescence detection (FluoStar, BMG Labtech, Offenburg, Germany) according to the manufacture’s protocol using the ViaLightTM plus kit (Lonza, Verviers, Belgium). Determination of the cellular oxygen consumption rate (OCR) For detection of changes in the mitochondrial respiration, HT-22 cells were seeded in XF96-well microplates (10,000 cells/well, Seahorse Bioscience) and were allowed to grow overnight. Then 3–5 mM glutamate was applied with or without TLCK and after 16 h of treatment the OCR was measured as previously described [54]. Briefly, the growth medium was replaced before the measurements with *180 ll of assay medium (with 4.5 g/L glucose as the sugar source, 2 mM glutamine, 1 mM pyruvate, pH 7.35) and cells were incubated for 60 min at 37 °C. Three baseline measurements were recorded before applying the different compounds. Oligomycin was added in Port A (20 ll) at a final concentration of 3 lM, FCCP (22.5 ll in Port B) at a concentration of 0.4 lM and Rotenone/Antimycin A (25 ll in Port C) at a concentration of 1 lM. Three measurements were performed after the addition of each compound (4 min mixing followed by 3 min measuring).

Statistical analysis All data are given as means ± standard deviation (SD). Analysis of variance (ANOVA) was performed followed by Scheffe´´ s post hoc test for statistical comparison between multiple groups. Calculations were performed with the WinStat standard statistical software (R. Fitch Software, Bad Krozingen, Germany). Acknowledgments We thank the excellent technical support by Mrs. Katharina Elsa¨sser and Eileen Daube and the support by our student Lucia von Wachter for the mitochondrial counting. We thank Wei Wan and Shuna Wang for their technical support. Furthermore, we thank Mrs. Emma Esser for careful editing of the manuscript and Roche Diagnostics GmbH for providing support with the xCELLigence system. Conflict of interests The authors declare that they have no conflict of interests.

References 1. Loo DT, Copani A, Pike CJ et al (1993) Apoptosis is induced by beta-amyloid in cultured central nervous system neurons. Proc Natl Acad Sci USA 90(17):7951–7955 2. Mattson MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1(2):120–129 3. Culmsee C, Landshamer S (2006) Molecular insights into mechanisms of the cell death program: role in the progression of neurodegenerative disorders. Curr Alzheimer Res 3(4): 269–283 4. Galluzzi L, Blomgren K, Kroemer G (2009) Mitochondrial membrane permeabilization in neuronal injury. Nat Rev Neurosci 10(7):481–494 5. Landshamer S, Hoehn M, Barth N et al (2008) Bid-induced release of AIF from mitochondria causes immediate neuronal cell death. Cell Death Differ 15(10):1553–1563 6. Grohm J, Plesnila N, Culmsee C (2010) Bid mediates fission, membrane permeabilization and peri-nuclear accumulation of mitochondria as a prerequisite for oxidative neuronal cell death. Brain Behav Immun 24(5):831–838 7. Galluzzi L, Vitale I, Abrams JM et al (2011) Molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death 2012. Cell Death Differ 19(1):107–120 8. Rodriguez J, Lazebnik Y (1999) Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev 13(24):3179–3184 9. Zou H (1999) An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274(17):11549–11556 10. Williams MS, Henkart PA (1994) Apoptotic cell death induced by intracellular proteolysis. J Immunol 153(9):4247–4255 11. Ferri KF, Kroemer G (2001) Organelle-specific initiation of cell death pathways. Nat Cell Biol 3(11):E255–E263 12. Guicciardi ME, Leist M, Gores GJ (2004) Lysosomes in cell death. Oncogene 23(16):2881–2890 13. Miao Q, Sun Y, Wei T et al (2008) Chymotrypsin B cached in rat liver lysosomes and involved in apoptotic regulation through a mitochondrial pathway. J Biol Chem 283(13):8218–8228 14. Saito Y, Kondo H, Hojo Y (2011) Granzyme B as a novel factor involved in cardiovascular diseases. J Cardiol 57(2):141–147 15. Roberg K (2001) Relocalization of cathepsin D and cytochrome c early in apoptosis revealed by immunoelectron microscopy. Lab Invest 81(2):149–158 16. Stoka V, Turk B, Schendel SL et al (2001) Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route. J Biol Chem 276(5):3149–3157 17. Cirman T, Oresic K, Mazovec GD et al (2004) Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem 279(5):3578–3587 18. Zhao K, Zhao X, Tu Y et al (2010) Lysosomal chymotrypsin B potentiates apoptosis via cleavage of Bid. Cell Mol Life Sci 67(15):2665–2678 19. Ruggiero V, Johnson SE, Baglioni C (1987) Protection from tumor necrosis factor cytotoxicity by protease inhibitors. Cell Immunol 107(2):317–325 20. Chow SC, Weis M, Kass GE et al (1995) Involvement of multiple proteases during fas-mediated apoptosis in T lymphocytes. FEBS Lett 364(2):134–138 21. Turgeon VL, Houenou LJ (1997) The role of thrombinlike (serine) proteases in the development, plasticity and pathology of the nervous system. Brain Res Brain Res Rev 25(1):85–95 22. Komatsu N, Oda T, Muramatsu T (1998) Involvement of both caspase-like proteases and serine proteases in apoptotic cell death

123

Apoptosis

23.

24.

25.

26. 27.

28.

29.

30.

31.

32.

33.

34.

35.

36. 37. 38. 39.

induced by ricin, modeccin, diphtheria toxin, and pseudomonas toxin. J Biochem 124(5):1038–1044 Hara A, Niwa M, Nakashima M et al (1998) Protective effect of apoptosis-inhibitory agent, N-tosyl-L-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage. J Cereb Blood Flow Metab 18(8):819–823 Mitsui C, Sakai K, Ninomiya T et al (2001) Involvement of TLCK-sensitive serine protease in colchicine-induced cell death of sympathetic neurons in culture. J Neurosci Res 66(4):601–611 Gong B, Chen Q, Endlich B et al (1999) Ionizing radiationinduced, bax-mediated cell death is dependent on activation of cysteine and serine proteases. Cell Growth Differ 10(7):491–502 Moffitt KL, Martin SL, Walker B (2007) The emerging role of serine proteases in apoptosis. Biochem Soc Trans 35(3):559–560 Dong Z, Saikumar P, Patel Y et al (2000) Serine protease inhibitors suppress cytochrome c-mediatedcaspase-9 activation and apoptosis during hypoxia-reoxygenation. Biochem J 347(Pt 3):669–677 Waterhouse NJ, Sedelies KA, Browne KA et al (2005) A central role for Bid in granzyme B-induced apoptosis. J Biol Chem 280(6):4476–4482 Aoshiba K, Yasuda K, Yasui S et al (2001) Serine proteases increase oxidative stress in lung cells. Am J Physiol Lung Cell Mol Physiol 281(3):L556–L564 Ding X, Patel M, Shen D et al (2009) Enhanced HtrA2/Omi expression in oxidative injury to retinal pigment epithelial cells and murine models of neurodegeneration. Invest Ophthalmol Vis Sci 50(10):4957–4966 Mattson MP (1998) Modification of ion homeostasis by lipid peroxidation: roles in neuronal degeneration and adaptive plasticity. Trends Neurosci 21(2):53–57 Tobaben S, Grohm J, Seiler A et al (2011) Bid-mediated mitochondrial damage is a key mechanism in glutamate-induced oxidative stress and AIF-dependent cell death in immortalized HT-22 hippocampal neurons. Cell Death Differ 18(2):282–292 Takahashi K (2010) Antioxidative action of N-?-tosyl-L-lysine chloromethyl ketone prevents death of glutathione-depleted cardiomyocytes induced by hydrogen peroxide. JBPC 01(03):164–171 Seo AY, Joseph A, Dutta D et al (2010) New insights into the role of mitochondria in aging: mitochondrial dynamics and more. J Cell Sci 123(15):2533–2542 Culmsee C, Krieglstein J (2007) Ischaemic brain damage after stroke: new insights into efficient therapeutic strategies. EMBO Rep 8(2):129–133 Ott M, Gogvadze V, Orrenius S et al (2007) Mitochondria, oxidative stress and cell death. Apoptosis 12(5):913–922 Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443(7113):787–795 Suen D, Norris KL, Youle RJ (2008) Mitochondrial dynamics and apoptosis. Genes Dev 22(12):1577–1590 Blomgran R, Zheng L, Stendahl O (2007) Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-

123

40.

41. 42. 43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

induced lysosomal membrane permeabilization. J Leukoc Biol 81(5):1213–1223 Castino R, Bellio N, Nicotra G et al (2007) Cathepsin D-Bax death pathway in oxidative stressed neuroblastoma cells. Free Radic Biol Med 42(9):1305–1316 Leist M, Ja¨a¨ttela¨ M (2001) Triggering of apoptosis by cathepsins. Cell Death Differ 8(4):324–326 Abraham MC, Shaham S (2004) Death without caspases, caspases without death. Trends Cell Biol 14(4):184–193 Hu Y, Huang M, Wang P et al (2013) Ucf-101 protects against cerebral oxidative injury and cognitive impairment in septic rat. Int Immunopharmacol 16(1):108–113 Rideout HJ, Zang E, Yeasmin M et al (2001) Inhibitors of trypsin-like serine proteases prevent DNA damage-induced neuronal death by acting upstream of the mitochondrial checkpoint and of p53 induction. Neuroscience 107(2):339–352 Wang Y, Luo W, Reiser G (2008) Trypsin and trypsin-like proteases in the brain: proteolysis and cellular functions. Cell Mol Life Sci 65(2):237–252 Solomon DH, O’Brian CA, Weinstein IB (1985) N-alpha-tosyl-Llysine chloromethyl ketone and N-alpha-tosyl-L-phenylalanine chloromethyl ketone inhibit protein kinase C. FEBS Lett 190(2):342–344 Kupfer A, Gani V, Jimenez JS et al (1979) Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone. Proc Natl Acad Sci 76(7):3073–3077 Saldeen J, Welsh N (1994) Interleukin-1b induced Activation of NF-jB in insulin-producing RINm5F cells is prevented by the protease inhibitor Na-\i[p-tosyl-L-lysine chloromethylketone. Biochem Biophy Res Commun 203(1):149–155 Kim H, Lee HS, Chang KT et al (1995) Chloromethyl ketones block induction of nitric oxide synthase in murine macrophages by preventing activation of nuclear factor-kappa B. J Immunol 154(9):4741–4748 Zhou Q, Salvesen G (1997) Activation of pro-caspase-7 by serine proteases includes a non-canonical specificity. Biochem J 324: 361–364 Frydrych I, Mlejnek P (2008) Serine protease inhibitors N-alphatosyl-L-lysinyl-chloromethylketone (TLCK) and N-tosyl-L-phenylalaninyl-chloromethylketone (TPCK) are potent inhibitors of activated caspase proteases. J Cell Biochem 103(5):1646–1656 Diemert S, Dolga AM, Tobaben S et al (2012) Impedance measurement for real time detection of neuronal cell death. J Neurosci Methods 203(1):69–77 Kazhdan I, Long L, Montellano R et al (2006) Targeted gene therapy for breast cancer with truncated Bid. Cancer Gene Ther 13(2):141–149 Gohil VM, Sheth SA, Nilsson R et al (2010) Nutrient-sensitized screening for drugs that shift energy metabolism from mitochondrial respiration to glycolysis. Nat Biotechnol 28(3):249–255