Lysosomal chymotrypsin induces mitochondrial fission in apoptotic cells by proteolytic activation of calcineurin

Apoptosis is a fundamental physiological process in mammals in which cells die by activating a suicide mechanism. The mitochondria are one of the major checkpoints in apoptotic regulation because they serve as sensors and amplifiers of cellular damage (Green and Kroemer, 2004). After mitochondrial outer membrane permeabilization (MOMP), the mitochondria release a number of factors that are critically involved in cell death signaling (Tait and Green, 2010). Bcl-2 family members are regarded as the key regulators of mitochondria-dependent apoptosis (Moldoveanu et al., 2014); however, dynamin-related protein 1 (Drp1), which orchestrates mitochondrial fission, also participates in apoptotic regulation by stimulating Bax oligomerization and thereby enhances MOMP (Montessuit et al., 2010); accordingly, the inhibition of Drp1 blocks mitochondrial fission and inhibits apoptosis (Cassidy-Stone et al., 2008). During mitochondrial fission, Drp1 assembles from the cytosol onto the mitochondria at focal sites of division, forming spiral chains around membrane constriction sites. It has been well documented that the phosphorylation/dephosphorylation of Drp1 may act as a molecular switch to “turn on” or “turn off”mitochondrial fission (Chang and Blackstone, 2010). The phosphorylation of Drp1 at Ser 656 by cyclicAMP-dependent protein kinase (PKA) induces mitochondrial elongation, whereas the dephosphorylation of Ser 656 by calcineurin promotes mitochondrial fragmentation (Cribbs and Strack, 2007). Calcineurin is a calciumand calmodulindependent phosphatase. In apoptotic cells, the fragmentation of depolarized mitochondria depends on Ca-evoked, calcineurin-mediated dephosphorylation of Drp1 at its conserved serine 637 site (Cereghetti et al., 2008). The importance of calcineurin in mitochondrial fission is also supported by the findings that an inhibitor of calcineurin (Cereghetti et al., 2010) or the use of a miRNA targeting calcineurin (Wang et al., 2011) regulates mitochondrial fission by modulating Drp1 dephosphorylation and translocation. In this study, we explored an uncanonical, lysosomal chymotrypsinmediated activation mechanism of calcineurin that leads to Drp1-mediated mitochondrial fission in apoptotic cells. Wehavepreviously reported that chymotrypsin is not only a digestive enzyme secreted by the pancreas but also expressed widely in rat tissues (Zhao et al., 2010) and cached in lysosomes (Miao et al., 2008). However, the expression and subcellular localization of chymotrypsin in cells with human origin remain unclear. Using immunofluorescence, we found that chymotrypsin was colocalized with the lysosomal marker protein LAMP1 in human neuroblastoma SH-SY5Y cells (Fig. 1A). Upon LeuLeuOMe treatment, which induced lysosomal membrane permeabilization (LMP) directly, lysosomal chymotrypsin was relocated to the cytosol. The induction of LMP triggered apoptosis in SH-SY5Y cells, which was evidencedby the releaseofmitochondrial cytochrome c (Fig. 1B), the cleavage of PARP by activated caspase 3 (Fig. 1C), and the increase in the percentage of apoptotic cells with sub-G1 DNA content (Fig. 1D). The pretreatment of cells with TPCK, which is a specific chymotrypsin inhibitor, effectively inhibited caspase 3 activation and prevented apoptosis, suggesting that lysosomal chymotrypsin may be responsible for the LMPtriggered apoptosis. To further confirm that the translocation of chymotrypsin to the cytosol was sufficient to induce apoptosis, we introduced recombinant human chymotrypsin into the cytosol of SH-SY5Ycells with theBioPorter reagent and found that the intracellular delivery of chymotrypsin significantly potentiated apoptosis, suggesting that chymotrypsin plays a proapoptotic role (Fig. 1E). We found that mitochondrial fission was an event that occurs downstream of LMP in early stage apoptotic cells. Confocal microscopic images indicated that the mitochondria tended to fuse when the lysosomes are intact, whereas 6-h LeuLeuOMe treatment, which induced LMP, ultimately led to mitochondrial fission (Fig. 1F). Pretreatment with TPCK partially blocked the fission of mitochondria (Fig. 1G and 1H); however, E64d and pepstatin A, two inhibitors of the lysosomal cathepsins, had no apparent effect on the LeuLeuOMe-induced mitochondrial fission and apoptosis (data not shown). These data suggested the involvement of chymotrypsin in mitochondrial fission during apoptosis. Mitochondrial fission depends on the translocation of cytoplasmic Drp1 to mitochondria, where it binds to Fis1, oligomerizes, and constricts the organelle, ultimately leading


SH-SY5Y human neuroblastoma cells obtained from ATCC (American Type Culture
Collection, Manassas, VA, USA) were cultured in DMEM/F12 (Life Technologies, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C with 5% CO 2 . To construct expression vectors containing full-length or truncated calcineurin, the cDNA for calcineurin was cloned by RT-PCR using human mRNA as the template. The cDNAs for full-length calcineurin and truncated calcineurin (1-410 lacking the C-terminal autoinhibitory domain) were subcloned into the mammalian expression vector pEGFP-C2 (Clontech, Mountain View, CA, USA). The shRNA targeting the calcineurin was provided by RiboBio (Guangzhou, China).
In some experiments SH-SY5Y cells were transfected using Fugene HD reagent (Promega, Madison, WI, USA) according to the manufacturer's instructions.

Expression of recombinant proteins
Recombinant human chymotrypsin was obtained as previously described (Zhao et al., 2010). Recombinant human calcineurin α was expressed and purified as previously described (Mondragon et al., 1997).

Intracellular delivery of recombinant chymotrypsin
Recombinant chymotrypsin was delivered to SH-SY5Y cells using the BioPORTER protein transfection reagent (Gene Therapy Systems; San Diego, CA, USA) as described previously (Zhao et al., 2010).

Immunofluorescence and confocal microscopy
SH-SY5Y neuroblastoma cells on glass coverslips were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100, and blocked for 1 h at room temperature with PBS containing 5% goat serum albumin. Cells were incubated with antibodies (anti-chymotrypsin, Santa Cruz Biotechnology, Santa Cruz, CA, USA; anti -Drp-1, Cell Signaling Technology, Danvers, MA, USA ) and then incubated with the appropriate secondary antibodies. To visualize mitochondria in living cells, cells were stained with MitoTracker Red CMXRos (Life Technologies) at 37°C for 30 min and washed twice with PBS. Nuclei were stained with DAPI (Sigma-Aldrich, St. Louis, MO, USA). Cells were visualized by Z-Stack imaging with a confocal microscope (Olympus FV1000; Tokyo, Japan) and processed using Fluoview software (Olympus). The length of mitochondria was measured using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) as described previously (Zhao et al., 2013).

Apoptosis assays
Cells were harvested by mild trypsinization, washed with cold PBS, and fixed with 70% ethanol. Cells were stained with propidium iodide (PI, Sigma-Aldrich, St. Louis, MO, USA), and the percentage of hypodiploid (apoptotic) cells was measured with a FACSCalibur flow cytometer.

In vitro cleavage of endogenous calcineurin
SH-SY5Y cells were lysed in ice-cold buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl) by sonication. After ultracentrifugation at 10,000×g for 10 min at 4°C, supernatants containing solubilized proteins were incubated with chymotrypsin at 37 °C for 1 h. Samples were resolved by SDS-PAGE and the specific cleavage of endogenous calcineurin was detected by immunoblotting with antibodies against Nor C-terminus of calcineurin, respectively.
Because calcineurin activity is enhanced in the presence of Mn 2+ , Mn 2+ was included in the assay buffer for conditions ii, iii and iv to aid detection.