Regulatory Mechanisms of Mitochondria Involved in Apoptosis, Necrosis and Autophagy in Anuclear Cells, Platelets

Mitochondria play an important role in mediating both apoptotic and necrotic cell death. Necrosis is a passive form of cell death, characterized by depletion of ATP, cell swelling, and the collapse of plasma membrane, etc. There are several features of apoptosis, including DNA fragmentation, chromatin condensation, and phosphatidylserine (PS) exposure, etc. In caspase-dependent apoptosis, cytokine ligands (such as [TNF-α or Fas-L) engage to membrane-bound death receptor, which can recruit DISC (death-inducing signaling complex). In mitochondrial pathway of apoptosis, it can be mediated by oligomerization of Bax/Bak, forming channels permit multiple proteins to release from mitochondria. In caspase-independent apoptosis, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) translocate from mitochondria to the nucleus and provoke DNA degradation. In the processes of apoptosis and necrosis, the mitochondrial permeability transition (mPT) leads to disruption of the mitochondrial membranes. mPT pore is thought to consist of a voltage-dependent anion channel, an adenine nucleotide translocator, cyclophilin D (CypD), and some other molecules. CypD and mPT are required for mediating Ca2+- and oxidative stress-induced cell necrosis. On the other hand, the regulatory process of autophagy (self-eating) involves the de-repression of the mammalian Target of Rapamycin (mTOR) Ser/Thr kinase. Two ubiquitin-like conjugation systems are part of the vesicle elongation process. One pathway involves the covalent conjugation of Atg (autophagic factor) 12 to Atg5. The second pathway involves the conjugation of phosphatidylethanolamine to LC3/Atg8 (LC3 is one of the mammalian homologues of Atg8). Lipid conjugation leads to the conversion of the soluble form of LC3 (named LC3I) to the autophagicvesicle-associated form (LC3II). LC3II is used as a marker of autophagy. Autophagosomes undergo maturation by fusion with lysosomes to create autolysosomes. Platelets, anuclear blood cells, play a central role in thrombotic and hemostatic processes. Intriguingly, apoptotic events have also been found in platelets. Platelets express several caspases, death receptors, and Bcl-2 families. Our preliminary results also revealed that platelet agonist (such as thrombin) exhibit a potent activity at inducing PS exposure, mitochondrial depolarization (Fig. 1), and stimulates caspase-9 and -3 activations (Fig. 2) in washed platelets. These apoptosis-like events suggest that apoptotic cytoplasmic apparatus might function without nuclear participation. In addition, NF-κB plays an important role in apoptosis. Under normal condition, IKKβ activates IκB and induces NF-κB nuclear translocation. Recently, we first demonstrate that stimulation of platelets, with amyloid β induces IKKβ phosphorylation (Fig. 3) and IκBα degradation (Fig. 4), it subsequent to allow 2 the NFκB (p65 and p50) translocation from cytosol into mitochondria (Fig. 5), which binds to mitochondrial DNA (mtDNA) (Fig. 6). It implies the transcription factors may function in the cellular compartments other then nucleus and may play a special role in anuclear cells. Evidences have showed that mitochondria play a crucial role in apoptosis, necrosis, and autophagy. However, the molecular mechanisms of apoptosis- and necrosis-relative events and the regulatory roles of mitochondria involved in these actions are still unclear in platelets. We therefore for the first time systematically examine these issues in this project, and further investigate whether autophagy may also occur in platelets. Specific aim 1: To determine the roles of Bax-associated signal cascades in platelet apoptosis (Fig. 7). Hypothesis 1: Bax may translocate from cytosol into mitochondrial, which induces mitochondrial permeability transition (mPT) and causes cytochrome c, Smac/Diablo, HtrA2/Omi, apoptosis-inducing factor (AIF), and endonuclease G (Endo G) releases, it subsequent to form the apoptosome (cytochrome c, apoptosis protease activating factor-1 (Apaf-1) and procaspase-9), and finally activates caspase-9 and -3 in platelets. 1.1. To study whether Bax translocated from cytosol into mitochondria occurs in human platelets. 1.2. To assess whether recombinant Bax (rBax) stimulates mPT in purified mitochondria in human platelets. 1.3. To investigate rBax-induced cytochrome c, Smac/Diablo, HtrA2/Omi, AIF, and Endo G releases in human platelets. 1.4. To determine rBax-induced mitochondrial DNA (mtDNA) fragmentation in platelets. 1.5. To study and compare the differences of mtDNA fragmentation, mPT, PS exposure, caspase-3 activation, and Smac/Diablo, HtrA2/Omi release in platelets isolated from Bax knockout (Bax-/-) and wild-type mice. Specific aim 2: To determine whether cyclophilin D-dependent mitochondrial permeability transition (mPT) regulates some necrotic but not apoptotic cell death in platelets (Fig. 7). Hypothesis 2: Cyclophilin D-deficient cells may be resistant to necrotic cell death induced by reactive oxygen species and Ca2+ overload, whereas normally die in response to various apoptotic stimuli, such as Bax. 2.1. To determine whether cyclophilin D plays a crucial role in regulation of mPT during platelet activation in washed human platelets. 2.2. To compare the differences of mitochondrial swelling, mPT, and cytochrome c 3 release stimulated by Ca2+ and H2O2 (stimulators of necrosis) or rBax (apoptotic factor) in washed human platelets. 2.3. To compare and analyze the differences of Ca2+-, H2O2- or rBax-induced mPT, cytochrome c release, PS exposure, and caspase-3 activation, respectively, in washed platelets from cyclophilin D knockout (CypD-/-) and wild-type mice. Specific aim 3: To determine whether translocated NF-κB regulates mitochondrial DNA (mtDNA)-encoded protein expression, contributing to platelet apoptosis (Fig. 7). Hypothesis 3: Stimulation of anuclear cells, platelets, induces IKKβ phosphorylation and IκBα degradation, it subsequent to allow the NF-κB translocation from cytosol into mitochondria, which participates in the regulation of mitochondrial function through transcriptional control of mitochondrial gene expression. 3.1. To determine the IKKβ phosphorylation and IκBα degradation in human platelets. 3.2. To determine NF-κB translocation from cytosol into mitochondria, and the translocated NF-κB binding to the NF-κB-like sequences in mtDNA in human platelets. 3.3. To study translocated NF-κB binding to mtDNA and subsequent inhibition of mtDNA-encoded protein expression (i.e., ND4, NADH dehydrogenase), following induces a series of apoptotic events (i.e., loss of mitochondrial membrane potential and caspase activation) in human platelets. Specific aim 4: To examine whether the machinery of autophagy occurs in human platelets (Fig. 7). Hypothesis 4: Platelets may express beclin-1, LC3 (Atg8), Atg5, and Atg12 mRNA and proteins; and H2O2 may induce beclin-1, Atg5, and Atg12 protein synthesis and stimulates the conversion of LC3I (soluble form) to LC3II (autophagicvesicle-associated form, a marker of autophagy). 4.1. To determine the beclin-1, LC3 (Atg8), Atg5, and Atg12 mRNA levels and protein expressions in human platelets. 4.2. To analyze whether H2O2 induces beclin-1, Atg5, and Atg12 protein synthesis, respectively, and stimulates the conversion of LC3I to LC3II in human platelets. 4.3 To observe autophagic vacuoles formation in H2O2-stimulated platelets by transmission electron microscope. Knowledge of these regulatory mechanisms could provide a rational basis for understanding the mitochondria involved in apoptosis, necrosis, and autophagy in anuclear cells, platelets.