Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

The Cytoskeletal Protein ��-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.Acid-sensing ion channels (ASICs)² are H⁺-gated members of the DEG/ENaC family (1–3). Members of this family contain cytosolic N and C termini, two transmembrane domains, and a large cysteine-rich extracellular domain. ASIC subunits combine as homo- or heterotrimers to form cation channels that are widely expressed in the central and peripheral nervous systems (1–4). In mammals, four genes encode ASICs, and two subunits, ASIC1 and ASIC2, have two splice forms, a and b. Central nervous system neurons express ASIC1a, ASIC2a, and ASIC2b (5–7). Homomeric ASIC1a channels are activated when extracellular pH drops below 7.2, and half-maximal activation occurs at pH 6.5–6.8 (8–10). These channels desensitize in the continued presence of a low extracellular pH, and they can conduct Ca²⁺ (9, 11–13). ASIC1a is required for acid-evoked currents in central nervous system neurons; disrupting the gene encoding ASIC1a eliminates H⁺-gated currents unless extracellular pH is reduced below pH 5.0 (5, 7).Previous studies found ASIC1a enriched in synaptosomal membrane fractions and present in dendritic spines, the site of excitatory synapses (5, 14, 15). Consistent with this localization, ASIC1a null mice manifested deficits in hippocampal long term potentiation, learning, and memory, which suggested that ASIC1a is required for normal synaptic plasticity (5, 16). ASICs might be activated during neurotransmission when synaptic vesicles empty their acidic contents into the synaptic cleft or when neuronal activity lowers extracellular pH (17–19). Ion channels, including those at the synapse often interact with multiple proteins in a macromolecular complex that incorporates regulators of their function (20, 21). For ASIC1a, only a few interacting proteins have been identified. Earlier work indicated that ASIC1a interacts with another postsynaptic scaffolding protein, PICK1 (15, 22, 23). ASIC1a also has been reported to interact with annexin II light chain p11 through its cytosolic N terminus to increase cell surface expression (24) and with Ca²⁺/calmodulin-dependent protein kinase II to phosphorylate the channel (25). However, whether ASIC1a interacts with additional proteins and with the cytoskeleton remain unknown. Moreover, it is not known whether such interactions alter ASIC1a function.In analyzing the ASIC1a amino acid sequence, we identified cytosolic residues that might bind α-actinins. α-Actinins cluster membrane proteins and signaling molecules into macromolecular complexes and link membrane proteins to the actincytoskeleton (for review, Ref. 26). Four genes encode α-actinin-1, -2, -3, and -4 isoforms. α-Actinins contain an N-terminal head domain that binds F-actin, a C-terminal region containing two EF-hand motifs, and a central rod domain containing four spectrin-like motifs (26–28). The C-terminal portion of the rod segment appears to be crucial for binding to membrane proteins. The α-actinins assemble into antiparallel homodimers through interactions in their rod domain. α-Actinins-1, -2, and -4 are enriched in dendritic spines, concentrating at the postsynaptic membrane (29–35). In the postsynaptic membrane of excitatory synapses, α-actinin connects the NMDA receptor to the actin cytoskeleton, and this interaction is key for Ca²⁺-dependent inhibition of NMDA receptors (36–38). α-Actinins can also regulate the membrane trafficking and function of several cation channels, including L-type Ca²⁺ channels, K⁺ channels, and TRP channels (39–41).To better understand the function of ASIC1a channels in macromolecular complexes, we asked if ASIC1a associates with α-actinins. We were interested in the α-actinins because they and ASIC1a, both, are present in dendritic spines, ASIC1a contains a potential α-actinin binding sequence, and the related epithelial Na⁺ channel (ENaC) interacts with the cytoskeleton (42, 43). Therefore, we hypothesized that α-actinin interacts structurally and functionally with ASIC1a.     

Mechanism of Sustained Activation of Ribosomal S6 Kinase (RSK) and ERK by Kaposi Sarcoma-associated Herpesvirus ORF45: MULTIPROTEIN COMPLEXES RETAIN ACTIVE PHOSPHORYLATED ERK AND RSK AND PROTECT THEM FROM DEPHOSPHORYLATION*

As obligate intracellular parasites, viruses exploit diverse cellular signaling machineries, including the mitogen-activated protein-kinase pathway, during their infections. We have demonstrated previously that the open reading frame 45 (ORF45) of Kaposi sarcoma-associated herpesvirus interacts with p90 ribosomal S6 kinases (RSKs) and strongly stimulates their kinase activities (Kuang, E., Tang, Q., Maul, G. G., and Zhu, F. (2008) J. Virol. 82 ,1838 -1850). Here, we define the mechanism by which ORF45 activates RSKs. We demonstrated that binding of ORF45 to RSK increases the association of extracellular signal-regulated kinase (ERK) with RSK, such that ORF45, RSK, and ERK formed high molecular mass protein complexes. We further demonstrated that the complexes shielded active pERK and pRSK from dephosphorylation. As a result, the complex-associated RSK and ERK were activated and sustained at high levels. Finally, we provide evidence that this mechanism contributes to the sustained activation of ERK and RSK in Kaposi sarcoma-associated herpesvirus lytic replication.The extracellular signal-regulated kinase (ERK)² mitogen-activated protein kinase (MAPK) signaling pathway has been implicated in diverse cellular physiological processes including proliferation, survival, growth, differentiation, and motility (1-4) and is also exploited by a variety of viruses such as Kaposi sarcoma-associated herpesvirus (KSHV), human cytomegalovirus, human immunodeficiency virus, respiratory syncytial virus, hepatitis B virus, coxsackie, vaccinia, coronavirus, and influenza virus (5-17). The MAPK kinases relay the extracellular signaling through sequential phosphorylation to an array of cytoplasmic and nuclear substrates to elicit specific responses (1, 2, 18). Phosphorylation of MAPK is reversible. The kinetics of deactivation or duration of signaling dictates diverse biological outcomes (19, 20). For example, sustained but not transient activation of ERK signaling induces the differentiation of PC12 cells into sympathetic-like neurons and transformation of NIH3T3 cells (20-22). During viral infection, a unique biphasic ERK activation has been observed for some viruses (an early transient activation triggered by viral binding or entry and a late sustained activation correlated with viral gene expression), but the responsible viral factors and underlying mechanism for the sustained ERK activation remain largely unknown (5, 8, 13, 23).The p90 ribosomal S6 kinases (RSKs) are a family of serine/threonine kinases that lie at the terminus of the ERK pathway (1, 24-26). In mammals, four isoforms are known, RSK1 to RSK4. Each one has two catalytically functional kinase domains, the N-terminal kinase domain (NTKD) and C-terminal kinase domain (CTKD) as well as a linker region between the two. The NTKD is responsible for phosphorylation of exogenous substrates, and the CTKD and linker region regulate RSK activation (1, 24, 25). In quiescent cells ERK binds to the docking site in the C terminus of RSK (27-29). Upon mitogen stimulation, ERK is activated by its upstream MAPK/ERK kinase (MEK). The active ERK phosphorylates Thr-359/Ser-363 of RSK in the linker region (amino acid numbers refer to human RSK1) and Thr-573 in the CTKD activation loop. The activated CTKD then phosphorylates Ser-380 in the linker region, creating a docking site for 3-phosphoinositide-dependent protein kinase-1. The 3-phosphoinositide-dependent protein kinase-1 phosphorylates Ser-221 of RSK in the activation loop and activates the NTKD. The activated NTKD autophosphorylates the serine residue near the ERK docking site, causing a transient dissociation of active ERK from RSK (25, 26, 28). The stimulation of quiescent cells by a mitogen such as epidermal growth factor or a phorbol ester such as 12-O-tetradecanoylphorbol-13-acetate (TPA) usually results in a transient RSK activation that lasts less than 30 min. RSKs have been implicated in regulating cell survival, growth, and proliferation. Mutation or aberrant expression of RSK has been implicated in several human diseases including Coffin-Lowry syndrome and prostate and breast cancers (1, 24, 25, 30-32).KSHV is a human DNA tumor virus etiologically linked to Kaposi sarcoma, primary effusion lymphoma, and a subset of multicentric Castleman disease (33, 34). Infection and reactivation of KSHV activate multiple MAPK pathways (6, 12, 35). Noticeably, the ERK/RSK activation is sustained late during KSHV primary infection and reactivation from latency (5, 6, 12, 23), but the mechanism of the sustained ERK/RSK activation is unclear. Recently, we demonstrated that ORF45, an immediate early and also virion tegument protein of KSHV, interacts with RSK1 and RSK2 and strongly stimulates their kinase activities (23). We also demonstrated that the activation of RSK plays an essential role in KSHV lytic replication (23). In the present study we determined the mechanism of ORF45-induced sustained ERK/RSK activation. We found that ORF45 increases the association of RSK with ERK and protects them from dephosphorylation, causing sustained activation of both ERK and RSK.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.