Oxidative stress and apoptosis are two key pathophysiological mechanisms underlying dopaminergic degeneration in Parkinson’s disease (PD). Recently, we identified that proteolytic activation of protein kinase C-delta (PKCδ), a member of the novel PKC family, contributes to oxidative stress-induced dopaminergic degeneration and that phosphorylation of tyrosine residue 311 (tyr311) on PKCδ is a key event preceding the PKCδ proteolytic activation during oxidative damage. Herein, we report that a non-receptor tyrosine kinase Fyn is significantly expressed in a dopaminergic neuronal N27 cell model. Exposure of N27 cells to the dopaminergic toxicant dieldrin (60 μM) rapidly activated Fyn kinase, PKCδ-tyr311 phosphorylation and proteolytic cleavage. Fyn kinase activation precedes the caspase-3-mediated proteolytic activation of PKCδ. Co-treatment with p60-tyrosine-specific kinase inhibitor (TSKI) almost completely attenuated dieldrin-induced phosphorylation of PKCδ-tyr311 and its proteolytic activation. Additionally, TSKI almost completely blocked dieldrin-induced apoptotic cell death. To further confirm Fyn’s role in the pro-apoptotic function of PKCδ, we adopted the RNAi approach. siRNA-mediated knockdown of Fyn kinase also effectively attenuated dieldrin-induced phosphorylation of PKCδ-tyr311, caspase-3-mediated PKCδ proteolytic cleavage, and DNA fragmentation, suggesting that Fyn kinase regulates the pro-apoptotic function of PKCδ. Collectively, these results demonstrate for the first time that Fyn kinase is a pro-apoptotic kinase that regulates upstream signaling of the PKCδ-mediated apoptotic cell death pathway in neurotoxicity models of pesticide exposure.
pesticides; oxidative stress; kinases; apoptosis; neurodegeneration
Mitochondria continuously undergo two opposing processes, fission and fusion. The disruption of this dynamic equilibrium may herald cell injury or death and may contribute to developmental and neurodegenerative disorders. Nitric oxide functions as a signaling molecule, but in excess it mediates neuronal injury, in part via mitochondrial fission or fragmentation. However, the underlying mechanism for nitric oxide–induced pathological fission remains unclear. We found that nitric oxide produced in response to β-amyloid protein, thought to be a key mediator of Alzheimer’s disease, triggered mitochondrial fission, synaptic loss, and neuronal damage, in part via S-nitrosylation of dynamin-related protein 1 (forming SNO-Drp1). Preventing nitrosylation of Drp1 by cysteine mutation abrogated these neurotoxic events. SNO-Drp1 is increased in brains of human Alzheimer’s disease patients and may thus contribute to the pathogenesis of neurodegeneration.
We recently demonstrated that PKCδ, an important member of the novel PKC family, is a key oxidative stress-sensitive kinase that can be activated by caspase-3-dependent proteolytic cleavage to induce dopaminergic neuronal cell death. We now report a novel association between α-synuclein (αsyn), a protein associated with the pathogenesis of Parkinson’s diseases (PD), and PKCδ, in which αsyn negatively modulates the p300 and NFκB dependent transactivation to down-regulate proapoptotic kinase PKCδ expression and thereby protects against apoptosis in dopaminergic neuronal cells. Stable-expression human wild-type αsyn at physiological levels in dopaminergic neuronal cells resulted in an isoform-dependent transcriptional suppression of PKCδ expression without changes in the stability of mRNA and protein or DNA methylation. The reduction in PKCδ transcription was mediated, in part, through the suppression of constitutive NFκB activity targeted at two proximal PKCδ promoter κB sites. This occurred independently of NFκB/IκBα nuclear translocation, but was associated with decreased NFκB-p65 acetylation. Also, αsyn reduced p300 levels and its histone acetyl-transferase (HAT) activity, thereby contributing to diminished PKCδ transactivation. Importantly, reduced PKCδ and p300 expression also were observed within nigral dopaminergic neurons in αsyn transgenic mice. These findings expand the role of αsyn in neuroprotection by modulating the expression of the key proapoptotic kinase PKCδ in dopaminergic neurons.
α-synuclein; PKCδ; apoptosis; Parkinson’s disease; p300; NFκB
Impaired regulation of mitochondrial dynamics, which shifts the balance towards fission, is associated with neuronal death in age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease. A role for mitochondrial dynamics in acute brain injury, however, has not been elucidated to date. Here, we investigated the role of dynamin-related protein 1 (Drp1), one of the key regulators of mitochondrial fission, in neuronal cell death induced by glutamate toxicity or oxygen–glucose deprivation (OGD) in vitro, and after ischemic brain damage in vivo. Drp1 siRNA and small molecule inhibitors of Drp1 prevented mitochondrial fission, loss of mitochondrial membrane potential (MMP), and cell death induced by glutamate or tBid overexpression in immortalized hippocampal HT-22 neuronal cells. Further, Drp1 inhibitors protected primary neurons against glutamate excitotoxicity and OGD, and reduced the infarct volume in a mouse model of transient focal ischemia. Our data indicate that Drp1 translocation and associated mitochondrial fission are key features preceding the loss of MMP and neuronal cell death. Thus, inhibition of Drp1 is proposed as an efficient strategy of neuroprotection against glutamate toxicity and OGD in vitro and ischemic brain damage in vivo.
neuronal cell death; oxidative stress; Drp1; mitochondrial fusion and fission; cerebral ischemia
We recently reported increased mitochondrial fission and decreased fusion, increased amyloid beta (Aβ) interaction with the mitochondrial fission protein Drp1, increased mitochondrial fragmentation, impaired axonal transport of mitochondria and synaptic degeneration in neurons affected by AD. In the present study, we extended our previous investigations to determine whether phosphorylated tau interacts with Drp1 and to elucidate mitochondrial damage in the progression of AD. We also investigated GTPase activity, which is critical for mitochondrial fragmentation, in postmortem brain tissues from patients with AD and brain tissues from APP, APP/PS1 and 3XTg.AD mice. Using co-immunoprecipitation and immunofluorescence analyses, for the first time, we demonstrated the physical interaction between phosphorylated tau and Drp1. Mitochondrial fission-linked GTPase activity was significantly elevated in the postmortem frontal cortex tissues from AD patients and cortical tissues from APP, APP/PS1 and 3XTg.AD mice. On the basis of these findings, we conclude that Drp1 interacts with Aβ and phosphorylated tau, likely leading to excessive mitochondrial fragmentation, and mitochondrial and synaptic deficiencies, ultimately possibly leading to neuronal damage and cognitive decline. Treatment designed to reduce the expression of Drp1, Aβ and/or phosphorylated tau may decrease the interaction between Drp1 and phosphorylated tau and the interaction between Drp1 and Aβ, conferring protection to neurons from toxic insults of excessive Drp1, Aβ and/or phosphorylated tau.
Mitochondria are the essential eukaryotic organelles that produce most cellular energy. The energy production and supply by mitochondria appear closely associated with the continuous shape change of mitochondria mediated by fission and fusion, as evidenced not only by the hereditary diseases caused by mutations in fission/fusion genes but also by aberrant mitochondrial morphologies associated with numerous pathologic insults. However, how morphological change of mitochondria is linked to their energy-producing activity is poorly understood. In this study, we found that perturbation of mitochondrial fission induces a unique mitochondrial uncoupling phenomenon through a large-scale fluctuation of a mitochondrial inner membrane potential. Furthermore, by genetically controlling mitochondrial fission and thereby inducing mild proton leak in mice, we were able to relieve these mice from oxidative stress in a hyperglycemic model. These findings provide mechanistic insight into how mitochondrial fission participates in regulating mitochondrial activity. In addition, these results suggest a potential application of mitochondrial fission to control mitochondrial reactive oxygen species production and oxidative stress in many human diseases.
Mutations in the mitochondrial fission factor GDAP1 are associated with severe peripheral neuropathies, but why the CNS remains unaffected is unclear. Using a Gdap1−/− mouse, Niemann et al. demonstrate that a CNS-expressed Gdap1 paralogue changes its subcellular localisation under oxidative stress conditions to also act as a mitochondrial fission factor.
The ganglioside-induced differentiation-associated protein 1 (GDAP1) is a mitochondrial fission factor and mutations in GDAP1 cause Charcot–Marie–Tooth disease. We found that Gdap1 knockout mice (Gdap1−/−), mimicking genetic alterations of patients suffering from severe forms of Charcot–Marie–Tooth disease, develop an age-related, hypomyelinating peripheral neuropathy. Ablation of Gdap1 expression in Schwann cells recapitulates this phenotype. Additionally, intra-axonal mitochondria of peripheral neurons are larger in Gdap1−/− mice and mitochondrial transport is impaired in cultured sensory neurons of Gdap1−/− mice compared with controls. These changes in mitochondrial morphology and dynamics also influence mitochondrial biogenesis. We demonstrate that mitochondrial DNA biogenesis and content is increased in the peripheral nervous system but not in the central nervous system of Gdap1−/− mice compared with control littermates. In search for a molecular mechanism we turned to the paralogue of GDAP1, GDAP1L1, which is mainly expressed in the unaffected central nervous system. GDAP1L1 responds to elevated levels of oxidized glutathione by translocating from the cytosol to mitochondria, where it inserts into the mitochondrial outer membrane. This translocation is necessary to substitute for loss of GDAP1 expression. Accordingly, more GDAP1L1 was associated with mitochondria in the spinal cord of aged Gdap1−/− mice compared with controls. Our findings demonstrate that Charcot–Marie–Tooth disease caused by mutations in GDAP1 leads to mild, persistent oxidative stress in the peripheral nervous system, which can be compensated by GDAP1L1 in the unaffected central nervous system. We conclude that members of the GDAP1 family are responsive and protective against stress associated with increased levels of oxidized glutathione.
animal models; Charcot-Marie-Tooth disease; mitochondria; axonal transport; demyelinating disease
PTEN-induced putative kinase 1 (PINK1) and Parkin act in a common pathway to regulate mitochondrial dynamics, the involvement of which in the pathogenesis of Parkinson's disease (PD) is increasingly being appreciated. However, how the PINK1/Parkin pathway influences mitochondrial function is not well understood, and the exact role of this pathway in controlling mitochondrial dynamics remains controversial. Here we used mammalian primary neurons to examine the function of the PINK1/Parkin pathway in regulating mitochondrial dynamics and function. In rat hippocampal neurons, PINK1 or Parkin overexpression resulted in increased mitochondrial number, smaller mitochondrial size and reduced mitochondrial occupancy of neuronal processes, suggesting that the balance of mitochondrial fission/fusion dynamics is tipped toward more fission. Conversely, inactivation of PINK1 resulted in elongated mitochondria, indicating that the balance of mitochondrial fission/fusion dynamics is tipped toward more fusion. Furthermore, overexpression of the fission protein Drp1 (dynamin-related protein 1) or knocking down of the fusion protein OPA1 (optical atrophy 1) suppressed PINK1 RNAi-induced mitochondrial morphological defect, and overexpression of PINK1 or Parkin suppressed the elongated mitochondria phenotype caused by Drp1 RNAi. Functionally, PINK1 knockdown and overexpression had opposite effects on dendritic spine formation and neuronal vulnerability to excitotoxicity. Finally, we found that PINK1/Parkin similarly influenced mitochondrial dynamics in rat midbrain dopaminergic neurons. These results, together with previous findings in Drosophila dopaminergic neurons, indicate that the PINK1/Parkin pathway plays conserved roles in regulating neuronal mitochondrial dynamics and function.
Mitochondrial dynamics play a crucial role in the pathobiology underlying Alzheimer’s disease (AD) and Parkinson’s disease (PD). Although a complete scientific understanding of these devastating conditions has yet to be realized, alterations in mitochondrial fission and fusion, and in the protein complexes that orchestrate mitochondrial fission and fusion, have been well established in AD- and PD-related neurodegeneration. Whether fission/fusion disruption in the brain is a causal agent in neuronal demise or a product of some other upstream disturbance is still a matter of debate; however, in both AD and PD, the potential for successful therapeutic amelioration of degeneration via mitochondrial protection is high. We here discuss the role of mitochondrial dynamics in AD and PD and assess the need for their therapeutic exploitation.
Alzheimer’s disease; DLP-1; Drp-1; Fis1; fission; fusion; Mfn1; Mfn2; mitochondrial dynamics; neurodegeneration; Parkinson’s disease; therapeutics
Mitochondrial dynamics and mitophagy are recognized as two critical processes underlying mitochondrial homeostasis. Morphological and bioenergetic characterization of the life cycle of an individual mitochondrion reveals several points where fusion, fission, and mitophagy interact. Mitochondrial fission can produce an impaired daughter unit that will be targeted by the autophagic machinery. Mitochondrial fusion, on the other hand, may serve to dilute impaired respiratory components and thereby prevent their removal. The inverse dependency of fusion and mitophagy on membrane potential allows them to act as complementary rather than competitive fates of the daughter mitochondrion after a fission event. We discuss the interplay between mitochondrial dynamics and mitophagy in different tissues and in different disease models under both stress-induced and steady-state conditions. Antioxid. Redox Signal. 14, 1939–1951.
Excessive nitrosative and oxidative stress is thought to trigger cellular signaling pathways leading to neurodegenerative conditions. Such redox dysregulation can result from many cellular events, including hyperactivation of the N-methyl-d-aspartate-type glutamate receptor, mitochondrial dysfunction, and cellular aging. Recently, we and our colleagues have shown that excessive generation of free radicals and related molecules, in particular nitric oxide species (NO), can trigger pathological production of misfolded proteins, abnormal mitochondrial dynamics (comprised of mitochondrial fission and fusion events), and apoptotic pathways in neuronal cells. Emerging evidence suggests that excessive NO production can contribute to these pathological processes, specifically by S-nitrosylation of specific target proteins. Here, we highlight examples of S-nitrosylated proteins that regulate misfolded protein accumulation and mitochondrial dynamics. For instance, in models of Parkinson's disease, these S-nitrosylation targets include parkin, a ubiquitin E3 ligase and neuroprotective molecule, and protein-disulfide isomerase, a chaperone enzyme for nascent protein folding. S-Nitrosylation of protein-disulfide isomerase may also be associated with mutant Cu/Zn superoxide dismutase toxicity in amyotrophic lateral sclerosis. Additionally, in models of Alzheimer's disease, excessive NO generation leads to the formation of S-nitrosylated dynamin-related protein 1 (forming SNO-Drp1), which contributes to abnormal mitochondrial fragmentation and resultant synaptic damage. Antioxid. Redox Signal. 14, 1479–1492.
Mitochondria are essential cytoplasmic organelles, critical for cell survival and death. Recent mitochondrial research revealed that mitochondrial dynamics – the balance of fission and fusion in normal mitochondrial dynamics – is an important cellular mechanism in eukaryotic cell and is involved in the maintenance of mitochondrial morphology, structure, number, distribution, and function. Research into mitochondria and cell function has revealed that mitochondrial dynamics is impaired in a large number of aging and neurodegenerative diseases, and in several inherited mitochondrial diseases, and that this impairment involves excessive mitochondrial fission, resulting in mitochondrial structural changes and dysfunction, and cell damage. Attempts have been made to develop molecules to reduce mitochondrial fission while maintaining normal mitochondrial fusion and function in those diseases that involve excessive mitochondrial fission. This review article discusses mechanisms of mitochondrial fission in normal and diseased states of mammalian cells and discusses research aimed at developing therapies, such as Mdivi, Dynasore and P110, to prevent or to inhibit excessive mitochondrial fission.
The protein kinase C (PKC) family of serine/threonine kinases regulates diverse cellular function, including cell death, proliferation and survival. In particular, PKCδ governs the cellular homeostatic response against hypoxic stress. Autophagy, a lysosome-dependent degradative pathway, and apoptosis are two fundamental cellular pathways that respond to stress conditions, such as hypoxia, oxidative stress, and nutrient starvation. Recently, we uncovered a novel role for PKCδ in the early stage of hypoxic response where PKCδ activates autophagy by promoting JNK1-mediated Bcl-2 phosphorylation and dissociation of the Bcl-2/Beclin 1 complex. Whereas acute hypoxic stress promotes autophagy, we have previously reported that prolonged hypoxic stress caused the cleavage of PKCδ by caspase-3, resulting in the nuclear translocation of a constitutively active catalytic fragment of PKCδ, PKCδ-CF. Moreover, PKCδ-CF also serves a feed-forward function for the reciprocal PKCδ and caspase-3 proteolytic activation. Here, we discussed the requirement for PKCδ and JNK1 for hypoxia-induced autophagy, and the kinetic relationship among Bcl-2/Beclin 1 interaction, caspase-3 activation and the steady-state level of Beclin 1 during hypoxic exposure. Based on these results, we propose a model for understanding the PKCδ-dependent crosstalk mechanisms between autophagy and apoptosis, both induced by hypoxic stress. These findings collectively support a pivotal role for PKCδ in regulating hypoxic stress with hitherto unappreciated significance.
PKCδ; JNK1; Bcl-2; Beclin 1; caspase-3; hypoxia; autophagy; apoptosis
Aberrant activation of Cdk5 has been implicated in the process of neurodegenerative diseases such as Alzheimer's disease (AD). We recently reported that S-nitrosylation of Cdk5 (forming SNO-Cdk5) at specific cysteine residues results in excessive activation of Cdk5, contributing to mitochondrial dysfunction, synaptic damage, and neuronal cell death in models of AD. Furthermore, SNO-Cdk5 acts as a nascent S-nitrosylase, transnitrosylating the mitochondrial fission protein Drp1 and enhancing excessive mitochondrial fission in dendritic spines. However, a molecular mechanism that leads to the formation of SNO-Cdk5 in neuronal cells remained obscure. Here, we demonstrate that neuronal nitric oxide synthase (NOS1) interacts with Cdk5 and that the close proximity of the two proteins facilitates the formation of SNO-Cdk5. Interestingly, as a negative feedback mechanism, Cdk5 phosphorylates and suppresses NOS1 activity. Thus, together with our previous report, these findings delineate an S-nitrosylation pathway wherein Cdk5/NOS1 interaction enhances SNO-Cdk5 formation, mediating mitochondrial dysfunction and synaptic loss during the etiology of AD.
nitrosative stress; Cyclin-dependent kinase 5; nitric oxide; neuronal NO synthase; transnitrosylation
Maintaining proper mitochondrial length is essential for normal mitochondrial function in neurons. Mitochondrial fragmentation has been associated with neuronal cell death caused by a variety of experimental toxic stressors. Despite the fact that oxidative stress is a hallmark of neurodegenerative conditions and aging and the resulting activation of p53 is believed to contribute to the neuropathology, little is still known regarding changes in mitochondrial morphology in p53-dependent neuronal death. Therefore, we specifically addressed the relationship between genotoxic stress, p53 activation and the regulation of mitochondrial morphology in neurons. In cultured postnatal mouse cortical neurons, treatment with the DNA damaging agent camptothecin (CPT) resulted in elongated mitochondria, in contrast to fragmented mitochondria observed upon staurosporine and glutamate treatment. In fibroblasts, however, CPT resulted in fragmented mitochondria. CPT treatment in neurons suppressed expression of the mitochondrial fission protein Drp1 and the E3 ubiquitin ligase parkin. The presence of elongated mitochondria and the declines in Drp1 and parkin expression occurred prior to the commitment point for apoptosis. The CPT-induced changes in Drp1 and parkin were not observed in p53-deficient neurons, while p53 overexpression alone was sufficient to reduce the expression of the two proteins. Elevating Drp1 and parkin expression prior to CPT treatment enhanced neuronal viability and restored a normal pattern of mitochondrial morphology. The present findings demonstrate that genotoxic stress in neurons results in elongated mitochondria in contrast to fission induced by other forms of stress, and p53-dependent declines in Drp1 and parkin levels contribute to altered mitochondrial morphology and cell death.
The loss or injury of neurons associated with oxidative and nitrosative redox stress plays an important role in the onset of various neurodegenerative diseases. Specifically, nitric oxide (NO), can affect neuronal survival through a process called S-nitrosylation, by which the NO group undergoes a redox reaction with specific protein thiols. This in turn can lead to the accumulation of misfolded proteins, which generally form aggregates in Alzheimer’s, Parkinson’s, and other neurodegenerative diseases. Evidence suggests that S-nitrosylation can also impair mitochondrial function and lead to excessive fission of mitochondria and consequent bioenergetic compromise via effects on the activity of the fission protein dynamin-related protein 1 (Drp1). This insult leads to synaptic dysfunction and loss. Additionally, high levels of NO can S-nitrosylate a number of aberrant targets involved in neuronal survival pathways, including the antiapoptotic protein XIAP, inhibiting its ability to prevent apoptosis.
The mitochondrial signaling complex PKA/AKAP1 protects neurons against
mitochondrial fragmentation and cell death by phosphorylating and inactivating
the mitochondrial fission enzyme Drp1.
Mitochondrial shape is determined by fission and fusion reactions catalyzed by
large GTPases of the dynamin family, mutation of which can cause neurological
dysfunction. While fission-inducing protein phosphatases have been identified,
the identity of opposing kinase signaling complexes has remained elusive. We
report here that in both neurons and non-neuronal cells, cAMP elevation and
expression of an outer-mitochondrial membrane (OMM) targeted form of the protein
kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected
network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes
mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression
approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1)
as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo.
According to epistasis studies with phosphorylation site-mutant dynamin-related
protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a
conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1
promote both mitochondrial elongation and neuronal survival. Phenocopied by a
mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the
disassembly step of its catalytic cycle, accumulating large, slowly recycling
Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a
mitochondrial reticulum, which protects neurons from diverse insults.
Mitochondria, the cellular powerhouse, are highly dynamic organelles shaped by
opposing fission and fusion events. Research over the past decade has identified
many components of the mitochondrial fission/fusion machinery and led to the
discovery that mutations in genes coding for these proteins can cause human
neurological diseases. While it is well established that mitochondrial shape
changes are intimately involved in cellular responses to environmental
stressors, we know very little about the mechanisms by which cells dynamically
adjust mitochondrial form and function. In this report, we show that the
scaffold protein AKAP1 brings the cAMP-dependent protein kinase PKA to the outer
mitochondrial membrane to protect neurons from injury. The PKA/AKAP1 complex
functions by inhibiting Drp1, an enzyme that mechanically constricts and
eventually severs mitochondria. Whereas active, dephosphorylated Drp1 rapidly
cycles between cytosol and mitochondria, phosphorylated Drp1 builds up in
inactive mitochondrial complexes, allowing mitochondria to fuse into a
neuroprotective reticulum. Our results suggest that altering the balance of
kinase and phosphatase activities at the outer mitochondrial membrane may
provide the basis for novel neuroprotective therapies.
Mitochondrial abnormalities have been documented in Alzheimer’s disease and related neurodegenerative disorders, but the causal relationship between mitochondrial changes and neurodegeneration, as well as the specific mechanisms promoting mitochondrial dysfunction, are not clear. Here we find that expression of human tau results in elongation of mitochondria in both Drosophila and mouse neurons. Elongation is accompanied by mitochondrial dysfunction and cell cycle-mediated cell death, which can be rescued in vivo by genetically restoring the proper balance of mitochondrial fission and fusion. We have previously demonstrated that stabilization of actin by tau is critical for neurotoxicity of the protein. Here we demonstrate a conserved role for actin and myosin in regulating mitochondrial fission, and show that excess actin stabilization inhibits association of the fission protein DRP1 with mitochondria leading to mitochondrial elongation and subsequent neurotoxicity. Our results thus identify actin-mediated disruption of mitochondrial dynamics as a direct mechanism of tau toxicity in neurons in vivo.
High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions.
Methodology and Principal Findings
Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21Cip1/WAF1. This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21Cip1/WAF1 at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic.
Conclusions and Significance
These observations disclose PKCδ as negative regulator of p21Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.
One of the main causes of cardiovascular complications in diabetes is the hyperglycemia-induced cell injury, and mitochondrial fission has been implicated in the apoptotic process. We investigated the role of mitochondrial fission in high glucoseinduced cardiovascular cell injury.
We used several types of cultured mouse, rat, and bovine cells from the cardiovascular system, and evaluated mitochondrial morphology, reactive oxygen species (ROS) levels and apoptotic parameters in sustained high glucose incubation. Adenoviral infection was used for the inhibition of the fission protein DLP1.
We found that mitochondria were short and fragmented in cells incubated in sustained high glucose conditions. Under the same conditions, cellular ROS levels were high and cell death was increased. We demonstrated that the increased level of ROS causes mitochondrial permeability transition (MPT), phosphatidylserine exposure, cytochrome c release, and caspase activation in prolonged high glucose conditions. Importantly, maintaining tubular mitochondria by inhibiting mitochondrial fission in sustained high glucose conditions normalized cellular ROS levels and prevented the MPT and subsequent cell death. These results demonstrate that mitochondrial fragmentation is an upstream factor for ROS overproduction and cell death in prolonged high glucose conditions.
These findings indicate that the fission-mediated fragmentation of mitochondrial tubules is causally associated with enhanced production of mitochondrial ROS and cardiovascular cell injury in hyperglycemic conditions.
Proteolytic cleavage and subsequent activation of protein kinase C (PKC) δ is required for apoptosis induced by a variety of genotoxic agent, including UV radiation. In addition, overexpression of the constitutively active PKCδ catalytic fragment (PKCδ-cat) is sufficient to trigger Bax activation, cytochrome c release, and apoptosis. While PKCδ is a key apoptotic effector, the downstream target(s) responsible for the mitochondrial apoptotic cascade are not known. We found that expression of the active PKCδ-cat in HaCaT cells triggers a reduction in the anti-apoptotic protein Mcl-1, similar to UV radiation. The down-regulation of Mcl-1 induced by PKCδ-cat was not at the mRNA level but was due to decreased protein half-life. Overexpression of Mcl-1 protected HaCaT cells from both UV and PKCδ-cat-induced apoptosis and blocked the release of cytochrome c from the mitochondria, indicating that Mcl-1 down-regulation was required for apoptosis signaling. Indeed, down-regulation of Mcl-1 with siRNA slightly increased the basal apoptotic rate of HaCaT cells and dramatically sensitized them to UV or PKCδ-cat-induced apoptosis. HaCaT cells with down-regulated Mcl-1 had higher activated Bax protein, as measured by Bax cross-linking, indicating that Mcl-1 down-regulation is sufficient for Bax activation. Finally, recombinant PKCδ could phosphorylate Mcl-1 in vitro, identifying Mcl-1 as a direct target for PKCδ. Overall our results identify Mcl-1 as an important target for PKCδ-cat that can mediate its pro-apoptotic effects on mitochondria to amplify the apoptotic signaling induced by a wide range of apoptotic stimuli.
Manganese (Mn) exposure causes Manganism, a neurological disorder similar to Parkinson’s disease. However, the cellular mechanism by which Mn induces dopaminergic neuronal cell death remains unclear. In the present study, we sought to investigate the key downstream apoptotic cell signaling events that contribute to Mn-induced cell death in mesencephalic dopaminergic neuronal (N27) cells. Mn exposure induced a dose-dependent increase in neuronal cell death in N27 cells. The cell death was accompanied by sequential activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 activation, and DNA fragmentation, but not caspase-8 activation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers Mn-induced apoptosis. Notably, Mn treatment proteolytically activated protein kinase Cδ (PKCδ), a member of a novel class of protein kinase C. The caspase-3 specific inhibitor Z-DEVD-FMK significantly blocked PKCδ cleavage and its kinase activity, indicating that caspase-3 mediates the proteolytic activation. Co-treatment with the PKCδ inhibitor rottlerin or the caspase-3 inhibitor Z-DEVD-FMK almost completely blocked Mn-induced DNA fragmentation. Additionally, N27 cells expressing a catalytically inactive PKCδK376R protein (PKCδ dominant negative mutant) or a caspase cleavage resistant PKCδD327A protein (PKCδ cleavage resistant mutant) were found to be resistant to Mn-induced apoptosis. To further establish the proapoptotic role of PKCδ, RNAi-mediated gene knockdown was performed. siRNA suppression of PKCδ expression protected N27 cells from Mn-induced apoptotic cell death. Collectively, these results suggest that caspase-3-dependent proteolytic activation of PKCδ plays a key role in Mn-induced apoptotic cell death.
Neuronal cell death via apoptosis or necrosis underlies several devastating neurodegenerative diseases associated with aging. Mitochondrial dysfunction resulting from oxidative or nitrosative stress often acts as an initiating stimulus for intrinsic apoptosis or necrosis. These events frequently occur in conjunction with imbalances in the mitochondrial fission and fusion equilibrium, although the cause and effect relationships remain elusive. Here, we demonstrate in primary rat cerebellar granule neurons (CGNs) that oxidative or nitrosative stress induces an N-terminal cleavage of optic atrophy-1 (OPA1), a dynamin-like GTPase that regulates mitochondrial fusion and maintenance of cristae architecture. This cleavage event is indistinguishable from the N-terminal cleavage of OPA1 observed in CGNs undergoing caspase-mediated apoptosis (Loucks et al., 2009) and results in removal of a key lysine residue (K301) within the GTPase domain. OPA1 cleavage in CGNs occurs coincident with extensive mitochondrial fragmentation, disruption of the microtubule network, and cell death. In contrast to OPA1 cleavage induced in CGNs by removing depolarizing extracellular potassium (5K apoptotic conditions), oxidative or nitrosative stress-induced OPA1 cleavage caused by complex I inhibition or nitric oxide, respectively, is caspase-independent. N-terminal cleavage of OPA1 is also observed in vivo in aged rat and mouse midbrain and hippocampal tissues. We conclude that N-terminal cleavage and subsequent inactivation of OPA1 may be a contributing factor in the neuronal cell death processes underlying neurodegenerative diseases, particularly those associated with aging. Furthermore, these data suggest that OPA1 cleavage is a likely convergence point for mitochondrial dysfunction and imbalances in mitochondrial fission and fusion induced by oxidative or nitrosative stress.
Mitochondrial dynamics; Apoptosis; Aging; Complex I; Reactive oxygen species; Nitric oxide; Caspases
During atherogenesis, excess amounts of low-density lipoproteins (LDL) accumulate in the subendothelial space where they undergo oxidative modifications. Oxidized LDL (oxLDL) alter the fragile balance between survival and death of vascular smooth muscle cells (VSMC) thereby leading to plaque instability and finally to atherothrombotic events. As protein kinase C δ (PKCδ) is pro-apoptotic in many cell types, we investigated its potential role in the regulation of VSMC apoptosis induced by oxLDL. We found that human VSMC silenced for PKCδ exhibited a protection towards oxLDL-induced apoptosis. OxLDL triggered the activation of PKCδ as shown by its phosphorylation and nuclear translocation. PKCδ activation was dependent on the reactive oxygen species generated by oxLDL. Moreover, we demonstrated that PKCδ participates in oxLDL-induced endoplasmic reticulum (ER) stress-dependent apoptotic signaling mainly through the IRE1α/JNK pathway. Finally, the role of PKCδ in the development of atherosclerosis was supported by immunohistological analyses showing the colocalization of activated PKCδ with ER stress and lipid peroxidation markers in human atherosclerotic lesions. These findings highlight a role for PKCδ as a key regulator of oxLDL-induced ER stress-mediated apoptosis in VSMC, which may contribute to atherosclerotic plaque instability and rupture.
PKCδ; oxidized low-density lipoproteins; apoptosis; ER stress; vascular smooth muscle cells
Glutamate-induced cytotoxicity is partially mediated by enhanced oxidative stress. The objectives of the present study are to determine the effects of glutamate on mitochondrial membrane potential, oxygen consumption, mitochondrial dynamics and autophagy regulating factors and to explore the protective effects of selenium against glutamate cytotoxicity in murine neuronal HT22 cells. Our results demonstrated that glutamate resulted in cell death in a dose-dependent manner and supplementation of 100 nM sodium selenite prevented the detrimental effects of glutamate on cell survival. The glutamate induced cytotoxicity was associated with mitochondrial hyperpolarization, increased ROS production and enhanced oxygen consumption. Selenium reversed these alterations. Furthermore, glutamate increased the levels of mitochondrial fission protein markers pDrp1 and Fis1 and caused increase in mitochondrial fragmentation. Selenium corrected the glutamate-caused mitochondrial dynamic imbalance and reduced the number of cells with fragmented mitochondria. Finally, glutamate activated autophagy markers Beclin 1 and LC3-II, while selenium prevented the activation. These results suggest that glutamate targets the mitochondria and selenium supplementation within physiological concentration is capable of preventing the detrimental effects of glutamate on the mitochondria. Therefore, adequate selenium supplementation may be an efficient strategy to prevent the detrimental glutamate toxicity and further studies are warranted to define the therapeutic potentials of selenium in animal disease models and in human.