Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1⁺ or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.     

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ_m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ_m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H₂O₂-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ_m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ_m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.     

Dopamine Selectively Sensitizes Dopaminergic Neurons to Rotenone-Induced Apoptosis

Among various types of neurons affected in Parkinson’s disease, dopamine (DA) neurons of the substantia nigra undergo the most pronounced degeneration. Products of DA oxidation and consequent cellular damage have been hypothesized to contribute to neuronal death. To examine whether elevated intracellular DA will selectively predispose the dopaminergic subpopulation of nigral neurons to damage by an oxidative insult, we first cultured rat primary mesencephalic cells in the presence of rotenone to elevate reactive oxygen species. Although MAP2⁺ neurons were more sensitive to rotenone-induced toxicity than type 1 astrocytes, rotenone affected equally both DA (TH⁺) neurons and MAP2⁺ neurons. In contrast, when intracellular DA concentration was elevated, DA neurons became selectively sensitized to rotenone. Raising intracellular DA levels in primary DA neurons resulted in dopaminergic neuron death in the presence of subtoxic concentrations of rotenone. Furthermore, mitochondrial superoxide dismutase mimetic, manganese (III) meso-tetrakis (4-benzoic acid) porphyrin, blocked activation of caspase-3, and consequent cell death. Our results demonstrate that an inhibitor of mitochondrial complex I and increased cytosolic DA may cooperatively lead to conditions of elevated oxidative stress and thereby promote selective demise of dopaminergic neurons.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.     

Subcellular expression and neuroprotective effects of SK channels in human dopaminergic neurons

Small-conductance Ca²⁺-activated K⁺ channel activation is an emerging therapeutic approach for treatment of neurological diseases, including stroke, amyotrophic lateral sclerosis and schizophrenia. Our previous studies showed that activation of SK channels exerted neuroprotective effects through inhibition of NMDAR-mediated excitotoxicity. In this study, we tested the therapeutic potential of SK channel activation of NS309 (25 μM) in cultured human postmitotic dopaminergic neurons in vitro conditionally immortalized and differentiated from human fetal mesencephalic cells. Quantitative RT-PCR and western blotting analysis showed that differentiated dopaminergic neurons expressed low levels of SK2 channels and high levels of SK1 and SK3 channels. Further, protein analysis of subcellular fractions revealed expression of SK2 channel subtype in mitochondrial-enriched fraction. Mitochondrial complex I inhibitor rotenone (0.5 μM) disrupted the dendritic network of human dopaminergic neurons and induced neuronal death. SK channel activation reduced mitochondrial membrane potential, while it preserved the dendritic network, cell viability and ATP levels after rotenone challenge. Mitochondrial dysfunction and delayed dopaminergic cell death were prevented by increasing and/or stabilizing SK channel activity. Overall, our findings show that activation of SK channels provides protective effects in human dopaminergic neurons, likely via activation of both membrane and mitochondrial SK channels. Thus, SK channels are promising therapeutic targets for neurodegenerative disorders such as Parkinson''s disease, where dopaminergic cell loss is associated with progression of the disease.     

PACAP protects neuronal differentiated PC12 cells against the neurotoxicity induced by a mitochondrial complex I inhibitor, rotenone

In vivo and in vitro studies have suggested a neuroprotective role for Pituitary adenylate cyclase activating polypeptide (PACAP) against neuronal insults. Here, we showed that PACAP27 protects against neurotoxicity induced by rotenone, a mitochondrial complex I inhibitor that has been implicated in the pathogenesis of Parkinson's disease (PD). The neuroprotective effect of PACAP27 was dose-dependent and blocked by its specific receptor antagonist, PACAP6-27. The effects of PACAP27 on rotenone-induced cell death were mimicked by dibutyryl-cAMP (db-cAMP), forskolin and prevented by the PKA inhibitor H89, the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PACAP27 administration blocked rotenone-induced increases in the level of caspase-3-like activity, whereas could not restore mitochondrial activity damaged by rotenone. Thus, our results demonstrate that PACAP27 has a neuroprotective role against rotenone-induced neurotoxicity in neuronal differentiated PC12 cells and the neuroprotective effects of PACAP are associated with activation of MAP kinase pathways by PKA and with inhibition of caspase-3 activity; the signaling mechanism appears to be mediated through mitochondrial-independent pathways.     

Activation of Srk1 by the mitogen-activated protein kinase Sty1/Spc1 precedes its dissociation from the kinase and signals its degradation

Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. The Schizosaccharomyces pombe SAPK Sty1/Spc1 orchestrates general changes in gene expression in response to diverse forms of cytotoxic stress. Here we show that Sty1/Spc1 is bound to its target, the Srk1 kinase, when the signaling pathway is inactive. In response to stress, Sty1/Spc1 phosphorylates Srk1 at threonine 463 of the regulatory domain, inducing both activation of Srk1 kinase, which negatively regulates cell cycle progression by inhibiting Cdc25, and dissociation of Srk1 from the SAPK, which leads to Srk1 degradation by the proteasome.     

DJ-1 Protects Dopaminergic Neurons against Rotenone-Induced Apoptosis by Enhancing ERK-Dependent Mitophagy

Loss-of-function mutations in the gene encoding the multifunctional protein, DJ-1, have been implicated in the pathogenesis of early-onset familial Parkinson's disease (PD), suggesting that DJ-1 may act as a neuroprotectant for dopaminergic (DA) neurons. Enhanced autophagy may benefit PD by clearing damaged organelles and protein aggregates; thus, we determined if DJ-1 protects DA neurons against mitochondrial dysfunction and oxidative stress through an autophagic pathway. Cultured DA cells (MN9D) overexpressing DJ-1 were treated with the mitochondrial complex I inhibitor, rotenone. In addition, rotenone was injected into the left substantia nigra of rats 4 weeks after injection with a DJ-1 expression vector. Overexpression of DJ-1 protected MN9D cells against apoptosis, significantly enhanced the survival of nigral DA neurons after rotenone treatment in vivo, and rescued rat behavioral abnormalities. Overexpression of DJ-1 enhanced rotenone-evoked expression of the autophagic markers, beclin-1 and LC3II, while transmission electron microscopy and confocal imaging revealed that the ultrastructural signs of autophagy were increased by DJ-1. The neuroprotective effects of DJ-1 were blocked by phosphoinositol 3‐kinase and the autophagy inhibitor, 3-methyladenine, and by the ERK pathway inhibitor, U0126. Confocal imaging revealed that the size of p62-positive puncta decreased significantly in DJ-1 overexpression of MN9D cells 12 h after rotenone treatment, suggesting that DJ-1 reveals the ability to clear aggregated p62 associated with PD. Factors that control autophagy, including DJ-1, may inhibit rotenone-induced apoptosis and present novel targets for therapeutic intervention in PD.     

Mitochondrial chaperone HSP-60 regulates anti-bacterial immunity via p38 MAP kinase signaling

Mitochondria play key roles in cellular immunity. How mitochondria contribute to organismal immunity remains poorly understood. Here, we show that HSP-60/HSPD1, a major mitochondrial chaperone, boosts anti-bacterial immunity through the up-regulation of p38 MAP kinase signaling. We first identify 16 evolutionarily conserved mitochondrial components that affect the immunity of Caenorhabditis elegans against pathogenic Pseudomonas aeruginosa (PA14). Among them, the mitochondrial chaperone HSP-60 is necessary and sufficient to increase resistance to PA14. We show that HSP-60 in the intestine and neurons is crucial for the resistance to PA14. We then find that p38 MAP kinase signaling, an evolutionarily conserved anti-bacterial immune pathway, is down-regulated by genetic inhibition of hsp-60, and up-regulated by increased expression of hsp-60. Overexpression of HSPD1, the mammalian ortholog of hsp-60, increases p38 MAP kinase activity in human cells, suggesting an evolutionarily conserved mechanism. Further, cytosol-localized HSP-60 physically binds and stabilizes SEK-1/MAP kinase kinase 3, which in turn up-regulates p38 MAP kinase and increases immunity. Our study suggests that mitochondrial chaperones protect host eukaryotes from pathogenic bacteria by up-regulating cytosolic p38 MAPK signaling.     

Increase in anti-apoptotic molecules,nucleolin, and heat shock protein 70, against upregulated LRRK2 kinase activity

Leucine-rich repeat kinase 2 (LRRK2) is involved in Parkinson’s disease (PD) pathology. A previous study showed that rotenone treatment induced apoptosis, mitochondrial damage, and nucleolar disruption via up-regulated LRRK2 kinase activity, and these effects were rescued by an LRRK2 kinase inhibitor. Heat-shock protein 70 (Hsp70) is an anti-oxidative stress chaperone, and overexpression of Hsp70 enhanced tolerance to rotenone. Nucleolin (NCL) is a component of the nucleolus; overexpression of NCL reduced cellular vulnerability to rotenone. Thus, we hypothesized that rotenone-induced LRRK2 activity would promote changes in neuronal Hsp70 and NCL expressions. Moreover, LRRK2 G2019S, the most prevalent LRRK2 pathogenic mutant with increased kinase activity, could induce changes in Hsp70 and NCL expression. Rotenone treatment of differentiated SH-SY5Y (dSY5Y) cells increased LRKK2 levels and kinase activity, including phospho-S935-LRRK2, phospho-S1292-LRRK2, and the phospho-moesin/moesin ratio, in a dose-dependent manner. Neuronal toxicity and the elevation of cleaved poly (ADP-ribose) polymerase, NCL, and Hsp70 were increased by rotenone. To validate the induction of NCL and Hsp70 expression in response to rotenone, cycloheximide (CHX), a protein synthesis blocker, was administered with rotenone. Post-rotenone increased NCL and Hsp70 expression was repressed by CHX; whereas, rotenone-induced kinase activity and apoptotic toxicity remained unchanged. Transient expression of G2019S in dSY5Y increased the NCL and Hsp70 levels, while administration of a kinase inhibitor diminished these changes. Similar results were observed in rat primary neurons after rotenone treatment or G2019S transfection. Brains from G2019S-transgenic mice also showed increased NCL and Hsp70 levels. Accordingly, LRRK2 kinase inhibition might prevent oxidative stress-mediated PD progression.

Abbreviations: 6-OHDA: 6-hydroxydopamine; CHX: cycloheximide; dSY5Y: differentiated SH-SY5Y; g2019S tg: g2019S transgenic mouse; GSK/A-KI: GSK2578215A kinase inhibitor; HSP70: heat shock protein 70; LDH: lactose dehydrogenase; LRRK2: leucine rich-repeat kinase 2; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; myc-GS LRRK2: myc-tagged g2019S LRRK2; NCL: nucleolin; PARP: poly(ADP-ribose) polymerase; PD: Parkinson’s disease; PINK1: PTEN-induced putative kinase 1; pmoesin: phosphorylated moesin at t558; ROS: reactive oxygen species     

Rho1 GTPase and PKC Ortholog Pck1 Are Upstream Activators of the Cell Integrity MAPK Pathway in Fission Yeast

In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.     

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.     

Parkin Protects Dopaminergic Neurons against Microtubule-depolymerizing Toxins by Attenuating Microtubule-associated Protein Kinase Activation

Mitogen-activated protein kinases, originally known as microtubule-associated protein (MAP) kinases, are activated in response to a variety of stimuli. Here we report that microtubule-depolymerizing agents such as colchicine or nocodazole induced strong activation of MAP kinases including JNK, ERK, and p38. This effect was markedly attenuated by parkin, whose mutations are linked to Parkinson disease (PD). Our previous study has shown that parkin stabilizes microtubules through strong interactions mediated by three independent domains. We found that each of the three microtubule-binding domains of parkin was sufficient to reduce MAP kinase activation induced by microtubule depolymerization. The ability to attenuate microtubule depolymerization and the ensuing MAP kinase activation was abrogated in B-lymphocytes and fibroblasts derived from PD patients with parkin mutations such as exon 4 deletion. Such mutations produced truncated parkin proteins lacking any microtubule binding domain and prevented parkin from protecting midbrain dopaminergic neurons against microtubule-depolymerizing toxins such as rotenone or colchicine. Consistent with these, blocking MAP kinase activation in midbrain dopaminergic neurons by knocking down MAP kinase kinases (MKK) significantly reduced the selective toxicity of rotenone or colchicine. Conversely, overexpression of MAP kinases caused marked toxicities that were significantly attenuated by parkin. Thus, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins such as rotenone by stabilizing microtubules to attenuate MAP kinase activation.Mitogen-activated protein kinases are a superfamily of kinases that include the extracellular signal-related kinases (ERK1/2),² Jun N-terminal kinases (JNK1/2/3), and p38 proteins (p38α/β/γ/δ) in mammalian species (1). Initially, MAP kinase stood for microtubule-associated protein kinase because microtubule-associated proteins such as MAP2 are excellent substrates of MAP kinases (2, 3). Previous studies have shown that a significant portion of ERK is associated with microtubules (4). It has also been shown that JNK1 is required for the maintenance of microtubule integrity in neurons through controlling the phosphorylation states of MAP2 and MAP1B (5). Phosphorylation of tau, an axon-enriched MAP, by p38δ promotes microtubule assembly (6). All MAP kinases are proline-directed kinases, preferring serine or threonine residues followed by proline. The abundance of such sites on many microtubule-associated proteins suggests that MAP kinases play critical roles in regulating the phosphorylation states of MAPs; hence, the dynamic properties of microtubules.The selective loss of dopaminergic (DA) neurons in substantia nigra is the pathological hallmark of Parkinson disease and directly contributes to its locomotor symptoms. Nigral DA neurons project to striatal target areas with very long axons, which rely on microtubules to transport dopamine vesicles over long distances. Our previous studies have shown that these dopaminergic neurons are particularly vulnerable to microtubule-depolymerizing agents including rotenone (7), an environmental toxin that causes PD-like symptoms and pathologies in animal models (8). Microtubule depolymerization disrupts vesicular transport, which significantly elevates oxidative stress due to increased oxidation of cytosolic dopamine leaked from vesicles (7). On the other hand our previous studies have shown that parkin, a protein-ubiquitin E3 ligase linked to Parkinson disease, strongly binds to microtubules (9) through redundant, high affinity interactions mediated by three independent domains (10). In addition, parkin increases the ubiquitination and degradation of both α- and β-tubulin (9), whose complex folding reactions are prone to produce misfolded intermediates (11). These results suggest that parkin plays an important role in maintaining the stability and normal functions of microtubules, which are critically involved in the survival of nigral DA neurons (12).In the present study we examined the impact of parkin on MAP kinase activation induced by microtubule depolymerization. Our results showed that MAP kinases, including JNK, ERK, and p38, were activated by microtubule-depolymerizing agents such as colchicine and nocodazole. This effect was greatly attenuated by overexpression of wild-type parkin or any one of its three microtubule binding domains. The ability of parkin to suppress microtubule depolymerization and the ensuing MAP kinase activation was abrogated in PD patients with parkin mutations such as exon 4 deletion, which produced a truncated protein lacking any microtubule-binding domain. This mutation also prevented parkin from protecting dopaminergic neurons against the selective toxicity of microtubule-depolymerizing toxins such as rotenone or colchicine. Blocking MAP kinase activation by small interfering RNA (siRNA) of MAP kinase kinases significantly reduced the selective toxicity of rotenone or colchicine, whereas overexpression of MAP kinases produced toxicities that were significantly reduced by parkin. Together, the results suggest that parkin protects midbrain dopaminergic neurons against microtubule-depolymerizing PD toxins by stabilizing microtubules to rein in MAP kinase activation.     

Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.     

Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism

Metformin is widely used as a hypoglycemic agent for the treatment of type 2 diabetes. Both metformin and rotenone, an inhibitor of respiratory chain complex I, suppressed glucose-6-phosphatase (G6pc), a rate limiting enzyme of liver glucose production, mRNA expression in a rat hepatoma cell line accompanied by a reduction of intracellular ATP concentration and an activation of AMP-activated protein kinase (AMPK). When yeast NADH-quinone oxidoreductase 1 (NDI1) gene was introduced into the cells, neither inhibition of ATP synthesis nor activation of AMPK was induced by these agents. Interestingly, in contrast to rotenone treatment, G6pc mRNA down-regulation was observed in the NDI1 expressing cells after metformin treatment. Since NDI1 can functionally complement the complex I under the presence of metformin or rotenone, our results indicate that metformin induces down-regulation of G6pc expression through an inhibition of complex I and an activation of AMPK-independent mechanism.     

Rotenone Induces Cell Death of Cholinergic Neurons in an Organotypic Co-Culture Brain Slice Model

In Alzheimer and Parkinson’s disease cell death of cholinergic and dopaminergic neurons are characteristic hallmarks, respectively. It is well established that rotenone, an inhibitor of complex I of the electron transport chain, induces cell death of dopaminergic neurons, however, not much is known on the effects of rotenone on cholinergic neurons. The aim of the present study was to evaluate the effects of rotenone on cholinergic neurons in an organotypic in vitro brain co-slice model. When co-cultures were treated with 10 μM rotenone for 24 h a significantly decreased number of cholinergic neurons was found in the basal nucleus of Meynert but not in the dorsal striatum. This cell death exhibited apoptotic DAPI-positive malformed nuclei and enhanced TUNEL-positive cells. In summary, inhibition of complex I of the electron transport chain may play a role in neurodegeneration of cholinergic neurons.     

Involvement of CaM kinase II in gonadotropin-releasing hormone-induced activation of MAP kinase in cultured hypothalamic neurons

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons. There is accumulating evidence that GnRH neurons have GnRH receptors and that the autocrine action of GnRH activates MAP kinase. In this study, we found that KN93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases), inhibited the GnRH-induced activation of MAP kinase in immortalized GnRH neurons (GT1-7 cells). Immunoblot analysis indicated that the CaM kinase IIdelta2 isoform (CaM kinase IIdelta2) and synapsin I were expressed in GT1-7 cells. GnRH treatment rapidly increased phosphorylation of synapsin I at serine 603, a specific phosphorylation site for CaM kinase II, suggesting that GnRH treatment rapidly activated CaM kinase IIdelta2. In addition, when we stably overexpressed CaM kinase IIdelta2 in GT1-7 cells, the activation of MAP kinase was strongly enhanced. These results suggest that CaM kinase IIdelta2 was involved in the GnRH-induced activation of MAP kinase in GT1-7 cells.     

Oxidative stress in mitochondria

In mitochondria, oxidative phosphorylation and enzymatic oxidation of biogenic amines by monoamine oxidase produce reactive oxygen and nitrogen species, which are proposed to cause neuronal cell death in neurodegenerative disorders, including Parkinson’s and Alzheimer’s disease. In these disorders, mitochondrial dysfunction, increased oxidative stress, and accumulation of oxidation-modified proteins are involved in cell death in definite neurons. The interactions among these factors were studied by use of a peroxynitrite-generating agent, N-morpholino sydnonimine (SIN-1) and an inhibitor of complex I, rotenone, in human dopaminergic SH-SY5Y cells. In control cells, peroxynitrite nitrated proteins, especially the subunits of mitochondrial complex I, as 3-nitrotyrosine, suggesting that neurons are exposed to constant oxidative stress even under physiological conditions. SIN-1 and an inhibitor of proteasome, carbobenzoxy-l-isoleucyl-γ-t-butyl-l-analyl-l-leucinal (PSI), increased markedly the levels of nitrated proteins with concomitant induction of apoptosis in the cells. Rotenone induced mitochondrial dysfunction and accumulation and aggregation of proteins modified with acrolein, an aldehyde product of lipid peroxidation in the cells. At the same time, the activity of the 20S β-subunit of proteasome was reduced significantly, which degrades oxidative-modified protein. The mechanism was proved to be the result of the modification of the 20S β-subunit with acrolein and to the binding of other acrolein-modified proteins to the 20S β-subunit. Increased oxidative stress caused by SIN-1 treatment induced a decline in the mitochondrial membrane potential, ΔΨm, and activated mitochondrial apoptotic signaling and induced cell death in SH-SY5Y cells. As another pathway, p38 mitogen-activated protein (MAP) kinase and exracellular signal-regulated kinase (ERK) mediated apoptosis induced by SIN-1. On the other hand, a series of neuroprotective propargylamine derivatives, including rasagiline [N-propargyl-1(R)aminoindan]and (−)deprenyl, intervened in the activation of apoptotic cascade by reactive oxygen species-reactive nitrogen species in mitochondria through stabilization of the membrane potential, ΔΨm. In addition, rasagiline induced antiapoptotic Bcl-2 and glial cell line-derived neurotrophic factor (GDNF) in SH-SY5Y cells, which was mediated by the ERK-nuclear factor (NF)-κB pathway. These results are discussed in relation to the interaction of oxidative stress and mitochondria in the regulation of neuronal death and survival in neurodegenerative diseases.