Hepatic LRP-1 plays an important role in amyloidosis in Alzheimer's disease mice: Potential role in chronic heavy alcohol feeding.

BACKGROUND: Hepatic lipoprotein receptor-related protein 1 (LRP-1) plays a central role in peripheral amyloid beta (Aß) clearance, but its importance in Alzheimer's disease (AD) pathology is understudied. Our previous work showed that intragastric alcohol feeding to C57BL/6 J mice reduced hepatic LRP-1 expression which correlated with significant AD-relevant brain changes. Herein, we examined the role of hepatic LRP-1 in AD pathogenesis in APP/PS1 AD mice using two approaches to modulate hepatic LRP-1, intragastric alcohol feeding to model chronic heavy drinking shown by us to reduce hepatic LRP-1, and hepato-specific LRP-1 silencing. METHODS: Eight-month-old male APP/PS1 mice were fed ethanol or control diet intragastrically for 5 weeks (n = 7-11/group). Brain and liver Aß were assessed using immunoassays. Three important mechanisms of brain amyloidosis were investigated: hepatic LRP-1 (major peripheral Aß regulator), blood-brain barrier (BBB) function (vascular Aß regulator), and microglia (major brain Aß regulator) using immunoassays. Spatial LRP-1 gene expression in the periportal versus pericentral hepatic regions was confirmed using NanoString GeoMx Digital Spatial Profiler. Further, hepatic LRP-1 was silenced by injecting LRP-1 microRNA delivered by the adeno-associated virus 8 (AAV8) and the hepato-specific thyroxine-binding globulin (TBG) promoter to 4-month-old male APP/PS1 mice (n = 6). Control male APP/PS1 mice received control AAV8 (n = 6). Spatial memory and locomotion were assessed 12 weeks after LRP-1 silencing using Y-maze and open-field test, respectively, and brain and liver Aß were measured. RESULTS: Alcohol feeding reduced plaque-associated microglia in APP/PS1 mice brains and increased aggregated Aß (p < 0.05) by ELISA and 6E10-positive Aß load by immunostaining (p < 0.05). Increased brain Aß corresponded with a significant downregulation of hepatic LRP-1 (p < 0.01) at the protein and transcript level, primarily in pericentral hepatocytes (zone 3) where alcohol-induced injury occurs. Hepato-specific LRP-1 silencing significantly increased brain Aß and locomotion hyperactivity (p < 0.05) in APP/PS1 mice. CONCLUSION: Chronic heavy alcohol intake reduced hepatic LRP-1 expression and increased brain Aß. The hepato-specific LRP-1 silencing similarly increased brain Aß which was associated with behavioral deficits in APP/PS1 mice. Collectively, our results suggest that hepatic LRP-1 is a key regulator of brain amyloidosis in alcohol-dependent AD.

Alcohol as a Modifiable Risk Factor for Alzheimer's Disease-Evidence from Experimental Studies.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment and memory loss. Epidemiological evidence suggests that heavy alcohol consumption aggravates AD pathology, whereas low alcohol intake may be protective. However, these observations have been inconsistent, and because of methodological discrepancies, the findings remain controversial. Alcohol-feeding studies in AD mice support the notion that high alcohol intake promotes AD, while also hinting that low alcohol doses may be protective against AD. Chronic alcohol feeding to AD mice that delivers alcohol doses sufficient to cause liver injury largely promotes and accelerates AD pathology. The mechanisms by which alcohol can modulate cerebral AD pathology include Toll-like receptors, protein kinase-B (Akt)/mammalian target of rapamycin (mTOR) pathway, cyclic adenosine monophosphate (cAMP) response element-binding protein phosphorylation pathway, glycogen synthase kinase 3-ß, cyclin-dependent kinase-5, insulin-like growth factor type-1 receptor, modulation of ß-amyloid (Aß) synthesis and clearance, microglial mediated, and brain endothelial alterations. Besides these brain-centric pathways, alcohol-mediated liver injury may significantly affect brain Aß levels through alterations in the peripheral-to-central Aß homeostasis. This article reviews published experimental studies (cell culture and AD rodent models) to summarize the scientific evidence and probable mechanisms (both cerebral and hepatic) by which alcohol promotes or protects against AD progression.

Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis.

UNLABELLED: Although necrosis in the acetaminophen (APAP) model is known to be regulated by c-Jun NH2-terminal kinase (JNK) through interaction with mitochondria, the role of necroptosis through receptor-interacting proteins 1 and 3 (RIPK1 and RIPK3) has also been suggested. Our aim was to determine the relationship between these two mechanisms of cell death. To verify the participation of RIPK1, we used antisense knockdown and confirmed protection comparable to the RIPK1 inhibitor, necrostatin, in vivo and in vitro. However, we found no evidence that RIPK3 is expressed in primary mouse hepatocytes under basal conditions or after APAP and RIPK3(-/-) mice were not protected. RIPK3 was exclusively expressed in nonparenchymal cells. RIPK1 knockdown protected RIPK3(-/-) mice to the same extent as wild-type mice, underscoring the independent role of RIPK1. We confirmed that necroptosis is not involved in APAP toxicity by using mixed lineage kinase domain-like protein (MLKL) knockout mice, which were not protected from APAP. Next, we addressed whether there is interplay between RIPK1 and JNK. RIPK1 knockdown decreased the level of JNK activation and translocation to mitochondria and abrogated subsequent translocation of dynamin-related protein 1 (Drp1). Interestingly, APAP induced translocation of RIPK1 to mitochondria, which was unaffected by knockdown of the mitochondrial JNK docking protein, Sh3 homology 3 binding protein 5 (Sab). CONCLUSION: RIPK1 participates in APAP-induced necrosis upstream of JNK activation whereas RIPK3 and MLKL are dispensable, indicating that necroptosis does not contribute to APAP-induced necrosis and RIPK1 has a unique, independent role.

Protein kinase C (PKC) participates in acetaminophen hepatotoxicity through c-jun-N-terminal kinase (JNK)-dependent and -independent signaling pathways.

UNLABELLED: This study examines the role of protein kinase C (PKC) and AMP-activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP cytotoxicity despite sustained c-jun-N-terminal kinase (JNK) activation. Broad-spectrum PKC inhibitor treatment enhanced p-AMPK levels and AMPK regulated survival-energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p-AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro-31-8245 treatment in mice up-regulated p-AMPK levels, increased autophagy (i.e., increased LC3-II formation, p62 degradation), and protected against APAP-induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. CONCLUSION: PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury.

Respiratory substrates regulate S-nitrosylation of mitochondrial proteins through a thiol-dependent pathway.

S-Nitrosylation is a reversible post-translational modification on cysteinyl thiols that can modulate the function of redox-sensitive proteins. The S-nitrosylation of mitochondrial proteins has been shown to regulate various mitochondrial activities involved in energy-transducing systems and mitochondrion-driven apoptosis. In isolated rat brain mitochondria, we demonstrate that mitochondrial protein S-nitrosylation is regulated by respiratory substrates (glutamate/malate) through a thiol-dependent pathway. Mitochondrial proteins become susceptible to S-nitrosoglutathione (GSNO)-induced S-nitrosylation in mitochondria with an oxidized environment (low glutathione (GSH), NADH, and NADPH, and high GSSG, NAD(+), and NADP(+)) caused by isolation of mitochondria using a discontinuous Percoll gradient. Activation of mitochondrial respiration by respiratory substrates leads to increased NAD(P)H and GSH levels, which in turn reduces mitochondrial S-nitrosylated proteins. 1-Chloro-2,4-dinitrobenzene (CDNB), which depletes mitochondrial GSH and inhibits the thioredoxin-thioredoxin reductase system, prevented the denitrosylation of mitochondrial proteins caused by respiratory substrate treatment. Using biotin-switch coupled with LC-MS/MS, several mitochondrial proteins were identified as targets of S-nitrosylation including adenine nucleotide translocase (ANT) and voltage-dependent anion channel (VDAC), important components of the mitochondria permeability transition pore (MPTP), as well as ATP synthase. The S-nitrosylation of ATP synthase by GSNO was found to inhibit its activity. These findings emphasize the importance of respiratory substrates in regulating S-nitrosylation through a thiol-dependent (GSH and/or thioredoxin) pathway, with implications for mitochondrial bioenergetics and mitochondrion-driven apoptosis.

Dynamic adaptation of liver mitochondria to chronic alcohol feeding in mice: biogenesis, remodeling, and functional alterations.

Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD(+) and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD(+), the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver.

c-Jun N-terminal kinase (JNK)-dependent acute liver injury from acetaminophen or tumor necrosis factor (TNF) requires mitochondrial Sab protein expression in mice.

Sustained JNK activation plays a critical role in hepatotoxicity by acetaminophen or GalN/TNF-α. To address the importance of JNK translocation to mitochondria that accompanies sustained activation in these models, we assessed the importance of the expression of a potential initial target of JNK in the outer membrane of mitochondria, namely Sab (SH3 domain-binding protein that preferentially associates with Btk), also known as Sh3bp5 (SH3 domain-binding protein 5). Silencing the expression of Sab in the liver using adenoviral shRNA inhibited sustained JNK activation and mitochondrial targeting of JNK and the upstream MKK4 (MAPK kinase 4), accompanied by striking protection against liver injury in vivo and in cultured hepatocytes in both toxicity models. We conclude that mitochondrial Sab may serve as a platform for the MAPK pathway enzymes and that the interaction of stress-activated JNK with Sab is required for sustained JNK activation and toxicity.

Comparative studies between the murine immortalized brain endothelial cell line (bEnd.3) and induced pluripotent stem cell-derived human brain endothelial cells for paracellular transport.

Brain microvascular endothelial cells, forming the anatomical site of the blood-brain barrier (BBB), are widely used as in vitro complements to in vivo BBB studies. Among the immortalized cells used as in vitro BBB models, the murine-derived bEnd.3 cells offer culturing consistency and low cost and are well characterized for functional and transport assays, but result in low transendothelial electrical resistance (TEER). Human-induced pluripotent stem cells differentiated into brain microvascular endothelial cells (ihBMECs) have superior barrier properties, but the process of differentiation is time-consuming and can result in mixed endothelial-epithelial gene expression. Here we performed a side-by-side comparison of the ihBMECs and bEnd.3 cells for key paracellular diffusional transport characteristics. The TEER across the ihBMECs was 45- to 68-fold higher than the bEnd.3 monolayer. The ihBMECs had significantly lower tracer permeability than the bEnd.3 cells. Both, however, could discriminate between the paracellular permeabilities of two tracers: sodium fluorescein (MW: 376 Da) and fluorescein isothiocyanate (FITC)-dextran (MW: 70 kDa). FITC-dextran permeability was a strong inverse-correlate of TEER in the bEnd.3 cells, whereas sodium fluorescein permeability was a strong inverse-correlate of TEER in the ihBMECs. Both bEnd.3 cells and ihBMECs showed the typical cobblestone morphology with robust uptake of acetylated LDL and strong immuno-positivity for vWF. Both models showed strong claudin-5 expression, albeit with differences in expression location. We further confirmed the vascular endothelial- (CD31 and tube-like formation) and erythrophagocytic-phenotypes and the response to inflammatory stimuli of ihBMECs. Overall, both bEnd.3 cells and ihBMECs express key brain endothelial phenotypic markers, and despite differential TEER measurements, these in vitro models can discriminate between the passage of different molecular weight tracers. Our results highlight the need to corroborate TEER measurements with different molecular weight tracers and that the bEnd.3 cells may be suitable for large molecule transport studies despite their low TEER.

Tunneling nanotube formation promotes survival against 5-fluorouracil in MCF-7 breast cancer cells.

Tunneling nanotubes (TNTs) are F-actin-based open-ended tubular extensions that form following stresses, such as nutritional deprivation and oxidative stress. The chemotherapy agent 5-fluorouracil (5-FU) represents a significant stressor to cancer cells and induces thymidine deficiency, a state similar to nutritional deprivation. However, the ability of 5-FU to induce TNT formation in cancer cells and potentially enhance survival has not been explored. In this study, we examined whether 5-FU can induce TNT formation in MCF-7 breast cancer cells. Cytotoxic doses of 5-FU (150-350 µm) were observed to significantly induce TNT formation beginning at 24 h after exposure. TNTs formed following 5-FU treatment probably originated as extensions of gap junctions as MCF-7 cells detach from cell clusters. TNTs act as conduits for exchange of cellular components and we observed mitochondrial exchange through TNTs following 5-FU treatment. 5-FU-induced TNT formation was inhibited by over 80% following treatment with the F-actin-depolymerizing agent, cytochalasin B (cytoB). The inhibition of TNTs by cytoB corresponded with increased 5-FU-induced cytotoxicity by 30-62% starting at 48 h, suggesting TNT formation aides in MCF-7 cell survival against 5-FU. Two other widely used chemotherapy agents, docetaxel and doxorubicin induced TNT formation at much lower levels than 5-FU. Our work suggests that the therapeutic targeting of TNTs may increase 5-FU chemotherapy efficacy and decrease drug resistance in cancer cells, and these findings merits further investigation.

Modulation of hepatic amyloid precursor protein and lipoprotein receptor-related protein 1 by chronic alcohol intake: Potential link between liver steatosis and amyloid-ß.

Heavy alcohol consumption is a known risk factor for various forms of dementia and the development of Alzheimer's disease (AD). In this work, we investigated how intragastric alcohol feeding may alter the liver-to-brain axis to induce and/or promote AD pathology. Four weeks of intragastric alcohol feeding to mice, which causes significant fatty liver (steatosis) and liver injury, caused no changes in AD pathology markers in the brain [amyloid precursor protein (APP), presenilin], except for a decrease in microglial cell number in the cortex of the brain. Interestingly, the decline in microglial numbers correlated with serum alanine transaminase (ALT) levels, suggesting a potential link between liver injury and microglial loss in the brain. Intragastric alcohol feeding significantly affected two hepatic proteins important in amyloid-beta (Aß) processing by the liver: 1) alcohol feeding downregulated lipoprotein receptor-related protein 1 (LRP1, ∼46%), the major receptor in the liver that removes Aß from blood and peripheral organs, and 2) alcohol significantly upregulated APP (∼2-fold), a potentially important source of Aß in the periphery and brain. The decrease in hepatic LRP1 and increase in hepatic APP likely switches the liver from being a remover or low producer of Aß to an important source of Aß in the periphery, which can impact the brain. The downregulation of LRP1 and upregulation of APP in the liver was observed in the first week of intragastric alcohol feeding, and also occurred in other alcohol feeding models (NIAAA binge alcohol model and intragastric alcohol feeding to rats). Modulation of hepatic LRP1 and APP does not seem alcohol-specific, as ob/ob mice with significant steatosis also had declines in LRP1 and increases in APP expression in the liver. These findings suggest that liver steatosis rather than alcohol-induced liver injury is likely responsible for regulation of hepatic LRP1 and APP. Both obesity and alcohol intake have been linked to AD and our data suggests that liver steatosis associated with these two conditions modulates hepatic LRP1 and APP to disrupt Aß processing by the liver to promote AD.

Regulation of mitochondrial glutathione redox status and protein glutathionylation by respiratory substrates.

Brain and liver mitochondria isolated by a discontinuous Percoll gradient show an oxidized redox environment, which is reflected by low GSH levels and high GSSG levels and significant glutathionylation of mitochondrial proteins as well as by low NAD(P)H/NAD(P) values. The redox potential of brain mitochondria isolated by a discontinuous Percoll gradient method was calculated to be -171 mV based on GSH and GSSG concentrations. Immunoblotting and LC/MS/MS analysis revealed that succinyl-CoA transferase and ATP synthase (F(1) complex, α-subunit) were extensively glutathionylated; S-glutathionylation of these proteins resulted in a substantial decrease of activity. Supplementation of mitochondria with complex I or complex II respiratory substrates (malate/glutamate or succinate, respectively) increased NADH and NADPH levels, resulting in the restoration of GSH levels through reduction of GSSG and deglutathionylation of mitochondrial proteins. Under these conditions, the redox potential of brain mitochondria was calculated to be -291 mV. Supplementation of mitochondria with respiratory substrates prevented GSSG formation and, consequently, ATP synthase glutathionylation in response to H(2)O(2) challenges. ATP synthase appears to be the major mitochondrial protein that becomes glutathionylated under oxidative stress conditions. Glutathionylation of mitochondrial proteins is a major consequence of oxidative stress, and respiratory substrates are key regulators of mitochondrial redox status (as reflected by thiol/disulfide exchange) by maintaining mitochondrial NADPH levels.

Silencing glycogen synthase kinase-3beta inhibits acetaminophen hepatotoxicity and attenuates JNK activation and loss of glutamate cysteine ligase and myeloid cell leukemia sequence 1.

Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3beta (GSK-3beta) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury ( approximately 1 h). The silencing of GSK-3beta, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3alpha (GSK-3alpha), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3beta affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3beta decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3beta translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3beta reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3beta also resulted in an inhibition of the early phase (0-2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3beta is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver.

Role of nitric oxide-mediated glutathionylation in neuronal function: potential regulation of energy utilization.

Excessive generation of nitric oxide radical (NO*) in neuroinflammation, excitotoxicity and during age-related neurodegenerative disorders entails the localized and concerted increase in nitric oxide synthase(s) expression in glial cells and neurons. The aim of the present study was to assess the biological significance of the impact of NO* on the cell's thiol status with emphasis on S-glutathionylation of targeted proteins. Exposure of primary cortical neurons or astrocytes to increasing flow rates of NO* (0.061-0.25 microM/s) resulted in the following. (i) A decrease in GSH (glutathione) in neurons accompanied by formation of GSNO (S-nitrosoglutathione) and GSSG (glutathione disulfide); neurons were far more sensitive to NO* exposure than astrocytes. (ii) A dose-dependent oxidation of the cellular redox status: the neuron's redox potential increased approximately 42 mV and that of astrocytes approximately 23 mV. A good correlation was observed between cell viability and the cellular redox potential. The higher susceptibility of neurons to NO* can be partly explained by a reduced capacity to recover GSH through lower activities of GSNO and GSSG reductases. (iii) S-glutathionylation of a small subset of proteins, among them GAPDH (glyceraldehyde-3-phosphate dehydrogenase), the S-glutathionylation of which resulted in inhibition of enzyme activity. The quantitative analyses of changes in the cell's thiol potential upon NO* exposure and their consequences for S-glutathionylation are discussed in terms of the distinct redox environment of astrocytes and neurons.

Signal transduction pathways involved in drug-induced liver injury.

Hepatocyte death following drug intake is the critical event in the clinical manifestation of drug-induced liver injury (DILI). Traditionally, hepatocyte death caused by drugs had been attributed to overwhelming oxidative stress and mitochondria dysfunction caused by reactive metabolites formed during drug metabolism. However, recent studies have also shown that signal transduction pathways activated/inhibited during oxidative stress play a key role in DILI. In acetaminophen (APAP)-induced liver injury, hepatocyte death requires the sustained activation of c-Jun kinase (JNK), a kinase important in mediating apoptotic and necrotic death. Inhibition of JNK using chemical inhibitors or knocking down JNK can prevent hepatocyte death even in the presence of extensive glutathione (GSH) depletion, covalent binding, and oxidative stress. Once activated, JNK translocates to mitochondria, to induce mitochondria permeability transition and trigger hepatocyte death. Mitochondria are central targets where prodeath kinases such as JNK, prosurvival death proteins such as bcl-xl, and oxidative damage converge to determine hepatocyte survival. The importance of mitochondria in DILI is also observed in the Mn-SOD heterozygous (+/-) model, where mice with less mitochondrial Mn-SOD are sensitized to liver injury caused by certain drugs. An extensive body of research is accumulating suggesting a central role of mitochondria in DILI. Drugs can also cause redox changes that inhibit important prosurvival pathways such as NF-kappaB. The inhibition of NF-kappaB by subtoxic doses of APAP sensitizes hepatocyte to the cytotoxic actions of tumor necrosis factor (TNF). Many drugs will induce liver injury if simultaneously treated with LPS, which promotes inflammation and cytokine release. Drugs may be sensitizing hepatocytes to the cytotoxic effects of cytokines such as TNF, or vice versa. Overall many signaling pathways are important in regulating DILI, and represent potential therapeutic targets to reduce liver injury caused by drugs.

Mitofusin-2 mediates doxorubicin sensitivity and acute resistance in Jurkat leukemia cells.

Mitochondria oscillate along a morphological continuum from fragmented individual units to hyperfused tubular networks. Their position at the junction of catabolic and anabolic metabolism couples this morphological plasticity, called mitochondrial dynamics, to larger cellular metabolic programs, which in turn implicate mitochondria in a number of disease states. In many cancers, fragmented mitochondria engage the cell with the biosynthetic capacity of aerobic glycolysis in service of proliferation and progression. Chemo-resistant cancers, however, favor remodeling dynamics that yield fused mitochondrial assemblies utilizing oxidative phosphorylation (OXPHOS) through the electron transport chain (ETC). In this study, expression of Mitofusin-2 (MFN-2), a GTPase protein mediator of mitochondrial fusion, was found to closely correlate to Jurkat leukemia cell survival post doxorubicin (DxR) assault. Moreover, this was accompanied by dramatically increased expression of OXPHOS respiratory complexes and ATP Synthase, as well as a commensurate escalation of state III respiration and respiratory control ratio (RCR). Importantly, CRISPR knockout of MFN-2 resulted in a considerable decrease of doxorubicin (DxR) median lethal dose compared to a treated wildtype control, suggesting an important role of mitochondrial fusion in chemotherapy sensitivity and acute resistance.

c-Jun N-terminal kinase regulates mitochondrial bioenergetics by modulating pyruvate dehydrogenase activity in primary cortical neurons.

This study examines the role of c-jun N-terminal kinase (JNK) in mitochondrial signaling and bioenergetics in primary cortical neurons and isolated rat brain mitochondria. Exposure of neurons to either anisomycin (an activator of JNK/p38 mitogen-activated protein kinases) or H2O2 resulted in activation (phosphorylation) of JNK (mostly p46(JNK1)) and its translocation to mitochondria. Experiments with mitochondria isolated from either rat brain or primary cortical neurons and incubated with proteinase K revealed that phosphorylated JNK was associated with the outer mitochondrial membrane; this association resulted in the phosphorylation of the E(1alpha) subunit of pyruvate dehydrogenase, a key enzyme that catalyzes the oxidative decarboxylation of pyruvate and that links two major metabolic pathways: glycolysis and the tricarboxylic acid cycle. JNK-mediated phosphorylation of pyruvate dehydrogenase was not observed in experiments carried out with mitoplasts, thus suggesting the requirement of intact, functional mitochondria for this effect. JNK-mediated phosphorylation of pyruvate dehydrogenase was associated with a decline in its activity and, consequently, a shift to anaerobic pyruvate metabolism: the latter was confirmed by increased accumulation of lactic acid and decreased overall energy production (ATP levels). Pyruvate dehydrogenase appears to be a specific phosphorylation target for JNK, for other kinases, such as protein kinase A and protein kinase C did not elicit pyruvate dehydrogenase phosphorylation and did not decrease the activity of the complex. These results suggest that JNK mediates a signaling pathway that regulates metabolic functions in mitochondria as part of a network that coordinates cytosolic and mitochondrial processes relevant for cell function.

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis.

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.

Obesity and steatosis promotes mitochondrial remodeling that enhances respiratory capacity in the liver of ob/ob mice.

We investigated if obesity/steatosis promotes mitochondrial remodeling in the liver of ob/ob mice (an obesity model). Liver mitochondria from ob/ob mice (21 weeks with significant steatosis) had ~ 2-fold increases in state III respiration compared with control (C57BL/6J, C57BL/6NJ) for all respiratory substrates examined (glutamate/malate, succinate, octanoate, and glycerol 3-phosphate). A corresponding 2-fold increase in the expression of respiratory complexes (I, IV, and V) and other respiratory proteins (glycerol phosphate dehydrogenase-2 and medium-chain acyl-coenzyme A dehydrogenase) occur in liver mitochondria of mature ob/ob mice. Conversely, respiration in liver mitochondria from young ob/ob mice (6 weeks) does not differ from control with any respiratory substrates examined. Overall, mitochondrial remodeling that enhances respiration increases with obesity/steatosis in the liver of ob/ob mice.

Mitochondrial remodeling in the liver following chronic alcohol feeding to rats.

The feeding of alcohol orally (Lieber-DeCarli diet) to rats has been shown to cause declines in mitochondrial respiration (state III), decreased expression of respiratory complexes, and decreased respiratory control ratios (RCR) in liver mitochondria. These declines and other mitochondrial alterations have led to the hypothesis that alcohol feeding causes "mitochondrial dysfunction" in the liver. If oral alcohol feeding leads to mitochondrial dysfunction, one would predict that increasing alcohol delivery by intragastric (IG) alcohol feeding to rats would cause greater declines in mitochondrial bioenergetics in the liver. In this study, we examined the mitochondrial alterations that occur in rats fed alcohol both orally and intragastrically. Oral alcohol feeding decreased glutamate/malate-, acetaldehyde- and succinate-driven state III respiration, RCR, and expression of respiratory complexes (I, III, IV, V) in liver mitochondria, in agreement with previous results. IG alcohol feeding, on the other hand, caused a slight increase in glutamate/malate-driven respiration, and significantly increased acetaldehyde-driven respiration in liver mitochondria. IG feeding also caused liver mitochondria to experience a decline in succinate-driven respiration, but these decreases were smaller than those observed with oral alcohol feeding. Surprisingly, oral and IG alcohol feeding to rats increased mitochondrial respiration using other substrates, including glycerol-3-phosphate (which delivers electrons from cytoplasmic NADH to mitochondria) and octanoate (a substrate for beta-oxidation). The enhancement of glycerol-3-phosphate- and octanoate-driven respiration suggests that liver mitochondria remodeled in response to alcohol feeding. In support of this notion, we observed that IG alcohol feeding also increased expression of mitochondrial glycerol phosphate dehydrogenase-2 (GPD2), transcription factor A (TFAM), and increased mitochondrial NAD+-NADH and NADP+-NADPH levels in the liver. Our findings suggest that mitochondrial dysfunction represents an incomplete picture of mitochondrial dynamics that occur in the liver following alcohol feeding. While alcohol feeding causes some mitochondrial dysfunction (i.e. succinate-driven respiration), our work suggests that the major consequence of alcohol feeding is mitochondrial remodeling in the liver as an adaptation. This mitochondrial remodeling may play an important role in the enhanced alcohol metabolism and other adaptations in the liver that develop with alcohol intake.

Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis.

Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.