IRE1 signaling increases PERK expression during chronic ER stress.

The Unfolded Protein Response (UPR) is an essential cellular process activated by the accumulation of unfolded proteins within the Endoplasmic Reticulum (ER), a condition referred to as ER stress. Three ER anchored receptors, IRE1, PERK and ATF6 act as ER stress sensors monitoring the health of the ER. Upon detection of ER stress, IRE1, PERK and ATF6 initiate downstream signaling pathways collectively referred to as the UPR. The overarching aim of the UPR is to restore ER homeostasis by reducing ER stress, however if that is not possible, the UPR transitions from a pro-survival to a pro-death response. While our understanding of the key signaling pathways central to the UPR is well defined, the same is not true of the subtle signaling events that help fine tune the UPR, supporting its ability to adapt to varying amplitudes or durations of ER stress. In this study, we demonstrate cross talk between the IRE1 and PERK branches of the UPR, wherein IRE1 via XBP1s signaling helps to sustain PERK expression during prolonged ER stress. Our findings suggest cross talk between UPR branches aids adaptiveness thereby helping to support the plasticity of UPR signaling responses.

Increased phosphorylation of HexM improves lysosomal uptake and potential for managing GM2 gangliosidoses.

Tay-Sachs and Sandhoff diseases are genetic disorders resulting from mutations in HEXA or HEXB, which code for the α- and ß-subunits of the heterodimer ß-hexosaminidase A (HexA), respectively. Loss of HexA activity results in the accumulation of GM2 ganglioside (GM2) in neuronal lysosomes, culminating in neurodegeneration and death, often by age 4. Previously, we combined critical features of the α- and ß-subunits of HexA into a single subunit to create a homodimeric enzyme known as HexM. HexM is twice as active as HexA and degrades GM2 in vivo, making it a candidate for enzyme replacement therapy (ERT). Here we show HexM production is scalable to meet ERT requirements and we describe an approach that enhances its cellular uptake via co-expression with an engineered GlcNAc-1-phosphotransferase that highly phosphorylates lysosomal proteins. Further, we developed a HexA overexpression system and functionally compared the recombinant enzyme to HexM, revealing the kinetic differences between the enzymes. This study further advances HexM as an ERT candidate and provides a convenient system to produce HexA for comparative studies.

Lyssavirus phosphoproteins increase mitochondrial complex I activity and levels of reactive oxygen species.

We have previously demonstrated that serine residues at positions 162 and 166 of the rabies virus (RABV) phosphoprotein (P) are critical for oxidative stress induced by CVS in cultured cells. We have now evaluated the P of two street RABV variants and Mokola (MOK) virus. The P of these viruses, like CVS, induces an increase in complex I activities and reactive oxygen species levels in transfected cells. Although the sequence homology of P is only 45% with MOK (higher for street viruses) and CVS, serine residues are conserved at positions 162 and 166, suggesting their potential importance in oxidative stress.

Serine residues at positions 162 and 166 of the rabies virus phosphoprotein are critical for the induction of oxidative stress in rabies virus infection.

Our previous work in a mouse model of experimental rabies showed neuronal process (dendrites and axons) degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 (CVS) strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases mitochondrial complex I activity and reactive oxygen species (ROS) production. Expression of a peptide from amino acid 139-172 of the CVS phosphoprotein (P) increased complex I activity and ROS generation similar to expression of the entire P. Site-directed mutational analyses illustrated the importance of the 145-151 and 157-169 regions of P and that serine residues at 162 and 166 are important single amino acid sites. Two CVS recombinant viruses with serine to alanine mutations at positions 162 (A162r) and 166 (A166r) did not increase complex I activity or ROS generation and also did not induce axonal swellings or inhibit axonal growth in DRG neurons. RABV infection is a mitochondrial disorder initiated by interaction of the RABV P and complex I; S162 and S166 are critical sites in the P for this interaction. The resulting mitochondrial dysfunction produces oxidative stress in neurons causing acute degenerative changes affecting neuronal processes resulting in a severe and fatal clinical disease. This information will be important for the future development of novel therapies for rabies.

Rabies virus phosphoprotein interacts with mitochondrial Complex I and induces mitochondrial dysfunction and oxidative stress.

Our previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion neurons infected with challenge virus standard (CVS)-11 strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases reactive oxygen species (ROS) production and mitochondrial Complex I activity vs. mock infection. We have hypothesized that a RABV protein targets mitochondria and triggers dysfunction. Mitochondrial extracts of mouse neuroblastoma cells were analyzed with a proteomics approach. We have identified peptides belonging to the RABV nucleocapsid protein (N), phosphoprotein (P), and glycoprotein (G), and our data indicate that the extract was most highly enriched with P. P was also detected by immunoblotting in RABV-infected purified mitochondrial extracts and also in Complex I immunoprecipitates from the extracts but not in mock-infected extracts. A plasmid expressing P in cells increased Complex I activity and increased ROS generation, whereas expression of other RABV proteins did not. We have analyzed recombinant plasmids encoding various P gene segments. Expression of a peptide from amino acid 139-172 increased Complex I activity and ROS generation similar to expression of the entire P protein, whereas peptides that did not contain this region did not increase Complex I activity or induce ROS generation. These results indicate that a region of the RABV P interacts with Complex I in mitochondria causing mitochondrial dysfunction, increased generation of ROS, and oxidative stress.

Mitochondrial dysfunction in rabies virus infection of neurons.

Infection with the challenge virus standard-11 (CVS) strain of fixed rabies virus induces neuronal process degeneration in adult mice after hindlimb footpad inoculation. CVS-induced axonal swellings of primary rodent dorsal root ganglion neurons are associated with 4-hydroxy-2-nonenal protein adduct staining, indicating a critical role of oxidative stress. Mitochondrial dysfunction is the major cause of oxidative stress. We hypothesized that CVS infection induces mitochondrial dysfunction leading to oxidative stress. We investigated the effects of CVS infection on several mitochondrial parameters in different cell types. CVS infection significantly increased maximal uncoupled respiration and complex IV respiration and complex I and complex IV activities, but did not affect complex II-III or citrate synthase activities. Increases in complex I activity, but not complex IV activity, correlated with susceptibility of the cells to CVS infection. CVS infection maintained coupled respiration and rate of proton leak, indicating a tight mitochondrial coupling. Possibly as a result of enhanced complex activity and efficient coupling, a high mitochondrial membrane potential was generated. CVS infection reduced the intracellular ATP level and altered the cellular redox state as indicated by a high NADH/NAD+ ratio. The basal production of reactive oxygen species (ROS) was not affected in CVS-infected neurons. However, a higher rate of ROS generation occurred in CVS-infected neurons in the presence of mitochondrial substrates and inhibitors. We conclude that CVS infection induces mitochondrial dysfunction leading to ROS overgeneration and oxidative stress.

Role of nuclear factor-κB in oxidative stress associated with rabies virus infection of adult rat dorsal root ganglion neurons.

Recent studies in an experimental model of rabies showed major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult rat dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 strain of rabies virus (CVS) showed axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress and reduced axonal growth compared to that of mock-infected DRG neurons. We have evaluated whether nuclear factor (NF)-κB might act as a critical bridge linking CVS infection and oxidative stress. On Western immunoblotting, CVS infection induced expression of the NF-κB p50 subunit compared to that of mock infection. Ciliary neurotrophic factor, a potent activator of NF-κB, had no effect on mock-infected rat DRG neurons and reduced the number of 4-HNE-labeled puncta. SN50, a peptide inhibitor of NF-κB, and CVS infection had an additive effect in producing axonal swellings, indicating that NF-κB is neuroprotective. The fluorescent signal for subunit p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 h postinfection (p.i.), there was a significant increase in the nucleus/cytoplasm ratio, indicating increased transcriptional activity of NF-κB, perhaps as a response to stress. At both 48 and 72 h p.i., there was significantly reduced nuclear localization of NF-κB. CVS infection may induce oxidative stress by inhibiting nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-κB activity. Further investigations are needed to gain a better understanding of the basic mechanisms involved in the oxidative damage associated with rabies virus infection.

Role of oxidative stress in rabies virus infection.

Recent studies in an experimental model of rabies indicated that there are major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult mouse dorsal root ganglion (DRG) neurons are a good in vitro model for studying the mechanisms involved in rabies virus-induced degeneration of neurites (axons) because, unlike other neuronal cell types, these neurons are fairly permissive to rabies virus infection. DRG neurons infected with the challenge virus standard-11 (CVS) strain of rabies virus show axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress, and also reduced axonal growth in comparison with mock-infected DRG neurons. Treatment with the antioxidant N-acetyl cysteine prevented the reduction in axonal outgrowth that occurred with CVS infection. The axonal swellings with 4-HNE-labeled puncta were found to be associated with aggregations of actively respiring mitochondria. We postulate that rabies virus infection likely induces mitochondrial dysfunction resulting in oxidative stress and degenerative changes involving neuronal processes. This mitochondrial dysfunction may be the result of either direct or indirect effects of the virus on the mitochondrial electron-transport chain or it may occur through other mechanisms. Further investigations are needed to gain a better understanding of the basic mechanisms involved in the oxidative damage associated with rabies virus infection. This information may prove helpful in the design of future therapeutic effects for this dreaded ancient disease.

Role of oxidative stress in rabies virus infection of adult mouse dorsal root ganglion neurons.

Rabies virus infection of dorsal root ganglia (DRG) was studied in vitro with cultured adult mouse DRG neurons. Recent in vivo studies of transgenic mice that express the yellow fluorescent protein indicate that neuronal process degeneration, involving both dendrites and axons, occurs in mice infected with the challenge virus standard (CVS) strain of rabies virus by footpad inoculation. Because of the similarities of the morphological changes in experimental rabies and in diabetic neuropathy and other diseases, we hypothesize that neuronal process degeneration occurs as a result of oxidative stress. DRG neurons were cultured from adult ICR mice. Two days after plating, they were infected with CVS. Immunostaining was evaluated with CVS- and mock-infected cultures for neuron specific beta-tubulin, rabies virus antigen, and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE) (marker of lipid peroxidation and hence oxidative stress). Neuronal viability (by trypan blue exclusion), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and axonal growth were also assessed with the cultures. CVS infected 33 to 54% of cultured DRG neurons. Levels of neuronal viability and TUNEL staining were similar in CVS- and mock-infected DRG neurons. There were significantly more 4-HNE-labeled puncta at 2 and 3 days postinfection in CVS-infected cultures than in mock-infected cultures, and axonal outgrowth was reduced at these time points in CVS infection. Axonal swellings with 4-HNE-labeled puncta were also associated with aggregations of actively respiring mitochondria. We have found evidence that rabies virus infection in vitro causes axonal injury of DRG neurons through oxidative stress. Oxidative stress may be important in vivo in rabies and may explain previous observations of the degeneration of neuronal processes.

Regulation of apoptosis in fibroblast-like synoviocytes by the hypoxia-induced Bcl-2 family member Bcl-2/adenovirus E1B 19-kd protein-interacting protein 3.

OBJECTIVE: Rheumatoid arthritis (RA) synovial hyperplasia is related in part to a resistance to apoptosis exhibited by fibroblast-like synoviocytes (FLS). Since hypoxia is a regulator of apoptosis, and since RA synovium is hypoxic, we conducted this study to examine the effects of hypoxia on the Bcl-2 pathway and the role this may play in regulating apoptosis in FLS. METHODS: Synovium samples from RA patients, osteoarthritis (OA) patients, and normal subjects were used for immunohistologic assessments and for generating FLS lines in vitro. FLS were stimulated under conditions of hypoxia (1% O(2)) and using 100 microM CoCl(2) to simulate the effects of severe hypoxia. Changes in the gene expression profile of FLS were evaluated using microarrays and were confirmed by quantitative polymerase chain reaction (PCR). Changes in protein expression were detected by Western blotting. The effect of transient transfection with a BNIP3 plasmid on the apoptosis of FLS was evaluated in the presence and absence of cytokines. RESULTS: Gene expression profiling demonstrated that BNIP3 was unique among the BCL2 family, in that it was induced by hypoxia in FLS. Quantitative PCR indicated a 2-3-fold induction of BNIP3 messenger RNA, and Western blotting showed a 3-5-fold increase in the 30-kd Bcl-2/adenovirus E1B 19-kd protein-interacting protein 3 (BNIP-3) monomer. BNIP-3 was widely expressed in RA synovium and was prominent in FLS from the lining layer. Overexpression of BNIP3 increased FLS apoptosis under hypoxic conditions, an effect that was inhibited by tumor necrosis factor alpha and interleukin-1beta. CONCLUSION: The proapoptotic protein BNIP-3 is induced in FLS by hypoxia and is widely expressed in RA synovium, but its proapoptotic effects may be inhibited in vivo by proinflammatory cytokines. Since overexpression of BNIP3 in FLS increases apoptosis, this may provide a novel approach for controlling synovial hyperplasia in RA.

Elevated K-ras activity with cholestyramine and lovastatin, but not konjac mannan or niacin in lung--importance of mouse strain.

Our previous work established that hypocholesterolemic agents altered K-ras intracellular localization in lung. Here, we examined K-ras activity to define further its potential importance in lung carcinogenesis. K-ras activity in lungs from male A/J, Swiss and C57BL/6 mice was examined. For 3 weeks, mice consumed either 2 or 4% cholestyramine (CS), 1% niacin, 5% konjac mannan (KM), or were injected with lovastatin 25mg/kg three or five times weekly (Lov-3X and Lov-5X). A pair-fed (PF) group was fed the same quantity of diet consumed by the Lov-5X mice to control for lower body weights in Lov-5X mice. After 3 weeks, serum cholesterol was assayed with a commercial kit. Activated K-ras protein from lung was affinity precipitated with a Raf-1 ras binding domain-glutathione-S-transferase fusion protein bound to glutathione-agarose beads, followed by Western blotting, K-ras antibody treatment, and chemiluminescent detection. Only KM reduced serum cholesterol (in two of three mouse strains). In C56BL/6 mice treated with Lov-3X, lung K-ras activity increased 1.8-fold versus control (p=0.009). In normal lung with wild-type K-ras, this would be expected to be associated with maintenance of differentiation. In A/J mice fed 4% CS, K-ras activity increased 2.1-fold (p=0.02), which might be responsible for the reported enhancement of carcinogenesis in carcinogen-treated rats fed CS. KM feeding and PF treatment had no significant effects on K-ras activity. These data are consistent with the concept that K-ras in lung has an oncogenic function when mutated, but may act as a tumor suppressor when wild-type.

Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein.

Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST-RBD protein induction temperature from the original 37 to 30 degrees C produced a consistently greater yield of fusion protein. To improve stability of the GST-RBD beads so as to perform large-scale experiments, 0.1% NaN(3) was added. NaN(3)-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST-RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST-RBD beads to at least 24 days, and enhanced detection sensitivity.

Increased K-ras protein and activity in mouse and human lung epithelial cells at confluence.

Although K-ras is frequently mutated in lung adenocarcinomas, the normal function of K-ras p21 in lung is not known. In two mouse (E10 and C10) and one human (HPL1D) immortalized lung cell lines from peripheral epithelium, we have measured total K-ras p21 and active K-ras p21-GTP during cell proliferation and at growth arrest caused by confluence. In all three cell types, total K-ras p21 increased 2- to 4-fold at confluence, and active K-ras p21-GTP increased 10- to 200-fold. It was estimated that 0.03% of total K-ras p21 was in the active GTP-bound state at 50% confluence, compared with 1.4% at postconfluence. By contrast, stimulation of proliferation by serum-containing medium did not involve K-ras p21 activation, even though a rapid, marked activation of both Erk1/2 and Akt occurred. At confluence, large increases, up to 14-fold, were seen in Grb2/Sos1 complexes, which may activate K-ras p21. In sum, increased protein expression and activity of K-ras p21 are associated with growth arrest, not with proliferation, in mouse and human lung cell lines.