Proteasome Composition in Cytokine-Treated Neurons and Astrocytes is Determined Mainly by Subunit Displacement.

In this study, we investigated if subunit displacement and/or alterations in proteasome biosynthesis are responsible for the changes in the levels of constitutive proteasomes (c-20S), immunoproteasomes (i-20S) and the activators PA28 and PA700 in neurons and astrocytes cultured with a cytokine mixture (IFN-γ/TNF-α/IL-1ß). Exposure of both cell types to cytokines for 24 h increases mRNA and protein expression of the i-20S-specific subunit ß5i and PA28α/ß, and leads to a decline in the amount of the c-20S-specific subunit ß5. Since ß5 mRNA levels are unchanged by the cytokine treatment, it is fair to conclude that displacement of constitutive ß-subunits with inducible ß5i subunits is likely the mechanism underlying the decrease in c-20S. As expected, the increase in the amount of the IFN-γ-inducible subunits coincides with elevated expression of phospho-STAT-1 and interferon regulatory factor-1 (IRF-1). However, inhibition of NF-κB signaling in cytokine-treated astrocytes reduces IRF-1 expression without affecting that of i-20S, c-20S and PA28. This suggests that STAT-1 is capable of increasing the transcription of i20S-specific subunits and PA28α/ß by itself. The lack of a decrease in proteasome ß5 mRNA expression is consistent with the fact that Nrf1 (Nfe2l1) and Nrf2 (Nfe2l2) levels are not reduced by pro-inflammatory cytokines. In contrast, we previously found that there is a significant Nrf1 dysregulation and reduced ß5 mRNA expression in the spinal cords of mice with experimental autoimmune encephalomyelitis (EAE). Thus, there are stressors in EAE, other than a pro-inflammatory environment, that are not present in cytokine-treated cells.

Decreased levels of constitutive proteasomes in experimental autoimmune encephalomyelitis may be caused by a combination of subunit displacement and reduced Nfe2l1 expression.

The goal of this study was to determine if subunit displacement and/or alterations in proteasome biosynthesis could explain the changes observed in the levels of constitutive proteasomes (c-20S) and immunoproteasomes (i-20S) in the spinal cords of mice with experimental autoimmune encephalomyelitis (EAE). To this end, EAE was induced in C57BL/6 mice by immunization with MOG35-55 peptide. Spinal cords were collected at different times during the disease course and used for western blotting, RNA analysis, and immunohistochemistry. The results show that, as expression of i-20S and the activator PA28 rise in EAE, there is a concomitant decline in that of c-20S at the mRNA and protein level. These changes are observed in neurons and astrocytes but not in oligodendrocytes. The increased amounts of the i-20S-specific subunit ß5i and PA28α/ß in EAE correlate with the levels of interferon-γ and its downstream effectors p-signal transducer and activator of transcription 1 and interferon regulatory factor-1, but not with those of nuclear factor kappa-light-chain-enhancer of activated B cells. This suggests that the signal transducer and activator of transcription 1/interferon regulatory factor-1 pathway is solely responsible for the induction of these subunits. The decrease in the mRNA and protein levels corresponding to the c-20S-specific subunit ß5 may also be due to reduced expression of the nuclear factor (erythroid-derived 2)-like-1 (Nrf1 or Nfe2l1), specifically Nrf1α and Nrf1ß. Low Nfe2l1 mRNA expression is unlikely caused by reduced mammalian target of rapamycin signaling but could be the result of diminished pre-B-cell leukemia homeobox-1 transcription factor levels. Together, these findings suggest that a combination of subunit displacement and reduced Nrf1 expression may be responsible for c-20S impairment in EAE. The present work provides insights into the dynamics of proteasome expression in the CNS of EAE mice and is the first to explore Nrf1 signaling in an inflammatory demyelinating disorder.

Reduced expression of the ferroptosis inhibitor glutathione peroxidase-4 in multiple sclerosis and experimental autoimmune encephalomyelitis.

Glutathione peroxidase 4 (GPx4) is the only enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor. GPx4 is the major inhibitor of ferroptosis, a non-apoptotic and iron-dependent programmed cell death pathway, which has been shown to occur in various neurological disorders with severe oxidative stress. In this study, we investigate whether GPx4 expression is altered in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). The results clearly show that mRNA expression for all three GPx4 isoforms (cytoplasmic, mitochondrial and nuclear) decline in multiple sclerosis gray matter and in the spinal cord of MOG35-55 peptide-induced EAE. The amount of GPx4 protein is also reduced in EAE, albeit not in all cells. Neuronal GPx4 immunostaining, mostly cytoplasmic, is lower in EAE spinal cords than in control spinal cords, while oligodendrocyte GPx4 immunostaining, mainly nuclear, is unaltered. Neither control nor EAE astrocytes and microglia cells show GPx4 labeling. In addition to GPx4, two other negative modulators of ferroptosis (γ-glutamylcysteine ligase and cysteine/glutamate antiporter), which are critical to maintain physiological levels of glutathione, are diminished in EAE. The decrease in the ability to eliminate hydroperoxides was also evidenced by the accumulation of lipid peroxidation products and the reduction in the proportion of the docosahexaenoic acid in non-myelin lipids. These findings, along with presence of abnormal neuronal mitochondria morphology, which includes an irregular matrix, disrupted outer membrane and reduced/absent cristae, are consistent with the occurrence of ferroptotic damage in inflammatory demyelinating disorders.

The Role of Calpain and Proteasomes in the Degradation of Carbonylated Neuronal Cytoskeletal Proteins in Acute Experimental Autoimmune Encephalomyelitis.

The present study was designed to investigate the role of calpain and the proteasome in the removal of oxidized neuronal cytoskeletal proteins in myelin basic protein-induced experimental autoimmune encephalomyelitis (EAE). To this end, EAE rats received a single intrathecal injection of calpeptin or epoxomicin at the first sign of clinical disease. Forty-eight hours later, animals were sacrificed and lumbar spinal cord segments were dissected and used for biochemical analyses. The results show that calpain and proteasome activity is specifically, but partially, inhibited with calpeptin and epoxomicin, respectively. Calpain inhibition causes an increase in total protein carbonylation and in the amount of neurofilament proteins (NFPs), ß-tubulin and ß-actin that were spared from degradation, but no changes are seen in the oxidation of any of three NFPs. By contrast, proteasome inhibition has no effect on total protein carbonylation or cytoskeletal protein degradation but increases the amount of oxidized NFH and NFM. These results suggest that while the proteasome may contribute to removal of oxidized NFPs, calpain is the main protease involved in degradation of neuronal cytoskeleton and does not preferentially targets oxidized NFPs species in acute EAE. Different results were obtained in a cell-free system, where calpain inhibition rises the amount of oxidized NFH, and proteasome inhibition fails to change the oxidation state of the NFPs. The later finding suggests that the preferential degradation of oxidized NFH and NFM in vivo by the proteasome occurs via the 26S and not the 20S particle.

Mechanism of Protein Carbonylation in Glutathione-Depleted Rat Brain Slices.

This study was conducted to further our understanding about the link between lipid peroxidation and protein carbonylation in rat brain slices incubated with the glutathione (GSH)-depletor diethyl maleate. Using this in vitro system of oxidative stress, we found that there is a significant lag between the appearance of carbonylated proteins and GSH depletion, which seems to be due to the removal of oxidized species early on in the incubation by the mitochondrial Lon protease. Upon acute GSH depletion, protein carbonyls accumulated mostly in mitochondria and to a lesser degree in other subcellular fractions that also contain high levels of polyunsaturated lipids. This result is consistent with our previous findings suggesting that lipid hydroperoxides mediate the oxidation of proteins in this system. However, these lipid hydroperoxides are not produced by oxidation of free arachidonic acid or other polyunsaturated free fatty acids by lipooxygenases or cyclooxygenases. Finally, γ-glutamyl semialdehyde and 2-amino-adipic semialdehyde were identified by HPLC as the carbonyl-containing amino acid residues, indicating that proteins are carbonylated by metal ion-catalyzed oxidation of lysine, arginine and proline residues. The present findings are important in the context of neurological disorders that exhibit increased lipid peroxidation and protein carbonylation, such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

Nrf2-dysregulation correlates with reduced synthesis and low glutathione levels in experimental autoimmune encephalomyelitis.

This study investigates the possible mechanism(s) underlying glutathione (GSH) deficiency in the mouse spinal cord during the course of myelin oligodendrocyte glycoprotein35-55 peptide-induced experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of multiple sclerosis. Using the classical enzymatic recycling method and a newly developed immunodot assay, we first demonstrated that total GSH levels (i.e. free GSH plus all its adducts) are reduced in EAE, suggesting an impaired synthesis. The decline in the levels of this essential antioxidant tripeptide in EAE coincides temporally and in magnitude with a reduction in the amount of γ-glutamylcysteine ligase, the rate-limiting enzyme in GSH synthesis. Other enzymes involved in GSH biosynthesis, whose genes also contain antioxidant-response elements, including glutathione synthetase, cystine/glutamate antiporter, and γ-glutamyl transpeptidase (γ-GT) are diminished in EAE as well. Low levels of γ-glutamylcysteine ligase, glutathione synthetase, and γ-GT are the consequence of reduced mRNA expression, which correlates with diminished expression of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in both the cytosol and nucleus. Interestingly, the low Nrf2 expression does not seem to be caused by increased degradation via Kelch-like ECH-associated protein 1-dependent or Kelch-like ECH-associated protein 1-independent mechanisms (such as glycogen synthetase kinase-3ß activation), or by reduced levels of Nrf2 mRNA. This suggests that translation of this important transcription factor and/or other still unidentified post-translational processes are altered in EAE. These novel findings are central toward understanding how critical antioxidant and protective responses are lost in inflammatory demyelinating disorders.

Type 5 adenylyl cyclase increases oxidative stress by transcriptional regulation of manganese superoxide dismutase via the SIRT1/FoxO3a pathway.

BACKGROUND: For reasons that remain unclear, whether type 5 adenylyl cyclase (AC5), 1 of 2 major AC isoforms in heart, is protective or deleterious in response to cardiac stress is controversial. To reconcile this controversy we examined the cardiomyopathy induced by chronic isoproterenol in AC5 transgenic (Tg) mice and the signaling mechanisms involved. METHODS AND RESULTS: Chronic isoproterenol increased oxidative stress and induced more severe cardiomyopathy in AC5 Tg, as left ventricular ejection fraction fell 1.9-fold more than wild type, along with greater left ventricular dilation and increased fibrosis, apoptosis, and hypertrophy. Oxidative stress induced by chronic isoproterenol, detected by 8-OhDG was 15% greater, P=0.007, in AC5 Tg hearts, whereas protein expression of manganese superoxide dismutase (MnSOD) was reduced by 38%, indicating that the susceptibility of AC5 Tg to cardiomyopathy may be attributable to decreased MnSOD expression. Consistent with this, susceptibility of the AC5 Tg to cardiomyopathy was suppressed by overexpression of MnSOD, whereas protection afforded by the AC5 knockout (KO) was lost in AC5 KO×MnSOD heterozyous KO mice. Elevation of MnSOD was eliminated by both sirtuin and MEK inhibitors, suggesting both the SIRT1/FoxO3a and MEK/ERK pathway are involved in MnSOD regulation by AC5. CONCLUSIONS: Overexpression of AC5 exacerbates the cardiomyopathy induced by chronic catecholamine stress by altering regulation of SIRT1/FoxO3a, MEK/ERK, and MnSOD, resulting in oxidative stress intolerance, thereby shedding light on new approaches for treatment of heart failure.

Adenylyl cyclase type 5 protein expression during cardiac development and stress.

Adenylyl cyclase (AC) types 5 and 6 (AC5 and AC6) are the two major AC isoforms expressed in the mammalian heart that mediate signals from beta-adrenergic receptor stimulation. Because of the unavailability of isoform-specific antibodies, it is difficult to ascertain the expression levels of AC5 protein in the heart. Here we demonstrated the successful generation of an AC5 isoform-specific mouse monoclonal antibody and studied the expression of AC5 protein during cardiac development in different mammalian species. The specificity of the antibody was confirmed using heart and brain tissues from AC5 knockout mice and from transgenic mice overexpressing AC5. In mice, the AC5 protein was highest in the brain but was also detectable in all organs studied, including the heart, brain, lung, liver, stomach, kidney, skeletal muscle, and vascular tissues. Western blot analysis showed that AC5 was most abundant in the neonatal heart and declined to basal levels in the adult heart. AC5 protein increased in the heart with pressure-overload left ventricular hypertrophy. Thus this new AC5 antibody demonstrated that this AC isoform behaves similarly to fetal type genes, such as atrial natriuretic peptide; i.e., it declines with development and increases with pressure-overload hypertrophy.

Genetic analysis of attractin homologs.

Attractin (ATRN) and Attractin-like 1 (ATRNL1) are highly similar type I transmembrane proteins. Atrn null mutant mice have a pleiotropic phenotype including dark fur, juvenile-onset spongiform neurodegeneration, hypomyelination, tremor, and reduced body weight and adiposity, implicating ATRN in numerous biological processes. Bioinformatic analysis indicated that Atrn and Atrnl1 arose from a common ancestral gene early in vertebrate evolution. To investigate the genetics of the ATRN system and explore potential redundancy between Atrn and Atrnl1, we generated and characterized Atrnl1 loss- and gain-of-function mutations in mice. Atrnl1 mutant mice were grossly normal with no alterations of pigmentation, central nervous system pathology or body weight. Atrn null mutant mice carrying a beta-actin promoter-driven Atrnl1 transgene had normal, agouti-banded hairs and significantly delayed onset of spongiform neurodegeneration, indicating that over-expression of ATRNL1 compensates for loss of ATRN. Thus, the two genes are redundant from the perspective of gain-of-function but not loss-of-function mutations.

Cell cycle progression and activation of Akt kinase are required for insulin-like growth factor I-mediated suppression of apoptosis in granulosa cells.

Ovarian follicle development is dependent on growth factors that stimulate cell proliferation and act as survival factors to prevent apoptosis of follicle cells. We examined the mechanism of the protective effect of IGF-I against Fas ligand-induced apoptosis of granulosa cells and its relationship to cell proliferation. IGF-I activated both the phosphoinositide 3'-OH kinase (PI3K) and the MAPK pathways. Experiments using specific inhibitors of these pathways showed that protection by IGF-I was mediated by the PI3K pathway and not the MAPK pathway. Recombinant adenoviruses were used to test whether the downstream target of PI3K activation, Akt kinase, was required for protection against apoptosis. Expression of dominant negative Akt prevented protection by IGF-I whereas expression of constitutively active Akt (myrAkt) mimicked the effect of IGF-I. Treatment with IGF-I, or expression of myrAkt, increased progression from G(0)/G(1) to S phase of the cell cycle whereas expression of dominant negative Akt inhibited G(0)/G(1) to S phase progression and prevented the stimulatory effect of IGF-I. We tested whether cell cycle progression was required for protection from apoptosis using the cyclin-dependent kinase-2 inhibitor roscovitine, which blocks cells at the G(1)/S transition. Roscovitine prevented the protective effect of IGF-I and myrAkt expression against apoptosis. Therefore, activation of Akt is not sufficient to protect granulosa cells from apoptosis in the absence of cell cycle progression. In summary, IGF-I protects granulosa cells from apoptosis by activation of the PI3K/Akt pathway. This protective effect can occur only when progression from G(1) to S phase of the cell cycle regulated by the PI3K/Akt pathway is unperturbed.