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.

Y-39983, a selective Rho-kinase inhibitor, attenuates experimental autoimmune encephalomyelitis via inhibition of demyelination.

OBJECTIVE: Rho-associated kinase (ROCK) is a serine/threonine kinase and a major downstream effector of the small GTP-binding protein, Rho. Rho-ROCK triggers an intracellular signaling cascade that controls actin cytoskeleton and is essential for cell motility and adhesion, neurite outgrowth and retraction. In chronic disabling disease, multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), demyelination and axonal damage are the major pathological changes contributing to neurological disability. We investigated the protective effect of a specific ROCK inhibitor, Y-39983, on demyelination and axonal damage in chronic EAE. METHODS: Western blotting for myelin proteins, electron microscopy and solochrome cyanine staining was performed to evaluate demyelination while neurofilament proteins and cytoskeletal proteins including ß-actin and ß-tubulin were used to determine axonal damage in a chronic mouse model of EAE treated with Y-39983. RESULTS: Y-39983 significantly suppressed clinical symptoms of EAE and prevented its relapse while increasing the amount of myelin proteins. No significant changes in neurofilaments and cytoskeletal proteins were observed compared with control EAE mice. The inhibition of demyelination by Y-39983 was confirmed by solochrome cyanine staining and electron microscopy. To further study the effect of Y-39983 on demyelination in EAE, we tested three major ROCK substrates, including myosin light chain phosphorylation, LIMK2 and collapsin response mediator protein-2. The activity of these molecules was decreased in EAE animals treated with Y-39983. CONCLUSION: The inhibitory effect of Y-39983 on demyelination is probably due to the inactivation of ROCK substrates, which are important for neurite outgrowth, growth cone collapse and demyelination of oligodendrocytes.

Changes in 20S subunit composition are largely responsible for altered proteasomal activities in experimental autoimmune encephalomyelitis.

We recently reported that the proteasomal peptidase activities are altered in the cerebellum of mice with myelin oligodendrocyte glycoprotein (MOG) peptide-induced experimental autoimmune encephalomyelitis (EAE). To determine whether these fluctuations are caused by proteasome activation/inactivation and/or changes in the levels of individual ß subunits, we characterized the proteasome subunit composition by western blotting. The results show that the rise in proteasomal peptidase activity in acute EAE correlates with an augmented expression of inducible ß subunits whereas the decline in activity in chronic EAE correlates with a reduction in the amount of standard ß subunits. Using pure standard (s) and immuno (i) 20S particles for calibration, we determined that the changes in the levels of catalytic subunits account for all of the fluctuations in peptidase activities in EAE. The i-20S and s-20S proteasome were found to degrade carbonylated ß-actin with similar efficiency, suggesting that the amount of protein carbonyls in EAE may be controlled by the activity of both core particles. We also found an increase in proteasome activator 11S regulatory particle and a decrease in inhibitor proteasome inhibitor with molecular mass of 31 kDa levels in acute EAE, reflecting a response to inflammation. Elevated levels of 19S regulatory particle and 11S regulatory particle in chronic EAE, however, may occur in response to diminished proteasomal activity in this phase. These findings are central towards understanding the altered proteasomal physiology in inflammatory demyelinating disorders.

Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis.

Carbonylated (oxidized) proteins are known to accumulate in the cerebral white matter (WM) and gray matter (GM) of patients with multiple sclerosis (MS). Although oxidative stress is necessary for carbonyl generation, it is the failure of the degradation systems that ultimately leads to the build-up of carbonylated proteins within tissues. In this study, we measured the activity of the 20S proteasome and other proteolytic systems in the cerebral WM and GM of 13 MS patients and 13 controls. We report that the activities of the three peptidases of the 20S proteasome (i.e. chymotrypsin-like, caspase-like and trypsin-like) in both MS-WM and MS-GM are greatly reduced. Interestingly, neither the amount of proteasome nor the levels of the catalytic subunits (ß1, ß2, and ß5) are diminished in this disease. Proteins containing Lys-48 poly-ubiquitin also accumulate in MS tissues, indicating failure of the 26S proteasome as well. Levels of the regulatory caps 11S α and 19S are also lower in MS than in controls, suggesting that the activity of the more complex proteasomes may be reduced further. Finally, the activities of other proteases that might also remove oxidized proteins (calpain, cathepsin B, mitochondrial LonP) are not lessened in MS. Together, these studies suggest that direct inactivation of proteolytic centers in the 20S particle and/or the presence of specific inhibitors is the underlying cause of proteasomal dysfunction in MS.

Reduced proteasomal activity contributes to the accumulation of carbonylated proteins in chronic experimental autoimmune encephalomyelitis.

We have recently shown that several carbonylated proteins, including glial fibrillary acidic protein, ß-actin and ß-tubulin, accumulate within cerebellar astrocytes during the chronic phase of myelin-oligodendrocyte glycoprotein (MOG)(35-55) peptide-induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. As protein carbonyls cannot be repaired and there is less oxidative stress in chronic than in acute EAE, we hypothesized that the accumulation of carbonylated proteins in these animals may be due to a defect in the degradation of the modified proteins. Alternatively, oxidized proteins in chronic EAE mice may be more resistant to proteolysis. Using lipopolysaccharide-stimulated astrocytes and several protease inhibitors we identified the 20S proteasome as the proteolytic system responsible for the elimination of most oxidized proteins. We also discovered that the chymotrysin-like and caspase-like activities of the 20S proteasome are impaired in chronic EAE, while the amount of proteasome was unchanged. Proteasome failure in these animals was confirmed by the build-up of ubiquitinated proteins, mostly within astrocytes. In a cell-free system, carbonylated proteins from EAE mice with acute and chronic disease seem to be equally sensitive to proteasomal degradation. Altogether, the results support the notion that diminished activity of the 20S proteasome is a major contributor to the accumulation of carbonylated proteins in astrocytes of chronic EAE mice.

Accumulation of protein carbonyls within cerebellar astrocytes in murine experimental autoimmune encephalomyelitis.

Recent work from our laboratory has implicated protein carbonylation in the pathophysiology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The present study was designed to determine the changes in protein carbonylation during disease progression and to identify the target cells and modified proteins in the cerebellum of EAE animals, prepared by active immunization of C57/BL6 mice with MOG(35-55) peptide. In this model, protein carbonylation was maximal at the peak of the disease (acute phase), to decrease thereafter (chronic phase). Double-immunofluorescence microscopy of affected cerebella showed that carbonyls accumulate in white matter astrocytes and to a lesser extent in microglia/macrophages, in both the acute and the chronic phase. Surprisingly, T cells, oligodendrocytes, and neurons were barely stained. By 2D oxyblot and mass spectrometry, ß-actin, ß-tubulin, GFAP, and HSC-71 were identified as the major targets of carbonylation throughout the disease. Using a pull-down/Western blot method, we found a significant increase in the proportion of carbonylated ß-actin, ß-tubulin, and GFAP in the chronic phase but not in the acute phase. These results suggest that as disease progresses from the inflammatory to the neurodegenerative phase there may be an inappropriate removal of oxidized cytoskeletal proteins. Additionally, the extensive accumulation of carbonylated GFAP in the chronic phase of EAE may be responsible for the abnormal shape of astrocytes observed at this stage.

Identification of major S-nitrosylated proteins in murine experimental autoimmune encephalomyelitis.

Nitrosative stress has been implicated in the pathophysiology of several CNS disorders, including multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). We have recently shown that protein nitrosothiols (PrSNOs) accumulate in the brain of MS patients, and there is indirect evidence that PrSNO levels are also increased in EAE. In this study we sought to identify the major PrSNOs in the spinal cord of EAE animals prepared by active immunization of C57/BL6 mice with MOG(35-55) peptide. For this purpose, PrSNOs from control and EAE mice at various disease stages were derivatized with HPDP-biotin, and the biotinylated proteins were isolated with streptavidin-agarose. Proteins from total and streptavidin-bound fractions were then analyzed by Western blotting using antibodies against the major S-nitrosylated substrates of CNS tissue. With this approach we found that the proportion of S-nitrosylated neurofilament proteins, NMDA receptors, alpha/beta-tubulin, beta-actin, and GAPDH is increased in EAE. Other potential substrates either were not S-nitrosylated in vivo (HCN3, HSP-72, CRMP-2, gamma-actin, calbindin) or their S-nitrosylation levels were unaltered in EAE (Na/K ATPase, hexokinase, glycogen phosphorylase). We also discovered that neuronal specific enolase is the major S-nitrosylated protein in acute EAE. Given that S-nitrosylation affects protein function, it is likely that the observed changes are significant to the pathophysiology of inflammatory demyelination.

Increased carbonylation, protein aggregation and apoptosis in the spinal cord of mice with experimental autoimmune encephalomyelitis.

Previous work from our laboratory implicated protein carbonylation in the pathophysiology of both MS (multiple sclerosis) and its animal model EAE (experimental autoimmune encephalomyelitis). Subsequent in vitro studies revealed that the accumulation of protein carbonyls, triggered by glutathione deficiency or proteasome inhibition, leads to protein aggregation and neuronal cell death. These findings prompted us to investigate whether their association can be also established in vivo. In the present study, we characterized protein carbonylation, protein aggregation and apoptosis along the spinal cord during the course of MOG (myelin-oligodendrocyte glycoprotein)(35-55) peptide-induced EAE in C57BL/6 mice. The results show that protein carbonyls accumulate throughout the course of the disease, albeit by different mechanisms: increased oxidative stress in acute EAE and decreased proteasomal activity in chronic EAE. We also show a temporal correlation between protein carbonylation (but not oxidative stress) and apoptosis. Furthermore, carbonyl levels are significantly higher in apoptotic cells than in live cells. A high number of juxta-nuclear and cytoplasmic protein aggregates containing the majority of the oxidized proteins are present during the course of EAE. The LC3 (microtubule-associated protein light chain 3)-II/LC3-I ratio is significantly reduced in both acute and chronic EAE indicating reduced autophagy and explaining why aggresomes accumulate in this disorder. Taken together, the results of the present study suggest a link between protein oxidation and neuronal/glial cell death in vivo, and also demonstrate impaired proteostasis in this widely used murine model of MS.

Protein carbonylation and aggregation precede neuronal apoptosis induced by partial glutathione depletion.

While the build-up of oxidized proteins within cells is believed to be toxic, there is currently no evidence linking protein carbonylation and cell death. In the present study, we show that incubation of nPC12 (neuron-like PC12) cells with 50 µM DEM (diethyl maleate) leads to a partial and transient depletion of glutathione (GSH). Concomitant with GSH disappearance there is increased accumulation of PCOs (protein carbonyls) and cell death (both by necrosis and apoptosis). Immunocytochemical studies also revealed a temporal/spatial relationship between carbonylation and cellular apoptosis. In addition, the extent of all three, PCO accumulation, protein aggregation and cell death, augments if oxidized proteins are not removed by proteasomal degradation. Furthermore, the effectiveness of the carbonyl scavengers hydralazine, histidine hydrazide and methoxylamine at preventing cell death identifies PCOs as the toxic species. Experiments using well-characterized apoptosis inhibitors place protein carbonylation downstream of the mitochondrial transition pore opening and upstream of caspase activation. While the study focused mostly on nPC12 cells, experiments in primary neuronal cultures yielded the same results. The findings are also not restricted to DEM-induced cell death, since a similar relationship between carbonylation and apoptosis was found in staurosporine- and buthionine sulfoximine-treated nPC12 cells. In sum, the above results show for the first time a causal relationship between carbonylation, protein aggregation and apoptosis of neurons undergoing oxidative damage. To the best of our knowledge, this is the first study to place direct (oxidative) protein carbonylation within the apoptotic pathway.