Long-term administration of morphine specifically alters the level of protein expression in different brain regions and affects the redox state

Abstract We investigated the changes in redox state and protein expression in selected parts of the rat brain induced by a 4 week administration of morphine (10 mg/kg/day). We found a significant reduction in lipid peroxidation that mostly persisted for 1 week after morphine withdrawal. Morphine treatment led to a significant increase in complex II in the cerebral cortex (Crt), which was accompanied by increased protein carbonylation, in contrast to the other brain regions studied. Glutathione levels were altered differently in the different brain regions after morphine treatment. Using label-free quantitative proteomic analysis, we found some specific changes in protein expression profiles in the Crt, hippocampus, striatum, and cerebellum on the day after morphine withdrawal and 1 week later. A common feature was the upregulation of anti-apoptotic proteins and dysregulation of the extracellular matrix. Our results indicate that the tested protocol of morphine administration has no significant toxic effect on the rat brain. On the contrary, it led to a decrease in lipid peroxidation and activation of anti-apoptotic proteins. Furthermore, our data suggest that long-term treatment with morphine acts specifically on different brain regions and that a 1 week drug withdrawal is not sufficient to normalize cellular redox state and protein levels.

IV  Lucie Hejnova et al.
Table S1:   Protein profiling in rat brain after morphine treatment  XI  Protein profiling in rat brain after morphine treatment  XV   Protein profiling in rat brain after morphine treatment  XIX

Continued
May play a role in cell-cell or cell-ligand interactions during synaptogenesis and other events in the Component of the cytochrome c oxidase. the last enzyme in the mitochondrial electron transport chain which drives oxidative Involved in vitamin D transport and storage P26644 Actin-binding protein involved in the contractile system of striated and smooth muscles and the cytoskeleton of non-Involved in cell adhesion.cell motility.opsonization.wound healing.and maintenance of protein involved in the contractile system of striated and smooth muscles and the cytoskeleton of non-The major constituent of the PSD, member of the NMDAR signaling complex in excitatory Integral membrane protein associated with caveolae ND, not detected; a, the difference in protein expression levels was more than 2× between the M28 and M28/W groups;-, unchanged protein expression between samples.Protein profiling in rat brain after morphine treatment  V TableS2: Qualitative changes in protein expression /W Component of the cytochrome c oxidase. the last enzyme in the mitochondrial electron transport chain which drives oxidative phosphorylation P10688 Phospholipase C-delta-1 Plcd1 M28, M28/W The production of the second messenger molecules diacylglycerol and inositol 1,4,5/W Mitochondrial serine transporter that mediates transport of serine into mitochondria , high-affinity amino acid transporter that mediates the uptake of L-glutamate and also L-aspartate and D-aspartate A0A0G2K1R5 CaM kinase-like vesicle-associated protein Camkv CON Pseudokinase of the CaMK.synaptic protein crucial for dendritic spine maintenance /W Hydrolysis of proteins to small peptides in the presence of ATP and magnesium Q5UAJ6 Cytochrome c oxidase subunit 2 COX2 M28, M28/W Component of the cytochrome c oxidase, the last enzyme in the mitochondrial electron transport chain which drives oxidative phosphorylation P10688 Phospholipase C-delta-1 Plcd1 M28, M28/W The production of the second messenger molecules diacylglycerol and inositol 1/W Mitochondrial serine transporter that mediates transport of serine into mitochondria A0A0G2K1R5 CaM kinase-like vesicle-associated protein Camkv CON Pseudokinase of the CaMK.synaptic protein crucial for dendritic spine /W Component of the cytochrome c oxidase, the last enzyme in the mitochondrial electron transport chain which /W Plays an important role in the organization of the cytoskeleton A0A0G2K1R5 CaM kinase-like vesicle-associated protein Camkv CON Pseudokinase of the CaMK.synaptic protein crucial for dendritic spine maintenance P63219 Guanine nucleotide-binding protein G(I)/G(S)/G(/W Component of the cytochrome c oxidase, the last enzyme in the mitochondrial electron transport chain which /W Myosin regulatory subunit that plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity via its phosphorylation.Implicated in cytokinesis.receptor capping/W Survival-promoting peptide promotes survival of neurons and displays phosphatase activity.It may bind IgG VIII  Lucie Hejnova et al.

Figure S1 :
Figure S1: Typical Ponceau S staining and Western blots for detection of complex V. Proteins (10 μg protein per lane) in samples from cortex, hippocampus (Hippo), striatum (Striat) and cerebellum (Cereb) were separated using SDS-PAGE (10% gel, at constant 200 V for 45 min.)and transferred to a nitrocellulose membrane (at constant voltage 100 V for 90 min.).The total amount of proteins bound to the membranes was stained with Ponceau S (a).Complex V of the respiratory chain was detected with Total OXPHOS Rodent WB antibody diluted 1:200,000 (a) or 1:50,000 (c), and (d).

Figure S2 :
Figure S2: Typical Ponceau S staining and Western blots for detection of complex IV.Proteins (10 μg protein per lane) in samples from cortex, hippocampus (Hippo), striatum (Striat) and cerebellum (Cereb) were separated using SDS-PAGE (10 % gel, at constant 200 V for 45 min.)and transferred to a nitrocellulose membrane (at constant voltage 100 V for 90 min.).The total amount of proteins bound to the membranes was stained with Ponceau S (a)-(c).Complex IV of the respiratory chain was detected with Total OXPHOS Rodent WB antibody diluted 1:50,000 (d)-(f).

Figure S3 :
Figure S3: Typical Ponceau S staining and Western blots for detection of complex III.Proteins (10 μg protein per lane) in samples from cortex, hippocampus (Hippo), striatum (Striat) and cerebellum (Cereb) were separated using SDS-PAGE (10% gel, at constant 200 V for 45 min.)and transferred to a nitrocellulose membrane (at constant voltage 100 V for 90 min.).The total amount of proteins bound to the membranes was stained with Ponceau S (a)-(c).Complex III of the respiratory chain was detected with Total OXPHOS Rodent WB antibody diluted 1:50,000 (d)-(f).

Figure S5 :
Figure S5: Typical Ponceau S staining and Western blots for detection of complex I. Proteins (10 μg protein per lane) in samples from cortex, hippocampus (Hippo), striatum (Striat) and cerebellum (Cereb) were separated using SDS-PAGE (10% gel, at constant 200 V for 45 min.)and transferred to a nitrocellulose membrane (at constant voltage 100 V for 90 min.).The total amount of proteins bound to the membranes was stained with Ponceau S (a) and (b).Complex I of the respiratory chain was detected with Total OXPHOS Rodent WB antibody diluted 1:10,000 (c)-(e).

Table S3 :
David analysis -Biological processes

Table S4 :
David analysis -Cellular components