Neuroadaptations in Human Chronic Alcoholics: Dysregulation of the NF-κB System

Anna Ökvist is with Karolinska Institute, Sofia Johansson is with Karolinska Institute, Alexander Kuzmin is with Karolinska Institute, Igor Bazov is with Karolinska Institute, Roxana Merino-Martinez is with Karolinska Institute, Igor Ponomarev is with UT Austin, R. Dayne Mayfield is with UT Austin, R. Adron Harris is with UT Austin, Donna Sheedy is with University of Sydney, Therese Garrick is with University of Sydney, Clive Harper is with University of Sydney, Yasmin L. Hurd is with Mount Sinai School of Medicine, Lars Terenius is with Karolinska Institute, Tomas J. Ekström is with Karolinska Institute, Georgy Bakalkin is with Karolinska Institute and Uppsala University, Tatjana Yakovleva is with Karolinska Institute and Uppsala University.Background -- Alcohol dependence and associated cognitive impairments apparently result from neuroadaptations to chronic alcohol consumption involving changes in expression of multiple genes. Here we investigated whether transcription factors of Nuclear Factor-kappaB (NF-κB) family, controlling neuronal plasticity and neurodegeneration, are involved in these adaptations in human chronic alcoholics. Methods and Findings -- Analysis of DNA-binding of NF-κB (p65/p50 heterodimer) and the p50 homodimer as well as NF-κB proteins and mRNAs was performed in postmortem human brain samples from 15 chronic alcoholics and 15 control subjects. The prefrontal cortex involved in alcohol dependence and cognition was analyzed and the motor cortex was studied for comparison. The p50 homodimer was identified as dominant κB binding factor in analyzed tissues. NF-κB and p50 homodimer DNA-binding was downregulated, levels of p65 (RELA) mRNA were attenuated, and the stoichiometry of p65/p50 proteins and respective mRNAs was altered in the prefrontal cortex of alcoholics. Comparison of a number of p50 homodimer/NF-κB target DNA sites, κB elements in 479 genes, down- or upregulated in alcoholics demonstrated that genes with κB elements were generally upregulated in alcoholics. No significant differences between alcoholics and controls were observed in the motor cortex. Conclusions -- We suggest that cycles of alcohol intoxication/withdrawal, which may initially activate NF-κB, when repeated over years downregulate RELA expression and NF-κB and p50 homodimer DNA-binding. Downregulation of the dominant p50 homodimer, a potent inhibitor of gene transcription apparently resulted in derepression of κB regulated genes. Alterations in expression of p50 homodimer/NF-κB regulated genes may contribute to neuroplastic adaptation underlying alcoholism.This work was supported by grants from the AFA Forsäkring to AK, YLH, TJE and GB, the Research Foundation of the Swedish Alcohol Retail Monopoly (SRA) and Karolinska Institutet to AK, TJE and GB, and the Swedish Science Research Council and the Swedish National Drug Policy Coordinator to GB. The Australian Brain Donor Programs NSW Tissue Resource Centre was supported by The University of Sydney, National Health and Medical Research Council of Australia, Neuroscience Institute of Schizophrenia and Allied Disorders, National Institute of Alcohol Abuse and Alcoholism and NSW Department of Health.Waggoner Center for Alcohol and Addiction Researc

Synaptic plasticity in drug abuse disorders : Studies of the human post-mortem brain

Drug addiction is a chronic disorder characterized by craving and compulsive drug use despite adverse consequences and high rates of relapse during periods of abstinence. Therapeutic interventions for most addiction disorders are limited today, partly because the underlying neurobiology is still unknown. A growing body of evidence indicates that synaptic plasticity contributes to the development and persistence of addiction, however, most research have focused on rodent animal models and very limited knowledge exists about the effects of drugs of abuse on the glutamatergic system in the human brain. The aim of this thesis was therefore to gain deeper insight into the neurobiology of drugs of abuse, including alcohol, heroin and cocaine directly in the human brain relevant to synaptic plasticity in key neuronal circuits relevant for the development and persistence of addiction. In the first study we examined the gene expression profile of sixteen endogenous control genes in the prefrontal and motor cortex of alcoholics. The results demonstrated differences in gene expression stability between the prefrontal and motor cortex as well as region-specific-alterations in several genes normally used as reference genes between alcoholic and controls. These observations implicate the importance of selecting proper genes for normalization when performing gene expression studies. Next we investigated whether the NF-kappaB system was altered in the prefrontal and motor cortex of alcoholics. The results revealed a reduced DNA-binding activity of the NF-kappaB and p50 homodimer in the prefrontal cortex of alcoholics that was coupled to a reduction in RELA mRNA levels. NF-kappaB has been implicated in synaptic plasticity and memory consolidation, thus it is tempting to speculate that decreased NF-kappaB function could lead to a disruption of learning and memory formation, or effect alcohol-induced associative memory reconsolidation often linked to relapse. Third, we examined the effect of alcohol consumption on modulators of synaptic strength (synaptophysin) and executors of glutamate release in the prefrontal and motor cortex. We observed increased synaptophysin I levels in the prefrontal cortex of alcoholics compared to controls, while levels of predominant members of the synaptic vesicular machinery important for glutamate release were unaltered. These results suggest a role for synaptophysin in the alcohol dependence associated enduring neuroplasticity in the prefrontal cortical glutamate circuitry. Finally, we evaluated glutamatergic receptors and their associated scaffolding proteins in the amygdala and striatum of heroin, cocaine and polysubstance (heroin/cocaine) abusers. The findings revealed region-specific disturbances in glutamatergic systems tightly coupled to PSD-95 and Homer in human drug abusers indicting an abberant regulation of glutamatergic signaling and function. In conclusion, we have demonstrated disturbances in several key mechanisms underlying synaptic plasticity/function in the human brain of drug abusers that are in line with research findings from animal models. Altogether these findings emphasize pathology of neuroplasticity as a common feature in addiction disorders

Correlation Between Pellet Reduction and Some Blast Furnace Operation Parameters

Godkänd; 2006; 20070416 (ysko

Correlation Between Pellet Reduction and Some Blast Furnace Operation Parameters

Godkänd; 2006; 20070416 (ysko

Blast furnace pellet properties under different reduction conditions

Tests in the LKAB Experimental Blast Furnace (EBF) were carried out under different pre-set process conditions. The choice of injection coal, an HV coal and an LV coal, affected the in-furnace conditions, which was assumed to contribute to the differences in reduction degree in pellets taken out with the upper shaft probe. A higher pellet reduction degree was attained during operation with the HV coal compared to injection of the LV coal. The differences in pellet reduction degree receded through the shaft and no correlation between pellet reduction degree and pre-set process conditions was observed in samples taken out with the lower shaft probe. For the HV coal, a higher pellet strength and an increase in Fe and C losses with the top gas were observed compared to operation with the LV coal. Blast-furnace-simulating laboratory reduction for simulated PCR, based on measurements in the EBF, was carried out. The increase in reduction potential and temperature level resulting from an increase in simulated PCR compensated for the decrease in reduction time between the simulated low and high PCR. Laboratory reduction under isothermal conditions showed an increased reduction rate at increased temperature as well as with an increased H2 content in the reduction gas.Godkänd; 2008; 20080821 (ulle

Blast furnace pellet textures during reduction and correlation to strength

The blast furnace is the most common means of producing hot metal. As the amounts of reduction agents increases, which influence in-furnace conditions such as ascending gas properties, temperature profiles and the ore-to-coke ratio, new demands are put on the iron-bearing material in terms of both reducibility and mechanical strength. To investigate the possibilities to use the Pellet Multi Press (PMP) equipment for compression strength measurements of reduced pellets and to gain a deeper understanding of the correlation between pellet texture and strength, an initial study of pellets taken from the LKAB Experimental Blast Furnace (EBF) was conducted. Furthermore, the pellet pieces generated after compression tests were characterized using light optical microscopy. In order to correlate the texture of pellet pieces to the pellet texture prior to breakage, a characterization of the chronological pellet texture development during reduction in the EBF was performed. The original pellet texture remained in the beginning of reduction and differences receded through the EBF shaft as wustite and Feme, was formed. Occurrence of Feme, in the pellet texture increased the compression strength, while less reduced and less sintered textures showed the reverse effect. So far, the results from compression strength tests indicate that disintegration of pellets takes place at a reaction front, at the transition between different texture types of iron oxide or at the location of a visible surface crack.Validerad; 2010; 20101112 (ysko)</p

Effects of a novel disulfide bond and engineered electrostatic interactions on the thermostability of azurin.

Identification and evaluation of factors important for thermostability in proteins is a growing research field with many industrial applications. This study investigates the effects of introducing a novel disulfide bond and engineered electrostatic interactions with respect to the thermostability of holo azurin from Pseudomonas aeruginosa. Four mutants were selected on the basis of rational design and novel temperature-dependent atomic displacement factors from crystal data collected at elevated temperatures. The atomic displacement parameters describe the molecular movement at higher temperatures. The thermostability was evaluated by optical spectroscopy as well as by differential scanning calorimetry. Although azurin has a high inherent stability, the introduction of a novel disulfide bond connecting a flexible loop with small alpha-helix (D62C/K74C copper-containing mutant), increased the T(m) by 3.7 degrees C compared with the holo protein. Furthermore, three mutants were designed to introduce electrostatic interactions, K24R, D23E/K128R, and D23E/K128R/K24R. Mutant K24R stabilizes loops between two separate beta-strands and D23E/K128R was selected to stabilize the C-terminus of azurin. Furthermore, D23E/K128R/K24R was selected to reflect the combination of the electrostatic interactions in D23E/K128R and K24R. The mutants involving electrostatic interactions had a minor effect on the thermostability. The crystal structures of the copper-containing mutants D62C/K74C and K24R have been determined to 1.5 and 1.8 A resolution. In addition the crystal structure of the zinc-loaded mutant D62C/K74C has also been completed to 1.8 A resolution. These structures support the selected design and provide valuable information for evaluating effects of the modifications on the thermostability of holo azurin

Dysregulation of cell death machinery in the prefrontal cortex of human alcoholics

In human alcoholics, the cell density is decreased in the prefrontal cortex (PFC) and other brain areas. This may be due to persistent activation of cell death pathways. To address this hypothesis, we examined the status of cell death machinery in the dorsolateral PFC in alcoholics. Protein and mRNA expression levels of several key pro- and anti-apoptotic genes were compared in post-mortem samples of 14 male human alcoholics and 14 male controls. The findings do not support the hypothesis. On the contrary, they show that several components of intrinsic apoptotic pathway are decreased in alcoholics. No differences were evident in the motor cortex, which is less damaged in alcoholics and was analysed for comparison. Thus, cell death mechanisms may be dysregulated by inhibition of intrinsic apoptotic pathway in the PFC in human alcoholics. This inhibition may reflect molecular adaptations that counteract alcohol neurotoxicity in cells that survive after many years of alcohol exposure and withdrawal