Mathias Lundberg

Cell surface adherence and endocytosis of protein transduction domains.

Protein transduction domains (PTD), such as the HIV TAT and the herpes simplex virus VP22 proteins, are reported to translocate across the membranes of mammalian cells. The mechanism of PTD membrane translocation has largely remained elusive, but recent studies suggest that the reported PTD translocation is due to a fixation artifact. We have constructed and expressed the PTDs VP22, TAT, polyarginine, and polylysine fused to the green fluorescent protein to visualize these proteins in both living and fixed cells. The investigated PTDs strongly adhered to the surface of living cells and were internalized by constitutive endocytosis. No cytosolic or nuclear import of the proteins was detected. In contrast, the PTD-GFP fusion proteins were redistributed to the cytosol and nucleus directly after fixation. Our findings suggest that the PTDs only mediate cell surface adherence, a property shared with many other positively charged macromolecules. The cell surface adherence results in endocytosis and accumulation of proteins in endosomes. We suggest that the biological effects observed for PTD fusion proteins are due to cell surface interactions and internalization of the proteins into cells by classical endocytosis.

Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells

Fluorescence microscopy of A549 cells stained with a glutathione (L‐γ‐glutamyl‐Lcysteinylglycine, GSH)‐specific polyclonal antibody displayed uniform staining of the perinuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol‐reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)‐dependent inhibition of GSH synthesis. However, confocal visualization of antibody‐stained A549 cells in the z‐plane revealed the majority of the perinuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z‐plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z‐section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near‐complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z‐plane. The antibody also provided initial visualization of the compartmentalization of protein‐GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti‐GSH antibody‐stained Jurkat T lymhocytes, we also demonstrated population variations in the cellular compliment of GSH and protein‐GSH mixed disulfides, formed in response to diamide. In addition, we showed cell‐cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

The thiol antioxidant N‐acetyl‐ l‐cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL‐2 and up‐regulation of the IL‐2 receptor. The 120‐kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen‐specific T cell clones (TCC). TCC were stimulated with anti‐CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down‐regulation of IL‐4, IL‐5 and IFN‐γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL‐4. Furthermore, they down‐regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20u2003m m NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down‐regulation of IL‐4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL‐4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases.

Thioredoxin-1 and protein disulfide isomerase catalyze the reduction of similar disulfides in HIV gp120

HIV-1 enters cells via interaction of the viral glycoprotein gp120, the host cell surface receptor CD4 and the co-receptors CCR5 or CXCR4. For entry, gp120 undergoes conformational changes that depend on the reduction of one or more disulfides. Previous studies indicate that protein disulfide isomerase (PDI), thioredoxin-1 (Trx1), and glutaredoxin-1 (Grx1) catalyze gp120 reduction, but their specific disulfide targets are not known. Here, it was demonstrated that PDI and Trx1 have similar gp120 disulfide targets as determined by labeling after reduction, but with some pattern differences, including overall stronger labeling with Trx1 than with PDI. Furthermore, uneven labeling of the residues of a disulfide may reflect altered accessibility by conformational changes upon the reduction process. Since both PDI and Trx1 may be involved in viral entry, compounds that target the host redox system or the viral gp120 were tested in vitro to investigate whether redox regulation is a target for anti-HIV therapy. Carbohydrate binding agents (CBAs), previously shown to bind gp120 and inhibit HIV entry, were now demonstrated to inhibit gp120 disulfide reduction. Auranofin, an inhibitor of thioredoxin reductase 1 (TrxR1), also showed inhibitory activity towards HIV infection, although close to its cytotoxic concentration. Our results demonstrate that both the host redox system and the viral surface glycoproteins are of interest for the development of new generations of anti-HIV therapeutics.

PACAP Protects Adult Neural Stem Cells from the Neurotoxic Effect of Ketamine Associated with Decreased Apoptosis, ER Stress and mTOR Pathway Activation.

Ketamine administration is a well-established approach to mimic experimentally some aspects of schizophrenia. Adult neurogenesis dysregulation is associated with psychiatric disorders, including schizophrenia. The potential role of neurogenesis in the ketamine-induced phenotype is largely unknown. Recent results from human genetic studies have shown the pituitary adenylate cyclase-activating polypeptide (PACAP) gene is a risk factor for schizophrenia. Its potential role on the regulation of neurogenesis in experimental model of schizophrenia remains to be investigated. We aimed to determine whether ketamine affects the viability of adult neural stem cells (NSC). We also investigated whether the detrimental effect mediated by ketamine could be counteracted by PACAP. NSCs were isolated from the subventricular zone of the mouse and exposed to ketamine with/without PACAP. After 24 hours, cell viability, potential involvement of apoptosis, endoplasmic reticulum (ER) stress, mTOR and AMPA pathway activation were assessed by quantitative RT-PCR and Western blot analysis. We show that ketamine impairs NSC viability in correlation with increased apoptosis, ER stress and mTOR activation. The results also suggest that the effect of ketamine occurs via AMPA receptor activation. Finally, we show that PACAP counteracted the decreased NSC viability induced by ketamine via the specific activation of the PAC-1 receptor subtype. Our study shows that the NSC viability may be negatively affected by ketamine with putative importance for the development of a schizophrenia phenotype in the ketamine induced animal model of schizophrenia. The neuroprotective effect via PAC-1 activation suggests a potentially novel pharmacological target for the treatment of schizophrenia, via neurogenesis normalization.

Pituitary Adenlylate Cyclase Activating Peptide Protects Adult Neural Stem Cells from a Hypoglycaemic milieu

Hypoglycaemia is a common side-effect of glucose-lowering therapies for type-2 diabetic patients, which may cause cognitive/neurological impairment. Although the effects of hypoglycaemia in the brain have been extensively studied in neurons, how hypoglycaemia impacts the viability of adult neural stem cells (NSCs) has been poorly investigated. In addition, the cellular and molecular mechanisms of how hypoglycaemia regulates NSCs survival have not been characterized. Recent work others and us have shown that the pituitary adenylate cyclase-activating polypeptide (PACAP) and the glucagon-like peptide-1 receptor (GLP-1R) agonist Exendin-4 stimulate NSCs survival against glucolipoapoptosis. The aim of this study was to establish an in vitro system where to study the effects of hypoglycaemia on NSC survival. Furthermore, we determine the potential role of PACAP and Exendin-4 in counteracting the effect of hypoglycaemia. A hypoglycaemic in vitro milieu was mimicked by exposing subventricular zone-derived NSC to low levels of glucose. Moreover, we studied the potential involvement of apoptosis and endoplasmic reticulum stress by quantifying protein levels of Bcl-2, cleaved caspase-3 and mRNA levels of CHOP. We show that PACAP via PAC-1 receptor and PKA activation counteracts impaired NSC viability induced by hypoglycaemia. The protective effect induced by PACAP correlated with endoplasmic reticulum stress, Exendin-4 was ineffective. The results show that hypoglycaemia decreases NSC viability and that this effect can be substantially counteracted by PACAP via PAC-1 receptor activation. The data supports a potential therapeutic role of PAC-1 receptor agonists for the treatment of neurological complications, based on neurogenesis impairment by hypoglycaemia.

Methodological Aspects of ELISA Analysis of Thioredoxin 1 in Human Plasma and Cerebrospinal Fluid

Thioredoxin-1 (Trx1) is a protein antioxidant involved in major cellular processes. Increased plasma levels of Trx1 have been associated with human diseases suggesting that Trx1 is a marker for oxidative stress with putative clinical use. However, the reported mean levels of Trx1 in the control cohorts vary a hundred-fold between studies (0.8–87 ng/ml), possibly due to methodological differences between the capture ELISA used in the different studies. The aim of this study was to investigate methodological aspects related to the ELISA measurement of Trx1. ELISAs utilizing different capture and detection combinations of antibodies to Trx1 and as well as recombinant human (rh) Trx1 standards from two sources were characterized. The different ELISAs were subsequently used to measure Trx1 in human plasma and cerebrospinal fluid samples (CSF) from healthy donors and from patients with various neurological diagnoses. The Trx1 standards differed in their content of monomeric and oligomeric Trx1, which affected the ELISAs composed of different antibody combinations. Thus, the levels of Trx1 determined in human plasma and CSF samples varied depending on the antibody used in the ELISAs and on the rhTrx1 standard. Furthermore, the relevance of preventing interference by heterophilic antibodies (HA) in human plasma and CSF was investigated. The addition of a HA blocking buffer to human samples drastically reduced the ELISA signals in many samples showing that HA are likely to cause false positive results unless they are blocked. In conclusion, the study shows that the design of a Trx1 ELISA in regards to antibodies and standards used has an impact on the measured Trx1 levels. Importantly, analyses of human plasma and CSF without preventing HA interference may obscure the obtained data. Overall, the results of this study are crucial for the improvement of future studies on the association of Trx1 levels with various diseases.

Is trichloroacetic acid an insufficient sample quencher of redox reactions

The global protein thiol pool has been reported to play a major role in the defense against oxidative stress as a redox buffer similar to glutathione. The present study uses a novel method to visualize cellular changes of the global protein thiol pool in response to induced oxidative stress. Unexpectedly, the results showed an uneven distribution of protein thiols in resting cells with no apparent change in their level or distribution in response to diamide as has been reported previously. Further analysis revealed that thiol pool oxidation is artificially high due to insufficient activity of the widely used sample quencher trichloroacetic acid (TCA). This suggests that previously published articles based on TCA as a quencher should be interpreted with caution as TCA could have caused similar artifacts. Overall, the results presented here question the major role for the global thiol pool in the defense against oxidative stress. Instead our hypothesis is that the fraction of proteins involved in response to oxidative stress is much smaller than previously anticipated in support of a fine-tuned cell signaling by redox regulation.

Exendin-4 Protects Neural Progenitor Cells from Glucolipoapoptosis

Type 2 diabetic and obese patients are under high risk to prematurely develop neurological complications such as stroke and Alzheimer’s disease. Interestingly, type 2-diabetes impairs adult neurogenesis in rodent animal models and this impairment has been suggested to play a role in the brain complications of this disease. Recent work from us and others showed that the treatment with the Glucagon-Like Peptide 1 Receptor (GLP-1R) agonist Exendin-4 stimulates adult neurogenesis in rodents. nBased on these findings we have raised the hypothesis that Exendin-4 may counteract the detrimental effects induced by diabetes in neural stem/progenitor cells. The aim of this study was to investigate whether Exendin-4 protect neural progenitor cells from glucolipotoxicity and to analyse if the regulation of apoptosis may be involved in the Exendin-4 protecting effect. Murine neural progenitor cells were exposed to high palmitate and glucose, which characterize diabetic glucolipotoxicity, in presence/absence of Exendin-4. To determine whether neural progenitor cells proliferation was impacted by the Exendin-4 treatment, [3H] thymidine incorporation experiments were also performed. The expression of apoptosis key players, such as cleaved-caspase 3 and Bcl-2, were evaluated by western blotting. We show that Exendin-4 counteracts the impaired neural progenitor cell viability induced by glucolipotoxicity. nCell proliferation was not influenced by the Exendin-4 treatment. The protective effect induced by Exendin-4 correlated with decreased apoptosis. In addition, the Exendin-4 protective effect was completely abolished by using the GLP- 1R antagonist Ex-9-39, indicating that the protective effect by Exendin-4 was GLP-1R-mediated. In conclusion, we show a direct survival effect of GLP-1R activation on neural progenitor cells challenged by diabetic-like conditions. The results support a potential therapeutic role of GLP-1R agonists, based on neurogenesis stimulation, for the treatment of the neurological complications in Type 2-diabetes and obesity.

Healthy Adolescent Performance With Standardized Scoring Tables for the MATRICS Consensus Cognitive Battery: A Multisite Study

Abstract Objective The aim of this study was to develop standardized scores and scoring tables for test performance in healthy adolescents for the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) Consensus Cognitive Battery (MCCB) for each year from 11 to 19 years of age, by sex, with T scores and percentile ranks. Methods A total of 502 healthy participants (aged 11–19 years) from 7 cohorts from Ireland, Norway, Sweden, and United States, were included in this multisite study. Regression-predicted means for the MCCB tests, except the social cognition subtest, were calculated using the MCCB test scores as outcome variables and age, age2, sex, age × sex as predictors. The regression-predicted means for each combination of age and sex were added with the residuals from the entire cohort to yield the expected distribution of that group. Age effects were examined using regression models with age and age2 as predictors. Sex differences were examined using Student’s t-tests. Results Significant positive age effects were found for all tests, except for the Brief Visuospatial Memory Test, revised (BVMT-R; measure of visual learning). Females performed significantly better than males on BACS Symbol coding (measure of speed of processing) and BVMT-R, while males performed significantly better than females on NAB Mazes (measure of reasoning and problem solving). Based on the regression-predicted distributions of scores, 19 standardized scoring tables for each test and domain were created. Conclusions With the results from this study, we have developed an accessible standardized data set of healthy adolescent test performance for the MCCB.