In vivo white matter microstructure in adolescents with early-onset psychosis: a multi-site mega-analysis.

Emerging evidence suggests brain white matter alterations in adolescents with early-onset psychosis (EOP; age of onset <18 years). However, as neuroimaging methods vary and sample sizes are modest, results remain inconclusive. Using harmonized data processing protocols and a mega-analytic approach, we compared white matter microstructure in EOP and healthy controls using diffusion tensor imaging (DTI). Our sample included 321 adolescents with EOP (median age = 16.6 years, interquartile range (IQR) = 2.14, 46.4% females) and 265 adolescent healthy controls (median age = 16.2 years, IQR = 2.43, 57.7% females) pooled from nine sites. All sites extracted mean fractional anisotropy (FA), mean diffusivity (MD), radial diffusivity (RD), and axial diffusivity (AD) for 25 white matter regions of interest per participant. ComBat harmonization was performed for all DTI measures to adjust for scanner differences. Multiple linear regression models were fitted to investigate case-control differences and associations with clinical variables in regional DTI measures. We found widespread lower FA in EOP compared to healthy controls, with the largest effect sizes in the superior longitudinal fasciculus (Cohen's d = 0.37), posterior corona radiata (d = 0.32), and superior fronto-occipital fasciculus (d = 0.31). We also found widespread higher RD and more localized higher MD and AD. We detected significant effects of diagnostic subgroup, sex, and duration of illness, but not medication status. Using the largest EOP DTI sample to date, our findings suggest a profile of widespread white matter microstructure alterations in adolescents with EOP, most prominently in male individuals with early-onset schizophrenia and individuals with a shorter duration of illness.

Intracranial and subcortical volumes in adolescents with early-onset psychosis: A multisite mega-analysis from the ENIGMA consortium.

Early-onset psychosis disorders are serious mental disorders arising before the age of 18 years. Here, we investigate the largest neuroimaging dataset, to date, of patients with early-onset psychosis and healthy controls for differences in intracranial and subcortical brain volumes. The sample included 263 patients with early-onset psychosis (mean age: 16.4 ± 1.4 years, mean illness duration: 1.5 ± 1.4 years, 39.2% female) and 359 healthy controls (mean age: 15.9 ± 1.7 years, 45.4% female) with magnetic resonance imaging data, pooled from 11 clinical cohorts. Patients were diagnosed with early-onset schizophrenia (n = 183), affective psychosis (n = 39), or other psychotic disorders (n = 41). We used linear mixed-effects models to investigate differences in intracranial and subcortical volumes across the patient sample, diagnostic subgroup and antipsychotic medication, relative to controls. We observed significantly lower intracranial (Cohen's d = -0.39) and hippocampal (d = -0.25) volumes, and higher caudate (d = 0.25) and pallidum (d = 0.24) volumes in patients relative to controls. Intracranial volume was lower in both early-onset schizophrenia (d = -0.34) and affective psychosis (d = -0.42), and early-onset schizophrenia showed lower hippocampal (d = -0.24) and higher pallidum (d = 0.29) volumes. Patients who were currently treated with antipsychotic medication (n = 193) had significantly lower intracranial volume (d = -0.42). The findings demonstrate a similar pattern of brain alterations in early-onset psychosis as previously reported in adult psychosis, but with notably low intracranial volume. The low intracranial volume suggests disrupted neurodevelopment in adolescent early-onset psychosis.

Challenges and opportunities in the diagnosis and treatment of early-onset psychosis: a case series from the youth affective disorders clinic in Stockholm, Sweden.

Early-onset psychosis is linked to adverse long-term outcomes, recurrent disease course, and prolonged periods of untreated illness; thus highlighting the urgency of improving early identification and intervention. This paper discusses three cases where initial emphasis on psychosocial treatments led to diagnostic and therapeutic delays: (1) a 15-year-old misdiagnosed with emotionally unstable personality disorder and autism, who improved on bipolar medication and antipsychotics; (2) another 15-year-old misdiagnosed with autism, who stabilized on lithium and antipsychotics, subsequently allowing for gender dysphoria evaluation; (3) a 9-year-old autistic boy incorrectly treated for ADHD, who recovered with appropriate antipsychotic treatment. These cases illuminate the vital importance of adhering to a diagnostic hierarchy, prioritizing diagnostic utility, and conducting longitudinal evaluations to facilitate early targeted treatment of psychotic symptoms in early-onset psychosis. Adherence to such strategies can minimize delays in managing early-onset psychosis and improve long-term prognoses.

Caudate nucleus volume in medicated and unmedicated patients with early- and adult-onset schizophrenia.

The caudate nucleus is a part of the striatum, and striatal hyperdopaminergia is considered central to the pathophysiology of schizophrenia. How caudate volume is affected in schizophrenia and what role antipsychotics play remains unclear. In early-onset schizophrenia (EOS), where psychosis emerges during a neurodevelopmentally critical phase, the caudate may exhibit a heightened vulnerability to the effects of antipsychotic medications. We hypothesized effects of both antipsychotic medication use and age of onset on caudate in schizophrenia. We included adult patients with EOS (n = 83) and adult-onset schizophrenia (AOS) (n = 246), adult healthy controls (HC, n = 774), adolescent patients with non-affective psychosis (n = 56) and adolescent HC (n = 97). We obtained T1-weighted MRI scans using a 1.5T Siemens scanner and General Electric 3T scanners. In our main analysis, we tested for main and interaction effects of diagnosis and current antipsychotic medication use on caudate volume. Adult patients with EOS (p < 0.001) and AOS (p = 0.002) had both larger caudate than HC. Age of onset (EOS/AOS) interacted with antipsychotic use (p = 0.004) which was associated with larger caudate in EOS (p < 0.001) but not in AOS (p = 0.654). Conversely, among medicated patients only, EOS had larger caudate than AOS (p < 0.001). No other subcortical structures showed differences between medicated EOS and AOS. Medicated adolescent patients with non-affective psychosis and medicated adult patients with EOS showed similar caudate volumes. The results may indicate a schizophrenia-related and a medication-induced caudate increase, the latter restricted to patients with EOS and possibly occurring already in adolescence shortly after disease onset.

Development of an ELISA displaying similar reactivity with reduced and oxidized human Thioredoxin-1 (Trx1): The plasma level of Trx1 in early onset psychosis disorders.

The plasma level of human thioredoxin-1 (Trx1) has been shown to be increased in various somatic diseases and psychiatric disorders. However, when comparing the reported plasma levels of Trx1, a great inter-study variability, as well as variability in study outcomes of differences between patients and control subjects has been observed, ultimately limiting the possibility to make comparative analyses. Trx1 is a highly redox active protein prone to form various redox forms, e.g. dimers, oligomers or Trx1-protein complexes. We have recently shown that ELISA systems may vary in reactivity to various Trx1 redox forms. The primary aim of the present study was to develop an ELISA system with similar reactivity to various Trx1 redox forms. By evaluating a panel of novel monoclonal antibodies (mAbs), in various paired combinations, three ELISA systems were generated, with observed large variability in reactivity to various Trx1 redox forms. Importantly, an ELISA system (capture mAb MT17R6 and detection mAb MT13X3-biotin), was identified that displayed similar reactivity to oxidized and DTT reduced Trx1. The ELISA system (MT17R6/MT13X3-biotin), was subsequently used to analyze the level of Trx1 in plasma from patients (<18 years) with early onset psychosis disorders (EOP). However, no significant (p > 0.7) difference in plasma Trx1 levels between patients with EOP (n = 23) and healthy age matched controls (HC) (n = 20) were observed. Furthermore, reliable measurement was shown to be dependent on the establishment of platelet poor plasma samples, enabled by rigorous blood sample centrifugation and by efficient blocking of potentially interfering heterophilic antibodies. In conclusion, we report the design and characterization of a Trx1 ELISA system with similar reactivity to various Trx1 redox forms. Importantly, data indicated that generated ELISA systems show large variability in reactivity to various redox forms with ultimate impact on measured levels of Trx1. Overall, results from this study suggests that future studies may be strongly improved by the use of Trx1 ELISA systems with characterized specificity to various redox forms.

Aberrant default mode connectivity in adolescents with early-onset psychosis: A resting state fMRI study.

Abnormal default mode network (DMN) connectivity has been found in schizophrenia and other psychotic disorders. However, there are limited studies on early onset psychosis (EOP), and their results show lack of agreement. Here, we investigated within-network DMN connectivity in EOP compared to healthy controls (HC), and its relationship to clinical characteristics. A sample of 68 adolescent patients with EOP (mean age 16.53 ± 1.12 [SD] years, females 66%) and 95 HC (mean age 16.24 ± 1.50 [SD], females 60%) from two Scandinavian cohorts underwent resting state functional magnetic resonance imaging (rsfMRI). A group independent component analysis (ICA) was performed to identify the DMN across all participants. Dual regression was used to estimate spatial maps reflecting each participant's DMN network, which were compared between EOP and HC using voxel-wise general linear models and permutation-based analyses. Subgroup analyses were performed within the patient group, to explore associations between diagnostic subcategories and current use of psychotropic medication in relation to connectivity strength. The analysis revealed significantly reduced DMN connectivity in EOP compared to HC in the posterior cingulate cortex, precuneus, fusiform cortex, putamen, pallidum, amygdala, and insula. The subgroup analysis in the EOP group showed strongest deviations for affective psychosis, followed by other psychotic disorders and schizophrenia. There was no association between DMN connectivity strength and the current use of psychotropic medication. In conclusion, the findings demonstrate weaker DMN connectivity in adolescent patients with EOP compared to healthy peers, and differential effects across diagnostic subcategories, which may inform our understanding of underlying disease mechanisms in EOP.

Preclinical atherosclerosis in adolescents with psychotic or bipolar disorders investigated with carotid high-frequency ultrasound.

OBJECTIVE: Early-onset psychosis (EOP) and bipolar disorder (EOBP) (at <18 years of age), are associated with an increased future risk of cardiovascular disease (CVD) and premature death. Yet it is unknown whether the arteries show visible signs of atherosclerosis in EOP and EOBP. This study investigated whether having EOP or EOBP was associated with detectable signs of preclinical atherosclerosis. METHOD: By using 22 MHz high-frequency ultrasound, different layers of the arterial wall of the left common carotid artery (LCCA) were assessed in 77 individuals with EOP (n = 25), EOBP (n = 22), and in age-matched healthy controls (n = 30). Conventional CVD confounders were included in the analyses. RESULTS: Adolescents with EOP and EOBP, compared to controls, had a significantly thicker LCCA intima thickness (0.132 vs. 0.095 mm, p < .001) and intima/media ratio (0.24 vs. 0.17 p < .001). There was a nonsignificant intima difference between EOP and EOBP. Conventional CVD risk factors did not explain the association between EOP/EOBP and intima thickness. In the group of EOP/EOBP, there was a significant correlation between the dose of current antipsychotic medication and intima thickness; however, the correlation was attenuated to a nonsignificant level when adjusted for global function. CONCLUSIONS: Adolescents with EOP or EOBP had an increased LCCA intima thickness, interpreted as a sign of preclinical atherosclerosis. Global function of the disorders was the strongest determinant of intima thickness. The findings, if replicated, might have implications for long-term treatment of EOP and EOBP in order to reduce a future risk of CVD.

Clozapine protects adult neural stem cells from ketamine-induced cell death in correlation with decreased apoptosis and autophagy.

Adult neurogenesis, the production of newborn neurons from neural stem cells (NSCs) has been suggested to be decreased in patients with schizophrenia. A similar finding was observed in an animal model of schizophrenia, as indicated by decreased bromodeoxyuridine (BrdU) labelling cells in response to a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist. The antipsychotic drug clozapine was shown to counteract the observed decrease in BrdU-labelled cells in hippocampal dentate gyrus (DG). However, phenotypic determination by immunohistochemistry analysis could not reveal whether BrdU-positive cells were indeed NSCs. Using a previously established cell model for analysing NSC protection in vitro, we investigated a protective effect of clozapine on NSCs. Primary NSCs were isolated from the mouse subventricular zone (SVZ), we show that clozapine had a NSC protective activity alone, as evident by employing an ATP cell viability assay. In contrast, haloperidol did not show any NSC protective properties. Subsequently, cells were exposed to the non-competitive NMDA-receptor antagonist ketamine. Clozapine, but not haloperidol, had a NSC protective/anti-apoptotic activity against ketamine-induced cytotoxicity. The observed NSC protective activity of clozapine was associated with increased expression of the anti-apoptotic marker Bcl-2, decreased expression of the pro-apoptotic cleaved form of caspase-3 and associated with decreased expression of the autophagosome marker 1A/1B-light chain 3 (LC3-II). Collectively, our findings suggest that clozapine may have a protective/anti-apoptotic effect on NSCs, supporting previous in vivo observations, indicating a neurogenesis-promoting activity for clozapine. If the data are further confirmed in vivo, the results may encourage an expanded use of clozapine to restore impaired neurogenesis in schizophrenia.

Autoantibodies to the N-Methyl-D-Aspartate Receptor in Adolescents With Early Onset Psychosis and Healthy Controls.

BACKGROUND: Autoantibodies to the N-methyl-D-aspartate receptor (NMDAR-Abs) in autoimmune encephalitis have been associated with prominent psychiatric symptoms. The aims of the present study are to identify the prevalence of NMDAR-Abs in adolescents with early onset psychosis disorders (EOP) and healthy controls (HC) and examine its clinical significance. METHOD: Plasma samples were acquired from 46 adolescent EOP patients and 69 age- and sex matched HC, and assessed for the presence of immunoglobulin G NMDAR-Abs. All participants underwent psychiatric evaluation, neurological examination and head magnetic resonance imaging. RESULTS: NMDAR-Abs were detected in three of 46 (6.5%) EOP patients and in two of 69 (2.9%) HC. One NMDAR-Abs EOP patient presented with unusual psychopathology and minor T1 weighted lesions of vasculopathological origin located bi-frontally and in the basal ganglia, and had a recent diagnosis of a separate autoimmune disease. One NMDAR-Ab HC displayed a T2 weighted FLAIR hyperintensity lesion in the left frontal lobe. The remaining three NMDAR-Ab participants were two EOP patients without neurological or radiological findings, and one HC without any clinical findings. CONCLUSIONS: We report that a small number of EOP patients and HC have NMDAR-Abs with a similar frequency in both groups. The presence of the antibodies was not associated with any distinctive clinical or radiological features. Detection of the antibodies had no diagnostic implication, and a positive NMDAR antibody test must be carefully interpreted and reviewed within the individual clinical context.

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides.

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Ralpha receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.

Inhibition of the thioredoxin system by PX-12 (1-methylpropyl 2-imidazolyl disulfide) impedes HIV-1 infection in TZM-bl cells.

Human immunodeficiency virus (HIV-1) entry is initiated by the binding between the viral envelope glycoprotein gp120 and the host receptor CD4, and followed by reduction of structural disulfides of gp120 and CD4. The host thioredoxin-1 (Trx1) efficiently reduces disulfides of gp120 and CD4 in vitro, and recently CD4-dependent HIV-1 entry was shown to be inhibited by anti-Trx1-antibodies, indicating a central role for Trx1. 1-methylpropyl-2-imidazolyl disulfide (PX-12) is a reversible inhibitor of the Trx1 system that may also cause a slow irreversible thioalkylation of Trx1. It was developed as an antitumor agent, however, the current study aimed to determine if it also has an anti-HIV-1 effect. We show that PX-12 has anti-HIV-1(IIIB) activity in TZM-bl cells, in fact, no virus was detected inside the cells in the presence of 10 µM PX-12. Moreover, PX-12 inhibited the enzymatic activity of Trx1 and the Trx1-dependent disulfide reduction of gp120. Microtubule polymerization and formation of acetylated microtubules were also inhibited, activities shown to be required for HIV-1 life cycle propagation. In conclusion, our data strengthens the notion that the early steps of the HIV-1 life cycle depends on the Trx1 system and indicate that the Trx1 system may be a rational drug target for HIV-1 treatment.

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

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.

Somatic symptoms in adolescence as a predictor of severe mental illness in adulthood: a long-term community-based follow-up study.

BACKGROUND: Somatic symptoms are common and costly for society and correlate with suffering and low functioning. Nevertheless, little is known about the long-term implications of somatic symptoms. The objective of this study was to assess if somatic symptoms in adolescents with depression and in their matched controls predict severe mental illness in adulthood by investigating the use of hospital-based care consequent to different mental disorders. METHODS: The entire school population of 16-17-year-olds in the city of Uppsala, Sweden, was screened for depression in 1991-1993 (n = 2300). Adolescents with positive screenings (n = 307) and matched non-depressed controls (n = 302) participated in a semi-structured diagnostic interview for mental disorders. In addition, 21 different self-rated somatic symptoms were assessed. The adolescents with depression and the matched non-depressed controls were engaged in follow-up through the National Patient Register 17-19 years after the baseline study (n = 375). The outcome measures covered hospital-based mental health care for different mental disorders according to ICD-10 criteria between the participants' ages of 18 and 35 years. RESULTS: Somatic symptoms were associated with an increased risk of later hospital-based mental health care in general in a dose-response relationship when adjusting for sex, adolescent depression, and adolescent anxiety (1 symptom: OR = 1.63, CI 0.55-4.85; 2-4 symptoms: OR = 2.77, 95% CI 1.04-7.39; ≥ 5 symptoms: OR = 5.75, 95% CI 1.98-16.72). With regards to specific diagnoses, somatic symptoms predicted hospital-based care for mood disorders when adjusting for sex, adolescent depression, and adolescent anxiety (p < 0.05). In adolescents with depression, somatic symptoms predicted later hospital-based mental health care in a dose-response relationship (p < 0.01). In adolescents without depression, reporting at least one somatic symptom predicted later hospital-based mental health care (p < 0.05). CONCLUSIONS: Somatic symptoms in adolescence predicted severe adult mental illness as measured by hospital-based care also when controlled for important confounders. The results suggest that adolescents with somatic symptoms need early treatment and extended follow-up to treat these specific symptoms, regardless of co-occurring depression and anxiety.

Glutathionylation of beta-actin via a cysteinyl sulfenic acid intermediary.

BACKGROUND: Cysteinyl residues in actin are glutathionylated, ie. form a mixed disulfide with glutathione, even in the absence of exogenous oxidative stress. Glutathionylation inhibits actin polymerization and reversible actin glutathionylation is a redox dependent mechanism for regulation of the cytoskeleton structure. The molecular mechanism that mediates actin glutathionylation in vivo is unclear. RESULTS: We have studied glutathionylation of alpha- and beta-actin in vitro using an enzyme-linked immunosorbant assay with a monoclonal anti-glutathione antibody. alpha- and beta-actin were both glutathionylated when incubated with reduced glutathione (GSH) combined with diamide as a thiol oxidant. However, beta-actin was also glutathionylated by both glutathione disulfide (GSSG) and GSH in the absence of diamide whereas alpha-actin was poorly glutathionylated by GSH or GSSG. Glutathionylation of beta-actin by GSSG is likely to be mediated by a thiol-exchange mechanism whereas glutathionylation by GSH requires thiol oxidation. beta-actin glutathionylation by GSH was inhibited by arsenite and dimedone suggesting that the mechanism involved formation of a cysteinyl sulfenic acid residue in beta-actin. CONCLUSION: We conclude that glutathionylation of beta-actin may occur via spontaneous oxidation of a cysteinyl residue to a sulfenic acid that readily reacts with GSH to form a mixed disulfide. We also show that the reactivity and oxidation to a reactive protein thiol intermediary differ between different actin isoforms.

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.

Lipid-mediated protein delivery of suicide nucleoside kinases.

Nucleoside kinases from several species are investigated as suicide genes for treatment of malignant tumors by combined gene/chemotherapy. In the present study, we have investigated a novel strategy where nucleoside kinase proteins are directly delivered to cells without delivery of genetic material. We used a mix of a trifluoroacetylated lipopolyamine and dioleoyl phosphatidylethanolamine (BioPorter) to form protein-lipid complexes containing either recombinant herpes simplex virus type-1 thymidine kinase or Drosophila melanogaster multisubstrate deoxyribonucleoside kinase. We showed that the nucleoside kinase containing protein-lipid complexes was imported into human osteosarcoma and Chinese hamster ovary cell lines by endocytosis and that the enzymes were delivered to the cytosol and nucleus. The nucleoside kinases imported into the cell lines retained enzymatic activity, and the cells treated with the enzyme-lipid complexes showed increased sensitivity to nucleoside analogues, such as ganciclovir, (E)-5-(2-bromovinyl)-2'-deoxyuridine, and 1-beta-D-arabinofuranosylthymine. Our results show that direct delivery of suicide gene proteins to cells may be an alternative approach to conventional suicide gene therapy strategies.

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.

The Cellular Thioredoxin-1/Thioredoxin Reductase-1 Driven Oxidoreduction Represents a Chemotherapeutic Target for HIV-1 Entry Inhibition.

BACKGROUND: The entry of HIV into its host cell is an interesting target for chemotherapeutic intervention in the life-cycle of the virus. During entry, reduction of disulfide bridges in the viral envelope glycoprotein gp120 by cellular oxidoreductases is crucial. The cellular thioredoxin reductase-1 plays an important role in this oxidoreduction process by recycling electrons to thioredoxin-1. Therefore, thioredoxin reductase-1 inhibitors may inhibit gp120 reduction during HIV-1 entry. In this present study, tellurium-based thioredoxin reductase-1 inhibitors were investigated as potential inhibitors of HIV entry. RESULTS: The organotellurium compounds inhibited HIV-1 and HIV-2 replication in cell culture at low micromolar concentrations by targeting an early event in the viral infection cycle. Time-of-drug-addition studies pointed to virus entry as the drug target, more specifically: the organotellurium compound TE-2 showed a profile similar or close to that of the fusion inhibitor enfuvirtide (T-20). Surface plasmon resonance-based interaction studies revealed that the compounds do not directly interact with the HIV envelope glycoproteins gp120 and gp41, nor with soluble CD4, but instead, dose-dependently bind to thioredoxin reductase-1. By inhibiting the thioredoxin-1/thioredoxin reductase-1-directed oxidoreduction of gp120, the organotellurium compounds prevent conformational changes in the viral glycoprotein which are necessary during viral entry. CONCLUSION: Our findings revealed that thioredoxin-1/thioredoxin reductase-1 acts as a cellular target for the inhibition of HIV entry.

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

Fluorescence microscopy of A549 cells stained with a glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the peri-nuclear 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 peri-nuclear 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 lymphocytes, 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.

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.