Benny Björkblom

The Arabidopsis DJ-1a protein confers stress protection through cytosolic SOD activation

Mutations in the DJ-1 gene (also known as PARK7) cause inherited Parkinsons disease, which is characterized by neuronal death. Although DJ-1 is thought to be an antioxidant protein, the underlying mechanism by which loss of DJ-1 function contributes to cell death is unclear. Human DJ-1 and its Arabidopsis thaliana homologue, AtDJ-1a, are evolutionarily conserved proteins, indicating a universal function. To gain further knowledge of the molecular features associated with DJ-1 dysfunction, we have characterized AtDJ-1a. We show that AtDJ-1a levels are responsive to stress treatment and that AtDJ-1a loss of function results in accelerated cell death in aging plants. By contrast, transgenic plants with elevated AtDJ-1a levels have increased protection against environmental stress conditions, such as strong light, H2O2, methyl viologen and copper sulfate. We further identify superoxide dismutase 1 (SOD1) and glutathione peroxidase 2 (GPX2) as interaction partners of both AtDJ-1a and human DJ-1, and show that this interaction results in AtDJ-1a- and DJ-1-mediated cytosolic SOD1 activation in a copper-dependent fashion. Our data have highlighted a conserved molecular mechanism for DJ-1 and revealed a new protein player in the oxidative stress response of plants.

Propidium monoazide combined with real-time quantitative PCR underestimates heat-killed Listeria innocua.

The combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) significantly overestimated the fraction of viable Listeria innocua as compared to plate counts and confocal fluorescence microscopy. Our data imply that PMA-qPCR must be used with caution as an analytical tool for the differentiation between viable and dead bacteria.

Reactive Oxygen Species-Mediated DJ-1 Monomerization Modulates Intracellular Trafficking Involving Karyopherin β2

ABSTRACT Mutations in DJ-1 are a cause of recessive, early-onset Parkinsons disease (PD). Although oxidative stress and mitochondrial integrity have been implicated in PD, it is largely unknown why neurons degenerate. DJ-1 is involved in oxidative stress-mediated responses and in mitochondrial maintenance; however, its specific function remains vague. Here we show that DJ-1 exhibits neuronal dynamic intracellular trafficking, with dimeric/monomeric cycling modulated by the oxidative environment. We demonstrate that oxidative stress enhances monomerization of wild-type cytosolic DJ-1, leading to nuclear recruitment. The pathogenic DJ-1/E163K variant is unable to homodimerize but is retained in the cytosol upon wild-type DJ-1 heterodimerization. We found that this wild-type/pathogenic heterodimer is disrupted by oxidative stress, leading to DJ-1/E163K mitochondrial translocation. We further demonstrated that endogenously expressed wild-type DJ-1 is imported into neuronal nuclei as a monomer and that nucleo-cytoplasmic transport is oxidative stress mediated. We identified a novel proline-tyrosine nuclear localization signal (PY-NLS) in DJ-1, and we found that nuclear monomeric DJ-1 import is mediated by an oxidative stress-dependent interaction with karyopherin β2. Our study provides evidence that oxidative stress-mediated intracellular trafficking of DJ-1, mediated by dynamic DJ-1 dimeric/monomeric cycling, is implicated in PD pathogenesis.

Metabolomic screening of pre-diagnostic serum samples identifies association between α- and γ-tocopherols and glioblastoma risk

Glioblastoma is associated with poor prognosis with a median survival of one year. High doses of ionizing radiation is the only established exogenous risk factor. To explore new potential biological risk factors for glioblastoma, we investigated alterations in metabolite concentrations in pre-diagnosed serum samples from glioblastoma patients diagnosed up to 22 years after sample collection, and undiseased controls. The study points out a latent biomarker for future glioblastoma consisting of nine metabolites (γ-tocopherol, α-tocopherol, erythritol, erythronic acid, myo-inositol, cystine, 2-keto-L-gluconic acid, hypoxanthine and xanthine) involved in antioxidant metabolism. We detected significantly higher serum concentrations of α-tocopherol (p=0.0018) and γ-tocopherol (p=0.0009) in future glioblastoma cases. Compared to their matched controls, the cases showed a significant average fold increase of α- and γ-tocopherol levels: 1.2 for α-T (p=0.018) and 1.6 for γ-T (p=0.003). These tocopherol levels were associated with a glioblastoma odds ratio of 1.7 (α-T, 95% CI:1.0-3.0) and 2.1 (γ-T, 95% CI:1.2-3.8). Our exploratory metabolomics study detected elevated serum levels of a panel of molecules with antioxidant properties as well as oxidative stress generated compounds. Additional studies are necessary to confirm the association between the observed serum metabolite pattern and future glioblastoma development.

DJ-1 is a redox sensitive adapter protein for high molecular weight complexes involved in regulation of catecholamine homeostasis

&NA; DJ‐1 is an oxidation sensitive protein encoded by the PARK7 gene. Mutations in PARK7 are a rare cause of familial recessive Parkinsons disease (PD), but growing evidence suggests involvement of DJ‐1 in idiopathic PD. The key clinical features of PD, rigidity and bradykinesia, result from neurotransmitter imbalance, particularly the catecholamines dopamine (DA) and noradrenaline. We report in human brain and human SH‐SY5Y neuroblastoma cell lines that DJ‐1 predominantly forms high molecular weight (HMW) complexes that included RNA metabolism proteins hnRNPA1 and PABP1 and the glycolysis enzyme GAPDH. In cell culture models the oxidation status of DJ‐1 determined the specific complex composition. RNA sequencing indicated that oxidative changes to DJ‐1 were concomitant with changes in mRNA transcripts mainly involved in catecholamine metabolism. Importantly, loss of DJ‐1 function upon knock down (KD) or expression of the PD associated form L166P resulted in the absence of HMW DJ‐1 complexes. In the KD model, the absence of DJ‐1 complexes was accompanied by impairment in catecholamine homeostasis, with significant increases in intracellular DA and noraderenaline levels. These changes in catecholamines could be rescued by re‐expression of DJ‐1. This catecholamine imbalance may contribute to the particular vulnerability of dopaminergic and noradrenergic neurons to neurodegeneration in PARK7‐related PD. Notably, oxidised DJ‐1 was significantly decreased in idiopathic PD brain, suggesting altered complex function may also play a role in the more common sporadic form of the disease.

Xenobiotic- and Serum-Free Culture of Oral Mucosal Epithelial Cells on Contact Lenses

Abstract Purpose/aim: Cultured autologous oral mucosal epithelial cells (OMECs) have proven useful in the treatment of ocular surface disorders. This study is the first to investigate the potential of expanding OMEC in a xenobiotic- and serum-free medium using therapeutic contact lenses (CLs) as a substrate and carrier. Materials and methods: Porcine OMEC were seeded on laminin-coated lotrafilcon A therapeutic CLs with the density of 8 × 104 cells/lens and cultured in a defined serum and xenobiotic-free medium. Confocal immunofluorescence microscopy was used to analyze the following: (1) cellular morphology by using rhodamine-phalloidin staining of F-actin, (2) phenotype by applying antibodies against the progenitor cell marker p63 and the putative stem cell marker ABCG2 and (3) cell viability by using propidium iodide and Hoechst 33342 dual staining. Results: Porcine OMEC attached well to the CLs, and cell-to-cell contacts were evident. After three days in culture, the OMEC displayed a confluent monolayer with uniform cobblestone morphology, whereas stratified cultures with 2–3 layers were formed after six days. No significant difference in expression of p63 was observed after three-day culture (79.4 ± 14.8%) compared with six-day culture (60.3 ± 18.9%). ABCG2 expression in the basal cell layer was 6.3 ± 1.0% and 4.8 ± 1.8% after three- and six-day culture, respectively. The basal layer viability of cultured OMECs was 99.3 ± 0.2% and 82.8 ± 1.1% after three and six days culture, respectively. Conclusions: The use of therapeutic CLs has potential as a substrate and carrier for OMEC cultured in a xenobiotic- and serum-free culture system.

Statistical loadings and latent significance simplify and improve interpretation of multivariate projection models

Multivariate projection methods are unique in being both multivariable by combining many variables into stronger predictive features (latent variables), and multivariate for being able to model systematic variation both related and orthogonal to an observed response. Orthogonal partial least squares (OPLS) is a versatile multivariate projection method for analysis of correlation, discrimination and effect changes. However, currently OPLS is not fully using its multivariate potential since orthogonal systematic variation is not considered in model interpretation, resulting in univariate interpretation of variable significance. We present a strategy for improved interpretation of OPLS models based upon a post-hoc linear regression analysis that can be used with or without the orthogonal OPLS score(s) as a covariate to make the interpretation multivariate or univariate respectively. By selecting the observed response y or estimated response yhat as a one of the factors in the linear regression the results are related to either of the OPLS loadings w or p. Furthermore, converting the OPLS loading values to statistical t-values creates a direct link to statistical significance. Finally, by applying three different Boolean loadings W, P and W∧P variable significance can be summarized based on three criteria. W and P reveal if the values in w or p respectively are outside the statistical limits with W∧P being the logical conjunction of W and P (significant if outside limits in both W and P). Two examples are used to verify the proposed strategy. First, a synthetic example, simulating a mix of mass spectra, and second a clinical metabolomics study of a dietary intervention. In the simulated example we show that multivariate interpretation gives higher accuracy for estimation of true differences, mainly due to higher true positive rate. Furthermore, we highlight how application of W∧P for summarizing variable significance leads to higher accuracy. For the metabolomics example, we show that a more detailed interpretation, i.e. larger number of significant metabolites of relevance, is obtained using the multivariate interpretation. In summary, the suggested strategy provides means for facilitated interpretation of OPLS models, beyond univariate statistics, and offers a multivariate tool for discovery of biomarker patterns, i.e. latent biomarkers.