Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Presenilin-1 Delta E9 Mutant Induces STIM1-Driven Store-Operated Calcium Channel Hyperactivation in Hippocampal Neurons

Presenilins regulate calcium homeostasis in the endoplasmic reticulum, and dysregulation of intracellular calcium has been implicated in the pathogenesis of Alzheimer disease. Elevated presenilin-1 (PS1) holoprotein levels have been detected in postmortem brains of patients carrying familial Alzheimer disease (FAD) PS1 mutations. This study examines the effect of the FAD presenilin mutant that lacks the ninth exon (PS1 ?E9) and does not undergo endoproteolysis on store-operated calcium (SOC) entry. Significant enhancement of SOC channel activation was detected by electrophysiological measurements in hippocampal neurons with PS1 ?E9 mutant expression. Here, we show that (i) the hyperactivation of SOC channels is mediated by the STIM1 sensor and can be attenuated by STIM1 knockdown or 2-aminoethoxydiphenyl borate application, (ii) the STIM2 is not involved in pathological changes of SOC entry, (iii) the pathological SOC entry demonstrates properties of both TRPC and Orai subunit composition, and (iiii) transgenic Drosophila flies with PS1 ?E9 expression in the cholinergic neuron system show short-term memory loss, which can be abolished by 2-aminoethoxydiphenyl borate feeding.     

<Emphasis Type="Italic">Salmonella enterica</Emphasis> subspecies <Emphasis Type="Italic">enterica</Emphasis> serovar Choleraesuis in a wild boar population in Germany

Salmonella (S.) enterica subspecies enterica serovar Choleraesuis, the swine-adapted serovar is found rarely in Western European countries including Germany. However, the regional laboratory of the federal state Thuringia in Germany examined diseased wild boars routinely also for the occurrence of Salmonella organisms. Between 2006 and 2008, only the serovar S. Choleraesuis was islolated from 24 animals, three strains isolated from domestic pigs were included. In order to detect a possible epidemiological context, the strains of S. Choleraesuis were characterised by macrorestriction and plasmid analysis, repetitive sequence PCR, antimicrobial testing and determining the biochemical profile. A combination of all methods enabled the identification of five epidemiological groups. Two groups were detected in the same territory but three other discriminative groups were predominant in different regions. S. Choleraesuis strains of the different epidemiological groups circulate in wild boar populations in the corresponding regions. However, it could be concluded that both natural barriers like mountains and artificial barriers like arterial roads may cause the separation of wild boar populations and as a result also the respective S. Choleraesuis organisms. The occurrence of the identical epidemiological groups in wild boars and domestic pigs indicates the possible mutual exposure of the pathogen. To avoid risks for human and domestic pig health regular inspection of meat from wildlife by official veterinarians and advice of hunters and persons who prepare and consume wild boar meat should be enhanced.     

Human septin 3 on chromosome 22q13.2 is upregulated by neuronal differentiation

An expression sequence tag identified in a screen for genes upregulated by retinoic acid induced neuronal differentiation of the human teratocarcinoma cell line Ntera2/D1 was found in close genomic proximity to a region of high sequence homology to the septin subfamily of GTPase genes. We could show that the tag corresponds to the 3' untranslated region of this novel gene named septin 3 and cloned three isoforms A (2191 bp), B (4378 bp), and C (1896 bp) from human Ntera2/D1 cDNA. We present the genomic localization and organization on chromosome 22q13.2, a chromosomal hot spot for translocations implicated in leukemia. Interestingly, MSF the closest paralog of septin 3 is a fusion partner in a therapy-related acute myeloid leukemia. Quantitative PCR confirmed the upregulation of the putative septin by neuronal differentiation and northern blotting showed only one band corresponding to sep3B with a neurospecific expression pattern in adult human tissues.     

Interaction of Bcl-2 with the Autophagy-related GABAA Receptor-associated Protein (GABARAP): BIOPHYSICAL CHARACTERIZATION AND FUNCTIONAL IMPLICATIONS*

Apoptosis and autophagy are fundamental homeostatic processes in eukaryotic organisms fulfilling essential roles in development and adaptation. Recently, the anti-apoptotic factor Bcl-2 has been reported to also inhibit autophagy, thus establishing a potential link between these pathways, but the mechanistic details are only beginning to emerge. Here we show that Bcl-2 directly binds to the phagophore-associated protein GABARAP. NMR experiments revealed that the interaction critically depends on a three-residue segment (EWD) of Bcl-2 adjacent to the BH4 region, which is anchored to one of the two hydrophobic pockets on the GABARAP molecule. This is at variance with the majority of GABARAP interaction partners identified previously, which occupy both hydrophobic pockets simultaneously. Bcl-2 affinity could also be detected for GEC1, but not for other mammalian Atg8 homologs. Finally, we provide evidence that overexpression of Bcl-2 inhibits lipidation of GABARAP, a key step in autophagosome formation, possibly via competition with the lipid conjugation machinery. These results support the regulatory role of Bcl-2 in autophagy and define GABARAP as a novel interaction partner involved in this intricate connection.     

The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress

The mouse hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Addition of extracellular glutamate depletes the cells of glutathione (GSH) by blocking the glutamate−cystine antiporter system x_c⁻. GSH is the main antioxidant in neurons and its depletion induces a well-defined program of cell death called oxytosis, which is probably synonymous with the iron-dependent form of non-apoptotic cell death termed ferroptosis. Oxytosis is characterized by an increase of reactive oxygen species and a strong calcium influx preceding cell death. We found a significant reduction in store-operated calcium entry (SOCE) in glutamate-resistant HT22 cells caused by downregulation of the Ca²⁺ channel ORAI1, but not the Ca²⁺ sensors STIM1 or STIM2. Pharmacological inhibition of SOCE mimicked this protection similarly to knockdown of ORAI1 by small interfering RNAs. Long-term calcium live-cell imaging after induction of the cell death program showed a specific reduction in Ca²⁺-positive cells by ORAI1 knockdown. These results suggest that dysregulated Ca²⁺ entry through ORAI1 mediates the detrimental Ca²⁺ entry in programmed cell death induced by GSH depletion. As this detrimental Ca²⁺ influx occurs late in the course of the cell death program, it might be amenable to therapeutic intervention in diseases caused by oxidative stress.     

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.     

Optical coherence tomography in parkinsonian syndromes

Background/Objective

Parkinson''s disease (PD) and the atypical parkinsonian syndromes multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and corticobasal syndrome (CBS) are movement disorders associated with degeneration of the central nervous system. Degeneration of the retina has not been systematically compared in these diseases.

Methods

This cross-sectional study used spectral-domain optical coherence tomography with manual segmentation to measure the peripapillar nerve fiber layer, the macular thickness, and the thickness of all retinal layers in foveal scans of 40 patients with PD, 19 with MSA, 10 with CBS, 15 with PSP, and 35 age- and sex-matched controls.

Results

The mean paramacular thickness and volume were reduced in PSP while the mean RNFL did not differ significantly between groups. In PSP patients, the complex of retinal ganglion cell- and inner plexiform layer and the outer nuclear layer was reduced. In PD, the inner nuclear layer was thicker than in controls, MSA and PSP. Using the ratio between the outer nuclear layer and the outer plexiform layer with a cut-off at 3.1 and the additional constraint that the inner nuclear layer be under 46 µm, we were able to differentiate PSP from PD in our patient sample with a sensitivity of 96% and a specificity of 70%.

Conclusion

Different parkinsonian syndromes are associated with distinct changes in retinal morphology. These findings may serve to facilitate the differential diagnosis of parkinsonian syndromes and give insight into the degenerative processes of patients with atypical parkinsonian syndromes.