Philipp Albrecht

Induction of Nrf2 and xCT are involved in the action of the neuroprotective antibiotic ceftriaxone in vitro.

In amyotrophic lateral sclerosis, down‐regulation of the astrocyte‐specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine‐antiporter system xc−. Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system xc− and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2‐related factor 2), a known inducer of system xc−, and the specific xc− subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone‐stimulated changes in Nrf2, system xc−, and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell‐derived human motor neurons. We conclude that ceftriaxone‐mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system xc− than to excitatory amino acid transporter induction.

Effects of dimethyl fumarate on neuroprotection and immunomodulation

BackgroundNeuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects.FindingsWe determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays.ConclusionsThese results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.

Mechanisms of Oxidative Glutamate Toxicity: The Glutamate/Cystine Antiporter System xc¯ as a Neuroprotective Drug Target

The glutamate/cystine antiporter system x(c)- transports cystine into cells in exchange for the important neurotransmitter glutamate at a ratio of 1:1. It is composed of a specific light chain, xCT, and a heavy chain, 4F2, linked by a disulfide bridge. Both subunits are localized prominently in the mouse and human brain especially in border areas between the brain and periphery including vascular endothelial cells, ependymal cells, choroid plexus, and leptomeninges. Glutamate exported by system x(c)- is largely responsible for the extracellular glutamate concentration in the brain, whereas the imported cystine is required for the synthesis of the major endogenous antioxidant, glutathione. System x(c)- thus connects the antioxidant defense with neurotransmission and behavior. Disturbances in the function of system x(c)- have been implicated in nerve cell death due to increased extracellular glutamate and reduced intracellular glutathione. In vitro, inhibition of cystine import through system x(c)- leads to cell death by a mechanism called oxidative glutamate toxicity or oxytosis, which includes depletion of intracellular glutathione, activation of 12-lipoxygenase, accumulation of intracellular peroxides, and the activation of a cyclic guanosine monophosphate (cGMP)-dependent calcium channel towards the end of the death cascade. Cell death caused by oxytosis is distinct from classical apoptosis. In this contribution, we discuss the function of system x(c)- in vitro and in vivo, the role of xCT as an important but due to its dual role probably ambivalent drug target, and the relevance of oxytosis as an in vitro assay for the identification of novel neuroprotective proteins and signaling pathways.

Optical Coherence Tomography in Parkinsonian Syndromes

Background/Objective Parkinsons 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.

The APOSTEL recommendations for reporting quantitative optical coherence tomography studies.

Objective: To develop consensus recommendations for reporting of quantitative optical coherence tomography (OCT) study results. Methods: A panel of experienced OCT researchers (including 11 neurologists, 2 ophthalmologists, and 2 neuroscientists) discussed requirements for performing and reporting quantitative analyses of retinal morphology and developed a list of initial recommendations based on experience and previous studies. The list of recommendations was subsequently revised during several meetings of the coordinating group. Results: We provide a 9-point checklist encompassing aspects deemed relevant when reporting quantitative OCT studies. The areas covered are study protocol, acquisition device, acquisition settings, scanning protocol, funduscopic imaging, postacquisition data selection, postacquisition data analysis, recommended nomenclature, and statistical analysis. Conclusions: The Advised Protocol for OCT Study Terminology and Elements recommendations include core items to standardize and improve quality of reporting in quantitative OCT studies. The recommendations will make reporting of quantitative OCT studies more consistent and in line with existing standards for reporting research in other biomedical areas. The recommendations originated from expert consensus and thus represent Class IV evidence. They will need to be regularly adjusted according to new insights and practices.

Charcot-Marie-Tooth disease CMT4A: GDAP1 increases cellular glutathione and the mitochondrial membrane potential

Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.

Stromal interaction molecule 1 (STIM1) is involved in the regulation of mitochondrial shape and bioenergetics and plays a role in oxidative stress

Background: Store-operated Ca2+ entry is regulated by the sensor STIM1 and the channel ORAI1. Results: Deficiency alters mitochondrial shape and increases mitochondrial activity resulting in increased susceptibility to oxidative stress and cell death by nuclear translocation of apoptosis-inducing factor. Conclusion: Store-operated Ca2+ entry regulates mitochondrial function and vulnerability. Significance: STIM1 plays a role in oxidative stress by regulating mitochondrial function. Calcium ions are involved in a plethora of cellular functions including cell death and mitochondrial energy metabolism. Store-operated Ca2+ entry over the plasma membrane is activated by depletion of intracellular Ca2+ stores and is mediated by the sensor STIM1 and the channel ORAI1. We compared cell death susceptibility to oxidative stress in STIM1 knock-out and ORAI1 knockdown mouse embryonic fibroblasts and in knock-out cells with reconstituted wild type and dominant active STIM1. We show that STIM1 and ORAI1 deficiency renders cells more susceptible to oxidative stress, which can be rescued by STIM1 and ORAI1 overexpression. STIM1 knock-out mitochondria are tubular, have a higher Ca2+ concentration, and are metabolically more active, resulting in constitutive oxidative stress causing increased nuclear translocation of the antioxidant transcription factor NRF2 triggered by increased phosphorylation of the translation initiation factor eIF2α and the protein kinase-like endoplasmic reticulum kinase PERK. This leads to increased transcription of antioxidant genes and a high basal glutathione in STIM1 knock-out cells, which is, however, more rapidly expended upon additional stress, resulting in increased release and nuclear translocation of apoptosis-inducing factor with subsequent cell death. Our data suggest that store-operated Ca2+ entry and STIM1 are involved in the regulation of mitochondrial shape and bioenergetics and play a role in oxidative stress.

Retinal layer segmentation in multiple sclerosis: a systematic review and meta-analysis

BACKGROUND Structural retinal imaging biomarkers are important for early recognition and monitoring of inflammation and neurodegeneration in multiple sclerosis. With the introduction of spectral domain optical coherence tomography (SD-OCT), supervised automated segmentation of individual retinal layers is possible. We aimed to investigate which retinal layers show atrophy associated with neurodegeneration in multiple sclerosis when measured with SD-OCT. METHODS In this systematic review and meta-analysis, we searched for studies in which SD-OCT was used to look at the retina in people with multiple sclerosis with or without optic neuritis in PubMed, Web of Science, and Google Scholar between Nov 22, 1991, and April 19, 2016. Data were taken from cross-sectional cohorts and from one timepoint from longitudinal studies (at least 3 months after onset in studies of optic neuritis). We classified data on eyes into healthy controls, multiple-sclerosis-associated optic neuritis (MSON), and multiple sclerosis without optic neuritis (MSNON). We assessed thickness of the retinal layers and we rated individual layer segmentation performance by random effects meta-analysis for MSON eyes versus control eyes, MSNON eyes versus control eyes, and MSNON eyes versus MSON eyes. We excluded relevant sources of bias by funnel plots. FINDINGS Of 25 497 records identified, 110 articles were eligible and 40 reported data (in total 5776 eyes from patients with multiple sclerosis [1667 MSON eyes and 4109 MSNON eyes] and 1697 eyes from healthy controls) that met published OCT quality control criteria and were suitable for meta-analysis. Compared with control eyes, the peripapillary retinal nerve fibre layer (RNFL) showed thinning in MSON eyes (mean difference -20·10 μm, 95% CI -22·76 to -17·44; p<0·0001) and in MSNON eyes (-7·41 μm, -8·98 to -5·83; p<0·0001). The macula showed RNFL thinning of -6·18 μm (-8·07 to -4·28; p<0·0001) in MSON eyes and -2·15 μm (-3·15 to -1·15; p<0·0001) in MSNON eyes compared with control eyes. Atrophy of the macular ganglion cell layer and inner plexiform layer (GCIPL) was -16·42 μm (-19·23 to -13·60; p<0·0001) for MSON eyes and -6·31 μm (-7·75 to -4·87; p<0·0001) for MSNON eyes compared with control eyes. A small degree of inner nuclear layer (INL) thickening occurred in MSON eyes compared with control eyes (0·77 μm, 0·25 to 1·28; p=0·003). We found no statistical difference in the thickness of the combined outer nuclear layer and outer plexiform layer when we compared MSNON or MSON eyes with control eyes, but we found a small degree of thickening of the combined layer when we compared MSON eyes with MSNON eyes (1·21 μm, 0·24 to 2·19; p=0·01). INTERPRETATION The largest and most robust differences between the eyes of people with multiple sclerosis and control eyes were found in the peripapillary RNFL and macular GCIPL. Inflammatory disease activity might be captured by the INL. Because of the consistency, robustness, and large effect size, we recommend inclusion of the peripapillary RNFL and macular GCIPL for diagnosis, monitoring, and research. FUNDING None.

Phosphoinositide 3-Kinases Upregulate System xc− via Eukaryotic Initiation Factor 2α and Activating Transcription Factor 4 – A Pathway Active in Glioblastomas and Epilepsy

AIMS Phosphoinositide 3-kinases (PI3Ks) relay growth factor signaling and mediate cytoprotection and cell growth. The cystine/glutamate antiporter system xc(-) imports cystine while exporting glutamate, thereby promoting glutathione synthesis while increasing extracellular cerebral glutamate. The aim of this study was to analyze the pathway through which growth factor and PI3K signaling induce the cystine/glutamate antiporter system xc(-) and to demonstrate its biological significance for neuroprotection, cell growth, and epilepsy. RESULTS PI3Ks induce system xc(-) through glycogen synthase kinase 3β (GSK-3β) inhibition, general control non-derepressible-2-mediated eukaryotic initiation factor 2α phosphorylation, and the subsequent translational up-regulation of activating transcription factor 4. This pathway is essential for PI3Ks to modulate oxidative stress resistance of nerve cells and insulin-induced growth in fibroblasts. Moreover, the pathway is active in human glioblastoma cells. In addition, it is induced in primary cortical neurons in response to robust neuronal activity and in hippocampi from patients with temporal lobe epilepsy. INNOVATION Our findings further extend the concepts of how growth factors and PI3Ks induce neuroprotection and cell growth by adding a new branch to the signaling network downstream of GSK-3β, which, ultimately, leads to the induction of the cystine/glutamate antiporter system xc(-). Importantly, the induction of this pathway by neuronal activity and in epileptic hippocampi points to a potential role in epilepsy. CONCLUSION PI3K-regulated system xc(-) activity is not only involved in the stress resistance of neuronal cells and in cell growth by increasing the cysteine supply and glutathione synthesis, but also plays a role in the pathophysiology of tumor- and non-tumor-associated epilepsy by up-regulating extracellular cerebral glutamate.

Retinal Neurodegeneration in Wilson’s Disease Revealed by Spectral Domain Optical Coherence Tomography

Background/Objective In addition to cirrhosis of the liver, Wilson’s disease leads to copper accumulation and widespread degeneration of the nervous system. Delayed visual evoked potentials (VEPs) suggest changes to the visual system and potential structural changes of the retina. Methods We used the latest generation of spectral domain optical coherence tomography to assess the retinal morphology of 42 patients with Wilson’s disease and 76 age- and sex-matched controls. We measured peripapillary retinal nerve fiber layer (RNFL) thickness and total macular thickness and manually segmented all retinal layers in foveal scans of 42 patients with Wilson’s disease and 76 age- and sex-matched controls. The results were compared with VEPs and clinical parameters. Results The mean thickness of the RNFL, paramacular region, retinal ganglion cell/inner plexiform layer and inner nuclear layer was reduced in Wilson’s disease. VEPs were altered with delayed N75 and P100 latencies, but the N140 latency and amplitude was unchanged. An analysis of the laboratory parameters indicated that the serum concentrations of copper and caeruloplasmin positively correlated with the thickness of the outer plexiform layer and with N75 and P100 VEP latencies. Conclusion Neuronal degeneration in Wilson’s disease involves the retina and changes can be quantified by optical coherence tomography. While the VEPs and the thickness of the outer plexiform layer appear to reflect the current copper metabolism, the thicknesses of the RNFL, ganglion cell/inner plexiform layer, inner nuclear layer and the total paramacular thickness may be the best indicators of chronic neuronal degeneration.