Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress

Teriflunomide (TFN) limits relapses in relapsing-remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria

Teriflunomide preserves peripheral nerve mitochondria from oxidative stress-mediated alterations

Mitochondrial dysfunction is a common pathological hallmark in various inflammatory and degenerative diseases of the central nervous system, including multiple sclerosis (MS). We previously showed that oxidative stress alters axonal mitochondria, limiting their transport and inducing conformational changes that lead to axonal damage. Teriflunomide (TFN), an oral immunomodulatory drug approved for the treatment of relapsing forms of MS, reversibly inhibits dihydroorotate dehydrogenase (DHODH). DHODH is crucial for de novo pyrimidine biosynthesis and is the only mitochondrial enzyme in this pathway, thus conferring a link between inflammation, mitochondrial activity and axonal integrity. Here, we investigated how DHODH inhibition may affect mitochondrial behavior in the context of oxidative stress. We employed a model of transected murine spinal roots, previously developed in our laboratory. Using confocal live imaging of axonal mitochondria, we showed that in unmanipulated axons, TFN increased significantly the mitochondria length without altering their transport features. In mitochondria challenged with 50 µM hydrogen peroxide (H2O2) to induce oxidative stress, the presence of TFN at 1 µM concentration was able to restore mitochondrial shape, motility, as well as mitochondrial oxidation potential to control levels. No effects were observed at 5 µM TFN, while some shape and motility parameters were restored to control levels at 50 µM TFN. Thus, our data demonstrate an undescribed link between DHODH and mitochondrial dynamics and point to a potential neuroprotective effect of DHODH inhibition in the context of oxidative stress-induced damage of axonal mitochondria

Automated vs manual delineations of regions of interest- a comparison in commercially available perfusion MRI software

Abstract Background In perfusion magnetic resonance imaging a manual approach to delineation of regions of interest is, due to rater bias and time intensive operator input, clinically less favorable than an automated approach would be. The goal of our study was to compare the performances of these approaches. Methods Using Stroketool, PMA and Perfscape/Neuroscape perfusion maps of cerebral blood flow, mean transit time and Tmax were created for 145 patients with acute ischemic stroke. Volumes of hypoperfused tissue were calculated using both a manual and an automated protocol, and the results compared between methods. Results The median difference between the automatically and manually derived volumes was up to 210 ml in Perfscape/Neuroscape, 123 ml in PMA and 135 ml in Stroketool. Correlation coefficients between perfusion volumes and radiological and clinical outcome were much lower for the automatic volumes than for the manually derived ones. Conclusions The agreement of the two methods was very poor, with the automated use producing falsely exaggerated volumes of hypoperfused tissue. Software improvements are necessary to enable highly automated protocols to credibly assess perfusion deficits.</p

Treatment of Chronic Experimental Autoimmune Encephalomyelitis with Epigallocatechin-3-Gallate and Glatiramer Acetate Alters Expression of Heme-Oxygenase-1.

We previously demonstrated that epigallocatechin-3-gallate (EGCG) synergizes with the immunomodulatory agent glatiramer acetate (GA) in eliciting anti-inflammatory and neuroprotective effects in the relapsing-remitting EAE model. Thus, we hypothesized that mice with chronic EAE may also benefit from this combination therapy. We first assessed how a treatment with a single dose of GA together with daily application of EGCG may modulate EAE. Although single therapies with a suboptimal dose of GA or EGCG led to disease amelioration and reduced CNS inflammation, the combination therapy had no effects. While EGCG appeared to preserve axons and myelin, the single GA dose did not improve axonal damage or demyelination. Interestingly, the neuroprotective effect of EGCG was abolished when GA was applied in combination. To elucidate how a single dose of GA may interfere with EGCG, we focused on the anti-inflammatory, iron chelating and anti-oxidant properties of EGCG. Surprisingly, we observed that while EGCG induced a downregulation of the gene expression of heme oxygenase-1 (HO-1) in affected CNS areas, the combined therapy of GA+EGCG seems to promote an increased HO-1 expression. These data suggest that upregulation of HO-1 may contribute to diminish the neuroprotective benefits of EGCG alone in this EAE model. Altogether, our data indicate that neuroprotection by EGCG in chronic EAE may involve regulation of oxidative processes, including downmodulation of HO-1. Further investigation of the re-dox balance in chronic neuroinflammation and in particular functional studies on HO-1 are warranted to understand its role in disease progression

Effects of EGCG alone and in combination with a suboptimal dose of GA in established EAE.

<p>A: Disease severity of control, GA, EGCG and combination therapy group. Analysis includes data from three independent experiments (Mann-Whitney test). B: Mean clinical scores of animals are shown. Data are given as mean ± SEM. Cumulative disease activity is represented as the area under the curve of control, GA, EGCG and combination therapy group (Kruskal-Wallis). C: Proliferation of MOG-specific CD4⁺ T cells at day 62 after immunization. CFSE-labeled lymph node cells were incubated for 72h with MOG (50μg/ml). As a positive control, cells were cultured with 3 μg/ml anti-CD3 antibody and 2.5 μg/ml anti-CD28 antibody. For the negative control, cells were incubated alone, in the absence of antigen. To assess cell division cells were stained with anti-CD4 Alexa Fluor 647 and analyzed by flow cytometry (ANOVA). *p<0.05, **p<0.01, ***p<0.001.</p

The combined application of GA and EGCG treatment does not alter the iron chelator activity but enhanced HO-1 expression compared to EGCG.

<p>A: Soluble iron content in blood serum was quantified day 26 after immunization by using a modification of the ferrozine-based assay. B: Relative mRNA expression of HO-1 in cerebellar and cerebral regions of the CNS of mice included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130251#pone.0130251.g003" target="_blank">Fig 3</a> treated with EGCG alone, or in combination with GA. (t-test) *p<0.05.</p

Addition EGCG therapy leads to decreased expression of heme oxygenase-1 (HO-1).

<p>A: Relative mRNA expression of HO-1 in cerebral and cerebellar regions as well as spinal cords of mice treated with vehicle control, EGCG alone, or in combination with GA. (ANOVA). B: Relative expression of HO-1 in mice treated with EGCG compared to mice treated with EGCG+GA C: Relative expression of HO-1 in mice treated with EGCG compared to control vehicle treated animals (t-test).</p