Investigating the Mitoprotective Effects of S1P Receptor Modulators Ex Vivo Using a Novel Semi-Automated Live Imaging Set-Up.

In multiple sclerosis (MS), mitochondrial alterations appear to contribute to disease progression. The sphingosine-1-phosphate receptor modulator siponimod is approved for treating secondary progressive MS. Its preceding compound fingolimod was shown to prevent oxidative stress-induced alterations in mitochondrial morphology. Here, we assessed the effects of siponimod, compared to fingolimod, on neuronal mitochondria in oxidatively stressed hippocampal slices. We have also advanced the model of chronic organotypic hippocampal slices for live imaging, enabling semi-automated monitoring of mitochondrial alterations. The slices were prepared from B6.Cg-Tg(Thy1-CFP/COX8A)S2Lich/J mice that display fluorescent neuronal mitochondria. They were treated with hydrogen peroxide (oxidative stress paradigm) ± 1 nM siponimod or fingolimod for 24 h. Afterwards, mitochondrial dynamics were investigated. Under oxidative stress, the fraction of motile mitochondria decreased and mitochondria were shorter, smaller, and covered smaller distances. Siponimod partly prevented oxidatively induced alterations in mitochondrial morphology; for fingolimod, a similar trend was observed. Siponimod reduced the decrease in mitochondrial track displacement, while both compounds significantly increased track speed and preserved motility. The novel established imaging and analysis tools are suitable for assessing the dynamics of neuronal mitochondria ex vivo. Using these approaches, we showed that siponimod at 1 nM partially prevented oxidatively induced mitochondrial alterations in chronic brain slices.

Preventing Axonal Sodium Overload or Mitochondrial Calcium Uptake Protects Axonal Mitochondria from Oxidative Stress-Induced Alterations.

In neuroinflammatory and neurodegenerative disorders such as multiple sclerosis, mitochondrial damage caused by oxidative stress is believed to contribute to neuroaxonal damage. Previously, we demonstrated that exposure to hydrogen peroxide (H2O2) alters mitochondrial morphology and motility in myelinated axons and that these changes initiate at the nodes of Ranvier, where numerous sodium channels are located. Therefore, we suggested that mitochondrial damage may lead to ATP deficit, thereby affecting the efficiency of the sodium-potassium ATPase and eventually leading to sodium overload in axons. The increased intra-axonal sodium may revert the axonal sodium-calcium exchangers and thus may lead to a pathological calcium overload in the axoplasm and mitochondria. Here, we used the explanted murine ventral spinal roots to investigate whether modulation of sodium or calcium influx may prevent mitochondrial alterations in myelinated axons during exogenous application of H2O2 inducing oxidative stress. For that, tetrodotoxin, an inhibitor of voltage-gated sodium ion channels, and ruthenium 360, an inhibitor of the mitochondrial calcium uniporter, were applied simultaneously with hydrogen peroxide to axons. Mitochondrial shape and motility were analyzed. We showed that inhibition of axonal sodium influx prevented oxidative stress-induced morphological changes (i.e., increase in circularity and area and decrease in length) and preserved mitochondrial membrane potential, which is crucial for ATP production. Blocking mitochondrial calcium uptake prevented decrease in mitochondrial motility and also preserved membrane potential. Our findings indicate that alterations of both mitochondrial morphology and motility in the contexts of oxidative stress can be counterbalanced by modulating intramitochondrial ion concentrations pharmacologically. Moreover, motile mitochondria show preserved membrane potentials, pointing to a close association between mitochondrial motility and functionality.

Imaging and analysis of neuronal mitochondria in murine acute brain slices.

BACKGROUND: Mitochondrial alterations are common to many inflammatory, degenerative as well as metabolic diseases. However, due to the vulnerability of mitochondria in explanted tissue, there is a general lack of ex vivo models, especially of CNS tissue, that preserve mitochondria and allow investigation of mitochondrial dynamics. NEW METHODS: Here, we present a model of acute hippocampal slices to study neuronal mitochondria ex vivo. We used two-photon microscopy to image CFP fluorescent neuronal mitochondria in B6. Cg-Tg(Thy1-CFP/COX8A)S2Lich mice brain slices. To define the optimal processing and culturing conditions, we compared mitochondrial morphology and motility with three different sets of slicing and incubation solutions. The investigation of mitochondrial dynamics was performed on deconvoluted images. For morphological investigation, images were segmented into three different categories according to the shape of mitochondria, while motility was investigated using semi-automated tracking. RESULTS: The imaging of acute brain slices by two-photon microscopy represented a suitable tool to monitor neuronal mitochondria ex vivo. We observed that mitochondrial dynamics were better preserved in slices incubated with HEPES aCSF, maintaining elongated rod-shaped morphology and the motility. COMPARISON WITH EXISTING METHODS: We showed for the first time a method that allows live imaging of mitochondria and its quantification, while the existing in vitro protocol are not suitable to investigate mitochondria in live tissue. CONCLUSION: We have established the best incubation conditions and microscopy tools to investigate living mitochondria in acute slices. We showed that preventing initial swelling with HEPES and addition of glucose, pyruvate, ascorbate and thiourea preserved mitochondria in adult brain slices, which could be monitored by two-photon microscopy.

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.

A Systematic Review of Transcriptional Dysregulation in Huntington's Disease Studied by RNA Sequencing.

Huntington's disease (HD) is a chronic neurodegenerative disorder caused by an expansion of polyglutamine repeats in exon 1 of the Huntingtin gene. Transcriptional dysregulation accompanied by epigenetic alterations is an early and central disease mechanism in HD yet, the exact mechanisms and regulators, and their associated gene expression programs remain incompletely understood. This systematic review investigates genome-wide transcriptional studies that were conducted using RNA sequencing (RNA-seq) technology in HD patients and models. The review protocol was registered at the Open Science Framework (OSF). The biomedical literature and gene expression databases, PubMed and NCBI BioProject, Array Express, European Nucleotide Archive (ENA), European Genome-Phenome Archive (EGA), respectively, were searched using the defined terms specified in the protocol following the PRISMA guidelines. We conducted a complete literature and database search to retrieve all RNA-seq-based gene expression studies in HD published until August 2020, retrieving 288 articles and 237 datasets from PubMed and the databases, respectively. A total of 27 studies meeting the eligibility criteria were included in this review. Collectively, comparative analysis of the datasets revealed frequent genes that are consistently dysregulated in HD. In postmortem brains from HD patients, DNAJB1, HSPA1B and HSPB1 genes were commonly upregulated across all brain regions and cell types except for medium spiny neurons (MSNs) at symptomatic disease stage, and HSPH1 and SAT1 genes were altered in expression in all symptomatic brain datasets, indicating early and sustained changes in the expression of genes related to heat shock response as well as response to misfolded proteins. Specifically in indirect pathway medium spiny neurons (iMSNs), mitochondria related genes were among the top uniquely dysregulated genes. Interestingly, blood from HD patients showed commonly differentially expressed genes with a number of brain regions and cells, with the highest number of overlapping genes with MSNs and BA9 region at symptomatic stage. We also found the differential expression and predicted altered activity of a set of transcription factors and epigenetic regulators, including BCL6, EGR1, FOSL2 and CREBBP, HDAC1, KDM4C, respectively, which may underlie the observed transcriptional changes in HD. Altogether, our work provides a complete overview of the transcriptional studies in HD, and by data synthesis, reveals a number of common and unique gene expression and regulatory changes across different cell and tissue types in HD. These changes could elucidate new insights into molecular mechanisms of differential vulnerability in HD. Systematic Review Registration: https://osf.io/pm3wq.

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.

MR Elastography-Based Assessment of Matrix Remodeling at Lesion Sites Associated With Clinical Severity in a Model of Multiple Sclerosis.

Magnetic resonance imaging (MRI) with gadolinium based contrast agents (GBCA) is routinely used in the clinic to visualize lesions in multiple sclerosis (MS). Although GBCA reveal endothelial permeability, they fail to expose other aspects of lesion formation such as the magnitude of inflammation or tissue changes occurring at sites of blood-brain barrier (BBB) disruption. Moreover, evidence pointing to potential side effects of GBCA has been increasing. Thus, there is an urgent need to develop GBCA-independent imaging tools to monitor pathology in MS. Using MR-elastography (MRE), we previously demonstrated in both MS and the animal model experimental autoimmune encephalomyelitis (EAE) that inflammation was associated with a reduction of brain stiffness. Now, using the relapsing-remitting EAE model, we show that the cerebellum-a region with predominant inflammation in this model-is especially prone to loss of stiffness. We also demonstrate that, contrary to GBCA-MRI, reduction of brain stiffness correlates with clinical disability and is associated with enhanced expression of the extracellular matrix protein fibronectin (FN). Further, we show that FN is largely expressed by activated astrocytes at acute lesions, and reflects the magnitude of tissue remodeling at sites of BBB breakdown. Therefore, MRE could emerge as a safe tool suitable to monitor disease activity in MS.

Product of serum calcium and phosphorus (Ca × PO4) as predictor of cardiovascular disease risk in predialysis patients.

OBJECTIVES: The mortality rate of chronic kidney disease (CKD) patients is very high due to cardiovascular diseases (CVD) which cannot be fully justified by traditional CVD markers. Since, mineral bone disorder is common in CKD, product of serum calcium and phosphorus (Ca × PO4) can be a predictor of future CVD. So, our study aims to assess the utility of higher Ca × PO4 in prediction of CVD risk in predialysis CKD patients. DESIGN AND METHODS: 150 CKD patients defined by NKF-KDOQI guideline not undergoing dialysis were recruited. Anthropometric and electrocardiographic parameters were recorded. We evaluated CVD risk by: i) Biochemical CVD markers, ii) NCEP ATP III guideline postulated risk factors and iii) Framingham risk scores. RESULTS: Higher Ca × PO4 is associated with presence of Left Ventricular Hypertrophy, oxidative stress, microinflammation, hyperhomocysteinemia, hypercholesterolemia, hypertriglyceridemia and increased LDLc. Compared to cases with Ca × PO4 <55 mg2/dL2, cases with ≥55 mg2/dL2 had relative risk (RR) of 1.82 (95% CI 1.25-2.64) for CVD, 3.24 (95% CI 2.37-4.41) for stroke and 2.43 (95% CI 1.37-4.31) for coronary heart disease (CHD). Moreover, compared to lowest quartile of Ca x PO4, the highest quartile group had RR of 2.13 (95% CI 1.06-4.28) for CVD, 2.61(95% CI 1.80-3.75) for stroke and 2.84 (95% CI 1.15-7.0) for CHD. CONCLUSION: In predialysis patients, higher Ca × PO4 is independent predictor of CVD risk.