While lactate shuttle theory states that glial cells metabolize glucose into lactate to shuttle it to neurons, how glial cells support axonal metabolism and function remains unclear. Lactate production is a common occurrence following anaerobic glycolysis in muscles. However, several other cell types, including some stem cells, activated macrophages and tumor cells, can produce lactate in presence of oxygen and cellular respiration, using Pyruvate Kinase 2 (PKM2) to divert pyruvate to lactate dehydrogenase. We show here that PKM2 is also upregulated in myelinating Schwann cells (mSC) of mature mouse sciatic nerve versus postnatal immature nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. While the structure of myelin sheath was preserved, mutant mice developed a peripheral neuropathy. Peripheral nerve axons of mutant mice failed to maintain lactate homeostasis upon activity, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not altered but axonal mitochondria transport was slowed down, muscle axon terminals retracted and motor neurons displayed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment, which diverts pyruvate to mitochondrial oxidative phosphorylation, further aggravated motor dysfunction in mutant mice. Thus, lactate production through PKM2 enzyme and aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

Corneal blindness is the fourth leading cause of blindness worldwide. Since corneal epithelium is constantly renewed, non-integrative gene transfer cannot be used to treat corneal diseases. In many of these diseases, the tear film is defective. Tears are a complex biological fluid secreted by the lacrimal apparatus. Their composition is modulated according to the context. After a corneal wound, the lacrimal gland secretes reflex tears, which contain growth factors supporting the wound healing process. In various pathological contexts, the tear composition can support neither corneal homeostasis nor wound healing. Here, we propose to use the lacrimal gland as bioreactor to produce and secrete specific factors supporting corneal physiology. In this study, we use an AAV2/9-mediated gene transfer to supplement the tear film. First, we demonstrate that a single injection of AAV2/9 is sufficient to transduce all epithelial cell types of the lacrimal gland efficiently and widely. Second, we detect no adverse effect after AAV2/9-mediated nerve growth factor expression in the lacrimal gland. Only a transitory increase in tear flow is measured. Remarkably, AAV2/9 induces an important and long-lasting secretion of this growth factor in the tear film. Altogether, our findings provide a new clinically applicable approach to tackle corneal blindness.

A large number of peripheral neuropathies, among which are traumatic and diabetic peripheral neuropathies, result from the degeneration of the myelin sheath, a process called demyelination. Demyelination does not result from Schwann cell death but from Schwann cell dedifferentiation, which includes reprograming and several catabolic and anabolic events. Starting around 4 h after nerve injury, activation of MAPK/cJun pathways is the earliest characterized step of this dedifferentiation program. Here we show, using real-time in vivo imaging, that Schwann cell mitochondrial pH, motility and calcium content are altered as soon as one hour after nerve injury. Mitochondrial calcium release occurred through the VDAC outer membrane channel and mPTP inner membrane channel. This calcium influx in the cytoplasm induced Schwann-cell demyelination via MAPK/c-Jun activation. Blocking calcium release through VDAC silencing or VDAC inhibitor TRO19622 prevented demyelination. We found that the kinetics of mitochondrial calcium release upon nerve injury were altered in the Schwann cells of diabetic mice suggesting a permanent leak of mitochondrial calcium in the cytoplasm. TRO19622 treatment alleviated peripheral nerve defects and motor deficit in diabetic mice. Together, these data indicate that mitochondrial calcium homeostasis is instrumental in the Schwann cell demyelination program and that blocking VDAC constitutes a molecular basis for developing anti-demyelinating drugs for diabetic peripheral neuropathy.

The voltage-dependent anion channel (VDAC), the most abundant protein on the outer mitochondrial membrane, is implicated in ATP, ion and metabolite exchange with cell compartments. In particular, the VDAC participates in cytoplasmic and mitochondrial Ca2+ homeostasis. Notably, the Ca2+ efflux out of Schwann cell mitochondria is involved in peripheral nerve demyelination that underlies most peripheral neuropathies. Hexokinase (HK) isoforms I and II, the main ligands of the VDAC, possess a hydrophobic N-terminal structured in α-helix (NHKI) that is necessary for the binding to the VDAC. To gain further insight into the molecular basis of HK binding to the VDAC, we developed and optimized peptides based on the NHKI sequence. These modifications lead to an increase of the peptide hydrophobicity and helical content that enhanced their ability to prevent peripheral nerve demyelination. Our results provide new insights into the molecular basis of VDAC/HK interaction that could lead to the development of therapeutic compounds for demyelinating peripheral neuropathies.

Mitochondrial dysfunction has been implicated in many diseases including cancer, cardiovascular and neurodegenerative diseases1,2. The voltage-dependant anion channel 1 (VDAC1) is the most abundant protein found in the outer mitochondrial membrane. VDAC1 functions as a gatekeeper and is considered as a multi-functional porin involved in cell survival and cell death3. VDAC1 mediates energy production as well as ions and metabolic exchanges between the mitochondria and other cellular compartments, thus modulating mitochondrial permeability. VDAC1 is also involved in the cellular Ca2+ homeostasis by its capacity to transport Ca2+ in and out of mitochondria4. In addition, VDAC1 interacts with multiple proteins implicated in neurodegeneration process5 as well as with pro-apoptotic and anti-apoptotic regulating proteins, and in particular Hexokinase isoforms I and II, its main ligand. Hexokinase I (HK I) is found in brain tissues and is known as the “guardian of the mitochondria”. HK possess a hydrophobic N-terminal structured in α-helix that is necessary for the binding of HK to VDAC6. The interaction between HK and VDAC1 has attracted much interest, and several studies have proposed different models of binding to VDAC17,8. However, the precise mechanism of binding remains undefined up to now. In order to give some insights to the nature of HK binding to VDAC1, we have developed new peptide analogs of HK I based on Hexokinase I N-terminal sequence. In this study, the in-vitro activity of the peptides was studied, the peptide helical content was investigated by circular dichroism and NMR, and peptide proteolytic stability was assessed. (1) Shoshan-Barmatz, V.; Shteinfer-Kuzmine, A.; Verma, A. VDAC1 at the Intersection of Cell Metabolism, Apoptosis, and Diseases. Biomolecules 2020, 10 (11). https://doi.org/10.3390/biom10111485.(2) Abramov, A. Y.; Berezhnov, A. V.; Fedotova, E. I.; Zinchenko, V. P.; Dolgacheva, L. P. Interaction of Misfolded Proteins and Mitochondria in Neurodegenerative Disorders. Biochem Soc Trans 2017, 45 (4), 1025–1033. https://doi.org/10.1042/BST20170024.(3) Shoshan-Barmatz, V.; Ben-Hail, D. VDAC, a Multi-Functional Mitochondrial Protein as a Pharmacological Target. Mitochondrion 2012, 12 (1), 24–34. https://doi.org/10.1016/j.mito.2011.04.001.(4) Shoshan-Barmatz, V.; Krelin, Y.; Shteinfer-Kuzmine, A. VDAC1 Functions in Ca2+ Homeostasis and Cell Life and Death in Health and Disease. Cell Calcium 2018, 69, 81–100. https://doi.org/10.1016/j.ceca.2017.06.007.(5) Magri, A.; Messina, A. Interactions of VDAC with Proteins Involved in Neurodegenerative Aggregation: An Opportunity for Advancement on Therapeutic Molecules. Curr Med Chem 2017, 24 (40), 4470–4487. https://doi.org/10.2174/0929867324666170601073920.(6) Xie, G. C.; Wilson, J. E. Rat Brain Hexokinase: The Hydrophobic N-Terminus of the Mitochondrially Bound Enzyme Is Inserted in the Lipid Bilayer. Arch Biochem Biophys 1988, 267 (2), 803–810. https://doi.org/10.1016/0003-9861(88)90090-2.(7) Rosano, C. Molecular Model of Hexokinase Binding to the Outer Mitochondrial Membrane Porin (VDAC1): Implication for the Design of New Cancer Therapies. Mitochondrion 2011, 11 (3), 513–519. https://doi.org/10.1016/j.mito.2011.01.012.(8) Abu-Hamad, S.; Zaid, H.; Israelson, A.; Nahon, E.; Shoshan-Barmatz, V. Hexokinase-I Protection against Apoptotic Cell Death Is Mediated via Interaction with the Voltage-Dependent Anion Channel-1: MAPPING THE SITE OF BINDING. J. Biol. Chem. 2008, 283 (19), 13482–13490. https://doi.org/10.1074/jbc.M708216200.

Charcot-Marie-Tooth disease 1 A (CMT1A) results from a duplication of the PMP22 gene in Schwann cells and a deficit of myelination in peripheral nerves. Patients with CMT1A have reduced nerve conduction velocity, muscle wasting, hand and foot deformations and foot drop walking. Here, we evaluate the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9) expressing GFP and shRNAs targeting Pmp22 mRNA in animal models of Charcot-Marie-Tooth disease 1 A. Intra-nerve delivery of AAV2/9 in the sciatic nerve allowed widespread transgene expression in resident myelinating Schwann cells in mice, rats and non-human primates. A bilateral treatment restore expression levels of PMP22 comparable to wild-type conditions, resulting in increased myelination and prevention of motor and sensory impairments over a twelve-months period in a rat model of CMT1A. We observed limited off-target transduction and immune response using the intra-nerve delivery route. A combination of previously characterized human skin biomarkers is able to discriminate between treated and untreated animals, indicating their potential use as part of outcome measures.

Summary Whether glial cells use a particular metabolism to support axonal metabolism and function remains controversial. We show here that the deletion of PKM2, an enzyme essential for the Warburg effect, in mature myelinating Schwann cells (mSC) leads to a deficit of lactate in these cells and in peripheral nerves, and to motor defects despite no alteration of the myelin sheath. When electrically stimulated, peripheral nerve axons of mSC-PKM2 mutant mice failed to maintain lactate homeostasis, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not changed but axonal mitochondria transport was altered, muscle axon terminals retracted and motor neurons showed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment further aggravated axonal malfunction in mutant mice. Thus, cancer-like Warburg effect is essential in mSC for the long-term maintenance of peripheral nerve axons physiology and function. One Sentence Summary Lactate-dependent axons rely on Warburg effect in Schwann cells.

Charcot-Marie-Tooth disease 1A (CMT1A) results from a duplication of the gene leading to an excess of PMP22, a deficit of myelination and an instability of the myelin sheath in peripheral nerves. Patients present with reduced nerve conduction velocity, muscle waste, hand and foot deformations and foot drop walking problems. As gene silencing therapy has been shown to be effective in other monogenic neurological disorders, we evaluated the safety and efficacy of recombinant adeno-associated viral vector serotype 9 (AAV2/9)-based gene therapy for CMT1A. AAV2/9-mediated delivery of eGFP and shRNAs targeting PMP22 mRNA in the sciatic nerve allowed widespread gene expression in myelinating Schwann cells in mouse, rat and nonhuman primate. The treatment restored wild-type PMP22 level, increased myelination and prevented motor and sensory impairment over 12 months in a rat model of CMT1A. Intra-nerve injection limited off-target transduction and immune response to barely detectable levels. A combination of previously characterized human skin biomarkers successfully discriminated treated animals from their untreated littermate controls indicating their potential use as part of outcome measures in future clinical trials. Our results support intra nerve injection of AAV2/9 as an effective strategy for the treatment of CMT1A as well as other demyelinating CMT diseases.

Affiliations: 1 INM, INSERM, Université de Montpellier, 80 Rue A. Fliche, 34090 Montpellier, France. 2 Aix-Marseille University, INSERM, MMG, 13385 Marseille, France 15 3 Departments of Clinical Neuroscience and Neuroscience, Karolinska Intitutet, Stockholm, Sweden. 4 Département de Biochimie et Génétique, Centre Hospitalier Universitaire, Angers, France. 5 Equipe Mitolab, MITOVASC, CNRS 6015, INSERM U1083, Université d'Angers, Angers, France. 20

Summary: Whether glial cells use a particular metabolism to support axonal metabolism and function remains controversial. We show here that the deletion of PKM2, an enzyme essential for the Warburg effect, in mature myelinating Schwann cells (mSC) leads to a deficit of lactate in these cells and in peripheral nerves, and to motor defects despite no alteration of the myelin 30 sheath. When electrically stimulated, peripheral nerve axons of mSC-PKM2 mutant mice failed to maintain lactate homeostasis, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not changed but axonal mitochondria transport was altered, muscle axon terminals retracted and motor neurons showed cellular stress. Further reducing lactate availability through dichloroacetate treatment definitely aggravated axonal malfunction in 35 mutant mice. Thus, cancer-like Warburg effect is essential in mSC for the long-term maintenance of peripheral nerve axons physiology and function.

Overweight and obesity are known to impact male fertility and are commonly associated with abdominal obesity and metabolic disorders. The association between abdominal obesity or metabolic syndrome with male reproduction has not been fully investigated. Moreover, many factors may interfere with the evaluation of the impact of metabolic syndrome on male fertility. Thus, tobacco is known to alter the spermatic parameters and phenomena linking smoking with metabolic syndrome are therefore complex. The main objective of this study has been to investigate the potential association of metabolic syndrome with male idiopathic infertility given smoking status. The data of this study concerned infertile (n = 96) and fertile (n = 100) men under 45 years of age who have been recruited in the ALIFERT case-control study. Body mass index and waist circumference were measured. Serum triglycerides, cholesterol (total, high density lipoprotein, and low density lipoprotein cholesterol) and fasting blood glucose were assayed. Metabolic syndrome has been diagnosed in the presence of at least three of the following criteria: increased waist circumference, high triglycerides, fasting glucose or arterial blood pressure and low high density lipoprotein cholesterol. The present study reports that infertile men are in poorer health condition compared to fertile men and are more often smokers. The results of this study suggested metabolic syndrome and smoking to be independent risk factor for idiopathic infertility. Metabolic syndrome and smoking should systematically be checked at the beginning of medical care in infertile males and personal and multifaceted coaching should be proposed to deal jointly with smoking and metabolic disorders. NCT01093378 ALIFERT. Registered: March 25, 2010.

MOTIVATION:In the continuously expanding omics era, novel computational and statistical strategies are needed for data integration and identification of biomarkers and molecular signatures. We present Data Integration Analysis for Biomarker discovery using Latent cOmponents (DIABLO), a multi-omics integrative method that seeks for common information across different data types through the selection of a subset of molecular features, while discriminating between multiple phenotypic groups.RESULTS:Using simulations and benchmark multi-omics studies, we show that DIABLO identifies features with superior biological relevance compared with existing unsupervised integrative methods, while achieving predictive performance comparable to state-of-the-art supervised approaches. DIABLO is versatile, allowing for modular-based analyses and cross-over study designs. In two case studies, DIABLO identified both known and novel multi-omics biomarkers consisting of mRNAs, miRNAs, CpGs, proteins and metabolites.AVAILABILITY AND IMPLEMENTATION:DIABLO is implemented in the mixOmics R Bioconductor package with functions for parameters' choice and visualization to assist in the interpretation of the integrative analyses, along with tutorials on http://mixomics.org and in our Bioconductor vignette.SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.

Mitochondria are critical for the function and maintenance of myelinated axons notably through Adenosine triphosphate (ATP) production. A direct by-product of this ATP production is reactive oxygen species (ROS), which are highly deleterious for neurons. While ATP shortage and ROS levels increase are involved in several neurodegenerative diseases, it is still unclear whether the real-time dynamics of both ATP and ROS production in axonal mitochondria are altered by axonal or demyelinating neuropathies. To answer this question, we imaged and quantified mitochondrial ATP and hydrogen peroxide (H2O2) in resting or stimulated peripheral nerve myelinated axons in vivo, using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. We found that ATP and H2O2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H2O2 productions but with different dynamics: ROS production peaked shortly and transiently after the stimulation while ATP production increased gradually for a longer period of time. In neuropathic MFN2R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, elevated H2O2 production was largely sustained. Finally, inducing demyelination with lysophosphatidylcholine resulted in a reduced level of ATP while H2O2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity.

Objective. We tested the discriminatory capacity of diffusion-weighted magnetic resonance imaging (DWI) and its potential as an objective measure of treatment response to tumor necrosis factor inhibition in ankylosing spondylitis (AS). Methods. Three cohorts were studied prospectively: (1) 18 AS patients with Bath Ankylosing Spondylitis Disease Activity Index > 4, and erythrocyte sedimentation rate > 25 and/or C-reactive protein > 10 meeting the modified New York criteria for AS; (2) 20 cases of nonradiographic axial spondyloarthritis (nr-axSpA) as defined by the Assessment of Spondyloarthritis international Society (ASAS) criteria; and (3) 20 non-AS patients with chronic low back pain, aged between 18 and 45 years, who did not meet the imaging arm of the ASAS criteria for axSpA. Group 1 patients were studied prior to and following adalimumab treatment. Patients were assessed by DWI and conventional magnetic resonance imaging (MRI), and standard nonimaging measures. Results. At baseline, in contrast to standard nonimaging measures, DWI apparent diffusion coefficient (ADC) values showed good discriminatory performance [area under the curve (AUC) > 80% for Group 1 or 2 compared with Group 3]. DWI ADC values were significantly lower posttreatment (0.45 ± 0.433 before, 0.154 ± 0.23 after, p = 0.0017), but had modest discriminating capacity comparing pre– and posttreatment measures (AUC = 68%). This performance was similar to the manual Spondyloarthritis Research Consortium of Canada (SPARCC) scoring system. Conclusion. DWI is informative for diagnosis of AS and nr-axSpA, and has moderate utility in assessment of disease activity or treatment response, with performance similar to that of the SPARCC MRI score.

Systems biology approaches, leveraging multi-omics measurements, are needed to capture the complexity of biological networks while identifying the key molecular drivers of disease mechanisms. We present DIABLO, a novel integrative method to identify multi-omics biomarker panels that can discriminate between multiple phenotypic groups. In the multi-omics analyses of simulated and real-world datasets, DIABLO resulted in superior biological enrichment compared to other integrative methods, and achieved comparable predictive performance with existing multi-step classification schemes. DIABLO is a versatile approach that will benefit a diverse range of research areas, where multiple high dimensional datasets are available for the same set of specimens. DIABLO is implemented along with tools for model selection, and validation, as well as graphical outputs to assist in the interpretation of these integrative analyses (<http://mixomics.org/>).

Mitochondria are critical for the function and maintenance of myelinated axons notably through ATP production. A by-product of this activity is reactive oxygen species (ROS), which are highly deleterious for neurons. While ROS and metabolism are involved in several neurodegenerative diseases, it is still unclear how axonal activity or myelin modulates ATP and ROS production in axonal mitochondria. We imaged and quantified mitochondrial ATP and hydrogen peroxide (H 2 O 2 ) in resting or stimulated peripheral nerve myelinated axons in vivo , using genetically-encoded fluorescent probes, two-photon time-lapse and CARS imaging. ATP and H 2 O 2 productions are intrinsically higher in nodes of Ranvier even in resting conditions. Axonal firing increased both ATP and H 2 O 2 productions but with different dynamics. In neuropathic MFN2 R94Q mice, mimicking Charcot-Marie-Tooth 2A disease, defective mitochondria failed to upregulate ATP production following axonal activity. However, H 2 O 2 production was dramatically sustained. Mimicking demyelinating peripheral neuropathy resulted in a reduced production of ATP while H 2 O 2 level soared. Taken together, our results suggest that ATP and ROS productions are decoupled under neuropathic conditions, which may compromise axonal function and integrity.

Rapid advances in technology have led to a wealth of large-scale molecular omics datasets. Integrating such data offers an unprecedented opportunity to assess molecular interactions at multiple functional levels and provide a more comprehensive understanding of the biological pathways involved in different diseases subgroups. However, multiple omics data integration is a challenging task due to the heterogeneity in the different platforms used. There is a need to address the complex and correlated nature of different data-types, in order to identify a robust and reliable multi-omics signature that can predict a phenotype of interest. We introduce a novel multivariate dimension reduction method for multiple omics integration, classification and identification of a multi-omics molecular signature. DIABLO - Data Integration Analysis for Biomarker discovery using a Latent component method for Omics studies, models the correlation structure between omics datasets, resulting in an improved ability to associate biomarkers across multiple functional levels to phenotypes of interest. We demonstrate the capabilities of DIABLO using simulated data and studies of breast cancer and asthma, integrating up to four types of omics datasets to identify relevant biomarkers, while still retaining competitive classification and predictive performance compared to existing methods. Our statistical integrative framework can benefit a diverse range of research areas with varying types of study designs, as well as enabling module-based analyses. Importantly, graphical outputs of our method assist in the interpretation of such complex analyses and provide significant biological insights.

The advent of high throughput technologies has led to a wealth of publicly available 'omics data coming from different sources, such as transcriptomics, proteomics, metabolomics. Combining such large-scale biological data sets can lead to the discovery of important biological insights, provided that relevant information can be extracted in a holistic manner. Current statistical approaches have been focusing on identifying small subsets of molecules (a 'molecular signature') to explain or predict biological conditions, but mainly for a single type of 'omics. In addition, commonly used methods are univariate and consider each biological feature independently. We introduce mixOmics, an R package dedicated to the multivariate analysis of biological data sets with a specific focus on data exploration, dimension reduction and visualisation. By adopting a systems biology approach, the toolkit provides a wide range of methods that statistically integrate several data sets at once to probe relationships between heterogeneous 'omics data sets. Our recent methods extend Projection to Latent Structure (PLS) models for discriminant analysis, for data integration across multiple 'omics data or across independent studies, and for the identification of molecular signatures. We illustrate our latest mixOmics integrative frameworks for the multivariate analyses of 'omics data available from the package.