Inflammation, Oxidative Stress, Senescence in Atherosclerosis: Thioredoxine-1 as an Emerging Therapeutic Target.

Atherosclerosis is a leading cause of cardiovascular diseases (CVD) worldwide and intimately linked to aging. This pathology is characterized by chronic inflammation, oxidative stress, gradual accumulation of low-density lipoproteins (LDL) particles and fibrous elements in focal areas of large and medium arteries. These fibrofatty lesions in the artery wall become progressively unstable and thrombogenic leading to heart attack, stroke or other severe heart ischemic syndromes. Elevated blood levels of LDL are major triggering events for atherosclerosis. A cascade of molecular and cellular events results in the atherosclerotic plaque formation, evolution, and rupture. Moreover, the senescence of multiple cell types present in the vasculature were reported to contribute to atherosclerotic plaque progression and destabilization. Classical therapeutic interventions consist of lipid-lowering drugs, anti-inflammatory and life style dispositions. Moreover, targeting oxidative stress by developing innovative antioxidant agents or boosting antioxidant systems is also a well-established strategy. Accumulation of senescent cells (SC) is also another important feature of atherosclerosis and was detected in various models. Hence, targeting SCs appears as an emerging therapeutic option, since senolytic agents favorably disturb atherosclerotic plaques. In this review, we propose a survey of the impact of inflammation, oxidative stress, and senescence in atherosclerosis; and the emerging therapeutic options, including thioredoxin-based approaches such as anti-oxidant, anti-inflammatory, and anti-atherogenic strategy with promising potential of senomodulation.

Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration.

NAD+ depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD+ was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD+ to NR prompted us to probe the effects of NAD+ and NR in protection against excitotoxicity. Here, we show that intracortical administration of NR but not NAD+ reduces brain damage induced by NMDA injection. Using cortical neurons, we found that provision of extracellular NR delays NMDA-induced axonal degeneration (AxD) much more strongly than extracellular NAD+ Moreover, the stronger effect of NR compared to NAD+ depends of axonal stress since in AxD induced by pharmacological inhibition of nicotinamide salvage, both NAD+ and NR prevent neuronal death and AxD in a manner that depends on internalization of NR. Taken together, our findings demonstrate that NR is a better neuroprotective agent than NAD+ in excitotoxicity-induced AxD and that axonal protection involves defending intracellular NAD+ homeostasis.-Vaur, P., Brugg, B., Mericskay, M., Li, Z., Schmidt, M. S., Vivien, D., Orset, C., Jacotot, E., Brenner, C., Duplus, E. Nicotinamide riboside, a form of vitamin B3, protects against excitotoxicity-induced axonal degeneration.

NAD+ acts on mitochondrial SirT3 to prevent axonal caspase activation and axonal degeneration.

In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.

A new model and precious tool to study molecular mechanisms of macrophage aging.

The accumulation of senescent cells, characterized by a senescence-associated secretory phenotype (SASP), contributes to chronic inflammation and age-related diseases (ARD). During aging, macrophages can adopt a senescent-like phenotype and an altered function, which promotes senescent cell accumulation. In the context of aging and ARD, controlling the resolution of the inflammatory response and preventing chronic inflammation, especially by targeting macrophages, must be a priority. Aging being a dynamic process, we developed a model of in vitro murine peritoneal macrophage aging. Our results show that macrophages cultured for 7 or 14 days exhibit a senescence-like phenotype: proliferation decrease, the levels of cyclin-dependent kinase inhibitors p16INK4A and p21CIP1 and of pro-inflammatory SASP components (MCP-1, IL-6, IL-1ß, TNF-α, and MMP-9) increase, phagocytosis capacity decline and glycolytic activity is induced. In our model, chronic treatment with CB3, a thioredoxin-1 mimetic anti-inflammatory peptide, completely prevents p21CIP1 increase and enables day 14 macrophages to maintain proliferative activity.We describe a new model of macrophage aging with a senescence-like phenotype associated with inflammatory, metabolic and functional perturbations. This model is a valuable tool for characterizing macrophage aging mechanisms and developing innovative strategies with promising therapeutical purpose in limiting inflammaging and ARD.

Cognitive and hippocampal effects of adult male mice exposure to environmentally relevant doses of phthalates.

We recently showed that chronic exposure of adult male mice to environmental doses of DEHP alone or in a phthalate mixture altered blood brain barrier integrity and induced an inflammatory profile in the hippocampus. Here, we investigate whether such exposure alters hippocampus-dependent behavior and underlying cellular mechanisms. Adult C57BL/6 J male mice were continuously exposed orally to the vehicle or DEHP alone (5 or 50 µg/kg/d) or to DEHP (5 µg/kg/d) in a phthalate mixture. In the Morris water maze, males showed reduced latencies across days to find the platform in the cue and spatial reference memory tasks, regardless of their treatment group. In the probe test, DEHP-50 exposed males displayed a higher latency to find the platform quadrant. In the temporal order memory test, males exposed to DEHP alone or in a phthalate mixture were unable to discriminate between the most recently and previously seen objects. They also displayed reduced ability to show a preference for the new object in the novel object recognition test. These behavioral alterations were associated with a lowered dendritic spine density and protein levels of glutamate receptors and postsynaptic markers, and increased protein levels of the presynaptic synaptophysin in the hippocampus. Metabolomic analysis of the hippocampus indicated changes in amino acid levels including reduced tryptophan and L-kynurenine and elevated NAD + levels, respectively, a precursor, intermediate and endproduct of the kynurenine pathway of tryptophan metabolism. Interestingly, the protein amounts of the xenobiotic aryl hydrocarbon receptor, a target of this metabolic pathway, were elevated in the CA1 area. These data indicate that chronic exposure of adult male mice to environmental doses of DEHP alone or in a phthalate mixture impacted hippocampal function and structure, associated with modifications in amino acid metabolites with a potential involvement of the kynurenine pathway of tryptophan metabolism.

Behavior, Neural Structure, and Metabolism in Adult Male Mice Exposed to Environmentally Relevant Doses of Di(2-ethylhexyl) Phthalate Alone or in a Phthalate Mixture.

BACKGROUND: We have previously shown that chronic exposure of adult male mice to low doses of di(2-ethylhexyl) phthalate (DEHP) altered male sexual behavior and induced down-regulation of the androgen receptor (AR) in the neural circuitry controlling this behavior. OBJECTIVES: The cellular mechanisms induced by chronic exposure of adult male mice to low doses of DEHP alone or in an environmental phthalate mixture were studied. METHODS: Two-month-old C57BL/6J males were exposed orally for 8 wk to DEHP alone (0, 5, or 50µg/kg/d) or to DEHP (50µg/kg/d) in a phthalate mixture. Behavior, dendritic density per 50-µm length, pre-/postsynaptic markers, synapse ultrastructure, and bioenergetic activity were analyzed. RESULTS: Mice exposed to DEHP either alone or in a phthalate mixture differed in mating, emission of ultrasonic vocalizations, and the ability to attract receptive females in urinary preference tests from control mice. Analyses in the medial preoptic area, the key hypothalamic region involved in male sexual behavior, showed lower dendritic spine density and protein levels of glutamate receptors and differences in other postsynaptic components and presynaptic markers between the treated groups. Ultrastructural observation of dendritic synapses by electron microscopy showed comparable morphology between the treated groups. Metabolic analyses highlighted differences in hypothalamic metabolites of males exposed to DEHP alone or in a phthalate mixture compared to control mice. These differences included lower tryptophan and higher NAD+ levels, respectively, a precursor and end product of the kynurenine pathway of tryptophan metabolism. The protein amounts of the xenobiotic aryl hydrocarbon receptor, one of the targets of this metabolic pathway and known negative regulator of the AR, were higher in the medial preoptic area of exposed male mice. DISCUSSION: Differences in behavior of male mice exposed to environmental doses of phthalates were associated with differences in neural structure and metabolism, with possibly a key role of the kynurenine pathway of tryptophan metabolism in the effects mediated by these substances. https://doi.org/10.1289/EHP11514.

Glioblastoma cell motility depends on enhanced oxidative stress coupled with mobilization of a sulfurtransferase.

Cell motility is critical for tumor malignancy. Metabolism being an obligatory step in shaping cell behavior, we looked for metabolic weaknesses shared by motile cells across the diverse genetic contexts of patients' glioblastoma. Computational analyses of single-cell transcriptomes from thirty patients' tumors isolated cells with high motile potential and highlighted their metabolic specificities. These cells were characterized by enhanced mitochondrial load and oxidative stress coupled with mobilization of the cysteine metabolism enzyme 3-Mercaptopyruvate sulfurtransferase (MPST). Functional assays with patients' tumor-derived cells and -tissue organoids, and genetic and pharmacological manipulations confirmed that the cells depend on enhanced ROS production and MPST activity for their motility. MPST action involved protection of protein cysteine residues from damaging hyperoxidation. Its knockdown translated in reduced tumor burden, and a robust increase in mice survival. Starting from cell-by-cell analyses of the patients' tumors, our work unravels metabolic dependencies of cell malignancy maintained across heterogeneous genomic landscapes.

Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics.

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases, including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of an active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics show that these new compounds, such as LJ2 or its specific isomers LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. In agreement with the established role of Casp2 in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~5,500,000 M-1 s-1), and the most selective inhibitor, LJ3a, has close to a 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that the spatial configuration of Cα at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit Casp2-mediated S1P cleavage and thus SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with ß-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

Identification of First-in-Class Inhibitors of Kallikrein-Related Peptidase 6 That Promote Oligodendrocyte Differentiation.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system (CNS) that causes severe motor, sensory, and cognitive impairments. Kallikrein-related peptidase (KLK)6 is the most abundant serine protease secreted in the CNS, mainly by oligodendrocytes, the myelin-producing cells of the CNS, and KLK6 is assumed to be a robust biomarker of MS, since it is highly increased in the cerebrospinal fluid (CSF) of MS patients. Here, we report the design and biological evaluation of KLK6's low-molecular-weight inhibitors, para-aminobenzyl derivatives. Interestingly, selected hit compounds were selective of the KLK6 proteolytic network encompassing KLK1 and plasmin that also participate in the development of MS physiopathology. Moreover, hits were found noncytotoxic on primary cultures of murine neurons and oligodendrocyte precursor cells (OPCs). Among them, two compounds (32 and 42) were shown to promote the differentiation of OPCs into mature oligodendrocytes in vitro constituting thus emerging leads for the development of regenerative therapies.

Phosphorylation and transcriptional activity regulation of retinoid-related orphan receptor alpha 1 by protein kinases C.

Retinoid-related orphan receptor alpha1 (RORalpha1) is a member of the nuclear receptor superfamily. It is highly expressed in CNS particularly in the cerebellum. Absence of this transcription factor in mice leads to several abnormalities, such as cerebellar atrophy linked to Purkinje cell death and impaired differentiation. A major role of RORalpha1 in neuronal survival is the control of reactive oxygen species homeostasis. RORalpha1 is a constitutively active receptor, but its regulation is yet not well known. Protein kinase C (PKC) also plays a major role in neuronal survival and differentiation, suggesting its possible involvement in post-translational modifications and regulation of RORalpha1 transcriptional activity. To test this hypothesis, we over-expressed the human isoform of this nuclear receptor in cortical neurons and COS-7 cells, which were then treated with different effectors acting on PKC activity. We showed for the first time that conventional PKCs induce phosphorylation and inhibition of RORalpha1 activity. We also investigated mitogen-activated protein kinase/extracellular signal-regulated kinase (1/2) involvement in this effect. Our results bring new insights into the control of RORalpha1 activity and highlight its importance in further investigations of the mechanisms involved in neuronal cell death in neurodegenerative diseases.

Acute amnestic encephalopathy in amyloid-ß oligomer-injected mice is due to their widespread diffusion in vivo.

Amyloid-ß (Aß) oligomers are the suspected culprit as initiators of Alzheimer's disease (AD). However, their diffusion in the brain remains unknown. Here, we studied Aß oligomers' dissemination and evaluated their in vivo toxicity. Wild-type mice were injected with 50 pmol of synthetic Aß oligomers (of different size) in the hippocampus. Oligomers diffused largely in the brain as soon as 1 hour and up to 7 days after injection. A transient encephalopathy with memory impairment was induced by this unique injection. The immunoreactivity of the postsynaptic marker PSD95 was diffusely decreased. Similar results (both on memory and PSD95 immunoreactivity) were obtained with delipidated and high molecular weight oligomers (>50 kDa) but not with smaller assemblies. Tau hyperphosphorylation was observed in the oligomer-injected brains. Finally, fos immunostaining was increased in Aß-derived diffusible ligands-injected mice, suggesting neuronal hyperactivity. Rapid and widespread diffusion of Aß oligomers was demonstrated in vivo and associated with decreased synaptic markers and memory deficits which gives new insight to the pathogenicity of Aß.

Expression of phosphoenolpyruvate carboxykinase gene in human adipose tissue: induction by rosiglitazone and genetic analyses of the adipocyte-specific region of the promoter in type 2 diabetes.

Cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) is the key enzyme in glyceroneogenesis, an important metabolic pathway in adipocytes for reesterification of fatty acids during fasting. Dysregulation of glyceroneogenesis could play a role in the increase in plasma non-esterified fatty acids that accompanies type 2 diabetes. In rodent adipocyte transcription of the PEPCK-C gene is induced by thiazolidinediones (TZDs) through an element, named PCK2, in its promoter. PCK2 binds a peroxisome proliferator activated receptor gamma (PPARgamma), retinoid X receptor alpha (RXRalpha) heterodimer. We demonstrated that in cultured human subcutaneous adipose tissue explants, PEPCK-C specific activity and mRNA were induced by 1 microM of the TZD rosiglitazone, respectively, about twofold in 8 h and fivefold in 5 h. Using gel shift experiments, we show that this effect is likely to involve the human PCK2 (hPCK2) element, which binds a protein complex that contains PPARgamma and RXRalpha. We analyzed hPCK2 (position -1031 to -1015 base pairs) and nearby sequences in the PEPCK-C promoter in 403 subjects with type 2 diabetes and 123 non-diabetic controls. The sequence of hPCK2 was not polymorphic, but we detected two C/T single nucleotide polymorphisms (SNPs), in complete linkage disequilibrium, at positions -1097 and -967 bp. Allele and genotype frequencies were not significantly different in patients and controls. However, our results suggest co-dominant effects of C and T-alleles on fasting plasma glucose and glycosylated hemoglobin A1c levels in obese type 2 diabetic patients.

Is there a single mechanism for fatty acid regulation of gene transcription?

Besides their role as energetic molecules, fatty acids (FAs) also act as signals involved in regulating gene expression. This review focuses on a few examples of FA regulation. The hepatic lipogenic enzyme, fatty acid synthase (FAS) is negatively regulated by polyunsaturated FAs (PUFAs) which suppress sterol regulatory element-binding protein 1 (SREBP 1) gene expression and nuclear content in hepatocytes, thereby reducing FAS gene transcription. It was proposed recently that this reduction in SREBP 1 was the result of a PUFA-induced antagonism of ligand-dependent activation of the liver X nuclear receptor (LXR), known to be an inducer of the SREBP 1 gene. In contrast, several genes are turned on by long-chain (LCFAs) and nonmetabolized FAs in a physiologically relevant manner. These include the acyl-CoA oxidase (AOX), the liver carnitine palmitoyltransferase 1 (L-CPT 1) and the liver fatty acid binding protein (L-FABP). While induction of AOX gene transcription appears to be PPARalpha-dependent, that of the L-CPT 1 gene seems disconnected from PPAR activation. Results obtained in preadipocytes and in intestine cells are in support of a key role played by the beta/delta isoform of PPAR in LCFA induction of the FABP gene. Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is stimulated by unsaturated and nonmetabolized LCFAs specifically in adipocytes. Our results reported here support the notion that the mechanisms by which PPARgamma activators and FAs induce transcription of the PEPCK gene are distinct. Altogether these data argue that several FA effects are PPAR-independent. Evidences suggesting that other transcription factors might be involved are debated. It seems now clear that depending upon the cell-specific context and the target gene, FAs can take very different routes to alter transcription.

ß-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network.

INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level. RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (µFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied ß-amyloid (Aß) peptides locally to the different cellular compartments. We observed that Aß application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aß deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aß peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aß stress at the network level we developed new µFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aß application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration. CONCLUSION: Thanks to µFD-based reconstructed neuronal networks we evaluated the distant effects of local Aß stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aß production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aß can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

Evidence for selective induction of phosphoenolpyruvate carboxykinase gene expression by unsaturated and nonmetabolized fatty acids in adipocytes.

Polyunsaturated fatty acids (PUFAs) and 3-thia fatty acids are hypolipidemic and decrease insulin resistance in Type II diabetic animals. To exert such an action, these FAs could decrease adipose tissue lipolysis or increase esterification. Glyceroneogenesis is an important metabolic pathway in adipocytes for re-esterification of FAs originating from lipolysis and in hepatocytes for triacylglycerol synthesis during fasting. Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) plays a key role in this pathway. Here we show that the PUFA docosahexaenoic acid (DHA) stimulates PEPCK mRNA in glucose-deprived adipose tissue explants from fed rats and in 3T3-F442A differentiated adipocytes. This effect is maximum at 3 h, stable up to at least 11 h of treatment, and affects the transcription of the gene. PEPCK mRNA half-life is not affected. Among a series of adipocyte transcripts, only the adipocyte lipid binding protein mRNA is also increased by DHA, although later than the PEPCK mRNA and at a much lower extent. DHA has no effect on PEPCK gene expression in the H4IIE hepatoma cells in which this gene is responsive to other inducers like cAMP. This lack of effect is not due to a failure of DHA to act in H4IIE cells since it induces the carnitine palmitoyltransferase 1 (CPT-1) mRNA. Therefore, the DHA effect appears to be cell-selective. Results of experiments using either tetradecylthio acetic acid and alpha-bromopalmitate, two nonmetabolized Fas, or a series of inhibitors of FA metabolism show that the FA effect on PEPCK mRNA is not due to a product of its metabolism. Hence, polyunsaturated and nonmetabolized FAs stimulate adipose PEPCK, therefore potentially enhancing glyceroneogenesis and reducing FA output. This mechanism could participate in the hypolipidemic action of PUFAs.