Tumor-associated macrophages and microglia (TAMs) are highly abundant myeloid cells in gliomas, with their phenotype and immune response determined by ontogeny and microenvironment. TAMs display distinctive transcriptional programs according to the IDH mutation status but the underlying regulatory mechanisms remain largely unknown. Herein, we uncovered that CD11B+ myeloid cells in human IDHmut gliomas exhibit DNA hypermethylation predominantly at distal enhancers. This hypermethylation was linked to decreased expression of genes involved in inflammatory responses and glycolytic metabolism, and the inactivation of transcription factors that regulate the microglial response to environmental stimuli. Prolonged exposure of human primary microglia to D-2-hydroxyglutarate (D-2HG) inhibits TET-mediated 5mC oxidation, resulting in a reduced accumulation of global 5hmC levels. We confirmed high 5mC/5hmC ratios at lineage-specific enhancers, by analyzing CpGs at single-base resolution. D-2HG-treated microglia show reduced proinflammatory capacity and enhanced oxidative phosphorylation, consistent with the remodeled enhancer landscape. Conversely, depletion of D-2HG following treatment of a glioma patient with a mutant IDH inhibitor unleashed an activating response in microglia, as assessed by snRNA-seq. Our findings provide a mechanistic rationale for the hyporesponsive state of microglia in IDHmut gliomas and support the concept that oncometabolites may disrupt the function of immune cells residing in the tumor microenvironment. ### Competing Interest Statement L.A.S. is a scientific advisor and co-founder of Cellintec L.L.C., which had no role in this research. M.L.S. is an equity holder, scientific co-founder, and advisory board member of Immunitas Therapeutics. The other authors declare no competing or financial interests.

Abstract BACKGROUND Resident microglia are highly plastic myeloid cells. Their phenotype and immune response are determined by the microenvironment composition. Although microglial cells are highly abundant in IDH-mutated (IDHm) gliomas, how this particular environment modulates the transcriptional programs and cell states of these cells remain largely uncharacterized. Herein we investigated whether and how D-2HG, the oncometabolite produced by the IDH mutation and released into the tumor microenvironment, impinges upon the phenotypic and functional properties of resident microglia. MATERIAL AND METHODS We analyzed bulk DNA methylome (Methylation EPIC array) and transcriptome of CD11b+magnetic-purified fractions from IDHm (n= 25) and IDH-wildtype (n=11) gliomas, as well from normal brain tissues (n=6). To determine direct effects of D-2-hydroxyglutarate (D-2HG), the oncometabolite produced by IDHm glioma cells, we optimized human primary microglial cultures obtained from glioma or drug-resistant epilepsy surgery. Microglia exposed to 5mM D-2HG or vehicle for 14 days were analyzed with DNA methylome (LC-MS/MS, EPIC arrays and single-base resolution approaches) and transcriptome (RNA-seq). Using snRNA-seq, we analyzed microglial transcriptome from a paired sample of one IDHm glioma patient before and after treatment with the IDH-inhibitor vorasidenib, which is known to deplete tumor D-2HG levels. RESULTS We uncovered that CD11b+ myeloid cells in human IDHm gliomas exhibit DNA hypermethylation predominantly at enhancer elements. Hypermethylation was linked to decreased expression of genes involved in inflammatory responses and glycolytic metabolism, as well as inactivation of transcription factors that regulate microglial responses to environmental stimuli. Prolonged exposure of human primary microglia to D-2HG inhibited TET-mediated 5mC oxidation, resulting in a reduced accumulation of global 5hmC levels. We observed high 5mC/5hmC ratios particularly at lineage-specific enhancers. Consistent with the remodeled enhancer landscape, D-2HG-treated microglia showed reduced proinflammatory capacity and enhanced oxidative phosphorylation. Conversely, depletion of D-2HG levels following vorasidenib treatment in an IDHm glioma patient was associated with overexpression of genes related to microglial activation. CONCLUSION Our findings provide a mechanistic rationale for the hyporesponsive state of microglia in IDHm gliomas and support the hypothesis that oncometabolites such as D-2HG may disrupt the function of immune cells residing in the tumor microenvironment.

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-1β, 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.

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.

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

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.

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 ( k 3 / K i ~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 C α 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 β-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

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.

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.

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.

Amyloid-beta (A beta) oligomers are the suspected culprit as initiators of Alzheimer's disease (AD). However, their diffusion in the brain remains unknown. Here, we studied A beta oligomers' dissemination and evaluated their in vivo toxicity. Wild-type mice were injected with 50 pmol of synthetic A beta 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 A beta-derived diffusible ligandseinjected mice, suggesting neuronal hyperactivity. Rapid and widespread diffusion of Ab oligomers was demonstrated in vivo and associated with decreased synaptic markers and memory deficits which gives new insight to the pathogenicity of A beta. (C) 2015 Elsevier Inc. All rights reserved.

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 β-amyloid (Aβ) peptides locally to the different cellular compartments. We observed that Aβ 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 Aβ deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aβ peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aβ 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. Aβ 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 Aβ 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 Aβ production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aβ can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

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.

The brain is a complex structure comprising many different neuronal populations making specific and polarized connections. The establishment of synaptic contacts are key events in brain development, and conversely, synaptic degeneration is an early and seminal process in various acute syndromes and neurodegenerative diseases eg, Alzheimer's disease (AD). The molecular mechanisms involved in both synaptic and axonal degeneration remain to be elucidated. Importantly, since neurons are highly polarized cells, both chronic and acute brain injuries induce localized insults that targets subparts of neurons and/or neuronal networks. However, the “distal” consequences of such focal aggression on the neuronal integrity are still unknown. Moreover, recent studies reveal that neurodegenerative hallmarks in AD (beta amyloid accumulation and tau phosphorylation) progress both spatially and temporally following specific neuronal pathways. This calls for studies analyzing acute and chronic neurodegenerative processes in neuronal networks. In this study, we use a new micro-fluidic culture platform that enables the in vitro reconstruction of oriented neuronal networks with two or three different interconnected neuronal populations (cortex, striatum and hippocampus) each one being individually accessible. We used these reconstructed networks to model i) acute cortical axon severing or ii) the effect offocalized Aß deposition on a restricted area of the network to evaluate their consequences on distant axonal and synaptic integrity. Our results show that both, cortical axon severing and cortical amyloid deposition leads to the propagation of an anterograde degenerative signal involving distant axo-dendriticsynaptic disconnection. Interestingly, this synaptic collapse is associated with an altered glutamatergic neurotransmission that culminates in trans-synaptic degenerative events in the connected post-synaptic neurons. Comparing the vulnerability of different post synaptic neurons, we test whether insults restricted to presynaptic cortical neurons leads to the propagation of degeneration along preferentialneur onal pathways. In conclusion our work aims at assessing the cellular and molecular mechanisms involved in the progression of degenerative events along neuro-anatomical pathways.

Retinoid-related orphan receptor alpha 1 (ROR alpha 1) 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 ROR alpha 1 in neuronal survival is the control of reactive oxygen species homeostasis. ROR alpha 1 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 ROR alpha 1 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 ROR alpha 1 activity. We also investigated mito-gen-activated protein kinase/extracellular signal-regulated kinase (1/2) involvement in this effect. Our results bring new insights into the control of ROR alpha 1 activity and highlight its importance in further investigations of the mechanisms involved in neuronal cell death in neurodegenerative diseases.